diff --git a/.gitignore b/.gitignore new file mode 100644 index 0000000..6f38bdf --- /dev/null +++ b/.gitignore @@ -0,0 +1,3 @@ +node_modules +test/dataset/out +.cache/* \ No newline at end of file diff --git a/index.js b/index.js new file mode 100644 index 0000000..3c76e6c --- /dev/null +++ b/index.js @@ -0,0 +1,121 @@ +/* global module */ +/* jslint node: true */ +/* jslint indent: 2 */ +'use strict'; + +var teeft = require('rd-teeft'), + async = require('async'), + path = require('path'), + fs = require('fs'); + +var jLouvain = require('./lib/jLouvain.js'); + +var myObject = {}; + +myObject.indexAll = function(directory, output, cb) { + var result = {}; // Regroup keywords by document Id + fs.readdir(directory, function(err, filenames) { + if (err) return cb(err); // I/O Errors + async.each(filenames, function(filename, callback) { + var filePath = path.join(directory, filename); + fs.readFile(filePath, 'utf-8', function(err, res) { + if (err) return callback(err); // I/O Errors + var docId = path.basename(filename, ('.txt')); + result[docId] = teeft.index(res).keywords; + callback(); + }); + }, function(err) { + if (err) return cb(err); // I/O Errors + // write data + fs.writeFile(output || './cache/indexAll.json', JSON.stringify(result), 'utf-8', function(err, res) { + if (err) return cb(err); + return cb(null, result); + }); + }); + }); +}; + +myObject.graphs = {} + +myObject.graphs.docToDoc = function(keywords, options, cb) { + if (!options) options = {}; + var terms = {}, // Each key is a term, his value is the list of documents containing it + documents = Object.keys(keywords), // List of document Ids + result = { + 'nodes': [], + 'links': [] + }, + edges = [], // [{'source': '', 'target': '', 'weight': 0}, ...] + nodes = [], // ['id', ...] + matrix = {}, // Matrix of "doc-doc" links (sparse matrix) + output = options.output || './cache/docToDoc.json', + minLinkValue = options.minLinkValue || 0; + // Construction of terms Object + for (var i = 0; i < documents.length; i++) { + var doc = documents[i]; + for (var j = 0; j < keywords[doc].length; j++) { + var term = keywords[doc][j].term; + if (!terms[term]) terms[term] = []; + terms[term].push(i); + } + } + // Construction of matrix Object + for (var key in terms) { + // Fill it with values + for (var i = 0; i < terms[key].length - 1; i++) { + var idDoc1 = terms[key][i]; + for (var j = i + 1; j < terms[key].length; j++) { + var idDoc2 = terms[key][j], + ids = [idDoc1, idDoc2], + id = { + 'min': Math.min(ids[0], ids[1]), + 'max': Math.max(ids[0], ids[1]) + }; + // Only half of it will be fill! + if (!matrix[id.min + ',' + id.max]) { + matrix[id.min + ',' + id.max] = 0; + } + matrix[id.min + ',' + id.max]++; + } + } + } + // Construction of matrix of links doc-doc + for (var key in matrix) { + var ids = key.split(','); + if (matrix[key] > minLinkValue) { + edges.push({ + 'source': ids[0], + 'target': ids[1], + 'weight': matrix[key] + }); + result.links.push({ + 'source': ids[0], + 'target': ids[1], + 'value': matrix[key] + }); + } + } + // Construction of Nodes object + for (var i = 0; i < documents.length; i++) { + nodes.push(i); + result.nodes.push({ + 'id': i, + 'value': documents[i], + 'group': 0 + }); + } + // Create the "community" + var community = jLouvain().nodes(nodes).edges(edges), + res = community(); + // Affect community for each node + for (var key in res) { + result.nodes[key].group = res[key]; + } + // write data + fs.writeFile(output, JSON.stringify(result), 'utf-8', function(err, res) { + if (err) return cb(err); + return cb(null, result); + }); +}; + +module.exports = myObject; \ No newline at end of file diff --git a/lib/jLouvain.js b/lib/jLouvain.js new file mode 100644 index 0000000..1181efe --- /dev/null +++ b/lib/jLouvain.js @@ -0,0 +1,398 @@ +/* + Author: Corneliu S. (github.com/upphiminn) + This is a javascript implementation of the Louvain + community detection algorithm (http://arxiv.org/abs/0803.0476) + Based on https://bitbucket.org/taynaud/python-louvain/overview + */ +var jLouvain = function () { + //Constants + var __PASS_MAX = -1; + var __MIN = 0.0000001; + + //Local vars + var original_graph_nodes; + var original_graph_edges; + var original_graph = {}; + var partition_init; + + //Helpers + function make_set(array) { + var set = {}; + array.forEach(function (d, i) { + set[d] = true; + }); + + return Object.keys(set); + } + + function obj_values(obj) { + var vals = []; + for (var key in obj) { + if (obj.hasOwnProperty(key)) { + vals.push(obj[key]); + } + } + + return vals; + } + + function get_degree_for_node(graph, node) { + var neighbours = graph._assoc_mat[node] ? Object.keys(graph._assoc_mat[node]) : []; + var weight = 0; + neighbours.forEach(function (neighbour, i) { + var value = graph._assoc_mat[node][neighbour] || 1; + if (node === neighbour) { + value *= 2; + } + weight += value; + }); + + return weight; + } + + function get_neighbours_of_node(graph, node) { + if (typeof graph._assoc_mat[node] === 'undefined') { + return []; + } + + var neighbours = Object.keys(graph._assoc_mat[node]); + + return neighbours; + } + + + function get_edge_weight(graph, node1, node2) { + return graph._assoc_mat[node1] ? graph._assoc_mat[node1][node2] : undefined; + } + + function get_graph_size(graph) { + var size = 0; + graph.edges.forEach(function (edge) { + size += edge.weight; + }); + + return size; + } + + function add_edge_to_graph(graph, edge) { + update_assoc_mat(graph, edge); + + var edge_index = graph.edges.map(function (d) { + return d.source + '_' + d.target; + }).indexOf(edge.source + '_' + edge.target); + + if (edge_index !== -1) { + graph.edges[edge_index].weight = edge.weight; + } else { + graph.edges.push(edge); + } + } + + function make_assoc_mat(edge_list) { + var mat = {}; + edge_list.forEach(function (edge, i) { + mat[edge.source] = mat[edge.source] || {}; + mat[edge.source][edge.target] = edge.weight; + mat[edge.target] = mat[edge.target] || {}; + mat[edge.target][edge.source] = edge.weight; + }); + + return mat; + } + + function update_assoc_mat(graph, edge) { + graph._assoc_mat[edge.source] = graph._assoc_mat[edge.source] || {}; + graph._assoc_mat[edge.source][edge.target] = edge.weight; + graph._assoc_mat[edge.target] = graph._assoc_mat[edge.target] || {}; + graph._assoc_mat[edge.target][edge.source] = edge.weight; + } + + function clone(obj) { + if (obj === null || typeof(obj) !== 'object') + return obj; + + var temp = obj.constructor(); + + for (var key in obj) { + temp[key] = clone(obj[key]); + } + + return temp; + } + + //Core-Algorithm Related + function init_status(graph, status, part) { + status['nodes_to_com'] = {}; + status['total_weight'] = 0; + status['internals'] = {}; + status['degrees'] = {}; + status['gdegrees'] = {}; + status['loops'] = {}; + status['total_weight'] = get_graph_size(graph); + + if (typeof part === 'undefined') { + graph.nodes.forEach(function (node, i) { + status.nodes_to_com[node] = i; + var deg = get_degree_for_node(graph, node); + + if (deg < 0) + throw 'Bad graph type, use positive weights!'; + + status.degrees[i] = deg; + status.gdegrees[node] = deg; + status.loops[node] = get_edge_weight(graph, node, node) || 0; + status.internals[i] = status.loops[node]; + }); + } else { + graph.nodes.forEach(function (node, i) { + var com = part[node]; + status.nodes_to_com[node] = com; + var deg = get_degree_for_node(graph, node); + status.degrees[com] = (status.degrees[com] || 0) + deg; + status.gdegrees[node] = deg; + var inc = 0.0; + + var neighbours = get_neighbours_of_node(graph, node); + neighbours.forEach(function (neighbour, i) { + var weight = graph._assoc_mat[node][neighbour]; + + if (weight <= 0) { + throw "Bad graph type, use positive weights"; + } + + if (part[neighbour] === com) { + if (neighbour === node) { + inc += weight; + } else { + inc += weight / 2.0; + } + } + }); + status.internals[com] = (status.internals[com] || 0) + inc; + }); + } + } + + function __modularity(status) { + var links = status.total_weight; + var result = 0.0; + var communities = make_set(obj_values(status.nodes_to_com)); + + communities.forEach(function (com, i) { + var in_degree = status.internals[com] || 0; + var degree = status.degrees[com] || 0; + if (links > 0) { + result = result + in_degree / links - Math.pow((degree / (2.0 * links)), 2); + } + }); + + return result; + } + + function __neighcom(node, graph, status) { + // compute the communities in the neighb. of the node, with the graph given by + // node_to_com + var weights = {}; + var neighboorhood = get_neighbours_of_node(graph, node);//make iterable; + + neighboorhood.forEach(function (neighbour, i) { + if (neighbour !== node) { + var weight = graph._assoc_mat[node][neighbour] || 1; + var neighbourcom = status.nodes_to_com[neighbour]; + weights[neighbourcom] = (weights[neighbourcom] || 0) + weight; + } + }); + + return weights; + } + + function __insert(node, com, weight, status) { + //insert node into com and modify status + status.nodes_to_com[node] = +com; + status.degrees[com] = (status.degrees[com] || 0) + (status.gdegrees[node] || 0); + status.internals[com] = (status.internals[com] || 0) + weight + (status.loops[node] || 0); + } + + function __remove(node, com, weight, status) { + //remove node from com and modify status + status.degrees[com] = ((status.degrees[com] || 0) - (status.gdegrees[node] || 0)); + status.internals[com] = ((status.internals[com] || 0) - weight - (status.loops[node] || 0)); + status.nodes_to_com[node] = -1; + } + + function __renumber(dict) { + var count = 0; + var ret = clone(dict); //deep copy :) + var new_values = {}; + var dict_keys = Object.keys(dict); + dict_keys.forEach(function (key) { + var value = dict[key]; + var new_value = typeof new_values[value] === 'undefined' ? -1 : new_values[value]; + if (new_value === -1) { + new_values[value] = count; + new_value = count; + count = count + 1; + } + ret[key] = new_value; + }); + + return ret; + } + + function __one_level(graph, status) { + //Compute one level of the Communities Dendogram. + var modif = true; + var nb_pass_done = 0; + var cur_mod = __modularity(status); + var new_mod = cur_mod; + + while (modif && nb_pass_done !== __PASS_MAX) { + cur_mod = new_mod; + modif = false; + nb_pass_done += 1 + + graph.nodes.forEach(function (node, i) { + var com_node = status.nodes_to_com[node]; + var degc_totw = (status.gdegrees[node] || 0) / (status.total_weight * 2.0); + var neigh_communities = __neighcom(node, graph, status); + __remove(node, com_node, (neigh_communities[com_node] || 0.0), status); + var best_com = com_node; + var best_increase = 0; + var neigh_communities_entries = Object.keys(neigh_communities);//make iterable; + + neigh_communities_entries.forEach(function (com, i) { + var incr = neigh_communities[com] - (status.degrees[com] || 0.0) * degc_totw; + if (incr > best_increase) { + best_increase = incr; + best_com = com; + } + }); + + __insert(node, best_com, neigh_communities[best_com] || 0, status); + + if (best_com !== com_node) { + modif = true; + } + }); + new_mod = __modularity(status); + if (new_mod - cur_mod < __MIN) { + break; + } + } + } + + function induced_graph(partition, graph) { + var ret = {nodes: [], edges: [], _assoc_mat: {}}; + var w_prec, weight; + //add nodes from partition values + var partition_values = obj_values(partition); + ret.nodes = ret.nodes.concat(make_set(partition_values)); //make set + graph.edges.forEach(function (edge, i) { + weight = edge.weight || 1; + var com1 = partition[edge.source]; + var com2 = partition[edge.target]; + w_prec = (get_edge_weight(ret, com1, com2) || 0); + var new_weight = (w_prec + weight); + add_edge_to_graph(ret, {'source': com1, 'target': com2, 'weight': new_weight}); + }); + + return ret; + } + + function partition_at_level(dendogram, level) { + var partition = clone(dendogram[0]); + for (var i = 1; i < level + 1; i++) { + Object.keys(partition).forEach(function (key, j) { + var node = key; + var com = partition[key]; + partition[node] = dendogram[i][com]; + }); + } + + return partition; + } + + + function generate_dendogram(graph, part_init) { + if (graph.edges.length === 0) { + var part = {}; + graph.nodes.forEach(function (node, i) { + part[node] = node; + }); + return part; + } + var status = {}; + + init_status(original_graph, status, part_init); + var mod = __modularity(status); + var status_list = []; + __one_level(original_graph, status); + var new_mod = __modularity(status); + var partition = __renumber(status.nodes_to_com); + status_list.push(partition); + mod = new_mod; + var current_graph = induced_graph(partition, original_graph); + init_status(current_graph, status); + + while (true) { + __one_level(current_graph, status); + new_mod = __modularity(status); + if (new_mod - mod < __MIN) { + break; + } + + partition = __renumber(status.nodes_to_com); + status_list.push(partition); + + mod = new_mod; + current_graph = induced_graph(partition, current_graph); + init_status(current_graph, status); + } + + return status_list; + } + + var core = function () { + var status = {}; + var dendogram = generate_dendogram(original_graph, partition_init); + + return partition_at_level(dendogram, dendogram.length - 1); + }; + + core.nodes = function (nds) { + if (arguments.length > 0) { + original_graph_nodes = nds; + } + + return core; + }; + + core.edges = function (edgs) { + if (typeof original_graph_nodes === 'undefined') + throw 'Please provide the graph nodes first!'; + + if (arguments.length > 0) { + original_graph_edges = edgs; + var assoc_mat = make_assoc_mat(edgs); + original_graph = { + 'nodes': original_graph_nodes, + 'edges': original_graph_edges, + '_assoc_mat': assoc_mat + }; + } + + return core; + + }; + + core.partition_init = function (prttn) { + if (arguments.length > 0) { + partition_init = prttn; + } + return core; + }; + + return core; +} + +module.exports = jLouvain; \ No newline at end of file diff --git a/package.json b/package.json new file mode 100644 index 0000000..8273b24 --- /dev/null +++ b/package.json @@ -0,0 +1,21 @@ +{ + "name": "ez-indexation", + "version": "1.0.0", + "description": "Script permettant d'indexer le contenu d'un répertoire", + "main": "index.js", + "scripts": { + "test": "mocha -t 120000" + }, + "repository": { + "type": "git", + "url": "https://git.istex.fr/git/kieffer/tdm-utils.git" + }, + "author": "istex", + "license": "ISC", + "dependencies": { + "async": "^2.1.4", + "mocha": "^3.2.0", + "rd-tu": "git+ssh://vsistex.intra.inist.fr:22222/istex/rd-tu.git", + "rd-teeft": "git+ssh://vsistex.intra.inist.fr:22222/istex/rd-teeft.git" + } +} diff --git a/test/dataset/in/data/dataset.json b/test/dataset/in/data/dataset.json new file mode 100644 index 0000000..3cefc27 --- /dev/null +++ b/test/dataset/in/data/dataset.json @@ -0,0 +1,27 @@ +{ + "indexAll": [{ + "label": "Devrait indexer tout le corpus", + "arguments": { + "directory": "./test/dataset/in/resources/corpus/", + "output": "./test/dataset/out/indexAll.json" + }, + "result": { + "equal": null + } + }], + "graphs": { + "docToDoc": [{ + "label": "Devrait créer le graph doc2doc", + "arguments": { + "filePath": "./test/dataset/out/indexAll.json", + "options": { + "output": "./test/dataset/out/docToDoc.json", + "minLinkValue": 1 + } + }, + "result": { + "equal": null + } + }] + } +} \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_000B8DFD0BDAA98015E637CFBF02826F25DB705E.txt b/test/dataset/in/resources/corpus/Clean_000B8DFD0BDAA98015E637CFBF02826F25DB705E.txt new file mode 100644 index 0000000..3863585 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_000B8DFD0BDAA98015E637CFBF02826F25DB705E.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 93210.1093/gerona/62.9.932 Journal of Gerontology: Biological Sciences Reduction in Glutathione Peroxidase 4 Increases Life Span Through Increased Sensitivity to Apoptosis Ran Qitao 1 3 Liang Hanyu Ikeno Yuji 1 3 Qi Wenbo Prolla Tomas A. Roberts L. Jackson II 5 Wolf Norman VanRemmen Holly 1 3 Richardson Arlan 1 3 1Department of Cellular and Structural Biology, and 2Barshop Institute for Longevity and Aging Studies, the University of Texas Health Science Center at San Antonio. 3Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, San Antonio. 4Departments of Genetics & Medical Genetics, University of Wisconsin-Madison. 5Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee. 6Department of Pathology, University of Washington, Seattle. Address correspondence to Arlan Richardson, PhD, Barshop Institute for Longevity and Aging Studies, 15355 Lambda Drive, San Antonio, TX 78245-3207. E-mail: richardsona@uthscsa.edu 9 2007 62 9 932 942 19 6 2007 1 5 2007 Copyright 2007 by The Gerontological Society of America 2007 Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme that plays an important role in detoxification of oxidative damage to membrane lipids. Because oxidative stress is proposed to play a causal role in aging, we compared the life spans of Gpx4 heterozygous knockout mice (Gpx4+/− mice) and wild-type mice (WT mice). To our surprise, the median life span of Gpx4+/− mice (1029 days) was significantly longer than that of WT mice (963 days) even though the expression of Gpx4 was reduced approximately 50% in all tissues of Gpx4+/− mice. Pathological analysis revealed that Gpx4+/− mice showed a delayed occurrence of fatal tumor lymphoma and a reduced severity of glomerulonephritis. Compared to WT mice, Gpx4+/− mice showed significantly increased sensitivity to oxidative stress-induced apoptosis. Our data indicate that lifelong reduction in Gpx4 increased life span and reduced/retarded age-related pathology most likely through alterations in sensitivity of tissues to apoptosis. hwp-legacy-fpage 932 hwp-legacy-dochead RESEARCH ARTICLE REACTIVE oxygen species (ROS), such as superoxide and hydrogen peroxide, are constantly generated in aerobic organisms by a variety of pathways; however, the major source of the production of ROS is mitochondria. Although ROS at physiological concentrations may be essential for normal cellular functions, such as cell signaling, excessive amounts of ROS can be detrimental because ROS can cause oxidative damage to lipids, protein, and DNA. Due to the presence of allylic hydrogens, polyunsaturated fatty acids, which are found predominantly in membranes of cells other than adipocytes, are especially vulnerable to attack by ROS (1). The resultant lipid hydroperoxides can be detrimental to cells in two ways. First, lipid hydroperoxides can impair membrane fluidity and function of membrane proteins, which could compromise the function of cells. Second, lipid hydroperoxides can undergo iron- and oxygen-mediated chain-breaking lipid peroxidation to generate reactive aldehydes such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) (2), which can attack other cellular targets, such as proteins and DNA, thereby propagating the initial damage in cellular membranes to other macromolecules and to other parts of cells. Therefore, lipid peroxidation appears to be a primary mechanism in the injury and demise of cells in response to oxidative stress. The glutathione peroxidases (Gpxs) are a group of selenoproteins that catalyze the reduction of peroxides generated by ROS at the expense of glutathione (3). Four selenium-containing Gpxs have been identified. Gpx1 is the most abundant Gpx and is ubiquitously expressed. Gpx2 is expressed in the gastrointestinal tract. Gpx3 is a plasma form of Gpx. Gpx4, known as phospholipid hydroperoxide glutathione peroxidase, is ubiquitously expressed and is a key enzyme in the detoxification of lipid hydroperoxides (2). All Gpxs can reduce hydrogen peroxide, alkyl peroxides, and fatty acid hydroperoxides; however, Gpx4 also reduces hydroperoxides in lipoproteins and complex lipids such as those derived from cholesterol, cholesteryl esters, and phospholipids. Although Gpx4 is a ubiquitously expressed enzyme, its activity makes up only a small fraction of total cellular Gpx activity in somatic tissues (3). Because of its small size and large hydrophobic surface, Gpx4 can interact with complex lipids in membranes and thereby detoxify membrane lipid hydroperoxides (4). The other major pathway for removing lipid peroxides from membranes is through the coupled actions of phospholipase A2 (PLA2) and Gpx1 (5): PLA2 first excises the fatty acid hydroperoxide from phospholipid hydroperoxide in the membrane, and Gpx1 then reduces the fatty acid hydroperoxide to alcohol and water. Based on kinetic modeling, Gpx4 is estimated to be much more efficient at removing phospholipid hydroperoxides than the PLA2–Gpx1 pathway because the affinity of Gpx4 to membrane phospholipid hydroperoxides is more than 104-fold greater than that of PLA2 (6). Thus, Gpx4 is considered the primary enzymatic defense system against oxidative damage to cellular membranes (3). Indeed, studies using mice deficient in Gpx4 and transgenic mice overexpressing Gpx4 have shown that Gpx4 is an essential enzyme and plays a critical role in antioxidant defense. For example, the homozygous null mutation of Gpx4 in mice is embryonic lethal (7,8), and cells from heterozygous Gpx4 knockout mice show increased lipid peroxidation and more cell death after exposure to oxidizing agents (9). Transgenic mice overexpressing Gpx4 are more resistant to oxidative insults (10), and cortical neurons from Gpx4 transgenic mice are more resistant to β-amyloid cytotoxicity (11). In addition, Gpx4 has been shown to play an important role in regulating apoptosis, and the attributed mechanism appears to be Gpx4's activity in regulating the oxidation of cardiolipin (CL), a mitochondrial membrane lipid that is rich in polyunsaturated fatty acids (12). One of the most popular theories of aging is the Free Radical or Oxidative Stress Theory of Aging. The basis of this theory is that cells exist in a chronic state of oxidative stress resulting from an imbalance between pro-oxidants and antioxidants. Because of this imbalance, which occurs as a consequence of aerobic metabolism, an accumulation of oxidative damage is proposed to occur with age in a variety of macromolecules within the cell. This steady-state accumulation of oxidative damage is thought to be an important mechanism underlying aging, age-related increases in pathology, and the progressive decline in the functional efficiency of various cellular processes (13,14). Among the macromolecules, polyunsaturated fatty acids in lipids and lipoproteins are the most prone to oxidative damage; therefore, membrane lipid peroxidation is believed to be especially pivotal in aging (15,16). The critical role of lipid peroxidation in aging is supported by studies showing increased levels of lipid peroxidation in aged animals (17,18), as well as by studies showing that calorie restriction, a manipulation known to retard aging, attenuates the age-related increase in lipid peroxidation (17,19). However, whether the modulation of membrane oxidation has a direct effect on life span is not known. Because of the high specificity and efficiency of Gpx4 in removing lipid hydroperoxides from membranes, animal models with altered levels of Gpx4 are expected to show altered levels of lipid peroxidation and, based on the Oxidative Stress Theory of Aging, to show altered aging and/or life spans. We have shown previously that Gpx4 heterozygous knockout (Gpx4+/−) mice have about a 50% reduction in Gpx4 levels in all tissues, which is consistent with a gene-dosage effect (7). Therefore, the Gpx4+/− mouse appears to be an ideal model to test directly the effect of increased membrane lipid peroxidation on aging. If lipid peroxidation plays an essential role in aging, one would predict that Gpx4+/− mice, which are more prone to lipid peroxidation of membranes, will have a shortened life span. In this study, we compared the life spans and age-related pathologies between Gpx4+/− mice and wild-type (WT) mice. Our results demonstrate that Gpx4+/− mice did not have a shortened life span compared to WT mice. In fact, Gpx4+/− mice had an increased median survival, and age-related pathology was retarded or reduced in the Gpx4+/− mice. Methods Animals Gpx4+/− mice, heterozygous for a targeted mutation in the Gpx4 gene, were originally generated in the 129 background (7). The mice used in this study were backcrossed 10 times to C57BL/6 mice. All procedures were approved by the Institutional Animal Care and Usage Committee at the University of Texas Health Science Center at San Antonio and the South Texas Veterans Health Care System, Audie L. Murphy Division. The colony of Gpx4+/− mice used for this study was generated by breeding male Gpx4+/− mice to female WT C57BL/6 mice purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were genotyped at 4–5 weeks of age by polymerase chain reaction (PCR) analysis of DNA obtained from tail clips as previously described (7). The mice were maintained under barrier conditions in a temperature-controlled environment. For the life-span studies, 50 male WT and 50 male Gpx4+/− mice born between January 2003 and February 2003 were housed four animals per cage starting at 2 months of age, and were fed a commercial mouse chow (Teklad Diet LM485; Harlan Teklad, Madison, WI) ad libitum. Mice assigned to survival groups were allowed to live out their life span. In other words, there was no censoring in either the WT or the Gpx4+/− mice when measuring survival. Life spans for Gpx4+/− mice and WT mice were determined by recording the age of spontaneous death of male Gpx4+/− and WT mice. The mean, median, 10% (the mean life span of longest-lived 10% animals), and maximum (the age of death for the longest-lived mouse in the cohort) life spans were calculated from the survival data for each genotype. Cataract Assessment To assess cataract formation, cataracts were read using a handheld slit lamp at a 30° angle after dilation with 1% tropicamide. Both eyes were scored on an opacity scale of 0, 1, 2, 3, or 4, with 4 representing complete lens opacity as described previously (20). Measurement of Gpx4 Protein Level and Oxidative Damage Tissues were collected from male mice and frozen immediately in liquid nitrogen. The tissues were stored at −80°C until used to measure enzyme activities or level of oxidative damage. Levels of Gpx4, Gpx1, catalase, manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/ZnSOD) were determined as previously described (10). Plasma and liver levels of F2-isoprostanes were determined as described by Morrow and Roberts (21). Briefly, blood was collected from the inferior vena cava of anesthetized animals, and then the liver was removed and immediately frozen in liquid nitrogen for storage at −80°C. F2-isoprostanes were extracted and quantified by gas chromatography-mass spectrometry (GC-MS) using the internal standard, 8-iso Prostaglandin F2α-d4 (Cayman Chemical, Ann Arbor, MI), which was added to the samples at the beginning of extraction to correct the yield of the extraction process. The amount of F2-isoprostanes in liver was expressed as picograms of 8-Iso-PGF2α per milligram of total liver protein, and the amount of F2-isoprostanes in plasma was expressed as picograms of 8-Iso-PGF2α per milliliter of plasma. F4-neuroprostanes in cerebral cortexes, which were dissected out and immediately frozen in liquid nitrogen, were extracted and determined as described by Roberts and colleagues (22). Briefly, after separation by thin layer chromatography, F4-neuroprostanes were quantified by GC/MS. The amount of F4-neuroprostanes in brain was expressed as nanograms per gram of total brain protein. Pathological Analysis All 50 Gpx4+/− mice in the survival group were subjected to end-of-life pathological analysis. We were unable to analyze three of the animals in the WT survival cohort because autolysis was too severe to obtain pathological data; therefore, 47 WT mice were subjected to end-of-life pathological analysis. After spontaneous death, mice were necropsied for gross pathological lesions. Organs and tissues were excised and preserved in 10% buffered formalin. The fixed tissues were processed conventionally, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin–eosin. For each mouse, a list of pathological lesions was constructed that included both neoplastic and nonneoplastic diseases. Based on these histopathological data, the tumor burden, disease burden, and severity of each lesion in each mouse were assessed (23). The severity of neoplastic and nephrologic lesions was assessed using the grading system previously described (24,25). For example, glomerulonephritis was graded in order of increasing severity: Grade 0: no lesions; Grade 1: minimal change in glomeruli (minimal glomerulosclerosis); Grade 2: Grade 1 with a few (< 10) casts in renal tubules; Grade 3: Grade 1 with > 10 casts in renal tubules; and Grade 4: Grade 3 with interstitial fibrosis. The probable cause of death for each mouse was determined by the severity of diseases found by necropsy. For neoplastic diseases, cases that had Grade 3 or 4 lesions were categorized as death by neoplastic lesions. For nonneoplastic diseases, cases that had a severe lesion, e.g., Grade 4, associated with other histopathological changes (pleural effusion, ascites, congestion and edema in lung) were categorized as death by nonneoplastic lesions. In > 90% of the cases, there was agreement by two pathologists. In cases in which there was not agreement or in which no one disease was considered severe enough, the cause of death was evaluated as undetermined. Measurement of Apoptosis Male mice, age 26–29 months, received an intraperitoneal injection of diquat dissolved in saline at a dose of 50 mg/kg. This dose is nonlethal, i.e., all of the mice survived this dose of diquat. Six hours after injection, the livers were collected for use in the assays described below. A small piece of liver tissue was fixed in 10% buffered formalin and was embedded in paraffin for the measurement of liver apoptosis. Another piece of liver tissue was homogenized in ice-old buffer I (250 mM mannitol, 75 mM sucrose, 500 μM EGTA, 100 μM EDTA, and 10 mM HEPES, pH 7.4) supplemented with a protease inhibitor cocktail. The homogenates were centrifuged at 600 × g for 10 minutes at 4°C to pellet nuclei and unbroken cells. The resultant supernatant was then centrifuged at 10,000 × g for 15 minutes at 4°C to obtain the mitochondrial pellet. The supernatant was further centrifuged at 100,000 × g for 60 minutes at 4°C to yield the cytosol. The mitochondrial pellets were washed once in buffer I containing 0.2% (wt/vol) bovine serum albumin (BSA) and twice in buffer I without BSA. The mitochondrial pellets obtained were used for CL peroxidation. The cytosols obtained were used to measure cytochrome c (cyt. c) release. Apoptotic cell counts in the liver were determined in situ by the presence of double-strand DNA breaks observed in paraffin-embedded tissue sections using an in situ oligo ligation (ISOL) kit (Chemicon International, Temecula, CA) with oligo B, according to the manufacturer's instructions. Compared to a conventional TdT-mediated dUTP nick-end labeling (TUNEL) assay, the ISOL assay uses a hairpin oligonucleotide probe to detect more specific DNA fragmentation caused by apoptosis, avoiding randomly damaged DNA (26). Slides were visualized under light microscopy, and the number of positive cells was determined in 10 random fields at ×400 magnification for each liver. Data are expressed as the mean of the ratio of the number of positive cells to the total number of cells in all 10 random fields. Release of cyt. c from mitochondria was detected by measuring the levels of cyt. c in cytosol of liver using Western blots. Briefly, cytosolic proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as described above, and the levels of cyt. c were detected using an anti-cyt. c antibody (sc-13560; Santa Cruz Biotechnology, Santa Cruz, CA). The intensities of the bands on the blot were quantified with ImageQuant 5.0 (Molecular Dynamics, Sunnyvale, CA) and normalized to a cytosolic loading control (IκB-α) as we have described previously (10). CL peroxidation was measured by 10-N-nonyl-Acridine Orange (NAO; Molecular Probes, Eugene, OR) binding to mitochondria as previously described by Petit and colleagues (27), with modifications. To avoid any potential effect of mitochondrial membrane potential on NAO binding (28), mitochondria were not energized with substrates in these experiments. Briefly, freshly isolated liver mitochondria were resuspended in buffer A (125 mM KCl, 10 mM HEPES, 5 mM MgCl2, and 2 mM K2HPO4, pH 7.4). The mitochondrial protein concentration was determined by the Bradford method. An equal amount of mitochondria (10 μg of mitochondrial protein) was added to 200 μL of 20 μM NAO, incubated for 5 minutes, then centrifuged at 30,000 × g for 5 minutes. Free dye in the supernatant was determined by measuring absorbance at 495 nm, and the NAO bound to the mitochondria was calculated as the total NAO minus free NAO. Results Based on the Oxidative Stress Theory of Aging and the known function of Gpx4 in the protection of cells against oxidative damage, we predicted that a reduced level of Gpx4 would lead to accelerated aging, as shown by a shortened life span. To test if this prediction was valid, we compared life spans of Gpx4+/− and WT mice. As shown in Figure 1, the life span of Gpx4+/− mice was not reduced compared to that of WT mice. In fact, Gpx4+/− mice had an increased life span. Compared to that of WT mice, the median life span of Gpx4+/− mice was increased by 7%, from 963 days to 1029 days, and this increase is statistically significant. The mean life span of Gpx4+/− mice also was longer, about 5% longer than the mean life span of WT mice; however, this difference did not reach statistical significance. The survival curves for these two groups of mice came together at around 1100 days of age, and no difference in mean life span of the top 10% longest-lived animals was observed. The age-dependent mortality rates for Gpx4+/− mice and WT mice, as determined by mathematical models described by Pletcher and colleagues (29), were not significantly different (data not shown). We also compared body weights of Gpx4+/− mice and WT mice because it is well established that reduced food consumption, which is shown by reduced body weight, leads to increased life span. As shown in Figure 2A, there was no difference in body weight between Gpx4+/− and WT mice from 8 weeks of age up to 24 months of age. Thus, there is no evidence that the Gpx4+/− mice are living longer because of reduced caloric intake. Interestingly, the Gpx4+/− mice showed less of a decrease in body weight than WT mice starting at 24 months of age, suggesting that the loss in body weight, which is common in aging, was retarded in the Gpx4+/− mice. To determine whether other parameters that change with age are altered in the Gpx4+/− mice, the appearance of cataracts in WT and Gpx4+/− mice at 25 months of age was determined because the development of cataracts is often used as a biological marker of aging (20). As shown in Figure 2B, there was no difference in cataract development between WT and Gpx4+/− mice. In our previous study (7), we demonstrated that the expression of Gpx4 (messenger RNA and protein) was reduced approximately 50% in 4- to 6-month-old Gpx4+/− mice, in comparison to age-matched WT mice. However, we were concerned that the lack of predicted effect on survival of Gpx4+/− mice might be due to an up-regulation of the wild-type allele of Gpx4 gene in older Gpx4+/− mice. In other words, the Gpx4+/− mice might not be deficient in Gpx4 as they aged. To determine if this is true, we measured Gpx4 protein levels in old Gpx4+/− mice and WT mice. As shown in Figure 3, the levels of Gpx4 protein in tissues from 26- to 29-month-old Gpx4+/− mice were reduced by approximately 50%, which is similar to what we had observed in young Gpx4+/− mice (7). Therefore, the Gpx4+/− mice have reduced expression of Gpx4 throughout their life span. Although our previous study found no differences in other major antioxidant defense enzymes in young Gpx4+/− mice, we were also concerned that the compensatory up-regulation of other antioxidant defense enzymes with age might neutralize the effect of Gpx4 deficiency on survival in Gpx4+/− mice. Thus, we measured levels of other major antioxidant defense enzymes such as Gpx1, MnSOD, and Cu/ZnSOD in old Gpx4+/− and WT mice and found no significant differences in these enzymes between the Gpx4+/− and WT mice (data not shown). Therefore, there was no compensatory up-regulation of other major antioxidant defense enzymes. To determine whether the reduced Gpx4 level in Gpx4+/− mice led to increased oxidative damage, we compared the levels of lipid peroxidation in old Gpx4+/− and WT mice. Isoprostanes and neuroprostanes are groups of prostaglandin-like compounds that arise from free radical attack on membrane phospholipids. Quantification of F2-isoprostanes has emerged as an accurate measurement of lipid peroxidation in vivo (30), and we showed that the level of F2-isoprostanes increases with age in plasma and other tissues of rats and is reduced by caloric restriction (31). Figure 4, A and B, shows the levels F2-isoprostanes in plasma and liver of Gpx4+/− mice and WT mice. Although the levels of F2-isoprostane were slightly higher in old Gpx4+/− mice than in old WT mice, the differences were not significant. We also observed no statistically significant difference in DNA oxidation in liver tissue between Gpx4+/− mice and WT mice (data not shown). We also measured the levels F4-neuroprostane in the brains of Gpx4+/− and WT mice. F4-neuroprostanes are derived from oxidized docosahexaenoic acid, whereas F2-isoprostanes are derived from oxidized arachidonic acid. Docosahexaenoic acid is more prone to oxidation than arachidonic acid and is highly enriched in brain (22). We observed a 52% increase in F4-neuroprostanes levels in brains of old Gpx4+/− mice over old WT mice, and this increase is statistically significant. To determine whether the increase in the life span of the Gpx4+/− mice was related to changes in the incidence of pathological lesions, we conducted a comprehensive pathological analysis on the mice in the survival groups after they died. As shown in Table 1, the probable causes of death for the WT and Gpx4+/− mice were similar: Approximately 55%–60% of the WT and Gpx4+/− mice died of neoplastic diseases, and the Gpx4+/− mice showed no reduction in occurrence of fatal tumors compared to WT mice. As expected for mice in the C57BL/6 background, the majority of fatal tumors in both WT and Gpx4+/− mice was lymphoma. Interestingly, the Gpx4+/− mice that died from lymphoma had a longer median age than the WT mice that died from lymphoma (963 days vs 928 days). In addition, whereas 37.5% of Gpx4+/− mice that died from lymphoma were older than 1026 days, only 16.7% of WT mice that died from lymphoma died at ages above 1026 days. To determine whether the incidence of death from lymphoma in the survival groups were different as a function of age, we followed the incidence of death when the majority of mice began to die, i.e., after 900 days of age, when the difference in survival is greatest. As shown in Figure 5A, the age of this group of Gpx4+/− mice is higher than the age of the group of WT mice, indicating that the occurrence of fatal lymphoma was delayed in Gpx4+/− mice. The major nonneoplastic pathology observed in WT and Gpx4+/− mice was glomerulonephritis. About 20% of WT mice in the survival group died of glomerulonephritis, whereas none of the Gpx4+/− mice in the survival group died of glomerulonephritis. To determine whether the reduced deaths from glomerulonephritis in the Gpx4+/− mice occurred because of changes in the initiation and/or development/progression of the disease, we compared the total incidence and severity of glomerulonephritis in mice in the survival groups. Our data indicated that 42 (of 47) WT mice and 47 (of 50) Gpx4+/− mice had glomerulonephritis, indicating that the total incidence of glomerulonephritis was similar for Gpx4+/− mice and WT mice. However, as shown in Figure 5B, the severity of glomerulonephritis was significantly reduced in Gpx4+/− mice. Therefore, it appears that the progression/development of glomerulonephritis was suppressed in Gpx4+/− mice. The data in Table 1 also show that death from acidophilic macrophage pneumonia (AMP) was higher for the Gpx4+/− mice than for the WT mice, but that the difference between the two groups is not statically significant. Interestingly, 90% of the deaths from AMP occurred late in the life span of the Gpx4+/− mice (after 1044 days of age) and where the survival curves of the Gpx4+/− and WT mice are shown to merge. Thus, the lack of an increase in the maximum survival of the Gpx4+/− mice could be due to a greater propensity of the old Gpx4+/− mice to die from AMP. Increased apoptosis could remove damaged cells that could otherwise give rise to increased cancer or pathology (32). Because Gpx4 is shown to play an important role in regulating apoptosis (12), it is possible that the delayed occurrence of fatal lymphoma and reduced severity of glomerulonephritis were due to altered apoptosis in tissues of Gpx4+/− mice. Therefore, we compared the induction of apoptosis by oxidative stress in old Gpx4+/− mice and WT mice when differences in survival and pathology were observed. When injected intraperitoneally, diquat, a superoxide generator, affects primarily liver (33). As shown in Figure 6A, apoptotic cell levels were low in untreated animals, and no difference in levels of apoptotic cells was observed in untreated old Gpx4+/− mice and WT mice. However, after diquat treatment, Gpx4+/− mice had significantly more apoptotic cells than their WT counterparts had, indicating that old Gpx4+/− mice were more sensitive to diquat-induced apoptosis. Release of cyt. c from the mitochondria is a critical early event in the initiation of the intrinsic pathway (mitochondrial pathway) of apoptosis (34), and studies show that Gpx4 can regulate the intrinsic pathway of apoptosis by altering cyt. c release from mitochondria (10,35). Therefore, we also compared cytosolic cyt. c levels in livers from old Gpx4+/− and WT mice (Figure 6B). As shown in Figure 6C, without treatment, there was no statistically significant difference in cytosolic levels of cyt. c between Gpx4+/− mice and WT mice. Diquat treatment resulted in an increase in cytosolic cyt. c levels in both Gpx4+/− mice and WT; however, cytosolic levels of cyt. c were significantly higher in livers of Gpx4+/− mice than in livers of WT mice, indicating increased release of cyt. c from mitochondria in Gpx4+/− mice. To determine the mechanism underlying the effect of Gpx4 deficiency on the induction of apoptosis by oxidative stress, we measured CL oxidation. CL is a phospholipid that localizes exclusively in mitochondria, primarily the inner membrane, and binds to cyt. c. Due to its high content of polyunsaturated fatty acids, CL is easily oxidizable, and peroxidized CL loses its ability to bind to cyt. c, leading to cyt. c release from mitochondria (36). Gpx4 is shown to rapidly reduce CL hydroperoxide (CLOOH) to its alcohol derivative (CLOH), which can bind cyt. c as well as CL, thereby preventing cyt. c release from mitochondrial inner membrane (37). To determine whether the level of CL peroxidation was altered in Gpx4+/− mice, we measured levels of CLOOH in Gpx4+/− and WT mice by the NAO binding assay. NAO is a lipophilic dye that has a 30-fold greater affinity for CL over other anionic phospholipids such as phosphatidylserine and phosphatidylinositol, and does not bind phosphatidylcholine and phosphatidylethanolamine (27). NAO has been extensively used for visualization and quantification of CL in isolated mitochondria as well as in living cells (27,38–40). However, a recent study (41) questioned the specificity of NAO binding to CL in living cells. To avoid any possible interference from other lipid components, we used isolated liver mitochondria because mitochondria have only trace levels of phosphatidylserine and phosphatidylinositol; thus, the observed NAO binding would come from CL. CL peroxidation was measured by the loss of NAO binding to the mitochondria because the fluorochrome has no affinity for CL hydroperoxide (38,42,43). As shown in Figure 6D, NAO binding was reduced in mitochondria from untreated, old Gpx4+/− mice compared to old WT mice; however, this decrease was not statistically significant. Diquat treatment resulted in a significant decrease in NAO binding to mitochondria isolated from both Gpx4+/− and WT mice, showing an increase in CL peroxidation. However, NAO binding was significantly reduced in the Gpx4+/− mice compared to the WT mice, indicating that old Gpx4+/− mice had increased levels of CL peroxidation after diquat treatment. Discussion According to the popular Oxidative Stress Theory of Aging, the steady-state accumulation of oxidative damage plays an important role in the biological mechanism underlying aging, leading to age-related increases in pathology as well as to progressive declines in the functional efficiency of cells/tissues (13,14). Polyunsaturated fatty acids in lipids and lipoproteins are especially prone to oxidative damage, and an increase in lipid peroxidation is shown to occur with increasing age (17,18). The age-related increase in oxidative damage to membrane lipids could be physiologically important to an organism because lipid peroxidation is shown to play an important role in a variety of cellular processes, e.g., inactivation of membrane enzymes, alterations in functions of ion channels, collapse of membrane potential, and reduced mitochondria functions such as respiration (44). To study the role of lipid peroxidation to membranes in aging, we used a unique mouse model, Gpx4+/− mice, which we recently generated and characterized. Gpx4 is an antioxidant enzyme in the Gpx family. Whereas all Gpxs, including Gpx4, reduce hydrogen peroxide, alkyl peroxides, and fatty acid hydroperoxides, Gpx4 is unique in that it also reduces hydroperoxides in lipoproteins and complex lipids such as those derived from cholesterol, cholesteryl esters, and phospholipids. Therefore, Gpx4 is thought to play a key role in protecting membrane lipids from oxidative damage (3). We showed that knockout mice null for Gpx4 die during early embryonic development and that Gpx4+/− mice, which have only one WT allele of the Gpx4 gene, appear normal but have approximately 50% less Gpx4 in all tissues compared to WT mice. Gpx4+/− mice and cells are more sensitive to oxidative stress (9). Because the levels of Gpx1 and catalase, two major antioxidant defense enzymes involved in reducing H2O2 and free fatty acid hydroperoxides, are similar in Gpx4+/− mice and WT mice (7), and because Gpx1 and catalase are at much higher levels than Gpx4 in all tissue except testes, the cells/tissues of Gpx4+/− mice are only deficient in their ability to protect membrane lipids from peroxidation. Therefore, these mice are ideal for studying the role of lipid peroxidation of membranes in aging and other biological processes. Based on the Oxidative Stress Theory of Aging and the potential physiological importance of membrane lipid peroxidation in cellular functions, we hypothesized that the Gpx4+/− mice would show accelerated aging, i.e., reduced life span. In contrast, if lipid peroxidation of membranes plays no role in aging, we would see no difference between the life spans of the Gpx4+/− and WT mice. We were surprised when we observed an increase in life span (which was significant for the median life span) in the Gpx4+/− mice. One might argue that the 7% increase in median life span, although statistically significant, is not impressive compared to the 15%–30% increase in life span found in studies of other genetically altered mouse models (reviewed in 45). However, it should be noted that the median survival that we observed for the Gpx4+/− mice (1029 days or 34.3 months) is extremely long for C57BL/6 mice and is longer than that of many of the long-lived mutant mouse models previously reported, e.g., Igf1r+/− mice (median life span of about 25.4 months) (46), FIRKO mice (median life span of 33.4 months) (47), transgenic mice overexpressing catalase in mitochondria (median life span of about 31.0 months) (48), GHR/BP−/− mice (median life span of 31.4 months) (49), p66sch−/− mice (median life span of 32.4 months) (50), and Klotho mice (median life span of about 33.2 months) (51). Therefore, we believe that the modest increase in life span of the Gpx4+/− mice is important because it results in one of the longest-lived genetic mutant mouse models generated to date. The life span of Gpx4+/− mice appears to be inconsistent with the Oxidative Stress Theory of Aging, i.e., increased oxidative stress should reduce life span, not extend life span. Previously, our group showed that Sod2+/− mice, which have reduced expression of MnSOD in all tissues and increased levels of oxidative damage to DNA, had an identical life span as control, WT mice (52). The life-span data of Gpx4+/− and Sod2+/− mice show that a deficiency in one component of the antioxidant defense does not directly lead to accelerated aging in mice. The increase in the median survival of Gpx4+/− mice was most likely a result of altered pathology. Our pathological analysis of mice in the survival groups revealed two significantly altered pathologies that may contribute to increased median survival of the Gpx4+/− mice: delayed occurrence of fatal lymphoma and reduced severity of glomerulonephritis. Lymphoma is a major fatal neoplasm in C57BL/6 mice, the incidence of which increases with age (53). Our pathological data indicate that the age-related increase in occurrence and/or progression of lymphoma was delayed in Gpx4+/− mice, i.e., the Gpx4+/− mice died of fatal lymphoma at older ages than did the WT mice. We also observed that glomerulonephritis, an age-associated nephropathy, was reduced in Gpx4+/− mice. Our data suggested that the reduction in fatal glomerulonephritis in Gpx4+/− mice was due to the suppression in the progression/development, but not the initiation, of glomerulonephritis. Hence, does the reduced Gpx4 expression increase life span and retard/reduce pathology by slowing down aging? Even though we observed that reduced expression of Gpx4 delayed the terminal loss of body weight associated with aging (54), our data would tend to indicate that aging is not altered because neither the 10% survival, maximum survival, age-dependent mortality rate nor cataract development was altered in the Gpx4+/− mice. One potential explanation for the improved pathology is an increase in apoptosis that would remove damaged/abnormal cells. Gpx4 appears to play an important role in regulating apoptosis. For example, the overexpression of Gpx4 inhibits the induction of apoptosis by a variety of oxidizing agents (55–57). Additionally, Gpx4 alters apoptosis through the intrinsic pathway by its ability to rapidly reduce hydroperoxides in CL, a mitochondrial inner membrane phospholipid that binds to cyt. c. Because of its high content (80%–90%) of linoleic acid (58), CL is vulnerable to free radical oxidation, and peroxidation of CL leads to cyt. c release from mitochondria. Using monolayers of CL, Nomura and colleagues (37) showed that oxidation of CL to CLOOH decreased cyt. c binding. Gpx4 can rapidly reduce CLOOH to CLOH (37), and the resultant CLOH can bind cyt. c as well as nonoxidized CL (37,58,59). In our previous study with Gpx4 transgenic mice exposed to diquat, we showed that liver from mice overexpressing Gpx4 had reduced levels of apoptosis and decreased cyt. c release from mitochondria, indicating that Gpx4 also plays an important role in regulating apoptosis in vivo (10). In this study, we showed that diquat treatment induced more apoptotic cells in livers of Gpx4+/− mice than in WT mice. In addition, the Gpx4+/− mice had increased cyt. c release from mitochondria and increased levels of mitochondrial CL peroxidation compared to WT mice after diquat treatment. Therefore, Gpx4+/− mice appear to have increased sensitivity to stress-induced apoptosis compared to WT mice. Increased apoptosis has been proposed to have both beneficial and detrimental effects on aging in mammalian systems (60). On one hand, increased apoptosis in postmitotic cells, such as neurons and cardiomyocytes, could be detrimental. On the other hand, increased apoptosis could serve as an important cellular defense mechanism by maintaining genetic stability through the elimination of damaged and dysfunctional cells (61). Altered sensitivity to apoptosis is known to alter the occurrence of tumors, such as lymphoma (32). Therefore, increased sensitivity to apoptosis likely contributed to the delayed occurrence of fatal lymphoma in Gpx4+/− mice. Increased apoptosis also could be beneficial in preventing glomerulonephritis through mechanisms such as the deletion of infiltrating leukocytes and restoration of normal glomerular structure (62). Therefore, at the present time, we believe the most likely explanation for the increased life span of the Gpx4+/− mice is through reduced pathology in certain cells/tissues because of increased sensitivity to apoptosis. Our observations on aging in Gpx4+/− mice appear to be in contrast to what has been reported for p66shc−/− mice. p66shc is shown to act as an oxidoreductase to generate ROS in mitochondria and activate the mitochondrial pathway of apoptosis by enhancing cyt. c release from mitochondria (63). Migliaccio and colleagues (50,64) showed that the life span of p66shc−/− mice was increased by 30% over control WT mice, and the increase in life span in p66shc−/− mice was correlated with reduced apoptosis in murine embryonic fibroblasts isolated from p66shc−/− mice after oxidative stress and increased survival of p66shc−/− mice after paraquat injection (50). In contrast, we showed in this study that the median life span of Gpx4+/− mice was increased by 7%, and we showed previously that murine embryonic fibroblasts from Gpx4+/− mice had increased apoptosis after oxidative stress and that Gpx4+/− mice had reduced survival after γ irradiation (7,9). Therefore, increased sensitivity to apoptosis (in Gpx4+/− mice) and reduced sensitivity to apoptosis (in p66shc−/− mice) are both correlated with increased life spans. As previously noted (60), the role of apoptosis in aging is very complex. The status of reduced or increased apoptosis in different cells and tissues could affect age-related pathology differently. We showed that the occurrence of fatal lymphoma was delayed in Gpx4+/− mice and that Gpx4+/− mice also had retarded glomerulonephritis. Unfortunately, there were no data on the pathology of the p66shc−/− mice in the life-span study (50), so we can not compare age-related pathology between Gpx4+/− mice and p66shc−/− mice. Decision Editor: Huber R. Warner, PhD Figure 1. Life span of wild-type (WT) mice and glutathione peroxidase 4 heterozygous knockout (Gpx4+/−) mice. Kaplan–Meier survival curves are shown for 50 WT (open diamond) and 50 Gpx4+/− (solid circle) mice. The mean (± standard error of the mean [SEM]), median, maximum, and top 10% (± SEM) survival of male WT and Gpx4+/− mice were determined from the age at death as described in the Methods section. *p <.05, as determined by median two-sample test Figure 2. Body weights and cataracts of wild-type (WT) mice and glutathione peroxidase 4 heterozygous knockout (Gpx4+/−) mice. A, Body weights of 12 WT and 12 Gpx4+/− mice were determined at indicated intervals throughout their lives. Data are expressed as mean ± standard error of the mean (SEM). The difference in the rates of weight loss between WT and Gpx4+/− mice is statistically significant (p =.001, as determined by mixed-effect linear model). B, Cataract formation was assessed in WT and Gpx4+/− mice as described in the Methods section. Data are expressed as mean (± SEM) for 33 WT mice and 33 Gpx4+/− mice Figure 3. Levels of glutathione peroxidase 4 (Gpx4) in old wild-type (WT) mice and Gpx4+/− mice. Levels of Gpx4 protein in various tissues of 26- to 29-month-old WT and Gpx4+/− mice were determined by Western blots as described in the Methods section. Data are expressed as mean ± standard error of the mean of data obtained from four mice of each genotype. The differences between WT and Gpx4+/− mice are statistically significant for all tissues (p <.05, as determined by the Student t test) Figure 4. Levels of lipid peroxidation WT and glutathione peroxidase 4 heterozygous knockout (Gpx4+/−) mice. Levels of F2-isoprostanes in the plasma (A) and liver (B), and F4-neuroprostanes in brain (C) of 26- to 29-month-old WT and Gpx4+/− mice were determined as described in the Methods section. Data are expressed as mean ± standard error of the mean for four mice of each genotype (*p <.05, as determined by Student's t test) Figure 5. Age related pathology in wild-type (WT) mice and glutathione peroxidase 4 heterozygous knockout (Gpx4+/−) mice. A, Percentage of mice dying from fatal lymphoma (WT and Gpx4+/−) between 920 and 1092 days of age, when the difference in the survival of WT and Gpx4+/− mice was greatest. The age difference between Gpx4+/− mice and WT mice is statistically significant as determined by log-rank test (p <.05). B, Severity of glomerulonephritis at end of life in WT and Gpx4+/− mice was determined as described in the Methods section. Data are expressed as mean ± standard error of the mean for 50 WT mice and 47 Gpx4+/− mice (*p <.05, as determined by Student's t test) Figure 6. Apoptosis in old wild-type (WT) mice and glutathione peroxidase 4 heterozygous knockout (Gpx4+/−) mice. Mice that were 26–29 months of age were treated with diquat (50 mg/kg) for 6 hours, and the following parameters were measured in the livers of WT (open bars) and Gpx4+/− (solid bars), as described in the Methods section. A, Level of apoptosis before (control) and after diquat treatment. B, Photograph of a representative Western blot showing cytochrome c (cyt. c) release into the cytosol. C, Quantification of cyt. c release as determined from Western blots before (control) and after diquat treatment. D, Cardiolipin peroxidation as measured by mitochondrial bound NAO before (control) and after diquat treatment. All values are expressed as mean ± standard error of the mean of data obtained from four mice. *p <.05 level Table 1. End-of-Life Pathology for WT and Gpx4+/− Mice. WT Gpx4 +/− Neoplasm 26 30     Lymphoma 18 24     Others 8 6 Nonneoplasm 14 12     Glomerulonephritis 10* 0     AMP 3 10     Others 1 2 Undetermined 7 8 Total 47 50 Notes: The numbers of fatal neoplasm and fatal nonneoplasm in glutathione peroxidase 4 heterozygous knockout (Gpx4+/−) mice (n = 50) and in wild type (WT) mice (n = 47) in the survival groups were determined as described in the Methods section. AMP, acidophilic macrophage pneumonia. *p <.01, as determined by Fisher's exact test. This study was supported by a Reserve Educational Assistance Program (REAP) and a Merit Award (HVR) from the Department of Veteran Affairs; by National Institutes of Health grants P01 AG19316, P01AG020591, R37GM42056; and by the San Antonio Nathan Shock Aging Center (1P30-AG13319). We thank Clyde Alex McMahan and John Cornell for help with statistical analysis in this study. Drs. Ran, Liang, and Ikeno contributed equally to this work. References 1 Porter NA, Caldwell SE, Mills KA. 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Antioxid Redox Signal.2006;8:600-608. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_00A8976B96F96209925FEEE6B144540CB9536724.txt b/test/dataset/in/resources/corpus/Clean_00A8976B96F96209925FEEE6B144540CB9536724.txt new file mode 100644 index 0000000..535c54c --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_00A8976B96F96209925FEEE6B144540CB9536724.txt @@ -0,0 +1 @@ +amjepidajeAmerican Journal of Epidemiology1476-62560002-9262Oxford University Press10.1093/aje/kwp426ORIGINAL CONTRIBUTIONSA Prospective Study of Albuminuria and Cognitive Function in Older AdultsThe Rancho Bernardo StudyJassalSimerjot K.*Kritz-SilversteinDonnaBarrett-ConnorElizabeth*Correspondence to Dr. Simerjot K. Jassal, VA San Diego Healthcare System, 3350 La Jolla Village Drive, Division of GIM/G, MC 111N, San Diego, CA 92161 (e-mail: sjassal@ucsd.edu).Editor's note: An invited commentary on this article appears on page 287, and the authors’ response is published on page 290.122010812010171327728612520091102009American Journal of Epidemiology Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2010.2010Chronic kidney disease is a risk factor for cognitive impairment. Albuminuria is an early manifestation of chronic kidney disease and a marker of endothelial dysfunction and vascular risk. Results of prior studies of albuminuria and cognitive function are contradictory. The authors studied 1,345 community-dwelling women and men in southern California (mean age, 75 years) at a 1992–1996 research clinic visit, when urine albumin/creatinine ratio (ACR) was measured in spot morning urine and cognitive function was evaluated by using the Mini-Mental State Examination Trail-Making Test B, and category fluency test. An ACR of ≥30 mg/g was found in 17% of women and 15% of men in 1992–1996. Analysis of covariance was used to compare cognitive function score by categorical ACR. Between 1999 and 2002, 759 participants returned for repeat cognitive function testing. For men, but not women, baseline albuminuria, but not estimated glomerular filtration rate, was associated with reduced cognitive function at follow-up on all tests (P's < 0.05). An ACR of ≥30 mg/g was associated with greater annual decline in Mini-Mental State Examination and category fluency scores. Albuminuria may be an easily measured marker predicting future cognitive function decline. Results imply a common underlying mechanism affecting the renal and cerebral microvasculature.agedalbuminuriacognitiondementiaThe association between chronic kidney disease and cardiovascular disease is well established (1). Both share common risk factors (diabetes and hypertension) (2, 3) and pathophysiology (inflammation and endothelial dysfunction) (4, 5). End-stage renal disease patients experience increased cerebrovascular accidents, subclinical ischemic cerebrovascular disease, and cognitive decline (6–10).The prevalence of diabetes and chronic kidney disease is increasing (11). Albuminuria is an early manifestation of chronic kidney disease (12, 13), usually in diabetes; it is a marker of endothelial dysfunction and vascular risk (14) and is a risk factor for cardiovascular disease (15), but its association with cognitive function remains uncertain. Although several studies of the association between cognitive function and estimated glomerular filtration rate (eGFR) exist (16–21), few have examined albuminuria as an exposure (22–26); only one was prospective (24).A study investigating the association of this modifiable stage of chronic kidney disease with cognitive decline is timely and important. The purpose of the present study was to examine the longitudinal association of urine albumin/creatinine ratio (ACR) with cognitive function change in a population-based sample of older, community-dwelling adults.MATERIALS AND METHODSParticipantsParticipants were community-dwelling women and men aged 51–98 years from the Rancho Bernardo Study. In 1972–1974, 82% of all adults aged 30 years or older living in this southern California community enrolled in a study of heart disease risk factors. Participants were Caucasian, well educated, and middle class. In 1992–1996, 1,781 participated in a research clinic visit, when albuminuria was measured; 1,429 (80%) completed 3 cognitive function tests. After exclusion of 11 individuals without urine samples, 12 younger than 50 years of age, and 61 who had a history of stroke, there remained 821 women and 524 men who had albuminuria and tests of cognitive function in 1992–1996. Of these, 461 women and 298 men had repeat cognitive function testing at a follow-up visit in 1999–2002, a mean of 6.6 (range, 4.5–9.5) years later (Figure 1). Of the 3,004 participants invited to the 1999–2002 visit, 25% (758/3,004) did not respond and 37% (1,105/3,004) refused the invitation (8% (252/3,004) were deceased; 9% (265/3,304) had moved away; and 20% (588/3,004) were unable to return for other reasons, such as poor health or caregiving responsibilities), leaving 38% (1,141/3,004) who were seen in the follow-up clinic.Figure 1.Selection of participants for a study of albuminuria and cognitive function in older adults, Rancho Bernardo, California. In 1992–1996, 1,429 participants attended a research clinic visit and completed 3 cognitive function tests. After exclusion of 11 individuals without urine samples, 12 who were younger than age 50 years, and 61 with a history of stroke, 821 women and 524 men remained who had urine samples for albuminuria testing and tests of cognitive function in 1992–1996. Of these participants, 461 women and 298 men returned for a follow-up visit and had repeat cognitive function testing in 1999–2002.This study was approved by the Human Subjects Protection Program of the University of California, San Diego. All participants were ambulatory and gave written informed consent.ProceduresAt the 1992–1996 clinic visit, standard self-administered questionnaires were used to define age, education, health habits (smoking, alcohol consumption, and exercise), medical history, and current medications. Height and weight were measured by using a regularly calibrated stadiometer and balance-beam scale with participants wearing light clothing and no shoes. Body mass index was calculated as weight in kilograms divided by height in meters squared. Systolic and diastolic blood pressures were measured twice in seated participants after a 5-minute rest by a trained nurse using the Hypertension Detection and Follow-up Program protocol (27). Ankle brachial index was measured by a trained nurse taking the highest systolic blood pressure in each leg (posterior tibial artery) and dividing by the highest systolic blood pressure in each arm (brachial artery). If the lowest of these 2 ankle brachial indices was ≤0.90, participants were classified as having peripheral arterial disease.A blood sample was obtained by venipuncture after a requested overnight fast (12–16 hours). A single, clean-catch, untimed morning urine sample (usually the second void) was collected. Fasting plasma glucose was measured by the glucose oxidase method and hemoglobin A1C (HbA1C) by high performance liquid chromatography; fasting plasma cholesterol, triglyceride, and high density lipoprotein and low density lipoprotein cholesterol levels were measured in a Centers for Disease Control and Prevention certified lipid research clinic laboratory. Total cholesterol and triglyceride levels were measured by enzymatic techniques using an ABA-200 biochromatic analyzer (Abbott Laboratories, Abbott Park, Illinois). High density lipoprotein cholesterol was measured according to the standardized procedures of the lipid research clinics manual (28); low density lipoprotein cholesterol was calculated by using the Friedewald formula (29). Serum creatinine was measured by SmithKline Beecham Clinical Laboratories (King of Prussia, Pennsylvania). Urine samples were shipped to the National Institutes of Health laboratory in Phoenix, Arizona. Urine albumin was measured by using the Behring Nephelometer BNA (Dade Behring GmbH, Marburg, Germany). The lower limit of detection of the assay was 6.8 mg/dL; values <6.8 mg/dL were assigned a value of 6.7 mg/dL. The interassay coefficient of variance was 4.5%. Urine creatinine was measured by the kinetic alkaline picrate method using the Ciba-Corning Express (Corning, Medfield, Massachusetts). Diabetes was defined according to the 1999 World Health Organization criteria (30)—fasting plasma glucose ≥126 mg/dL (7 mmol/L), a physician diagnosis of diabetes, or use of diabetes-specific medication (oral or insulin).Statistical analysisAt both the 1992–1996 and 1999–2002 clinic visits, 3 standardized tests, chosen to assess diverse domains of cognitive function with demonstrated reliability and validity (31), were administered by a trained interviewer: the Mini-Mental State Examination (MMSE), Trail-Making Test B (Trails B), and a category fluency test. The MMSE is a global test of orientation, registration, attention, calculation, language, and recall (32, 33). It is used to screen for incipient dementia, has limited sensitivity for change in cognitive function (34), and is influenced by education, with lower sensitivity for detecting cognitive dysfunction in those with a college education or higher (33). It is scored on a scale of 0 to 30, with dementia suspected for scores <24. Trails B (from the Halstead-Reitan Neuropsychological Test Battery) is a test of visuomotor tracking and attention (35) in which participants identify alternating patterns of letters and numbers in sequence over 300 seconds. It is scored by the time required to complete the test. Lastly, the Animals Naming Category Fluency test is a test of cognitive flexibility and executive function in which participants are asked to name as many animals as possible in 1 minute (36). The score is the number of correctly named animals; repetitions, variations, and intrusions (words other than names of animals, e.g., apple) are not counted. For the MMSE and category fluency test, higher scores indicate better performance; for Trails B, a lower score indicates better performance. Recommended cutoff values indicative of poor performance obtained from the Alzheimer's Disease Research Center of the University of California, San Diego, were as follows: MMSE <24, Trails B ≥132, and category fluency <12.Depressed mood was assessed by using the Beck Depression Inventory, a self-administered questionnaire asking participants to choose statements that best describe their feelings. Higher score indicates greater depressed mood, and a score ≥12 suggests clinical depression (37).ACR was calculated as follows: ACR (mg/g) = Urine albumin (mg/dL)/Urine creatinine (g/dL). It was categorized as ≥30 mg/g (albuminuria) or <30 mg/g (normal) (38).eGFR was calculated by using the abbreviated Modification of Diet in Renal Disease study equation (39, 40): eGFR (mL/minute/1.73 m2) = 186 × (serum creatinine (mg/dL))−1.154 × (age)−0.203 × (0.742 if female) × (1.210 if black). It was categorized as ≥60 mL/minute/1.73 m2 (normal to mildly decreased) or <60 mL/minute/1.73 m2 (moderately to severely decreased).Annual change in cognitive function was calculated as follows: Annual change in score = (Score at follow-up – Score at baseline)/Years between visits.Analyses were stratified by sex because of known differences in kidney and cognitive function between women and men. All measures were normally distributed except ACR, which required log transformation. A univariate general linear model (analysis of covariance) was used to compare mean values for continuous variables by sex or by ACR or eGFR category; the chi-square statistic was used to compare differences in prevalence for categorical variables. Medians for ACR were calculated.Three analytic strategies were used to assess the cross-sectional and longitudinal association between kidney function and cognitive function: 1) multiple linear regression to assess the association between logACR at baseline and scores on the MMSE, Trails B, and category fluency tests and annual change in scores; 2) logistic regression to assess the association between logACR and categorical poor performance on cognitive function tests; and 3) analysis of covariance to compare cognitive function scores by categorical ACR (<30 mg/g vs. ≥30 mg/g). Analyses were repeated by using eGFR as a continuous or categorical (≥60 mL/minute/1.73 m2 vs. <60 mL/minute/1.73 m2) variable.Covariates significantly associated with both predictor and outcome variables by correlation coefficients were used to create parsimonious multivariate models. Multivariable models were adjusted for age, systolic blood pressure, HbA1C, education (some college or more vs. no college), strenuous exercise 3 or more times per week (yes/no), consumption of alcoholic beverages 3 or more times per week (yes/no), and current estrogen use (yes/no) by women, and additionally for eGFR, Beck Depression Inventory, and use of antihypertensive and lipid-lowering medications. Inclusion of body mass index, low density lipoprotein cholesterol, current smoking (yes/no), and peripheral arterial disease (yes/no) did not materially change the results; these variables were therefore not included in the final model. Longitudinal analyses of cognitive function score on the MMSE, Trails B, or category fluency test at the 1999–2002 visit were adjusted for score on the same test at the 1992–1996 visit.All P values are 2 sided. SPSS software (SPSS Base 15.0 for Windows; SPSS Inc., Chicago, Illinois) was used for all analyses.RESULTSAt baseline in 1992–1996, the mean age of women and men was similar—74.9 (standard deviation, 9.1) years and 74.5 (standard deviation, 8.6) years (P = 0.44), respectively. There were significant corresponding sex differences (P < 0.01 for all) in mean eGFR (64.9 mL/minute/1.73 m2 vs. 68.7 mL/minute/1.73 m2), median ACR (13.4 mg/g vs. 9.7 mg/g), and scores on the MMSE (28.1 vs. 27.8), Trails B (135.7 vs. 126.4), and category fluency (17.0 vs. 18.1) tests; women performed better than men on the MMSE but worse on Trails B and category fluency. Systolic blood pressure, HbA1C, body mass index, low density lipoprotein cholesterol, Beck Depression Inventory, education, exercise, and alcohol consumption also differed significantly by sex (Table 1).Table 1.Baseline Characteristics of Participants in a Prospective Study of Albuminuria and Cognitive Function, Rancho Bernardo, California, 1992–1996aWomen (n = 821)Men (n = 524)P ValueMean (standard deviation)    Age, years74.9 (9.1)74.5 (8.6)0.44    Body mass index, kg/m224.7 (4.1)26.2 (3.6)<0.001    Systolic blood pressure, mm Hg140.3 (22.4)137.5 (20.2)0.02    HbA1C, %4.3 (0.6)4.3 (0.7)0.04    Low density lipoprotein cholesterol, mg/dL128.0 (34.4)123.5 (30.4)<0.01    Serum creatinine, mg/dL0.9 (0.2)1.2 (0.25)<0.001    eGFR, mL/minute/1.73 m264.9 (15.7)68.7 (16.7)<0.001    ACR, mg/gb13.4 (8.4–22.1)9.7 (6.4–19.4)<0.01    Beck Depression Inventory score5.9 (4.6)4.8 (4.0)<0.001    MMSE score28.1 (2.0)27.8 (2.3)<0.01    Trails B score135.7 (61.1)126.4 (56.4)<0.01    Category fluency score17.0 (4.7)18.1 (5.0)<0.001%    Some college or more63.981.5<0.001    Exercise ≥3×/week69.277.7<0.01    Alcohol consumption ≥3×/week39.855.9<0.001    Current smoking7.25.30.18    Current estrogen use40.7n/an/a    Current lipid-lowering medication use10.59.20.41    Current antihypertensives use34.138.10.22    Diabetes13.618.10.03Abbreviations: ACR, urine albumin/creatinine ratio; eGFR, estimated glomerular filtration rate; HbA1C, hemoglobin A1C; MMSE, Mini-Mental State Examination; n/a, not applicable; Trails B, Trail-Making Test B.aExcept for ACR, P values were obtained from analysis of variance for normally distributed values and the chi-square statistic for categorical variables.bValues are expressed as median (interquartile range). P values were obtained from the Wilcoxon rank sum test for this skewed variable.In 1992–1996, 17% of women and 15% of men had albuminuria (ACR ≥30 mg/g); 42% of women and 27% of men had moderately to severely impaired kidney function by eGFR (<60 mL/minute/1.73 m2). A majority (56%; n = 754) had preserved kidney function by both measures, and only 8% (n = 108) had impaired kidney function by both measures. However, 28% (n = 375) had an eGFR of <60 mL/minute/1.73 m2 but an ACR of <30mg/g, and 8% (n = 105) had an eGFR of ≥60 mL/minute/1.73 m2 but an ACR of ≥30 mg/g (Figure 2), suggesting these measures assess different spectrums of kidney disease.Figure 2.Prevalence (%) of impaired kidney function by estimated glomerular filtration rate (eGFR) and urine albumin/creatinine ratio (ACR) at the baseline research clinic visit in 1992–1996, Rancho Bernardo, California.The predictors, outcomes, and most covariates all differed significantly by sex. Testing revealed a significant ACR-by-sex interaction (P = 0.01). Therefore, all analyses were sex specific. As shown in Table 2, men and women with an ACR of ≥30 mg/g versus <30 mg/g were older, had a higher systolic blood pressure and HbA1C, were less likely to drink alcohol regularly, and were more likely to use antihypertensives. Women with an ACR of ≥30 mg/g versus <30 mg/g were also less likely to report regular exercise, were more likely to have diabetes or depressed mood, and had worse scores on the MMSE, Trails B, and category fluency tests. At follow-up, more men and women had poor cognitive function according to the MMSE and category fluency tests than at baseline, but fewer scored poorly on Trails B (Figure 3). Mean differences in cognitive function test scores from the 1992–1996 to the follow-up visit for women and men, respectively, were as follows: MMSE −1.0 (standard deviation, 2.1) and −0.9 (standard deviation, 1.9); Trails B −15.7 (standard deviation, 55.1) and −8.4 (standard deviation, 46.0) (unexpectedly improved); and category fluency −1.8 (standard deviation, 4.3) and −1.6 (standard deviation, 4.5). Cross-sectional analyses using multiple linear regression, logistic regression, or analysis of covariance adjusted for age and other known covariates indicated that albuminuria was not associated with any of the 3 measures of cognitive function at baseline in either sex.Table 2.Comparison of Baseline Characteristics of Study Subjects by Urine ACR Category, Rancho Bernardo, California, 1992–1996aWomen (n = 821)Men (n = 524)ACR <30 (n = 686)ACR ≥30 (n = 135)P ValueACR <30 (n = 446)ACR ≥30 (n = 78)P ValueMean    Age, years74.178.7<0.00174.177.2<0.01    Body mass index, kg/m224.924.20.0626.226.40.67    Systolic blood pressure, mm Hg138.6147.8<0.001135.7147.9<0.001    HbA1C, %4.24.4<0.014.34.6<0.01    Low density lipoprotein cholesterol, mg/dL128.5126.00.44123.6123.40.96    Serum creatinine, mg/dL0.91.0<0.011.11.3<0.001    eGFR, mL/minute/1.73 m265.562.30.0370.160.9<0.001    ACR, mg/gb11.444.7<0.0018.460.6<0.001    Beck Depression Inventory score5.736.63<0.054.685.600.06    MMSE score28.227.9<0.0527.927.60.33    Trails B score131.4155.5<0.001124.7136.00.10    Category fluency17.315.7<0.00118.217.40.21%    Some college or more63.665.80.7582.475.80.22    Exercise ≥3×/week71.159.3<0.0178.075.60.66    Alcohol consumption ≥3×/week41.728.9<0.0158.347.30.01    Current smoking7.37.41.005.45.11.00    Current estrogen use41.337.00.39n/an/an/a    Current lipid-lowering medication use10.98.10.4410.13.80.09    Current antihypertensives use36.757.0<0.00139.767.9<0.001    Diabetes12.121.5<0.0117.323.10.26Abbreviations: ACR, albumin/creatinine ratio; eGFR, estimated glomerular filtration rate; HbA1C, hemoglobin A1C; MMSE, Mini-Mental State Examination; Trails B, Trail-Making Test B.aExcept for ACR, P values were obtained from analysis of variance for normally distributed values and the chi-square statistic for categorical variables.bValues are expressed as median. P values were obtained from the Wilcoxon rank sum test for this skewed variable.Figure 3.Prevalence (%) of poor cognitive function—Mini-Mental State Examination (MMSE) score <24, Trail-Making Test B (Trails B) score ≥132, and category fluency test score <12—in 1992–1996 and 1999–2002, Rancho Bernardo, California.In contrast, prospective analyses using analysis of covariance showed that albuminuria (ACR ≥30 mg/g vs. <30 mg/g) in 1992–1996 was associated with worse cognitive function on all tests (MMSE, Trails B, and category fluency) in 1999–2002 (Table 3) and with greater annual decline in MMSE and category fluency in men but not women, even after adjusting for all covariates (P’s ≤ 0.05 for all) (Table 4). However, there was an unexpected trend toward improvement in Trails B score (a decrease) among men with an ACR of ≥30 mg/g versus <30 mg/g (P = 0.06) (Table 4). Prospective multivariable analyses using multiple linear regression or logistic regression showed similar trends but no significant association.Table 3.Adjusted Longitudinal Comparisons of Mean Cognitive Function Scores at Study Follow-up in 1999–2002 by Baseline Urine ACR Category in 1992–1996,a Rancho Bernardo, CaliforniaWomen (n = 461)Men (n = 298)ACR <30 (n = 406)ACR ≥30 (n = 55)P ValueACR <30 (n = 263)ACR ≥30 (n = 35)P ValueMMSE    Unadjusted27.627.80.5427.726.4<0.01    Age adjusted27.628.00.1827.626.8<0.05    All adjustedb27.728.00.3627.726.60.02Trails B    Unadjusted129.5150.60.03114.8152.5<0.01    Age adjusted130.8141.70.21117.1133.50.12    All adjustedb129.1133.40.62112.5132.9<0.05Category fluency    Unadjusted16.116.50.6217.816.0<0.05    Age adjusted16.116.80.2417.716.80.31    All adjustedb16.216.60.5718.015.60.01Abbreviations: ACR, albumin/creatinine ratio; MMSE, Mini-Mental State Examination; Trails B, Trail-Making Test B.aResults of analysis of covariance.bAdjusted for baseline age, systolic blood pressure, hemoglobin A1C, education (some college), strenuous exercise 3 or more times per week, alcoholic beverage consumption 3 or more times per week, baseline score on cognitive function test, current estrogen use (women only), estimated glomerular filtration rate, Beck Depression Inventory score, antihypertensive medication use, and lipid-lowering medication use.Table 4.Adjusted Longitudinal Comparisons of Mean Annual Change in Cognitive Function Scores Between Baseline in 1992–1996 and Follow-up in 1999–2002 by Baseline Urine ACR Category in 1992–1996,a Rancho Bernardo, CaliforniaWomen (n = 461)Men (n = 298)ACR <30 (n = 406)ACR ≥30 (n = 55)P ValueACR <30 (n = 263)ACR ≥30 (n = 35)P ValueMMSE    Unadjusted−0.16−0.120.42−0.12−0.270.01    Age adjusted−0.16−0.100.15−0.13−0.230.09    All adjustedb−0.15−0.100.38−0.11−0.280.01Trails B    Unadjusted−2.20−4.210.11−0.99−3.98<0.05    Age adjusted−2.38−3.620.28−1.11−2.910.19    All adjustedb−2.45−3.160.59−0.87−3.990.06Category fluency    Unadjusted−0.28−0.280.96−0.23−0.470.07    Age adjusted−0.28−0.270.94−0.24−0.400.24    All adjustedb−0.29−0.270.82−0.22−0.550.04Abbreviations: ACR, albumin/creatinine ratio; MMSE, Mini-Mental State Examination; Trails B, Trail-Making Test B.aResults of analysis of covariance.bAdjusted for baseline age, systolic blood pressure, hemoglobin A1C, education (some college), strenuous exercise 3 or more times per week, alcoholic beverage consumption 3 or more times per week, baseline score on cognitive function test, current estrogen use (women only), estimated glomerular filtration rate, Beck Depression Inventory score, antihypertensive medication use, and lipid-lowering medication use.To determine whether these findings were the result of the established association between diabetes and cognitive function, analyses were repeated by excluding 208 participants with diabetes (30); doing so did not materially change the results. To ensure that results were not driven by macroalbuminuria, analyses were repeated by excluding 21 participants who had an ACR of ≥300 mg/g, which also did not materially change the results. To further understand sex differences, prospective analyses were repeated by using sex-specific cutoffs for albuminuria (women: normal <25 mg/g (n = 649; 79%), albuminuria ≥25 mg/g (n = 173; 21%); men: normal <17 mg/g (n = 374; 71%), albuminuria ≥17 mg/g (n = 150; 29%) (41) but did not materially change the results for either men or women. To determine whether sex differences in cognitive function might vary by baseline estrogen use, analyses were repeated by stratifying women by baseline estrogen status; no association remained between albuminuria and cognitive decline in women who were or were not current estrogen users at baseline. Analyses were repeated after additionally adjusting for body mass index, low density lipoprotein cholesterol, and current smoking or by peripheral arterial disease, without a material change in results. We also attempted to stratify by median age (<75 or ≥75 years) and by peripheral arterial disease to determine whether results were modified by age or peripheral arterial disease; however, sample sizes were too small for results to be interpretable.Similar prospective analyses were performed by using eGFR as the exposure variable instead of ACR. There were no significant differences by baseline eGFR category in mean score on any cognitive function test at follow-up, or in mean annual change in any score, for either sex.To determine whether the absent cross-sectional ACR association and the strong, positive, prospective association were explained by survival or nonresponse bias, we compared differences in baseline characteristics between those who attended only the baseline visit and those who attended both visits. Compared with those who attended only the baseline visit, at baseline, those who attended both visits were healthier as evidenced by statistically significant differences (P < 0.05) in nearly every baseline measure shown in Table 1: they were younger; had a lower systolic blood pressure, HbA1C, and ACR and a higher eGFR; were more likely to report regular exercise; and performed better on all baseline measures of cognitive function than those who attended the baseline visit only, documenting selective loss of older, less healthy, and more cognitively impaired participants. This truncation would be expected to reduce any true increased risk. Nonresponse did not differ significantly by sex, making it unlikely to explain the sex differences.DISCUSSIONIn this study of older, community-dwelling adults, categorically defined albuminuria was not associated with cognitive function at baseline. However, for men only, it was associated with worse cognitive function 6.6 years later.Significant differences were observed when ACR was used as a categorical but not a continuous predictor, suggesting that the ≥30 mg/g cutpoint based on American Diabetes Association (30) and National Kidney Foundation/Kidney Disease Outcomes Quality Initiative (41) guidelines may carry prognostic value. This level of albuminuria has been associated with other microvascular complications of diabetes including diabetic retinopathy (42) and suggests that cognitive decline may result from microvascular disease in the brain.In this study, a significant association between albuminuria and cognitive function was found in longitudinal but not cross-sectional analyses. This finding is consistent with 2 prior studies in this cohort, which reported a longitudinal, but not cross-sectional, association between glucose tolerance status or HbA1C and cognitive function over a 4-year follow-up (3, 43). One possible explanation for this disparity is that, since albuminuria is an early predictor of kidney disease (usually preceding a decline in eGFR) and an early marker of endothelial dysfunction, it may signal the early stage of a process resulting in vascular disease and cognitive decline over the 6.6-year follow-up period.To our knowledge, the observed sex differences in the association between albuminuria and cognitive function have not been reported previously, and the reasons for them are unknown. They were not explained by sex differences in age, baseline test performance, or exogenous hormone use and are unlikely to be due to endogenous sex hormones, because higher endogenous estrogen levels have been associated with greater decline in category fluency in this cohort (44). Known sex differences in cognitive function include better performance by women on tests of visuospatial, visuoconceptional, and mental control function and better performance on verbal tasks (45); women also have a lower modified MMSE score than men do in populations with disparities in educational resources (46). Rancho Bernardo women were less likely than men to have attended college (63.9% vs. 81.5%, P < 0.001) and had lower baseline scores on Trails B and category fluency; consequently, they may have been functioning at a lower level than men, thereby blunting the ability to observe potential differences by albuminuria.Of note, a lower proportion of both men and women in our study had poorer Trails B test scores at the 1999–2002 visit than the 1992–1996 visit, and average score on this test improved among those who completed the test at both visits. This finding likely reflects a practice effect, with improvement on repeat testing, as reported previously in this (47) and other (48, 49) cohorts. Alternatively, the selective loss of those with the poorest cognitive function excluded those with the poorest Trails B test performance at baseline.To our knowledge, 5 prior papers have investigated the association between albuminuria and cognitive function (22–26); only 1 was prospective (24), and none reported sex-specific data. Two papers from the National Health and Nutrition Examination Survey cohort reported the cross-sectional association between microalbuminuria and a single measure of cognitive function in more than 2,000 participants (22, 23). In multiply adjusted analyses, participants with microalbuminuria had significantly lower Weschler digit symbol substitution scores compared with those without microalbuminuria; after additional adjustment for cardiovascular disease and risk factors, this association was no longer significant except in the subset with peripheral arterial disease (22, 23). Another cross-sectional study assessed dementia and albuminuria in 2,316 Cardiovascular Health Cognition Study participants (25). Albuminuria was associated with increased odds of dementia (1.6, 95% confidence interval: 1.2, 2.3) independent of heart disease, risk factors, and eGFR. A cross-sectional study by Weiner et al. (26) evaluated 335 participants from the Nutrition, Aging, and Memory in Elders Study; those with albuminuria had poorer performance on several cognitive tests but no difference in MMSE scores or memory. Albuminuria was also associated with increased white matter hyperintensity volume on magnetic resonance imaging (26).To our knowledge, the only published prospective study of the association between albuminuria and cognitive function was by Abbatecola et al. (24), who studied 140 elderly (mean age, 86 years) nondemented participants with impaired glucose tolerance. Baseline 24-hour urine albumin excretion rate was measured 3 times, and cognitive function was assessed by the MMSE, Verbal Fluency, Digit Span forward and backward, and Trail-Making Tests A and B at baseline and 12 months. Baseline albuminuria predicted increased risk of poor cognition after adjustment for baseline MMSE score, age, education, body mass index, smoking, depression, drug intake, postprandial glucose, and systolic blood pressure (relative risk = 1.8, 95% confidence interval: 1.1, 2.1). Our prospective study differs in its larger size, use of spot ACR, 6.6-year follow-up, and sex-specific analyses. Abbatecola et al.’s use of three 24-hour urine samples would be expected to better classify albuminuria, but their small, shorter (1 year) study was probably not powered to reveal sex differences.ACR may be a marker of systemic vascular endothelial dysfunction common to the kidney and brain, resulting from advanced glycated end products or inflammation. This possibility is supported by studies showing an association of albuminuria with carotid intimal medial thickness (50) and pulse wave velocity (51) and the association of markers of inflammation and hemostasis (52) with both albuminuria (53) and cognitive decline (54). Furthermore, the study by Abbatecola et al. (24) showed that, while the longitudinal association between baseline albuminuria and poor cognition 1 year later persisted in multivariable analyses adjusted for intimal medial thickness, it did not persist after pulse wave velocity was added (relative risk = 1.1, 95% confidence interval: 1.0, 1.2), and the authors concluded that the association might be mediated by the effects of vascular stiffness and endothelial dysfunction on cerebral blood flow.Several limitations and strengths of this study should be noted. ACR was measured with a single, untimed spot urine sample, but this method has been shown to correlate well with 24-hour urine albumin excretion rates (55), and any misclassification would be expected to bias results toward the null, reducing the observed association. Only 3 cognitive function tests were performed at both visits; thus, cognitive assessment may be limited. Another limitation was our inability to stratify analyses by age group or peripheral arterial disease because of the lack of power resulting from small sample sizes in some groups. Because the Rancho Bernardo cohort is mostly white, well educated, and affluent, findings may not be generalizable to other ethnic groups or to those of different socioeconomic or educational backgrounds. Selection bias and loss to follow-up, as in any study of the elderly, are additional limitations. The strengths of this study are its large size, prospective design, long follow-up, assessment of several domains of cognitive function, and use of sex-specific analyses in a cohort very well characterized for directly measured covariates.In conclusion, men with albuminuria had worse cognitive function and greater cognitive decline as assessed by 2 of 3 cognitive tests over a 6.6-year interval. This association was not explained by eGFR or diabetes. Progression of albuminuria may be slowed and cardiovascular outcomes delayed with interventions that control blood pressure, especially with blockade of the renin-angiotensin system (56). Clinical trials are necessary to determine whether interventions targeting albuminuria can prevent cognitive decline among older men with this early marker of kidney disease.AbbreviationsACRalbumin/creatinine ratioeGFRestimated glomerular filtration rateHbA1Chemoglobin A1CMMSEMini-Mental State ExaminationTrails BTrail-Making Test BAuthor affiliations: Division of General Internal Medicine and Geriatrics, Department of Medicine, University of California, San Diego, California (Simerjot K. Jassal); VA San Diego Healthcare System, San Diego, California (Simerjot K. Jassal); and Division of Epidemiology, Department of Family and Preventive Medicine, University of California, San Diego, La Jolla, California (Donna Kritz-Silverstein, Elizabeth Barrett-Connor).This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (grant DK31801), the National Institute on Aging (grant AG07181), and the National Institutes of Health and the National Institute on Aging (grant R01AG028507).This work was presented at the American Heart Association's 49th Cardiovascular Disease Epidemiology and Prevention Annual Conference 2009 in association with the Council on Nutrition, Physical Activity and Metabolism, Palm Harbor, Florida, March 10–14, 2009.Conflict of interest: none declared.1.GoASChertowGMFanDChronic kidney disease and the risks of death, cardiovascular events, and hospitalizationN Engl J Med200435113129613052.CherubiniALowenthalDTParanEHypertension and cognitive function in the elderlyAm J Ther20071465335543.KanayaAMBarrett-ConnorEGildengorinGChange in cognitive function by glucose tolerance status in older adults: a 4-year prospective study of the Rancho Bernardo study cohortArch Intern Med200416412132713334.OztürkCOzgeAYalinOOThe diagnostic role of serum inflammatory and soluble proteins on dementia subtypes: correlation with cognitive and functional declineBehav Neurol20071842072155.ZulianiGCavalieriMGalvaniMMarkers of endothelial dysfunction in older subjects with late onset Alzheimer's disease or vascular dementiaJ Neurol Sci.20082721-21641706.SeligerSLGillenDLLongstrethWTJrElevated risk of stroke among patients with end-stage renal diseaseKidney Int20036426036097.KhatriMWrightCBNickolasTLChronic kidney disease is associated with white matter hyperintensity volume: the Northern Manhattan Study (NOMAS)Stroke20073812312131268.SehgalARGreySFDeOreoPBPrevalence, recognition, and implications of mental impairment among hemodialysis patientsAm J Kidney Dis199730141499.MaderoMGulASarnakMJCognitive function in chronic kidney diseaseSemin Dial2008211293710.MurrayAMCognitive impairment in the aging dialysis and chronic kidney disease populations: an occult burdenAdv Chronic Kidney Dis200815212313211.XueJLMaJZLouisTAForecast of the number of patients with end-stage renal disease in the United States to the year 2010J Am Soc Nephrol200112122753275812.MogensenCEChristensenCKVittinghusEThe stages in diabetic renal disease. 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Mitochondrial Dysfunction Contributes to the Age-Associated Reduction in VO2max Hagen Jason L. 1 Krause Daniel J. 2 Baker David J. 1 2 Fu Ming Hua 3 Tarnopolsky Mark A. 4 5 Hepple Russell T. 1 2 1Faculty of Kinesiology 2Faculty of Medicine, University of Calgary, Alberta, Canada. 3Departments of Kinesiology 4Pediatrics 5Medicine, McMaster University, Hamilton, Ontario, Canada. Address correspondence to Russell T. Hepple, PhD, Faculty of Kinesiology, University of Calgary, 2500 University Dr. NW, Calgary, AB, Canada T2N 1N4. E-mail: hepple@ucalgary.ca 11 2004 59 11 1099 1110 25 7 2004 31 3 2004 The Gerontological Society of America 2004 Although mitochondrial DNA damage accumulates in aging skeletal muscles, how this relates to the decline in muscle mass-specific skeletal muscle aerobic function is unknown. We used a pump-perfused rat hind-limb model to examine maximal aerobic performance (V̇O2max) in young adult (YA; 8–9-month-old), late middle aged (LMA; 28–30-month-old) and senescent (SEN; 36-month-old) Fischer 344 × Brown Norway F1-hybrid rats at matched rates of convective O2 delivery (QO2). Despite similar muscle QO2 during a 4-minute contraction bout, muscle mass-specific V̇O2max was reduced in LMA (15%) and SEN (52%) versus YA. In plantaris muscle homogenates, nested polymerase chain reaction revealed an increased frequency of mitochondrial DNA deletions in the older animals. A greater reduction in the flux through electron transport chain complexes I–III than citrate synthase activity in the older animals suggests mitochondrial dysfunction consequent to mitochondrial DNA damage with aging. These results support the hypothesis that a reduced oxidative capacity, due in part to age-related mitochondrial dysfunction, contributes to the decline in aerobic performance in aging skeletal muscles. hwp-legacy-fpage 1099 hwp-legacy-dochead RESEARCH ARTICLE AGING is associated with a general decline in physiological function that leads to increased morbidity and mortality. Among the most well-known changes in the exercise response is a reduction in maximal oxygen consumption (V̇O2max) with increasing age (1–3), a change that is intimately tied to impaired mobility with aging (4). Whereas an age-related reduction in convective O2 delivery (blood flow × arterial oxygen content) has been a primary explanation for the reduction in V̇O2max with aging (5,6), the role contributed by an intrinsic reduction in skeletal muscle aerobic function has only recently been established (7). Specifically, in these latter experiments it was shown that late middle aged rat skeletal muscles exhibit a lower mass-specific V̇O2max than young adult muscles even when perfused at a similar rate of convective O2 delivery (arterial O2 content × blood flow), revealing an impairment at one or more points in the movement of O2 from blood to cytochrome oxidase in the muscle mitochondria (7). Aging is associated with significant alterations in skeletal muscle, such as reduced muscle mass (8,9) and reduction of the activity of some mitochondrial enzymes (10–12), although not all studies are in agreement with this latter point (e.g., 13). Whereas these alterations in skeletal muscle are influenced by reduced levels of physical activity with aging, a portion of these changes are believed to be a consequence of the biological process(es) of aging (14–18). In this respect, the oxidative stress theory of aging states that some of the physiological decrements typical of increasing age can be ascribed to the life long accumulation of intracellular damage induced by the generation of free radicals (19). In particular, it is significant that the mitochondrial genome is more prone to oxidative damage because of its location (attached to the inner mitochondrial membrane), a lack of histone proteins, and less efficient repair mechanisms than nuclear DNA, rendering it some 16-fold more susceptible to oxidative damage than nuclear DNA (20). Thus, it has been argued that oxidative damage over time leads to mitochondrial DNA mutation deletions that result in dysfunctional mitochondria, and that skeletal muscle is one tissue that is particularly susceptible to this phenomenon (15,16). Since skeletal muscle V̇O2max is a function of an interaction between O2 supply and mitochondrial oxidative capacity (21), it stands to reason that mitochondrial dysfunction, due to oxidative damage, would also be a contributor to the reduction in V̇O2max with increasing age. To address this issue, we examined changes in skeletal muscle function and biochemistry in young adult (8- to 9-month-old), late middle-aged (28- to 30-month-old), and senescent (36-month-old) Fischer 344 × Brown Norway F1-hybrid (F344BN) rats. We hypothesized that under similar conditions of skeletal muscle convective O2 delivery, mass-specific V̇O2max of the skeletal muscles in senescent animals would be even lower than seen previously in skeletal muscles from late middle aged animals compared to young adult animals (7). Furthermore, we hypothesized that the decline in muscle mass–specific V̇O2max with increasing age would be associated with genetic (e.g., mitochondrial DNA deletions) and biochemical (greater decrease in activity of a biochemical pathway containing mitochondrial DNA-encoded peptides versus a nuclear encoded mitochondrial enzyme) evidence of mitochondrial dysfunction consequent to accumulation of oxidative damage. Methods Animals All experiments were conducted after obtaining the approval of the University of Calgary Animal Care Committee. Three groups of specific pathogen-free male F344BN rats were obtained from the National Institute on Aging to represent young adult (8- to 9-month-old; n = 13), late middle-aged (28- to 30-month-old; n = 18), and senescent (36-month old; n = 9) animals, based on previously published survival curves for this strain of rat (22), in a relative comparison to survival curves for humans (23). Rats were housed two per cage (with filter bonnets) in the Faculty of Medicine vivarium at the University of Calgary for a minimum of 1 week prior to experiments (12:12 hour light/dark cycle, 22°C), and were provided water and Purina rat chow ad libitum. As described previously (7), necropsies were performed postexperiment to detect any abnormalities or lesions within each animal. The internal assessment involved an examination of the internal organs and tissues looking for specific identifiable lesions (24,25). To prevent contamination of the data by the presence of disease, animals demonstrating tissue abnormalities or lesions were excluded from the data set, as recommended by NIA guidelines (24). Note that one 28-month-old animal and three of the 36-month-old animals were excluded on this basis (large tumors in each case). As such, the final data set comprises n = 13 (8- to 9–month-old), 17 (28- to 30-month-old), and 6 (36-month-old) animals. Physical Activity The amount of voluntary physical activity over a 72-hour period was quantified in four of the 8-month-old, four of the 28- to 30-month-old [both published previously in (26)], and all six of the 36-month-old animals. As described previously (26), this involved placing individual rats in a standard cage (21 × 20 × 42 cm) that was resting on two aluminum lever arms instrumented with strain gauges at their fulcrum. Measurements from each rat were collected online using a data acquisition system (Dataq DI-700; Dataq Instruments, Inc., Akron, OH) connected to a laptop computer running Windaq Pro+ software (Dataq Instruments). Data analysis of physical activity recordings was performed off-line with Matlab (ver. 6.5; The MathWorks, Inc., Natick, MA) and involved integrating the time during which the voltage signal was elevated above the baseline voltage associated with small movements, such as shifts in body weight and grooming behavior (baseline determined for each animal individually). As such, these measurements are taken as an indicator of locomotor activity. As noted previously (26), the first 2 hours of recording were discarded for each animal to exclude the exploratory behavior associated with transfer to a new environment (27). Surgical Procedures After anesthetizing the animal with Pentobarbital Sodium (i.p. 75 mg/kg), the right iliac artery and vein were ligated and the right gastrocnemius–plantaris–soleus muscle group was removed, trimmed free of fat and connective tissue, and weighed. The right plantaris muscle from 10 of the 8- to 9-month-old, 11 of the 28- to 30-month-old, and all 6 of the 36-month-old animals was frozen in liquid nitrogen and stored at −70°C for subsequent morphological (data not shown) and/or biochemical (below) analyses. Following this, 12 of the 8–9-month-old, 10 of the 28–30-month-old, and all 6 of the 36-month-old animals were prepared for surgical perfusion of the left hind limb (21,28). Note that the metabolic and contractile data during hind-limb perfusion for eight of the 8- to 9-month-old animals and seven of the 28- to 30-month-old animals were part of a recently published study (7) (Table 1). Preparation for hind-limb perfusion and muscle contractions began with isolating the left sciatic nerve in the avascular space between the biceps femoris muscles. The inferior gluteal nerve was then severed to prevent stimulation of the upper hind-limb muscles, and the sciatic nerve was cut proximally in preparation for electrical stimulation via a platinum hook electrode. The Achilles tendon was cut, with a portion of the calcaneous intact, and secured by 1.0 silk thread to a force transducer (FT-10; Grass Instruments, Quincy, MA). The animal was then placed on a heating pad and moved to a stereological base plate where a metal clamp attached around the proximal femur and another clamp attached to the ankle was secured to immobilize the distal hind limb during force measurements. Catheters were inserted into the iliac artery (22 ga) and vein (20 ga) and advanced into the respective femoral artery and vein to initiate perfusion to the hind limb. Ligatures were placed around the iliac artery inside the abdominal wall (immediately proximal to the inguinal ligament) and around the femoral vein to secure the arterial and venous catheters in place. The hind limb was wrapped in warm saline soaked gauze, saran wrap (encompassing a thermistor probe connected to a heat lamp), and aluminum foil to maintain a muscle temperature of 37°C. The perfusion medium consisted of bovine erythrocytes reconstituted in a Krebs–Henseleit buffer (pH 7.4) containing 4.5% bovine serum albumin, bovine erythrocytes (hematocrit 42%), 5 mM glucose, 100 mU/mL insulin, and 0.15 mM pyruvate. Hematocrit (∼42%) was verified by direct observation in centrifuged capillary tubes and yielded an average hemoglobin concentration of 14.3 ± 0.2 g/dL. Prior to entering the hind limb, the perfusion medium was gassed with 95% O2 and 5% CO2 using an oxygenator (4 L flask containing 7 m of silastic tubing), yielding an average arterial O2 content of 20.7 ± 0.2 % by volume. Flow was controlled using a peristaltic pump (Gilson Minipuls 3; Gilson, Inc., Middleton, WI), with flow verified after each experiment by timed blood collection through the arterial catheter. A pressure transducer (PT-300; Grass Instruments) was placed at the height of the hind limb for determination of total perfusion pressure during perfusion conditions. Experimental Protocol Perfusion rate was incrementally increased to elicit a flow-induced vasodilatory response until the desired rate of perfusion was achieved (∼30 min). Note that the desired flow rate was selected to permit matching of mass-specific blood flow to the contracting muscles between age groups, as done previously (7). Briefly, this involved measuring the mass of the gastrocnemius–plantaris–soleus muscle group in the contralateral (right) hind limb and using this value to select the rate of perfusion on the pump that would yield a similar rate of mass-specific muscle blood flow for each animal. Tetanic stimulation, elicited by square wave pulses (200 ms trains at 100 pulses/s, each 0.2 ms in duration), was used to elicit muscle contractions at a frequency of 60 tetani/minute for 4 minutes, since this frequency yields the highest V̇O2 for this preparation (29). Muscle length and voltage (∼7 volts) were adjusted to yield maximum force development. Blood samples were drawn anaerobically every 30 s during contractions from the arterial blood and venous effluent and were analyzed for PO2, PCO2, O2 saturation (SO2), [hemoglobin], and [lactate] [note: lactate was only measured in a subset of animals; see our companion paper (30)] by a blood gas analyzer (Stat Profile M3; Nova Biomedical; Waltham, MA). Blood oxygen content was calculated using the formula: [O2] × SO2 × 1.39 + 0.003 × PO2. V̇O2 across the hind limb was determined from the product of blood flow and the arteriovenous O2 content difference, with V̇O2max taken as the sample having the lowest venous percent O2 saturation (arterial O2 content and blood flow were held constant). Normalization Procedures As demonstrated by Gorski and colleagues (28), total perfused muscle mass in the hind limb in this preparation includes all of the hind-limb muscles (hamstrings, quadriceps, gastrocnemius, plantaris, soleus, tibilias anterior, and remaining tibial muscles) except the gluteal muscles (c.f. 29). The total perfused muscle mass was measured in all of the 36-month-old animals, and in three each from the 8- to 9-month-old and 28- to 30-month-old animals. Using this data, the relationship between total perfused muscle mass and the total contracting muscle mass [the latter was measured in all animals and includes the gastrocnemius–plantaris–soleus muscle group, tibialis anterior, and remaining tibial muscles (28)] was determined for each age group, and total perfused muscle mass was estimated from these relationships in the remaining animals. Total noncontracting perfused muscle mass was calculated as the difference between the total perfused muscle mass and the total contracting muscle mass and comprises 72 ± 1%, 71 ± 1%, and 80 ± 2% of the total perfused muscle mass in the 8- to 9-month-old, 28- to 30-month-old, and 36-month-old animals, respectively. As done previously (7,29), since most of the V̇O2 measured at rest is contributed by these noncontracting tissues, V̇O2 during contractions was normalized to the mass of the contracting muscles after subtracting the V̇O2 contributed by the noncontracting tissues (calculated based upon values observed at rest). Blood Flow Distribution As described previously (21), following the contraction bout ∼280,000 colored microspheres (15 μm diameter, Dye Tak; Triton Technology, Nottinghamshire, U.K.) were drawn from a known stock concentration and injected slowly (while reducing the pump output to maintain perfusion pressure) into a side arm port situated proximal to the arterial catheter. Saline (2 mL) was slowly infused immediately after the microsphere infusion to ensure that all microspheres reached the hind limb. The gastrocnemius, plantaris, and soleus muscles were excised and heated in a water bath (60°C) in centrifuge tubes containing 4 M KOH until each muscle was digested (≥60 min). The content of each centrifuge tube, along with a reference sample (stock solution), was individually filtered through 8 μm pore membranes (Whatman Nucleopore, Clifton, NJ) to trap the microspheres. The membranes were placed in microcentrifuge tubes containing 1 ml of N, N-dimethyl-formamide to release the color of the spheres. Absorbance of the resulting solution was analyzed using a spectrophotometer (Ultrospec 2100 Pro; Biochrom, Berlin, Germany) after 10 minutes at a wavelength of 448 nm to determine the number of microspheres in each sample (calculated based on the regression equation provided by the lot manufacturer). The blood flow to the gastrocnemius–plantaris–soleus muscle group was determined as the product of the total hind-limb blood flow and the proportion of microspheres found in the gastrocnemius–plantaris–soleus muscle group. Similarly, muscle convective O2 delivery was calculated as the product of the blood flow to the gastrocnemius–plantaris–soleus muscle group and the arterial O2 content. As done previously (7), O2 extraction across the contracting muscles was estimated as the quotient of the mass-specific V̇O2max and blood flow to the gastrocnemius–plantaris–soleus muscle group. Biochemistry The flux through mitochondrial electron transport chain complexes I–III [which contains 8 of the 13 polypeptides encoded by the mitochondrial genome (31)] and citrate synthase activity (entirely nuclear encoded) was determined using spectrophotometric methods. Muscles were thawed on ice and placed in a glass homogenizer at 4°C in phosphate buffer (pH 8.0) containing 0.05 M Tris–HCl and 0.67 M sucrose, and the resulting muscle homogenates were stored at −70°C until assayed. As done previously (7,21), to assess the flux through complexes I–III, homogenates were thawed on ice and the rate of reduction of cytochrome-c at 38°C was followed spectrophotometrically at 550 nm (Biochrom Ultrospec 2100 Pro) after the addition of 20 μl of homogenate to 1.0 M phosphate buffer (pH 8.0), 0.1 M NaN3, 1% aqueous cytochrome-c, 0.01 M NADH, and bringing the total volume to 1 ml with double distilled H2O. Each sample was measured in triplicate and the average activity over the linear portion of the absorbance versus time relationship was used to represent the flux through complexes I–III (21). Citrate synthase activity was measured according to the method of Srere (32), with the exception that the homogenizing medium used in this assay was the same as that used in preparing samples for measuring the flux through complexes I–III described above (such that both enzyme pathways could be determined from the same homogenate sample). Briefly, each homogenate was thawed on ice and then spun at 600 g for 3 minutes. These samples were then diluted by adding homogenizing medium to reach a 1:22 dilution. Each sample was vortexed for 20 seconds, sonicated in an ice bath for 10 minutes, and refrozen at −20°C; this was repeated 3 times. The final dilution was then performed by adding 1.9 ml of homogenizing medium to 0.1 ml homogenate to yield a final dilution of 1:440. Citrate synthase activity was determined by measuring the rate of production of the mercaptide ion spectrophotometrically at 412 nm (Biochrom Ultrospec 2100 Pro) after the addition of 20 μl of the homogenate to 3 mM Acetyl CoA, 100 mM Tris buffer (pH 8.0), 1 mM DTNB, and 5 mM oxaloacetate at 38°C. Each sample was measured in triplicate, and citrate synthase activity was determined from the average activity over the linear portion of the absorbance versus time relationship. As done previously (7,21), the protein content was assessed for each homogenate by the Biuret method such that the flux through complexes I–III and citrate synthase activity could be expressed relative to total muscle protein. The ratio of the flux through complexes I–III and citrate synthase activity was used as an index of mitochondrial “health,” with dysfunctional mitochondria being characterized by a disproportionately lower flux through complexes I–III versus citrate synthase activity (i.e., lower ratio). Mitochondrial DNA Analysis Mitochondrial DNA deletions were assessed in homogenates of plantaris muscle used in biochemical analyses (above) from four of the 8- to 9-month-old, four of the 28- to 30–month-old, and five of the 36-month-old animals. Note that the primers used spanned the region of the mitochondrial genome containing 10 of the 13 mitochondrial DNA-encoded components of the electron transport chain: ND3, ND4L, ND4, ND5, and ND6 in complex I; cytochrome b in complex III; COX II and COX III in complex IV; and ATP6 and ATP8 of complex V. Total DNA was recovered by adding 1 M Tris–HCL (pH 8.0), 0.5 M EDTA, and 20% SDS to each homogenate to give a final concentration of 10 mM, 0.1 M, and 0.5%, respectively. Pancreatic RNAase (400 μg/ml) was then added to each homogenate and incubated at 37°C for 1 hour. Proteinase K (100 μg/ml) was added to each homogenate and incubated at 50°C for 3 more hours. DNA was subsequently extracted with phenol, and quantified using spectrophotometry, according to the method described by Sambrook and Russell (33). Following DNA quantification, a stock DNA solution of 50 ng/ul was prepared and used for polymerase chain reaction (PCR). Mitochondrial DNA was amplified from the DNA stock using the PCR Takara Ex Taq hot start version kit (Takara Bio Inc., Japan) and the following forward and reverse primers: 5′-TCCCTTCACTAGGGTTAAAAACCGA-3′ (forward); 5′-GGCGGAATGTTAAGCTGCGTTGT-3′ (reverse), with 30 thermocycles, consisting of 30 seconds at 94°C, 25 seconds at 60°C, and 90 seconds at 72°C. The primary PCR product was then further amplified using nested PCR, to determine deletions within the mitochondrial DNA itself. This was achieved using the Takara hot start version PCR kit (as described above) and the following forward and reverse primers: 5′-CCGGCCGCCTAAACCAAGCTACAGT-3′ (forward); 5′-TGGGGTGGGGTGTTGAGGGGGTTAG-3′ (reverse), with 30 thermocycles, consisting of 30 seconds at 94°C, 25 seconds at 66°C, and 90 seconds at 72°C. Intact and deleted mitochondrial DNA was then photographed under UV light following gel electrophoresis (1% agarose gel with ethidium bromide, 160 volts for 1.5 hours). Total amplifiable mitochondrial DNA was estimated from the optical density of the largest bands in each lane using image analysis software (Sigmascan Pro 5.0; SPSS, Inc., Chicago, IL) calibrated for optical density units. Statistical Analysis Values are reported as means ± standard error (SE). Differences between 8-, 28- to 30-, and 36-month-old animals were assessed by Student's t test (ratio of the flux through complex I–III and citrate synthase activity; combined older groups), one-way analysis of variance (ANOVA) (i.e., animal characteristics, physical activity, perfusion conditions, contractile and metabolic responses, total amount of amplifiable mitochondrial DNA), or a two-way ANOVA (i.e., muscle × age for blood flow distribution) with a Bonferroni post hoc test. The relationship between distal hind-limb muscle mass and mass-specific V̇O2max was assessed by linear regression. The α was set at.05. Results Animal Characteristics The descriptive data of the animals are found in Table 2. Despite a greater body mass, the mass of the gastrocnemius–plantaris–soleus muscle group was progressively reduced (8- to 9–mo old > 28- to 30-mo old > 36-mo old) in the older animals. As a result, the mass of the gastrocnemius–plantaris–soleus muscle group relative to the whole body mass in the 28- to 30-month-old (0.44 ± 0.01%) and 36-month-old (0.23 ± 0.02%) animals was significantly lower than that in the 8- to 9-month-old animals (0.62 ± 0.01%). The degree of muscle atrophy varied between muscles and accelerated between late middle age and senescence in each muscle examined. Specifically, the soleus muscle mass was well maintained up to 28–30 months of age but declined 40% by 36 months of age versus 8- to 9-month-old animals. In contrast, plantaris muscle mass declined 13% by 28–30 months of age and 52% by 36 months of age, and the gastrocnemius muscle declined 19% by 28–30 months of age and 60% by 36 months of age versus 8- to 9-month-old animals. Physical Activity The voluntary physical activity levels measured over a 70-hour period in an instrumented cage were progressively reduced as a function of age and, like muscle mass, this was exacerbated between late middle age and senescence. As shown previously (26), whereas the 8- to 9-month-old animals were active for 3.6 ± 0.3 hours, the 28- to 30-month-old animals were active for 2.5 ± 0.1 hours. The current results show that physical activity was further reduced in the 36-month-old animals (1.3 ± 0.2 h; p <.05). Perfusion Conditions Note that there was a surgical error with one of the 8- to 9-month-old animals and one of the 36-month-old animals and, as such, contractile and metabolic measurements during hind-limb pump-perfusion are based on 11 animals in the 8- to 9-month-old group and 5 animals in the 36-month-old group. The mass of the contracting muscles (gastrocnemius muscle, plantaris muscle, soleus muscle, tibialis anterior muscle, and remaining deep tibial muscles) was significantly reduced with increasing age. The net perfusion pressure (difference between total perfusion pressure and the pressure required to overcome the resistance of the perfusion system) was lower in the 36-month-old animals versus the 28- to 30- and 8- to 9-month-old animals (Table 3). Blood flow to the whole hind limb was less in the 36-month-old and 28- to 30-month-old versus the 8- to 9-month-old animals. However, because the muscle mass was lower in the older animals (see above), muscle mass-specific blood flow to the gastrocnemius–plantaris–soleus muscle group was not different between the 8- to 9-month-old, 28- to 30-month-old, and 36-month-old animals. Similarly, blood flow distribution between the gastrocnemius, plantaris, and soleus muscles was not different between groups (Figure 1). Finally, there was no difference in the muscle mass-specific convective O2 delivery between age groups (Figure 2), showing that muscle convective O2 delivery was well matched between groups. Metabolic Responses When expressed in absolute terms, the hind-limb resting V̇O2 in the 36-month-old animals was lower compared with the 8- to 9-month-old and 28- to 30-month-old animals. However, when expressed relative to the total estimated perfused muscle mass, the resting V̇O2 was not different between groups (Table 4; p =.085), which is similar to our previous observations in young adult and late middle-aged rats (7). Detailed treatment of the force measurements made in a subset of these animals during the contraction bout can be found in our companion paper (30). The force at V̇O2max was progressively lower in the 28- to 30-month-old and 36-month-old animals compared with the 8- to 9-month-old animals. Whether expressed in absolute terms (Table 4) or normalized to the mass of the contracting hind-limb muscles, V̇O2max in the 28- to 30- and 36-month-old animals was significantly less than the 8- to 9-month-old animals, despite very similar rates of convective O2 delivery (Figure 2). In this regard, the difference in V̇O2max between 8- to 9-month-old and 28- to 30-month-old animals (15%) was slightly less than that observed previously in a smaller number of animals (22%) (7). The estimated gastrocnemius–plantaris–soleus muscle group oxygen extraction at V̇O2max was significantly lower in the 36-month-old compared with the 28- to 30-month-old and 8- to 9-month-old animals. Mitochondrial DNA Analyses Whereas there were no mitochondrial DNA deletions seen in the 8- to 9-month-old animals, there was a progressive increase in the frequency of mitochondrial DNA deletions in 28- to 30-month-old and 36-month-old animals (Figure 3). Similarly, the optical density of the 7591 bp bands on the PCR gel were 40% lower in the 36-month-old animals compared with young adult animals (Figure 4), suggesting a reduced amount of intact total amplifiable mitochondrial DNA in the senescent animals. Muscle Oxidative Capacity There was a progressive reduction (8- to 9-month old > 28- to 30-month old > 36-month-old rats) in the flux through electron transport chain complexes I–III with increasing age. In contrast, citrate synthase activity was not different between 8- to 9-month-old and 28- to 30-month-old animals, but was significantly reduced in 36-month-old versus 8- to 9-month-old animals (Figure 5). In comparing the relative changes between these two enzyme pathways with aging, the 36-month-old animals were pooled with the 28- to 30-month-old animals because the number of animals in the 36-month-old group was not large enough to yield sufficient statistical power and there were no significant differences in the ratio of the two enzyme pathways between these two groups. In this comparison, the flux through complexes I–III was reduced to a greater extent than citrate synthase activity in the older animals, such that the ratio of the two enzyme pathways (complex I–III:citrate synthase) was 36% lower in homogenates prepared from the plantaris muscle of the old (28- to 30-month-old and 36-month-old rats combined: 2.5 ± 0.3) compared with the 8-month-old (3.9 ± 0.7) rats. Discussion The objective of this study was to test the hypothesis that the decline in skeletal muscle mass-specific V̇O2max with aging is due, in part, to mitochondrial dysfunction consequent to accumulation of damage to the mitochondrial genome. To this end, we examined contractile and metabolic responses in the distal hind-limb muscles of young adult, late middle aged, and senescent rats using an in situ pump-perfused hind-limb preparation to permit matching of skeletal muscle convective oxygen delivery between groups. Our results showed that the decline in muscle mass-specific V̇O2max previously reported between young adulthood and late middle age (7) accelerates between late middle age and senescence in rat distal hind-limb skeletal muscles perfused at similar rates of convective oxygen delivery. Whereas the decline in voluntary physical activity previously reported between young adulthood and late middle age (26) also accelerated between late middle age and senescence, the decline in muscle oxidative capacity in the older animals (28- to 30- and 36-month-old animals combined) was characterized by a relatively greater decline in flux through electron transport chain complexes I–III than citrate synthase activity in homogenates prepared from the plantaris muscle. As such, these latter results suggest that the decline in oxidative capacity was not entirely explained by the decline in physical activity, but was also affected by age-associated mitochondrial dysfunction. Consistent with this interpretation, there was an increased frequency of mitochondrial DNA deletions and a reduced amount of intact total amplifiable mitochondrial DNA in homogenates prepared from the plantaris muscle with aging. As such, our results support the hypothesis that a portion of the decline in skeletal muscle V̇O2max with aging is due to mitochondrial dysfunction consequent to age-associated mitochondrial DNA damage. Critique of the Model The animal model used in our studies is the F344BN rat, which was developed by the National Institute on Aging for aging research. This rat strain lives considerably longer and exhibits fewer tissue pathologies at any given absolute age than either of the parental strains (Fischer 344 or Brown Norway) (34). The ages of the animals in the current study were chosen to represent young adult (8- to 9-month-old), late middle-aged (28- to 30-month-old), and senescent (36-month-old) animals, based on published survival characteristics for this strain of rat (22). Although maturational differences between humans and rats prevent an exact comparison, if we use survival characteristics as the basis for our comparison, the human equivalents are roughly 20 years of age (young adult), 60 years of age (late middle aged), and 80 years of age (senescent) (23). Previous studies of the F344BN rat have shown that it exhibits a progressive loss of muscle with aging (35,36), like humans (37–39). In this regard, there was a 17% decrease in the mass of the distal hind-limb muscles (i.e., total contracting muscle mass; Table 2) between young adult and late middle age, and a decrease of 53% between young adult and senescence in our study. Based upon Lexell's data examining changes in cross-sectional area in whole vastus lateralis muscle from human cadavers aged 15–83 years (38), this degree of muscle mass loss is very similar to that observed in humans between young adulthood and late middle age (∼22%), and between young adulthood and senescence (∼50%). With the caveat that this comparison is confined to examples of locomotor skeletal muscles, not only is the degree of muscle atrophy similar with aging in the F344BN rat and humans, but also an accelerated loss of muscle after late middle age is apparent in both species. Since a decline in physical activity is known to affect skeletal muscle mass and function, it is also noteworthy that, like humans, there is a decline in voluntary physical activity with aging in F344BN rats. Lastly, we have reported an accumulation of mitochondrial DNA deletions in homogenates prepared from aged muscles compared to that obtained from young adult muscles in rats. This is consistent with prior studies showing that skeletal muscle from both rats and humans accumulate mitochondrial DNA deletions with aging (18,40,41). Thus, on the basis of these comparisons, the F344BN rat is a useful model for providing insight into the effects of aging in human skeletal muscle. Based upon the higher mass-specific blood flows observed in the distal hind-limb muscles of young adult rats during treadmill running (42), it is likely that the highest V̇O2 attained for these muscles is greater in vivo than seen with the in situ pump-perfused model used in our study. In this respect, the high degree of fatigue seen with our electrical stimulation protocol [see Figure 1 in companion paper (30)], and the resulting intracellular perturbation, might be considered to compromise the aerobic metabolic response. However, in previous experiments in our laboratory where we used a gradual increase in contraction frequency, despite a more gradual fatigue of the stimulated muscles, this did not affect the maximal V̇O2 attained (29). We have since observed that this is also true in 8- to 10-month-old and 35-month-old F344BN rats (R.T. Hepple, unpublished observations). As such, the V̇O2max values observed in this study are the maximum achievable for these perfusion conditions (29), and, therefore, can be used to gain insight into the factors that contribute to the decline in skeletal muscle aerobic function with aging. Further to this point, whereas it is not technically feasible to control convective O2 delivery in human studies, the pump-perfused rat hind-limb preparation is well suited to this purpose. Reasons for Reduced V̇O2max With Aging Although a decline in whole body V̇O2max with increasing age is well established (1,3,43), the causes of this decline remain to be clarified. The physiological determinants of V̇O2max during whole body exercise in young adult humans or animal models involve the coordinated actions of multiple systems, beginning at the lung and ending with cytochrome oxidase in the mitochondria of contracting skeletal muscles. Although a comprehensive review of the evidence is beyond the scope of this manuscript, alterations in O2 delivery (44–47) and mitochondrial oxidative capacity (48,49) affect V̇O2max in a near-proportional manner. Most recently, it was shown that when reduced oxygen delivery was combined with reduced mitochondrial oxidative capacity the reduction in V̇O2max was greater than either intervention performed independently, revealing an interaction between oxygen delivery and mitochondrial oxidative capacity in determining V̇O2max (21). The implication of this last point is that even though maximal mitochondrial oxidative capacity may appear to be in relative excess of that required in vivo at V̇O2max, it apparently still exerts an influence on V̇O2max (21). Based on the preceding evidence, it is likely that a reduced capacity at several points in the chain of events linking oxygen transport and oxygen utilization contributes to reduce V̇O2max during whole body exercise with aging. In this regard, whereas a reduced cardiac output (5,6,50) and maldistributed blood flow (51–53) are thought to impair V̇O2max with aging, few studies have addressed the contribution that alterations within the contracting muscles play in this response. Consistent with prior evidence from whole body exercise responses in humans suggesting that skeletal muscle changes (e.g., a reduced oxidative capacity) contribute to the decline in V̇O2max with aging (54,55), a reduced ability to use oxygen was recently observed in skeletal muscles of late middle aged rats (7), and is further supported by our current findings. Specifically, by using a pump-perfused rat hind-limb preparation to achieve similar rates of convective oxygen delivery to the contracting muscles across age groups (where blood flow and its distribution was not different between groups), the lower V̇O2max observed in the skeletal muscles of the late middle aged [current results and (7)] and senescent animals (current results) can be attributed to differences in the movement of oxygen from the blood to the tissues. As such, the factors that could be involved in the lower V̇O2max seen in the aged muscles include the structural (i.e., capillary number) and functional (i.e., erythrocyte hemodynamics) capillary surface area, myoglobin concentration, and mitochondrial oxidative capacity. It is, therefore, pertinent that the anatomical capillarization is not compromised in distal hind-limb skeletal muscles of late middle aged (56) or senescent rats (57). Similarly, the erythrocyte hemodynamics exhibit minor changes in resting spinotrapezius muscles of older rats (58), although the hemodynamics during muscular contractions remain to be examined. In addition, a prior study reported no change in myoglobin concentration between the ages of 9 months and 25 months in the gastrocnemius muscle of Sprague-Dawley rats (59). This leaves changes in mitochondrial oxidative capacity as a likely contributor to the decline in V̇O2max seen in the aged muscles. Flux Through Complexes I–III and V̇O2max The current results show that the flux through electron transport chain complexes I–III was reduced by 43% and 60% in 28- to 30-month-old and 36-month-old rats, respectively. We chose to examine the flux through electron transport chain complexes I–III as a marker of mitochondrial oxidative capacity for two reasons. Firstly, we (21), and Terjung's group (48,49), have previously shown that acute reduction in the flux capacity of this pathway (using the complex III inhibitor, myxothiazol) results in a proportional reduction in V̇O2max, showing that this pathway plays an important part in determining maximal aerobic function in skeletal muscles. Secondly, this enzyme pathway reflects the biochemical consequences of age-associated alterations in the mitochondrial genome because it contains several mitochondrial DNA-encoded peptides (31). As seen in Figure 6, where we have plotted our previous results using myxothiazol to acutely reduce the flux capacity through complexes I–III together with the current results, it is apparent that whereas the senescent animals fall on the line depicting the relationship between V̇O2max and the flux capacity through complexes I–III, the late middle-aged animals have a considerably higher V̇O2max than would be predicted by this relationship. [In our previous study (21), V̇O2max values were normalized to the mass of the gastrocnemius–plantaris–soleus muscle group, rather than the mass of the entire contracting distal hind-limb muscles (as done in the current study). Thus, we have estimated the mass of the distal hind-limb muscles for our previous results based upon the relationship between the mass of the gastrocnemius–plantaris–soleus muscle group and the entire distal hind-limb muscles in 49 animals studied in our laboratory (total distal mass = 302 + {1.660 × gastrocnemius–plantaris–soleus muscle group mass}; r2 =.84, p <.001), and used these values to normalize our prior V̇O2max data (21) in the same manner used in the current study.] Thus, on the basis of the aforementioned points, the decline in flux through this biochemical pathway must be contributing to the decline in muscle mass-specific V̇O2max with aging observed in our study. The dissociation of V̇O2max and the flux capacity through complexes I–III in the late middle aged animals may indicate some compensation at this age. Alternatively, because the relationship between V̇O2max and the flux through electron transport chain complexes I–III was derived from experiments utilizing the complex III inhibitor, myxothiazol (21), it is possible that complex III activity in the late middle aged group is reduced in proportion to V̇O2max, but that a relatively greater decline in complex I is obscuring this relationship. Indeed, previous studies have shown that complex I often exhibits the greatest degree of dysfunction with aging (60–62), lending support to this explanation. The fact that the senescent animals fall on the line predicted by the relationship between V̇O2max the flux through complexes I–III suggests that the decline in complex I and complex III activity in senescence is more proportional. Further studies using assessment of the activity of individual components of the electron transport chain are required to address these possibilities. Effects of Aging and Physical Inactivity on Mitochondrial Oxidative Capacity The accumulation of mitochondrial DNA damage (e.g., due to the accumulated effects of oxidative stress with aging) has been linked to a decreased mitochondrial electron transport chain function in tissues exhibiting a high aerobic metabolic activity [e.g., skeletal muscle, cardiac muscle, neurons (16,31,63)]. Whereas a decline in skeletal muscle oxidative capacity with aging has been well documented (10–12); with noted exceptions (13,64), distinguishing between a decline which is due to reduced physical activity versus that which is due to aging processes (such as mitochondrial DNA damage) requires consideration of mitochondrial biochemistry. Although physical inactivity leads to a reduced oxidative capacity, the activity of individual mitochondrial enzymes relative to one another in a given volume of mitochondria remains constant across wide differences in mitochondrial content when comparing physically active versus sedentary animals (65). In contrast, aging has been shown to cause a relatively greater loss in the activity of mitochondrial enzymes that contain mitochondrial DNA-encoded polypeptides (i.e., electron transport chain complexes I, III, IV, and V) than mitochondrial enzymes that are entirely nuclear encoded (e.g., complex II) (62,66). Furthermore, this dissociation in the activities of mitochondrial enzymes is thought to be the result of aging-associated damage to the mitochondrial genome [secondary to oxidative stress (18,19)], which causes impaired function of the complexes containing polypeptides encoded by the affected regions (16,18,67). Thus, although the observed reduction in voluntary physical activity likely is contributing to a reduction in oxidative capacity with aging in our study, the relatively greater reduction in the flux through electron transport chain complexes I–III than citrate synthase activity suggests that a portion of the reduction in oxidative capacity with increasing age is due to mitochondrial dysfunction. Müller-Hocker and colleagues were among the first to show that, in extraocular muscles obtained from elderly subjects, fibers exhibiting very low activities of cytochrome oxidase also had very high levels of mitochondrial DNA mutations (68). More recently, mitochondrial DNA deletions have been found in cytochrome oxidase–deficient fibers from aged human limb skeletal muscles (16) and aged rat limb skeletal muscles (67). Indeed, the better maintained proportionality of mitochondrial enzymes with aging seen in long-term caloric restricted animals (62) is consistent with the lower incidence of mitochondrial DNA damage seen with caloric restriction (66,69). Furthermore, a recent study showed that generation of mice with defective mitochondrial DNA polymerase results in a markedly shortened life span, in conjunction with an earlier age-associated accumulation of mitochondrial DNA damage and cytochrome oxidase deficient cells in heart (70). Supporting the idea that mitochondrial DNA mutations are linked to mitochondrial dysfunction, we observed an age-associated increase in the frequency of deletions in mitochondrial DNA extracted from homogenates of the plantaris muscle in conjunction with biochemical evidence of mitochondrial dysfunction. Studies from patients with mitochondrial myopathies suggest that the ratio of mutant to normal mitochondrial DNA (mitochondrial heteroplasmy) within individual cells needs to be quite high (≥60% mutation loads) to affect electron transport chain function (71–73). Given that our measurements were made on whole muscle (not individual cells), it is likely that despite a relatively low deletion load detected in the late middle-aged group, some individual cells may have reached levels necessary to impair electron transport chain function. The marked reduction in the amount of intact total amplifiable mitochondrial DNA in the senescent animals could limit mitochondrial gene expression and thus, compromise mitochondrial biogenesis in the aged muscles (74). In this latter regard, a reduced amount of mitochondrial DNA in skeletal muscle with aging has been observed in humans (75,76) and rats (74), a change thought to result from exhaustion of mitochondrial DNA repair mechanisms consequent to age-associated acceleration in mitochondrial DNA damage (75). This explanation is supported by the current results showing that a reduced amount of intact amplifiable mitochondrial DNA coincides with a dramatic increase in mitochondrial DNA deletions in the senescent muscles. We hypothesized that the decrease in oxidative capacity resulting from production of dysfunctional electron transport chain complexes with aging contributes to a reduction in the muscle oxidative capacity, and thus, impairs the muscle's aerobic function. In support of this hypothesis, we observed that muscle mass-specific V̇O2max was progressively reduced with increasing age even when provided with similar levels of convective oxygen delivery; a difference that is due in large part to the lower mitochondrial oxidative capacity. Since our results suggest that a portion of the decline in oxidative capacity can be attributed to mitochondrial dysfunction, at least a portion of the decline in V̇O2max must also be due to mitochondrial dysfunction. Our results are similar in this regard to a recent study of creatine-stimulated respiration in single permeabilized skeletal muscle fibers from aged humans which also concluded that mitochondrial dysfunction contributed to reduce skeletal muscle aerobic function with aging (77). Future studies using strategies for limiting mitochondrial dysfunction with aging (e.g., through dietary manipulations such as caloric restriction) should prove useful in further testing for a link between oxidative stress, mitochondrial dysfunction, and impaired aerobic function of aging skeletal muscles. Summary Our current results expand upon our previous study showing reduced ability of late middle aged skeletal muscles to use O2 even when provided similar levels of convective O2 delivery as young adult muscles (7), and show that this decline is accelerated between late middle age and senescence. These results reveal an impairment at one or more points in the flux of O2 from blood to cytochrome oxidase in myocyte mitochondria in aged muscles. In this regard, the relatively greater decline in the flux through electron transport chain complexes I–III than citrate synthase activity with aging is indicative of mitochondrial dysfunction that is the result of age-related mitochondrial DNA damage. Consistent with this notion, not only was there a progressive increase in mitochondrial DNA deletions between late middle age and senescence, there was also a dramatic decrease in the intact total amount of amplifiable mitochondrial DNA in senescent muscles. Therefore, our results support the hypothesis that a portion of the decline in skeletal muscle mass-specific V̇O2max is due to age-associated mitochondrial dysfunction. Decision Editor: James R. Smith, PhD Figure 1. Blood flow distribution in the gastrocnemius–plantaris–soleus muscle group. SOL = soleus muscle, PLA = plantaris muscle, GAS = gastrocnemius muscle, Combined = gastrocnemius–plantaris–soleus muscle group as a whole. Note that n = 11 for the 8- to 9-month-old group, n = 10 for the 28- to 30-month-old group, and n = 5 for the 36-month-old group. Values are means ± SE Figure 2. Convective O2 delivery (QO2) and V̇O2max measurements during contraction bout. Note that n = 11 for the 8- to 9-month-old group, n = 10 for the 28- to 30-month-old group, and n = 5 for the 36-month-old group. Values are means ± SE. *p <.05 versus other groups Figure 3. Ethidium bromide–stained agarose gel of intact (arrow at 7591 bp) and fragmented mitochondrial DNA extracted from homogenates of plantaris muscles of 8- to 9-month-old, 28- to 30–month-old, and 36-month-old F344BN rats. Far right lane is a marker denoting the size of mitochondrial DNA deletion products. Note the presence of deleted regions of mitochondrial DNA in both the 28- to 30-month-old and 36-month-old animals but not in the 8- to 9-month-old animals Figure 4. Optical density of the 7591 bp bands (representing intact mitochondrial DNA) on the agarose gel (see 
 Figure 3) in 8- to 9-month-old (n = 4), 28- to 30-month-old (n = 4), and 36-month-old (n = 5) F344BN rats. Results show a reduced amount of intact total amplifiable mitochondrial DNA in the 36-month-old animals. Values are means ± SE. *p <.05 versus other groups Figure 5. The flux through mitochondrial electron transport complexes I–III and citrate synthase activity in homogenates prepared from the plantaris muscle. Units refer to the rate of appearance of reduced cytochrome-c (Complex I–III), or the appearance of the mercaptide ion (citrate synthase). Note that n = 11 for the 8- to 9-month-old group, n = 11 for the 28- to 30-month-old group, and n = 6 for the 36-month-old group. Values are means ± SE. *p <.05 versus other groups; †p <.05 versus 8- to 9-month-old group Figure 6. Relationship between mass-specific V̇O2max and the flux through electron transport chain complexes I–III. Note that the mass-specific convective O2 delivery (arterial O2 content × blood flow) was not different between groups (mean: 550 ± 19 μmol/min/100 g). Control myxo study = control animals from a previous study (21); 0.1 μM myxo = group treated with 0.1 μM myxothiazol to inhibit complex III activity from a previous study (21) Table 1. Summary of Animals Common to the Current Study and Two of Our Previous Studies. Current Study Previous Studies 8-Month-Old Group V̇O2 CSa and Flux I–III V̇O2max (7) Flux I–III (56) 1 X X 2 X X X X 3 X X X X 4 X X X X 5 X X X X 6 X X 7 X X X X 8 X X 9 X X 10 X 11 X X 12 X X 13 X X 28–30-Month-Old Group V̇O2max CSa and Flux I–III V̇O2max Previously (7) Flux I–III Previously (56) 1 X 2 X 3 X 4 X X 5 X 6 X X 7 X X 8 X X X X 9 X X 10 X X 11 X X X X 12 X X 13 X X 14 X X 15 X X 16 X 17 X X Note: CSa = citrate synthase activity; Flux I–III = the flux through electron transport chain complexes I–III. “X” denotes an animal included in the specified study. Table 2. Animal Characteristics. Age Body Mass (g) Sol Mass (mg) Plan Mass (mg) Gastroc Mass (mg) Gas–Plan–Sol Mass (mg) 8–9 Months 428 ± 7* 166 ± 4 394 ± 7 2131 ± 34 2691 ± 42 28–30 Months 516 ± 16 162 ± 6 345 ± 8* 1727 ± 41* 2235 ± 52* 36 Months 504 ± 24 100 ± 4* 191 ± 10* 854 ± 112* 1085 ± 120* Notes: N = 13 for the 8–9-month-old group; N = 17 for the 28–30-month-old group; N = 6 for the 36-month-old group. Sol = soleus muscle; Plan = plantaris muscle; Gastroc = gastrocneminus muscle; Gas–Plan–Sol = gastrocnemius–plantaris–soleus muscle group. Values are means ± SE. *p <.05. Table 3. Perfusion Conditions. Age 8–9 Months Old 28–30 Months Old 36 Months Old Total contracting muscle mass (g) 4.72 ± 0.08 3.92 ± 0.12* 2.13 ± 0.23* Total perfusion pressure (Torr) 145 ± 5 125 ± 3* 103 ± 6* Net perfusion pressure (Torr) 93 ± 4 81 ± 3† 68 ± 3† Total hind-limb blood flow (ml/min) 11.5 ± 0.3 9.9 ± 0.2* 7.3 ± 0.7* Notes: Total contracting muscle mass = the gastrocnemius–plantaris–soleus muscle group; tibialis anterior muscle and remaining deep tibial muscles; total perfusion pressure = pressure through the perfusion tubing and the rat hind limb; net perfusion pressure = the difference between the total perfusion pressure and the pressure required to overcome the resistance of the perfusion tubing. N = 11 for the 8–9-month-old group; N = 10 for the 28–30-month-old group; N = 5 for the 36-month-old group. Values are means ± SE. *p <.05 versus other groups; †p <.05 versus 8–9-month-old group. Table 4. Contractile and Metabolic Responses. Age 8–9 Months Old 28–30 Months Old 36 Months Old Hind-limb resting V̇O2 (μmol/min) 6.0 ± 0.4 5.9 ± 0.4 3.5 ± 0.5* Hind-limb resting V̇O2 (μmol/min/100 g) 35 ± 2 42 ± 3 32 ± 4 Force at V̇O2max (N) 16 ± 1 9 ± 1* 5 ± 1* V̇O2max (μmol/min) 25.2 ± 0.9 18.5 ± 0.7* 7.2 ± 0.9* V̇O2max (μmol/min/100 g) 440 ± 16 372 ± 20* 211 ± 38* Peak O2 extraction (%) 84 ± 7 73 ± 7 32 ± 3* Notes: N = 11 for the 8–9-month-old group; N = 10 for the 28–30-month-old group; N = 5 for the 36-month-old group. Values are means ± SE. *p <.05 versus other groups. The authors thank Chelsey Wyrostock for her assistance in conducting the biochemical assessments of mitochondrial oxidative capacity and Adrzej Stanos for his help in designing the system for measuring voluntary physical activity. Funding was provided by an operating grant from the Canadian Institutes of Health Research (MOP 57808). Dr. Russell T. Hepple was supported by a Canadian Institutes of Health Research Institute of Aging New Investigator Award. 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Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF). Am J Hum Genet.1992;51:1187-1200. 73 Sciacco M, Bonilla E, Schon EA, DiMauro S, Moraes CT. Distribution of wild-type and common deletion forms of mtDNA in normal and respiration-deficient muscle fibers from patients with mitochondrial myopathy. Hum Mol Genet.1994;3:13-19. 74 Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome C oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem.2000;275:3343-3347. 75 Kopsidas G, Zhang C, Yarovaya N, et al. Stochastic mitochondrial DNA changes: bioenergy decline in type I skeletal muscle fibres correlates with a decline in the amount of amplifiable full-length mtDNA. Biogerontology.2002;3:29-36. 76 Welle S, Bhatt K, Shah B, Needler N, Delehanty JM, Thornton CA. Reduced amount of mitochondrial DNA in aged human muscle. J Appl Physiol.2003;94:1479-1484. 77 Tonkonogi M, Fernstrom M, Walsh B, et al. Reduced oxidative power but unchanged antioxidative capacity in skeletal muscle from aged humans. Pflugers Arch.2003;446:261-269. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_00B38A11A5905E74AC6ACC070C451E2AA5C80608.txt b/test/dataset/in/resources/corpus/Clean_00B38A11A5905E74AC6ACC070C451E2AA5C80608.txt new file mode 100644 index 0000000..ae4725d --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_00B38A11A5905E74AC6ACC070C451E2AA5C80608.txt @@ -0,0 +1 @@ +designdesignJournal of Design History1741-72790952-4649Oxford University Press10.1093/jdh/epl026ArticlesSeeing Is Believing: Reflections on Video Oral Histories with Chinese Graphic DesignersJo IshinoCatherineUniversity of Minnesota DuluthE-mail: cishino@d.umn.eduWinter2006194Special Issue: Oral Histories and Design319331© The Author [2006]. Published by Oxford University Press on behalf of The Design History Society. All rights reserved.2006This paper presents an overview of the historical development of video oral history, and the place it has achieved in the USA, including a report on the outcomes and studies conducted by the Smithsonian Institution, in conjunction with the Alfred P. Sloan Foundation from 1986–92. It demonstrates how effective video oral history can be in documenting how graphic designers situate their works within the larger, global visual culture. As a case study, I reflect on video interviews conducted in Beijing with three generations of Chinese designers in 2004, which draw on my experience in art directing television newscasts in order to highlight parallels between video oral history and news documentaries.Chinese designersdesign historygraphic designhistory of technologyoral historyvideo oral historyIntroduction: Oral history in the USAThe introduction of the audiotape recorder in the 1950s into the print-embedded culture of US oral history (and the overarching discipline of history itself ) brought new issues to the fore.1 Early oral historians would make handwritten notes during an interview and summarize their findings in a written report.2 The accepted view at the time was of the historian as an analytical, dispassionate, empirical observer derivative of the Germanic school of history.3The core of the conundrum lies in the role of communications in the history of history. The historical profession has always been structured around the medium of the written word. Writing and history have always been synonymous, as the ‘deep structure’ of the practice of history.4With the advent of audio recordings, inflection and nuance of meaning became audible and present. Concerns arose in the process of transcribing the audiotapes to written transcripts. Should denotations of a speaker's audible and intended communications be indicated when transferring the audio to written transcripts?5 At the time, it seemed as though the very authenticity of the original interview seemed to be at issue with the advent of this new technology. This dilemma was resolved, however, with the agreement that unsaid meanings could be found by returning to the original voice recording or by reading through the multiple pages of transcripts and uncovering meanings embedded in the conversation between the interviewer and interviewee.6 When audio-recording equipment became digital and formed a stable archival record, it also became less expensive, lightweight, compact and easier to use. As a result of all these new attributes, audiotapings became adopted as standard practice for oral history gathering. Therefore, the earlier qualms about the use of audio-recording devices in conducting oral history interviews were outweighed by the convenience of being able to record accurately and preserve the ‘dialogic encounter’ between narrator and interviewer in its totality for historical posterity.Most recently, the practice of audio oral history in the USA has become popularized and mainstreamed by an organization called StoryCorp,8 which set up a recording booth in New York City's Grand Central Station to facilitate the practice whereby anyone could easily interview their ’friends or loved ones‘. The results have been archived in the American Folklife Center in the Library of Congress and some are being broadcast weekly on National Public Radio.9 Subsequently, a second StoryBooth was set up in lower Manhattan with special access for families and friends remembering those lost on 9/11. The project has continued onto the national highways and towns of the USA.10Video oral history in the USASince 1948, the first oral history centre in the USA, established at Columbia University,11 sought to expand the aims of its archive of prominent individuals with orally based, contextual biographies for future historians. Its founder, Allan Nevins, wrote in his 1938 preface to The Gateway to History:… we have only the most scattered and haphazard agencies for obtaining a little of the immense mass of information about the more recent American past … which might come fresh and direct from men once prominent in politics, in business, in their professions, and other fields …12Nonetheless, the generation of the 1960s and 1970s was able to benefit from new technological recording capabilities such as audio as well video recorders. Furthermore, they sought to be more inclusive in their archival aims so as to involve the collection of local, ethnic and regional people's life stories.13In 1967, at the First National Colloquium on Oral History, Louis Shores urged the audience to utilize the emergence of new technology in the audiovisual dimension to strengthen their collections. He argued that the ability ‘to capture voice, movement and presence’ was warranted, especially for those who were ‘demonstrating a particular skill, technique or ability’ and for those who ‘interacted with their environment in a particular manner’.14 Oral histories conducted in the early 1980s for the Schomburg Center for the New York Public Library also used video. As its Director James Briggs Murray argued, ‘… more of a person can be perceived from seeing moods, expressions on a screen than can possibly be picked up from … a voice recording or … letters on a … transcript …’.15 By the 1970s and early 1980s, videotaping oral histories had become a regular practice, and the focus was on developing the use of computerized research aids and personal computers.16In order to scientifically test some of the current assumptions surrounding video history, an intensive study, The Smithsonian Videohistory Program (SVP),17 was launched to test the use of this medium from 1986 to 1992, underwritten by a five-year grant from the Alfred P. Sloan Foundation of New York. There were over 300 participants, more than 250 hours of videotape recorded on twenty-two different subjects about the history of science and technology. The experiment was entitled, ‘Science in the National Life’, with participation by eighteen historians to prove that ‘scholars, rather than video producers, would define and drive the process of production; and in the spirit of scientific inquiry’.18 To begin with, the SVP committee decided not to try and conform to the industry standards of broadcast TV and filmed documentaries, rather they looked more towards testing the medium of video as a viable research tool solely for historical uses. Other parameters to be outlined were the ease and capability to archive and retrieve the collected visual material. They were insistent that the medium not be the sole impetus for research, rather the visual output should be an additive component to enhance and illustrate findings. Other practical issues were the appropriate means of documenting their final results, procedures for collaborating with outside specialists who could assist them technically with video technology and how to set up their on-site technical facilities. The Program also wanted to clarify the methods and means of consulting advice from social scientists and material cultural academics. In the end, the SVP reported their findings by stating:We found that video is generally most useful when recording the interaction of people with objects, places, or other people, when capturing personality and ‘body language’, and when exploring a process or documenting the function of artifacts.19Nonetheless, apart from revealing issues of methodology and providing an expanded knowledge-based resource, the end results had to be clearly understood and integrated into the original premise and research project. Therefore, the Program defined video history as ‘the video recording of visual information as primary historical evidence and involv[ing] a historian in shaping the original inquiry’.20 The merger of the medium and the content was ideally synthesized into one end-product, thus upholding later claims that the video outcomes were not only archival records for future historians to use but also historical entities themselves. Since the SVP's experiments ended, there has been no parallel, large systematic study of the role of video history.American oral historians have embraced the medium but the literature remains sketchy.21 As historian Dan Snipe notes:… video seems to inhabit some sort of twilight zone: many oral historians … accept the value and … even use it …7… the discussion of the relationship between oral history and moving images is only beginning. … it is the historical methodology most open to multiple modes of communication …… increasing recognition and use of moving images can help it [oral history] push both inquiry and historical communication even farther.To this end, the time has come for sustained, systematic description, discussion, and analysis of how moving images can work as an integral dimension of oral historical practice.22One year after the SVP had ended, the official account A Practical Introduction to Video History: The Smithsonian Institution and Alfred P. Sloan Foundation Experiment was published. Reviewing its claims, Susan Hamovitch points out one potential problem she saw throughout the report:Ethnography, spontaneous shooting, documentary editing were ostensibly not part of the SVP. However, they crept in. … this almost scientific documentation, the recurring theme of conflict between an accurate rendering of history and an artificially manipulated product. … historians and producers were concerned with capturing emotion and spontaneity—making an exciting video—as any documentary film producer.23She cites Brien Williams as ‘one of the video producers … [who] comes closest to a verdict on the place of video-history, classifying it somewhere between an archival record and broadcast television’.24Michael Frisch, a critical figure in advancing the validity of oral history work in the USA, contended in 1990 that what oral historians produced was also ‘the object, not merely the method of history’.25 He also underlined the Program's findings stating that he understood ‘film and television as a media [sic] … and assumes the importance and relevance of film and video to oral history for documentation’.26Oral history and newscastsThe process of creating an oral history project has many parallels with news-gathering methods. In fact, the first president of the Oral History Association in the USA, Louis Starr, began his career as a journalist.27 Additionally, the first lengthy oral history project conducted by Nevins was ‘Broadcast Pioneers’ on the early years of radio (1950–52).28 He writes:The planners [of oral history] had a connection with journalism, and saw it in the daily obituary columns proof that knowledge valuable to the historian, novelist, sociologist, and economist was daily perishing forever without yielding any part of their riches.29Construction and presentation of an oral history are an important part of the historian's project, as the present Director of the Bancroft Library's Regional Oral History Office at the University of California, Berkeley, Richard Cándida Smith, observes:The choice of how to organize and present interview material must follow the author's sense of … [the] way to communicate [the] story and argument. … to provide readers with information, an argumentative point of view, or human-interest stories … the author must anticipate the confusions of the original text and answer the queries that readers might address to the speaker … a vibrant human being oral historians have come to know, respect and admire. … an author must convey to readers, almost none of whom will ever personally meet the interviewee by sharing his … response to interviewees and their stories [to a] much broader public to get a glimmer of the effect the narrator can have on others.30Although referring to written publications, the precepts of what Smith proposes could readily apply to TV news journalism practices, the medium of videotape and its visual narrative outcomes. Ideally, the ethical and moral responsibility of the journalist is to allow the key points of an interviewee to be communicated directly to the viewer. Also, he or she must carefully consider the pertinent questions the viewers would want to ask,31 as well as how to convey the context, meaning and the ‘tele-visual presence’ of the interviewee and their ‘story’.Smith goes on to make the following point that further parallels the news-editing process and protocol in creating a story for TV broadcast:Rearrangement … heightens drama and emotion, making for a better read, but from a more purely intellectual perspective, its primary functions are to help the reader see as clearly as possible choices that narrators recall having faced in the past along with emotional and practical consequences.32This editing is standard practice in newsrooms.33 The goal is to depict a story as succinctly and accurately as is viable within the given constraints of a newscast.Additionally, Smith points to the primary dilemma ‘facing authors of … oral history work [as] not one of techniques but of principles’. He concludes:My goal … for presentation is to refocus attention onto the circuits of meaning that constitute public communication. Exchange of perspectives characterizes oral speech. Publication of oral history work may involve an inevitable alienation from the source but if such work perpetuates dialogue, if it opens up new ways of looking at social relations and the roots of the present in the past retains, it remains faithful to the inner logic of oral history.34Consequently, in 2004, attending the first Alliance Graphique Internationale (AGI) Congress to be held in China, I brought my mini-DV video camera equipment in order to document this historic event, more as an ‘embedded’ visual journalist (and educator, to expose my cloistered students to another design culture) [1], and to further continuing research in making video oral histories of graphic designers.35Fig 1 Pagoda and Satellite, rooftop of Taiwan Hotel, Wangfujing Dajie (Street), Beijing 2004. Photograph, C. IshinoThe coverage surrounding the New China, its free-market economy, exponential manufacturing growth and exploding consumer culture in the media were unavoidable [2]. The AGI Congress would undoubtedly affect the country's design sensibility, especially given that the design enterprise itself is so linked to cultural changes and events.36 As graphic design historian Philip Meggs has noted:The immediacy and ephemeral nature of graphic design, combined with its link with the social, political and economic life of its culture, enable it to more closely express the Zeitgeist of an epoch than many other forms of human expression.37Fig 2 One World Department Store, Dengshixikou (Street), Beijing, 2004. Photograph, C. IshinoMoreover, the AGI Congress schedule had conveniently set aside a ‘Chinese Design Day’ devoted to design and designers from Hong Kong, Macao and the Peoples' Republic of China (PRC). All were to present their regions' design work.38 I would be a first-hand witness able to document key graphic designers in China showing their work, in many cases for the first time to a global professional audience.As had been my practice in my TV news days, I sought to determine what ‘the story’ might be, as well as researching which designers and designs to include, leading to the decision to conduct interviews with three generations of designers, at various stages of their careers in order to document perceptions of change. Aged between 20 and 70 years, each designer would have distinct perspectives on the PRC's rapid economic growth, particularly as these changes placed extraordinary demands on design and designers locally.39Moreover, as had been noted:… since China's social and political reforms began in 1979, there have been three generations in Chinese design. Until the early 1990s, the first generation still created mainly handmade work as there were few computers in the country. The second embraced the Macintosh and began to absorb international design influences. The third … grew up in the age of the Internet with access to information not available to earlier Chinese designers. They have an international outlook … share the ‘independent DIY spirit’ found in young designers the world over.40I sensed that the PRC designers must have felt the pressure of history to broaden their design identity and sensibilities, especially given their previously isolated state.41 In brief, I wanted to ask the fundamental questions of a news-breaking reporter in the world of graphic design.Case study: Beijing designersYu Bingnan (b. 1933)42 was my first interviewee. He was the initiator and main organizer of the AGI annual conference in Beijing. In 1992, Yu Bingnan was the first PRC designer to become a member of AGI, nominated by the ‘father of Hong Kong design’ Henry Steiner.43 He was currently a Professor at the Academy of Art and Design at Tsinghua University [3]. Since Yu Bingnan had practised before and after the ‘free-market’ economy, I was hoping he would provide me with a broad perspective on his country's design. (Moreover, I knew from my news experience that given his seniority, his consent would pave the way for the others to follow.) For consistency and editing purposes, the interview location was constant.Fig 3Yu Bingnan, 1998, Poster: Taiwan Image, Chinese Character: FamilyYu Bingnan, a lean man, dressed austerely in a dark shirt and trousers, was reminiscent, to an outsider to Chinese culture, of a Politburo leader, as often shown of the National Congress Party of China meetings in the US mass media. A stereotypical representation revealing my own cultural visual references:… we secure answers to … questions in split-second observations of each other's appearance as composed of body movements, facial expressions, gestures, clothing, and adornment. … clothing is usually perceived before voice can be heard or gestures and facial expressions seen. Thus clothing and adornment, as they modify appearance, become a universal, primary, nonverbal communication system. It is a complex intentional system learned by all members of any culture at an early age.44Throughout our interview, he seemed formal and reserved, which seemed to match his high status within the AGI Congress gathering,45 yet at times, he would lean forward as if to emphasize a key point.46 He would pause before answering,47 but in accordance with advice to Westerners doing business with China: ‘Silence can be a virtue …, so don't be dismayed if there are periods of silence in your … business conversations. It is a sign of politeness and of contemplation’.48What follows is an excerpted and paraphrased translation of the interview through an interpreter.49CI: What is the difference between design today and twenty years ago?YB: Today graphic design in China is more professional and mature. Yet our design is at an early stage of its development and potential. Right now, our graphic design level is finally capable of setting up a dialogue with our design counterparts worldwide and on a more equal standing. I know these exchanges are helping with our progress in graphic design.CI: Do you find Chinese designers looking to Europe and America?YB: Chinese find their own way, they look back to their own tradition and education. They combine the professionalism of visual language and universal language to create their own originality.CI: What do you think the relationship between Hong Kong, Mainland China and Taiwan design is today?YB: Ten years ago, the level in Hong Kong, Macao and Taiwan was much better. This has changed with informal and international exchange. The Mainland China group is doing very well, so there is not much difference now. The localities do express their own visual languages and still share commonalities.CI: What do you see as the future of PRC design?YB: Younger and more brilliant designers are emerging. There is a lot of potential for commerciality. There is professionalism in the near future. Younger designers are more sensitive to new and better designs than the older generation. But with their [desire for] individualism, they must remember their culture.CI. What do you think about designers in US?YB. I have lots of respect for American designers. I have grown to appreciate the high levels of professionalism and individualism, expressed through the visual language of our counterparts in the USA. We could create more links, so more exchanges and greater understanding could take place between us. Hopefully, more exchanges like the one we are having now will happen, which will expand the visual language of American and Chinese designers so a more peaceful world will come.Throughout our interview, Yu Bingnan conveyed his view that with the formation of the ‘New China’, design in both East and West is being transformed by more open communication.50The second interview51 was with Min Wang (b. 1956),52 since 2003 Dean of the School of Design at the Central Academy of Fine Arts (CAFA) in Beijing, the only fine arts school supported by the PRC's Central Government. Wang had been raised and educated in the PRC. At the age of 27 years, he left to study graphic design in Germany (1983) and the USA (1986), eventually lecturing on graphic design to graduate students at his alma mater, Yale University, from 1989 to 1998. While teaching, he also worked for Adobe Systems in various senior design roles. Min Wang was among the first to use ‘Illustrator’ software to design Chinese characters or radicals for Japanese users of Adobe's fonts. He had helped create the visual presentation that won Beijing the bid for the 2008 Olympic games. For the oral history project, he represented the crossover generation, who had gone to America for higher education. He had lived and worked there for 17 years, then returned home in 2003, when the free-market economy took hold. Wang's background uniquely qualified him as the ideal bicultural and bilingual designer addressing both Eastern and Western design sensibilities [4].Fig 4 Min Wang, 2004, Poster: Chinese Studies at Stanford UniversityWang spoke in English during the interview.53 In his dress and manner, Wang was not unlike the Eastern Seaboard intellectuals from Ivy League schools, an appearance he must have adopted while teaching at Yale for almost a decade.54 As with Yu Bingnan, my questions focused on the experience of change; Min Wang's replies arise out of his experience of the sharp contrasts between the USA and PRC, which he understood I could share.CI: Why did you leave China to get your higher education?MW: China was closed. I couldn't see what was going on outside. I wanted help to become a designer and design educator, so I went overseas to Germany and the USA.CI: How is China different from when you left?MW: The change is enormous, incredible, hard to describe … very different when I left in 1983. There were no highways. We had few cars. There was no branding. Not even much color printing. The term, logo, would have been misunderstood then.CI: Why did you decide to come back to China to become a teacher and a Dean at CAFA?MW: After 25 years of change, China changed from a closed society to an open market society. … CAFA is central to fine arts and design education too in China today. I want to contribute to China and [its] change, to make good designers, improve society and peoples' lives.CI: What do you think is important for your students to learn or know as future designers?MW: Change is constant. To give perspective from both sides—Western and China. Don't just imitate design from Europe or America. Use own culture with strong Chinese character, color and identity.CI: How would you describe the Chinese identity?MW: Hard to describe. Hard to choose words. Not just use elements from Chinese art or culture. Have much more to bring to international platform. Be first compatible with Chinese people and understand their identity. Help others understand Chinese and at the same time Chinese identity.CI: How would you describe design in China today?MW: The need for design has increased tremendously. There are many more designers today and so many schools. Twenty years ago, there were about ten schools offering design programs. Now there are 1400, by conservative estimates. There is a big need for design educators.CI: How would you differentiate the design between Hong Kong, Taiwan and Beijing?MW: There is a lot of differentiation …55 Actually they have helped Mainland Chinese designers and the schools in the past 20 years to transform from a closed society into today's society. They all contribute to today's Chinese design.CI: What do you perceive the role of design in Mainland China?MW: Graphic design is one of the best occupations and most popular. I would say most desirable position for young kids. Better paid, relatively speaking. They make art and make money and make design. And design is art. They want to be designers. And there is a big need. It's a good thing to be a designer in China.CI: What would you say to US designers about the future of Mainland Chinese design?MW: Right now I would say, it's very exciting in the design field. Graphic designers and design in China are in a stage which could lead to a big leap forward. Very soon, even today, you can see a lot of young designers, very smart, talented … doing impressive work and you will see this more and more.His enthusiastic responses gave the distinct impression of how he was caught up in the reinvigoration of his homeland. Perhaps, having lived outside the country for nearly twenty years, in contrast to Yu Bingnan, Wang was more focused on the role he might be able to play in China's future through his direct participation in the PRC's national design policy, as well as CAFA's design pedagogy, which was supported by outstanding technical facilities (better than those in the USA) and a new, dedicated building. Also, as a second-generation designer and educator, Wang was cognizant that while it was important for his students to compete in the international design arena, it was also crucial they maintain a sense of their distinct ‘Chinese-ness’ as Yu Bingnan had emphasized in his interview.The interview with Song Xiewei (b. 1963)56 was mediated once more through the interpreter. A self-described artist/designer/tutor/associate professor, Song teaches at CAFA. He has his own eponymous independent design practice and was treated by the young designers at the conference as a celebrity. Conceivably, Song had attained this status because he was among the first in the PRC to implement a studio model of working that was thriving and highly successful. Nationally and internationally, he had won scores of prestigious design awards.57 His exalted lifestyle involves frequent travels throughout Europe, Canada and the USA. Song Xiewei seemed to epitomize design success in the New China. On Chinese Design Day, he was selected by the host committee to present the work of emerging PRC designers including some of CAFA's former students. At the conference, he was also a key figure.His designer penthouse studio overlooking Beijing was evidence of his success.58 During the interview, students were shooting flash pictures and asking for his autograph to be penned on their shirts. Song, too, had signatures scrawled across his white, open collared and loose flapping dress shirt, but his were of world-renowned designers in attendance at the conference. Song was the leader of this young group of designers, many of whom also worked in his home and campus studios. He appeared to set the tone and behaviour for them, which they gladly followed. Song's hairstyle was a shoulder length, layered cut, very modern and hip as well as unusual for men on the streets of Beijing. Also, unlike my two earlier interviewees, Song smiled frequently.59CI: How is design different from when you started over twenty years ago?60SX: In the past, we focused on functionality, the basic elements of design. Today, with the advancement of high-end information technology, our way of living and even our way of thinking has been greatly changed. Currently, our design is focused on the humanistic side and individualist expression in our work.Young designers and students' work expresses the visual language of the era of high tech. They grew up in it, they depend on it. They are more in the modern era—cross cultural and international. Consider their education. They couldn't survive with a traditional educational background.High tech, IT, affects my work and I learn it from young designers, who feel quite natural with it. We seek a higher designer economy in China—quantity, larger and bigger. But it's not like that today. … The significance of designers and role they should play, [should be] comprehensive, impact everyday life, to every corner.CI: How would you compare and contrast the design of Mainland China, Taiwan and Hong Kong?SX: There is a great deal of difference in the design of these three places. The design in Mainland China has been conceptually infused with a strong sense of Chinese cultural context. But also, of late, we have been undergoing a lot of modernization. The design in Hong Kong is highly successful commercially. And to the extent that a Chinese cultural influence is in their work, I can see they are much more advanced than us in Mainland China. As for design in Taiwan, to their credit, they have returned to their cultural traditions of Chinese and Asian culture.CI: Any advice for young designers?SX: Try and focus on the process of design and the details. Give more consideration to the concepts, creativity and new ideas related to high-end information technology. By doing this, they can become more contemporary, create good concepts and strong market potential. Also they should keep in mind, the impact of local and traditional cultures; where they originate. Without considering their locality, paradoxically, they cannot become international in their scope of design creation.CI: What would you say about US design?SX: American design has been more advanced than in China, due to its extensive history in the industry. They have a huge market in China at the moment. But still, there is room for progress in the US and China.CI: Tell me about the space you have created here [at his studio penthouse, where all the interviews took place]:SX: I designed my own studio space. I wanted to make all the elements controversial. In the middle is a cement box; it's anti-decoration. The outside is the inside. The exterior space here is for use by designers. As the interior space can be very busy [with work], I wanted to create a space they could relax, be in nature with the bamboo trees, the pond and running water, the rock garden, fresh air and so on.Unlike my two previous interviewees, Song Xiewei seemed to be more an avant-garde practitioner in his outlook and work61 than a teacher or administrator [5]. Also, he was the most outspoken about his personal opinions on design, and Song's aspirations for Chinese design having a powerful role in the world, allied most closely with the international designers' aims for design at this Congress.Fig 5Song Xiewei, 2002, Calendar in ‘Half Book’ format: ‘Above the surface, Beyond the border’The final set of interviews was with a young, group practice of designers, Liu Zhizhi (b. 1975), He Jun (b. 1977) and Jiang Su (b. 1977).62 All were graduates of CAFA, under the tutelage of Song Xeiwei. All three were dressed in the most current, hip-hop style of global youth culture. Liu Zhizhi's English-speaking girlfriend acted as interpreter. The works of the designers they most admired were from Japan, Germany, Holland and England.CI: What do you think of Hong Kong and Taiwanese designers?Answer: They are way ahead of the game compared to us. Taiwan designers put more traditional elements into their work, so visually the work ends up looking more Chinese. Hong Kong designers have a style more like the Western63 designers, but they keep some of the Chinese sensibilities. Their work looks like it's undergoing a transitional change from Eastern to Western design. Taiwan designers use traditional Chinese elements more accurately and profoundly than the Mainland or Hong Kong designers. Currently, we, Mainland designers, choose to follow the Western design more.CI: What do you think the future of PRC is?Answer: There is a bright future in design. [People] consume design as they do other products.CI: How would you describe the state of PRC design?Answer: We have confidence in ourselves. We can make it better.CI: What advice would you give to other designers?Answer: First have confidence in [one's] self. [Try to] gain techniques in a short period [of time]. Never give up, just keep doing it.CI: What do you love about design?Answer: [We] discover new things and create new things everyday. [We] are responsible for ourselves and our own work.CI: Anything you'd like to say to US designers? What about the state of PRC design today?Answer: We very much like the work of young American designers. They seem to be doing very well. Comparatively speaking, it seems as if their career paths are relatively clearer and smoother than ours in China. We can work together!These young designers were much more informal and uninhibited in their demeanour. They exuded youthful confidence and optimism, unabashed in their desire to follow the Western (as opposed to international or modern) way of design which provided a way to express themselves, and have the freedom to evolve in their own independent manner, unlike the preceding generations, who believed in maintaining the dialectic of the West and the East in their design work. This is where the third generation stood apart. The first generation was working to gain international attention by creating exchanges. The second one was interested in gaining global stature by winning design awards. This third generation felt able and confident enough to invite others to work with them.64Cross-cultural interviewingAs Valerie Yow has commented:Generally, there is more open communication when age, gender, class and race are the same, but in any interviewing situation the interviewer must be conscious of the ways in which these basic social attributes impinge. Sensitivity in interpersonal relations and respect create the climate most conducive to a productive interview.65As a second-generation, American-born woman of Japanese descent,66 in interviewing three generations of Beijing designers, all of whom were men, I became cognizant of my bicultural background as well my gender. I was conscious of not only Japan's long, conflicted history with China, battling one another for land and power, but also the troublesome relationship the USA had with the PRC, for example, on issues regarding Taiwan. As a person who straddles both Asian and Western cultures, I was sensitive to how these intercultural issues might affect my interview.67 As Edward T. Hall, cultural anthropologist and cross-cultural researcher, states:I am convinced that much of our difficulty with other people in other countries stems from the fact that so little is known about cross-cultural communication … Formal training in the language, history, government, and customs of another nation is only the first step in a comprehensive program.68Debates in cross-cultural communication69 highlighted how my upbringing70 inadvertently affected these interviews, as did my role as an educator attentive to the ‘colonizer's gaze’ and visual multiculturalism.71A recent visual anthropologists' revisionist critique of Margaret Mead's ethnographic films72 is not unlike what occurred when I worked in the news media in the late 1980s and early 90s when US reporters were airlifted into war zones with little background information on the country they were assigned to cover.73When we view culture as such an infinitely complex mesh of intertwined behaviors … in constant movement … we further realize that in today's rapidly shrinking world, due to a growing intercultural awareness and communication among countries, it is becoming more and more difficult to define individual cultures in themselves, we understand that the term communication can almost never represent an absolute concept.74ConclusionThese three generations of Beijing designers represented the transformations of their country in which the swift development of the free-market economy propelled them into the midst of the global design sphere. Generational notions of regional design similarities and differences reflected the impact of past political sovereignties and economic systems. The first two generations upheld the importance of maintaining their national visual identity, while continuing to reach out to international designers to develop and modernize their own work. In contrast, the third generation, already steeped in modernization, stood certain in wanting to work with, though not necessarily learn from, the West.Using videotape to document these designers, I was able to capture not only their tele-visual presence, the animated spirit of each person as well as their expressions but also their dress, hairstyles and the dialogic encounter itself. Also, as a motion and video designer, I was able to apply my background in TV news to create my video narratives with more visually descriptive and informative framing to convey the context and experience of the recordings. Designer, Hillman Curtis, who has been creating MTV-type video portraits of graphic designers75 has noted how… It's a whole different story creating video for the Web, because you have to have information, telling your story in a tighter timeframe than broadcast … the Web is the first truly global medium. No borders. … You can't rely on words to get your message across in different countries and motion is just one more tool that can help you. … It's a universal language. It transcends text-based communications. … many people in the industry feel this union of video and the Web will become the standard.76The significance of Web-based dissemination and access has become key. After a visit to China in 2005, design writer Rick Poynor posted his view that:If there is cause for hope … a new form of public space … from a Chinese point of view. … Large quantities of information are now able to break through the traditional system of information control … The Internet gives the Chinese an anonymous platform for opinions that cannot otherwise be expressed freely. … Digital images … are helping to create a new, more democratic order in Chinese society.77In closing, graphic design's visual oral history has taken another leap forward bringing in its wake a new set of methodological issues—not only with video streaming but also with other digital authoring and duplication devices such as DVDs.78Simply put I would argue the essence of graphic design communication is visual. This ever-changing craft and practice is bound and expanded by technological advances. The history of its work and people should also move ahead and be documented as moving imagery which itself becomes an analytic category. What better medium than digital, streaming video to create a visual oral history of its makers? Digital moving imagery is the most all-encompassing archival form of documentation and distribution currently available, enabling video oral designs historians to create living histories of designers, their work processes, interactions and environments, and to disseminate this globally.1D. K. Dunaway, ‘The Interdisciplinary of Oral History: Introduction’ in D. K Dunaway & W. K. Baum (eds.), Oral History: An Interdisciplinary Anthology, 2nd edn., Alta Mira Press, Walnut Creek, 1996, p.16.2B. Tuchman, ‘Distinguishing the Significant from the Insignificant’, in D. K. Dunaway & W. K. Baum, op. cit., p. 97.3V. R. Yow, Recording Oral History, 2nd edn., Alta Mira Press, Walnut Creek, 2005, p. 4.4D. Sipe, ‘The Future of Oral History and Moving Images’, in R. Perks & A. Thomson (eds.), The Oral History Reader, Routledge Press, New York, 1998, p. 379.5S. Page, ‘The Invisible Participant: The Role of the Transcriber’, Interpreting Meaning in Oral History Research for The Power of Oral History: Memory, Healing and Oral History, 2002, XII International Oral History Conference, p. 5.6D. K. Dunaway, op. cit., p. 8.7D. Sipe, op. cit., p. 384.8See the StoryCorps project: http://www.storycorps.net/about/, accessed August 2006.9See http://www.npr.org/templates/story/story.php?storyId=4516989, accessed August 2006.10StoryMobile founder, David Isay, a former radio broadcaster, in response to my query about using video, replied, ‘No, as I believe the voice is the soul of a person’.11A. Nevins, ‘Oral History: How and Why it was Born’, in D. K. Dunaway & W. K. Baum, op. cit., p. 29–38.12A. Nevins, The Gateway to History (1938) quoted by L. Starr, ‘Oral History’, in D. K. Dunaway & W. K. Baum, op. cit., p. 44.13D. K. Dunaway, op. cit., p. 7.14L. Starr, op. cit, p. 1–2.15T. A. Schorzman (ed.), A Practical Introduction to Videohistory: The Smithsonian Institution and Alfred P. Sloan Foundation Experiment, Krieger Publishing Company, Malabar, Florida, 1993, p. 2.16D. K. Dunaway, op. cit., p. 7.17Ibid. ‘Preface’, viii.18S. Hamovitch, ‘A Practical Introduction: The Smithsonian and Alfred P. Sloan Foundation Experiment’ (Reviews), Historical Journal of Film, Radio and Television, March 1995, available on http://www.findarticles.com/p/articles/mi_m2584/is_n1_v15/ai_16922331, accessed May 2006.19T. A. Schorzman, op. cit., p. 21. The quote goes on to state ‘Video is least helpful in historical research when people talk about abstract concepts, which is better left for audio or written text’—a view this paper refutes.20T. A. Schorzman, op. cit., ‘Preface’, p. vii.21K. Howarth, Oral History: A Handbook, Sutton Publishing Limited, Phoenix Mill, 1998, pp. 160–70.22D. Sipe ‘The Future of Oral History and Images’, in R. Perks & A. Thomson (eds.), op. cit., p. 387–8.23S. Hamovitch, op. cit.24Ibid.25R. Perks & A. Thomson, op. cit., ‘Introduction’, p. 2–3.26M. Frisch, ‘A Shared Authority: Essays on the Craft and Meaning of Oral and Public History’, in R. Perks & A. Thomson, op. cit., p. 384–5.27L. Starr, op. cit., p. 39.28L. Starr, op. cit., p. 45.29A. Nevins, op. cit., p. 31.30R. C. Smith, ‘Publishing Oral History: Oral Exchange and Print Culture’, in T. Charlton, L. Myers & R. Sharpless (eds.), Oral History Handbook, Alta Mira Press, Walnut Creek, 2006, ch. 13, p. 5–6.31‘Mr. MacNeil and Mr. Lehrer, in their passion to avoid adversarial or advocacy journalism, do not draw conclusions. They only ask questions.’ J. Corry on The MacNeil/Lehrer NewsHour, The New York Times, 1 August 1985.32R. C. Smith, op. cit. p. 9.33For more information on video editing, see chapter in book by H. Zettl, ‘Structuring the Four-Dimensional Field: Continuity Editing and Complexity Editing’, in Sight, Sound, Motion: Applied Media Aesthetics, Thompson/Wadsworth, Belmont, 2005.34R. C. Smith, op. cit. p. 18.35See Godard, Kidd & Danziger video clips http://www.catherineishino.org, portfolio, oral histories. Other interviewees include Keith Goddard, Victor Margolin, Janet and Peter Good.36For broader overview of cultural changes and impact on graphic design in China, see article by W. S. Wong, ‘Detachment and unification: a Chinese graphic design history in Greater China since 1979 ’, Design Issues, vol. 17, no. 4, Autumn 2001, MIT Press, Boston, p. 51–71; and book by S. Minick and J. Ping, Chinese Graphic Design in the Twentieth Century, Van Nostrand Reinhold, New York, 1990.37P. B. Meggs, ‘Preface to the First Edition’, in A History of Graphic Design, 3rd edn., John Wiley & Sons, Inc., New York, 1998, p. xiii.38See W. S. Wong's paper, ‘In search of a new graphic design frontier in China: establishing the “Chinese-ness” of international style’, available on http://www.idemployee.id.tue.nl/g.w.m.rauterberg/conferences/CD_doNotOpen/ADC/final_paper/081.pdf, accessed August 2006. Also, W. S. Wong, Ph.D., Curator, Lubalin Curatorial Fellow, Herb Lubalin Study Center of Design and Typography, Chinese Graphic Design towards the International Sphere, 2000, The Cooper Union School of Art, New York, September 2001, available on http://www.cooper.edu/art/lubalin/cgd/, accessed 4 August 2006.39For influence of economic changes on Chinese design see article by C. Dillnot, ‘Which way will the dragon turn? Three scenarios for design in China over the next half-century ’, p. 5–20, and T. Fry, ‘The futherings of Hong Kong’, p. 71–82, Design Issues, vol. 19, no. 3, Summer 2003, MIT Press, Boston.40Ou Ning, one of the show's curators, ‘Get it Louder’ exhibition in Shenzhen, in southern China, notes that, ‘When doing the selection, we especially avoided those works that use Chinese elements on purpose’. In his mid-thirties, he is the very model of a restlessly mobile, boundary-breaking contemporary designer person, working as a writer, music promoter and graphic designer … and founder of U-thèque, an independent film and video organization. R. Poynor, http://www.designobserver.com/archives/002505.html, accessed July 2006.41For more on designers’ identity and work see online article by W. S. Wong, ‘In search of a new graphic design frontier in China: establishing the “Chinese-ness’’ of international style’—http://www.idemployee.id.tue.nl/g.w.m.rauterberg/conferences/CD_doNotOpen/ADC/final_paper/081.pdf—from paper given at the Asian Design International Conference, Tsukuba, 2003, and published in the Journal of the Asian Design International Conference, vol. 1 and The 6th Asian Design International Conference Proceedings (CD-Rom). Also, see H. Clark ‘Introduction’, p. 1–3, and her conversation with B. D. Leong, ‘Culture-based knowledge towards a new design thinking,’ p. 48–58, Design Issues, vol. 19, no. 3, Summer 2003, MIT Press, Boston.42Bingnan is the vice president of Icograda and an honour legate of the International Trademark Center. In 1956, he graduated from LuXun Academy of Fine Arts, attaining his Master's degree from the Hochschule fur Grafik und Buchkunst Leipzig in Germany in 1962.43For more information on Henry Steiner, see http://www.commarts.com/CA/feaint/china/110_china.html, accessed August 2006.44F. Poyatos (ed.), ‘Clothing as Nonverbal Communication’, in Cross-Cultural Perspectives in Nonverbal Communication, C. J. Hoegrefe, Toronto, 1988, p. 292–4. Also see M. Argyle, Bodily Communication, Methuen and Co Ltd, London, 1975.45‘… most Chinese maintain an impassive expression when speaking.’ See Kwintessential, culture and language experts, available on http://www.kwintessential.co.uk/resources/global-etiquette/china-country-profile.html, accessed July 2006.46‘Posture is important [in China] so don’t slouch …,’ R. E. Axtell, Gestures: The Do's and Taboos of Body Language Around the World, John Wiley and Sons, Inc., New York, 1991, p. 172.47‘Non-verbal signals are used to manage much the same range of situations and relationships in all cultures, and these in turn are similar to those found in animal societies’. M. Argyle, op. cit., p. 94–5.48R. E. Axtell, op. cit., p. 173.49Interpreter Jin Hua is the International Deputy Director of CAFA, educated in his English-speaking skills in Australia.50See W. S. Wong's paper, op. cit.51For a video clip of his interview, please see http://www.catherineishino.org, portfolio, oral histories, min wang.52For Min Wang's work, see http://www.a-g-i.org/about/member_work.php?id=474&nationalgroup=China&country_code=cn, accessed June 2006.53We knew the same colleagues, and were familiar with each other's companies.54‘… cross-cultural observers can trace the “trickle down” adaptation by … metamorphosing, cultures of clothing from dominant cultures. Study of culture diffusion patterns indicates that men are the first to adopt clothing artifacts from … an alien … culture.’ F. Poyatos, op. cit., p. 301.55For the sake of not repeating essentially the same answer that Yu Bingnan's gave, I have given only what Wang contributed.56For biographical information on Song Xiewei, see http://www.cipb.org/introenall.htm, accessed June 2006.57For list of awards see The History of Chinese Graphic Design: Designing in the New Century, available on http://www.cipb.org/introenall.htm.58To see article and inside view of his studio, see http://www.21stcentury.com.cn/article.php?side=4224, accessed June 2006.59‘Nonverbal communication can be used as international, intercultural and interracial language. … Whether in the United States, China, or South America, people seem to have the same general meaning for a smile.’ L. A. Samovar, R. E. Porter & N. C. Jain, Understanding Intercultural Communication, Wadsworth Publishing Company, Belmont, 1981, p. 160.60‘Each participant will take an opportunity to dominate the floor for lengthy periods. … Be patient and listen. There could be subtle messages being transmitted that would assist you …’ See Kwintessential, op. cit.61See http://www.a-g-i.org/about/member_work.php?id=450&nationalgroup=China&country_code=cn, accessed June 2007.62I have not separated out each designer's response, but combined them.63Note the use of ‘Westerner’ versus ‘International’ in the two previous interviews. I believe this indicative of the Euro-Ameri-centric attraction the mass exportation of entertainment industry has on youth culture worldwide. In fact, entertainment is America's largest moneymaking export.64For more information on young Chinese designers, see http://www.designobserver.com/archives/002505.html and http://www.getitlouder.com/, accessed August 2006.65V. R. Yow, op. cit., p. 169–79.66My grandparents were born in Japan (b. 1880s), and emigrated to the USA (1890–1910). Both my parents were born in southern California (b. 1920s). I was born and primarily raised in the Midwest (b. 1952). My father is a professor of cultural anthropology. My mother practises and teaches Ikebana, the Japanese art of flower arranging, and has attained highest level of certification. I have lived briefly in Japan.67For more on intercultural communications, see F. Poyatas, Perspectives in Nonverbal Communication: Studies in Cultural Anthropology, Social Psychology, Linguistics, Literature, and Semiotics, Pergamon Press, Oxford, 1983.68Edward T. Hall quoted in Samovar, R. E. Porter, & N. C. Jain, op. cit., p. 154.69For more on nonverbal interactions between nations, see Geert Hofstede's IBM study on the web, available on http://feweb.uvt.nl/center/hofstede/page3.htm, accessed August 2006. Also see European Commission backed website, http://www.media-net-works.de/, ‘The aim of the project Intercultural ICT-mediated Communication competencies as a key to enable participation in a network society’.70‘[A] person who has spent a significant part of his or her developmental years outside the parents’ culture. The third culture kid builds relationships to all of the cultures, while not having full ownership in any. Although elements from each culture are assimilated into the third culture kid's life experience, the sense of belonging is in relationship to others of the same background.’ Dr Ruth Useem, 2001. For more information on ‘Third culture kids’, see http://www.tckworld.com/useem/art5.html, accessed August 11, 2006; this article was first published in NewsLinks—the newspaper of International Schools Services, vol. 13, no. 4, March 1994, Princeton.71See L. Lippard, Mixed Blessings: New Art in a Multicultural America, NW Norton, New York, 1990; L. Lippard, Lure of the Local, New Press, New York, 1997; S. Sontag, On Photography, Farrar, Straus and Giroux, New York, 1977.72J. Collier, Jr & M. Collier, Visual Anthropology: Photography as a Research Method, University of New Mexico Press, Albuquerque, 1986. ‘Foreword’, pp. xiii–xvii. ‘Film and video’, ch. 11, pp. 139–49.73For more information, see D. Schechter, Global Policy Forum, 777 UN Plaza, Suite 3D, New York, available on http://www.globalpolicy.org/security/issues/iraq/media/2004/1109spin.htm, accessed 11 August 2006, and http://www.zmag.org/schechteraudio.html.74F. Poyatas, op. cit., p. 13.75For more on video portraits of graphic designers, see http://www.aiga.org/content.cfm/artistvideoseries, accessed July 2006.76Available on http://www.cmykmag.com/magazine/issues/013/articles/agency/agency_1a.html, accessed July 2006.77R. Poynor, Design Observer, 05.05.05, available on http://www.designobserver.com/archives/002505.html, accessed August 2006.78‘… liberating power to DVD piracy, which has broken down cultural isolation by allowing the Chinese cheap access to previously unavailable films.’ R. Poynor, op. cit. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_00B80AFF7872C69437FF8BF5E05572DF671ED4B1.txt b/test/dataset/in/resources/corpus/Clean_00B80AFF7872C69437FF8BF5E05572DF671ED4B1.txt new file mode 100644 index 0000000..342c50e --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_00B80AFF7872C69437FF8BF5E05572DF671ED4B1.txt @@ -0,0 +1 @@ +amjepidajeAmerican Journal of Epidemiology1476-62560002-9262Oxford University Press10.1093/aje/kwq215ORIGINAL CONTRIBUTIONSPopulation-Level Impact of Osteoporotic Fractures on Mortality and Trends Over Time: A Nationwide Analysis of Vital Statistics for France, 1968–2004ZiadéNellyJouglaEricCosteJoël**Correspondence to Dr. Joël Coste, Biostatistics and Epidemiology Unit, Pavillon Saint-Jacques, Hôpital Cochin, 27 rue du Faubourg Saint-Jacques, 75674 Paris Cedex 14, France (e-mail: coste@cochin.univ-paris5.fr).1510201012820101728942951432010962010American Journal of Epidemiology © The Author 2010. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2010Osteoporotic fractures are one of the leading causes of death in the elderly population, but mortality may have been reduced by the advances in management and prevention during recent decades. The authors analyzed the population-level impact of these fractures on mortality in France from 1968 to 2004. About 20 million death certificates registered in metropolitan France from 1968 to 2004 were analyzed. Osteoporotic fractures were identified by using a previously developed methodology. Age-specific and standardized mortality rates were calculated by site of fracture and sex, and time trends were evaluated. Associated causes of death were compared between the extreme periods of the study by the observed/expected pairs method; 440,890 (2.2%) death certificates reported an osteoporotic fracture. Osteoporotic fractures overall, particularly hip and skull fractures, declined by half during the study period, exceeding the decline in general mortality and resulting in fracture-deceased subjects being older. However, pelvis, vertebral, and rib fractures became more frequent. Associated causes of death increased with time, except for decubitus ulcers, indicating a change in the pattern of the death process. Despite a 50% decline, osteoporotic fractures still have a significant impact on mortality. The pattern of the death process has changed, with an increased role for comorbidities.cause of deathdeath certificatesfractures, boneInternational Classification of Diseasesmortalityosteoporosisvital statisticsOsteoporotic fractures are one of the leading causes of death in the elderly population (1) and a major contributor to the burden of disease (2). This burden is expected to increase as life expectancy increases (3), and it is a growing issue in all aging societies (4, 5).Treatments have been developed to prevent osteoporotic fractures and related mortality. Hip prosthesis, becoming widespread in the 1970s, reduced complications due to rapid mobilization and was further improved by effective anticoagulation and better anesthetic procedures (6–8). More recently, medical treatments have been widely used to reduce the incidence of osteoporotic fractures; they include postmenopausal hormonal replacement therapy (9) and bone-protective agents such as bisphosphonates (10).Despite the growing importance of osteoporotic fractures and the advances in their management, few studies have addressed the consequences for mortality in large populations or the changes in these consequences. Most available information comes from variously assembled cohorts, followed for short periods, and assessing mortality after a fracture event (11–15).Assessing the impact of osteoporotic fractures on the mortality of the entire population (i.e., how frequently osteoporotic fractures contribute to death) is valuable from a public health perspective. Nationwide vital registration systems provide consistent, relevant, and nationwide data, and these systems are available, exhaustive, low cost, and suitable for international comparisons. This approach requires identification and analysis of causes of death reported on death certificates and coded according to the International Classification of Diseases (ICD), following rules issued by the World Health Organization.We have developed a methodology (16) for estimating the contribution of osteoporotic fractures to mortality in the general population, based on the analysis of cross-classifications of essential components related to current definitions of osteoporotic fractures (age, sex, site, and mechanism of fracture). Here, we use this methodology to investigate the impact of osteoporotic fractures on mortality in France between 1968 and 2004. We also examined changes in the patterns of associated causes of death during the same period.MATERIALS AND METHODSData sourceMortality data were obtained from the Center of Epidemiology on Medical Causes of Death (“CépiDc”; INSERM), where all French death certificates are collected, checked for quality, and coded. All data from 1968 to 2004 for metropolitan France were analyzed. Variables including sex, age at death, and all coded causes of death were recorded.Death certification and codingIn France, death certification is mandatory and necessarily performed by a medical doctor. Death certificates are exhaustive, and the data since 1968 are available (17). The certificate is established according to World Health Organization recommendations, and the medical cause of death section consists of 2 parts. The first is used for reporting the sequence of events leading to death, with the underlying cause stated last. The second part reports the other contributing causes of death (“associated causes of death”). The medical information provided by the French death records is detailed; a mean of 3.1 causes was reported for each death in 2003 (17).Coding consists of attributing a digital code to each cause of death, according to the latest revision of the ICD, and then selecting the underlying cause according to World Health Organization rules. During the study period, 3 changes were made to the French coding system. First, the ICD was revised twice: The Eighth Revision was used from 1968 to 1978 (ICD-8), the Ninth Revision from 1979 to 1999 (ICD-9), and the Tenth Revision from year 2000 (ICD-10). Second, since the year 2000, 80% of death certificates have been coded by software that also attributes the underlying cause. The knowledge base included in the French software uses the Mortality Medical Data System decision tables of the National Center for Health Statistics, US Centers for Disease Control and Prevention. Third, the number of coded contributing causes was increased from 1 (1968–1978) to 2 (1979–1999) and then to be exhaustive (since 2000).Two types of ICD codes are used to describe injuries: one describes the nature of the injury or injury code (I-code, e.g., hip fracture), and the other describes the mechanism of the injury (E-code, e.g., fall). According to ICD rules, the mechanism of injury (E-code) is always selected as the underlying cause of death and is the one routinely published. If the certifying medical doctor omits an E-code, a nonspecific code is automatically assigned (X59 in ICD-10) and selected as the initial cause of death.Method for identification of an osteoporotic fracture-related deathWe previously developed a multiple-step method to estimate the contribution of osteoporotic fractures to mortality in the general population (16). Current definitions of osteoporotic fractures were identified, and their components were individualized and translated into an operational definition using age, sex, site, and mechanism of fracture. The main results were, first, the identification of an age threshold of 70 years, after which fractures are reported with increasing frequency. Second, the patterns of rates for men and women were similar. Third, a similar pattern of rates was found for all sites of fracture, including skull fractures, suggesting that all fractures, and not merely classical “osteoporotic” fractures, should be considered. Fourth, the mechanism of injury was useful for categorizing fractures as osteoporotic, by allowing the exclusion of high-energy fractures. According to current definitions of osteoporotic fractures, high-energy fractures should be excluded and only “fragility-related” fractures considered. However, certifying medical doctors may omit the mechanism of fracture; we adjusted for this underdeclaration of the mechanisms of fractures by calculating a corrective factor for each site of fracture, by sex. Fractures occurring between 20 and 30 years of age are obviously nonosteoporotic, so the sensitivity of the high-energy code for detecting high-energy fractures (or corrective factor) was calculated as the number of reports of the high-energy code divided by the total number of fractures in this age category. High-energy fractures were multiplied by this site- and sex-specific corrective factor to control for their underestimation. The estimated number of osteoporotic fractures was, thus, all fractures occurring after the age of 70 years minus the corrected number of high-energy fractures.The method was developed for ICD-10, so I-codes and E-codes in ICD-8 and ICD-9 were identified (Web Table 1; this information is described in the first of 2 supplementary tables referred to as ‘‘Web Table’’ in the text and posted on the Journal’s website (http://aje.oupjournals.org/).). Sites of fractures were individualized as follows: hip, vertebrae, pelvis, ribs, skull, multiple, and other. Bridge-coding tables allowed the estimation of the comparability ratios between ICD versions (18). We performed a detailed dissection of the codes and multiple simulations, comparing the numbers of mentions of the related causes of death in the years bracketing ICD modifications: 1974–1978, 1979–1983, 1995–1999, and 2000–2004. Some discrepancies were found. First, fractures of the pelvis were well individualized in ICD-8 and ICD-9 but were grouped with lumbar spine fractures in ICD-10, artificially decreasing mentions for pelvis fractures and increasing those for spine fractures over time. Second, an E-code for unspecified mechanism of fracture was included in the falls section in ICD-8 and ICD-9 (E887) but not in ICD-10 (X59: exposure to unspecified factor), artificially decreasing the reporting of falls over time. On the basis of this analysis, fractures of the lumbar spine and pelvis were grouped together because this minimized variation associated with the transition from ICD-9 to ICD-10, and the change of classification of the unspecified E-code E887/X59 was taken into account.Table 1.Standardized Mortality Rates per 100,000 Persons Through the Study Period, by Sex and Site of Osteoporotic Fracture, France, 1968–2004a1968–19721973–19781979–19841985–19891990–19941995–19992000–2004% ChangebMen    Hip153.24162.12139.54108.0986.2574.6870.37−54.08    Pelvis + vertebrae3.996.887.866.657.357.587.9599.24    Skull4.856.164.252.773.152.41.8−63.01    Ribs2.975.055.364.614.254.385.7295.52    Multiple1.411.621.611.120.930.771.720.83    Others27.2529.9729.4422.919.8918.8221.95−19.47    All193.72211.8188.05146.12121.82108.63109.49−43.48    External causesc372.5368.27387.6397.33362.08339.57268.34−27.96    General mortalityd8,888.58,274.637,852.757,873.937,033.156,6876,302.01−29.05Women    Hip294.79304.9240.54196.51142.8109.8102.38−65.27    Pelvis + vertebrae5.699.0110.39.529.389.779.9675.01    Skull3.483.453.123.022.821.931.79−48.63    Ribs1.482.682.682.692.382.453.74152.21    Multiple2.32.412.72.181.531.564.1982.34    Others45.0250.9750.3542.213634.342.04−6.62    All352.76373.43309.69256.13194.9159.8164.1−53.48    External causesc361.78368.03347.88341.84310.29209.46176.05−51.34    General mortalityd6,637.046,244.725,893.975,875.375,476.015,246.214,949.3−25.43aMortality rates are related to external causes of death, and general mortality rates are included for comparison.bComparison of mortality rates between 1968–1972 (reference) and 2000–2004. All changes were statistically significant (Pchi-square < 0.0001), except for multiple fractures in men (P = 0.10).cNumber of death certificates for subjects over 70 years of age reporting any external cause of death × 100,000/general population over 70 years of age.dTotal number of death certificates for subjects aged over 70 years × 100,000/general population over 70 years of age.We also verified that the previously identified thresholds were also valid for ICD-8 and ICD-9. Applying the same methodology, we found that the age threshold of 70 years can still be used. Because the high-energy threshold used to calculate the corrective factor allowing exclusion of high-energy fractures varied with time, it was calculated for 5-year periods for each sex and site of fracture (Web Figure 1) (http://aje.oupjournals.org/).Figure 1.Standardized mortality rates, by fracture site, France, 1968–2004. Continuous line for men and dashed lines for women. A, all fractures (right axis scale: all-cause mortality rate, represented in open squares for men and open circles for women); B, hip; C, pelvis and vertebrae; D, ribs; E, others; and F, skull.Death ratesCrude death rates were calculated annually by site, sex, and age, using demographic data provided by the National Institute for Statistics and Economic Studies for estimating the denominator. Age-adjusted mortality rates were calculated by using a direct method of standardization to account for differences in age composition and the French reference population of 1990 (a census year situated near the middle of the study period). Age-specific death rates were calculated for osteoporotic fractures by 5-year age group (70–74, 75–79, 80–84, 85–89, 90–94, and older than 94 years).Statistical analysisStandardized mortality rates were compared between the beginning (1968–1972) and the end (2000–2004) of the study period with a χ2 test (19). The trends of age-specific death rates were estimated by Poisson regression analysis (20), using the “85–89 years” age group as reference. The mean age at death was calculated yearly by using death certificates with osteoporotic fractures and compared with the mean age at death for the entire population after the age of 70 years; trends in the difference were studied by linear regression.Measurements of the association between osteoporotic fractures and other causes of death used the ratio method of Israel et al. (21): The ratio of the number of observed pairs of causes of death to the number of expected pairs of causes was calculated, based on the assumption of independence. An observed/expected ratio (O/E ratio) greater (less) than 1 indicates that more (fewer) deaths with paired causes were reported than would be expected by chance if the paired causes were independent. Chi-square statistics were used to test the null hypothesis H0: O/E ratio = 1. Two time periods were compared: 1968–1972 and 2000–2004, by site of fracture. For each O/E ratio, 95% confidence intervals were defined and, in the absence of an appropriate statistical test, the overlap of these intervals was used to assess statistical significance.RESULTSA total of 19,989,760 death certificates were registered in metropolitan France between 1968 and 2004. Of these, 440,890 (2.2%) reported an osteoporotic fracture, and 306,245 (69% of osteoporotic fractures and 1.5% of all death certificates) reported a hip fracture. During the period 2000–2004, 46,849 (1.78%) death certificates reported an osteoporotic fracture.Fewer men than women died with osteoporotic fractures, with a standardized mortality sex ratio (men/women) of 0.61 for the entire study period. This ratio was not stable across sites between 1968 and 2004; it varied from 0.52 to 0.69 for hip, from 0.70 to 0.80 for pelvis and vertebrae, from 2.06 to 1.52 for ribs, and from 1.41 to 1.02 for skull, thus reducing the gap between men and women.Hip fractures were the most frequently reported on death certificates followed by pelvis and vertebrae, skull, ribs, and multiple fractures. Standardized mortality rates changed through the study period and between the different sites (Table 1). There was a statistically significant halving of osteoporotic fractures overall (−43.5% in men and −53.5% in women) between 1968 and 2004, mainly due to decreasing reports of hip and skull fractures. The pattern was, however, similar for most sites: a small increase early in the study period and a subsequent continuous decline from the mid-1970s (Figure 1). This decline paralleled but exceeded both the 25%–29% decline in general mortality and the decline in external causes-related mortality (in France, mainly road traffic accidents).Although the decline in skull fractures was substantial, they constitute only a minority of all fractures (1.49% between 1968 and 2004), and their contribution to the general trend was small. Unlike hip and skull fractures, reports of pelvis and vertebral fracture increased gradually with time. Rib and “other” fractures increased initially and then stabilized in the 1980s (the sharp increase in the year 2000 is largely an artifact of the exhaustive coding of causes from that date).A statistically significant estimate was found for the third (cubic) power of year, indicating a polynomial—and not linear—trend over time. There were 2 inflexion points: an increase in mortality rates for all fractures at the beginning of the study period, followed by a rapid decrease that slowed slightly at the end.Age-specific, 5-year mortality rates associated with osteoporotic fractures were calculated (Figures 2 and 3). For all osteoporotic fractures, mortality rates decreased for all age categories, most markedly in younger age groups. Poisson regression estimates for decreasing trends with time were statistically significant for hip and “other” fractures in all age groups and for skull fractures in men younger than 85 years (Web Table 2). The increasing trends for pelvis, vertebrae, and rib fractures were apparently more pronounced in the older age groups (not statistically significant except for pelvis and vertebrae fractures in women).Table 2.Ratios of Actual to Expected Number of Pairs of Causes of Death Including Low-Energy Fractures, for ICD Chapters and Selected Causes, France, 1968–1972 and 2000–2004a1968–19722000–2004All Deaths, no.Observed, no.Expected, no.Observed/ Expected Ratio95% Confidence IntervalAll Deaths, no.Observed, no.Expected, no.Observed/ Expected Ratio95% Confidence IntervalICD chapters    Infectious and parasitic diseases23,551257837.000.310.27, 0.35140,1683,6035,139.170.700.68, 0.72    Neoplasms274,6441,0149,760.780.100.10, 0.11518,5574,73319,012.570.250.24, 0.26    Diseases of the blood6,11171217.180.330.26, 0.4144,0831,2231,616.280.760.72, 0.81    Endocrine diseases105,8882,0973,763.230.560.53, 0.58252,6307,4909,262.520.810.79, 0.83    Diseases of the nervous system90,5432,0053,217.880.620.06, 0.65313,77710,99111,504.440.960.94, 0.97    Diseases of the circulatory system848,8589,90730,168.210.330.32, 0.331,059,49126,33838,845.580.680.67, 0.69    Diseases of the respiratory system205,0962,7847,289.060.380.37, 0.40428,16211,17515,698.290.710.70, 0.73    Diseases of the digestive system87,0035183,092.070.170.15, 0.18169,0943,4266,199.730.550.53, 0.57    Diseases of the genitourinary system37,8754031,346.070.300.27, 0.33142,1553,6375,212.020.700.68, 0.72    Diseases of the musculoskeletal system11,040209392.360.530.46, 0.6140,8081,7661,496.021.181.13, 1.24    Diseases of skin and subcutaneous tissue12,4121,031441.122.342.20, 2.4854,5233,2811,999.051.641.59, 1.70    Others not classified elsewhere485,3089,36317,247.730.540.53, 0.55971,22928,00835,609.510.790.78, 0.80Selected causes of death (ranked by association with osteoporotic fractures in 2000–2004)    Decubitus ulcer10,6011,010376.762.682.52, 2.8544,6492,9901,637.031.831.76, 1.89    Pulmonary embolism14,675246521.550.470.41, 0.5357,1292,9582,094.601.411.36, 1.46    Gastrointestinal ulcer6,76041240.250.170.12, 0.239,013357330.461.080.97, 1.20    Dementia19,205484682.540.710.65, 0.77113,8444,4744,174.021.071.04, 1.11    Rheumatoid arthritis3,76157133.670.430.32, 0.545,363201196.631.020.89, 1.17    Ischemic cardiopathy141,2787345,020.990.150.14, 0.16157,6205,0435,779.040.870.85, 0.90    Pyelonephritis1,0381236.890.330.17, 0.544,546134166.680.800.67, 0.95    Hypertensive diseases25,256320897.590.360.32, 0.40173,1524,9576,348.510.780.76, 0.80    Pneumonia42,8211,0171,521.850.670.63, 0.71153,4414,0685,625.820.720.70, 0.75    Gastrointestinal hemorrhage1,574855.940.140.06, 0.2628,9546641,061.580.630.58, 0.67    Renal failure7,19146255.570.180.13, 0.24115,3142,6554,227.920.630.60, 0.65    Diabetes47,0561,0691,672.360.640.60, 0.6868,7081,4922,519.140.590.56, 0.62    Malignant neoplasms253,1938908,998.420.100.09, 0.11428,3313,57415,704.490.230.22, 0.24Total no. of times reported2,272,97580,7814,253,093155,937Total no. of death certificates1,630,36051,1221,930,42350,171Abbreviation: ICD, International Classification of Diseases.aNinety-five percent confidence intervals for the observed/expected ratio are given in a separate column. An observed/expected ratio of >1 indicates a positive association with low-energy fractures.Figure 2.Age-specific mortality rates for men on a log10 scale, France, 1968–2004. Diamonds, ages 70–74 years; squares, ages 75–79 years; triangles, ages 80–84 years; dashed line, ages 85–89 years (reference); crosses, ages 90–94 years; circles, ages >94 years. All interactions between time and age groups are significant (P < 0.05) except for pelvis and vertebrae, ribs, and skull fractures in the >90-year age groups. A, all fractures; B, hip; C, pelvis and vertebrae; D, ribs; E, others; and F, skull.Figure 3.Age-specific mortality rates for women on a log10 scale, France, 1968–2004. Diamonds, ages 70–74 years; squares, ages 75–79 years; triangles, ages 80–84 years; dashed line, ages 85–89 years (reference); crosses, ages 90–94 years; circles, ages >94 years. All interactions between time and age groups are significant (P < 0.05) except for the pelvis and vertebrae (90–94-year age group), ribs (>90-year age groups), and skull fractures. A, all fractures; B, hip; C, pelvis and vertebrae; D, ribs; E, others; and F, skull.The mean age at death increased continuously. The mean age at death associated with osteoporotic fractures increased more (P < 0.0001) than that for general mortality in both sexes (with a difference of 2.30 years for men and 1.77 years for women) (Figure 4).Figure 4.Mean age at death, France, 1968–2004. Osteoporotic fracture-related mortality is represented with continuous lines for men and dashed lines for women; the all-cause mortality rate is represented by open squares (men) and open circles (women).The number of causes of death for death certificates reporting osteoporotic fracture doubled from an average of 1.6 causes per certificate in 1968–1972 to 3.1 in 2000–2004 (contrasting with 1.4 in 1968–1972 and 2.2 in 2000–2004 for all certificates). The incidence of comorbidities in cases of fracture-related deaths thus increased substantially.Diseases of the circulatory system were the comorbidities most frequently associated with osteoporotic fractures (19.4% of death certificates in 1968–1972 and 53.2% in 2000–2004). The most common were ischemic cardiopathy and hypertensive diseases. Other frequent comorbidities were diabetes, pneumonia, and decubitus ulcer in 1968–1972 and pneumonia, renal failure, and dementia in 2000–2004 (Table 2). An O/E ratio greater than 1, indicating a positive association with osteoporotic fractures, was found for diseases of the musculoskeletal system, decubitus ulcer, pulmonary embolism, and dementia in the period 2000–2004. All associations (O/E ratios) increased with time, a trend also found for all sites studied individually (data not shown), with 2 exceptions: Decubitus ulcer, which was significant in 1968–1972, decreased significantly with time for all sites, and the diseases of the respiratory system (including pneumonia) decreased only for rib fracture-associated deaths.DISCUSSIONWe analyzed nearly 20 million death certificates covering 37 years and identified a significant contribution of osteoporotic fractures to nationwide mortality. Osteoporotic fractures were reported on 440,890 death certificates (2.2% during the entire period), supporting the established belief that they represent a major public health problem. By comparison, during the same period, diabetes, renal failure, and septicemia were mentioned on 430,967, 312,881, and 222,665 death certificates, respectively.Between 1968 and 2004, the mortality rates associated with osteoporotic fractures were reduced by about half, in both sexes and in all age groups, largely due to changes associated with hip and skull fractures. This decrease was apparent despite the increase in the number of coded causes of death per certificate, indicating a true significant decline. The decline exceeded that of general mortality, translating into a significant increase over time in the mean age at death from osteoporotic fractures. However, mortality rates associated with fractures at some sites increased. In some cases (rib and multiple fractures), this was a consequence of the change in coding rules with the transition to ICD-10 in 2000 and the increase of the total number of coded causes; in others (pelvis and vertebrae), it was not. These sites represented a minority of the total, such that these increases had little impact on osteoporotic fracture-associated mortality overall.The decline in the incidence of and mortality associated with hip fractures has been reported in large cohort studies (22, 23) and posthospitalization follow-up studies (15, 22). However, none of these studies fully explains the causes of the decrease in mortality. Our study allows various hypotheses concerning management and behavioral changes to be considered. During recent decades, temporally compatible with the observed falling mortality rates, surgical management and postsurgical care of hip fractures have clearly improved (7, 8). The use of antiosteoporotic treatments, initially hormone replacement therapy, increased steeply in France in the late 1980s and early 1990s (9), but this can explain only a small part of the change because the decrease started in the mid-1970s and affected both women and men (albeit less so). The decrease in hip fracture rates reported elsewhere was similarly much too large to be explained by antiosteoporotic medication (22, 24). Other possible causes include changes in smoking habits, improved general health, calcium and vitamin D supplementation, regular exercise, awareness of falls, and moderating alcohol intake (clearly documented in France) (25). The absence of age- and sex-specific differences in mortality trends is in favor of a more general cause, for example, improved postsurgical management and behavioral changes, rather than a medication-related effect. One potential bias should be addressed. Increased awareness of the severity of osteoporotic fractures and promotion of their medical management during the later years of the study period (10) may have increased the frequency of reporting of some fracture types (vertebral and pelvis fractures). The consequent increased reporting of less severe sites not traditionally associated with high risk of mortality may have decreased the observed general mortality rates associated with fractures. This bias was controlled for by analyzing each site of fracture separately. The observed trends in mortality rates were clearly site specific. Wrist fractures, clearly related to osteoporosis in all awareness campaigns, were seldom identified in our sample, indicating no artificial effect on the physicians’ certification habits. There was a 50% reduction of mortality rates associated with hip and skull fractures over the study period. Hip fracture may lead to death in the immediate postfracture period, so failure to report it on death certificates is less likely, even early during the study period.The skull site is not traditionally associated with osteoporotic fractures. However, our data suggest that some skull fractures, occurring at an older age and not associated with a high-energy mechanism, follow a pattern similar to that of all other osteoporotic fractures. Therefore, skull fractures were included in the analysis, but their contribution to the overall trends is small (1.49% of all osteoporotic fractures).Interestingly, we found an increase with time for all comorbidities associated with osteoporotic fractures at the moment of death, with 2 exceptions. For decubitus ulcer, the association weakened, reflecting a better mobility after surgery, and for diseases of the respiratory system, the association weakened in rib fracture deaths only. Similar increases in comorbidities have been observed by others (22), suggesting a change in mortality patterns, with comorbidities increasingly contributing to the death process, as fractured subjects at risk of death became older and frailer. Some causes of death were less associated with fractures than expected. The values are consistent throughout the study period, so medical certification errors are unlikely to be the cause. Some causes of death may “compete” at 2 levels. The first is true competition, leading to different mortality patterns (26). Patterns of mortality may differ according to age, sex, and time period, with one main pattern associating neoplasms and cardiovascular and digestive diseases in those aged 45–84 years and another pattern represented by injury and poisoning. The second level is an artificial competition in the mind of a certifying medical doctor. If death is associated with a “serious” condition, such as cancer, severe infection, or cardiovascular disease, all other causes, and not only fractures, are underdeclared (the cause is serious enough, and the certifying physician does not need to seek other associated causes).Our study has several strengths including the analysis of a very large set of death certificates exhaustively covering a large population over a long period (1968–2004). Nevertheless, it has some limitations. Diagnosis errors, unavailable medical records at the time of certification, and information missing from death certificates have all been documented (27). However, our study did not aim to estimate all osteoporotic fractures leading to death in France but, rather, it attempts to identify cases in which the certifying physician considered the fracture to have contributed to death. Moreover, underreporting is probably less frequent for injury-related causes of deaths such as fractures than for chronic medical conditions. Another limitation is that death certificate data represent only mortality rapidly following a fracture. Although the risk remains elevated up to 10 years following a hip fracture (12), the highest risk of mortality is immediately after the fracture event (4, 11). In a recent systematic review (11), excess mortality after a hip fracture ranged from 5.9% (among patients aged 50–74 years) to 50% during the first year after fracture and remained elevated for many years. One-quarter to one-third of the mortality occurred during the first month after fracture, around half within 3 months, and 70% within 6 months. The mechanism of fractures was poorly documented in our study. In particular, low-energy mechanisms were seldom reported. Work with the Swedish population-based register showed that low-energy trauma was responsible for 53% of fractures in those over 50 years of age and for 80% in those over 75 years of age between 1993 and 2004 (13). A better understanding of injury mechanisms is necessary for preventive interventions, and better reporting of mechanisms on the death certificate would be valuable. Coding the causes of death by using the ICD may lead to errors and inaccuracies. However, this is unlikely for fracture data, because there is no ambiguity in ICD codes for such injuries.In conclusion, we found a significant decline of reports of osteoporotic fractures on death certificates between 1968 and 2004, starting in the 1970s, in both genders and all age groups. This was driven mainly by the decline of hip and skull fractures and coincided with better surgical and postsurgical management and general behavioral changes. Nevertheless, the impact of osteoporotic fractures on mortality remains significant, and the trends through time were opposite for the sites pelvis, vertebrae, and ribs. Finally, the mortality pattern changed, suggesting an increasing role for comorbidities in the death process as subjects become older and frailer.AbbreviationsICDInternational Classification of DiseasesICD-8ICD-9, and ICD-10, International Classification of Diseases, Eighth, Ninth, and Tenth Revisions, respectivelyO/E ratioobserved/expected ratioAuthor affiliations: Research unit APEMAC, EA 4360, Nancy-Université, Université Paris-Descartes, Université Metz Paul Verlaine, Paris, France (Joël Coste, Nelly Ziadé); Centre d’épidémiologie sur les causes médicales de décès (CépiDc), National Institute for Health and Medical Research, Le Vésinet, France (Eric Jougla); and Faculté de Médecine, Université Saint-Joseph, Beirut, Lebanon (Nelly Ziadé).Conflict of interest: none declared.1.GenantHKCooperCPoorGInterim report and recommendations of the World Health Organization Task-Force for OsteoporosisOsteoporos Int19991042592642.JohnellOKanisJAJonssonBThe burden of hospitalised fractures in SwedenOsteoporos Int20051622222283.CummingsSRMeltonLJEpidemiology and outcomes of osteoporotic fracturesLancet20023599319176117674.JohnellOKanisJEpidemiology of osteoporotic fracturesOsteoporos Int200516suppl 2S3S75.OinumaTSakumaMEndoNSecular change of the incidence of four fracture types associated with senile osteoporosis in Sado, Japan: the results of a 3-year surveyJ Bone Miner Metab201028155596.ScheerlinckTHaentjensPFractures de l'extrémité supérieure du fémur chez l'adulte. (In French)2003Paris, FranceScientific and Medical Publishing Elsevier SAS7.HandollHHFarrarMJMcBirnieJHeparin, low molecular weight heparin and physical methods for preventing deep vein thrombosis and pulmonary embolism following surgery for hip fracturesCochrane Database Syst Rev.20002CD0003058.HandollHHSherringtonCMobilisation strategies after hip fracture surgery in adultsCochrane Database Syst Rev.20071CD0017049.RingaVJaussentIGuégenRTrends in the use of hormone replacement therapy in eastern France between 1986 and 1993Eur J Public Health19999430030510.StaffordRSDrielingRLHershALNational trends in osteoporosis visits and osteoporosis treatment, 1988–2003Arch Intern Med2004164141525153011.AbrahamsenBvan StaaTArielyRExcess mortality following hip fracture: a systematic epidemiological reviewOsteoporos Int200920101633165012.BliucDNguyenNDMilchVEMortality risk associated with low-trauma osteoporotic fracture and subsequent fracture in men and womenJAMA2009301551352113.BergströmUBjörnstigUStenlundHFracture mechanisms and fracture pattern in men and women aged 50 years and older: a study of a 12-year population-based injury register, UmeåSweden. Osteoporos Int20081991267127314.VestergaardPRejnmarkLMosekildeLHas mortality after a hip fracture increased?J Am Geriatr Soc.200755111720172615.RobertsSEGoldacreMJTime trends and demography of mortality after fractured neck of femur in an English population, 1968–98: database studyBMJ2003327741877177516.ZiadéNJouglaECosteJUsing vital statistics to estimate the population-level impact of osteoporotic fractures on mortality based on death certificates, with an application to France(2000–2004). BMC Public Health. 2009;9:344. (doi:10.1186/1471-2458-9-344)17.PavillonGLaurentFCertification et codification des causes médicales de décès. (In French)Bull Epidémiol Hebd200330/3113413818.PavillonGBoileauJRenaudGBridge coding ICD9-ICD10 and effects on French mortality data. 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(Accessed November 2, 2009)21.IsraelRARosenbergHMCurtinLRAnalytical potential for multiple cause-of-death dataAm J Epidemiol1986124216117922.BrauerCACoca-PerraillonMCutlerDMIncidence and mortality of hip fractures in the United StatesJAMA2009302141573157923.LeslieWDO'DonnellSJeanSTrends in hip fracture rates in CanadaJAMA2009302888388924.AbrahamsenBVestergaardPDeclining incidence of hip fractures and the extent of use of anti-osteoporotic therapy in Denmark 1997–2006Osteoporos Int201021337338025.PyöräläETrends in alcohol consumption in Spain, Portugal, France and Italy from the 1950s until the 1980sBr J Addict199085446947726.CosteJBernardinEJouglaEPatterns of mortality and their changes in France (1968–99): insights into the structure of diseases leading to death and epidemiological transition in an industrialised countryJ Epidemiol Community Health2006601194595527.SwiftBWestKDeath certification: an audit of practice entering the 21st centuryJ Clin Pathol2002554275279 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_00BEC90FD19C3AEBDD8BF7110F0AD4C5E756BA8B.txt b/test/dataset/in/resources/corpus/Clean_00BEC90FD19C3AEBDD8BF7110F0AD4C5E756BA8B.txt new file mode 100644 index 0000000..68f690c --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_00BEC90FD19C3AEBDD8BF7110F0AD4C5E756BA8B.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press 9808410.1093/geronb/55.3.P131 Journal of Gerontology: Psychological Sciences Age-Related Differences in the Use of Contextual Information in Recognition Memory A Global Matching Approach Bayen Ute J. a Phelps Matthew P. b Spaniol Julia a aDepartment of Psychology, The University of North Carolina at Chapel Hill bDepartment of Psychology, Malone College, Canton, Ohio Ute J. Bayen, Department of Psychology, The University of North Carolina, Davie Hall, CB#3270, Chapel Hill, NC 27599 E-mail: ubayen@unc.edu. 1 5 2000 55 3 P131 P141 10 12 1999 31 8 1998 The Gerontological Society of America 2000 Age differences in the processing of contextual information were investigated using the Item, associated Context, and Ensemble (ICE) model (K. Murnane, M. P. Phelps, and K. Malmberg 1999), a general global matching model of recognition memory. In two experiments, young and older adults studied words in environmental contexts and were tested in both the same and different contexts. Patterns of context effects for hit rate, false alarm rate, and d′ suggest that older adults process associated context, but have difficulties integrating items and context into an ensemble. Thus, older adults appear to have a specific, rather than a general, deficit in processing contextual information. A deficiency in ensemble processing may be responsible for the prevalent finding that older adults show poorer recognition memory performance than young adults. hwp-legacy-fpage P131 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: Toni C. Antonucci, PhD ONE of the best-established results of research in the area of human memory and aging is that older adults' performance on episodic-memory tasks is generally lower than that of young adults (for reviews see Craik and Jennings 1992; Kausler 1994; A. D. Smith 1996). Episodic memory is a term coined by Tulving 1972 that denotes memory for specific events. An episode is defined by the context (e.g., time, environment, mood state) in which information is encoded. For example, if you try to remember whether you had muffins for breakfast this morning, it is not sufficient for you to know what muffins are or that you usually eat muffins, rather you have to remember if muffins occurred in the particular context of your breakfast this morning. Because successful performance on episodic-memory tasks requires the encoding and retrieval of the context in which information was acquired, gerontologists have suggested that older adults' lower performance on such tasks might be attributable to age-related differences in the processing of context information. This hypothesis was first proposed in the early 1980s (Burke and Light 1981; Craik and Byrd 1982; Rabinowitz, Craik, and Ackerman 1982) and has since inspired a number of researchers to investigate the role of context processing in age differences on episodic-memory tasks. In this article we examine the effects of changes in environmental context between learning and test on recognition memory in young and older adults. Although age differences in recall are generally larger than age differences in recognition (Craik and McDowd 1987), statistically significant age differences in recognition were found in most studies (for a review, see Kausler 1994). We propose that older adults have a specific, rather than a general, deficit in processing contextual information in recognition tasks. More specifically, we argue that older adults process contextual information in a nonintegrative manner as efficiently as younger adults, but have difficulties integrating context information with to-be-remembered items. A specific deficit in integrating to-be-remembered items with their surrounding context may provide an explanation for the prevalent finding that older adults show poorer recognition memory performance than young adults. One line of research that investigates the issue of context processing in older adults is research on aging and source memory, which involves direct questions about the source of information or the context in which information was acquired (e.g., Bayen and Murnane 1996). In a source-memory paradigm, participants study information in different contexts and are later asked to remember the context in which particular pieces of information were learned. According to the results of this research, under most circumstances older adults show lower performance on such tasks than young adults (see a meta-analysis by Spencer and Raz 1995), indicating that older adults' memory for context is impaired. Another line of research investigates the facilitative effects of context memory on memory for the content of episodes. If, in comparison to young adults, older adults are less adept at encoding and retrieving contextual information, then they are expected to show less encoding specificity. One form of encoding specificity (Tulving and Thompson 1973), a context effect, refers to the effect that memory performance is better the more similar contextual conditions are between encoding and retrieval. If older adults have difficulties encoding or retrieving context, their memory for items learned in a particular context should benefit less than that of young adults from matching contextual conditions at encoding and retrieval. Several studies have compared context effects in young and older adults. Using recognition tasks, Naveh-Benjamin and Craik 1995 found that older adults benefit at least as much as their younger counterparts when environmental context at encoding matches that at test. An absence of age differences in environmental-context effects on recognition was also reported by Vakil, Melamed, and Even 1996. Similarly, Schramke and Bauer 1997 found age-independent context effects of physiological states on free recall, and Light, LaVoie, Valencia-Laver, Albertson Owens, and Mead 1992 reported increased repetition priming in both young and older adults with matching versus nonmatching acquisition and test modalities. Park and collaborators also did not find age differences in the effects of encoding specificity using picture recognition (Park, Puglisi, Smith, and Dudley 1987; Park, Puglisi, and Sovacool 1984) and word recall (Puglisi, Park, Smith, and Dudley 1988). However, Park, Smith, Morrell, Puglisi, and Dudley 1990 as well as A. D. Smith, Park, Earles, Shaw, and Whiting 1998 asserted that older adults do show an impairment in context processing when a task requires self-initiated integration of item and context. They concluded this from their findings of larger age differences in cued-recall when items and their contextual cues were unrelated to each other than when they were related to each other. In a study by Earles, Smith, and Park 1996, older adults benefited from instructions to integrate item and context information, but not as much as a comparison group of young adults. It thus appears that older adults do encode and retrieve context information, but that they have problems with the integration of item and context information. In our research, we have tested this hypothesis using a qualitative, formal theory of recognition memory that allows us to disentangle possible age differences in different forms of context processing. Although formal models are available that give sophisticated accounts of the role of context in episodic-memory tasks such as recognition and recall tasks, none of these models have thus far been used to shed light on the issue of age differences in episodic memory. A model that is particularly suitable for cognitive-aging research is the Item, associated Context, and Ensemble (ICE) model developed by Murnane, Phelps, and Malmberg 1999, because it specifies two different kinds of context processing, namely, the processing of context separate from an item (associated context), and the formation of an integration of item and context information (an ensemble). Depending on which kind of context processing participants use, different patterns of context effects are predicted in recognition tasks. We first describe the characteristics of the ICE model and summarize prior research on young adults that has validated this model. We then derive predictions regarding age differences in different forms of context processing and present two experiments to support our hypotheses. The ICE Model of Recognition Memory The ICE model (Murnane et al. 1999) is a general global matching model of recognition memory, in that it incorporates the common, general characteristics of the set of specific global matching models. Examples of global matching (or "global activation") models of recognition are SAM (Gillund and Shiffrin 1984), MINERVA2 (Hintzman 1988), TODAM2 (Murdock 1993), and the Matrix model (Humphreys, Bain, and Pike 1989). For a review of global matching models see Clark and Gronlund 1996. In a recognition task, participants are presented with a list of items and are later asked which items from a test list appeared on the earlier study list. Global matching models assume that recognition of items in such a task is based on the activation of a large number of memory representations. These representations are activated in response to a retrieval cue. For example, let us assume a research participant has studied a list of words, and then receives an old–new recognition test in which he or she has to indicate if a test item (e.g., "table") had appeared on the study list or not. The participant forms a retrieval cue of test-item information and test context and matches this cue against the representations of all items and their contexts in memory. The degree to which a memory representation is activated by the retrieval cue depends on the similarity between the information in the cue and the information stored in memory. These component activations are combined (i.e., summed) to yield a global match, which forms the basis for the recognition judgment. The higher the global match, the greater the probability of an "old" response. According to ICE, item information (I) plays a role in recognition as well as two theoretical forms of context information. These two forms of context are associated context (C), and ensemble (or integration) of item and context (E). These three components (I, C, and E) can each contribute to the global match. Consider the example of the word "rose" presented in the picture of a living room for a recognition test. In this example, any information encoded about "rose" that is not related to the living room context is item information (I), any information encoded about the picture of the living room that is not related to roses is associated-context information (C). Ensemble information (E) is a third and distinct type of information produced by integrating item and associated-context information. For example, you might integrate item and context into an ensemble by thinking of your significant other giving you a rose in the living room. ICE makes predictions about context effects on different measures of recognition, including hit rate, false alarm rate, and the discrimination measure d′. Hit rate (HR) is the proportion of old items identified as old. False alarm rate (FAR) is the proportion of new items called old. Measures of discrimination (such as d′) are composite measures of recognition memory that index an individual's ability to distinguish old from new items, independent of response biases. In our paradigm, context effects are obtained in a list-learning experiment with an old–new recognition test in which items are either tested in the same context in which they were studied or in a different context that the participant did not see during study. For example, if the word "bear" was presented on a yellow background on the study list, it is either presented on the same yellow background at test or on a different background, such as a red background. Similarly, distractor items are either tested in a context that the participant experienced during the learning phase, or in a new, different context that was not presented during the learning phase of the experiment. A context effect occurs when HR, FAR, or d′ in same-context testing is larger than HR, FAR, or d′, respectively, in different-context testing. The magnitude of a context effect is calculated by subtracting different-context scores from same-context scores. The predicted patterns of context effects are different depending on which information is stored in memory and used in the retrieval cue, either I and C only, or I, C, and E. The formal derivations of the following predictions from the ICE model can be found in Murnane and collaborators (1999). ICE predicts that if only item information (I) and associated-context information (C) are used in the global match, then the global match for target items tested in the same context is higher than the global match for targets tested in a different context. Consequently, context effects are expected on HR. Also, the global match for distractors tested in a context that the participant has experienced during encoding ("same context") is predicted to be higher than the global match for distractors presented in a new ("different") context. Thus, context effects are also predicted on FAR. Because an associated-context match increases the global match to both targets and distractors, it is not likely to enhance discrimination as measured by d′. Thus, if only I and C are used, we do not predict a context effect on d′ (see Appendix, Note 1). If, on the other hand, people use item information (I), associated-context information (C), and an ensemble (E) in the global match, then the ensemble is an additional source of match for targets tested in the same context. Consequently, the global match for targets tested in the same context is increased compared with the case in which no ensemble is used. The global match for distractors tested in a formerly presented context, however, is not increased compared with the no-ensemble case, because an ensemble cue formed from a distractor does not match any ensembles stored in memory. Because an ensemble match increases the global match to targets but not to distractors, it contributes to enhanced recognition memory as indicated by the discrimination measure d′. Thus, when I, C, and E are used, context effects are predicted in HR, FAR, and d′. In a series of seven experiments with young college students, Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995 varied simple visual contexts (as defined by foreground color, background color, and location on a computer screen) of words at study and test and found context effects on both HR and FAR. In the majority of conditions, no context effects on d′ appeared. This research suggests that simple visual context is not conducive to the formation of an ensemble of item and context information. Thus, when simple visual contexts are used, only item information (I) and associated-context information (C) are encoded in memory and in the retrieval cue. Murnane and collaborators (1999) found the pattern of results predicted by ICE when I, C, and E contribute to the global match. They used a contextual manipulation that they referred to as rich visual context (i.e., pictures of scenes like those in Fig. 1) and samples of young adult participants. Their results indicate that young adults encode rich visual context as both associated context and as context integrated with items into an ensemble. Unlike simple visual context, rich visual context is rich in meaningful content that can be integrated with meaningful items (i.e., words) into an ensemble. In summary, we predict on the basis of the ICE theory and prior research (Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995; Murnane et al. 1999) that if only I and C are encoded in memory and used as retrieval cues, context effects will be observed in HR and FAR, but not in d′. If, however, I, C, and E are used, context effects are predicted for HR, FAR, and d′. In our research, we used the ICE model to shed light on possible age-related differences in context processing in recognition tasks. On the basis of prior research that has found evidence that older adults encode and retrieve context information in episodic memory (Light et al. 1992), we hypothesized that older adults process associated context (C) as effectively as do young adults. Prior research also indicates, however, that older adults have difficulties with the self-initiated integration of item and context information (Park et al. 1990; A. D. Smith et al. 1998). Within the framework of the ICE theory, this translates into an age-related impairment in the formation of an ensemble of item and context (E). We hypothesize that older adults have difficulties forming an ensemble from item and context information. Thus, in situations in which young adults use all three components of the ICE model (i.e., I, C, and E), older adults use only I and C. Because it is the ensemble match that clearly contributes to enhanced recognition memory performance (i.e., discrimination between targets and distractors), an impairment in ensemble formation is a possible explanation for the prevalent findings of age-related differences in recognition memory. Overview of the Experiments To test our hypothesis that older adults use associated-context information in recognition tasks but have difficulties forming ensembles, we performed two experiments. In both of them, participants were presented with words in particular environmental contexts, which was followed by an old–new recognition test in which items were tested either in the same context in which they had been presented at learning or in a different context. Our goal was to study age differences under conditions that reveal the default mode of context processing, that is, how contextual information is processed when conditions do not elicit complex strategies for remembering or for context integration. Therefore, we used an incidental learning procedure and an orienting task that did not explicitly require the formation of ensembles. An important rationale for investigating context processing under these sorts of conditions is that they mirror the conditions under which most episodic memories are formed in everyday life. Another reason for doing so is that it allows us to begin to answer the more basic question of how these populations tend, by default, to process contextual information before considering (in future research) the more complex issue of how they may be able to process context more effectively when they are instructed to do so. In Experiment 1, we used simple visual contexts (combinations of foreground color, background color, and screen location of words), which as shown by Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995 do not foster the formation of an ensemble of item and context information in young adults. In this task, observed context effects are solely based on item and associated-context information (I and C). If older adults process associated-context information, they should show a pattern of context effects similar to that of young adults. That is, both age groups should show context effects in both HR and FAR, but not in d′. In Experiment 1, both age groups should show this same pattern of context effects. For Experiment 2, we used a design similar to that of Experiment 1, with the difference that rich visual contexts (i.e., pictures of scenes) were used instead of simple visual contexts. Murnane and collaborators (1999) found that young adults encode associated context and integrate item and context into ensembles when rich visual context is manipulated between study and test. If, as our hypothesis predicts, older adults do not form and/or use an ensemble to the extent that young adults do, then young and older adults should show different patterns of context effects in this rich-context experiment. The data pattern for older adults in this experiment should be similar to the data pattern for young adults under conditions of simple visual contexts. That is, older adults should show context effects on HR and FAR, but not on d′. On the other hand, young adults in Experiment 2 (rich visual contexts) should, in replication of results reported by Murnane and collaborators (1999), show context effects on HR, FAR, and d′. According to Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995, an experimental design has to satisfy certain methodological conditions to make the above predictions of the ICE model applicable. First, the same-versus-different-context variable must be manipulated within participants and within the recognition-test list (as opposed to between participants or lists) to minimize the likelihood that different response criteria are adopted in response to different overall levels of activation in the two test-context conditions. Such differences in response criteria could mask context effects on HR and FAR (for a detailed discussion of these issues, see Murnane and Phelps 1993). Second, the experiment should use what Murnane and Phelps 1994 refer to as an AB-X paradigm, as opposed to an AB-A paradigm. In an AB-X paradigm, the "different" test context is a context that was never experienced during study. In an AB-A paradigm, the "different" context was experienced during study, but is different from the context in which the test item was experienced during study. Murnane and Phelps 1994 formal derivations show that for an AB-X design, ICE predicts context effects on HR and FAR, whereas no or very small context effects are predicted if items are tested in an AB-A design. These different predictions have found empirical support (Murnane and Phelps 1994). We have designed two experiments in accordance with the methodological recommendations given by Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995. That is, we have used AB-X designs in which the test-context variable is manipulated within participants and within the test list. Experiment 1 In this experiment we used simple visual contexts. We predicted that the same pattern of context effects would emerge for both young and older adults. Both age groups should show context effects on HR and FAR; neither age group should show a context effect on d′. This pattern of results is predicted if both age groups use associated context (C) in their recognition judgments, but not an ensemble of item and context (E). Method Participants. A total of 104 adults participated in this experiment. Of these, 52 were young adult undergraduate students at The University of North Carolina at Chapel Hill (23 men, 29 women). They were between the ages of 18 and 24 years (mean age 19.7 years) and were recruited from introductory psychology courses. They received class credit as compensation for their participation in the experiment. Fifty-two of the participants were older adults (18 men, 34 women) between the ages of 59 and 83 years (mean age 71.0 years), resided in communities in central North Carolina, and were recruited through newspaper advertisements as well as flyers on community bulletin boards and in Senior Centers. They received payment for their participation in the study. All participants were native speakers of English. Mean years of education were 13.3 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 0.85\) \end{document}) for young adults and 15.4 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 2.57\) \end{document}) for older adults, a significant difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(102)\ =\ 5.79\) \end{document}, p < .0005. The mean performance in raw score units on the 30-point Gardner–Monge (Gardner and Monge 1977) vocabulary test was 15.4 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 3.67\) \end{document}) for young adults and 23.0 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 4.56\) \end{document}) for older adults, also a significant difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(101)\ =\ 9.27\) \end{document}, p < .0005. One young adult achieved only three points in the vocabulary test. This participant's response pattern indicated that she had not taken this test seriously, and her vocabulary test score was therefore not included in the mean reported above. In a near-vision test, all participants could fluently read printed text in 7-point font size. On a 4-point overall health rating scale with the options "poor," "fair," "good," and "excellent," 86% of the young adults and 98% of the older adults rated their health as good or excellent. None of the participants rated their health as poor. Persons who, in a phone interview, reported a history of heart attack, stroke, diabetes, emphysema, Parkinson's disease, brain trauma, or alcoholism were excluded from participation in the study. Individuals who reported being colorblind were also excluded. The data from two young adults were not logged because of technical failure. We excluded participants who did not give responses within the maximum response time of 5 s in four or more of the 64 recognition-test trials. Five participants were excluded for this reason: One older adult did not respond in 11 of the trials, 2 older adults did not respond in 5 of the trials, 1 young adult did not respond in 8 of the trials, and 1 young adult did not give any responses in the memory test. All excluded participants were replaced by additional participants. Design. The design was a 2 × 2 mixed factorial with age group (young vs. older) as a between-subjects variable, and test context (same as learning context vs. different-from-learning context) as a within-subjects variable. Materials. Stimulus items were 96 high-frequency nouns (Francis and Kucera 1982). These items were presented on color monitors in 24-point-size lower-case letters. Two different combinations of item color, screen color, and screen location served as learning and test contexts for the items. In Context A, light-green items were presented in the upper left-hand corner of a magenta computer screen. In Context B, yellow items were presented in the lower right-hand corner of a blue screen. Procedure. Each experimental session included 1 or 2 older participants or between 1 and 4 young participants. After signing consent forms and taking a brief vision test, participants were directed to individual computer booths. They were informed that they would see word pairs on the computer screen, one pair at a time, and for each pair were supposed to rate on a 5-point scale how related they thought the two words were. Following the experiments by Murnane and collaborators (Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995; Murnane et al. 1999), we presented word pairs at study, instead of single words, to reduce interitem associations across study trials and cross-context encoding of items. For each trial, two words were randomly drawn from the pool of 96 nouns. Participants were not informed of the upcoming memory test. The purpose of the relatedness judgment task was to assure that participants focused their attention on and processed the items on the study list. A practice list of 8 word pairs was presented first. Participants were then presented with a study list of 32 word pairs. The two members of each word pair on the practice and study lists were presented side-by-side. Half of the participants of each age group studied the word pairs in Context A, and the other half studied them in Context B. Participants saw each word pair for 5 s, then the words disappeared and the sentence "Please enter your relatedness judgment now" appeared in the middle of the computer screen. This sentence served as a signal for participants to rate the relatedness of the word pair by pressing the number keys 1 through 5 on the computer keyboard (with 1 indicating least related, and 5 indicating most related). The next word pair appeared immediately after a response had been entered for the previous one or 5 s after the disappearance of the previous pair, whichever occurred first. The presentation order of the word pairs was randomized by participant. After completion of the relatedness judgments, participants were informed of the upcoming recognition test. For this test, the computer keys "D" and "K" were labeled with stickers "YES" and "NO." The assignment of computer keys to response options was counterbalanced across participants. Participants were instructed to use the digits of their right and left hands to hit the two response keys. They were furthermore instructed to hit the "YES" key if the test item had previously been presented on the study list and to hit the "NO" key if the test item had not appeared on the study list. Participants were asked to respond as fast and as accurately as possible to each test item. The recognition test was preceded by six practice trials after which participants had the opportunity to ask the experimenter questions. Again, this opportunity was rarely used. The test word disappeared immediately after the participant responded, and the next trial started after an intertrial interval of 150 ms during which a black, blank screen appeared. If a participant did not respond within 5 s after appearance of a test word, the word disappeared, and the next test trial started. Error feedback was not provided. Thirty-two target items and an equal number of distractor items were presented on the recognition test. The order of test-item presentation was randomized by participant. From each of the 32 word pairs that had been presented at study, one item was randomly selected to serve as a target item in the recognition test, with the constraint that half of the target items had been the left member of a studied word pair, and the other half had been the right member of a pair. For each participant, items from the word pool that had not been presented on the study list served as distractor items in the recognition test. Half of the target items and half of the distractor items were tested in the same context the participant saw at learning; the other half was tested in the other context, that is, a context different from the learning context. The assignment of test items to the two test contexts was randomized by participant. After completion of the memory test, participants received a computerized version of the Gardner-Monge (Gardner and Monge 1977) vocabulary test. IBM-compatible personal computers controlled the screen presentation and response collection for the entire experiment as well as the vocabulary test. After completion of the vocabulary test, participants filled out a paper-and-pencil health-and-education questionnaire and were debriefed. Results and Discussion Table 1 shows HR, FAR, d′s, and context effects for Experiment 1. d′ is the discrimination measure based on signal detection theory (SDT; Green and Swets 1966) and measures an individual's ability to distinguish old from new items. We chose d′ over other available discrimination measures (for a review, see Macmillan and Creelman 1991) because according to global matching theories, the output from memory in response to recognition test items lies along a continuum of familiarity or match strength. SDT makes this same assumption. However, we also analyzed our data with alternative measures of old–new discrimination, namely, HR − FAR, the discrimination measure of two-high threshold theory (Egan 1958; Snodgrass and Corwin 1988) and the nonparametric discrimination measure A′ (Macmillan and Creelman 1990; Pollack and Norman 1964). Analyses with these two alternative measures yielded the same patterns of results as did d′ for both our experiments. For both experiments, we calculated mean d′ scores from individual participant d′s. For the calculation of d′, HR of 1.00 were adjusted to 0.95, and FAR of 0.00 were adjusted to 0.05. This adjustment scheme follows Murnane and colleagues 1999. Another common adjustment scheme is to convert rates of 1.00 and 0.00 to 1 − 1/(2N) and 1/(2N), respectively (Macmillan and Creelman 1991). That is, 16 hits (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HR}\ =\ 1.00\) \end{document}) are converted to 15.5 hits (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HR}\ =\ 0.97\) \end{document}), and 0 false alarms (FAR = 0.00) are converted to 0.5 false alarms (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{FAR}\ =\ 0.03\) \end{document}). Analysis of our data from both experiments with this latter adjustment scheme yielded the same patterns of results that were found with Murnane and collaborators' (1999) adjustment scheme. A context effect occurs when HR, FAR, or d′ in same-context testing is larger than HR, FAR, or d′, respectively, in different-context testing. The magnitude of a context effect is calculated by subtracting different-context scores from same-context scores. Planned comparisons in the form of paired t tests were conducted to test if observed context effects on HR, FAR, and d′ were statistically significant. We chose a conventional alpha level of .05 for all statistical tests reported in this article. According to a priori power analyses, each of the one-tailed t tests we report has a statistical power of .81 to detect a medium effect size (d = .5, see Cohen 1988, and Appendix, Note 2). For young adults, both the expected context effect on HR and the expected context effect on FAR were statistically significant, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(51)\ =\ 1.88,\ p\ =\ .033,\ \mathrm{and}\ t(51)\ =\ 3.31,\ p\ =\ .001\) \end{document}, respectively. As predicted, a reliable d′context effect was not observed. As expected, older adults showed a significant context effect on FAR, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(51)\ =\ 4.17,\ p\ =\ .0005\) \end{document}. The context effect on HR was not significant for this age group. According to our predictions, the older group did not show a higher d′ under same-context test conditions compared with different-context test conditions. Related to the (unexpected) absence of a context effect on HR for this age group, d′ was higher in the different-context condition than the same-context condition. Older adults thus showed an unexpected negative context effect. For young adults, our results replicate those reported by Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995 and Murnane and collaborators (1999). Our results, as well as theirs, indicate that young adults use item information (I) and associated context (C), but not an ensemble of item and context (E) in a recognition task with simple visual contexts. We predicted that with simple contexts, the pattern of context effects would be the same for young and older adults, that is, both groups should show context effects on HR and FAR, and no context effect on d′. This was confirmed by the significant context effect for FAR, and the absence of a positive context effect on d′ in the older age group. The context effect on FAR is predicted by ICE from the use of associated context (C). However, if older adults are using associated context, they should also show a context effect on HR, which was not observed. The issue of associated-context use by older adults was further addressed in Experiment 2. We predicted that in Experiment 1, the second form of context postulated by ICE, namely, the ensemble of item and context, would not be used by either young or older adults. This prediction was confirmed. Use of an ensemble would have resulted in a context effect on d′ (i.e., larger d′ for same than different context), which we did not observe in either age group. These results replicate prior research on young adults regarding the lack of ensemble processing when simple visual context is manipulated (Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995; Murnane et al. 1999) and extend them to older adults. Experiment 2 The purpose of Experiment 2 was two-fold. One purpose was to provide further evidence for the use of associated context by older adults that had been suggested by the context effect on FAR in Experiment 1. The second purpose was to test the hypothesis that older adults have difficulties integrating item and context information into an ensemble. In Experiment 2 we used rich visual contexts (i.e., pictures of scenes) which according to Murnane and collaborators (1999), lead to ensemble formation in young adults. Experiment 2 thus allows a comparison of ensemble formation in young and older adults. The design and procedure of Experiment 2 were similar to those of Experiment 1. Again, participants performed a relatedness-judgment task on word pairs and, subsequently, received a set of old–new recognition tests. Words were tested either in the same context they had been studied in or in a different context. The major difference between the two experiments is that in Experiment 1 items were presented in simple visual contexts (i.e., combinations of foreground color, background color, and screen location), whereas rich visual contexts (i.e., pictures of scenes) were used in Experiment 2. We predicted that with rich visual contexts young adults would use both associated context and ensembles and, therefore, show context effects on HR, FAR, and d′, thus replicating the results of Murnane and collaborators (1999). With regard to older adults, we predicted that they would use associated context, but would have difficulties integrating context information with the to-be-remembered item information into an ensemble. Therefore, older adults were predicted to show context effects on HR and FAR, but not on d′. Method Participants. Fifty-two young adults and 52 older adults participated in Experiment 2. Of the young adults, 40 were undergraduate students at The University of Memphis, and 12 were undergraduate students at The University of North Carolina at Chapel Hill. Twenty of the young participants were men and 32 were women. They were between the ages of 18 and 25 years (mean age 19.8 years), were recruited from introductory psychology courses, and received class credit for their participation. The older adults (17 men and 35 women) were between the ages of 60 and 84 years (mean age 69.4 years), lived in the community, and were recruited through newspaper advertisements and community organizations in the city of Memphis. They received payment for their participation. All participants were native speakers of English. Mean years of education were 13.8 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 1.1\) \end{document}) for young adults and 14.7 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 2.5\) \end{document}) for older adults, a significant difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(102)\ =\ 2.5,\ p\ =\ .014\) \end{document}. The mean performance in raw score units on the 30-point Gardner-Monge (Gardner and Monge 1977) vocabulary test was 12.7 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 3.22\) \end{document}) for young adults and 18.8 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(SD\ =\ 6.17\) \end{document}) for older adults, also a significant difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(102)\ =\ 6.31\) \end{document}, p < .0005. In a near-vision test, all participants were able to read text in 7-point size print. On the health rating scale, 7% of the young adults and 90% of the older adults rated their health as good or excellent. None of the participants rated their health as poor. Health criteria for participation in the experiment were the same as for Experiment 1. The data from 5 older adults were excluded from analyses for the following reasons: (a) One participant did not understand instructions for the relatedness judgment, (b) 1 participant reported that she could not clearly read the words presented in one of the learning contexts, (c) 3 participants did not give a recognition response within the given time limit in four or more of the trials (two in four trials, and one in eight trials), and (d) 1 young participant could not complete the experiment because of technical failure. These participants were replaced by additional participants. Design. Like Experiment 1, Experiment 2 also had a 2 × 2 mixed factorial design with age (young vs. older) as a between-subjects variable, and test context (same vs. different) as a within-subjects variable. Materials. Stimulus items were the same 96 high-frequency nouns that were also used in Experiment 1 (Francis and Kucera 1982). They were presented in 22-point-size lower-case black letters. Four colorful pictures of scenes served as rich visual learning contexts: a living room, an airplane, a bus, and a street. These same pictures served as test contexts in same-context conditions. A picture of a classroom served as the context in all different-context test trials. The pictures had been drawn with Microsoft Paintbrush for Windows. An example is given in Fig. 1. Procedure. Between 1 and 4 participants were tested in each session. Participants were given a consent form and a brief vision test and were directed to individual computer booths. As in Experiment 1, participants were instructed to judge the relatedness of word pairs. A practice list of 8 word pairs preceded a study list of 32 word pairs. The word pairs on the study list were equally divided among four learning contexts such that eight word pairs were presented in each context. Assignment of word pairs to contexts was randomized by participant. The words of each pair appeared on top of each other (with two spaces between the words) within an object in the scene (e.g., on the side of the bus or on the TV in the living room). The word pairs were presented within their picture context for 5 s, after which the sentence "Enter your relatedness judgment" was presented to prompt the relatedness judgment. The next study trial began after participant response or after the prompt had been presented for 5 s. In the incidental single-item, old–new recognition test, the labeling and assignment of computer keys to response options was the same as in Experiment 1. Again, participants were asked to respond to each test item as fast and as accurately as they could. The recognition test was preceded by eight practice trials. The test word disappeared immediately after the participant responded or after 5 s. The next test trial started after an intertrial interval of 2 s filled with a black, blank screen. No error feedback was provided. Thirty-two target items and an equal number of distractor items appeared in random order on the recognition test. Target items were randomly drawn from the word pairs that had appeared on the study list with the constraint that half of the target items had appeared as the first member of a studied word pair, whereas the other half of the target items had been the second member of a pair. Distractor items were new items that had not appeared on the study list. Half of the target items were tested in the same context they had been studied in (same-context test condition); the other half were tested in a new context that the participants had not seen during learning (different-context test condition). Half of the distractor items were assigned randomly and in equal numbers to the four learning contexts (same-context test condition). The remaining distractor items were tested in the new context (different-context test condition). Assignment of items to either same- or different-context test conditions was randomized by participant. Screen presentation and response collection for the entire experiment was controlled by IBM-compatible personal computers. After completion of the experiment, participants took a computerized version of the Gardner-Monge (Gardner and Monge 1977) vocabulary test and a paper-and-pencil health-and-education questionnaire. Results and Discussion HR, FAR, d′, and context effects for Experiment 2 are presented in Table 2 . Planned comparisons were conducted in the form of paired t tests to test if observed context effects on HR, FAR, and d′ were statistically significant. Young adults showed a significant context effect on HR, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(51)\ =\ 4.37\) \end{document}, p < .0005. The FAR context effect was in the predicted direction, but did not reach statistical significance, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(51)\ =\ 1.13,\ p\ =\ .132\) \end{document}. As predicted, a context effect was found in d′, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(51)\ =\ 2.26,\ p\ =\ .014\) \end{document}, in the younger group. Older adults showed a significant context effect on HR, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(51)\ =\ 2.08,\ p\ =\ .021\) \end{document}, as well as on FAR, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(t(51)\ =\ 1.94,\ p\ =\ .029\) \end{document}, confirming our predictions. As also predicted for this age group, there was not a reliable context effect on d′. The pattern of context effects observed for young adults indicates that they use associated-context information (C) as well as ensemble information (E) when rich visual context is manipulated. This pattern of results replicates that found by Murnane and collaborators. (1999) in two experiments that used rich visual contexts in a total of five experimental conditions. Although the context effect on FAR of .03 that we observed in our experiment did not reach significance, it is similar in size to the context effects on FAR reliably found by Murnane and collaborators (1999). These context effects range from .03 to .07 (weighted mean of .05). Older adults in our experiment showed context effects on HR and FAR, but no context effect on d′. These findings indicate that older adults use item information (I) and associated context (C), but have difficulties in using an ensemble of item and context (E) in the recognition task. General Discussion We derived a complex pattern of predictions from the ICE model and, with very few exceptions, the results of the experiments confirmed those predictions. The results of our research support the hypothesis that older adults encode and use context information in recognition, but do not integrate item and context information as well as young adults do. In two experiments, we investigated age differences in environmental-context effects on recognition and interpreted results based on predictions of the ICE model of recognition memory (Murnane et al. 1999). ICE postulates that three kinds of activation (or match) may play a role in recognition: activation of item information (I), activation of context information that is associated with item information (C), and activation of an ensemble or integration of item and context information (E). Context effects on HR, FAR, and d′ are predicted when I, C, and E are activated. Context effects on HR and FAR, but not on d′, are predicted when only I and C are activated. In Experiment 1, with simple visual contexts, patterns of context effects showed that young adults used I and C. Results suggested that older adults may have also used I and C. This suggestion was confirmed in Experiment 2. In this experiment, which employed rich visual contexts, young adults used I, C, and E, whereas older adults used I and C, but not E. In conclusion, older adults encode and use associated context but have difficulties with the integration of item and context information compared with young adults. It is informative to compare our context effects with those observed in prior studies that used similar paradigms with young adults. With simple visual contexts (Experiments 1), we obtained context effects on HR and FAR of .03 and .05, respectively (Experiment 1), for young adults. These effects are comparable to those found in nine conditions of Murnane and collaborators' (1999) experiments that are comparable to our simple-context experiments in terms of type of context, presentation time, orienting task, and number of item presentations (Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995; Murnane et al. 1999). For these conditions, the weighted mean size of context effects on HR and FAR was .06 and .05, respectively. Context effects in this experimental paradigm are usually small, but reliable. The small size of context effects is not surprising given that in both Murnane and collaborators' experiments, as well as in our experiment, only a small part of the overall context was manipulated. That is, only the appearance of the computer screen was manipulated, whereas virtually all other aspects of the context (room, location in room, presence of experimenter, silence, and so forth) remained constant throughout the experiment. With regard to d′, Murnane and collaborators (1999) reported context effects from 5 young adult participant groups that saw rich visual contexts at study and at test. These context effects were statistically significant and ranged between .12 and .21 (weighted mean of .16). In our Experiment 2, we found a comparatively large d′ context effect of .24 for young adults. Thus, we created conditions in this experiment that were very conducive to young adults' use of ensemble information. Yet, under these conditions, our older adults did not show a context effect on d′. The absence of a context effect for older adults with rich contexts corresponds to the absence of context effects that Murnane and collaborators (1999) found with young adults and simple contexts, that is under conditions when young adults do not use an ensemble. The nonsignificant context effect of .03 we found for older adults in Experiment 2 (rich contexts) corresponds very closely to the nonsignificant context effect of .02 found with young adults and simple contexts by Murnane and collaborators (1999) and to the absence of context effects with young adults and simple contexts in six prior experiments by Murnane and Phelps 1993, Murnane and Phelps 1995. Thus, our older adults show a pattern of context effects in Experiment 2 that is predicted when participants do not make use of ensembles. These results provide strong support for the hypothesis that older adults have difficulties integrating item and context information into an ensemble. It is the integration of item and context that allows the discrimination of old and new items in recognition memory, because an ensemble match increases the global match in response to targets but not to distractors. The lack of integration of item and context by older adults thus offers an explanation for age-related differences in recognition memory performance. By using a model that distinguishes between different kinds of context processing in recognition tasks, we can show that a general theory that older adults have difficulties in processing contextual information is likely to be incorrect. Our data suggest that older adults use associated context as effectively as young adults do. This finding concurs with conclusions by a number of other researchers (Light et al. 1992; Naveh-Benjamin and Craik 1995; Vakil et al. 1996). Our data further suggest that the particular problem of older adults is the integration of item and context, a conclusion that was also reached by Park and colleagues 1990 and by A. D. Smith and colleagues 1998 in experiments in which recall paradigms were used. We have noted that the mechanisms specified in the ICE theory are based on familiarity processes alone, not on recollective processes. We have to consider the possibility that our participants drew on recollective processes in addition to familiarity-based processes when making recognition judgments. In recall tasks, which are based on recollective processes, context effects are found with great consistency regardless of the kinds of contexts used (see S. M. Smith 1988, for a review). Thus, if recollective processes played a large role in our recognition task, context effects on d′ would have been expected in the simple-context experiment (Experiment 1). No such context effects are predicted by the familiarity-based ICE theory. Context effects were not found in either our simple-context experiment or numerous simple-context experiments by Murnane and collaborators (Murnane and Phelps 1993, Murnane and Phelps 1994, Murnane and Phelps 1995; Murnane et al. 1999). Thus, although we cannot exclude the possibility that participants used recollective in addition to familiarity-based processes in our tasks, the pattern of results with regard to context effects can be explained with familiarity-based processes alone. Some researchers in the area of memory and aging have suggested that recollective processes are impaired in older adults, whereas familiarity-based processes remain intact (e.g., Jennings and Jacoby 1993, Jennings and Jacoby 1997). The age differences we found in Experiment 2 (rich contexts), however, are explained by a familiarity-based recognition model. We, therefore, conclude that older adults can be disadvantaged in familiarity-based processes if these processes are based on information that requires integration on the part of the participant. Our results are in line with those of the many studies reporting age differences in source memory. Performance on most source-memory tasks requires the integration of item and context information. So does the formation of an ensemble. Other authors have argued that older adults' performance on source-memory tasks is impaired, because such tasks require explicit context retrieval in a direct test of context (Light et al. 1992; Naveh-Benjamin and Craik 1995; Vakil et al. 1996). In our experiments, context was tested in an indirect manner, and age differences did appear that can be explained by differences in ensemble formation. The crucial variable determining if age differences are found is thus not the directness or indirectness of context tests. The crucial variable appears to be the necessity of integration of item and context information. Although the focus of our study was to reveal age differences in basic memory processes, our findings also have practical implications. If older adults' problem with episodic-memory tasks lies in a lack of integration of item and context information, the training of such integration might alleviate some of the everyday memory problems encountered by persons of advanced age. Our study has several limitations that have to be addressed in future research. Although the ICE theory is a general theory of context-dependent recognition and encompasses environmental as well as semantic context, the theory has thus far only been tested with environmental contexts. It remains to be examined if in a paradigm that employs semantic rather than environmental contexts, older adults also show problems of item-context integration. Hess 1984 found context effects for young and older adults when items were presented in semantically related contexts, but context effects for young adults only when items were presented in semantically unrelated contexts. These findings suggest that older adults' integration of item and context information is facilitated by semantic relatedness, a hypothesis that can be tested with greater precision using the ICE model. Future research should also address whether conditions can be created under which older adults are more likely than in our present paradigm to integrate item and environmental context information into an ensemble. Last but not least, future research needs to speak to possible explanations for the observed age-related impairment in item-context integration. Our results are in line with a reduced-processing-resource account of cognitive aging (Craik and Byrd 1982), which postulates that age differences in episodic-memory (and other cognitive tasks) can be attributed to age-related differences in working memory or processing speed (Light 1991; Park et al. 1996; Salthouse 1996). The formation of an ensemble at encoding and retrieval may require additional processing resources that may not be available to older adults. Therefore, these adults base their recognition judgments on item (I) and associated context (C) alone. Younger adults might have more processing resources in the form of higher working-memory capacity or higher processing speed, allowing them to form and use an ensemble in addition to item and associated-context information. It remains to be determined if individual differences in older adults' ensemble formation can be explained by differences in measures of processing resources. Our research offers an explanation for age-related differences in recognition memory derived from ICE, a model of recognition memory that emphasizes integrative and nonintegrative processing of contextual information. As indexed by their use of associated context, older adults engaged in nonintegrative processing of contextual information as effectively as young adults did. However, as indicated by their deficit in ensemble formation compared with that of young adults, older adults showed difficulties in self-initiated integration of item and context information. Older adults appear to have a specific, rather than a general, deficit in processing contextual information. Their difficulty with ensemble formation may explain why older adults tend to show poorer performance on recognition-memory tasks than young adults. Note 1. ICE can predict present or absent context effects on d′ when only item and associated-context information contribute to the global match. Which prediction is made depends on properties of the activation function used to combine item and associated-context match (i.e., whether or not the effect of associated-context match on the global match depends on the magnitude of the item match with which it is combined) and on the magnitude of match-mismatch differences achieved in a particular experimental paradigm. See Murnane and collaborators (1999) for a more thorough discussion of this issue and evidence that associated context is not likely to produce d′ context effects in the experimental paradigms used in this research. Note 2. The power analyses reported in this article were computed by means of the GPOWER program (Faul and Erdfelder 1992; see Erdfelder, Faul, and Buchner 1996). Table 1. Hit Rates (HR), False Alarm Rates (FAR), d′, and Context Effects for Young and Older Adults in Experiment 1 Same context Different context Context effect M SD M SD M SD Young adults HR .86 .11 .83 .10 .03 .12 FAR .23 .16 .18 .13 .05 .11 d′ 1.97 .61 1.99 .60 −.02 .52 Older adults HR .73 .17 .72 .18 .01 .17 FAR .29 .17 .22 .15 .07 .12 d′ 1.29 .64 1.52 .48 −.23 .63 Note: n = 52 for both young and older adults. Table 2. Hit Rates (HR), False Alarm Rates (FAR), d′, and Context Effects for Young and Older Adults in Experiment 2 Same context Different context Context effect M SD M SD M SD Young adults HR .84 .12 .76 .14 .08 .13 FAR .19 .17 .16 .12 .03 .17 d′ 2.06 .69 1.82 .71 .24 .75 Older adults HR .72 .20 .68 .22 .04 .15 FAR .31 .19 .27 .20 .04 .15 d′ 1.24 .62 1.21 .51 .03 .59 Note: n = 52 for both young and older adults. Figure 1. Example of a rich visual context used in Experiment 2. The research reported in this article was supported by a Junior Faculty Development Award, a University Research Council Grant, and a Cognitive Science Research Seed Grant from The University of North Carolina at Chapel Hill, as well as a Project Grant from the Institute on Aging of The University of North Carolina System. Preparation of this article was supported in part by Research Grant AG17456 from the National Institute on Aging. Parts of this research were presented at the 38th Annual Meeting of the Psychonomic Society, Philadelphia, November 1997. We thank Andreas Enneking, Destiny Shellhammer, Alicia Hill, Jill Zukerman, Erica Snider, Jamie Newman, Victoria Pomeroy, and Richard Carroll for assistance with participant recruitment and data collection, and appreciate the help of the Chatham County and Chapel Hill Senior Centers. We thank Kevin Murnane for many discussions of the ICE model. Bayen U. J., Murnane K., 1996. 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Aging, Neuropsychology, and Cognition 1:30-36. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A0498C99B46F843C42F5B958D8446771E22F144.txt b/test/dataset/in/resources/corpus/Clean_0A0498C99B46F843C42F5B958D8446771E22F144.txt new file mode 100644 index 0000000..9ead8de --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A0498C99B46F843C42F5B958D8446771E22F144.txt @@ -0,0 +1 @@ +intimmintimmInternational Immunology1460-23770953-8178Oxford University Press10.1093/intimm/dxn129ORIGINAL RESEARCH PAPERSElevated non-specific immunity and normal Listeria clearance in young and old vitamin D receptor knockout miceBruceDanny1WhitcombJames P.2AugustAvery1McDowellMary Ann2CantornaMargherita T.11Department of Veterinary and Biomedical Science, Center for Immunology and Infectious Disease, Pathobiology Graduate Program, Pennsylvania State University, University Park, PA 16802, USA2Eck Institute for Global Health and Infectious Diseases, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USACorrespondence to: M. T. Cantorna; E-mail: mxc69@psu.eduTransmitting editor: S. M. Hedrick2200915122008212113122317200812112008© The Japanese Society for Immunology. 2008. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org20091,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and the vitamin D receptor (VDR) are important regulators of autoimmunity. The effect of the VDR on the ability of mice to fight a primary or secondary infection has not been determined. Young and old VDR knockout (KO) mice were able to clear both primary and secondary infections with Listeria monocytogenes. However, the kinetics of clearance was somewhat delayed in the absence of the VDR. Memory T cell development was not different in young VDR KO and wild-type (WT) mice; however, old VDR KO mice had significantly less memory T cells than their WT counterparts but still mounted an adequate immune response as determined by the complete clearance of L. monocytogenes. Although the primary and secondary immune responses were largely intact in the VDR KO mice, the old VDR KO mice had increased cytokines and antibody responses compared with the old WT mice. In particular, old VDR KO mice had elevated antigen non-specific antibodies; however, these magnified immune responses did not correspond to more effective Listeria clearance. The increased antibody and cytokine responses in the old VDR KO mice are consistent with the increased susceptibility of these mice to autoimmunity.agingantibodies1,25-dihydroxyvitamin D3Listeria monocytogenesIntroductionThe active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to have an important role in regulating the immune system. The effects of vitamin D can be observed in many cells of the immune system and the vitamin D receptor (VDR) is found in myeloid and lymphoid cells in resting and activated states (1). It has been shown that 1,25(OH)2D3 suppresses T cell proliferation and decreases the production of the Th1 cytokines IFN-γ, IL-2 and tumor necrosis factor (TNF)-α (2, 3). Th1-driven autoimmune diseases including experimental multiple sclerosis and experimental inflammatory bowel disease are suppressed by in vivo treatment with 1,25(OH)2D3 (4, 5) and 1,25(OH)2D3 inhibits the listeriacidal activity and oxidative burst of IFN-γ-stimulated macrophage in vitro (6), indicating that 1,25(OH)2D3 is an important regulator of Th1-mediated immune responses.In the absence of the VDR, the immune system develops normal numbers and subsets of T cells and B cells. NKT cell development, however, is blocked in the VDR knockout (KO) mice (7). VDR KO mice have heightened IFN-γ-mediated immune responses and decreased induction of Th2 response. For example, VDR KO mice are more susceptible to experimental inflammatory bowel disease, have heightened IFN-γ responses and decreased IL-4 responses and are more resistant to the Th2 disease experimental allergic asthma (8, 9). VDR KO mice develop larger granulomas when infected with Shistosoma mansoni [Th2 dominated, (8)] and have decreased Leishmania major (Th1 dominated) parasite burdens compared with wild-type (WT) controls (10). However, in vitro killing of Listeria by infected macrophages is not affected by deletion of the VDR (6). The VDR KO mice thus have heightened IFN-γ-mediated immune responses and decreased induction of Th2 responses.Listeria monocytogenes is a gram-positive intracellular bacterium that causes infections when ingested in contaminated food. This bacterium is commonly used as a model for studying immune responses to intracellular pathogens. IFN-γ, IL-12 and CD8 T cells are critical for clearance of L. monocytogenes infections (11). Both MHC class I and class II are required for the response to Listeria since deletion of either gene leads to more severe infection, reduced IFN-γ production and increased severity of granulomatous lesions (12). While IL-10 is required for the immune response to Listeria, reduction in this cytokine early during infection leads to more rapid clearance of the bacteria (13, 14). CD8+ T cell memory is reduced in the absence of IL-10, accompanied by increased susceptibility to secondary infections, supporting an important role for IL-10 in the response to this disease (15). During secondary L. monocytogenes infection, CD8+/CD44high T cells are responsible for early onset of specific immune responses (16). IFN-γ is required for sterilizing immunity to secondary infection and this is partially dependent on IL-12 production, and while IFN-γ-deficient mice are extremely susceptible to primary infections, they can produce CD8+ T cell-mediated responses during secondary infection (17, 18). In addition, IL-12 deficiency leads to decreased effector responses during primary infection and increased memory following immunization (17). Protection from L. monocytogenes is dependent on classical Th1-driven cytokine responses with induction of memory CD8 T cells for sterilizing immunity.Aging results in a decrease in antigen-specific immunity to new antigens. The elderly are more susceptible to infectious diseases including L. monocytogenes infection due to a decline in their ability to specifically generate anti-bacterial immunity (19). Increased susceptibility to infections in the elderly has been attributed to reduced T cell proliferation and decreased IL-2 production (20, 21). The number of mature splenic B cells also decreases with age, and reduced titers of antibodies are made following infection (21, 22). Although the aged immune response is reduced in response to new antigenic challenges, there is an increase in self-reactivity (23).As 1,25(OH)2D3, and in particular the VDR, is an important regulator of Th1-mediated immune responses, we have determined the role of the VDR on the ability of mice to mount an immune response to L. monocytogenes. Our experiments show that young VDR KO mice exhibited a lag in the clearance of L. monocytogenes during primary challenge of the mice. The lag in clearance occurred early post-infection when the VDR KO mice produced significantly higher levels of IL-10 and IFN-γ compared with WT mice. Memory CD8 and CD4 T cell responses, as well as antigen-specific IgG1 and IgG2c developed normally in the young VDR KO and WT mice. The aged VDR KO mouse also effectively cleared L. monocytogenes from the spleen with largely the same kinetics of the aged WT mouse. However, following a primary infection, aged VDR KO mice had slower kinetics of clearance in the liver compared with WT mice. By contrast, antigen-specific as well as total IgG1 and IgG2c were significantly higher following secondary infection in aged VDR KO mice. The aged VDR KO mice produced more IFN-γ despite the fact that the number of memory CD4 and CD8 T cells producing IFN-γ were lower in the mice compared with WT. These results indicate that expression of the VDR is not required for host resistance to L. monocytogenes in either young or old mice. Instead, old VDR KO mice have heightened cytokine and antibody responses that did not correlate with the ability to clear the infection.Materials and methodsMiceFemale C57BL/6 (WT) and VDR KO mice were bred and maintained at the Pennsylvania State University (University Park, PA, USA). The mice were 2 months (young) or 19 months (old) at the initiation of the experiments. All experimental procedures were approved by the Office of Research Protection's Institutional Animal Care and Use Committee (Pennsylvania State University).Listeria monocytogenes and immunizationTransgenic recombinant L. monocytogenes expressing ovalbumin (OVA) [gift from Hao Shen, University of Pennsylvania, PA, USA (15)] was incubated overnight in blood heart infusion (BHI) broth. For primary infections, mice received 5 × 106 colony-forming unit (CFU) intra-peritoneal injections of Listeria and were euthanized 2, 4 and 10 days later. For secondary challenge, mice received a low dose of 1 × 104 CFU for primary exposure, and then 30 days later were challenged with 5 × 106 CFU and euthanized 1, 2 and 4 days later.Assessment of bacterial burdenSpleens and livers of infected animals were harvested and weighed and the tissue was homogenized in 1 ml sterile PBS. CFUs were determined by plating 10-fold dilutions on BHI agar plates and the colonies were counted 48–72 h later.Flow cytometrySplenocytes were collected and after lysis of the RBCs were stimulated 10–12 h with phorbol 12-myristate 13-acetate (0.1 μg ml−1) and ionomycin (0.5 μg ml−1); brefeldin A (10 μg ml−1) was added for the final 6 h. Cells were stained with anti-CD44 (PE), anti-CD4 (ECD) and anti-CD8 (PE–Cy5), fixed with 4% PFA, permeabilized with 0.1% saponin and stained with anti-IFN-γ (FITC), all from BD PharMingen (San Diego, CA, USA). Analyses were performed on a FC500 bench top cytometer (Beckman Coulter, Miami, FL, USA). Data were evaluated with WinMDI 2.9 software (Scripps Institute, La Jolla, CA, USA).Cytokine productionSplenocytes (5 × 106) were incubated 72 h in RPMI-1640 complete media alone or with additional stimulation with OVA (100 μg ml−1) and supernatants were collected 72 h later. ELISAs were performed for cytokines IFN-γ, IL-10, IL-12 and TNF-α (kits purchased from BD Biosciences). The limits of detection were 40 pg ml−1 IFN-γ, 30 pg ml−1 IL-10, 35 pg ml−1 IL-12 and 100 pg ml−1 TNF-α.Antibody titersSerum from infected mice was serially diluted and incubated in 96-well plates coated with 1 × 106 Listeria for 2 h. Plates were washed three times, probed with HRP-conjugated anti-IgG2c or anti-IgG1 (BD PharMingen) followed by 3.3′,5.5′-tetramethylbenzidine substrate (BD Biosciences) and analyzed on a HTS 7000 BioAssay Reader (PerkinElmer, Norwalk, CT, USA).Statistical analysisResults are expressed as the mean ± standard error of the mean. Experiments in young mice were performed twice with four mice per group and at each time point. Representative data from one experiment are shown. Experiments in old mice were performed once due to the high costs associated with housing mice for 19 months. Analysis was performed using unpaired t-tests and analysis of variances (Prism 4, GraphPad software, San Diego, CA, USA). Statistical significance is considered to be a value of P ≤0.05.ResultsListeria monocytogenes is cleared from young VDR KO mice following both primary and secondary infectionsThe clearance of L. monocytogenes was tracked in the spleen and liver of young VDR KO and WT mice. Except for day 2, post-infection weights of the spleen and liver of VDR KO and WT mice following primary infection were not significantly different (Supplementary Table S1, available at International Immunology Online). Two days following primary infection, WT and VDR KO mice had equal numbers of bacteria in the spleen (even when the organ weights are taken into account, Supplementary Table S1, available at International Immunology Online, and Fig. 1A). The kinetics of bacterial clearance in the spleen was faster in the WT compared with the VDR KO mice (Fig. 1A). In the liver, VDR KO mice had more Listeria than WT mice at day 4 (Fig. 1A). All VDR KO and WT mice had completely cleared the bacteria in the liver by day 10 post-infection and both groups of mice completely cleared the primary infection by day 14 post-infection (data not shown).Fig. 1.Bacterial CFU in the spleen and liver of young WT and VDR KO mice following primary and secondary infections with Listeria monocytogenes. (A) CFU in the spleen and liver following primary infection with 5 × 106 L. monocytogenes. (B) CFU in the spleen and liver following primary infection with 1 × 104 L. monocytogenes and secondary infection 30 days later with 5 × 106 L. monocytogenes. Values are mean ± standard error of the mean of four individual mice at each time point and one of two representative experiments. VDR KO value is significantly different than the corresponding WT value, *P ≤ 0.05, **P ≤ 0.001.To determine if there were any differences in the recall response, young WT and VDR KO mice were infected with 104 Listeria organisms and then re-challenged 30 days later with 5 × 106 L. monocytogenes. The spleens of the VDR KO mice were significantly heavier at 1 and 2 days post-infection than their WT counterparts (Supplementary Table S1, available at International Immunology Online). The liver was also heavier at day 2 post-infection (Supplementary Table S1, available at International Immunology Online). As expected, clearance of the secondary challenge was faster than clearance of the primary infection in the WT mice (compare Fig. 1A and B). However, as in the primary infection, the kinetics of the response in VDR KO mice was delayed, with more L. monocytogenes recoverable from the spleen (significant) and liver (not significant) of VDR KO mice at 2 days post-infection than WT mice (Fig. 1B). Since the bacterial numbers are calculated per gram of tissue and as noted the spleen and liver of the VDR KO mice were larger than the WT at day 2 post-infection, the differences in bacterial burden at this time point between WT and VDR KO mice are amplified.Cytokine and antibody responses of young VDR KO and WT mice following primary and secondary infection with L. monocytogenesTo examine the cytokine response, splenocytes were removed from VDR KO and WT mice cultured alone or re-stimulated in vitro with OVA, following a primary infection with L. monocytogenes, since the bacteria carry a transgene expressing OVA (15). In the absence of re-stimulation, splenocytes produced only low levels of cytokines in all groups (data not shown). OVA stimulation of naive splenocytes did not induce cytokine production (data not shown). Because the spleens of both the WT and VDR KO mice still contained Listeria, the stimulation with OVA was in effect done in the presence of Listeria, therefore inducing production of cytokines from innate immune cells and early T cell responses. Stimulation of the VDR KO and WT splenocytes with OVA induced the same amounts of IFN-γ at all time points except day 4, when VDR KO splenocytes produced significantly higher amounts of IFN-γ than those from WT mice (data not shown for days 2 and 10; Fig. 2A). These early differences may reflect the higher numbers of Listeria present in the VDR KO spleen at these time points. Serum IFN-γ levels were also measured following a primary Listeria infection. At all time points except day 2 post-infection, the levels of IFN-γ in the serum was below the limits of detection. At day 2 post-infection, WT mice had 1240 ± 64 pgml−1 and VDR KO mice had 1665 ± 306 pgml−1 IFN-γ in the serum, which was not significantly different. Production of IL-12 was similar in VDR KO and WT cultures, while IL-10 production was significantly increased in VDR KO cultures at all time points (Fig. 2A; day 2 and day 10 not shown). By contrast, the kinetics and the amounts of IFN-γ, IL-12 and IL-10 generated by OVA-stimulated splenocytes of young WT and VDR KO mice after secondary challenge were comparable at all time points (Fig. 2B). Low levels of IFN-γ were detected in the sera at day 1 post-secondary infection but were not different between WT and VDR KO mice with Listeria (data not shown). We also determined Listeria-specific IgG1 and IgG2c antibody titers in the sera of young VDR KO and WT mice 4 days after the secondary infection and there was no significant difference in the titers of Listeria-specific IgG1 and IgG2c antibodies between the two groups (Fig. 2C).Fig. 2.Peripheral immune responses following primary and secondary infection of young WT and VDR KO mice with Listeria monocytogenes. (A) IFN-γ, IL-12 and IL-10 production from whole splenocytes following primary infection and in vitro stimulation with OVA at 4 days post-infection. (B) IFN-γ, IL-12 and IL-10 production following secondary L. monocytogenes infection and in vitro stimulation with OVA. (C) Listeria-specific antibody responses in young VDR KO and WT mice following secondary L. monocytogenes infection. Values are mean ± standard error of the mean of four individual mice and one of two representative experiments. IFN-γ and IL-10 values from VDR KO mice were significantly higher than WT mice, P < 0.05.Development of memory CD4 and CD8 T cells in young VDR KO mice and WT miceThe memory T cell responses of young VDR KO and WT mice were measured following secondary infection with L. monocytogenes. Splenocytes from WT and VDR KO mice were stained for CD4, CD8 and the memory marker CD44. There were similar percentages of memory and naive CD4+ and CD8+ T cells in WT and VDR KO mice (Table 1). The percentage of IFN-γ-producing cells was determined in CD8+/CD44high and CD4+/CD44high T cells (Fig. 3). The percentages of IFN-γ-secreting memory CD8+ and CD4+ T cells were similar at all time points post-infection in the VDR KO and WT mice (Fig. 3B).Table 1.Memory T cells in young and old VDR KO and WT miceWT (%)VDR KO (%)YoungCD8+/CD44high50 ± 542 ± 6CD4+/CD44high61 ± 463 ± 5OldCD8+/CD44high26 ± 531 ± 16CD4+/CD44high42 ± 1533 ± 7Values are the mean ± standard error of the mean of n = 4.Fig. 3.IFN-γ secretion by memory T cells in young VDR KO and WT mice after secondary exposure to Listeria monocytogenes. Splenocytes were stained for IFN-γ, CD8, CD4 and CD44 following 1–4 days post-secondary infection. (A) CD8+/CD44high (top) or CD4+/CD44high (bottom) T cells were gated and the histograms showing staining for IFN-γ-producing cells presented. The solid peak shows the isotype control staining and IFN-γ expression of VDR KO (black line) and WT (gray line). (B) Average percentage of IFN-γ-producing CD8/CD44high (top) and CD4/CD44high (lower) T cells in young VDR KO and WT mice. Values are mean ± standard error of the mean of four individual mice. Experiments were repeated twice.Old VDR KO and WT mice clear L. monocytogenes infectionTo determine if age affects the ability of VDR null mice to clear Listeria infection, 19-month-old female (old) VDR KO and WT mice were used for the next set of infections. Similar experiments were performed where old WT and VDR KO mice were infected with Listeria as done for the young mice. The spleens and livers of old VDR KO mice infected with Listeria weighed significantly more than their old WT counterparts at the early time points (day 2 and day 4, Supplementary Table S2, available at International Immunology Online). The CFUs per gram of spleen were the same in the old VDR KO and WT mice at day 2 and day 4 post-infection. However, old VDR KO mice had spleens that were twice as big as the old WT spleens and therefore had larger bacterial burdens at day 2 and day 4 post-infection (Fig. 4A and Supplementary Table S2, available at International Immunology Online). By day 10 post-infection, old WT and VDR KO mice cleared L. monocytogenes primary infections from the spleen (Fig. 4A). This was in contrast to the young VDR KO and WT mice, where L. monocytogenes was recoverable at day 10 post-primary infection (Fig. 1A). However, old WT and VDR KO mice showed persistent low grade colonization of the liver following a primary infection (Fig. 4A). Further analysis revealed that the liver of the old VDR KO mice was larger and had significantly more L. monocytogenes at day 2 and day 4 following primary infection, but not at day 10, than the old WT mice (Fig. 4A). Old VDR KO and old WT mice were able to completely clear the primary infection from the liver by 21 days post-infection (data not shown).Fig. 4.Bacterial CFU in the spleen and liver of old WT and VDR KO mice following primary and secondary infections with Listeria monocytogenes. (A) CFU in the spleen and liver following primary infection with 5 × 106 L. monocytogenes. (B) CFU in the spleen and liver following primary infection with 1 × 104 L. monocytogenes and secondary infections 30 days later with 5 × 106 L. monocytogenes. Values are mean ± standard error of the mean of three to four individual mice at each time point. VDR KO value is significantly different than the corresponding WT value, *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.As described previously for young mice, additional old VDR KO and WT mice were challenged twice, first with a low dose and then with the challenge dose of L. monocytogenes. We found that in contrast to the results from young mice, secondary challenge of old WT and VDR KO mice did not result in faster clearance of L. monocytogenes compared with the primary infection (compare Fig. 4A and B). There were no differences in the weights of the spleens and livers from VDR KO and WT mice at any time points post-infection (Supplementary Table S2, available at International Immunology Online). The number of L. monocytogenes in the spleen of old mice at day 4 post-secondary infection was not different than at day 4 post-primary infection (Fig. 4). A similar picture was apparent in the livers of old WT mice where the numbers of L. monocytogenes were the same after 1, 2 or 4 days post-secondary and primary infection (Fig. 4). In addition, old VDR KO mice had fewer CFUs in the liver following a secondary Listeria infection compared with the primary infection (Fig. 4). However, there was 100% clearance of L. monocytogenes in the spleens and livers of all old VDR KO and WT mice by 21 days post-infection (data not shown).Old VDR KO mice have heightened cytokine responses compared with old WT miceThe cytokine response of the old mice was determined following culture alone or re-stimulation in vitro with OVA. Cultures not stimulated with OVA produced undetectable amounts of cytokine. In addition to the OVA, all cultures contained Listeria organisms that had not yet been cleared from the spleens of either the WT or the VDR KO mice prior to the day 10 time point. Splenocytes from old WT and VDR KO mice produced similar levels of IFN-γ and IL-10 at all time points following primary infection. However, the IFN-γ response of the old mice (maximum of 6 ng ml−1) was less than that of young mice (maximum of 40 ng ml−1) following a primary L. monocytogenes infection (compare Figs 2A and 5A). At day 2 post-infection, old WT mice had 206 ± 56 pg ml−1 and old VDR KO mice had 176 ± 90 pg ml−1 IFN-γ in the serum, which was not significantly different. At other times post-infection, IFN-γ was undetectable. At 10 days post-infection, the IL-12 response of VDR KO mice remained high while the WT IL-12 response decreased significantly (Fig. 5A).Fig. 5.Cytokine production following primary and secondary infection of old WT and VDR KO mice with Listeria monocytogenes. Old WT and VDR KO mice were evaluated for cytokine production following primary and secondary L. monocytogenes infection. (A) IFN-γ, IL-12 and IL-10 production from whole splenocytes following primary infection and in vitro stimulation with OVA. (B) IFN-γ, IL-12 and IL-10 production following secondary L. monocytogenes infection and in vitro stimulation with OVA. Values are the mean ± standard error of the mean of n = 3–4 individual mice at each time point. VDR KO value is significantly different than the corresponding WT value, *P ≤ 0.05, **P ≤ 0.001.Secondary infection of old WT mice with L. monocytogenes resulted in a blunted IFN-γ response that was also reflected in the IL-12 response (Fig. 5B). IL-10 responses of stimulated splenocytes from old WT mice were extremely variable but also appeared to show little change following secondary challenge (Fig. 5B). The levels of IL-12 and IL-10 were not significantly different between the two groups although at 2 days post-infection, the VDR KO splenocytes trended toward secreting higher levels of these cytokines. Low levels of IFN-γ were detected in the sera at day 1 post-secondary infection but were not different between WT and VDR KO mice with Listeria (data not shown). Old VDR KO mice secreted significantly more IFN-γ at 2 days post-infection than old WT mice (Fig. 5B). In addition, the old VDR KO mice produced more IFN-γ 2 days post-infection than young VDR KO mice (compare Figs 2B and 5B).Old VDR KO mice produce high levels of antibodyAntibody responses following secondary L. monocytogenes infection were measured in the old mice. Consistent with the literature, old WT mice showed a decreased ability to mount a recall response and produced less antigen-specific IgG1 and IgG2c than young WT mice (compare Figs 2C and 6A; P = 0.0002 and P = 0.052, respectively). Similarly, old VDR KO mice produced significantly less IgG1 than young VDR KO mice (compare Figs 2C and 6A; P = 0.0001). However, at all time points tested, old VDR KO mice produced significantly more Listeria-specific IgG1 and IgG2c compared with WT mice (Fig. 6A). To determine if the VDR KO mice had elevated levels of Listeria non-specific antibodies as well, the total amounts of IgG1 and IgG2c were also measured in old VDR KO and WT mice. We found that old VDR KO mice produced 2-fold more total IgG1 and 5-fold more total IgG2c than old WT mice (Fig. 6B).Fig. 6.B cell and T cell memory function in old VDR KO and WT mice following secondary challenge with Listeria monocytogenes. (A) Listeria-specific IgG1 and IgG2c responses following secondary challenge with L. monocytogenes. (B) Total IgG1 and IgG2c in old mice. C) Average percentage of IFN-γ-producing CD8/CD44high and CD4/CD44high T cells in old VDR KO and WT mice. Values are the mean ± standard error of the mean of n = 3–4 individual mice per time point. VDR KO value is significantly different than the corresponding WT value, *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.Memory T cell response in old WT and VDR KO miceAnalysis of the percentage of memory CD4+ and CD8+ T cells (CD8+/CD44high or CD4+/CD44high) in the spleens of old WT and VDR KO mice revealed that there was no significant difference (Table 1). In addition, there were no differences in the number of splenocytes, CD4+ or CD8+ T cells in WT and VDR KO mice (data not shown). However, old WT mice had higher percentages of CD8+ and CD4+ memory T cells that produced IFN-γ compared with old VDR KO mice (Fig. 6C). Old WT mice had higher percentages of IFN-γ-producing CD44high-expressing T cells than their younger counterparts (compare Figs 3B and 6C at all time points except day 4 in CD4+ T cell compartment). Old VDR KO mice had higher percentages than young VDR KO mice early but not later post-infection (compare Figs 3B and 6C).DiscussionVDR KO mice mount a strong primary and secondary immune response to L. monocytogenes infection that is adequate to completely clear infection in both young and old VDR KO mice. However, young VDR KO mice exhibited delayed L. monocytogenes clearance (compared with young WT mice) that was likely the result of higher levels of both IL-10 and IFN-γ early post-infection. IL-10 and IFN-γ have opposing effects on growth and clearance of L. monocytogenes (13, 18, 24). The early secretion of IL-10 (likely due to macrophage or other innate cells) in the VDR KO host likely reduced the efficacy of the IFN-γ response, leading to delayed kinetics in clearing infections in the spleen and liver. Interestingly, the generation of memory T and B cell responses occurred unimpeded in the VDR KO mice and therefore the observed delayed kinetics of clearance in young VDR KO versus young WT mice was less pronounced in the secondary challenge.Acquired immunity decreases with age and although old VDR KO and WT mice took longer than young mice to clear a primary and secondary L. monocytogenes infection, they eventually resolved the infections. The age-related delay in clearance of a primary infection was associated with reduced amounts of IFN-γ secreted by the old mice. The increased secretion of IFN-γ by the VDR KO mice following secondary infection may explain the ability of these mice to clear the infection with kinetics that were more similar to WT mice. In addition, although the VDR KO mice had a lower frequency of memory cells secreting IFN-γ, they were able to secrete significantly more IFN-γ following stimulation, suggesting that the VDR KO memory T cells secreted more IFN-γ per cell.Antibody responses, both antigen specific and non-specific, were higher in old VDR KO than WT mice. However, the high amounts of antibody produced in the old VDR KO mice did not correspond to improvements in host resistance to L. monocytogenes. Overall, the primary and secondary immune response of old VDR KO mice was as effective as that of old WT mice for clearance of L. monocytogenes. The high amounts of cytokines coupled with the large amounts of antibodies produced in the old VDR KO mice suggest that non-specific immune responses and autoimmunity are higher in the absence of the VDR. The heightened immune responses in old VDR KO mice are consistent with the previously described susceptibility of the VDR KO mice to experimental autoimmunity (8, 25).Based on the effects of 1,25(OH)2D3 on Th1-driven immunity and the heightened IFN-γ and IL-12 responses in the VDR KO host, it is surprising that the VDR KO mice cleared Listeria infections with slower kinetics than their WT counterparts. NKT cells have been shown to be important innate immune cells during L. monocytogenes infection; NKT cell-deficient CD1d KO mice exhibit decreased liver IFN-γ production and increased susceptibility to Listeria infection (26). Approximately 30% of the liver mononuclear cells from WT mice are NKT cells, compared with only 6% NKT cells in VDR KO mice (7). In addition, the remaining NKT cells in the VDR KO mice are of an immature phenotype and produce low levels of cytokines including IFN-γ (7). Therefore, VDR KO mice are essentially NKT cell deficient, suggesting that the delayed kinetics of L. monocytogenes clearance following primary infection is likely a result of low amounts of NKT cell produced IFN-γ, which would predominately affect the response in the liver. 1,25(OH)2D3 differentially regulates IFN-γ production depending on the cell type that produces it. Paradoxically, 1,25(OH)2D3 enhances IFN-γ production from NKT cells and inhibits IFN-γ production from Th1 cells (3, 7), indicating a cell type-specific IFN-γ regulation by 1,25(OH)2D3. It appears that early during an immune response, 1,25(OH)2D3 induces IFN-γ production by NKT cells, followed by inhibition of IFN-γ along with other Th1-mediated immune responses.The role of vitamin D, 1,25(OH)2D3 and the VDR in B cell development and function has not been extensively studied. B cells express the VDR depending on how they are stimulated and from where they are recovered (27). 1,25(OH)2D3 has been reported to act as a vaccine adjuvant at mucosal sites and to induce antigen-specific IgA (28, 29). More recently, 1,25(OH)2D3 has been shown to inhibit B cell proliferation in vitro depending on when it is added to the culture (27). In addition, memory B cell and plasma cell generation is inhibited by 1,25(OH)2D3 in cultures of human cells (27). Moreover, 1,25(OH)2D3 has been shown to suppress experimental systemic lupus in the MRL/MpJ mouse that is a model of a B cell-mediated autoimmune disorder (30). While we find that B cells from VDR KO mice generated normal amounts of Listeria-specific antibody in young mice, old VDR KO mice produced significantly higher levels of the IFN-γ-driven IgG2c and IL-4/IL-5-driven IgG1. In addition, there were high amounts of antigen-specific and total antibodies in the old VDR KO mice. Increased amounts of antibodies in old VDR KO mice suggest a role for the VDR and 1,25(OH)2D3 as a negative regulator of B cell antibody responses, and vitamin D may play a role in limiting the development of self-reactivity that occurs with aging.Our data indicate that the expression of the VDR is not required for clearance of L. monocytogenes following either primary or secondary infection, although young VDR KO mice showed delayed kinetics of clearance that corresponded to early production of IL-10. There was a decline in the percentage of memory cells that developed with the age of the mice and VDR KO mice had fewer IFN-γ-producing memory T cells than WT mice. IFN-γ production, however, was higher in cells from old VDR KO mice than old WT mice. Consistent with the increased susceptibility of the VDR KO mice to various autoimmune diseases, old VDR KO mice over-produced IFN-γ, IL-12 and both antigen-specific and non-specific antibodies. 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Immunol.200413538030DelucaHFCantornaMTVitamin D: its role and uses in immunologyFASEB J.2001152579 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A09B7578E5BE7EF5247A2C22CE6D267F465B0C3.txt b/test/dataset/in/resources/corpus/Clean_0A09B7578E5BE7EF5247A2C22CE6D267F465B0C3.txt new file mode 100644 index 0000000..9b32dad --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A09B7578E5BE7EF5247A2C22CE6D267F465B0C3.txt @@ -0,0 +1 @@ +herhealedHealth Education Research1465-36480268-1153Oxford University Press10.1093/her/cyn026ORIGINAL ARTICLESMoving from theory to practice: implementing the Kin KeeperSM Cancer Prevention ModelWilliamsK. P.1*MullanP. B.2TodemD.31Obstetrics, Gynecology and Reproductive Biology, Michigan State University, East Lansing, MI 48824, USA2Medical Education, University of Michigan Medical School, Ann Arbor, MI 48109, USA3Epidemiology, Michigan State University, East Lansing, MI 48824, USA*Correspondence to: K. P. Williams. E-mail: karen.williams@ht.msu.edu420092952008242343356Published by Oxford University Press 2008.2009This paper presents the rationale and findings of a feasibility and process study of the Kin KeeperSM Cancer Prevention Intervention. An observational cohort study design was implemented with African-American women in synergistic female family relationships. Community health workers (CHWs) from two Michigan public health programs recruited women to serve as ‘kin keepers’ who in turn recruited their female family members. In total, 161 kin keepers and female family members were sampled. Trained CHWs led kin keepers and family members in learning about breast cancer. Data methods included baseline and post-training administration of a breast cancer literacy assessment, post-training focus groups and review of personal action plans. To validate the feasibility of the process, a linear mixed-effects regression with 97% power was identified and differences in pre–post scores were detected at 5% significance level. Adjusting for family random effects, breast cancer literacy scores increased for all participants recruited (P-value = 0.0004) suggesting that the process was feasible. Analysis of focus groups and action plans indicated that participants valued the instruction and planned to act upon it. This experience with kin keepers and their families offers encouragement that the theoretical model and its community-based delivery can continue to enhance scholarship dedicated to ameliorating health care disparities.IntroductionThis paper presents the rationale and findings of a feasibility study of the Kin KeeperSM cancer prevention program, which began as a theoretical model positing that African-American female family relationships are synergistic such that empowerment or self-efficacy education for individual women could engage other female family members. Assembling to learn about breast cancer prevention and early detection would empower African-American female family members [1]. In this model, community health workers (CHWs), who serve as a liaison between the family and the health care system and have a relationship of trust with one of the family members, serve as a point of access, recruiting women from their public health program catchment area to serve as ‘kin keepers’. These kin keepers, in turn, identify other female family members. The trained CHWs lead the kin keepers and their families in learning about their individual and familial risks for breast cancer. They also learn about screening guidelines, how to perform a breast self-examination (BSE), what to expect when obtaining clinical breast examinations (CBEs) and mammograms and how to share this education with their female family members. The kin keeper's home serves as the safe learning environment for this education.Thus far, approaches to cancer education, prevention and screening involved using the influences of either the health care provider or community-based programs. Although some of these programs may not have been presented as cancer literacy programs per se, they have promoted health literacy in one form or another. For example, community-based interventions render technical assistance by disseminating knowledge regarding cancer prevention and available services. Some interventions are attached to ongoing programs in the community where employment skills are being offered, fostering self-help that leads to empowerment, which is highly valued in the African-American community [2]. As it relates to the Kin KeeperSM model and specific to this study, empowerment is a process and an outcome [3]. It influences women to consider changing their behavior (a process), including the degree of effort they invest in achieving change and the long-term maintenance of behavioral change [4]. The empowerment that the women experience builds capacity (an outcome), which often extends to other areas of their lives [5]. Effective community-based interventions take a variety of forms and locations, from being housed at a local clinic to forming a partnership with a local community-based organization [6–16]. A common theme of these approaches is the use of African-American women as employees or volunteers of the clinic or program. Programs that work with African-American faith-based organizations, such as churches, are also considered community-based approaches. Researchers found that faith-based organizations and religious practices influence African-American women's decisions about cancer prevention and screening behaviors [17–19]. Some investigators have begun to expand their community-based research by designing multigenerational or life span cancer control programs involving various stakeholders, such as middle school and high school children, churches and established senior citizen programs [20].Another factor that influences cancer prevention behaviors of African-American women is the health care provider. Provider-focused approaches are of particular importance because they address issues of health care or access to health services for African-American women. Researchers have sought to investigate the influence that physicians or other health providers have on decisions by African-American women to engage in cancer prevention behaviors [15, 21–25]. Provider recommendation is probably one of the few accepted and undisputed predictors of mammography screening [26]. It is the strongest predictor of whether older African-American women will get screened [27]. Although provider recommendations are a strong predictor of behaviors related to cancer prevention [28], physicians often forget to discuss prevention in busy clinics providing primary care for chronic disease management [29]. One approach to overcoming this barrier has been to use reminders as cues to action. Such strategies have been effective in increasing breast cancer screening rates by 20% at reasonable costs [29, 30].Culture is a vital component of health communications and often serves as a barrier between African-American women and cancer screening [31–34]. ‘A group's culture influences their knowledge, attitudes, and personal practices, which affects their responses to health information’ [34]. Therefore, health literacy must be understood and addressed in the context of culture and language [35]. ‘Culture values influence the understanding that people bring to the acquisition of new knowledge and skills’ [36]. Understanding the sociocultural basis of variables that influence self-care practice and the influence of culture on language, interventions that are effective and culturally sensitive can be designed and implemented [36]. Friedell et al. [37] found that many individuals with limited literacy, rather than reading about the disease, more often obtained information about cancer from family and others who have had experiences with a late-stage diagnosis. Additionally, research studies have indicated that African-Americans tend to be more oriented towards relationship to kinship [38, 39] and possess a strong oral tradition [40, 41]. As a result, African-American women turn to their family members for guidance on cancer matters. Therefore, education of all family members is essential in order to eliminate the sharing of false information.These community-based programs and provider initiatives have met with varying degrees of success and have provided us with insights into working with these women, who have only recently gained attention as research participants [42–44]. Thus far, we have learned that we must adapt our research to the population we are studying, rather than adopt a one-size-fits-all approach. We have learned that building trusted relationships with the research team makes a difference in our ability to recruit African-American women into our studies [45]. We have also learned that tailoring interventions that integrate cultural aspects of the population being investigated are also effective. On the other hand, we have learned that even effective community-based approaches have limitations. They cast a narrow net, often leaving out women who are not connected with a community-based organization or missing women who are not plugged into the health care system through insurance and regular visits. They are often low-income women who do not have the opportunity to learn about cancer risks or low-cost or free mammograms or how to conduct a BSE. The research that has so far been brought to bear on African-American women and their cancer prevention behaviors has shown us that there is no one way to do things nor is there a quick fix for engaging this population. However, there is a lack of understanding of the role of the African-American family, specifically female family members, in influencing cancer prevention practices among other females in the family.Theoretical framework for Kin KeeperSMThe Kin KeeperSM model follows the human ecological perspective, which views an individual woman, her health and development and the health and development of her family in the context of physical, psychological, social and cultural environments [46, 47]. Environments are often described as nested and are depicted as concentric circles from proximal to distal, including individual, family and community. Environments can enhance or limit the potential for health and positive growth and development. An ecological perspective calls attention to relationships between persons and their family and community and transactions that occur among them. These transactions are dynamic and develop over time. To be successful in reducing cancer disparities and improving health-related behaviors, health promotion and prevention programs must identify and address the individual, family and community factors and the relationships among such factors that influence cultural and lifestyle practices of African-American women [48].Kin KeeperSM uses a conceptual framework based on the premise that the natural ways that African-American women communicate various health messages to females in their families (mother, daughter(s), grandmother(s), sister(s) and aunt(s)) can be used to influence them to engage in cancer prevention and screening behaviors. Building on this generational and cultural behavior, the Kin KeeperSM was developed as a women's health advocacy intervention model that naturally integrates health literacy. It uses various indigenous lay health models along with community development principles of capacity building [49]. The rationale for this model is as follows: (i) women serve as point persons for their own and their families’ health and well-being by making most of the decisions for health care; (ii) African-American female family members have the ability to influence each other to conduct regular BSE, CBE and mammograms at the appropriate time and (iii) when cancer prevention options, such as a chemoprevention trial, are presented in a non-intimidating environment, such as the home, by a trusted person, such as a CHW, who serves as a liaison between the woman and the healthcare system, African-American women will be more likely to be actively engaged in their health. The Kin KeeperSM model makes two assertions: (i) understanding individual and familial cancer risk factors will influence early detection behaviors [5] and (ii) the model helps African-American women make the connection between cancer risk factors, family cancer history and cancer detection behavior. The model helps to explain various dynamic factors—the community, the provider and the family—that influence African-American women to participate in cancer prevention and screening practices [50].The Kin KeeperSM model uses CHWs (also known as lay health advisors, community health workers, indigenous community health workers, community health advisors, lay health home visitors and health aides), who work in community settings and serve as connectors between health care consumers and providers, to promote health among medically underserved groups [51]. They promote healthy living by providing education about how to prevent disease and injury and helping community residents understand and access formal health and human service systems [52]. Using CHWs has been particularly relevant among underserved and older African-American women, who often seek advice from female family members [53, 54]. The literature is also clear that the use of CHWs is effective in connecting hard-to-reach women with health care services [53, 55–58]. Million-Underwood et al. [59] found that when African-Americans were provided with information regarding the benefits of participating in screening and treatment trials, they expressed a great willingness to participate. CHWs facilitate the understanding of the benefits of prevention and early detection. Like the patient navigators who act as patient advocates [60] in the Kin KeeperSM model, CHWs assist women in navigating the health care system for preventive services and provide them with additional resources.Although existing empirical research provides important lessons about cancer control outreach to African-Americans [61, 62], the continuing disparity of breast cancer provides evidence of the need for innovative cancer control models for prevention and early detection. Between 2000 and 2003, African-American women in the United States suffered a 36% higher death rate from breast cancer than Caucasian women [63, 64]. This disparity persists despite African-American women's lower breast cancer incidence rate. The best controllable predictor of a favorable outcome for breast cancer is early stage diagnosis. A major factor associated with earlier diagnosis is use of early detection screening services, including mammography and CBE. To be most effective, services like mammography must be received on a regular, recurring basis at recommended frequencies.Our study combined three forms of scholarship: theory, research integration and application [65]. We conducted an observational cohort study, using the Kin KeeperSM Cancer Prevention Intervention. In this paper, we report the results of the initial implementation of the Kin KeeperSM model and potential implications.MethodsThis study was conducted in partnership with two community-based public health programs in Michigan. In one program, each Medicaid-eligible woman deemed at risk for less favorable pregnancy outcomes was assigned a CHW through Maternal and Infant Support Services (M/ISS). Their participation in the M/ISS can range from 2 months prenatal through 12 months postpartum. The other community-based program, Village Health Worker Program (VHWP), focuses on educating and mobilizing high-risk groups to receive screening for diabetes and cardiovascular disease, using CHWs and the social networking method of hosting house parties [66]. An advantage of working with both public health programs is that women in these programs are accustomed to learning about risk and benefits related to their bodies. Therefore, they could be ready to become more empowered and increase their personal capacity by learning about early breast cancer screening, the types of screenings and the guidelines for age/risk-appropriate screenings. Michigan State University's Institutional Review Board for the study of human subjects approved this study.Research participantsIn this manuscript, we focus on the 161 kin keepers and family members recruited through the efforts of our CHWs. Of these women, 49% were aged ≥40 years and therefore met the American Cancer Society's guidelines for annual mammograms. Further descriptive statistics on the women's socio-demographic characteristics are shown in Table I.Table I.Socio-demographics of participants by public health programVariableHealth programVHWP (within-row %) [within-column %]M/ISS (within-row %) [within-column %]Total [within-column %]Age (years)    <3010 (29) [10]24 (71) [39]34 [21]***    30–3935 (75) [35]12 (26) [19]47 [29]    40–4917 (65) [17]9 (35) [15]26 [16]    ≥5037 (69) [37]17 (32) [27]54 [34]Education    College degree or higher15 (65) [15]8 (35) [13]23 [14]**    Some college28 (58) [29]20 (42) [32]48 [30] High school diploma or General Education Development50 (69) [52]22 (31) [35]72 [45]    Less than high school4 (25) [4]12 (75) [19]16 [10]Income    <$10 00015 (40) [15]23 (61) [40]38 [24]***    $10 000–$19 99943 (75) [44]14 (25) [24]57 [37]    $20 000–$39 99927 (67) [28]13 (33) [22]40 [26]    ≥$40 00013 (62) [13]8 (38) [14]21 [13]Employment status    Full time or part time38 (50) [38]38 (50) [60]76 [47]***    Unemployed or laid off19 (59) [19]13 (41) [21]32 [20]    Retired20 (91) [20]2 (9) [3]22 [14]    Stopped working due to disability4 (40) [4]6 (60) [10]10 [6]    Homemaker8 (73) [8]3 (27) [5]11 [7]    Self-employed10 (91) [10]1 (9) [2]11 [7]Marital status    Married22 (65) [23]12 (35) [19]34 [22]    Single/never married45 (52) [48]41 (48) [65]86 [55]    Separated9 (75) [10]3 (25) [5]12 [8]    Divorced11 (69) [12]5 (31) [8]16 [10]    Widowed7 (79) [7]2 (22) [3]9 [6]Health insurance type    Plan from employer49 (77) [50]15 (23) [25]64 [41]*    Plan purchased directly5 (100) [5]0 (0) [0]5 [3]    Medicare/Medicaid31 (47) [32]35 (53) [59]66 [42]    No coverage13 (59) [13]9 (41) [15]22 [14]Kin keeper or family member    Kin keeper32 (59) [32]22 (41) [37]54 [34]    Family member69 (65) [68]38 (36) [63]107 [66]Ever had self breast exam    Yes98 (67) [100]49 (33) [80]147 [93]***    No0 (0) [0]12 (100) [20]12 [8]Ever had clinical breast exam    Yes92 (65) [96]50 (35) [82]142 [90]***    No4 (27) [4]11 (73) [18]15 [10]Had clinical breast exam last year    Yes84 (69) [87]37 (31) [60]121 [76]***    No13 (34) [13]25 (66) [40]38 [24]Ever had a mammogram    Yes51 (62) [53]31 (38) [50]82 [52]    No46 (60) [47]31 (40) [50]77 [48]Had a mammogram last year    Yes45 (68) [47]21 (32) [34]66 [42]    No51 (55) [53]41 (45) [66]92 [58]Two-sided asymptotic likelihood ratio test: ***P-value <0.001, **P-value <0.01, *P-value <0.05.Community health workersNine CHWs employed by one of two public health programs—the M/ISS or VHWP—served as paid CHWs for the current study. They were selected by their respective supervisor based on their experience as CHWs, their effectiveness with their clients and their willingness to work on this project. These CHWs each had more than 8 years experience, including recruiting their clients for university-based research projects. They all completed 16 hours of Kin KeeperSM Cancer Prevention Intervention training on topics including breast and cervical cancer prevention and early detection and how to implement the Kin KeeperSM model. From her caseload of ∼30 women, each CHW was responsible for enrolling a minimum of five African-American women to serve as kin keepers. This involved arranging two home visits with family members whom the kin keeper had identified, performing two follow-ups with individual family members (mailing a 6-month postcard reminder and conducting a 12-month in-person interview), making appropriate referrals and completing follow-up documents. Although some CHWs failed to meet the recruitment goal, others recruited more than five kin keepers.Kin keeper eligibilityTo be eligible, women had to be African-American, aged ≥18 years and participants in one of the two community health programs. Only after giving informed consent could the public health client be recruited into the study and take on the role of a kin keeper. As a kin keeper she was asked by her CHW to (i) complete a survey identifying the African-American family lineage of her mother and father and paternal and maternal grandparents and (ii) invite adult bloodline female family members (mother, grandmothers, sisters and aunts) to two home visitations to learn about breast cancer prevention and early detection screening. This proved to be a challenge for some CHWs with clients who self-identified as African-American but whose parents or grandparents included a non-African-American. The kin keeper also agreed to assist the CHW, if necessary, in locating family members for the 12-month interviews. The kin keeper received a stipend for her participation and for recruiting her family. A total of 54 women participated as kin keepers.Family criteriaUp to five individual family members, including the kin keeper, were eligible for inclusion in the educational sessions as a family unit. At least two members were to be between ages 35 and 64 and thus eligible for free/low-cost high-risk breast cancer screening programs. For participating as a family unit, the group received a restaurant gift certificate. In addition, each research participant received a gift bag with various breast cancer learning materials. Our sample consisted of 54 families and 161 individuals (see Fig. 1).Fig. 1.Study participant recruitment and setting.Data collectionQuantitative data were collected using three instruments: a socio-demographic questionnaire, a pre/post-breast cancer literacy assessment and a personal action plan. We collected qualitative data through the use of focus groups.Pre/post-breast cancer literacy assessmentThe breast cancer literacy assessment measures a woman's functional understanding of breast cancer [67]. It is a 16-item scale administered in a pre- and post-test format. It uses a response format that combines multiple choice and true/false responses. Its three domains are (i) general cancer awareness, (ii) breast cancer knowledge and (iii) breast cancer prevention and control. The CHW reads each question aloud to the participants, thereby enabling each person to independently complete the assessment regardless of her reading level. The assessment takes about 10 minutes to complete. The instrument formative study has been reported [67]. In this study, we present findings on pre–post test scores only as validation that our implementation process is feasible and effective, as reflected by increases in test scores.Personal action planIn the Kin KeeperSM model, women have an opportunity to establish a personal action plan related to breast cancer screening. Women complete this with the assistance of the CHW and then refer back to it during the 12-month follow-up visit. The CHW keeps a copy, the participant retains a copy and a copy remains in the research office.Focus groupsFollowing the second home visit, we conducted two focus groups, one with five CHWs and the other combining kin keepers and family members in a group of six. This enabled us to elicit exploratory and confirmatory perceptions [68]. Both sets of participants were associated with the M/ISS program. Each CHW was asked to recruit two participants to be in the family focus group session. All focus group participants, including the CHWs, were given a cash stipend for their participation. Funding limitations prevented us from conducting focus groups with the VHWP that was located 160 miles from the M/ISS program.We asked the kin keepers/family members questions focusing on their opinion of the overall program and about the unique features of this program, such as the CHW reading all the materials at the home visits, the personal action plans and their experiences discussing their goals with other members in their family. In the CHWs’ focus group session, we asked them about their perception of how their families received/liked the intervention, what they learned from the research project and things they would change. Each focus group session lasted ∼1 hour. An observer took notes while a professional transcriber recorded all responses and comments. To protect confidentiality, each participant was identified numerically rather than by name in the transcriptions.ImplementationThe Kin KeeperSM educational session took place during family home visits. The CHW and kin keeper together scheduled the first family visit. The second family visit was scheduled by the group during the first visit, when all were in attendance at the kin keeper's home. Both visits were scheduled for evenings or weekends. The second visit occurred 1–3 weeks after the first one. Each visit lasted ∼1.5 hours. Before and after each visit, all family members completed the breast cancer literacy assessment.During the first visit, after each family member completed the assessment, the CHW instructed the family on the benefits of CBE and mammograms, what to expect in the clinic and individual and familial risks. Participants learned how to perform a BSE with the use of breast models. Each person then completed the assessment again. At the second visit, after the assessment, the CHW reinforced key information presented during the first visit and cleared up any misunderstandings. Women were able to practice their BSE technique. At the end of this visit, the CHW assisted individuals in developing a personal action plan.Statistical methodsTo describe the participants, we performed descriptive analyses and used chi-square tests. To establish the feasibility of the Kin KeeperSM Cancer Prevention Intervention and the implementation process, we calculated participant drop-out rates between the first and second home visits. We also analyzed participants’ understanding of key concepts and screening and prevention practices through their responses to the breast cancer literacy assessment. To validate the feasibility of the Kin KeeperSM Cancer Prevention Intervention, we performed a paired t-test of the pre/post-test scores, then controlled for covariates using a random-effects mixed model with the difference in pre- and post-test scores as the dependent variable. Explanatory variables included in the model were the public health program (M/ISS or VHWP) and the woman's kin keeper status (i.e. kin keeper or other family member). Interactions between these explanatory variables were also assessed.Responses from members of the same family are likely to be correlated because two individuals within the same family will have responses that are more similar than two individuals in different families. Ignoring this dependence is apt to yield incorrect standard errors [69]. To address this, we used the principle that similarity in the responses within a family may be conceptualized as due to unobserved random family-level variables. Therefore, a family random-effects term was added to the mean model. This adjusts for heterogeneity of families, which can be due to unmeasured family-level random effects (predispositions).A simple linear mixed-effects model (also referred to as the intercept-only model) to analyze the differenced breast cancer literacy scores denoted as yijklfor a woman l in the family i, participating in the program j and of kin keeper status k can be formulated as yijkl = δ+Fi + εijkl, where δ is the mean score difference, Fi is the random family effect and εijkl is the measurement error. It is assumed that the random effect Fi and the measurement error εijkl are independent and distributed as central normal distributions with variances σF2 and σ2, respectively.The fixed-effects parameter δ captures the overall change over time across all levels of the kin keeper status and the health program. If the null hypothesis δ = 0 is rejected at 5% significance level, we can then conclude that there is some evidence from the data of a difference between the pre- and post-intervention scores with adjustment for the family random effects. The model can also be extended to assess whether the kin keeper status or the health program affects the change. The model is formulated as yijkl = δ0 + βj + γ k + Fi + εijkl. Tests on βs and γs assess the effect of the kin keeper status and the health program on the outcome difference. This model can also examine the interaction between explanatory variables. The estimates of these mixed models are used to compute the posterior power by simulation to show that the study has adequate power to detect differences observed in the data.Qualitative analysisThe focus group transcripts and observation notes were discussed, analyzed and validated by consensus in accordance with grounded qualitative theory [70]. Investigators independently read transcripts and extracted key comments associated with knowledge and experiences with breast cancer prevention and early detection using pragmatic and semantic content analysis [70]. The latter focuses on the meaning of the statement, the former on why something was said. Observer notes were compared with the categories for investigators’ independent coding. Using the grounded theory of analytic process provided a context for understanding the practicality of the Kin KeeperSM model.ResultsResults of the bivariate analyses using the chi-square tests showed that age, education and income were significantly different between women from the two health program groups (see Table I). Similar findings are listed in Table I concerning their self-reported breast examination behaviors.To assess feasibility of the Kin KeeperSM Cancer Prevention Intervention, we calculated the overall participant drop-out rate and found a very low rate of 3%. The drop-out rate for women recruited through the M/ISS health program was 5%, while that for women recruited through the VHWP was 2%. The low drop-out rates suggest that the Kin KeeperSM Cancer Intervention and the implementation process are feasible and capable of retaining participants over time. Results of the two-sample t-tests for the pre/post-test scores show that there was a significant increase in test scores. Detailed results by health program and by kin keeper/family member categories are shown in Table II. The increase in scores across the board suggests that our process was indeed feasible and effective for different categories of women.Table II.Individuals' matched (paired t-test) pre/post-projects breast cancer literacy scoresPaired differencesMean difference in percentage scoreStandard deviationStandard error of the mean95% CI upper95% CI lowerdfP-valueVHWPa (n = 100)4.311.71.1711.9896.63699<0.001M/ISS (n = 60)15.316.12.08211.14619.47959<0.001Kin keepers (n = 54)10.015.22.0745.79514.11353<0.001Family members (n = 106)7. 714.11.3724.94410.386105<0.001Overall (N = 160)8.414.51.1476.17310.702159<0.001CI, confidence interval.aVHWP—Diabetes Cardiovascular ProgramTo validate the result that the Kin KeeperSM Cancer Intervention was feasible, we assessed changes in cancer literacy scores using random-effects mixed models. While results from these models could be construed as assessing program intervention effect, here we use them solely to validate the feasibility of the Kin KeeperSM model. If the women's cancer literacy scores did not increase (while controlling for fixed and random effects), then we would have inferred that the practicality and feasibility of the Kin KeeperSM model were questionable. Results are shown in Table III. As predicted, no significant differences were detected between the cancer literacy scores of kin keepers and family members, nor were there any significant interactions between kin keeper status and community-based public health programs. Controlling for random effects of between-subject correlations, the cumulative improvement in mean scores was 5.9% following the implementation of the Kin KeeperSM model. The post-implementation breast cancer literacy scores are significantly higher than the initial scores at 5% significance level (P-value = 0.0004) when family random effects are accounted for. In the main-effects model, the increase in cancer literacy scores is significant (P-value = 0.0377). While all participating women increased their breast cancer literacy scores over time, women in the M/ISS program appeared to increase their scores more compared with women in the VHWP program. Interaction between the community-based public health program variable and kin keeper status was assessed and found to be non-significant at the 5% level (P-value = 0.6667). The increase in scores across the board suggests that our finding of the Kin KeeperSM Cancer Prevention Intervention as feasible was valid.Table III.Random-effects models: parameter estimates and inferencesEffectsIntercept-only modelMain-effects modelMain-effects with interactionsEstimateStandard errorP-valueEstimateStandard errorP-valueEstimateStandard errorP-valueIntercept0.0590.0150.00040.0590.0150.00040.0940.0270.0009Health program (VHWP)—−0.0650.0370.0377−0.0710.0340.0397Kin keeper status (kin keeper)—0.0220.0200.26130.0120.0320.7132Program × Kin keeper status (kin keeper and VHWP)——0.0180.0410.6667Family random-effect variance (σ2F)0.00800.0027—0.00730.0026—0.0070.003—Error term variance (σ2)0.0140.0020—0.0140.0019—0.0140.002—Power analysisWith use of the intercept-only model, yijkl = δ + Fi + εijkl, there is a power of 97% to detect the difference of 0.059 on the log-scale between the pre- and post-breast cancer scores at the 5% level of significance. The statistical package SAS version 9.1 (SAS Institute, Inc., Cary, NC) was used for fitting mixed-effects models (PROC MIXED) and calculating the non-centrality parameter and the related numbers of degrees of freedom for the power calculation. A P-value of <0.05 was selected to denote statistical significance.Qualitative dataAs was anticipated with the Kin KeeperSM model, women did share their breast cancer screening goals with their family. Examples from the focus group responses corroborate this element of the design. KF103: ‘My sisters and I discussed our goals and that [BSE] was one of our goals:… to check more often since the three of us had a bout with a lump in the breast, … check ourselves much more often than what we did in the past.’ Women in the study particularly appreciated instruction in how to properly perform a BSE. CHW 104: ‘Once we got past the myths of mammograms and all that type of stuff, the education played its part. They were amazed. A lot of them were surprised that they were doing their BSE the wrong way. So they enjoyed that.’ CHW 105: ‘All my families really enjoyed it. We had a ball with all the information and especially the hands-on stuff’.Although women readily included both BSE and CBE in their actions plans, their responses to open-ended questions about the frequency of these examinations suggested their uncertainty about the required frequency as well as the need for a revised form to make it easier to use. Most women (88%) indicated they would conduct a monthly BSE. This health behavior is not dependent on health care access. However, even with two home visits and a cancer literacy score of ≥75 for all participating women, 10% of women who cited conducting a BSE as their goal were confused about how often they should perform a BSE. For example, a few indicated ‘daily’ and two indicated ‘over a 100 times a month’. American Cancer Society guidelines call for 80 of the 161 women to have mammograms annually due to age (≥40 years). Even though 82 women had had a mammogram at some time, only 66 had mammograms in the past year and only 54 were aged ≥40 years, implying that 67.5% of women aged ≥40 years received needed mammograms. The issue of frequency is also raised by the fact that 10% of the 93 women who included mammography in their personal actions plan were confused about how often they should get a mammogram.DiscussionTo teach women about the salient aspects of breast cancer prevention and early detection, we implemented an innovative family-focused intervention, Kin KeeperSM, which builds on the generational and cultural behavior of African-American women [71]. We theorized that the existing ways in which African-American women communicate with other females in their family could be used to empower the family unit to engage in cancer prevention and screening behavior. Using the ability of CHWs to access vulnerable African-American women in their communities, we demonstrated that a dynamic recruitment protocol worked to reach other women in the family who were not connected to the public health program. The successful track record that CHWs have had in public health in building trust with women who have been marginalized and connecting them to health services [72, 73] is why they are an integral part of the Kin KeeperSM model. Women of the same family were willing to attend two meetings to discuss breast health and wellness and their individual and familial risks, as well as learn about screening guidelines. The family setting was a safe one for family discussions about personal goals and familial risks and a source of screening motivation. The low drop-out rates suggest that the Kin KeeperSM Cancer Intervention and the implementation process are feasible and capable of retaining participants over time.The breast cancer literacy assessment baseline scores, compared with post-intervention assessment scores, showed that women increased their functional understanding of breast cancer prevention and early detection. In their personal action plans, most women set attainable goals for themselves. Data from the focus group sessions suggested that women saw value in learning the proper techniques of BSE. The increase in scores across the board suggests that our process was indeed feasible and effective for different categories of women.Currently, the predominant current approaches for cancer education, prevention and screening have focused on using the influences of either the health care provider [26–30, 74] or community-based programs [6–13, 15, 19]. As mentioned, these approaches cast a narrow net, often leaving out women without connections to a community-based organization or missing women without connections to the health care system through insurance or regular visits. Low-income women who do not have the opportunity to learn about cancer risks, low-cost screening services or how to conduct a BSE may be left particularly vulnerable by traditional outreach approaches. By design, the Kin KeeperSM accounts for the diversity that exists within African-American families, such as income and access to health care information [71]. Working with two extremely different public health programs, we demonstrated the implementation of the life span approach and its appeal in a familial setting. As we look toward the expanded utility of Kin KeeperSM as a model for education and recruitment of women into clinical trials and genetic studies, family lineage will be important to identify for familial cancer registries.We observed that some CHWs were challenged by our kin keeper eligibility criteria, which required that parents and grandparents be African-American and that at least two family members who live in the defined community be between ages 35 and 64 years. As a result, data collection was extended by 3 months. Other than redefining the community to include the county, which is considered part of our public health programs’ service area, we retained the criteria. We deliberately focused on the African-American family rather than multiracial families that include African-Americans to gain more insight into the social, economic and heterogeneity among African-Americans that could facilitate development of more tailored screening services.CHWs and program staff alike also cited the design of the personal action plans as something that needed to be revamped. A routine protocol meeting with the CHWs and the research coordinator revealed that the formatting was confusing, leading some women to complete the section on ‘future goals’ and ‘accomplished goals’ in the same setting. In addition, some of the older women were so moved by the intervention that they set goals that clearly were not in line with any guidelines, such as performing a BSE ‘100 times in a month’.LimitationsCommunity-based research methods caution against generalizations or one-size-fits-all recommendations, which could be viewed as a limitation. 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National Center for Primary Care Morehouse School of Medicine; October 200373.SheppardVBWilliamsKPRichardsonJTWomen's priorities for lay health home visitors: implications for eliminating health disparities among underserved womenJ Health Soc Policy2005183193574.MandelblattJSYabroffKREffectiveness of interventions designed to increase mammography use: a meta-analysis of provider-targeted strategiesCancer Epidemiol Biomarkers Prev19998975967 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A1670B30286350C48609FA300538420273A1FFB.txt b/test/dataset/in/resources/corpus/Clean_0A1670B30286350C48609FA300538420273A1FFB.txt new file mode 100644 index 0000000..2813c87 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A1670B30286350C48609FA300538420273A1FFB.txt @@ -0,0 +1 @@ +ageingageingAge and AgeingAge Ageing0002-07291468-2834Oxford University Press10.1093/ageing/afq022afq022Research PaperHandgrip strength as a predictor of functional, psychological and social health. A prospective population-based study among the oldest oldD. G. Taekema et al.Handgrip strength as a predictor of health declineTaekemaDiana G.12GusseklooJacobijn3MaierAndrea B.14WestendorpRudi G. J.14de CraenAnton J. M.141Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, The Netherlands2Department of Geriatrics, Rijnstate Hospital, Arnhem, The Netherlands3Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, The Netherlands4Netherlands Consortium for Healthy Aging, Leiden University Medical Center, Leiden, The NetherlandsAddress correspondence to: D. G. Taekema. Department of Gerontology and Geriatrics, Leiden University Medical Center, C2-R-133, PO Box 9600, 2300 RC Leiden, The Netherlands. Tel: +31 71 526 6640; Fax: +31 71 524 8159. Email: d.g.taekema@lumc.nl52010103201039333133715920092012200929122009© The Author 2010. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org2010Oxford University PressBackground: muscle wasting is associated with a detrimental outcome in older people. Muscle strength measurements could be useful as part of a clinical evaluation of oldest old patients to determine who are most at risk of accelerated decline in the near future.Objective: this study aimed to assess if handgrip strength predicts changes in functional, psychological and social health among oldest old.Design: the Leiden 85-plus Study is a prospective population-based follow-up study.Subjects: five-hundred fifty-five, all aged 85 years at baseline, participated in the study.Methods: handgrip strength was measured with a handgrip strength dynamometer. Functional, psychological and social health were assessed annually. Baseline data on chronic diseases were obtained from the treating physician, pharmacist, electrocardiogram and blood sample analysis.Results: at age 85, lower handgrip strength was correlated with poorer scores in functional, psychological and social health domains (all, P < 0.001). Lower baseline handgrip strength predicted an accelerated decline in activities of daily living (ADL) and cognition (both, P ≤ 0.001), but not in social health (P > 0.30).Conclusion: poor handgrip strength predicts accelerated dependency in ADL and cognitive decline in oldest old. Measuring handgrip strength could be a useful instrument in geriatric practice to identify those oldest old patients at risk for this accelerated decline.Keywordsdisabilityelderlyhandgrip strengthhealthsarcopeniaIntroductionMuscle wasting is a dominant feature of old age and is commonly referred to as sarcopenia. Estimates of the prevalence of sarcopenia range depending on the definition from 18% to over 60% in the general population of the oldest old [1]. Due to a rapid growth of the number of oldest old in our societies, sarcopenia will become a common health problem [1].Muscle wasting is associated with detrimental outcome in the elderly, such as disability and mortality [2]. Several recent cross-sectional studies have shown associations between muscle strength and physical fitness, disability or cognition [3–6]. A number of prospective studies have described the association of handgrip strength and health decline in the elderly, predominantly describing its association with functional disability [7–12] and mortality [13, 14]. A limited number of studies report on the associations between muscle strength and cognition [15–17].All these associations raise the question about the value of muscle strength as a potential predictor of declining health in the oldest old. Muscle strength measurements could be useful as part of a clinical evaluation of the oldest old patients in determining which patients are most at risk of accelerated decline in the near future. Therefore we have studied the impact of muscle weakness on three health domains, functional, psychological and social health. Handgrip strength was used as a proxy for muscle strength in this study [3].MethodsParticipants and proceduresThe Leiden 85-plus Study is a community-based prospective follow-up study of inhabitants of the city of Leiden, The Netherlands. Enrolment took place between 1997 and 1999. All inhabitants, including nursing home residents, who reached the age of 85 were eligible to participate. There were no selection criteria on health or demographic characteristics [18]. In total 599 persons participated, 87% of all eligible inhabitants. At baseline, a research nurse visited participants at their place of residence. During these visits, socio-demographic characteristics including gender, marital status and living situation were recorded, performance tests were conducted, blood samples collected and an electrocardiogram (ECG) was recorded. The medical history was obtained from the general practitioner or nursing home physician. Follow-up visits were performed annually. The Medical Ethical Committee of the Leiden University Medical Center approved the study. All participants gave informed consent. In case of severe cognitive impairment, a guardian gave informed consent.Handgrip strengthAt ages 85 and 89, handgrip strength was measured with a Jamar hand dynamometer (Sammons Preston Inc., Bolingbrook, IL). The participant was asked to stand up and hold the dynamometer in the dominant hand with the arm parallel to the body without squeezing the arm against the body. The width of the handle was adjusted to the size of the hand to make sure that the middle phalanx rested on the inner handle. The participant was allowed to perform one test trial. After this, three trials followed and the best score was used for analysis. Handgrip strength was expressed in kilogrammes (Kg). Only handgrip strength measurements that were assessed as reliable by the research nurse were included in the analysis. The research nurse judged the effort according to the following criteria: refusal of participation, physical impairment, cognitive impairment, inability to follow the instructions and technical difficulties.Items of functional healthCompetence in daily functioning was measured with the Groningen Activity Restriction Scale (GARS) [19]. The GARS is a questionnaire that assesses disabilities in competence in the area of nine basic activities of daily living (ADL) and nine instrumental activities of daily living (IADL). A sum score was calculated separately for ADL and IADL. Hence, each sum score ranged from nine (competent in all activities) to 36 (unable to perform any activity without help). Walking speed was assessed with a standardised 6-m walking test as used in other longitudinal ageing studies [20]. Participants were requested to walk 3 m back and forth as quickly as possible. The use of a walking aid was allowed during this test. The time for the walking test was measured in seconds.Items of psychological healthCognitive functioning was measured with the Mini-Mental State Examination (MMSE) [21]. The 15-item Geriatric Depression Scale (GDS-15) was used as a screening instrument for depression [22]. As the validity and reliability of the GDS-15 may be reduced in subjects with impaired cognitive function, this questionnaire was restricted to those with MMSE scores above 18 points (n = 482) [23, 24].Items of social healthSocial functioning was measured with the Time Spending Pattern questionnaire (TSP). The TSP lists involvement in social and leisure activities leading to a sum score for these activities [25]. The questionnaire consists of 23 items (e.g. bathing a spouse, cycling, gardening, reading a book or watching television) scored from 0 (no activities) to 4 (participating in activity on a daily basis).Feelings of loneliness were annually assessed by the Loneliness Scale [26], an 11-item questionnaire especially developed for use in elderly populations. Scale scores range from 0 (absence of loneliness) to 11 (severe loneliness). The Loneliness Scale was also restricted to those with MMSE scores above 18 points.Potential confoundersData on common chronic diseases were obtained from the general practitioner, pharmacist's records, ECG and blood sample analysis [27].Chronic diseases included stroke, angina pectoris, myocardial infarction, intermittent claudication, peripheral arterial surgery, diabetes mellitus, obstructive pulmonary disease, malignancy and arthritis. Multi-morbidity was defined as the sum score of these somatic diseases.Statistical analysisBaseline cross-sectional associations at age 85 were assessed between tertiles of handgrip strength and items of health, using ANOVA (Analyses of variance). Handgrip strength was ranked and divided into tertiles for men and women separately.The prospective association between handgrip strength and the items of health was analysed with linear mixed models. The flexibility of mixed models makes them the preferred choice for the analysis of repeated-measures data [28]. The used models included estimates for ‘handgrip strength’, ‘time’ and ‘handgrip strength * time’. The estimate for ‘handgrip strength’ indicates the baseline association between handgrip strength and health item scores (presented in Table 3 as ‘baseline difference’). This estimate indicates the change in health items per kilogramme increase in handgrip strength. The estimate for ‘time’ indicates the annual change in performance for those participants with mean handgrip strength levels (presented in Table 3 as ‘annual change’). The estimate for ‘handgrip strength * time’ indicates the accelerated annual decline in health items per kilogramme decrease of handgrip strength at baseline (presented in Table 3 as ‘accelerated decline’). All estimates were adjusted for gender, height, weight and income. Estimates were standardised per kilogramme change of grip strength by using the formula: (individual handgrip strength − mean handgrip strength in study population).SPSS 16.0 for Windows was used for all analyses. P-values < 0.05 were considered statistically significant.ResultsParticipants characteristicsReliable scores for handgrip strength were available for 555 (92.6%) participants at age 85. At baseline, there were 44 non-completed handgrip strength measurements due to refusal to participate (n = 3), physical impairment (n = 17), cognitive impairment (n = 9), inability to follow instructions (n = 5) and other reasons (n = 10). There were 73 (12.9%) participants with an MMSE score ≤ 18 points, being indicative of cognitive impairment. Depressive symptoms (GDS score ≥ 4 points) were present in 114 (20.5%) of the participants. The other baseline characteristics of the study population are shown in Table 1.Table 1Characteristics of participants (n = 555) at baseline (85 years)Men (%)194 (35.0)Widowed (%)314 (56.6)Living arrangements  Independent (%)319 (57.5) Sheltered (%)156 (28.1) Institutionalised (%)80 (14.4)General health  ≥3 chronic diseasesa (%)135 (24.3) Body mass index <20 (%)24 (4.5)Functional health domain (median, ITR)b  ADL disability (points)c10 (9.0–11.0) IADL disability (points)c17 (13.8–22.0) Walking speed (seconds)d11.6 (9.4–14.2)Psychological health domain (median, ITR)  Cognition (MMSE, points)e26 (24–28) Depression (GDS, points)f2 (1–3)Social health domain (median, ITR)  Time spending pattern (points)g48 (43–51) Loneliness (points)h1 (0–2)aChronic diseases included stroke, angina pectoris, myocardial infarction, intermittent claudication, peripheral arterial surgery, diabetes mellitus, obstructive pulmonary disease, malignancy and arthritis.bITR, intertertile range.cGARS, possible scores range from 9 to 36 points (best to worst).d6-m walking test, scores ranged from 4.16 to 76.47 s (best to worst).eMMSE, possible scores range from 0 to 30 points (worst to best).fGDS-15, possible scores range from 0 to 15 points (best to worst).gTSP, possible scores range from 0 to 92 points (worst to best).hde Jong-Gierveld Loneliness Scale, possible scores range from 0 to 11 points (best to worst).Functional, psychological, and social health domainThe cross-sectional analyses at age 85 of functional, psychological and social items of health are shown in Table 2 for each tertile of handgrip strength. Lower handgrip strength was significantly correlated with poorer health item scores at baseline (Table 2, all P ≤ 0.03).Table 2Items of health according to handgrip strength tertiles at age 85DomainHandgrip strengtha Highest tertileMiddle tertileLowest tertileP for trendb34–54 kg men20–33 kg men10–27 kg men21–32 kg women17–20 kg women1–16 kg women n = 194n = 177n = 184Functional health     ADL-disability (points)c10.2 (0.2)11.1 (0.2)14.1 (0.5)<0.001 IADL-disability (points)c21.8 (0.8)18.3 (0.5)23.6 (0.7)<0.001 Walking speed (seconds)d10.9 (0.7)14.8 (0.7)18.8 (1.1)<0.001Psychological health     Cognition (points)e26.3 (0.3)25.4 (0.3)22.3 (0.5)<0.001 Depression (points)f2.4 (0.3)2.4 (0.2)3.0 (0.2)<0.001Social health     Time spending pattern (points)g50.3 (0.5)48.3 (0.5)44.3 (0.5)<0.001 Loneliness (points)h1.6 (0.2)1.5 (0.2)2.1 (0.2)0.03aData presented as mean (SE, standard error). Handgrip strength was ranked and divided into tertiles for men and women separately.bANOVA.cGARS, possible scores range from 9 to 36 points (best to worst).d6-m walking test, scores ranged from 4.16 to 76.47 s (best to worst).eMMSE, possible scores range from 0 to 30 points (worst to best).fGDS-15, possible scores range from 0 to 15 points (best to worst).gTSP, possible scores range from 0 to 92 points (worst to best).hde Jong-Gierveld Loneliness Scale, possible scores range from 0 to 11 points (best to worst).To analyse the prospective association between baseline handgrip strength and changes in the various health domains, we used linear mixed models (Table 3). In line with the cross-sectional results, we confirmed the association between handgrip strength and health item scores at baseline as indicated by ‘baseline difference’. Over time all health items, except loneliness, declined as indicated by the estimate ‘annual change’. Finally, we assessed if lower handgrip strength at baseline predicted an accelerated decline in the health domains as assessed by the estimate ‘accelerated decline’. Where such an estimate is significant, this indicates a predictive relationship in the model. Lower handgrip strength predicted an accelerated decline in ADL-disability in the functional health domain (0.02 points increase in GARS score per kilogramme loss of handgrip strength, P ≤ 0.001) and cognition in the psychological health domain (0.01 points decline in MMSE score per kilogramme loss of handgrip strength, P = 0.001), but not in other items of health (all P > 0.30). Additional adjustments for baseline MMSE and GDS scores did not change the prospective results of the functional health items. The prospective results of the psychological health items did not change after adjustment for baseline scores of ADL disability, IADL disability and walking speed. The results of the social health items were not influenced by adjustment for baseline functional health items or baseline psychological items.Table 3Changes in items of health according to handgrip strength at 85 (per kg)aDomainBaseline differenceAnnual changeAccelerated decline Estimate (SE)bP-valueEstimate (SE)bP-valueEstimate (SE)bP-valueFunctional health       ADL-disability (points)c−0.27 (0.04)<0.0011.28 (0.05)<0.001−0.02 (0.01)<0.001 IADL-disability (points)c−0.46 (0.05)<0.0012.25 (0.06)<0.0010.01 (0.01)0.385 Walking speed (seconds)d−0.50 (0.08)<0.0010.35 (0.17)0.040.01 (0.02)0.471Psychological health       Cognition (points)e0.25 (0.04)<0.001−0.75 (0.04)<0.0010.01 (0.004)0.001 Depression (points)f−0.08 (0.02)<0.0010.29 (0.03)<0.0010.002 (0.003)0.626Social health       Time spending pattern (points)g0.40 (0.05)<0.001−1.38 (0.06)<0.001−0.004 (0.01)0.560 Loneliness (points)h−0.05 (0.02)<0.0010.02 (0.02)0.2520.001(0.002)0.968aLinear mixed model adjusted for gender, height, weight, income and multi-morbidity, n = 555. The estimate for ‘baseline difference’ indicates the baseline association between handgrip strength and health item scores. The estimate for ‘annual change’ indicates the annual change in performance for those participants with mean handgrip strength levels. The estimate for ‘accelerated decline’ indicates the accelerated annual change in health items per kilogramme change of handgrip strength at baseline.bSE, standard error.cGARS, possible scores range from 9 to 36 points (best to worst).d6-m walking test, scores ranged from 4.16 to 76.47 s (best to worst).eMMSE, possible scores range from 0 to 30 points (worst to best).fGDS-15, possible scores range from 9 to 36 points (best to worst).gTSP, possible scores range from 0 to 92 points (worst to best).hde Jong-Gierveld Loneliness Scale, possible scores range from 0 to 11 points (best to worst).DiscussionThe aim of the present study was to explore if handgrip strength predicts decline in functional, psychological and social health in the oldest old. Our findings show that lower handgrip strength predicted an accelerated decline in ADL-disability and cognition, and thus contributes to increasing dependency in old age.To our knowledge, our study is the first to report on the prospective associations between handgrip strength and three health domains in a cohort of oldest old participants. A number of other studies have reported on prospective associations between handgrip strength and functional ability, or cognition in the elderly, but the mean age of participants in these prospective studies was younger and none of these included all three health domains [7–12, 15–17]. Some of these studies were limited to men [7, 8] or women [12, 13].We confirmed the predictive value of handgrip strength in the functional health domain in oldest old participants. No predictive association was found between handgrip strength and IADL disability, which had been shown to be a predictor in Japanese community-dwelling elderly in participants aged 65 years and older [9]. For walking speed, we could not confirm a predictive value of handgrip strength, which were associated with each other in a comprehensive cross-sectional study [3].For the association between muscle strength and functional health, one would expect that interventions aimed at improving muscle strength are beneficial. A recent review [29] has assessed the effect of resistance training on physical functioning in subjects over 60 years old. High intensity strength training, three times a week, significantly improved muscle strength, and was associated with improvement in physical ability.Our finding that low handgrip strength predicts accelerated cognitive decline has been reported by others, but again in younger study participants [15–17]. Changes in handgrip strength did not predict changes in depression, possibly because of a process of psychological adaptation during ageing in elderly people [30].In the social health domain, no predictive association was found with the item loneliness. This might be explained by the fact that the need for care results in regular contact with caregivers, thereby stimulating psychological well being as suggested by a cross-sectional Scandinavian study among elderly nursing home residents [31].This study has several key strengths to studying consequences of sarcopenia in elderly people. The Leiden 85-plus Study is a longitudinal population-based cohort study with extensive measures for health and functioning. Therefore the results can be generalised to the western population of oldest old. Furthermore, the longitudinal design with repeated measurements of diverse items of health allowed us to demonstrate a temporal association.A possible weakness of our study could be that our participants appear to be relatively fit. For very frail elderly people, measuring handgrip strength might be difficult and the results could not be applicable to this group. But, only 44 (7.3%) measurements of handgrip strength were excluded from our study because these were deemed unreliable. Of which, 31 (5.2%) were the result of physical or cognitive impairment. We don't know if our participants are fitter compared to other oldest old. Comparison of participant characteristics of the Leiden 85-plus Study with other prospective studies on ageing is difficult because of age differences and different methodology. The Newcastle 85-plus Study started in 2006 [32] and is comparable in design to the Leiden 85-plus Study. The characteristics of the subjects from the Newcastle pilot study are similar to our study participants with regard to living arrangements, cognitive ability and depressive symptoms [33]. Another weakness of the study could be that the questionnaires on depression and loneliness were limited to those participants without cognitive decline which could have underestimated the associations between handgrip strength and the psychological indicators. However, only 73 (13%) of the participants were excluded due to an MMSE score of 18 points or lower. One could also argue that the chosen health domains are indirectly related to one another; however, further adjustment of the linear mixed model for this possible confounding did not change the results.Functional measurements, as walking or gait speed, chair stand test and balance, have also been shown to predict functional limitations of the lower body [12, 20, 34], and cognition [35] in older subjects. As yet it is unclear whether muscle strength or functional measurements are the stronger predictor, and which causal pathways are involved. An advantage of handgrip strength could be that it is easy to use in clinical practice.We conclude that poor handgrip strength is a predictor of accelerated dependency in ADL and cognitive decline in oldest old. Based on these findings, we conclude that measuring handgrip strength could be a useful instrument in geriatric clinical practice to identify those oldest old patients at risk for accelerated decline in ADL ability and cognition.Key pointsPoor handgrip strength predicts accelerated dependency in ADL and cognitive decline in oldest old.Measuring handgrip strength can be useful to identify those oldest old patients at risk for future decline.Handgrip strength measurement is an easy to use instrument in clinical geriatric practice.Conflicts of interestNone.This study was supported by unrestricted grants from the Netherlands Organisation of Scientific Research (ZonMw), the Ministry of Health, Welfare and Sports, the Netherlands Genomics Initiative/Netherlands Organization for scientific research and the Netherlands Consortium for Healthy Aging. (NGI/NWO; 05040202 and 050-060-810 NCHA).References1DohertyTJInvited review: aging and sarcopeniaJ Appl Physiol2003951717272FisherALOf worms and women: sarcopenia and its role in disability and mortalityJ Am Geriatr Soc2004521185903LauretaniFRussoCRBandinelliSAge-associated changes in skeletal muscles and their effect on mobility: an operational diagnosis of sarcopeniaJ Appl Physiol2003951851604JeuneBSkyttheACournilAHandgrip strength among nonagenarians and centenarians in three European regionsJ Gerontol A Biol Sci Med Sci200661707125HasegawaRIslamMMLeeSCThreshold of lower body muscular strength necessary to perform ADL independently in community-dwelling older adultsClin Rehabil200822902106TakataYAnsaiTSohIPhysical fitness and cognitive function in an 85-year-old community-dwelling populationGerontology200854354607GiampaoliSFerrucciLCecchiFHand-grip strength predicts incident disability in non-disabled older menAge Ageing19992828388RantanenTGuralnikJMFoleyDMidlife hand grip strength as a predictor of old age disabilityJAMA1999281558609IshizakiTWatanabeSSuzukiTPredictors for functional decline among nondisabled older Japanese living in a community during a 3-year follow-upJ Am Geriatr Soc20008424910RantanenTAvlundKSuominenHMuscle strength as a predictor of onset of ADL dependence in people aged 75 yearsAging Clin Exp Res20021410511Al SnihSMarkidesKSRayLHandgrip strength and mortality in older Mexican AmericansJ Am Geriatr Soc2002501250612OnderGPenninxBWFerrucciLMeasures of physical performance and risk for progressive and catastrophic disability: results from the Women's Health and Aging StudyJ Gerontol A Biol Sci Med Sci20056074913RantanenTVolpatoSFerrucciLHandgrip strength and cause-specific and total mortality in older disabled women: exploring the mechanismJ Am Geriatr Soc2003516364114Al SnihSMarkidesKSOttenbacherKJHand grip strength and incident ADL disability in elderly Mexican Americans over a seven-year periodAging Clin Exp Res200416481615ChristensenHKortenAEMackinnonAJAre changes in sensory disability, reaction time, and grip strength associated with changes in memory and crystallized Intelligence? A longitudinal analysis in an elderly community sampleGerontology2000462769216Alfaro-AchaAAl SnihSRajiMAHandgrip strength and cognitive decline in older Mexican AmericansJ Gerontol A Biol Sci Med Sci2006618596517BuchmanASWilsonRSBoylePAGrip strength and the risk of incident Alzheimer's diseaseNeuroepidimiology200729667318Bootsma-van der WielAvan ExelEde CraenAJMA high response is not essential to prevent selection bias: results from the Leiden 85-plus StudyJ Clin Epidemiol20025511192519KempenGIMiedemaIOrmelJThe assessment of disability with the Groningen Activity Restriction Scale. Conceptual framework and psychometric propertiesSoc Sci Med19964316011020GuralnikMSimonsickEMFerruciLA short physical performance battery assessing lower extremity function: association with self reported disability and prediction of mortality and nursing home admissionJ Gerontol199449M859421FolsteinMFFolsteinSEMcHughPR“Mini-Mental State”. A practical method for grading the cognitive state of patients for the clinicianJ Psychiatr Res1975121899822YesavageJABrinkTLRoseTLDevelopment and validation of a geriatric depression screening scale: a preliminary reportJ Psychiatr Res198217374923TombaughTNMcIntyreNJThe Mini-Mental State Examination. A comprehensive reviewJ Am Geriatr Soc1992409223524van ExelEde CraenAJRemarqueEJInteraction of atherosclerosis and inflammation in elderly subjects with poor cognitive functionNeurology200361169570125van EijkLActivity and Well-being in the Elderly1997The NetherlandsUniversity of GroningenThesis26de Jong-GierveldJKamphuisFThe development of a Rasch-type loneliness scaleAppl Psychol Measur198592899927Bootsma-van der WielAGusseklooJde CraenAJMCommon chronic diseases and general impairments as determinants of walking disability in the oldest-old populationJ Am Geriatr Soc20025014051028GueorguievaRKrystalJHMove over ANOVA: progress in analyzing repeated-measures data and its reflection in papers published in the Archives of General PsychiatryArch Gen Psychiatry200461310729Liu CJ, Latham NK. Progressive resistance strength training for improving physical function in older adults. Cochrane Database Syst Rev 2009, Issue 330von FaberMBootsma-van der WielAvan ExelESuccessful aging in the oldest old. Who can be characterized as successfully aged?Arch Intern Med2001161269470031DragesetJThe importance of activities of daily living and social contact for loneliness: a survey among residents in nursing homesScand J Caring Sci200418657132AdamsonAJCollertonJDaviesKNutrition in advanced age: dietary assessment in the Newcastle 85+ studyEur J Clin Nutr200963S61833CollertonJBarrassKBondJThe Newcastle 85+ study: biological, clinical and psychosocial factors associated with healthy ageing: study protocolBMC Geriatr200771434VasunilashornSCoppinAKPatelKVUse of the Short Physical Performance Battery Score to predict loss of ability to walk 400 meters: analysis from the in CHIANTI StudyJ Gerontol A Biol Sci Med Sci200964223935DeshpandeNMetterEJBandinelliSGait speed under varied challenges and cognitive decline in older persons: a prospective studyAge Ageing20093850914 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A1715B0CA87E21607FA6D059A503BB7FD04BC30.txt b/test/dataset/in/resources/corpus/Clean_0A1715B0CA87E21607FA6D059A503BB7FD04BC30.txt new file mode 100644 index 0000000..6a6306a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A1715B0CA87E21607FA6D059A503BB7FD04BC30.txt @@ -0,0 +1 @@ +]>HEARES3476S0378-5955(00)00098-810.1016/S0378-5955(00)00098-8Elsevier Science B.V.Fig. 1ABR responses to various intensities of 2 kHz logon bone conducted stimuli delivered to the intact skull, to the skull in the presence of a craniotomy (bone) and directly on the exposed brain. The amplitude calibration bars represent 0.5 μV. Thr.: threshold.Fig. 2The instantaneous acceleration response of skull bone and of brain (craniotomy) to the 2 kHz logon bone conducted stimuli (at 120 dB pe SPL) applied to the skull in the presence of a craniotomy (bone) and directly to the brain (craniotomy). Note that no acceleration could be picked up from the skull when the stimulus was applied on the brain (craniotomy), while clear vibrations of the brain (craniotomy) were recorded in response to bone vibrator-induced vibrations of the skull.Table 1The mean (±S.D.) thresholds, latencies and amplitudes of wave 1 of the ABR to bone vibrator stimuli (logons at 2 and 8 kHz) applied to the intact skull, to skull bone after a small craniotomy had been made and directly to the exposed brain, through the craniotomyLogon 2 kHzIntact skullCraniotomyBoneBrainΔ(bone−brain)Threshold X7084.693.2−8.64±S.D.510.49.08.09P<0.01Latency W1 X1.241.521.380.14±S.D.0.050.080.060.07P<0.001Amplitude W1 X4.962.552.200.35±S.D.1.641.311.590.93NSLogon 8kHzThreshold X7082.786.8−4.09±S.D.4.56.17.27.01NSLatency W1 X1.131.261.190.07±S.D.0.050.110.070.10P<0.05Amplitude W1 X5.023.283.080.21±S.D.1.251.060.740.56NSAlso shown are the differences between these values on skull bone and on brain, along with their statistical (paired t-test) evaluation. ABR wave 1 latencies and amplitudes are to a stimulus intensity (setting on the EP system) of 120 dB pe SPL. There was no difference in ABR threshold to air conducted (earphone) stimulation between pre- and post-craniotomy states.Bone conduction experiments in animals – evidence for a non-osseous mechanismSharonFreemanaJean-YvesSichelbHaimSohmera*sohmer@md2.huji.ac.ilaDepartment of Physiology, Hebrew University, Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, IsraelbDepartment of Otolaryngology/Head and Neck Surgery, Hadassah University Hospital, Jerusalem, Israel*Corresponding author. Tel.: +972 (2) 6758385; Fax: +972 (2) 6439736AbstractBone conducted stimuli are used to differentiate between conductive and sensori-neural hearing loss. It has been thought that the main route for the transfer of vibratory energy from the point of application of the bone vibrator on the skull to the inner ear is completely osseous. An additional mechanism may play a prominent role. In rats, a bone vibrator was applied to the skull and also directly on the brain, after removing bone (a craniotomy), exposing the brain. Auditory nerve-brainstem evoked response (ABR) could be elicited not only with the vibrator on bone, but also with the vibrator directly on the brain. Similar results were obtained in guinea-pigs and fat sand rats. Noise masked this ABR. Extensive removal of skull bone did not alter the ABR to bone-conducted stimuli delivered to the exposed brain. Experimental elimination of the ossicular chain inertial mechanism and of the occlusion effect did not greatly alter the bone conduction response. A reduction in the fluid volume of the cranial cavity induced threshold elevations of the bone conducted ABR but not of the air conducted ABR. These findings can be interpreted as evidence that the ‘classical’ bone conduction mechanisms should be modified to include a major pathway for cochlear excitation which is non-osseous: when a bone vibrator is applied to the skull, the bone vibrations may induce audio-frequency sound pressures in the skull contents (brain and cerebro-spinal fluid) which are then communicated by fluid channels to the fluids of the inner ear.KeywordsBone conductionAuditory nerve-brainstem evoked responseVibratorSkullCerebro-spinal fluidCochlea1IntroductionBone conduction stimulation of the cochlea is a widely used clinical test to differentiate between a conductive hearing loss (where air conducted thresholds are elevated while bone conduction thresholds are normal) and a sensori-neural (inner ear-auditory nerve) hearing loss (both air- and bone-conducted thresholds are elevated to the same extent). Recent animal experiments conducted in this laboratory, along with a review of the literature have led us to the conclusion that even though determination of bone conduction thresholds is an integral part of the clinical test battery for the successful differentiation between conductive and sensori-neural hearing losses, the mechanism of bone conduction is quite complex and requires further clarification. This is particularly true with respect to the exact pathway of vibratory energy transmission from the point of application of the bone vibrator on the skull to the inner ear, which then leads to neural excitation.Major contributions to the mechanism of bone conduction have been made by von Bekesy (1932,1960), Barany (1938), Wever and Lawrence (1954), Kirikae (1959) and Tonndorf (1966,1968), Tonndorf and Tabor (1962). Bekesy and Wever and Lawrence maintain that the primary vibratory energy pathway from the point of contact of the bone vibrator on the skull to the ear is completely osseous; i.e. as transverse waves conducted along the skull bones. Tonndorf has added the possibility that a small amount of energy may be conducted as surface waves along the skin and soft tissues of the head and, in addition, that some energy may pass through the interior of the skull as pressure and/or translation waves and act directly on the cochlea through the so-called ‘third window’, e.g. the cochlear aqueduct (Tonndorf, 1966). Nevertheless Barany (1938) and Schneider (1959) concluded that the skull contents are not involved in bone conduction. These latter pathways have not been evaluated and are considered to be of minor importance.It is thought that the response of the inner ear following the skull and cochlear shell vibrations is initiated by three basic mechanisms, all based on osseous pathways. 1.According to the translatory (or inertial) mode of bone conduction, as the cochlear bony shell is vibrated, there is an inertial lag between cochlear shell vibration and that of the ossicular chain. Thus relative motion is set up between the cochlear shell and the ossicular chain. This mechanism has been emphasized by Barany (1938), by Kirikae (1959) and by Wever and Lawrence (1954). It is thought that this pathway is more important at lower frequencies (Kirikae, 1959; Tonndorf, 1966). An inertial component between the cochlear shell and the cochlear fluids has also been suggested. This induces alternating pressure changes across the basilar membrane, exciting the cochlea.2.In the compressional mode of bone conduction (modified by Tonndorf, 1968 – to the ‘distortional’ mode of bone conduction), the skull vibrations are propagated to the temporal bone and cause distortion of the bony cochlear shell. This causes fluid displacements in and out of the cochlear windows, with basilar membrane displacement and excitation. Kirikae (1959) and Tonndorf (1966) maintain that this is a major pathway at higher frequencies.3.In addition, particularly when the external auditory meatus is blocked, an additional, occlusion mechanism is present, whereby the vibrations of the skull radiate into the occluded meatus, producing aerial waves which then act on the tympanic membrane like any other aerial stimulus.Upon reaching the ear, these waves (surface, transverse and pressure) interact with each other. The final stimulus transferred to the cochlea is a vectorial integration of these pathways of wave energy transfer across and along the skull and its excitation will depend on the vectorial summation of all the modes of bone conduction, depending on their relative magnitudes and phases. It is possible that each of these mechanisms is more effective at different frequencies. The inner ear response is finally initiated by the same transduction mechanism as in air conduction (von Bekesy, 1932,1960). Thus, experimental modification of any one of the vibratory pathways from the point of contact of the stimulating vibrator on the skull to the ear will lead to alterations in cochlear excitation, either increase or decrease, depending on the relative phase and magnitude of the deleted component and hence in the final bone conduction response of the ear.A series of animal experiments conducted in this laboratory has provided evidence that an additional major pathway involved in the transmission of skull-induced vibrations to the inner ear is by a completely non-osseous route. In this mechanism, the skull vibrations (distortions) induce audio-frequency pressure variations in the brain/cerebro-spinal fluid (CSF) which are then transmitted by the fluid communications between the CSF and the inner ear fluids directly to the inner ear. A similar conclusion has been reached from the results of experiments in humans and these are reported separately (Sohmer et al., 2000).2Materials and methodsThese experiments were conducted on rats, guinea pigs and fat sand rats (Psammomys obesus), anesthetized with intraperitoneal injections of pentobarbital (rats and sand rats: 60 mg/kg; guinea pigs: 45 mg/kg). Body temperature was maintained (36.5–37.5°C) throughout the experiment. All experiments were conducted in accordance with the guidelines published by the Hebrew University–Hadassah Medical School Animal Care and Use Committee.The auditory stimuli used consisted of either air conducted (earphone or the bone vibrator suspended in the air over the skull, but not in contact with it) or bone conducted, alternating polarity clicks, or alternating polarity logons at frequencies of 2 and 8 kHz. The different frequency stimuli were used in order to study the possible frequency dependence of bone conduction mechanisms, as suggested by Tonndorf (1966). The bone-conducted stimuli were delivered by means of a standard clinical bone vibrator (Radioear B-71) to which a flat-headed screw was attached with acrylic dental cement in order to reduce the area of contact between the bone vibrator and the small head of the animal. The skin of the scalp was excised, and the scalp muscles were retracted. Thus there was no possibility of transmission of vibrations as surface waves along the skin and soft tissues of the head. In the first three experiments, a 90 g weight was attached to the bone vibrator and it was applied directly to the skull, suspended so that its pressure on the skull was that of its own weight (26 g) and the 90 g weight (116 g). This weight was too large for the experiments involving placement on exposed brain (craniotomies), so that in all the remaining experiments, the bone vibrator was applied to all sites, suspended with its own weight (There was no difference in threshold when the bone vibrator was applied to the skull with a force of 26 or 116 g). Thus in all experiments, the effective transmission of the vibrations to the head of the animals was uniform across animals and the transmission to the exposed brain through the craniotomy was also uniform across animals, though probably somewhat different from that to the skull. In most experiments, the external auditory canals were filled with plasticine in order to induce a conductive hearing loss so that air-conducted stimulation by the bone vibrator was severely reduced.The response of the cochlea to these stimuli under the various experimental conditions was evaluated by recording the auditory nerve-brainstem evoked response (ABR) with needle (Grass) electrodes in the skin, somewhat caudal to the vertex, with respect to the chin. The ground electrode was in a forepaw. Special attention was paid to the threshold of the ABR, defined as the lowest intensity stimulus, from a maximum setting of 120 dB pe SPL to which an ABR response could be recorded (in 5 dB steps). A setting of 120 dB pe SPL on the evoked response system used delivers a bone-conducted click stimulus 40 dB above threshold in rats (Geal-Dor et al., 1993). The latency and amplitude of the first wave of the ABR at 120 dB pe SPL were also evaluated. This wave represents the compound action potential of those auditory nerve fibers synchronously activated by the stimulus. The auditory stimuli and the ABR responses were generated and analyzed by a Microshev 4000 evoked response system.Experiment A: comparison between ABR thresholds elicited with bone vibrator on intact skull, on the skull in the presence of a small craniotomy and directly on the exposed brain in the region of the craniotomy. These experiments were conducted on 11 rats. In each, ABR threshold and the latency and amplitude of the first ABR wave (120 dB pe SPL) were first determined in response to the bone vibrator (using 2 and 8 kHz logon stimuli) applied directly on the intact skull, slightly rostral to the bregma (the intersection of the four main sutures, perpendicular to each other, on the rat skull). A small craniotomy (approximately 1 cm in diameter) was then made caudal to the bregma. Usually the dura was torn during this procedure and there was a loss of some CSF. The ABR threshold, wave 1 amplitude and latency to the bone vibrator on the skull, in the same region as before (caudal to the craniotomy), were again determined, in the presence of this craniotomy. Finally, the bone vibrator was applied directly to the exposed brain and the ABR measurements were repeated. Thresholds were alternatively and repeatedly measured with the bone vibrator on the skull and on the brain. Differences in ABR threshold, amplitude and latency across these experimental conditions were then statistically evaluated (paired t-tests). Similar experiments were conducted on two guinea pigs and on three fat sand rats. In one rat, a Bruel and Kjaer accelerometer (type 4393) was applied to the skull and used to measure the amplitude of the skull vibrations induced by the bone vibrator when it was applied to the skull and when it was applied to the exposed brain (craniotomy). This was repeated in a guinea pig and in addition, the accelerometer was also applied to the exposed brain (craniotomy) and the bone vibrator applied to the skull in order to measure the vibrations of the brain induced by vibration of the skull.Experiment B: the effect of the size of the craniotomy on ABR threshold, amplitude and latency to bone vibrator stimulation directly on the skull. These experiments were conducted on four rats and two guinea pigs. The bone vibrator was always applied to the skull – initially to the intact skull and then a small (2 mm) craniotomy was produced and ABR measurements were made with the bone vibrator applied to the skull. Then the craniotomy was enlarged further in one or two stages, and ABR repeated each time. ABR threshold, amplitude and latency were evaluated at each stage. In several rats, the bone vibrator was applied to the brain also in the presence of craniotomies of various sizes, including maximal removal of skull bone (calvarium).Experiment C: the effect of the presence of a craniotomy on ABR air-conducted thresholds. In eight rats, air-conducted ABR thresholds (in one rat using an earphone and in seven, using the bone vibrator suspended over the head of the rat, but not in contact with the skull) were determined in the absence of a craniotomy (intact skull) and in the presence of craniotomies. In the experiment with the earphone, the external canals were not blocked with plasticine. ABR threshold across these experimental conditions (presence and absence of a craniotomy) was evaluated.Experiment D: the effect of eliminating the ossicular chain inertial component and of the external meatus occlusion mechanism on ABR threshold, amplitude and latency. These experiments were conducted on six fat sand rats (P. obesus) since they have a unique middle-inner ear anatomy, including a large bulla cavity with a thin-walled inner ear so the cochlea and the three semicircular canals bulge into the middle ear cavity, facilitating experimental manipulations. Initially, ABR threshold to bone conduction stimulation was measured in the intact animal. The cochlea on one side was surgically destroyed and the experiment was conducted on the remaining intact ear. Cyanoacrylate glue was then applied to the middle ear cavity, immobilizing the stapes footplate, and cementing (immobilizing) the ossicular chain to the walls of the bulla cavity of the temporal bone. The external canal was filled with plasticine. ABR threshold, amplitude and latency to bone vibrator stimulation (click) on the skull in the intact ear were then compared to that in the same ear following the experimental manipulations of blocking the ossicular chain inertia and filling the external auditory meatus. Finally, a craniotomy was made and ABR responses were recorded with the bone vibrator on the skull and repeated with the bone vibrator directly on the brain. In four of the animals the middle ear fixation procedure was carried out prior to the craniotomy, while in the remaining two animals, it was performed after the craniotomy.Experiment E: the effect of attempts to reduce intracranial fluid volume on bone conduction thresholds. In seven rats with intact skulls (no craniotomy), ABR thresholds to air conducted and bone-conducted 2 kHz logon stimuli were recorded. The air conduction thresholds were recorded at the beginning (control) and at the end of the experiment. The bone conduction thresholds were recorded before (control) and several times after injecting i.p. a bolus of about 2 cc of a 25% (hypertonic) solution (2 g/kg) of mannitol. This induces a reduction of intracranial water content (of the brain and of the CSF) and is therefore used therapeutically to reduce intracranial pressure (Donato et al., 1994; Treib et al., 1998). If there was no effect on bone-conducted thresholds or if the effect was temporary (in most cases), additional boluses were administered, with 30 min between injections. In this way, there were 13 injections of mannitol in these seven rats.3ResultsExperiment A: Fig. 1 shows the ABR recordings in a typical rat in response to several intensities of bone-conducted stimuli (2 kHz logon), from a maximum instrument setting of 120 dB pe SPL, down to threshold. In the left column, the bone vibrator was placed on the (still) intact skull; in the middle column, the vibrator was on skull bone in the presence of a craniotomy and in the right column, the bone vibrator was placed directly on the exposed brain, through the craniotomy. As can be seen, clear ABR responses could be recorded when the vibrator was applied directly to the brain, through the craniotomy. These ABR responses could be masked by broad band noise presented by a speaker (air-conducted). The lowest threshold was obtained when the vibrator was on the intact skull.Table 1 shows the mean results obtained in the rats studied in this way. The lowest ABR thresholds, shortest latencies and largest amplitudes were obtained in rats when the bone vibrator was applied to the intact skull. This was true both for 2 and 8 kHz (low and high frequencies) logon stimuli. In the presence of a craniotomy, ABR thresholds were consistently elevated, latencies increased and amplitudes decreased. For the 2 kHz logon, the threshold was significantly higher when the bone vibrator was applied to the exposed brain compared to when it was on the bone. With respect to the 8 kHz logon, there was no significant difference between the thresholds (vibrator on bone or vibrator on brain, both in the presence of the craniotomy).In the guinea pigs and in the fat sand rats, clear ABR responses were also obtained in response to bone vibrator stimulation directly to the brain, through a craniotomy.An accelerometer placed on the skull was clearly able to record the vibrations induced by a bone vibrator applied to the skull about 15 mm distant both in the intact skull and in the presence of a craniotomy. However when the bone vibrator was applied directly to the exposed brain through the craniotomy, the accelerometer, at maximum sensitivity, was unable to record any vibrations of the skull. Identical results were obtained in a guinea pig and furthermore the accelerometer on the exposed brain recorded large vibrations that were induced by the application of the bone vibrator on the skull (see Fig. 2).Experiment B: the size of the craniotomy had a consistent effect on the ABR threshold to bone vibrator stimuli applied to the bone. As the craniotomy was successively enlarged, the bone vibrator on bone ABR thresholds became more and more elevated, reaching a maximum elevation of about 15 dB. However, the bone vibrator on brain ABR thresholds was not further elevated by the size of the craniotomy (starting with the minimal size of craniotomy required to place the vibrator on the brain without making contact with the surrounding bone – a diameter of about 1 cm).Experiment C: on the other hand, the size of the craniotomy did not affect the ABR threshold to air-delivered stimuli (earphone or bone vibrator suspended above the skull) with plasticine in the external auditory canal, showing that cochlear function was not compromised by the induction and presence of these craniotomies. Furthermore the mean air-conducted ABR threshold (±S.D.) in seven rats using the bone vibrator as an air-conducted sound source (suspended above the skull, with plasticine in the external auditory canal) was 115.0±7.1 dB pe SPL before the craniotomy and 113.9±8.1 dB pe SPL after the craniotomy (difference not significant) and significantly higher than those recorded with the bone vibrator on the skull or brain. This provides confirmation that the ABR thresholds obtained with the bone vibrator on bone or on brain were not in response to air-conducted sounds.Experiment D: in three sand rats, following contralateral destruction of the cochlea, the ossicles, the stapes footplate in the oval window and the bulla cavity of the opposite temporal bone were fixed into one solid mass (cyanoacrylate), without obstructing the round window. In these animals, the ABR threshold to stimulation of the intact skull was 5–10 dB better compared to that prior to fixation. The persistence of these responses in the presence of cyanoacrylate shows that this glue in the middle ear is not toxic to the ear, at least for the duration of the experiment. After the craniotomy, ABR could still be recorded in these animals in response to application of the bone vibrator to the skull and also directly to the brain, with thresholds on the brain 5–10 dB better than on the skull. Post mortem observation of the middle ears confirmed that the ossicles and middle ear cavity were one solid mass and the round window was not obstructed. In the remaining three animals, the round window was also obstructed by the glue, limiting its vibration. In these cases, ABR threshold with the bone vibrator on the brain was elevated by 25–30 dB compared with the threshold prior to middle ear ossicular and window immobilization.Experiment E: following the administration of mannitol to seven rats with intact skulls, the ABR threshold to air-conducted 2 kHz logon stimuli was temporarily lowered (improved) by 3.93±3.4 dB compared to the pre-mannitol state. This threshold improvement was statistically significant (P<0.025; paired t-test). At the same time, when the bone vibrator was applied to the skull, ABR thresholds became elevated following the mannitol injection compared to the baseline threshold in the same animals. In most cases, the bone conduction thresholds recovered spontaneously about 30 min after the mannitol bolus injection, so that multiple boluses could be given to the same animal. In no case did the bone conduction threshold improve following mannitol administration. The mean bone conduction threshold elevation was 7.5±3.82 dB compared to the baseline bone conduction threshold and this elevation was significant (P<0.005; paired t-test).4DiscussionThe major result of these experiments is that ABR responses to vibratory (bone conduction) stimuli can be elicited in rats, in guinea pigs and in fat sand rats when the bone vibrator is applied directly to the exposed brain, and not only with the bone vibrator on the skull. The waveform of the ABR was similar in these two conditions. The ABR recorded with the bone vibrator on the brain could be masked by white noise, presented by an earphone, confirming the cochlear origin of the responses.Since the accelerometer on the skull could not detect bone vibrations when the vibrator was directly on the brain in the craniotomy, it is not likely that direct vibration of the brain and its fluid induced vibrations of the skull. Furthermore, consideration of the impedances of the tissues makes it unlikely that brain-fluid vibrations would be able to induce vibrations of overlying skull, or of the bony wall of the cochlear shell. On the other hand, vibration stimuli delivered to the skull induced clear vibrations of the underlying brain.In these experiments (for example, Experiment A) vibrations were induced by placing the bone vibrator on skull bone and directly on the brain through a craniotomy. However the load impedances of these tissues (bone and brain) are not identical and it is possible that the accelerations of brain induced by the vibrator on the brain could be greater than those of bone induced by the vibrator on bone. These cannot be simply measured since the source impedance of these two tissues with respect to the measuring accelerometer also differ. In fact the acceleration measured on brain in response to vibration of brain was smaller than that of skull bone in response to vibration of skull bone (detailed results not presented). In any case, the magnitude of the skull bone vibrations induced by the bone vibrator on the skull was sufficient to give rise to large vibrations of the underlying brain. On the other hand, the magnitude of the vibrations induced in the overlying bone by the bone vibrator on the brain were below the sensitivity of the accelerometer (see Experiment A).In addition, the presence of a craniotomy and its size did not cause threshold elevations in the ABR to air-delivered stimulation, showing that the cochlea was not affected by the craniotomies. Also the extent of the craniotomy did not affect the ABR threshold when the bone vibrator was applied directly to the brain.Finally, ABR responses to the bone conduction stimulation could still be obtained even when the relevant classical mechanisms thought to be involved in transmission of vibratory energy from the point of application of the vibrator to the final cochlear ABR response had been eliminated by experimental manipulations:1.The bone vibrator was applied directly to the skull following retraction of skin and muscle from over the skull. In this way, transmission as surface waves along the soft tissues of the skull was eliminated.2.In addition, the external auditory meatus was completely filled with plasticine, so that the occlusion affect was greatly reduced, since the effective external ear cavity was then very small.3.Also the ossicular chain was fixed to the middle ear cavity of the temporal bone, preventing relative motion between the cochlear shell and the ossicular chain. This would eliminate the ossicular chain inertial component of bone conduction (Experiment D), which has been thought by Barany (1938), Wever and Lawrence (1954) and Kirikae (1959) to be the major mechanism of bone conduction. Stenfelt et al. (2000), based on studies on a dry human skull, also suggest that ossicular chain inertia has a major influence on bone conduction. It was not possible in the present study to completely eliminate the cochlear shell distortional component but it is probable that this mechanism would not be activated by direct stimulation of the brain. Stenfelt et al. (2000) also conclude that compression-distortion of the cochlear shell is of minor importance.In spite of the successive experimental removal of the classical bone conduction mechanisms, ‘bone conduction’ responses could still be obtained. The ABR responses were much larger in amplitude than those obtained in response to the air conduction stimulus generated by the bone vibrator. Similar results to those obtained here in experiment D had previously been seen in rats (Geal-Dor et al., 1993) and now confirmed in fat sand rats. In this situation, only the cochlear shell distortional–compressional mechanism of bone conduction remained and even this was largely removed when a relatively wide craniotomy was made and the bone vibrator was applied directly to the brain. Although ABR thresholds to bone vibrator on bone were then elevated by 12–14 dB, the ABR thresholds to bone vibration on brain were not affected by even maximal removal of calvarium. This latter result thus shows that skull bone may not even be necessary for the induction of ABR responses to ‘bone-conducted’ stimuli, as long as the bone vibrator is applied directly to the brain. Following removal of the ossicular chain inertia mechanism, bone conduction ABR thresholds improved by 5–10 dB compared to those before immobilization. This could be due to the presence of a small inertial bone conduction component of opposite phase with respect to the major fluid pathway component. Then ossicular immobilization, removing this inertial component, could give rise to a larger response, i.e. threshold improvement.Thus, it is likely that when the bone vibrator is applied directly to the brain, it induces audio-frequency pressure vibrations in the contents of the skull, probably in the CSF, which are then communicated via fluid channels directly to the cochlea, leading to cochlear excitation. The results of the experiments involving the apparent reduction in CSF volume by mannitol administration in rats with intact skulls (Experiment E) are highly relevant here. The small air conduction ABR threshold improvements (3.93 dB) show that cochlear function was slightly improved by this manipulation, probably related to the increase in cochlear blood flow shown following mannitol injection in animals (Larsen et al., 1982; Goodwin et al., 1984; Baldwin et al., 1992). At the same time, the bone conduction thresholds in the same animals became elevated following the presumed reduction in intracranial fluid (including CSF) volume. Thus if one considers the air conduction threshold improvements, it is likely that the simultaneous bone conduction threshold elevations were even slightly greater than those actually recorded. This provides strong support for the suggestion that the skull content components which are involved in the transfer of a major part of the vibratory energy from the skull to the inner ear fluids, are the fluid contents of the cranial cavity, especially CSF. This is further confirmation of the involvement of a fluid pathway in bone conduction stimulation in the intact animal, and that the results in rats with craniotomies are not artifacts due to the craniotomy. It is therefore suggested that in bone conduction stimulation with an intact skull, the audio-frequency vibrations of the skull induce audio-frequency pressure alternations of the skull contents (CSF), which are conveyed by fluid pathways directly to the inner ear fluid spaces. In addition, one or more of the classical bone conduction mechanisms vectorially sum with this major fluid mechanism, exciting the cochlea.It had been thought that the final cochlear response to a bone conduction stimulus represents the vectorial summation of the energy pathways between the point of vibrator application on the skull and the cochlea (surface, transverse and pressure waves), and the vectorial summation of the inner ear modes of bone conduction response (inertial, occlusion and distortional-compression). If this were the case, then the successive removal of each these components in the experimental manipulation in these experiments, should have been accompanied by major changes in the ABR waveform and threshold. This was not the case, providing further evidence that the major (dominant) mechanism in the initiation of the bone conduction response is the fluid pathway described, with smaller contributions from the other classical mechanisms.Why are the ABR thresholds to bone vibrator on bone stimuli elevated (12–14 dB; in addition, longer latencies and lower amplitudes) in the presence of a craniotomy? This is not due to the craniotomy (fluid leak) adversely affecting the cochlea since air conduction thresholds were not changed following the craniotomy. It is possible that the craniotomy, reducing the amount of bone on the skull, may have attenuated, for example, a smaller contribution from the transverse wave (propagation along skull bone) mechanism of bone conduction to the final response. If so, in the intact skull, the contribution from the fluid mechanism and the contribution from the transverse wave mechanism would have had to be in phase and therefore summating, so that the removal of the distortional mechanism would induce a reduction in response amplitude, that is a threshold elevation. However it is more likely that the craniotomy causes a dissipation (damping) of the CSF pressure waves induced by the skull vibrations and the larger the craniotomy, the greater the dissipation. This is similar to the explanation for the finding that the magnitude of the CSF pressure alternations related and time locked to the heart (pulse) and the respiration are dependent on the intracranial pressure such that as this pressure becomes elevated, the pulse and respiratory waves become larger in amplitude (Bering, 1955; Dunbar et al., 1966). The loss of some CSF through the craniotomy reducing CSF pressure, may also be responsible for part of this threshold elevation. Furthermore these CSF pulse pressures measured in a patient in a lateral ventricle by Bering (1955) with an electronic manometer were larger when the valve connecting to an open bore manometer in the opposite lateral ventricle was closed. This was ascribed to the damping effect of the open bore manometer.In similar experiments in humans (Sohmer et al., 2000), the bone vibrator was placed on the fontanelle in neonates and on a previous craniotomy in patients. The auditory thresholds obtained (ABR to clicks in neonates and audiometric to pure tones in patients) were similar to those obtained in response to bone vibrator placements over intact skull in the same patients. In these cases the brain and CSF were covered by dura and skin and it is likely that these overlying tissues led to less dissipation of the vibratory energy which had been induced by the bone vibrator in the intracranial contents (brain and CSF). On the other hand, in the animal experiments reported here, the experimental craniotomy was not covered so that when the bone vibrator was placed on the brain in the craniotomy, the dissipation was in the immediate surrounding of the bone vibrator and already maximal. However, when the bone vibrator was placed in the same animals on nearby intact bone, the region of dissipation was further away, with intact bone lying between the site of the bone vibrator and the craniotomy. This could lead to less dissipation in the latter situation, explaining the better ABR thresholds obtained with the vibrator on bone compared to those with vibrator on brain. Increasing the size of the craniotomy would then be accompanied by further dissipation and more elevated thresholds to stimulation of bone.The fluid communications which are probably involved in the transfer of pressure changes from the CSF to the inner ear could include the cochlear aqueduct which communicates between the subarachnoid space of the posterior cranial fossa and scala tympani of the basal turn of the cochlea and perhaps also perivascular (e.g. inferior cochlear vein) and perineural spaces (e.g. in the internal auditory meatus) (Sando et al., 1971; Palva et al., 1979; Schuknecht and Reisser, 1988). The vestibular aqueduct may also be involved (Marchbanks and Reid, 1990; Kitahara et al., 1994; Konradsson et al., 1994; Yoshida and Uemura, 1991). In fact in man the diameters of vestibular aqueduct apertures communicating between the endolymph in the saccule and the subarachnoid space are larger than the corresponding apertures of the cochlear aqueduct (Anson et al., 1965). These fluid channels need not be involved in bulk flow of CSF or perilymph or endolymph across these fluid spaces. They need only transmit audio-frequency pressures from one fluid space to another. This is similar to the cardiac and respiratory pressure pulses present in the CSF which could also be recorded in the inner ear and whose origin is intracranial (Martinez, 1968; Carlborg et al., 1982; Bohmer, 1993). Further experimentation is required in order to determine which fluid channel(s) is (are) involved.In conclusion, this study in small experimental animals has contributed to the elucidation of the complex mechanisms involved in bone conduction stimulation of the ear. It seems that a major mechanism involves skull vibrations which give rise to alternating CSF pressure. These are communicated by fluid channels from the CSF directly to the inner ear fluids. Contributions from the ‘classical’ bone conduction mechanisms (surface waves, ossicular chain inertia and skull distortions) vectorially sum with these CSF pressure waves, exciting the cochlea. Since each of these mechanisms, including the fluid one suggested here, bypass the outer and middle ears, bone conduction stimulation is still a valid means for the differentiation between conductive and sensori-neural hearing losses. Even though the basic physiological mechanism of bone conduction is better understood as a result of these experiments, further clarification is required.ReferencesAnson et al., 1965B.J.AnsonJ.A.DonaldsonR.L.WarpehaT.R.WinchThe vestibular and cochlear aqueducts: their variational anatomy in the adult human earLaryngoscope75196512031223Baldwin et al., 1992D.L.BaldwinK.A.OhlsenJ.M.MillerA.L.NuttallCochlear blood flow and microvascular resistance changes in response to hypertonic glycerol, urea, and mannitol infusionsAnn. Otol. Rhinol. Laryngol.1011992168175Barany, 1938E.BaranyA contribution to the physiology of bone conductionActa. Oto-Laryngol.26Suppl.19381223von Bekesy, 1932G.von BekesyZur Theorie des Horens bei der Schallaufnahme durch KnochenleitungAnn. Phys.131932111136von Bekesy, 1960von Bekesy, G., 1960. In: Wever, E.G. (Ed.), Experiments in Hearing. McGraw-Hill, New York.Bering, 1955E.A.BeringChoroid plexus and arterial pulsation of cerebrospinal fluidArch. Neurol. 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(Stockh.)981984403412Kirikae, 1959I.KirikaeAn experimental study on the fundamental mechanism of bone conductionActa Oto-Laryngol.145Suppl.19591111Kitahara et al., 1994M.KitaharaM.SuzukiA.KodamaEquilibrium of inner and middle ear pressureActa Oto-Laryngol. (Stockh.)510Suppl.1994113115Konradsson et al., 1994K.S.KonradssonA.H.CarlborgJ.C.FarmerJr.B.I.CarlborgPerilymph pressure during hypobaric conditions – cochlear aqueduct obstructedActa. Oto-Laryngol. (Stockh.)11419942429Larsen et al., 1982H.C.LarsenC.AngelborgE.HultcrantzCochlear blood flow related to hyperosmotic solutionArch. Otorhinolaryngol.2341982145150Marchbanks and Reid, 1990R.J.MarchbanksA.ReidCochlear and cerebrospinal fluid pressure: their inter-relationship and control mechanismsBr. J. 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Res.14620008188Stenfelt et al., 2000S.StenfeltB.HakanssonA.TjellstromVibration characteristics of bone conducted sound in vitroJ. Acoust. Soc. Am.1072000422431Tonndorf, 1966J.TonndorfBone conduction. Studies in experimental animalsActa Otolaryngol. (Stockh.)213Suppl.19661132Tonndorf, 1968J.TonndorfA new concept of bone conductionArch. Otolaryngol.871968595600Tonndorf and Tabor, 1962J.TonndorfJ.R.TaborClosure of the cochlear windows: its effect upon air- and bone-conductionAnn. Otol. Rhinol. Laryngol.711962529Treib et al., 1998J.TreibS.C.BeckerM.GrauerA.HaassTranscranial Doppler monitoring of intracranial pressure therapy with mannitol, sorbitol and glycerol in patients with acute strokeEur. Neurol.401998212219Wever and Lawrence, 1954Wever, E.G., Lawrence, M., 1954. Physiological Acoustics. Princeton University Press, Princeton, NJ.Yoshida and Uemura, 1991M.YoshidaT.UemuraTransmission of cerebrospinal fluid pressure changes to the inner ear and its effect on cochlear microphonicsEur. Arch. Otorhinolaryngol.2481991139143 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A21D78022E6A3BC92DE1B85C1CBAAD488CD7036.txt b/test/dataset/in/resources/corpus/Clean_0A21D78022E6A3BC92DE1B85C1CBAAD488CD7036.txt new file mode 100644 index 0000000..c541ea7 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A21D78022E6A3BC92DE1B85C1CBAAD488CD7036.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 010048TG10.1093/geront/41.6.799 RESEARCH ARTICLE E-mail Versus Conventional Postal Mail Survey of Geriatric Chiefs Raziano Donna Brady MD a Jayadevappa Ravishankar PhD b Valenzula Duaré c Weiner Mark MD d Lavizzo-Mourey Risa MD, MBA e aDepartment of Medicine, Division of Internal Medicine, University of Pennsylvania, Philadelphia bDepartment of Medicine, Leonard Davis Institute on Health Economics, and Institute on Aging, University of Pennsylvania, Philadelphia cSchool of Arts and Sciences, University of Pennsylvania, Philadelphia dDepartment of Medicine, University of Pennsylvania, Philadelphia eDivision of Geriatric Medicine and Institute on Aging, University of Pennsylvania, Philadelphia Donna Brady Raziano, MD, Fellow, Geriatric Medicine, Department of Medicine, University of Pennsylvania, Ralston-Penn Center, 3615 Chestnut Street, Philadelphia, PA 19104. E-mail: Donna.Raziano@uphs.upenn.edu. 1 12 2001 41 6 799 804 23 7 2001 19 2 2001 The Gerontological Society of America 2001 Purpose: This study compared the response time, response rate, and cost of two types of survey administration techniques: e-mail/web-based versus conventional postal mail. The main aim of the survey was to collect descriptive information on the existence of Acute Care for Elders units and their characteristics by surveying geriatric division chiefs. Design and Methods: Two randomized cohorts of geriatric division chiefs were formed to receive a survey either by electronic mail (n = 57) or by conventional postal mail (n = 57). If there was no response to the initial mailing, two follow-up mailings were sent to both groups using the original modality; a third follow-up was performed using the alternative modality. For each group, response rate and response time were calculated. The average total cost was computed and compared across two groups. Results: The aggregate response rate was 58% (n = 31) for the e-mail group versus 77% (n = 44) for the postal mail group. The overall average response time was shorter in the e-mail group, 18 days compared with 33 days for the conventional postal mailing group. The cost comparison showed that average cost was $7.70 for the e-mail group, compared to $10.50 per response for the conventional mail group. Implications: It appears that although the web-based technology is gaining popularity and leads to lower cost per response, the conventional postal method of surveying continues to deliver a better response rate among the geriatric medicine division chiefs. The web-based approach holds promise given its lower costs and acceptable response rate combined with the shorter response time. Electronic mail Mail surveys Survey techniques hwp-legacy-fpage 799 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: Laurence G. Branch, PhD Recently, Internet-based survey techniques have been gaining popularity for health care research and medical applications (Houston and Fiore 1998; Jones and Pitt 1999; Lakeman 1997; Schleyer and Forrest 2000; Thomas, Stamler, Lafreniere, and Dumula 2000). The Internet provides a new survey technique to the geriatric research community in targeting the geriatric populations in a more cost-effective way. Despite the growing enthusiasm for Internet-based surveys (Lakeman 1997; Schleyer and Forrest 2000), the geriatric medical community seems to have fallen behind in the effective use of this technique. The current literature indicates that when using conventional survey techniques, such as postal surveys, researchers can expect a response rate ranging from 70% to 75% (Davis 1999; Jones and Pitt 1999). On the other hand, the response rates for electronic mail surveys have been found to vary from 34% to 76% (Eley 1999; Jones and Pitt 1999). These numbers, however, should be viewed with caution because there is a paucity of substantial comparison studies, and none are noted in the geriatric research community. Additionally, the nature of the sample or the target group, the conditions under which the survey was administered, and the content of the survey can have profound implications on response rate and effectiveness of Internet-based survey techniques (Houston and Fiore 1998; Jones and Pitt 1999; Lakeman 1997; Schleyer and Forrest 2000). Table 1 displays some of the benefits and drawbacks of a postal survey versus an e-mail/web-based survey (Houston and Fiore 1998; Jones and Pitt 1999; Lakeman 1997; Schleyer and Forrest 2000; Thomas et al. 2000). We compared the attributes of an e-mail/web-based survey to a conventional postal survey. This study was part of a larger project on Acute Care for Elders (ACE) units and the main aim of the survey was to collect descriptive information on the existence of ACE units and their characteristics. We present a comparison of the response rate, response time, and administration costs between electronic mail and conventional postal mail surveys, targeted toward the geriatrics programs and division chiefs across the country. Methods The study population consisted of 114 chiefs of established geriatric medical divisions and geriatric teaching programs across the United States. The names, mailing addresses, and e-mail addresses for the study population were obtained by reviewing the following sources: the American Geriatrics Society (AGS) board of directors and task force committees, American Directory of Geriatric Academic Programs (ADGAP), and Geriatric Education Centers (GEC). Two groups, each containing 57 institutions, were formed by random assignment of the study population. The institutions were numbered from 1 to 114; then, with the help of random number tables, an institution was assigned to either e-mail group (group 1) or the conventional postal mailing group (group 2). The process was repeated until each group had 57 institutions. We obtained e-mail addresses for chiefs in group 1 via the following sources: Internet institution directory sites, professional medical society directories of the AGS, American College of Physicians, The Gerontological Society of America, GEC lists, ADGAP program directories, and the Association of American Medical Colleges Graduate Medical Education directory. We were able to obtain e-mail addresses for 40 chiefs by using these methods. For the remaining 13 chiefs, whose e-mail could not be obtained by these methods, we obtained e-mail addresses from their office assistants without disclosing the study. Four individuals who did not have an e-mail address were excluded from the e-mail survey group; thus, the adjusted denominator for group 1 was 53. For group 2 (n = 57), the most recent postal mailing addresses from the sources mentioned were used. Survey Design The survey instrument was designed to determine the prevalence and characteristics of ACE units among academic geriatric programs. The survey (see the Table A1 1) consisted of 17 multiple-choice questions, including questions on the existence, size, length of stay, and patient characteristics of the ACE unit and demographics. To develop the web-based version of the survey, Claris Home Page 3.0 was used to format the survey with Hyper Text Markup Language (HTML). Functional testing of the web page was successful on Netscape and Internet explorer platforms on both Macintosh and Windows-based computers. Each question was reviewed for readability and ease of downloading. Downloading took only a few seconds, and the survey took about 5 minutes to complete. List boxes, radio buttons, and check boxes were provided because none of the questions were open ended; the format was simple, with consistent positioning of check buttons that made concentrating on the questions easy. The preliminary draft of the survey was reviewed within the Division of Geriatric Medicine, University of Pennsylvania. The faculty was asked to comment on the various aspects of the survey, such as question format, layout, wording, and duration. We responded by revising some of the questions for their content and omitting one question altogether. Survey Administration Respondents in the e-mail group received a letter stating the purpose of the survey and our contact information. The survey could be accessed through the e-mail letter as an attachment in an HTML format and/or as a hyper link to our survey on the World Wide Web. The Uniform Resource Locator address for the location of the survey was not stated in the letter, but an access button was created for convenience and to restrict unsolicited responses. Thus, the e-mail group could respond to the survey by completing and returning the attachment, by accessing and completing the survey on web page, or by printing out the survey and mailing back the completed survey via postal mail or fax. In total, three follow-ups were done for the nonrespondents. For those who did not respond to the first e-mail survey request, surveys were re-sent twice at 5-day intervals. The third and final follow-up for the nonrespondents was performed through conventional postal mail (the alternative method). Respondents in the conventional postal mail group received an introductory letter similar to group 1. The chiefs received a hard copy of the survey and a self-addressed, stamped return envelope. They were also given an option to return the survey via fax. For nonrespondents in group 2, three follow-ups were done in total. They were re-sent the postal mail survey twice, allowing about 20 days between each attempt. The third and final follow-up for the nonrespondents was performed by e-mail (the alternative method). It is important to note here that the allocated time space between subsequent follow-ups is different for the two groups. We had to take into account the longer institutional processing time for conventional mail. For each group, response rate and response time were calculated. The response time was defined as the number of days between the date the survey was sent out and the date it was received. Hence, the first mailing and subsequent follow-ups were treated as discrete events. The overall costs included labor, supplies, and postal costs. Costs of the final follow-up by the alternative method were not included. The average total cost was computed and compared across two groups. Finally, paired t-test and chi-square statistics were used to compare the average group response rate and response time. Results The e-mail group had four individuals with either incorrect or no e-mail address, thus reducing the total number of participants in this group to 53. Although these individuals were sent a postal mail survey, their responses were excluded for the purpose of this study. The first attempt received maximum responses in both groups, with a 39% response rate in the e-mail group and a 63% response rate in the conventional postal mailing group. For both groups, the response rate showed a decreasing trend during the second and third follow-ups. Key findings are summarized in Table 2 . Response Rate There was a significant (p < .001) difference in aggregate response rate of all three attempts, with 58% for the e-mail group and 77% for the conventional mail group. For the e-mail group, the total number of responses for the three attempts was 31 out of 53. Of these, 23 responses were returned via e-mail. Eight respondents (26%) in the e-mail group preferred to print out the survey and responded either through postal mail or fax. However, after the third follow-up attempt through conventional postal mailing follow-up, the total response rate was increased to 83% (n = 44) for the e-mail group. For the conventional mail group, total number of responses for the first three mailings was 44 out of 57 (77%). The third and final follow-up attempt, using e-mail, resulted in increasing the total responses to 46 (81%). Following the completion of the study, the e-mail nonresponders were contacted to better understand the nonresponder behavior and influencing factors. Individuals who did not respond to all three e-mail attempts (n = 6), but completed a conventional postal mail survey, were contacted personally by telephone and asked why they did not respond via e-mail. The reasons reported included a higher level of comfort with the conventional mail survey (n = 1), unavailability of e-mail accounts (n = 2), and lack of technical savvy with the Internet and attachments (n = 3). Therefore, in group 1, in addition to four for whom we could not identify an e-mail address, three more individuals had but did not use e-mail accounts. Thus, approximately 12% of the e-mail group was inaccessible electronically. Response Time There was a significant difference in the response time between the two groups (p < .001). The average response time for the e-mail group was 18 days, with 30% of the responses arriving within 24 hours after request. On the other hand, for the conventional postal mailing group, the average response time was 33 days. The first response from the conventional postal mailing group was received on the 15th day. This suggests that e-mail surveys are capable of generating much quicker responses compared with conventional postal surveys. For the e-mail group, the average electronic response time for the first attempt was 3.3 days. The average electronic response time for the second attempt was 4.7 days and for the third attempt was 2.3 days. For the conventional postal group, the average response time for the first mailing was 21 days. The average response time for the second mailing was 15.5 days and for the third mailing was 21 days. Cost Analysis We calculated average cost per response for both groups. The total costs included the labor costs involved in survey preparation, follow-up, survey administration, data input, stationary cost, and postage cost. We derived these costs based on the cost estimates for our institution letterhead, large and regular mailing envelopes, hard copies of the survey, and postage for both our mailing of the envelopes and prepaid return envelopes. Long-distance telephone calls and Internet connection costs were not included. The labor cost differed for each activity: The development of an Internet-based e-mail survey required a research assistant with reasonable computer skills at $40.00 per hour, whereas for all other activities (data input, postal mailing survey administration, and survey preparation) the cost was $20.00 per hour. The average cost per response was $10.50 for conventional postal mail group and $7.70 for the e-mail group. Thus, the conventional postal mail survey costs were 27% higher than the e-mail survey. This higher cost of a conventional postal mail survey can be attributed to the extra costs of stationary, postage, administration, and follow-up. However, for longitudinal surveys and surveys of large populations, cost of survey administration and follow-ups can be reduced significantly by using e-mail/web-based survey techniques. Table 3 presents the cost analysis for both groups. For the e-mail group, the task of survey preparation was more expensive compared to administration and follow-up costs. It required 2 hours at the rate of $40 per hour for a computer professional to set up the web-based survey. The survey administration, follow-up, and data input costs were small ($60 in total) for the e-mail group. On the other hand, the task of survey preparation accounted for only a small portion of the total cost for the conventional postal mailing group, with $17 for institutional letterheads and other stationary. In this case, survey administration, follow-up, and data input were the more expensive items. Survey administration required a total of $197 ($57 in postage, 2 hours of labor at the rate of $20 per hour, and $100 for obtaining the ADGAP address list). Survey follow-up cost was $150 for two follow-up attempts, and an additional $100 was required for data input. The costs of the fourth attempt by the alternative method were not included in the cost analysis. However, it should be noted that costs are extremely sensitive to the complexity and the length of the survey. A lengthier survey would increase the administration, follow-up, and data input costs significantly in group 2. Actual sending of the e-mail surveys did not take as long as preparing and sending the postal mail surveys, but it did take much longer to sort out problems with incorrect e-mail addresses, redirects, and miscommunication via e-mail messaging and other incidentals. Discussion Although the postal mail survey had a higher response, e-mail questionnaires are a promising way to survey the division chiefs of geriatric medicine because of lower response time and costs. Currently there are many reports available that describe the methods involved in conducting an e-mail or web-based survey. However, little knowledge exists about the effectiveness of this technique among geriatric chiefs and the barriers faced in implementing these techniques. Earlier studies have indicated the ways in which e-mail or web-based surveys could be used across different health professionals. Researchers at Temple University, Philadelphia, developed a web-based survey with 22 questions to study the use of the Internet in clinical practice. They recruited 450 dentists from an e-mail address list to receive this web-based survey. Participants were sent one e-mail, and a maximum of three e-mail follow-ups were sent to the nonresponders. E-mail response rates, strictly defined as an e-mail only, were 32.9%, 50.2%, 57.1%, and 64.4% for the chronological attempts. The addition of alternative responses, such as fax or mail, raised total response rate to 74.2%. The cost comparison indicated almost 50% reduction in total cost for e-mail surveys compared with conventional mail surveys ($1,916 compared with a hypothetical postal cost of $3,092). A minimum of 275 e-mail responses were recommended to break even on the costs of e-mail and to make it a more economical method than postal mail (Schleyer and Forrest 2000). This study differs from our study because recruited participants were established e-mail users. The process of obtaining e-mail addresses through mailing address lists was extremely labor-intensive, despite the availability of a number of Internet search engines. This finding was less of an issue in other studies, such as the dental study described, and suggests that geriatrics is lagging other medical subspecialties in creating searchable electronic databases of its physicians. The existence of such a database would have made the electronic surveys even more cost-effective. A study of university staff done in the United Kingdom compared the response timing and rates using electronic and paper methods. E-mail health surveys that required web-based submission were sent to 200 people, and 100 other people received traditional postal surveys. The postal mail survey received a response rate of 72%, followed by 32% with respect to e-mail and 19% for the World Wide Web. Although e-mail and web-based surveys were quicker and cheaper by half, the authors concluded that the higher response rate made postal mail more preferable (Jones and Pitt 1999). Caution must be exercised, because the overall cost is extremely sensitive to the type and scale of the target population. If the Internet usage of the target population is low, a great deal of effort would be expended trying to locate e-mail addresses for people who are not reachable electronically; among those who could be reached, response rates could be poor as a result of inadequate Internet skills. An experiment comparing the response rates and response time for different approaches to mailing highlighted the strengths and weakness of e-mail surveys. In this study, the respondents recruited from the Internet received postal mail or e-mail surveys. The respondents were randomly assigned to one of five groups. Group 1 consisted of regular mail with no prenotification, no incentives, and no reminders (n = 202); group 2 consisted of regular mail with prenotification, incentives, and reminders (n = 107); and groups 3 (n = 60) and 4 (n = 122) were e-mail replications of groups 1 and 2. The final group (n = 172) is an international group, otherwise the same as group 4. The results showed that e-mail surveys had much lower response time (2–3 days vs 3 weeks) and higher response rates (40% in group 1 vs 45% in group 3). In the prenotified groups the rates increased to 63% and 83%, respectively, and were much less expensive compared to conventional mail surveys (Mehta and Sivadas 1995). None of the previously reported survey research has directly compared the postal mail and the electronic web-based mail in a random selection method in the geriatric community. In addition, all previous studies utilized established e-mail address lists, implying that the potential respondents were known to have both a working e-mail address and Internet access. Open recruitment of participants as well as guaranteed Internet users as participants has commonly occurred in other published research (Eley 1999; Mehta and Sivadas 1995). Our study differs in this respect since we had no prior knowledge of the computer skill level of the participants or their level of Internet knowledge. The higher response rate for the conventional mail group was countered by a longer response time in our study. On the other hand, the administration of the e-mail surveys resulted in faster responses and was more cost-effective albeit with a lower overall response rate. It should be noted that, from the first three attempts, we received 31 responses from the e-mail group and 44 responses from the conventional mail group. The electronic response was acceptable but still not as high as expected. The response rate of 58% for the e-mail group is less than other published results that indicate a response rate closer to 70% (Lakeman 1997; Mehta and Sivadas 1995). This discrepancy may have been due to the fact that our target population is unlike the other research populations that used existing Internet or e-mail user lists. Also, it is important to note that our response rates, response time, and related cost for the chiefs may not be generalized to other physicians. To minimize sampling bias in our small preselected population, randomization of survey recipients was used to make the two groups comparable. Despite moderate response rates, the rapid turnaround time could make e-mail the superior method for eliciting data in the future as more and more geriatric chiefs access electronic mail accounts. In tune with earlier findings, the electronic mail response rate for our survey was comparatively lower. We conclude that, even with this limitation, e-mail should be the preferred method if a prompt response is a primary goal. There may also be better control over the respondents and quality of the data with e-mail. Especially if traditional postal mailing is used as a salvage step, the e-mail/web-based survey follow-up reminders were easier and more cost-effective. The cost saving associated with electronic surveys must be weighed against the poorer expected response rate. Practice Concepts The Forum Book Reviews Table 1. Comparison of E-mail/Web-Based and Conventional Postal Mail Survey E-mail/Web-Based Survey Conventional Postal Mail Survey Can be cost-effective in established e-mail users. Targeted population is more difficult to monitor for receipt and completion of survey. Data are collected in electronic format, thus minimizing data input effort. Requires more effort in terms of data input. Survey administration is less costly. Survey administration and follow-up are costly. Hypertext markup language can be designed to suit preferences of the respondent. It is more time intensive. Accessibility is restricted to populations that have access to Internet or e-mail. Wide range of population accessibility. Requires some basic skills of computer. Basic computer skills are not necessary. Table 2. Response Rate and Response Time E-mail/Web-Based (n = 53)Consecutive Attempts Conventional Postal Mail (n = 57) Consecutive Attempts Measure First Second Third Fourth First Second Third Fourth Total response 21 7 3 13 36 5 3 2 Average response time (in days) 3.3 4.7 2.3 21 15.5 21 Response rate for each attempt 39% 22% 10% 50% 63% 23% 19% 12% (21/53) (7/32) (3/29) (13/26) (36/57) (5/21) (3/16) (2/13) Total average response time 18 Days 33 Days Mode of response E-mail = 52% E-mail = 3.5% Mail = 45% Mail = 96.5% Fax = 3% Fax = 0 Table 3. Cost Analysis Types E-mail Group (n = 53) Postal Mail Group (n = 57) Survey preparation $80 $17 Survey administration cost $120 $197 Survey follow-up cost $40 $150 Data input cost 00 $100 Total cost $240 $464 Average cost per response $7.70 (n = 31) $10.50 (n = 44) Table A1. Institute on AgingDivision of Geriatric MedicineDepartment of MedicineUniversity of PennsylvaniaNATIONAL ACE UNIT SURVEY 1. Name of the hospital _____________________________________________________________________________ 2. Address _________________________________________________________________________ Zip: _________ 3. Location of the hospital □ Rural □ Urban □ Suburban 4. Is your hospital (check all that apply)? □ For Profit □ Non Profit □ Community Hospital □ Community Teaching Hospital □ University based Teaching □ Non-teaching 5. Do you have an Acute Care for Elderly (ACE) unit? □ Yes □ No If answer to question 5 is NO, you may STOP here but please return the survey 6. Year ACE unit was established: _ _ _ _ 7. Is your ACE unit limited to community dwelling (e.g., non-nursing home) patients only? □ Yes □ No 8. What is the average daily census on the ACE unit? □ <10 □ 10-15 □ 16-20 □ 21-25 □ >25 9. Do you have telemetry beds within your ACE unit? □ Yes □ No 10. Average length of stay in days ________________ 11. Patients average age group □ 60-65 □ 66-70 □ 71-75 □ 76-80 □ >80 12. Are patients from the following settings routinely admitted to your ACE unit (Check all that apply)? □ Nursing Home □ Surgical Floor □ Emergency Room □ Non-ICU medical unit □ Living at home 13. Please check the top three diagnoses in your ACE unit 1 2 3 Congestive Heart Failure ____ ____ ____ Pneumonia & Respiratory infection ____ ____ ____ Kidney & Urinary Tract Infection ____ ____ ____ Metabolic disorders ____ ____ ____ Mental status change ____ ____ ____ Gastrointestinal hemorrhage ____ ____ ____ Sepsis ____ ____ ____ Cerebrovascular events ____ ____ ____ Other ________________________________________ ____ ____ ____ 14. Are the following health care professionals allowed to admit patients to your ACE unit (Check all that apply)? □ Attending in Geriatric Medicine □ Attending in Internal Medicine □ Attending in Family Practice □ Attending in Surgery □ Attending in Psychiatry □ Attending in Rehabilitation Medicine □ Community Physicians 15. What is the average nurse to patient ratio in your ACE unit? □ 1:4 □ 1:5 □ 1:6 □ 1:7 □ 1:8 □ 1:9 □ 1:10 16. Which of the following does your ACE unit have? (Check all that apply) □ Pharmacist □ Physical Therapist □ Occupational therapist □ Social worker □ Geriatric psychiatrist □ Speech pathologist □ Nutrition/dietitian specialist 17. Would you be interested in participating in a randomized control trial to evaluate the outcomes of patients admitted to ACE units? □ Yes □ No THANK YOU Support for this study was provided by an American Federation on Aging research geriatric fellowship. Davis R. N., 1999. Web-based administration of a personality questionnaire: Comparison with traditional methods. Behavior Research Methods Instruments:31-572. Eley S., 1999. Nutrition research using electronic mail. British Journal of Nutrition 81:413-416. Houston J. D., Fiore D. C., 1998. Online medical surveys: Using the Internet as a research tool. MD Computing 15:116-120. Jones R., Pitt N., 1999. Health surveys in the workplace: Comparison of postal, email and World Wide Web methods. Occupational Medicine (Oxford) 49:556-558. Lakeman R., 1997. Using the Internet for data collection in nursing research. Computers in Nursing 15:269-275. Mehta R., Sivadas E., 1995. Comparing response rates and response content in mail versus electronic mail surveys. Journal of the Market Research Society 37:429-439. Schleyer T. K. L., Forrest J. L., 2000. Methods for the design and administration of web-based surveys. Journal of the American Medical Information Association 7:416-425. Thomas B., Stamler L. L., Lafreniere K., Dumula R., 2000. The Internet: An effective tool for nursing research with women. Computers in Nursing 18:13-18. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A27E6DBA4C3F47D4148B5D4F21DC29251C2A054.txt b/test/dataset/in/resources/corpus/Clean_0A27E6DBA4C3F47D4148B5D4F21DC29251C2A054.txt new file mode 100644 index 0000000..d417aca --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A27E6DBA4C3F47D4148B5D4F21DC29251C2A054.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 53910.1093/geront/45.4.539 PRACTICE CONCEPTS The Exercise Plus Program for Older Women Post Hip Fracture: Participant Perspectives Resnick Barbara PhD, CRNP, FAAN, FAANP 1 Orwig Denise PhD 2 Wehren Lois PhD, MD 2 Zimmerman Sheryl PhD 3 Simpson Marjorie MS, CRNP 1 Magaziner and Jay PhD 2 Address correspondences to Barbara Resnick, PhD,CRNP, FAAN, FAANP, University of Maryland, School of Nursing, 655 West Lombard Street, Baltimore, MD 21201. E-mail: bresnick@umaryland.edu 8 2005 45 4 539 544 11 10 2004 1 3 2004 The Gerontological Society of America 2005 Purpose: The purpose of this study was to explore the experiences of older women post hip fracture who were exposed to a motivational intervention, the Exercise Plus Program, intended to increase adherence to exercise. Design and Methods: This study used a naturalistic inquiry. We interviewed a total of 70 older women, 12 months post hip fracture, using an interview guide asking about issues related to willingness to exercise. All interviews were tape recorded and transcribed verbatim. Results: Analysis of the semi-structured interviews revealed 44 codes that were categorized and reduced to 14 major themes. Eleven of these themes focused on components that facilitated participation in exercise and included: (a) real and expected benefits; (b) visual cues and knowing what to do; (c) simplicity; (d) individualized care; (e) verbal encouragement to exercise; (f) regular schedule; (g) confidence (i.e., self-efficacy); (h) determination; (i) social support; (j) reciprocity; and (k) goal identification. The remaining three themes reflected what decreased the participants' willingness to exercise: (a) unpleasant sensations; (b) constraints to exercise; and (c) getting back to baseline. Implications: The findings provide some support for the benefit of the Exercise Plus Program. Lessons learned in the Exercise Plus Program can be translated to the development of other motivational interventions to help engage older adults in exercise, particularly those individuals who recently experienced an acute event, such as a hip fracture. Exercise Motivation Self-efficacy Outcome expectations hwp-legacy-fpage 539 hwp-legacy-dochead RESEARCH ARTICLE It is estimated that more than 350,000 older adults experience a hip fracture each year (Agency for Health Care Policy and Research, 1999). Typically those who sustain hip fractures are female and age 80 and older; they commonly incur significant morbidity and mortality associated with the fracture (Van Balen, Steyerberg, Cools, Polder, & Habbema, 2002; Magaziner et al., 2000). For those who have sustained a hip fracture, regular exercise (resistive and/or aerobic) has been reported to improve mobility, increase walking speed, improve quadriceps strength, and increase weight-bearing ability (Gill et al., 2003; Sherrington, Lord, & Herbert, 2004). Unfortunately, despite the potential benefits of exercise, is it difficult to get older women to initiate exercise activity, and helping them adhere to an exercise regime is even more challenging (Resnick & Spellbring, 2000). It is essential, therefore, to consider the most appropriate ways to motivate these individuals to exercise regularly. The purpose of this study was to explore the experiences of older women exposed to a home-based self-efficacy motivational intervention (The Exercise Plus Program) used to increase adherence to exercise post hip fracture. The theory of self-efficacy suggests that the stronger the individual's self-efficacy and outcome expectations, the more likely it is that he or she will initiate and persist with a given activity. Self-efficacy expectations are the individuals' beliefs in their capabilities to perform a course of action to attain a desired outcome; outcome expectations are the beliefs that a certain consequence will be produced by personal action. Efficacy expectations are appraised by four mechanisms (Bandura, 1997): (a) enactive mastery experience, or successful performance of the activity of interest; (b) verbal persuasion, or verbal encouragement given by a credible source that the individual is capable of performing the activity of interest; (c) role-modeling or self-modeling; and (d) physiological and affective states such as exhilaration, pain, fatigue, or anxiety associated with a given activity. The Exercise Plus Program is an innovative approach to motivating older women to exercise in that it addresses all four of these mechanisms and focuses specifically on strengthening outcome expectations as well as self-efficacy expectations. Table 1 provides an overview of this program. The Exercise Plus Program The Exercise Plus Program was implemented by certified exercise trainers. We visted participants two to three times per week for the first 2 months of the intervention, and this was progressively decreased to once per month during the one-year study duration. We encouraged participants to perform five exercise sessions (strength training and aerobic) per week for approximately 30 min durations each. We provided education and verbal encouragement using an investigator-developed “Exercise After Your Hip Fracture” booklet that reviewed the benefits of exercise post hip fracture and ways to overcome the barriers to exercising. We established short (weekly) and long-term goals with participants and gave incentive gifts when weekly goals were achieved. We provided ongoing verbal encouragement toward goal achievement and revised goals as needed. We addressed physiological sensations and affective states (e.g., pain, fear, and associated anxiety) during each interaction, and we implemented standardized techniques to decrease unpleasant sensations. We provided participants with a picture of themselves exercising, an exercise calendar, and a large-print individually tailored write-up of the exercises to expose them to self-modeling techniques. Methodology Design This study used a naturalistic, or constructivist, inquiry (Crabtree & Miller, 1992). We completed a single open-ended interview with all participants who were exposed to the Exercise Plus Program. The study was approved by the Institutional Review Board at the University of Maryland, and we obtained informed consent prior to data collection. Interviews were done by two geriatric nurse practitioners in the individual's current residence (e.g., home, long-term-care facility) or by telephone. The interviews lasted approximately 20–30 min, were audiotaped, and transcribed verbatim. The major focus of the interviews was to explore which aspects of the Exercise Plus Program had an influence on the participant's willingness to engage in exercise. Sample The study included the first 70 women who completed the Exercise Plus Program, were reachable by telephone, and had not withdrawn from the original clinical trial in which the Exercise Plus Program was being evaluated. Criteria for eligibility to participate in the Exercise Plus Program included living in the community prior to hip fracture, receiving some type of surgical repair of the fracture (e.g., open reduction internal fixation, hemiarthroplasty), being able to walk at least 50 feet, having a Mini Mental State Exam (MMSE) score of ≥20, and having no medical problems that would put the individual at risk for exercising alone in the home setting, such as Parkinson's Disease, myocardial infarction within the past 6 months, or long-term use of coumadin. Participation in the qualitative interview was not based on adherence to the exercise program and the interviewers were blind to any of the outcome data related to physical activity. All eligible women we contacted were willing to participate in the qualitative interview. The average age (±SD) of the women who participated in the qualitative interviews was 80.9 ± 6.0 and the majority was Caucasian (96%) and widowed (66%). A smaller number were married (27%), divorced (6%), or never married (1%). Based on the Yale Physical Activity Survey (DiPietro, Caspersen, Ostfeld, & Nadel, 1993), these women engaged in.74 ± 1.35 hr per week of exercise and 33.48 ± 28.53 hr per week of physical activity before the hip fracture and 2.10 ± 1.95 hr per week of exercise and 24.63 ± 22.17 hr per week of physical activity one year post hip fracture. The participants described their overall health as good, very good, or excellent (73%). We conducted the majority of the interviews by telephone (97%), and we completed the remaining interviews in home settings or long-term-care facilities. Data Analysis We completed data analysis was basic content analysis (Crabtree & Miller, 1992), starting with the first interview. The analysis began with “in vivo” coding (Dowd, 1991), which involves using the informants' own words to capture a particular idea. The following is an example of “in vivo” coding in which the code identified was determination: “… I was just determined to do them.” Credibility and Confirmability of the Qualitative Data Credibility of the data refers to the believability, fit, and applicability of the findings to the phenomena under study (Lincoln & Guba, 1985). The data in this study were collected over a 3-year period so that the individual codes identified early in the study by the first participants were confirmed with subsequent study participants, and findings were presented to other members of the research team and five women who had sustained a hip fracture. All reviewers supported the themes as credible. Confirmability of the data was supported by a second researcher who reviewed the coded data and definitions of the codes and categories. She agreed with the coding and provided some additional comments to further explain and understand behavior. Results Analysis of the semistructured interviews revealed 41 codes that were categorized and reduced to 14 major themes (Table 2). Eleven of these themes focused on components that facilitated participation in exercise and included: (a) real and expected benefits; (b) visual cues and knowing what to do; (c) simplicity; (d) individualized care; (e) verbal encouragement to exercise; (f) regular schedule; (g) confidence (i.e., self-efficacy); (h) determination; (i) social support; (j) reciprocity; and (k) goal identification. The remaining three themes reflected what decreased the participants' willingness to exercise: (a) unpleasant sensations; (b) constraints to exercise; and (c) getting back to baseline. Real and Expected Benefits The older women in this study described several real and expected benefits of exercise that influenced their willingness to engage in exercise. The physical benefits included a general belief that the exercise was “good for me,” would improve bone density, endurance, and prevent stiffness; emotional benefits included a generalized feeling of well-being, an emotional lift, and a feeling of accomplishment. These benefits were expressed by responses such as: “Exercise builds bone density”; “I hope it will build my bones so nothing else will break”; “The exercise just generally makes me feel better”; “There is an emotional lift to doing the exercise.” Visual Cues and Knowing What to Do The participants reported that being told what exercises to do, how to do them, and when to do them was very helpful. One participant described this as a “recipe,” and once she had this recipe and knew what to do with it, she could continue to exercise on her own. The participants also reported that visual cues, such as the calendar and exercise booklet provided in the Exercise Plus Program, helped them to remember to exercise. These benefits were expressed by responses such as: “Having the booklet with the exercise helped. I would open that up and do them”; “I plan to continue to keep a calendar and write it down when I exercise. If I don't write it down I know I can let something slide for a couple of days.” Simplicity Similar to the idea of knowing what to do, the participants also described the importance of the simplicity of the program recommended. The exercises that were perceived by the participants to be more complicated were not performed. One respondent, for example, stated: “There was a book with more elaborate things which I did not do as well. The stepping up and the reaching were hard to do and I was not as good at them. I did them when I could get through them but they were really just too complicated.” Individualized Care The participants described the warmth, kindness, and caring they experienced from the trainers as having an important influence on their willingness to participate in the exercise program. The participants appreciated the way in which the trainer individualized the exercise program so that they didn't feel pushed or threatened. Individualized care was described by one respondent as follows: “I think the trainers were very caring people, and we were very compatible. There was no time that I was resisting what they were asking me to do. They were good at recognizing what I could and couldn't do.” Verbal Encouragement In addition to individualized care, participants felt that verbal encouragement given by the trainers helped motivate them to exercise. One example of verbal encouragement described was as follows: “They [the trainers] encouraged me. They taught me about the benefits of exercise and encouraged me to do it. I wasn't too interested in the beginning but they helped me believe that it was important.” Regular Schedule Participants stated that keeping a regular schedule was as an important way in which to adhere to an exercise program. Conversely, participants recognized that not scheduling the exercise during a specific time of the day often meant the exercises did not get done. One respondent described the importance of the regular schedule as follows: “I just think I need to get myself on a schedule and do the exercises in the morning before I do anything else. Otherwise there is always something else that comes up and happens. Someone calls and wants me to do something.” Confidence The participants described how practice with the exercise helped build their confidence and beliefs that they were able to exercise at this level and to do so consistently. A few participants reported that prior exercise successes helped them believe they could exercise again. A respondent described this by stating: “Doing the activities with her helped me believe that I could do them when alone.” Determination Several of the participants described their underlying personalities as having an influence on their willingness to initiate and adhere to the exercise program. They described themselves as determined individuals, and when challenged with the hip fracture they were determined to recover and regain prior function. Determination was expressed as follows: “I was just determined to do them, and I was determined to walk. I was determined to do everything for myself that I could. I just knew that it was the best way to get well.” Social Support Social supports outside of the exercise trainer were not included as part of the Exercise Plus Program. Some participants reported, however, that social supports served as sources of security during exercise sessions, providing verbal encouragement and holding them accountable for exercising. One respondent described the social support she got from her family to exercise: “I have two sons, one especially who is very athletic. He calls me twice a week to prod me along. He wants me to try getting up out of the chair without holding on. He does push to keep me exercising!” Reciprocity Participants described their willingness to complete the exercises recommended by the trainers because of a desire to reciprocate the kindness and support they experienced from these trainers. Exercising with and for the trainer was something they could give back. Respondents alluded to the concept of reciprocity through statements such as: “I wanted to be able to tell her I was doing them [the exercises]”; “I just liked her so much as a person”; “I wanted to do what she wanted me to.” Goal Identification The participants reported that identification of goals gave them something to work toward. Goals included returning to baseline function, maintaining function, or being able to ambulate without an assistive device. Some participants described working toward time-related exercise goals (i.e., minutes exercised). One respondent described her goal, and the importance of goal identification, in the following way: “My goal was to be able to keep one foot in front of the other. The trainer told me that if I stop exercising I would be back to where I started in 2 weeks. I thought, I have gotten to this point I can't quit. They said no, no you can't! You tell yourself you have to keep it up.” Unpleasant Sensations The most common reason reported by the participants for not adhering to the exercise program was the unpleasant sensations associated with exercise. The most frequently reported unpleasant sensations included the anxiety associated with fear of falling or getting hurt, feeling short of breath, fatigue, and having pain. Fear, for example, was described by one respondent as: “I was able to do the exercise when there was someone with me. We walked in the building no problem. It was just doing it by myself I was scared. I was really scared of falling. I have never been scared before but I am now. It wasn't enough time with the trainer to overcome that. I think if I worked some more I could do that.” Constraints to Exercise The women in this study identified time and environment as two common constraints to exercise. Specifically, a few women indicated that due to family, social, medical, or caregiving responsibilities they did not have time to exercise. Some of the women reported they didn't have a sufficient space to exercise. One respondent expressed time-related constraints: “I tried to do them [the exercises] but I am really so busy. I have a husband and children. Sometimes I am called on for a part-time job if there is an emergency. Somebody was always wanting me to carry them someplace.” Getting Back to Baseline One of the surprisingly common themes noted in this study was the belief among many of the women that once they got back to baseline function they didn't need to exercise anymore. Respondents made statements such as: “I feel much better. My hip is doing better, and I didn't think I needed to do it anymore.” Implications for Practice Although final quantitative data are not yet available to evaluate the direct benefit of the motivational components of the Exercise Plus Program, the feedback from participants indicates that all four sources of efficacy enhancement facilitate motivation to exercise post hip fracture: (a) the confidence gained from repeatedly exercising and recognition of the positive outcomes associated with exercise; (b) verbal encouragement from the trainers; (c) self-modeling and cues from the written materials; and (d) eliminating unpleasant sensations associated with exercise. All influenced the participants' willingness to engage in the exercise program. The sources may benefit older adults in other situations as well. Building Confidence Through Actual Performance Performance of the recommended exercise program with participants helped the individuals learn and feel confident in knowing what exercises to do and how to do them correctly. Although certified exercise trainers taught the hip fracture participants the exercise program, older adults in the community exposed to “lay” trainers have reported similar positive effects when lay trainers provide the exercise program (Resnick, Vogel, & Luisi, in press). The Power of Words: Verbal Encouragement Central to the effectiveness of verbal encouragement was the participants' perception that the trainers cared about them. Caring was demonstrated by the repetitive nature of the interactions and the ongoing encouragement to exercise, the setting of individualized goals, and the review of progress toward those goals. Additionally, as has previously been reported (Collins, Lee, Albright, & King, 2004; Wilcox, Bopp, Oberrecht, Kammermann, & McElmurray, 2003), participants indicated that social supports provided verbal encouragement to exercise and are important sources of motivation for both initiation and maintenance of exercise activities. Self-Modeling: Cueing to Exercise The participants indicated that the materials for cueing to exercise offered in the Exercise Plus Program were useful. The written materials served as a reminder to exercise and also helped the participants feel secure that they were doing exercises that would be of benefit to them. The posters and written information about exercise were noted to be simple and easy to use, and the participants repeatedly indicated that “simple was best!” Addressing Barriers to Exercise: Physiological Feedback and Affective States The hip fracture participants indicated that fear, shortness of breath, fatigue, and pain were barriers to exercise. Although the Exercise Plus Program attempts to address and relieve these symptoms, it is likely that the interventions were not always successful. Fear of falling is particularly challenging to resolve in older adults post hip fracture (McKee et al., 2002), and aggressive attempts must be made to eliminate this fear and associated anxiety. In addition, we learned from participants post hip fracture that returning to baseline function was a barrier to continuing to exercise. Once resumption of function was achieved, these individuals felt that they no longer needed to exercise. Anticipating this response after an acute event such as a hip fracture and reinforcing the importance of long-term adherence to exercise should be considered. Personality: The Central Core of Motivation Personality, or determination, was noted by participants to be central to motivation to exercise. Within social cognitive theory, from which the theory of self-efficacy is derived, personality interacts with the environment and behavior or experiences. Health care providers should recognize that although personality is an important component of motivation, interventions can be implemented to effectively motivate individuals who may not be self-directed or determined to exercise. Conclusion Data from this qualitative study identified 14 themes that are commonly experienced by older women enrolled in an exercise program following a hip fracture. Further study will be needed to assess the association between these themes and actual level of participation in routine exercise. Of particular interest is the impact of “getting back to baseline function” and the continuation (or discontinuation) of a regular exercise program. Specific motivational interventions should be developed and tested to address this barrier. This type of intervention may be useful following other acute events in which older adults revert to prior behavior once baseline health and function are achieved. Given that the sample was selected based on specific inclusion criteria, these results may not be relevant to all hip fracture patients. Our findings do provide some support for the Exercise Plus Program and some insight into how these interventions can be utilized. Specifically, we recommend that providers focus on the benefits of exercise; provide clear and simple guidelines for what exercise activity to engage in; teach the individual(s) how to perform the exercise; establish individualized goals; provide cues to remember what exercises to do and when to do them; give ongoing encouragement to exercise and reinforce progress toward goals; celebrate the benefits associated with exercise; explore ways to overcome barriers to exercise (particularly unpleasant sensations); and encourage involvement of social supports. Most importantly, these interventions must be implemented in a way in which the participant feels truly cared for and cared about. Use of such interventions is likely to help older women post hip fracture to not only initiate an exercise program but to remain engaged in exercise over time. This work was supported by the National Institutes of Health NIA Grant RO1 AG17082-01, Principal Investigator Barbara Resnick, PhD,CRNP, FAAN, FAANP, and the National Institutes of Health NIA Grant R37 AG09901, Principal Investigator Jay Magaziner, PhD, MSHyg. The work was also supported by the Claude D. Pepper Center at the University of Maryland (Health, Exercise, and Rehabilitation) and was funded by Grant P60 AG12583 from the National Institues on Aging. 1 Organizational Adult Health, School of Nursing, University of Maryland, Baltimore. 2 Deptartment of Epidemiology and Preventative Medicine, School of Medicine, University of Maryland, Baltimore. 3 School of Social Work, University of North Carolina, Chapel Hill. Decision Editor: David E. Biegel, PhD Table 1. Description of the Exercise Plus Program. Exercise Plus Program Description Exercise component Exercise intervention designed for women post hip fracture that combines a warm up and cool down, 2 days per week of resistive exercises and 3 days per week of aerobic exercise. Plus component Trainer teaches the individual, using the “Exercise After Your Hip Fracture Booklet” about the benefits of exercise and how to overcome barriers to regular exercise. Trainer teaches the exercise intervention, individualized to ability and needs of the participant, and provides posters describing the program and/or written material. Trainer identifies weekly short-term goals and a long-term goal related to exercise, provides verbal encouragement related to goal achievement, and a weekly prize if goals are achieved. Trainer gives a weekly written prescription on a goal form to remind individual of what exercises to do and when to do them. Trainer reviews unpleasant sensations related to exercise and provides interventions to decrease these unpleasant sensations. Trainer takes pictures of the individual exercising at intervals to demonstrate progress over the 12 months. Trainer places weekly telephone calls in months 7 to 12 when visits are decreased to once a month. Weekly aspects of the motivational intervention are implemented via telephone contact (i.e., goal setting, verbal encouragement, education about exercise). Notes: A more detailed description and associated tools of the Exercise Plus Program are available from the first author on request. Table 2. Themes and Associated Codes. Theme Associated Codes Real and expected Physical benefit benefits of exercise Emotional benefit Feeling of accomplishment General benefit Something to look forward to Good for me Outcome expectation Visual cues and knowing Knowing what to do what to do Visual cues Exercise definitions Realistic exercise guidelines Convenient cues Simplicity of the program Simple activities Easy to follow Individualized care Encouragement Kindness Positive reinforcement Verbal encouragement to exercise Verbal encouragement to exercise Regular schedule Regular schedule Convenience Set visits Confidence and self-efficacy Building confidence Self-efficacy Security Determination Unwillingness Self-determination Lack of determination Social supports Social supports Reciprocity Reciprocity Accountability Goal identification Recovery of function Losing gains Sliding backwards Unpleasant sensations Fear Boredom Physical discomfort Shortness of breath Fatigue Perceived constraints Environment to exercise Expectations from others Getting back to baseline Returning to baseline function References Agency for Health Care Policy and Research. (1999). Inpatient hospital statistics, 1996 (Pub no. 99-0034). Rockville, MD: Author. Bandura, A., (1997). Self-efficacy: The exercise of control. New York: W.H. Freeman and Company. Collins, R., Lee, R. E., Albright, C. L., & King, A. C., (2004). Ready to be physically active? The effects of a course preparing low-income multiethnic women to be more physically active. Health Education and Behavior, 31, 47-64. Crabtree, B., & Miller, W., (1992). Doing qualitative research. Newbury Park, CA: Sage Publications. DiPietro, L., Caspersen, A., Ostfeld, M., & Nadel, E., (1993). A survey for assessing physical activity among older adults. Medicine and Science in Sports and Exercise, 25, 628-642. Dowd, T., (1991). Discovering older women's experiences of urinary incontinence. Research in Nursing and Health, 14, 179-186. Gill, T. M., Baker, D. I., Gottschalk, M., Gahbauer, E. A., Charpentier, P. A., & de Regt, P. T., et al (2003). A prehabilitation program for physically frail community-living older persons. Archives of Physical Medicine and Rehabilitation, 84, 394-404. Lincoln, Y., & Guba, E., (1985). Naturalistic inquiry. Newbury Park, CA: Sage Publications. Magaziner, J., Hawkes, W., Hebel, R., Zimmerman, S. I., Fox, K. M., & Dolan, M., et al (2000). Recovery from hip fracture in eight areas of function. Journal of Gerontology: Medical Sciences, 55A, M498-M507. McKee, K. J., Orbell, S., Austin, C. A., Bettridge, R., Liddle, B. J., & Morgan, K., et al (2002). Fear of falling, falls efficacy, and health outcomes in older people following hip fracture. Disability Rehabilitation, 24, 327-333. Resnick, B., & Spellbring, A., (2000). Understanding what motivates older adults to exercise. Journal of Gerontological Nursing, 26, 34-42. Resnick, B., Vogel, A., & Luisi, D., in press. Motivating minority older adults to exercise. Cultural Diversity and Ethnic Minority Psychology.. Sherrington, C., Lord, S. R., & Herbert, R. D., (2004). A randomized controlled trial of weight-bearing versus non-weight-bearing exercise for improving physical ability after usual care for hip fracture. Archives of Physical Medicine and Rehabilitation, 85, 710-716. Van Balen, R., Steyerberg, E. W., Cools, H. J., Polder, J. J., & Habbema, J. D., (2002). Early discharge of hip fracture patients from hospital: Transfer of costs from hospital to nursing home. Acta Orthopedics Scandinavia, 73, 491-495. Wilcox, S., Bopp, M., Oberrecht, L., Kammermann, S. K., & McElmurray, C. T., (2003). Psychosocial and perceived environmental correlates of physical activity in rural and older African American and white women. Journal of Gerontology: Psychological Sciences, 58B, P329-P337. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A3696EBAEC49E050E097CDBA921DBEE4B61CEA3.txt b/test/dataset/in/resources/corpus/Clean_0A3696EBAEC49E050E097CDBA921DBEE4B61CEA3.txt new file mode 100644 index 0000000..241969b --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A3696EBAEC49E050E097CDBA921DBEE4B61CEA3.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 46910.1093/geront/44.4.469 KNOWLEDGE AND PERCEPTIONS ABOUT PAIN Improving Nursing Home Staff Knowledge and Attitudes About Pain Jones Katherine R. RN, PhD, FAAN 1 Fink Regina RN, PhD, FAAN 2 Pepper Ginny RN, PhD, FAAN 3 Hutt Evelyn MD 4 Vojir Carol P. PhD 5 Scott Jill RN, PhD 6 Clark Lauren RN, PhD 5 Mellis Karen BSM 5 Address correspondence to Katherine Jones, RN, PhD, FAAN, School of Nursing, Yale University, 100 Church St. S., Box 9740, New Haven, CT 06536-0740. E-mail: katherine.jones@yale.edu 8 2004 44 4 469 478 27 1 2004 7 7 2003 The Gerontological Society of America 2004 Purpose: Effective pain management remains a serious problem in the nursing home setting. Barriers to achieving optimal pain practices include staff knowledge deficits, biases, and attitudes that influence assessment and management of the residents' pain. Design and Methods: Twelve nursing homes participated in this intervention study: six treatment homes and six control homes, divided evenly between urban and rural locations. Three hundred licensed and unlicensed nursing home staff members completed written knowledge and attitude surveys at baseline, and 378 staff members completed the surveys after intervention implementation. Results: Baseline results revealed notable knowledge deficits in the areas of pharmacology, drug addiction and dependence, side effect management, and nonpharmacologic management-strategy effectiveness. Significant differences were noted by job title (registered nurse/licensed practical nurse/certified nursing assistant). Case studies displayed a knowledge application problem, with nurses often filtering resident pain reports through observed resident behaviors. The intervention led to significant improvement in knowledge scores in some, but not all, the treatment homes. Perceived barriers to effective pain management showed a significant decline across all study nursing homes. Implications: Knowledge deficits related to pain management persist in nursing homes. An interactive multifaceted educational program was only partially successful in improving knowledge across settings and job categories. Attitudes and beliefs appear more difficult to change, whereas environmental and contextual factors appeared to be reducing perceived barriers to effective pain management across all participating nursing homes. Pain Nursing homes Staff knowledge Attitudes hwp-legacy-fpage 469 hwp-legacy-dochead RESEARCH ARTICLE Pain is common among nursing home residents, although it is often underreported, underassessed, and undertreated (Ferrell, 1995; Slyk, 1999; Stein & Ferrell, 1996). The American Geriatrics Society (AGS) Panel on Persistent Pain in Older Persons (2002) estimated that 45–80% of nursing home residents have substantial pain. Studies have documented that 25–26% of those with daily pain received no analgesia (Bernabei et al., 1998; Won, Lapane, Gambassi, Bernabei, Mor, & Lipsitz, 1999). A high prevalence of dementia, sensory impairments, and disability in the nursing home population makes assessment and management of pain more difficult (AGS, 2002). The consequences of poor pain management include sleep deprivation, poor nutrition, depression, anxiety, agitation, decreased activity, delayed healing, and lower overall quality of life (Ferrell, 1995; Ferrell, Ferrell, & Rivera, 1995; Herr, 2002; Parmelee, Katz, & Lawton, 1991; Stein, 2001). Lack of knowledge about pain and its treatment remains an important barrier to effective pain management. Clinicians are unsure about the characteristics of chronic pain and the appropriate use of pain medications in the older adult population (Brockopp, Brockopp, Warden, Wilson, Carpenter, & Vandeveer, 1998; McCaffery & Ferrell, 1997). Pain may be expected to increase with aging, fostering a belief that one should learn to “live with it” (Brown & Williams, 1995; Ferrell, 1996). These beliefs inhibit older adults from reporting pain and decrease clinician readiness to treat pain (Davis, Hiemenz, & White, 2002). Staff Knowledge and Attitudes About Pain Staff knowledge deficits result from limited or absent curriculum time devoted to pain management (Weissman, 1988) and inadequate information about pain treatment in textbooks (Ferrell, McCaffery, & Rhiner, 1992). Coyne and colleagues (1999) tested levels of knowledge about pain in hospital nurses and concluded that significant knowledge gaps exist. Mobily and Herr (1996) noted deficiencies in pain management knowledge among both nurses and physicians in the nursing home setting. Beliefs and attitudes are also important in shaping how people respond to pain (Parmelee, 1997; Strong, Ashton, & Chant, 1992). Nurses tend to undervalue residents' reports of pain and employ the most conservative approaches to pain management. Nurses' choice of analgesia may not correspond with the level of pain intensity reported by the patient (Coyne, Smith, Stein, Hieser, & Hoover, 1998). Nurses have a high degree of concern about addiction (Lander, 1990) and also worry about sedation, depression, and constipation, increasing their reluctance to medicate older adults with opioids (Herr & Mobily, 1997). Improving Knowledge and Attitudes Staff must be knowledgeable about pain and pain medications to dispel the many resident and family myths and misconceptions surrounding this topic (Herr, 2002). However, success with pain education programs has been inconsistent (Allcock, 1996; Bookbinder et al., 1996; Breitbart, Rosenfeld, & Passik, 1998; de Ronde, de Wit, van Dam, van Campen, den Hartog, & Klievink, 2000; Francke, Luiken, de Schepper, Abu-Saad, & Grypdonch, 1997; Titler et al., 1994; Wallace, Graham, Ventura, & Burke, 1997). A pain education program may increase nurses' knowledge of pain management but may be insufficient to change actual pain management behavior (Mobily & Herr, 1996) or pain outcomes (Carr & Thomas, 1997; Lloyd & McLauchlan, 1994). Staff attitudes and beliefs, resident and physician factors, and organizational issues may exert pressures that prevent more effective practices from being implemented or sustained over time. This article describes the results of a study to improve pain practices in nursing homes. Baseline and postintervention data included staff knowledge and attitudes about pain and its treatment in the nursing home setting, perceived barriers to effective pain practices within the nursing homes, and case studies requiring documentation of pain intensity and selection of a pain management strategy. Methods An intervention study to improve pain practices in nursing homes was funded by the Agency for Healthcare Research and Quality (AHRQ). The study aims included (a) improving staff knowledge and attitudes about pain, (b) improving pain policies and practices, and (c) developing and testing a multifaceted intervention to improve pain assessment and management in nursing homes. The study sample included 12 nursing homes: 6 located in urban areas and 6 located in rural areas of the state. The nursing homes, ranging in size from 65 to 180 beds, were both not for profit and for profit. The intervention was implemented in six nursing homes (three urban/three rural), whereas the rest served as control sites. Intervention The intervention was multifaceted and included educational and behavioral components. The educational aspects of the intervention included a comprehensive pain resource binder (given to all nursing homes in the study), four 30-min staff development sessions, a 23-min staff training video that included three resident vignettes describing three pain types (neuropathic, visceral, and somatic), single pain “factoids” for posting in visible areas of the nursing home, a 7-min resident educational video and pamphlet (both in English and in Spanish), and a continuing education seminar for physicians. Educational material was based on contents of the AGS (1998) and American Medical Directors Association (AMDA, 1999) clinical practice guidelines for pain in the elderly as well as the American Pain Society Principles of Analgesic Use for Acute and Cancer Pain (American Pain Society, 1999). The behavioral aspects of the intervention included designation of a three-member internal pain team (IPT), IPT development of a pain vital sign, and site visits with discussion of feedback reports, pain rounds, and consultations. The four educational sessions, which focused on pain assessment, pharmacologic management, pain communication, and integrative case analyses, were scheduled once every 5 weeks and delivered multiple times during the day, from early morning to late afternoon. The sessions were presented by members of the grant team (pain clinical nurse specialist, nurse–pharmacologist, and experts in communication and quality improvement). Although these sessions were targeted primarily at nursing staff, other employees were invited to attend. These educational sessions were followed by five site visits and meetings with the IPT, conducted primarily by the team pain specialist who was often accompanied by the principal investigator or geriatrician. The site visits were focused on working with the IPT, consisting of a certified nursing assistant (CNA), licensed practical nurse (LPN), and third member designated by the nursing home, but the IPTs were soon expanded to include the director of nursing (DON) and staff development coordinator. Pain consultations were provided by the team pain specialist and the physician during site visits and also via telephone between visits. The research team's physician and nurse–pharmacologist designed and conducted the physician seminar, which was scheduled at a time and location selected by the relevant physician groups. They used case vignettes from the facility and from the physicians' and researchers' personal practices to discuss assessing pain in cognitively impaired residents, prescribing an analgesic appropriate to the type and intensity of pain, equi-analgesic opioid dosing, and preventing and managing common analgesic side effects in the elderly. A list of physicians, mid-level providers, and consulting pharmacists who care for residents at each of the intervention facilities was obtained from the DON. The physician member of the research team contacted the facility's medical director at least twice to discuss the purpose of the seminar, seek advice about its scheduling, ask the director to prepare one or two examples of difficult pain management issues in the facility for discussion, and encourage attendance. Written invitations to the mealtime seminar were sent via mail. Food, continuing education credit, and points that could be used to lower malpractice insurance premiums were offered to those who attended. The physician seminars were targeted at the primary care physicians who regularly admitted residents to the relevant treatment nursing homes. Instruments The multidisciplinary research team modified and expanded two existing surveys of pain knowledge and attitudes (University of Wisconsin and City of Hope) (City of Hope, 2002) to align them with the geriatric pain management guidelines (AGS, 1998; AMDA, 1999) and the nursing home environment. The resulting survey included 36 knowledge items (true/false), 21 attitude items (5-point Likert scale ranging from strongly agree to strongly disagree), 2 short case studies requiring pain assessment and treatment decisions, and 14 possible barriers to effective pain management (four levels ranging from very important to not important at all). Items on the knowledge questionnaire included pain myths and misconceptions, pain assessment practices, pharmacologic and nonpharmacologic management, and concepts of addiction/tolerance. Pain and geriatrics experts reviewed the items for content validity. A version appropriate for CNAs was developed that used simpler language (eighth grade level) and excluded the medication management items. A Spanish version of the CNA questionnaire was also developed by a certified translator, back-translated by a bilingual team member, and verified by a native Spanish speaker on the research team. Overall internal consistency (KR-20) reliabilities of the knowledge test of licensed/professional and CNA surveys were.61 and.71, respectively, levels that are adequate for newly developed research measures (Nunnally & Bernstein, 1994). The attitude questions were developed by a subgroup of the investigators, who first identified key themes to be covered. These included religious traditions and beliefs about pain and suffering, beliefs about cultural and gender differences in the expression of pain, attitudes about why residents might complain about pain (e.g., to get attention), and how staff might respond to complaints about pain. The final attitude scales included general pain biases and attitudes; general beliefs about aging; beliefs and attitudes about the role of religion, culture, and gender; pain medication attitudes; and communication issues. Overall internal consistency (Cronbach's α) reliability for the attitude survey was.70. The barrier items were grouped into resident and family, physician, staff, and organizational categories. Staff barriers included knowledge deficits and communication issues. Physician barriers included knowledge deficits and reluctance to order opioids. Resident barriers included inability to report pain and reluctance to take pain medication. Organizational barriers included lack of drug availability and concerns about regulatory oversight. Respondents were asked to note how important each barrier was in their specific nursing home. Cronbach's α reliabilities for these subscales ranged from a minimum of.71 (organizational barriers) to a maximum of.87 (staff barriers). Reliability for the overall 14-item barrier scale was.93. Procedures Working with the DONs and staff development coordinators, the investigators distributed surveys and consent forms to nursing home staff during mandatory staff meetings or special staff development sessions. Baseline surveys were administered in February 2001; postintervention surveys were administered in November/December 2002. The implementation of the intervention began in January/February 2002 and lasted 9 months. Surveys were voluntary and anonymous, although each staff member was asked to create a unique identifier known only to the research participant for the purpose of matching baseline and postintervention surveys. The investigators collected completed surveys and signed consent forms at the end of each meeting. Small gifts (e.g., pens, Post-It Notes) and snacks were offered as incentives to participate in the survey process. Five staff members attending the baseline meeting and 20 attending the postintervention meeting declined to participate or completed only a part of the survey before returning it. Analysis Nested factorial analyses of variance (ANOVAs) and generalized linear models (GLMs) with generalized estimating equations (GEEs) were used to test the first aim of the study. This approach acknowledges the fact that respondents are not truly independent of each other. More specifically, staff members are clustered within the individual nursing homes that are either receiving the intervention or serving as controls. Independent variables included group (experimental/control), job title (registered nurse [RN], LPN, CNA), and change across time (baseline [round 1]/post intervention [round 2]). Those participants with repeated data (n = 43) were omitted from reported analyses, as were the other job titles (n = 74). One treatment nursing home (n = 50) was also omitted after failing to complete the intervention owing to major internal upheavals throughout the course of the study. Although, overall, 678 staff members returned surveys (300 at baseline and 378 post intervention), owing to the two reasons just mentioned as well as a variable amount of information missing on the independent variables, the sample size for analysis was 432 surveys: 176 at baseline and 256 post intervention. Results Table 1 displays the demographics of the analysis sample. As noted above, only 43 staff members completed the survey at both time periods, reflecting the high staff-turnover rates in nursing homes. Respondents from both time periods were predominantly female, White, and middle-aged and worked days. The largest single job category was CNA, and the largest minority/ethnic group was Hispanic/Latino (slightly <20% and reflective of Colorado population percentages). The percentage of CNA respondents increased significantly post intervention, whereas the percentage of RNs declined. Respondent tenure in the nursing homes averaged 5–6 years. This was a convenience sample, surveying only those staff members who attended the staff meetings on the date of survey administration and who agreed to sign a consent form. Knowledge Scores Mean treatment home knowledge scores increased from 69% to 71%, whereas mean control nursing home knowledge scores essentially did not change (from 68% to 67%). These results are somewhat misleading because of the difference in the distribution of job titles between the two data collection periods. The postintervention sample used for analysis contained many more unlicensed personnel than the baseline sample (62% of total vs 49% of total), driving the average postintervention scores down. For the overall analysis sample, RN knowledge scores increased from 75.4% to 78.7%, LPN knowledge scores increased from 69.1% to 73.8%, and CNA knowledge scores increased from 64.6% to 65.4%. ANOVA revealed that there was not a significant overall improvement in staff knowledge in the treatment homes after our intervention was implemented. There were significant overall differences in knowledge across the job titles (p <.001). Variability in the nursing homes within the treatment and control groups and very small numbers of staff in some cells precluded achievement of significant results at the intervention group level. Inability to perform repeated measures analysis due to the small number of participants who completed both surveys was also a barrier. Staff across all the nursing homes demonstrated similar knowledge deficits, which were not improved substantially after the intervention. There was underestimation of the potential effectiveness of nonpharmacologic measures such as distraction techniques and the ability of residents to sleep and interact socially while experiencing moderate to severe pain. Many staff members were unaware of safe and effective analgesia dosing levels and schedules for different types of analgesics. They were also uninformed about drug addiction, physical dependence and tolerance, and which types of laxatives are safest for the elderly. Case Studies Two case studies were included to assess the application of knowledge in a real-life resident pain scenario (see Appendix). Both cases presented the same demographic and treatment data; they differed only in described behavior of the resident when reporting level of pain to the nurse. Case A included no obvious pain behaviors, whereas Case B had a marked stereotypic response to pain. In Case A at baseline, 61% of the analysis sample recorded the resident's pain level below the resident-reported 7 on a scale of 0–10. In Case B at baseline, 37% of the analysis sample rated the resident's pain level above the resident-reported 7, whereas 18% rated this resident's pain level below 7. Recording a pain level incongruent with resident pain reports appeared to be a problem for all staff, although the CNAs were more likely to record a pain level different from that reported by the resident. In Case A, 76% of the CNAs at baseline said the pain level should be recorded as <7 contrasted with 47% of the professional staff. In Case B, 30% of the CNAs at baseline said the pain level should be recorded as below 7 compared with 5% of the professional staff. These percentages improved trivially post intervention. We also examined the change in responses for both cases after the intervention was implemented in the treatment homes. For Case A, treatment home RNs showed improvement across rounds, but this did not reach statistical significance as the majority had already rated pain correctly. Treatment home LPNs showed significant improvement (p =.028) across rounds, with the majority rating pain below 7 at baseline and at 7 post intervention. CNAs in treatment homes also showed significant improvement (p =.045) across rounds, as 20% more CNAs rated pain as 7 post intervention than at baseline. The majority still rated the pain level below 7, however. The control group participants' ratings of pain for this case showed mixed results over the study period; CNAs had the most improvement, although the majority underreported the resident's pain. For Case B, treatment home RNs did not change significantly, and 89% overall rated pain correctly. Treatment home LPNs showed significant improvement (p =.013) across the intervention period. Half had overrated pain at baseline; post intervention, 81% rated pain correctly. Although 12% more treatment home CNAs rated pain correctly post intervention, the change was not statistically significant. The majority still rated pain incorrectly. None of the changes for control group participants was significant. GLM/GEE analytic strategies with pain assessment coded as correct (a response of “7”) or incorrect (all other nonmissing responses) in the analysis sample showed significant job title and treatment group differences. For Case A (p =.012), RNs were 2.6 times more likely than LPNs and 8.7 times more likely than CNAs to choose the correct pain level. LPNs were 3.3 times more likely than CNAs to choose the correct pain level. Treatment group staff members generally were 2.5 times more likely to choose the correct pain level than control group staff members (p =.014). For Case B (p =.011), RNs were 2.1 times more likely to choose the correct pain level than LPNs and 8.0 times more likely than CNAs. LPNs were 3.8 times more likely to choose the correct pain level than CNAs. Treatment group staff members generally were 4.0 times more likely to choose the correct pain level than control staff members (p =.002). Respondents (excluding the CNAs) were also asked to choose a management strategy for the two scenarios. Responses were classified as conservative if the treatment selected was less intensive than the previous dose (ongoing assessment), unchanged if the treatment was similar in intensity to the previous dose (ibuprofen 400 mg now, oxycodone 5 mg now, oxycodone 10 mg in 1 hr), and aggressive if the recommended treatment exceeded the previous dose (oxycodone 10 mg now). At baseline, treatment approaches similar to previous dose were more frequently selected for Case A (with the modal response being to medicate with ibuprofen). At baseline, more than half of the analysis sample (50.6%) chose more aggressive therapy for Case B (with the modal response being to medicate with oxycodone 10 mg now). There were no differences in approach between RNs and LPNs. Analysis of patterns between treatment and control home nurses for Case A showed that even though more treatment home RNs selected aggressive treatment post intervention, the improvement was not significant. Control home RNs did as well or better. For Case B, although the majority of treatment home RNs changed to an aggressive treatment strategy post intervention, the shift was not statistically significant. RNs in the control homes showed a similar but smaller association between time period and aggressiveness of the therapy. LPNs showed essentially no improvement patterns over time for either case. Analysis with GLM/GEE of the aggressiveness of the management strategy showed treatment group differences only for Case A (p =.002). Treatment group staff members generally were 2.5 times more likely to have chosen the most aggressive strategy than were control group staff members. Staff Attitudes Table 2 presents the results of the staff attitudes and beliefs section of the survey. The items with the most negative responses were as follows:Residents might believe that pain and suffering are necessary based on religious beliefs.Some cultural groups are more emotional in their response to pain.Residents/families have a right to expect total pain relief.I want to be known as a person who doesn't complain about pain.Some residents exaggerate their complaints to get attention.Residents are embarrassed to tell their nurses they are hurting. The intervention did not have much effect on staff attitudes between baseline and postintervention periods. ANOVA showed that there were no significant differences in overall attitudes between treatment and control nursing homes after the intervention period. However, there were job classification differences (p <.001). Multiple comparison procedures revealed that CNA attitudes were significantly different from those of RNs and LPNs. Scores for the CNAs indicated a higher likelihood of having beliefs that could interfere with optimal pain management. Barriers Staff perceived a high level of barriers to effective pain management within their nursing homes prior to the intervention phase of the study. As shown in Tables 3 and 4, the percentage of staff reporting little or no importance of the barriers increased markedly between the two data collection periods, and the average barrier scale score (higher numbers reflect less perceived importance) improved over the intervention period (p <.001). This finding was true of both treatment and control homes and probably was related to contextual and environmental factors, including the implementation of the Centers for Medicare and Medicaid Services Nursing Home Compare Report Card in Colorado and the presence of study data collectors in all the nursing homes every 3 months. Discussion The multifaceted intervention implemented in this study did not significantly improve staff knowledge in the treatment homes, although there was notable improvement in staff knowledge across the nursing job titles. Because of variation in success of implementing our intervention across the individual nursing homes, we failed to achieve statistical significance, but knowledge scores did improve in selected nursing homes, and attitude and perceived barriers scores moved in the right direction. A major challenge in this study was the high turnover rates among all the staff, leading to a completely new sample of staff members responding to the survey at the postintervention time period. These staff all received different exposure to the intervention, diminishing its potency. Qualitative analysis of study field notes showed that, in general, those nursing homes with more stable staffing, especially of the DON, administrator, and staff development coordinator, as well as stronger leadership commitment and involvement in the project had greater gains in knowledge and positive attitudes than those homes with less stable staffing and less involved leadership. Attendance at the staff training sessions varied across the nursing homes, with the more successful homes mandating attendance and facilitating coverage so staff members could leave the unit for the sessions. The treatment nursing homes in rural locations had the largest number of physicians attending the continuing medical education seminar (overall attendance across all six treatment homes ranged from 4% to 70% of invited providers). Two of these homes had consistent leadership involvement and no turnover in key positions and showed the greatest improvement in knowledge over time. The remaining rural treatment home had improvement only in LPN knowledge scores. This facility had repeated administrator turnover, an invisible staff development coordinator, DON turnover, little commitment to the project, and poor attendance at the training sessions. One urban treatment home that showed substantial improvement for RN and CNA job categories was Joint Commissions for the Accreditation of Healthcare Organizations (JCAHO) accredited and experienced turnover of the DON position only; however, few physicians attended the continuing education seminar, and attendance at the training sessions was low. The remaining urban nursing home showed little change in knowledge scores across the three job categories. This facility experienced administrator and staff development coordinator turnover, had poor attendance at the physician continuing education seminar, and observed conflict between the CNAs and nursing staff. On the other hand, the two control nursing homes that showed a significant improvement in RN knowledge both had volunteered to work closely with the state quality improvement organization on their pain initiative during the last year of this study. To achieve sustainable quality improvements in nursing homes, the retention and leadership problems must be addressed. In addition, intervention strategies that are succinct and increase the ability of the nursing home to deliver its own educational programs periodically as new staff come on board would probably be more successful. Better strategies to encourage physicians to increase their knowledge of pain management in the elderly are also required. Staff must be knowledgeable about pain and pain medications to dispel the many resident and family myths and misconceptions surrounding this topic (Herr, 2002). Results of the staff knowledge survey reiterated the findings of surveys in other settings: Knowledge deficits regarding pain assessment and management prevail. Lack of adequate knowledge is a critical factor in the suboptimal pain management documented in large proportions of the nursing home population. Effective pain assessment and management strategies must be based on scientific knowledge and research and systematically applied in patient care delivery (Agency for Health Care Policy and Research, 1992). Without such a foundation, pain treatment will be sporadic, ineffective, or both (Coyne et al., 1999). Existing evidence-based clinical practice guidelines for pain management are apparently not being utilized. None of the nursing homes in our study had clinical practice guidelines for pain management on the units. The inclusion of pain as a quality measure for both short-term and long-term residents in the Centers for Medicare and Medicaid Services Nursing Home Report Card is intended to provide an incentive for nursing homes to improve their practices in this area (Department of Health and Human Services, Centers for Medicare and Medicaid Services, n.d.). However, more aggressive assessment and identification of pain lead to higher pain scores on the report card, a possible disincentive to mount serious pain management programs. In addition, receiving a deficiency from the state related to pain may also serve as an incentive to engage more seriously in pain improvement programs. Our data indicate that two important aspects of pain management require particular educational attention. Pharmacologic pain management is one area where there are significant knowledge deficits. Items related to appropriateness and side effects of medications were most frequently missed. There was also confusion about the concepts (definitions) of addiction, dependence, and tolerance. The case study analyses also revealed a reluctance to use aggressive pain management strategies, even in the face of reported severe pain and observed pain behaviors. The use of nonpharmacologic strategies such as massage, positioning, and distraction also needs increased attention. The 1998 and 2002 updated AGS pain guidelines consider educational programs about pain management an essential element of training and orientation programs for all employees and affiliated professionals in long-term care facilities. Nursing assistants and other direct-care staff should receive training and mentoring in pain recognition. A special emphasis needs to be placed on programs for the unlicensed staff, as they have lower knowledge levels and more negative attitudes and beliefs than the licensed staff yet spend an average of 2 hr/day, in contrast to the 45 min/day spent by licensed nurses, with each resident (Kramer, Eilertsen, Lin, Martau, & Hutt, 2000). They therefore have a much greater opportunity to observe behavior changes that could be indicative of pain. The study team, however, noted little involvement of CNAs in care planning, rounds, or reports, and their input was infrequently solicited and sometimes ignored. Any educational interventions that are provided need to be designed so they can be offered on an ongoing basis in the nursing home and delivered with interactive, easy-to-understand, and quick-to-use material. Increased staff knowledge by itself may be insufficient to achieve substantial practice changes as demonstrated in our case studies, which showed significant improvement in pain assessment by the nursing home staff but not in aggressiveness of pain treatment. Shifts in attitudes and beliefs are essential in the following areas:Pain is a necessary and acceptable part of aging;Residents from ethnic minority groups experience and report their pain in a more emotional manner; andHealth care providers know more about the pain experience than the person reporting the pain. This will require alterations in commonly held stereotypes about pain and aging. Our research team has developed a 7-min video about pain, in English and Spanish, that can be shown to new residents and to family members on resident council and family education nights. Several of our nursing homes show this video to new staff members during orientation. Although the tools for effective pain management have long been available, the best method for disseminating this knowledge remains elusive. This study demonstrates that interactive educational programs may improve knowledge regarding pain management in some nursing homes and within some job titles, but they might not be sufficient to change clinical management behaviors. A multifaceted intervention has to overcome multiple barriers in the nursing home setting. In the case of pain, knowledge and attitudes held by staff, residents, and physicians must all be addressed. The research translation literature indicates that strategies such as self-administered audits of key metrics, quality collaboratives, and designation of internal change champions may be more successful in changing clinical practices. However, different dissemination strategies work for different populations and in different settings. Little work has been carried out in the nursing home setting, so the evidence base for effective translation strategies is limited at this point. Rantz and colleagues (2001) have found that performance feedback reports and on-site consultations have been successful for achieving quality improvement in their sample of nursing homes. But organizational readiness, willingness, and capacity to change must also be considered. Constant turnover of administrators and staff makes implementation of quality improvement interventions difficult and sustainability of improvement over time even more challenging. Appendix Case Studies Case A. Ida is a 78 year old woman whose only medical problems are osteoarthritis and some memory loss. She received 5 mg of oxycodone 3 hours ago. As you enter her room, she smiles at you and continues talking and joking with her daughter. Your assessment reveals the following information: alert and unsedated; BP = 120/80; HR = 80; R = 18. She rates her pain as a “7” and describes it as achy, throbbing and unchanged in the last 3 hours. Circle the number that best represents your assessment of Ida's pain to be marked on her medical record. (scale 0–10 provided). Her current orders for analgesia are oxycodone 5–10 mg PO q 3–4 hours PRN for pain and ibuprofen 400 mg PO TID PRN pain. Of the following, check what is best for the nurse to administer at this time:no analgesia at this time; continue to assess5 mg oxycodone now10 mg oxycodone now10 mg oxycodone in one houribuprofen 400 mg now Case B. Same case, except with the following substitution: As you enter her room, she lies quietly in bed, grimaces, and guards her hip as she turns in bed. This work was supported by Grant U18-HS11093 from the Agency for Healthcare Research and Quality to the School of Nursing, University of Colorado Health Sciences Center (principal investigator Katherine Jones). 1 School of Nursing, Yale University, New Haven, CT. 2 University of Colorado Hospital, Denver. 3 School of Nursing, University of Utah, Salt Lake City. 4 School of Medicine, University of Colorado Health Sciences Center, Denver. 5 School of Nursing, University of Colorado Health Sciences Center, Denver. 6 School of Nursing, University of Missouri, Columbia. Decision Editor: Linda S. Noelker, PhD Table 1. Demographics of Nursing Home Staff Respondents With Nursing Job Titles. Variable Baseline Post Intervention Age (y) n = 148 n = 208     Mean ± SD 41.4 ± 12.8 39.4 ± 12.2     Range 17–71 18–73 Sex n = 176 n = 251     Female (%) 93 93 Ethnicity/race (%) n = 169 n = 236     White 75 74     Hispanic 18 19     Other 7 7 Job title (%) n = 176 n = 256     RN 25 14     LPN 26 24     Nursing assistant 49 62 Tenure (y)     Current job title n = 167 n = 238     M ± SD 9.7 ± 9.2 9.4 ± 9.1     Current facility n = 161 n = 239     M ± SD 6.0 ± 7.1 4.8 ± 7.2     Nursing home care n = 165 n = 232     M ± SD 10.6 ± 8.8 10.3 ± 9.4 Shift (%) n = 176 n = 247     Days 52 46     Evenings 19 26     Nights 12 10     Other 17 18 Notes: RN = registered nurse; LPN = licensed practical nurse. Table 2. Staff Attitude and Belief Changes by Time and Treatment/Control Group. Baseline (% D/SD) Post Intervention (% D/SD) Attitude Item n Treatment Control n Treatment Control Older people will bore you to death talking about pain. 175 88 87 254 74 75 I want to be known as person who doesn't complain. 175 38 34 252 25 24 Older women complain about pain more than men. 172 58 54 254 47 52 Men are supposed to be brave—not show pain. 175 96 91 253 86 83 Some residents exaggerate pain to get attention. 174 33 47 254 59 36 Even when resident is in pain, hard to get someone to pay attention. 169 84 80 254 76 70 Residents are embarrassed to tell staff they are hurting. 173 53 56 254 44 48 Some cultural groups are more emotional in their pain response. 171 30 22 254 16 25 Residents may believe pain and suffering are necessary. 172 29 22 254 21 21 Learning to live with pain builds character. 174 93 92 251 84 80 Life is painful—no getting around that. 174 72 77 252 65 57 Suffering purifies ourselves for life to come. 174 92 89 252 85 76 If residents get around, have to question their pain. 173 93 84 250 81 76 Good residents avoid talking about pain. 174 94 94 250 84 85 Easier for residents to put up with pain than side effects. 171 85 86 252 73 69 Pain medication should be given only when pain is severe. 173 88 87 252 87 80 More experience with pain leads to better tolerance of pain. 171 92 83 252 82 80 Residents should experience discomfort before next dose. 173 94 88 252 84 79 Residents/families have right to expect total relief. 172 27 32 250 36 35 Notes: D/SD = disagree/strongly disagree with the statement. Table 3. Barrier Changes by Time and Treatment/Control Group. Baseline (% Little/No Importance) Post Intervention (% Little/No Importance) Barrier n Treatment Control n Treatment Control Resident reluctance to report pain 172 20 25 252 61 65 Resident inability to report pain 173 12 8 252 40 48 Physician reluctance to prescribe 172 20 25 242 65 63 Nurse reluctance to administer 170 30 35 251 74 69 Resident reluctance to take 172 25 26 250 63 58 Inadequate time to assess 171 30 28 251 71 68 Inadequate communication 171 29 21 252 76 73 Inadequate staff knowledge 169 32 22 251 81 71 Inadequate physician knowledge 171 25 22 250 76 66 Influence state and federal regulations 171 19 24 244 67 66 Availability of drugs 168 37 37 238 83 77 Resident fear of side effects 171 45 37 249 79 74 Nurses' concern about side effects 171 37 34 247 78 74 Family's concern about side effects 171 30 22 249 69 66 Notes: % Little/No Importance = percentage of staff agreeing that a specific barrier is of little or no importance in their facility. Table 4. Changes in Grouped Barrier Scores Between Baseline and Post Intervention. Type of Barrier Baseline (SD) n Post Intervention (SD) n Resident/family* 1.9 (0.6) 174 2.7 (0.6) 254 Staff* 2.0 (0.8) 173 3.0 (0.7) 254 Physician* 1.8 (0.8) 173 3.0 (0.9) 253 Organizational* 2.0 (0.8) 171 3.0 (0.9) 251 Overall* 1.9 (0.6) 174 2.9 (0.6) 254 *p < 001. References Agency for Health Care Policy and Research. (1992). Acute pain management: Operative or medical procedures and trauma. In Clinical practice guidelines. Rockville, MD: U.S. Department of Health and Human Services. Allcock, N., (1996). Factors affecting the assessment of postoperative pain: A literature review. Journal of Advanced Nursing, 24, 1144-1151. American Geriatrics Society Panel on Chronic Pain in Older Persons. (1998). The management of chronic pain in older persons. Journal of the American Geriatrics Society, 46, 635-651. American Geriatrics Society Panel on Persistent Pain in Older Persons. (2002). 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Swanson (Eds.), Advances in gerontological nursing: Chronic illness and the older adult (pp. 82–111). New York: Springer Publishing. Kramer, A. M., Eilertsen, T. B., Lin, M., Martau, J., & Hutt, E., (2000). Synthesis of findings on effects of staffing on quality of care. In M. Feuerberg (Ed.), Appropriateness of minimum nurse staffing ratios in nursing homes: Phase one (pp. 12-1–12-15). Baltimore: Health Care Financing Administration. Lander, J. L., (1990). Fallacies and phobias about addiction and pain. British Journal of Addiction, 85, 803-809. Lloyd, G., & McLauchlan, A., (1994). Nurses' attitudes towards management of pain. Nursing Times, 90, 40-43. McCaffery, M., & Ferrell, B., (1997). Nurses' knowledge of pain assessment and management: How much progress have we made? Journal of Pain and Symptom Management, 14, 175-188. Mobily, P. R., & Herr, K. A., (1996). Barriers to managing residents' pain. Contemporary Long Term Care, 19, 60A-60B. Nunnally, J. C., & Bernstein, I. 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Strong, J., Ashton, R., & Chant, D., (1992). The measurement of attitudes towards and beliefs about pain. Pain, 48, 227-236. Titler, M. G., Moss, L., Greiner, J., Alpen, M., Jones, G., & Olson, K., et al (1994). Research utilization in critical care: An exemplar. Clinical Issues, 5, 124-132. Wallace, K. G., Graham, K. M., Ventura, M. R., & Burke, R., (1997). Lessons learned in implementing a staff education program in pain management in the acute care setting. Journal of Nursing Staff Development, 13, 24-31. Weissman, D. E., (1988). Cancer pain education: A call for role models. Journal of Clinical Oncology, 6, 1793-1794. Won, A., Lapane, K., Gambassi, G., Bernabei, R., Mor, V., & Lipsitz, L. A., (1999). Correlates and management of nonmalignant pain in the nursing home. Journal of the American Geriatrics Society, 47, 936-942. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A40CB48BC7DDEB336E57BE89F888B04EE4BED39.txt b/test/dataset/in/resources/corpus/Clean_0A40CB48BC7DDEB336E57BE89F888B04EE4BED39.txt new file mode 100644 index 0000000..bac9072 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A40CB48BC7DDEB336E57BE89F888B04EE4BED39.txt @@ -0,0 +1 @@ +annoncannoncAnnals of Oncology1569-80410923-7534Oxford University Press10.1093/annonc/mdm087original articlesepidemiologyUp-to-date survival estimates and historical trends of cutaneous malignant melanoma in the south-east of The Netherlandsde VriesE12*HoutermanS2Janssen-HeijnenMLG2NijstenT3van de SchansSAM2EggermontAMM4CoeberghJWW121Department of Public Health, Erasmus MC University Medical Centre Rotterdam, Rotterdam2Eindhoven Cancer Registry, Comprehensive Cancer Centre South, Eindhoven3Department of Dermatology, Erasmus MC University Medical Centre Rotterdam, Rotterdam4Department of Surgical Oncology, Erasmus MC University Medical Centre Rotterdam, Rotterdam, The Netherlands*Correspondence to: Dr E. de Vries, Department of Public Health, Erasmus MC University Medical Centre Rotterdam, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Tel: +31-10-4087730; Fax: +31-10-4638475; E-mail: e.devries@erasmusmc.nl62007134200718611101116158200623120071222007© 2007 European Society for Medical Oncology2007Background: We present survival outcomes of patients registered in the Dutch population-based Eindhoven Cancer Registry (ECR).Patients and methods: Data on patients diagnosed with a melanoma between 1980 and 2002 were obtained from the ECR. Data on vital status up to 1 January 2005 were obtained, up-to-date survival rates were calculated using period analysis. Multivariate analyses were carried out using Cox proportional hazards model.Results: Ten-year crude survival rates were 82% for women and 60% for men (P < 0.05). Thin melanomas (Breslow thickness ≤ 2.0 mm) had 5-year crude survival rates >74%, for melanomas >4.0 mm these rates were <65% (P < 0.05). In the early 1980s, 5-year relative survival rates were 84% and 62% for young (<60 years) women and men, and 66% and 69%, respectively, for the elderly (aged 60+). In the period 2000–2002, these rates had improved to >90% for females and to >72% for males. Multivariate analyses showed increased hazard ratios with increasing age and Breslow thickness, being male, having a melanoma on the trunk or unknown sites and having a nodular melanoma.Conclusions: Despite the absence of improvements in treatment options for melanoma, survival improved significantly, except for elderly males.crude survivalcutaneous malignant melanomaperiod analysispopulation-basedrelative survivalintroductionIncidence and survival rates of cutaneous malignant melanoma (melanoma) have been increasing markedly over the past decades in Caucasian populations [1–3]. There is, however, a shortage of up-to-date population-based survival statistics of melanoma patients. Most available survival estimates are for patients diagnosed up to 1996 [1–6]; very few recent population-based estimates are available [7].Clinicians and policy makers should use the most recent available information about survival of newly diagnosed cancer patients; however, almost all available survival estimates are based on patients diagnosed up to 1996. A new method of survival analysis, the so-called period analysis [8], results in more up-to-date long-term survival rates than the traditional methods for estimating survival rates [9–11]. This is especially important when there have been recent improvements in survival, as is the case for melanoma [12].In this study, we evaluated the trends in incidence and survival of cutaneous malignant melanoma using information from the Eindhoven Cancer Registry (ECR). In order to provide up-to-date survival statistics, we estimated relative and crude survival rates of patients diagnosed with cutaneous melanoma by means of period analysis, stratifying or correcting for age, Breslow thickness, site and histogenetic subtype. As there are gender differences in both incidence and survival statistics, all analyses were stratified by sex. Multivariate analyses were carried out to simultaneously control for all these factors and investigate their effects on survival.patients and methodsThe ECR collects data on all patients with newly diagnosed cancer in the southeastern part of The Netherlands. The registry serves a population of 2.4 million inhabitants. The area offers good access to specialised medical care in 10 general hospitals and two large radiotherapy institutes. In the last 25 years, the number of dermatologists in the region increased from ∼30–45. Until 1988, the Registry only collected data on patients from the eastern part of the region, after which another part (the west) was also included. Data on vital status, available until 1 January 2005, was obtained from the hospital records and the death registry of the Central Bureau for Genealogy (that registers all deceased in The Netherlands via the municipal civil registries). Incidence rates were age standardised using the European standard population, resulting in European standardised sates.Distribution of melanoma cases diagnosed in the period 1980–2002 by sex, age, body site and histogenetic type were calculated, excluding noncutaneous sites. Trends in incidence were estimated by calculating the estimated annual percentage change (EAPC). EAPC's were calculated by fitting a regression line to the natural logarithm of the rates (y) using calendar year (x) as regressor variable, i.e. y = mx + b; the EAPC = 100 (em − 1). One EAPC value was calculated for the whole period 1980–2002 because visual inspection of trends in incidence rates in this period showed no clear changes in the trends, making join point analyses unnecessary.Survival times were calculated from the date of diagnosis of primary melanoma until the date of death and considered censored for patients who were alive at their last date of follow-up. In addition to crude survival rates, relative survival rates were calculated. Relative survival is an estimation of disease-specific survival, as it is corrected for the background mortality in the general population within the same age- and sex groups. As different (sub)types of cancer often occur in patient groups with different age structures, comparisons between patient groups, registries or countries, should use relative rather than crude survival estimates. The relative survival is calculated as the crude survival rate among cancer patients divided by the expected survival rate from the general population with the same sex and age structure [14]. Standard errors were calculated according to Greenwood's method [15].In order to provide up-to-date estimates of long-term relative survival for a recent time period (1992–2002), period analysis was used. This means that all observations included in the analyses are left-truncated at the beginning of the period of interest in addition to being right censored at its end. The method of period analysis is described in detail elsewhere [8, 16, 17]. Relative survival estimates for patients diagnosed in earlier time periods were made using traditional cohort-based analysis. Relative survival rates were calculated with the SAS computer package (SAS Institute Inc., Cary, NC 1999), using a publicly available macro[18].We analysed relative survival by the known prognostic factors that were available from the cancer registry: age at diagnosis, sex, body site, histogenetic subtype and Breslow thickness of the melanomas. We subdivided the patients into young (<60 years) and elderly (60 years and older) melanoma patients and carried out all analyses separately for men and women. Body site was subdivided into head and neck, arms, trunk, legs, other and unknown body sites. Histogenetic subtype was divided into superficial spreading melanomas (SSM), nodular melanomas (NM), lentigo maligna melanomas and other melanomas, which included all other types of melanomas and melanomas of which histogenetic subtype was unknown. We made a subdivision into melanomas with a Breslow thickness thinner than 1.0 mm, between 1.01 and 2.0 mm, 2.01–4.0 mm and >4.0 mm. Eleven per cent of male and 7% of female melanoma patients had an unknown Breslow thickness.Multivariate analyses were carried out using the Cox proportional hazards model, including the variables year of diagnosis, age and Breslow thickness as continuous variables, sex, body site and histogenetic subtype in the same categories as used for the stratified univariate analyses described above.resultsA total of 1644 male and 2372 female melanoma patients were included in the analyses (Table 1). Incidence of melanoma has been increasing rapidly. Current and past incidence rates per 100 000 person-years by sex, age, body site, histogenetic subtype and Breslow thickness of the melanomas are given in Table 1. Overall, increases in incidence during the period 1980–2002 were 4.8% for men and 4.3% for women. Strong increases (>4.5%) were observed for melanomas on the trunk (both sexes), melanomas on the arms (women), SSM and NM (both sexes), melanomas with a thickness of ≤1.0 mm (both sexes) and for melanomas with a thickness >1.0 mm (men).Table 1.Age-standardised incidence rates (European standard population) per 100 000 person-years of melanoma patients diagnosed between 1980 and 2002 in the Eindhoven Cancer Registry regionEuropean standardised rateTotal (N)EAPC (%)P value1980–19841985–19891990–19941995–19992000–2002Males    Total5.56.48.310.812.216444.8<0.001Age (years)    <604.54.75.88.18.310643.80.16    60+11.315.621.225.032.95805.4<0.001Body site    Trunk1.82.63.44.95.27036.7<0.001    Head and neck1.11.21.42.02.62984.1<0.001    Arm1.30.91.41.42.02603.70.01    Leg0.81.21.41.91.82774.3<0.001    X0.50.50.60.70.71062.40.02Histogenetic subtype    SSM0.71.73.04.45.06098.4<0.001    NM0.11.01.32.02.226812.5<0.001    LMM0.10.10.20.10.4322.80.21    Other4.73.63.84.24.67350.30.713Breslow thickness (in mm)    ≤1.01.31.52.34.04.35307.9<0.001    1.01–2.01.31.21.52.53.13644.9<0.001    2.01–4.01.41.42.42.02.23594.50.012    >4.00.81.20.91.41.62094.50.009    X0.71.11.21.01.11822.00.121Females    Total7.89.411.314.915.023724.3<0.001Age (years)    <607.08.39.813.113.016783.50.09    60+12,015,019,024,225,86944.10.01Body site    Trunk0.91.73.04.33.958510.5<0.001    Head and neck1.50.81.21.61.32621.80.30    Arm1.71.92.33.23.25025.4<0.001    Leg0.81.21.41.91.89502.8<0.001    X0.20.30.40.50.4730.60.77Histogenetic subtype    SSM0.63.25.36.88.310229.9<0.001    NM0.60.91.51.41.82677.0<0.001    LMM0.10.10.20.20.4482.80.24    Other6.55.24.36.44.51035−0.40.68Breslow thickness (in mm)    ≤1.02.13.24.97.48.210647.9<0.001    1.01–2.02.22.82.93.23.35673.60.004    2.01–4.01.61.92.02.32.04011.70.084    >4.01.00.90.71.01.01840.30.821    X0.90.70.90.90.51560.00.999EAPC, estimated annual percentage change; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma; Other, other and unspecified melanomas; X, unknown.Median age at diagnosis was higher for men (53 years) than women (49 years). Sixty-nine per cent of women and 54% of men were diagnosed with a relatively thin melanoma (Breslow thickness ≤ 2.0 mm). Among females, but not males, a strong shift to earlier diagnosis seems to have occurred; increases are only seen in the categories with thinner Breslow thickness, whereas no significant increases were observed for the categories with a Breslow thickness ≥2.0 mm (Table 1).Ten-year crude survival rates were 82% [95% confidence interval (CI) 78% to 86%] for females and 60% (54% to 66%) for males. Twenty-year relative survival rates were 12%–13% units higher than the crude survival rates: 93% (89% to 98%) for females and 72% (65% to 79%) for males (Figure 1).Figure 1.Relative and crude survival rates of melanoma cases diagnosed in the Eindhoven Cancer Registry region (period analysis 2000–2002).Ten-year crude and relative survival of head and neck melanomas were lower than that of other body sites among males, but only reached significance for crude survival, after taking the background mortality into account (relative survival) this difference was not longer significant (Table 2). Five-year survival rates of melanomas in the head and neck area and on the trunk were significantly higher for females than males; among males NMs exhibited a significantly worse survival than the other histogenetic subtypes.Table 2.Sex-specific 5- and 10-year crude and relative survival rates of melanoma patients diagnosed in the Eindhoven Cancer Registry region (period analysis 2000–2002)5-year relative survival rates10-year crude survival ratesCrude(95% CI)Relative(95% CI)Crude(95% CI)Relative(95% CI)Males    Overall69(64–74)76(71–81)60(54–66)72(65–79)Age    <60 (ref)76(71–81)78(73–84)71(65–77)74(68–81)    60+56a(47–65)72a(61–83)36a(24–48)64(42–86)Site    Arm (ref)81(70–92)86(75–97)70(55–85)78(61–95)    Trunk73(66–80)78(71–85)68(61–75)79(70–88)    Head and neck55a(43–67)66(52–81)40a(26–54)Na    Leg79(69–89)86(75–97)70(56–84)81(65–97)Histogenetic subtype    SSM (ref)80(74–86)86(79–93)75(67–83)86(77–95)    NM55a(43–67)60a(47–73)47a(32–62)41a(28–54)    LMMNaNaNaNa    Other66a(59–73)73(65–81)58a(49–67)70(59–81)Breslow thickness (in mm)    ≤1.0 (ref)89(85–93)98(91–105)87(80–94)101(93–109)    1.01–2.074a(63–85)80(68–92)62a(48–76)71a(55–87)    2.01–4.057a(41–73)63a(46–80)Na–Na–    >4.0NaNaNa–Na–Females    Overall89(86–92)94(91–97)82(78–86)93(89–98)Age    <60 (ref)94(91–97)95(93–98)91(88–94)93(90–96)    60+78a(71–85)93(85–101)59a(47–71)94(75–113)Site    Arm (ref)89(83–95)94(88–100)84(76–92)96(87–105)    Trunk89(84–94)91(86–96)86(80–92)91(85–98)    Head and neck88(79–97)102(92–112)62(46–78)89(66–113)    Leg92(88–96)96(92–100)86(80–92)95(88–102)Histogenetic subtype    SSM (ref)93(90–96)97(94–100)86(80–92)96(90–102)    NM88(80–96)97(88–106)74(59–89)89(72–106)    LMMNaNaNaNa    Other85a(80–90)89a(84–94)80(74–86)91(85–98)Breslow thickness (in mm)    ≤1.0 (ref)96(93–99)100(97–103)93(89–97)102(98–106)    1.01–2.084a(75–93)88(79–97)81(71–91)90(79–101)    2.01–4.081(68–94)90(75–105)Na–Na–    >4.065a(48–82)70a(51–89)Na–Na–aSignificantly different (P < 0.05) from (ref) category.Na, not sufficient cases left to calculate survival rates (i.e. <10 cases surviving).CI, confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma; other, other and unspecified melanomas.Trends in 5- and 10-year relative survival over time are depicted in Figure 2. Since the early 1980s, survival of melanoma has improved substantially, except for elderly males, whose survival rates remained stable. In the early 1980s, 5-year relative survival was 84% (95% CI 77% to 91%) and 62% (51% to 73%) for young (<60 years of age) females and males, and 66% (46% to 86%) and 69% (42% to 96%) for elderly (aged 60 and over) females and males, respectively. This improved to >90% for females of all ages [young females 95% (92% to 98%) and elderly females 93% (85% to 101%)] in the period 2000–2002. Five-year relative survival rates in 2000–2002 for young and elderly males were 78% (73% to 83%) and 72% (61% to 83%), respectively. These increases were statistically significant for females only and borderline significant for young males. Ten-year relative survival rates also increased substantially for females, reaching significance for the age group <60 years (Figure 2).Figure 2.Trends in 5- and 10-year relative survival 1980–2002, Eindhoven Cancer Registry region, by sex and age group, with 95% confidence intervals (RS, relative survival. **The number of men surviving >5 years was too small to calculate 10-year relative survival rates in the period 1980–1989).The multivariate analyses showed that, taking into account tumour-specific characteristics such as body site, histogenetic subtype and Breslow thickness, the survival of melanoma patients has improved over the years (Table 3). For every more recent year of diagnosis, the hazard ratio of dying decreased with 4% compared with the previous year. These analyses also show that being male or having a melanoma on the trunk, having a NM or a melanoma with higher Breslow thickness significantly increased the hazard of death (Table 3).Table 3.Results of the multivariate Cox regression analyses for melanoma patients diagnosed between 1993 and 2002 in the population-based Eindhoven Cancer RegistryVariableHR(95% CI)Year of diagnosis    Continuous (increasing)0.96(0.92–1.00)Age    Continuous1.05(1.04–1.05)Sex    Female (ref)1.00    Male2.00(1.60–2.49)Site    Arms (ref)1.00    Trunk1.52(1.10–2.10)    Head and neck1.34(0.94–1.91)    Legs1.14(0.82–1.60)Histogenetic subtype    SSM (ref)1.00    NM2.01(1.51–2.68)    LMM0.78(0.33–1.81)    Other1.65(1.28–2.13)Breslow thickness    Continuous1.14(1.11–1.17)HR, hazard ratio; CI, confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma.discussionWe reported up-to-date population-based survival rates of melanoma patients diagnosed in the southeastern Netherlands. Despite the absence of major improvements in melanoma treatment [19, 20], survival rates have improved substantially over the past two decades, except for elderly men. It is unclear why elderly men did not benefit from the improvements that were accomplished for the other groups, although it is known that females and younger males are more sensitive for sun-safe messages [21] and therefore, melanomas are probably detected earlier in this group. These improvements in survival rates are surprising as marked increases in incidence were still seen in the melanomas with high Breslow thickness and NM. Earlier detection seemed to have occurred among females, where there were no significant increases in the group of melanomas with a Breslow thickness of ≥4.0 mm, whereas strong increases were observed in this category among males. Contrary to the United States, where Welch et al. [22] observed that the increases in incidence was largely the result of increased diagnostic scrutiny rather than real increases in the disease, the increases in melanoma incidence in the ECR population were not confined to early-stage cancer and mortality did increase [23].Relative survival estimates were always higher than crude survival statistics, due to the fact that the crude survival rates are corrected by the background mortality of the population. This is clearly illustrated in the case of head and neck melanomas, and melanomas occurring in people aged ≥60 years, where we observed differences of >20 per cent units. This phenomenon is caused by the fact that these are populations of elderly patients, in which the background mortality is high, hence, mortality due to melanoma only contributes a small part to the overall mortality.The observed survival rates are higher than most reported by other studies, especially for females [1–4, 24]. This is mostly due to the fact that we used more recent data and a method which produces more up-to-date estimates of survival [8, 16, 17]. Most other studies used traditional methods to calculate melanoma survival rates, resulting in estimates that represent survival rates of previous generations of melanoma patients, rather than the most current estimates. The available, but mostly outdated, population-based data on melanoma survival showed substantial differences in survival between countries [4] with marked improvement of 5-year survival rates over time [1, 3]. The most recently reported European average 5-year relative survival for melanoma patients diagnosed between 1990 and 1994 was 85% for women and 75% for men [4]. Five-year relative survival rates for Australians diagnosed with melanoma between 1992 and 1997 were 90% for males and 94.6% for females [25]. Surveillance, Epidemiology and End Results (SEER) statistics (United States) for whites diagnosed with melanoma between 1996 and 2002 were 90.3% (males) and 93.4% (females) [26]. Most European population-based studies used traditional methods for survival analysis; 5-year crude survival estimates being between 85% and 87% for women and 80% for men [2, 3], the most recent 5-year relative survival rates come from Yorkshire, England, and were 81.5% for women and 70% for men [7]. Five-year crude survival rates in the cohort used by the American Joint Committee on Cancer were only 73% and in the SEER registry 89% [27]. The American Joint Committee on Cancer (AJCC) melanoma staging system was designed to determine criteria to be used in the tumour–node–metastasis classification and stage grouping. The criteria were meant to be ‘relevant to current clinical practice’, ‘regularly incorporated in clinical trials’ and the required data should be ‘sufficiently easy for tumour registrars to identify in medical records to code staging information’ [28]. The staging system was tested in the AJCC cohort which included patients diagnosed as early as 1940 and used cohort analysis to estimate survival rates [24]. Moreover, the AJCC cohort was not population-based but rather influenced by varying referral patterns and clinical trial entry criteria to tertiary medical centers and cooperative groups. These features might have skewed the patients distribution overall compared with the general population [24]. These characteristics of the AJCC cohort and analyses cause the presented survival rates to be largely outdated and not representative of the general population. In this study, we presented population-based data of all patients diagnosed in the ECR region. The survival rates predicted by the AJCC were already proven to underestimate survival in the USA population [27], as they are now proven to do for this Dutch population.Although women had higher incidence rates of melanoma than males, their survival rates were significantly better: ∼20% higher 5- and 10-year relative survival rates than men. This gender difference in survival has been reported previously and is currently not well understood [1–7]. Part of the difference was probably due to the fact that compared with men women were younger at diagnosis and were more often diagnosed with thin melanomas and of the SSM type [29]. However, even melanomas of the same Breslow thickness and histopathological subtype and trunk and head and neck melanomas exhibited better relative survival rates for females than males. The multivariate analysis showed that, after correcting for all these factors, the hazard of death for male melanoma patients was significantly higher than for women. Therefore, besides a younger age, better stage at diagnosis and more favourable histogenetic subtypes, other factors are likely to be responsible for this superior survival of women. This phenomenon deserves further investigation.Most [6, 24, 30], but not all [5], previous studies showed a worse survival for patients with trunk melanomas. In this study, absolute and relative survival rates of patients with trunk melanomas were not significantly worse compared with survival of patients with melanomas on other body sites. However, after taking all available prognostic factors into account simultaneously in the multivariate analyses, trunk melanomas had a significantly increased hazard ratio.NMs had a worse survival rates than SSM among males only, the reason for this remains unclear. It probably has to do with more favourable other tumour characteristics among women. In multivariate analyses, NMs showed an increased hazard ratio. Few other studies reported survival rates by histogenetic subtype, usually being worse for nodular than for SSMs [5, 7].Unfortunately, no information was available on ulceration, which is an important prognostic factor. Should we have been able to include ulceration into the multivariate models, the results would likely have changed towards an important effect of ulceration and a somewhat less strong effect of Breslow thickness alone.In conclusion, survival of melanoma in the south-east of The Netherlands has improved over the past decades, mainly in the 1980s. Especially for women with thin melanomas, the prospects are very good. Secondary prevention activities seem to have been effective for females only. Ways should be found to better reach the elderly males, because in this group many life-years can theoretically be gained by improving early detection.The authors would like to thank the ECR for the data collection and data disposition. This study was funded by the Comprehensive Cancer Centre South.1.SmithJAEWhatleyPMRedburnJCGroupEWImproving survival of melanoma patients in Europe since 1978Eur J Cancer199834219722032.MacKieRMBrayCAHoleDJIncidence of and survival from malignant melanoma in Scotland: an epidemiological studyLancet20023605875913.KölmelKFKulleBLippoldASeebacherCSurvival probabilities and hazard functions of malignant melanoma in Germany 1972–1996, an analysis of 10433 patients. Evolution of gender differences and malignancyEur J Cancer200238138813944.SantMAareleidTBerrinoFEUROCARE-3: survival of cancer patients diagnosed 1990–94-results and commentaryAnn Oncol200314Suppl 5V61V1185.KemenyMMBuschEStewartAKMenckHRSuperior survival of young women with malignant melanomaAm J Surg19981754374446.GillgrenPBrattstromGFrisellJEffect of primary site on prognosis in patients with cutaneous malignant melanoma. A study using a new model to analyse anatomical locationsMelanoma Res2005151251327.DowningANewton-BishopJAFormanDRecent trends in cutaneous malignant melanoma in the Yorkshire region of England; incidence, mortality and survival in relation to stage of disease, 1993–2003Br J Cancer200695191958.BrennerHGefellerOAn alternative approach to monitoring cancer patient survivalCancer199678200420109.BrennerHHakulinenTLong-term cancer patient survival achieved by the end of the 20th century: most up-to-date estimates from the nationwide Finnish cancer registryBr J Cancer20018536737110.BrennerHSodermanBHakulinenTUse of period analysis for providing more up-to-date estimates of long-term survival rates: empirical evaluation among 370,000 cancer patients in FinlandInt J Epidemiol20023145646211.BrennerHLong-term survival rates of cancer patients achieved by the end of the 20th century: a period analysisLancet20023601131113512.HoutermanSJanssen-HeijnenMLvan de Poll-FranseLVHigher long-term cancer survival rates in southeastern Netherlands using up-to-date period analysisAnn Oncol20061770971213.dos Santos SilvaICancer Epidemiology: Principles and Methods1999Barcelona, SpainTHAY S.L14.HakulinenTAbeywickramaKHA computer program package for relative survival analysisComput Programs Biomed19851919720715.GreenwoodMA Report on the Natural Duration of Cancer1926LondonMinistry of Health HMSO16.BrennerHGefellerODeriving more up-to-date estimates of long-term patient survivalJ Clin Epidemiol19975021121617.BrennerHGefellerOHakulinenTPeriod analysis for ‘up-to-date’ cancer survival data: theory, empirical evaluation, computational realisation and applicationsEur J Cancer20044032633518.BrennerHGefellerOHakulinenTA computer program for period analysis of cancer patient survivalEur J Cancer20023869069519.KavanaghDHillADDjikstraBAdjuvant therapies in the treatment of stage II and III malignant melanomaSurgeon2005324525620.AtallahEFlahertyLTreatment of metastatic malignant melanomaCurr Treat Options Oncol2005618519321.DevosSABaeyensKVan HeckeLSunscreen use and skin protection behavior on the Belgian beachInt J Dermatol20034235235622.WelchHGWoloshinSSchwartzLMSkin biopsy rates and incidence of melanoma: population based ecological studyBr Med J2005331751548123.de VriesECoeberghJWMelanoma incidence has risen in EuropeBr Med J200533169824.BalchCMSoongSJGershenwaldJEPrognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer melanoma staging systemJ Clin Oncol2001193622363425.Australian Institute of Health and Welfare (AIHW) and Australasian Association of Cancer Registries (AACR)Cancer survival in Australia, 2001. Part 1:National summary statistics. AIHW cat. no. CAN 132001Canberra, AustraliaAustralian Institute of Health and Welfare26.RiesLHarkinsDKrapchoMSEER Cancer Statistics Review, 1975–20032006Bethesda, MDNational Cancer Institute27.GimottyPABotbylJSoongSJGuerryDA population-based validation of the American Joint Committee on Cancer melanoma staging systemJ Clin Oncol2005238065807528.BalchCMBuzaidACSoongSJFinal version of the American Joint Committee on Cancer staging system for cutaneous melanomaJ Clin Oncol2001193635364829.de VriesEHoutermanSCoebergJWDifferent site-distribution does not explain gender differences in survival of melanomaBMJ2006http://bmj.bmjjournals.com/cgi/eletters/332/7548/987#135142 (16 March 2007, date last accessed)30.ScogginsCRRossMIReintgenDSGender-related differences in outcome for melanoma patientsAnn Surg2006243693698discussion 698–700 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A4B22382E95826F025262E6FF2DF24ED9A32D18.txt b/test/dataset/in/resources/corpus/Clean_0A4B22382E95826F025262E6FF2DF24ED9A32D18.txt new file mode 100644 index 0000000..39e7b1f --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A4B22382E95826F025262E6FF2DF24ED9A32D18.txt @@ -0,0 +1 @@ +intimmintimmInternational Immunology1460-23770953-8178Oxford University Press10.1093/intimm/dxm152ORIGINAL RESEARCH PAPERSTissue-specific expression of B-cell translocation gene 2 (BTG2) and its function in T-cell immune responses in a transgenic mouse modelTerraRafik1LuoHongyu1QiaoXiaoying1WuJiangping121The Laboratory of Immunology, Centre hospitalier de l'Université de Montréal, Notre-Dame Hospital, Pavilion DeSève, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4M1, Canada2Nephrology Service, Centre hospitalier de l'Université de Montréal, Notre-Dame Hospital, Pavilion DeSève, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4M1, CanadaCorrespondence to: J. Wu; E-mail: jianping.wu@umontreal.caTransmitting editor: A. Falus32008141200820331732615920077122007© The Japanese Society for Immunology. 2008. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org2008B-cell translocation gene 2 (BTG2) belongs to the anti-proliferative gene family. According to previous in vitro studies, BTG2 overexpression leads to delayed cell cycling. We investigated BTG2 expression during mouse ontogeny and its immune and circadian functions in this study. In situ hybridization showed that BTG2 was expressed at high levels in the central nervous system, liver, stomach, thymus, spleen, skin, adrenal gland, pituitary gland and salivary glands during embryonic days (E10–E17), postnatal days (P1 and P10) and adult stages. Expression was observed in organs and tissues from adult mice with and without a robust proliferation program. Thus, the gene might have important functions that are both related and unrelated to proliferation. BTG2 expression was induced after in vitro T-cell receptor stimulation in T cells using anti-CD3 antibodies. However, transgenic (Tg) mice with actin promoter-driven expression of BTG2 showed normal in vitro and in vivo T-cell responses, such as thymus development, T-cell activation marker expression, T-cell proliferation and migration, as well as in vivo delayed-type hypersensitivity reactions. Although BTG2 was expressed in the suprachiasmatic nucleus and pineal gland in the brain, BTG2 Tg mice had no abnormal circadian behavior. Our data on BTG2 expression during ontogeny provide useful clues for the further investigation of BTG2 function. Additional studies are warranted to examine its role in immune and other systems.BTG2circadian rhythmin situ hybridizationT cellstransgenic mouseIntroductionB-cell translocation gene 2 (BTG2) belongs to the anti-proliferative gene family, which consists of 19 members. BTG2 contains a BTG-A box and a BTG-B box in its protein sequence (1), which are highly conserved among species. According to immunohistochemistry, BTG2 is expressed in various organs and tissues, with high levels detected in the lung, kidney, intestine, pancreas and prostate (2). BTG2 is located in the cytoplasm with a short half-life of ∼15 min (3). Its expression is induced by growth factors (4) or DNA-damaging processes (5). BTG2 seems to function as an adaptor, interacting with diverse molecules such as protein arginine methyltransferase PRMT1, homeobox protein Hoxb9, carbonylate repressor-associated factor Caf1, cyclin B1-associated kinase Cdc2, PKC-α binding protein PICK1, an ER-α coactivator CCR4 and peptidyl prolyl cis/trans isomerase Pin-1. Based mostly on in vitro-forced expression studies, BTG2 has been shown to inhibit G1/S transition (1), induce G2/M arrest (1), augment apoptosis (1), enhance retinoic acid-induced differentiation of hematopoietic cells (6) and suppress thymocyte expansion (7). Reduced BTG2 expression is observed in some malignant tumors, suggesting its role as a tumor suppressor gene (8). However, these in vitro or correlative findings have not been confirmed in a BTG2 null-mutant model in vivo. BTG2−/− mice are viable and fertile without a documented increase of malignancy (9). Their T- and B-cell development seems to be normal according to cell subpopulations in the thymus and spleen. The prominent phenotype of these mice is vertebrae malformation, underscoring the importance of this gene product in development and bone pattern formation.In our ongoing study to investigate mechanisms of T-cell activation, we compared gene expression on resting versus activated T cells using DNA microarray. BTG2 was among genes differentially expressed after TCR ligation of T cells. It was thus selected for further study on its expression during ontogeny, employing in situ hybridization (ISH). BTG2 transgenic (Tg) mice were generated to explore the function of this gene in the immune system.MethodsIn situ hybridizationFull-length 2583-pb BTG2 cDNA in pSPORT1 (clone H3059F09 from National Institute of Aging, Bethesda, MD, USA) was employed as a template for sense and anti-sense riboprobe synthesis, using SP6 and T7 RNA polymerase for both 35S-UTP and 35S-CTP incorporation (10).Tissues were frozen in -35°C isopentane and kept at -80°C until sectioned. ISH was performed on 10-μm thick cryostat-cut slices, as outlined previously (10). Anatomic ISH was conducted with X-ray films.Northern blot analysisA partial BTG2 cDNA fragment (clone H3059F09 from the National Institute of Aging, USA; mouse 15K cDNA clone sets; positions 1–2145 bp, according to the sequence of NM007570 in GeneBank) was labeled with a digoxin (Dig)-labeling kit from Roche Applied Sciences (Laval, Quebec, Canada). Total RNA was extracted from cultured cells with Trizol (Invitrogen, Burlington, Ontario, Canada). RNA (20 μg per lane) was resolved in 0.9% formaldehyde agarose gel and transferred to N-Hybond nylon membranes (Amersham Biosciences, Baie d'Urfe, Quebec, Canada). The membranes were hybridized with Dig-labeled probes, and the signals were revealed by a Dig detection kit (Roche Applied Sciences). Band intensities of 18S and 28S ribosomal RNA were indicators of even RNA loading.Generation of BTG2 Tg miceThe 2145-bp mouse full-length BTG2 cDNA was excised from pSPORT1 with SmaI/XbaI, and cloned into the BamHI (blunted)/XbaIs sites in vector pAC, between the human β-actin promoter and β-actin polyA signals. The resulting construct was named pAC-BTG2. The 7.4-kb ClaI/ClaI fragment containing the β-actin promoter, BTG2 cDNA and β-actin polyA signal was excised and injected into fertilized C3H × C57BL/6 eggs. Genotyping of the Tg mice was first performed by Southern blot analysis. Tail DNA of the founders (10 μg per each) was digested with PstI and resolved by 1% agarose gel electrophoresis. The DNA was transferred onto N+ Hybond nylon membranes after denaturation. A 4.2-kb band specific to the transgene was detected by the same Dig-labeled BTG2 probe as was employed for northern blot analysis. Subsequent genotyping was undertaken by PCR. The 5′ and 3′ primers were AGGTGGCTTCGTCTCTCTTGCTTT and GAATGCAATTGTTGTTGGTAACTTG, respectively, for detection of a 550-bp band. The following PCR conditions were employed: 94°C × 5 min, 1 cycle; 94°C × 1 min, 55°C × 1 min, 72°C × 1 min, 30 cycles; 72°C × 5 min, 1 cycle.Real-time reverse transcription–PCRBTG2 mRNA in Tg and wild type (WT) cells was measured by real-time reverse transcription (RT)–PCR; the 5′ and 3′ primers were TGAGCGAGCAGAGACTCAAGGTTT and ACAGCGATAGCCAGAACCTTTGGA, respectively. A 112-bp product was detected with the following amplification program: 95°C × 15 min, 1 cycle; 94°C × 15 s, 55°C × 30 s, 72°C × 30 s, 40 cycles.β-actin mRNA levels were measured as internal controls; the 5′ and 3′ primers were TGGTACCACAGGCATTGTGAT and TGATGTCACGCACGATTTCCCT, respectively, with the same amplification program as for BTG2 mRNA.Real-time PCR was performed in triplicate, and the signal ratios of BTG2/β-actin represented the normalized expression levels of BTG2.ImmunofluorescenceThe cytofix/cytoperm kit (Pharmingen, San Diego, C, USA) was used for intracellular BTG2 staining. WT and Tg thymocytes and splenocytes were fixed and permeabilized with 250 μl Cytofix/Cytoperm solution for 20 min at 4°C. Cells were washed twice with 1 ml of Perm/Wash buffer and stained for 30 min in 100 μl Perm/Wash buffer with anti-BTG2 polyclonal antibody (Cedarlane laboratories, Hornby, ON, Canada). After two washes, cells were stained with the secondary antibody (FITC-anti-rabbit Ig F(ab′)2; Chemicon, Australia). Cells were then centrifuged on glass slides using a Cytospin (Shandon, Pittsburg, PA, USA). The cells were visualized under a fluorescent microscope.Flow cytometryThe expression of CD3, CD4, CD8, CD19, CD25, CD44, CD69, Thy1.2 and B220 in thymocytes, splenocytes and lymph node (LN) cells was analyzed by flow cytometry as described in our previous publication (11). Different stages of double-negative (DN) subpopulations of adult thymocytes were analyzed by CD25 and CD44 staining on lineage-negative cells (12), which were gated with a mAb cocktail against CD3ε, CD11b, CD45R, B220, Ly6C, Ly6G, GR-1 and TER-119. Annexin V staining was conducted according to a procedure described earlier (11). All antibodies as well as annexin V–FITC were purchased from BD Pharmingen (San Diego, CA, USA), Cedarlane Laboratories (Hornby, Ontario, Canada) and Beckman Coulter (Mississauga, Ontario, Canada). Cells were analyzed on a Coulter flow cytometer with Expo32 software.T- and B-cell proliferationThymocytes and spleen T and B cells were purified, cultured and stimulated according to established methods (11). Cell proliferation was measured by 3H-thymidine uptake.Delayed-type hypersensitivity assayMice were first primed by painting their shaved abdominal skin with FITC. On day 6, ear thickness was measured, and the ears were then challenged by FITC painting. Ear thickness was remeasured after 24 h on day 7, and any increases were registered. This method has been described elsewhere (11).In vitro LN cell migration assayIn vitro migration assays were performed in Transwell chambers (6.5 mm in diameter, 5 μm pore size; Costar Corp., Cambridge, MA, USA), as detailed in our previous publication (13). The lower chamber contained 600 μl serum-free medium in the presence of stromal cell derived factor (SDF)-1α (80 ng ml−1) from R & D Systems (Minneapolis, MN, USA). The upper chamber held 100 μl serum-free medium containing 0.3 × 106 T cells. The Transwell ensemble was then incubated at 37°C for 2 h, and T cells migrating into the lower chamber were counted by flow cytometry. All the samples were in duplicate.ResultsBTG2 expression during ontogenyWe employed ISH to map the BTG2 expression pattern during ontogeny, starting from embryonic day 10 (E10) and in adult organs, using an anti-sense probe derived from BTG2 cDNA.BTG2 was expressed in the whole primordial nervous system at mid-gestation on E10 and E12 (Fig. 1A and B); its signals were very high in the mesencephalon, neural tube, spinal cord, myencephalon, primordial cerebellum, cranial flexure and optic chiasma. During the perinatal period from E17 to postnatal day 1 (P1), and the early postnatal stage at P10, BTG2 expression was reduced in the nervous system in general, but high signals were detected in a few brain regions, such as the ventricular zone, olfactory turbinates, olfactory lobe and retina (Fig. 1D–F). Biphasic BTG2 expression in the liver was noted. The first peak of expression was evident in the hepatic primordium on E12 and E15 (Fig. 1C), followed by a decline from E17 to P1 (Fig. 1D and E); the second expression peak was seen on P10 (Fig. 1F). BTG2 expression in the thymus and pulmonary tissue was induced at late gestation on E17 and remained high on P10 (Fig. 1D and F), at which time, expression in the spleen was also obvious (Fig. 1F). The skin and stomach wall started to show high BTG2 expression on P10.Fig. 1.BTG2 expression during ontogeny according to ISH. X-ray autoradiography (dark field) of BTG2 ISH in mice from E10 to P10 is shown (A–F). Hybridization with the sense (S) probe is presented as control (F'). MC, mesencephalon; NT, neural tube; EM, embryonic membranes; MyC, myencephalon; Cb, cerebellum promordium; CF, cranial flexure; SpC, spinal cord; Li, liver, hepatic primordium; Och, optic chiasma; TC, telencephalon; AD, anterior part of the diencephalon; M, striated muscles; Lu, lung; VZ, ventricular zone; OL, primordial olfactory lobe; OT, olfactory turbinates; Th, thymus; Smax, submaxillary gland; Ret, retina; H, heart; Int, intestine; K, kidney; NOE, neuroolfactive epithelium; DG, dentate gyrus (part of the hippocampal formation in the brain); Sk, skin; Sp, spleen; St, stomach. Magnification scales and ages are indicated.BTG2 mRNA in adult tissuesIn the adult central nervous system (Fig. 2A–C), significantly concentrated BTG2 mRNA was present in a few specialized structures, such as the ventricular zone, which is important in maintaining brain stem-cell population; the suprachiasmatic nucleus and pineal gland, both of which are involved in circadian rhythm regulation; the dentate gyrus, which is part of the hippocampal formation; the substantia nigra, which regulates involuntary motor activity; the olfactory lobe (Fig. 2C) and the spinal cord (Fig. 2D). In the endocrine system, both the intermediate and anterior lobes but not the posterior lobe of the pituitary gland were highly labeled (Fig. 2E); the signals were very high in both the cortex and medulla of the adrenal gland (Fig. 2F). Elevated BTG2 mRNA levels were also found in the glandular and non-glandular regions of the stomach (Fig. 2M), submaxillary gland and sublingual gland (Fig. 2N). Intermediate levels of the BTG2 signal were detected in the kidney medulla, pulmonary artery (Fig. 2J), lung (Fig. 2K) and liver (Fig. 2O).Fig. 2.BTG2 expression in adult organs according to ISH. X-ray autoradiography (dark field) of ISH are shown. (A) and (B): sagittal brain sections. SC, suprachiasmatic nuclei; PinGl, pineal gland; Cal, CAl region in the hippocampus; ChPl, choroid plexus; DG, dentate gyrus; Hb, habenula; SN, substantia nigra. (C) Longitudinal brain section. VZ, ventricular zone; DG, dentate gyrus; Cx, cerebral cortex; Cb, cerebellum. (D) Spinal cord. DH, dorsal horn; VH, ventral horn. (E) Pituitary gland. IL, intermediate lobes; AL, anterior lobes; PL, posterior lobe. (F) Adrenal gland. Cx, cortex; Me, medulla. (G) Thymus. Cx, cortex; Me, medulla. (H) Spleen. WP, white pulp; RP, red pulp. (I) Lymph nodes. (J) Heart. LV, left ventricles; RV, right ventricles; PA, pulmonary arteries; LN, small lymph nodules. (K) Lung. (L) Kidney. Cx, cortex; Me, medulla. (M) Stomach. GI, glandular regions; N GI, non-glandular regions. (N) Salivary glands. Smax, submaxillary gland; SL, sublingual gland. (O) Liver. (P) Northern blot analysis of BTG2 expression in thymocytes. Thymocytes were activated by Con A (1.5 μg ml−1), and spleen T cells were activated by soluble anti-CD3 (0.1 μg ml−1). The duration of activation is indicated. Bands of 28S and 18S ribosomal RNA are presented to show RNA loading. (Q) Real-time RT–PCR analysis of BTG2 expression in T cells. Spleen T cells were activated by soluble anti-CD3 (0.1 μg ml−1). The duration of activation is indicated. Means ± SD of ratios of BTG2 versus β-actin signals are shown.BTG2 mRNA expression in adult lymphoid organs and T cellsAdult lymphoid organs, such as the thymus cortex (Fig. 2G), spleen white pulp (Fig. 2H) and LN (Fig. 2I), contained high levels of BTG2 mRNA.BTG2 expression during in vitro T-cell activation was investigated by northern blot analysis for thymocytes and real-time RT–PCR for spleen T cells. We found that the constitutive BTG2 expression in unstimulated thymocytes and spleen T cells was relatively low, compared with activated cells (Fig. 2P and 2Q). Thymocytes and spleen T cells were stimulated in vitro with Con A and anti-CD3 (clone 2C11), respectively. Thymocytes were harvested at 5 and 24 h and spleen T cells, at 0 h, 30 min, 1, 2 and 4 h. As shown in Fig. 2P, one discrete band representing BTG2 mRNA above the 18S ribosome marker was detected in unstimulated cells; its intensity increased after 5 h followed by a decline at 24 h. In spleen T cells, BTG2 mRNA levels rose drastically after 30 min, and the high levels were maintained for at least 2 h (Fig. 2Q).Generation of BTG2 Tg miceTo investigate BTG2 functions not necessarily restricted to the immune system, Tg mice expressing human β-actin promoter-driven BTG2 were generated. The plasmid construct for Tg mice generation is illustrated in (Fig. 3A). Two Tg founders (lines 17352 and 17657) were established. Line 17657 was selected for further experimentation because of its higher transgene copy number. PCR was performed for routine genotyping of Tg mice; Tg but not WT tail DNA displayed a 550-bp band (Fig. 3B). Increased BTG2 mRNA expression in resting Tg thymocytes, LN cells and splenocytes was confirmed by real-time RT-PCR (Fig. 3C). BTG2 protein expression levels in Tg thymocytes and splenocytes were increased compared with WT cells, according to immunofluorescence (Fig. 3D). BTG2 Tg mice were fertile and manifested no gross anomalies upon visual inspection.Fig. 3.Generation and characterization of BTG2 Tg mice. (A) pAC-BTG2 construct for Tg mice generation. The 7.4-kb ClaI/ClaI fragment was used for microinjection. (B) Genotyping of BTG2 Tg founder tail DNA by PCR. The 428-bp band specific to the BTG2 transgene is indicated by an arrow. (C) Real-time RT–PCR of BTG2 mRNA from thymocytes, spleen and LN T cells. Means ± SD of ratios of BTG2 versus β-actin signals from three pairs of Tg and WT mice are shown. (D) Immunofluorescent staining of BTG2 in WT versus Tg thymocytes and splenocytes. Permeabilized thymocytes and splenocytes were stained with rabbit anti-BTG2 antibody followed by the FITC–anti-rabbit antibody, and signals were registered by fluorescent microscopy.BTG2 Tg mice presented normal thymocyte, splenocyte and LN cell subpopulationsBTG2 expression was drastically induced in thymocytes and T cells after their activation. This implies that the constitutive BTG2 levels in these cells are not sufficient for the cell activation program. The effect of BTG2 overexpression was studied in these cells using Tg mice to assess whether the augmentation of its constitutive level would disturb the development and function of these cells. Thymus and spleen size and cellularity in BTG2 Tg mice were similar to those in WT mice (Fig. 4A). Flow cytometry analysis demonstrated that B-cell percentage (CD19+B220+) in the spleen and LN of Tg and WT mice was comparable (Fig. 4B). No apparent difference was observed in the percentages of CD4+CD8+ double-positive (DP), CD4+ single-positive (SP) and CD8+ SP subpopulations in the Tg versus WT thymus (Fig. 4C). CD4- versus CD8-cell ratios in the spleen and LN of Tg mice were comparable to those in WT controls (Fig. 4C).Fig. 4.Subpopulations of lymphocytes in BTG2 Tg lymphoid organs. (A) Cellularity of the thymus and spleen. Tg mice and their WT littermates were compared for their thymus and spleen cellularity (n = 5). Means ± SD are shown. (B) DN thymocyte subpopulations. Thymocytes from Tg and WT mice were isolated and stained with lineage (Lin) markers (CD3ε, CD8β, TCRβ, CD11b, CD45R, B220, Ly6C, Ly6G, TER-119), CD25 and CD44 for three-color flow cytometry. Lin- cells were gated and analyzed for their CD25 and CD44 expression. The percentages of DN1, DN2, DN3 and DN4 subpopulations are indicated. Representative data from three pairs of Tg and WT thymi are shown. (C) T-cell subpopulations in the thymus, spleen and LN. CD4 and CD8 T-cell populations in the Tg and WT thymus, spleen and LN were analyzed by two-color flow cytometry. The percentages are indicated. The experiments were repeated more than three times, and representative data are shown. (D) B-cell population in lymphoid organs. The B-cell population in the spleen and LN was analyzed by B220 and CD19 in two-color flow cytometry. The experiments were repeated three times, and representative data are shown.We wondered whether BTG2 overexpression might interfere with early thymocyte development. Fast-proliferating DN thymocytes from Tg and WT thymi were further divided into DN1 (CD44+CD25-Lin-), DN2 (CD44+CD25+Lin-), DN3 (CD44-CD25+Lin-) and DN4 (CD44-CD25-Lin-) subpopulations by flow cytometry, and compared, but, no discernible difference was evident (Fig. 4D).In vitro Tg lymphocyte functionCompared with WT T cells, expression of the T-cell activation markers CD25, CD69 and CD44 was normal in Tg T cells stimulated by anti-CD3 or phorbol myristate acetate (PMA) plus ionomycin (Fig. 5A). Tg T-cell proliferation stimulated by anti-CD3, anti-CD3 plus anti-CD28 or PMA plus ionomycin was similar to that of WT T cells (Fig. 5B). Tg B cells also proliferated similarly to WT B cells under stimulation by IL-4 plus CD40L or IL-4 plus LPS (Fig. 5C). Tg LN cells chemotaxis toward SDF-1α showed no abnormality compared with WT cells, according to in vitro Transwell assays (Fig. 5D).Fig. 5.Tg T- and B-cell functions in vitro. (A) C69, CD25 and CD44 expression on activated Tg T cells. Tg and WT T cells were stimulated overnight by solid phase anti-CD3 (4 μg ml−1; concentration used for coating) or anti-CD3 plus anti-CD28 (0.57 and 2.86 μg ml−1, respectively). CD69, CD25 and CD44 expression on Thy-1.2-gated T cells was measured by two-color flow cytometry. Shadowed areas represent cells cultured in medium. (B) Tg T-cell proliferation. Tg and WT spleen T cells were stimulated by soluble PMA (10 nM) plus ionomycin (0.1 μg ml−1), solid phase anti-CD3 (4 μg ml−1) or solid phase anti-CD3 (0.57 μg ml−1) plus anti-CD28 (2.86 μg ml−1) as indicated. The cells were pulsed with 3H-thymidine 16 h before harvesting. 3H-thymidine uptake of the cells was measured at 48 and 72 h. The samples were in triplicate, and means ± SD of cpm are shown. (C) Tg B-cell proliferation. Spleen non-T cells were stimulated with IL-4 (20 ng ml−1) plus CD40L (0.1 μg ml−1) or IL-4 (20 ng ml−1) plus LPS (10 μg ml−1). 3H-thymidine uptake was measured in triplicate at 48, 72 and 96 h after the initiation of culture. Means ± SD of cpm are shown. (D) Tg LN cell migration toward SDF-1α. Means ± SD of the percentage of LN cell migration toward SDF-1α in the lower Transwell chamber measured by flow cytometry. The samples were in duplicate. The experiments in this figure were repeated three times, and representative data are shown.Apoptosis in Tg thymocytes and T cellsThe reported in vitro apoptosis induced by forced BTG2 expression led us to examine apoptosis in the T-cell compartment. Due to positive and negative selection, there is vigorous proliferation accompanied by massive apoptosis in the thymus. However, in freshly isolated thymocytes, there was no significant difference between all the stages examined, i.e. DN, DP and SP (Fig. 6A). We wondered whether the difference would only be revealed if external apoptotic signals were applied. Thus, Tg and WT thymocytes were treated with FasL and FasL plus cycloheximide, but no difference in their apoptosis was noted (Fig. 6B). The Tg and WT spleen T-cell apoptosis triggered by FasL was also similar (Fig. 6C). Thus, Tg BTG2 overexpression did not seem to affect apoptosis of these cells.Fig. 6.Tg thymocytes and T-cell apoptosis. (A) Apoptosis of freshly isolated Tg thymocytes. Freshly isolated Tg and WT thymocytes were stained with annexin V and developmental markers (Lin, CD25, CD44, CD4 and CD8) and analyzed by three-color flow cytometry. The percentages of annexin V-positive cells are indicated. (B) Apoptosis of Tg thymocytes stimulated by FasL and cycloheximide (CHX). Tg and WT thymocytes were stimulated with FasL (0.2 μg ml−1) or FasL plus CHX (0.5 μM) for 20 h. The cells were then stained with annexin V and analyzed by flow cytometry. (C) Apoptosis of Tg spleen T cells stimulated by FasL. Tg and WT spleen T cells were stimulated with FasL (0.2 μg ml−1) for 20 h, and their apoptosis was analyzed by annexin V staining in flow cytometry. The experiments in this figure were repeated twice, and representative data are shown.In vivo Tg T-cell functionT-cell function in vivo was evaluated according to delayed-type hypersensitivity (DTH), a T-cell-dependent reaction. In the response measured 1 week after FITC skin priming, Tg and WT mice exhibited similar percentages of ear thickness increment (Fig. 7).Fig. 7.Normal DTH in Tg mice. Tg mice and their WT littermates were tested for DTH. Ear thickness was measured three times, and mean values were registered. The percentage increase in ear thickness of each mouse was calculated as follows: % increase = (mean ear thickness 24 h after ear painting with FITC – mean ear thickness before ear painting)/mean ear thickness before ear painting. Horizontal bars represent the group means.BTG2 Tg mice displayed normal circadian rhythmOur ISH analysis demonstrated that BTG2 mRNA was expressed at high levels in the suprachiasmatic nucleus and pineal gland, both of which are involved in circadian rhythm regulation. The circadian activity of Tg mice was examined with the use of running wheels. The mice were kept under 12-h light and 12-h dark cycles. Tg and WT all followed precise activity rhythms, i.e. running the wheels only during the dark cycles, and no anomaly was observed (Fig. 8).Fig. 8.Tg mice presented normal circadian activity. Tg and their WT littermates were housed in cages with running wheels with 12-h light/dark cycles. Their running activities in terms running speed (meter per minute) were recorded from day 2 to day 6. The results of two WT (black and gray solid lines) and two BTG2 Tg (black and gray dotted lines) mice are shown.DiscussionPrevious in vitro studies showed that BTG2 overexpression hinders cell-cycle progress. Although BTG2 null-mutant mice have been generated, there are no reports describing abnormal cell cycling in BTG2−/− cells. Additional studies are required to elucidate whether BTG2 function is primarily in cell-cycle control. In this study, we took two approaches to this end. First, ISH was employed to map its expression during ontogeny and in adulthood. Subsequently, Tg mice with actin promoter-driven universal BTG2 expression were generated to explore the function of this gene, with emphasis on the immune system and T-cell proliferation.According to our ISH study, BTG2 was expressed in several organs and tissues containing fast-proliferating cells, such as the thymus, skin and stomach. Northern blotting also indicates that its expression was augmented 5 h after thymocyte activation and 30 min after spleen T cells activation, which was in the beginning of G0 to G1 transition. Such findings suggest that this gene probably has functions other than damping cell-cycle progress. This notion is further supported by the fact that many particular regions in the central nervous system, where regulation of proliferative activity is not required, also express high BTG2 levels. Thus, BTG2 likely has function outsides the cell-cycling program.BTG2 expression in the WT suprachiasmatic nucleus and pineal gland raised the possibility that this gene is involved in circadian regulation. However, Tg mice had normal circadian activity based on running wheel tests. They also adapted rapidly to forced 12-h delay of light/dark cycles, as did WT mice (data not shown). As actin-promoter-driven BTG2 overexpression does not affect the circadian rhythm, it will be interesting to examine circadian activity in BTG2−/− mice.In adulthood, relatively high-level BTG2 transcription occurred in the adrenal cortex and medulla, pointing to its possible involvement in steroid and catecholamine synthesis and/or secretion.BTG2 was expressed at high levels in the thymus, starting on E17, and its expression there remained high in adulthood. The spleen white pulp and LN also expressed BTG2 levels higher than neighboring tissues. However, when examined quantitatively using northern blotting and real-time PCR, it was found that resting thymocytes and spleen T cells had basal BTG2 expression, and such expression was rapidly augmented 3- to 4-fold after activation. This implies that the basal BTG2 expression is not sufficient to cope with the requirement of activated T cells. This prompted us to investigate whether basal BTG2 overexpression in thymocytes and resting T cells, using a Tg approach, would disturb their differentiation and function. Our Tg overexpression approach was also prompted by Konrad's observation that BTG2/TIS21 overexpression using a retroviral expression system in thymocyte progenitors led to delayed progression of thymocytes at the DN2 and DN3 stages and at the DN to DP transition in fetal thymus organ culture and OP9-DL1 co-culture model (7). However, when the gene was overexpressed in Tg mice, according to all the tests we performed in vitro and in vivo, T cells seemed to show no anomaly in terms of their development, activation, proliferation, migration and in vivo DTH. There could be several explanations for such observations. Most likely, endogenous BTG2 expression was more than sufficient for T-cell functions; therefore, the increased expression in Tg T cells minimally further influenced the function of this gene. It is also possible that the 2- to 5-fold rise in BTG2 expression in Tg thymocytes and T cells was not sufficient to disturb T-cell biology. Indeed, Konrad and Zuniga-Pflucker (7) have observed delayed progression of thymocytes at the DN stages and at DN to DP transition after BTG2 overexpression in thymocytes. The reason we did not observe such a delay is probably due to that the expression in our system was not sufficiently high, compared with Konrad's system (7). Lastly, we cannot rule out that there are undiscovered abnormal functions in immune as well as in other systems of Tg mice. In any case, such negative results are not findings that could be predicted a priori.The human genome project revealed far fewer genes than expected. This leads to the speculation that not only the gene products but also their expression levels are important in modulating cell functions. The ISH reveals gene expression levels in constitutive status, but we really do not know what should be considered as high or low expression until all the stimulated status of cells are tested. Therefore, unbiased investigation using overexpression and knockdown/knockout should be conducted to elucidate functions of genes concerned. We cannot determine a priori which one of these approaches will reveal discernable phenotype, and both approaches are justified, as attempted here in this study using BTG2 Tg overexpression.FundingCanadian Institutes of Health Research (CIHR, MOP57697 and MOP69089); the Kidney Foundation of Canada; the Heart and Stroke Foundation of Quebec; the Juvenile Diabetes Research Foundation, USA (1-2005-197); the J.-Louis Levesque Foundation to J.W. Group grants from Genome Canada/Quebec, the CIHR for New Emerging Teams in Transplantation, and Fonds de la recherche en santé du Québec (FRSQ) for Transfusional and Hemovigilance Medical Research to J.W.; CIHR (MOP79565) to H.L; National Scholarship to J.W.; Postdoctoral training grant from the FRSQ and CHIR/Rx&D-Wyeth Pharmaceuticals research fellowship program to R.T.AbbreviationsBTG2B-cell translocation gene 2DNdouble negativeDPdouble positiveDTHdelayed-type hypersensitivityEembryonic dayISHin situ hybridizationLinlineageLNlymph nodePpostnatal dayPMAphorbol myristate acetateRTreverse transcriptionSDFstromal cell derived factorSPsingle positiveTgtransgenicWTwild typeThe authors thank Ovid Da Silva for his editorial assistance, Martin Marcinkiewicz for ISH analysis and the Core Facility of the New Emerging Team of Transplantation for DNA microarray analysis.1LimIKTIS21 (/BTG2/PC3) as a link between ageing and cancer: cell cycle regulator and endogenous cell death moleculeJ. Cancer Res. Clin. Oncol.20061324172MelamedJKernizanSWaldenPDExpression of B-cell translocation gene 2 protein in normal human tissuesTissue Cell200234283VarnumBCReddySTKoskiRAHerschmanHRSynthesis, degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3J. Cell. Physiol.19941582054FletcherBSLimRWVarnumBCKujubuDAKoskiRAHerschmanHRStructure and expression of TIS21, a primary response gene induced by growth factors and tumor promotersJ. Biol. Chem.1991266145115RouaultJPFaletteNGuehenneuxFIdentification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathwayNat. Genet.1996144826PasseriDMarcucciARizzoGBtg2 enhances retinoic acid-induced differentiation by modulating histone H4 methylation and acetylationMol. Cell. Biol.20062650237KonradMAZuniga-PfluckerJCThe BTG/TOB family protein TIS21 regulates stage-specific proliferation of developing thymocytesEur. J. Immunol.20053530308DuriezCFaletteNAudoynaudCThe human BTG2/TIS21/PC3 gene: genomic structure, transcriptional regulation and evaluation as a candidate tumor suppressor geneGene20022822079ParkSLeeYJLeeHJB-cell translocation gene 2 (Btg2) regulates vertebral patterning by modulating bone morphogenetic protein/smad signalingMol. Cell. Biol.2004241025610MarcinkiewiczMBetaAPP and furin mRNA concentrates in immature senile plaques in the brain of Alzheimer patientsJ. Neuropathol. Exp. Neurol.20026181511LuoHYuGTremblayJWuJEphB6-null mutation results in compromised T cell functionJ. Clin. Invest.2004114176212TerraRLouisILe BlancROuelletSZuniga-PfluckerJCPerreaultCT-cell generation by lymph node resident progenitor cellsBlood200510619313ShiGWuYZhangJWuJDeath decoy receptor TR6/DcR3 inhibits T cell chemotaxis in vitro and in vivoJ. Immunol.20031713407 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A4E19B432818D9F6227886B00F569BEE54867F8.txt b/test/dataset/in/resources/corpus/Clean_0A4E19B432818D9F6227886B00F569BEE54867F8.txt new file mode 100644 index 0000000..514396c --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A4E19B432818D9F6227886B00F569BEE54867F8.txt @@ -0,0 +1 @@ +]>MAD2369S0047-6374(00)00176-710.1016/S0047-6374(00)00176-7Elsevier Science Ireland LtdFig. 1Box-plot of the incorporation of tritiated thymidine into submandibular glands in vivo with age and treatment. Results are expressed as counts per min per g gland of six animals. Effect of treatment: significant differences (5%) compared with WO and SAL are marked with (*), compared with SAL with (+) by one-way ANOVA; effect of age: significant differences (5%) compared with 3 m are marked with (†) by one-way ANOVA.Fig. 2Box-plots of submandibular flow rates with age and treatment. Results are expressed as μl/min per gland (a) and μl/min/g gland (b). Values represent six to eight animals in each group. Effect of treatment: significant differences (5%) compared with WO and SAL are marked with (*), compared with SAL with (+) by one-way ANOVA. Effect of age: significant differences (5%) compared with 3 m are marked with (†), compared with 12 m with (¥) by one-way ANOVA.Fig. 3Box-plots of (a) the protein concentration (μg/ml), and (b) of the total amount of protein secreted (μg) with age and treatment. Results represent the values of six to eight animals in each group. Effect of age: significant differences (5%) compared with 3 m are marked with (†) by one-way ANOVA.Fig. 4Submandibular saliva protein analyzed on 10% SDS polyacrylamide gels with a representative sample (15 μg of protein) of each of the three age and treatment groups (Coomassie blue staining). Pre-stained molecular standards (Std) were; phosphorylase B, 130 kDa; bovine serum albumin, (BSA) 80 kDa; ovalbumin, 49.5 kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa; lysozyme, 18.5 kDa.Fig. 5Box-plots of (a) the salivary EGF-concentration (mg/ml), and of (b) the salivary EGF content per mg protein secreted (μg/mg). The results express the values of six animals in each group. Effect of treatment: significant differences (5%) compared with SAL are marked with (+), compared with WO with (≠) by one-way ANOVA; effect of age; significant differences (5%) compared with 3 and 12 m are marked with (#), compared with 3 m with (†), compared with 12 m with (¥) by one-way ANOVA.Fig. 6Box-plot of the salivary peroxidase (a) and lysozyme (b) content per mg protein secreted (ng/mg) with age and treatment. The results represent the values of six animals in each group. Effect of treatment: significant differences (5%) compared with WO and SAL are marked with (*), compared with SAL with (+) by one-way ANOVA; effect of age: significant differences (5%) compared with 3 m are marked with (†), compared with 12 m with (¥) by one-way ANOVA.Fig. 7Box-plot of the RNA content in submandibular glands with age and treatment. The results are expressed as μg per g gland of six to nine animals in each group. Effect of age; significant differences (5%) compared with 3 m are marked with (†) by one-way ANOVA.An analysis of submandibular salivary gland function with desipramine and age in female NIA Fischer 344 ratsMarkus MKollera*mmkoller@zzmk.unizh.chRichard ACowmanbMichael GHumphreys-BehercdPhilip JScarpacedeaDepartment of Oral and Maxillofacial Surgery, Claude Denson Pepper Center for Research on Oral Health in Aging, University of Florida, Gainesville, FL 32610, USAbDental Research Unit, Department of Veterans Affairs Medical Center, Miami, FL 33125, USAcDepartment of Oral Biology, University of Florida, Gainesville, FL 32610, USAdDepartment of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USAeGeriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Gainesville, FL 32608, USA*Present address: Center for Dental and Oral Medicine, Clinic for Geriatric and Special Care Dentistry, University of Zurich, P.O. Box 322, CH-8028 Zurich, Switzerland. Tel.: +41-1-6343341; fax: +41-1-6344319AbstractDry mouth is one of the major side effects of cyclic antidepressants, which are still a dominating group of psychotherapeutic drugs used in the treatment of depression. In this study we analyzed the effects of 28 day tricyclic antidepressant administration and the reversibility of this treatment following a 15 day washout period on different parameters in submandibular gland function in aging rats. We postulated that desipramine would decrease gland function, accented with age, and delay recovery in senescent animals. In contrast to body weight, desipramine had no effect on glandular wet weight. While glandular DNA synthesis was changed with age and treatment, the concentration of total membrane and soluble proteins was not affected. Flow rate was significantly changed with age, but desipramine increased salivary flow in the youngest animals only. Neither age nor treatment influenced salivary protein concentrations, but the total amount of proteins secreted, revealed perturbation with age. SDS- polyacrylamide gel analysis revealed changes in protein expression with treatment and age. Desipramine decreased epidermal growth factor (EGF) levels in all age groups, but increased the secretion of peroxidase and lysozyme. Analysis of total RNA showed a pronounced decrease with age. These data indicate that desipramine has profound effects on submandibular salivary gland function.KeywordsDesipramineEGFLysozymePeroxidaseSubmandibular glandSaliva1IntroductionThe primary function of salivary glands is to secrete saliva, a fluid composed of water, electrolytes, and various multifunctional proteins. The autonomic sympathetic and parasympathetic receptor systems regulate salivary gland secretion via a co-ordinated sequence of signal transduction and intracellular signalling events (Looms et al., 1998; Ambudkar, 2000). Saliva is an essential fluid for the health of human teeth and oral mucosal surfaces, maintains microbial balance, and supports other oral functions. The basic protective mechanism mediated by saliva is bacterial clearance. But saliva also contains many antibacterial, antifungal and antiviral substances. Salivary glands and saliva are a part of the mucosal immune system (Tomasi and Plaut, 1985). Besides the predominant immunoglobulin secretory IgA, the oral cavity is protected, as well by many salivary nonimmunoglobulin antimicrobial molecules such as lysozyme, lactoferrin, and peroxidases (Mandel and Ellison, 1985). Many of these antimicrobial factors interact with each other and depend on salivary flow rate (Mandel and Ellison, 1985).Pharmaceutical side effects, systemic diseases, and radiation therapy are reported to be the most common causes of acquired salivary gland dysfunction (Mandel, 1980), leading to many subjective complaints (Fox et al., 1987) and dysfunction related objective findings (Gråhn et al., 1988; Atkinson and Fox, 1992). With age, an increasing number of people have medical conditions and/or use therapeutics that may significantly affect salivary gland physiology and the oral ecology. Tricyclic antidepressants, the basic group of amine uptake inhibitors, have a wide pharmacological spectrum displaying α1-adrenoceptor antagonistic, antiserotonin, antimuscarinic, and antihistaminic properties. Unwanted side effects are common (Trindade et al., 1998), and become more common with age (Tümer et al., 1992). One of the most reported ailments of tricyclic antidepressant therapy is dry mouth (Fox et al., 1985; Remick, 1988). Decreased salivary flow rate (Wu and Ship, 1993), increased salivary proteins and electrolytes (Blackwell et al., 1980; von Knorring and Mörnstad, 1981; Mörnstad et al., 1986), and changes in oral microflora were found in humans (Parvinen et al., 1984). This may compromise oral and systemic health, and overall quality of life. Salivary gland derived health problems pose a significant disease burden for the host.As in humans, submandibular flow rate seems not to be reduced in aging rats (Koller et al., 1992), while a progressive age-related decline in the rates of secretory protein synthesis in submandibular glands has been observed (Baum et al., 1983). Epidermal growth factor (EGF) was found to decrease with age, as well (Koller et al., 1992). Catecholamine responsiveness declines with age and is primarily due to a decrease in β-adrenergic signal transduction (Scarpace et al., 1991). In peripheral tissue, for the most part, the density of β-adrenoceptors is maintained with age, whereas the ability to activate adenyl cyclase declines (Scarpace et al., 1991). Tricyclic antidepressants may indirectly (central nervous system), and/or directly (salivary glands) reduce salivary gland function as a result of their action on modulating neural transmission. It has been shown (Scarpace et al., 1992, 1993), that signal transduction in submandibular glands was perturbed after chronic administration of desipramine. This drug down-regulated β-adrenergic receptors at all ages, but receptor recovery after drug withdrawal was prolonged in the brain of aged animals (Greenberg et al., 1985). In addition, we recently reported that desipramine led to changes in protein secretion, what may have negatively influenced oral health in aging rats (Koller et al., 2000).In this study, we investigated physiological and biochemical changes in submandibular gland secretory function in vivo with age and the long-term administration of the tricyclic antidepressant desipramine. This drug is a relatively selective inhibitor of the reuptake of norepinephrine and has weak effects on serotonergic neuronal activity. Therefore we expected (a) that submandibular flow rate would decrease temporarily with treatment; (b) that total protein secretion, as well as the content of specific proteins (EGF, peroxidase, lysozyme) in submandibular saliva would be depressed transitorily, and (c) that age would modulate the treatment-related changes.2Materials and methods2.1AnimalsPathogen-free female, Fischer 344 NIA rats of 3, 12 and 24 months of age were purchased from Harlan Sprague-Dawley (Indianapolis, USA) under contract with the National Institute of Aging (NIA). Animals were housed individually in 7×10 in. stainless steel micro-isolated cages, in a room maintained at 25°C. No clinical evidence of infectious illness was detected. All animals were maintained on Purina rat chow and water ad libitum and exposed to 12-h light–12-h dark cycle starting at 07:00 h. Animals were acclimatized for a minimum of 10 day before starting the experiments. At the beginning of the experimental period the young animals weighed 176–200 g, the middle age group rats 216–261 g, and the oldest ones 310–352 g.2.2Experimental designAnimals received a daily intraperitoneal (i.p. between 07:00 and 09:00 h) injection of desipramine or saline (10 mg/kg body weight) for 28 days. One half of each group was sacrificed on the third day after the last dose (DMI and SAL groups), the other half after a washout (WO) period of 15 days (DMI/WO and SAL/WO groups). Body weight was measured weekly, the final weight was monitored at the day of the experiment.2.3Saliva collection and flow rateThe same person between 09:00 and 11:00 h, using two animals per day, always carried out saliva collections. The rats were deprived of food the day before the experiment at 16:00 h. Animals were anesthetized i.p. (sodium pentobarbital, 50 mg/kg) and tracheotomized. The submandibular duct of the animal's left side was canulated with calibrated (lumen and length) drawn PE-10 plastic tubes. To stimulate salivary flow, pilocarpine HCl (Sigma Chemical, USA; 5 mg/kg, i.p.) was administered; to induce discharge of stored proteins isoproterenol (Sigma Chemical, USA; 5 mg/kg, i.p.) was chosen. These agents were dissolved in 37°C isotonic saline. The initial drop of saliva was discarded and subsequent flow was collected for 30 min into pre-weighed plastic tubes kept on ice. The sample tubes were reweighed to obtain a gravimetric estimate of the volume of saliva, assuming that 1 μl of saliva equals 1 mg. Flow rate was calculated as μl/min per gland and corrected for glandular wet weight and/or body weight. Saliva was stored at −80°C until analyzed. Sodium ethylene diamine tetraacetate (EDTA 1%, pH 8.6) was added to submandibular saliva (100 μl/ml saliva) to prevent the formation of a calcium-protein precipitate (Abe et al., 1980).2.4Salivary proteinsSamples were aliquoted to avoid repetitive freezing and thawing, assayed at one time, and in duplicate, to minimize error. Proteins were analyzed at one time by the method of Bradford (1976), using bovine plasma gamma globulin as the standard.For EGF estimation the volume of total saliva was brought up to 1 ml with sterile water. The glacial acetic acid (58 μl) was added, the solution cooled on ice for 30 min and then centrifuged at 4°C for 30 min in an Eppendorf centrifuge. The supernatant was transferred to a new tube, frozen at −80°C and then lyophilized. The remaining proteins were resuspended in 200 μl sterile water. Then 100 μl were added to 100 μl of human placental microvilli membranes and [125I]-labeled human EGF. Following incubation at 37°C for 2 h the membrane was diluted in phosphate buffered saline (PBS), centrifuged for 20 min at 7000×g and the radiolabel associated with the membrane determined in a gamma counter. This method of membrane binding competition is independent of species origin for the EGF source (Booth et al., 1980).Salivary peroxidase was assayed by the ABTS [2,2′-azinobis (3-ethylbenzothiozaline-6-sulfonic acid)] method of Shindler et al. (1976). The μmol substrate oxidized per min at 20°C was calculated from the absorbance change at 412 nm using the extinction coefficient for the radical cation produced. The salivary activity values were compared with a plot of activity versus ng protein prepared using purified bovine milk lactoperoxidase (Sigma Chemical, USA) as the standard. Salivary peroxidase concentrations were expressed as ng peroxidase per μl saliva.Lysozyme was assayed by the lysoplate method of Greenwald and Moy (1976) using low sulfur content agarose, and Micrococcus lysodeikticus as the indicator bacterium. Plates were incubated in a humidity chamber at 35°C for 48 h. The diameter of zone clearing was measured to the nearest 0.1 mm in both a vertical and horizontal direction. The mean zonal diameter values were compared with a plot of zone clearing versus ng protein prepared using grade I hen egg-white lysozyme (Sigma Chemical, USA) as the standard. Lysozyme concentrations were expressed as ng lysozyme per μl saliva.2.5Membrane preparation, cellular proteins, RNA contentAt sacrifice, the circulatory system was perfused with 20 ml of cold saline. The two submandibular glands were excised and dissected free of lymph nodes, fat, and fascia, and the sublingual glands removed. The wet weight was determined on an analytical balance, and the tissues immersed in 0.9% NaCl at 4°C. All subsequent steps were carried out at 4°C. The glands were finely minced in 20 volumes of 0.25 M sucrose, 1 mM MgCl2, and 5 mM Tris–HCl (pH 7.4). To avoid protein degradation, protease inhibitors were added to the mixture (1 μM leupeptin, 100 μM benzamidine, and 100 μM phenylmethylsulfonylfluoride-PMSF final concentrations). Preparations were disrupted by a Tekmar Tissuemizer (Cincinnati, USA) for 20 s and subsequently homogenized with 10 strokes of a motor-driven, teflon-tipped pestle at moderate speed. The homogenate was passed through two layers of cheese cloth, and centrifuged at 48 000×g for 15 min. The resultant pellet was resuspended in 20 volumes of 8 mM MgCl2, 0.08 mM ascorbic acid, the above mentioned protease inhibitors, and 50 mM HEPES (N-hydroxyethyl piperazine-N′-2′ ethane sulfonic acid, pH 7.4). Protein assays for total membrane and supernatant were determined by the above mentioned method of Bradford (1976).Submandibular gland RNA was isolated as described by Chomczynski and Sacchi (1987). One gland was homogenized in 2 ml of denaturing solution consisting of 4 M guanidinium thiocyanate. 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol were added followed by 0.35 ml of 3 M sodium acetate, pH 5.2, 5 ml of phenol (water saturated), 1 ml of chloroform-isoamyl alcohol mixture (49:1) to extract the proteins from nucleic acid. The suspension was mixed and cooled on ice for 15 min and centrifuged at 10 000×g for 20 min at 4°C. The aqueous phase containing the RNA was transferred to a new tube, mixed with 5 ml of isopropanol, and then placed at −20°C for 1 h to precipitate RNA. Sedimentation at 10 000×g for 20 min at 4°C was again performed. The resulting RNA pellet was dissolved in 0.3 ml of the denaturing solution and precipitated with 0.1 ml 3 M sodium acetate (pH 5.2) and 0.4 ml isopropanol at −20°C for 1 h. After centrifugation in an Eppendorf centrifuge for 10 min at 4°C the RNA pellet was resuspended in 1 ml of 99% ethanol, sedimented, vacuum dried (10 min), and dissolved in 100 ml DEP-treated water and 0.5% SDS. The RNA content of all samples was estimated at one time by absorption at a wavelength of 260 nm and expressed as μg/g gland.2.6DNA synthesisIn vivo DNA synthesis was determined in submandibular glands by the incorporation of [3H]-thymidine into DNA by the injection of radiolabel (50 μCi per 100 g) 1 h before pentobarbital anesthesia and 4 h prior to sacrificing. Samples (100 μl) for [3H]-thymidine incorporation were removed prior to membrane centrifugation, and analyzed by scintillation counting using 10 ml of Fisher premixed nonaqueous scintillation cocktail (Sci Vers 5X20-4).2.7Polyacrylamide gel electrophoresisSubmandibular saliva was subjected to electrophoresis on a 1.5 mm thick 10% sodium dodecyl-sulfate polyacrylamide gel (SDS/PAGE) using a modified Tris–glycine system of Laemmli, as described by Pugsley and Schnaitman (1979). Gels were fixed and stained by a modification of the method of Fairbanks et al. (1971) as described by Humphreys-Beher and Wells (1984). Samples for gels were made up at 1.0 mg/ml of sample buffer. 15 μg of protein per well was used for gel electrophoresis.2.8Statistical analysisThe null hypotheses, no change with age and desipramine treatment, were tested by two way analysis of variance (ANOVA). When the main effect was significant, subgroups were examined by one-way ANOVA. Body weight was analyzed by repeated measures ANOVA. The Bonferroni/Dunn multiple comparison procedure was carried out to determine significant differences among the means following a significant one- and two-way ANOVA. No significant differences between the two saline groups were detected for all data. Therefore, we did not differentiate between the two saline groups in the final analysis.3Results3.1Body weight, organ weight and DNA-synthesisLong-term desipramine administration significantly reduced body weight throughout the experimental period between control and drug treated animals in all age groups (P<0.0001), with the greatest loss occurring in senescent rats (around 80 g). The animals gradually regained weight after the cessation of treatment. By day 15 post-treatment, body weight was comparable to that of control animals in the youngest age group only. A significant interaction between the parameters age and treatment was observed (P<0.0001).Comparing the control groups, the wet gland weight significantly increased between 3 and 12 m (P<0.0001), while desipramine administration had no major effect on glandular weight as on cortex weight in any age group. The histosomatic index, the organ to body weight ratio, was significantly decreased with treatment in the oldest animals only (P<0.0001).With regard to the general state of the glandular cells we found, that the DNA synthesis, as measured by the incorporation of tritiated thymidine (counts per min/g gland), was significantly influenced by treatment (P<0.0001) and age (P<0.0001), with no significant interaction (Fig. 1). While DNA synthesis was significantly decreased with desipramine in the two younger age groups (P≤0.0010), the control animals showed a significant increase between 3 and 12 month of age (P=0.0002). Recovery from drug effects was significantly delayed in senescent rats (P=0.0076).3.2Flow rateSubmandibular flow rate (Fig. 2), expressed as μl/min per gland, was significantly affected by age (P=0.0013). Correction for differences in glandular wet weight (μl/min/g gland) and/or for body weight (μl/min/g gland per kg, μl/min per gland per kg) eliminated the age related effect (P=0.5099, 0.0435, 0.7598, respectively). Correction for body weight only revealed a treatment related effect (P=0.0004). With desipramine, significant changes in submandibular flow rate were observed in the youngest animals only. Comparing the control groups, submandibular flow rate significantly increased with age (P=0.0017) when expressed as volume per time, but significantly decreased with age when correcting for glandular wet weight and body weight (P=0.0118).3.3ProteinsNo significant age- and treatment-related changes in protein concentrations (μg/μl) were detected for total membrane (structural proteins) and supernatant (soluble proteins). For the ratio structural/soluble proteins, a significant age-related effect was noted between 3 and 12 month old animals (P=0.0099). Subgroup analysis revealed no significant differences.Salivary protein concentrations (μg/μl) were not significantly changed with age and treatment, but the three age groups reacted differently to the drug regimen (Fig. 3). While in young and adolescent animals a decrease with desipramine occurred (27 and 40%, respectively), senescent rats showed a treatment-related increase of 45%. Recovery during the 15 day washout period was quite complete in all age groups (108, 85 and 102%). Correction for the observed changes in flow rate (total proteins secreted in 30 min) demonstrated significant age-related changes (P=0.0007). Again, the three age groups showed different effects with treatment. While 3 and 12 month old animals showed depressed protein secretion with the administration of the antidepressant (30 and 27%), total proteins secreted were elevated with treatment in senescent rats (30%). Protein concentrations in control animals revealed a steady increase with age, being significant when comparing 3 and 24 month old animals (P=0.0073), while total proteins secreted showed a steady, but not significant increase with age.On SDS/PAGE gels, loaded with a representative sample of each of the three treatment and age groups (15 μg of salivary proteins), protein expression differed with treatment and age (Fig. 4). In the Coomassie blue-stained gel, changes were detected in the bands around 36 kDa. The synthesis of these proteins was most pronounced in the treatment groups. An unusual protein (around 49 kDa) was detected in the desipramine group of 24 month old animals.Estimating the concentration (mg/ml) of EGF (Fig. 5) tested the secretory capacity of the submandibular granular tubule cells. Both, age and treatment revealed significant changes in the salivary EGF concentration (P<0.0001), and the interaction age×treatment was significant, as well (P<0.0211). Comparing the control groups, submandibular saliva of 12 month old animals contained 40% more EGF per ml saliva than the saliva of the youngest animals (P=0.1282), 24 month old rats showed a decrease of 81% compared with the adolescent rats (P=0.0004). All age groups showed decreased EGF concentrations with desipramine, significant in the middle age group only (P<0.0001). Basing the content of EGF on 1 mg of total protein in the sample (μg/mg) revealed a quite similar pattern for age (P<0.0001) and treatment (P<0.0140), and the interaction was still significant (P=0.0367).The content of peroxidase in submandibular saliva (Fig. 6), expressed as ng/mg protein secreted, was significantly affected by age (P=0.0237) and treatment (P=0.0206), with a significant interaction between the two parameters (P=0.0439). With desipramine administration, an increase was noted in all age groups, significant in 12 month old animals (P=0.0007). Recovery was incomplete in this age group only (P=0.0036). Comparing the controls, saliva of adolescent rats showed a significant lower peroxidase content than in the youngest age groups tested (P≤0.0054).The immunoprotein lysozyme showed similar effects with age (P=0.0002) and treatment (P=0.0021) as peroxidase (Fig. 6). The increase with desipramine was significant in the oldest age group only (P=0.0021). The control animals showed no age-related effect.Total RNA (Fig. 7), expressed as μg/g gland, was significantly affected by age (P=0.0213), but not by treatment (P=0.3440). In control animals, RNA decreased from 3 to 12 month of age by 17% and by 16% from 12 to 24 month old animals.4DiscussionIn the brain, desipramine blocks the reuptake of norepinephrine, leading to desensitization of the postjunctional β-adrenergic receptors, involving both a rapid attenuation of the responsiveness of adenylate cyclase and a slower downregulation of receptor number (Hausdorff et al., 1990). In submandibular glands of young rats, we observed desensitization of isoproterenol-stimulated activity, unchanged post-receptor signal transduction, and complete recovery after a 15 day washout period (Scarpace et al., 1992). With age, desipramine led to down-regulation in receptor number and attenuated receptor stimulated adenylate cyclase activity in submandibular glands (Scarpace et al., 1993). In addition, drug attenuated G-protein linked post receptor adenylate cyclase activity persisted through the 15 day washout period. In 12 month old rats, submandibular receptor up-regulation and adenylate cyclase supersensitivity were observed. Therefore, the animals in the present study probably also experienced receptor downregulation and adenylate cyclase desensitization, and the revealed alterations in submandibular protein secretion are, at least partially, the consequences of these changes in the signal transduction cascade changes present in all ages.It is well known, that most exocrine protein secretion occurs subsequent to β-adrenoreceptor activation. In this study we confirmed, that long-term administration of aging rats to the tricyclic antidepressant desipramine results in changes in salivary protein synthesis. A stimulating effect on the biosynthesis and secretion of different specific salivary proteins (e.g. bands around 36 kDa, lysozyme, peroxidase) was observed in all age groups. To maintain stable levels of total proteins secreted, the excretion rate of other proteins must have been depressed with desipramine. This was the case for the secretion of EGF in all the age groups. In a parallel study with the same drug regimen we showed unaffected salivary lactoferrin contents with treatment (Koller et al., 2000). Thyroid hormone is part of the regulatory mechanisms in salivary gland function (Tumilasci et al., 1986; Johnson and Kalu, 1988; Rousseau et al., 1998), e.g. it regulates the synthesis of EGF (Rall et al., 1985; Gubits et al., 1986; Johnson et al., 1987). In addition, serum levels of tetra- and triiodothyronine are influenced by treatment with antidepressants (Dagogo-Jack, 1995). Therefore, the detected changes in EGF secretion with desipramine may have indirectly influenced submandibular gland physiology in this study. As growth factor and cytokine receptors, many steroid hormones and their receptors found in salivary cells play a major regulatory role in salivary gland proliferation and function (Baum and Wellner, 1999; Purushotham and Humphreys-Beher, 1995), the decline in serum levels of thyroid hormones may have influenced cellular DNA synthesis and specific protein synthesis in this study. In the brain, chronic antidepressant therapy has been shown to induce changes in the function of protein kinase C (PKC), cyclic AMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase (Popoli et al., 2000). Therefore, as protein phosphorylation is an obligate step for most signaling pathways, desipramine may directly affect salivary gland proliferation and inactivate certain enzymes through the inactivation of important second messenger signaling pathway components (Purushotham and Humphreys-Beher, 1995).Differences in the induction of specific proteins with desipramine may be due to either nonspecific interaction with β-adrenergic signal transduction or differences in the induction of protein synthesis in different glands (Bedi, 1993). Additionally, there may be a differential control of secretion of proteins synthesized by ductal and acinar cells. While the secretion of many ductal proteins, such as EGF, appears to be initiated by α-adrenergic receptors (Wallace and Partlow, 1976; Orstavik and Gautvik, 1977; Hirata and Orth, 1980), the synthesis and secretion of acinar cell products is regulated by β-adrenergic receptors (Johnson and Cortez, 1988). Therefore, in submandibular glands, desipramine may affect not only β-adrenergic signal transduction (Scarpace et al., 1992, 1993), but β-adrenergic and muscarinic signaling, as well. Hanft and Gross (1990) showed, that rat cortical α1a- and α1b-adrenoceptors are regulated in a subtype-selective manner by desipramine (Hanft and Gross, 1990). This mechanism may influence secretory functioning of peripheral organs, as well. Additionally, desipramine administration may alter other neurotransmitter receptors involved in salivary secretory events as vasoactive intestinal polypeptide (VIP), tachykinin and purine receptors.The appearance of structurally altered proteins (Danner and Holbrook, 1990) is a prominent age-related change observed in various organs and tissues. These changes affect cellular contents, as well as functional properties of glandular proteins. Inanaga et al. (1988) observed unusual proteins in parotid saliva of 8- and 15-month-old rats similar to those we detected in submandibular saliva in senescent rats treated with the antidepressant desipramine.Saliva, having antimicrobial and growth-stimulating factors simultaneously, and the oral microbiota are the main factors determining oral health. Salivary antibody and non-immunoglobulin antimicrobial factors (e.g. lysozyme, peroxidase) maintain ecological balance (Gråhn et al., 1988). Peroxidase and lysozyme are only minor constituents of submandibular saliva. The changes in their content with treatment may have only a minor influence on oral microbiota in rats, but may partially reflect the reported increased incidence of gingivitis with desipramine treatment (Koller et al., 2000).Contrary to our expectations, considering the antagonistic action of desipramine on α1-adrenergic and muscarinic-cholinergic receptors in the brain (Potter et al., 1991), significant increased submandibular flow rates were detected with desipramine administration in young animals. One can speculate, that the detected increase in flow rate was caused by a positive feedback mechanism through the muscarinic-cholinergic, α1-adrenergic and substance P signal transduction system due to the observed decreased protein concentration.In contrast to human studies (Fernstrom and Kupfer, 1988), long-term treatment of rats with tricyclic antidepressants caused decreased food intake and a related weight loss (Nobrega and Coscina, 1987; Orthen-Gambill and Salomon, 1990). Long-term desipramine administration altered food and water intake (Durcan et al., 1988). After an initial decrease, body weight progressively returned towards pretreatment levels. This was explained by a rapid suppressive effect (acute central action of desipramine), and an adaptive effect during treatment (changes in feeding associated brain regions). This confirms our results, as body weight, after an initial decrease (1–2 weeks), was quite stable during the drug regimen in the two younger age groups. The decrease in the oldest animals lasted until the end of treatment and recovery was incomplete. As mentioned before, food intake, mastication, and long-term desipramine administration influenced serum levels of tetra- and triiodothyronine. Therefore, the loss in body weight and related changes in food and water intake with treatment may have indirectly influenced submandibular gland function in this study.In conclusion, the complexity of psychotropic drug actions, and variations in the absorption and excretion of these compounds and their metabolites, make precise predictions of their effects on salivary gland function difficult. The correlation between receptor activation and intracellular responses is often not predictable. Various signaling pathways seem to intersect and crosstalk, to modify and influence the biological outcome of the extracellular signal. 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Res.891981313320Wallace and Partlow, 1976L.J.WallaceL.M.Partlowα-adrenergic regulation of secretion of mouse saliva rich in nerve growth factorProc. Natl. Acad. Sci. USA73197642104214Wu and Ship, 1993A.J.WuJ.A.ShipA characterization of major salivary gland flow rates in the presence of medications and systemic diseaseOral. Surg. Oral. Med. Oral. Pathol.761993301306 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A4F4C3C727FEC1AC4A4B690A68F1584EF2363E1.txt b/test/dataset/in/resources/corpus/Clean_0A4F4C3C727FEC1AC4A4B690A68F1584EF2363E1.txt new file mode 100644 index 0000000..ff07445 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A4F4C3C727FEC1AC4A4B690A68F1584EF2363E1.txt @@ -0,0 +1 @@ +toxscitoxsciToxicological Sciences1096-09291096-6080Oxford University Press10.1093/toxsci/kfq034SYSTEMS TOXICOLOGYAcute Exposure to Ozone Exacerbates Acetaminophen-Induced Liver Injury in MiceIbrahim AiboDaher*†BirminghamNeil P.†LewandowskiRyan*MaddoxJane F.†‡RothRobert A.†‡GaneyPatricia E.†‡WagnerJames G.*†HarkemaJack R.*1*Department of Pathobiology and Diagnostic Investigation†Center for Integrative Toxicology‡Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 488241To whom correspondence should be addressed at Department of Pathobiology and Diagnostic Investigation, Michigan State University, 212 Food Safety and Toxicology Bldg., East Lansing, MI 48824. Fax: (517) 353-9902. E-mail: harkemaj@msu.edu.52010122010115126728561020091812010© The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org2010Ozone (O3), an oxidant air pollutant in photochemical smog, principally targets epithelial cells lining the respiratory tract. However, changes in gene expression have also been reported in livers of O3-exposed mice. The principal aim of the present study was to determine if acute exposure to environmentally relevant concentrations of O3 could cause exacerbation of drug-induced liver injury in mice. Overdose with acetaminophen (APAP) is the most common cause of drug-induced liver injury in developed countries. In the present study, we examined the hepatic effects of acute O3 exposure in mice pretreated with a hepatotoxic dose of APAP. C57BL/6 male mice were fasted overnight and then given APAP (300 mg/kg ip) or saline vehicle (0 mg/kg APAP). Two hours later, mice were exposed to 0, 0.25, or 0.5 ppm O3 for 6 h and then sacrificed 9 or 32 h after APAP administration (1 or 24 h after O3 exposure, respectively). Animals euthanized at 32 h were given 5-bromo-2-deoxyuridine 2 h before sacrifice to identify hepatocytes undergoing reparative DNA synthesis. Saline-treated mice exposed to either air or O3 had no liver injury. All APAP-treated mice developed marked centrilobular hepatocellular necrosis that increased in severity with time after APAP exposure. O3 exposure increased the severity of APAP-induced liver injury as indicated by an increase in necrotic hepatic tissue and plasma alanine aminotransferase activity. O3 also caused an increase in neutrophil accumulation in livers of APAP-treated animals. APAP induced a 10-fold increase in the number of bromodeoxyuridine-labeled hepatocytes that was markedly attenuated by O3 exposure. Gene expression analysis 9 h after APAP revealed differential expression of genes involved in inflammation, oxidative stress, and cellular regeneration in mice treated with APAP and O3 compared to APAP or O3 alone, providing some indications of the mechanisms behind the APAP and O3 potentiation. These results suggest that acute exposure to near ambient concentrations of this oxidant air pollutant may exacerbate drug-induced liver injury by delaying hepatic repair.ozoneacetaminophenhepatotoxicitymiceO3 is the principal oxidant pollutant in photochemical smog. Approximately half of the U.S. population lives in areas that persistently exceed the U.S. EPA’s National Ambient Air Quality Standard for this highly reactive and irritant gas (U.S. EPA, 2008). Short- and long-term exposures to high ambient concentrations of O3 have been linked to adverse health outcomes that include increases in both morbidity and mortality from respiratory causes (Bell et al., 2004; Jerrett et al., 2009; Katsouyanni et al., 1995). Though numerous studies in laboratory animals and human subjects have documented the toxic effects of inhaled O3 on the lung, much less is known about its effects on extrapulmonary organs like the liver (U.S. EPA, 2008).Recently, using global gene expression analyses, investigators found that livers of C57BL/6 mice acutely exposed to inhaled O3 had significant downregulation of gene families related to lipid, fatty acid, and carbohydrate metabolisms that were consistent with systemic cachexic responses to exposure (Last et al., 2005). Transcription of several messenger RNAs (mRNAs)–encoding enzymes of xenobiotic metabolism was also decreased in livers of these O3-exposed mice. Since several interferon (IFN)-dependent hepatic genes were downregulated with O3 exposure, the investigators suggested that IFN may act as the signaling molecule between the lung and liver. Interestingly, mice exposed to O3 have prolonged pentobarbital sleeping time (Graham et al., 1981) also suggesting impairment of hepatic drug metabolism.To our knowledge, no studies investigating the potential hepatotoxic interactions of inhaled environmental pollutants and commonly used therapeutic drugs have been reported. In the present study, we investigated the acute effects of inhaled high ambient concentrations of O3 on drug-induced liver injury in mice caused by a widely used antipyretic/analgesic agent, acetaminophen (APAP). APAP is one of the most commonly used nonprescription drugs in the world, and although remarkably safe within therapeutic doses, it has a relatively narrow therapeutic window. Indeed, APAP overdose is a commonly reported cause of liver failure in the United States (Larson et al., 2005). Like in humans, mice receiving an overdose of APAP develop acute liver injury that is characterized pathologically by centrilobular hepatocellular degeneration and necrosis with elevated blood activity of liver enzymes, such as alanine aminotransferase (ALT) (Jemnitz et al., 2008; Tee et al., 1987).Commonly reported risk factors for APAP-induced liver injury include chronic alcohol use as well as the concurrent intake of some medicinal agents (e.g., isoniazid, phenytoin, zidovudine) (McClements et al., 1990; Shriner and Goetz, 1992). Environmental pollutants have also been recognized as risk factors in pulmonary, cardiovascular, and metabolic conditions, such as type II diabetes (Gent et al., 2003; Morris et al., 1995; O’Neill et al., 2005). More recently, it has been reported that inhalation of ambient air particulates promotes systemic and liver oxidative stress in mice (Araujo et al., 2008). Though these risk factors are well documented, the potential interactive effects of inhaled air pollutants, like O3, with APAP have not been investigated.The principal aim of the present study was to determine if acute exposure to environmentally relevant concentrations of O3 could cause exacerbation of ongoing drug-induced liver injury in mice. APAP is the most widely used hepatotoxicant for experimental studies of drug-induced liver injury. A known hepatotoxic dose of APAP in mice was used to ensure centrilobular hepatocellular injury prior to the start of O3 exposure. We report for the first time that a single near ambient exposure to O3 exacerbates APAP-induced hepatic injury in mice, resulting in more severe hepatocellular necrosis and attenuation of early hepatic repair mechanisms.MATERIALS AND METHODSLaboratory animals.Pathogen-free male C57BL/6J mice (8–10 weeks of age; the Jackson Laboratory, Bar Harbor, ME) were used in this study. Mice were housed in polycarbonate cages on heat-treated aspen hardwood bedding (Nepco-Northeastern Product Corp., Warrensburg, NY). Boxes were covered with filter bonnets, and animals were provided free access to food (Harlan Teklad Laboratory Rodents 22/5 diet, Madison, WI) and water. Mice were maintained in Michigan State University (MSU) animal housing facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and according to the National Institutes of Health guidelines as overseen by the MSU Institutional Animal Care and Use Committee. Rooms were maintained at temperatures of 21°C–24°C and relative humidities of 45–70%, with a 12-h light/dark cycle starting at 7:30 A.M.Experimental protocol: APAP treatment and O3 exposures.Mice were randomly divided into 12 groups, each consisting of six animals. They were given an intraperitoneal (ip) injection of 0 (saline vehicle) or 300 mg/kg APAP (Sigma Chemical Co., St Louis, MO) in 20 ml/kg saline. Animals were fasted 14 h before the administration of APAP at 4:00 A.M.. The dose of 300 mg/kg APAP was chosen based on the results of a pilot study in our laboratory that found mice treated with this dose, but not 150 mg/kg, developed centrilobular hepatocellular necrosis as identified by histopathology with corresponding elevations in plasma ALT activity.Two hours after APAP administration, mice were exposed to 0 (air), 0.25, or 0.5 ppm O3 for 6 h (Fig. 1). Most of the bioactivation of APAP and formation of the toxic APAP metabolite (N-acetyl-p-benzo-quinone imine) in the livers of mice are completed within the first 2 h after administration (Jollow et al., 1973; Muldrew et al., 2002). Therefore, the selected exposure time for O3 was after the bioactivation phase of APAP-induced hepatotoxicity. Mice were killed 9 or 32 h after APAP (1 or 24 h after O3 exposure, respectively). Data analysis in animals given 0.25 ppm was limited to morphological evaluations (liver necrosis and 5-bromo-2-deoxyuridine bromodexyuridine [BrdU] immunostaining) and plasma ALT activity at the later time (32 h) (Fig. 1).FIG. 1.Experimental design. Eight- to 10-week-old C57BL/6 male mice were given 0 (saline) or 300 mg/kg APAP and then exposed to O3 (0, 0.25, or 0.5 ppm) for 6 h. Mice were sacrificed at 9 or 32 h after APAP injection (1 or 24 h after O3 exposure, respectively).Mice were housed individually and exposed to O3 in stainless steel wire cages, whole-body inhalation exposure chambers (H-1000; Lab Products, Maywood, NJ). O3 was generated with an OREC 03V1-O ozonizer (O3 Research and Equipment Corp., AZ) using compressed air as a source of oxygen. Total airflow through the exposure chambers was 250 l/min (15 chamber air changes per hour). The concentration of O3 within chambers was monitored during the exposure using Dasibi 1003 AH ambient air O3 monitors (Dasibi Environmental Corp., Glendale, CA). Two O3 sampling probes were placed in the middle of the ozone chambers, 10–15 cm above cage racks. Airborne concentrations during the inhalation exposures were 0.26 ± 0.02 or 0.53 ± 0.01 ppm (mean ± SEM) for O3 chambers and 0.02 ± 0.009 ppm for air chambers.Animal necropsies and microscopic and biochemical analyses.Two hours before sacrifice, mice euthanized at the 32-h time were given BrdU (50 mg/kg; Fisher Scientific, Fair Lawn, NJ) ip. At the time of necropsy, mice were anesthetized with an ip injection of sodium pentobarbital (50 mg/kg; Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI), the abdominal cavity was opened, and blood was collected from the abdominal vena cava in heparinized tubes (BD Microtainer, Franklin Lakes, NJ). Animals were then killed by exsanguination.The liver was removed from the abdominal cavity, and the left liver lobe was fixed in 10% neutral buffered formalin (Fisher Scientific) for light microscopic examination and morphometric analyses. The caudate liver lobe from each mouse was removed and placed in RNAlater (Qiagen, Valencia, CA) at 4°C for 24 h and then stored at −20°C for gene expression analyses using real-time PCR. The remaining liver lobes were frozen and stored at −80°C for biochemical analysis of inflammatory cytokines, glutathione, and thiobarbituric acid–reactive substances (TBARS).After collection of liver samples, hemidiaphragms were punctured to allow collapse of right and left lung lobes, and the thoracic cavity was opened for the removal of the trachea and heart-lung en bloc. After the trachea was cannulated, the heart-lung block was excised, and the lungs were gently lavaged twice with 0.9 ml of sterile saline. Approximately 75–90% of the intratracheally instilled saline was recovered as bronchoalveolar lavage fluid (BALF) from the lavaged lung lobes and immediately placed on ice until further analysis.Cellular and biochemical analyses of BALF.Total cell counts in the collected BALF from each mouse were determined using a hemocytometer. Cytological slides prepared by centrifugation at 40g for 10 min using a Shandon cytospin 3 (Shandon Scientific, Sewickley, PA) were stained with Diff-Quick (Dade Behring, Newark, DE). Differential counts of neutrophils, eosinophils, macrophages, and lymphocytes were assessed on a total of 200 cells. Remaining BALF was centrifuged at 240g for 15 min to collect the supernatant fraction, which was stored at −80°C for later biochemical analysis.Flow cytometric analyses for inflammatory cytokines.BALF supernatants were assayed for inflammatory cytokines that included interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), IL-6, monocyte chemotactic protein-1 (MCP-1), IL-12, keratinocyte-derived chemokine (KC), and IL-10. Plasma cytokine concentrations for KC, TNF-α, MCP-1, and IL-6 were also determined. All cytokine kits were purchased as Flex Set reagents or as a preconfigured cytometric bead array kit (BD Biosciences, San Jose, CA). Cytokines analysis was performed using an FACSCalibur flow cytometer (BD Biosciences). Briefly, 50 μl of BALF or plasma was added to the antibody-coated bead complexes and incubation buffer. Phycoerythrin-conjugated secondary antibodies were added to form sandwich complexes. After acquisition of sample data using the flow cytometer, cytokine concentrations were calculated based on standard curve data using FCAP Array software (BD Biosciences).Liver tissues designated for similar cytokine analyses were suspended in PBS at 4°C and homogenized on ice. Homogenates were then centrifuged at 17,000g for 10 min at 4°C. Fifty microliters of the resulting supernatant was collected and assayed for IL-6, MCP-1, KC, TNF-α, and IL-10 by flow cytometry as described above.Plasma ALT assay.Blood collected at the time of necropsy was used to evaluate plasma ALT activity spectrophotometrically using Infinity ALT reagents purchased from Thermo Electron Corp. (Louisville, CO).Liver tissue processing for light microscopy and immunohistochemistry.Transverse sections from the middle of the left liver lobe were embedded in paraffin, cut at a thickness of 5 μm, and stained with hematoxylin & eosin (H&E) for routine histopathological examination and morphometric analyses. Other tissue sections were histochemically stained with periodic acid–Schiff (PAS) staining and counterstained with hematoxylin to identify intracellular glycogen.Routine immunohistochemical techniques were used for hepatocellular detection of nuclear BrdU, hepatic infiltration of neutrophils, and hepatocellular expression of hypoxia-inducible factor-1 alpha (HIF-1α). Briefly, liver sections were deparaffinized in xylene and rehydrated through descending grades of ethanol and immersed in 3% hydrogen peroxide to block endogenous peroxides. Sections were incubated with normal sera to inhibit nonspecific proteins (normal horse, rabbit, or goat sera for BrdU, neutrophils, or HIF-1α immunostaining, respectively; Vector Laboratories Inc., Burlingame, CA) followed by specific dilutions of primary antibodies (1:40, monoclonal mouse anti-BrdU antibody, BD Biosciences; 1:2500, monoclonal rat anti-neutrophil antibody, AbD Serotec, Raleigh, NC; and 1:200, polyclonal rabbit anti-HIF-1α, Novus Biologicals, Littleton, CO). Tissue sections were subsequently covered with secondary biotinylated antibodies, and immunostaining was developed with the Vector RTU Elite ABC kit (BrdU and HIF-1α; Vector Laboratories Inc.) or the RTU Phosphatase-labeled Streptavidin kit (neutrophils; Kirkegaard Perry Labs, Gaithersburg, MD) and visualized with Vector Red (neutrophils; Vector Laboratories Inc.) or 3,3′-diaminobenzidine (BrdU or HIF-1α; Sigma Chemicals) chromogens. Slides were counterstained with Gill 2 hematoxylin (Thermo Fisher, Pittsburgh, PA).Morphometric analyses of liver.BrdU-stained and unstained hepatocellular nuclei were counted in 10 medium power fields (×200) for each animal, starting with a randomly selected field and evaluating every third field. The hepatocellular labeling index (LI; % of hepatocytes undergoing DNA synthesis) was determined by counting the number of BrdU-labeled cells divided by the total number of hepatocytes and multiplying by 100.Hepatic neutrophil accumulation was assessed by averaging the numbers of neutrophils in 10 medium power fields (×200) in each slide. Analyzed fields were selected in an unbiased manner with a random start and counting every third field. Neutrophils were identified by positive immunohistochemical staining with the neutrophil-specific antibody and their polymorphologic nuclear profiles.Hepatocellular degeneration/necrosis in sections from the left liver lobe was quantified using standard morphometric methods that were similar to those previously described in detail (Yee et al., 2000). Briefly, H&E-stained liver sections from the left liver lobe were visualized with an Olympus BX-40 light microscope (Olympus Corp., Lake Success, NY) coupled with a 3.3-megapixel digital color camera (Qimaging, Surrey, British Columbia, Canada). Images at a magnification of ×200 were evaluated employing a 168-point lattice grid overlaying fields of hepatic parenchyma to determine (1) the total area of liver analyzed, (2) the area of degenerative/necrotic hepatic parenchyma, and (3) the area of normal parenchyma. The area of each object of interest (e.g., lesion) was calculated using the following expression (Cruz-Orive, 1982):Distance between points was 13 μm. Accordingly, the area represented by each point was 511 μm2. Section from the liver of each mouse was systematically scanned using adjacent nonoverlapping microscopic fields. The first image field analyzed in each section was chosen randomly. Thereafter, every third field was evaluated (∼10–14 fields evaluated/section). The measured fields represented ∼65% of the total area of each liver section. Percent lesion area was estimated based on the following formula:Quantitative real-time reverse transcription-PCR for hepatic gene expression.The caudate liver lobe was isolated and placed in RNAlater (Qiagen) and kept at 4°C for at least 24 h and then transferred to −20°C until processed. Total RNA was extracted using RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). Briefly, tissues were homogenized in RLT buffer containing β-mercaptoethanol with a 5-mm Rotor-Sator Homogenizer (PRO Scientific, Oxford, CT) and centrifuged at 12,000g for 3 min. Samples were then treated with Rnase-free Dnase, Rnase-free buffer, and water on the column for 30 min (Qiagen). Eluted RNA was diluted 1:5 with Rnase-free water and quantified using a GeneQuant Pro spectrophotometer (BioCrom, Cambridge, UK).Reverse transcription (RT) reaction was performed using High Capacity cDNA Reverse Transcription Kit reagents (Applied Biosystems, Foster City, CA) and a GeneAmp PCR System 9700 Thermocycler PE (Applied Biosystems). Each RT reaction was run in 5 μl of sample with 20 μl of cDNA Master Mix prepared according to the manufacturer's protocol (Applied Biosystems).Expression analyses of isolated mRNA were performed by quantitative real-time PCR using individual animal's cDNA with the ABI PRISM 7900 HT Sequence Detection System using Taqman Gene Expression Assay reagents (Applied Biosystems). The cycling parameters were 48°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s followed by 60°C for 1 min. Individual data are reported as fold change of mRNA in experimental samples compared to the saline/air control group. Real-time PCR amplifications were quantified using the comparative Ct method normalized to the mean of two endogenous controls (18S and glyceraldehyde 3-phosphate dehydrogenase). The cycle number at which each amplified product crosses the set threshold represented the Ct value. The amount of target gene normalized to the mean of the endogenous reference genes was calculated by subtracting the endogenous reference Ct from the target gene Ct (ΔCt). Relative mRNA expression was calculated by subtracting the mean ΔCt of the treated samples from the ΔCt of the control samples (saline treated and air exposed) (ΔΔCt). The absolute values of the comparative expression level (fold change) was then calculated by using the formula: Fold change = 2−ΔΔCt.Glutathione assay.To determine hepatic concentrations of oxidized glutathione (GSSG) and total glutathione (reduced plus oxidized GSH and GSSG, respectively), median lobes of the liver (preserved at −80°C) were homogenized in cold buffer (0.4M 2-(N-morpholino)ethanesulfonic acid, 0.1M phosphate, and 2mM EDTA, pH 6). Homogenates were centrifuged at 10,000g for 15 min at 4°C, and the supernatants were collected and deproteinated. For deproteination, an equal volume of tissue sample and metaphosphoric acid were mixed and centrifuged. The supernatant was carefully pipetted and mixed with triethanolamine. The total glutathione concentration was then assayed as recommended by the manufacturer (Cayman Chemical Co., Ann Arbor, MI). GSSG concentration was determined after derivatization of GSH with 2-vinylpyridine. Sample absorbance was determined at 405 nm, and the total or oxidized glutathione concentrations in liver homogenates was assessed by comparison of absorption to standard curves.TBARS assay.Lipid peroxidation in the liver was estimated using a commercially available kit according to the manufacturer's recommendations and malonaldehyde as a standard (TBARS kit; Cayman Chemical Co.). Liver tissue was homogenized on ice in RIPA Buffer and Proteases Inhibitor (Thermo Scientific, Rockford, IL). Homogenates were centrifuged at 1600g, and the supernatant was collected and used to detect malonaldehyde and TBARS adducts in acidic conditions and under high temperature (100°C). Absorbance was measured at 530 nm.Statistical analyses.Data were reported as mean ± SEM. Differences among groups were analyzed by a one- or two-way ANOVA followed by Student-Newman-Keuls post hoc test. When normality or variance equality failed, a Kruskal-Wallis ranked test was conducted. All analyses were performed using SigmaStat software (SigmaStat; Jandel Scientific, San Rafael, CA). Significance was assigned to p values less than or equal to 0.05.RESULTSNo unscheduled deaths occurred in any groups of this study, and all mice were sacrificed at the scheduled posttreatment times described above. No mortality was observed in any group of APAP, O3, or APAP/O3 mice held for 96 h prior to sacrifice (data not shown).Inflammatory Responses Reflected in BALFSaline treatment/O3 exposure (O3-alone or SAL/O3 group) did not cause changes in BALF total inflammatory cell number at any time compared to saline treatment/air exposure (controls or SAL/air group) (Figs. 2A and 2B). As compared to control mice, animals that were administered APAP and were exposed to either air (APAP/air) or O3 (APAP/0.25 ppm O3 or APAP/0.5 ppm O3) had a time-dependent statistical increase in the number of total inflammatory cells in the BALF at 9 and 32 h after APAP administration (Figs. 2A and 2B). Though not statistically significant, there was a trend for greater total inflammatory cells in the lungs of the APAP and 0.5 ppm O3-coexposed mice compared to APAP alone (Figs. 2A and 2B). No difference in total inflammatory cell number was detected between APAP-alone and APAP/0.25 ppm O3 groups at 32 h (Fig. 2B).FIG. 2.Inflammatory cell accumulation in BALF of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air), 0.25 (32 h only), or 0.5 ppm O3 for 6 h. Nine (A, C, and E) or 32 h (B, D, and F) after APAP administration, mice were sacrificed and lungs were lavaged with saline as described in detail in the text. The numbers of inflammatory cells per milliliter in the recovered BALF are graphically presented as total inflammatory cells (A and B), neutrophils (C and D), and macrophages (E and F). Bars represent group means ± SEM (n = 6). a, significantly different from saline/air group; b, significantly different from saline/0.5 ppm O3 group; and c, significantly different from saline/0.25 ppm O3 (p ≤ 0.05).Pulmonary inflammatory cell responses, as reflected in the BALF, were due to increases in alveolar macrophages and/or neutrophils (Figs. 2C–F). O3 alone did not cause changes in neutrophil or macrophage number in BALF at any time (Figs. 2C–F). At 9 and 32 h after APAP treatment, mice exposed to APAP alone or with O3 had significant increases of alveolar macrophages (Figs. 2E and 2F). This response was somewhat greater at 32 h compared to 9 h. On the other hand, the number of neutrophils in BALF was not affected by APAP at 9 h (Figs. 2C and 2D). At 32 h, only APAP/O3-coexposed mice had marked increases in neutrophil numbers in BALF (Fig. 2D), indicating a potentiating effect.BALF supernatants were analyzed by flow cytometry using cytometric bead array technology for exposure-induced changes in several inflammatory cytokines (IL-1, TNF-α, IL-10, IFN-γ, IL-6, MCP-1, IL-12, and KC). Most of these cytokines were not significantly changed at either examined time (data not shown), with the exception of IL-6 and MCP-1. APAP or O3 alone did not cause an increase in IL-6 at 9 h and only a minimal increase occurred at 32 h with the APAP treatment (Figs. 3A and 3B). APAP/O3 coexposure resulted in a significant increase of IL-6 in BALF compared to either substance alone at 9 h but not at 32 h after APAP administration (Figs. 3A and 3B). At the early time, MCP-1 was undetectable in the BALF of mice from any of the groups (Fig. 3C). O3 exposure caused a slight, but significant, elevation of MCP-1 at 32 h after APAP treatment (Fig. 3D). MCP-1 in BALF was similarly and significantly elevated in APAP alone- or APAP/O3-coexposed mice 32 h after APAP (Fig. 3D).FIG. 3.IL-6 (A and B) and MCP-1 (C and D) protein concentrations in the BALF of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine or 32 h after APAP administration, mice were sacrificed and lungs were lavaged with saline as described in detail in the text. The amount of IL-6 and MCP-1 in the recovered BALF are graphically presented. Bars represent group means ± SEM (n = 6). a, significantly different from saline/0.5 ppm O3 group; b, significantly different from APAP/air group; and c, significantly different from saline/air group (p ≤ 0.05).Inflammatory Cytokine Concentrations in PlasmaExposure-related changes in plasma cytokines were restricted to IL-6, MCP-1, and KC. Changes in plasma IL-6 reflected those in BALF with only APAP/O3 coexposure inducing significant elevation in IL-6 concentration and only at 9 h after treatment (Figs. 4A and 4B). O3 alone–exposed mice had no plasma MCP-1 change at any time compared to controls. APAP alone caused significant elevation of plasma MCP-1 concentration 9 h after treatment that was not observed in the APAP/O3 coexposure group (Fig. 4C). At 32 h, APAP alone– and APAP/O3-coexposed mice had similar increases in plasma MCP-1 compared to controls (Fig. 4D).FIG. 4.IL-6 (A and B), MCP-1 (C and D), and KC (E and F) protein concentrations in plasma of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine or 32 h after APAP administration, animals were sacrificed and blood collected and analyzed as described in detail in the text. Bars represent group means ± SEM (n = 6). a, significantly different from saline/0.5 ppm O3 group; b, significantly different from APAP/air group; and c, significantly different from saline/air group (p ≤ 0.05).O3 exposure did not change the plasma concentrations of KC, a neutrophil chemokine (Figs. 4E and 4F). KC was significantly elevated only in APAP/O3-coexposed mice at 9 h after APAP treatment (Fig. 4E). At the later time, plasma KC returned to control levels in APAP/O3-coexposed mice, but there was a significant elevation in KC concentration in the plasma of mice given APAP alone (Fig. 4F).Histopathology and Morphometric Assessment of Liver InjurySaline-treated mice exposed to either air or O3 had no hepatic histopathology at either time postexposure (Figs. 5A and 5B). All APAP-treated mice developed hepatic centrilobular necrosis at 9 (data not shown) and 32 h (Figs. 5C and 5D). This drug-induced liver lesion increased in severity with time after APAP administration. Inhalation exposures to either 0.25 or 0.5 ppm O3 markedly increased the APAP-induced centrilobular necrosis at 32 h (Fig. 5D) but not at 9 h after APAP administration (24 and 1 h after O3 exposure, respectively). At the later time, APAP- and O3-coexposed mice had expanded areas of centrilobular necrosis compared to APAP alone–treated mice. These expanded areas were rimmed by a distinctive layer of enlarged hepatocytes with highly vacuolated cytoplasm and pyknotic nuclei. This one to two cell layer of hepatocytes undergoing ballooning degeneration separated the conspicuous centrilobular areas of coagulative necrosis from the normal midzonal and periportal hepatocytes. Morphometrically, APAP-treated mice exposed to 0.25 or 0.5 ppm O3 had 1.46 or 1.62 times increase, respectively, in hepatocellular necrosis compared to APAP alone–treated mice (Fig. 5E).FIG. 5.Hepatic histopathology/morphometry: liver damage induced by APAP and/or O3 exposure 32 h after APAP. Light photomicrographs of liver sections from mice treated with saline/air (A), saline/0.5 ppm O3 (B), APAP/air (C), or APAP/0.5 ppm O3 (D). All tissue sections are stained with H&E. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (filtered air), 0.25, or 0.5 ppm O3 for 6 h. Thirty-two hours after APAP administration, mice were euthanized and liver tissues were processed for light microscopy. No histopathology is evident in the livers of control or O3-exposed mice (A and B, respectively). Centrilobular hepatocellular necrosis (solid arrow) surrounding the central vein (CV) is present in the liver of APAP/air mouse (C). Increased amount of hepatocellular necrosis (solid arrow) circumscribed with hepatocytes undergoing ballooning vacuolar degeneration (stippled arrow) is present in the liver section of the APAP/0.5 ppm O3 mouse (D). Graphic representation of the morphometric determinations of the amounts of hepatocellular injury is presented in (E). Bars represent the group means ± SEM (n = 6). a, significantly different from APAP/Air group (p ≤ 0.05).No changes in plasma ALT activity, a marker of hepatocellular injury in circulating blood, were detected in O3 alone–exposed mice as compared to control mice (Figs. 6A and 6B). As expected, plasma ALT activity was significantly elevated in APAP-treated mice at 9 or 32 h after administration (Figs. 6A and 6B). At the early time, no significant differences in ALT activity were observed between APAP alone– and APAP/O3-coexposed mice (Fig. 6A). At 32 h after APAP injection, however, ALT activity was significantly greater in APAP/O3-coexposed mice as compared to APAP/air-exposed mice (Fig. 6B). This finding was reflected in differences in the extent of the centrilobular lesions between these groups. As mentioned previously, no mortality was observed in any group up to 96 h after APAP administration. By 96 h, APAP-induced hepatic lesions were markedly attenuated (minimal severity) or completely resolved, and plasma ALT concentrations had returned to normal in both the APAP/air-exposed and the APAP/O3-coexposed mice (data not shown).FIG. 6.Plasma ALT activity in APAP and/or O3-exposed mice 9 h (A) and 32 h (B) after APAP. Nine or 32 h after APAP administration, animals were sacrificed and plasma collected and analyzed for ALT activity as described in detail in the text. Bars represent group means ± SEM (n = 6). a, significantly different from saline/air group; b, significantly different from saline/0.25 ppm O3 group; c, significantly different from saline/0.5 ppm O3; and d, significantly different from APAP/air group (p ≤ 0.05).No neutrophilic accumulation was observed in livers of O3 alone–exposed mice at either time postexposure (Figs. 7B, 7E, and 7F). In APAP-treated mice, neutrophilic accumulation was observed predominantly within the areas of hepatocellular degeneration and necrosis (Figs. 7C and 7D). APAP/air and APAP/O3 groups had similar numbers of neutrophils in the liver as determined by morphometric analyses at 9-h posttreatment (Fig. 7E). At 32 h, the numbers of neutrophils in the livers of APAP/O3 mice were not significantly greater compared to APAP/air mice (Fig. 7F).FIG. 7.Liver neutrophil infiltration in APAP- and/or O3-exposed mice after APAP. Light photomicrographs of liver sections from mice treated 32 h earlier with saline/air (A), saline/0.5 ppm O3 (B), APAP/air (C) and APAP/0.5 ppm O3 (D). Tissue sections were immunohistochemically stained for infiltrating neutrophils (red chromagen; arrows) and counterstained with hematoxylin as described in detail in the text. Morphometric determinations of the numeric cell density of neutrophils in the hepatic parenchyma are graphically presented in (E and F). Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Bars in (E and F) represent group means ± SEM (n = 6). a, significantly different from saline/air group and b, significantly different from saline/O3 group (p ≤ 0.05); CV, central vein.Hepatocellular Regeneration and HypoxiaBrdU was administered to mice euthanized 32 h after APAP to identify hepatocytes undergoing DNA synthesis (S phase of the cell cycle). O3 exposure alone had no effect on hepatocellular BrdU incorporation (Figs. 8A and 8E). APAP treatment caused a marked increase of BrdU immunopositive nuclei, which was dose dependently reduced by coexposure with O3 (Figs. 8C–E). O3 (0.25 and 0.5 ppm) completely blocked the APAP-induced increase in BrdU incorporation in the liver (Fig. 8E).FIG. 8.Hepatocellular proliferation (DNA synthesis) in APAP- and/or O3-exposed mice 32 h after APAP. Light photomicrographs of liver sections from mice treated with saline/air (A), saline/0.5 ppm O3 (B), APAP/air (C), and APAP/0.5 ppm O3 (D). Tissues were immunohistochemically stained for nuclear incorporation of BrdU (brown chromagen; arrows) in hepatocytes undergoing DNA synthesis (cells in S phase of cell cycle). Morphometric determinations of the LI (%) of BrdU-labeled hepatocytes is graphically presented in (E). Mice were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air), 0.25, or 0.5 ppm O3 for 6 h. Two hours before sacrifice, mice were injected ip with BrdU. In (E), bars represent group means ± SE (n = 6). a, significantly different from saline/air group and b, significantly different from APAP/O3 groups (p ≤ 0.05). PV, portal vein; CV, central vein.At 32 h, no change in glycogen or HIF-1α staining was seen in mice exposed to O3 alone compared to controls (Figs. 9A1–2 and 9B1–2). At the same time, necrotic areas in the livers of APAP-treated mice were surrounded by a one to two cell thick layer of glycogen-depleted hepatocytes (Fig. 9A3). Interestingly, in APAP/O3-coexposed mice, the ballooning degeneration of hepatocytes was located in this layer of glycogen depletion and appeared to be the targeted tissue for the O3-induced expansion of APAP-induced liver injury (Fig. 9A4). APAP hepatotoxicity was accompanied by an increase in hepatocellular HIF-1α, a key transcription factor that mediates cellular response to hypoxia (Pouyssegur et al., 2006; Semenza, 2003). HIF-1α accumulation in APAP-treated mice was consistently found in the cytoplasm and less frequently in the nucleus of hepatocytes located in glycogen-depleted areas (junction of centrilobular necrotic zone and healthy parenchyma) (Fig. 9B3). In the APAP/O3 group, few hepatocellular nuclei, located primarily at the periphery of the necrotic zone, had HIF-1α accumulation (Fig. 9B4).FIG. 9.Intracellular glycogen (A) and HIF-1α (B) staining 32 h after APAP. Light photomicrographs of liver sections from mice given saline/air (A1 and B1), saline/0.5 ppm O3 (A2 and B2), APAP/air (A3 and B3), and APAP/0.5 ppm O3 (A4 and B4). Mice were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. In (A), all tissue sections were histochemically stained with PAS for glycogen (purple stain) as described in detail in the text. In (B), all tissue sections were immunohistochemically stained for HIF-α (dark brown chromagen) as described in detail in the text. nH, normal hepatocytes; HH, hypertrophic hepatocytes circumscribing areas of hepatocellular necrosis (asterisk); black arrows in (A) indicate hepatocellular vacuolar degeneration of HH; arrows in (B) indicate cytoplasmic or nuclear localization of HIF-α (solid and stippled arrows, respectively); CV, central vein. In APAP-treated mice exposed to air or O3 (A3 and A4, respectively), there is loss of PAS-stained glycogen in the areas of centrilobular necrosis as well as in hypertrophic hepatocytes (HH). In (A4), some of the HH are undergoing vacuolar degeneration. In (B), no HIF-α is present in liver sections (B1 and B2) from saline/air control mouse and saline/0.5 ppm O3 mouse, respectively. In the liver section from the APAP/air mouse (B3), HH circumscribing the areas of centrilobular necrosis (asterisk) contain cytoplasmic and/or nuclear HIF-α (solid arrow and stippled arrow, respectively). In the liver section from the APAP/O3 mouse, there is a loss of HIF-α in the HH undergoing vacuolar degeneration and necrosis.Relative Gene Expression and Protein Concentration of Inflammatory Cytokines in the LiverO3 exposure alone did not cause relative gene expression changes in chemokines KC or MIP-2 in the livers at any time postexposure (Figs. 10A–D). This correlated with the lack of neutrophil liver accumulation in these mice. APAP treatment or APAP/O3 coexposure caused significant increases in relative expression of KC and MIP-2 genes 9 h after APAP (Figs. 10A and 10C). KC protein concentration was also elevated in APAP/air and APAP/O3 groups at both 9 and 32 h after APAP (Figs. 11A and 11B). At 9 h, no differences were observed in mRNA expression of KC between APAP/air and APAP/O3 groups (Fig. 10A). At the same time, APAP/O3-coexposed mice had approximately three times the MIP-2 mRNA expression of the APAP-alone group (Fig. 10C). Relative gene expression of these chemokines declined to levels similar to those of controls at 32 h after APAP (Figs. 10B and 10D).FIG. 10.KC (A and B), MIP-2 (C and D), and MCP-1 (E and F) gene expression in livers of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine or 32 h after APAP administration, mice were sacrificed and liver samples were analyzed by quantitative real-time RT-PCR as described in detail in the text. Bars represent the group means ± SEM (n = 6) of the fold change in mRNA expression relative to that of the saline/air control group. a, significantly different from saline/air group; b, significantly different from saline/0.5 ppm O3 group; and c, significantly different from APAP/air group (p ≤ 0.05).FIG. 11.KC (A and B), MCP-1 (C and D), and IL-6 (E and F) protein concentrations in livers of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine (A, C, and E) or 32 h (B, D, and F) after APAP administration, mice were sacrificed and liver samples were analyzed as described in detail in the text for protein concentrations. Bars represent group means ± SEM (n = 6). a, significantly different from saline/air group; b, significantly different from saline/0.5 ppm O3 group; and c, significantly different from APAP/Air group (p ≤ 0.05).O3 exposure alone had minimal effects on relative mRNA expression or protein concentration of MCP-1 in the liver at any time (Figs. 10E and 10F and Figs. 11C and 11D). APAP treatment alone caused a significant increase in MCP-1 relative expression or protein concentration over control levels 9 h after its administration (Figs. 10E and 11C). In comparison, 0.5 ppm O3 exposure caused a more than fivefold reduction in APAP-induced increase in the mRNA or protein concentration of MCP-1 (Figs. 10E and 11C). At 32 h, both APAP/air and APAP/O3 groups had significant increases in MCP-1 mRNA expression and protein concentration (Figs. 10F and 11D).O3 exposure had no effect on IL-6 or plasminogen activator inhibitor-1 (PAI-1) relative expression at any time (Figs. 12A–D). APAP treatment on the other hand resulted in significantly elevated hepatic IL-6 and PAI-1 mRNA at 9 or 32 h after its administration (Figs. 12A and 12B). O3 coexposure tended to reduce the increases in IL-6 and PAI-1 mRNA caused by APAP, but this reduction reached statistical significance at the early time only (Figs. 12A and 12B). In agreement with these gene expression data, IL-6 protein was increased in the liver by APAP, but not APAP/O3, at 9 h only after APAP treatment (Figs. 11E and F).FIG. 12.IL-6 (A and B) and PAI-1 (C and D) genes expression in livers of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine (A and C) or 32 h (B and D) after APAP administration, mice were sacrificed and liver samples were analyzed by quantitative real-time RT-PCR as described in detail in the text. Bars represent the group means ± SEM (n = 6) of the fold change in mRNA expression relative to that of the saline/air control group. Data are expressed as mean ± SE (n = 6). a, significantly different from saline/air group; b, significantly different from APAP/air group; and c, significantly different from saline/O3 group (p ≤ 0.05).Regeneration-Related Gene Expression in Liver TissueO3 or APAP alone caused an increase in liver mRNA expression of the cyclin-dependent kinase inhibitor P21, at the early time postexposure (Fig. 13A). At the same time, APAP/O3 coexposure resulted in significantly greater expression of P21 mRNA expression compared to either APAP or O3 (Fig. 13A). At 9 h, suppressor of cytokine signaling 3 (SOCS3) expression was decreased by O3 exposure but increased by APAP treatment (Fig. 13C). APAP/O3 group had no statistically significant increase in expression of SOCS3 as compared to APAP-alone group (Fig. 13C). At 32 h, APAP-alone and APAP/O3 mice had similar increases in the expression of P21 and SOCS3 as compared to control mice (Figs. 13B and 13D).FIG. 13.P21 (A and B) and SOCS3 (C and D) genes expression in APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine (A and C) or 32 h (B and D) after APAP administration, mice were sacrificed and liver samples were analyzed by quantitative real-time RT-PCR as described in detail in the text. Bars represent the group means ± SEM (n = 6) of the fold change in mRNA expression relative to that of the saline/air control group. a, significantly different from saline/air group; b, significantly different from saline/0.5 ppm O3 group; and c, significantly different from APAP/air group (p ≤ 0.05).Liver Oxidative Damage (Antioxidant Genes, Glutathione, and TBARS Assays)Heme oxygenase-1 (HO-1), metallothionein-1 (MT-1), and the catalytic subunit of glutamate-cysteine ligase (GCLC) were evaluated as markers of oxidative stress. For all three of these antioxidant genes, mRNA expression was significantly elevated with APAP treatment, whereas only MT-1 was increased with O3 exposure 9 h after APAP (Figs. 14A, 14C, and 14E). At 9 h, APAP/O3 coexposure resulted in significant increase of MT-1 expression above APAP or O3 levels (Fig. 14A). At the later time (32 h), expression of these genes declined in APAP/air or APAP/O3 groups as compared to the early time, and APAP/O3-coexposed mice had less or comparable mRNA expression compared to APAP/air-treated mice (Figs. 14B, 14D, and 14F).FIG. 14.MT-1 (A and B), HO-1 (C and D), and GCLC (E and F) gene expression in livers of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine (A, C, and E) or 32 h (B, D, and F) after APAP administration, mice were sacrificed and liver samples were analyzed by quantitative real-time RT-PCR as described in detail in the text. Bars represent the group means ± SEM (n = 6) of the fold change in mRNA expression relative to that of the saline/air control group. a, significantly different from saline/air group; b, significantly different from saline/0.5 ppm O3 group; and c, significantly different from APAP/air group (p ≤ 0.05).To explore further O3 exacerbation of APAP-induced liver toxicity and the role of oxidative stress, we evaluated concentrations of GSH and GSSG. At 9 h, total glutathione concentration was greater in APAP-alone and APAP/O3 exposure groups than in control animals (Fig. 15A). At the same time, O3-exposed mice had less total glutathione concentration in the liver compared to control mice (Fig. 15A). Interestingly, O3-alone and APAP/O3 groups had more GSSG than control and APAP-alone groups, respectively (Fig. 15B).FIG. 15.Total (A) or oxidized (B) glutathione and TBARS (C and D) concentrations in livers of APAP- and/or O3-exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. Nine (A, B, and C) or 32 h (D) after APAP administration, mice were sacrificed and liver samples were processed for analytical determination of glutathione (GSH and GSSG) and TBARS concentrations by standard assays described in detail in the text. Bars represent the group means ± SEM (n = 6). a, significantly different from saline/air group; b, significantly different from saline/0.5 ppm O3 group; and c, significantly different from APAP/air group (p ≤ 0.05).APAP-induced changes in the levels of GSH and GSSG were time dependent, as have been previously reported by others. It has been shown that APAP treatment in mice, at a similar dose used in our study, causes depletion of hepatic GSH within the first hour after administration (Muldrew et al., 2002) but GSH recovers to pretreatment levels by 2 h (Saito et al., 2009). Therefore, the APAP-induced elevations in hepatic GSH observed in the present study were probably a rebound effect from an early depletion of GSH.We also evaluated lipid peroxidation levels in the livers using TBARS assay. Mice coexposed to APAP and O3 had greater concentrations of TBARS at the early time postexposure relative to APAP alone–treated mice (Fig. 15C). O3 alone–exposed mice had no significant increases in the concentration of TBARS compared to controls (Fig. 15C). By 32 h, TBARS concentrations in APAP-alone and APAP/O3 groups were less than that of their respective control groups (Fig. 15D). These results also indicate that APAP has a time-dependent effect on TBARS in the livers of mice. It has previously been shown in mice that APAP treatment increases hepatic TBARS levels that peak at around 2–3 h after administration (Amimoto et al., 1995). At 6-h posttreatment, the concentration of TBARS decline from peak levels. The early time in our study (9-h post-APAP) was probably on the recovery side of this response, accordingly. As to the later time point, it is likely that the antioxidant mechanisms induced by APAP (as suggested by gene expression data above) were probably responsible for the decrease in the levels of TBARS in the livers of APAP-treated groups.DISCUSSIONTo our knowledge, this is the first study to examine the pulmonary and systemic effects of APAP/O3 coexposure in laboratory animals. Acute exposure to 0.25 or 0.5 ppm O3 alone did not cause pulmonary inflammation in the mice of our study, as evidenced by BALF analysis. In contrast, APAP treatment alone did cause acute inflammation of the lung, and this drug-induced pulmonary response was exacerbated by O3 coexposure. The most remarkable finding, however, was that a single 6-h inhalation exposure to O3 resulted in exacerbation of APAP-induced liver injury. After a hepatotoxic dose of APAP, exposure of mice to 0.5 ppm O3 resulted in a 60% increase in hepatocellular necrosis and an 80% decrease in hepatocellular regeneration as compared to mice treated with APAP alone. How a single acute exposure of O3 caused such marked enhancement of this drug-induced liver injury in mice is unknown.It is unlikely that this potentiation of APAP-induced liver injury caused by O3 exposure was due to changes in APAP metabolism since most, if not all, of the hepatic bioactivation of APAP and formation of the toxic APAP metabolite would have occurred in the first couple of hours after administration (Jollow et al., 1973; Muldrew et al., 2002). Furthermore, others have reported that hepatic levels of several isoforms of cytochrome P450s, including those involved in bioactivation of this drug, are unchanged or downregulated, rather than upregulated, after inhalation exposure to O3 (Last et al., 2005).It is also not likely that O3 caused direct injury to the liver since it is one of the most reactive chemicals known, and others have demonstrated that when inhaled it reacts quickly with airway surface lining fluid and is converted to secondary lipid ozonation products (Miller, 1995; Pryor, 1992; Pryor et al., 1995). These secondary products (e.g., aldehydes, hydroxyhydroperoxides etc) are thought to be the principal toxicants responsible for O3’s toxicity to epithelial cells lining the airway surfaces. Therefore, O3 could not have entered the systemic circulation to be transported from the lung to the liver resulting in direct hepatotoxicity.Though the present study was not designed to definitively determine how acute inhalation exposure to O3 caused exacerbation of APAP-induced liver injury, the results of our biochemical and molecular analyses suggest some plausible hypotheses that will have to be addressed in future studies.One possible hypothesis is that the O3-induced enhancement of APAP hepatotoxicity is due in part to increased oxidative stress in the liver (i.e., more injurious oxidant-free radicals than protective antioxidants). Goldstein et al. (1978) suggested that extrapulmonary effects of O3 are related to lipid oxidation products, particularly malonaldehyde released after the interaction of O3 with airway epithelial cell membrane fatty acids. Interestingly, oxidative stress has also been suggested to play a key role in the progression of APAP-induced liver injury, specifically through induction of mitochondrial permeability transition pore formation (Jaeschke et al., 2003). It is plausible that the systemic increase in oxidative stress induced by inhaled O3 may have been partially responsible for the exacerbation of both APAP-induced liver injury observed in our study.In the present study, APAP/O3 coexposure resulted in significant expression of the oxidative stress–responsive genes, MT-1, HO-1, and GCLC in the liver. We also found that O3 exposure in either APAP- or saline-treated mice caused significant increases of hepatic GSSG, another indicator of oxidative stress (i.e., oxidation of GSH). In addition, lipid peroxidation, an indicator of oxidant-induced cellular injury, was biochemically evident in the livers of O3-exposed mice.The greatest measured response in antioxidant gene expression in the liver of APAP/O3-coexposed mice was MT-1. Metallothioneins, including MT-1, are cysteine-rich proteins with various protective roles, including antioxidant properties (Kang, 2006). Both APAP and O3 have been shown to induce increases of these proteins in the liver and lung of mice, respectively (Johnston et al., 1999; Wormser and Calp, 1988). In addition, mice lacking MT-1 and MT-2 are more sensitive to APAP or O3 toxicity compared to their wild-type counterparts (Inoue et al., 2008; Liu et al., 1999). Last et al. (2005) also reported that mice exposed to O3 exhibited greater hepatic expression of MT-1 compared to air-exposed animals.The antioxidant effects of MTs have been ascribed to their abundant cysteine moieties and direct scavenging properties of phenoxyl or hydroxyl radicals and superoxide anions as demonstrated by in vitro studies (Schwarz et al., 1994). Alternatively, the effect of MTs could be due to antioxidant properties of zinc released from MTs by reactive oxygen species (Powell, 2000; Schwarz et al., 1994). Owing to these roles, it is probable that the greater induction of MT-1 in the combined APAP and O3 coexposure was in response to enhanced oxidative stress.With immunohistochemical analysis, we found that mice given APAP had conspicuous accumulation of HIF-1α in hepatocytes immediately surrounding the centrilobular areas of necrosis. These same hepatocytes had concurrent loss of cytoplasmic glycogen as demonstrated by PAS histochemistry. It was these HIF-1α–overexpressing glycogen-depleted hepatocytes at the edge of the drug-induced necrotic regions that appeared microscopically to be further altered (e.g., ballooning degeneration and necrosis) by acute coexposure to O3. It is known that APAP directly targets mitochondria, inhibiting oxidative phosphorylation and compromising ATP synthesis with subsequent induction of glycolysis (Kon et al., 2004). APAP also induces HIF-1α accumulation in a hypoxia-unrelated oxidative stress–dependent fashion (James et al., 2006). In addition, HIF-1α induction results in decreased oxidative phosphorylation and stimulation of glycolysis in the liver of mice (Denko, 2008). Thus, glycogen depletion in the perinecrotic hepatocytes in APAP-treated mice might have been mediated by HIF-1α in our study.O3 inhalation is also known to alter cardiopulmonary function by increasing breathing frequency and pulmonary resistance and decreasing tidal volume, forced vital capacity, heart rate, and mean arterial pressure (U.S. EPA, 2008). Though speculative, it is possible that changes in cardiopulmonary function during O3 exposure could have compromised oxygen delivery to the APAP-induced areas of hepatic injury leading to additional tissue hypoxia and further injury. Interestingly, other studies in mice have recently demonstrated that chronic intermittent hypoxia can cause lipid peroxidation in the liver and exacerbate APAP-induced hepatic injury (Savransky et al., 2007). It is therefore reasonable that the APAP-induced loss of glycogen and increased HIF-1α in these hepatocytes might have made these cells more susceptible to O3 toxicity. Additional studies designed to examine pulmonary and cardiovascular function as well as blood gas parameters in mice treated with APAP and exposed to O3 will be needed to adequately investigate this possible pathogenesis.In the present study, PAI-1 expression was increased in the livers of APAP-treated mice similar to that reported by others (Bajt et al., 2008; Ganey et al., 2007; Reilly et al., 2001). In contrast, APAP/O3-coexposed mice in our study had markedly less PAI-1 liver expression as compared to mice given APAP alone. PAI-1 inhibits plasminogen activators involved in the formation of plasmin and as such inhibits fibrinolysis in mice (Bajt et al., 2008). In mice deficient in PAI-1, APAP caused greater plasma ALT activity, hepatocellular necrosis, and reduced hepatocellular regeneration. Like these PAI-1–deficient animals, the APAP/O3-coexposed mice in our study had greater hepatocellular injury and impaired hepatocellular repair (i.e., reduction in BrdU LI). One explanation for the role of PAI-1 in APAP/O3 cotoxicity could be that reduced PAI-1 led to early fibrinolysis in these animals, which then resulted in an ischemia/reperfusion-like mechanism.It has been reported that P21 mRNA was increased in APAP-treated PAI-1–deficient mice (Bajt et al., 2008). In our study, APAP/O3-coexposed mice had reduced expression of PAI-1 compared to APAP alone and an increase in the expression of P21 mRNA. P21 halts the cell cycle in the G1 phase by inhibiting the activity of cyclinE/cdk2 complexes (Weinberg and Denning, 2002). Therefore, it is possible that the enhanced liver pathology in APAP/O3-coexposed mice might be due in part to an O3-induced reduction in PAI-1, which in turn caused an increase in P21 that led to impairment of hepatocellular regeneration.As mentioned above, the results of our study clearly demonstrated that O3 exposure significantly impaired reparative hepatocellular regeneration in APAP-treated mice. This suggested to us that the O3 enhancement of APAP-induced liver injury may be due in part to reduced repair mechanisms. Mehendale and collaborators have proposed that the severity of acute chemical–induced liver toxicity is strongly dependent upon tissue repair processes (Soni et al., 1999). They have shown that cotreatment with small doses of hepatotoxicants (e.g., chlordecone and carbon tetrachloride, CCl4) can cause synergistic toxicity by inhibition of tissue repair (Soni and Mehendale, 1998). The differential expression of several genes in the livers of APAP/O3-coexposed mice in our study suggests some possible mechanisms by which O3 exposure might have compromised the hepatocellular regeneration after APAP-induced injury. For example, IL-6 gene and protein expression in the livers of APAP/O3-coexposed mice was significantly reduced compared to that in mice treated with APAP alone. This correlated with the marked reduction in hepatocellular BrdU labeling (reduced DNA synthesis). IL-6 is known to be an essential protein for the initial phases of hepatocellular regeneration, transitioning cells from the G0 to the G1 phase of the cell cycle (Fausto et al., 2006; Taub, 2004).Cressman et al. (1996) have shown that after partial hepatectomy, mice deficient in IL-6 had greater hepatocellular injury and reduced reparative regeneration as compared to IL-6–sufficient hepatectomized mice. IL-6–deficient mice treated with CCl4 had greater hepatocellular damage and decreased number of hepatocytes in the S phase of the cell cycle as compared to IL-6–sufficient mice (Kovalovich et al., 2000). Pretreatment of IL-6–deficient mice with IL-6 significantly reduced CCl4-induced liver injury and restored the reparative induction of DNA synthesis. We found that O3 inhalation caused impairment of hepatocellular repair following APAP-induced injury. Though the downregulation of hepatic IL-6 expression may be responsible for impaired liver regeneration 32 h after APAP, there may be other mechanisms involved.Other potential candidates responsible for the impaired regeneration in APAP/O3-coexposed mice are P21 and MCP-1. In APAP/O3-coexposed mice, expression of P21 was increased relative to mice given APAP or O3 alone. Activation of P21 after DNA damage is known to delay or arrest the cell cycle (Garner and Raj, 2008). APAP treatment or O3 exposure causes DNA damage in the liver or lung, respectively (Bornholdt et al., 2002; Hongslo et al., 1994; Ito et al., 2005; Ray et al., 1990). In the present study, mice treated with APAP or exposed to O3 alone had greater hepatic P21 expression than saline-treated and air-exposed control mice. APAP/O3-coexposed mice had even greater P21 expression compared to mice receiving only one of these chemical agents. Though the level of DNA damage in the liver was not measured in this study, the marked increase in P21 expression in the livers of coexposed mice might have been due to increased DNA damage, which is known to lead to hepatic cell death (Corcoran and Ray, 1992).Another interesting finding in this study was the effect of APAP and O3 on the expression of the inflammatory chemokine MCP-1 in the liver. APAP treatment caused a significant increase in the expression of this chemokine, but O3 exposure caused a marked reduction in MCP-1 expression. This reduction was associated with enhanced liver toxicity and defective hepatocellular regeneration responses. MCP-1 has been shown to be involved in cell regeneration and tissue repair in various tissues after different types of induced cell injury (Kim et al., 2003; Low et al., 2001; Shireman et al., 2007). Mice lacking MCP-1 or the receptor for MCP-1 have been reported to be more (Hogaboam et al., 2000) or similarly (Dambach et al., 2002) sensitive to APAP-induced hepatotoxicity compared to their wild-type counterparts. Interestingly, mice lacking the receptor for MCP-1 also had decreased reparative DNA synthesis in a murine model of arterial injury (Kim et al., 2003). It is not known how a reduction of MCP-1 could lead to impaired cell regeneration, but the role of MCP-1 in the O3 enhancement of APAP-induced hepatotoxicity should be explored in future studies.In conclusion, we found that a single 6-h inhalation exposure of mice to high ambient concentrations of O3 caused marked enhancement of APAP-induced hepatotoxicity in mice. The present study was not designed to determine the underlying mechanism(s) responsible for this observed systemic effect caused by this common oxidant air pollutant. Several biochemical and molecular markers of oxidative stress were elevated in the livers of APAP/O3-coexposed mice compared to mice that received only APAP or O3 alone. In addition, we found that concurrent with the enhancement of hepatotoxicity, O3 also caused a marked attenuation of normal increases in DNA synthesis necessary for hepatocellular regeneration and repair in response to chemical-induced liver injury. This finding suggests a possible role for impaired cellular regeneration and enhanced toxicity in the livers of coexposed mice. Though it is unclear how inhalation of this highly reactive gas could enhance chemical-induced liver injury, several endogenous hepatic proteins such as IL-6, HIF-1α, PAI-1, P21, and MCP-1 have been identified as potentially playing important roles in this effect.These results in mice also suggest that exposures to high ambient concentrations of O3 and other air pollutants (e.g., particulate matter) may pose a risk to people with preexisting chemical-induced and other liver diseases. Further studies are needed not only to understand the biological mechanisms underlying this systemic effect of inhaled O3 but also to determine the smallest doses of O3 and APAP at which these synergistic responses in the liver occur. 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Pharmacol.19873610411052U.S. Environmental Protection Agency (U.S. EPA)Air Quality Criteria for O3 and Related Photochemical Oxidants (Final)2008EPA 600/R-05/004-aF-cF, Research Triangle Park, NCWeinbergWCDenningMFP21Waf1 control of epithelial cell cycle and cell fateCrit. Rev. Oral. Biol. Med.200213453464WormserUCalpDIncreased levels of hepatic metallothionein in rat and mouse after injection of acetaminophenToxicology198853323329YeeSBKinserSHillDABartonCCHotchkissJAHarkemaJRGaneyPERothRASynergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotalineToxicol. Appl. Pharmacol.2000166173185 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A4FE23B6D5F32F54BAEB91487AE8D0C856194F6.txt b/test/dataset/in/resources/corpus/Clean_0A4FE23B6D5F32F54BAEB91487AE8D0C856194F6.txt new file mode 100644 index 0000000..d5c166a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A4FE23B6D5F32F54BAEB91487AE8D0C856194F6.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 41310.1093/gerona/60.4.413 Journal of Gerontology: Biological Sciences National Institute on Aging Microarray Facility—Resources for Gerontology Research Nadon Nancy L. 1 Mohr David 2 Becker Kevin G. 3 1Office of Biological Resources and Resource Development, National Institute on Aging, Bethesda, Maryland. 2The Johns Hopkins University Microarray Facility, Baltimore, Maryland. 3Gene Expression and Genomics Unit, National Institute on Aging Gerontology Research Center, Baltimore, Maryland. Address correspondence to Nancy L. Nadon, PhD, Office of Biological Resources and Resource Development, National Institute on Aging, 7201 Wisconsin Ave., GW2C231, Bethesda MD 20892. E-mail: nadonn@nia.nih.gov 4 2005 60 4 413 415 23 11 2004 28 9 2004 Copyright 2005 by The Gerontological Society of America 2005 The use of DNA microarrays allows rapid, large-scale analyses of changes in gene expression. This article introduces the National Institute on Aging Microarray Facility, developed to provide DNA microarrays to the biogerontology research community and to promote the use of this new technology. hwp-legacy-fpage 413 hwp-legacy-dochead RAPID COMMUNICATION MICROARRAYS, hybridization platforms containing large numbers of complementary DNA (cDNA) clones or gene-specific oligonucleotides, have revolutionized gene expression analysis. They allow analysis of expression of from hundreds to thousands of genes in a single hybridization experiment. These high-throughput analyses have contributed enormously to discoveries that might never have been made in the single-gene analysis approach. Arrays have increased the pace of discovery in the following areas of cancer research: cancer development, normal aging, and pathological development (1,2). The DNA Array Unit of the National Institute on Aging Gerontology Research Center (NIA GRC) has focused on the development of microarrays on nylon membranes. Membrane-based hybridization technology has a well-established place in modern molecular biology laboratories. Any laboratory doing standard hybridization protocols, such as Northern and Southern blotting, has the basic expertise and equipment required to use membrane microarrays. Radioactive labeling reagents are highly sensitive, widely available, and relatively inexpensive. Membrane arrays have some advantages over chips or glass arrays, particularly for laboratories that do not want to make a significant investment in new technology and equipment. Both probes and membranes can be reused, reducing the cost of replicating experiments (3). Although membrane arrays do not lend themselves to two-color probing that can be done on glass arrays, analytical software for membrane arrays is as powerful as that used with glass arrays. Large numbers of membranes can be hybridized at a time, increasing throughput, sample sizes, and thus overall experimental design. Membrane arrays, and the reagents to use them, are quite often less costly than commercially available array formats. To make membrane arrays more accessible to the research community, the NIA Array Unit established the NIA Microarray Facility to supply nylon membrane cDNAs at low cost. The website for the NIA Microarray Facility, located at Johns Hopkins University, is: http://www.daf.jhmi.edu/microarray/index.htm. Mouse cDNA Microarray The mouse 17K cDNA microarray contains 16,897 features, which include approximately 12,341 unique genes. The mouse array was based on the original 15K mouse cDNA clone set developed from mouse embryos at the NIA (4,5). Approximately 1700 genes were added to the original set to produce the 17K sequenced, verified clone set for production of this array format. The NIA mouse 17K array represents a large general mouse array similar to other large cDNA array collections. Although it is not a whole genome array, it contains a set of clones from embryonic cells and tissues from approximately 34 unique cDNA libraries, including specific libraries from neonatal tissues. Gene identification numbers in the 15K mouse clone set are available from the NIA Microarray Facility website (from the downloads link), and are also posted at the NIA Laboratory of Genetics website (http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html). The mouse cDNA microarrays are now available. Figure 1 shows a representative set of NIA mouse 17K membranes. Each microarray consists of two nylon membranes, each approximately the size of a 96-well culture plate, or 8 cm × 12 cm. Earlier versions of these arrays have been used productively in many model systems, including murine models of drug abuse (6,7) and murine developmental models (8,9). Human cDNA Microarrays Human cDNA microarrays have been developed and are currently available. This human array consists of full-length cDNA clones generated by the National Cancer Institute's Mammalian Gene Collection (10) (http://mgc.nci.nih.gov/). These clones have been completely sequenced and have been specifically chosen to represent the full-length open reading frame of each representative gene. The human cDNA arrays developed from these clones consist of 9600 features, representing approximately 6424 unique genes. This is printed on one 8 cm × 12 cm filter. Figure 2 shows a representative example of the human NIA membrane. Gene Expression Analysis Using the NIA Microarrays The range of biological applications, laboratory use, data collection, and analysis strategies using this array format is conceptually similar to most other array formats. Array hybridization is straightforward and requires minimal training time. Radioactive labeling for these arrays with 33P-α-dCTP requires a relatively low amount of input RNA (5 μg) of total RNA per sample, with no nucleic acid amplification, (i.e., RNA amplification). This quantity is particularly amenable to small tissue samples. Using this approach, we have utilized membrane arrays in this format quite extensively in studies of mouse embryogenesis, in in vitro cell line studies, and in studies of drug toxicity, normal aging, caloric restriction, and Alzheimer's disease, among many others. These arrays produce single channel data, similar to Affymetrix Genechip arrays or those using Illumina BeadChips. This produces primary intensity values, which are normalized and ultimately compared against other experimental samples to produce ratio data. The data are then analyzed using approaches and bioinformatic tools very similar to most other microarray formats. There are no specific plans for a central repository for data from these arrays. It is recommended that data should be deposited in a public microarray repository such as the National Center for Biotechnology Information's Gene Expression Omnibus (NCBI GEO). The data-tagging process in GEO should allow identification and retrieval of all information derived from this series of arrays. Information and Support Resources The NIA Microarray Facility website contains several links to information useful in the design and execution of experiments using the membrane arrays. The downloads link takes the viewer to information on the gene identification numbers of the clone sets used on the arrays. The images link presents views of hybridized arrays. The FAQ (frequently asked questions) link provides answers to questions often encountered by users new to nylon array hybridization and additional information on the versatility of the nylon arrays. The references link lists references that demonstrate the range of work possible with nylon arrays. There are protocols for preparing probes for hybridization, probe labeling, and reusing the membranes on the protocols link. All protocols listed were developed, tested, and used extensively by the NIA Array Unit. One of the benefits of radiolabeled hybridization of nylon membranes is that several options for data management and analysis already exist. The analysis link provides information on analysis of the array data, including information on analytical software and how to use it. The analysis link includes links to many free analysis programs that can be downloaded from the Web. Assistance with the use of NIA arrays or purchasing arrays is available from the contacts link and may also be sought by e-mailing the authors. Pertinent addresses areKevin Becker, PhD: beckerk@grc.nia.nih.govNancy Nadon, PhD: nadonn@nia.nih.govNIA Microarray Facility home page: http://www.daf.jhmi.edu/microarray/index.htm. Decision Editor: James R. Smith, PhD Figure 1. Typical images from the National Institute on Aging Mouse 17KA and 17KB filters Figure 2. Typical image from the Human MGC 9.6K filter References 1 Fan J, Yang X, Wang W, Wood WH, Becker KG, Gorospe M. Global analysis of stress-regulated mRNA turnover by using cDNA arrays. Proc Natl Acad Sci U S A.2002;99:10611-10616. 2 Sawiris GP, Sherman-Baust CA, Becker KG, Cheadle C, Teichberg D, Morin PJ. Development of a highly specialized cDNA array for the study and diagnosis of epithelial ovarian cancer. Cancer Res.2002;62:2923-2928. 3 Donovan DM, Becker KG. Double round hybridization of membrane based cDNA arrays: improved background reduction and data replication. J Neurosci Methods.2002;118:59-62. 4 Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, et al. Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc Natl Acad Sci U S A.2000;97:9127-9132. 5 Kargul GJ, Dudekula DB, Qian Y, Lim MK, Jaradat SA, Tanaka TS, et al. Verification and initial annotation of the NIA mouse 15K cDNA clone set. Nat Genet.2001;28:17-18. 6 Xie T, Tong L, McCann UD, Yuan J, Becker KG, Mechan AO, et al. Identification and characterization of metallothionein-1 and -2 gene expression in the context of (+/−)3,4-methylenedioxymethamphetamine-induced toxicity to brain dopaminergic neurons. J Neurosci.2004;24:7043-50. 7 Noailles PA, Becker KG. Wood WH 3rd, Teichberg D, Cadet JL. Methamphetamine-induced gene expression profiles in the striatum of male rat pups exposed to the drug in utero. Brain Res Dev Brain Res.2003;147:153-162. 8 Buttitta L, Tanaka TS, Chen AE, Ko MS, Fan CM. Microarray analysis of somitogenesis reveals novel targets of different WNT signaling pathways in the somitic mesoderm. Dev Biol.2003;258:91-104. 9 Suemizu H, Aiba K, Yoshikawa T, Sharov AA, Shimozawa N, Tamaoki N, et al. Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells. Dev Biol.2003;253:36-53. 10 Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, Collins FS, et al. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci U S A.2002;99:16899-16903. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A6084B9AED0D5F646B9D4A5156F477A2897AE62.txt b/test/dataset/in/resources/corpus/Clean_0A6084B9AED0D5F646B9D4A5156F477A2897AE62.txt new file mode 100644 index 0000000..adb9f86 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A6084B9AED0D5F646B9D4A5156F477A2897AE62.txt @@ -0,0 +1 @@ +geronageronaJournals of Gerontology Series A: Biomedical Sciences and Medical Sciences1758-535X1079-5006Oxford University Press10.1093/gerona/glq147Journal of Gerontology: BIOLOGICAL SCIENCES65TH ANNIVERSARY CELEBRASTIOBN ARTICLEVasoprotective Effects of Life Span-Extending Peripubertal GH Replacement in Lewis Dwarf RatsUngvariZoltan1GautamTripti1KonczPeter1HenthornJim C.1PintoJohn T.2BallabhPraveen2YanHan1MitschelenMatthew1FarleyJulie1SonntagWilliam E.1CsiszarAnna11Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City2Departments of Biochemistry, Anatomy, and Cell Biology, New York Medical College, ValhallaAddress correspondence to Anna Csiszar, MD, PhD, Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma HSC, 975 N. E. 10th Street—BRC 1303, Oklahoma City, OK 73104. Email: anna-csiszar@ouhsc.eduDecision Editor: Rafael de Cabo, PhD1120101608201065A111145115617520101972010© The Author 2010. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2010In humans, growth hormone deficiency (GHD) and low circulating levels of insulin-like growth factor 1 (IGF-1) significantly increase the risk for cerebrovascular disease. Genetic growth hormone (GH)/IGF-1 deficiency in Lewis dwarf rats significantly increases the incidence of late-life strokes, similar to the effects of GHD in elderly humans. Peripubertal treatment of Lewis dwarf rats with GH delays the occurrence of late-life stroke, which results in a significant extension of life span. The present study was designed to characterize the vascular effects of life span-extending peripubertal GH replacement in Lewis dwarf rats. Here, we report, based on measurements of dihydroethidium fluorescence, tissue isoprostane, GSH, and ascorbate content, that peripubertal GH/IGF-1 deficiency in Lewis dwarf rats increases vascular oxidative stress, which is prevented by GH replacement. Peripubertal GHD did not alter superoxide dismutase or catalase activities in the aorta nor the expression of Cu-Zn-SOD, Mn-SOD, and catalase in the cerebral arteries of dwarf rats. In contrast, cerebrovascular expression of glutathione peroxidase 1 was significantly decreased in dwarf vessels, and this effect was reversed by GH treatment. Peripubertal GHD significantly decreases expression of the Nrf2 target genes NQO1 and GCLC in the cerebral arteries, whereas it does not affect expression and activity of endothelial nitric oxide synthase and vascular expression of IGF-1, IGF-binding proteins, and inflammatory markers (tumor necrosis factor alpha, interluekin-6, interluekin-1β, inducible nitric oxide synthase, intercellular adhesion molecule 1, and monocyte chemotactic protein-1). In conclusion, peripubertal GH/IGF-1 deficiency confers pro-oxidative cellular effects, which likely promote an adverse functional and structural phenotype in the vasculature, and results in accelerated vascular impairments later in life.Oxidative stressGH deficiencyGH replacementVasoprotectionIGF-1 deficiencyDISRUPTION of the insulin/insulin-like growth factor 1 (IGF-I) pathway increases life span in invertebrates. However, effects of decreased growth hormone (GH)/IGF-I signaling in mammals remain controversial. The cardiovascular system is an important target organ for GH and IGF-1, and it is well documented that in human patients, growth hormone deficiency (GHD) and low circulating levels of IGF-1 significantly increase the risk for cardiovascular and cerebrovascular diseases (1–6). Although there are mouse models extant in which disruption of GH/IGF-1 signaling is associated with lifespan extension (eg, Ames (7) and Snell dwarf mice (8,9) and male mice heterozygous for the deletion of the IGF-1 receptor (10)), other rodent models with compromised GH/IGF-1 signaling do not exhibit a longevity phenotype (eg, female mice heterozygous for the deletion of the IGF-1 receptor (10) and genetically GH/IGF-1–deficient strain of Lewis rats (11)). The Lewis dwarf rat is a useful model of human GHD as Lewis dwarf rats have normal pituitary function except for a selective genetic GH deficiency and they mimic many of the pathophysiological alterations present in human GHD patients (including mild cognitive impairment (12)). Importantly, GH deficiency in Lewis rats significantly increases the incidence of late-life strokes (11), similar to the effects of GHD in elderly humans. It is significant that short-term peripubertal treatment of GH-deficient Lewis dwarf rats with GH decreases the incidence and delays the occurrence of late-life stroke, which results in a significant (∼14%) extension of life span (11). These findings suggest that peripubertal levels of GH and IGF-I are important for developing an adequate tissue architecture, maintaining vascular health, and delaying the development of age-related cardiovascular diseases. Although previous studies have established that GH/IGF-1 deficiency in adult patients and laboratory animals results in deleterious effects on vascular endothelial and smooth muscle cells (13–16), there is little or no information available on vascular alterations either in childhood GHD patients or animal models of peripubertal GHD.The available data demonstrate that in adult patients and laboratory animals, the GH/IGF-1 axis is an important regulator of vascular redox homeostasis (13,17,18), although the specific mechanisms are not well understood. Previously, we demonstrated that in the vasculature of adult GH/IGF-1–deficient Ames dwarf mice, antioxidant enzymes are downregulated, which is associated with increased vascular reactive oxygen species (ROS) levels and impaired endothelial function (13). The present study was designed to characterize the effects of peripubertal GH/IGF-1 deficiency on vascular redox homeostasis, the abundance and activity of antioxidant enzymes in the vascular wall, the local IGF-1 system, and the expression of inflammatory mediators in the cerebrovasculature, using Lewis dwarf rats as a model system. Moreover, we also determined whether life span-extending peripubertal GH replacement exerts vasoprotective effects in Lewis dwarf rats.METHODSAnimalsIn the present study, we used male Lewis rats that are heterozygous or homozygous for the spontaneous autosomal recessive dw-4 mutation, which causes a decrease in GH secretion from the pituitary gland (19). Lewis dwarf (dw-4/dw-4) rats have chronically low levels of GH and IGF-1 and make an excellent animal model of childhood-onset GHD (19). These rats have a spontaneous mutation that results in decreased GH secretion from the pituitary beginning around postnatal Day 26 (9,19,20). Female heterozygous (dw-4/−) Lewis rats were bred with male homozygous Lewis dwarf rats (dw-4/dw-4) to generate heterozygous (dw-4/−) offspring with a normal phenotype (“control”, n = 6) or homozygous rats (dw-4/dw-4) with a dwarf phenotype (“dwarf”). Classification as control or dwarf was based on their body weight as well as the serum IGF-1 levels at 33 days of age. Pubertal status was determined by age and the associated changes in growth and IGF-1 levels. Beginning on Day 35, dwarf rats were divided into two experimental groups: (a) early-onset GHD given saline (n = 6) and (b) GH replete with GH administered beginning at 5 weeks of age and continued throughout the experimental period of 30 days (termed “GH replete,” n = 6). Saline or GH (300 μg recombinant porcine GH; Alpharma, Victoria, Australia) was injected subcutaneously twice daily. The heterozygous rats were used as controls and given saline injections twice daily from 5 weeks of age to the end of the experimental period. Rats had access to food and water ad libitum and were housed in pairs in the barrier facility of the University of Oklahoma Health Sciences Center. Animals were fed standard rodent chow (PicoLab Rodent Diet 20 from Purina Mills [Richmond, IN], containing 20% protein by mass and 24% protein by caloric content). All studies were approved by the Institutional Animal Care and Use Committee.Measurement of Serum IGF-1Rats were anesthetized with isofluorane, and serum was obtained via tail bleed. Total IGF-1 levels in serum were determined as previously described (12,20–22).Measurement of Vascular O2− ProductionProduction of O2− in the aortic wall was determined using dihydroethidium (DHE), an oxidative fluorescent dye, as we previously reported (23,24). In brief, vessels were incubated with DHE (3 × 10−6 mol/L; at 37°C for 30 minutes). The vessels were then washed three times, embedded in optimal cutting temperature media, and cryosectioned. Red fluorescent images were captured at 20× magnification and analyzed using the Metamorph imaging software as reported (13). Four entire fields per vessel were analyzed with one image per field. The mean fluorescence intensities of DHE-stained nuclei in the endothelium and medial layer were calculated for each vessel. Thereafter, the intensity values for each animal in the group were averaged.8-Isoprostane AssayTotal tissue 8-isoprostane content was measured in aorta homogenates using the 8-isoprostane EIA kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's guidelines.Determination of Endogenous Glutathione and Ascorbate Using high-performance liquid chromatography Electrochemical DetectionConcentrations of redox-active GSH and ascorbate were measured in aorta homogenates using a Perkin-Elmer high-performance liquid chromatography equipped with an eight-channel amperometric array detector as described (25). In brief, 10 mg aliquots of tissue samples were washed with ice-cold phosphate-buffered saline and homogenized in 5% (w/v) metaphosphoric acid. Samples were centrifuged at 10,000g for 10 minutes to sediment protein, and the supernatant fraction was stored for analysis of redox-sensitive compounds. Precipitated proteins were dissolved in 0.1 N NaOH and stored for protein determination by a spectrophotometric quantitation method using BCA reagent (Pierce Chemical Co., Rockford, IL). Concentrations of GSH and ascorbic acid in supernatant fractions were determined by injecting 5 μL aliquots onto an Ultrasphere 5 u, 4.6 × 250 mm, C18 column and eluting with mobile phase of 50 mM NaH2PO4, 0.05 mM octane sulfonic acid, and 1.5% acetonitrile (pH 2.62) at a flow rate of 1 mL/min. The detectors were set at 200, 350, 400, 450, 500, 550, 600, and 700 mV, respectively. Peak areas were analyzed using ESA, Inc. (Chelmsford, MA) software, and concentrations of GSH and ascorbate are reported as nanomoles per milligram protein.Antioxidant Enzyme ActivitiesActivity of antioxidant enzymes and endothelial nitric oxide synthase (eNOS) in aorta homogenates was measured using the Glutathione Peroxidase Assay Kit (#703102), Catalase Assay Kit (#707002), Superoxide Dismutase Assay Kit (#706002), and NOS Activity Assay Kit (#781001) according to the manufacturer's guidelines (Cayman Chemical Company).Quantitative Real-Time RT-PCRA quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) technique was used to analyze messenger RNA (mRNA) expression of SOD1, SOD2, glutathione peroxidase 1, catalase, Sirt1, eNOS, the Nrf2/antioxidant response element target genes NQO1 and GCLC, components of the local IGF-1 system (IGF-1, IGF-1R, insulin-like growth factor–binding protein [IGFBP] 1, IGF-BP2, IGFBP3, and IGFBP4), and inflammatory markers (including tumor necrosis factor alpha, interluekin-6, interluekin-1β, inducible nitric oxide synthase, intercellular adhesion molecule 1, and monocyte chemotactic protein-1) in the middle cerebral arteries, as previously reported (26–29). In brief, total RNA was isolated with a Mini RNA Isolation Kit (Zymo Research, Orange, CA) and was reverse transcribed using Superscript III RT (Invitrogen , Carlsbad, CA) as described previously (26,30). A real-time RT-PCR technique was used to analyze mRNA expression using a Strategen MX3000, as reported (30). Amplification efficiencies were determined using a dilution series of a standard vascular sample. Quantification was performed using the efficiency-corrected ΔΔCq method. The relative quantities of the reference genes GAPDH, hypoxanthine phosphoribosyltransferase 1, and β-actin were determined, and a normalization factor was calculated based on the geometric mean for internal normalization. Oligonucleotides used for quantitative real-time RT-PCR are listed in Table 1. Fidelity of the PCR reaction was determined by melting temperature analysis and visualization of the product on a 2% agarose gel.Table 1.Oligonucleotides for Real-Time Reverse Transcriptase–Polymerase Chain ReactionmRNA TargetsDescriptionSenseAntisenseSOD2Mn-SODAGGCAAACGGGTTTCTGAGTGAGCTTGTCAGGGAAAGGGTGTCSOD1Cu,Zn-SODCTCAGGAGAGCATTCCATCATTGAATCACACCACAAGCCAAGCGpx-1Glutathione peroxidase 1TAGGTCCAGACGGTGTTCGATACCAGGAATGCCTTAGGCatalaseGCCTCACCAGTAATCATCGATCCAAACAGAAGTCCTAAGCeNOSEndothelial nitric oxide synthaseTTCCTGACAATCTTCTTCAGAGAGTTCTTAAATCGGNQO1NAD(P)H:quinone oxidoreductase 1TGGGATATGAATCAGGGAGAGGAGAGGTAACTAATAGCAACAAGGCLCGlutamate-cysteine ligase, catalytic subunit1GCTTTCTCCTACCTGTTTCTTGTGGCAGAGTTCAGTTCCGSirt1Sirtuin (silent mating type information regulation 2 homolog) 1CGCCTTATCCTCTAGTTCCTGTGCGGTCTGTCAGCATCATCTTCCIGF-1Insulin-like growth factor 1 (somatomedin C)GGTGGTTGATGAATGGTTGCCTAGACGAGATTATTAGATTIGFRInsulin-like growth factor 1 receptorGTAACAATCTATTCACAAGCTATTAAGAGCTGTCATTIGFBP1Insulin-like growth factor–binding protein 1CTCCACATGCTGCTTGATGAAGAGCTGAGATGGTAGTATTIGFBP2Insulin-like growth factor–binding protein 2TGTATTTATATTTGGAAAGAGATCTATTAGAAGCAGGAACIGFBP3Insulin-like growth factor–binding protein 3GGAGAGAATCACCTGTTTGTTACATCACCTAGCATCTIGFBP4Insulin-like growth factor–binding protein 4CAAGAGGTCTGAGTGTAGTGTAGTATGTGGTCAATAGATTNFαTumor necrosis factor alphaAACCACCAAGCAGAGGAGCTTGATGGCGGAGAGGAGIL-6Interleukin 6TACCCCAACTTCCAATGCGATACCCATCGACAGGATIL-1βInterleukin 1 betaCAGCAATGGTCGGGACATAGGTAAGTGGTTGCCTiNOSInducible nitric oxide synthaseTCCCGAAACGCTACACTCAATCCACAACTCGCTICAM-1Intercellular adhesion molecule 1CACAGCCTGGAGTCTCCCCTTCTAAGTGGTTGGAAMCP-1Monocyte chemotactic protein-1GCATCAACCCTAAGGACTTCAGGGCATCACATTCCAAATCACACHmox1Heme oxygenase 1 (HO-1)GGCTGTGAACTCTGTCTCGGCATCTCCTTCCATTCCHPRTHypoxanthine phosphoribosyltransferase 1AAGACAGCGGCAAGTTGAATCAAGGGACGCAGCAACAGACGAPDHGlyceraldehyde-3-phosphate dehydrogenaseCCAAGGAGTAAGAAACCCTTGATGGTATTCGAGAGAAGGβ-actinBeta actin (ACTB)GAAGTGTGACGTTGACATACATCTGCTGGAAGGTGData AnalysisGene expression data were normalized to the respective control mean values. Statistical analyses of data were performed by Student's t test or by two-way analysis of variance followed by the Tukey post hoc test, as appropriate. p < .05 was considered statistically significant. Data are expressed as means ± standard error of the mean, unless otherwise indicated.RESULTSSerum IGF-1 Levels and Body WeightIGF-1 levels were measured in serum obtained from tail blood before and after the 30-day experimental period. Table 2 shows that at the end of the experimental period, control and dwarf GH-replete rats had significantly higher serum IGF-1 levels compared with the untreated dwarf rats (p ≤ .05, each), indicating that twice daily administration of GH to the dwarf rats normalized serum IGF-1.Table 2.Changes in Body Mass and Serum IGF-1 Levels of Homozygous Control Rats, GHD Dwarf Rats, and GH-Treated Dwarf RatsSerum IGF-1, After Treatment (ng/mL)Body Mass, Before Treatment (g)Body Mass, After Treatment (g)Gain in Body Mass (g)Control970 ± 96113 ± 8274 ± 11161 ± 8GHD464 ± 51*84 ± 5*170 ± 7*86 ± 6*GH replete849 ± 11086 ± 5*238 ± 11*152 ± 8Notes: See Methods section for description of experimental groups. Serum level of IGF-1 did not differ between the GHD and GH-replete groups (322 ± 69 and 325 ± 62 ng/mL, respectively) before GH treatment. Data are mean ± standard deviation.*p < .05 vs respective controls. GH = growth hormone; GHD = growth hormone deficiency; IGF-1 = insulin-like growth factor 1.The control and GH-replete rats expressed the typical phenotype of adequate GH levels, indicated by comparable increases in body weight during the experimental period, whereas untreated dwarf rats gained significantly less weight than the control group (Table 2).Vascular Oxidative StressRepresentative fluorescent images of cross sections of DHE-stained aortas isolated from control, dwarf, and GH-replete rats are shown in Figure 1A–C. Analysis of nuclear DHE fluorescent intensities showed that, compared with vessels from control rats, O2− production was significantly increased in aortas of dwarf rats (Figure 1D). Vascular O2− generation was significantly reduced by GH treatment (Figure 1D). Vascular 8-isoprostane content also tended to increase in dwarf rats, yet, the difference did not reach statistical significance (Figure 1E). Consistent with the presence of GHD-related oxidative stress, aortic GSH content and ascorbate concentrations were significantly reduced in dwarf rats (Figure 2E and F). GH treatment normalized GSH content in the aorta of GH-replete dwarf rats (Figure 2E).Figure 1.Representative images showing red nuclear dihydroethidium (DHE) fluorescence, representing cellular O2− production, in sections of aortas of control rats (Panel A), growth hormone–deficient (GHD) dwarf rats (Panel B), and growth hormone (GH)–treated dwarf rats (Panel C). For orientation purposes, overlay of DHE signal and green autofluorescence of elastic laminae is also shown (right panels; lu: lumen, m: media, and ad: adventitia). Original magnification: 20×; scale bar: 100 μm. Panel D: summary data for nuclear DHE fluorescence intensities. Data are mean ± standard error of the mean. *p < . 05 vs control, #p < .05 vs GHD. Panel E: total tissue isoprostane content in the aortas of homozygous control rats, GHD rats, and GH-treated dwarf rats. Data are mean ± standard error of the mean (n = 5–6 in each group).Figure 2.High-performance liquid chromatography amperometric analysis of glutathione (GSH) content in homogenates of the aortas of control rats (Panel B), growth hormone–deficient (GHD) dwarf rats (Panel C), and growth hormone (GH)–treated dwarf rats (Panel D). The peak elution times and patterns of oxidation of a standard mixture of 10 nmol/mL cysteine (Peak 1), 10 nmol/mL ascorbate (Peak 2), and 10 nmo/L GSH (Peak 3) are shown in panel A. See Methods section for further details on analytical conditions. Bar graphs represent summary data for GSH (Panel E) and ascorbate (Panel F) content in aortic segments of control, GHD, and GH-replete dwarf rats. Data are normalized to protein content and expressed as mean ± standard error of the mean. *p < . 05 vs control, #p < .05 vs GHD (n = 5–6 in each group).Antioxidant Enzyme ActivitiesIn dwarf rats and GH-replete animals, we did not detect any significant changes in superoxide dismutase, catalase, and glutathione peroxidase enzyme activities as compared with controls (Figure 3A–C).Figure 3.Superoxide dismutase activity (Panel A), catalase activity (Panel B), and glutathione peroxidase activity (Panel C) in homogenates of aortas from control rats, growth hormone–deficient (GHD) dwarf rats and growth hormone (GH)–treated dwarf rats. Data are mean ± standard error of the mean (n = 5–6 in each group).Vascular Expression of Antioxidant EnzymesA quantitative real-time RT-PCR technique was used to analyze mRNA expression of major antioxidant enzymes in branches of the middle cerebral artery. We found that expression of Mn-SOD, Cu,Zn-SOD, and catalase (Figure 4A–C) did not differ among the three groups. By contrast, expression of glutathione peroxidase 1 was significantly decreased in the cerebral arteries of dwarf rats and was significantly increased by GH treatment (Figure 4D)Figure 4.Quantitative real-time -polymerase chain reaction data showing messenger RNA (mRNA) expression of Cu,Zn-SOD (A), Mn-SOD (B), catalase (C), and glutathione peroxidase-1 (D) in branches of the middle cerebral arteries of control rats, growth hormone–deficient (GHD) dwarf rats, and growth hormone (GH)–treated dwarf (GH-replete) rats. Data are mean ± standard error of the mean. *p < .05 vs control, #P < .05 vs GHD (n = 5–6 in each group).Vascular Expression of Nrf2 TargetsCerebral arteries of dwarf rats exhibited a significantly reduced expression of the known Nrf2 targets GCLC and NQO1 compared with vessels from control animals. These changes were normalized by GH treatment (Figure 5A and B). Expression of SIRT-1 in dwarf and GH-replete animals showed similar pattern, SIRT1 being downregulated in arteries of dwarf rats and upregulated by GH repletion (Figure 5C).Figure 5.Quantitative real-time reverse transcriptase–polymerase chain reaction data showing messenger RNA (mRNA) expression of GCLC (A), NQO1 (B), and SIRT1 (C) in branches of the middle cerebral arteries of control rats, growth hormone–deficient (GHD) dwarf rats, and growth hormone (GH)–treated dwarf (GH-replete) rats. Data are mean ± standard error of the mean. *p < .05 vs control, #p < .05 vs GHD (n = 5–6 in each group).Vascular eNOS Expression and ActivityExpression of eNOS mRNA in the middle cerebral arteries (Figure 6A) and nitric oxide synthase activity in the aortas (Figure 6B) were not statistically different among the three groups.Figure 6.Quantitative real-time reverse transcriptase–polymerase chain reaction data (Panel A) showing messenger RNA (mRNA) expression of endothelial nitric oxide synthase (eNOS) in branches of the middle cerebral arteries of control rats, growth hormone–deficient (GHD) dwarf rats, and growth hormone (GH)–treated dwarf (GH-replete) rats. Panel B depicts nitric oxide synthase activity in homogenates of aortas isolated from control, GHD, and GH-treated dwarf rats. Data are mean ± standard error of the mean. (n = 5–6 in each group)Vascular Expression of Components of the Local IGF-1 SystemExpression of IGF-1 (Figure 7A) was similar in the middle cerebral arteries of control and dwarf rats, whereas expression of insulin-like growth factor 1 receptor was significantly increased in vessels of dwarf rats (Figure 7B). Expression of IGFBP1, IGFBP2, and IGFBP4 (Figure 7C, D, and F) was unchanged in arteries of dwarf animals. Expression of IGFBP3 tended to be decreased in dwarf arteries as compared with controls, yet the difference did not reach statistical significance (Figure 7E).Figure 7.Quantitative real-time reverse transcriptase–polymerase chain reaction data showing messenger RNA (mRNA) expression of insulin-like growth factor 1 (IGF-1; A), insulin-like growth factor1 receptor (IGFR; B), insulin-like growth factor–binding protein (IGFBP) 1 (C), IGFBP2 (D), IGFBP3 (E), and IGFBP4 (F) in branches of the middle cerebral arteries of control rats and growth hormone–deficient (GHD) dwarf rats. Data are mean ± standard error of the mean. *p < .05 vs control (n = 5–6 in each group).Vascular Expression of Inflammatory MarkersWe found that mRNA expression of the inflammatory markers tumor necrosis factor alpha, interluekin-6, interluekin-1β, inducible nitric oxide synthase, Intercellular adhesion molecule 1, and monocyte chemotactic protein-1 in the middle cerebral arteries was statistically not different among the three groups (Figure 8).Figure 8.Quantitative real-time reverse transcriptase–polymerase chain reaction data showing messenger RNA (mRNA) expression of tumor necrosis factor alpha (TNFα; A), interleukin (IL)-1β (B), IL-6 (C), monocyte chemotactic protein (MCP)-1 (D), intercellular adhesion molecule (ICAM)-1 (E), and inducible nitric oxide synthase (iNOS; F) in branches of the middle cerebral arteries of control rats, growth hormone (GH)-replete, and growth hormone–deficient (GHD) dwarf rats. Data are mean ± standard error of the mean (n = 5–6 in each group).DISCUSSIONThe levels of GH and IGF-1 vary substantially throughout the life span. Postnatal GH and IGF-1 levels are low but increase to higher concentrations immediately before puberty and then progressively decline with increasing age (31–33). Late-life changes in the endocrine milieu, including the age-related decline of GH/IGF-1 axis, have led to the formulation of a number of neuroendocrine theories of aging, which aim to explain peripheral aging on the basis endocrine dysregulation (34). Recent findings provide key evidence that changes in peripubertal GH and IGF-1 levels can also modulate peripheral aging (11), especially in the vascular system.Deficits of GH in childhood can be due to a congenital deficiency (incidence: 1 in every 3,800 live births) or acquired in childhood due to hypothalamic–pituitary tumors. Previous studies focused on the deleterious effects of GHD on systemic cardiovascular risk factors in adults but provided little information on vascular effects directly affected by peripubertal GHD. Here, we demonstrate for the first time that peripubertal GH/IGF-1 deficiency in Lewis dwarf rats results in significant changes in vascular redox homeostasis. Measurements of DHE fluorescence (Figure 1A–D) as well as measurements of tissue isoprostane (Figure 1E), GSH, and ascorbate content (Figure 2) suggest that peripubertal GH/IGF-1 deficiency increases vascular oxidative stress. Because rats can synthesize ascorbate, we interpret the simultaneous decline in tissue ascorbate (35) and GSH levels as a sign of GHD-induced oxidative stress rather than a sign of dietary vitamin deficiency. Importantly, a relatively brief peripubertal exposure of Lewis dwarf rats to GH prevented or attenuated most of the aforementioned pro-oxidative alterations (Figures 1and 2). These in vivo findings are in complete agreement with the observations that in vitro treatment with GH and/or IGF-1 also reduces ROS production in cultured endothelial cells (13,36). It is significant that the same peripubertal treatment of young Lewis dwarf rats with GH was previously reported to significantly postpone the development of age-associated cerebrovascular disease and increase life span (11). Taken together, these observations raise the possibility that levels of GH present in the circulation during a brief transitional period starting around puberty and ending in young adulthood are likely to influence vascular pathology later in life (11) by modulating vascular redox homeostasis. We speculate that GHD-induced increased oxidative stress during development likely modulates the activity of various redox-sensitive pathways (eg, mitogen activated protein kinases), which affect cerebromicrovascular architecture, the composition of extracellular matrix, and/or elicit epigenetic changes in vascular endothelial and smooth muscle cells, which will promote the development of cerebrovascular pathologies later in life. Importantly, if GHD persists in adulthood, it will likely exert a progressive deleterious effect on the vasculature (13,37–39). The clinical emphasis on treating childhood-onset GHD currently focuses on the somatic role of GH in increasing body size (mainly height) during puberty (40,41). Continued GH treatment during the important transition period between adolescence and adulthood is important for the normal development of bone and muscle in adulthood (20–30 years old) (40,41). In light of the aforementioned findings, it would be interesting to determine in follow-up studies how peripubertal GH replacement affects late-life cardiovascular morbidity and mortality in humans.The underlying mechanisms for elevated ROS production in the cardiovascular system of GH/IGF-1–deficient rats are likely multifaceted. Previous studies suggest that GH/IGF-1 deficiency is associated with downregulation of vascular expression of major cellular antioxidant enzymes in adult Ames dwarf mice (13). In the present study, peripubertal GH/IGF-1 deficiency did not alter superoxide dismutase or catalase activities in the aorta nor the expression of Cu,Zn-SOD, Mn-SOD, and catalase in the cerebral arteries of dwarf rats. In contrast, cerebrovascular expression of glutathione peroxidase 1 was significantly decreased in dwarf vessels, and this effect was reversed by GH treatment. Glutathione peroxidase activity also tended to decrease in aortas of dwarf rats, but the difference did not reach statistical significance. The hitherto hypothetical concept, named mitochondrial hormesis, suggests that low levels of oxidative stress may have beneficial effects on the aging process by upregulating antioxidants. It is significant that in the vasculature free radical detoxification pathways driven by the transcription factor NF-E2-related factor 2 (Nrf2) play an important role in maintaining cellular redox homeostasis under conditions of oxidative stress (42). Nrf2 triggers expression of genes, including NAD(P)H:quinone oxidoreductase 1 (NQO1, a key component of the plasma membrane redox system and γ-glutamylcysteine ligase [GCLC]), via the antioxidant response element, which attenuate cellular oxidative stress. The second interesting finding of the present study is that peripubertal GH/IGF-1 deficiency significantly decreases expression of both of these Nrf2 target genes in the cerebral arteries, showing that adaptive induction of antioxidant enzymes in dysfunctional in Lewis dwarf rats. Because the plasma membrane redox system is responsible for the maintenance of the redox status of ascorbate, we posit that downregulation of NQO1 may contribute to GHD-induced decreases in ascorbate levels in the vascular wall of dwarf rats (Figure 2F). Because GCLC is the rate-limiting enzyme for GSH synthesis, it is likely that this downregulation contributes to the decline of vascular GSH content in these animals.Recent studies demonstrate a cross talk between IGF-1 signaling and the prosurvival factor SIRT1 (43,44), both of which regulate cellular redox status, including mitochondrial ROS production in endothelial cells (45). The finding that in dwarf rats, vascular expression of SIRT1 is downregulated, whereas it is induced in GH-replete animals raises the possibility that this pathway also may contribute to the pro-oxidative effects of peripubertal GHD. Previously, we found that mitochondrial ROS generation is increased in arteries of Ames dwarf mice (13); thus, future studies are warranted to determine whether downregulation of SIRT1 is associated with an increased mitochondrial oxidative stress in blood vessels of rats with peripubertal GHD. eNOS is an important downstream target of both SIRT1 (46) and IGF-1 (13) that confers multifaceted vasoprotective effects. Importantly, low IGF-1 levels were shown to be associated with downregulation of eNOS both in the vasculature of adult Ames dwarf mice (13) and arteries of aged rats (26). The findings that cerebrovascular expression of eNOS and nitric oxide synthase activity in the aortas are unaffected by peripubertal GH/IGF-1 deficiency in dwarf rats give support to the view that young organisms have greater ability to adapt to changes in the endocrine milieu than older animals.In addition to its endocrine function, IGF-1 also exerts its biologic effects via autocrine/paracrine pathways, and recent studies suggest that circulating IGF-1 levels and tissue expression of components of the local IGF-1 system are not always correlated (47–49). We found that peripubertal GH/IGF-1 deficiency did not affect the expression of IGF-1 in the middle cerebral arteries but upregulated insulin-like growth factor 1 receptor, which likely represents an adaptive mechanism resulting from IGF-1 deficiency. Expression of IGFBP1, IGFBP2, and IGFBP4 did not change in the wall of dwarf arteries. As expected, cerebrovascular expression of IGFBP3 tended to decrease in the dwarf rats because this is regulated by circulating GH levels. In that regard, it is significant that in humans, there is a close correlation between peripubertal serum levels of IGF-1 and IGFBP3 (31) and that GH deficiency in childhood is characterized by decreased levels of IGFBP3 (50).Recent studies suggest that IGF-1 confers anti-inflammatory vascular effects (17) and that short-term GH treatment attenuates age-related inflammation, including upregulation of tumor necrosis factor alpha, in the cardiovascular system of senescence-accelerated mice (51). Human GH/IGF-1–deficient dwarfs at adulthood are known to develop premature atherosclerosis in the cerebral arteries. Importantly, in Lewis dwarf rats, peripubertal GH replacement, sufficient to raise IGF-1 levels to that of normal animals during adolescence, significantly delays cerebrovascular alterations associated with old age and increases life span (11). To determine whether peripubertal GH/IGF-1 deficiency promotes vascular inflammation during adolescence, we assessed the expression of various inflammatory markers in the middle cerebral arteries of Lewis dwarf rats. We found that expression of proinflammatory cytokines, chemokines, and adhesion molecules was unaffected either by peripubertal GH/IGF-1 deficiency or GH treatment (Figure 8). These data provide additional support for the view that young organisms have greater ability to adapt to changes in circulating GH/IGF-1 than older animals.In conclusion, peripubertal GH/IGF-1 deficiency results in a pro-oxidative cellular environment, which likely promotes an adverse functional and structural phenotype in the vasculature that contributes to the development of stroke later in life. Peripubertal GH treatment of Lewis dwarf rats prevents these adverse vascular effects during a critical developmental time window, which contributes to the delay of stroke thereby extending the life span of these animals. Additional studies are warranted to further characterize the effects of peripubertal GH treatment on age-related structural and functional cerebrovascular alteration in Lewis rats. 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Gerontol200641441742950.MaghnieMStrigazziCTinelliCGrowth hormone (GH) deficiency (GHD) of childhood onset: reassessment of GH status and evaluation of the predictive criteria for permanent GHD in young adultsJ Clin Endocrinol Metab19998441324132851.FormanKVaraEGarciaCAriznavarretaCEscamesGTresguerresJACardiological aging in SAM model: effect of chronic treatment with growth hormoneBiogerontology2009113275286 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A635767165CF0B1C572765F29B32BC5A22E1471.txt b/test/dataset/in/resources/corpus/Clean_0A635767165CF0B1C572765F29B32BC5A22E1471.txt new file mode 100644 index 0000000..2055c0b --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A635767165CF0B1C572765F29B32BC5A22E1471.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 122110.1093/gerona/59.12.1221 Journal of Gerontology: Biological Sciences Semirandom Sampling to Detect Differentiation-Related and Age-Related Epigenome Remodeling Russanova Valya R. Hirai Tazuko H. Howard Bruce H. National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland. Address correspondence to Bruce Howard, MD, NICHD, NIH, Bldg. 31, Rm. 2A25, 6 Center Dr., Bethesda, MD 20892. E-mail: howard@helix.nih.gov 12 2004 59 12 1221 1233 1 10 2004 3 8 2004 The Gerontological Society of America 2004 With completion of the human genome project, patterns of higher order chromatin structure can be easily related to other features of genome organization. A well-studied aspect of chromatin, histone H4 acetylation, is examined here on the basis of its role in setting competence for gene activation. Three applications of a new hybrid genome sampling–chromatin immunoprecipitation strategy are described. The first explores aspects of epigenome architecture in human fibroblasts. A second focuses on chromatin from HL-60 promyelocytic leukemia cells before and after differentiation into macrophage-like cells. A third application explores age-related epigenome change. In the latter, acetylation patterns are compared in human skin fibroblast chromatin from donors of various ages. Two sites are reported at which observed histone H4 acetylation differences suggest decreasing acetylation over time. The sites, located in chromosome 4p16.1 and 4q35.2 regions, appear to remodel during late fetal–early child development and from preadolescence through adult life, respectively. hwp-legacy-fpage 1221 hwp-legacy-dochead RESEARCH ARTICLE HIGHER order chromatin structure plays an integral role in the control of gene expression (1–3). Central to chromatin structure are histone modifications that serve to recruit regulatory proteins and remodeling complexes (4–9). The latter facilitate or retard further modifications, and ultimately regulate the recruitment of the transcription apparatus. Cumulatively, such mechanisms give rise to patterns of histone modification extending across the genome. Pattern features may be localized to tens of base pairs, but may also extend over hundreds of kilobases or even encompass entire chromosomes (10–13). Specialized chromatin domains are thought to form the basis for epigenetic memory in yeast and Drosophila systems (14–17). In vertebrates and plants, such domains act in conjunction with DNA methylation to store epigenetic information (18–20). Much of what we know concerning the role of chromatin in transcriptional regulation derives from well-studied gene loci (12,13,21–23). These are most often described in the context of development or differentiation pathways. Paradigms for developmentally linked chromatin remodeling and epigenetic memory are provided by studies on Drosophila homeodomain genes by Polycomb and Trithorax group gene products, as well as X chromosome dosage compensation (24). More recently, the genetically tractable Arabidopsis system is providing interesting examples of chromatin-mediated gene control (25,26). In vertebrates, epigenetic processes are notably evident in developmental regulation of the chicken beta-globin locus (12,13,21,24,27–29), X chromosome inactivation, and imprinting (19,30,31). T-cell differentiation also turns out to be a process associated with striking changes in chromatin and DNA methylation profiles (11,32). With completion of human and mouse genome assemblies, a more general picture of epigenetic processes should become available. An emerging goal is to describe chromatin and DNA methylation patterns, which, taken together, form the epigenome for a given cell type. Experimental approaches that are or should soon become feasible in mammalian systems have been reported in recent work with Saccharomyces cerevisiae (33,34). One productive strategy in yeast relates gene expression profiles to chromatin regions through the use of microarrays. The relatively small size of the S. cerevisiae genome renders this practical. By contrast, the much greater complexity of mammalian genomes makes it difficult to obtain comparable array coverage. At the resolution of bacterial artificial chromosomes (BACs), a complete tiled representation of the human genome has been reported (35). To query smaller domains, reasonable alternatives are to construct microarrays restricted to small chromosomes, promoter-proximal regions, or CpG islands (36–43). The high cost and long lead time associated with microarrays have prompted several alternative strategies (44–46). In this article, we describe epigenome mapping using a complementary approach related to differential display (47,48). The approach allows general sampling of mammalian genomes and has been implemented in three ways. First, a screen was done to gain an overall profile of histone H4 acetylation patterns in chromatin from normal human fibroblasts. The results are consistent with large alternating regions of high- and low-acetylation distributed along the chromosome arms, as has been reported previously (24). Second, a screen was performed to compare histone H4 acetylation patterns in undifferentiated versus macrophage-like HL-60 cells. This revealed regions with substantial shifts to be relatively infrequent, being on the order of 0.5% of the epigenome. Third, the genome sampling approach was used to search for locus-specific acetylation differences in chromatin from human fibroblasts taken at different passage levels or donor ages. This search was successful, revealing sites that appear to report age-related epigenome change near the chromosome 4p and 4q termini. Methods Cell Culture Normal human skin fibroblasts from donors of different ages were obtained from the Coriell collection. [HSC172 human lung embryo fibroblasts (49) were a gift from Sam Goldstein.] Fetal cells used were: AG04392A, 16 weeks, population doubling level (PDL) 17; AG04449, 12 weeks, PDL 11; AG04431B, 15 weeks, PDL 18; AG04451, 16 weeks, PDL 13; AG04525, 17 weeks, PDL17. Cells from preadolescents were: GM03348D, 10 years, passage 9; GM2036A, 11 years, passage 14; GM00500, 10 years, passage 10; GM1582A, 11 years, passage 9. Cells from young adults were: AG07720B, 23.9 years, PDL 16; AG07719A, 27.9 years, PDL 16; AG04441B, 29.4 years, PDL 16; AG13153, 30.2 years, PDL 5. Cells from old adults were: GM08401, 75 years, passage 2; GM09918, 78 years, passage 10; GM00288B, 64 years, passage 10. Fibroblasts were grown in Dulbecco's modified Eagle medium (MEM) (Gibco/BRL, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS) or Eagle's MEM with Earle's salts with 15% FSC, 2 mM glutamine, and antibiotics (50 U of penicillin/ml and 50 mg of streptomycin/ml). Chinese hamster–human hybrid cell lines Y162-11C and X86T2 were grown as published (50). HL-60 cells were propagated in RPMI media, 10% fetal calf serum. Differentiation was induced by 12-O-tetradecanoylphorbol-13 acetate (TPA) treatment (51). Preparation of Nuclei and Chromatin HSC172 cells were grown to confluency, and, in most experiments, incubated for 18 hours with 20 mM sodium butyrate to inhibit histone deacetylase activity. HL-60 cells were not treated with butyrate prior to harvesting. HSC172 nuclei and histone-H1-depleted chromatin were prepared by a procedure described previously (52). Nuclei from HL-60 cells were isolated according to (51). Micrococcal nuclease concentrations were titrated in order to obtain chromatin fragments with a median length of 1500 bp. All solutions used during preparation of nuclei contained 10 mM sodium butyrate. Immunoprecipitation of Acetylated Chromatin Antibody against acetylated H4 was prepared by immunizing rabbits with chemically acetylated histone H4 (22,53). Affinity purification was accomplished with acetylated bovine serum albumin (BSA) coupled to BrCN Sepharose and characterized by enzyme-linked immunosorbent assay (ELISA) and Western blotting as described previously (53). Affinity-purified antibody (70 μg) was incubated with magnetic beads coated with antirabbit IgG (Dynal Biotech, Brown Deer, WI) as described by the manufacturer. Chromatin fragments (25 μg measured as DNA at 260 nm) were incubated with antibody-coated beads by rotating overnight at 4°C in 50 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 10 mM Na-butyrate, and proteinase inhibitor cocktail (22,53). Supernatant from the first magnetic separation combined with the first wash was taken as the unbound chromatin fraction. Beads were washed six times with 1 ml 150 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 10 mM Na-butyrate, proteinase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Samples were transferred to new tubes prior to a final wash. The bound acetylated chromatin was eluted by two incubations in 100 μl 1% SDS, 1% beta-mercaptoethanol for 15 minutes at 37°C. DNA was purified by proteinase K digestion, phenol-chloroform extraction and ethanol precipitation in the presence of 40 μg/ml glycogen, or alternatively was purified using Qiagen PCR (polymerase chain reaction) purification kits (Valencia, CA). The concentration of DNA was determined fluorimetrically. The typical yield from 25 μg chromatin was 0.5–1 μg bound DNA. PCR Amplification Phosphoglycerate kinase (PGK) sequence primers (54):5′GGTCTCGCACATTCTTCACGTC, 3′GCAAGGAACCTTCCCGACTTAGHuman α-satellite sequence primers (55):5′TCAGAAACTTCTTTGT, 3′GAATGCTTCTGTCTAGFirst intron of the human ribosomal protein S-14 gene (56):5′CTGTTGCTGATTGGTTAGGC, 3′TGAAGGAGAGAAGACTGGAG PCR amplification was performed in a 20 μl reaction mix containing 1–3 ng DNA, 1× buffer (PerkinElmer, Wellesley, MA), 1.5 or 2.5 mM MgCl, 2 μM deoxynucleotides, 0.2 μM primers, 1 unit Taq Polymerase (Boehringer-Mannheim/Roche), and 2 μCi 33P-dCTP (NEN, DuPont, Wilmington, DE). The cycling parameters were: 5 minutes at 94°C, followed by 25 cycles of 15 seconds at 94°C, 15 seconds at the annealing temperature, and 15 seconds at 72°C. For longer PCR products, the nucleotide concentrations were increased to 10 μM and each cycling step was 30 seconds. For α-satellite PCR, the number of cycles was decreased to 15. Due to the high guanine plus cytosine content in the PGK promoter sequence, 10% glycerol and 3.5% formamide were included in the reaction buffer. As a check for linearity, reactions containing 1 or 3 ng DNA were performed in triplicate. PCR products were separated by 5% or 6% polyacrylamide gel electrophoresis (PAGE) containing Tris/borate/EDTA (TBE) and 7.5 M urea. Quantification was performed by phosphoimager scanning. Real-time PCR was performed using SYBR Green Fast-start PCR kits (Qiagen) and a LightCycler instrument (Roche). One ng of each sample was analyzed in triplicate together with 0.1–3 ng input DNA used for standard curve calculation. Genome Sampling—Chromatin Immunoprecipitation (gsChIP) DNA samples purified from bound (acetylated), unbound (under-acetylated), and input chromatin fractions were analyzed by PCR using combinations of random primer pairs similar to those utilized previously for RNA differential display (47,48). Each primer contains a 12-nucleotide arbitrary sequence at the 3′ end and 10 invariant nucleotides including a Hind III site at the 5′ end. PCR reactions were performed essentially as described above, except that 25 μM dNTP and 1.25 μM of each primer were used. The cycling parameters were: initial denaturation step at 94°C for 5 minutes, 4 cycles of 45 seconds at 94°C, 60 seconds at 39°C to 45°C and 60 seconds at 72°C, followed by 25 cycles of 45 seconds at 94°C, 60 seconds at 60°C, and 60 seconds at 72°C (47) (Gene Hunter, Inc., Nashville, TN). PCR products were separated on 6% DNA sequencing gels. After autoradiography, the film and corresponding gel were aligned and fractions of interest excised. Eluted DNAs were reamplified and the resulting PCR products were purified on low-melting agarose gel for direct sequence analysis (Applied Biosystems, Foster City, CA). Primary sequences were aligned to the human genome using the National Center for Biotechnology Information (NCBI) blast utility to obtain candidate loci. Specific primers were then designed for these loci and used for quantitative or real-time PCR. Statistical and Sequence Analysis Mean difference test (MDT) values were calculated using Mathematica version 5 software (Wolfram Research, Inc., Champaign, IL). Fisher's Exact Test was used as implemented by O. Langsrud at http://www.matforsk.no/ola/index.html. Distributions of interspersed repeat sequences were determined using the RepeatMasker utility (A. F. A. Smit and P. Green, “RepeatMasker” at http://repeatmasker.genome.washington.edu). This study utilized the high-performance computational capabilities of the Biowulf PC/Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov). Results Antibody Specificity and Control Studies An affinity-purified polyclonal antiacetyl-lysine antibody, acLHA, was employed throughout for immunofractionation (12,53). This antibody primarily recognizes multiacetylated forms of histone H4 (Figure 1A). For chromatin immunoprecipitation (ChIP), oligonucleosomes were separated into bound and nonbound fractions, extracted to recover the corresponding DNA fragment preparations, and then adjusted to equal concentrations for PCR amplification and analysis. With this procedure, loci packaged in more highly acetylated domains than average are represented preferentially in the bound fraction, and the converse is true for loci packaged in under-acetylated regions. Initial experiments focused on the establishment of positive controls. Oligonucleosomes were prepared from two hamster lines, Y162-11C and X86T2 cells; these lines carry a single active human X chromosome or a single inactive X chromosome, respectively (50,54). Immunofractionation was performed and chromatin structure was probed at the X-chromosome-linked phosphoglycerate kinase 1 (PGK1) locus, known to be associated with euchromatin in the Y162-11C cell line, and with heterochromatin in the X86T2 cell line. As shown in Figure 1B, oligonucleosomes derived from the active X chromosome appeared, as expected, predominantly in the bound fraction, whereas identically prepared material from the inactive X chromosome distributed with a bias to the unbound fraction. In additional controls prepared from human normal fibroblast cells, α-satellite repeat sequences (55) were represented preferentially in the unbound fraction; conversely, the ribosomal protein S-14 first intron, localized within a housekeeping gene and near an early-firing origin of DNA replication (47,56) (and thus likely to reside in a euchromatin region), was efficiently precipitated by the anti-acetylated histone H4 antibody. Linking Genome Sampling With ChIP Having established differential immunoprecipitation at several known loci, we next modified this approach to obtain a more general picture of H4 acetylation states across the human genome. Oligonucleosomes were prepared from normal, that is, nonimmortalized, human fetal lung (HSC172) fibroblasts (57). As before, pools of anti-acetyl H4 bound and nonbound oligonucleosomes were extracted and the DNA content normalized prior to PCR reactions. Arbitrary pairs of DNA primers were used with four low-stringency cycles followed by 25 standard cycles, that is, conditions similar to those employed for RNA differential display (47,48). An example is shown in Figure 2A of the products obtained. In general, triplicate PCR reactions for each primer pair–DNA combination proved very useful as a means to distinguish reproducible from nonreproducible products. A series of reactions was performed with 16 arbitrary primer pairs, yielding more than 1000 reproducible products. Bound to nonbound ratios of bands from the first eight primer pair sets, plotted according to the presumed histone H4 acetylation level, exhibit a roughly normal distribution (Figure 2B). From the gels used to generate these initial data, 91 bands were excised, reamplified, and subjected to DNA sequence analysis, yielding informative sequence information in 65 cases. Of these, 45 were selected for further study, including 17 loci potentially representing highly acetylated regions and 14 candidates for very under-acetylated regions. An additional 14 loci were taken as controls. The positions of the selected loci are plotted onto build 34 of the human genome in Figure 2C. The candidates for both the highly acetylated and under-acetylated regions distribute along the chromosome arms, consistent with semirandom sampling of the epigenome. Confirmation by Real-Time PCR and Local Mapping Deviations from a 1:1 distribution in the differential display protocol might reflect one or more unexpected artifacts. To address this issue, we designed independent specific-primer sets for 26 single-copy loci. Real-time PCR was then performed to compare bound and nonbound material, yielding high-quality data in 20 cases. Several additional loci were confirmed by standard PCR verified through calibration for linearity with respect to DNA input. In all but one instance, the results and the original differential display results were in agreement. Quantification of the real-time PCR data for the confirmed loci is shown in Figure 3A. From these and additional experiments (see below), we conclude that candidates generated by genomic differential display are correctly identified in almost all instances. An important question is whether genome sampling as described here predominantly detects very local chromatin states, for example, limited to one or two nucleosomes, or provides marker loci for larger regions of atypical chromatin. Evidence in support of the latter possibility was provided by walking across ≈20 kb (kilobase) regions flanking two loci, h40 and h90, characterized respectively by under-acetylation or high-acetylation. The under-acetylated locus, h40, is flanked by loci having similar acetylation levels (Figure 3C). The highly acetylated locus h90 (Figure 3B) lies just upstream from the transcription start point of the trichorhinophalangeal syndrome I (TRPS1) gene; it is also adjacent to a CpG island. Each position checked, extending from h90 across the CpG island and the transcription start site for TRPS1, exhibits elevated acetylation. This is consistent with previous reports that CpG islands and their flanking regions are frequently over-acetylated (12,58,59). Regional Transcripts and CpG Islands The human genome is believed to be composed of gene-rich and gene-poor regions, the latter often referred to as gene deserts (60). We asked whether the loci identified in this study might show a nonrandom distribution with respect to local gene density or expression levels. Taking the gsChIP-identified subsets characterized by low and high acetylation, respectively, overlapping or nearby genes are observed more frequently in the latter (4/14 vs 14/16 closer than 50 kb; p =.0015, Fisher's exact test; see also Table 1). Related to gene distribution are additional parameters, including both the presence or absence of nearby CpG islands and the number of regional expressed sequence tags (ESTs) documented by the human genome project. To analyze these parameters, flanking regions were extended to include a total of 125 kb (the human genome assembly build 34 provided finished sequences for all but one locus). Strong statistical relations were observed, with under-acetylated loci lying uniformly within EST-poor regions relative to highly acetylated loci (p =.007, MDT; see Figure 4A). Genome regions flanking under-acetylated loci are devoid of CpG islands, whereas such islands are common in the regions flanking highly acetylated loci (p =.002, MDT; Figure 4B). A question that can be asked is whether the CpG and EST distributions observed are merely those that would be seen by landing at random points across the human genome. To address this issue, an additional set of 44 loci was included, representing randomly chosen chromosome positions centered within 125 kb regions. ESTs associated with such regions show wide variation in abundance, similar to highly acetylated loci. By contrast, comparisons between under-acetylated and random subsets reveal highly significant differences in both EST abundance (p =.0001, MDT) and CpG island frequency (p =.002, MDT; not shown). Differentiation-Associated Chromatin Remodeling Of considerable value would be a general means to identify regions of local chromatin remodeling in association with changes in differentiation state. In vertebrates, such remodeling has been described in detail through studies on the chicken beta-globin and human immunoglobulin gene loci. We asked whether gsChIP applied at an intermediate scale would be sufficiently sensitive to detect chromatin state changes associated with the human HL-60 promyelocyte-to-macrophage differentiation program. PCR reactions were performed with 27 sets of arbitrary primers taking oligonucleosomes from promyelocytic cells and in parallel from phorbol ester-treated macrophage-like cells. The display patterns obtained were greater than 99% identical, perhaps surprising given the morphological differences between these respective cell types. Still, three loci were identified at which changes in acetylation level accompany differentiation. These changes were confirmed using independent sets of specific primers and real-time PCR (Figure 5A). Additional confirmation was obtained by mapping alterations in chromatin acetylation across a region spanning ≈ 30 kb (Figure 5B). Further characterization remains to be done concerning the size of the regions apparently remodeled in association with differentiation, as well as concerning potential impacts on the expression levels of nearby genes. However, these experiments confirm that chromatin changes occurring in the context of a simple differentiation program can be discovered by semirandom genome sampling as described here. Age-Related Chromatin Remodeling A subject of major interest has been to what extent postnatal development and adult life span in vertebrates are accompanied by age-related epigenome remodeling. This has been extensively investigated with respect to DNA methylation (61), but very little is known concerning locus-specific histone modifications. We realized that gsChIP could be of special value in the latter context, as the requisite first step to find such changes is a relatively unbiased search approach. Human HSC lung embryo fibroblasts were compared at early-passage and late-passage levels (19–27 PDL and 55–60 PDL, respectively). PCR reactions were performed with 16 sets of arbitrary primers, yielding several candidate loci at which histone acetylation appeared to vary. One of these, h9, exhibited decreasing histone H4 acetylation as cells progressed from early passage to midpassage, that is, substantially before the onset of cellular senescence (Figure 6A and unpublished results). The h9 band was recovered and sequenced, revealing it to be located in the chromosome 4p16.1 region, approximately 11 Mb from the 4p terminus (position 10,967,271 in human genome build 34). We postulated that altered acetylation at h9 might also be evident on examination of fetal and postnatal fibroblasts harvested at comparable early-passage levels. Comparisons were done using the appropriate arbitrary primer pair to generate the h9 band, but substituting chromatin preparations from several fetal skin fibroblast lines and skin fibroblasts derived from young adults. The results obtained confirmed this idea (Figure 6B and unpublished results). Either very small or no differences at h9 were observed in experiments comparing chromatin from young and old adults (not shown). To search for epigenome remodeling during subsequent life span, chromatin was prepared and pooled from preadolescents versus old adults (ages 10–11 years vs 64–78 years). PCR reactions were done with 40 sets of arbitrary primer pairs. As in previous comparisons, the gsChIP patterns obtained were almost identical. However, one striking difference was observed at a site termed here p14. Sequencing of the p14 band identified it as being derived from the chromosome 4q35.2 region (position 188,927,735), about 3 Mb from the 4q telomere. While the p14 sequence overlaps a Tigger 1 element belonging to the MER2 repetitive sequence family, there is sufficient divergence from other such sequences in human genome build 34 for the identification to be unambiguous. However, it cannot be excluded that another Tigger 1 repeat element of equal or greater sequence similarity remains to be integrated into a subsequent human genome assembly. Discussion Much remains to be learned at the genome level concerning histone modification patterns and their remodeling in association with differentiation pathways, development, and aging. Here we describe the application of a genome sampling strategy coupled with chromatin immunoprecipitation to explore such aspects of epigenome structure. Several points of a technical nature apply in interpreting the results. The antibody used throughout for chromatin immunoprecipitation recognizes multiacetylated forms of histone H4 (22,53). These forms are abundant; however, it cannot be excluded that in sampling many genome loci, the antibody might recognize other multiacetylated proteins at a small number of sites. The sampling of genome loci as implemented here should be considered as semirandom. While a very large number of chromosome positions should be accessible, extended blocks of low-complexity sequence, simple tandem repeats, and other repeat arrays are necessarily under-represented. A major component of the human and other mammalian genomes is composed of interspersed LINE (long interspersed nuclear elements), SINE (short interspersed nuclear elements), and retrovirus-like elements. This being so, we expected a substantial percentage of sampled loci to overlap partially or completely with repeat sequences. Fortunately, it turns out that unambiguous genome assignments can be made in the great majority of cases. Reamplified fragments containing repetitive elements often contain junctions with unique sequence or adjacent repeats that facilitate identification. At other loci, repetitive elements are sufficiently diverged from other copies for the assignment to be straightforward. We routinely searched flanking genome regions for partial matches to the arbitrary primers used in the original PCR reaction, and these were confirmed for all loci listed in the study. For future work, the data compiled should be useful in setting search parameters. Within the 12 bp variable regions, minimum primer annealing corresponded to matches at the two 3′ terminal bases, with no more than six mismatches at the remaining positions; in addition, the sum of mismatches for the two primer variable regions did not exceed nine (unpublished observations). Finally, for candidate loci residing within moderately conserved repetitive elements, the entire genome was searched to exclude the occurrence of other elements having equal or better matches to the primers (unpublished data). As mentioned in the introduction, other approaches to map the epigenome are being pursued (34,36–46,62). How efficient is semirandom sampling in comparison to these alternative approaches? For small scale experiments, it is easy to implement gsChIP and to obtain results rapidly. For more extensive sampling, automated liquid handling instruments are certainly desirable. High-end sampling should be comparable to microarrays constructed from PCR products with a layout of approximately 32,000 positions. A simple calculation indicates that 40 arbitrary primers, taken as all combinations, suffice to carry out 780 distinct PCR sampling reactions. Based on our experience, each reaction yields reproducible information for 50–100 loci, thus providing sampling information for ≈ 40,000–80,000 chromosome positions. This is attractive in terms of the investment required, at least relative to PCR-based microarrays, where the cost would include 80,000–160,000 primers and fabrication of the arrays themselves. In terms of flexibility, the same or similar arbitrary primer sets should be usable for any higher eukaryotic genome or combination of genomes of interest. The fraction of a given genome that is accessible by semirandom sampling is a somewhat difficult issue to evaluate, since it depends in part on the average size of the chromatin regions in which the characterized loci reside. Based on results here and in the research literature, a conservative estimate for distinguishable histone H4 acetylation domains is at least 20 kb. Taking this value, a medium scale sampling with arbitrary primers—for example 100 primer pairs—should cover roughly 5% of the human genome. This remains a relatively small fraction; however, a common goal will be to obtain seed examples of differentiation-linked chromatin changes. Taking the above numbers and a differentiating cell type in which ≈100 complex sequence regions undergo remodeling, the probability of discovering at least a few such changes is in fact quite high (>99% in the absence of unexpected sources of bias). Such seed examples, in addition to their intrinsic interest, should frequently provide the basis for bioinformatics-based queries to search for additional instances. Beyond a description and comments on the potential of gsChIP, this article provides three practical examples of its utility. With respect to static epigenome architecture, a finding of some interest is the comparative homogeneity of regions flanking under-acetylated loci, both with respect to CpG islands and to ESTs. The contrast is valid for comparisons to regions near highly acetylated loci as well as to regions flanking randomly selected chromosome positions. CpG islands near genes are highly acetylated, presumably due to associated transcription factors that recruit histone acetyltransferase complexes (12,58,59). Likewise, polymerase complexes typically require multiple acetyltransferases for efficient assembly. Thus, the under-acetylated loci identified here seem most likely due to a lack of nearby entry sites for histone acetyltransferase complexes. An important application of gsChIP is to identify regions of chromatin remodeling associated with differentiation programs. Along these lines, there is scant information at the genome level, and much valuable information to be gained. We chose HL-60 as a model system in which homogeneous populations of prepromyelocytes growing in suspension can be readily separated from adherent macrophage-like derivatives. Nuclei of the latter have somewhat more compact nuclei than their parent cells at the level of microscopy; nevertheless, it was uncertain whether regions exhibiting a change in acetylation would be common or, at the other extreme, too rare for detection. It is notable that remodeling events turn out to be infrequent but detectable, and that a moderate-sized screen consisting of 27 primer pairs was adequate to reveal these events. Our initial motivation to develop gsChIP was in fact based on a desire to search for age-related epigenome remodeling. X-chromosome reactivation is well described in mouse models (63–66), but to date has been detected in human cells only at relatively low levels (67,68). Likewise, most CpG island silencing with age occurs in too small a fraction of cells to have been detectable by the approach described here (61). The identification of two candidate chromatin loci exhibiting age-related differences in histone H4 acetylation suggests that this form of remodeling may occur in a larger fraction of cells than the previously observed epigenetic changes. How many comparable loci might be found by genome sampling remains to be seen; however, as noted above, it seems likely that a relatively small fraction of the epigenome has been effectively queried so far. An important question is whether mapping of the regions flanking the h9 and p14 sites will reveal extended areas of change, as is found at the L28 locus in association with HL-60 differentiation. Before addressing this question, we chose to develop a much more efficient set of techniques for building regional epigenome maps. These techniques, together with results relating to the h9 and p14 loci, are described elsewhere (70). Decision Editor: James R. Smith, Jr. Figure 1. Control experiments to verify experimental approach. A: Specificity of the antibody against acetylated histone H4. Total histones were isolated from HeLa cells treated with butyrate, separated by Triton X-100-urea PAGE (polyacrylamide gel electrophoresis), and analyzed by Western blotting. Left panel: incubation with anti-acetyl-H4; right panel: gel stained with Coomassie blue. Histone types as well as acetylated forms of H4 are indicated. B: Controls for chromatin immunoprecipitation (ChIP) procedure. Chromatin fragments were separated into bound and unbound fractions using an antibody against acetylated histone H4. DNA was purified and polymerase chain reaction performed using primers specific for the ribosomal protein S-14 gene [1], alpha-satellite [2], or the human PGK1 gene [3 and 4]. Templates for samples 3 and 4 derived from inactive and active human X chromosomes, respectively. In each comparison, total DNA concentrations were equalized in the samples from bound and unbound fractions. Gels were analyzed by PhosphorImager scanning (Amersham Biosciences/GE Healthcare, Milwaukee, WI) and data were quantified from 3–6 independent experiments. Results are expressed as ratios between bound (B) and nonbound (NB) levels set to a log scale Figure 2. Highly acetylated and under-acetylated sites identified in human fibroblasts. A: genome sampling/chromatin immunoprecipitation (gsChIP) gel. DNA samples from bound (B) and nonbound (NB) chromatin fractions were subjected to polymerase chain reaction (PCR) using pairs of arbitrary primers. Products were separated by 6% sequencing PAAG for direct comparison. Triplicate PCR reactions are represented for each DNA template. H = product from highly acetylated locus (mostly bound); L = products from under-acetylated loci (mostly nonbound). B: Distribution of bands from 8 primer pairs. Approximately 850 reproducible and clearly separated bands were counted from autoradiography films. Bands were grouped according to B/NB ratios and plotted to show presumed acetylation distributions. C: Locations of identified loci along chromosomes. Chromosome lengths and positions depicted are based on build 34 of the human genome. Centromeres are drawn as gray circles. Loci shown as open triangles = hyperacetylated; black triangles = under-acetylated; gray triangles = near equal or variable distribution between bound and unbound fractions Figure 3. Verification and mapping of genome sampling/chromatin immunoprecipitation (gsChIP) sites. A: Real-time polymerase chain reaction (PCR) values for loci estimated by gsChIP to be under-acetylated (L) or highly acetylated (H). DNA was eluted from gel bands, sequenced, and loci mapped on the human genome. Real-time PCR was performed with locus-specific primers using SYBR Green Fast-start PCR kits on a LightCycler instrument (Roche). One ng of each sample was analyzed in triplicate with 0.11 to 3 ng input DNA used for standard curve preparation. Shown are B/NB ratios set to a log scale. B and C: Mapping across representative under-acetylated (h40) and highly acetylated (h90) regions. Primer pairs distributed across ≈20 Kb regions flanking h40 (C) and h90 (B) were used in real-time PCR assays performed in triplicate. ARs (amplified regions) are shown to scale. LINEs, SINEs and LTRs are shaded black for conserved elements and gray for less conserved repeats. TRPS1 = trichorhinophalangeal syndrome I gene; LINE = long interspersed nuclear element; SINE = short interspersed nuclear element; LTR = long-terminal repeat Figure 4. Distributions of ESTs and CpG islands in 125 kb regions flanking underacetylated and highly acetylated loci. A: EST values were taken from Unigen maps for build 34 of the human genome sequence. B: CpG islands were scored as defined by (69). EST = expressed sequence tag Figure 5. Local chromatin remodeling associated with HL-60 differentiation. A. Control and TPA-treated HL-60 cells were compared by genome sampling/chromatin immunoprecipitation (gsChIP). Real-time polymerase chain reaction (PCR) data are shown for three loci exhibiting differential amplification (Unstable) and for control loci (Stable). B: Acetylation levels across ≈25 kb region flanking locus L28. Black triangles show position of original locus. Fold change values are indicated below Figure 6. Local chromatin differences between fibroblasts related to passage level or donor age. A: Comparison of chromatin fractions derived from early and late passage HSC172 human lung embryo fibroblasts. Segment of genome sampling/chromatin immunoprecipitation (gsChIP) gel pattern is shown. Arrow indicates h9 product. Early passage corresponds to population doubling level (PDL) 19; Late, PDL 60. B = bound; NB = nonbound material. Total DNA concentrations in B and NB fractions were equalized prior to polymerase chain reaction (PCR) amplification. B: Comparison of materials derived from fetal and young adult skin fibroblasts. Segment of gsChIP gel with h9 product indicated by arrow. Fetal skin fibroblasts were derived from a 16-week-old embryo (AG04392A, PDL 17). Young adult fibroblasts were from individuals of age range 24 to 30 yr (see Methods). C: Comparison of chromatin preparations derived from preadolescent and old donors (see Methods). Segment of gsChIP gel with p14 product indicated by arrow Table 1. Summary Analysis of Under-Acetylated and Highly Acetylated Regions. Locus chrState* Chr Pos(bld34) Overlapping Gene or Gene <50 kb Distant (Comment)† mRNA/ESTs‡ cpgi§ h04 L X 88290116 No 1 0 h06 L 20 41570598 Yes (intron) 33 0 h07 L 7 69872430 No 3 0 h12 L 3 68524375 No 36 0 h14 L 16 73870316 No 6 0 h22 L 4 35884816 No 61 0 h32 L 14 70474677 Yes (intron) 68 0 h37 L 12 53458013 No 0 0 h38 L 1 187764975 Yes (30 kb from 3′ end) 30 0 h39 L 4 94550476 Yes (intron) 10 0 h40 L 14 24220310 No 2 0 h44 L 4 164503214 No 3 0 h46 L 10 112798270 No 2 0 h80 L 2 156242152 No 1 0 h11 H 17 39441746 Yes (10 kb from 3′ end; 27 kb from 5′ end) 286 0 h49 H 6 1431101 No 6 3 h53 H 3 130157067 Yes (intron) 1157 2 h55 H 5 57455101 No 8 0 h56 H 4 99823816 Yes (2 kb from 5′end; 28 kb from 3′end) 499 0 h57 H 2 99803409 Yes (exon) 176 1 h58 H 5 96166432 Yes (intron; 4 kb from 3′ end) 853 3 h59 H 18 21144397 Yes (intron) 87 1 h64 H 2 207029045 Yes (43 kb from 3′ end) 150 0 h68 H 4 2665818 Yes (intron) NA NA h69 H 15 34680715 Yes (intron) 80 0 h70 H 8 73063517 Yes (25 kb from 5′ end) 67 1 h74 H 2 120755398 Yes (intron) 89 0 h77 H 22 24918468 Yes (intron) 115 1 h78 H 2 191721964 Yes (4.5 kb from 5′ end) 202 1 h82 H 5 95793101 Yes (7 kb from 3′ end) 101 0 h90 H 8 116633475 Yes (intron) 110 2 Notes: *chrState. † This field and fields to right refer to 125 Kb regions flanking indicated loci. ‡ Human UniGene cluster map. § Number of CpG islands. 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Mapping development-related and age-related chromatin remodeling by a high throughput ChIP-HPLC approach. J Gerontol Biol Sci.2004;59A:1234-1243. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A6B6A0DB1B5CC83B000DD5EBE21EF4C45E8F9DB.txt b/test/dataset/in/resources/corpus/Clean_0A6B6A0DB1B5CC83B000DD5EBE21EF4C45E8F9DB.txt new file mode 100644 index 0000000..d1a4cd4 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A6B6A0DB1B5CC83B000DD5EBE21EF4C45E8F9DB.txt @@ -0,0 +1 @@ +AMGP61489S1064-7481(12)61489-410.1097/00019442-200008000-00009American Association for Geriatric PsychiatryTABLE 1Subject demographicsElectroconvulsive TherapyPharmacotherapySignificance (P)n3939NSAge, years83.69 ±3–8383.43 ±3–26NSGender (female/male)36/334/5NSRace (white/black)38/137/2NSMedications4.64 ±2.034.18±2.57NSMedical diagnoses2.89 ± 1.63.08 ± 1.2NSCardiac diagnoses1424NSLength of stay42.51 ±20.9223–36± 11.490.001ASA score2.28±0.502.10±0.51NSNote: ASA = American Society of Anesthesia “medical acuity” score.TABLE 2ComplicationsCategoryElectroconvulsive TherapyPharmacotherapySignificance (P)Falls24NSCardiovascular060.013Confusion/Neurologic105NSGastrointestinal040.027Pulmonary10NSMetabolic00NSTotal complications1319NSPatients with complications, n1016NSNote: Several patients in each group had more than one complication.TABLE 3Cardiac historyElectroconvulsive TherapyPharmacotherapySignificance (P)Hypertension1918NSArteriosclerotic cardiovascular disease67NSMyocardial infarction22NSCongestive heart failure36NSTotal cardiac diagnoses3033NSPatients with cardiac history, n2424NSNote: Several patients in each group had more than one cardiac diagnosis.TABLE 4Outcome, n (percent)Electroconvulsive TherapyPharmacotherapySignificance (P)Good30 (76.9)13 (33.3)0.001Moderate9 (23.1)22 (56.4)0.003Poor04 (10.3)0.06Overall0.003Regular ArticlesElectroconvulsive Therapy in Old-Old PatientsDavid T.ManlyM.D.*Stanley P.OakleyJr.M.D.Richard M.BlochPh.D.Department of Psychiatric Medicine, East Carolina University School of Medicine, Greenville, North Carolina*Address correspondence to Dr. Manly, Park Ridge Hospital, P.O. Box 1569, Fletcher, NC 28732The authors compared falls, cardiovascular factors, confusion, gastrointestinal, pulmonary, and metabolic side effects for “old-old” (>75 years) patient groups treated with either electroconvulsive therapy (ECT) or pharmacotherapy. A subset of a pharmacotherapy patient group was selected to match for age, sex, and diagnosis in a case-control design. Side effects were recorded from each selected patient's medical record and compared between groups. Patients receiving ECT showed fewer cardiovascular and gastrointestinal side effects. Patients receiving ECT had longer lengths of stay and more favorable outcomes. Overall, there was a tendency for ECT to result in fewer side effects and better treatment outcomes. ECT appears to be relatively safe and more effective than pharmacotherapy for major depressive disorders in old-old patients.Major depression is a common and treatable cause of morbidity and mortality in elderly patients that is often underdiagnosed and undertreated in primary care settings and nursing homes. Studies in the past have shown that the use of electroconvulsive therapy (ECT) in major depression increases with age and that patients over 65 receive a disproportionately high share of ECT compared with younger patients.1 There are controversies over the safety of ECT in elderly and medically ill patients, and questions have been raised as to whether it is more effective than pharmacotherapy. As the population in the United States continues to age, the use and safety of ECT in elderly patients will be an increasingly important clinical issue. The most rapidly growing subgroup of our population is the “old-old”, generally defined as 75 years of age and older. Population projections for the period between 1980 and 2040 estimate the general population will grow 41%, whereas those over 65 will grow 160%, and those 75 and older will grow 268%.2Most studies of the treatment of depression have included patients of all ages and have often actually excluded the elderly patient with medical complications. Therefore, few studies have specifically addressed the treatment of depression in elderly patients.3,4 This is particularly true for the “old-old”, frail elderly patients, and those with concomitant medical or neurological disorders. A recent study addressing the use of ECT in old-old patients retrospectively compared 39 patients age 80 and older with 42 younger patients age 65 through 80. The study found ECT to be relatively well tolerated, even in the older age-group. Confusion was the most common complication in both age-groups. The older age-group had more cardiovascular problems and falls, reflecting higher medical acuity, as measured by American Society of Anesthesia (ASA) scores, number of medical problems, and number of cardiac medications. Outcome was better in the younger group, but the older patients did well, with at least moderate improvement noted in 85%.5The current study extends these findings by directly comparing two groups of old-old patients suffering from major depression. One of these age- and sex-matched groups was treated pharmacologically, and the other was treated with ECT. We compared therapeutic outcomes and complications.METHODSWe conducted a retrospective chart review of inpatients at a university medical center with both private and academic psychiatric services. Charts were selected from the years 1987 to 1993 for patients 75 years of age and older whose discharge diagnoses included a major affective disorder (unipolar or bipolar depression) and who had received ECT. From these, data were obtained regarding age, gender, medical diagnoses, number and type of medications, number and laterality of ECT (if available), complications, and outcome. On the basis of matching age, gender, and discharge diagnosis of major affective disorder, a comparison group of records was sequentially identified for patients who had been treated psychopharmacologically without ECT. The same data elements were gathered from these charts. Diagnoses for all patients had been determined by an attending psychiatrist with DSM-III-R criteria as recorded in the discharge notes.6 Because all charts meeting ECT and age criteria were selected for the experimental group, and “computer-applied matching criteria” selected the comparison group, group selection bias was minimized.ECT was administered 2 or 3 times per week by use of a brief pulse device (Mecta SR1). Lead placements were bilateral in 19 patients, right-unilateral in 9, both bi- and unilateral in 9, and not noted in 2 patients.Medical status was assessed independently by one of the investigators (DM or SO) using the American Society of Anesthesiology (ASA) 5-point rating scale.7 Each selected chart was reviewed for notations of complications, which were defined as any unexpected event requiring intervention or a change in treatment plan. All complications were classified into one of six categories: cardiovascular, pulmonary, neurologic (including confusion), falls, gastrointestinal, and metabolic. Complications did not include the expected physiological responses to ECT, such as transient hypertension, tachycardia, or postictal confusion immediately after the stimulus. The results of treatment were evaluated by use of a global rating scale used by previous investigators.5,8,9 A good outcome was defined as a complete resolution of symptoms with a return to premorbid baseline and with no or only minor residual sequelae that do not interfere with social functioning. A moderate outcome was defined as some improvement, but with residual symptoms. A poor outcome was defined as no improvement or worsening of condition and level of functioning.The frequencies of various treatment complications and cardiac histories for each group were compared by use of the Fisher's exact test statistic. Other comparisons between groups, such as ASA scores or length of hospital stay, utilized standard t-tests for independent groups.10RESULTSTable 1 shows subject group characteristics. The ECT and pharmacological control populations are well matched for age, gender, and race. The two groups were also medically comparable. ASA scores did not differ between groups (t[76] = 1.56; P = 0.12). The majority of patients in both groups had scores of 2 or 3. No patients in either group had a score greater than 3. The average number of medications (4.6 vs. 4.2) and average number of medical diagnoses (3.1 vs. 2.9) were also similar between groups. However, the groups did differ in terms of the overall length of hospital stay, with the ECT group requiring an average of 19 more days of hospitalization than the medication-only control group (t[76] = 4.84; P = <0.001).The complications in both groups are noted in Table 2. The only neurological complication in the ECT group was confusion, which turned out to be common in the control group as well. Ten patients with ECT experienced confusion, whereas six experienced confusion in the control group (Fisher's P = 0.2 [NS]).Gastrointestinal side effects were noted only in the psychopharmacologically treated group (Fisher's P = 0.027). These included nausea/vomiting, constipation, and dry mouth. All except dry mouth were associated with the use of tricyclic antidepressants.The most common complication in the control group was cardiovascular in nature. Six pharmacologically treated patients experienced cardiovascular complications. Interestingly, no patients in the ECT group were noted to have cardiac complications (Fisher's P = 0.013), although, as shown in Table 3, there was no difference in the frequency of cardiac diagnoses. Overall, there were more complications in the control group, with 16 patients out of 39 having complications as compared with only 10 out of 39 in the ECT group, although this difference did not reach statistical significance.Table 4 shows the outcome data for the two treatment groups. Significantly more ECT patients had a good outcome (76.9%) than the pharmacologically treated group (33.3%; Fisher's P<0.001). In contrast, more patients in the pharmacologically treated group achieved only a moderate outcome (56.4% vs. 23.1%; Fisher's P = 0.003) whereas four of the pharmacologically treated group had a poor outcome (10.3%; Fisher's P = 0.06). It should be noted that no ECT-treated patients had a poor outcome in this study.DISCUSSIONTreatment of depression in elderly patients is frequently complicated by comorbid medical conditions and polypharmacy. These issues are particularly prominent in “old-old” patients, a group that has been inadequately studied in the past. In fact, old-old patients have frequently been excluded from treatment studies because of their age and frequent comorbidity. Our study was undertaken to evaluate the safety and efficacy of ECT in this group. Our hypothesis was that ECT would be found to be more effective, with fewer side effects than pharmacologic treatment in hospitalized old-old patients with major affective disorders. This study confirms our hypothesis and extends the result of Cattan et al.5 Our study is particularly useful in that ECT-treated patients were compared with an age- and gender-matched, pharmacologically treated group of comparable medical status.Length of stay was considerably longer in the ECT-treated group, 41.5 days compared with 23.3 days in the medically-treated control subjects. This finding is consistent with several previous studies showing significantly longer length of stays in ECT-treated inpatients.11–14 In one study of 101 elderly (more than 65 years old) patients with depression, 46 patients who received ECT were hospitalized for 43.7 days as compared with 55 patients not treated with ECT, who were hospitalized for only 24.4 days.11 A recent retrospective naturalistic study12 of geriatric patients hospitalized for depression found that the length of stay for ECT patients was twice that of patients treated without ECT (48 days vs. 24 days). Furthermore, they found that when ECT was the treatment of choice initially, the length of stay was 40 days. However, when ECT was used secondarily, following a medication trial, the length of stay increased to 56 days. Yet another study13 found the mean length of time between admission and the first ECT was 21.4 days. This suggests that significant factors in the longer lengths of stay for ECT patients are medication trials prior to ECT, weaning off medications, and medical evaluation and stabilization of elderly patients before ECT. Furthermore, it has become common practice to initiate maintenance antidepressant medication after the completion of ECT, which can further extend the length of stay.The incidence of complications noted in Table 2 is in agreement with similar studies. Confusion was the most common complication, with 10 of 39 ECT patients (25.6%) experiencing confusion and 7.6% of patients experiencing other medical complications. Our overall complication rate was 33%. Mulsant et al.13 reviewed the literature, citing 14 studies with a total of over 1,000 geriatric patients (age 65 and older) treated with ECT and found the incidence of confusion to be about 10% and also found 6% of patients having some other medical complications during ECT. The same investigators performed a prospective naturalistic study of elderly inpatients and found a complication rate very similar to ours. They found that 31% of patients suffered confusion, and 7% had some other medical complication. Cattan et al. found confusion to be the most common complication, occurring in 45% of patients age 65–80 and in 59% of patients over the age of 80. It should be noted that one possible factor in the discrepancy in the rate of confusion between our study and Cattan et al.5 is that patients in Cattan's study were treated with a sine-wave ECT apparatus, which is known to cause significantly greater confusion.15,16 Individual studies show a wide range of incidence of complications, ranging from none to over 50%. The most common complications seem to be cardiovascular, confusion, and falls. Our study differs in that we had no cardiovascular complications noted within our ECT population. As shown in Table 3, both groups had the same frequency of cardiac diagnoses (24/39) and so were equally at risk of cardiac side effects. The lack of cardiac complications, although surprising, may be, in part, due to the fact that we did not include transient phenomena, which should be considered “normal” physiological concomitants of ECT, such as transient hypertension, sinus tachycardia, or occasional PVCs. A recent retrospective study by Gormley et al.17 also found no cardiac complications in a review of 93 courses of ECT in 67 patients over the age of 75.In this study we used a retrospective naturalistic design. Several notable weaknesses are inherently possible in this design. The nonblinded nature of the data-gathering could have resulted in bias in the assessment of the outcome. Furthermore, the difference in length of stay could have led to an overestimation of the response rate to ECT due to the fact that patients were evaluated at different points in time. Also, when data are collected retrospectively, the evaluation of complications depends on the documentation of the attending physician at the time of occurrence. Subtle biases in the recognition or documentation of side effects of ECT could, therefore, account for some of the findings in the current study. Patient selection biases must also be considered. Patients viewed as likely to have side effects may be less likely to be given ECT in the first place. The fact that this study replicates and extends similar ECT side-effect advantages in elderly patients5 suggests that at least local biases in terms of selection, recognition, or documentation are less likely to have accounted for these findings. A final methodological consideration involves the possibility that multiple comparisons may have taken advantage of chance differences between groups. The low level of various types of side effects was a relatively consistent pattern for ECT and makes it highly unlikely that the findings reflect a consistent multiple-comparison effect.Our study supports a dramatic positive effect of ECT in the treatment of affective disorders, even in the old-old age-group. Our results are in agreement with the study by Rubin and colleagues11 that found a 98% response rate, with 45 of 46 patients showing a moderate-to-good response to ECT, as measured by the Beck Depression Inventory, Global Depression Scale, and nonblinded global assessment. Mulsant et al.'s study13 using the Ham-D scale in elderly patients demonstrated that 100% had at least a slight degree of improvement, with two-thirds of patients showing at least a 50% reduction in Ham-D scores.Our study confirms that ECT is both safe and extremely effective in geriatric patients with severe depression and extends earlier results to the old-old population over 75 years of age. If carefully monitored, even “old-old” patients can be safely treated with ECT. Further prospective studies using objective outcome measures such as the Ham-D18 and documentation of complications are indicated to elaborate upon these findings.References1JWThompsonRDWeinerCPMeyersUse of ECT in the United States in 1975, 1980, and 1986Am J Psychiatry1511994165716612DPRiceDemographic realities and projections of an aging populationSAndreopoulosJJognessHealth Care for Aging Society1989Church LivingstoneNew York15453SIFinkelEfficacy and tolerability of antidepressant therapy in the old-oldJ Clin Psychiatry57suppl5199623284CSalzmanLSchneiderBLebowitzAntidepressant treatment of very old patientsAm J Geriatr Psychiatry1199321295RACattanPPBarryGMeadElectroconvulsive therapy in octogenariansJ Am Geriatr Soc3819907537586American Psychiatric AssociationDiagnostic and Statistical Manual of Mental Disorders3rd Edition1987American Psychiatric AssociationWashington, DCRevised7American Society of AnesthesiologistsNew classification of physical statusAnesthesiology2419631118WJBurkeJLRuthefordCFZorumskiElectroconvulsive therapy and the elderlyCompr Psychiatry2619854804869WJBurkeEJRubinCFZorumskiThe Safety of ECT in geriatric psychiatryJ Am Geriatr Soc35198751652110StatMost 32 for Windows1996DataMost CorporationSalt Lake City, UT11EHRubinDAKinsherfSAWehrmanResponse to treatment of depression in the old and very oldJ Geriatr Psychiatry Neurol41991657012RAPhilibertLRichardsCFLynchEffect of ECT on mortality and clinical outcome in geriatric unipolar depressionJ Clin Psychiatry56199539039413BHMulsantJRosenJEThorntonA prospective naturalistic study of electroconvulsive therapy in late-life depressionJ Geriatr Psychiatry Neurol4199131314DKoesslerBSFogelElectroconvulsive therapy for major depression in the oldest oldAm J Geriatr Psychiatry11993303715RAbramsElectroconvulsive Therapy2nd Edition1992Oxford University PressNew York16HASackeimElectroconvulsive therapy in late-life depressionCSalzmanClinical Geriatric Psychopharmacology3rd Edition1998Williams & WilkinsBaltimore, MD26230917NGormleyCCullenLWaltersThe safety and efficacy of electroconvulsive therapy in patients over age 75Int J Geriatr Psychiatry13199887187418MHamiltonDevelopment of a rating scale for primary depressive illnessBr J Soc Clin Psychol61967278296 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A72A907018F1E19ADF967141A8A455B7DEC7891.txt b/test/dataset/in/resources/corpus/Clean_0A72A907018F1E19ADF967141A8A455B7DEC7891.txt new file mode 100644 index 0000000..2c15be3 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A72A907018F1E19ADF967141A8A455B7DEC7891.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 23810.1093/geront/46.2.238 ASSISTED LIVING An Empirical Typology of Residential Care/Assisted Living Based on a Four-State Study Park Nan Sook PhD 1 Zimmerman Sheryl PhD 2 3 Sloane Philip D. MD 2 4 Gruber-Baldini Ann L. PhD 5 Eckert J. Kevin PhD 6 Address correspondence to Nan Sook Park, PhD, School of Social Work, The University of Alabama, Box 870314, Tuscaloosa, AL 35487-0314. E-mail: npark@bama.ua.edu 4 2006 46 2 238 248 24 10 2005 21 3 2005 Copyright 2006 by The Gerontological Society of America 2006 Purpose: Residential care/assisted living describes diverse facilities providing non–nursing home care to a heterogeneous group of primarily elderly residents. This article derives typologies of assisted living based on theoretically and practically grounded evidence.Design and Methods: We obtained data from the Collaborative Studies of Long-Term Care, which examined 193 assisted living facilities in four states: Florida, Maryland, New Jersey, and North Carolina. By using mixture modeling, we derived typologies in five different ways, based on: structure; process; resident case-mix; structure and process; and structure, process, and resident case-mix. Results: Although configurations of typologies varied as a function of criterion variables used, common themes emerged from different cluster solutions. A typology based on resident case-mix yielded a five-cluster solution, whereas a typology based on structure, process, and resident case-mix resulted in six distinct clusters. Medicaid case-mix/psychiatric illness and high resident impairment were two clusters identified by both strategies. Implications: Because of the wide variation in structure, residents, and services within assisted living facilities, typologies such as those described here may be useful in clinical practice, research, and policy. To the extent that public payment defines its own cluster, the potential for inequities in care merits careful attention. Typology Residential care Assisted living Cluster analysis Long-term care hwp-legacy-fpage 238 hwp-legacy-dochead RESEARCH ARTICLE Residential care/assisted living (RC/AL) facilities have become a popular housing option for older Americans. It is estimated that there are more than 35,000 RC/AL facilities in the United States, which provide services to as many as 1 million Americans (Ball et al., 2000); however, the number varies depending on how RC/AL is defined (Hawes, Phillips, Rose, Holan, & Sherman, 2003). Although RC/AL facilities share the commonality of housing people who need assistance in non-nursing home settings, they provide extremely diverse care to a heterogeneous group of people. For example, facilities range in size from a few beds to hundreds of beds; serve residents across a wide spectrum of ages, functional abilities, and health problems; and provide services that vary widely in comprehensiveness (Zimmerman, Sloane, & Eckert, 2001). Because the range of variability is so much broader for RC/AL than for nursing homes, it is difficult to talk about these facilities as if they were monolithic, which in part hinders research efforts (Golant, 2004; Zimmerman et al., 2003). Developing a typology is especially useful when a group is heterogeneous and when classification systems have not been established (Everitt, Landau, & Leese, 2001), which is certainly the case with RC/AL. Typology development involves a process in which the group is classified into meaningful subgroups that share similar characteristics. In fact, typologies have already been used to identify common patterns in long-term care, allowing researchers to describe and understand the configurations of settings that commonly exist for older adults (Lawton, 2001; Timko & Moos, 1991). The majority of these studies, however, are limited to classifying nursing homes, particularly dementia care units (Davis et al., 2000; Gold, Sloane, Mathew, Bledsoe, & Konanc, 1991; Grant, 1998; Grant, Kane, & Stark, 1995; Holmes & Teresi, 1994; Sloane, Lindeman, Phillips, Moritz, & Koch, 1995). A few studies have addressed RC/AL settings (e.g., Hawes, Phillips, & Rose, 2000; Timko & Moos), but none have studied the entire range of RC/AL across multiple states. In developing and describing typologies, researchers have commonly employed the structure–process–outcome framework (Donabedian, 1978, 1980, 1988). In this framework, structure refers to the capacity of the facility to provide care, which includes physical amenities (e.g., safety features) and human resources (e.g., the staff-to-resident ratio). Process is how the facility delivers care, which encompasses the activities that occur between care providers and residents (e.g., the provision of organized activity programs). Outcome is the effect of care received. Davis and colleagues (2000) evaluated specialized dementia programs in RC/AL settings based on two structure-related variables: facility size (i.e., small vs large) and administrative relationships with other facilities (i.e., affiliation with other RC/AL facilities); they derived five types. Hawes and colleagues (2000) classified a national sample of 1,251 larger RC/AL facilities based on reported consumer preferences for two process-related characteristics (i.e., privacy and service); they identified four types. Although both of these studies were able to parsimoniously present similarities and differences across clusters, they were limited by a focus on few variables to differentiate the types of care settings. One component of care that both studies overlooked was resident case-mix, which is likely to be closely related to the structure of care (Wunderlich, Sloan, & Davis, 1996). For instance, facilities consisting of high proportions of residents who are functionally dependent may require more resources and staff than those caring for less impaired individuals. Indeed, Timko and Moos (1991) examined the configurations of different facilities for older adults from a national sample of 235 nursing homes, RC/AL facilities, and congregate-care apartments. They found that both facility characteristics (e.g., level of care, ownership, and size) and resident case-mix (e.g., social resources, functional ability, and gender) contributed to the social climate of facilities. They contended, in essence, that resident case-mix gauges the overall demands on the staff, which in turn affects facility dynamics. As RC/AL prospered under the recent building boom (i.e., it has accounted for more than 80% of new projects in the senior-housing industry), the heterogeneity in the field has increased (Adler, 1998). Consequently, as recognized by a national task force, it has become increasingly difficult to even agree on a definition of RC/AL (Assisted Living Workgroup, 2003). Being able to delineate the subtypes of facilities that exist should facilitate understanding, discussion, research, policy, and practice. Therefore, this article derives typologies of RC/AL by using criteria from structure, process, and resident case-mix domains. In doing so, it uses a more comprehensive set of variables than has been applied in the past. We used mixture modeling, a special case of cluster analysis developed in the latent variable framework, in order to classify facilities, and we evaluated resultant typologies based on statistical, theoretical, and practical significance. Methods Sample and Design We derived data for this study from the Collaborative Studies of Long-Term Care (CS–LTC), which was funded by the National Institutes of Health to describe the structure, process, and outcomes of care across a diversity of RC/AL settings (Zimmerman et al., 2001). The CS–LTC broadly defined RC/AL as “facilities or discrete portions of facilities, licensed by the state at a non-nursing-home level of care, that provide room, board, 24-hour oversight, and assistance with activities of daily living” (Zimmerman et al., 2001, p. 120). This is the most inclusive and most commonly accepted definition of RC/AL. The CS–LTC enrolled 2,078 residents in 193 facilities from representative regions across four states: Florida, Maryland, New Jersey, and North Carolina. Baseline data were collected from October 1997 to November 1998. After consultation with national experts and a review of state regulations and current research, the CS–LTC selected these states in consideration of geographical proximity (to enable onsite data collection) and variability (their RC/AL policies reflected the diversity of the field). A purposive sample of counties (sampling regions) was selected within each state based on three criteria: (a) at least 15% of each type of the state's RC/AL facilities were in the region; (b) rural and urban diversity was reflected in terms of demographic and health-service characteristics; and (c) the region represented the state. To ensure that all facility types meeting the definition of RC/AL were included, the CS–LTC stratified facilities into three types: facilities with fewer than 16 beds, traditional “board-and-care” facilities with 16 or more beds, and “new-model” facilities with 16 or more beds. New-model facilities with 16 or more beds were defined through a pilot study as having been built after January 1, 1987, and having either two or more different private-pay monthly rates, 20% or more residents requiring assistance in transfer, 25% or more residents who are incontinent daily, or either a registered nurse or licensed practical nurse on duty at all times (Zimmerman et al., 2003). A total of 113 smaller facilities and 40 of each of the other facility types were included in the CS–LTC. Administrators of the 193 facilities were interviewed onsite to gather facility-level information. Detailed information about CS–LTC methods can be obtained elsewhere (Zimmerman et al., 2001). Variables Used in Typology Development The CS–LTC offered rich information with which to develop typologies. We organized the variables under study according to the domains of structure, process, and resident case-mix. Data were not available to incorporate outcomes in the analyses. Table 1 presents definitions of the facility-level variables used in these analyses. We included the following facility-level measures of structure in these analyses: facility size (bed capacity); years in operation; ownership (proprietary vs not for profit); affiliation with other facilities (RC/AL facilities, nursing homes, hospitals, or continuing care retirement communities); staffing (presence/absence of registered nurse or licensed practical nurse, total number of licensed nursing hours or aide-care hours, and aide turnover rate); percentage of private rooms; and environmental quality. We measured the latter by using the Therapeutic Environment Screening Survey for Residential Care (Sloane, Zimmerman, & Walsh, 2001), an observational instrument from which a composite indicator of quality, the Assisted Living Environmental Quality Scale, can be derived. The Assisted Living Environmental Quality Scale summarizes environmental quality with respect to several domains, such as maintenance, safety, cleanliness, and privacy. In addition, neighborhood attractiveness was a rating of whether the surrounding area was in good or bad repair/maintenance. We evaluated process of care by using eight scales from the Policy and Program Information Form (POLIF) of the Multiphasic Environmental Assessment Procedure (Moos & Lemke, 1996). POLIF has been used in other studies, which have found it to discriminate between different long-term care settings and the types of residents they serve (Zimmerman et al., 2001). The eight POLIF scales included in these analyses evaluated admission policies, acceptance of problem behaviors, policy choice, policy clarity, provision of privacy, resident control, overall provision of services, and availability of social and recreational activities. The range of Cronbach's alphas for the subscales was.69–.84, showing moderately high internal consistency of the items (Moos & Lemke; Zimmerman et al., 2003). We scored each POLIF scale as a range from 0%–100%, with higher percentages indicating endorsement of more items. The analyses in this article excluded two measures (provision of health services and admission policies specific to ADL functioning), which were subsets of other measures, and so were highly correlated. We used six variables describing a range of resident characteristics and case-mix in typology development: percentage of residents on Medicaid; percentage of residents who required assistance taking care of their own appearance; percentage of residents who needed help getting in and out of bed; percentage of residents who had been diagnosed with dementia; percentage of residents who had a diagnosis of mental or psychiatric illness; and percentage of residents who had behavioral symptoms. An administrator for the whole facility reported these variables (i.e., we did not derive them from resident-level data). Analyses We first examined the distribution of individual variables (e.g., mean, standard deviation) and patterns of missing values. Next, we conducted mixture cluster analyses (Muthén & Muthén, 1998–2001) separately for structure (S), process (P), and resident case-mix (R). Subsequently, we used a combination of structure and process variables as the criterion variables (SP), and, lastly, we ran a combination of structure, process, and resident case-mix (SPR) for the final model. We examined the configurations of clusters (i.e., which clusters were comparable to others and which variables were driving the differences) and fit indices at each step. Mixture modeling (i.e., mixture cluster analysis), a special case of cluster analysis, presumes that “unobserved heterogeneity” in the sample explains variability among observed variables (Muthén, 2001; Muthén & Muthén, 1998–2001). The heterogeneity is assumed to be latent or unobserved, hence to be inferred from the data. Applying this assumption to the current study, we can say that the diversity of RC/AL is due to the unobserved heterogeneity of the settings that may be explained through the chosen criterion variables from structure, process, and resident case-mix domains. The assumptions and procedures of cluster analysis resemble exploratory factor analysis in that both approaches can be used as data-reduction techniques. The notable difference is that factor analysis takes a variable-centered approach, whereas cluster analysis takes a case-centered approach (Muthén & Muthén, 2000). Mixture cluster analysis assumes that latent variables are categorical, whereas observed variables can be either categorical or continuous; this assumption is suited to the current study. Mixture cluster analysis is also versatile in terms of handling missing data, whereas missing values are problematic in traditional cluster analysis. That is, mixture modeling using the Mplus program uses all observations available in the data and estimates the parameters through a maximum likelihood estimator (Muthén & Muthén, 1998–2001). In the present analyses, we determined the best-cluster solutions through the largest loglikelihood values using expectation-maximization algorithms, highest entropy (i.e., an index of classification quality), and lowest Bayesian Information Criterion (BIC) values (Muthén, 2001; Muthén & Muthén, 1998–2001; Schwartz, 1978), as well as high posterior probabilities (i.e., probabilities for cases to be in their respective class; Muthén & Muthén, 2000). Once we had classified the facilities into clusters based on each domain of criterion variables (S, P, or R) and on a combination of the domains (SP or SPR), we used chi-square tests and one-way analysis of variance (ANOVA) in order to examine associations between cluster types by facility and resident characteristics. In ANOVA, we made additional tests to compare groups on a post-hoc basis. We conducted paired comparisons by using Fisher's least significant difference procedure (where F tests were significant at α =.05). Finally, we examined patterns of cluster membership across the resultant five typologies (e.g., whether facilities belonging to the same cluster in the SP typology stayed together in the SPR typology). We calculated kappa statistics among different combinations of the five typologies, reflecting the degree to which different observers or, in this case, methods, classify a particular case in the identical category (Stokes, Davis, & Koch, 2000). We performed mixture cluster analyses by using Mplus 2.1 (Muthén & Muthén, 1998–2001), chi-square tests and ANOVA by using SPSS 11 (SPSS, 1991–2000), and kappa statistics by using SAS 8.2 (SAS Institute, 1999–2001). Results Descriptive Statistics Table 2 shows the means and standard deviations or percentages of the 26 variables used for typology development. We examined missing patterns across variables and facilities; we excluded 4 facilities with excessive missing data (i.e., more than 70% of the values missing) from the analyses, resulting in 189 facilities for which data were available. Facilities that were included (n = 189) and excluded (n = 4) were not significantly different with respect to facility size, years in business, proprietary status, affiliation with other facilities, percentage of private rooms, environmental quality, or resident-level functional and cognitive impairment, psychiatric illness, or behavioral symptoms. Study facilities had an average of 28 beds (range 4–190, the mean being reduced by the disproportionate number of smaller facilities) and had been in business for an average of 14 years. The majority of the facilities were for profit (83%), and roughly 24%–33% were affiliated with other facilities or operated as part of a chain with other RC/AL facilities. About one half of the sample had a registered nurse or licensed practical nurse on duty, who worked an average of two hours per resident per week. In addition, personal care aides worked roughly 11 hours per resident per week. Annual aide turnover was 69%. More than one half of the rooms were private (66%), and most of the facilities (82%) were located in attractive or very attractive neighborhoods. Facilities varied in their environmental quality scores (range 5–26), with an average score of approximately 17. Facilities tended to have few restrictions for admitting residents; however, some were reluctant to admit those with behavior problems. Generally, facilities scored moderately on policies involving individual freedom, institutional order, and social/recreational activities. With respect to resident case-mix, approximately 15% of residents across facilities were on Medicaid. More residents required help in taking care of themselves than they did transferring (47% vs 16%, respectively). A small proportion of residents had a history of mental illness (12%) or required attention because of behavioral symptoms (9%). Administrators reported that, on average, one third of their residents had dementia. Typologies We derived typologies by using five different combinations of criterion variables based on structure (S); process (P); resident case-mix (R); structure and process (SP); and structure, process, and resident case-mix (SPR). Although each approach derived distinct clusters of RC/AL depending on the criterion variables, the S, P, and SP models proved of limited utility because their results were dominated by facility size. Therefore, we present here only the R and SPR models, because they provide more unique and comprehensive information than the S, P, and SP models. Further, the kappa value between the R typology and SPR typology was 0.26, suggesting slight agreement (p <.01) and the utility of displaying both. Cluster Solutions Based on Resident Case-Mix Characteristics (R) Table 3 shows the means and standard deviations of the variables in the five clusters that were derived from the six facility-level resident characteristics, as well as the results of ANOVA or chi-square tests for each variable. Fit measures of mixture modeling favored a five-cluster solution in that values of loglikelihood, BIC, entropy, and average posterior probabilities indicated reasonably good fit compared with other cluster solutions (log likelihood = −4,633; BIC = 9,475; entropy = 0.87). The five clusters were distinguishable by their criterion variables (i.e., resident case-mix); accordingly, all ANOVA tests for the criterion variables were highly significant at p <.05. Four variables in the structure domain (i.e., years in business, nursing, environment, and privacy) were differentiated by this clustering as well, as were two variables in the process domain (i.e., overall admission policies and privacy). We describe each cluster briefly here: Cluster R-1 (22 facilities) was distinguished by a high proportion of residents on Medicaid and with mental/psychiatric illness, a moderate level of residents who required self-care assistance, poorer environmental quality, and a lower level of privacy. Cluster R-2 (25 facilities) was characterized by high proportions of physically impaired residents (53% requiring help in transfer, compared with 4%–28% in other facility types). Although it was not significant, dementia was common (61%), but the rate for behavioral symptoms was low (6%). Cluster R-3 (8 facilities) contained a very high proportion of residents with behavioral symptoms (81%, compared with less than 10% in all other clusters), many of whom had dementia (64%). Also, 28% of residents in this cluster required help in transfer. There were more licensed nursing hours in this cluster than in any other (5.2 hours/resident/week vs 0.70–2.05 hours/resident/week). Cluster R-4 (59 facilities) housed residents who were significantly less impaired in physical function (12% and 4% required help in self care and transfer, respectively, vs 44%–80% and 11%–53%, respectively, in other clusters). Facilities in this cluster had the strictest admission policies (60% vs 76%–85% in other facilities), tended to be older, and provided more privacy (nonsignificant). Cluster R-5 (72 facilities) tended to be mid range across most variables. Cluster Solutions Based on Structure, Process, and Resident Case-Mix Characteristics (SPR) A six-cluster solution was the best fitting model for the SPR analyses (log likelihood = −16,231; BIC = 33,416; entropy = 0.95). Table 4 shows this cluster solution, including means and standard deviations of variables by cluster type and results of test statistics and least significant difference multiple comparisons. We describe each cluster briefly here: Cluster SPR-1 (14 facilities) offered the fewest services (37% vs 48%–71% in the other clusters, p <.05). Facilities in this cluster scored moderately high in the provision of privacy (65% vs 40%–50% in four of the other clusters). These facilities tended to be older, and residents in this cluster were the least functionally impaired, although differences were not statistically significant. Cluster SPR-2 (25 facilities) was characterized by higher percentages of individuals on Medicaid (68% vs 3%–49% in other clusters) and with a history of mental and psychiatric illness (19%, which was higher than all clusters except Cluster SPR-4). Cluster SPR-3 (57 facilities) had a higher proportion of residents with functional, cognitive, and behavioral impairments than any other cluster. Privacy and service provision were moderately high. Cluster SPR-4 (54 facilities) tended to include large facilities (mean bed capacity = 66 vs 9–16 for other clusters). These facilities had the highest environmental quality score (21 vs 13–16 for other clusters) and scored highest in terms of policy choice, privacy, services, and social activities. Cluster SPR-5 (7 facilities) had the highest aide turnover rates (600% vs 37%–125% in other clusters), reported the highest proportion of residents with mental illness, and had many residents on Medicaid. Cluster SPR-6 (32 facilities) was not statistically different than other clusters in any one variable, but it did have the fewest licensed nursing hours, lowest aide turnover, least policy clarity, least resident control, and lowest Medicaid case-mix. Discussion We examined a large, diverse, multistate sample of RC/AL facilities in order to determine the extent to which the facilities could be clustered into distinct subtypes based on facility-level structure (S), process (P), and resident case-mix (R) characteristics. This study is one of the few that has developed typologies focusing on RC/AL, and the only one that includes such a broad and theoretically and empirically justified set of variables. The four states under study (Florida, Maryland, New Jersey, and North Carolina) were well-suited to this exploration because they all have RC/AL regulations that refer to two or more types of housing and service, and they all provide Medicaid funding (Mollica, 2001). The four states also exhibited relevant differences in that New Jersey was the most advanced in articulating regulations and allowing for extensive services and retention, whereas Maryland was just beginning to develop regulations at the time of data collection (Zimmerman et al., 2001). Facility size was an overriding factor in three of the five subtypes (S, P, and SP). Much has been written about the importance of facility size in the field of RC/AL, with key concepts including the homelike-ness of “mom and pop” styles of care and the dangers of impersonalization and institutionalization in larger models of care (Kane & Wilson, 2001; Morgan, Gruber-Baldini, Eckert, & Zimmerman, 2004). Thus, in order to better understand the RC/AL field, we endeavored to identify criterion variables other than facility size. The typology based on the resident case-mix (R) variables was successful in this regard, identifying five distinctly different facility clusters involving a large variety of variables. Variables that significantly differentiated the clusters included facility age, nursing care, environmental quality, privacy, and resident case-mix (i.e., Medicaid, self-care, transfer, dementia, mental/psychiatric illness, and behavioral symptoms). Clearly, the analytic process was successful in differentiating types of facilities based on the types of residents they serve. Furthermore, there is some indication that the structure and process of care within clusters was consistent with resident needs and/or the challenges they presented, indicating that RC/AL facilities were organized around both resident need and market conditions. For example, in Cluster R-3, high cognitive impairment and behavioral symptoms were associated with more nursing care; in Cluster R-4, low resident impairment was associated with high privacy (structure and process) and stricter admission policies (process). Two very similar clusters were identified using both the R and the SPR strategies: high Medicaid, high mental/psychiatric illness; and high resident ADL impairment. Unfortunately, findings of the present study also indicate that high Medicaid, high mental/psychiatric illness was paired with a poor physical environment and low privacy, raising issues about a link between public costs and quality of care. Although scholars argue that higher quality is not necessarily associated with higher costs (Davis, 1991; Mukamel & Spector, 2000), the current findings suggest that this may not be the case with regard to environmental quality. Thus, older adults who are economically disadvantaged may be forced to live in poorer physical environments with limited options. However, these data do not report the actual experiences and outcomes of care in such a cluster. Although one study did report a higher risk of hospitalization in facilities with poorer environments (Zimmerman, Gruber-Baldini, Hebel, Sloane, & Magaziner, 2002), further research is necessary to characterize the linkage between environmental quality and other outcomes. Another notable finding about the SPR model is that the large facilities formed a cluster that carried with it better scores on physical environment quality, privacy, availability of services, and policy choice. This cluster parallels the privacy/service typology identified by Hawes and colleagues (2003), but because it is but one of six clusters, the scope of analysis used here is more comprehensive. The aggregation of large facilities into such a cluster is in line with the notion that large facilities have more resources, activities, and services than smaller facilities (Morgan, Eckert, & Lyon, 1995; Weihl, 1981). The question of whether larger size involves a tradeoff, such as more impersonalization, warrants consideration, but cannot be answered with these data. The typologies developed in this study expand on what has been done by other researchers, most of which relates to nursing home care. Gold and colleagues (1991) derived a typology of 55 nursing homes through unstructured narratives. By using eight criterion variables related to physical environment, staff/resident interaction, and staff and administrator attitudes toward residents, they identified eight clusters with an indication of good or bad type. Although this qualitatively driven typology suggests differences related to quality, the study is limited in generalizability to nursing homes for memory-impaired older adults. Grant (1998) developed an empirical typology of 390 units in 123 nursing homes through cluster analysis, a similar procedure to that used in the present study. By using seven care attributes including physical environment, resident activity participation, and staff training, he obtained six clusters with a varying combination of care attributes. Grant's typology offered a useful way of classifying dementia care in nursing homes, but, like the typology identified by Gold and colleagues, used a narrow range of variables and was limited to nursing homes. Timko and Moos (1991) developed a typology through cluster analysis using a sample of 235 nursing homes, residential care facilities, and congregate apartments. Six distinct types of facilities emerged from the seven physical and social environment variables. Their study is one of the few that included RC/AL, but it was not specific to RC/AL. Thus, although all of these typology studies provide insights into understanding the configuration of long-term-care facilities using quantitative or qualitative methods, the present study is unique with respect to its focus on a broad range of RC/AL facilities and criterion variables and its use of resident case-mix and an innovative analytic method using a latent variable approach. Nevertheless, it is important to note that the present study has several limitations. First, although we based clustering variables on theoretical and empirical evidence, the procedure of clustering facilities into several groups may be criticized as being data driven. Indeed, one caution about cluster analysis procedures is that the analytic procedures tend to be structure imposing rather than structure seeking (Aldenderfer & Blashfield, 1984). Replication of these results by applying the same procedures to a different data set of RC/AL facilities would strengthen findings (Milligan & Cooper, 1987). A second limitation involves both sampling and policy issues. This study was based in four states, which may limit generalizability, because state regulations affect RC/AL philosophy, admission and retention criteria, and service provision (Mollica, 2001). However, although the facilities under study were not necessarily representative of all facilities across the country, they certainly were representative of the four study states and, most likely, of common facility types nationally. Third, the resident case-mix data were reported by facility administrators, rather than compiled by a detailed listing of all facility residents. Thus, an unknown possibility of reporting bias may have affected the results. Despite these limitations, however, this typology is a helpful first step in both elucidating different models of care and providing clear criteria by which to differentiate them. Furthermore, this analytic strategy can be replicated in other states for purposes of verification or to add to what is known. In closing, cluster solutions must be examined in terms of their practicality and relevance (Everitt et al., 2001; Muthén & Muthén, 2000). From a face-validity perspective, many of these clusters parallel distinct subtypes discussed informally by research staff involved in data collection. Some facilities with higher resident ADL and cognitive impairment may be skimming off of the nursing home market and so would be required to provide more intense services. Others, such as those with a mixed resident population, may require that the facilities contract with health professionals to meet their occasional needs for health care. The clusters do not, however, recognize the heterogeneity that exists within the clusters, related to variables that were not under study. That point notwithstanding, the two models identified both the clustering of financing (Medicaid) and mental illness, and of high ADL impairment. These variables are relevant to state regulations regarding the allocation of public dollars, as well as to the level of resident disability likely to be served by RC/AL across the nation. Additional tests of relevance would include a study of the relationship between facility type and outcomes such as resident quality of life, health status, and health care utilization. Thus, although the findings reported here have immediate usefulness in describing and differentiating the range of RC/AL facilities (and perhaps in guiding care provision and helping families and professionals match potential residents with facilities), the ultimate value of the typologies requires their replication in other regions and their application to longitudinal studies of resident outcomes. Finally, continued study is necessary to test the stability and consistency of the cluster solutions within and across states that maintain different regulatory systems and that may well be characterized through the configuration of the typologies. This research was supported by Grants RO1 AG13871, RO1 AG13863, and K02 AG00970 from the National Institute on Aging. We acknowledge and note appreciation for the cooperation of the facilities, residents, and families participating in the Collaborative Studies of Long-Term Care. We give special thanks to Drs. Richard Barth, Natasha Bowen, Shenyang Guo, Lucinda Roff, and Ellen Csikai, who provided invaluable comments on an earlier version of this article. 1 School of Social Work, The University of Alabama, Tuscaloosa. 2 Cecil G. Sheps Center for Health Services Research, University of North Carolina at Chapel Hill. 3 School of Social Work, University of North Carolina at Chapel Hill. 4 Department of Family Medicine, University of North Carolina at Chapel Hill. 5 Department of Epidemiology and Preventive Medicine, University of Maryland, Baltimore County. 6 Department of Sociology and Anthropology, University of Maryland, Baltimore County. Decision Editor: Linda S. Noelker, PhD Table 1. Description of Facility-Level Variables Used in Typology Development. Variables Description Structure domain The capacity of the facility to provide care     Facility size Bed capacity     Years in business Years of facility operation     Ownership Proprietary status (1 = for profit; 0 = not for profit)     Affiliated with other facilities Whether facility is affiliated with a nursing home, a hospital, or a continuing care retirement community (1 = yes; 0 = no)     RC/AL chain Whether facility is in a chain with other RC/AL facilities (1 = yes; 0 = otherwise)     RN or LPN Whether facility has RNs or LPNs on duty (1 = yes; 0 = no)     Licensed nursing care hours The number of RN and LPN hours per week per resident     Aide care hours The number of nursing aide hours per week per resident     Aide turnover Annual nursing aide turnover rate     Facility environment Facility environmental quality measured with the Assisted Living Environmental Quality Scale (AL-EQS), with a higher score indicating a better environment     Private rooms Percent of private rooms     Neighborhood How attractive the neighborhood in which facility is located is rated (1 = attractive; 0 = not attractive) Process domain The way care is delivered (number of items and internal consistency of subscales)     Overall admission policies The admission expectations related to resident characteristics (24 items; α =.84)     Acceptance of problem behavior The extent to which the facility tolerates behavior problems exhibited by residents (16 items; α =.79)     Policy choice The degree to which residents individualize their routines (17 items; α =.76 )     Policy clarity The extent to which behavioral expectations and facility rules are well defined (10 items; α =.69)     Provision of privacy The extent to which privacy is given to residents (9 items; α =.83)     Resident control The degree to which residents are able to influence facility administration and policy (24 items; α =.80)     Overall provision of services The extent to which the facility offers health and supportive services (20 items; α =.80)     Provision of social and recreational services The degree to which organizational activities are available (13 items; α =.79) Resident case-mix domain Facility-level resident characteristics     Medicaid residents % of residents on Medicaid     Residents requiring help in self care % of residents who require assistance taking care of their own appearance     Residents requiring help in transfer % of residents who need help getting in and out of bed     Residents with a dementia diagnosis % of residents who are diagnosed with dementia     Residents with history of mental or psychiatric illness % of residents who have a diagnosis of mental or psychiatric illness     Residents with behavioral symptoms % of residents who require attention because of behavioral symptoms Notes: RC/AL = residential care/assisted living; RN = registered nurse; LPN = licensed practical nurse. Dichotomous variables are so indicated; other variables are continuously scaled. Table 2. Distribution of Variables Used in Typology Development. Variable M (SD) or % Structure     Bed capacity (range 4–190) 28.49 (35.24)     Years in business (range 0.17–148) 13.95 (17.04)     Proprietary ownership 82.54%     Affiliation         Affiliated with NH, hospital, or CCRC 23.94%         RC/AL chain 32.98%     Staffing         Have a RN or LPN 47.09%         Licensed nursing care hours per resident per week (range 0–20) 1.96 (3.42)         Aide care hours per resident per week (range 0–74) 10.66 (9.03)         Aide turnover, annual (range 0–80) 68.92%     Environment quality         Facility environment (range 5–26)a 16.50 (5.13)         % Private rooms (range 0–100) 66.01 (32.43)         Attractive neighborhood 82.01% Process     Requirements for residents         Overall admission policies (range 0–100) 72.58 (18.38)         Acceptance of problem behavior (range 0–100) 33.88 (21.52)     Individual freedom or institutional order         Policy choice (range 10.53–89.47) 50.24 (15.69)         Policy clarity (range 0–100) 58.72 (23.95)         Provision of privacy (range 22.22–100) 56.87 (21.69)         Resident control (range 0–64) 28.22 (16.22)     Provision of service and activities         Overall provision of services (range 14.81–92.59) 58.39 (17.00)         Provision of social or recreational activities (range 0–96.15) 50.71 (22.46) Resident case mix (all ranges 0–100)     % Medicaid residents 14.19 (23.52)     Functional status         % Require help in self-care 46.51 (30.50)         % Require help in transfer 15.97 (20.40)         % Demented 33.81 (27.05)         % History of mental or psychiatric illness 12.45 (17.55)         % Behavioral symptoms 8.68 (17.56) Notes: NH = nursing home; CCRC = continuing care retirement community; RC/AL = residential care/assisted living; RN = registered nurse; LPN = licensed practical nurse. For the table, N = 189. Four facilities with excessive missing values were excluded. Some variables have more missing values than others. The missing values for staffing-related variables range from 32%–54%. Percent of private rooms has 35% missing. For process variables, the missing values range from 2%–6%. For facility-level resident variables, missing values are distributed evenly (4%–6%) across variables except % Medicaid residents, of which 22% of the values are missing. aFacility environment was measured with the Assisted Living Environmental Quality Score, which ranged from 5–26, with higher scores indicating better environment. Table 3. Cluster Solutions Based on Resident Case-Mix Variables. Variable Cluster R-1 High Medicaid, Mental, or Psychiatric Illness; Poor Environment; Low Privacy (n = 22) Cluster R-2 High ADL Impairment, Cognitive Impairment (n = 25) Cluster R-3 High Cognitive Impairment, Behavioral Symptoms, Nursing Care (n = 8) Cluster R-4 Low Impairment; Older Facilities; High Privacy; Strict Admission (n = 59) Cluster R-5 Moderate Structure, Process, Case mix (n = 72) Test Statistic and Group Comparisonsa Structure     Bed capacity 14.14 (10.72) 20.80 (22.66) 13.38 (8.23) 35.07 (44.50) 30.53 (33.29) F = 2.3 (1: 4,5)     Years in business 11.50 (8.60) 11.76 (11.86) 8.50 (5.32) 21.36 (25.01) 9.72 (10.16) F = 4.7** (4: 1,2,3,5)     Proprietary ownership 1.00 (0.00) 0.88 (0.33) 0.88 (0.35) 0.75 (0.44) 0.81 (0.40) χ2 = 42.2***     Affiliated with hospital or CCRC 0.14 (0.35) 0.28 (0.46) 0.13 (0.35) 0.28 (0.45) 0.25 (0.44) χ2 = 70.9***     RC/AL chain 0.27 (0.46) 0.28 (0.46) 0.50 (0.53) 0.19 (0.40) 0.47 (0.50) χ2 = 25.2***     Have RN or LPN 0.42 (0.51) 0.35 (0.49) 0.63 (0.52) 0.47 (0.50) 0.51 (0.50) χ2 = 90.1***     Licensed nursing hours per resident per week 0.70 (1.18) 1.77 (3.26) 5.15 (6.84) 1.91 (3.46) 2.05 (3.14) F = 2.5* (3: 1,2,4,5)     Aide care hours per resident per week 8.14 (5.46) 12.33 (7.00) 12.96 (7.48) 6.85 (5.75) 13.58 (11.33) F = 4.8** (1: 5); (4: 2,5)     Aide turnover 0.91 (1.14) 0.69 (1.02) 0.77 (0.81) 0.64 (1.39) 0.66 (0.68) F = 0.2     Environment (AL-EQS) 12.77 (5.06) 17.60 (3.89) 17.88 (3.56) 17.10 (5.16) 16.78 (5.23) F = 3.8** (1: 2,3,4,5)     Proportion private room 0.38 (0.35) 0.66 (0.31) 0.67 (0.39) 0.72 (0.32) 0.69 (0.29) F = 4.0** (1: 2,3,4,5)     Attractive neighborhood 0.73 (0.46) 0.76 (0.44) 0.88 (0.35) 0.86 (0.35) 0.83 (0.38) χ2 = 3.8 Process     % Admission policies 77.57 (15.57) 84.67 (11.01) 79.17 (11.14) 60.36 (21.39) 75.68 (13.41) F = 13.2*** (4: 1,2,3,5); (2: 5)     % Acceptance of problem behaviors 27.88 (18.70) 42.27 (23.15) 41.67 (27.08) 30.77 (18.44) 34.86 (22.73) F = 2.0 (2: 1,4)     % Policy choice 41.95 (12.08) 50.82 (15.64) 49.78 (16.62) 52.35 (14.37) 51.01 (17.21) F = 1.9 (1: 4,5)     % Policy clarity 57.27 (20.57) 63.24 (22.28) 65.14 (13.36) 58.77 (25.24) 56.53 (25.46) F = 0.5     % Privacy 40.74 (12.34) 54.11 (20.73) 48.61 (15.64) 65.09 (21.51) 56.81 (22.13) F = 6.0*** (1: 2,5); (4: 1,2,3,5)     % Resident Control 30.51 (17.69) 32.23 (14.15) 20.24 (15.60) 29.79 (15.76) 25.91 (16.67) F = 1.5     % Total no. of services 57.58 (12.30) 64.10 (18.41) 57.87 (20.18) 58.92 (16.11) 55.94 (17.85) F = 1.1 (2: 5)     % Social and recreational activities 41.78 (19.98) 61.63 (19.40) 52.40 (14.96) 43.74 (22.65) 54.58 (22.10) F = 4.7** (1: 2,5); (4: 2,5) Resident case mix     % Residents on Medicaid 70.45 (17.61) 6.79 (10.42) 3.57 (6.56) 7.84 (13.14) 6.71 (10.28) F = 115.5*** (1: 2,3,4,5)     % Residents require help in self-care 44.43 (28.10) 80.37 (15.65) 76.79 (17.99) 11.70 (11.25) 60.45 (15.90) F = 108.1*** (1: 2,3,4,5); (4: 2,3); (5: 2,3,4)     % Residents require help in transfer 11.04 (12.07) 53.26 (16.53) 27.83 (32.33) 3.77 (8.69) 12.96 (11.29) F = 67.7*** (1: 2,3,4); (2: 3,4,5); (3: 4,5); (4: 5)     % Residents demented 24.69 (23.62) 61.37 (25.26) 64.38 (21.26) 15.01 (16.77) 39.10 (23.00) F = 27.5*** (1: 2,3,5); (2: 4,5); (3: 4,5); (4: 5)     % Residents mental or psychiatric illness 34.83 (31.14) 11.82 (13.13) 19.58 (24.33) 7.90 (10.35) 8.64 (10.67) F = 14.3*** (1: 2,3,4,5); (3: 4)     % Residents with behavioral symptoms 2.83 (5.63) 6.24 (7.53) 80.89 (17.99) 3.41 (5.72) 7.59 (9.41) F = 161.1*** (1: 3,5); (2: 3); (3: 4,5); (4: 5) Notes: CCRC = continuing care retirement community; RC/AL = residential care/assisted living; RN = registered nurse; LPN = licensed practical nurse; AL-EQS = Assisted Living–Environmental Quality Score. Data presented are means; standard deviations are presented parenthetically. Three facilities without information on the six resident case-mix variables were excluded, resulting in n = 186 facilities. Bold statistics are those that significantly differentiate that cluster from all others. Depending on the results from multiple comparisons, it is possible to have multiple significant clusters on a variable. Each cluster was labeled to identify those characteristics that significantly differentiate it from the others or had a comparatively high mean score (even if not significant from all others). *p <.05; **p <.01; ***p <.001. aChi-square tests for categorical variables show only test statistics. A statistically significant ANOVA resulted in multiple comparisons, as shown. Table 4. Cluster Solutions Based on Structure, Process, and Resident Case-Mix Variables. Variable Cluster SPR-1 Moderately Low Service; High Privacy (n = 14) Cluster SPR-2 High Medicaid, Mental, or Moderately High Psychiatric Illness (n = 25) Cluster SPR-3 High Impairment; Moderate Privacy; Moderately High Service (n = 57) Cluster SPR-4 Large Facilities; Good Environment; High Privacy, Choice, Service (n = 54) Cluster SPR-5 High Staff Turnover, Medicaid, Mental or Psychiatric Illness (n = 7) Cluster SPR-6 Lower Nursing Care, Aide Turnover, Policy Clarity, Resident Control, Medicaid Case Mix (n = 32) Test Statistic and Group Comparisonsa Structure     Bed capacity 11.21 (4.46) 13.56 (10.25) 16.25 (15.10) 66.46 (44.78) 11.14 (9.49) 9.25 (5.82) F = 32.7*** (4: 1,2,3,5,6)     Years in business 34.50 (24.81) 11.04 (6.23) 10.56 (12.11) 14.86 (22.60) 21.71 (14.95) 10.00 (6.51) F = 6.0*** (1: 2,3,4,6)     Proprietary ownership 0.64 (0.50) 1.00 (0.00) 0.96 (0.19) 0.52 (0.50) 1.00 (0.00) 1.00 (0.00) χ2 = 59.8***     Affiliated with hospital or CCRC 0.07 (0.27) 0.24 (0.44) 0.14 (0.35) 0.57 (0.50) 0.00 (0.00) 0.00 (0.00) χ2 = 48.6***     RC/AL chain 0.14 (0.36) 0.44 (0.51) 0.51 (0.50) 0.38 (0.49) 0.00 (0.00) 0.00 (0.00) χ2 = 31.6***     Have RN or LPN 0.11 (0.33) 0.41 (0.50) 0.45 (0.50) 0.98 (0.15) 0.00 (0.00) 0.00 (0.00) χ2 = 77.5***     Licensed nursing hours per resident per week 0.50 (1.14) 0.59 (1.13) 2.93 (4.95) 3.35 (2.73) 0.13 (0.19) 0.06 (0.22) F = 6.2*** (1: 3,4); (2: 3,4); (6: 3,4)     Aide care hours per resident per week 1.87 (3.42) 7.65 (4.95) 13.86 (11.89) 9.49 (5.05) 6.85 (10.81) 12.81 (8.80) F = 4.7*** (1: 3,4,6); (2: 3,4,6)     Aide turnover 1.25 (1.06) 0.64 (0.76) 0.80 (0.90) 0.50 (0.44) 6.00 (2.83) 0.37 (0.56) F = 22.3*** (5: 1,2,3,4,6); (3: 6)     Environment (AL-EQS) 15.36 (4.88) 13.28 (4.89) 15.68 (4.07) 20.93 (3.83) 14.14 (6.07) 14.00 (4.13) F = 17.7*** (2: 3); (4: 1,2,3,5,6)     Proportion private room 0.91 (0.16) 0.39 (0.32) 0.65 (0.29) 0.88 (0.21) 0.14 (0.17) 0.52 (0.29) F = 16.5*** (1: 2,3,5,6); (3: 2,4,5,6); (4: 2,5,6); (5: 6)     Attractive neighborhood 0.86 (0.36) 0.76 (0.44) 0.77 (0.42) 0.91 (0.29) 0.57 (0.53) 0.84 (0.37) χ2 = 7.5 Process     % Admission policies 35.12 (16.80) 78.09 (16.82) 82.37 (10.51) 70.14 (15.58) 58.33 (13.61) 74.45 (12.91) F = 28.4*** (1: 3,4,5); (3: 4,5,6); (4: 2,5); (5: 2,6)     % Acceptance of problem behaviors 28.23 (14.36) 26.67 (18.86) 40.22 (24.13) 38.12 (21.55) 23.33 (15.06) 25.63 (17.28) F = 3.6** (2: 3,4); (6: 3,4)     % Policy choice 47.74 (12.12) 41.75 (11.94) 48.16 (14.64) 62.18 (14.94) 49.12 (5.44) 42.11 (12.57) F = 12.6*** (3: 2,4,5,6); (4: 1,2,5,6)     % Policy clarity 36.09 (19.09) 59.33 (16.99) 58.05 (17.31) 81.14 (12.92) 42.67 (23.50) 34.69 (20.58) F = 36.8*** (1: 2,3,4); (2: 4,5,6); (3: 5,6); (4: 5,6)     % Privacy 65.08 (14.36) 40.28 (11.73) 44.08 (13.59) 82.29 (13.61) 40.74 (9.07) 49.60 (14.45) F = 59.0*** (1: 2,3,4,5,6); (2: 6); (4: 2,3,5,6)     % Resident control 20.76 (15.23) 28.30 (17.36) 25.67 (16.29) 38.63 (12.79) 26.77 (8.78) 19.01 (13.06) F = 8.9*** (4: 1,2,3); (6: 2,3,4)     % Total no. of services 37.04 (9.85) 59.85 (11.94) 57.99 (17.12) 71.10 (12.52) 49.67 (12.47) 47.77 (12.25) F = 20.2*** (1: 2,3,4,5,6); (4: 2,3,5); (6: 2,3,4)     % Social and Recreational Activities 26.37 (19.13) 38.00 (20.06) 60.21 (17.13) 67.08 (12.59) 32.05 (17.82) 30.74 (14.64) F = 35.6*** (2: 1,3,4); (3: 1,4,5,6); (4: 1,5,6) Resident case mix     % Residents on Medicaid 9.31 (14.65) 67.64 (20.45) 7.78 (10.39) 5.02 (9.07) 49.17 (18.73) 3.46 (8.41) F = 97.2*** (2: 1,3,4,5,6); (5: 1,3,4,6)     % Residents require help in self-care 7.35 (13.58) 41.57 (28.04) 67.34 (21.52) 42.27 (29.79) 8.89 (9.81) 45.61 (27.04) F = 17.2*** (1: 2,3,4,6); (3: 2,4,5,6); (5: 2,4,6)     % Residents require help in transfer 0.38 (1.41) 9.58 (12.72) 25.95 (21.92) 14.84 (21.90) 3.33 (8.16) 15.18 (18.46) F = 5.9*** (1: 3,4,6); (3: 2,4,5,6)     % Residents demented 6.34 (9.43) 24.40 (23.30) 50.14 (25.45) 31.62 (24.26) 11.11 (20.18) 32.40 (26.44) F = 10.8*** (1: 2,3,4,6); (3: 2,4,5,6); (5: 4,6)     % Residents with mental or psychiatric illness 10.45 (10.23) 24.65 (27.50) 10.97 (14.10) 7.49 (10.81) 42.50 (32.00) 8.67 (11.28) F = 8.7*** (2: 1,3,4,5,6); (5: 1,3,4,6)     % Residents with behavioral symptoms 4.33 (6.68) 2.76 (5.39) 18.56 (27.61) 5.73 (8.67) 2.78 (6.80) 3.81 (6.79) F = 5.9*** (3: 1,2,4,5,6); (4: 5) Notes: CCRC = continuing care retirement community; RC/AL = residential care/assisted living; RN = registered nurse; LPN = licensed practical nurse; AL-EQS = Assisted Living–Environmental Quality Score. For the table, N = 189. Table data presented are means; standard deviations are shown parenthetically. Bold statistics are those that significantly differentiate that cluster from all others. Depending on the results from multiple comparisons, it is possible to have multiple significant clusters on a variable. Each cluster was labeled to identify those characteristics that significantly differentiate it from the others or had a comparatively high or low mean score (even if not significant from all others). *p <.05; **p <.01; ***p <.001. aChi-square tests for categorical variables show only test statistics. A statistically significant ANOVA resulted in multiple comparisons, as shown. References Adler, S., (1998). Get ready for consolidation: Today's product in the pipeline will build tomorrow's big players. Contemporary Long Term Care, 21, (8), 39-45. Aldenderfer, M. S., & Blashfield, R. K., (1984). Cluster analysis. Beverly Hills, CA: Sage. Assisted Living Workgroup. (2003). Assuring quality in assisted living: Guidelines for federal and state policy, state regulation, and operations. Washington, DC: Author. Ball, M. M., Whittington, F. J., Perkins, M. M., Patterson, V. L., Hollingsworth, C., & King, S. V., et al (2000). Quality of life in assisted living facilities: Viewpoints of residents. Journal of Applied Gerontology, 19, 304-325. Davis, K. J., Sloane, P., Mitchell, C. M., Preisser, J., Grant, L., & Hawes, M. C., et al (2000). Specialized dementia programs in residential care settings. The Gerontologist, 40, 32-42. Davis, M. A., (1991). On nursing home quality: A review and analysis. Medical Care Review, 48, 129-166. Donabedian, A., (1978). The quality of medical care. Science, 200, 856-864. Donabedian, A., (1980). Explorations in quality assessment and monitoring. Vol. 1, The definition of quality and approaches to its assessment. Ann Arbor, MI: Health Administration Press. Donabedian, A., (1988). The quality of care: How can it be assessed? Journal of the American Medical Association, 260, 1743-1748. Everitt, B. S., Landau, S., & Leese, M., (2001). Cluster analysis (4th ed.). New York: Oxford University Press. Golant, S. M., (2004). Do impaired older persons with health care needs occupy U.S. assisted living facilities? An analysis of six national studies. Journal of Gerontology: Social Sciences, 59B, S68-S79. Gold, D. T., Sloane, P. D., Mathew, L. J., Bledsoe, M. M., & Konanc, D. A., (1991). Special care units: A typology of care settings for memory-impaired older adults. The Gerontologist, 31, 467-475. Grant, L. A., (1998). Beyond the dichotomy: An empirical typology of Alzheimer's care in nursing homes. Research on Aging, 20, 569-592. Grant, L. A., Kane, R. A., & Stark, A. J., (1995). Beyond labels: Nursing home care for Alzheimer's disease in and out of special care units. Journal of the American Geriatrics Society, 43, 569-576. Hawes, C., Phillips, C. D., & Rose, M., (2000). High service or high privacy assisted living facilities, their residents, and staff: Results from a national survey. Beachwood, OH: Myers Research Institute. Hawes, C., Phillips, C. D., Rose, M., Holan, S., & Sherman, M., (2003). National survey of assisted living facilities. The Gerontologist, 43, 875-882. Holmes, D., & Teresi, J., (1994). Characteristics of special care units in the northeast five-state survey: Implications of different definitional criteria. Alzheimer Disease and Associated Disorders, 8, (Suppl. 1), S97-S105. Kane, R. A., & Wilson, K. B., (2001). Assisted living at the crossroads: Principles for its future. Portland, OR: Jessie F. Richardson Foundation. Lawton, M. P., (2001). The physical environment of the person with Alzheimer's disease. Aging and Mental Health, 5, (Suppl. 1), S56-S64. Milligan, G. W., & Cooper, M. C., (1987). Methodology review: Clustering methods. Applied Psychological Measurement, 11, 329-354. Mollica, R. L., (2001). State policy and regulations. In S. Zimmerman, P. D. Sloane, & J. K. Eckert (Eds.), Assisted living: Needs, practice, and policies in residential care for the elderly (pp. 9–33). Baltimore, MD: The Johns Hopkins University Press. Moos, R. H., & Lemke, S., (1996). Evaluating residential facilities: The multiphasic environmental assessment procedure. Thousand Oaks, CA: Sage. Morgan, L. A., Eckert, J. K., & Lyon, S. M., (1995). Small board and care homes: Residential care in transition. Baltimore: Johns Hopkins University Press. Morgan, L. A., Gruber-Baldini, A. L., Eckert, J. K., & Zimmerman, S., (2004). Policy and research for small assisted living facilities. Journal of Aging & Social Policy, 16, (4), 1-16. Mukamel, D. B., & Spector, W. D., (2000). Nursing home costs and risk-adjusted outcome measures of quality. Medical Care, 38, 78-89. Muthén, B. O., (2001). Latent variable mixture modeling. In G. A. Marcoulides & R. E. Schumacker (Eds.), New developments and techniques in structural equation modeling (pp. 1–33). Mahwah, NJ: Erlbaum. Muthén, B. O., & Muthén, L. K., (2000). Integrating person-centered and variable-centered analyses: Growth mixture modeling with latent trajectory classes. Alcoholism: Clinical and Experimental Research, 24, 882-891. Muthén, L. K., & Muthén, B. O., (1998). Mplus user's guide (2nd ed.). Los Angeles: Authors. SAS Institute. (1999). SAS system for Windows [Computer software]. Cary, NC: Author. Schwartz, G., (1978). Estimating the dimension of a model. Annals of Statistics, 6, 461-464. Sloane, P. D., Lindeman, D. A., Phillips, C., Moritz, D. J., & Koch, G., (1995). Evaluating Alzheimer's special care units: Reviewing the evidence and identifying potential sources of study bias. The Gerontologist, 35, 103-111. Sloane, P. D., Zimmerman, S., & Walsh, J. F., (2001). The physical environment. In S. Zimmerman, P. D. Sloane, & J. K. Eckert (Eds.), Assisted living: Needs, practice, and policies in residential care for the elderly (pp. 173–197). Baltimore: Johns Hopkins University Press. SPSS. (1991). SPSS for Windows [Computer software]. Chicago, IL: Author. Stokes, M. E., Davis, C. S., & Koch, G. G., (2000). Categorical data analysis using the SAS system (2nd ed.). Cary, NC: SAS Institute. Timko, C., & Moos, R. H., (1991). A typology of social climates in group residential facilities for older people. Journal of Gerontology: Social Sciences, 46, S160-S169. Weihl, H., (1981). On the relationship between the size of residential institutions and the well-being of residents. The Gerontologist, 21, 247-250. Wunderlich, G. S., Sloan, F. A., & Davis, C. K., (1996). Nursing staff in hospitals and nursing homes: Is it adequate?. Washington, DC: National Academy Press. Zimmerman, S., Gruber-Baldini, A. L., Hebel, J. R., Sloane, P. D., & Magaziner, J., (2002). Nursing home facility risk factors for infection and hospitalization: Importance of RN turnover, administration, and social factors. Journal of the American Geriatrics Society, 50, 1987-1995. Zimmerman, S., Gruber-Baldini, A. L., Sloane, P. D., Eckert, J. K., Hebel, J. R., & Morgan, L. A., et al (2003). Assisted living and nursing homes: Apples and oranges? The Gerontologist, 43, (Special Issue II), 107-117. Zimmerman, S., Sloane, P. D., & Eckert, J. K., (2001). Assisted living: Needs, practice, and policies in residential care for the elderly. Baltimore: Johns Hopkins University Press. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A786A5064D35A35A1DDE38EE56DFE6B7F40BAC7.txt b/test/dataset/in/resources/corpus/Clean_0A786A5064D35A35A1DDE38EE56DFE6B7F40BAC7.txt new file mode 100644 index 0000000..d489a6d --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A786A5064D35A35A1DDE38EE56DFE6B7F40BAC7.txt @@ -0,0 +1 @@ +]>EXG5341S0531-5565(00)00153-410.1016/S0531-5565(00)00153-4Elsevier Science Inc.Fig. 1The plasma concentrations of glucose, immunoreactive glucagon (IRG), immunoreactive insulin (IRI), epinephrine, and norepinephrine after microinjection of saline (1μl; ○) or neostigmine (5×10−8mol; •) into 3rd cerebral ventricle. Values are means±SE for 7–22 rats. Significant differences between subjects and saline-treated controls at identical times (at least p<0.05).Fig. 2Relative contributions of the pathways to hyperglycemia induced by the injection of neostigmine into the central nervous system.Fig. 3The plasma concentrations of glucose, epinephrine, and norepinephrine after intrahippocampal injection of 5×10−8, 5×10−9mol of neostigmine, saline, or coinjection of 5×10−8mol of neostogmine and atropine.Fig. 4The concentrations of epinephrine(A), dopamine(B), and norepinephrine(C) in DAT, VD, and ND groups, respectively. One way ANOVA shows significant differences among the three groups (p=0.0261); epinephrine level in DAT subjects was significantly lower than that in ND by Scheffe's post hoc test (p=0.0332).Table 1Number of subjects, mean age, mean arterial pressure, and MMSE scores in DAT, VD, and ND groupsDATVDNDNumber of subjects662821Mean age (mean±S.D.)82.49±7.7782.86±5.8682.95±7.77Mean atrial pressure (mean±S.D.) (mmHg)92.64±10.9994.74±10.3194.56±12.09Mean score of MMSE (mean±S.D.)7.96±6.495.29±5.1924.52±2.86Table 2Plasma concentrations of epinephrine, norepinephrine, dopamine, glucose, and insulin in DAT, VD, and ND groupsDATVDNDEpinephrine (pg/ml)18.83±13.1124.93±20.4531.52±29.83Norepinephrine (pg/ml)286.25±172.89333.29±221.77357.14±259.69Dopamine (pg/ml)7.08±8.9213.61±22.5014.33±21.36Glucose (mg/dl)88.81±8.1889.86±9.6289.57±7.19Insuline (uU/ml)5.31±1.685.33±2.265.09±1.64The metabolism of plasma glucose and catecholamines in Alzheimer's diseaseHUmegakia*umegaki@tsuru.med.nagoya-u.ac.jpNTamayaaTShinkaibAIguchiaaDepartment of Geriatrics, Nagoya University Graduate School of Medicine, 65 Tsuruma-Cho, Showa-Ku, Nagoya, Aichi, 466-8550, JapanbDepartment of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-3 Sakae-Cho, Itabashi-Ku, Tokyo, 173-0015, Japan*Corresponding author. Tel.: +81-52-744-2365; fax: +81-52-744-2371AbstractSeveral lines of evidence suggest that the cholinergic system in the hippocampus plays a pivotal roll in regulating the peripheral metabolism of glucose and catecholamines. The injection of cholinergic stimulators including neostigmine, the acetylcholine esterase inhibitor, into the third ventricle or the hippocampus induces the elevation of glucose or catecholamines in plasma in rats. Under stress conditions, release of acetylcholine in the hippocampus increases, which coincides with the elevation of plasma glucose and catecholamines. Age-related reduction in responsivity of the cholinergic system in the hippocampus has been well documented. The intrahippocampal neostigmine injection induces significantly attenuated responses in plasma glucose and catecholamines in rats, the finding suggested that changes in cholinergic system activity in the hippocampus could result in alteration of the peripheral metabolism of glucose and catecholamines. In Alzheimer's disease (AD), the most common type of dementia, degeneration of the hippocampal cholinergic system is one of the most robust pathological features. Measurement of plasma catecholamines during a fasting state in the groups of AD subjects, vascular dementia subjects, and non-demented control subjects showed significantly lower plasma epinephrine levels in the AD subjects.KeywordsHippocampusAcetylcholineAgingStressInsulinDementia1IntroductionThe close regulation of plasma glucose and catecholamines is essential to maintenance of life. Under acute stress, the plasma levels of glucose and catecholamines become elevated, and these reactions coincide with the activation of several brain regions including the hippocampus. Several studies have reported that acute stress induced acetylcholine (ACh) release in the hippocampus (Gilad, 1987; Imperato et al., 1991; Mizuno and Kimura, 1997). Tajima et al. (1996) also found that restraint stress induced the increase of ACh release in the hippocampus by using microdialysis, noting the elevation of plasma levels of glucose, epinephrine, and norepinephrine in rats.The stress reportedly affects the central nervous system (CNS). Chronic and acute stress has been shown to induce several changes in the CNS (Sapolsky, 1997; McEwen, 1999). Chronic stress has been shown to be associated with the alteration of synaptic terminal structures in the hippocampus (Magarinos et al., 1997). Several lines of evidence indicate that stress and glucocorticoids affect memory performance (Wolkowitz et al., 1997; de Quervain et al., 1998; Kim and Yoon, 1998; Sandi, 1998). Lupien et al. (1998) demonstrated that elevated cortisol, the major stress hormone, produced hippocampal atrophy and memory deficits in the human model. However, it is largely unknown how changes in CNS affect stress responses.Chemical stimulation of the CNS with various substances has been reported to induce hyperglycemia (Iguchi et al., 1984, 1985, 1986, 1988, 1991). These substances include adrenergic, cholinergic, histamine, neuropeptides such as bombesin, thyrotropin-releasing hormone, and 2-deoxyglucose. Iguchi et al. (1988) compared the potency to induce hyperglycemia of several substances, and reported that the muscarinic subtype of cholinergic receptors play an important role in CNS-mediated glucoregulation. Injection of neostigmine, the choline esterase inhibitor, into the third ventricle or hippocampus induces the increase of glucose and catecholamines in plasma, and these peripheral reactions resemble stress responses. Activity in the CNS, including the hippocampus, and particularly in the cholinergic system, must be involved in the regulation of glucose and catecholamines in plasma.Age-related changes in the hippocampal cholinergic system have been studied extensively. The overall findings remain controversial, although, several studies showed that the input of the cholinergic system into the hippocampus was attenuated with aging. We observed attenuated responses in plasma glucose and catecholamines after injection of neostigmine into the hippocampus of aged rats. This finding suggested that age-related changes in hippocampal formation could be associated with peripheral metabolic changes.Alzheimer's disease (AD) is one of the most common types of dementia. Pathologies in the AD brain include the substantial degeneration of several regions, including the cortex and the hippocampus (Jack et al., 1998). Degeneration of the cholinergic system in the hippocampus is one of the earliest and most severe features of AD pathology. Based on findings that strongly suggest the involvement of the hippocampus in the regulation of plasma glucose and catecholamines, we hypothesized that the metabolism of these substances could be altered.2Stimulation of the cholinergic system in CNSIguchi et al. (1986) reported that injection of neostigmine, the ACh esterase inhibitor, into the third ventricle of rats caused a dose-dependent increase of glucose, epinephrine, norepinephrine, and glucagon concentration in plasma (Fig. 1). This effect probably occurred through increase of endogenous ACh in the CNS, given that neostigmine inhibits ACh esterase, which degrades ACh. Neostigmine-induced hyperglycemia was prevented by coadministration of atropine, suggesting that this response was mediated by cholinergic receptors of muscarinic subtype.The mechanism of these reactions has not yet been fully elucidated, however, the hypothesized mechanism is given below. The secretion of epinephrine from the adrenal medulla is under neuronal control. Increase of epinephrine occurs probably via activation of the neurons regulating the adrenal medulla, which is triggered by the signals associated with the activation of the cholinergic system in the hippocampus. Most of the norepinephrine in plasma is spillover from the sympathetic nervous terminals. Increase of norepinephrine after neostigmine injection into the CNS should reflect the activation of the sympathetic nervous system. In the mechanism of neostigmine-induced elevation of plasma glucose, at least four possible pathways were hypothesized. First, the secreted epinephrine may directly act on the hepatic release of glucose. Second, epinephrine may induce the release of glucagon and the suppression of insulin secretion in the pancreas. Third, direct neuronal control in the pancreas causes glucagon secretion. Fourth, direct innervation in the liver activates the glucose release. The relative contribution to induction of hyperglycemia was 22% for glucagon, 29% for epinephrine, and 49% for other factors including direct neuronal innervation in the liver (Fig. 2) (Iguchi et al., 1988).Uemura et al. (1989) studied the involvement of the cholinergic system in the hippocampus in CNS-mediated glucoregulation. Intrahippocampal injection of neostigmine also caused the significant increase of plasma glucose and catecholamines in a dose-dependent manner. Coinjection of atropine into both the hippocampus and the ventromedial hypothalamus suppressed this reaction (Iguchi et al., 1991). This finding suggested that the activation of muscarinic receptors in the hippocampus induced the increase of plasma glucose and catecholamines partly through, at least in, the activation of muscarinic cholinergic receptors in the ventromedial hypothalamus.These findings, taken together, strongly suggested that the hippocampus plays a pivotal role in CNS-mediated regulation of plasma glucose, catecholamines, and other hormones.3Age-associated change in the response of the cholinergic systemAge-related changes in the cholinergic system in the CNS have been well investigated (Decker, 1987). However, data regarding the age-related changes of input of cholinergic neurons into the hippocampus are controversial. Age-related changes in the activity of choline acetyltransferase, the synthetic enzyme of ACh, the content of ACh in the hippocampus, or the number of cholinergic neurons in the basal forebrain, the origin of cholinergic innervation, have been inconsistently found. The responsivity of the cholinergic system in the hippocampus, however, has been consistently suggested to demonstrate age-related changes. ACh synthesis, stimulation-induced release of ACh, and the electrophysiological response of postsynaptic neurons are diminished during the process of aging. Lippa et al. (1980, 1981, 1985) have extensively studied the reduced responsivity of aged hippocampal neurons by using in vivo recording. Other studies using hippocampal slice preparations have reported diminished neuronal responsivity to ACh application (Segal, 1982; Haigler et al., 1985). Mizuno and Kimura (1997) reported attenuated hippocampal ACh release in aged rats by using microdialysis.Based on these findings, we hypothesized that reduced responsivity of hippocampal neurons to ACh could result in changes in the peripheral metabolism. We injected neostigmine into the hippocampus of both aged (24MO) and young (6MO) Fischer 344 rats (n=20 and n=25, respectively), and measured the levels of glucose, epinephrine, and norepineprine in plasma after injection. The results (Fig. 3) indicated attenuated responses of plasma glucose and catecholamines by stimulation of the cholinergic system in the hippocampus of the aged rats (unpublished data).This finding suggested that the age-associated decrease of activity in the hippocampal cholinergic system could result in alteration of the peripheral metabolism.4Metabolism of catecholamines and glucose in plasma in Alzheimer's diseaseThe metabolic alterations in the brain of Alzheimer's disease have been investigated extensively (Meier-Ruge and Bertoni-Freddari, 1996; Yamaguchi et al., 1997), and the alteration of glucose metabolism in the brain is involved in the impairment of cognitive function in this disease. However, metabolic changes in the body characteristic of this disease have been less clear. One of the robust features in Alzheimer's disease is degeneration in the hippocampus, particularly in the cholinergic system. Based on findings indicating that the hippocampal cholinergic system is closely associated with the peripheral metabolism, we hypothesized that impairment of the cholinergic system in Alzheimer's disease could lead to changes in the peripheral metabolism.Several studies have focused on the plasma glucose metabolism in Alzheimer's disease, though, their results are inconsistent. Landin et al. (1993) and Bucht et al. (1983) reported finding lower fasting plasma glucose in subjects with Alzheimer's disease than in non-demented subjects, whose finding contrasted with those in other reports, which failed to confirm this difference (Winograd et al., 1991; Kilander et al., 1993). In some studies, hyperinsulinemia and reduced insulin sensitivity were found in both fasting states and during glucose tolerance testing (Bucht et al., 1983; Fujisawa et al., 1991; Razay and Wilcock 1994; Craft et al., 1998), though, other reports indicated no difference between the level of insulin in AD subjects and that in controls (Winograd et al., 1991; Kilander et al., 1993).Other studies have focused on the level of catecholamines in AD. Findings in these studies are also markedly inconsistent. Elrod et al. (1997) reported that level of norepinephrine in the plasma of DAT subjects increased. Conversely, a study done by Vitiello et al. (1993) reported a trend toward decreased levels of plasma norepinephrine and epinephrine in DAT subjects.We previously investigated the level of plasma catecholamines, glucose, and insulin in fasting state in AD, vascular dementia (VD), and non-demented (ND) female subjects (Umegaki et al., 2000). The three groups in this study were age-matched (AD=82.49±7.77, VD=82.86±5.86, ND=82.95±7.77), and the subjects were non-smokers (Table 1); those who had chronic obstructive pulmonary diseases, diabetes mellitus, neurodegenerative disease, or abnormal thyroid function were excluded. What we found in this study was that AD subjects, though not VD subjects, had significantly lower plasma epinephrine levels than non-demented subjects (AD=18.83±13.11, ND=31.52±29.83pg/ml). The other catecholamines investigated, norepinephrine and dopamine, showed lower levels in AD subjects, although the difference did not reach significance (Fig. 4, Table 2). The VD and AD subjects recruited for this study had similar scores on the Mini-Mental State Examination, which is the most well-established assessment test for cognitive function (Folstein et al., 1975) (Table 1). Consequently, the low plasma epinephrine appears to be specific for AD; not for demented states in general. No differences in fasting plasma glucose and insulin were found among these three groups. Recently, Craft et al. (1998) reported that apolipoprotein E genotypes are closely related to the fasting plasma insulin level. AD subjects who are apolipoprotein E-e4 homozygotes had a normal plasma insulin level whereas AD subjects who are not e4 homozygotes had an elevated plasma insulin level. The apoE4 genotype of the subjects we recruited should be investigated. The mechanism behind the low-plasma epinephrine level in AD subjects remains to be elucidated, although we could find the reason in the impaired hippocampal cholinergic system. The cholinergic system in the hippocampus might regulate the basal tone of epinephrine release from the adrenal medulla as well as epinephrine release during the stimulated state.5ConclusionData from animal studies show that the cholinergic system in the hippocampus is deeply involved in the regulation of plasma glucose and catecholamines. 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Psychiatry621997596600 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A88DF3F6AF324FE2B0D8BE63446457B1FEE862B.txt b/test/dataset/in/resources/corpus/Clean_0A88DF3F6AF324FE2B0D8BE63446457B1FEE862B.txt new file mode 100644 index 0000000..8d68790 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A88DF3F6AF324FE2B0D8BE63446457B1FEE862B.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 6810.1093/gerona/59.1.M68 Journal of Gerontology: Medical Sciences Nutritional Risk Predicts Quality of Life in Elderly Community-Living Canadians Keller Heather H. 1 Østbye Truls 2 Goy Richard 3 1Department of Family Relations and Applied Nutrition 3College of Social and Applied Human Sciences, University of Guelph, Ontario, Canada. 2Department of Community and Family Medicine, Duke University, Durham, North Carolina. 1 2004 59 1 M68 M74 17 1 2003 16 1 2003 The Gerontological Society of America 2004 Background. Although nutrition parameters have been linked to quality of life (QOL), few studies have determined if nutritional risk predicts changes in QOL over time in older adults. Methods. 367 frail older adults were recruited from 23 service agencies in the community. Baseline interview included nutritional risk as measured by SCREEN (Seniors in the Community: Risk Evaluation for Eating and Nutrition), as well as a wide variety of covariates. Participants were contacted every 3 months for 18 months to determine QOL as measured by three questions from the Behavioral Risk Factor Surveillance System (BRFSS), a general whole-life satisfaction question, and a general change in QOL question. “Good physical health days” from the BRFSS was the focus of bivariate and multivariate analyses, adjusting for influential covariates. Results. Seniors with high nutritional risk had fewer good physical health days and whole-life satisfaction at each follow-up point compared with those at low risk. In general, participants reported decreases in general QOL from baseline, with those in the moderate nutritional risk category most likely to report this change. Nutritional risk predicted change in good physical health days over time. Other important covariates include: gender, number of health conditions, perceived health, and age. Conclusions. Nutritional risk is an independent predictor of change in health-related QOL. The results also indicate a relationship between nutrition and the more holistic view of QOL. Evaluation studies of interventions for older adults need to include QOL measures as potential outcomes to further demonstrate the benefits of good nutrition. hwp-legacy-fpage M68 hwp-legacy-dochead RESEARCH ARTICLE LATELY, there has been a shift in the concept of healthy and successful aging from a focus on quantity or years of life to the quality of those years (1,2). For example, in a recent review (3), Drewnowski and Evans compared the goal of the Healthy People 2000 report, increasing life span, to that of the Healthy People 2010 report, increasing overall well-being and quality of life (QOL). This shift has brought to the forefront that a healthy lifestyle, and specifically food intake and physical activity, are important determinants of QOL and well-being (3,4). Although there is much discussion on what constitutes QOL (5–7) and on assertions that healthy eating is related to this outcome (3,4), there has been relatively little research to support this link (3,4,8). In the few studies done in this area, objective nutrition parameters have been associated with well-being scales, and the DETERMINE Checklist has been linked to a general measure of QOL (9,10). As there are potentially many mechanisms by which nutrition can influence QOL (7), it is important to determine the extent of this association and whether nutrition should be a target for intervention. Good nutrition ensures that the body has sufficient nutrients to maintain its function, prevent deficiency and the secondary symptoms of these deficiencies, and ensure that nutrient intake is “in balance” to delay the onset of chronic diseases associated with over-consumption. Good nutrition can therefore promote health and survival even in elderly individuals (11–13). Poor food intake, primarily as a result of decreased dietary energy intake, is common in the older adult (14,15). Although overt undernutrition is believed to be relatively rare (estimated at about 10%) in older community-living adults (16), nutritional risk, which precedes undernutrition, is more common, ranging from 25%–65% depending on the subgroup of elderly people assessed (17–20). The independent relationship between nutritional risk and health-related QOL has only just been demonstrated in multivariate analyses controlling for a wide variety of covariates (21). As QOL can change over time, this begs the question whether nutrition influences the trajectory of change or if it can be an important predictor of change in QOL over time. The purpose of this analysis was to demonstrate whether, and to what extent, nutritional risk, as measured by a valid and reliable screening tool, is an independent predictor of QOL changes over time in frail community-living seniors. Methods Participants To promote efficiency, frail seniors were recruited from agencies providing services to elderly persons (e.g., home care agencies, Meals-on-Wheels programs) in southwestern Ontario communities. To promote diversity (22) in the sample, 25 agencies were approached and, of these, 23 agencies participated, although some agencies dictated and controlled recruitment strategies to ensure confidentiality. Invited elderly people included: a) a sample of approximately 900 senior home care clients randomly chosen from the previous year's client lists (letters inviting clients to participate in the study were mailed by providers); b) Meals-on-Wheels clients (provided a letter of information with their delivered meals); c) all residents of selected supportive housing units (letters of invitation were mailed); d) clients at mobile foot-care clinics (staff provided an information letter to clients) who were aged 65 years or older and functionally dependent; e) clients at congregate dining sites and day-care programs (recruited in person by the coordinator); and f) seniors who were part of a peer visiting program (recruited by case-management staff). A total of 3263 individual information letters or invitations for participation were distributed. To be eligible, participants had to be dependent in at least one instrumental activity of daily living or basic activity of daily living, have adequate cognition to complete the consent form and study procedure, and speak English; 624 seniors volunteered for the study and 395 were eligible to participate. Of the original 395 participants, a small number (n = 28) of seniors did not complete the baseline interview due to illness (n = 11) or deciding that the study was too complex (n = 17); baseline interviews were completed for 367 eligible participants. Baseline Interview Process The study protocol included an interview with 1 of 5 trained research assistants, typically in the senior's home. Interviews were conducted between October 1998 and November 1999. The interview included 108 questions and took on average of 1 hour to complete. Structured, closed-ended questions were chosen from standard interviewer-administered survey schedules such as Statistics Canada (Survey of Veterans' Care Needs 1996/1997, Census 1996, Survey on Ageing and Independence (1985), National Population Health Survey (1996), AHEAD (1993), and the Aging in Manitoba Interview Schedule (1996). Covariates specific to this analysis are included in the appendix. Functional ability was assessed with 16 questions based on the Multidimensional Functional Assessment Questionnaire (23). The interviewer-administered version of SCREEN (Seniors in the Community: Risk Evaluation for Eating and Nutrition) was used to determine nutritional risk (24). This is a valid and reliable 15-item questionnaire with scores ranging from 0 to 60; each item has several possible response options ranging from 0 to 4, with higher scores indicating less risk (24). Compared with a dietitian's clinical judgment in 128 seniors, SCREEN was found to be sensitive (Se) and specific (Sp); sensitivity was higher at the 49/50 cut-point (Se = 94%, Sp = 32%) than the 45/46 cut-point (Se = 81%, Sp = 55%). Test–retest reliability of the self-administered version has also been demonstrated (r = 0.68, ICC [intraclass correlation coefficient] = 0.57) in 247 members of a seniors center (24). For these analyses, nutritional risk was categorized as either “low” (SCREEN score = 50–60), “moderate” (SCREEN score = 46–49), or “high” (SCREEN score ≤ 45). Health-related QOL was measured with several indicators. At baseline, a global “whole-life satisfaction” question was used (“How do you feel about your life as a whole?”) with potential options of “very dissatisfied” to “very satisfied.” This question was dichotomized for analysis (“very satisfied” to “neutral” vs “dissatisfied” to “very dissatisfied”). Questions from the Behavioral Risk Factor Surveillance System (BRFSS) were also used at baseline (25). These questions were chosen as they could be administered over the telephone for the follow-up (26): a) “How many days during the previous 30 days was your physical health not good because of injury or illness?”; b) “How many days during the previous 30 days was your mental health not good because of stress, depression, or problems with emotions?”; and c) “How many days during the previous 30 days did your physical or mental health prevent you from performing your usual activities, such as self-care, work or recreation?” Responses were reverse-coded to provide the number of days when physical health, mental health, or activities were not limited (25) and were referred to as “good” physical health days, and so forth (values were truncated at zero if the resulting number of days was negative). The whole-life satisfaction question and these BRFSS questions were also asked at each follow-up. A final general question was asked about the senior's perception of his or her whole quality of life at each follow-up: “Do you think that your quality of life has changed since you were first interviewed for this study?” (seniors were given a reminder of the date if necessary). If they responded “yes,” they were asked if this was a decrease or an increase in the quality of life that they were experiencing. Follow-Up Interviews Telephone follow-up with the participant or, in the event that the senior could not be reached, a family contact, was used to collect outcome data. Participants were phoned every 3 months by the project coordinator until the study end date, i.e.,18 months after their baseline interview. There were 11 dropouts after the baseline data collection. These participants were compared with those who were followed throughout the study period; there were no significant differences in demographic, health, or nutritional risk variables collected at baseline. Statistical Analyses Although data were available on several health-related QOL variables, the “good physical health days” variable from the BRFSS was believed to be the variable most readily influenced by nutritional risk, and was therefore the primary focus of analyses. Analyses were also completed on “good mental health days” and the combined measure of “no-bed days” as recommended (25), but will not be presented in detail. Bivariate analyses were used to compare nutritional risk by mean number of good physical health days at each time point. Repeated measures analysis using the number of good physical health days for each time point (baseline, 3, 6, 9, 12, 15, and 18 months) using PROC MIXED (SAS Institute, Inc., Cary, NC) assessed whether nutritional risk was an independent covariate of QOL over time. The Residual Maximum Likelihood estimation method was used, and the Type III test for fixed effects is presented with estimated least square means for number of good physical health days. A summary measure, number of good physical health days in 12 months, was also created, by summarizing the answers to this question for the first 12 months of the follow-up period (baseline *1.5 + 3 month *3 + 6 month *3 + 9 month *3 + 12 month *1.5). Although somewhat skewed, residuals were normally distributed and multiple linear regression analyses were conducted using this measure. Significant bivariate associations that did not exhibit collinearity were chosen to demonstrate the independent effect of nutritional risk on number of good physical health days in 12 months. The general whole-life satisfaction question asked at baseline and at each follow-up point is plotted by nutritional risk to demonstrate the change over time by risk group. Finally, bivariate analyses were completed on the senior's report of change in QOL from baseline. The proportion of seniors believing that their QOL changed and the proportion who reported that it had decreased are presented for the three nutritional risk groups (low, moderate, high). SPSS (version 10.0; SPSS, Inc., Chicago, IL) and SAS (version 8.0) were used for all data management and statistical analyses. Results Three hundred sixty-seven seniors completed the baseline assessment. Almost three quarters (73.6%) of respondents were female, and the average age was 79.3 years (SD [standard deviation] 7.9 years; range, 54–100 years). The majority of participants, particularly women, were widowed (58.6% total; 36.1% of men and 66.3% of women) or lived alone (74.1% total; 59.8% men and 79.3% women). Almost half (49.9%) had a high school diploma. Participants on average reported 5.6 health problems, and 46.6% perceived their health to be fair to poor. Twenty-seven of the participants (7.4%) died during the follow-up period and 46 (12.5%) were institutionalized. About one third (31.3%) had SCREEN scores between 50 and 60 and were considered low nutritional risk, 24.3% were moderate risk, and 44.4% were high risk. Good physical health days ranged from 0 to 30 for baseline and follow-up time points. Means ranged from 20.4 to 22.3 (SD 11.1–12.9), although the data were skewed as the medians ranged from 28.5 to 30. The mean “total good physical health days in 12 months” was 262.6 (±107.11), although the median was 307.5. Over half (57%) had fewer than 320 good health days during 12 months. When low/moderate nutritional risk was compared with high risk for the mean number of good physical health days per 12 months, significant associations were noted (p <.05). The mean and standard deviation for each time point were compared between those at low/moderate nutritional risk and those at high risk and are presented in Table 1. The slight increase in the mean number of good health days noted in the high nutritional risk participants over time was probably related to those seniors leaving the study due to death or institutionalization. Table 2 presents the seniors' perception of whether their QOL had changed since baseline. The proportion that reported a change and, of those who changed, reported a decrease, are presented for the three nutritional risk groups. In general, the proportion that reported a change in QOL from baseline increased over time for all nutritional groups, although the greatest change was seen for the moderate nutritional risk group (15.1% at 3 months to 47.8% at 18 months). Of those who reported a change in QOL, more than 50% reported a decrease from baseline, which progressed to approximately 75% by the end of the study follow-up, regardless of nutritional grouping. High-risk seniors who reported a change appeared to have a higher proportion who reported a decrease in QOL at 3 months and consistently throughout the follow-up period, as compared with the other groups. Table 3 presents the bivariate associations between select covariates and number of good physical health days (dichotomized at 320/321). Associations were in the expected direction for all covariates. Those seniors who reported fewer good physical health days had poorer perceived health, more nutritional risk, more pain, poorer vision, were more likely to be female, had poorer total functional ability, more physician visits, and increased use of medications and reported health conditions. Figure 1 presents the unadjusted mean rating for the global whole-life satisfaction question over time with scores ranging from 1 (very dissatisfied) to 5 (very satisfied) for two nutritional risk groups, low/moderate, as compared to high nutritional risk. As can be seen from the graph, those with high nutritional risk had a consistently lower mean rating for this question at baseline and at each follow-up time point as compared to those with low/moderate nutritional risk. For both groups, there was a decrease in the rating of whole life satisfaction over time. Tables 4 and 5 present the multivariate analyses determining if nutritional risk (low/moderate vs high risk) was independently associated with good physical health days. Both analyses identified nutritional risk to be an important and significant covariate. In Table 4, on average, over time, those with high nutritional risk had 2.2 fewer good physical health days per month than their low/moderate risk counterparts. Those who perceived their health to be good to excellent and men also reported a higher number of good physical health days. Age and age-squared were also significant covariates with good physical days decreasing over time with age. [The squared variable was included in this analysis, as it was hypothesized that the relationship between age and QOL would not be linear, when plotted good health days decreased to the age of 81 and then increased; results not shown.] Those with more health conditions also had fewer good physical health days over time. The interpretation of the results from the multiple linear regression analyses noted in Table 5 is similar. Respondents with high nutrition risk had fewer good physical health days over the 12-month follow-up period; ß demonstrates the number of good physical health days per year for each unit change of the covariate, thus, those at high nutritional risk had 31 fewer good physical health days than their low-risk counterparts over the year, or approximately 2.5 days per month. Discussion There has been little work done to examine QOL over time in community-based samples of frail seniors (3). This is the first report to have used a valid screening instrument to determine if nutritional risk was associated with self-reported QOL over time in frail older adults (3). Although a convenience sample was used and this work should be considered preliminary, the results support the contention that nutrition is an important predictor of QOL. Specifically, these analyses have identified that QOL over time, whether measured with a global whole-life satisfaction question or reported good physical health days, declines over a relatively short period (18 months) in frail older adults. Nutritional risk, which is believed to precede poor nutritional health, morbidity, and mortality (27), is a predictor of this decline in QOL (Tables 4 and 5). Although there appears to be a similar pattern to the decline in whole-life satisfaction when level of nutritional risk is considered (Figure 1), those at moderate nutritional risk may have a faster rate of decline than seniors at low or high risk (Table 2). When the concept of health-related QOL is measured by good physical health days, significant covariates in addition to nutritional risk include medical conditions, medications, functional status, self-perceived health, pain, falls, and gender. The few previous studies that have been presented in this area demonstrate an association between nutrition parameters or indicators and QOL (9,10,29,30). There are also a few reports that suggest there is no link (31) between nutrition and health-related QOL, although poor measurement of nutrition parameters may explain the lack of effect (4). The different indicators of QOL used in this analysis (including results not shown on good mental health days and no-bed days) provide support for the idea that nutrition has a multidimensional influence on QOL (3,28), although more research is required to determine how nutrition is integrated into the QOL concept (3). From the results, it appears that there is a strong relationship between nutritional risk and the “physical” portion of QOL. Amarantos and colleagues (11) suggest that this health-related QOL (subjective sense of physical and/or mental well-being) is directly associated with nutritional well-being, as nutrition can influence transport proteins, hormones, muscle mass, and functional ability. Nutritional risk as measured with an Australian version of the DETERMINE Checklist in a large sample of Australian women found risk to be associated with measures of physical and mental health and health service utilization measures (32). Multivariate analyses have found an independent effect of protein undernutrition and obesity on SF-36 scores, and these scores are predictive of death (29). These analyses suggest a direct link between nutrition and health-related QOL. Others have found independent associations between nutrition parameters and health and functional measures (33,34), further corroborating this direct link between nutrition and the physical portion of QOL. Finally, a longitudinal study identified the DETERMINE Checklist to predict functional disability and depression, but not mortality, 1 year later in community-dwelling seniors (35). As food intake is not just the physical act of eating food but also involves sociological and cultural aspects of eating, the broader view of QOL, frequently termed “life satisfaction,” also has the potential to be influenced by nutrition (11,36). It can even be argued that good food and happy meals are an integral part of QOL. Crude analyses of whole-life satisfaction and self-reported change in general QOL over time suggest that these more-holistic measures are also influenced by nutritional risk. These results suggest that nutrition also has the potential to influence QOL as viewed from a psychological and/or interpersonal perspective, in addition to the physical portion of QOL. In a randomized trial of megestrol acetate in institutionalized seniors, improvement in physiological markers for anorexia of aging (appetite, weight) was shown to improve mood, well-being, and an “enjoyment list” (37). The proposed mechanism was improved appetite with use of the drug, improved weight, improved prealbumin, and thus improved sense of well-being and QOL. Breakfast consumption was found to be associated with less depression, less emotional stress, and lower levels of perceived stress in persons aged 20–79 years (38). However, as these data were cross-sectional, it was unclear if skipping meals was predictive of these measures of well-being or whether affect influences eating habits. The potential pathway of positive well-being, affecting what is consumed and thus nutritional status, has been further explored (39). In 107 older women, self-esteem, perceived health, health self-determinism, and demographics were significantly associated with nutrition health-promoting behaviors when data were collected at one point in time (39). Finally, in an attempt to determine the predictive relationship between nutrition and well-being, Balacombe and colleagues (30) measured well-being (Philadelphia Geriatric Center Morale Scale) and body mass index (BMI) in 31 older adults on admission to hospital and 3 months later. A positive linear relationship was found between well-being and BMI at follow-up, suggesting that changes in BMI lead to changes in well-being. However no independent effect was found when other variables were considered, suggesting that BMI does not have a direct influence on well-being. Amarantos and colleagues (11) further suggested that malnutrition can influence the inclusive life satisfaction by contributing to social disability, which may result from physical and mental limitations. Anorexia may contribute to less enjoyment from food intake and lead to other social problems (11). Conclusion Although the mechanisms between nutritional risk, food intake, nutritional status, and QOL will continue to evolve with our greater understanding of these concepts, it is clear from this analysis that there is a predictive aspect of nutritional risk on QOL measures taken over time in frail community-living seniors. The literature and this analysis appear to support a direct link between nutrition and the more physical health-related QOL, but the relationship between nutrition and the broadly defined QOL may be indirect or direct. In either case, improving nutritional well-being should be viewed as an important mechanism for improving QOL in older adults. Future nutrition intervention studies in elderly individuals should include QOL measures as viable outcomes. Appendix: Covariates Used in Analysis Age; sex; number of reported health problems and medications; current perceived health status as compared with others their own age (excellent to poor) and as compared with one's own health 1 year ago (better, same, worse); smoking and alcohol use at least monthly; usually pain free (yes/no); occurrence of at least one fall inside or outside the home in the past 6 months; vision and hearing (excellent to poor); number of social visits in the past week; size of social network and satisfaction with level of social contact (wants more, wants less, wants the same); self-reported frequency of depression (“never” to “all of the time”); satisfaction with life as a whole (“very satisfied” to “very unsatisfied”); and current household income. Functional dependence in basic and instrumental activities of daily living, mobility (stair-climbing, walking 1 block, using mobility aides), and transportation was also assessed with 16 questions based on the Multidimensional Functional Assessment Questionnaire (23); previously been in the Canadian Study of Health and Aging. For each of these activities, total dependence for completion of the task was scored as 0, partial dependence scored as 1, and complete independence scored as 2 (23). Thus, higher scores indicated greater independence; the 7 Activities of Daily Living, 6 Instrumental Activities of Daily Living, and 3 mobility/transportation scores were added together for a Total Function Score (range 0–32). Figure 1. Whole-life satisfaction over time by nutritional risk. SCREEN = Seniors in the Community: Risk Evaluation for Eating and Nutrition Table 1. Average “Good Physical Health Days” by Nutritional Risk Grouping for Each Follow-Up Time Point. Mean “Good Physical Health Days” Period Low/Moderate Nutritional Risk High Nutritional Risk Baseline 24.4 ± 9.7 16.6 ± 13.1 3 months 22.6 ± 11.5 16.4 ± 12.7 6 months 21.7 ± 12.4 18.6 ± 13.3 9 months 23.0 ± 11.2 16.7 ± 13.6 12 months 22.9 ± 11.0 19.9 ± 12.3 15 months 24.0 ± 10.2 20.3 ± 12.2 18 months 25.0 ± 8.8 18.4 ± 13.2 Table 2. Self-Reported Change in General Quality of Life From Baseline by Nutritional Risk. Follow-Up Period Level of Nutritional Risk 3 Months %* (n) 6 Months % (n) 9 Months % (n) 12 Months % (n) 15 Months % (n) 18 Months % (n) Low risk (SCREEN 50–60), n = 115     Changed 19.1 (21) 24.5 (26) 25.2 (26) 24 (23) 33 (31) 30 (27)     Decreased as % of changed 52.4 73.1 70.4 87 80.6 75 Moderate risk (SCREEN 46–49), n = 89     Changed 15.1 (13) 18.8 (15) 27.3 (21) 23.4 (18) 27 (20) 47.8 (33)     Decreased as % of changed 57.1 62.5 90.5 68.4 75 72.7 High risk (SCREEN ≤ 45) n = 163     Changed 32.9 (50) 38.7 (55) 41.5 (54) 37.1 (46) 39.1 (45) 42 (47)     Decreased as % of changed 59.6 73.7 76.4 74.5 74.5 72.3 Notes: *Drop outs and those who left the study (death, institutionalization) for each time point at each level of risk are excluded when calculating percentages. SCREEN = Seniors in the Community: Risk Evaluation for Eating and Nutrition. Table 3. Bivariate Associations With Good Physical Health Days Over the First 12 Months of the Study. Covariate Good Physical Health Days <320 in 12 Months (n = 106) Good Physical Health Days ≥320 in 12 Months (n = 80) Mean ± SD Age 77.4 (7.4) 79.2 (8.6) Number of medical conditions 6.3 (2.6) 4.1 (2.2)*** Number of medications 6.7 (3.9) 4.3 (3.6)*** Hours of all household help in past week 6.5 (10.6) 4.9 (5.1) Number of physician visits in past 6 months 5.2 (8.2) 2.9 (3.9)* Number of nights in hospital in past 6 months 22.5 (30.9) 12.0 (9.2) SCREEN† 44.6 (6.4) 48.9 (5.8)*** Total Function Score‡ (ADL + Mobility + IADL) 19.7 (4.9) 22.3 (5.0)** Proportion Wants more contact with family/friends 41.5 31.3 Good/excellent perceived health 44.3 77.5*** Health better or same as previous year 60.4 86.3*** Nutritional risk (SCREEN ≤ 45) 51.9 22.5*** Pain prevents activities 43.4 10.0*** Fall inside home in past 6 months 30.2 13.8** Gender (male) 21.7 37.5* Good vision 60.4 76.3* Notes: *Significant at p =.05; **significant at p =.01 to.001; ***significant at p <.001. †Possible range: 0 (highest risk)–60 (lowest risk). ‡Possible range: 0 (complete dependence)–32 (complete independence). SD = standard deviation; ADL = Activities of Daily Living; IADL = Instrumental Activities of Daily Living; SCREEN = Seniors in the Community: Risk Evaluation for Eating and Nutrition. Table 4. Predictors of Good Physical Health Days Per Month During Follow-Up: Final Multivariate Model for Repeated Measures Analysis (n = 367). Covariate Coefficient Estimate (Days/Month) Standard Error of Estimate Least Squares Mean (Days) p Value Gender −3.1 0.62 Female: 19.86 Male: 22.96 .0001 Nutritional risk (SCREEN) 2.19 0.61 Low risk: 22.51High risk: 20.31 .0004 Perceived health −4.19 0.63 Poor: 19.33 Good/excellent: 23.49 .0001 Age (per year) −1.36 0.53 N/A .01 Age × Age (per year2) 0.0085 0.0034 N/A .01 Number of health conditions −0.78 0.12 N/A .0001 Notes: −2 Residual Log Likelihood = 12385; Chi2 = 8.44; p =.004; Residual Maximum Likelihood estimation method, nonlinear time. SCREEN = Seniors in the Community: Risk Evaluation for Eating and Nutrition. Table 5. Predictors of Good Physical Health Days Per Year: Final Multiple Linear Regression Model (n = 186). Covariate ß Standard Error Standardized ß p Value Age (per year) −.19 0.85 −0.14 .82 Gender (male) 37.9 14.9 0.16 .01 Nutritional risk (SCREEN ≤45) −31.0 14.5 −.14 .03 Number of health conditions −10.5 2.8 −.26 <.001 Perceived health (good to excellent) 63.34 14.8 0.29 <.001 Notes: R2 =.32; F = 16.9; p <.0001. SCREEN = Seniors in the Community: Risk Evaluation for Eating and Nutrition. The authors thank the senior participants, service provider organizations, interviewers (L. Boudreau, D. Dewaal, P. Vanderkooy, S. Chamberlain, R. Davidson), and the project coordinator (J. McKenzie) for being instrumental to this study. Also, the authors acknowledge the support of M. Pattillo, the Region of Halton, and the Regional Geriatric Program of Central West Ontario. SCREEN (Seniors in the Community: Risk Evaluation for Eating and Nutrition) is a copyrighted program. 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Screening for nutritional risk among community-dwelling elderly on Prince Edward Island. Can J Pub Health.. 1998;89:342-346. 20 Coulston AM, Craig L, Coble Voss A. Meals-on-wheels applicants are a population at risk for poor nutritional status. J Am Diet Assoc.. 1996;96:570-573. 21 Keller HH. Nutrition and health related quality of life in frail older adults. J Nutr Health Aging. In press. 22 Hicks Patrick J, Pruchno RA, Rose MS. Recruiting research participants: a comparison of the costs and effectiveness of five recruitment strategies. Gerontologist.. 1998;38:295-302. 23 Fillenbaum GG. Multidimensional Functional Assessment of Older Adults: the Duke Older Americans Resources and Services Procedures. Hillsdale, New Jersey: L. Erlbaum Associates Publishing; 1988. 24 Keller HH, McKenzie JD, Goy RE. Construct validation and test-retest reliability of the seniors in the community: risk evaluation for eating and nutrition questionnaire. J Gerontol Med Sci.. 2001;56A:M552-M558. 25 CDC. Quality of life as a new public health measures—Behavioral Risk Factor Surveillance System, 1993. Morbid Mortal Wk Rpt.. 1994;43:375-380. 26 CDC. Health-related quality-of-life measures-United States, 1993. Morbid Mortal Wk Rpt.. 1994;44:195-200. 27 Reuben DB, Greendale GA, Harrison GG. Nutrition screening in older persons. J Am Geriatr Soc.. 1995;43:415-425. 28 Padilla GV. The role of nutrition in quality of life. Diet Curr.. 1990;17:5-8. 29 Kalantar-Zadeh K, Kopple JD, Block G, Humphreys MH. Association among SF36 quality of life measures and nutrition, hospitalization, and mortality in hemodialysis. J Am Soc Nephrol.. 2001;12:2797-2806. 30 Balacombe NR, Ferry PG, Saweirs WM. Nutritional status and well-being. Is there a relationship between body mass index and the well-being of older people? Curr Med Res Opin.. 2001;17:1-7. 31 Greenlund KJ, Giles WH, Keenan NL, Croft JB, Mensah GA. Physician advice, patient actions, and health-related quality of life in secondary prevention of stroke through diet and exercise. Stroke.. 2002;33:565-571. 32 Patterson AJ, Young AF, Powers JR, Brown WJ, Byles JE. Relationships between nutrition screening checklists and the health and well-being of older Australian women. Publ Health Nutr.. 2002;5:654-671. 33 Jensen GL, Friedmann JM, Coleman CD, Smiciklas-Wright H. Screening for hospitalization and nutritional risks among community-dwelling older persons. Am J Clin Nutr.. 2001;74:201-205. 34 Jensen GL, Kita K, Fish J, Heydt D, Frey C. Nutrition risk screening characteristics of rural older persons: relation to functional limitations and health care charges. Am J Clin Nutr.. 1997;66:819-828. 35 Boult C, Krinke UB, Flood Urdangarin C, Skarin V. The validity of nutritional status as a marker for future disability and depressive symptoms among high risk older adults. J Am Geriatr Soc.. 1999;47:995-999. 36 Wahlqvist ML, Saviage GS. Interventions aimed at dietary and lifestyle changes to promote healthy aging. Eur J Clin Nutr.. 2000;54:(Suppl 3): S148-S156. 37 Yeh S, Wu SY, Levine DM, Parker TS, Olson JS, Stevens MR, Schuster MW. Quality of life and stimulation of weight gain after treatment with megestrol acetate: correlation between cytokine levels and nutritional status, appetite in geriatric patients with wasting syndrome. J Nutr Health Aging.. 2000;4:246-251. 38 Smith AP. Breakfast and mental health. Int J Food Sci Nutr. 1998;49:397-402. 39 Lucas JA, Orshan SA, Cook F. Determinants of health-promoting behavior among women ages 65 and above living in the community. Sch Inq Nurs Pract. 2000;14:77-100. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A89791A360A8439F0449791867011A5815E637F.txt b/test/dataset/in/resources/corpus/Clean_0A89791A360A8439F0449791867011A5815E637F.txt new file mode 100644 index 0000000..e75d8d4 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A89791A360A8439F0449791867011A5815E637F.txt @@ -0,0 +1 @@ +jleojleorgThe Journal of Law, Economics, & Organization1465-73418756-6222Oxford University Press10.1093/jleo/ewn023Regular ArticlesProfessionals or Politicians: The Uncertain Empirical Case for an Elected Rather than Appointed JudiciaryChoiStephen J.*New York UniversityGulatiG. Mitu**Duke UniversityPosnerEric A.***University of Chicago*New York University, School of Law, 40 Washington Square South, New York, NY 10012, USA. Email: stephen.choi@nyu.edu.**Duke University, School of Law, Room 4026, Science Drive and Towerview Road, Durham, NC 27708, USA. Email: gulati@law.duke.edu.***School of Law, 1111 East 60th Street, Chicago, IL 60637, USA. Email: eposner@uchicago.edu.08201005112008262290336© The Author 2008. Published by Oxford University Press on behalf of Yale University. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org2010Conventional wisdom holds that appointed judges are superior to elected judges because appointed judges are less vulnerable to political pressure. However, there is little empirical evidence for this view. Using a data set of state high court opinions, we construct measures for three aspects of judicial performance: effort, skill, and independence. The measures permit a test of the relationship between performance and the four primary methods of state high court judge selection: partisan election, non-partisan election, merit plan, and appointment. Appointed judges write higher quality opinions than elected judges do, but elected judges write more opinions, and the evidence suggests that the large quantity difference makes up for the small quality difference. In addition, elected judges are not less independent than appointed judges. The results suggest that elected judges focus on providing service to the voters, whereas appointed judges care more about their long-term legacy as creators of precedent.If the state has a problem with judicial impartiality, it is largely one the state brought upon itself by continuing the practice of popularly electing judges.Justice O'Connor, concurring in Republican Party of Minn. v. White, 536 U.S. 765, 792 (2002).1IntroductionJustice O'Connor's backhanded put-down of Minnesota's elected judiciary reflects the conventional wisdom among lawyers and scholars that judges should be appointed by elected officials or independent commissions and should not be elected themselves (Geyh 2003; Tarr 2003). The conventional wisdom reflects a deeply rooted conviction that voters are too unsophisticated to evaluate judges and candidates for judicial office. When judges use campaign contributions to finance simple-minded television commercials, conflict of interest is layered on public confusion. However, this conviction is hardly self-evident. In a system that uses judicial appointments, nothing forces the appointing official to select judges on the basis of their legal ability; cronyism is common. And, as the literature on voting shows, ordinary people use various strategies for evaluating candidates whose qualifications they do not fully understand. For example, they rely on party endorsements and newspaper editorials, and the give-and-take of the campaign.1 And when many people participate in a decision-making process, aggregation of information occurs, which can produce more accurate results than when the decision is made by only one person.The relative merits of appointment and electoral systems are an empirical question, but what exactly should be tested? Empirical work on this topic focuses on judicial independence, the willingness of a judge to vote against the ideological interests of the party of the elected official who appointed her or of the party to which she belongs. However, independence captures only a part of the judge's role. Judges are supposed to be independent but not arbitrary: a judge who votes against her party may still make bad decisions. And an independent judge who is lazy will not resolve many cases or will resolve them poorly. The measures of independence that have been used in the literature imply that the best judicial system would be one in which Democratic judges voted in favor of Republican interests and Republican judges voted in favor of Democratic interests. It is as though empirical studies of central banks focused exclusively on whether central banks made decisions that contradicted the expressed desires of the government and not on whether their decisions were correct, as a matter of monetary policy. Central bank independence is important but a central bank that always decided the opposite of what the government sought would not necessarily be a good one. The same can be said about judges.To test the conventional wisdom that appointed judges are better than elected judges, we use a tripartite definition of judicial quality—productivity, opinion-quality, and independence. Productivity refers to the number of opinions a judge writes in a particular time period such as a year. The more opinions a judge writes, the more disputes she has resolved—and dispute resolution is the chief function of the judge. Opinion-quality refers to the opinion's reasoning. Better-reasoned opinions explain to the parties why they won or lost, but more important, they provide guidance to future judges who face similar cases, and to people and businesses who want to avoid litigation in the first place. And independence refers to the willingness of judges to follow the law rather than the interests of political parties.The independent variable of interest is the selection method for high court judges. In 12 states, judges are appointed by governors (or, in few instances, legislatures). At the opposite extreme, judges in nine states run for election—and reelection—as members of political parties. In between, there are two systems that combine partisan and nonpartisan elements. In 16 states, merit commissions are used: typically, an independent commission provides nominees whom the governor may appoint, whereas a retention election is used at the end of a judge's term (rather than a competitive election). In 13 states, nonpartisan elections are held: the public votes but judges are not permitted to advertise themselves as members of particular political parties.A small empirical literature has investigated the relationship between selection systems and judicial characteristics in the states. As noted, the literature has focused on judicial independence and not other attributes of quality (Cann 2007; McLeod 2007; Shepherd 2007) and suggests that appointed judges are more independent than elected judges. Our tests of independence produce more complicated results and do not favor either system in a clear way. As for overall quality, again the literature assumes that appointed judges are better—albeit, with little attempt to measure quality (Cann 2007).2 We find that elected judges are more productive. And although appointed judges write opinions that are cited more often, the difference is small and outweighed by the productivity difference. In other words, in a given time period, the product of the number of opinions authored and citations-per-opinion is higher for elected judges than for appointed judges.After discussing our results, we attempt an explanation for why elected judges might differ in these ways from appointed judges. Elected judges look like politicians, whereas appointed judges look like professionals. Professionals care about their reputation among a national community of like-minded professionals, whereas politicians care about their reputation in the local community of lay voters and politicians. Appointed judges thus labor to write opinions that will be admired, whereas elected judges try to satisfy as many litigants as possible by dispensing quick but adequate justice. Our evidence does not prove that elected judges are superior to appointed judges, but it casts doubt on the conventional wisdom.2Theory2.1The Determinants of Judicial QualityBeginning with the legal realists, scholars and even judges themselves have speculated about the motives of judges, and whether judges decide cases by applying the law in a neutral fashion or in a manner that reflects personal or political views, or both (e.g., Newman 1984; Cross 2005). Because American judges have the power to strike down laws, the early controversy about judges' motives led to a debate about the proper role of judges in the constitutional system. If judges are ideologically motivated, then their power to strike down laws sits uneasily with democratic commitments; if they are not, or if their ideological motives are constrained, then judicial review has many attractive properties, including possibly the ability to prevent majorities from exploiting minorities or otherwise supporting bad law.3In recent years, this debate has reemerged in the framework of an agency model, which treats the judiciary or individual judges as agents, and the public or particular elected officials as the principals (e.g., Maskin and Tirole 2004). Agency models warn that agents, unless properly selected, monitored, and rewarded, will not act in the interests of principals. In the context of the judiciary, political institutions need to be designed to ensure that people with preferences similar to that of the public are selected to be judges and that judges be given the right incentives to decide cases according to the law.With respect to selection, judges should be impartial and competent, rather than partisan, ideological, eccentric, or incompetent. What selection mechanism will ensure that good, rather than bad, people are selected to be judges and that they will remain good after they become judges? Elections might ensure that people with mainstream views become judges, but the electorate may not be able to evaluate a potential judge's legal ability and other technical qualifications, let alone whether she is misleading the electorate in terms of her future fidelity to what is best for the populace. Appointments might result in competent and politically mainstream judges; however, elected officials might prefer to use judgeships as patronage positions. Unhappiness with these two extremes has led, in many states, to reliance on commissions, combinations of appointments and elections, and other mechanisms.With respect to incentives, judges should, in principle, face sanctions if they decide cases poorly and rewards if they decide cases well. However, if rewards and sanctions are used, someone must apply them, and if that person has political power, judges might be afraid to decide cases impartially. The federal system avoids this risk by giving judges lifetime tenure on good behavior, but the danger with such a system is that it allows judges to decide cases badly or in a partisan fashion, without fear of sanction. Most state systems attempt to constrain judges by forcing them to undergo reelection or a reappointment process, but the danger is that judges will decide cases in partisan fashion so as to avoid a partisan sanction.The empirical literature on judicial behavior has focused primarily on federal judges, and especially on the US Supreme Court (George 1998). One vein of this literature establishes that judges' voting behavior reflects partisan or ideological preferences, at least to some extent. Judges who are Republicans or who are supported, at time of appointment, by conservative media, tend to vote in an ideologically conservative way; a corresponding bias characterizes Democrats (Segal and Spaeth 2002). This work confirms that unconstrained judges do not necessarily decide cases impartially and casts doubt on the assumption that appointment systems are necessarily best. Another vein in the literature shows that structural and institutional features influence and constrain judges' incentives to vote once on the bench. For example, perhaps because of whistleblower or group polarization effects, federal judges vote less ideologically when the panel is split by political party than when it is not (Cross and Tiller 1998; Sunstein et al. 2004).A smaller literature on the state courts has come to similar results. Brace and various coauthors find correlations between voting and ideology, analogous to the federal court studies (e.g., Brace et al. 2001; Brace et al. 2006). Tabarrok and Helland (1999) find that tort awards are higher in electoral states than in nonelectoral states. They argue that their results reflect the stronger incentives of elected judges to redistribute wealth from out-of-state corporations to in-state voters and to please the local trial bar (see also Helland and Tabarrok 2002). Hanssen (1999) finds more litigation in nonelectoral states than in electoral states. He argues that high court judges in nonelectoral states have more independence and therefore are under less pressure to decide cases consistently. Greater uncertainty about the law generates more litigation. Hanssen (2000) finds that state bureaucracies are larger in nonelectoral states than in electoral states, which he attributes to defensive efforts by the agencies to protect themselves from less politically constrained judges. Pinello (1995) finds that appointed judges are more likely to favor criminal defendants than are elected judges. Besley and Payne (2006) find that employment discrimination claims are more numerous in electoral states than in nonelectoral states, which they argue shows that elected judges are more likely to rule in favor of employees than are appointed judges. Berkowitz and Clay (2006) find that the quality of state courts, as measured by surveys of senior attorneys at wealthy companies, is positively correlated with non-partisan judicial retention procedures. Shepherd (2007) focuses on the political party of “retention agents”—those people who decide whether a judge will be retained or not—and finds that judges (of whatever party) are more likely to vote in favor of traditional Republican interests when retention agents are Republicans and are more likely to vote in favor of traditional Democratic interests when retention agents are Democrats. The effect is larger when the retention process is electoral than when it involves reappointment. An early study by Landes and Posner (1980) finds that citations (including federal and out-of-state citations) of state supreme court opinions are uncorrelated with the selection system.The literature, taken as a whole, provides evidence that selection and retention institutions influence judicial outcomes—by influencing who becomes a judge, or how judges decide cases, or both. The literature also confirms that judges are influenced by political factors. The literature largely skirts our topic—whether elected judges are, overall, better or worse than appointed judges—but offers tantalizing hints. The study of Tabarrok and Helland (1999) implies that elected judges are better agents of their constituents than are appointed judges; the social problem they identify is due to the federal structure of the country and the overlapping jurisdictions of states. Hanssen's (1999) finding that electoral pressures force state judges to be more consistent implies that elected judges are better—more consistent opinions, all else equal, are better than inconsistent opinions. Berkowitz and Clay (2006) do use a measure of overall judicial quality based on the views of senior attorneys at wealthy companies, but, as they acknowledge (pp. 412–13), the views of these senior attorneys are hardly unbiased. The study of Landes and Posner (1980) is closest to ours, but they do not look at productivity and independence, and their study has several methodological limitations.4 Our results are quite different from theirs.Following the literature, we envision the relationship between the public and the judiciary as an agency relationship. The optimal selection mechanism minimizes agency costs. Judges expend unobservable effort to decide cases. Agency costs can take two forms: laziness (resulting in bad opinions or few opinions) and ideological self-indulgence (biased opinions). The optimal selection mechanism screens out judges with a strong preference for laziness or ideological self-indulgence and/or punishes judges who are observably lazy or ideologically self-indulgent.Which type of selection mechanism is more likely to perform this function? The advantage of electoral systems is that, in principle, the public can select judges who appear energetic and politically neutral, and it can vote out of office judges whose judicial activity reflects laziness and political bias. The appointments system adds an extra decision-maker layer (typically the state governor), possibly dampening the public's ability to monitor judges. If appointed judges perform badly, the public would need to vote out of office the governor responsible for appointing the judges and reappointing bad judges, but the public would need to take account of other aspects of the governor's performance as well. On the other hand, if governors benefit from a well-functioning judicial system (because the business constituency is happy, for example) and are better able to monitor judges than is the public (because the governor and his or her staff are more sophisticated), then appointed judges might perform better than elected judges. So theory does not clearly point in either direction.We do propose a more specific hypothesis, however. It seems reasonable to assume that in more populous states, the more dispersed public (the principal) would have more difficulty monitoring judges (the agents). The reason is as the number of monitors increases, the incentive to free ride on monitoring (which is a collective good) increases. By contrast, governors in larger states would not have more difficulty monitoring judges (though the public's ability to punish governors for failing to appoint and monitor judges effectively might be blunted). Thus, we predict that elected judges perform better in small states than in large states, whereas there is no or less difference in the performance of appointed judges in large and small states.2.1Judicial Quality and Judicial Selection Mechanisms2.1.1Judicial Quality.We use three measures of judicial quality.Productivity: Judges have some discretion over how many opinions they write. Judges who write slowly will write fewer opinions. Judges who are lazy and wish to avoid writing concurrences and dissents, will also write fewer opinions. We measure a judge's productivity using the total number of opinions she writes during our sample time period (1998–2000), including majority, concurring, and dissenting opinions.Productivity is a measure of effort, but is also a measure of quality because, all else equal, more opinions settle a greater number of legal disputes and resolve more legal issues. However, not all else is equal. A judge who writes more opinions might devote less time to each opinion, so that quality suffers. So productivity is at best only a partial measure of quality.Citations: We assume, consistently with the rapidly expanding literature on judicial citations, that citations are a measure of quality (e.g., Landes et al. 1998; Cross and Lindquist 2006; Lott 2005; Baker 2007). Better opinions are cited more frequently than worse opinions. An opinion is cited frequently because it resolves a legal question or identifies some new legal problem or represents an advance in the law or simply clarifies doctrine. We focus on out-of-state citations because this allows us to control for the possibility that in-state citations reflect local legal customs or conventions. To check for robustness, we further subdivide citations into federal, state, and law review citations.5Independence: Judges have the duty to enforce the law impartially, without regard to the legally irrelevant characteristics of the litigants or the goals of political parties. We thus assume that better judges are more independent. Some studies (e.g., Shepherd 2007) measure independence by the propensity of a judge to vote against interests associated with her party—for example, a Republican judge voting in favor of an employee and against an employer. This measure of independence focuses exclusively on the vote in the case and does not take into account the direction of the opinion's reasoning. It would, for example, code an opinion that decides a particular case against an employee but creates precedent through its reasoning that would assist later employee suits, as Republican. And then there is the question of whether coding a vote against an employee and for the employer, regardless of the facts of the case, gets at the Republican/Democrat distinction at all. To avoid the foregoing pitfalls, we look directly at when judges decide to write opinions against judges of the same or opposite party. We posit that a judge who writes several dissents against majority opinions authored by judges of the same party (or majority opinions against dissents of same party judges) is more independent than a judge who rarely dissents and never against a judge of the same party.Our measure of independence is imperfect, and we address its problems and alternatives subsequently. For now, we note two problems. First, the measure can be distorted by personal animosities. Personal animosities might cause judges to refuse to join opinions as often as they otherwise would (Choi and Gulati 2004). Second, there might be judges who are excessively partisan. These judges, because they view their co-partisan colleagues as not partisan enough, may end up dissenting a lot against the moderate judges from their own party. Such behavior—extremely partisan behavior—may then be interpreted as independence under our measure.2.1.2Selection Mechanisms.State judicial selection mechanisms can be divided into several ways. The literature has not arrived at a consistent methodology, and our approach differs from those of other researchers. Nonetheless, the approaches are roughly consistent, and we test ours for robustness. We divide judicial selection mechanisms into four categories (Table 1).Table 1.Selection SystemsAppointedMerit SelectionNon-Partisan ElectionPartisan ElectionConnecticutAlaskaGeorgiaaAlabamaaDelawareArizonaaIdahoArkansasHawaiiColoradoaKentuckyaIllinoisaMassachusettsaIowaLouisianaaNorth CarolinaaMaineIndianaaMichiganaNew MexicoNew HampshireKansasMinnesotaaPennsylvaniaaNew JerseyaMarylandaMississippiTexasaNew YorkaMissouriaMontanaWest VirginiaRhode IslandNebraskaNorth DakotaVermontOklahomaNevadaSouth CarolinaSouth DakotaOhioaVirginiaaUtahOregonWyomingWashingtonaCaliforniaaWisconsinaFloridaaTennesseeaaIndicates that the state is in the top half of our sample states ranked by population in 1997.Governor or Legislative Appointment: In 12 states, judges are appointed by the governor or (in South Carolina and Virginia) the legislature. Gubernatorial appointments usually require the consent of the upper house of the legislature or the participation of a special commission such as an executive council. In most of these states, judges serve a term (ranging from 6 to 14 years) and then may be reappointed in the same manner. In Massachusetts, New Hampshire, and Rhode Island, judges enjoy lifetime or near-lifetime tenure.Merit Plan: In 16 states, judges are nominated by a non-partisan commission and then appointed by the governor. Judges serve a term and then are subject to a retention election, where they run alone, and voters can either approve another term or vote against them. Terms vary but on the whole are less than those in appointment states.Non-partisan Election: In 13 states, judges run for election. Their political affiliations are not listed on the ballot, and so voters, unless specifically informed, do not know a candidate's political party. These judges serve a term and then may run for reelection. The terms range from 6 to 10 years.Partisan Election: In nine states, judges run for election as a member of a political party. They serve a term in the range of 6–10 years for the most part and then may run for reelection.Readers might be skeptical about whether voters care much about judicial elections and use the elections as an opportunity to reward good judges and punish bad judges. Hall (2001) finds that only 8.3% of state supreme court judges seeking reelection between 1980 and 1994 were defeated. Hall, nonetheless, reports a great deal of variation across time and selection systems. In partisan elections, judges during this period were defeated 18.8% of the time; in 1994, 36% of them were defeated. And judges' electoral success appears to hinge on their ideological similarity to voters. “The fact of the matter … is that supreme court justices face competition, that is, by two of three measures, equivalent if not higher to that for the US House” (Hall 2001: 319). (For further discussion, see Dudley 1997; Aspin 1999; Geyh 2003).We should note that each state has a unique system; the categorizations suppress a great deal of variation. For example, the governor of Massachusetts appoints nominees of a judicial selection commission, whereas the governor of Maine makes appointments subject to confirmation by the Senate. Massachusetts judges serve until the age of 70; Maine judges have 7 year terms, at the end of which they may be reappointed by the governor, again subject to Senate confirmation. These differences have led to different coding practices in the literature, with some authors focusing on retention (e.g., Shepherd 2007) and others on selection procedures (e.g., Besley and Payne 2006); some using only two categories, others using multiple categories, and so forth.Our categorization is similar to that of the other authors. The main concern is that if selection is relatively non-partisan and retention is relatively partisan, our selection variable will be misleading. Fortunately, it appears that the degree of partisanship tends to be the same at selection and retention decisions, and indeed tenure length is negatively correlated with the partisanship of the selection process (see Table 3, below).2.2HypothesisConventional wisdom is that appointed judges are better than elected judges. If so, appointed judges should have higher productivity, citation numbers, and independence. The existing empirical literature provides some support for the hypothesis that appointed judges are more independent. Our analysis adds an analysis of productivity and citation numbers in addition to a new measure of independence to get at the underlying question of whether appointed judges are of higher quality than elected judges.3Data Description3.1The Data SetWe examine the decisions of all the judges of the highest court of every state for the years 1998, 1999, and 2000. The District of Columbia is excluded because of its anomalous position. Two states—Texas and Oklahoma—have two highest courts, one with jurisdiction over civil appeals and the other with jurisdiction over criminal appeals. We, in effect, treat these courts as separate state courts: so we have 52 “states.”Each court has a certain number of seats, but we count judges, rather than seats, so if turnover occurs, a court will have more judges than seats, and, if some seats are left empty, there could be fewer judges than seats. Our data set contains 408 judges, about eight per court. The average judge spent 2.65 of the 3 years in our sample period on the court. And each judge wrote on average about 67 opinions over his or her time in office.We organize the data in three ways. For productivity, we run judge-year level regressions. Each observation is a judge for a particular year; there are 1082 observations—that is, the product of 408 and 2.65. For citations, we run opinion-level regressions to allow us to assess the factors that lead to citations to specific majority opinions. There are 27,596 opinion observations in our data set—consisting of 19,499 majority opinions (70.7%), 5669 dissenting opinions (20.5%), and 2428 concurring opinions (8.8%). For independence, we run judge-level regressions on data pooled from 1998 to 2000, and hence there are 408 observations. We assume that a judge's independence does not change over the 1998–2000 time period and use pooled data to obtain as large a sample as possible of opposing opinions with which to construct our independence measure. In many of our regressions, the actual number of observations is lower as a result of inadequate data for variables of interest.3.2Measures of Judicial Quality3.2.1Productivity.Productivity is measured by total number of opinions written for any given year, including dissents and concurrences (Total Opinions). Our least productive judge wrote two opinions in one year,6 whereas our most productive judge wrote 83 opinions in one year. The mean was 25.6 opinions per year. Table 2 provides productivity data arranged by type of selection system.Table 2.Measures of Judicial PerformanceElection PartisanElection Non-PartisanMeritAppointedProductivity measure    Total Opinions28.629.623.620.9a    Majority Opinions17.619.318.016.7    Dissenting Opinions6.97.73.73.0aQuality measure    Outside Federal Court0.1220.0960.1340.211a    Other State Court0.5050.5520.6310.657a    US Supreme Court0.0060.0060.0090.006    Outside State Citations0.6320.6530.7740.872a    Law Review Citations2.0161.6921.842.273Independence measure    Independence−0.057−0.025−0.030−0.028    Independence_Indicator0.4550.3540.4330.435The productivity measure is the average number of opinions per judge per year in the designated category. Total Opinions includes majority, dissenting, and concurring opinions. The quality measure is the average number of citations per opinion. Outside Federal Court includes all citations from a federal district or circuit court located in a circuit that does not contain the state in question. Other State Court includes all citations from state courts outside of the state in question. US Supreme Court includes all citations from the US Supreme Court. Outside State Citations is the sum of Outside Federal Court + Other State Court + US Supreme Court. All citations are from the LEXIS Shepard's database and are tracked up until January 1, 2007. Law Review Citations are for law reviews as tracked by the LEXIS Shepard's database (until January 1, 2007). Independence is defined as the Opposite_Pool—Opposite_Party. Opposite_Party is the number of opposing opinions written against a judge of the opposite party divided by the number of opposing opinions written against a judge of either the opposite or same party from 1998 to 2000. Opposite Pool is the total number of majority opinions authored by an opposite party judge divided by the total number of majority opinions authored by either an opposite or same party judge from 1998 to 2000. Independence_Indicator is defined as 1 if Independence is greater or equal to zero and 0 otherwise. Only judges for whom we could identify a political party were included in the analysis. We exclude judges from states where all judges in our sample were of the same political party from the analysis (Georgia, Maryland, New Mexico, South Carolina, South Dakota).aThe t-test of difference in means for Election Partisan and Appointed Judge is significant at the <1% level.Table 3.Tenure DataNumber of statesMeanStandard deviationMinimumMaximumPartisan Election96.72.43.511.1Non-Partisan Election147.11.54.69.1Merit Selection1710.04.54.019.1Appointed129.23.15.014.9Total528.53.43.519.1Tenure is defined as the average tenure of high court judges for the state in question, measured as of the spring of 1997 (from Hanssen 1999, table 1).3.2.2Opinion Quality.Our primary quality variable is the number of out-of-state citations to a particular opinion by a particular judge (Outside State Citations). We also look at narrower measures, such as law review citations and outside federal court citations.7The best measure of quality is citation by an outside court—including another state court or outside federal court. Inside state and home federal court citations are driven to large extent by precedent. Looking at only outside citations allows us to examine those citations where judges have greater discretion to pick which opinions to cite. Judges are citing these outside opinions because they are helpful, not because they have precedential force. Because of this discretion, an outside circuit citation represents a better indication of which opinions judges deem of higher quality. Table 2 provides citation data, categorized by selection system.3.2.3Independence.Our independence measure focuses on the tendency of judges to write opinions that disagree with co-partisans when the pool of judges provides opportunities to do so.8 We define an “opposing opinion” as either a majority opinion when a dissent exists or a dissent when a majority exists. We assume that a judge exhibits independence when she writes an opposing opinion against a co-partisan.We obtain a measure of the political party for each judge in our sample. We looked to three sources of information on party membership. First, we searched NEXIS and the Internet (using Google) for any news reports on the political affiliation of each judge. Second, we searched for information on political contributions at the opensecrets.org website.9 We used the political party of the donee candidate as a proxy for the political party of judges who contributed. Where a judge contributed to candidates from more than one political party, we did not use the Opensecrets data to assign a political affiliation to the judge. Third, we used the party of the governor (if any) who appointed the judge as a proxy for the judge's political party. In most of the cases where we had multiple sources of information on political party, the party was consistent across these sources. Where we found no data on the judge's political affiliation or the judge’s affiliation was neither a Democrat nor a Republican (but was instead an Independent), we ignored the judge for purposes of calculating the Independence measure. When our three sources reported different parties, we gave first priority to the party identified through our NEXIS and Internet searches, second to the party identified in the opensecrets.org database, and third to the party of the appointing governor. In our sample, 220 judges were classified as a Democrat and 170 as a Republican (with 16 no data or Independent party judges). Of the 390 judges classified as either a Democrat or Republican, 35 (or 8.97%) had a conflict in our three methods of determining political affiliation.We define Opposite_Party as the number of opposing opinions written, by the judge of interest, against a judge of the opposite party divided by the number of opposing opinions written against a judge of either party from 1998 to 2000. This variable measures propensity to side with co-partisans. Not all opposing opinions are driven by the ideology of the opposing judges. A judge who dissents at random would dissent 70% of the time against an opposite party judge if the background pool of majority opinions consisted of 70% opposite party authored opinions. To take into account the background pool of opinions, we define Opposite Pool as the total number of majority opinions authored by an opposite party judge divided by the total number of majority opinions authored by either an opposite or same party judge (not including the judge in question) from 1998 to 2000.We define Independence as Opposite_Pool minus Opposite_Party. A more negative Independence score corresponds to a judge who writes opposing opinions against opposite party judges more frequently than the background pool of majority opinions authored by opposite party judges. Conversely, a more positive Independence score corresponds to an authoring judge who writes opposing opinions less frequently against opposite party judges compared with the background pool of opinions (and thus more frequently against co-partisans). We treat a more positive Independence score as indicative of a more independent judge.10Table 2 reports summary statistics on our Independence measure. Only judges for whom we could identify a political party were included in the analysis, and only judges from states that had a mixture of judges from different political parties were included.11Two problems may affect our Independence measure. First, consider the extreme case where all judges on a particular state high court are all of the same political party (say all Republican). In this case, our Independence measure will equal zero since Opposite_Party will equal Opposite_Pool (and both will equal zero since there are no Democrat-authored opinions). Our Independence measure in Table 2 excludes judges who come from states with no variation in political party among judges for this reason. But, by the same token, we lose data.Second, even where all judges are not of the same political party in a state, if an imbalance exists, the range of the Independence variable will vary. Consider two Republican judges. One is in a state with 90% of the majority opinions written by Democrats and the other is in a state with 10% of the majority opinions written by Democrats. For the first, Independence can range from −0.1 to +0.9. For the second, Independence can range from −0.9 to +0.1. So the second judge could have a much more negative Independence score than the first judge simply because the range is shifted over.To address these problems, we create a version of the Independence variable that is less dependent on the political makeup of a court. Independence_Indicator is defined as 1 if Independence is greater or equal to zero and 0 otherwise. The indicator variable addresses the range problem but also throws out information: it suggests judges subject to nonpartisan elections are less independent than the other types, who are about the same. The last row of the Independence panel of Table 2 reports on the mean of Independence_Indicator. None of the differences in mean Independence_Indicator levels among the varying selection systems are statistically significant.In our multivariate tests, we also consider the possibility that the act of writing a dissenting opinion (even against opposite-partisan judges) can be a greater display of independence than writing a majority opinion (even against co-partisan dissenters). Judges who write dissents display independence in their willingness to write critically of the opinions of their colleagues.4Multivariate TestsWe estimate a number of multivariate regression models to assess the relationship of several key independent variables, including the judicial selection mechanism, and our measures of judicial quality (used as the dependent variables in our models). We also include a number of common control variables in our models.4.1Selection MechanismsOur main interest is the relationship between the selection mechanism used by states to select and retain judges and our dependent variable measures of judicial productivity, quality, and independence. Table 1 describes the selection systems for the different states. In all the states, the selection mechanism long predates our data pool and thus mitigates concerns about endogeneity—that states adopted new mechanisms in response to changes in judicial quality. Electoral systems can be traced back to the Jacksonian era. The switch to alternative systems generally occurred during the Progressive Era, and the process was more or less complete by the 1970s. Recent changes, with the exception of South Carolina, have been marginal (Hanssen 1999; Besley and Payne 2006, table 1).To check for robustness, we test the importance of judicial tenure data—the average tenure (meaning actual service, not de jure term length) of high court judges by state, as of the spring of 1997 (from Hanssen 1999, table 1) (Tenure). The Tenure data does not come from our data set: we do not have information on the tenure of the judges, many of whom are still in office. We provide summary statistics on the relationship between Tenure and the different judge selection mechanisms in Table 3.Note from Table 3 that mean tenure rises as the involvement of the public in the selection of judges falls. We have two clusters—election systems and low tenure, and appointment/merit plan systems and high tenure.The advantage of the Tenure variable is that it allows us to compare the different selection mechanisms along a common metric: the length of time that a judge expects to remain in office. Only a few appointment states have lifetime tenure, so even in systems where the public lacks the power to select judges, judges may be “punished”: elected officials or commissions unhappy with the performance of judges can refuse to reappoint them or support their reappointment. Thus, tenure indicates the vulnerability of the judge to later retention decisions more directly than do the selection mechanisms. Judges with longer tenure face less retention pressure, whereas judges with shorter tenure face greater retention pressure. Tenure allows us to measure the real effects of the retention mechanisms while allowing us to rely less on the jure rules that might be evaded in local legal practice.However, problems exist with the Tenure variable. If a judge's performance displeases the retention decision maker, the judge may be deprived of an additional term, in which case she ends up with a short tenure. Judges in equilibrium may respond to the threat of a frequent retention decisions by catering to the preferences of the retention decision maker. Thus, a judge with a long tenure could represent either a judge who rarely faces a retention decision or, alternatively, a judge who does face frequent retention decisions but who in equilibrium is adept at satisfying the preferences of the retention decision maker. The strong correlation between Tenure and state selection systems that provide for longer (if not lifetime) tenure leads us to discount this latter possibility. But because of these potential problems with Tenure, we use Tenure only as a robustness check of our selection system results.4.2Control VariablesOur multivariate models of judicial productivity, quality, and independence include a common set of control variables. First, the multivariate models include judge level controls (referred to as “Judge Controls”).We include an indicator variable for whether the judge was the chief judge of the high court (Chief Judge). A judge who is chief judge may have less time to author opinions. The chief judge may also command greater respect and receive greater numbers of citations as a result for her opinions. Alternatively, the chief may be able to assign herself the more important opinions and garner more citations that way (Langer 2003). We include the number of years between 1998 and the year in which the judge received her law degree (Post Law-School Experience) and the number of years the judge has been on the high court (Court Experience). More experienced judges may decide opinions with greater skill, leading to more citations. We include variables for whether a judge retired within 1 year or less (Retirement within 1 year) or in exactly 2 years, 3 years, or 4 years (Retirement in 2 Years; Retirement in 3 years; Retirement in 4 years)—judges who retire soon have little to lose from deciding badly.We also include a number of variables specific to the background of the individual judge. These include the age of the judge (Age), an indicator variable for the whether the judge is female (Female), and an indicator variable for whether the judge's primary experience before becoming a judge was in private practice (Private Practice). We include an indicator variable for whether the judge raised funds relating to election campaign expenditures for the current year (Election Spending). Lastly, we include the PAJID score for each judge as developed by Brace et al. (2000). These scores locate judges on a political continuum from highly conservative (0) to highly liberal (100).It is possible that the selection system may filter out and select for specific types of judges. Including judge-level controls separate from our selection system variables may therefore understate the effect of the selection system variables. On the other hand, the judge-specific variables may have an effect independent from the selection system. For example, those selecting judges may only care marginally about a judge's age. However, the age of a judge may nonetheless directly affect productivity and our other measures of judicial performance. Including age separately allows us to account for this separate influence. We alternatively include and exclude our judge-level controls in our multivariate models.Second, we include court level controls that attempt to capture differences among the state high courts that might account for judicial performance (referred to as “Court Controls”). We include measures for the average high court associate justice salary (Adjusted Associate Justice Salary) and the average partner salary in the state (Adjusted Partner Salary). The salary variables are adjusted for the cost of living for the metro area in which the high court is located in the state. Higher paying states may attract higher quality judges. Perhaps judges work harder if their salary will decline (or not rise much) if they are not retained. We include an indicator variable for whether the judges on the high court remained the same throughout our sample time period from 1998 to 2000 (Stable Court) and the size of the bench during the 1998–2000 period (Number of Active Judges on Bench). We include an indicator variable for whether the judges in a specific court do not face mandatory retirement (No Mandatory Retirement).As a measure of resources available to high court judges, we include the average number of clerks per judge for the 1998–2000 period (Number of Clerks Per Judge) and an indicator variable for whether the clerks are tenured for at least 1 year (Long-Term Clerk). To capture the opportunity cost of being a law clerk, the difference between the average salary of an entering associate at law firm in that state and the law clerk salary is used (Law Clerk Opportunity Cost). Judges may also act differently if facing a high workload, particularly if an intermediate appellate level court does not exist to help with the workload. We include the log of the number of trial cases in the state measured in 1998 (ln(Number of Trial Cases in the State)) and an indicator variable for the presence of an intermediate appellate court (Intermediate Appellate Court). Specific court rules may affect the workload facing judges, thereby affecting the level of judicial output. We include an indicator variable for whether judges face a mandatory publication rule (Mandatory Publication). The variables for the number of clerks, clerk tenure, clerk opportunity cost, the size of the bench, the number of trial cases, and the existence of an intermediate appellate court may all influence a judge's choice to devote time to any specific case.12Finally, to mediate the effects of state-level differences on our results, we include variables relating to the characteristics of each state (referred to as “State Controls”). Differences in overall state population (ln(Population)), gross state product (ln(Gross State Product)), and crime rates (Crime Index) may lead to different mixes of cases and judicial responses to these cases. Likewise, the median age of the population (Median Age of Population) and state median per capita income (State Median Income) as measured in the 2000 US Census may affect the mix of cases and judicial response. Because previous research suggests that state judges are influenced by judges in neighboring states (Harris 1985) and because larger neighboring states might produce different types of cases, we include a variable for the aggregate population of border states (ln(Border Population)). We also include a measure of the age of the state (State Age). Older states have longer judicial traditions and hence possibly a more sophisticated jurisprudence on which modern judges can draw. State Age controls for the possibility that modern judges are cited more often outside of the state just because they can draw on the older and more sophisticated jurisprudence of their particular state. We include the fraction of the population comprised of blacks as obtained from the 2000 Census (Black Population Fraction). Greater racial heterogeneity may produce greater complexity in the mix of cases that go to the state high court and affect a judge's attitudes toward such cases. The background ideology of the citizens of a state may affect the preferences of high court judges.Table 4 contains summary statistics for the control variables.Table 4.Summary StatisticsElection partisanElection non-partisanMeritAppointedJudge controlsAdjusted Associate Justice Salary (1000s)112.661104.014107.78998.085aAdjusted Partner Salary (1000s)226.086200.874213.275183.316aChief Judge0.1450.1410.1730.160Court Experience6.5957.5239.4358.454aPost-Law School Experience29.82030.05431.83032.773aRetirement within ≤1 year0.2500.1370.1220.181cRetirement in 2 years0.0950.0560.0570.076Retirement in 3 years0.0550.0560.0630.059Retirement in 4 years0.0550.0650.0660.067Age57.09057.33758.83959.378aFemale0.2050.3070.1900.298bPrivate Practice0.8400.8660.8570.735aElection Spending0.1150.1470.0770.000aPAJID Score34.19237.87132.19247.849aSummary statistics reported for judge-year level data. See definitions in the Appendix.Court controlsStable Court0.1110.2860.2350.333Number of Active Judges8.8898.2147.2947.250cNo Mandatory Retirement0.2220.2860.2940.250Long-Term Clerk0.5560.5710.5630.250Number of Clerks Per Judge2.3652.1782.0591.958Law Clerk Opportunity Cost34.72123.87128.23234.004Trial Cases in the State (1000s)4106.6991391.6211650.8351612.924cIntermediate Appellate Court0.8890.7860.8820.583Mandatory Publication0.2220.2140.2350.083Total Cases Filed3208.1251506.9231651.4381670.167Total Cases Granted563.143556.400532.909570.400Mandatory Civil Jurisdiction0.2500.3570.3130.583Mandatory Criminal Jurisdiction0.0000.3570.3130.583aSummary statistics reported for state level data. See definitions in the Appdix.State controlsState Age163.111152.643131.765192.833State Population (mill.)8.9564.4685.5724.332cPopulation in Border States (millions)28.71823.28922.24318.164cCrime Index4889.9084773.0465104.3854087.724Median Age of Population35.32235.26434.88836.658cGross State Product (billions)295.829145.766178.841163.272State Median Income (1000s)26.07326.87427.70231.942bBlack Pop. Fraction0.1290.1090.0730.100Citizen Ideology Score47.47743.23438.94661.398aSummary statistics reported for state level data. See definitions in the Appendix.aThe t-test of difference in means for Election Partisan and Appointed Judge is significant at the <1% level.bThe t-test of difference in means for Election Partisan and Appointed Judge is significant at the 5% level.cThe t-test of difference in means for Election Partisan and Appointed Judge is significant at the 10% level.4.3ProductivityOur first multivariate model focuses on judicial productivity. We estimate the following equation on judge year-level data using an ordinary least squares regression with robust standard errors clustered by state:Our model relates the log of the total number of opinions authored by a judge in any given year to three indicator variables for Partisan Election, Non-Partisan Election, and Merit Plan states. The three indicator variables use Appointed states as the baseline. The regression model includes year-level Judge, Court, and State Controls described above (see Appendix for definitions). We introduce the three sets of control variables separately (Models 1–3) and together (Model 4) to assess the effect of the controls on the relationship between the selection system and judicial output of opinions. The model also includes year fixed effects. Table 5 reports results.Table 5.ProductivityIndependent variablesModel 1Model 2Model 3Model 4Election Partisan0.2840.346+0.511*0.616**(1.46)(1.87)(2.32)(3.67)Election Nonpartisan0.2520.1700.533**0.291*(1.49)(0.97)(3.26)(2.03)Merit Plan0.0910.0870.581**0.284+(0.72)(0.58)(3.09)(1.76)Chief Judge−0.176**−0.146**(−3.29)(−2.80)Court Experience0.015*0.012**(2.65)(2.72)Post-Law School Experience0.0040.007(0.56)(1.39)Retirement within ≤1 year−0.280**−0.304**(−3.10)(−4.37)Retirement in 2 years−0.113−0.105(−1.08)(−1.42)Retirement in 3 years−0.110−0.099(−1.18)(−1.55)Retirement in 4 years−0.168*−0.072(−2.01)(−1.00)Age−0.001−0.001(−0.20)(−0.20)Female−0.091−0.032(−1.18)(−0.54)Private Practice−0.014−0.053(−0.17)(−0.63)Election Spending−0.038−0.021(−0.59)(−0.39)PAJID Score0.0010.003+(0.97)(−1.80)Adjusted Associate Justice Salary0.009*0.007(2.23)(1.64)Adjusted Partner Salary−0.001−0.001(−0.99)(−0.88)Stable Court0.366*0.331*(2.32)(2.31)Number of Active Judges0.011−0.030(0.26)(−1.05)No Mandatory Retirement−0.307+−0.249+(−1.88)(−1.98)Long-Term Clerk−0.144−0.053(−1.04)(−0.50)Number of Clerks Per Judge0.0930.042(0.97)(0.53)Law Clerk Opportunity Cost−0.005−0.009*(−1.10)(−2.56)ln(Trial Cases in the State)−0.197*−0.132+(−2.42)(−1.89)Intermediate Appellate Court0.0460.053(0.20)(0.24)Mandatory Publication0.1340.205+(0.75)(1.82)State Age0.004*0.004*(2.06)(2.16)ln(State Population)0.976+1.934**(1.96)(3.50)ln(Population in Border States)−0.211**−0.278**(−3.64)(−4.84)Crime Index0.000*0.000(−2.62)(−1.54)Median Age of Population−0.012−0.035(−0.33)(−0.95)ln(Gross State Product)−1.049+−1.921**(−1.93)(−3.43)State Median Income0.0200.054+(0.70)(1.97)Black Pop. Fraction1.287+0.367(1.94)(0.51)Citizen Ideology Score0.001−0.004(0.28)(−0.64)Constant2.889**3.529**6.714**10.518**(10.38)(7.30)(3.44)(4.60)Year Fixed EffectsYesYesYesYesN101910531074998Adjusted R20.04650.10720.16580.2578Dependent variable is ln(1 + Total Opinions). The t-statistics (in parentheses) are calculated using robust standard errors clustered by state. Variable definitions are in the Appendix.+Coefficient significant at the 10% level or less.*Coefficient significant at the 5% level or less.**Coefficient significant at less than the 1% level.The results in Table 5 contradict the hypothesis that judges subject to more partisan pressure are less productive; the opposite is the case. The coefficients on Election Partisan are positive and significant from the 10% to <1% levels in three of the four models. The results are less strong for Election Non-Partisan and Merit judges. In the models with only Judge (Model 1) and Court Controls (Model 2), the coefficients on Election Non-Partisan and Merit are positive but not significantly different from zero. In the models with State Controls alone (Model 3) and State Controls together with Judge and Court Controls (Model 4), the coefficients on Election Partisan, Election Non-Partisan and Merit are all positive and significant. In Model 4 (with all control variables), Partisan-elected judges are the most productive, followed by merit plan and nonpartisan judges. Appointed judges (the omitted, baseline case) are the least productive.13Our control variables largely make sense and are interesting in their own right. More highly paid judges are more productive, taking into account the opportunity cost of forgoing a practicing lawyer's salary—though the magnitude is small. Judges on a stable court are more productive, probably because collegial norms are stronger on stable courts. Chief judges are less productive, perhaps because they have administrative duties. Judges with more experience on the court are more productive; but judges become less productive as they approach retirement (particularly in the last year before retirement). Judges from states with a large population are more productive because they have more cases to decide, and, because of the greater activity and diversity of the population, more law to make (but with statistical significance in only one of two models). The coefficients on the presence of an intermediate appellate court and the crime index variables are either statistically insignificant or small in magnitude.14Why are judges subject to electoral pressures more productive? Perhaps productivity is used by voters as a signal of judicial competence, or by intermediaries, such as newspaper editorialists, bar associations, and parties. Judges who write few opinions, then, will be vulnerable in reelection campaigns.15 This would also explain why productivity declines as retirement nears, as reported in both Models 2 (Judge Controls only) and 4 (with all control variables).If elected judges respond to electoral pressures through increased productivity, one might predict that productivity may vary with the size of the state. The public in large states has more trouble monitoring judges; hence these judges would be less productive. To test the effect of state size, we start with Model 4 of Table 5 and include an indicator variable for whether the state is in the top half of states in terms of population size (Large State). We also include interaction terms between Large State and Election Partisan, Election Non-Partisan, and Merit Plan. Model 1 of Table 6 below reports our results.Table 6.Variations on Productivity ModelModel 1Model 2Model 3Model 4Dependent variableln(1+Total Opinions)In(1+Total Opinions in Low Salience Areas)ln(1+Total Opinions in High Salience Areas)ln(1+Total Opinions)Independent variablesElection Partisan1.219**0.600**0.665**(4.930)(2.890)(3.780)Election Non-Partisan0.738**0.332*0.099(3.180)(2.110)(0.510)Merit Plan0.651*0.331+0.059(2.240)(1.770)(0.310)Election Partisan × Large State−0.800*(−2.600)Election Non-Partisan × Large State−0.601+(−2.000)Merit Plan × Large State−0.461(−1.100)Large State0.181(0.540)Tenure−0.032+(−1.930)Constant8.975**10.059**9.310**10.124**(3.850)(3.730)(3.250)(4.640)Judge ControlsYesYesYesYesCourt ControlsYesYesYesYesState ControlsYesYesYesYesYear Fixed EffectsYesYesYesYesN998994934998Adjusted R20.28480.25750.19220.2416The t-statistics (in parentheses) are calculated using robust standard errors clustered by state. Variable definitions are in the Appendix.+Coefficient significant at the 10% level or less.*Coefficient significant at the 5% level or less.**Coefficient significant at less than the 1% level.Model 1 of Table 6 reports that the coefficient on Large State is not significantly different from zero. In contrast, the coefficients on the interaction terms between Large State and Election Partisan and Election Non-Partisan are negative and significant at the 5% and 10% levels, respectively. The sum of Large State + Election Non-Partisan × Large State is negative and significant at the 10% level; the sum of Large State + Election Partisan × Large State is negative and significant at the 5% level. Although judges from Election Non-Partisan and Election Partisan states are generally more productive, evidence exists that the amount of increased productivity diminishes in states with a large population—particularly for Election Partisan judges (but still remains positive compared with judges from Appointed states).16Production of published opinions may also vary with the public salience of the subject matter of the opinion. For hot button issues, judges may have more (or less) inclination to produce a published opinion. Judges in Election states, for example, may wish to shy away from publishing in controversial areas (such as capital punishment) to avoid angering constituents. On the other hand, judges eager to make a name for themselves may gravitate to publishing in more salient areas. To test the importance of public salience, we again start with Model 4 of Table 5 and estimate the model separately using as the dependent variable, alternatively, the log of the total number of opinions dealing with Low Salience and High Salience subject matter areas.As detailed in the Appendix, we classify opinions into 12 subject categories. The high salience categories are taken from Choi and Gulati (2008) and include Administrative Law, Capital Punishment, Church and State, First Amendment, and Property Rights cases. Following Epstein and Segal (2000), Choi and Gulati count news stories relating to the US Supreme Court on the front page of the New York Times to determine which subject categories are of public salience. Although the methodology uses the US Supreme Court, we assume that the general public will find the salient subject matters similarly high profile in the state court context. Models 2 and 3 of Table 6 report results.Judges in Election Partisan, Election Non-Partisan, and Merit Plan states are all more productive compared with Appointed judges for low salience opinions. However, only Election Partisan judges are more productive than Appointed Judges for high salience opinions. Election Partisan judges perhaps use high salience opinions to signal their political credentials to voters in the state. Appointed Judges and Election Non-Partisan judges do not have similar incentives.We next replace the three indicator variables for the state judge selection system with Tenure, our alternative measure of the pressure facing judges, focusing on the retention decision. Model 4 of Table 6 reports that the coefficient on Tenure is negative and significant at the 10% level. Judges who face frequent retention decisions (and presumably experience a shorter expected tenure) are more productive than judges who have the luxury of a longer expected tenure. These results are consistent with the results of the other productivity models above.4.4CitationsFor outside citations, we estimate the following equation for each majority opinion using ordinary least squares and robust standard errors clustered by judge:The model relates the number of outside state citations (Outside State Citations) for any specific majority opinion with three indicator variables for Election Partisan, Election Non-Partisan, and Merit Plan states. The three indicator variables use Appointed states as the baseline. The model includes the number of dissents written against the majority opinion in question (Number of Dissents). A majority opinion with one or more dissents may deal with more novel issues of law and generate more citations as a result. The model includes the number of west key pages (West Key Pages) as a rough measure of the legal importance of the opinion. Similarly the model includes the length of the opinion (Opinion Length); longer opinions are more likely to contain analysis that other judges may cite compared with shorter opinions, all other things being equal. We also include subject matter fixed effects for the 12 different subject matter categories, including Administrative, Attorney and Client, Capital Punishment, Church and State, Commercial, Criminal, Family, First Amendment, Labor, Property, Rights, and Torts (see Appendix for definitions). We use Other opinions as the baseline subject matter category.We include Judge, Court, and State Controls, as described earlier. We introduce the three set of control variables separately (Models 1–3) and together (Model 4) to assess the affect of the controls on the relationship between the selection system and judicial output of opinions. We also use year fixed effects. Table 7 reports the results from our multivariate outside citation model.Table 7.CitationsIndependent variablesModel 1Model 2Model 3Model 4Election Partisan−0.073*−0.084*−0.076+−0.040(−2.29)(−2.62)(−1.95)(−1.34)Election Non-Partisan−0.069−0.008−0.0500.008(−1.67)(−0.21)(−1.28)(0.28)Merit Plan−0.038−0.023−0.075−0.056(−1.23)(−0.73)(−1.66)(−1.66)Number of Dissents0.078**0.065**0.070**0.060**(4.88)(5.99)(6.15)(6.34)West Key Pages0.021*0.032**0.015*0.023**(2.53)(3.40)(2.11)(3.26)Opinion Length0.032**0.032**0.033**0.032**(11.26)(11.54)(12.65)(11.60)Constant0.056−0.171−1.646**−2.390**(0.76)(−1.55)(−3.12)(−5.77)Subject Matter CategoriesYesYesYesYesJudge ControlsYesNoNoYesCourt ControlsNoYesNoYesState ControlsNoNoYesYesYear Fixed EffectsYesYesYesYesN18,62319,15919,46118,321R20.12490.12570.13860.1341Dependent variable is ln(1+Outside Citations). The t-statistics (in parentheses) are calculated using robust standard errors clustered by state. Variable definitions are in the Appendix.+Coefficient significant at the 10% level or less.*Coefficient significant at the 5% level or less.**Coefficient significant at less than the 1% level.Our results are largely consistent with the hypothesis that judges subject to less partisan pressure write higher quality—more frequently cited—opinions. In the first three models, the coefficient on Election Partisan is negative and significant (ranging from the 5% to 10% level); the coefficient on Election Partisan is negative but insignificant in Model 4 however. The coefficients on Election Non-Partisan and Merit Plan are negative but not significantly different from zero across all of the models. Overall, and particularly compared with Election Partisan judges, the models are consistent with the view that Appointed judges write the best opinions. Also, majority opinions with dissents are cited more frequently, perhaps because the presence of a dissent indicates that the majority opinion makes new law. This reason might also explain why longer opinions are cited more often.17Why would appointed judges write better opinions than elected judges? One possibility is that the quality of an opinion, unlike the number of opinions written, is not observable to the public, so elected judges do not have a strong incentive to write high-quality opinions. With less pressure to produce, appointed judges might prefer to advance their influence and professional reputation by writing good opinions. Another possibility is that a system that selects for judges skilled at electioneering and politicking does not also necessarily select for judges skilled at authoring high-quality legal opinions.18To test the importance of large states, we start with Model 4 of Table 7 and include the Large State indicator variable as well as interaction terms between Large State and Election Partisan, Election Non-Partisan, and Merit Plan. Model 1 of Table 8 reports the results.Table 8.Variations on Citation ModelModel 1Model 2Model 3Model 4Dependent variableln(1+Outside Citations)ln(1+Outside Citations) for Low Salience Opinionsln(1+Outside Citations) for High Salience Opinionsln(1+Outside Citations)Independent variablesElection Partisan0.013−0.070*−0.029(0.26)(−2.15)(−0.65)Election Non-Partisan−0.013−0.0110.010(−0.26)(−0.34)(0.23)Merit Plan−0.090−0.067+−0.045(−1.48)(−1.74)(−1.19)Election Partisan × Large State−0.103*(−2.04)Election Non-Partisan × Large State−0.011(−0.19)Merit Plan × Large State0.035(0.43)Large State0.034(0.54)Tenure0.000(−0.12)Number of Dissents0.061**0.063**0.056+0.063**(6.46)(6.59)(1.98)(6.43)West Key Pages0.023**0.0090.069**0.023**(3.23)(1.31)(3.32)(3.17)Opinion Length0.032**0.034**0.016**0.031**(11.72)(11.02)(5.03)(11.99)Constant−2.279**−2.611**−0.197−2.041**(−5.04)(−5.89)(−0.27)(−4.61)Subject Matter CategoriesYesNoNoYesJudge ControlsYesYesYesYesCourt ControlsYesYesYesYesState ControlsYesYesYesYesYear Fixed EffectsYesYesYesYesN18,32116,996132518,321Adjusted R20.13740.11830.12160.1327Dependent variable is ln(1+Outside Citations). The t-statistics (in parentheses) are calculated using robust standard errors clustered by judge. Variable definitions are in the Appendix.+Coefficient significant at the 10% level or less.*Coefficient significant at the 5% level or less.**Coefficient significant at the 1% level.The most dramatic difference in the model with Large State and the interaction terms with Large State is the result for the citations to Election Partisan judges. The coefficient on Election Partisan is positive and not significantly different from zero. Opinions authored by judges from smaller states with partisan elections receive a similar number of citations to that of appointed judges. The relationship changes, however, for larger states. The coefficient on Election Partisan × Large State is negative and significant at the 5% level. Moreover, the sum of Large State + Election Partisan × Large State is negative and significantly different from zero at the 10% level. Outside state citations diminish for opinions written by Election Partisan judges in larger states compared with smaller states. Perhaps because judges in larger states cannot be as effectively monitored by electorates than judges in smaller states, their opinion quality is lower in larger states.To test the importance of public salience on opinion quality, we start with Model 4 of Table 7 and estimate the model solely for opinions in a low salience subject matter area and solely for opinions in a high salience area. Model 2 (low salience) and Model 3 (high salience) of Table 8 reports results. Model 2 reports that the coefficients for Election Partisan and Merit Plan are negative and significant at the 5% and 10% levels, respectively, for low salience opinions; the coefficient on Election Non-Partisan is not significantly different from zero. In contrast, the coefficients on Election Partisan, Election Non-Partisan, and Merit Plan are not significantly different from zero in Model 3 for high salience opinions. Judges from Election Partisan and Merit Plan states write lower quality opinions primarily for low salience subject matter areas.We lastly replace the three indicator variables for the state judge selection system with Tenure, our alternative measure of the pressure facing judges, focusing on the retention decision. Model 4 of Table 8 reports that the coefficient on Tenure is not significantly different from zero. Unlike our productivity model, we do not find evidence that judges that face more retention pressure differ from judges with less retention pressure in the quality of opinions produced.4.5Independence4.5.1Propensity to Dissent.To assess what factors correlate with a high propensity to write dissenting opinions, we estimate the following equation on judge-year level data using ordinary least squares and robust standard errors clustered by state:The regression model relates the number of dissenting opinions written in a given year (ln(1+Total Dissents)) with three indicator variables for Partisan Election, Non-Partisan Election, and Merit Plan states. The indicator variables use Appointed states as the baseline. The model includes year-level versions of the State Controls and Judge Controls as well as year fixed effects. Table 9 reports the model.Table 9.DissentsModel 1Model 2Dependent variableln(1+Total Dissents)ln(1+Total Dissents)Independent variablesElection Partisan0.927**(3.90)Election Non-Partisan0.769**(4.51)Merit Plan0.244(1.17)Tenure−0.056**(−3.03)Constant4.0054.942(1.31)(1.66)Year Fixed EffectsYesYesCourt ControlsYesYesState ControlsYesYesN998998Adjusted R20.22030.1835Dependent variable is ln(1+Total Dissents). The t-statistics (in parentheses) are calculated using robust standard errors clustered by state. Variable definitions are in the Appendix.+Coefficient significant at the 10% level or less.*Coefficient significant at the 5% level or less.**Coefficient significant at less than the 1% level.Model 1 of Table 9 indicates that elected judges, especially partisan-elected judges, dissent the most, with merit-plan judges in the middle. Appointed judges write the fewest dissenting opinions.Model 2 of Table 9 reports the results of our robustness test using Tenure as an alternative specification of how judges differ across the states with a focus on the retention decision. Note that the coefficient on Tenure in Model 2 is negative and significant at the <1% level. Judges who face the possibility of a short tenure (as in elected states) write more dissents.If dissenting indicates a willingness to express one's honest view, then our results suggest that elected judges are more independent. 194.5.2Propensity to Write Opinions against Co-Partisans (Independence).Given the shortcomings of focusing solely on the number of dissenting opinions as a measure of independence, we turn to our Independence measure. We estimate the following equation on pooled data from 1998 to 2000 using an ordinary least squares regression model with robust standard errors clustered by state:The regression model relates our Independence measure (Independence) based on pooled 1998 to 2000 data for each judge with three indicator variables for Non-Partisan Election, Partisan Election, and Merit Plan states. The three variables use Appointed states as the baseline. As a control for the subject matter composition of the pool of opinions, for each of the 12 subject matter categories (see Appendix for definitions) we compute the number of majority opinions that deal with the specific subject matter divided by the total number of majority opinions for the state in the 1998 to 2000 time period. We include this ratio for each subject matter in the model as pooled controls for subject matter in the state. The model includes pooled data version of the Judge, Court, and State controls as described above. We introduce the three set of control variables separately (Models 1–3) and together (Model 4) to assess the affect of the controls on the relationship between the selection system and judicial output of opinions. Table 10 reports results.Table 10.IndependenceIndependent variablesModel 1Model 2Model 3Model 4Election Partisan0.0100.023−0.075−0.043(0.29)(0.64)(−1.16)(−0.59)Election Non-Partisan−0.044−0.060+−0.127+−0.150(−1.34)(−1.85)(−1.85)(−1.60)Merit Plan−0.052−0.068*−0.147*−0.154+(−1.40)(−2.19)(−2.08)(−1.70)Constant−0.1971.360*1.639+1.882(−0.40)(2.34)(1.95)(1.49)Subject Matter CategoriesYesYesYesYesJudge ControlsYesNoNoYesCourt ControlsNoYesNoYesState ControlsNoNoYesYesYear Fixed EffectsYesYesYesYesN331345352324Adjusted R20.08780.09660.05290.1191Dependent variable is Independence. The t-statistics (in parentheses) are calculated using robust standard errors clustered by state. We exclude judges from states where all judges in our sample were of the same political party from the analysis (Georgia, Maryland, New Mexico, South Carolina, South Dakota). Variable definitions are in the Appendix.+Coefficient significant at the 10% level or less.*Coefficient significant at the 5% level or less.**Coefficient significant at less than the 1% level.In all four models of Table 10, the coefficient on Election Partisan is not significantly different from zero. Moreover the magnitude is small. Our Independence measure ranges from −0.804 to 0.737 for the sample judges. The coefficient for Election Partisan in Model 4 is equal to −0.043, representing only 2.8% of the range between the sample minimum and maximum. To understand this figure, imagine that an elected judge and an appointed judge sit on benches that are otherwise identical in terms of partisan composition: the elected judge is 2.8 percentage points more likely to disagree with an opposite party judge than an appointed judge is. Table 10 suggests that judges from Election Partisan states are not any less (or more) independent than judges from Appointed states.In contrast, the story is mixed for Election Non-Partisan and Merit Plan judges. In the Court control–only model (Model 2), the State control–only model (Model 3) and the model with Judge, Court, and State controls (Model 4), the coefficients on Election Non-Partisan and Merit Plan are negative and significant at levels ranging from 5% to 10% (in one case falling a bit short of the 10% level). Appointed judges appear more independent than judges from these other two types of states. The magnitude of the difference in Independence between Appointed and Election Non-Partisan and Merit Plan states is relatively large (compared with the difference between Appointed and Election Partisan judges). The coefficient for Election Non-Partisan is equal to −0.150 and the coefficient for Merit Plan is equal to −0.154 in Model 4, representing 9.7% and 10.0%, respectively, of the range between the sample minimum and maximum Independence scores.We consider the possibility that the act of writing a dissenting opinion (even against opposite partisan judges) can be a greater display of independence than writing a majority opinion (even against co-partisan dissenters). Judges who write dissents display independence in their willingness to write critically of the opinions of their colleagues. To see if those who dissent more frequently are also more independent, we add the total number of dissents written in our sample period for each judge (Total Dissents) and interaction terms between Total Dissents and Election Partisan, Election Non-Partisan, and Merit Plan to Model 4 of Table 10. We report the results in Table 11.Table 11.Variations on the Independence ModelModel 1Model 2Model 3Model 4Dependent variableIndependenceIndependenceIndependenceIndep_IndicatorIndependent variablesElection Partisan−0.031−0.0220.256**−0.710(−0.39)(−0.27)(3.08)(−0.76)Election Non-Partisan−0.148−0.1220.026−2.656**(−1.58)(−1.32)(0.34)(−2.59)Merit Plan−0.151−0.167+0.104−2.848*(−1.59)(−1.95)(1.43)(−2.56)Election Partisan × Total Dissents−0.004+(−1.85)Election Non-Partisan × Total−0.003Dissents(0.88)Merit Plan × Total Dissents−0.002(−0.88)Total Dissents0.004**(5.08)Election Partisan × Opensecrets−0.167+(−2.01)Election Non-Partisan × Opensecrets−0.194*(−2.15)Merit Plan × Opensecrets−0.123(−1.35)Opensecrets0.087(1.26)Election Partisan × Large State−0.177*(−2.71)Election Non-Partisan × Large State0.063(1.16)Merit Plan × Large State−0.103(−1.39)Large State−0.029(−0.49)Constant1.8621.8402.130+44.035**(1.33)(1.48)(1.77)(3.04)Subject Matter CategoriesYesYesYesYesJudge ControlsYesYesYesYesCourt ControlsYesYesYesYesState ControlsYesYesYesYesYear Fixed EffectsYesYesYesYesN324324324309Adjusted or Pseudo R20.11330.13540.13140.1942The t-statistics (in parentheses) are calculated using robust standard errors clustered by state. We exclude judges from states where all judges in our sample were of the same political party from the analysis (Georgia, Maryland, New Mexico, South Carolina, South Dakota). Variable definitions are in the Appendix.+Coefficient significant at the 10% level or less.*Coefficient significant at the 5% level or less.**Coefficient significant at less than the 1% level.Model 1 of Table 11 reports that the coefficients on Election Partisan, Election Non-Partisan, and Merit Plan are insignificant. The coefficient on Total Dissents is positive and significant at the <1% level. Those judges who write more dissents are also more independent under our Independence measure. In contrast, the coefficient on the interaction term between Election Partisan and Total Dissents is negative and significant at the 10% level. The sum of Total Dissents and Election Partisan × Total Dissents is also not significantly different from zero. Although judges who write more dissenting opinions generally receive a higher Independence score (particularly for Appointed State judges), this relationship does not hold true for judges in Election Partisan states.We posit that more ideologically intense judges will tend to act less independently when faced with the possibility of an election. To test this, we add an indicator variable for whether the judge contributed to a political candidate as tracked in the Opensecrets database (Opensecrets) as a proxy for political intensity to Model 4 of Table 10. We also add interaction terms between Open Secrets and the three indicator variables for the state selection systems. Model 2 of Table 11 reports that the interaction terms between Opensecrets and Election Partisan and Election Non-Partisan are both negative (indicating less independence) and significant at the 10% and 5% levels, respectively. Although more intensely political judges do not correlate significantly with a decreased independence score for Appointed and Merit Plan judges, we see a negative relationship between political intensity and independence for the elected judges.To test the importance of large states, we start with Model 4 of Table 10 and include the Large State indicator variable as well as interaction terms between Large State and Election Partisan, Election Non-Partisan, and Merit Plan. Model 3 of Table 11 reports the results. Now the coefficient on Election Partisan is positive and significant at the <1% level. For smaller states, judges from Election Partisan states are more independent than from Appointed states. This relationship changes however for larger states. The coefficient on Election Partisan × Large State is negative and significant at the 5% level. Moreover, the sum of the coefficients on Large State + Election Partisan × Large State is negative and significant at the <1% level. Judges from larger Election Partisan states are less independent than judges from smaller Election Partisan states. This result is consistent with our hypothesis that electorates in larger states might have more trouble monitoring judges than electorates in smaller states.20 In contrast, the coefficient on Large State alone is not significantly different from zero. Appointed judges in larger states are not more (or less) independent than appointed judges in smaller states.Our results are complicated and difficult to summarize, but our overall sense is that elected judges are more likely to dissent (suggesting more independence), and the two types of judges are otherwise roughly equally like to write against co-partisans (suggesting equal independence). Strongly partisan judges act in a more partisan way in electoral systems than in appointment systems, but it is not clear why this matters if independence for overall judicial activity is the same. What is clear is that the conventional wisdom that appointed judges are more independent than elected judges is a simplification and probably an exaggeration.4.6Productivity versus QualityGiven the ambiguity of our independence results, what's more important: productivity or quality? As a measure of the overall influence of a judge's opinions, we calculated the aggregate number of outside citations to the opinions written by each judge in 1 year (Aggregate Outside Citations). The average judge in a Partisan Election system in 1 year writes opinions cited (outside of both the state and home federal circuit) in the aggregate 11.3 times, whereas the average judge in an Appointed system writes opinions cited in the aggregate 15.0 times (difference significant at the <1% level). The numbers for Non-Partisan Election systems and Merit selection systems are 12.6 and 14.0, respectively. At a summary statistic level, the influence of appointed judges is significantly greater than that of elected judges despite the lower productivity on the part of appointed judges.To provide a multivariate test controlling for state and judge characteristics, we reestimate our judge productivity model (Model 4 of Table 5) on judge-year level data, replacing ln(1+Total Opinions) with ln(1+Aggregate Outside Citations) as the dependent variable. Unreported, the coefficient on Election Partisan is positive and significant at the <1% level, using Appointed as the base case. Judges from Election Partisan states have significantly higher levels of aggregate outside citations than do Appointed judges. The coefficients on Partisan Non-Election and Merit Plan are also positive and significant at the 5% level. In contrast with the summary statistic comparison, the multivariate test supports the view that the lack of productivity on the part of appointed judges diminishes the overall influence and quality of their total judicial output of opinions relative to Election Partisan judges. The differing results between the summary statistic comparison and the multivariate model at the very least call into question the conventional wisdom that appointed judges are superior to elected judges.We reestimate the multivariate model adding the Large State indicator variable and interaction terms between Large State and the judge selection system variables. Unreported, the coefficient on Large State is positive and significant at the <1% level. The sum of Large State and Large State × Partisan Election however is negative and significant at the <1% level.21 Large States have better overall judicial performance but this relationship reverses for Partisan Election states: judges in larger Partisan Election states perform more poorly than judges in smaller Partisan Election states. The reduction in performance for judges in larger Election Partisan states is consistent with our hypothesis that the public is less able to monitor judges through elections in large states.Our results highlight a problem with earlier studies that used only a single measure of judicial quality (such as judicial independence): if judicial quality is multidimensional, as it surely is, judges might choose to maximize along one dimension rather than another, depending on the incentives that they face. A study might show that “quality” is correlated with some institutional factor such as selection mechanism when, in fact, only a particular dimension of quality is correlated and overall quality is not. We return to this problem in Section 5.5Explaining the ResultsWe summarize our results in Table 12.Table 12.Selection Mechanisms and QualitySelection MechanismTenureProductivityIndependenceQualityElection PartisanLowerHighHigherLowElection Non-PartisanLowerMiddleLowerMiddleMerit PlanHigherMiddleLowerLowAppointedHigherLowHigherHighElected judges write more opinions, but their average citation counts per opinion are lower than those for appointed judges. From the average litigant's perspective, elected judges provide more justice to more people—if one takes the receipt of a written explanation for the court's decision as a component of justice. Appointed judges write fewer opinions, but those that are written tend to be higher quality opinions—they garner more citations. Fewer litigants are receiving this high-quality justice though. Judges in the different systems exhibit similar levels of independence, with perhaps somewhat lower scores for Non-Partisan Election judges and Merit Plan judges. Elected judges in small states perform better on all quality measures than elected judges in large states; size of state does not make a difference for appointed judges. What explains these results?We began with a simple agency model, which did not have clear predictions for whether elected or appointed judges would be superior, but did suggest that size of state would matter more for elected judges than for appointed judges, as our results confirm. We can get further insight from the multitasking model (Holmstrom and Milgrom 1991), which shows that if an agent is given two objectives (say, quality and productivity), and the activities that further only one of those objectives can be measured and monitored by the principal, then the agent will shirk on the hard-to-measure objective and invest in achieving the other.Judges in more partisan systems may write more opinions because raw productivity is observable. Judges who write few opinions, for example, can be easily criticized and, as we have seen, some judges use productivity as a campaign issue. And, as the size of the electorate increases, judges might feel less pressure to be productive because it is harder for the public to monitor judges' productivity. Quality is hard to observe. With respect to quality, the public may have more trouble monitoring judges than governors do; our citations results are consistent with this hypothesis. Also note that again, within electoral states, quality does matter in small states, and not in large states, consistent with the hypothesis that small electorates are better monitors than are larger electorates.As for independence, the propensity to vote with or against co-partisans is also hard to observe. If so, elected judges would have no more incentive to refrain from acting independently than appointed judges do. So it is, in the end, not surprising that the independence levels of elected and appointed judges are not clearly different. Interestingly, elected judges are more independent in small states compared with large states. This again suggests that size of state matters: electorates may disapprove of politically biased judges but be unable to monitor them if the electorate is too large.There is another interpretation of the data, one that focuses on selection rather than monitoring. It might be that the different systems attract different types of people to judgeships: selection matters more than incentives to behave once in office. In particular, electoral systems would seem to attract politicians, whereas appointment systems are more likely to attract professionals. Politicians want to satisfy the voting public, and this might mean deciding cases expeditiously and in great number. Professionals are more concerned about their reputation among other lawyers and judges and are more interested in delivering well-crafted opinions that these others will admire.Recall from the summary statistics presented in Table 4 above that, compared with Appointed judges, Elected judges make more campaign contributions; are paid less; are on less stable benches; and have shorter tenures. We also examined the law school that our sample judges attended. A little under 70% of our Election Partisan judges attended an in-state law school compared with only 33% of the Appointed judges (difference significant at the <1% level). In addition, the US News Ranking (measured as of 2005) of the law school for Election Partisan judges was on average equal to 57.9, whereas the mean ranking for Appointed judges was equal to 32.3 (difference significant at the <1% level). Election Partisan judges are more likely to have gone to a law school in the state in which they sit and are more likely to have gone to a lower-rank law school. They are, in short, more politically involved, more locally connected, more temporary, and less well educated than appointed judges. They are more like politicians and less like professionals. Politicians are likely to see their role as judges as predominantly one of resolving disputes (as many as possible), whereas professionals are more likely to see their role as advancing the law. This might explain the productivity and quality differences.One might think that politicians would be more concerned about party identification, and hence would be more likely to vote together than professionals are—but this is not the case, as our independence measures show. One simple explanation for our results is that for most cases, one's vote has no political salience. The vast majority of cases are unanimous. It is more important even for politician-judges to decide many cases correctly than to agree with their co-partisans. Political identification matters as a proxy for how one's view affects how one evaluates a case. That is why judges do tend to vote with co-partisans. But this general tendency does not produce different independence outcomes in electoral and appointment systems because independence for all but a tiny number of cases is unobservable (and therefore not a focus of judges under either type of selection system).In sum, a simple explanation for our results is that electoral judgeships attract and reward politically savvy people, whereas appointed judgeships attract more professionally able people (the selection argument). Elected judges try to decide a lot of cases because productivity is observable, whereas appointed judges write better opinions because they care more about their long-term reputation among professionals (the incentives/multitasking argument). It is possible that the politically savvy people might give the public what it wants—adequate rather than great opinions, issued in greater quantity and therefore (given the time constraint) greater average speed.6ConclusionWe began this project with the assumption that the data would demonstrate that appointed judges are better than elected judges. Our results suggest a more complicated story. It may be that elected judges are, indeed, superior to appointed judges. Or it may be that elected judges are superior to appointed judges in small states only and not necessarily in large states. At a minimum, the conventional wisdom needs to be reexamined.A full comparative evaluation of the systems would require more research. There has been much concern in recent years about the rise in the cost of election campaigns for state supreme court justices, and some evidence that supreme court justices are more likely to vote in favor of contributors and their interests (Goldberg et al. 2006; Liptak and Roberts 2006).22 However, by the same token campaign expenditures enhance public awareness and indicate that judges face real political competition, which might be a good thing. In addition, appointment systems have hidden costs, namely, that they can serve patronage purposes. Empirical examination of the relationship between campaign expenditures and judicial quality is a promising avenue for future research.Thanks to Brannon Denning, Herbert Kritzer, Bill Landes, Stefanie Lindquist, Tom Miles, Un Kyung Park, Richard Posner, Jonathan Wiener, and participants at presentations at the 2007 Empirical Legal Studies Conference, the University of Chicago Law School, NYU Law School and the Cumberland Law School for comments, and Nathan Richardson for helpful research assistance. Also many thanks to two anonymous referees and the editor, Luis Garicano.AppendixTable A1.Key Variable DefinitionsVariableDefinitionTotal OpinionsTotal number of majority, concurring, and dissenting opinions authored by a particular judge in 1 year (ranging from 1998 to 2000).Outside State CitationsTotal number of citations from (1) federal courts outside the circuit that includes the state in question and (2) courts in other states. Citations are measured in opinions authored up until January 1, 2007 (as tracked in the LEXIS Shepard's database).Opposite_PartyThe total number of opposing opinions written against an opposite party judge divided by the total number of opposing opinions written against either a judge of the opposite or same party as the judge in question for the 1998 to 2000 time period. Opposing opinions include dissents written against a majority opinion and majority opinions where a dissenting opinion exists.Opposite_PoolTotal number of majority opinions written by the high court judges of the opposite political party (from the perspective of the judge in question) divided by the total number of majority opinions written by judges of both the same and opposite parties from 1998 to 2000.IndependenceDefined as Opposite_Pool minus Opposite_Party. A more negative Independence score occurs when Opposite_Pool < Opposite Party, indicating an increased tendency to write an opposing opinion against an opposite party judge. Conversely, a more positive Independence score indicates a decreased tendency to write an opposing opinion against an opposite party judge.Election Non-PartisanIndicator variable equal to 1 if the state uses a non-partisan election to select high court justices and 0 otherwise.Election PartisanIndicator variable equal to 1 if the state uses a partisan election to select high court justices and 0 otherwise.Merit PlanIndicator variable equal to 1 if the state follows the Missouri Merit Plan or a variant (including the Tennessee Plan) to select High Court justices and 0 otherwise.TenureThe average tenure of high court judges for the state in question, measured as of the spring of 1997 (from Hanssen 1999, table 1).Number of DissentsIndicator Variable equal to 1 if the judge authoring an opinion is Republican and 0 otherwise.West Key PagesNumber of pages in an opinion associated with the West key pages section (as provided in the West reporter version of the opinion and tabulated on Westlaw).Opinion LengthNumber of pages from the start of the opinion to the end of the opinion as provided in the West reporter version of the opinion and tabulated on Westlaw. For majority opinions, we measured from where the authoring judge's actual opinion starts to the end of the majority opinion.Open SecretsIndicator Variable equal to 1 if the judge authoring the opinion in question has donated to a political candidate and 0 otherwise. Political contributions are tracked by www.opensecrets.org and include Federal Election Commission records of receipts from all individuals who contribute at least $200 from 1992 to 2006.Table A2.Judge-Level Variable DefinitionsVariableDefinitionChief JudgeFor year-level data, indicator variable equal to 1 if the judge in question is the chief judge of the court in the year in question and 0 otherwise. For pooled data, indicator variable equal to 1 if the judge in question is the chief judge of the court for any year from 1998 to 2000 and 0 otherwise.Court ExperienceFor year-level data, the difference between the year in question and the year the judge first joined the high court. For pooled data, the difference between 1998 and the year the judge first joined he high court (if the judge started on the court in 1998 or later court experience is set to 0).Post-Law School ExperienceThe difference between 1998 and the year the judge graduated law school.Retirement within ≤1 yearWe calculate the number of years to retirement as equal to the year of retirement—the year in question. Indicator variable equal to 1 if the number of years to retirement is equal to 1 year or less and 0 otherwise. For pooled data, we calculate the number of years to retirement as equal to the year of retirement—2000.Retirement in 2 yearsWe calculate the number of years to retirement as equal to the year of retirement—the year in question. Indicator variable equal to 1 if the number of years to retirement is equal to 2 years and 0 otherwise. For pooled data, we calculate the number of years to retirement as equal to the year of retirement—2000Retirement in 3 yearsWe calculate the number of years to retirement as equal to the year of retirement—the year in question. Indicator variable equal to 1 if the number of years to retirement is equal to 3 years and 0 otherwise. For pooled data, we calculate the number of years to retirement as equal to the year of retirement—2000.Retirement in 4 yearsWe calculate the number of years to retirement as equal to the year of retirement—the year in question. Indicator variable equal to 1 if the number of years to retirement is equal to 4 years and 0 otherwise. For pooled data, we calculate the number of years to retirement as equal to the year of retirement—2000AgeAge of the judge in years.FemaleIndicator variable equal to 1 if the judge is female and 0 if male.Private PracticeIndicator variable equal to 1 if the judge had private practice experience before becoming a judge and 0 otherwise.Election SpendingIndicator variable equal to 1 if the judge raised funds relating to election campaign expenditures for the current year and 0 otherwise.PAJID ScorePAJID score for each judge as developed by Brace et al. (2000). These scores locate judges on a political continuum from highly conservative (0) to highly liberal (100)Table A3.Court-Level Variable DefinitionsVariableDefinitionAdjusted Associate Justice SalaryFor year-level data, the associate justice salary reported in the prior year for the state (so 1997 for 1998 judge-level data) divided by a cost of living adjustment for the year in question measured for the metro area in which the high court of the state is located. For pooled data, the associate justice salary reported in 1997 divided by the cost of living adjustment for 1998 (in thousands of dollars)Adjusted Partner SalaryFor year-level data, the average partner salary reported for the year in question for the state divided by a cost of living adjustment for the year in question measured for the metro area in which the high court of the state is located. For pooled data, the average partner salary in 1998 divided by the cost of living adjustment for 1998 (in thousands of dollars).Stable CourtIndicator variable equal to 1 if the state high court justices stayed the same from 1998 to 2000 and 0 otherwise.Number of Active Judges on BenchNumber of judges who were active at any time from 1998 to 2000 for the state in question.No Mandatory RetirementIndicator variable equal to 1 if the judges on the state high court do not face mandatory retirement and 0 otherwise.Long-Term ClerkIndicator variable equal to 1 if state clerks are tenured for more than one year and 0 if tenure is 1 year or less.Number of Clerks Per JudgeAverage number of clerks per judge in the 1998 to 2000 time period.Law Clerk Opportunity CostThe difference between the average salary of an entering associate at law firm in that state and the law clerk salary (in thousands of dollars).Number of Trial Cases in the StateNumber of trial cases in the entire state in 1998 (in thousands).Intermediate Appellate CourtIndicator variable equal to 1 if the opinion is in opposition to the opinion of another judge in the same case and 0 otherwise. In the case of a dissenting opinion written by the judge in question, the opinion is treated as in active opposition to the majority opinion. In the case of a majority opinion by the judge in question, active opposition exists if the majority opinion is opposed by a dissenting opinion.Mandatory PublicationIndicator variable equal to 1 if judges on the state high court face a mandatory publication rule and 0 otherwise.Table A4.State-Level Variable DefinitionsVariableDefinitionState AgeAge of the state. For year-level data this is defined as the difference between the year in question and the year of admission of the state into the United States. For pooled data, this is defined as the difference between 1998 and the year of admission of the state into the United States.State PopulationFor year level data, the population of the state in millions measured in the year prior to the year in question (so the population in 1997 if the data year is 1998). For pooled data, the population of the state in millions measured for 1997.Border PopulationTotal population of all bordering states of the state in question (measured as of 1997 in millions).Crime IndexFor year level data, overall crime rate for the state (including property and violent crime) per 100,000 people from the FBI Crime Report for the year prior to the year in question. For pooled data, the overall crime rate measured for 1997.Gross State ProductGross State Product (measured as of 1998 in billion of dollars).Median Age of PopulationMedian age of state population (2000 US Census)State Median IncomeMedian per capita income of the state population (2000 US Census in thousands of dollars)Black Population FractionFraction of the population comprised of blacks as obtained from the 2000 Census.Citizen Ideology ScoreMeasure of citizen ideology based on election results in each district, which are then used to compute a state-wide average (ultimately based on interest group ratings of a given state's federal congressional delegation) (from Berry et al. 1998).Law Enforcement AgenciesNumber of law enforcement agencies in 2000 (from Reaves and Hickman 2000)Law Enforcement EmployeesNumber of full-time law enforcement employees in 2000 (from Reaves and Hickman 2000)ProsecutionsNumber of prosecutions in 2000 (from Rottman and Strickland 2000)Law-Related EmployeesNumber of law-related employees in 2000 (from Reaves & Hickman, 2000)Law-Related PayrollAnnual payroll for all law-related employees in 2000 (from Reaves and Hickman 2000)Law EstablishmentsNumber of law firms (from Reaves and Hickman 2000)Table A5.Subject Matter CategoriesVariableDefinitionAdministrativeReview of Agency/Government Decision making (not in another subject matter category). Also includes Government Actions (e.g., State suit to comply with state statute that does not fit in other categories); private actions suing state actors for negligence, etc (unless the case involves prisoner rights which is included in the “Criminal” category of cases).Attorney and ClientAttorney Misconduct; Attorney fees (unless fits in one of above categories); Disbarment; contempt of court order against attorneyCapital PunishmentCapital Punishment-related actions.Church and StatePledge of Allegiance; Funding for Private Religious Schools; Prayer in School; Ten Commandments.CommercialContracts; Insurance; Private arbitration; Creditor v. Debtor; Lessor-Lessee; Usury Laws; Franchise v. Franchisor; Employment Contractual Disputes; Corporate Law; Piercing the Corporate Veil; Tax; Bankruptcy; Enforcement of mechanics lien; Implied warrant of merchantability.CriminalSentencing Guidelines; Prisoners Rights; Murder; Rape; Drugs/Controlled Substances; Attorney-Client Privilege in Criminal Context; Grand Jury-related; Juvenile Criminals. Excludes Capital Punishment cases.FamilyDivorce; Adoption; Child Support; Probate/Inheritance.First AmendmentEmployment issues (excluding employment contractual disputes); ERISA; National Labor Relations Board (NLRB); Occupational Safety and Health Act (OSHA); Fair Labor Standards Act (FLSA); Wrongful Discharge; Labor Management Relations Act (LMRA); Family and Medical Leave Act (FMLA); Employee Benefits; Worker's Compensation claims; Retaliatory Discharge claims.LaborEmployment issues (excluding (1) employment contractual disputes that are not Workers Comp or state administrative wage rate related—these go to “Commercial” and (2) excluding discrimination-type claims that fit in “Civil Rights”); ERISA; NLRB; Occupational Safety and Health Act (OSHA); Fair Labor Standards Act (FLSA); Wrongful Discharge; Labor Management Relations Act (LMRA); Family and Medical Leave Act (FMLA); Employee Benefits; Worker's Compensation claims; Retaliatory Discharge claims; State Wage Rate ClaimsPropertyTakings claims; Zoning issues; Property rights; Property Licensing-Related or Permit-Related; Landlord-Tenant-Related.RightsRace Discrimination; Sex Discrimination; Affirmative Action; Civil Rights; Age Discrimination; Privacy; Handicap Discrimination; Abortion (Includes discrimination in employment context cases); Voting Rights-Voting RelatedTortsFederal Tort Related Act; Medical Malpractice; Products Liability; Wrongful Death; Libel; etc.OtherAll other cases.AbramowiczMichaelEmersonTiller“Judicial Citation of Legislative History: Contextual Theory and Empirical Analysis,”2005http://papers.ssrn.com/sol3/papers.cfm? abstract_id=725919. (accessed October27, 2008)AspinLarry“Trends in Judicial Retention Elections,”Judicature19998381BakerScott“Should We Pay Federal Circuit Judges More?,”1999unpublished manuscript, available at: http://papers.ssrn.com/sol3/papers.cfm?abstract_id=998395BerryWilliam DEvanJ RingquistRichardC FordingRussellL Hanson“Measuring Citizen and Government Ideology in the American States, 1960–93,”American Journal of Political Science19984232748Available from: http://www.uky.edu/%7Erford/Home_files/page0005.htmBesleyTimothyAbigailPayne“Implementation of Anti-Discrimination Policy: Does Judicial Discretion Matter?,”CEPR Discussion Paper20055211Available from: http://www.cepr.org/PUBS/NEW-DPS/dplist.asp?dpno=5211&action.x=0&action.y=0&action=ShowDPBhattacharyaMitaSmythRussell“The Determinants of Judicial Prestige and Influence: Some Empirical Evidence From the High Court of Australia,”Journal of Legal Studies200130223BibbyJohn FHolbrookThomas MVirginiaGrayRusselHansonHerbertJacob“Parties and Elections,”Politics in the American States.1999Washington, DCCongressional Quarterly BooksBickelAlexander MThe Least Dangerous Branch.1986New Haven, CTYale University PressBracePaulMelindaGann HallNeo-Institutionalism and Dissent in State Supreme CourtsJournal of Politics19905254———“Studying Courts Comparatively: The View from the American States,”Political Research Quarterly199548529BracePaulMelindaGann HallLauraLanger“Measuring the Preferences of State Supreme Court Judges,”Journal of Politics200062387413———“Placing State Supreme Courts in State Politics,”State Politics and Policy Quarterly2001181108BracePaulJeffYatesBoyeaBrent D“The Conditional Effects of Ideology and Institutional Structure on Judicial Voting in State Supreme Courts”ExpressO Preprint Series2006Working Paper 1831 Available from: http://law.bepress.com/expresso/eps/1831. (accessed October27, 2008)CannDamon“Beyond Accountability and Independence: Judicial Selection and State Court Performance,”Judicature200790226CanonBradley“The Impact of Formal Selection Processes on the Characteristics of Judges—Reconsidered,”Law and Society Review19726579CarringtonPaul DJudicial Independence and Democratic Accountability in Highest State CourtsLaw and Contemporary Problems19986179ChoiStephen JGulatiG Mitu“Choosing the Next Supreme Court Justice: An Empirical Ranking of Judge Performance,”Southern California Law Review20047823———“Bias in Judicial Citations: A New Window into the Behavior of Judges,”Journal of Legal Studies20083787CroleySteven P“The Majoritarian Difficulty: Elective Judiciaries and the Rule of Law,”University of Chicago Law Review199562689794CrossFrank B“Legal Process, Legal Realism, and the Strategic Political Effects of Procedural Rules,”2005unpublished manuscript Available from: http://papers.ssrn.com/sol3/papers.cfm?abstract_id=837665. (accessed October27, 2008)CrossFrankEmersonTiller H“Judicial Partisanship and Obedience to Legal Doctrine: Whistle-blowing on the Federal Court of Appeals,”Yale Law Journal1998107215576CrossFrankLindquistStefanie“Judging the Judges,”2006unpublished draftDamKenneth WThe Law-Growth Nexus: The Rule of Law and Economic Development.2006Washington, DCThe Brookings Institution PressDudleyRobert L“Turnover and Tenure on State High Courts: Does Method of Selection Make a Difference?,”The Justice System Journal1997191EpsteinLeeJeffreySegal A“Measuring Issue Salience,”American Journal of Political Science2000446683GeorgeTracey E“Developing a Positive Theory of Decision Making on the U.S. Court of Appeals,”Ohio State Law Journal1998581635GeyhCharles G“Why Judicial Elections Stink,”Ohio State Law Journal20036443GlickHenryEmmertCraig“Selection Systems and Judicial Characteristics: The Recruitment of State Supreme Court Justices,”Judicature198770228GoldbergDeborahHolmanCraigSanchezSamanthaThe New Politics of Judicial Elections.2006New York, NYBrennan CenterMelindaGann Hall“State Supreme Courts in American Democracy: Probing the Myths of Judicial Reform,”American Political Science Review200195315HanssenAndrew F“The Effect of Judicial Institutions on Uncertainty and the Rate of Litigation: The Election versus Appointment of State Judges,”Journal of Legal Studies19992820532———“Independent Courts and Administrative Agencies: An Empirical Analysis of the States,”Journal of Law, Economics and Organization20001653471———“On the Politics of Judicial Selection: Lawyers and the State Campaigns for the Merit Plan,”Public Choice20021107997———“Learning About Judicial Independence: Institutional Change in the State Courts,”Journal of Legal Studies200433431HarrisPeter“Ecology and Culture in the Communication of Precedent Among State Supreme Courts, 1870–1970,”Law and Society Review198619449HellandEricTabarrokAlex“The Effect of Electoral Institutions on Tort Awards,”American Law and Economics Review2002434170HirschlRanTowards Juristocracy: The Origins and Consequences of the New Constitutionalism.2004Cambridge, MAHarvard University PressHolmesStephenJonElsterRuneSlagstad“Precommitment and the Paradox of Democracy,”Constitutionalism and Democracy.1988Cambridge, UKCambridge University PressHolmstromBengtMilgromPaul“Multitask Principal-Agent Analyses: Incentive Contracts, Asset Ownership, and Job Design,”Journal of Law, Economics and Organization199172452La PortaRafaelFlorencioLópez-de-SilanesCristianPop-ElechesAndreiShleifer“Judicial Checks and Balances,”Journal of Political Economy200411244570LandesWilliam MPosnerRichard A“Legal Change, Judicial Behavior, and the Diversity Jurisdiction,”Journal of Legal Studies19809367LangerLauraJudicial Review in State Supreme Courts: A Comparative Study.2002AlbanyState University of New York PressLangerLauraMcMullenJodyRayNicholas PStrattonDaniel D“Recruitment of Chief Justices on State Supreme Courts: A Choice Between Institutional and Personal Goals,”Journal of Politics20036565675LindquistStefanie A“Bureaucratization and Balkanization: The Origins and Effects of Decision-Making Norms in the Federal Appellate Courts,”University of Richmond Law Review200741659LiptakAdamRobertsJanet“Campaign Cash Mirrors High Court's Rulings,”New York Times20062006, A1 October 1LottJohn R“The Judicial Confirmation Process: The Difficulty with Being Smart,”Journal of Empirical Legal Studies20052407MaskinEricTiroleJean“The Politician and the Judge: Accountability in Government,”American Economic Review200494103454McLeodAman L“Judicial Performance Review: A Balance Between Judicial Accountability and Public Accountability,”Fordham Urban Law Journal200734343NewmanJon O“Between Legal Principles and Neutral Principles: The Legitimacy of Institutional Values,”198472 California Law Review 200PinelloDaniel RThe Impact of Judicial-Selection Method on State-Supreme-Court Policy: Innovation, Reaction, and Atrophy.1995Westport, CTGreenwood PressReavesBrian AHickmanMatthew JThe Census of State and Local Law Enforcement Agencies.2000Washington, DCBureau of Justice StatisticsRottmanDavid BStricklandShauna MState Court Organization.2000Washington, DCBureau of Justice StatisticsSegalJeffrey ASpaethHarold JThe Supreme Court and the Attitudinal Model Revisited.2002New YorkCambridge University PressShepherdJoanna“The Influence of Retention Politics on Judges' Voting.”2007Available from: http://papers.ssrn.com/sol3/papers.cfm?abstract_id=997491. (accessed October27, 2008)SunsteinCass RDavidSchkadeEllmanLisa M“Ideological Voting on Federal Courts of Appeals: A Preliminary Investigation,”Virginia Law Review20049030154Symposium on Rethinking Judicial Selection: A Critical Appraisal of Appointive Selection for State Court JudgesFordham Urban Law Journal2007343TabarrokAlexanderHellandEric“Court Politics: The Political Economy of Tort Awards,”Journal of Law and Economics19994215788TarrGA“Selection of State Appellate Judges: Reform Proposals: Rethinking the Selection of State Supreme Court Justices,”Willamette Law Review20033914451Newspaper endorsements of judicial candidates are common.2There are a handful of empirical studies that do attempt to construct quality measures so as to evaluate judicial performance. These studies use surveys, measures of educational qualifications, or measures of experience as their dependent variables (Canon 1972; Glick and Emmert 1987; Cann 2007). Surveys may reflect the biases of the respondents as we discuss in the text. As for educational and professional qualifications, those look to be more appropriate as an independent variable rather than as a measures of judging quality. (As we discuss below, there are interesting differences overlooked in the literature: elected judges went to worse law schools but have stronger local ties than do appointed judges).3The vast literature cannot be described here. Holmes (1988) traces the history of this debate. Bickel (1986) is the source of the modern debate in constitutional law. Croley (1995) brings the debate to bear on state courts.4We use a larger sample, different variable definitions, and many more control variables. The Landes and Posner (1980) study is mainly about the federal courts. And their data come from a different era, before the modern concern about excessive political competition among state court justices fueled by campaign donations (Carrington 1998).5The problems with citations studies have been rehearsed elsewhere and we will not repeat them here (e.g., on the possibility of bias, see Bhattacharya and Smyth 2004; Abramowicz and Tiller 2005; Choi and Gulati 2007).6Probably because the judge left office early in the year, entered office late in the year, was sick during the year, or had administrative duties.7As a check, we also examine in-state and home federal circuit citations. We discuss the results of this robustness test later in the Article.8The variable is defined as follows: indep = (demopratio − opdisratio)*(republican==1)+ (repopratio −opdisratio)*(democrat==1), where demopratio is the fraction of majority opinions in the state written by a Democrat (and similarly for repopratio) and opdisratio is the fraction of opposing opinions written by the judge in question against a Democrat.9In the Opensecrets database, we searched for political contributions for each judge by first and last name in the state in which the judge sits on the high court. We also looked at the profession of each donor as provided by Opensecrets—counting only donations by persons with the same first and last name and who either listed their profession as on the state high court or who listed a law firm affiliation (where we were able to match the judge to the law firm through other sources).10Consider, for example, a Republican judge sitting on a high court in a state where the other judges are split 50–50 between Republican and Democrat judges and the pool of majority opinions written by other judges corresponds to this 50–50 split. Suppose our Republican judge authors 10 dissents and 20 majority opinions where there is a dissenting opinion. And suppose that 5 of the 10 dissents are authored against a Democrat judge and 15 of the 20 majority opinions face a dissent from a Democrat judge. In this case, Opposite_Party would equal (5+15)/(10+20) = 2/3. Opposite_Pool equals 1/2. Independence would then equal 1/2 – 2/3 = – 1/6. Because of the tendency of our Republican judge to write an opposing opinion more frequently against Democrats compared with the background pool of majority opinions, the Republican judge receives a negative Independence score.11States where all judges in our sample were of the same political party included Georgia, Maryland, New Mexico, South Carolina, and South Dakota.12We also construct indicator variables for whether the state high court has mandatory jurisdiction over civil (Mandatory Civil Jurisdiction) or criminal (Mandatory Criminal Jurisdiction) cases. We also total the number of petitions filed with the state high court (Total Cases Filed) and the number of petitions where the high court granted a hearing (Total Cases Granted). Both sets of variables relate to the workload facing a judge. Unfortunately, we lack information on these variables for all of our states. We therefore do not include them in our set of state control variables but instead use them for robustness tests as discussed later in the paper.13To assess whether any particular subset of the state controls is important in affecting the relationship between the judge selection system and productivity (a concern highlighted by the large coefficients for state population and gross state product, which are larger than the coefficients for the election variables), we ran several variations of Model 4 of Table 5 alternatively using the following subsets of our state controls: (1) State Age, (2) ln(State Population) and ln(Border Population), (3) Crime Index and Median Age of Population, (4) ln(Gross State Product) and State Median Income, (5) Black Pop. Fraction, and Citizen Ideology Score. The coefficient for Election Partisan is positive and significant ranging from the <1% to 10% levels for each variation except for the variation using the ln(Gross State Product) and State Median Income variables (where the coefficient for Election Partisan is positive but insignificant). In contrast, the coefficient for Election Non-Partisan is positive and significant only for the variation with state age. The coefficient for Merit Plan is not significantly different from zero in all of the variations. As with the models of Table 5, these variations are generally consistent with the result that Election Partisan judges are more productive than Appointed judges.To further test the importance of state size, we replaced ln(Population) in Model 4 of Table 5 with several alternate measures related to state size (in separate regressions) including the number of law enforcement agencies, the number of full-time law enforcement employees, the number of prosecutions, the number of law-related employees, the annual payroll for all law-related employees, and the number of law establishments for a particular state (all measured in 2000). The coefficients for Election Partisan, Election Non-Partisan, and Merit Plan are positive and significant (at either the 5% or <1% level) for each regression. The coefficient for each of our alternate measures of state size is positive, but significant only for the number of full-time law enforcement employees, the number of law-related employees, the annual payroll for all law-related employees, and the number of law establishments.We ran a series of placebo regressions to test whether state-specific unobservables may affect the relationship of our judge selection system variables and productivity. We randomized the observations in Model 4 into four equally sized groupings (to parallel the four selection systems in Model 4). We replaced Election Partisan, Election Non-Partisan, and Merit Plan with three of the groupings, using the fourth as the base category. We then estimated this randomized groupings model 100 times. The coefficient on the randomized Election Partisan variable was significant at the 5% level a total of 8 times (and at the 10% level a total of 15 times) out of 100 times. The coefficient on the randomized Election Non-Partisan variable was significant at the 5% level a total of 9 times (and at the 10% level a total of 11 times) out of 100 times. The coefficient on the randomized Election Non-Partisan variable was significant at the 5% level a total of 11 times (and at the 10% level a total of 18 times) out of 100 times. The relative infrequency of significant results from the randomized groupings coupled with the high level of significance of the results of Model 4 of Table 5 lead us to view Model 4 as correctly specified and the significant results for our judge selection variables in Model 4 as robust.14As a robustness test, we reestimated Model 4 of Table 5 with the addition, alternatively, of (1) the Total Opinions Filed with the high court, (2) the Total Opinions Granted a hearing before the high court, and (3) indicator variables for whether the high court has mandatory civil jurisdiction and mandatory criminal jurisdiction. We lack data on these variables for all our states. Nonetheless, unreported, we obtained largely the same qualitative results as in Model 4 in the three robustness models. Judges from Election Non-Partisan, Election Partisan, and Merit Plan states are more productive than Appointed judges. In the model with the addition of the Total Opinions Granted a hearing before the high court as an independent variable, however, the coefficient on Election Nonpartisan while positive is significant at only the 11% level.We also reestimated Model 4 using the log of 1 + the total number of majority opinions in any given year and, alternatively, the log of 1 + the total pages written for all opinions in any given year as dependent variables. Unreported, we obtained largely the same qualitative results as in Model 4 in both robustness models. Judges from Election Non-Partisan, Election Partisan, and Merit Plan states are more productive than Appointed judges (whether measured by total majority opinions or total pages written). In the majority opinion model, however, the coefficient on Election Non-Partisan while positive is not statistically significant.To test the individual importance of each year in our data set, we reestimated Model 4 separately for 1998, 1999, and 2000. In each model, the coefficient on Partisan Election is positive, significant, and greater in magnitude compared with the positive coefficients on Partisan Non-Election, and Merit Plan—consistent with the results in Model 4. The coefficients on Partisan Election and Partisan Non-Election are positive and significant at the 5% and 10% levels, respectively, while the coefficient on Merit Plan is insignificant for the 1998 model. Only the coefficient on Partisan Election is significant (at the 5% level and positive) for the 1999 model. The coefficients on Partisan Election, Partisan Non-Election, and Merit Plan are all positive and significant (ranging from the <1% to 5% level) for the 2000 model.Lastly, we reestimated Model 4 with the addition of indicator control variables for whether the state is a member of US Census region Midwest, Northeast, or South (using West as the base case). Because substantial collinearity exists between our judge selection system variables and our census region variables (e.g., almost all judges from the Northeast Census region are Appointed judges), we add census region variables as a robustness test only. Unreported, the coefficient on Election Partisan, Election Non-Partisan, and Merit Plan are all positive and significant at the <1% level, consistent with the results in Table 5.15Anecdotal evidence supports the hypothesis that elected judges feel pressure to be productive. Productivity is mentioned in newspaper endorsements and judicial evaluation materials from time to time. A clear example can be found in the reelection campaign materials of a Texas intermediate appellate court judge, which include a table with productivity statistics for a group of judges:For the fiscal year ending August 31, 2006, Third Court of Appeals Justice Bob Pemberton ranked #1 statewide among Texas' court of appeals judges in production of original appellate opinions on the merits. These results show that Justice Pemberton is the most productive appeals judge in Texas for original opinions and the Third Court of Appeals is the most productive of Texas' 14 courts of appeals.http://www.bobpemberton.com/2006/09/20/appeals_opinion_productivity/.16The sum of Election Partisan + Large State + Election Partisan × Large State is positive and significant at the 5.3% level. The sum of Election Nonpartisan + Large State + Election Nonpartisan × Large State is positive and insignificant however.17As a robustness test, we also reestimated Model 4 of Table 7 with the addition, alternatively, of (1) the Total Opinions Filed with the high court, (2) the Total Opinions Granted a hearing before the high court, and (3) indicator variables for whether the high court has mandatory civil jurisdiction and mandatory criminal jurisdiction. We lack data on these variables for all our states. Nonetheless, unreported, we obtained similar qualitative results as in the Models of Table 7. The coefficients on Election Nonpartisan, Election Partisan, and Merit Plan are negative in all the models. The coefficient for Election Partisan is generally (but not always) significant. The coefficient for Election Nonpartisan is insignificant. The coefficient for Merit Plan is significant (either at the 5% or 10% level). Judges from Election Partisan and Merit Plan states are generally cited less than Appointed judges in these variations of Model 4.18As a robustness test, we reestimated Model 4 of Table 7 using the log of one plus the number of law review citations to a majority opinion (ln(1+Law Review Citations)) as an alternative measure of opinion quality (and as the dependent variable in the model). Not reported, we obtain similar results as in the Models of Table 7. Judges from Election Partisan, Election Nonpartisan, and Merit Plan states produce opinions that are cited less by law reviews than judges from Appointed states. Unlike in Model 4, the coefficients for Election Partisan and Merit Plan are significant at the 5% level. The biggest drop off in the level of law review citations is for Election Partisan opinions.We also reestimated Model 4 with the use of ln(1+Same State Citations + Home Federal Citations) as the dependent variable. Same State Citations include all in-state citations; Home Federal Citations include citations from a federal court in the same circuit as the state in question. Unreported, the coefficients on Election Partisan and Election Non-Partisan are not significantly different from zero. Only the coefficient on Merit Plan is significant (at the 5% level); however, the coefficient is positive, indicating that Merit Plan judges produce opinions that are cited more by same state and home federal cases compared with Appointed judges. Same state citations are not driven by the same factors behind out of state citations; same state citations, for example, often occur because of the need to cite to precedent.To test the individual importance of each year in our data set, we reestimated Model 4 separately for 1998, 1999, and 2000. In all three year–specific models, the coefficients on Election Partisan and Election Nonpartisan are not significant. In two of the models (1998 and 2000), the coefficient on Merit Plan is negative and significant at the 5% and 10% levels.We reestimated Model 4 with the addition of indicator control variables for whether the state is a member of US Census region Midwest, Northeast, or South (using West as the base case). Unreported, the coefficient on Election Partisan and Merit Plan are negative and significant at the 10% and 5% levels, respectively. The coefficient on Election Non-Partisan, although negative, however, is not significantly different from zero.Lastly, we ran a series of placebo regressions to test whether state-specific unobservables may affect the relationship of our judge selection system variables and outside state citations. We randomized the observations in Model 4 of Table 7 into four equally sized groupings (to parallel the four selection systems in Model 4). We replaced Election Partisan, Election Nonpartisan, and Merit Plan with three of the groupings, using the fourth as the base category. We then estimated this randomized groupings model 100 times. The coefficient on the randomized Election Partisan variable was significant at the 5% level a total of 3 times (and at the 10% level a total of nine times) out of 100 times. The coefficient on the randomized Election Non-Partisan variable was significant at the 5% level a total of 6 times (and at the 10% level a total of eight times) out of 100 times. The coefficient on the randomized Election Non-Partisan variable was significant at the 5% level a total of five times (and at the 10% level a total of eight times) out of 100 times. The relative infrequency of significant results from the randomized groupings leads us to view Model 4 of Table 7 as correctly specified.19We also find a positive correlation between dissent activity and the size of the bench (i.e., the number of judges participating in cases during our period), which is consistent with Lindquist (2007), who finds the same result using federal appellate courts.20As discussed, our Independence measure suffers from a range problem. To address this, we reestimate Model 4 of Table 10 using Indep_Indicator as the dependent variable, set equal to 1 if Independence is greater or equal to 0 and set equal to 0 otherwise. The use of Indep_Indicator lessens the range problem but at the cost of less data. Model 4 of Table 11 reports the results. As with Model 4 of Table 10, the coefficient on Election Partisan is not significant while the coefficients on Election Non-Partisan and Merit Plan are negative and significant.As a robustness test, we reestimate Model 4 of Table 10 with the addition of indicator control variables for whether the state is a member of US Census region Midwest, Northeast, or South (using West as the base case). In the robustness model, none of the coefficients on Election Partisan, Election Non-Partisan, and Merit Plan are significantly different from zero, indicating relatively little difference among the different selection systems in terms of judicial independence.Lastly, we ran a series of placebo regressions to test whether state-specific unobservables may affect the relationship of our judge selection system variables and the Independence measure. We randomized the observations in Model 4 of Table 10 into four equally sized groupings (to parallel the four selection systems in Model 4). We replaced Election Partisan, Election Non-Partisan, and Merit Plan with three of the groupings, using the fourth as the base category. We then estimated this randomized groupings model 100 times. The coefficient on the randomized Election Partisan variable was significant at the 5% level a total of 3 times (and at the 10% level a total of four times) out of 100 times. The coefficient on the randomized Election Non-Partisan variable was significant at the 5% level a total of 7 times (and at the 10% level a total of 11 times) out of 100 times. The coefficient on the randomized Election Non-Partisan variable was significant at the 5% level a total of 5 times (and at the 10% level a total of seven times) out of 100 times. The relative infrequency of significant results from the randomized groupings leads us to view Model 4 of Table 10 as correctly specified.21The sum of Large State and Large State × Non-Partisan Election is positive and insignificant. The sum of Large State and Large State × Merit Plan is negative and insignificant.22Goldberg et al. (2006) identify Alabama, Illinois, Michigan, and Ohio as outliers—states where campaign contributions were much greater than in other states—as of 2000, the last year of our study. We checked the independence scores of judges in these states and found Alabama and Michigan had lower mean independence scores than the mean for all states, whereas Illinois and Ohio had higher scores. This is an interesting topic for future research, but as of now the data do not seem reliable and accessible enough to do a rigorous test. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A89D76726F97F949EB81FECCFFA9FD44EC7EB90.txt b/test/dataset/in/resources/corpus/Clean_0A89D76726F97F949EB81FECCFFA9FD44EC7EB90.txt new file mode 100644 index 0000000..9ff3350 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A89D76726F97F949EB81FECCFFA9FD44EC7EB90.txt @@ -0,0 +1 @@ +]>HEARES3680S0378-5955(01)00265-910.1016/S0378-5955(01)00265-9Elsevier Science B.V.Fig. 1Average difference plots (post-exposure minus pre-exposure DPOAEs) showing changes in DPOAE levels, in response to 55-dB SPL primary tones, for mice (n=8 ears of four mice) after a 10-min OBN exposure centered at 10 kHz, at 105 dB SPL. No change from the pre-exposure control measurements is indicated by the dotted line at ‘0’ on the ordinate. DPOAEs were maximally depressed immediately after the exposure (open squares), but partially recovered, particularly, over the lower frequency range by 1 day post-exposure (open diamonds). By 5 days following exposure (open circles), DPOAEs were essentially recovered to their pre-exposure levels. The control group of stress-only mice (n=4) showed no decreases in DPOAE levels (solid squares), along the dotted line at ‘0’, over the same measurement period. The dashed line around −20 to −30 dB, represents the NF of the combined measurement equipment and animal preparation, and delimits the maximal possible noise-induced loss in DPOAE levels. The shaded line above the abscissa centered at 10 kHz represents the frequency spectrum of the OBN exposure. In this plot and those of Figs. 2 and 3 , the error bars associated with each symbol represent SEM.Fig. 2Average DPOAE difference plots obtained with 55- (top: A,D), 65- (middle: B,E), or 75-dB (bottom: C,F) SPL primaries for mice (n=8 ears from four mice for each group), at 2 (left panels) and 31 days (right panels) following exposure to the OBN, for either 0.5 (open circles), 1 (filled squares), 3 (open diamonds), or 6 (filled circles) h. At 2 days post-exposure, increases in the duration of the OBN exposure produced significant increments in DPOAE loss (A,B,C), for each primary-tone level. However, permanent DPOAE loss essentially plateaued after 3 h of exposure, especially, after 31 days of recovery (D,E,F), for each primary-tone level. Note that, for the 1-h exposure (filled squares), DPOAE losses at 2 days post-exposure were generally less than the permanent reductions measured at 31 days post-exposure, thus suggesting that OHC function worsened during the recovery period.Fig. 3Growth of DPOAE loss as a function of the duration of exposure measured for GM frequencies at 7 kHz (f2=7.786 kHz) (top: A,D), 13.9 kHz (f2=15.6 kHz) (middle: B,E), or 24.2 kHz (f2=27.114 kHz) (bottom: C,F), with L1=L2=55 (asterisks), 65 (open circles), or 75 dB SPL (open squares), at either 2 (left panels) or 31 days (right panels) post-exposure. The 2-day loss data were best approximated by a linear-fit function, while the 31-day findings were more closely matched by a one-phase exponential [Y=Ymax(1−exp)−KX)] function. These differences in growth of loss rate may reflect, in part, different mechanisms involved in temporary versus permanent noise-induced reductions in DPOAE levels. Additionally, it is clear that DPOAEs elicited by 75-dB SPL primaries were generally less affected, particularly, at 31 days post-exposure, than those generated by the lower primary-tone levels. For each plot, the gray dashed lines, in ascending order, indicate the average NFs for the 55-, 65-, and 75-dB SPL stimuli, respectively.Fig. 4Low-power photomicrographs of mouse cochleas from a region of the cochlear duct that was approximately at a 45–60% distance from the apex of the cochlea, equivalent to the 11–18-kHz frequency region, which represents a location expected to be maximally damaged by the 10-kHz OBN exposure. Left panels (A,C,E) represent sections from a control ‘stress-only’ non-exposed mouse, while right panels (B,D,F) are from similar regions for a 6-h noise-exposed cochlea, at 1 month (31 days) post-exposure. In the noise-exposed ear (B), only about 25%, on average, of the OHCs remained in the damaged region of the cochlea, most of which were first-row OHCs. In the control cochlea (A), all three rows of OHCs are clearly visible. In this same region, the efferent innervation visualized using staining for AChE activity remained at 1 month following the OBN exposure (D,F) and was not visibly different from that for a stress-control specimen (C,E). The focus for these sections (C,D) was on the ISB, while the bottom panels (E,F) were focussed on the efferent synapses with the OHCs, in the same region of the cochlea. IHC=inner hair cells, OHC=outer hair cells for rows 1, 2, and 3, ISB=inner spiral bundle, bars=50 μm.Temporary and permanent noise-induced changes in distortion product otoacoustic emissions in CBA/CaJ miceAna E.Vázqueza*avazquez@med.miami.eduAnne E.LuebkeabGlen K.MartinabBrenda L.Lonsbury-MartinabaDepartment of Otolaryngology, University of Miami Ear Institute (M805), P.O. Box 016960, Miami, FL 33101-6960, USAbNeuroscience Program, University of Miami School of Medicine, Miami, FL, USA*Corresponding author. Tel.: +1 (305) 243-4641; Fax: +1 (305) 243-5552AbstractA number of studies have shown that the ear can be protected from sound over-exposure, either by activating the cochlear efferent system, or by sound ‘conditioning’ in which the role of the efferent system is less certain. To study more definitively the molecular basis of deliberately induced cochlear protection from excessive sounds, it is advantageous to determine, for an inbred mouse strain, a range of noise exposure parameters that effectively alter cochlear function. As an initial step towards this goal, young CBA/CaJ mice were exposed to a 105-dB SPL octave-band noise (OBN), centered at 10 kHz, for various lengths of time consisting of 10 min, or 0.5, 1, 3, or 6 h. Distortion product otoacoustic emissions (DPOAEs) at the 2f1−f2 frequency, in response to equilevel primary tones of low to moderate levels, were used to quantify the damaging effects of these sound over-exposures on cochlear function. In addition, staining for acetylcholinesterase (AChE) activity to assess for noise-induced changes in the pattern of efferent-nerve innervation to the cochlea was also performed in a subset of mice that were exposed to the longest-lasting 6-h OBN. The 10-min OBN resulted in only temporary reductions in DPOAE levels, which recovered to pre-exposure values within 5 days. Increasing the exposure to 0.5 h resulted in permanent DPOAE losses that, for low primary-tone levels, were still present at 31 days post-exposure. Additionally, the 1-h and longer exposures caused permanent reductions in DPOAEs for all test levels, which were measurable at 31 days following exposure. Light-microscopic observations restricted to the 11–18-kHz frequency region of the organ of Corti, for a subset of mice exposed to the 6-h OBN, uncovered a significant loss of outer hair cells (OHCs). However, despite the OHC loss in this region, the AChE activity associated with the related pattern of efferent innervation remained largely intact.KeywordsNoise damageCochleaDistortion product otoacoustic emissionMouseAcetylcholinesteraseEfferent innervationAbbreviationsAChE, acetylcholinesteraseAHL, age-related hearing lossCAP, compound action potentialCBA, CBA/CaJDSP, digital signal processorDPOAE, distortion product otoacoustic emissionFFT, fast Fourier transformOBN, octave-band noiseGM, geometric meanISB, inner spiral bundleOHC, outer hair cellNF, noise floorPBS, phosphate-buffered salineIHC, inner hair cellSD, standard deviationSEM, standard error of the mean1IntroductionThere has long been considerable interest in the fundamental processes that protect the ear from sound over-stimulation, especially with respect to the so-called conditioning effect, whereby prior exposure to a typically low-level sound protects the ear from a subsequent more traumatic exposure to an identical, but more intense sound. Although studied extensively, the basic processes underlying such purported protective effects are just beginning to be elucidated. One possibility, proposed by a number of investigators, is the potentially critical role played by the cochlear efferent system (Canlon et al., 1988; Franklin et al., 1991; Miyakita et al., 1992; Subramaniam et al., 1992; Pukkila et al., 1997). Although the significance of the role of the efferent system in the ‘conditioning’ phenomenon is still being debated, the function of the cochlea’s medial efferent pathway in protecting the ear from noise over-exposure is more firmly established (Cody and Johnstone, 1982; Liberman, 1991; Kujawa and Liberman, 1997; Yamasoba and Dolan, 1998). If such sound-conditioning effects could be reliably obtained in mice, a species commonly used as a molecular model of genetically based ear disorders, they would become ideal experimental subjects within which to pursue the fundamental basis of such cochlear protection.The earliest failed attempts (Fowler et al., 1995) to confirm sound conditioning in mice were unexpected given previous demonstrations of such protection in chinchillas (Clark et al., 1987), guinea pigs (Canlon et al., 1988), rabbits (Franklin et al., 1991), humans (Miyakita et al., 1992), and gerbils (Ryan et al., 1994). This unanticipated outcome in mice was likely due to the relatively low frequencies used, which consisted of narrow-band noises centered at 4.5 kHz, that tested only the extreme lower end of the mouse audibility range (see Ehret, 1974; Heffner and Masterton, 1980). More recently, using higher test and conditioning frequencies, ranging from 8 to 16 kHz, Yoshida and Liberman (2000) reported success in obtaining a protective conditioning effect of sound for CBA/CaJ (CBA) mice. In their study, either a 15-min or a 1-week training protocol reduced the extent of subsequent permanent damage, as measured by reductions in both distortion product otoacoustic emissions (DPOAEs) and compound action potentials (CAPs), compared to what would be expected in the absence of ‘conditioning’.Sun and Kim (1999) showed earlier that mice exhibit efferent-induced alterations in DPOAEs, similar to those described for the cat (Puria et al., 1996), guinea pig (Maison and Liberman, 2000), and rabbit (Luebke et al., 2000). In their paradigm, decreased or increased DPOAE levels were observed following the onset of primary tones, when the efferents innervating the outer hair cells (OHCs) were activated by ipsilaterally applied sounds. Thus, in mice, the contributions of the cochlear efferent system to sound conditioning can be assayed in a straightforward manner, and prior studies have even demonstrated that the efferent system contributes to the mouse’s cochlear response to sound over-exposure.Previous studies on the cochlear efferent system in other species have shown that efferent innervation is very robust, and is not easily affected by acoustic trauma, at least, as indicated by the presence of acetylcholinesterase (AChE) activity in efferent fibers, following the application of a traumatizing noise exposure (Rossi and Cortesina, 1965; Kokko-Cunningham and Ades, 1976). Such findings are consistent with the notion that the cochlear efferent system likely remains functional during sound exposure, and therefore, could play an active role during the actual noise over-stimulation process.Recently, Jimenez et al. (1999a) used DPOAEs to measure age-related changes in OHC activity in four inbred mouse strains, including CBA mice, and compared the susceptibility of these same strains to both temporary and permanent noise-induced cochlear dysfunction (Jimenez et al., 1999b, 2001). Their findings suggested that the cellular processes affected by a standard noise-exposure paradigm, that resulted in reversible reductions in DPOAEs, were not necessarily the same as those affected by a protocol that caused more permanent decreases in DPOAEs. In addition, because different mutant mouse strains exhibited unique exposure effects, the susceptibility of cochlear processes to noise over-stimulation appeared to be dependent on each strain’s specific genetic background (Jimenez et al., 1999b, 2001).In combination, the above findings suggest that by utilizing DPOAE measures, an ideal animal model can be established in the mouse to study the fundamental basis of an efferent-related protection and susceptibility to excessive noises at the OHC level. Mice have an advantage over other species, because genetically based variability can be reduced, or other specific defects introduced, by the selection of particular inbred strains as experimental subjects. In addition, because of the well-established existence of a number of inbred mutant strains, the mouse is one of the only laboratory species in which the genes responsible for the ear’s plasticity, in response to acoustic over-stimulation, can efficaciously be explored.Consequently, the present study was designed to explore the effects of various noise-exposure parameters on DPOAEs in the CBA mouse, with the aim of eventually developing an animal model in which the ear’s resistance, and/or susceptibility to over-exposure, could be explored at both the functional and molecular levels. Toward this end, CBA mice were exposed to one of several durations of a standardized octave-band noise (OBN) to identify the noise-exposure parameters that subsequently result in a range of both reversible and non-reversible changes in DPOAE levels. The effects of non-reversible noise damage on the innervation of the mouse’s cochlear efferent system were also studied by staining for AChE activity in a subset of mice with permanently altered ears.2Materials and methods2.1SubjectsSubjects were 2.5-month-old CBA mice, purchased from a commercial supplier (The Jackson Laboratory), and housed under routine vivarium conditions consisting of standard lighting (i.e. 12-h light/dark cycles) and free access to food and water. The mean noise level of the maintenance room in the vivarium, as measured by a noise-logging dosimeter (Quest M-27) over a representative work interval of 2 days, was about 60 dBA for 99.3% of the time, and approximately 70 dBA for the remaining time. During data collection, mice were anesthetized with an initial dose of ketamine hydrochloride (100 mg/kg) and xylazine (4 mg/kg), with maintenance doses (ketamine 50 mg/kg, xylazine 2 mg/kg) administered when needed, as indicated by twitching vibrissae. The primary study consisted of a within-subjects experimental design (i.e. pre- versus post-exposure comparisons), for five experimental exposure groups, each consisting of four mice (i.e. n=8 ears), along with a sixth ‘stress’ control group of four mice (i.e. n=8 ears). The control-group mice were subjected to the same manipulations as endured by the mice of the 10-min exposure group, but without the noise over-exposure episode. That is, they were handled and transported, anesthetized, tested at baseline for documentation of ‘pre-exposure’ DP-grams, placed in the exposure cage for 10 min, and then post-tested immediately following the mock ‘exposure’ session, and at 1 and 5 days ‘post-exposure’.2.2DPOAE measurementsThe primary functional measure was the 2f1−f2 DPOAE. Complete details of the mouse DPOAE-recording procedure have been described elsewhere (Jimenez et al., 1999a). Briefly, the f1 and f2 primary tones were generated by a dual-channel synthesizer (Hewlett-Packard 3326A) and attenuated using customized software that operated a digital signal processor (DSP) on-board a personal microcomputer system. The f1 and f2 primaries (f2/f1=1.25) were then presented over two separate earspeakers (Radio Shack, Realistic, Dual Radial Horn Tweeters), and delivered to the outer ear canal through an acoustic probe, fitted with a soft rubber tip (Etymotic, ER3-34 Infant Silicon Tip), where they were allowed to acoustically mix to avoid artifactual distortion. Ear-canal sound pressure levels, which were measured by an emissions microphone assembly (Etymotic Research, ER-10B+) embedded in the probe, were sampled, synchronously averaged and processed, using a fast Fourier transform (FFT), for geometric mean (GM) frequencies (i.e. (f1×f2)0.5) ranging from 5.6 to 19.7 kHz (i.e. f2=6.3–22.5 kHz) by the computer-based DSP board. Corresponding noise floors (NFs) were computed by averaging the levels of the ear-canal sound pressure for five frequency bins above and below the DPOAE-frequency bin (i.e. ±54 Hz).For test frequencies above 20.1 kHz, a computer-controlled dynamic-signal analyzer (Hewlett-Packard 3561A) was used. The related NFs were estimated by averaging the levels of the ear-canal sound pressure for the two FFT-frequency bins below the DPOAE frequency (i.e. for 3.75 Hz below the DPOAE). No artifactual DPOAEs were ever measured in a hard-walled cavity that approximated the size of the mouse outer ear canal. This receptacle was also used to calibrate the tonal stimuli by fitting a 1/4-in microphone at the end of the cavity that was opposite the emissions-probe assembly. For both stimulus protocols, DPOAEs were considered to be present when they were, at least, 3 dB above the NF.The primary measure in the form of the DP-gram described DPOAE levels as a function of the GM-test frequencies, and overall, these were obtained from 5.6 to 49.7 kHz (i.e. f2=6.3–54.2 kHz), in 0.1-octave increments. In the DPOAE plots below (Figs. 1 and 2), DP-grams were converted to difference functions by subtracting post-exposure functions from their corresponding pre-exposure, baseline counterparts.2.3Noise exposureAfter DPOAE baseline testing, mice were exposed to an OBN, centered at 10 kHz, at a level (RMS) of 105 dB SPL, for various lengths of time consisting of 10 min, or 0.5, 1, 3, or 6 h. Levels of the 2f1−f2 DPOAE were measured immediately after the over-stimulation episode for all mouse-exposure groups, and at 1 and 5 days post-exposure for the 10-min exposure group. For the remaining exposure groups, DPOAEs were measured at 2 and 31 days post-exposure. The OBN level was monitored with a 1/2-in microphone (ACO Pacific, AC07013), in combination with a precision sound-level meter (Quest 155). The OBN itself was generated by filtering (Frequency Devices 9002) a broadband white-noise signal, produced by a custom-made noise generator, and amplifying it with a stereo amplifier (NAD 3225 PE). The resulting OBN was presented by two direct-reflecting loudspeakers (Bose 901), that were controlled by an associated active sound equalizer. The resulting spectrum, when analyzed with the dynamic-signal analyzer in 1/3-octave bands, ranged from 8 to 15 kHz, with the maximum energy of 100 dB SPL, at 10 kHz. During the noise exposure, one awake mouse was placed into each compartment (12 cm wide) of a custom made, wire-mesh cage, that was divided into two subsections. For each free-field exposure session, two cages, containing a total of four mice, in individual compartments, with free access to food, were positioned in the center of a double-walled sound-isolation chamber, fitted with hard-reflecting surfaces, to achieve a homogeneous sound field.2.4Visualization of OHCs and efferent innervation patternsA subset of four mice exposed to the OBN for 6 h (n=8 ears), along with a counter set of four control, non-exposed mice (n=8 ears), were deeply anesthetized and systemically perfused, initially with phosphate buffered saline (PBS), and then with 4% paraformaldehyde in PBS. Next, each cochlea was dissected out and small perfusion holes placed in the apical and basal turns. The cochlea was then further fixed by gentle perfusion with a pipet that contained 4% paraformaldehyde in PBS, followed by a 2-h submersion in the latter solution. After fixation, the cochlea was decalcified by gentle stirring in a solution of 250 mM ethylenediaminetetraacetic acid, in PBS (pH 7.4), for 5 days. Prior to further processing, the decalcified cochlea was rinsed in distilled water three times.To aid in the visualization of OHCs at a high power of magnification under the light microscope, one cochlea from each mouse was lightly osmicated by gentle perfusion, and then immersed for 10 min in a 1% osmium-tetroxide solution in Dalton’s buffer, with 1 ml of 1.65% CaCl2 per 100 ml of buffer. The contralateral cochlea was stained for AChE activity. Visualization of AChE-positive fibers was accomplished using the method of Karnowsky and Roots (1964), in which the incubation solution was modified with 10−4 iso-tetra-isopropylpyrophosphoramide, using 2 mM acetylthiocholine iodide as the substrate. All control incubations carried out in the absence of substrate were negative. For all specimens, the stain was developed for 20 min at room temperature, and terminated when a brown precipitate corresponding to the inner spiral bundle (ISB) was observed under a dissecting microscope. Finally, both cochleas were embedded in plastic (araldite) and manually cut into wedge-like sections representing quarter turns of the cochlear duct following the methodology originally described by Bohne (1972) for the chinchilla, and later modified by Bohne and colleagues for the mouse (Ou et al., 2000a,b).The 11–18-kHz region of the organ of Corti of the osmium-stained cochlea, which is located approximately at a 45–60% distance from the apical extent of the cochlear duct (Ou et al., 2000a), was inspected by differential-interference contrast microscopy to determine the percentage of missing OHCs. The complete absence of the nucleus, while the plane of focus was changed from the base of the hair cell to the reticular lamina, served as the primary criterion for defining an OHC as missing. To document the histopathology identified for OHCs within the 11–18-kHz organ of Corti region, photomicrographic images were captured at a resolution of 1280×1024 pixels using a digital camera (Pixera PVC 100) mounted on a Nikon microscope (Microphot-SA). A similar region of the AChE strained cochlea was examined for amount and location of brown reaction product signifying the presence of AChE activity.2.5Data analysisFollowing collection of the emission data, DPOAE levels and their related NF levels were converted to ASCII text files, and then imported to a commercial database (Microsoft Corp., Excel 98, v. 7.0). A set of descriptive statistics was obtained, which included the computation of means, SD, and SEM. Curve fits to the post-exposure DPOAE data using mathematical functions were performed by commercially available software (GraphPad Software Inc., Prism, v. 2.0). All animal procedures were approved and monitored by the University of Miami’s Institutional Animal Care and Use Committee.3Results3.1Effects of noise over-exposure on DPOAEsThe difference DP-gram plotted in Fig. 1 illustrates the effects of the shortest, 10-min OBN exposure on DPOAE levels, elicited by the lowest-level primaries at L1=L2=55 dB SPL. This difference plot, devised by subtracting the levels of post-exposure DPOAEs from their pre-exposure, baseline counterparts, shows that immediately after the exposure (open squares), DPOAEs were reduced over the majority of the tested frequency range, except for the highest-test frequencies, that were greater than about 40-kHz GM frequency (f2>50.6 kHz). In the frequency region from 14.1 to 30 kHz, where the maximum noise-induced reductions in DPOAE levels were expected, i.e. at about one half to an octave or two above the 10-kHz OBN, emissions were clearly reduced to NF levels (dashed line). At 1 day post-exposure (open diamonds), DPOAE levels had partially recovered over the lower-frequency range, from 5.6 to 11 kHz. Moreover, by 5 days post-exposure (open circles), the emissions had essentially returned to their pre-exposure levels. Thus, after 5 days of recovery, it is apparent that DPOAEs from the mice exposed to the 10-min OBN, completely superimposed those obtained from their stress-control counterparts (filled squares), which received the same manipulations, but without being subjected to the actual OBN exposure. It is interesting to note that, within the frequencies encompassed by the exposure band (shaded bar along the abscissa), low-frequency DPOAEs recovered more rapidly than those at the more vulnerable higher frequencies. In addition, it is clear from Fig. 1 that variability in the after-effects of the OBN on DPOAE levels between mice, as reflected by the S.E.M. bars, were greatest for the frequencies most affected by the over-exposure episode. In summary, a 10-min OBN exposure resulted only in moderate, temporary cochlear dysfunction in CBA mice as measured by DPOAEs.In Fig. 2A–F, average DP-gram difference plots are shown for mice at 2 (A–C) and 31 days (D–F), following OBN exposures lasting either 0.5 (open circles), 1 (solid squares), 3 (open diamonds), or 6 h (solid circles), for each of the three primary-tone levels tested at L1=L2=55 (A, D), 65 (B, E), or 75 dB (C, F) SPL. While the 2 days post-exposure data (left panels) presumably represents a mixture of both the reversible and permanent components of over-exposure effects, DPOAEs obtained at 31 days post-exposure (right panels) reflect only the permanent consequences of the OBN over-stimulation.From the inspection of these functions, it appears that the 0.5-h OBN exposure (open circles), at 31 days post-exposure (Fig. 2D–F), was near the threshold for producing widespread permanent DPOAE dysfunction for the CBA mouse. That is, on average, the 0.5-h exposure resulted in a mild, permanent decrement in both mid- and high-frequency DPOAEs, at all levels of test stimulation. Clearly, for this exposure group, as shown in Fig. 2F, DPOAEs elicited by 75-dB SPL primaries exhibited near-normal levels at 31 days post-exposure, especially for frequencies <40 kHz. Also note, for the 0.5-h OBN exposure at 31 days of recovery, not unexpectedly, DPOAEs elicited by the lowest-level primaries at 55-dB SPL stimuli (Fig. 2D) exhibited the greatest degree of variability in the most affected frequency region (i.e. ∼13–17 kHz) as indicated by the large S.E.M. values.As might be anticipated, incrementing the exposure duration from 0.5 to 1, 3, or 6 h, significantly increased the DPOAE losses observed at 2 days post-exposure. For example, as illustrated in Fig. 2A–C, at 2 days post-exposure, the 6-h exposure (solid circles) always reduced DPOAEs to NF levels, while, depending upon the primary-tone level, the 1- (solid squares) and 3-h (open diamonds) exposures did not. Overall, at 2 days of recovery, as shown in Fig. 2A,B, increasing exposure duration produced an orderly increase in DPOAE loss, especially for those elicited by the 55- and 65-dB SPL primaries, respectively. In contrast, permanent losses at 31 days post-exposure, as shown in Fig. 2D–F, were much less dependent upon exposure duration. That is, although increasing exposure duration from 0.5 to 1 h substantially increased DPOAE losses, particularly for the lower-level primaries, as shown in Fig. 2D,E, further increases in duration produced a much smaller increment in permanent DPOAE loss. Overall, the amount of DPOAE loss at 2 days post-exposure was much more dependent upon exposure duration than was the permanent loss determined at 31 days. Thus, it is clear that the 3- versus 6-h exposures produced considerably different amounts of initial reductions in DPOAE levels, as illustrated, particularly, in Fig. 2B,C. However, at 31 days post-exposure, these unique exposure durations resulted in similar amounts of permanent loss as shown in Fig. 2D–F. Moreover, although considerable variability, as indicated by the SEM bars, is evident across the exposure groups, such intersubject differences were greater for DPOAEs elicited by the lower primary-tone levels (e.g. Fig. 2A,D), and for emissions measured at 31 days post-exposure, when permanent changes were apparent (Fig. 2D–F).Fig. 3 shows more clearly the above-noted differences between recovery functions obtained early during the recovery process, and those measured about 1 month later. Specifically, these plots show, for three representative test frequencies at 7 (A,D), 13.96 (B,E), and 24 kHz (C,F), differences in the growth of the post-exposure DPOAE loss as a function of the exposure durations of 0.5, 1, 3, and 6 h, at 2 (Fig. 3A–C) and 31 days (Fig. 3D–F) of recovery, for each of the three primary-tone levels tested at 55 (asterisks), 65 (open circles), and 75 dB (open squares) SPL. For example, the plots of Fig. 3B,E indicate the DPOAE loss computed for the GM test frequency of 13.96 kHz (f2=15.6 kHz), i.e. at approximately 1/2 an octave above the center frequency of the 10-kHz OBN exposure, where the noise-induced reductions in DPOAE levels were maximum. It is clear that the 2-day post-exposure rate of loss shown in Fig. 3B was approximated by a linear fit to the data (i.e. r2=0.3, 0.7, and 0.8, for the L1=L2=55-, 65-, and 75-dB SPL primaries, respectively). In contrast, the more permanent DPOAE losses at 31 days post-exposure illustrated in Fig. 3E were approximated more closely by an exponential function (i.e. r2=0.4, 0.5, and 0.3, for the L1=L2=55-, 65-, and 75-dB SPL primaries, respectively). These low correlations resulted primarily from the substantial variability of the noise-induced reductions in DPOAEs between the 0.5- and 1-h exposure groups. When measured at 2 days post-exposure, each hour of OBN over-stimulation resulted in approximately 6.1, 5.7, or 3.5 dB of DPOAE loss, for the 75-, 65, and 55-dB SPL primaries, respectively, until the DPOAEs reached their corresponding NF.The growth of permanent DPOAE loss measured at 31 days of recovery as a function of exposure duration for the two lower-level primaries, at 55 and 65 dB SPL, began to asymptote by 3 h of exposure, at the NF of the measurement equipment, which represented a loss of approximately 30 dB from pre-exposure baseline levels. For the three test-stimulus levels, longer exposures at 3 and 6 h produced less variable reductions in DPOAE levels. More specifically, for the higher primary tones tested at 75 dB SPL, the 0.5-h exposure produced no decrement in DPOAE levels, while for the 1-h and longer exposures, DPOAE loss began to asymptote at approximately 10 dB below pre-exposure levels. Consequently, the DPOAEs elicited by the 75-dB SPL primaries were much less affected by the OBN than those elicited by the lower-level primary tones. However, the pattern of DPOAE loss elicited by the 75-dB SPL primaries followed a similar, if not parallel-shifted, exponential function that described the eventual noise-induced effects.From the inspection of the plots of Fig. 3A,D, or C,F, for the GM test frequencies of 7 and 24 kHz, respectively, it is clear that the above-noted observations for the 13.96-kHz GM test frequency generalize to other test frequencies located both below and above the one expected to show the greatest loss at about half an octave above the center frequency of the exposure (i.e. at 13.96 kHz), even if the amount of DPOAE loss was considerably less (e.g. for 7 kHz).3.2Histological observationsIn general, there was little evidence of OHC loss in the apical turn of the cochlea for either the control or 6-h noise-exposed mice. However, in the basal turn, a segment of cochlear duct that was at about a 45–60% distance from the apex, and that clearly contained a region of OHC loss, was selected for more detailed study. As this area corresponded approximately to the 11–18-kHz region of the mouse hearing range, according to the computations of Ou et al. (2000a), the sensory cell damage was considered, most likely, to be due to the 10-kHz OBN exposure. Certainly, this particular cochlear region corresponded to areas of the mouse DP-grams of the present study that exhibited, at least, a 25-dB decrement in DPOAE levels measured with either 55- or 65-dB SPL primaries. For control mice, the three rows of OHCs in this upper basal-turn region were always visible and appeared normal, as shown in Fig. 4A, for a representative non-exposed mouse. In contrast, as illustrated in Fig. 4B for a 6-h exposed mouse, up to 70% of the OHCs were missing in the noise-exposed mice that were sacrificed about 1 month after the over-stimulation episode.To determine the influence of noise exposure on efferent innervation, four mice from each of the control and the 6-h exposed groups were examined histologically for AChE activity at 31 days post-exposure. In these cochleas, the most intense AChE activity was always observed in the nerve fibers of the ISB, with no apparent discontinuity in staining, even in regions expected to be severely damaged by the OBN exposure (i.e. over the 11–18-kHz region). This finding can be appreciated by comparing panels C and E of Fig. 4, showing light photomicrographs of an AChE-stained control mouse, with panels D and F displaying similar cochlear regions for a 6-h noise-treated mouse. Upon comparison of these sections, it is clear that there were no obvious differences in the amount of brown reaction product between the two non-exposure and exposure conditions.However, overall, there was more variability in the surface preparations of the noise-exposed mice stained for AChE activity. For example, less intense staining of the ISB was observed in some of the noise-damaged cochleas, along with a disrupted arrangement of the efferent endings on the OHCs, and less packed tunnel crossing fibers. In other preparations, where the efferent innervation seemed abnormal, the tunnel of Corti appeared narrow as compared to the appearance of this structure in more normal specimens. Importantly, none of the AChE-stained control mice showed these abnormalities. Considering that these histological data were obtained from the mice receiving the most severe 6-h exposures, that resulted in the greatest DPOAE losses, it is unlikely that many significant changes would have been observed in the efferent endings of mice exposed for the shorter time durations.4DiscussionThe present study was designed to examine the effects of a range of noise-exposure durations on the DPOAEs of CBA mice. The major aim was to identify over-stimulation paradigms that produced OBN-induced changes in exposed mice that could eventually be used to study, in detail, the protective effects of sound conditioning, and the potential role of the cochlear efferent system in such processes. The findings revealed that, for the 2.5-month-old CBA mouse, a 10-min exposure was capable of initially reducing, to NF levels, the range of testable DPOAE frequencies, in and above the frequency band of the 105-dB SPL, 10-kHz exposure. However, no permanent effects were observed 5 days later for this briefest exposure duration. Increasing the duration to 0.5 h appeared to be near the threshold for producing permanent DPOAE decrements, while exposures beyond 3 h did not appreciably increase the amount of DPOAE loss observed for the 3-h exposures themselves, especially after 31 days of recovery. Thus, the tested exposures defined a range of durations within which acoustic over-stimulations appropriate for sound conditioning can be selected, as well as ones relevant for subsequently producing permanently damaging effects on OHC functions.The present study used changes in DPOAE levels to evaluate cochlear function. Thus, the findings reflect functional alterations that were restricted to the OHC level of the peripheral auditory system. In contrast, most prior studies of noise-induced damage in CBA mice utilized electrophysiological potentials to estimate subsequent changes in hearing thresholds. For example, Henry (1982) measuring a CAP-like response in adult CBA mice, was first to demonstrate that a 5-min exposure to a 124-dB SPL OBN, ranging from 12 to 24 kHz, produced changes in such cochlear potentials that lessened with increasing age. Specifically, he showed that 60-day-old mice, representing an early post-puberty stage of development, exhibited the greatest noise-induced threshold elevations, whereas a progressively less severe effect was observed for older mice between the ages of 90 and 360 days.In contrast to the CAP measure used by Henry (1982), the majority of the more recent studies of noise-induced modifications of auditory responses in the CBA mouse have used the auditory brainstem response (ABR) to estimate changes in hearing thresholds. For example, in an extensive series of studies, Li and Borg and colleagues (Li and Borg, 1991; Li, 1992; Li et al., 1993) examined noise-induced damage in CBA mice using high-level exposures, at 120 dB SPL, that were lower in their frequency range, at 2–7 kHz, than the ones reported here. These investigators showed that susceptibility to noise damage in the CBA mice was maximal at 1 month of age, and decreased substantially by 3 months of age. Other results by Shone et al. (1991) also indirectly suggested that 6-month-old mice were more resistant to noise over-exposure. These investigators showed that a 45-min, 101-dB SPL exposure produced no permanent ABR threshold shifts. Finally, the findings that older mice are more resistant to noise damage is also supported in a recent study by Davis et al. (1999), in which 3–4-month-old CBA mice showed no permanent ABR threshold shifts following a 1-h exposure to a 110-dB SPL OBN, centered-weighted between 7 and 17 kHz.The DPOAE results reported here also support the notion of an age-dependent susceptibility to excessive sounds. Thus, the present findings of significant noise-exposure effects in 75-day-old mice (i.e. age=2.5 month) can be contrasted to those of Jimenez et al. (1999b, 2001), who showed, in slightly more mature 90-day-old CBA mice (i.e. age=3 months), that the same 10-kHz, 105-dB SPL, 1-h OBN exposure as used here did not result in any detectable permanent DPOAE losses.Recently, two studies by other investigators reported the effects on DPOAEs of exposing CBA mice to a similar OBN. Specifically, for 2.5-month-old CBA mice, Yoshida et al. (1999, 2000) described frequency-dependent permanent threshold shifts for CAPs and DPOAEs following a 2-h exposure to a 100-dB SPL OBN ranging from 8 to 16 kHz. The findings of these investigators are in agreement with the present results, thus, supporting an increased susceptibility to noise for young CBA mice. Moreover, although Yoshida et al. (1999) observed no OBN-induced loss of OHCs, they described a disarrangement of the stereocilia pattern on OHCs for mice that were terminated 7 days after the exposure. In the present study, mice were histologically examined at 1 month after the OBN exposure. Thus, it is possible that the longer survival time, or the longer duration of the 6-h exposure they experienced, accounts for the degeneration of the OHCs that was observed in the region of the noise-exposure band for these subjects. In all, it is important to take into account the combined findings of the present study and those of Jimenez et al. (1999b, 2001), that CBA mice have a relatively abrupt change in their susceptibility to noise between 2 and 3 months of age, when comparing the susceptibility of the CBA strain to other mouse strains, such as the 129/SvEv (Yoshida et al., 2000).The effects of noise over-exposure on the efferent-nerve innervation of the cochlea were studied previously in several experimental models. In an early study in guinea pigs by Rossi and Cortesina (1965), treatment with neomycin sulfate (20 mg/kg/day) combined with an acoustic trauma, in the form of an 8-h, 90-dB SPL pure-tone exposure, at either 0.512, 2.048, 4.096, or 6.144 kHz, for 60 days, completely destroyed the organ of Corti in the majority of subjects. However, at 18 months post-exposure, for guinea pigs in which portions of the organ of Corti remained, histologic examination showed that AChE activity was present. Although the animals exhibiting a complete destruction of the organ of Corti showed no AChE staining in the cochlear duct, AChE activity clearly remained in the intraganglionic spiral bundle, and within the canaliculi of the osseous spiral lamina. Further work by Strominger et al. (1995) in the chinchilla showed a complete disappearance of efferent fibers, but only in cochlear regions that had been entirely destroyed by exposure for 12 h to a 0.5-kHz OBN at 120 dB SPL. Again, in the ‘partial wipeout’ regions described by these authors, where remnants of the organ of Corti remained, AChE-positive fibers were still observed.To our knowledge, observations of AChE-positive fibers in noise-treated mice have not yet been reported. The apparent survival of efferents when the organ of Corti was incompletely damaged may be due to a small percentage of remaining OHC and supporting cells. These cells may help maintain an environment that supports survival of the efferent fibers. Alternatively, if, as discussed by Kokko-Cunningham and Ades (1976), the efferent fibers bifurcate at the level of the tunnel, based on light- and electron-microscopy observations, such branching would allow a fiber to synapse onto more than one OHC, thus, allowing the fiber to survive, even when a large percentage of the target cells were missing. In any case, it appears that maintenance of efferent innervation seems dependent on there being some remaining target cells, or the afferent fibers, onto which a portion of the efferent fibers are known to synapse. Thus, it appears that, during both the temporary and permanently damaging effects of sound exposure, efferents are present and may actively participate in the damage process.There is considerable evidence that different mechanisms are involved in the temporary versus permanent effects of noise damage (Nordmann et al., 2000), regardless of the methods used to assess the damaged ear. The findings at 2 days post-exposure, when temporary influences presumably dominate, that decreases in DPOAEs proceeded linearly as a function of exposure duration, whereas permanent losses developed in an exponential manner and peaked, also argues for significant differences between the temporary and permanent aspects of noise damage as assessed with DPOAEs. Similar conclusions regarding the differences between temporary and permanent DPOAE losses were reached when comparing the temporary effects of a brief 1-min pure-tone exposure to those produced by the 1-h OBN employed here, in normal CBA mice versus three strains of mutant mice exhibiting AHL (Jimenez et al., 1999b).A major observation of the present investigation was that DPOAEs evoked by high- as compared to low-level primaries behaved differently with respect to their sensitivity to noise damage. That is, DPOAEs evoked by high-level primary tones (Fig. 2F) were much less affected than those evoked by lower-level primaries (Fig. 2D). What is especially interesting with respect to this finding is the observation that the temporary effects of noise exposure can completely eliminate DPOAEs evoked by high-level primaries (e.g. Fig. 2C). However, when the driving potential across the OHC is reduced by the temporary effects of loop diuretics on the endocochlear potential, low-level DPOAEs are eliminated, while those evoked by 75-dB SPL primaries are completely unaffected (see Fig. 3 in Whitehead et al., 1992). Thus, the insensitivity of high-level DPOAEs does not necessarily pertain to temporarily damaging noise exposures. Perhaps, in this reversible situation, OHC stereocilia are damaged or detached from the tectorial membrane (Nordmann et al., 2000), so that no amount of acoustic stimulation can activate the non-linear aspects of OHC transduction to result in the production of DPOAEs. Similar results were also obtained when the effects of age-related hearing loss (AHL) were studied and compared in three strains of mice exhibiting AHL (Jimenez et al., 1999a). In these latter experiments, high-level primaries were just as effective as lower-level tones in detecting the progressive OHC losses. Again, it appears as if some aspect of OHC transduction is gradually eliminated during longer durations of noise over-exposure that prevents even high-level stimuli from eliciting DPOAEs.Finally, although noise exposure in humans tends to produce the greatest losses in hearing sensitivity at a half to an octave above the frequency band of the exposure (e.g. Taylor et al., 1965), the after-effects noted here at a half to two octaves above the 10-kHz OBN are considerably more extensive for mice. These latter observations have also been demonstrated by Henry (1982) and Ou et al. (2000b), who showed that mice sustained maximum noise-induced shifts in ABR thresholds for frequencies more than two octaves above the exposure band.In summary, the present results define noise-exposure parameters that produce either temporary or permanent decreases in DPOAE levels, indicative of a loss of OHC function in young 2.5-month-old CBA mice. For purposes of sound conditioning, it appears that exposures should be limited to 10–15 min, if levels as high as 105 dB SPL are employed. For more traumatic exposures, durations in the range of 1–3 h appear adequate. Based on the first reports of successful sound conditioning in mice (Yoshida et al., 1999; Yoshida and Liberman, 2000), the exposure parameters suggested here, at about 105 dB SPL for 10 min, are within the ranges needed to rapidly produce such training effects.Observations in the present work of the effects of noise over-exposure on efferent innervation demonstrated that the efferent fibers remain, even after severe damage to their OHC targets, although a much larger variability in the surface preparations of the noise-exposed mice stained for AChE activity was apparent than for control animals. In addition, less intense staining of the ISB was observed, along with, in some noise-damaged cochleas, a disrupted arrangement of the efferent endings and less packed tunnel crossing fibers. These features of some 6-h exposed cochleas were not observed in any of the control cochleas. Such observations are in agreement with earlier studies in guinea pig and chinchilla which showed that, only in regions where the organ of Corti was completely destroyed, did the efferent innervation degenerate (Rossi and Cortesina, 1965; Kokko-Cunningham and Ades, 1976; Strominger et al., 1995). Based upon the present findings in mice and those in other species, it appears that efferents could be active over a large range of noise-exposure conditions, consistent with a number of studies implicating the cochlear efferent system in protection of the ear during the exposure process. 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Res.141200097106 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A8A1BD90A23E7997DAB9BF9F99ACCC6F7C3D95E.txt b/test/dataset/in/resources/corpus/Clean_0A8A1BD90A23E7997DAB9BF9F99ACCC6F7C3D95E.txt new file mode 100644 index 0000000..7f72b76 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A8A1BD90A23E7997DAB9BF9F99ACCC6F7C3D95E.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press S41510.1093/geronb/62.6.S415 Journal of Gerontology: Social Sciences Perceptions of Body Weight Among Older Adults: Analyses of the Intersection of Gender, Race, and Socioeconomic Status Schieman Scott Pudrovska Tetyana Eccles Rachel 1Department of Sociology, University of Toronto, Canada. 2Department of Sociology, University of Wisconsin. 3Department of Sociology, University of Toronto, Canada. Address Correspondence to Scott Schieman, PhD, University of Toronto, Department of Sociology, 725 Spadina Avenue, Toronto, Ontario M5S 2J4, Canada. E-mail: scott.schieman@utoronto.ca 11 2007 62 6 S415 S423 30 7 2007 28 11 2006 Copyright 2007 by The Gerontological Society of America 2007 Objectives. We examine the effects of gender, race, and socioeconomic status (SES) on perceptions of body weight among older adults and the role of status-based differences in BMI in these processes. Methods. Data are derived from face-to-face interviews with 1,164 adults aged 65 years and older in the District of Columbia and two counties in Maryland in 2000-2001. Results. With “perceived appropriate weight” as the comparison group, multinomial logistic regression analyses indicate that white adults, women, and high-SES individuals are more likely than black adults, men, and low-SES individuals to describe themselves as overweight or obese. However, these disparities are observed only after statistically adjusting for race, gender, and SES disparities in BMI. Moreover, the positive effect of SES on the likelihood of reporting overweight or obese perceptions is strongest among black women. Among low SES individuals, white women are more likely than men and black women to describe themselves as obese (relative to the “perceived appropriate weight” category). Discussion. Our observations underscore the importance of taking SES contingencies into account when exploring race-gender differences in perceived body weight. This study further contributes to the literature by documenting the important suppression patterns associated with race, gender, and SES differences in BMI. hwp-legacy-fpage S415 hwp-legacy-dochead RESEARCH ARTICLE INDIVIDUALS maintain different perceptions about their own body weight. Some describe their weight as appropriate, whereas others feel as though they could or should lose a few (or many) pounds; others report the desire to add to their body weight. The central aim of this article is to examine whether there are systematic social status variations in these perceptions. Although the health effects of body mass index (BMI) and its social distribution are well documented, our research contributes to existing knowledge by documenting the intersection of gender, race, and social class—among a socioeconomically diverse sample of older adults—and the ways in which these dimensions of social stratification shape perceptions of body weight in late life. Specifically, we asked: Do women and men differ in their perceptions of their body weight? If so, do these differences vary by race and socioeconomic status (SES) net of relative body weight? The prevalence of people with high BMI scores (>30) has risen sharply in recent years (Hedley et al., 2004; McTigue, Garrett, & Popkin, 2002). Estimates from the National Center for Health Statistics (2003) indicate that the percentage of obese American adults increased from 13% to 30% between 1980 and 2000. Recent surveys document that roughly 34% of adult women and 28% of adult men are obese. Those rates increase to 62% and 67% for women and men, respectively, if one combines overweight and obese groups. Moreover, African Americans have a higher rate of obesity compared to other racial/ethnic groups in the United States (Denney, Krueger, Rogers, & Boardman, 2004; Flegal, Carroll, Ogden, & Johnson, 2002). The high level of obesity has emerged as a public health issue because of the associated risks of chronic diseases, functional impairments, mortality, and psychosocial difficulties (Carr & Friedman, 2005; Fabricatore & Wadden, 2004; Ferraro & Kelley-Moore, 2003; Flegal, Graubard, Williamson, & Gail, 2005; Hassan, Joshi, Madhavan, & Amonkar, 2003). Collectively, these health consequences and their links to medical care cost an estimated $75 billion annually in the United States (Finkelstein, Fiebelkorn, & Wang, 2003; Sturm, 2002). Although the prevalence of obesity peeks around ages 50 to 59 and tends to decline in later life, rates are increasing among older adults. Roughly 19.5% of adults aged 65 or older are obese, and that rate has increased from 12% in 1990 (Centers for Disease Control and Prevention, 2005). Roughly 13% of adults older than age 80 are obese (Himes, 2004). Obesity is associated with higher risks of health conditions such as arthritis, diabetes, hypertension, and functional limitations—conditions that are especially relevant in late life (Kahng, Dunkle, & Jackson, 2004; Thorpe & Ferraro, 2004). The Importance of Perceptions of Body Weight The study of perceived body weight is important because of the potential implications of perceived body weight for psychological, social, and behavioral outcomes—independently of and in combination with relative body weight. Social differences in perceived body weight are especially relevant given the well-documented racial/ethnic and SES disparities in obesity-related health conditions (Hassan et al., 2003). Moreover, the ways in which individuals evaluate their body weight inform the discussion of gender, race, and SES differences in health lifestyle decisions (i.e., diet, exercise) and persistent gender–race group disparities in health (Cockerham, 2005). Misperceptions may cause individuals to overlook potential problems, thwart motivation to engage in preventive behaviors, or even contribute to actions that cause new health problems (Kuchler & Variyam, 2003). The processes and consequences of perceptions about body weight connect to an array of psychosocial and mental health outcomes. For example, the rising levels of body dissatisfaction over the past 50 years have occurred in tandem with higher rates of eating disorders, especially among women (Feingold & Mazzella, 1998). To date, however, most studies about perceptions of body weight have examined college students or young adults (Fallon & Rozin, 1985; Milkie, 1999). Less is known about perceptions of weight in late life, perhaps because of an assumption that body issues diminish in importance among elders. To the contrary, evidence suggests that concerns about aging and its effect on body weight and physical appearance are common in late life, particularly among older women (Halliwell & Dittmar, 2003). Allaz, Bernstein, Rouget, Archinard, and Morabia (1998) found that body weight concerns rank second behind memory loss as a central issue among elders. Likewise, Clarke (2002) found that weight-related issues are a major source of dissatisfaction among older women; the increased difficulty of weight loss is highly frustrating. Dimensions of Social Stratification and Perceptions of Body Weight Of all of the dimensions of social stratification, gender is central because of its influence on social norms and meanings about physical appearance. Pervasive cultural ideals of female thinness, the stigma associated with being overweight, and traditional gender-role differences in the importance of attractiveness may contribute to women's generally lower levels of satisfaction with their body weight (Reboussin et al., 2000; Rodin, Silberstein, & Striegel-Moore, 1984; Ross, 1994). Studies have shown that women report a lower BMI as the “ideal” (Crawford & Campbell, 1999) and are more likely than men to perceive themselves as overweight even if they are of normal weight, whereas men are more likely to consider themselves as normal weight even if they are overweight (Chang & Christakis, 2003; Kuchler & Variyam, 2002, 2003). Race represents another core dimension of stratification that influences body weight issues. For example, African Americans are less likely than other racial/ethnic groups to describe themselves as “overweight” (Chang & Christakis, 2003). In addition, Black adults tend to underestimate their body weight and White adults tend to overestimate their weight (Bhuiyan, Gustat, Srinivasan, & Berensen, 2003; Kuchler & Variyam, 2002). Studies also suggest that gender and race intersect to shape perceptions about body weight and size. Compared to their White counterparts, Black adolescent girls and adult women report less social pressure to be slim (Kumanyika, Wilson, & Guilford-Davenport, 1993; Powell & Kahn, 1995), less dissatisfaction with body size and weight, a greater acceptance of overweight (Brown et al., 1998; Miller et al., 2000; Smith, Thompson, Raczynski, & Hilner, 1999), a preference for a larger body shape (Becker, Yanek, Koffman, & Bronner, 1999; Parnell et al., 1996), a higher BMI criterion for body image discrepancy (Fitzgibbon, Blackman, & Avellone, 2000), and a lower risk of eating disorders or unhealthy weight-control methods (Henriques, Calhoun, & Cann, 1996; Neff, Sargent, McKeown, & Jackson, 1997). These processes may be part of a “cultural economy” in which body size and image are salient sources of social status (Chang & Christakis, 2005). Some evidence, however, has indicated that exposure to unrealistic images of African American models is associated with lower levels of body satisfaction among African American undergraduate women (Frisby, 2004). Despite the valuable information provided by studies of younger samples, Himes (2004) contended that much remains unknown about race differences in late life. The results of the few studies of older adults have tended to replicate patterns observed among younger samples. In a study that compared Black and White women older than age 65, Stevens, Shiriki, Kumanyika, and Keil (1994) documented that overweight Black women are more satisfied with their weight, less likely to feel guilty after overeating, less likely to diet, and more likely to consider themselves attractive than overweight White women. They also found that, among women who are not overweight, White women are more likely than Black women to describe themselves as overweight and report a lower ideal body weight. Similarly, another study that focused on overweight and obese women aged 40 and older found that Black women are more likely than White women to report satisfaction with their body size (Anderson, Eyler, Galuska, Brown, & Brownson, 2002). Notably, most prior research has focused on comparisons among women only. Moreover, none that we could identify had explicitly assessed the interactive effects of gender, race, and SES. Evidence has consistently shown that higher SES women tend to have a lower risk of obesity than lower SES women, although the SES effects are less definitive among men (Langenberg, Hardy, Kuh, Brunner, & Wadsworth, 2003; Sundquist & Johansson, 1998; Wardle, Waller, & Jarvis, 2002). Despite higher SES individuals' lower risk of obesity, they tend to report a higher likelihood of feeling overweight (Chang & Christakis, 2003) and higher levels of dissatisfaction with their bodies than lower SES groups (Wardle & Griffith, 2001); this is especially true among women (Allaz et al., 1998; McLaren & Kuh, 2004; Ogden & Thomas, 1999). Although these patterns are paradoxical because higher SES women tend to exercise more, eat healthier, and respond faster to weight gain than low-SES women (Jeffery & French, 1996), they are consistent with the positive association between SES and the risk of eating disorders (Gard & Freeman, 1996; Hsu, 1996). Collectively, these ideas suggest the potential intersection of gender, race, and SES in shaping perceptions about body weight. Yet, few studies (if any) have explicitly tested for Gender × Race × SES interaction effects. One study of female college students, however, provides some clues. Molloy and Herzberger (1998) observed that although low-SES Black women are heavier than high-SES Black women and White women of all SES levels, low-SES Black women rated heavier body types as more attractive than did women in these other groups. Drawing from that research, we suspect that differences between elderly White and Black women in perceived body weight may be greatest at lower levels of SES. Conversely, at higher levels of SES, it is plausible that White and Black women share similar perceptions of their weight—net of gender and race disparities in BMI. In an extension of prior research, we contrast differences among women with an expectation of smaller observed differences between Black and White men in perceptions of weight across BMI and SES levels. An additional component of our analyses proposes that stratification-based differences in levels of BMI suppress differences in weight perceptions for the following reasons. African Americans have higher levels of BMI than White adults (Chang, 2006; Kahng et al., 2004). Overall, 77% of Black women are overweight, compared to about 57% of White women (Flegal et al., 2002). Given that SES is associated negatively with the likelihood of obesity, it is plausible that these patterns will suppress stratification-based differences in perceived weight. McLaren and Kuh (2004), for example, found that a negative association between SES and a measure of “weight esteem” among women emerged only after they statistically adjusted for BMI. This implies that, at the same level of BMI, women of higher SES tend to report more negative appraisals of their own weight compared to women of lower SES. Building off that research, we sought to elaborate these patterns by assessing if gender–race disparities in BMI level suppress gender–race differentials in perceived weight. For instance, White women's lower average level of BMI might suppress their overall tendency toward more negative appraisals of weight relative to other gender–race groups. Taken together, prior theoretical and empirical perspectives provide a basis for the following hypotheses: (a) Women are more likely than men to describe their weight as overweight or obese; (b) among women, White women are more likely than Black women to describe their weight as overweight or obese; (c) in addition, SES may modify these effects such that low-SES White and Black women are more different than high-SES White and Black women; (d) White women's lower BMI suppresses these associations such that, at the same level of BMI, White women will be more likely to describe themselves as overweight or obese; and (e) despite race differences in BMI, we see no compelling rationale for expecting substantial differences in perceptions of body weight between White and Black men. Methods Sample To test our ideas, we used data derived from face-to-face interviews in 2000–2001 with people 65 years and older residing in the District of Columbia and two adjoining Maryland counties, Prince George's and Montgomery. We selected those locales because of their social, racial, and economic diversity. Sample selection and recruitment began with the Medicare beneficiary files for the areas, which identified persons 65 years and older who were entitled to Medicare. Using gender and race information in the Medicare files, we randomly selected 4,800 names equally divided among the three locales, Black adults and White adults, and women and men, creating 12 groups that each contained 400 names. Our goal was to enlist a sample of 1,200 people living independently, with approximately 100 cases in each of the 12 groups. Approximately 65% of all eligible respondents who were contacted (1,741) agreed to participate, yielding a total sample of 1,167 cases. For these analyses, we excluded three cases that were missing responses to focal measures (N = 1,164). The age distribution within the four gender–race groups was similar to the population from the 2000 Census (available upon request). Measures Our question about perceptions of body weight asked, “How would you describe your present weight? Would you say it's about right, you should lose a few pounds, you should lose many pounds, or you should put on some weight?” To simplify the presentation, we refer to “it's about right” responses as perceived appropriate weight, “should lose a few pounds” responses as perceived overweight, “should lose many pounds” responses as perceived obese, and “should put on some weight” responses as perceived underweight. In regression analyses, we compare the appropriate weight category with the perceived overweight, obese, or underweight categories. We acknowledge that this perception implies behaviors related to losing or gaining weight. We infer that if one reports the desire to “lose many pounds” that he or she appraises his or her weight as excessive relative to some standard irrespective of whether that person's BMI is appropriate. We calculated BMI as weight in kilograms divided by the square of height in meters; this was based on self-reported height and weight. Following National Heart, Lung, and Blood Institute (1998) standards, we created the following categories: BMI < 20.0 = underweight, BMI 20.0 to 24.6 = normal weight, BMI 25.0 to 29.9 = overweight, and BMI ≥30.0 = obese. In the descriptive parts of our analyses, we used these categories. However, when we used BMI as a predictor in our analyses, we included it as continuous measure to simplify the presentation and interpretation of the findings. Separate analyses (not shown) found that categorical and continuous versions of BMI as a predictor measure yielded similar results. We coded gender as binary, with 1 for women and 0 for men; likewise, we coded race as binary, with 1 for African American adults and 0 for White adults. To test for Gender × Race differences, we multiplied gender by race and included this interaction term in our models. To measure SES, we standardized and then averaged education and income scores. The education categories were eighth grade or less, some high school but did not graduate, high school graduate or general equivalency diploma, specialized (vocational) training, some college but no degree earned, and college graduate or more (coded 1–6). The income item asked for total household income before taxes in the past year, including salaries for everyone in the household, money market funds, Social Security, pensions, real estate, or government entitlements. Response categories were less than $10,000, $10,000 to $19,999, $20,000 to $29,999, $30,000 to $39,999, $40,000 to $49,999, $50,000 to $59,999, $60,000 to $69,999, $70,000 to $79,999, $80,000 to $89,999, $90,000 to $99,999, and $100,000 or more. Analyses of education and income separately revealed redundancy in their predictive effects. Therefore, results are based on the combined SES index. A few additional comments about the SES index are warranted. First, the correlation between education and income was.54. The overall average education was high school, and the average income was in the $40,000 range. Some readers may wonder about the distribution of cases across SES and whether the data contained sufficient cases for adequate gender–race subgroup comparisons. For the purposes of additional analyses to assess this concern, we defined low SES as scores in the lowest quartile and high SES as scores in the highest quartile. In the low-SES group there were 93 Black men, 133 Black women, 46 White women, and 22 White men. In the high-SES group there were 61 Black men, 34 Black women, 71 White women, and 157 White men. More variation is always ideal, especially at the upper end of the SES scale for Black adults and at the bottom end of the SES scale for White adults. However, in no instance did data sparseness appear to be problematic for the estimates that we present in the Results section. Plan of Analyses We examined the associations between gender, race, and SES with BMI and perceived body weight as dependent measure. In the analysis of perceived weight, multinomial logistic regression models specified the likelihood of perceived overweight, obese, or underweight versus perceived appropriate weight (the comparison group). To test for suppression effects of BMI, we followed a two-step approach in which the first model excluded BMI and the second model adjusted for it. Suppression effects were evident if we observed an increase in the sizes of the gender, race, and SES coefficients once we included BMI in the model. To assess the potential modifying effects of SES, we multiplied SES by gender and race and included these interaction terms in the models. Modifying effects were evident if we observed that the differences between race–gender groups in perceptions of weight varied across levels of SES (Mirowsky, 1999). All analyses adjusted for age, coded in years; we observed no nonlinear age effects. Results Table 1 reports the unadjusted gender–race differences in BMI level. White women had lower average levels of BMI compared to Black women and men. When we categorized BMI into medically defined standards of underweight, normal weight, overweight, and obese, the following gender–race disparities emerged: (a) White women were more likely to have normal BMI levels compared to Black women and Black men; (b) White women were less likely than Black women, Black men, and White men to have overweight BMI; (c) Black women were more likely than White women, White men, and Black men to have obese BMI; and (d) White women were more likely to have underweight BMI compared to the other gender–race groups. Table 1 also reports unadjusted gender–race group differences in perceptions of body weight. Black women were the least likely to describe their weight as appropriate, but only the contrast with Black men was statistically significant. Likewise, Black women were most likely to describe their weight as obese, and the contrasts with Black men and White men were significant. It is important to note that in these unadjusted findings, there were no statistically significant gender–race differences in reporting overweight or underweight perceptions. (As we observed in later analyses, however, larger disparities emerged as statistically significant when we assessed SES variations.) Briefly, we also found that White men reported the highest level of SES, followed by White women, Black men, and Black women; each of these gender–race comparisons was statistically significant. Also, Black men had the lowest average age. It is important to note that BMI decreases with increasing age among older adults. Therefore, some of the unadjusted differences in BMI observed in Table 1 may have been attributable to age differences between the groups. In Table 2, we report unadjusted percentages of perceived weight versus actual BMI categories across gender–race groups. The patterns in the table suggest that Black adults, overall, were generally more likely than White adults to underestimate in their reporting (i.e., describe themselves as appropriate weight when in fact they were classified in overweight or obese BMI categories). In contrast, White women in particular were generally more likely than men and Black women to overestimate in their reporting (i.e., describe themselves as overweight when in fact they were classified as normal BMI). Supplementary analyses that used multinomial logistic regression techniques indicated that these race differences in underestimation and Gender × Race differences in overestimation were statistically significant at the p <.01 level (full results available upon request). Perceived Overweight Versus Perceived Appropriate Comparisons In Table 3, we report results from multinomial logistic regression analyses of perceived weight. For the dependent variable, we used perceived appropriate weight as the comparison group. The three columns of Model 1 show results prior to the statistical adjustment for BMI. The first column displays contrasts between perceived overweight versus perceived appropriate weight. The significant Race × Gender coefficient indicated that gender differences in perceived overweight depended on race. Specifically, without adjustments for BMI, only Black women appeared to differ significantly from men such that they were more likely to describe themselves as overweight. By contrast, none of SES contingencies were significant prior to controlling for BMI. In the first column of Model 2, statistical controls for BMI revealed that it had important suppression effects. The lower order gender, race, and SES disparities in perceptions about weight became substantially larger and statistically significant, indicating that White adults, women, and individuals with higher levels of SES were more likely to describe themselves as overweight than Black adults, men, and low-SES individuals—but only after we took into account race, gender, and SES disparities in BMI. That is, the overall gap between White women and the other gender–race groups widened because White women reported the lowest average BMI. However, the positive three-way interaction coefficient for the Race × Gender × SES term indicated that positive effect of SES was strongest among Black women; at the highest levels of SES, Black and White women converged in the likelihood of describing themselves as being overweight. Figure 1 shows predicted probabilities to illustrate these patterns. In sum, the suppression effects of BMI were critical. Specifically, the SES-contingent similarities and differences among across the race–gender groups became substantially more apparent—especially among women—once we took three patterns into account: (a) White women had the lowest average BMI; (b) Black women had the highest BMI; and (c) SES was associated negatively with BMI. Perceived Obese Versus Perceived Appropriate Comparisons The second column of Model 1 in Table 3 indicates that women were more likely than men to describe themselves as obese. In addition, however, the significant Race × Gender × SES coefficient indicated that differences between women and men varied by both race and SES. Specifically, at higher SES levels, Black women were more likely than White women to describe themselves as obese. Moreover, as we observed for overweight versus appropriate weight comparisons, these observations became even sharper once we adjusted for gender, race, and SES differences in BMI level. As shown in the second column of Model 2, the gender–race disparities in perceptions of being obese became greater. The significant Race × Gender × SES coefficient indicated that the probabilities associated with Black women's description of themselves as obese dramatically increased as SES increased. Figure 2 illustrates these predicted probabilities. Among low-SES individuals, White women were more likely than the other race–gender groups to describe themselves as obese. The likelihood of describing oneself as obese increased with SES for all race–gender groups. However, this increase was the strongest among Black women. Overall, these patterns once again underscored the importance of taking SES contingencies into account. Perceived Underweight Versus Perceived Appropriate Comparisons In Table 3, the third column in Model 1 displays contrasts between perceived underweight versus appropriate weight. Prior to the adjustment for BMI, the gender–race groups reported similar likelihoods of describing themselves as underweight. The third column in Model 2, however, shows that the gender disparities widened net of BMI. Women were less likely than men to describe themselves as underweight. Likewise, SES was associated negatively with the likelihood of describing oneself as underweight. Again, BMI suppression effects occurred because women and higher SES groups reported the lowest average BMI. Therefore, at the same level of BMI, women were more likely than men to describe themselves as underweight. Similarly, at the same level of BMI, high-SES individuals were less likely than low-SES individuals to describe themselves as underweight. Discussion Our observations underscore two previously undocumented themes: (a) Core dimensions of stratification—gender, race, and SES—intersect to shape perceptions of body weight; and (b) stratification-based differences in BMI levels suppress stratification-based differences in perceived body weight. In addition to extending prior research that has focused mainly on samples of adolescents and young women, we also contribute to this literature by documenting Gender × Race contrasts, SES contingencies, and the critical role of BMI as a suppressor. Prior theory and research has contended that norms about body image are most relevant for White women of higher SES. By contrast, research has often found Black women to be more satisfied with their weight and body size. Our findings challenge the uniformity of those claims and build off of previous work that has implied Gender × Race differences. For example, Black adolescents and adult women report less dissatisfaction with their body or weight than their White counterparts. Moreover, by illustrating the ways that gender–race comparisons are also contingent upon SES, we modify the conclusion that being overweight or obese is uniformly more acceptable among Black adults than White adults. We observed that high-SES Black women are more likely to describe themselves as obese even though SES is associated negatively with actual BMI level. Collectively, these findings reinforce the importance of examining the intersection of dimensions of all three forms of stratification—gender, race, and SES—in analyses of body weight. Moreover, the discovery of the suppression effects of BMI indicates that the same level of BMI has different implications for gender, race, and SES differences. Here, our findings parallel previous evidence that although low-SES Black women have a higher BMI than high-SES Black women and White women, they also tend to perceive heavier body types as more attractive than do women in those other groups (Molloy & Herzberger, 1998). It is important to note the possibility, however, that the perceptions of body weight may have less to do with attractiveness and more to do with health concerns or awareness. High SES may represent greater lifetime access to health information that has encouraged weight loss or a more conscientious approach to weight maintenance and a healthier lifestyle. If so, higher SES individuals, regardless of race, may be more prone to reporting a desire to lose weight than their lower SES peers. Thus, perceptions of weight may contain elements of health-related socialization processes, better access to information, or even social desirability influences. Misperceptions about the need to gain or lose weight can have deleterious consequences for the motivation to implement weight-change regimens and other health behaviors. These issues are important in late life because overweight, obesity, and underweight statuses can exacerbate or amplify problems with health conditions and functional limitations. Moreover, the psychosocial impact of perceptions of body discrepancies may represent an understudied stressor for older women. Although it is plausible that the cultural ideals associated with female thinness, the stigma associated with being overweight, and traditional gender-role differences in the importance of attractiveness diminish in later life, the evidence we cited earlier implies the opposite. Gender and race differences may persist into late life. As the population ages, weight-related concerns for elders may become an even more salient element of well-being (Himes, 2000, 2004). Nonetheless, it is important for research to consider whether overweight and obesity are associated differently with health risks in older adults. There is some controversy regarding the extent that overweight is positive or negative for health and mortality risk in late life. As we noted, weight gain or loss may be associated with the loss of muscle mass because of chronic diseases. More evidence is needed about the ways in which these processes are relevant for elders' subjective appraisals of body weight and their decisions about weight loss or gain. Several limitations of our study deserve mention: self-reports and selective survival. Numerous studies have utilized self-reports to measure BMI levels (Boardman, Saint Onge, Rogers, & Denney, 2005; Carr & Friedman, 2005; Chang, 2006; Chang & Christakis, 2005; Crosnoe & Muller, 2004; Eaton, Lowry, Brener, Galuska, & Crosby, 2005; Kuchler & Variyam, 2003), including the Centers for Disease Control and Prevention's Behavioral Risk Factor Surveillance System. Although researchers have argued that self-reports of body weight are highly correlated with scale indicators (e.g., Palta, Prineas, Berman, & Hannan, 1982; Stunkard & Albaum, 1981) and that the bias introduced in self-report data tends to be trivial (Palta et al., 1982), self-reports may be a limitation if elders systematically misrepresent their height and weight. Self-reported weight measures, although often close to objective measures, may be slight underestimates on both ends of the weight distribution (Ferraro & Kelley-Moore, 2003). A study of Australian adults aged 18 and older estimated that 26% of men and 21% of women underreported their weight by 4 pounds or more; likewise, 15% of men and 7% of women overreported their weight by 4 pounds or more (Crawford & Campbell, 1999). The age distribution of these estimates is unclear. Others have shown that individuals aged 65 and older are more likely to believe their weight is healthy or underweight when they are actually obese or overweight (Kuchler & Variyam, 2003). Errors due to underreporting may make estimates conservative (Chang, 2006). To systematically address concern about self-reported BMI, we conducted analyses of the effects of two scenarios that may have biased our estimates. The first scenario assumed that White adults overestimated their weight by 4 pounds, whereas Black adults underestimated their weight by 4 pounds. The second scenario assumed that White women overestimated their weight by 5 pounds, White men overestimated their weight by 2 pounds, Black women underestimated their weight by 5 pounds, and Black men underestimated their weight by 2 pounds. Although other scenarios are possible, we based these values on evidence cited previously. Regression results using BMI scores from these scenarios yielded only a few slight and often trivial changes in some estimates (available upon request). These observations should partially assuage concerns about self-reported BMI. Selective survival is another issue. Elders with extremely high or low BMI may have had a lower probability of being in our sample because of death, impairment, or institutionalization. Therefore, selection bias may have truncated the range of variation in BMI. If an elder loses weight because of a chronic condition, such as cancer, but remains in the normal weight BMI category, then he or she may still believe that it is necessary to put on a few pounds as a preemptive measure against weight loss or from the desire to regain weight that was lost because of illness. These ideas connect with another limitation of our study: There were too few cases in the underweight BMI and perceived weight groups. The distribution and implications of underweight (perceived and actual) are understudied domains. Most research has focused on obesity despite evidence that being underweight has deleterious effects on health (Flegal et al., 2005). Body weight concerns remain important into late life, especially among women. Moreover, perceptions about body weight can influence decisions about health lifestyle. Our study has sought to expand on current knowledge by documenting the ways in which gender, race, and SES intersect to influence these perceptions. Although our observations are generally consistent with the notion of women's dissatisfaction with their weight as a “normative discontent” (Rodin et al., 1984), our study has sought to qualify this claim by revealing important stratification differentials and the suppressor role of stratification-based differences in relative body weight. Decision Editor:Kenneth F. Ferraro, PhD Figure 1. Predicted probabilities of reporting perceived overweight (versus perceived appropriate weight) by gender–race group and SES. Predicted probabilities are based on body mass index and age-adjusted values from Model 2 in Table 3. Low SES = 10th percentile; high SES = 90th percentile; SES = socioeconomic status Figure 2. Predicted probabilities of reporting perceived obese (versus perceived appropriate weight) by gender–race group and SES. Predicted probabilities are based on body mass index and age-adjusted values from Model 2 in Table 3. Low SES = 10th percentile; high SES = 90th percentile; SES = socioeconomic status Table 1. Unadjusted Summary Statistics by Gender–Race Groups. Variable Total (N = 1,164) White Women (n = 293) Black Women (n = 291) White Men (n = 295) Black Men (n = 285) BMI (continuous) 26.85 25.53a,c 28.54b 26.09a,c 27.27a BMI (categorical)     Normal 35.40 43.69a,c 25.09b 40.68 31.93     Overweight 37.46 24.91a,b,c 36.43 46.10 42.46     Obese 22.25 18.43a 35.05b,c 12.20c 23.51     Underweight 4.90 12.97a,b,c 3.44 1.02 2.11 Perceptions of body weight     Perceived appropriate weight 36.51 36.18 29.21c 37.29 43.51     Perceived overweight 38.40 39.25 36.77 43.39 34.04     Perceived obese 18.56 19.11 26.46b,c 13.56 15.09     Perceived underweight 6.53 5.46 7.37 5.76 7.56 Socioeconomic status .003 .062a,b,c −.454b,c .565c −.174 Age 74.828 75.341c 74.828c 75.031c 72.954 Notes: Values for BMI (continuous), age, and socioeconomic status are means; values for BMI (categorical) and perceptions of body weight are percentages. BMI = body mass index. aSignificantly different from Black women (p <.05). bSignificantly different from White men (p <.05). cSignificantly different from Black men (p <.05). Table 2. Perceived Weight and Actual BMI Across Gender–Race Groups. Perception About Weight Normal BMI Overweight BMI Obese BMI Underweight BMI Perceived appropriate weight     Black men 66 46 12 17     Black women 59 27 8 50     White men 64 22 6 33     White women 52 15 2 71 Perceived overweight     Black men 20 44 39 0     Black women 23 54 32 0     White men 24 66 25 0     White women 41 56 39 3 Perceived obese     Black men 1 7 49 0     Black women 0 16 59 0     White men 1 10 69 0     White women 3 29 57 0 Perceived underweight     Black men 13 3 0 83     Black women 18 3 1 50     White men 11 1 0 67     White women 4 0 2 26 Note: Numbers in the table represent percentages of cases in each of the actual BMI categories. BMI = body mass index. Table 3. Results From Multinomial Logistic Regression Analyses Predicting Perceptions About Weight. Model 1 Model 2 Variable Overweight vs Appropriatea Obese vs Appropriatea Underweight vs Appropriatea Overweight vs Appropriatea Obese vs Appropriatea Underweight vs Appropriatea Race (Black = 1) −0.41/0.67 (.22) −0.11/0.90 (.31) −0.13/0.88 (.40) −0.76/0.47** (.26) −0.78/0.46 (.41) −0.01/0.99 (.41) Gender (women = 1) 0.09/1.10 (.22) 0.62/1.85* (.31) −0.24/0.79 (.39) 0.92/2.52*** (.26) 1.60/4.97*** (.41) −1.38/0.25** (.46) SES 0.26/1.29 (.19) 0.30/1.36 (.27) −0.50/0.61 (.34) 0.60/1.83** (.21) 1.10/3.01** (.35) −0.71/0.49* (.34) Race × Gender 0.81/2.24* (.32) 0.91/2.48* (.41) 0.68/1.97 (.59) 0.21/1.23 (.38) 0.11/1.11 (.54) 1.10/3.00 (.63) Race × SES −0.04/0.96 (.24) −0.25/0.78 (.34) 0.12/1.13 (.44) −0.18/0.83 (.28) −0.63/0.53 (.44) 0.52/1.69 (.46) Gender × SES −0.13/0.88 (.27) −0.51/0.60 (.36) 0.47/1.59 (.51) −0.23/0.79 (.31) −0.59/0.55 (.48) 0.54/1.72 (.51) Race × Gender × SES 0.40/1.50 (.37) 1.17/3.23* (.46) −0.06/0.95 (.67) 0.94/2.55* (.44) 1.99/7.29** (.63) −0.95/0.39 (.71) Age −0.08/0.93*** (.01) −0.12/0.89*** (.02) 0.04/1.04* (.02) −0.07/0.93*** (.01) −0.09/0.91*** (.02) 0.03/1.03 (.02) Body mass index — — — 0.44/1.56*** (.03) 0.74/2.09*** (.04) −0.44/0.64*** (.06) Likelihood ratio χ2 169.13 898.67 Intercept 5.72 7.66 −4.86 −6.35 −14.46 6.58 Notes: Numbers shown are logistic regression coefficients/relative risk ratios (SE). 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Socioeconomic status and weight control practices in British adults. Journal of Epidemiology and Community Health, 55,185-190. Wardle, J., Waller, J., Jarvis, M. J. (2002). Sex differences in the association of socioeconomic status with obesity. American Journal of Public Health, 92,1299-1304. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A98E34DA6C21FC9B5A313F5DB24D9FF9F13580A.txt b/test/dataset/in/resources/corpus/Clean_0A98E34DA6C21FC9B5A313F5DB24D9FF9F13580A.txt new file mode 100644 index 0000000..f8fe579 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A98E34DA6C21FC9B5A313F5DB24D9FF9F13580A.txt @@ -0,0 +1 @@ +ageingAge AgeingageingAge and AgeingAge Ageing0002-07291468-2834Oxford University Press20410.1093/ageing/afi204Case ReportsBuried Bumper Syndrome complicated by intra-abdominal sepsis WaltersGareth RameshP. MemonMuhammed Ibrahim Department of Care of the Elderly, Solihull Hospital, Heart of England Foundation Trust, Lode Lane, Solihull, West Midlands B91 2JL, UKAddress correspondence to P. Ramesh. Fax (+44) 0121 424 4611. Email: parthasarathy.ramesh@heartofengland.nhs.ukNovember20053466506513820052872005© The Author 2005. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org2005 There is growing evidence that enteral feeding tubes are associated with increased mortality and complication rates in patients with advanced dementia. Buried Bumper Syndrome is an uncommon, but well documented late complication of PEG placement. Our case report reinforces this recognised risk of PEG feeding in an elderly, cognitively impaired patient. percutaneous endoscopic gastrostomy dementia buried bumper syndrome hwp-legacy-fpage650cover-dateNovember 2005hwp-legacy-docheadCase Report Introduction Percutaneous Endoscopic Gastrostomy (PEG) is used increasingly for long-term enteral support in patients with dementia. However, numerous complications have been reported since its introduction in 1980 [1]. Buried Bumper Syndrome (BBS) is an uncommon, but well documented complication of PEG placement. We report a case of BBS complicated by a large intra-abdominal abscess. Case report An 81-year-old lady with advanced dementia was admitted to our hospital from a nursing home with vomiting and rigors. A PEG had been in situ for 3 years. Examination revealed a large, tender, right-sided abdominal mass. She was tachycardic and pyrexial with raised inflammatory markers. Ultrasound abdomen revealed a large intra-peritoneal abscess. Cultures grew enterococcal species and she was commenced on intravenous antibiotics. Gastroscopy and CT abdomen revealed migration of the PEG internal bumper out of the stomach. The patient was referred to the surgeons for urgent exploration and removal of PEG. She died two days after the procedure following a brainstem infarction. Discussion BBS is a serious complication of PEG tube insertion first described in 1988 [2, 3] and reported to occur in 0.3–2.4% of patients [4]. It is a late complication occurring up to 3 years post PEG insertion [5], but has been described at 21 days [6]. The internal bumper becomes lodged along the gastrostomy tract between the gastric wall and skin. Epithelialisation occurs and the bumper becomes covered with gastric mucosa. Diagnosis should be considered if abdominal pain, peri-tubular leakage, or inability to infuse feed occur. This can be confirmed endoscopically with the help of CT or contrast studies. Failure to recognise the syndrome has resulted in gastric perforation and gastrointestinal haemorrhage [6]. PEG feeding is commonly used to provide nutrition in demented patients. There is no evidence that this provides any improvement in nutritional state, functional capacity or survival and is actually a risk factor for developing aspiration [7]. Additionally, the quality of life of a patient with a PEG tube can be adversely affected once the tube is inserted [8]. This case report reinforces the risks of PEG feeding in advanced dementia. References 1. Gauderer MWl, Ponsky JL, Izant RJ. Gastrostomy without laparotomy: a percutaneous endoscopic technique. J Paediatric Surg 1980; 15 872–5. 2. Shallman RW, Norfleet RG, Hardache JM. Percutaneous endoscopic gastrostomy feeding tube migration and impaction in the abdominal wall. Gastrointest Endosc 1988; 34 367–68. 3. Gluck M, Levant JA, Drennan F. Retraction of Sacks-Vine gastrostomy tubes into the gastric wall: a report of seven cases. Gastrointest Endosc 1988; 34 215. 4. Venu RP, Brown RD. Pastika BJ Erickson LW. The buried bumper syndrome: a simple management approach in two patients. Gastrointest Endosc 2002; 56 582–84. 5. Ballester P, Ammori BJ. Laparoscopic removal and replacement of tube gastrostomy in the management of buried bumper syndrome. Int J Surg 2004; 5 2. 6. Anagnostopoulos GK, Kostopoulos P, Arvanitidis DM. Buried Bumper Syndrome with a fatal outcome, presenting early as gastrointestinal bleeding after percutaneous endoscopic gastrostomy placement. J Postgrad Med 2003; 49 325–27. 7. Finucane TE, Christmas C, Travis K. Tube feeding in patients with advanced dementia. JAMA 1999; 282 1365–70. 8. Monteleoni C, Clark E. Using rapid cycle quality improvement methodology to reduce feeding tubes in patents with advanced dementia. BMJ 2004; 329 491–94. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0A9EC47F214584BB63DD6D111877FD44F5D7B57A.txt b/test/dataset/in/resources/corpus/Clean_0A9EC47F214584BB63DD6D111877FD44F5D7B57A.txt new file mode 100644 index 0000000..141763a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0A9EC47F214584BB63DD6D111877FD44F5D7B57A.txt @@ -0,0 +1 @@ +toxscitoxsciToxicological Sciences1096-09291096-6080Oxford University Press10.1093/toxsci/kfm312ENDOCRINE TOXICOLOGYA Biologically Based Dose-Response Model for Dietary Iodide and the Hypothalamic-Pituitary-Thyroid Axis in the Adult Rat: Evaluation of Iodide DeficiencyMcLanahanEva D.*AndersenMelvin E.†FisherJeffrey W.*1*University of Georgia, Interdisciplinary Toxicology Program, Athens, Georgia 30602†The Hamner Institutes for Health Sciences, Division of Computational Biology, Research Triangle Park, North Carolina 277091To whom correspondence should be addressed at 206 Environmental Health Sciences Department, University of Georgia, Athens, GA 30602-2102. Fax: (706) 542-7472. E-mail: jwfisher@uga.edu.42008312008102224125321020071812200724122007© The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org2008A biologically based dose-response (BBDR) model was developed for dietary iodide and the hypothalamic-pituitary-thyroid (HPT) axis in adult rats. This BBDR-HPT axis model includes submodels for dietary iodide, thyroid-stimulating hormone (TSH), and the thyroid hormones, T4 and T3. The submodels are linked together via key biological processes, including (1) the influence of T4 on TSH production (the HPT axis negative feedback loop), (2) stimulation of thyroidal T4 and T3 production by TSH, (3) TSH upregulation of the thyroid sodium (Na+)/iodide symporter, and (4) recycling of iodide from metabolism of thyroid hormones. The BBDR-HPT axis model was calibrated to predict steady-state concentrations of iodide, T4, T3, and TSH for the euthyroid rat whose dietary intake of iodide was 20 μg/day. Then the BBDR-HPT axis model was used to predict perturbations in the HPT axis caused by insufficient dietary iodide intake, and simulation results were compared to experimental findings. The BBDR-HPT axis model was successful in simulating perturbations in serum T4, TSH, and thyroid iodide stores for low-iodide diets of 0.33–1.14 μg/day. Model predictions of serum T3 concentrations were inconsistent with observations in some cases. BBDR-HPT axis model simulations show a steep dose-response relationship between dietary intake of iodide and serum T4 and TSH when dietary iodide intake becomes insufficient (less than 2 μg/day) to sustain the HPT axis. This BBDR-HPT axis model can be linked with physiologically based pharmacokinetic models for thyroid-active chemicals to evaluate and predict dose-dependent HPT axis alterations based on hypothesized modes of action. To support continued development of this model, future studies should include time course data after perturbation of the HPT axis to capture changes in endogenous iodide, serum TSH, T4, and T3.iodideBBDR modelHPT axisthyroxineTSHpharmacokineticsThe hypothalamic-pituitary-thyroid (HPT) axis regulates many physiologic functions, including metabolism, growth, development, and reproduction. In humans, ingestion of insufficient or excessive amounts of dietary iodide, thyroid-active drugs, or exposure to thyroid-active environmental contaminants can perturb the HPT axis to varying degrees. If HPT alterations are severe enough or occur during a critical period of neurodevelopment, lifelong consequences may occur, such as learning deficits. Iodide deficiency, which leads to hypothyroidism, is the most preventable cause of mental retardation and brain damage throughout the world (Delange, 2001). Insufficient iodide intake is still prevalent in almost one-third of world population (Delange, 2001). Alterations in the HPT axis of laboratory animals are readily demonstrated using thyroid-active compounds or by altering the dietary intake of iodide (Brucker-Davis, 1998; Zoeller, 2007).The process of thyroid hormone formation is highly regulated. The thyroid gland actively sequesters iodide via the sodium (Na+)/iodide(I−) symporter (NIS), which is an indispensable component of thyroid hormones. Iodide is then available for incorporation and use in thyroid hormone production. The normal thyroid gland produces thyroxine (T4) in greater quantities than the biologically active hormone 3,5,3′-triiodothyronine (T3) (Greer et al., 1968). T4 and T3 are secreted from the thyroid gland into systemic circulation, where T4 can be metabolized to T3 in peripheral tissues by a family of enzymes called 5′-deiodinases. When circulating blood levels of T4 and T3 are low, the anterior pituitary gland produces more thyroid-stimulating hormone (TSH), a classical negative feedback loop. TSH, delivered by blood to the thyroid gland, binds to receptors on the plasma membrane of thyroid follicular cells. This receptor-TSH complex regulates second messenger cascades that stimulate thyroidal processes such as the increase in NIS expression and activity and increased production of thyroid peroxidase (TPO) and thyroglobulin (Tg) (Kogai et al., 2006). These orchestrated biochemical events ultimately allow for compensatory increases in thyroidal uptake of iodide and production and secretion of T4 and T3.Recently, several laboratories have reported on the potency of anions, which are environmental contaminants, to block thyroidal uptake of radiolabeled iodide in laboratory animals (perchlorate and nitrate, Tonacchera et al., 2004; Yu et al., 2002) and humans (perchlorate, Greer et al., 2002). The ability of perchlorate to alter the HPT axis in humans (e.g., increase serum TSH levels and decrease serum T4 levels) appears to be feeble under conditions of high dietary iodide intake (Crump et al., 2000; Téllez et al., 2005) and significant for mildly iodide-deficient women (Blount et al., 2006). In this paper, a biologically based dose-response (BBDR) model for the HPT axis in the adult rat is developed to evaluate iodide deficiency as a first step in understanding the relationship between dietary iodide intake, potency of anions to block thyroidal uptake of dietary iodide, and disruption of the HPT axis.Pharmacokinetic models have played an important role in understanding the quantitative aspects of the HPT axis. DiStefano and colleagues have published several kinetic papers on this topic. In particular, DiStefano et al. (1982) and DiStefano and Feng (1988) used a three-compartment rodent model for the thyroid hormones, T4 and T3, to estimate thyroid hormone production and metabolic clearance rates. Li et al. (1995) also used a compartmental approach to simulate the pulsatile release of TSH in humans. In 1996, Kohn et al. developed a rodent HPT axis submodel linked with a physiological model for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to evaluate TCDD-mediated induction of hepatic T4 metabolism and clearance. More recently, Dietrich et al. (2002) described the negative feedback of the HPT axis in humans and the pulsatile secretion of TSH. Mukhopadhyay and Bhattacharyya (2006) also described the pulsatile secretion of TSH in humans using time delays to relate production of T4 with TSH secretion. Physiologically based pharmacokinetic (PBPK) models for radiolabeled iodide (125I) and perchlorate in rodents and humans have been developed for different life stages (Clewell et al., 2003a,b; Merrill et al., 2003, 2005) to evaluate the impact of perchlorate on thyroidal uptake of 125I.Although scientists have constructed compartmental models to describe the HPT axis, published models were not found that take into account TSH production and dietary iodide (127I) linked to T4 and T3 formation and secretion. Thus, a quantitative BBDR-HPT axis model was developed to include the most informative serum hormones, namely, T4 and T3, and the signaling molecule, TSH, and dietary iodide. Features in the BBDR-HPT axis model include the active transport and regulation of iodide uptake into the thyroid by the NIS, T4/TSH negative feedback loop, TSH stimulation of thyroidal processes, extrathyroidal metabolism of T4 to form the biologically active T3, and recycling of metabolically derived iodide from extrathyroidal metabolism of thyroid hormones.MATERIALS AND METHODSThe BBDR-HPT axis submodels for the adult rat were constructed using simple model structures. The production of thyroid hormones (Equation 14) is controlled, in part, by the model predicted serum TSH concentration, and the maximal rate of active sequestration of iodide into the thyroid (Equation 2) is also controlled by the serum TSH concentration. This infers an instantaneous rate of change in protein synthesis without the use of delay functions or other equations to account for protein synthesis or degradation rates. This simple approach was adequate because BBDR-HPT axis model predictions were compared to experimental data under quasi steady-state conditions (days to months). This BBDR- HPT axis model was not validated against data to predict the initial onset of HPT axis disturbances for less than 24 h. Radiotracer time course data (supplementary data) were used to assist in obtaining preliminary parameter values for each subcompartment in the euthyroid adult rat. Other investigators have recently described endocrine systems, using serum levels of signaling molecules to control feedback loops such as the adult male rat hypothalamic-pituitary-gonadal (HPG) axis (Barton and Andersen, 1998) and the human HPG axis/menstrual cycle (Rasgon et al., 2003; Schlosser and Selgrade, 2000).Models were coded using acslXtreme version 2.4.0.11 (Aegis Technologies, Huntsville, AL) and solved with the Gear algorithm for stiff systems. Standardized units of nanomoles (nmol), liters (L), kilograms (kg), and hours (h) were used in the submodels. The approach for the development of the BBDR-HPT axis model was to first create simple and independent submodel structures (supplementary data) for radiolabeled iodide, radiolabeled TSH, radiolabeled T4, and radiolabeled T3 using radiotracer studies reported in literature for the adult rat. This provided several BBDR-HPT axis model parameter values, although sometimes preliminary. Additional data pertaining to the metabolism and excretion of T4 and T3 were used to guide model development.The submodels for iodide, TSH, T4, and T3 were linked as an interactive system to simulate the HPT axis in the euthyroid adult rat. The euthyroid steady-state BBDR-HPT axis model relied on dietary iodide as the only exogenous input. Finally, the calibrated euthyroid, iodide-sufficient adult rat BBDR-HPT axis model was tested for its ability to predict perturbations in the system under iodide-deficient conditions.Submodel Structure and Key EquationsIodide.Iodide was described as distributing into a volume of distribution (Vd) and thyroid gland (Fig. 1). Iodide is rapidly absorbed to the bloodstream from the digestive tract and quickly diffuses into extracellular spaces throughout the body. Iodide fate is largely determined by a competition between thyroidal sequestration and urinary excretion (Verger et al., 2001). Urinary excretion of iodide is described as a first-order clearance from the Vd. Uptake of iodide into the thyroid compartment is described assuming active uptake by the NIS and diffusion (Fig. 1).FIG. 1.BBDR-HPT axis model structure for the adult rat HPT axis is composed of submodels (shaded in gray) for dietary iodide, TSH, T4, and T3. Solid arrows () represent blood flows, bold arrows () within tissue compartments represent active uptake, diffusion limitation represented by solid arrows () within tissue compartments, metabolic links symbolized as dashed arrows () between submodels. Dashed and dotted arrow () represents the use of dietary iodide in thyroid hormone production, and the bold () arrows connecting model processes show control (stimulation or inhibition) by the compound. Details shown in the figure are as follows: formation of free iodide from T3 metabolism in the Vd and liver; formation of free iodide from T4 to T3 metabolism in the Vd and liver; loss of bound thyroidal iodide secreted as thyroid hormones; metabolism of T3 (metabolism to free iodide and fecal elimination of T3); deiodination of T4 in the liver to T3 and free iodide; phase II metabolism (conjugation of T4) and excretion into feces; TSH stimulation of NIS iodide uptake; TSH stimulation of organification of iodide, forming thyroid hormone precursors; TSH stimulation of thyroid hormone production; T4 negative feedback on TSH production.Free iodide enters the thyroid in two ways as follows: (1) active uptake by NIS and (2) diffusion via ion channels. NIS is a plasma membrane protein that actively transports two sodium molecules with one iodide molecule down the sodium ion gradient generated by sodium-potassium ATPases (Kogai et al., 2006). TSH has been shown to stimulate NIS mRNA production, NIS protein expression, and retention in the plasma membrane (Carrasco, 1993; Kogai et al., 1997, 2000; Levy et al., 1997; Riedel et al., 2001; Sherwin and Tong, 1974). Sherwin and Tong (1974) found that TSH-induced stimulation of iodide transport increased the rate of iodide uptake and did not affect the affinity (Kmi) of iodide for the transporter. Thus, iodide uptake into the thyroid via the NIS (rTNISi, nmol/h) and TSH-stimulated maximum NIS iodide transport rate (VmaxTiTSH, nmol/h) were described as follows:(1)(2)where Cvti (nmol/l) is the free concentration of iodide in thyroid blood, Kmi (nmol/l) is the affinity constant of iodide for the NIS, VmaxTi (nmol/h) is the maximum rate of NIS iodide uptake, CaTSH (nmol/l) is the serum concentration of TSH, and KNISTSH (nmol/l) is the serum concentration of TSH that results in half-maximal TSH stimulation of VmaxTi.Once iodide enters the thyroid by NIS-active uptake or diffusion, iodide is incorporated (organified) by binding to tyrosine residues present in Tg via a TPO-mediated mechanism (Degroot and Niepomiszcze, 1977). This bound fraction of iodide in this model represents the thyroidal iodide pool that is attached to Tg and the folding and formation of TH attached to the Tg backbone. TSH increases the expression of many genes involved in thyroid hormone synthesis, including Tg and TPO (Kogai et al., 2006). The rate of incorporation of iodide (rIB, nmol/h) into thyroid hormone precursors and TSH stimulation (VmaxBiTSH, nmol/h) of the organification process is described by:(3)(4)where CTFi (nmol/l) is the free concentration of iodide in the thyroid, Kbi (nmol/l) is the concentration of free iodide in the thyroid when binding rate is half maximal, VmaxBi (nmol/h) is the maximum rate of organification of iodide, and KbTSH (nmol/l) is the concentration of serum TSH that results in half-maximal TSH stimulation of VmaxBi.Loss of free iodide from the thyroid by outward diffusion was described using an estimated permeability cross-product (PATi), and loss of bound iodide as thyroid hormones is described in Equations 14–16. Thus, the thyroid tissue compartment for iodide was described for free (dATFi/dt, nmol/h), bound/thyroid hormone incorporated (dATBi/dt, nmol/h), and total (ATi, nmol) iodide by the following equations:(5)(6)(7)where rT4prod is the secretion rate of T4 from the thyroid (nmol T4/h, Equation 16), T4ieq is the molar fraction of iodide in a T4 molecule (0.6534), rT3prod is the secretion rate of T3 from the thyroid (nmol T3/h, Equation 15), and T3ieq is the molar fraction of iodide in a T3 molecule (0.5848).Thyroid-stimulating hormone (TSH), thyroxine (T4), and 3,5,3′-triiodothyronine (T3).TSH does not distribute into tissues, thus a one-compartment submodel for TSH was constructed using a Vd with a first-order clearance (Fig. 1). Each thyroid hormone submodel was developed with a Vd and liver compartment (Fig. 1). Bidirectional diffusion of T4 in the liver was included in the description of hepatic influx and efflux. T4 has also been shown to be actively transported into the liver by a high-affinity, low-capacity transporter, as well as a low-affinity, high-capacity transporter (Krenning et al., 1981). However, the rate of hepatic uptake of T4 (rLUT4, nmol/h) was simplified and described using a single Michaelis-Menten equation:(8)where VmaxT4LU is the maximal rate of active uptake of T4 into the liver (nmol/h), KmT4LU is the affinity constant for T4 active transport (nmol/l), and CvlT4 is the concentration of T4 in the liver venous blood (nmol/l). Since at least 99% of T4 is bound to serum proteins in rodents (Mendel et al., 1992), the submodel code was modified to reflect only free-serum T4 (1% of total serum T4) available for active transport and diffusion into the liver. Phase II saturable metabolism of T4 in the liver was described using Michaelis-Menten metabolism equations for glucuronidation (forming T4-glucuronide, T4-G) and type I 5′-deiodination of T4 (forming T3 and free iodide). The diffusion-limited liver blood compartment for T4 was described using the equations:(9)(10)where dALbT4/dt is the rate of change of T4 in the liver blood (nmol/h), QL is the blood flow to the liver (l/h), CaT4 is the arterial blood concentration of T4 perfusing the liver (nmol/l), PALT4 is the liver permeability area cross-product for T4 bidirectional diffusion (l/h), CvlT4 is the concentration of T4 in the liver venous blood (nmol/l), VLb is the volume of liver blood (l), and PLT4 is the T4 liver:blood partition coefficient (unitless).The liver tissue compartment for T4 was described as follows:(11)(12)where dALT4/dt (nmol/h) is the rate of change of T4 in the liver tissue, CLT4 (nmol/l) is the concentration of T4 in the liver, rLUT4 (nmol/h, Equation 8) is the rate of active uptake of T4 into the liver from liver blood, rDILT4 (nmol/h) is the Michaelis-Menten metabolic equation for rate of T4 conversion to T3 and free iodide by type I 5′-deiodinating enzymes, rUGTT4 (nmol/h) is also a saturable Michaelis-Menten metabolism equation for the rate of formation of T4-G formation, and VL (l) is the volume of the liver. T4 has also been shown to undergo other hepatic metabolic processes, such as sulfation (T4-S formation), which accounts for a small fraction (6%) of overall T4 metabolism (Rutgers et al., 1989), and T4-S is rapidly deiodinated in the liver (Visser et al., 1990). To account for the rest of the body metabolism of T4 to T3, a first-order metabolism of T4 was included as a loss from the Vd compartment.Similar to T4, transport of T3 into the liver compartment was described by bidirectional diffusion and active uptake by a transporter protein (Fig. 1). Experimental evidence for hepatic transporter uptake of T3 from blood suggests that T3 uptake is not saturable at physiological conditions (Blondeau et al., 1988); thus, the active uptake was described as a first-order process. Hepatic metabolism of T3 in the liver was also described as a first-order process, with the assumption that a percentage of the metabolized T3 is excreted in feces as T3 conjugates (T3-G, T3-S, etc.). The remainder is metabolized to free iodide, assuming T3 metabolism to T2 is the rate-limiting step in releasing free iodide. The fraction of T3 metabolism excreted in feces (FT3feces, 0.30) was fit to provide an approximation (26%) of the percent dose of T3 excreted in feces (4.9–54.9%; DiStefano and Sapin, 1987; DiStefano et al., 1993). First-order metabolism of T3 was included in the Vd to account for rest of body metabolism of T3 to T0, also assuming that T3 to T2 is the rate-limiting step.Linking the submodels to create a BBDR-HPT axis model.The submodels described in supplementary data for iodide, T4, T3, and TSH are linked as shown in Figure 1. All compartments for each submodel were assigned steady-state–derived masses at the onset of the simulations. The initial amounts of TSH, iodide, or thyroid hormones were established by running simulations to steady state with a dietary iodide intake of 20 μg/day. Dietary intake of iodide was assumed to take place over a 12-h period, with food/iodide consumption occurring during the night hours (7:00 P.M.–7:00 A.M.).TSH is secreted by the anterior pituitary and is found in systemic circulation. Briefly, the TSH one-compartment model (described in supplementary data) in the linked BBDR-HPT axis model was modified to include an endogenous production term (Equation 13). The production of TSH is based on the primary negative feedback loop of the thyroid axis; that is, adequate levels of serum thyroid hormones result in a normal secretion of TSH from the pituitary, but when serum thyroid hormone levels decrease, the feedback control is diminished and TSH production rate increases. Several researchers have shown a negative correlation between serum T4 and TSH concentrations (Fukuda et al., 1975; Pedraza et al., 2006; Riesco et al., 1977). This is a primary experimental observation reported by several laboratories and is used in the development of the negative feedback loop for the BBDR-HPT axis model. Since total serum T4 is a common measurement in most thyroid disruptor studies, as opposed to free T4, the TSH/T4 negative feedback loop was described using total serum T4 as shown in Equation 13. The empirical description of TSH production is regulated by the model-predicted total serum T4 concentration (CaT4). The complete equation used to determine the amount of TSH in the Vd was as follows:(13)where k0TSH (nmol/h) is the maximal production rate of TSH in the absence of T4, KT4inh (nmol/l) is the estimated concentration of T4 in the serum which results in half-maximal production rate of TSH, CaT4 (nmol/l) is the total T4 serum concentration, and CaTSH (nmol/l) is the TSH serum concentration calculated by dividing the integral of Equation 13 by VdTSH (l).The rate of overall thyroidal production (release of T4 and T3 from Tg backbone—stored thyroidal iodide) and secretion of thyroid hormones (T4 and T3) were determined by a fitted rate constant (kTSHIB) times the model predicted serum concentration of TSH and concentration of available thyroidal iodide in the form of hormone precursors:(14)where kTSHIB (l2/nmol/h) is a linear rate term, CaTSH (nmol/l) is the serum concentration of TSH, and CTBi (nmol/l) is the concentration of bound thyroidal iodide as thyroid hormone precursors. The proportion of thyroid hormones produced as T3 and T4 was then described as a fraction of the total production rate, using the following equations:(15)(16)where rT3prod (nmol/h) is the rate of thyroidal T3 production and rT4prod (nmol/h) is the rate of thyroidal T4 production. The ratio of T3/T4 secretion increases during iodide deficiency. To account for this, Equation 17 was derived from Pedraza et al. (2006), who collected experimental data on total thyroidal iodide stores and thyroidal T3/T4 ratios for different iodide intake rates. FT3 (unitless) is the fraction of overall thyroid hormone production within the thyroid that is T3 and was modeled as:(17)where ATi (μg) is the total amount of iodide in the thyroid, as calculated in Equation 7 and converted to micrograms. In iodide-deficient conditions, a shift from primarily T4 to T3 production in the thyroid occurs (Greer et al., 1968; Pedraza et al., 2006). This may be due to the increase in deiodination of T4 in the thyroid or simply the formation of less T4 because less iodide is needed to make T3. However, no instances have been reported where the thyroid synthesizes only T3 at the cost of zero T4 production. A MIN command was implemented in acslXtreme to ensure that the exponential FT3 function (Equation 17) did not exceed 0.90.Data sets used in steady-state euthyroid BBDR-HPT axis model calibration.Serum T4, and TSH, along with total thyroid iodide data from adult male Sprague-Dawley rats published by McLanahan et al. (2007) and serum T3 data (unpublished data) from our laboratory were used to calibrate the model for steady-state euthyroid conditions in the adult rat (320 g). It was also important to include liver T4 and T3 concentrations for calibration; however, there are few data sets with tissue concentrations of thyroid hormones. Liver T4 and T3 concentrations reported by Morreale de Escobar et al. (1994) in euthyroid adult female Wistar rats were used in BBDR-HPT axis model calibration. Additionally, the only study found to report measured free iodide serum concentrations was Eng et al. (1999) who reported data for euthyroid (control) adult male Sprague-Dawley rats.Model ParametersModel parameters were derived from the published literature whenever possible. Default assumptions for allometric scaling were employed. Thus, blood flows (Q), maximum velocities (Vmax)1 (An evaluation of literature for total thyroid iodide (127I) concentrations for the range of body weights simulated in this study (120–500 g) showed slight change in total amount of thyroidal 127I. The model parameter maintaining the stores in the thyroid is VmaxBci (Vmax for iodide incorporation into thyroid hormone precursors). Thus, to empirically describe total thyroid 127I concentrations, the value of VmaxBci was divided by BW0.75.), and permeability area cross-products (PA) were multiplied by body weight (BW)0.75 and clearance rates (Cl and kel) were divided by BW0.25. Volumes of distribution (Vd) were scaled linearly with BW.Physiological parameters.Growth equations developed by Mirfazaelian et al. (2007) were used to account for body weight changes for simulations that were longer than 1 month. Otherwise, the terminal body weight reported for the study was used in simulation. Blood flows and tissue volumes (V) were obtained from literature (Brown et al., 1997; Malendowicz and Bednarek, 1986; McLanahan et al., 2007). Physiological parameters are shown in Table 1.TABLE 1Physiological Parameters for the Adult RataParameterValueSourceTissue volumes    Liver, VLc (% BW)3.66Brown et al. (1997)    Liver blood, VLBc (% VL)21Brown et al. (1997)    Thyroid, VTc (% BW)0.005McLanahan et al. (2007)    Thyroid blood, VTBc (% VT)15.7Malendowicz and Bednarek (1986)Blood flows    Cardiac output, QCc (l/h/kg0.075)14.0Brown et al. (1997)    Liver, QLc (% QC)17.4Brown et al. (1997)    Thyroid, QTc (% QC)1.6bBrown et al. (1997)aBody weight (BW, kg) is not shown in this table because body weights reported in each study were used in model simulations.bHuman value.Literature-derived compound-specific parameters.When possible, compound-specific parameters for each submodel were derived from literature. Parameters for iodide, T4, T3, and TSH are shown in Table 2. Liver partition coefficients for T4 (PLT4, 1.27) and T3 (PLT3, 4.47) were determined from steady-state serum and liver concentrations reported by Escobar-Morreale et al. (1996) for female euthyroid, control rats. These values are similar to the values used by Kohn et al. (1996) for T4 and T3 liver partition coefficients (1.632 and 2.22, respectively) that were estimated from Kow values and the use of various regression equations.TABLE 2Compound-Specific ParametersParameterValueSourceVolume of distribution (% BW)    Iodide, Vdci50aVisual Fit    TSH, VdcTSH5.54Connors et al. (1984)    T4, VdcT415.6bKohn et al. (1996)    T3, VdcT318.6bDiStefano (1986)Partition coefficients (unitless)    T4—liver:blood, PLT41.27Escobar-Morreale et al. (1996)    T3—liver:blood, PLT34.47Escobar-Morreale et al. (1996)Permeability area cross-products (l/h/kg0.75)    T4—liver blood to liver tissue, PALcT40.0423Optimized    T3—liver blood to liver tissue, PALcT30.1699Optimized    Iodide—thyroid blood to thyroid tissue, PATci0.0001Merrill et al. (2003)Affinity constants (nmol/l)    Iodide—thyroid NIS, Kmi31519Gluzman and Niepomniszcze (1983); Merrill et al. (2003)    TSH—thyroid NIS, KTSHNIS0.949Optimized    Iodide—iodide organification in thyroid, Kbi244.59Optimized    TSH—iodide organification in thyroid, KbTSH733.98Optimized    T4—liver type I 5′-deiodinase, KmT4DI2300Leonard and Visser (1986)    T4—liver glucuronidation, KmT4UGT1 × 105Visser et al. (1993)    T4—liver uptake, KmT4LU650Blondeau et al. (1988)Maximum velocities (nmol/h/kg0.75)    Iodide—thyroid NIS, VmaxTci5738.267Optimized    Iodide—iodide organification in thyroid, VmaxBci1005.9cOptimized    T4—liver type I 5′-deiodinase, VmaxcT4DI19.89Optimized    T4—liver glucuronidation, VmaxcT4UGT3435.89Optimized    T4—liver uptake, VmaxcT4LU4384.73OptimizedT3—first-order liver uptake, kmT3LU (l/h)1.25OptimizedClearance values    Iodide—urinary excretion, ClUci (l/h/kg0.25)0.0046Optimized    TSH—Vd clearance, kelcTSH (/h/kg0.25)1.8899Lemarchand-Beraud and Berthier (1981)    T4—Vd metabolism, kelcT4 (/h/kg0.25)0.05dAbrams and Larsen (1973)    T3—Vd metabolism, kelcT3 (/h/kg0.25)0.12dAbrams and Larsen (1973)    T3—liver metabolism, kmetLcT3 (/h/kg0.25)3.65Optimized    T3—fraction of liver T3 metabolism excreted in feces, FT3feces (unitless)0.30Visually FitTSH/thyroid hormone production parameters    Thyroid hormone production constant, kTSHIB (l2/nmol/h)5 × 10−7Visually Fit    Maximum rate of TSH production in the absence of T4, kTSH0 (nmol/h/kg0.75)6Connors et al. (1984)    T4 concentration for half-maximal TSH production, Kinh_T4 (nmol/l)0.2OptimizedaIodide Vd used in the model was calculated by subtracting VTc from Vdci.bVd for T4 and T3 were calculated by subtracting VLc from VdcT4 and VdcT3, respectively.cScaled by dividing by BW0.75. See footnote 1.dCalculated from serum half-life using kel = ln 2/t1/2.The Vd for T4, T3, and TSH were obtained from literature, as shown in Table 2, and the volume of the liver was subtracted from the Vdc for T4 and T3. The Vd for T4 (VdcT4, 15.6% BW) was obtained from the thyroid hormone model developed by Kohn et al. (1996), whereas the Vd for T3 (VdcT3, 18.6% BW) was used as estimated by DiStefano (1986) with a simple compartmental model for T3. A TSH Vd (VdcTSH, 5.54% BW) was used as reported by Connors et al. (1984).Clearance terms to account for metabolism in the Vd for TSH, T4, and T3 were calculated from literature values using the relationship(18)where t1/2 (h) is the serum half-life of the compound reported as 0.3667 h for 125I-TSH (Lemarchand-Beraud and Berthier, 1981) and 6 and 12 h for T3 and T4, respectively (Abrams and Larsen, 1973).Affinity constants, Km(s), for metabolism and active transport of iodide and T4 were obtained from the literature (Table 2). The affinity constant for thyroid iodide transport by the NIS (Kmi) of 3.1 × 104 nmol/l was the average value reported by Gluzman and Niepomniszcze (1983), using radiolabeled iodide and euthyroid human and porcine thyroid cells. The affinity constant for active uptake of T4 into the liver (KmT4LU) of 650 nmol/l was reported by Blondeau et al. (1988) using rat hepatocytes. Michaelis-Menten saturable metabolism of T4 in the liver was described for the phase II glucuronidation and deiodination pathways. The saturable metabolism of T4, by type I 5′-deiodination, was described assuming that one molecule of T3 is formed and one molecule of free iodide is released for each molecule of T4 metabolized. Phase II metabolism of T4 (formation of T4-G) occurs by a reaction catalyzed by uridine diphosphate glucuronyl transferases. The Km value for the type I 5′-deiodinase metabolism of T4 (KmT4DI, 2300 nmol/l) was obtained from Leonard and Visser (1986) from in vitro metabolic studies, and the Km for the formation of T4-G (KmT4UGT, 1 × 105 nmol/l) was taken from Visser et al. (1993) in vitro studies in Wistar rat liver microsomes. For each of these saturable metabolic processes, the Km values were derived from the literature, and Vmax values were optimized to fit serum kinetics of T4 that resulted in values that were close to the literature reported radiotracer data for fraction of T4 metabolized to T3 (14–27%, DiStefano et al., 1982) and fraction of T4 excreted in feces (10–38%, DiStefano and Sapin, 1987; Nguyen et al., 1993).When submodels were combined to form the BBDR-HPT axis model, endogenous production of TSH was described as shown in Equation 13. The maximal rate of TSH production (k0TSH) was set to the maximum value (6 nmol/h) of TSH secretion reported by Connors et al. (1984) 14 days after thyroidectomy in adult female Sprague-Dawley rats (Table 2).Parameter optimization.Model parameters not available in literature were first optimized to fit each radiotracer data set (125I, 131I-T4, and 125I-T3, described in supplementary data); then when the models were linked to form the BBDR-HPT axis model, parameters were reoptimized to fit euthyroid, steady-state, iodide-sufficient (20 μg iodide/day) conditions. Volume of distribution for iodide (Vdci, 50% BW) and the linear rate term for thyroid hormone production (kTSHIB, 5 × 10−7 l2/nmol/h) were determined from visual fits. Global optimization was performed for model parameters in the BBDR-HPT axis model. During this optimization, all model parameters were optimized at steady state, for euthyroid and iodide-sufficient (20 μg I/day) conditions (serum and liver T4 and T3, serum TSH, serum-free iodide, and total thyroidal iodide). Additional data for metabolism were included in the optimization, as described previously. Optimization of model parameters was performed using acslXtreme Parameter Estimation version 2.4.0.11 (Aegis Technologies).Model Performance AnalysisThe predictive ability of the model was determined by calculating the area under the curve (AUC) ratios for each metric (serum T4, serum T3, serum TSH, and total thyroid iodide), as described by Gustafson et al. (2002). To determine the AUC predicted/measured (P/M) ratio, the BBDR-HPT axis model–predicted AUC was divided by the data-derived AUC for each of the four data sets (Figs. 2–4) tested under iodide-deficient conditions. The AUC P/M ratio calculation was:(19)where AUCpredicted is the model-predicted AUC for serum T4, T3, TSH, or total thyroid iodide and AUCexperimental is the corresponding data-derived AUC. Average AUC P/M ratios were calculated for each metric and overall model performance.FIG. 2.Short-term effects of feeding a low iodide diet (0.35 μg I/day) on serum thyroid hormones and total thyroid iodide of adult male HSD rats. Rats began a low iodide intake of approximately 0.35 μg I/day on day 0 and continued for 26 days (Riesco et al., 1977). Model simulations are represented by lines for serum T4 (, ng/ml), T3 (, ng/ml), TSH (, fold change), and total thyroid 127I (, μg). Data for serum T4 (□ ± SD), T3 (▪ ± SD), TSH (○), and total thyroid 127I (• ± SD) were adapted from Riesco et al. (1977).FIG. 3.Long-term effects of a low iodide diet (0.33 μg I/day) on serum thyroid hormones and total thyroid iodide of adult male (A) SA and (B) HSD rats. On day 0, rats began a low iodide intake of approximately 0.33 μg I/day and continued for 84 days (Okamura et al. 1981a). Model simulations are represented by lines for serum T4 (, ng/ml), T3 (, ng/ml), TSH (, fold change), and total thyroid 127I (, μg). Data were adapted from Okamura et al. (1981a) for serum T4 (▾ ± SD), T3 (▪ ± SD), TSH (○), and total thyroid 127I (• ± SD).FIG. 4.Long-term effects of a low iodide diet (1.14 μg I/day) on serum thyroid hormones and total thyroid 127I of adult male HSD rats. On day 0, rats were administered a low iodide diet providing 1.14 μg I/day and continued for 96 days (Okamura et al., 1981b). Model simulations are represented by lines for serum T4 (, ng/ml), T3 (, ng/ml), and total thyroid 127I (, μg). Data for serum T4 (▾ ± SD), T3 (▪ ± SD), and total thyroid 127I (• ± SD) were adapted from Okamura et al. (1981b).Sensitivity AnalysisAn analysis of model parameter sensitivity under steady-state conditions was determined for predicted serum concentrations of T4, T3, and TSH and total thyroidal iodide content. Normalized sensitivity coefficients (NSCs) were calculated that represent a fractional change in output corresponding to a fractional change in the parameter (Clewell et al., 2000; Merrill et al., 2003; Tornero-Velez and Rappaport, 2001). Model parameters were increased by 1% and the model executed using iodide-sufficient (20 μg/day) and iodide-deficient (1 μg/day) intakes. The NSCs were calculated using the equation:(20)where A equals the model prediction (serum T4, T3, TSH, or total thyroid iodide) with a 1% increase in parameter value, B is model prediction with original parameter value, C is parameter value increased by 1%, and D is original parameter value.Application of BBDR Model to Iodide DeficiencyStudies were available that provided weekly to monthly time-course information on iodide deficiency–induced HPT axis alterations (Okamura et al., 1981a,b; Riesco et al., 1977). In one case, recovery from iodide deficiency (Fukuda et al., 1975) was reported. These papers contained the most complete experimental data sets (iodide content of the diet, serum T4, T3, TSH, and total thyroid iodide). Many other studies prior to 1970 have been conducted; however, they were considered incomplete for modeling purposes. Average daily iodide intake was calculated by multiplying food consumption (20 g/day assumed when not reported for the study) by the iodide content in the diet (μg/g). To compare across studies, we have reported the intakes as micrograms iodide per day. The iodide deficiency data sets simulated using our BBDR-HPT axis model are briefly described below.Riesco et al. (1977) provided adult male Holtzman-Sprague-Dawley (HSD) (120 g) rats a low iodide diet resulting in intake of 0.3–0.4 μg I/day for a short-term iodide deficiency study. They determined serum T4, T3, TSH, and total thyroid iodide after 0, 2, 4, 6, 8, 11, 15, and 26 days of feeding the low iodide diet. An average intake of 0.35 μg I/day was used in model simulation. A longer time course for HPT response of rats maintained on a low iodide diet was reported by Okamura et al. (1981a). Adult male Simonsen Albino (SA) and HSD rats were divided by strain and provided a low iodide diet of 0.3–0.36 μg I/day (15–18 μg I/kg chow). Average intake of 0.33 μg I/day was used in model simulation. Measurements of serum T4, T3, TSH, and total thyroid iodide were obtained after 0, 14, 28, 56, and 84 days of feeding the low iodide diet. SA rats appeared to display a greater sensitivity or degree of HPT axis response to the low iodide diet than the HSD rats. In another study by Okamura et al. (1981b), they examined the opposing effects of iodide and nutritional deficiency, by administering two different low iodide diets (ICN Remington and Teklad Remington). The ICN Remington diet was not considered. Adult male HSD rats (139 g) were administered the Teklad Remington (57 ng I/g or 1.14 μg I/day, nutritionally adequate) diet and killed on days 19, 33, 63, and 96 of treatment for measurements of serum T4, T3, TSH, and total thyroid iodide. No baseline TSH levels were reported; thus, fold change in serum TSH levels were not used in this study. Fukuda et al. (1975) evaluated the recovery of the HPT axis in rats that were placed on iodide supplement after a low iodide diet. Adult male Sprague-Dawley rats (400–500 g) were placed on an iodide-deficient diet of 0.6 μg I/day (30 μg I/kg chow) for 7 months and then provided 2 or 8 μg I/day for 9 days in drinking water. The average iodide intake during the recovery period was 2.6 or 8.6 μg I/day. Serial blood samples were taken, and measurements of serum T4 and TSH were obtained 0, 1, 2, 3, 6, and 9 days during supplementation. A wide range of serum TSH concentrations were observed at the onset of iodide supplementation, and some serum T4 concentrations were reported as nondetectable. The data sets for the recovery period were expressed as percent of baseline.RESULTSDietary Iodide BBDR-HPT Axis Model—Model Calibration and Simulation of Steady-State Euthyroid, Iodide-Sufficient ConditionsWhen the radiotracer submodels were linked to create the BBDR-HPT axis model by including the production of thyroid hormones (Equations 14–17), metabolism of thyroid hormones, recycling of freed iodide, and the T4/TSH negative feedback loop (Equation 13), as shown in Figure 1, an adequate description of the euthyroid, steady-state iodide-sufficient (20 μg I/day) condition was not readily achieved. For example, predictions of serum iodide were too low, liver concentrations of T3 and T4 were too high, and serum T4 concentrations were too high, which resulted in under-predicted serum TSH concentrations (simulations not shown). Therefore, the submodel parameter values obtained to predict serum clearance kinetics of trace amounts of radiolabeled iodide, T4, T3, and TSH (supplementary data) were adjusted. This was not completely unexpected for describing endogenous masses of thyroid hormones, dietary iodide, and TSH. Thus, a global optimization of model parameters for the BBDR-HPT axis model was performed, and final model parameters are shown in Table 2. The calibrated steady-state euthyroid, iodide-sufficient model predictions for a 320-g rat are shown in Table 3. Total thyroid and free serum iodide, serum TSH, serum and liver T4, and serum and liver T3 model predictions fall within the range for normal rats reported in literature.TABLE 3Steady-State Iodide-Sufficient Model Predictions Compared to Laboratory DataHPT Axis IndexUnitsData ± SDModel PredictedTotal thyroidal iodideμg15 ± 3a17.5Free serum iodideμg/dl7.3 ± 0.3, 10 ± 1.4b9.7–15.6cSerum T4ng/ml40.6 ± 11.3a39.4Liver T4ng/g18.7, 23.2, 25.5d21.8Serum T3ng/ml0.46 ± 0.1e0.46Liver T3ng/g3.7, 4.9, 5.7d4.5Serum TSHng/ml6.5 ± 2.5a6.3a McLanahan et al. (2007).b Eng et al. (1999).c A range is reported because of the daily fluctuation predicted in serum iodide, resulting from the assumption that the rats consume chow 12 h of the 24-h day.d Morreale de Escobar et al. (1994).e Unpublished data.Iodide Deficiency HPT Axis SimulationsUsing the BBDR-HPT axis model parameter values, globally optimized for euthyroid iodide-sufficient steady-state conditions, the ability of the model to predict temporal changes in serum thyroid hormones (T4 and T3), TSH, and total thyroidal iodide was tested for iodide-deficient conditions. HPT axis disturbances caused by feeding an iodide-deficient diet of 0.35 μg I/day for 26 days (Riesco et al., 1977) is depicted in Figure 2 for adult male HSD rats. Serum T4 concentrations gradually decreased in a parallel fashion with thyroidal iodide stores, while only a slight change in serum T3 concentrations occurred. Serum TSH concentrations increased over 10-fold during the study period. After 15 days of administration of the low iodide diet, the thyroidal iodide stores were severely depleted. The BBDR-HPT axis model predictions of serum thyroid hormones were in agreement with observed values. The predicted thyroidal iodide stores were slightly overpredicted initially and near the end of the study. Serum TSH increases were predicted during the first 11 days and then moderately overpredicted by day 15. In severe iodide-deficient conditions, when thyroidal iodide stores were predicted to be below 1 μg, oscillations in serum TSH and T4 and thyroidal iodide occurred because of assumptions about dietary intake of iodide. Next, the capability of the BBDR-HPT axis model to predict changes during administration of 0.33 μg I/day administered for 84 days to adult male SA and HSD rats (Okamura et al., 1981a) was tested (Fig. 3). The SA strain (Fig. 3A) exhibited a greater sensitivity, shown by the rapid increase in TSH compared to the HSD strain (Fig. 3B). The BBDR-HPT axis model predicted the change in TSH better for the SA rats than the HSD rats. Serum T3 concentrations were predicted to be lower than suggested by the data.BBDR-HPT axis model simulations for an iodide-deficient diet of 1.14 μg I/day administered to adult male HSD (Okamura et al., 1981b) are depicted in Figure 4. The initial decrease in thyroidal iodide stores and the apparent recovery after 60 days suggests adaptive responses, such as the negative feedback loop. The BBDR-HPT axis model predictions also suggest this as evidenced by an increase in predicted thyroidal iodide stores and little decline in serum thyroid hormones after 25 days. At a dietary intake of 1 μg/day, this strain of adult rat has some ability to compensate for low iodide intake. Predictions of serum T3 were slightly underpredicted. Finally, the BBDR-HPT axis model was used to simulate recovery of the HPT axis in rats rendered iodide deficient for 7 months (Fukuda et al., 1975) with an average daily iodide intake of 0.6 μg/day (Fig. 5). On day 0 of the recovery phase, the rats were supplemented with iodide in drinking water to provide total intake of either 2.6 or 8.6 μg I/day. The BBDR-HPT axis model slightly overpredicted day 1 increases in serum T4 following iodide supplementation for both doses, while the remaining predicted serum T4 and TSH concentrations agreed with observations.FIG. 5.Recovery from iodide deficiency in adult male Sprague-Dawley rats fed a low iodide diet for 7 months. After 7 months on a low iodide diet (0.6 μg I/day), rats were supplemented with iodide to provide total intake of approximately 2.6 μg I/day (black) or 8.6 μg I/day (dark gray) beginning on day 0 and continuing for 9 days (Fukuda et al., 1975). Model simulations of serum T4 (solid lines) and TSH (dashed lines) are compared with recovery data modified from Fukuda et al. (1975) for serum T4 (•, 2.6 μg I/day; ○, 8.6 μg I/day) and serum TSH (▾, 2.6 μg I/day; ▿, 8.6 μg I/day). Data expressed as percent of baseline recorded at day 0.Model Performance AnalysisThe predictive ability of the model to describe iodide deficiency was evaluated using AUC P/M ratios described by Gustafson et al. (2002). A value near 1.0 indicates agreement between model predictions and data observations. The AUC P/M ratios ranged from 0.37 to 2.27 for four metrics (serum T4, T3, TSH, and total thyroid iodide) across four iodide deficiency data sets (Figs. 2–4). The average AUC P/M ratios for each metric were 0.77, 0.54, 1.52, and 1.47 for serum T4, T3, TSH, and total thyroidal iodide, respectively. Taken together, the overall average AUC P/M ratio for the BBDR-HPT axis model predictive performance was 1.08.Sensitivity AnalysisThe sensitivity analysis of the BBDR-HPT axis model was carried out for steady-state serum concentrations of T4, T3, and TSH, and total thyroid iodide content for an iodide-sufficient intake of 20 μg I/day and iodide-deficient intake of 1 μg I/day. None of the parameters are associated with NSCs greater than 1.0, suggesting that there is minimal amplification of error from the inputs to the model outputs (Clewell et al., 2000). Total amount of thyroidal iodide predictions were most affected by a 1% change in the volume of the thyroid (VTc) (NSC = 0.99) under iodide-sufficient conditions and an NSC of 0.98 under iodide-deficient conditions. The parameters that play a major role in the retention of thyroidal bound iodide were more sensitive under iodide-sufficient conditions (VmaxBci, 0.94 and KbTSH, −0.93) for the prediction of total thyroid iodide than iodide-deficient conditions (both less than 0.01). A 1% change in the thyroid hormone production constant (kTSHIB) also reflected similar sensitivity of the total amount of iodide in the thyroid with NSCs of −0.95 and −0.96 under iodide-sufficient and -deficient conditions, respectively. Evaluation of the sensitivity analysis shows that the basal rate of TSH production (k0TSH) is more influential under iodide-deficient conditions (total thyroid iodide, NSC = −0.73; serum TSH, NSC = 0.89) than iodide-sufficient conditions (total thyroid iodide, NSC = 0.01; serum TSH, NSC = 0.49). Overall, serum T4 and T3 were much less sensitive than total thyroid iodide and serum TSH to a 1% change in model parameters.DISCUSSIONThe intent of this research was to develop a first generation BBDR model for the HPT axis in the adult rat using serum thyroxine (T4) and TSH levels to control the TSH-mediated thyroidal uptake of dietary iodide and the production and secretion of thyroid hormones. This parsimonious approach represents the simplest model structure to describe the negative feedback loop and, for now, ignores protein synthesis rates. This approach was successful, with some exception, in describing HPT axis changes that occurred over days to months (steady-state conditions) for data sets from several laboratories. The dominant negative feedback control of T4 on TSH was described (Equation 13), along with the stimulation of TSH on thyroidal iodide uptake (Equations 1–2) and subsequent thyroid hormone production (Equation 14).Adult rats excrete approximately 95% of a daily iodide-sufficient intake (normal laboratory intake of 20 μg I/day) according to our model simulations. Urinary iodide levels arise from metabolism of thyroid hormones, as well as excess iodide provided in the diet. The normal adult rat stores 12–18 μg iodide (McLanahan et al., 2007), and model predictions estimate that rats utilize about 1.4 μg I/day in thyroid hormone production under normal, euthyroid conditions. Furthermore, under iodide-sufficient conditions, our model predicts that 85% of the daily T3 production is derived from T4 metabolism, with the remaining (15%) produced in the thyroid. This is in agreement with others who suggest that at least 80% of the daily T3 production occurs as a result of T4 metabolism in a euthyroid system (Burger, 1986).The BBDR-HPT axis model was tested for its ability to predict changes in serum T4, T3, TSH, and total thyroid iodide during administration of low iodide diets. The model predicted the temporal response (over days to months) for decreases in serum T4 and increases in serum TSH resulting from the lack of available iodide for thyroid hormone production in an acceptable manner with some exceptions. Across all studies, the predictions of serum T3 was less consistent with the experimental data compared to other predicted endpoints. However, our model does agree with literature data such that the percent of daily T3 production in the thyroid increases significantly under iodide-deficient conditions (Abrams and Larsen, 1973; Greer et al., 1968). The percent of overall T3 production in the thyroid is predicted to increase from 15% (iodide-sufficient 20 μg I/day intake) to 25% as iodide intake rate decreases to 1 μg/day and 45% at an iodide intake rate of 0.35 μg/day. Model predictions during steady-state iodide deficiency (1 μg I/day) suggest that the percent of daily iodide intake excreted in urine decreases to about 65% and only 0.67 μg of iodide is utilized in daily thyroid hormone production. Thyroid iodide stores are severely depleted to about 20% (2.8 μg) of euthyroid, iodide-sufficient values, resulting in a decrease of over 50% in serum T4 concentrations.Using the BBDR-HPT axis model to evaluate dietary intake of iodide under steady-state conditions, a sharp decline in serum T4 is predicted to occur when dietary intake is less than 2 μg I/day even in the presence of significant TSH stimulation of the thyroid (Fig. 6). Others have reported that laboratory rats require an iodide intake greater than 2 μg I/day to maintain euthyroid status (Pedraza et al., 2006).FIG. 6.Iodide dose-response plot for serum T4 and TSH. BBDR-HPT axis model was used to determine steady-state serum T4 and TSH concentrations over a wide range of iodide intakes, intakes ranged from insufficient (0–2 μg I/day) to sufficient (> 2 μg I/day).Comments on the First-Generation BBDR-HPT Axis ModelSeveral feature of the HPT axis were not included in the present model, for example, protein synthesis, thyroid-releasing hormone (TRH), or metabolites of thyroid hormones (reverse T3, T2, T1, or thyroid hormone conjugates other than T4-glucuronide). Physiological changes were not included that occur during long-term iodide deficiency and hypothyroidism. For example, structural changes in the thyroid (Colzani et al., 1999), increases in thyroid blood flow (Michalkiewicz et al., 1989), and altered biological activity of thyroid hormone–metabolizing enzymes (Janssen et al., 1994; Obregon, et al., 2005; Pedraza et al., 2006) were not accounted for in this model iteration. TRH, secreted by the hypothalamus, stimulates release of TSH from the pituitary, while circulating T4 and T3 inhibit both TRH and TSH synthesis and release (Fail et al., 1999; Simpkins et al., 1976). Our model does not explicitly describe TRH effect on TSH; however, the BBDR-HPT axis model relates serum T4 levels to the TSH output from the pituitary, which implicitly includes TRH effects. There are no time-course data for TRH changes under iodide deficiency or following exposure to thyroid active compounds; thus, it is not possible to adequately describe TRH mathematically for euthyroid, steady-state or perturbed systems.A challenge in the development of this model was relying on published HPT axis data sets that varied dramatically. For example, reported TSH values for adult male Sprague-Dawley rats range from 4.6 ± 0.49 ng/ml to 8.73 ± 0.81 ng/ml (McLanahan et al., 2007), approximately 15–20 ng/ml (Siglin et al., 2000), 327 ± 174 ng/ml (Okamura et al., 1981a), to a high of 440 ± 220 ng/ml (Lemarchand-Beraud and Berthier, 1981). Several factors may contribute to this variability including, time of sampling, weight of animal, and radioimmunoassay analytical method, and standards employed. Thus, in reporting our model results, we reported TSH as fold change to normalize and compare model simulations with more data sets. Most of the iodine deficiency studies occurred prior to 1990, and many methods for analysis of thyroid hormones, TSH, and iodide have evolved since their publication. Another significant concern is verification of iodide and iodine in the rat chow. This amount can vary significantly between batches of rodent chow (Naeije et al., 1978). Unfortunately, the actual iodide and iodine concentrations in rodent chow is not usually measured by laboratories.The development of this model was initiated with the ultimate goal of integrating it with PBPK models for thyroid toxicants to interpret dose-response characteristics of HPT axis–mediated toxicity. Thyroid toxicants are defined as compounds, which alter serum thyroid hormone and TSH concentrations (Zoeller and Tan, 2007). The role of dietary iodide intake and the ability of anions to disturb the HPT axis will be explored with this first generation model and then expanded to include thyroid active chemicals that act by other modes of action.Future studies to support continued development of the BBDR-HPT axis model should include time-course studies after perturbation of the HPT axis to capture changes in endogenous iodide, serum TSH, T4, and T3, and thyroid hormones in tissues, such as the liver and regions of the brain. Time scales for intra- and extrathyroidal changes in protein synthesis and activity need to be explored in further detail. To this end, Kogai et al. (1997) have demonstrated that changes in NIS mRNA in FRTL-5 cells occur much faster (6 h) than protein expression (36 h) in response to TSH exposure. In vitro studies are also needed to better understand endogenous synthesis rates of TSH and thyroid hormones and metabolic clearance rates.SUPPLEMENTARY DATAThe supplementary data includes details of the radiotracer submodel development for 125I, 125I-TSH, 125I-T3, and 131I-T4, as well as model simulations for these stand alone submodels. The submodels developed and presented in supplementary data were used to test the model structure and obtain preliminary model parameters for the linked BBDR-HPT axis model. The model parameters optimized for radiotracer submodels (Table 1S) were used with the model structures depicted in Figure 1S for simulations shown in Figure 2S. These model parameters were reoptimized to euthyroid, steady-state iodide-sufficient conditions in the dietary iodide BBDR-HPT axis combined model. Supplementary data are available online at http://toxsci.oxfordjournals.org/.FUNDINGUnited States Environmental Protection Agency Science to Achieve Results research grant (RD83213401-0); United States Environmental Protection Agency Science to Achieve Results Fellowship (FP-91679301-0 to E.D.M).The authors extend special thanks to Dr Kyung O. Yu for providing experimental data sets for use in radioiodide model development. Sincere thanks to Dr Jerry L. Campbell, Jr, for model review. The views expressed in the manuscript are those of the authors and do not represent official opinions of the United States Environmental Protection Agency. 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Toxicol.200737195210 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AA986A1CE477C8DF2AEA0A6838BAF13E143C9DE.txt b/test/dataset/in/resources/corpus/Clean_0AA986A1CE477C8DF2AEA0A6838BAF13E143C9DE.txt new file mode 100644 index 0000000..637ff1a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AA986A1CE477C8DF2AEA0A6838BAF13E143C9DE.txt @@ -0,0 +1 @@ +]>HEARES3195S0378-5955(99)00009-X10.1016/S0378-5955(99)00009-XElsevier Science B.V.Fig. 1Velocity responses of the BM for an applied current to the cochlea (average of 256 stimuli). Positive velocity values represent motion toward scala tympani. The rectangular pulse of current (400 μA, 1.5 ms duration) was passed between electrodes located in the scala tympani and the scala vestibuli of the first turn. (GP033, bead location on BM is at the zona arcuata.) The duration of the current is given by the bar graphic in the center of the figure. A: The polarity of the voltage applied to the electrodes was scala vestibuli positive. B: The polarity of the voltage was scala vestibuli negative.Fig. 2Displacement responses (positive values are toward the scala vestibuli) of the basilar membrane for current applied to the cochlea as described in the caption of Fig. 1. Data were collected from a bead located at the basilar membrane radial location of the outer pillar cell/first row of OHCs. Positive voltage applied to the scala vestibuli resulted in the response given by the dotted line. Negative voltage to the scala vestibuli gave a response given by the solid line. These responses are the average from 2048 stimuli. The unit of BM displacement is nanometer (nm). (GP033, bead location on BM is at the zona arcuata.)Fig. 3Input/output functions for BM displacement (peak value) as a function of the current delivered to the cochlea. Positive voltage to the scala vestibuli and negative voltage to the scala tympani resulted in motion toward the scala vestibuli for beads located on the pectinate area of the BM (outer pillar cell area (OPC), outer hair cell one area (OHC1), outer hair cell three area (OHC3)), while the bead on the arcuate area at the margin of the spiral osseous lamina (on the zona arcuata (ZA)) moved toward scala tympani. The functions are linear for the range of current tested. Current level is presented in μA.Fig. 4Basilar membrane (BM) velocity responses from four locations (spiral osseous lamina edge (SOL), outer hair cell one (OHC1), outer hair cell two (OHC2), Claudius cells) across the width of the BM. The stimulus was a 500 μA rectangular pulse (1.5 ms duration) applied to the basal turn starting at 1.0 ms (see bar) with scala vestibuli having a positive voltage and scala tympani a negative voltage. For the topmost trace and the second trace, arrows mark the occurrences of onset and offset pulses. These pulses correspond to BM displacement steps (see Fig. 2) at the initiation and termination of the current. The relative size of the onset and offset velocity pulses indicate the relative magnitude of BM displacement at the various radial locations which, in this animal (GP 036), are not of typical relative magnitude as shown in Fig. 5. Note the phase reversal of the velocity pulse between the SOL location on the zona arcuata and the other locations, which are all on the zona pectinata of the BM. Ringing transient responses in this less sensitive cochlea are smaller and less delayed than the more sensitive example of Fig. 1.Fig. 5The composite data from the animals in this study as given in Table 1. When a current of 400 μA was applied to the cochlea, displacement and peak velocity of the ringing responses both reached a maximum at the same radial location; near the outer pillar cell or the first row of OHCs. Filled circles and open squares are the mean and bars are ±1 S.D. from the mean. The numbers over each bar are the numbers of measurement animals in the mean calculation.Table 1The direction of BM displacement with current stimulationAnimal #dB loss at 18 kHznear SOLOPCOHC1OHC2OHC3Claudius cellsGP00820↓↓GP01023↓↓GP01525↓↓↓GP016>20↑↓GP01718↓GP02014↑↓↓↓GP0217↑↓GP02525↓↓↓GP026>40↓↓GP02828↑↓GP03246↓↑↑GP0339↑↓↓GP034>30↑↑↑GP03510↓GP036>30↑↓↓↓GP03711↑↓↓↓GP0398↑↓GP0468↑↓GP04712↑↓Direction trend⇑⇓⇓⇓⇓⇓⇑Various current levels ‘500 μA, scala tympani positive, scala vestibuli negative. ↑=displacement toward scala media; ↓=displacement toward scala tympani. SOL, spiral osseous lamina; OPC, outer pillar cell; OHC, outer hair cell.The radial pattern of basilar membrane motion evoked by electric stimulation of the cochleaAlfred L.Nuttallac*nuttall@ohsu.eduMengheGuobTianyingRenaaOregon Hearing Research Center, NRC04, Department of Otolaryngology/Head and Neck Surgery, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098, USAbDepartment of Otolaryngology, Zhujiang Hospital, The First Military Medical University, Guangzhou 510282,ChinacKresge Hearing Research Institute, The University of Michigan, 1301 East Ann Street, Ann Arbor, MI 48109-0506, USA*Corresponding authorAbstractElectric current applied to the cochlea can evoke in situ electromotile responses of the organ of Corti. These nonsound-generated responses can give insight into the mechanics of the organ as the putative forces produced by outer hair cells (OHC) must couple to the modes of vibration of the basilar membrane (BM). In this study, platinum-iridium wire electrodes were positioned into the scala vestibuli and scala tympani of the first cochlear turn in the guinea pig. Current (1.5 ms rectangular-shaped pulses) was applied to these electrodes at levels to 500 μA peak. A laser Doppler velocimeter was used to record the velocity or displacement of the basilar membrane at the tonotopic 18 kHz place via an opening into the scala tympani of the first cochlear turn. Beads were positioned across the width of the BM so that the velocity or displacement of the BM could be studied in the radial direction. It was found that the current pulses evoked linear displacements of up to 2 nm for current levels of 500 μA (higher levels were damaging to the organ of Corti). The pattern of motion across the width of the BM was such that maximum displacement and velocity was located near the first row of OHCs and the position of the outer pillar cell footplate. The BM motion was biphasic in that the zona arcuata moved in the opposite direction to that of the zona pectinata. The results of this study demonstrate that the level of force produced by OHCs is effective in moving the BM and that the distribution of force within the organ of Corti leads to a multimodal motion pattern of the BM for this experimentally artificial means of evoking OHC motion.KeywordsGuinea pigOtoacoustic emissionLaser Doppler velocimetryOuter hair cellElectromotility1IntroductionThe electromotility of outer hair cells (OHCs) has been characterized in a variety of in vitro studies beginning with the seminal paper by Brownell et al. (1985). It has been shown that the voltage sensitivity of somatic length change is about 2 nm/mV at a membrane resting potential of −70 mV (Santos-Sacchi, 1989). A measurable force from OHCs can be inferred when there is a dimension change of the organ of Corti (e.g., Zenner et al., 1992). It has also been determined that when operating under load, OHC elongation is considerably smaller than when a hair cell is unloaded (Hallworth, 1995).Little is known about the effects of any force or displacement that might result from OHC membrane polarization changes when OHCs are operating in their natural environment within the organ of Corti in vivo. According to current hypotheses the mechanical role of OHCs is that they could act as acoustic force amplifiers in a cycle-by-cycle positive or negative feedback amplifier fashion (Dallos, 1992; Mountain and Hubbard, 1989) or as active mechanical stiffness regulators (Dallos and He, 1997).The result of OHC force or displacement in the organ of Corti is not only dependent on their inherent mechanical properties but also on the structural elements of the organ that permit the delivery of force, or the coupling of stiffness, into the motion of the basilar membrane (BM) and ultimately the displacement of inner hair cell stereocilia. The OHCs are arranged in complex association with supporting cells, particularly Deiters’ cells and pillar cells. Models of cochlear mechanics that attempt to provide a deeper understanding of normal sensitivity and frequency tuning require incorporation of organ of Corti micromechanical features (e.g., Geisler, 1986; Steele and Taber, 1981). This is especially true for finite element modeling approaches where mechanical properties are needed for the various nodes in the model (Kolston and Ashmore, 1996; Zhang et al., 1997).In this study we examined the displacement and velocity of the BM that is produced by putative forces produced by OHCs when electrically stimulated. The measurements were made in the intact and living guinea pig organ of Corti. We observed that rectangular displacements of at least 1–2 nm could be elicited. The radial pattern of BM motion was complex (bi-directional) for a given polarity of electric polarization across the organ. The data are consistent with the hypothesis that the morphologically thin arcuate zone region of the basilar membrane is very compliant by comparison with the pectinate zone.2MethodsHealthy young adult pigmented guinea pigs weighing 250–400 g were used in this study. Animals were from the Murphy Breeding Laboratory or the Charles River Company and were housed in American Association for Accreditation of Laboratory Animal Care approved facilities. Experimental protocols were approved by the Committee on Use and Care of Animals at the University of Michigan and at the Oregon Health Sciences University. The animals were preanesthetized with an initial injection of pentobarbital (15 mg/kg i.p.) followed 15 min later by an injection of ketamine/xylazine (ketamine 40 mg/kg; xylazine 10 mg/kg i.m.). Supplemental doses of ketamine and xylazine were given on a schedule or as needed, judging by a slight withdrawal in response to toe pinch. Pentobarbital supplements (7 mg/kg i.p.) were given every 2 h.The animal’s rectal temperature was maintained at 38±18°C with a servo-regulated heating blanket. The cochlear temperature was additionally controlled by supplemental heat to the head from a lamp and a heated head holder. The electrocardiogram and heart rate were continuously monitored as an additional measure of anesthesia level and animal general condition. All presented data were collected from animals with a normal electrocardiogram.The guinea pig’s head was firmly fixed in a heated headholder that was mounted on a custom-made manipulator to allow positioning of the cochlea in the visual field of the BM velocity-measuring microscope. A tracheotomy was performed and a ventilation tube inserted into the trachea to insure free breathing. A ventral and postauricular surgical dissection exposed the auditory bulla on the left side and the external ear was removed (exposing the lateral 2/3 of the external auditory canal) to facilitate placement of the acoustic speculum. The bulla was widely opened to expose the cochlea and the middle ear muscle tendons were sectioned. An Ag-AgCl ball electrode with about 200–300 μm diameter was placed in the round window niche for recording of sound-evoked cochlear potentials. An Ag-AgCl ground electrode was inserted into neck soft tissue medial to the exposed bulla. The round window signal, relative to the neck ground electrode, was amplified 10 times by a P16 Grass amplifier and 100 times by a custom-designed AC amplifier. The amplified CAP signal from the round window was displayed on an oscilloscope and the N1 detection of 10 μV was used as a threshold criterion.An opening approximately 300 μm in diameter was made on the lateral wall of the scala tympani of the basal cochlear turn for measurements of the BM velocity. The surgical opening into the cochlea to observe BM motion was made by thinning the bone over the scala tympani of the basal turn with a knife and picking out the bone pieces with a small hook.Two electrodes were made of Teflon-insulated 75 μm diameter platinum-iridium wire for the cochlear electrical stimulation. One stimulation electrode was inserted inside the scala vestibuli through a 100 μm diameter hole made on the lateral wall of the scala vestibuli of the basal cochlear turn. The electrode was fixed and the hole was sealed with tissue cement. Another platinum-iridium electrode was positioned in the scala tympani through the opening on the lateral wall of the scala tympani. To evoke basilar membrane motion, a 1.5 ms electrical pulse was generated by a D/A converter and was delivered as an electrical stimulus to the cochlea by an optical-isolated constant current stimulator. The current level of the electrical stimulus was controlled by a programmable attenuator.After the electrodes were fixed in position and the animal’s head was maneuvered into position presenting the BM approximately in the horizontal plane, gold-coated glass beads (<20 μm diameter) were placed on the BM to serve as reflective objects for the laser beam of the laser Doppler velocimeter (Polytec OFV-1101).The radial positions of these beads across the width of the basilar membrane were random. Their radial locations were noted in relation to visible morphological features of the organ of Corti such as the OHCs, the spiral capillary of the BM and the osseous spiral lamina. Generally, it was possible to achieve more than two radial bead locations in a given experiment (see Table 1). The laser beam of the laser Doppler velocimeter was coupled into a compound microscope and the focused laser beam was directed at a selected glass microbead on the BM. Velocity signals from the instrument were analyzed and measured with the aid of a lock-in amplifier (Stanford Instruments SR530) and a spectrum analyzer (Stanford Instruments SR760). Experiments were accomplished on the top of a vibration isolation table and inside a double-walled soundproof booth. Details of the surgical method and the measurement of BM velocity can be found in Brown et al. (1983) and Nuttall et al. (1991).For the measurement of electrically evoked BM vibration, the velocity or displacement signal of the laser Doppler velocimeter was digitized by an A/D converter at the rate of 125 000 samples per second with a 4 ms time window synchronized with electrical stimuli without time delay. The laser Doppler velocimeter signal was averaged by custom software. A velocity or displacement waveform of the BM vibration could be obtained from the instrument. The directions and amplitudes of the BM step responses and of transient ringing responses were determined. The relationship between the vibration and the radial positions of the beads across the BM was observed.3ResultsThe results presented in this report are derived from 19 guinea pig experiments. Individual examples of typical responses are shown in the figures for the purpose of qualitatively conveying the nature of BM displacement and velocity. The purpose of this report is to describe the pattern of BM displacement that results from a mechanical force acting within the organ of Corti when the cochlea is stimulated electrically. Since electrically evoked BM responses are greatly reduced in cochleas of guinea pigs treated with ototoxic drugs to destroy OHCs, the mechanical force is likely due to the electromotile activity of these cells (Nuttall and Ren, 1995).The application of electric current pulses to the cochlea evokes BM motion. Fig. 1 illustrates the general form of this motion. The velocity responses of the BM are shown in response to 400 μA positive (Fig. 1A) and negative (Fig. 1B) current pulses of 1.5 ms duration. Initially, an onset transient occurs (a velocity spike), the direction of which is dependent on the polarity of the electric current. These velocity transients correspond to a displacement step of BM motion. An example of displacement steps is given in Fig. 2 (obtained from the same animal). The displacement waveforms are considerably more noisy than the velocity waveform, reflecting the animal motion artifact introduced by cardiovascular, respiratory, and skeletal muscle activity and the poorer signal-to-noise ratio of the interferometer for displacement measurements. In these experiments, using bipolar electrodes between the scala tympani and scala vestibuli, a positive voltage applied to the scala vestibuli relative to a negative voltage in scala tympani evoked motion of the BM near outer hair cells toward the scala media.Note in Fig. 1 that the velocity ‘spikes’ which occur at the beginning of each current pulse in Fig. 1A,B are approximately equal in size (height). This indicates that the displacement motion of the BM for the two polarities of stimulation was the same and therefore that the length of the putative OHC elongation or contraction was symmetrical. Fig. 2 illustrates the pattern of the actual displacement measured with amplitude-sensitive interferometry. The time-dependent shape of the displacement pattern in Fig. 2 was found to be variable between animals. In cochleas with poor sensitivity, the displacement pattern tended to be rectangular in shape. Sensitive cochleas tended to have displacement responses that had the appearance of being high pass filtered. We define the sensitive cochlea as the condition where the sound level required for a criterion response of the cochlear action potential (CAP) evoked by acoustic tone bursts (i.e., the 10 μV N1 threshold at 18 kHz, the best frequency of the BM measurement location) is within about 10 dB of normal. Most of the cochleas studied (Table 1) have greater loss than 10 dB but their sensitivity is generally better than would be the result of a complete loss of the cochlear amplifier (>40 dB loss). The time-dependent changes of the displacement were not systematically studied in this investigation.Following the onset of the displacement (or the onset velocity spike) a ringing response of the BM is observed. A second ringing response occurs following the offset of current. The polarity of the current (i.e., scala vestibuli positive related to scala tympani) alters the size of the ringing response. Positive current enhances the BM ringing motion while negative current decreases the response. The ringing responses are the local resonant vibration of the BM at the tonotopic best frequency (BF), which in this case is about 18 kHz. We have shown that these transient, ringing responses are equivalent to those that result from acoustic transient stimuli (clicks) and that they are the result of intracochlearly generated acoustic energy from OHCs stimulated by the current (Nuttall and Ren, 1995). That current modulates the transient ringing response has previously been reported by us (Nuttall and Dolan, 1993; Nuttall et al., 1995). The cause of the amplitude modulation of the ringing is not known but is possibly related to the influence of the current on OHC membrane polarization and changes in the gain of the cochlear amplifier.The symmetric displacements of the BM are linear functions of current in the range used in this study (up to 500 μA). Fig. 3 shows input/output curves for BM displacement for levels of current from 100 to 500 μA. The functions are approximately straight. The three lines plotted, however, illustrate that the amount of displacement is a function of the radial position across the BM. The highest sloped line is for a location at the junction of the zona arcuata and the zona pectinata areas of the BM and at the location of the OHCs.The radial pattern of velocity across the BM is illustrated by the data plotted in Fig. 4 for fortuitous distribution of glass measurement beads on the surface of the BM. The figure shows that relative displacement (as measured by the size of the initial velocity spike) and the ringing transient response are functions of the radial distance across the width of the BM. Fig. 5 graphs the mean (±1 S.D.) displacement and ringing response peak velocity (for positive current) responses of a number of animals in this study. Note that the maximum displacement and velocity motion of the BM is at a location close to the junction of zona arcuata and pectinata or near the first row of OHCs. The large variance of the data at the pillar cell, OHC1 and OHC2 locations, could be due to position assessment inaccuracies. Close to the margin of the spiral osseous lamina, the displacement reversed in direction. Therefore, there must be a transition point of no motion that is located somewhere between the outer pillar cell and the spiral osseous lamina. We were not able to define this point with any greater precision. Only in one animal were three beads fortuitously next to one another across the width of the zona arcuata. The center bead did have a ‘neutral’ motion while the other two had opposite phase. Since the three beads were touching, or nearly so, it is possible that they influenced the motion of one another.Fig. 5 also depicts that the radial position of maximum velocity of the ringing response corresponds to the position displacement response maximum. The junction between the anatomically different regions of the BM (zona arcuata and zona pectinata) is the location of large amplitude motion. Our results on the position of peak displacement appear similar to data reported by Nilsen and Russell (1998).Since only a few reflective beads are placed on the BM in a given animal, it is difficult to obtain information on all locations across the BM. It is therefore useful to examine the group data of all the measured guinea pigs in this report. The direction of BM motion is summarized in Table 1, which shows that the area near the osseous spiral lamina is opposite (in general) from that which occurs near OHCs when the cochlea is electrically stimulated by a rectangular pulse of current. The place of transition between the opposing motion patterns is near the junction of the zona arcuata and the zona pectinata.4DiscussionIn this study we have made an initial exploration of the micromechanics of the organ of Corti, in situ. The means to explore the mechanics is through the application of electric current to the cochlea. In an ear not stimulated by sound, electric current evokes motion of the BM. A putative elongation and force produced by the OHCs from the electrically induced polarization changes of their membranes is most likely responsible for moving cellular elements of the organ of Corti. A motion of the BM so induced is a demonstration that OHCs have the strength to displace the BM (acting against a stiffness in this case), and the pattern of the motion would reveal some properties about the transfer of the OHC-produced force within the structure of the organ of Corti.Xue et al. (1995) observed displacements between 5 and 10 nm for a current of 50 μA delivered into the scala media. This is a larger displacement level than we achieved and is possibly due to a more effective OHC membrane polarization by the scala media delivered current. Xue et al. (1995), using estimates of the electrical resistances of the scala media and OHCs, calculated that 50 μA would produce a membrane potential shift of 40 mV which would be depolarizing for positive voltage in the scala media. When current is applied across the cochlear duct (the method of this paper), electroanatomical considerations dictate that perhaps only 10% of the applied current would pass into scala media (Honrubia and Ward, 1968). The effective current would therefore be similar to that used by Xue et al. (1995).We observed that the BM displacement responses were linear over ±500 μA1 (Fig. 3). This linearity also suggests that the membrane potential shifts induced were small because the OHC length-to-membrane potential relationship is nonlinear (Evans et al., 1991; Santos-Sacchi, 1993). It is consistent that Hallworth (1995) has shown that mechanically loaded or unloaded isolated OHCs give linear displacements for estimated membrane potentials of up to 15 mV2. Moreover, we have observed linear electrically evoked otoacoustic emissions, a finding that implies linear tissue vibration (Ren and Nuttall, 1995; Ren et al., 1996; Ren, 1996).1The limiting factors of current delivery in our experiments are both biological and electrochemical. With the typical size platinum wire we use (3T), noticeable gassing occurs at the wire surface between 800 and 1200 μA. From the biological point of view, we have empirically determined in our experiments that damage to the cochlear amplifier causing reduced sensitivity occurs above 500 μA. The pathophysiology of the damage is not known at this time.2The 15 mV estimate was obtained from voltage divider model of the microchamber stimulated OHC in the Hallworth (1995) paper using the parameters: 4 μm radius and 30 μm length.The measured BM displacement is considerably less than that expected from studies of isolated OHCs and the estimated membrane potential shift induced in this study. The slope gains of isolated OHCs are between about 1 and 4 nm/mV and strongly dependent on resting membrane potential (Evans et al., 1991; Santos-Sacchi, 1989). Mammano and Ashmore (1993) have determined that current induced in situ displacements of OHCs of the cochlear apical turn cause up to a five times larger antiphasic movement of the reticula lamina than the BM. Since this antiphasic motion of the reticular lamina and the BM will be a function of the compliance of both structures, it may be possible that their displacement relationship is changed considerably in the base of the cochlea where the BM is as much as 100 times stiffer than in the apex (von Békésy, 1960). Nevertheless, the clear displacement of the BM indicates that OHCs can produce a physiologically relevant force (causing displacements of the same order as evoked by moderate-level sounds). Xue et al. (1995), based on BM point stiffness measurements from Olson and Mountain (1991), calculated the single OHC slope gain for force to be about 2 pN/mV, which is comparable to that obtained in other studies (Hallworth, 1995; Russell and Schauz, 1995).In the present study, we observed that the pattern of motion across the width of the organ of Corti is biphasic. These results extend our earlier report (Nuttall et al., 1995, 1997) and confirm the similar finding of Xue et al. (1993). The concept of a second mode of vibration across the width of the BM can be found in the speculations by von Békésy (1960) based on anatomical considerations. The OHCs are located in association with the thicker pectinate zone of the BM.The point stiffness of the pectinate and arcuate zones of the BM have been measured by Olson and Mountain (1994). They found a highly asymmetric stiffness distribution about the position of the outer pillar cell. The outer pillar cell location was about five times stiffer than the BM in the arcuate zone. They attribute this stiffness difference to cellular elements of the organ of Corti rather than the inherent properties of the two zones of the BM. The outer pillar cell may be responsible for the peak in stiffness at its radial location across the width of the BM. An antiphasic motion vibration mode of the BM could be achieved by the deflection and rotation of this cell. Antiphasic motion also could result from fluid pressure acting within an isovolumetric organ of Corti. Conservation of volume could require the relatively compliant arcuate zone of the BM to move in the opposite direction relative to the pectinate zone when the outer hair cells distend the reticular lamina and the BM in opposite directions.The stimulus protocol of this study (pulses) is sufficiently different from that of Xue et al. (1993, 1995) (sinusoids) that direct comparison of the results is difficult. Xue et al. (1993; their Fig. 4B) show the antiphasic motion of the BM in the gerbil as a vibration mode is present at high frequencies, and we also observe the phase of the two zones to be different in our stimulation protocol. Moreover, Xue et al. (1995) found a significant phase lead of the pectinate zone relative to the current at low frequencies. This was attributed either to velocity tracking of the length changes of OHCs or a phase shift of the OHC membrane potential. In the current study, the displacement responses of the BM were in phase with the current when the cochlea was insensitive but in cochleas with preserved sensitivity (i.e., a functional cochlear amplifier) the responses were sometimes velocity-like. The study of velocity-like responses is an area of future work.Although this was not specifically investigated in the current study, we have previously reported that both acoustic and electrical stimulation of the organ of Corti with sinusoids resulted in equivalent phase vs. frequency functions. In contrast Xue et al. (1995) found that electrical stimulation caused much smaller total phase shifts. The difference could be due to electrode configurations. However, our interpretation of the mode of stimulation of the local activity of the BM for high frequencies is that the current causes an intracochlear differential acoustic pressure by OHC-caused shape change of the organ of Corti that is equivalent to an external stimulus (Nuttall and Ren, 1995). In the current study we observed a related phenomenon where the ringing responses following onset or offset of a given polarity of current stimulation always had the same phase in the two zones even though the rectangular BM displacement due to the current was antiphasic. There was no phase shift of the ringing responses as a function of radial distance across the BM. We attribute this result to the forced oscillation of the two zones experiencing the same initial pressure.The direction of motion of the BM from a current applied across the cochlea with positive voltage applied to the scala vestibuli relative to the scala tympani is pectinate zone deflection toward the scala media. This direction is consistent with Xue et al. (1995) for positive voltage in the scala media. Previously we reported that this polarity moved the BM toward the scala tympani but that was for a reflective bead on the BM at the zona arcuata. We also interpreted ear canal condensation acoustic pressure to be correlated to BM motion toward scala tympani (Nuttall and Ren, 1995). However, this displacement was of the arcuate zone. We now observe that putative OHC contraction with applied current leads to motion of the pectinate zone of the BM toward scala media and results in rarefaction acoustic pressure in the ear canal, a result that is consistent with measurements by Xue et al. (1996).AcknowledgementsThis work was supported by NIH R01 DC00141 and by NIH Program Project P01 DC00078.ReferencesBrown et al., 1983M.C.BrownA.L.NuttallR.I.MastaIntracellular recordings from cochlear inner hair cells: effects of stimulation of the crossed olivocochlear efferentsScience22219836972Brownell et al., 1985W.E.BrownellC.R.BaderD.BertrandY.de RibaupierreEvoked mechanical responses of isolated cochlear outer hair cellsScience2271985194196Dallos, 1992P.DallosThe active cochleaJ. 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World Scientific, New Jersey. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AACD22F36FC79603A2EB3DE9699FD3BB6FBC6D0.txt b/test/dataset/in/resources/corpus/Clean_0AACD22F36FC79603A2EB3DE9699FD3BB6FBC6D0.txt new file mode 100644 index 0000000..bf8b1eb --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AACD22F36FC79603A2EB3DE9699FD3BB6FBC6D0.txt @@ -0,0 +1 @@ +]>NBA5129S0197-4580(97)00150-410.1016/S0197-4580(97)00150-4Elsevier Science Inc.Fig. 1Mean (±SE) number of errors made per trial across the seven days of acquisition of the spatial memory task. Aged rats made significantly more errors overall, and both young and aged rats showed significant learning during this period. In addition, a significant interaction effect of age and days of training suggest that aged rats learned the task at a slower rate than young rats.Fig. 2(LEFT) Scatter distribution showing the correlation between the log transform of spatial selectivity scores of CA1 hippocampal place neurons and the mean number of errors made per trial per day that each cell was recorded. The data presented reflects a summary of data across the entire acquisition period. Each dot represents the average specificity for all cells recorded within a given recording session. A significant correlation was observed between decreasing errors and increasing spatial selectivity for old (TOP), but not young (BOTTOM) rats. (RIGHT) Scatter distribution of the reliability scores of the same cells presented to the left. No relationship was found between reliability scores and errors in old rats; however, a marginally significant positive correlation was observed for young rats.Fig. 3Examples of the spatial distribution of CA1 place fields recorded from a representative neuron of an aged (TOP) and a representative neuron of a young (BOTTOM) animal on Day 1 (LEFT) and Day 7 (RIGHT) of acquisition of the spatial memory task. Dots indicated visited locations by the rat on the maze. The size of the circles is proportional to the local firing rate of the cell, and the vector indicated the direction of movement by the animal as the cell fired.Fig. 4Scatter distribution showing the correlation between the log transform of spatial selectivity (LEFT) and reliability (RIGHT) scores of CA1 hippocampal place neurons and the mean number of errors made per trial for memory-intact old rats (TOP) and memory-impaired old rats (BOTTOM). Only the memory intact old rats showed a significant correlation between choice accuracy and spatial selectivity scores, and between choice accuracy and spatial reliability. One interpretation of this pattern of results is that such representational reorganization facilitated accurate performance on the memory task.Fig. 5A comparison of the proportion change in in-field firing rates and out-of-field firing rates from Day 1 to Day 7 of acquisition training for those aged CA1 place cells that were held across the training period. It is clear that different patterns of change were observed and ultimately contributed to the correlations shown in Fig. 2.Fig. 6Scatter distribution showing the correlation between the log transform of spatial selectivity scores of hilar place neurons and the mean number of errors made per trial per day that each cell was recorded. The data presented reflects a summary of data across the entire acquisition period. Each dot represents the average specificity for all cells recorded within a given recording session. No significant correlations were observed between errors and spatial selectivity (LEFT) or reliability (RIGHT) for old (TOP) or young (BOTTOM) rats.Original ArticlesAge and Experience-Dependent Representational Reorganization During Spatial LearningMizumoriS.J.YMizumoriA*KalyaniAKalyaniAAPsychology Department, University of Utah, Salt Lake City, UT 84112, USA*Sheri J. Y. Mizumori, Psychology Department, University of Utah, Salt Lake City, UT 84112.AbstractPreviously, we found that aged rats showed a significant enhancement of hippocampal CA1 place cell spatial specificity, as well as a reduction of hilar place cell spatial specificity, during asymptote performance of a spatial memory task. Because such an age effect was not observed when animals performed a nonspatial task, the present study tested the hypothesis that the different patterns of spatial selectivity observed in memory and nonmemory tests reflected a redistribution of spatial representations that occurred in response to changing task demands. In the present experiment, after animals became familiar with the test environment and motor demands of performance on a radial maze, CA1 and hilar place cells were recorded as they learned a spatial memory task. CA1 place cells recorded from unimpaired old, but not impaired old or young, animals became more spatially selective as animals learned the task. Hilar spatial selectivity for both age groups was not significantly related to choice accuracy. These data support the hypothesis that at least a subpopulation of aged rats may benefit from reorganization of spatial representations in such a way that the normal age-related spatial learning deficit is attenuated.KeywordsPlace cellsHippocampusSpatial learningRepresentational neuroplasticityAgingThe relatively poor performance of aged rats on spatial memory tasks has been shown not to be due to general sensory or motor decline (e.g., [3, 6, 12, 29, 33]). Rather, arguments have been made for a select difficulty in acquiring new information, especially spatial information. Recent studies have shown that aged rats and monkeys do not show significant loss of cells in any of the hippocampal subregions examined (i.e., the granule cell layer, hilus, CA3, CA1, or subiculum; refs. [28, 30, 31]). Therefore, it is likely that a hippocampal contribution to the spatial learning decline with senescence reflects more subtle changes in hippocampal cell function. Indeed, there is evidence that the synaptic structure of hippocampal neurons undergoes age-associated changes that are correlated with the memory impairment (e.g., ref. [9]). The functional significance of such synaptic changes is presently unknown. However, it is not unreasonable to postulate that such intracellular alterations might change the manner in which information is coded by aged hippocampal neurons.A large proportion of hippocampal neurons (place cells) are known to selectively discharge as animals traverse circumscribed portions of environmental space, referred to as place fields [24, 25]. Hill and Best [11]showed that place fields emerge relatively quickly when placed in a new environment. This initial result was recently extended by Wilson and McNaughton [32]and Austin and colleagues [1], who showed that place coding by particular hippocampal neurons can undergo initially rapid, then more subtle long-term changes with continued exposure to a constant environment. The latter studies demonstrate that familiarity can influence place cell firing and that place field specificity is not merely a reflection of the current sensory environment.Previously, we reported that CA1 and hilar neurons recorded from aged rats show different organization of spatial (place) representation compared to young rats when well trained animals perform a spatial memory task [19]. Specifically, CA1 cells of aged animals showed enhanced spatial selectivity, and hilar cells demonstrated reduced spatial selectivity. The present study tested the hypothesis that this age-related representational reorganization is experience-dependent. Markus et al. [14]examined the relationship between various place field properties and continued performance on a forced choice (i.e., non memory) radial maze task. A significant relationship between choice accuracy and place cell properties was reported, but this conclusion was based on relatively few “errors” made by the rats. The present study tested more directly how changing the mnemonic requirements of a familiar and spatially complex environment might alter place cell firing not only in young but also aged animals.1Method1.1Subjects, Surgical Procedures, and Behavioral TrainingMale Fischer-344 rats (retired breeders from Harlan Sprague-Dawley Laboratory) were obtained at 9 months and 24 months of age. The rats were singly housed and allowed to adapt to laboratory conditions for 2 weeks before surgical implantation of recording electrodes. Mature, young rats were initially anesthetized with 33 mg/kg Nembutal (50 mg/mL), and aged rats with 25 mg/kg Nembutal, followed by supplements of 0.05 mL as needed. Electrodes were placed at the following stereotaxic coordinates (skull horizontal): (AP) -3.5–4.5 mm posterior to Bregma, (L) ± 2.5 mm lateral to the midsaggital suture, and (DV) about 1.5 mm below the dural surface for CA1 recordings, or 2.0 mm below the dural surface for hilar recordings. Two microdrives were implanted per animal, one over each hemisphere. A reference electrode (Teflon-coated stainless steel) was placed in corpus callosum: (AP) -6.5 mm; (L) 2.0 mm. A ground lead was soldered to a ground screw attached to the skull. Amphenol pin connectors attached to each electrode were inserted into a connector that became permanently attached to the rat’s head. After surgery, 0.1 mL Bicillin (300,000 units/mL) was administered into each hindleg to prevent infection.Animals were allowed to recover from surgery for 7 days. After this period, access to food was restricted to maintain animals at about 80% of their free feeding weights. The rats were then placed on an 8-arm radial maze and trained according to a forced-choice procedure (for details, see ref. [19]). Briefly, individual arms of the maze were presented to the rat sequentially and in pseudo random order such that the rat entered each maze arm once per trial. After animals were able to perform 8 such maze trials (2 min intertrial interval) within about an hour, and for 7 consecutive days, the recording electrodes were slowly advanced toward or through the hippocampus. When stable, well isolated single hippocampal complex-spike cells were obtained in either the CA1 or hilar regions, the maze training procedure was changed to a spatial memory procedure. That is, for each trial, following the individual presentation of the first 4 maze arms, all 8 arms were simultaneously presented and the rat was required to select those arms not previously entered that trial. The animals were not handled between choices 4 and 5, and there were no experimenter-imposed delays between choices 4 and 5. Reentries constituted errors. Again, animals performed 8 trials per day for 7 consecutive days. We attempted to record the same cell(s) throughout this spatial learning phase.We attempted to take run speed into account by calculating the amount of time spent per choice. This was done by taking the total time required to complete the task, divided by the number of choices made by the animal. This was determined for each trial, then averaged across trials to arrive at a score for each day of testing.1.2Unit Recording and Analysis ProceduresHippocampal single unit activity was recorded according to the stereotrode method described by McNaughton and colleagues [17]. Specifically, unit activity was independently recorded on two adjacent lacquer-coated tungsten wires (25 μm dia; California FineWire), which were twisted together, dipped in Epoxylite, and then baked. The electrode tips were gold plated to give final impedances of 50–150 kΩ (tested at 1 kHz). Each stereotrode pair of wires was threaded through a 30-gauge stainless steel tube, then mounted on a moveable microdrive [19–21]. A headstage was attached to a connecting socket on the rat’s head for all spatial learning recording sessions. The headstage contained 5 field effect transistors and an infrared LED for behavioral monitoring [20]. The incoming signals of the stereotrode pair were processed independently, with an amplification of 5–10 K, and filtered at 600–800 Hz (high pass) and 6 kHz (low pass). The analog signals were subsequently passed through a window discriminator such that a 1-ms sampling period by a DataWave system began when a signal from either channel surpassed a predetermined threshold. The maximum and minimum voltages of each signal, as well as the latencies of these values from the onset of the sampling period, were used to preliminarily isolate single spike generators within an otherwise multiunit record. More detailed off-line analyses were available to further isolate single units (e.g., template matching algorithms; ref. [19]).The final recording depths were determined relative to stratum pyramidale, which could be clearly identified by its electrophysiological properties. CA1 neurons were recorded in the first layer of complex-spike cells encountered, which was typically about 2000 μm below the cortical surface. Hilar complex-spike cells were typically encountered 700-1000 μm below CA1. For CA1-recorded animals, the electrodes entered CA1 only. For hilar-recorded animals, the electrodes passed through CA1 first, then the dentate region. The hilar group of cells likely included a combination of granule cells, hilar cells, and possibly CA4 cells. Whether a particular animal contributed CA1 or hilar data was determined somewhat randomly at the time of surgery. As a result, the order of recording was mixed. The stereotrodes were lowered through one hemisphere at a time; if well isolated units were not encountered in the first hemisphere, the stereotrodes were lowered through the second hemisphere.Spatially localized discharge by hippocampal CA1 and hilar neurons was quantified as in previous studies [19–21]. Briefly, the firing rates as the rat moved inward and outward on the 8 maze arms were calculated. The highest of these 16 rates was divided by the average of the remaining 15 to arrive at a spatial specificity score. It is important to note that our calculations incorporate not only the location component of spatial behavior but also directional movement. Both aspects of navigational behaviors are particularly characteristic of hippocampal place cells when tested on a radial maze. A cell was considered to have a place field if the cell had a specificity index greater than 2.0. Reliability of a place field was determined by counting the number of trials in which the in-field firing rate was higher than the firing rate on the remaining 7 maze arms. A directional score was calculated for each cell for each day it was recorded; the mean rate was calculated for times when the rat moved inward and outward on the arm associated with the highest rate (i.e., contained the place field). The highest of these two rates was divided by the lowest rate.2ResultsThe average number of errors made per trial on a given day of training was compared to a number of single unit firing properties of complex-spike units recorded on the same day. Initial, on-line classification of a complex-spike cell as either a CA1 or hilar neuron was confirmed by examination of the location and depth of the relevant electrode tract in histologic sections. All electrodes passed through dorsal hippocampus.Ten young and 10 old rats were trained on the forced choice task for 6.5 ± 1.5 days and 11.2 ± 1.6 days, respectively, before the 7 days of asymptote performance was observed. This difference in the number of days to required to perform the required 8 daily trials was marginally significant, F (1, 18) = 4.38, p = 0.05. Both young and old rats performed at asymptote on the forced choice task for at least 7 days prior to spatial memory training, if not longer. Such training began only when well isolated CA1 or hilar complex-spike cells were encountered. The total number of days of exposure to the test room before unit recording began was found not to vary significantly between the 4 groups: young CA1-21.8 ± 3.4 days; young hilus-18.2 + 1.5 days; old CA1-26.3 ± 2.3 days; old hilus-19.3 ± 1.2 days. No significant differences existed between age groups or recording sites.Fig. 1 shows that across the 7-day acquisition phase of spatial memory training, both age groups of rats showed significant learning, F (6, 18) = 52.18, p < 0.001 (repeated measures ANOVA). In addition, a significant age effect was observed, indicating that overall, the old rats performed worse than young rats, F (1, 18) = 6.38, p < 0.05. Furthermore, a significant interaction effect, F (6, 18) = 2.79, p < 0.02, revealed that aged rats learned the maze task at a slower rate than young rats. This pattern of statistical results replicates that reported previously [19]for young and old rats trained under identical conditions.A particular unit was considered to have been recorded across days if it was determined that its cluster parameter boundaries were comparable across days. Thus, 19% (5/26) of CA1 complex-spike cells and 25% (10/40) of hilar complex-spike cells recorded from young rats were deemed stable across the 7-days of acquisition training. About 56% (24/43) of CA1 complex-spike cells and 27% (4/15) of hilar complex-spike cells recorded from old rats were stable across all 7 test days. Other neurons were recorded for 2–6 days. The analysis described below was carried out separately for cells recorded across all 7 days and those that were recorded for less than 7 days. The latter group consisted of cells that were initially recorded on Day 1, then were lost before the 7 days of training were completed, as well as cells that emerged for the first time after Day 1. Unit-behavioral correlates or spike discharge patterns did not significantly differ depending on the duration of recording of a particular unit, or the day of first recording. Therefore, unless otherwise specified, individual recording sessions for each cell were treated as independent events for statistical purposes.The log values of the spatial selectivity scores of CA1 and hilar complex-spike cells were correlated with the number of errors made by young and old rats. Specifically, the mean specificity for all complex-spike cells recorded on a given day was correlated with the corresponding mean number of errors committed per trial on the same day. The results for CA1 neurons are shown in Fig. 2. CA1 complex-spike cells of young rats (n = 4) did not show spatially selective firing that was statistically correlated with the number of errors made (r = +0.17, df = 26, p > 0.10). However, reliability was marginally correlated with the number of errors made (r = +0.39, df = 26, p = 0.05). That specificity was not directly related to learning is consistent with our previous result [19]that spatial selectivity of young CA1 cells is not different for rats performing at asymptote on a nonspatial task vs. a spatial memory task. It is also consistent with the report by Markus et al. [14]that reliability, and not spatial selectivity, of CA1 place field firing is related to errors. However, it should be noted that the latter authors reported a negative relationship, whereas we found a marginally significant positive relationship, although visual inspection of the reliability data in Fig. 2 does not reveal a particularly striking positive correlation.In contrast to the results from young animals, place-specific firing of CA1 complex-spike cells recorded from old rats (n = 7) was significantly correlated with the number of errors made on the maze (r = −0.41, df = 50, p < 0.01). That is, for old, but not young rats, CA1 spatially-selective discharge become more location and/or directionally tuned as learning took place (as indicated by the reduction in errors). Similar to our past results, the mean spatial selectivity score was greater for old rats (young: 7.47 ± 1.9; old: 11.83 ± 2.6; p < 0.05) by the end of acquisition training on the memory task (See Fig. 3 for an example). This age-related increase in spatial selectivity may be due to a constriction of place fields over time because 1) the proportion of aged CA1 cells showing place fields did not change (Day 1 = 79% and Day 7 = 81%), and 2) the same relationship between increased spatial selectivity and reduced errors was observed when the analysis was restricted to those cells recorded across the entire 7-day acquisition period (r = −0.23, df = 158, p < 0.01). Furthermore, the average percent change in CA1 place field specificity from Day 1 to Day 7 was −19.0% ± 18.0% for young animals and +91.4% ± 39% for old animals. There were no effects of age on the reliability of CA1 place fields (p > 0.10), and no correlation between errors and place field reliability (r = −0.19, df = 50, p > 0.10).Many studies have shown that certain physiological functions change only for old rats that show a spatial memory impairment, but not for aged rats whose performance is comparable to young animals (e.g., see ref. [7]for review). Therefore, it was of interest to divide the aged animals into impaired and unimpaired groups to determine if the apparent reorganization of representations in CA1 is related to efficiency of performance. Because young animals made between 0.08 and 0.38 errors per trial by the end of acquisition training, aged animals who averaged more than 0.38 errors per trial at the end of the acquisition period were considered impaired. Fig. 4 presents separate analyses for the 4 impaired and 3 unimpaired aged rats who contributed CA1 data. A significant correlation between choice accuracy and spatial selectivity was observed only for the intact, unimpaired aged rats (r = −0.67, df = 20, p < 0.01). Impaired aged rats did not show such a correlation (r = −0.22; df = 29, p > 0.10). Unimpaired aged rats showed a marginal relationship between errors made and reliability (r = −0.43, df = 20, p = 0.05), although impaired old rats did not (r = +0.01, df = 29, p > 0.10).Spatial selectivity scores represent a comparison between in-field and out-of-field firing rates. Because we found a significant age effect on spatial selectivity scores for CA1 cells, it was of interest to determine whether this was due to changes in the in-field and/or out-of-field firing rates. No significant correlations were found for either young or old CA1 cells with respect to in-field or out-of-field firing rates. This raised the possibility that different patterns of firing rate changes occurred in the aged CA1 cells. Some cells may have increased in-field firing coincident with decreased out-of-field firing; others may have increased in-field firing only, or decreased out-of-field firing only. In all cases, the result would be increased spatial selectivity. An initial examination of these possibilities was performed by evaluating further those 24 aged CA1 place cells that were held for the duration of the acquisition period. Specifically, the proportion change in in-field firing rate and out-of-field firing rate from Day 1 and Day 7 was first calculated according to the following formula: (a − b)/(a + b), where a = Day 1 rates and b = Day 7 rates. Fig. 5 presents a scatterplot of the resultant indices of proportion change: positive values indicate increased firing by Day 7, and negative values indicate decreased firing by Day 7, for either in-field (x axis) or out-of-field (y axis) rates. These data show that although different patterns of change occurred in our old animals, the change was observed for both in- and out-of-field rates. Further, there was a general tendency for the in-field firing rate to change more than the out-of-field firing rate. This pattern supports the hypothesis that place fields of aged CA1 cells tended to become smaller.Fig. 6 shows that in contrast to what was observed for cells recorded in the CA1 region, place-specific firing by hilar complex-spike cells was not correlated with the number of errors made on the maze for either young (n = 6) or old (n = 3) rats (young: r = +0.18, df = 41, p > 0.10; old: r = −0.05, df = 16, p > 0.10), regardless of their memory status. Nevertheless, the mean spatial selectivity scores at the end of training showed the same age and experience-dependent pattern reported previously: 11.07 ± 4.6 and 5.08 ± 0.7 (p < 0.05) for young and old rats, respectively.A correlation has been suggested to exist between spike amplitude and spatial selectivity, with larger spikes being associated with greater selectivity [32]. It has also been suggested that spatial selectivity scores could be biased by the overall spontaneous firing rate of neurons [4]. These suggestions, together with the concern that the spike signal might have deteriorated over multiple recording sessions, prompted us to determine if the observed age-related change in CA1 place field spatial selectivity could be accounted for by age-dependent changes in spike amplitude and/or mean firing rate. Considering those cells that were recorded across all 7 acquisition training days, it was found that the average CA1 complex-spike cell amplitudes for Days 1 and 7 for young animals were 168.8 μV ± 21.5 μV and 165.8 μV ± 26.0 μV, respectively. CA1 complex-spike cell amplitudes for cells recorded from old animals on Days 1 and 7 were 170.7 μV ± 18.7 μV and 139.7 μV ± 15.1 μV, respectively. For both age groups, the change in amplitude over days was not statistically significant. Also, no age effects on spike amplitude were observed when all CA1 cells were considered together, regardless of the number of days a cell was recorded. A similar pattern of results was also obtained for hilar cells. Thus, a change in spike amplitude across days could not readily account for the age-related elevation of place specificity of CA1 complex-spike cells, nor the reduced specificity of hilar complex-spike cells. However, this pattern of results is entirely consistent with our finding that unit spatial selectivity and reliability of newly encountered cells did not differ from correlates of cells that were recorded over many days (see above).The above described spike amplitude data are congruent with the claim by McNaughton and colleagues [32]that a significant correlation exists between spike amplitude and spatial selectivity score. Such a correlation was observed for both CA1 and hilar cells of young rats when one correlated a given day’s specificity score with the average spike amplitude for that same cell on the same day (r = +0.24, df = 99, p < 0.02 and r = +0.26, df = 142, p < 0.01). A similar correlation was also observed for hilar cells of old rats (r = +0.37, df = 59, p < 0.01), but not for CA1 cells recorded from aged rats (r = −0.04, df = 230, p > 0.50). It is worth noting here that although spike amplitude changes per se cannot account for the correlation observed for the old CA1 place cells, it is not clear what one can say regarding the significant correlations found. Spike amplitude can vary for a number of reasons, such as distance from the electrode tip, health of the cell being recorded, and electrode impedance.Mean firing rates did not distinguish the two age groups for either CA1 complex-spike cells (young = 0.53 ± 0.05 Hz; old = 0.63 ± 0.13 Hz) or hilar complex-spike cells (young = 0.30 ± 0.05 Hz; old = 0.23 ± 0.07 Hz). However, mean rates were statistically correlated with spatial selectivity for both CA1 (r = −0.34, df = 99, p < 0.01) and hilar cells (r = −0.18, df = 142, p < 0.05) of young rats, but not for either cell type recorded from aged rats (r = −0.12, df = 230, p < 0.08 and r = −0.19, df = 59, p > 0.10). These findings support previous claims of a relationship between spatial selectivity and mean firing rate in young animals [4], and reveal that the age-specific response of CA1 and hilar neurons to acquisition of a spatial task cannot be accounted for by age-changes in mean firing rate per se.One consistent feature of hippocampal place cells recorded as animals perform the radial maze has been the directional nature of the spatial code [13, 16]. Because it has been reported that place fields do not have a significant directional component when rats traverse open arenas [22], it is possible that place fields become more directional when directed search is required [13]. If this were true, one might expect a correlation between directional bias of place fields and choice accuracy during acquisition of a spatial memory task. There was no correlation between the latter ratio and errors made (p > 0.10) for young or aged animals, nor was there a consistent change in directionality across the days of acquisition training for either young or old CA1 or hilar cells (p > 0.10). Comparison of directional indices revealed that CA1 and hilar complex-spike cells recorded from both young and old animals showed clear directional discharge, even on Day 1 of spatial memory training (young CA1: 7.78 ± 1.76; old CA1: 10.06 ± 3.04; young hilus: 6.10 ± 2.20; old hilus: 6.09 ± 2.12).Many reports in the literature show that aged rats typically require more time to perform tasks that require unrestrained navigation. The requirement for more time by old rats could be due to the fact that old rats move more slowly, spending less time in theta-related behaviors. To provide a preliminary evaluation of the relationship between run speed and spatial selectivity, we examined the correlation between spatial selectivity scores with the time required per choice. No statistically significant effects were observed for CA1 or hilar cells of young or old rats (for all, p > 0.10). Furthermore, run time per choice did not significantly vary across the 7 days of acquisition for either young or old animals (repeated measures ANOVA: for both, p > 0.10).3DiscussionTo address the issue of whether the apparent age-related reorganization of spatial representation in hippocampus [19]is due to the fact that, relative to young animals, old rat hippocampal neurons are differentially responsive to task demands, we attempted to correlate the number of errors made during the acquisition of a spatial task and measures of spatial encoding by hippocampal place cells. It was found that CA1 cells recorded from memory-intact old rats became more spatially selective as fewer errors were made on the maze. In contrast, CA1 cells of young animals or of memory-impaired old animals did not show such a correlation. Additionally, hilar place neurons from young or old rats did not show significant correlations between place field specificity and errors. This pattern of effects is consistent with our previous findings of no differences between young and old CA1 or hilar place cells when rats perform a nonspatial task, and significantly increased spatial selectivity of aged CA1 place fields during asymptote performance on a spatial memory task. The present data suggest that the age difference in CA1 place representation emerges as new (spatial memory) task demands are placed on the animal.During acquisition of the new spatial memory task, CA1 place field specificity in old rats was related to errors. This was not the case for aged rats performing at asymptote [19]. That acquisition and asymptote performance are differentially related to place field specificity in old rats suggests a different role for CA1 place field in spatial performance depending on whether animals are learning new spatial information or are using flexibly familiar spatial information.In contrast to what was observed for CA1 place cells, hilar place cell specificity or reliability were not related to choice accuracy. One could argue that the old hilar cells did not show such a correlation because there was a floor effect on spatial selectivity scores. This is probably unlikely because our previous work has shown that spatial selectivity scores of hippocampal neurons as low as 1.0 can be found. A different interpretation is that with age, there are neuropathological changes in the dentate gyrus that must be compensated for (perhaps in CA1) during new spatial learning.The pattern of effects observed in this study underscores the selective nature of the aging process on hippocampal function [2, 19]: there is not a general demise in information processing capacity by all hippocampal neurons. Rather, it appears that subpopulations of aged hippocampal cells have the capacity to increase or decrease the specificity of information codes depending on the behavioral context. This type of experience-dependent plasticity differs from that observed in young animals whose behavioral accuracy is reflected more in terms of changes in reliability, not specificity, of the spatial code. Nevertheless, for both young and old animals, the flexibility conferred by the dynamic nature of spatial codes allows experience and task demands to direct the organization of neural representations such that particular spatial strategies can be implemented.The possible spatial strategies an animal might use include ones that rely predominantly on global geometric environmental cues, specific environmental landmarks, or idiothetic (internally-generated) cues. In principle, use of any of these strategies could lead to significant learning. There is much experimental data supporting theories that suggest young adult rats use a combination of these strategies to achieve a consistently high level of performance across time. The hippocampus likely contributes to the functional operation of each one of the above spatial strategies because place fields have been shown to respond to changes in global environmental cues, specific landmark cues, and vestibular input [10, 15, 26, 32]. Not only do hippocampal place cells respond to changes in sensory input, but it is becoming more clear that hippocampal spatial representations of young rats also dynamically reflect the current behavioral context (e.g., [1, 19, 32]). We extend this view of hippocampal representation by hypothesizing that behavioral context determines the relative contribution of different spatial cognitive strategies to behavior of young animals, and that the particular strategy selected reflects the organization of spatial neural codes [19], i.e., the current configuration of neural representation across hippocampal subregions, and the current constellation of encoding properties. Thus, a given cell’s correlate may become more or less reliable depending on the relative weight of the message to be relayed. As young adult animals learn a spatial task, certain patterns of hippocampal discharge become reinforced at some level in the spatial learning system (not necessarily in hippocampus), leading to the observed correlation between errors and reliability of spatial codes.Old animals suffer from a variety of neuropathological changes across different brain structures, including hippocampus. Such changes may lead to a breakdown in the system that modulates the reliability of hippocampal spatial codes, which in turn leads to the development of compensatory information processing networks. The functional consequence of the development of such networks could be a greater reliance on other spatial coding parameters besides reliability, such as the spatial selectivity of cells. That such a change in the nature of hippocampal spatial representation could result in efficient performance is suggested by the results of Fig. 2, which demonstrates that only memory-intact aged rats showed the statistical correlation between errors and place field specificity. Impaired aged rats did not. Given that the hippocampus of old rats probably does not undergo a significant loss of neurons [28, 30], enhanced spatial selectivity by CA1 neurons more than likely reflects increased dendritic extent [27], a change in afferent input and/or biophysical/integrative properties of individual CA1 neurons.Not only did the old rats of this study show increased specificity of CA1 place fields during maze acquisition, but we again observed the result that hilar place fields are less spatially selective. Therefore, we would hesitate to conclude that only CA1 shows a neuroplastic response to learning in old rats. Rather, we suggest there is a reorganization of spatial representation among subpopulations of hippocampal place neurons. Given our presently limited understanding of the significance of place field size, one can only speculate on the significance of this apparent representational reorganization. It was suggested that the clearly different place field sizes of young CA1 and hilar neurons reflect the differential contribution of these substructures to hippocampal processing. Specifically, it may be that CA1 tends to show more broad place fields than hilar place cells because CA1 encodes more than sensory-defined spatial information. CA1 may also encode nonspatial (e.g., contextual) information. In this way, CA1 could provide the hippocampus with a general spatial referent, or framework, within which hilus can encode specific locations. With regard to the lifespan changes observed in aged animals, it is possible that the originally very selective hilus place fields lose spatial specificity because of the deafferentation of the dentate gyrus, which is known to occur [8]. Aged CA1 neurons then become more location-selective (perhaps with the support of direct entorhinal afferents [34]), as if to compensate for the lost hilar spatial codes. A functional implication of this series of events is that the aged hippocampus as a whole engages in less efficient and/or less flexible spatial context coding. One prediction of this interpretation is that aged rats should have more difficulty distinguishing changes in behavioral context. Consistent with this prediction is the finding that old rats have more difficulty learning a spatial task in a familiar room where a nonspatial task was previously performed than learning the same spatial task in a novel environment [19].In the face of past reports of reduced neuroplasticity in the hippocampus of old rats (e.g., ref. [5]), it may initially appear contradictory that here we argue that hippocampal neurons of at least some old rats engage in adaptive neural responses. Past work suggesting reduced plasticity generally evaluate dentate gyrus field potential responses to afferent stimulation. Our data are entirely consistent with these previous reports because we found hilar neurons to show lower place field specificity that is not related to errors. Therefore, to elaborate a point raised earlier, the sum of the neurophysiological data with performing old animals suggests that old age not only results in selective deterioration within the hippocampus, but also selective enhancement of cells’ relationships to behavior.With respect to experiments performed on young animals only, it is worth noting that recently it was reported that hippocampal place cells are more directional when directed search is required [13]. Here, we found that directional tuning did not change as animals learned a spatial task subsequent to learning a nonspatial task on the same apparatus. Together with the findings of Markus et al., these data suggest that the directional component of hippocampal neuron discharge is established upon initial exposure to a new environment and/or when new routes to reward must be learned. If the sensory environment or the routes to places of significance (e.g., chocolate milk reward) are unchanged, the directional code is constant. Because the memory demands of a behavioral situation play little or no role in determining the extent of directional bias of these cells, we suggest that the directional component of place field firing reflects knowledge of the general spatial framework, whereas the reliability or selectivity component reflects context-specific landmark information. The implication here is that individual hippocampal place neurons serve as perhaps one of many links between spatial reference frames, leading to integration of specific landmark cues with general environmental information.A final implication of this work is that it argues for a dynamic and more expansive neural systems approach to the study of the neurobiology of age-related behavioral change. That is, it should not be assumed that the declining function of one population of cells indicates that an entire structure is similarly deteriorating. Rather, given the powerfully dynamic responses of cortical neurons to experience or neural injury [18, 23], one should consider the possibility that there may be functional restructuring of information codes that allow animals to behaviorally compensate for age-related neuropathology.AcknowledgementsThis research was supported by National Institutes of Health Grant AG09299 to S.J.Y.M. We gratefully acknowledge the assistance of Karen Burk, Mitch Burt, Sonal Patel, and Leigh Hardy during behavioral testing and data analysis.References1K.B.AustinL.H.WhiteM.L.ShapiroShort- and long-term effects of experience on hippocampal place fieldsSoc. Neurosci. 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Lett.851988193198 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AAE31628A6182FD4870D74A34ED864E42FBF3E2.txt b/test/dataset/in/resources/corpus/Clean_0AAE31628A6182FD4870D74A34ED864E42FBF3E2.txt new file mode 100644 index 0000000..e166e9a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AAE31628A6182FD4870D74A34ED864E42FBF3E2.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press P14410.1093/geronb/61.3.P144 Journal of Gerontology: Psychological Sciences Short-Term Variability in Cognitive Performance and the Calibration of Longitudinal Change Salthouse Timothy A. Nesselroade John R. Berish Diane E. Department of Psychology, University of Virginia, Charlottesville. 5 2006 61 3 P144 P151 10 10 2005 15 6 2005 The Gerontological Society of America 2006 Recent studies have documented that normal adults exhibit considerable variability in cognitive performance from one occasion to another. We investigated this phenomenon in a study in which 143 adults ranging from 18 to 97 years of age performed different versions of 13 cognitive tests in three separate sessions. Substantial within-person variability was apparent across 13 different cognitive variables, and there were also large individual differences in the magnitude of within-person variability. Because people differ in the amount of short-term variability, we propose that this variability might provide a meaningful basis for calibrating change in longitudinal research. Correlations among the measures of within-person variability were very low, even after we adjusted for reliability, and there was little evidence that increased age was associated with a larger amount of within-person variability. hwp-legacy-fpage P144 hwp-legacy-dochead RESEARCH ARTICLE IT IS often assumed, at least implicitly, that people can be characterized as having a fixed level of a cognitive ability that can be accurately evaluated with a single assessment. However, to the extent that performance on cognitive tasks varies from one occasion to the next, as has been reported in numerous recent studies (e.g., Hertzog, Dixon, & Hultsch, 1992; Hultsch, MacDonald, Hunter, Levy-Bencheton, & Strauss, 2000; Li, Aggen, Nesselroade, & Baltes, 2001; Nesselroade & Salthouse, 2004; Rabbitt, Osman, Moore, & Stollery, 2001; Rapport, Brines, Axelrod, & Thiesen, 1997; Salinsky, Storzbach, Dodrill, & Binder, 2001; Salthouse & Berish, 2005; Shamni, Bosman, & Stuss, 1998), single assessments may represent only one of many possible levels of performance that could have been observed for that individual, and hence they are potentially misleading (cf. Hultsch & MacDonald, 2004; Nesselroade, 1991). Much of the prior research on within-person variability has focused on reaction time or other speed variables. These types of variables are often used as measures of transient states of arousal or alertness, and thus it is not surprising to find that they exhibit within-person variability. Only a few studies have investigated within-person variability with cognitive variables measured in terms of accuracy rather than in time or speed. In one such study, Hultsch and colleagues (2000) reported results on two memory tasks (word recognition and story recognition) across four occasions from 15 healthy older adults and 30 adults with dementia or osteoarthritis. In another study, Li and associates (2001) examined performance on three memory tasks (digit span, text memory, and spatial recognition) in 25 older adults across 25 sessions. Both studies reported substantial across-occasion variability for the measures of memory accuracy. However, in neither study was there any mention of how the test versions administered on different occasions were equated for difficulty, and therefore it is possible that some of the within-person (across-occasion) variability in those studies was attributable to differences in the difficulty of the versions. Only the study by Li and associates provided information about the reliability of the measures of within-person variability of cognitive performance, and the estimates were disappointingly low. That is, these researchers reported correlations between the variabilities computed across the first and the second half of the occasions, and between the variabilities computed across the odd- and even-numbered occasions, but both sets of correlations were quite low (i.e., Mdn = 0.20 and Mdn = 0.32, respectively). Finally, another limitation of the previous studies is that the sample sizes of normal healthy adults were fairly small, and only a narrow range of ages was represented. We designed the study reported here to address these limitations by using a moderately large sample of adults (N = 143) across a wide age range (18 to 97 years), who each performed a battery of 13 different cognitive tests on multiple occasions. Different test versions were administered on each occasion, but we carried out adjustments for version differences on the basis of data from another group of individuals who performed the versions in a counterbalanced order across sessions. There were two major issues of interest in this study. One concerned the magnitude of within-person variability in different measures of cognitive performance, and the implications that short-term variability might have for the interpretation of longitudinal change. Of particular interest is whether longitudinal change either could be difficult to detect or could be confused with short-term fluctuation, if the magnitude of short-term within-person variability is large relative to any systematic within-person change that may be occurring. To the extent that this might be the case, it is worth considering whether an individual's within-person variability might be used to help calibrate the magnitude of his or her change (Salthouse, Kausler, & Saults, 1986). Because some of the participants in this study had performed several of the tasks 3 years earlier, we were able to compare different ways of evaluating longitudinal change. The second major issue concerned the nature of within-person variability, particularly whether it is merely random noise or is a reflection of a meaningful individual difference characteristic that might be uniquely informative about the performance capabilities of the individual. One type of information relevant to this issue is the reliability of the within-person variability measures, because only if they were reliable would it be useful to characterize people as systematically differing in their degree of across-occasion variability. A second type of relevant information is the magnitude of correlations among measures of within-person variability for different cognitive variables. The rationale is that if the correlations were found to be moderately high, then the influences contributing to variability are unlikely to reflect measurement error or determinants that are specific to a particular variable. When investigating within-person variability, researchers need to consider two aspects: the number of occasions of measurement and the type of assessment in each occasion. For the first aspect, a minimum of two occasions is needed to evaluate within-person variability, but if a researcher is interested in attempting to determine the asymptotic level of performance, then 50 or more sessions may be required (e.g., Kliegl, Smith, & Baltes, 1989; Salthouse & Somberg, 1982). The optimum number of occasions obviously depends on the specific goals of the study, because if a researcher is interested in decomposing the variability in terms of factors related to learning, or to cyclical variations, then he or she will need a relatively large number of occasions. However, pragmatic considerations usually mean that there is a trade-off between the number of occasions available from each individual and the number of individuals with some estimate of within-person variability. Because our earlier work (Nesselroade & Salthouse, 2004) indicated that three occasions provided sufficient information to warrant comparisons of individual differences in measures of within-person variability, we had each participant in the current study perform the tests on three occasions. The second aspect that researchers need to consider in studies of within-person variability is how they can be confident that the assessments on different occasions are equivalent, and that any variation observed from one occasion to the next is not attributable to differences in the particular items or versions that are administered on a given occasion. This is seldom a concern with reaction-time tasks and certain memory tasks in which the trials or items are either identical or very similar, and are sampled randomly within and across occasions. However, version equivalence is more of a concern with tests of other types of cognitive abilities, and there are a number of possible solutions to this problem. One approach is to use exactly the same version of the tests on each occasion. Although this obviously eliminates version differences, it may lead to an underestimate of within-person variability if at least some of the performance on later occasions is determined by one's memory for specific items from earlier occasions, or to an overestimate if awareness of the repeated items leads to feelings of resentment or boredom. Another approach is to use different versions of the tests on each occasion, and merely assume that they are equivalent without any explicit evaluation. However, to the extent that the versions are not truly equivalent, some of the observed variability may be attributable to version differences rather than to fluctuations within the individual. What might be the ideal approach would be to use different versions on each occasion that have been precisely equated by means of a procedure such as item response theory. Although this method would ensure that none of the across-occasion variation is attributable to differences in the tests, researchers would need a considerable amount of data to determine the characteristics of each item prior to conducting the research of primary interest. A compromise approach, and the one we adopt in this study, is to use different versions of the tests on each occasion, but to collect data from another sample of individuals who performed the versions in counterbalanced order to allow the difficulty levels of the different versions to be adjusted statistically. Methods Participants Characteristics of the 143 participants, divided into three age groups for ease of description, are summarized in Table 1. An inspection of the table reveals that increased age was associated with slightly poorer self-ratings of health, but that there was no relation of age to the scaled scores for vocabulary, digit symbol, logical memory, or word recall. The means of the scaled scores ranged from 11.5 to 13.6, indicating that the participants were functioning well above the average levels of the nationally representative normative sample. However, the absence of significant relations between age and the scaled scores suggests that there was no confounding between age and degree of selectivity or representativeness relative to the normative sample. Materials The test battery consisted of 13 tests that we selected to represent four distinct cognitive abilities. We chose the tests, which are briefly described in Table 2, because prior research indicated that the measures were all reliable, and each had a strong loading on its respective ability factor in a confirmatory factor analysis (Salthouse, 2004; Salthouse & Ferrer-Caja, 2003). We developed alternate versions of the tests by using other items from the same tests, such as even-numbered items from the Advanced Ravens Progressive Matrices, or from similar tests, such as the Wechsler Abbreviated Scale of Intelligence (WASI; 1999) vocabulary and Neuropsychological Assessment Battery (Stern & White, 2003) story memory, or by creating new items (e.g., digit symbol, paper folding, spatial relations, letter comparison, pattern comparison, synonym vocabulary, antonym vocabulary, and picture vocabulary). In all cases the number of items in the new versions of the tests was the same as in the original versions, but there was no overlap of specific items across versions. We designated the three versions as O for the original version and as A and B for each of the two new versions, respectively. Procedure The three versions of the tests were administered on successive occasions to all participants in the order O-A-B. We scheduled the three occasions at the individual's convenience, but most occasions were at approximately the same time of day within a 2-week period. Depending on the individual's schedule, the sessions occurred across successive days or within an interval extending to as many as 10 weeks. Because the test versions could have differed in difficulty, we conducted a preliminary study in which 60 young adults performed the three versions of each test in different orders. That is, 10 individuals each performed the tests in the six possible orders (O-A-B, O-B-A, etc.). The results from the preliminary study revealed that there were significant version differences in most of the tests, and thus we used the following procedure to adjust the scores on the A and B versions of each test to approximately match the mean of the O version. First, we computed linear regression equations from the data in the preliminary study to predict the scores on the O version of the test from the score on either the A or the B version. Second, we used the intercept and slope parameters from these equations to create adjusted A and B scores for each participant in the current study. To illustrate, the regression equation predicting the digit symbol score on the O version from the score on the A version based on the data from the preliminary study was DSO = 13.45 + 0.83(DSA). The application of these parameters to a participant in the current study with a score of 75 on version A would therefore result in an adjusted version A score of 75.7, or 13.45 + 0.83(75). Our rationale for the adjustment procedure was that the results from a study in which the test versions were administered in counterbalanced order can be used to equate the difficulty of the new versions to that of the original version (see endnote). Results Table 3 contains means, standard deviations, and estimated (coefficient alpha) reliabilities of the original (O version) and adjusted (A and B versions) scores for each test. It can be seen that most of the reliability estimates were above.7, with the exception of the synonym and antonym measures for the A and B versions. Although not presented in Table 3, the correlations between the scores on different versions of the same test were all moderately high; the median was.74, and it was.88 after we applied the standard formula to adjust each correlation for unreliability of the scores. Figure 1 portrays the mean composite scores, which we created by averaging the z scores for the variables assumed to represent the same construct (see Table 3), as a function of age and session or version. Two points should be noted about the results in this figure. First, the patterns of age relations on the composite variables are very similar to those reported in many previous studies (e.g., Salthouse, 2004; Salthouse & Ferrer-Caja, 2003), as there is an increase followed by a slight decrease in measures of vocabulary but generally monotonic declines for the other measures. Second, the patterns were nearly identical for the composite scores based on the three versions administered in different sessions. This latter finding suggests that any version or session differences that might exist were fairly similar in each age group. Three ways of expressing within-person variability for the measures are presented in Table 4. The simplest method is with the standard deviation of the scores across the three occasions (versions). Although computationally straightforward, the standard deviation is not easily interpretable without a frame of reference. One such frame of reference is the variability apparent across people, and therefore a second method of expressing within-person variability is in terms of the ratio of the average within-person standard deviation to the between-person standard deviation on the first occasion. Values for this index of within-person variability are contained in the fourth column of Table 4. Another frame of reference is the variation associated with an individual difference variable such as age. The fifth column of Table 4 therefore expresses within-person variability relative to the differences associated with cross-sectional age variation for the measures with significant age correlations. Specifically, we computed the slope relating the score on the first occasion to years of age, and then we divided the slope into the average within-person standard deviation to determine the number of years of cross-sectional age difference corresponding to the average within-person standard deviation. To illustrate, the age regression slope for the digit symbol variable was −0.635, and when this number was divided into 5.4 it yielded a value of 8.5. The median within-to-between ratio across the 13 measures was 0.46, which indicates that the variation for a given individual from one occasion to the next is almost one half as much as the variation from one person to the next. (This comparison is based on standard deviations as the measure of variability because they are in the same units of measurement as the variable. A comparison based on variances would obviously yield much smaller ratios, because the variance is the square of the standard deviation.) For the 9 measures with negative age correlations, the average within-person standard deviation was equivalent to the amount of variation apparent in cross-sectional comparisons across a period ranging from 8 to 29 years, with a median of 18.2. The values in Table 4 indicate that there is considerable within-person variability in each of the measures of cognitive functioning. Moreover, the ratios of within-person to between-person variability were actually somewhat larger for the vocabulary, fluid cognition, and episodic memory variables than for the perceptual speed variables that are most similar to the types of variables examined in prior studies of within-person variability. Another point to note in this table is that people differ in the magnitude of their within-person variability. That is, the (between-person) standard deviation of the within-person (across-occasion) standard deviations, reported in the third column of Table 4, are all moderately large. The variation across occasions is therefore not simply a reflection of a situation in which everybody is affected to the same degree. Table 5 contains correlations of the within-person standard deviation and mean across the three occasions, and the correlations of these variables with age before and after control of the other variable. The values in the second column indicate that most of the correlations between an individual's mean and his or her across-session variability were negative, which indicates that better (higher) performance was associated with smaller across-session variability. An inspection of the third column in Table 5 reveals that increased age was associated with significantly larger within-person variability for five of the variables. However, in every case the correlation with age was substantially reduced, and no longer significantly different from zero, after we controlled the variation in the mean score by means of a semipartial correlation. Nine of the variables had significant (negative) correlations between age and the mean, and all were still statistically significant after we controlled the variation in the across-occasion standard deviation. This pattern suggests that increased age is associated with lower mean performance on many of the cognitive variables, but that any relations between age and the measure of within-person variability appear to be attributable to the relations both variables have with the mean. We investigated the question of whether people can be characterized as more or less variable across different types of cognitive tests by computing correlations of the within-person standard deviations for the 13 cognitive tests. The correlations ranged from −.15 to +.48, but the median was only.05, and it was still only.09 when we ignored the sign of the correlation. We also computed correlations after partialling age from both variables, and in subsamples with a narrower range of ages. The median age-partialled correlation was.05, and the median correlations for participants 18 to 39, 40 to 59, and 60 to 97 years of age were, respectively,.06, −.01, and.11. One possible reason for the low correlations is weak reliability of the within-person variability measures. We investigated this possibility by obtaining estimates of the reliability of the within-person standard deviations by using the standard deviations from scores on different pairs of sessions as the “items” in the coefficient alpha. That is, for each individual, we computed three standard deviations for each test variable based on the scores in sessions 1 and 2, the scores in sessions 1 and 3, and the scores in sessions 2 and 3, and we then treated these three standard deviations (each based on two scores) as items in the computation of the coefficient alpha. The reliability estimates derived in this manner ranged from.42 to.71, with a median of.59. Although these values are lower than the psychometric standard of.70, they nevertheless indicate that the measures of within-person variability were sufficiently high to sustain meaningful correlational patterns if they were to exist in the data. What is most important is that, when we adjusted the correlations among the within-person standard deviations for these estimates of reliability, they were still quite small, with a median of only.10. At least on the basis of these results, therefore, it does not appear that individuals who exhibit large across-occasion variability in one cognitive measure are any more likely than the average individual to exhibit large across-occasion variability in other cognitive measures. We also conducted an exploratory factor analysis on the within-person standard deviations. As we would expect from the low correlations, the factor analysis did not reveal much evidence of structure. The correlation matrix had six eigenvalues greater than one, and thus we extracted six factors (iterative principal axes) and rotated them by means of promax. There were three variables with loadings greater than.4 on the first factor (i.e., spatial relations, paper folding, and paired associates), two on the second factor (i.e., synonym vocabulary and antonym vocabulary), four on the third factor (i.e., vocabulary, logical memory, recall, and paired associates), two on the fourth factor (i.e., letter comparison and recall), and one each on the fifth (i.e., matrix reasoning) and sixth (i.e., pattern comparison) factors. The relatively large number of factors with modest loadings of the variables on each factor, and the diversity of the variables loading on each factor, suggests that there is little or no structure in the available measures of within-person variability. This finding is substantially different from that observed in analyses of the means, because there is clear evidence of a strong structure among the means of these variables (e.g., Salthouse, 2004; Salthouse & Ferrer-Caja, 2003). The other major issue of interest in this study concerned the relation between short-term variability and longitudinal change. Because people vary in the magnitude of short-term within-person variability, it is possible that the same absolute value of longitudinal change could have quite different meanings for different people. For some individuals the change might be well within their normal range of fluctuation, but for others it might represent an extreme value. We could examine this possibility in the current data because 18 of the participants (age at first assessment, M = 57.1) had performed the original version of six of the tests 3 years earlier. The tests common across the 2001 and 2004 assessments were Vocabulary, Picture Vocabulary, Digit Symbol, Logical Memory, Word Recall, and Paired Associates tests. This sample is too small for meaningful statistical analyses, but it is useful for illustrating the point that change in the original units of measurement may not have the same functional meaning for different people. The longitudinal patterns were generally similar for each variable and can be illustrated with the results from the Word Recall test. The values for individual participants on this variable are portrayed in Figure 2, with the solid circles indicating the 2001 score and the open circles with bars representing the mean and standard deviation, respectively, of the three scores in 2004. An inspection of the figure indicates that, for many of the individuals, the scores were higher at the later assessment. Although these gains could reflect true improvements in ability, we suspect that a substantial proportion of the performance gains are attributable to retest effects (e.g., Ferrer, Salthouse, Stewart, & Schwartz, 2004; Salthouse, Schroeder, & Ferrer, 2004). The bars around the 2004 (Time 2, or T2) estimates in the figure confirm the finding that there is variability in performance from one occasion to another within a short interval, and the variation in the size of the bars indicates that the degree of across-occasion variability varies across individuals. These results imply that the same absolute longitudinal change may not have the same meaning for everyone. That is, a given magnitude difference from 2001 to 2004 may be small relative to the typical fluctuation for someone with relatively large within-person variability, whereas it could be large for a person who is much less variable. This point can be illustrated by considering two individuals in Figure 2 who were 44 and 46 years of age in 2001. These two individuals had similar word recall scores of 38 and 36 in 2001, and similar means across the three assessments in 2004 of 40 and 39, respectively. However, their across-occasion standard deviations in 2004 were 0.7 and 4.9, respectively, which indicates that the slightly larger absolute difference for the 46-year-old individual (i.e., 3 vs 2) was actually substantially smaller than that of the 44-year-old when it is expressed in within-person standard deviation units (i.e., 0.6 vs 2.9). The preceding example suggests that, depending on the method used to calibrate change, different conclusions might be reached about the magnitude and correlates of change. In order to examine this issue more systematically, Table 6 contains information relevant to different methods of calibrating change for the six variables with longitudinal data. In addition to containing the means and between-person standard deviations for the 2001 (Time 1, or T2) score, the mean across the three T2 scores, and the standard deviation of the T2 scores, Table 6 also summarizes three ways of expressing the within-person change from T1 to T2. The simplest and most frequently used method of representing change is the difference between the relevant scores at each occasion (i.e., the mean of the T2 scores minus the T1 score) in the original units of measurement. A second method is the difference scaled in T1 between-person standard deviation units (e.g., Ivnik et al., 1999; Schaie, 1996), and a third method is the difference relative to each individual's T2 within-person standard deviation (Salthouse et al., 1986). An examination of the entries in Table 6 reveals that the different methods of evaluating change yield different estimates of the magnitude of change, and perhaps more importantly, of the magnitude of individual differences in change. To illustrate, the between-person standard deviation for the absolute difference in the paired associates variable was approximately one half the average 3-year change (i.e., 0.8 vs 1.5), but when we scaled change in within-person standard deviation units, the between-person standard deviation was almost double the average change (i.e., 8.4 vs 4.7). The rank ordering of individuals in terms of the amount of change can also differ according to method of assessment. In fact, the correlations between the absolute difference and the difference scaled in within-person standard deviation units were.66 for vocabulary,.81 for picture vocabulary,.71 for digit symbol,.49 for word recall,.75 for logical memory, and.24 for paired associates. Discussion The results of this study provide answers to the two major issues that motivated us to initiate the project. First, the short-term within-person variability in accuracy measures of cognitive functioning is substantial, and for many variables it is nearly one half as large as that for between-person variability. The existence of moderate to large within-person variability for each of the 13 cognitive variables implies that single assessments may not be very informative about an individual's true level of functioning. This further compounds the psychometric problems associated with evaluations of within-person change associated with normal aging, disease, trauma, or some type of intervention. Accuracy of measurement is sometimes evaluated with retest correlations because they indicate the stability of the ordering of individuals. However, it is important to note that retest correlations could be moderately large because of the substantial variation across people in their average levels, and yet there could still be considerable fluctuation around these levels. Indeed, this situation is apparent in Figure 2 because people maintain approximately the same relative positions from T1 to T2 (i.e., the 3-year stability coefficient for the word recall variable was.67, and the median across the six variables was.81), but the bars corresponding to within-person variability indicate that the assessments still exhibited substantial variability. The existence of within-person variability in cognitive functioning has important implications for the evaluation of change. Most contemporary researchers assess change in the original units of measurement, and thus they implicitly assume that the units have the same meaning for everyone. A number of researchers have attempted to evaluate individual change relative to the variability that exists across people (e.g., Ivnik et al., 1999; Schaie, 1996), but this is not ideal because between-person variability is only a crude approximation of within-person variability. Salthouse and colleagues (1986) proposed that researchers might obtain more sensitive assessments of change by expressing the change for each individual relative to his or her own across-occasion variability. This method is analogous to the computation of an effect size for each individual, and it shares the property that normal variability is taken into account when the magnitude of an effect is specified. Conclusions about the magnitude of change, about between-person variation in change, and about correlates of change will therefore vary depending on how the change is assessed. The optimal method to be used in assessing change will obviously depend on the specific question of interest. For example, a comparison in the absolute units of measurement may be more meaningful if the variable is scaled in a ratio level of measurement such as units of time, or if it represents progress toward an absolute criterion. However, change calibrated relative to each individual's within-person variability may be more meaningful if the goal is to investigate change in units that are functionally equivalent in different people. Although large individual differences in amount of within-person variability are evident in every variable in Tables 4 and 6 and in Figure 2, there were no significant correlations between age and the measures of within-person variability after we adjusted for influences associated with the mean. This pattern is similar to that recently reported by Salthouse and Berish (2005) in several analyses, and it seems to suggest that information about an individual's short-term variability may not have any unique predictive power beyond what is available from his or her mean level of performance. However, it should be noted that in samples of older adults, Hultsch and colleagues (2000) and Rabbitt and associates (2001) have reported that within-person variability in reaction time was correlated with level of performance in other cognitive tasks. Therefore, it may be the case that within-person variability only provides unique information with variables assessing performance speed rather than performance accuracy, or in samples of individuals likely to be experiencing substantial change in level of cognitive functioning. In our study there was also little evidence of structure in the measures of within-person variability either in the raw correlations or in the exploratory factor analysis, before or after we adjusted for reliability of the measures. This pattern suggests that influences contributing to across-occasion variability in these variables are specific to particular variables and are not shared across different variables, even those assumed to reflect the same cognitive ability. In other words, even though the reliability estimates suggest that within-person variability is not simply random fluctuation, there is no evidence in these data that people who exhibit high across-occasion variability for one cognitive variable exhibit high across-occasion variability for other cognitive variables. Li and associates (2001) also failed to find much evidence of structure among the measures of within-person variability for several memory measures assessed across 25 occasions. Important questions for future research are the determination of what is responsible for the across-occasion variability in cognitive performance, and why there are such weak relations among the within-person variability measures from different cognitive tasks. In conclusion, there is now considerable evidence that calls into question the adequacy of the classical notion of a fixed true score as an ideal focus of measurement efforts. Theoretical concepts and analytical methods should therefore reflect this shift of thinking if progress it to be made in describing, measuring, and explaining behavior and behavior change. Decision Editor: Thomas M. Hess, PhD Although it would have been desirable to use individuals of the same age and ability range as those in the primary study for this calibration study, college students were more readily available and could be compensated with credit towards a course requirement rather than with money. These young adults had somewhat higher average levels of performance than the age-heterogeneous sample in the primary study on many of the variables. However, most of the relations between the predictor (i.e., version A or B) and criterion (i.e., version O) variables in the primary sample were linear (i.e., for the linear relation, median R2 = was .532; for the quadratic relation, median R2 = .004; and for the cubic relation, median R2 = .002), indicating that it is reasonable to extrapolate from one region of the distribution to the entire distribution. Figure 1. Mean composite scores across the three sessions or versions as a function of age. Bars above and below each point are standard errors Figure 2. Scores in 2001 (solid circles), and means (open circles) and standard deviations (bars) across the three assessments in 2004 for individual participants on the Wechsler Memory Scale III word recall variable Table 1. Characteristics of Participants. Age Group Age Correlation 18–39 40–59 60–97 Variable M SD M SD M SD N 38 — 49 — 59 — — Age 27.5 5.7 51.9 4.9 70.8 9.1 — Proportion female .58 — .69 — .48 — −.10 Education 15.2 2.0 16.1 2.0 15.4 3.0 .03 Health 1.6 0.8 1.8 0.8 2.2 0.9 .30* Anxiety 13.4 3.3 12.3 2.6 12.1 2.6 −.19 Scaled scores     Vocabulary 13.5 2.3 12.9 2.3 12.8 2.9 −.18     Digit symbol 12.3 2.3 12.8 2.8 12.1 3.4 .00     Logical memory 11.5 2.7 12.1 2.0 12.0 3.0 .11     Word recall 13.5 3.5 13.6 3.1 13.6 3.3 −.02 Notes: Education is reported in years, and health was a self-rating on a scale ranging from 1 = excellent to 5 = poor. Anxiety is the average state anxiety score (Spielberger et al., 1970) across the three occasions. The scaled scores were based on the age-adjusted values in the WAIS III (Wechsler, 1997a) and WMS III (Wechsler, 1997b) that are scaled to have a mean of 10 and a standard deviation of 3. *p <.01. Table 2. Description and Source of Variables. Variable Description Source Vocabulary Provide definitions of words Wechsler (1997a) Picture vocabulary Name the pictured object Woodcock & Johnson (1990) Synonym vocabulary Select the best synonym of the target word Salthouse (1993) Antonym vocabulary Select the best antonym of the target word Salthouse (1993) Digit symbol Use a code table to write the correct symbol below each digit Wechsler (1997a) Letter comparison Same or different comparison of pairs of letter stringsa Salthouse & Babcock (1991) Pattern comparison Same or different comparison of pairs of line patternsa Salthouse & Babcock (1991) Matrix reasoning Determine which pattern best completes the missing cell in a matrix Raven (1962) Spatial relations Determine the correspondence between a 3-D figure and alternative 2-D figures Bennett et al. (1997) Paper folding Determine the pattern of holes that would result from a sequence of folds and a punch through folded paper Ekstrom et al. (1976) Logical memory No. of idea units recalled across three stories Wechsler (1997b) Free recall No. of words recalled across Trials 1–4 of a word list Wechsler (1997b) Paired associates No. of response terms recalled when presented with a stimulus term Salthouse et al. (1996) aThere are two separately timed parts to this test, and thus the scores on each part can be treated as separate items when computing estimates of reliability. Table 3. Adjusted Means, Between-Person Standard Deviations, and Estimated Reliabilities. Original A B Construct Variable M SD Reliability M SD Reliability M SD Reliability Vocabulary     Vocabulary 51.9 9.2 .92 55.2 6.9 .85 54.6 5.3 .81     Picture vocabulary 20.0 4.5 .84 17.9 2.8 .71 18.4 3.5 .76     Synonym vocabulary 7.7 2.3 .78 6.7 1.7 .65 6.9 1.9 .52     Antonym vocabulary 6.7 2.9 .84 5.9 2.0 .61 6.0 2.1 .58 Perceptual speed     Digit symbol 76.2 18.3 NA 83.0 15.8 NA 82.8 19.2 NA     Letter comparison 11.0 2.7 .89 11.9 1.8 .88 12.1 2.1 .81     Pattern comparison 17.0 4.2 .90 17.7 3.9 .90 18.5 3.9 .90 Fluid Cognition     Matrix reasoning 8.2 3.3 .79 10.6 2.2 .79 8.2 3.9 .81     Spatial relations 9.6 4.8 .87 13.8 2.1 .66 15.1 2.0 .70     Paper folding 6.6 2.8 .77 8.5 1.7 .74 7.8 2.7 .84 Episodic memory     Logical memory 43.9 9.2 .86 47.8 5.6 .84 48.6 6.9 .86     Word recall 36.8 6.4 .90 39.4 3.9 .91 38.7 4.2 .92     Paired associates 3.6 1.8 .82 4.5 0.7 .82 4.3 0.9 .89 Notes: For all variables except for the antonym vocabulary and matrix reasoning variables in version B, scores for versions A and B were adjusted with the regression equations derived from the results of the preliminary study. Reliability refers to coefficient alpha estimates of reliability. NA indicates that an estimate of reliability was not available. Table 4. Different Methods of Expressing Within-Person Variability. Within SD Variable M SD Within/ Between Age, in Years Vocabulary 3.5 2.4 .38 NA Picture vocabulary 2.1 1.1 .47 NA Synonym vocabulary 1.3 0.9 .57 NA Antonym vocabulary 1.5 0.9 .52 NA Digit symbol 5.4 2.9 .30 8.5 Letter comparison 1.0 0.6 .37 12.3 Pattern comparison 1.4 0.8 .33 9.2 Matrix reasoning 2.0 1.0 .61 18.2 Spatial relations 3.3 1.8 .69 27.3 Paper folding 1.5 0.8 .54 19.0 Logical memory 4.2 2.3 .46 29.2 Word recall 2.5 1.5 .39 16.3 Paired associates 0.8 0.5 .44 18.6 Notes: Within SD refers to the within-person standard deviation across the three occasions; within, between is the ratio of average within-person standard deviation to the between-person standard deviation on the first occasion; and years of age refers to the number of years of cross-sectional age difference on the first occasion corresponding to the within-person standard deviation. NA indicates that the estimate was not available because the variable did not have a significant negative correlation with age. Table 5. Correlations of Within-Person Variability and Mean Performance With Each Other and With Age Before and After Control of the Other Variable. Age Correlations Variable SD−M SD SD· M M M·SD Vocabulary −.70* .26* .14 −.18 .01 Picture vocabulary −.02 .17 .17 −.04 −.04 Synonym vocabulary −.20 −.01 .03 .19 .19 Antonym vocabulary −.14 .06 .06 −.01 .00 Digit symbol −.10 .13 .08 −.65* −.65* Letter comparison −.34* .21 .00 −.61* −.55* Pattern comparison −.12 −.08 .01 −.71* −.70* Matrix reasoning −.48* .18 −.18 −.65* −.57* Spatial relations −.78* .36* −.01 −.49* −.21* Paper folding −.58* .37* .06 −.54* −.35* Logical memory −.39* .03 −.13 −.39* −.38* Word recall −.60* .29* −.00 −.49* −.33* Paired associates −.70* .23* −.12 −.48* −.33* Notes: The age correlation for SD·M is the semipartial correlation between age and SD after controlling the variation in the mean, and the age correlation for M·SD is the semipartial correlation between age and mean after controlling for variation in the SD. *p <.01. Table 6. Retest Statistics. Variable T1 T2 M T2 SD Difference Difference (T1 SD) Difference (SD) Vocabulary 52.1 (13.2) 52.3 (6.2) 3.8 (3.2) 0.2 (8.8) 0.01 (.67) −0.2 (1.8) Picture vocabulary 19.7 (5.2) 18.4 (3.7) 2.8 (1.4) −1.3 (2.2) −0.25 (.43) −0.2 (2.4) Digit symbol 72.6 (18.3) 75.4 (16.7) 5.8 (3.6) 2.8 (7.0) 0.15 (.38) 1.4 (2.7) Word recall 33.9 (6.1) 36.9 (4.8) 2.5 (1.7) 3.0 (4.6) 0.50 (.76) 8.4 (28.8)a Logical memory 43.3 (10.9) 45.9 (7.1) 3.7 (2.0) 2.6 (5.5) 0.24 (.50) 1.0 (2.4) Paired associates 2.5 (1.6) 4.0 (1.1) 0.7 (0.5) 1.5 (0.8) 0.94 (.50) 4.7 (8.4) Notes: For the table, N = 18. T1 is the score at the 2001 assessment; T2 M is the mean across the three assessments in 2004; T2 SD is the within-person standard deviation across the three assessments in 2004; Difference is the T2 M − T1 difference; Difference (T1 SD) is the difference between T2 M and T1 in T1 between-person SD units; and Difference (SD) is the difference between T2 M and T1 in person-specific T2 SD units. Values in parentheses are between-person standard deviations. aThe presence of an extreme outlier inflated the between-person standard deviation because the value without the outlier was 2.3. This research was supported by the National Institute on Aging under Grant RO1 AG 19627 to T. A. Salthouse. We thank the following people for the scheduling and testing of participants and the entering and checking of data: James Darragh, Samantha Norton, Malaika Schiller, Sara Shelley, Rachel Spiotto, Catherine Thrasher, and David Yost. References Bennett, G. K., Seashore, H. G., Wesman, A. G. (1997). Differential Aptitude Test. San Antonio, TX: The Psychological Corporation. Ekstrom, R. B., French, J. W., Harman, H. H., Dermen, D. (1976). Manual for kit of factor-referenced cognitive tests. Princeton, NJ: Educational Testing Service. Ferrer, E., Salthouse, T. A., Stewart, W., Schwartz, B. (2004). Modeling age and retest processes in longitudinal studies of cognitive abilities. 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A., Somberg, B. L. (1982). Skilled performance: The effects of adult age and experience on elementary processes. Journal of Experimental Psychology: General, 111,176-207. Schaie, K. W. (1996). Intellectual development in adulthood: The Seattle Longitudinal Study. New York: Cambridge University Press. Shammi, P., Bosman, E., Stuss, D. T. (1998). Aging and variability in performance. Aging, Neuropsychology, and Cognition, 5,1-13. Spielberger, C. D., Gorsuch, R. L., Lushere, R. E. (1970). Manual for the State–Trait Anxiety Inventory—Form X. Palo Alto, CA: Consulting Psychologists Press. Stern, R. A., White, T. (2003). Neuropsychological Assessment Battery administration, scoring, and interpretation manual. Lutz, FL: Psychological Assessment Resources. WASI (Wechsler Abbreviated Scale of Intelligence) manual., San Antonio: The Psychological Corporation. Wechsler, D. (1997). Wechsler Adult Intelligence Scale (3rd ed.). San Antonio, TX: The Psychological Corporation. Wechsler, D. (1997). 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Allen, TX: DLM. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AAFC5FCF43CD0EAD64BB3079A31F08AA3DEC061.txt b/test/dataset/in/resources/corpus/Clean_0AAFC5FCF43CD0EAD64BB3079A31F08AA3DEC061.txt new file mode 100644 index 0000000..fef8cef --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AAFC5FCF43CD0EAD64BB3079A31F08AA3DEC061.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press 3610.1093/geronb/62.1.S36 Journal of Gerontology: Social Sciences Functional Limitations and Changes in Levels of Depression Among Older Adults: A Multiple-Hierarchy Stratification Perspective Schieman Scott Plickert Gabriele Department of Sociology, University of Toronto, Ontario, Canada. 1 2007 62 1 S36 S42 20 7 2006 15 12 2005 Copyright 2007 by The Gerontological Society of America 2007 Objectives. This study examined the effects of functional limitations on changes in levels of depression over time. A multiple-hierarchy stratification perspective framed the analyses of potential stratification-based contingencies of race, gender, and socioeconomic status. Methods. We derived data from a longitudinal study of adults aged 65 and older in the Washington, DC, metropolitan area over a 3-year period (2001–2003). We used ordinary least squares regression models in order to assess the focal associations across a set of social status interaction terms. Results. Changes in functional limitations were associated with changes in depression, but the patterns depended on interactions among race, gender, and socioeconomic status. Discussion. These observations contribute to research by blending ideas from double and triple jeopardy and differential vulnerability perspectives. Although analyses of multiple contingencies create analytical challenges, this complexity is needed to accurately specify the mental health effects of functional limitations. hwp-legacy-fpage S36 hwp-legacy-dochead RESEARCH ARTICLE THE stress process perspective identifies limitations in activities of daily living as among the most pernicious stressors in late life (Pearlin & Skaff, 1996). Levels of functional limitations are higher among older age groups (Long & Pavalko, 2004) and are associated positively with depression (Kelley-Moore & Ferraro, 2005; Yang & George, 2005). Moreover, limitations are distributed unequally across statuses such as gender, race, and socioeconomic status (SES; House, Lantz, & Herd, 2005). Although most research has focused on status variations in the levels of limitations, for this study we asked the following question: Do dimensions of stratification modify the effects of changes in limitations on changes in depression? In order to frame our analyses, we applied Clark and Maddox's (1992) multiple-hierarchy stratification perspective, which asserts that “it is useful in understanding health outcomes to consider income, education, gender, and race as independent but potentially interactive influences on changes in functional status over time” (p. S223). Social scientists have long been interested in the ways in which ascribed statuses intersect to influence health over the life course (McLeod & Nonnemaker, 1999). Of those statuses, race and gender are among the most influential because of their association with racism, sexism, and socioeconomic disadvantages (Williams, 2005). For example, the double jeopardy thesis posits that African Americans have worse health outcomes at all ages (Ferraro & Farmer, 1996)—but being African American and old has compounding effects (Clark & Maddox, 1992). This draws upon the cumulative disadvantage thesis, which posits that health disparities increase with age (Dannefer, 2003; O'Rand, 1996). Alternatively, some scholars contend that age is a leveler because it erodes the harmful effects of disadvantages, reducing the race-based health gap with age or even creating a reversal among the oldest-old population (Dowd & Bengtson, 1978). In terms of health, some scholars have identified elderly African Americans as a more robust group than their younger peers (Gibson, 1991) or a survival elite (Kelley-Moore & Ferraro, 2004). Others have described a disability crossover in which African Americans in old age surpass similarly aged White peers in health and physical functioning (Clark & Maddox, 1992; Johnson, 2000). According to Ferraro and Farmer (1996), “Another feature of the literature that merits further investigation is the extension of the concept of double jeopardy to triple jeopardy due to sexism” (p. 29). That is, inequalities associated with gender extend these ideas to the triple jeopardy hypothesis: African American women are most likely to experience the sharpest health declines during late life (Clark & Maddox, 1992). Yet, Clark and Maddox found that African American women had more modest physical health declines than African American men. Evidence on this matter remains inconclusive. For example, Manton (1980, 1988) contended that African American men who survive to old age do not have a lower life expectancy than White men. After age 75, in fact, African American men may have better life expectancy. Despite these advances in knowledge, the role of SES in triple jeopardy processes remains unclear. Researchers have attributed race-linked health disparities partly to SES disadvantages (Williams, 2005). We examined the extent that SES—in combination with gender and race—influences the association between changes in limitations and depression. In order to frame our ideas, we propose the resource vulnerability versus resource erosion hypotheses. The resource vulnerability hypothesis is derived from the differential vulnerability perspective, which implies that lower SES groups tend to experience more deleterious effects of stressors because they have fewer resources to avoid or manage stressors (McLeod & Nonnemaker, 1999). These ideas suggest that limitations are associated with greater increases in depression among low-SES elders. Moreover, the multiple-hierarchy stratification perspective includes race and gender variants of the SES-based resource vulnerability hypothesis. Clark and Maddox (1992) asserted that “the greater probability of entering old age in poverty and with fewer years of formal education exemplifies and may add to the impact of minority status on the experience of aging, it has been argued, particularly for minority women” (p. S222). Applying these ideas, the gender variant predicts that limitations are associated with the largest increase in depression among low-SES women; the race variant predicts that increased limitations have the strongest effect among low-SES African Americans. In contrast to the resource vulnerability view, it is plausible that high-SES groups have the most to lose with respect to health status. For example, Pampel and Rogers (2004) contended that “high socioeconomic status groups are harmed the most by unhealthy behaviors because, given their greater potential for good health, they have the most to lose from damaging lifestyles” (p. 307). African Americans and low-SES groups tend to experience lower life expectancy, higher morbidity and mortality, and worse health across the life course (Kelley-Moore & Ferraro, 2004). If Whites and high-SES groups are more likely to have experienced health advantages over the life course, then late-life exposure to limitations may be unexpected, unfamiliar, and challenging. By extension, although socioeconomic resources may enhance coping and reduce vulnerability to depression, limitations could undermine these resource benefits (the resource erosion hypothesis). Moreover, the resource erosion view includes race and gender variants, which researchers must consider simultaneously because White women have the highest life expectancy (80.0 years), followed by African American women (74.9), White men (74.8), and African American men (68.2) (Centers for Disease Control, 2002). Although women report poorer health than men on some measures, men die at younger ages from some conditions (Rieker & Bird, 2005). And although high-SES Whites tend to have better health than other race/ethnic groups over the life course, race may modify health patterns in late life. If elderly African Americans are indeed a healthier group than their White peers, then increased limitations are likely to be more depressing for high-SES Whites and least depressing for low-SES African American men. In sum, we expected that increases in limitations would be associated with increases in depression. The differential vulnerability hypothesis proposes that individuals with low status are more vulnerable to limitations, whereas higher status is protective against the distressing effects of increased limitations. The multiple-stratification perspective underscores the intersections of race, gender, and SES. For example, race and gender variants of the differential vulnerability thesis would predict that the depressing effects of limitations would be strongest among low-SES elders, especially African Americans and women; we labeled this the resource vulnerability hypothesis (the converse is the resource benefit, in which higher statuses have protective effects). By contrast, the resource erosion hypothesis would contend that increased limitations would erode the resource benefits of those who have traditionally enjoyed higher status advantages (i.e., high-SES Whites). Methods Sample The data in this sample derived from in-person interviews conducted in 2001–2002 with people 65 years and older residing in the District of Columbia and two adjoining Maryland counties (Prince George's and Montgomery). Sample selection and recruitment began with the Medicare beneficiary files for the three areas. In addition to the names of all people 65 years and older who are entitled to Medicare, the files provided information about the race and gender of each beneficiary. The next step entailed selection from the large pool of potential participants. To maximize the social and economic diversity, we randomly selected a total of 4,800 names equally divided among the three locales, African Americans and Whites, and women and men; this strategy created 12 groups, each containing 400 names. The goal was to enlist a sample of 1,200 people living independently, with approximately 100 in each of the 12 groups. Approximately 65% of eligible respondents (1,741) contacted agreed to participate, yielding 1,167 cases; eligible participants spoke English, lived in the community, and were able to independently complete the interview. Data collection occurred in three waves, each separated by approximately 12 months. Wave 1 interviews occurred during 2001–2002. For this article, we analyzed data from Waves 1 and 3 because of insufficient change in levels of depression between Waves 1 and 2. At Wave 3, which occurred approximately 2 years after the first interview, we reinterviewed 925 individuals (79%). We present results for individuals who were in Waves 1 and 3 and for whom we had complete responses to focal measures (N = 898). Measures The Appendix presents the specific items used to measure depression, functional limitations, and diseases. Depression items asked about symptoms in the past 7 days. We averaged the items to create the index (αT1 =.770; αT3 =.764). We selected these items from the longer version of the widely used Hopkins Symptom Checklist (Derogatis, Lipman, Rickels, Uhlenhuth, & Covi 1974). Studies document that the depression score is correlated with major depression as defined by Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 1994). We subtracted T1 scores from T3 scores in order to create the change in depression score. The functional limitations items asked participants about the extent to which they experienced difficulties performing nine different activities of living (Katz, Ford, Moskowitz, Jackson, & Jaffee, 1963). Three additional questions, selected from well-known indices (see McDowell & Newell, 1996), asked about scenarios that required physical effort. We standardized and averaged all items to create the limitations index; higher scores indicate more limitations (αT1 =.920, αT3 =.898). We subtracted T1 scores from T3 scores in order to assess change in limitations. In order to assess the joint effects of gender and race, we created three dummy variables (African American women, White women, and White men) with African American men as the contrast code because, as we described above, the literature identifies the experience of African American men in late life as the most different (Clark & Maddox, 1992; Johnson, 2000; Manton 1980, 1988). We created an SES index by averaging standardized education and household income (α =.700). We coded education from low to high: eighth grade or less (1), some high school but did not graduate, high school graduate or general equivalency diploma, specialized training, some college but no degree, and college graduate or more (6). Household income (before taxes in the past year) included the participant's best estimate of all sources of income for household members from salaries, money market funds, Social Security, pensions, real estate, or government entitlements. Eleven categories ranged from low (less than $10,000) to high ($100,000 or more). We coded marital status such that divorced, widowed, and never married persons were contrasted with married persons. We coded current/recent smoker as 1 if participants currently smoked or had smoked in the past 10 years, and 0 otherwise. We calculated body mass index as weight (kg) divided by height squared (cm2) based on the self-reported measures of weight (in pounds) and height (in inches). The disease index summed the number of health problems in the 5 years prior to the Wave 1 interview (see Appendix). In addition, the new diseases index summed the number of new diseases that occurred after the Wave 1 interview. Plan of Analysis Using ordinary least squares regression techniques, we tested a series of models: (a) the stability model, which included only Wave 1 depression as the sole predictor of change; (b) the additive model, which included gender–race groups, baseline and change in limitations, and controls (excluding the interaction terms); (c) the model including the Gender–race × Change in limitations terms; and (d) the inclusion of other health measures. We included the fourth model to ensure that other health statuses and conditions did not account for our focal associations. In addition, we examined these models separately for low (< median) and high (> median) SES groups. Separate analyses (not shown) examined three-way interactions between gender–race groups, limitations, and SES to confirm their statistically significant effects; however, presenting all possible interactions in one table is logistically complex. Thus we present results only for the low- versus high-SES groups. Following the procedures outlined by Mirowsky and Ross (2001), we used the difference score as the dependent variable (T3 depression minus T1 depression score) and adjusted for T1 depression in all models to control for regression to the mean. A common problem of longitudinal data analysis is that sample attrition may bias the results, especially if depression at baseline affects attrition. In order to account for this, we adjusted for the likelihood of attrition in all of our regression models. This adjustment took into account the probability that the baseline depression or any other of the T1 independent variables predicted attrition (Winship & Mare, 1992). Using logistic regression techniques, we found that baseline depression was unrelated to the odds of attrition. Limitations, low SES, and age, however, were associated positively with the odds of attrition; body mass index and diseases, paradoxically, were associated negatively with attrition. None of the other baseline variables were statistically significant. The model yielded a pseudo r-square of.05, indicating that almost all attrition was random with respect to the model's baseline measures (results available upon request). Results Table 1 presents summary statistics for all of the focal measures across SES and gender–race groups. Table 2 presents the findings for the low-SES group. Model 1 presents the stability model, showing that baseline levels of depression were associated negatively with change in depression. Model 2 indicates that baseline levels and changes in functional limitations were associated with increases in depression, and levels of depression increased more for White women compared to African American men. However, we observed significant Gender–race × Change in limitations interactions in Model 3, indicating that increases in limitations were associated with increases in depression. That pattern was stronger among White women and men compared to African American men; tests for model improvement showed that the inclusion of these interactions improved model fit (F = 2.99, p <.05). In sum, increases in limitations were associated with concurrent increases in depression for White women and men only. Moreover, as Model 4 shows, controlling for other health measures only slightly altered the interactions. Table 3 presents findings for the high-SES group. Model 1 indicates that baseline depression was associated negatively with change in depression over time. Model 2 indicates that baseline levels and changes in limitations were associated with increases in depression. In Model 3, we observed significant Gender–race × Change in limitations interactions that were different from those among low-SES elders. The negative White women × Change in limitations coefficient indicates that increases in limitations were associated with increases in depression more strongly among African American men compared to White women; however, tests for model improvement showed that the inclusion of these interactions marginally improved model fit (F = 2.17, p =.09). Adjustments for other health measures in Model 4 had little effect. In sum, we observed that increases in limitations were associated with increases in depression; these patterns depended on gender, race, and SES. Among low-SES elders, increases in limitations were associated with increases in depression among White men and women only; among high-SES elders, increases in limitations were associated with increases in depression among African American women, African American men, and White men only. We have presented observations separately by SES for ease of interpretation; however, analyses (not shown) of three-way interactions supported the findings presented in Tables 2 and 3 (White men: t = 2.83, p <.01; White women: t = 2.14, p <.05; full analyses available upon request). Discussion Although the patterning of levels of functional limitations across dimensions of social stratification is well established, we expanded the scope of research by documenting the status-contingent effects of limitations on changes in depression. Drawing upon stress process theory, we proposed that social strata yield differential vulnerabilities with respect to the effects of limitations. We also used the multiple-hierarchy stratification perspective to examine race, gender, and SES contingencies. This allowed us to identify resource vulnerability and resource erosion variants of the SES-based differential vulnerability thesis across race–gender groups. Among low-SES elders, we observed that increases in limitations were associated with concurrent increases in depression among White men and, to a lesser extent, White women. These observations are consistent with the resource erosion view in that Whites, irrespective of SES, may enjoy better health and fewer limitations over the life course than African Americans. With advancing age and increases in limitations, individuals may perceive threats to well-being as unfamiliar and unexpected. Moreover, the fact that limitations did not influence levels of depression among African Americans of low SES reinforces and extends the notion that elderly African Americans may be an especially robust group in terms of health and may reflect a survival elite. Our observations are also consistent with the idea of the disability crossover, in which African Americans in old age surpass similarly aged White peers in terms of health and physical functioning. According to Ferraro and Farmer (1996), “The advantages that majority persons held over minority persons may cease to be as important when all persons are confronted with the basic challenges to health and functional ability” (p. 28). Here, we identify the advantage with respect to the fact that, at the same level of increase in limitations, African Americans (especially men) reported lower levels of a concurrent increase in depression than White men and women. For lower SES individuals of minority status, being a member of the survivor elite may yield psychosocial benefits that counter the depressing consequences of impairment. We also found evidence consistent with the resource erosion view of SES. This view implies that high SES should buffer against the distressing effects of increasing limitations. The resource erosion dimension of increasing levels of limitations, however, may overwhelm the protective benefits of high SES. Sooner or later, even people who have enjoyed health advantages over the life course experience health problems or impairment (House et al., 2005). The question becomes: At the same level of limitations, what are the mental health effects for individuals who are familiar with prior health adversities (normative) compared to those who are unfamiliar with health stressors (non-normative)? The stability of health inequalities idea implies that the mental health impact of limitations is worse for people who have enjoyed health advantages over the life course—in this case, people with high SES. Thus, there may be an advantage resource margin that compresses during late-life in terms of morbidity and limitations (House et al., 2005), as well as the deleterious mental health correlates of limitations. Our findings among high-SES African American women, African American men, and White men are consistent with the resource erosion view: people with higher SES tend to experience more advantages with respect to health over the life course. For them, the sudden exposure to limitations may be unexpected, unfamiliar, and challenging. By contrast, our observations for high-SES White women are more consistent with the resource benefits view: higher SES is protective against the distressing effects of limitations. Several limitations of our study deserve brief mention. First, the small sample sizes of specific subgroups suggest caution in the interpretation of our estimates. Moreover, there may be reporting biases across these subgroups that we are unable to assess in the present study. The regional nature of the sample is also a potential limitation. On the one hand, the metropolitan District of Columbia area likely contains a slightly higher number of well-educated, wealthier individuals. This is particularly the case for elderly African American women and men in Prince George's County. On the other hand, this is also a potential strength of the data set, because it allows us to examine health and well-being processes among a group of African American elders that represents a solid cross-section of the socioeconomic spectrum. In addition, the low r-square suggests that much of the variance in changes in depression remains unexplained. This is hardly unique to this study, but it deserves brief mention here and attention in future investigations. Finally, the short duration of time between interviews limited the degree of change in focal measures. Future plans for additional interviews of these participants, however, should help to expand the scope of the present study to include growth-curve models of changes in functioning over a longer time span. Conclusion The stress process framework identifies functional limitations as a stressor in late life. We drew upon an array of theoretical and empirical views to propose that the increased limitations have different psychological effects that vary across social strata. Our observations underscore the necessity of using a multiple-hierarchy stratification perspective to examine race, gender, and SES contingencies to refine and extend current knowledge about the effects of limitations. Decision Editor: Kenneth F. Ferraro, PhD Table 1. Means or Proportions for All Study Variables Across Socioeconomic Status and Race–Gender Groups. Socioeconomic Status < Median Socioeconomic Status > Median Variable African American Women (n = 156) African American Men (n = 119) White Women (n = 112) White Men (n = 54) African American Women (n = 62) African American Men (n = 97) White Women (n = 117) White Men (n = 181) Change in depression −0.018 (0.552) −0.075 (0.634) 0.061 (0.472) 0.089 (0.440) 0.044 (0.512) 0.065 (0.365) 0.015 (0.532) 0.015 (0.442) Baseline depression 1.480 (0.526) 1.459 (0.603) 1.515 (0.610) 1.349 (0.416) 1.390 (0.476) 1.253b (0.375) 1.480c (0.504) 1.324 (0.403) Baseline functional limitations 0.186c (0.900) −0.057b (0.803) 0.283c (0.956) −0.231 (0.756) −0.037 (0.741) −0.345a (0.752) −0.215 (0.556) −0.294 (0.584) Change in functional limitations 0.088 (0.694) 0.160 (0.831) 0.132 (0.646) 0.132 (0.589) 0.081 (0.368) 0.061 (0.589) 0.052 (0.462) −0.011 (0.508) Age 73.750 (6.802) 73.403 (5.419) 76.152 (6.921) 75.556 (5.971) 72.661 (5.572) 72.082c (5.063) 73.632 (5.757) 74.376 (6.551) Married 0.244c 0.555a,b 0.330c 0.685 0.435c 0.814a,b 0.538c 0.790 Divorced/separated 0.224c 0.168 0.107 0.056 0.177c 0.062 0.120 0.050 Widowed 0.481c 0.244a,b 0.509c 0.204 0.371c 0.113a 0.248c 0.088 Never married 0.051 0.034 0.054 0.056 0.016 0.010 0.094 0.072 Current/recent smoker 0.141 0.319a,b,c 0.143 0.130 0.113 0.082 0.103 0.072 Body mass index 29.295b (5.264) 27.200a (4.551) 26.697 (6.525) 27.346 (3.852) 28.549b,c (6.182) 27.451b (3.826) 24.890 (4.581) 25.983 (3.342) Baseline diseases 2.256 (1.463) 2.176 (1.516) 2.357 (1.361) 2.148 (1.535) 2.274 (1.473) 1.918 (1.441) 2.068 (1.400) 1.796 (1.413) New diseases 1.686 (1.463) 1.387 (1.491) 1.616 (1.390) 1.444 (1.436) 1.290 (1.311) 1.165 (1.336) 1.350 (1.347) 1.155 (1.255) Notes: Standard deviations are shown in parentheses. aSignificantly different from African American women (p <.05). bSignificantly different from White women (p <.05). cSignificantly different from White men (p <.05). Table 2. Regression of Change in Depression Among Individuals in the Low-SES Group (n = 441). Variable Model 1 Model 2 Model 3 Model 4 Focal associations     Baseline depression −.557*** (.038) −.611*** (.039) −.622*** (.039) −.641*** (.040)     African American womena .044 (.055) .028 (.055) .025 (.057)     White womena .138* (.059) .115 (.060) .111 (.060)     White mena .129 (.072) .089 (.073) .086 (.073)     Baseline functional limitations .111*** (.027) .117*** (.027) .087** (.029)     Change in functional limitations .133*** (.031) .047 (.048) .045 (.049)     African American women × Change in functional limitationsa .085 (.070) .072 (.070)     White women × Change in functional limitationsa .162* (.081) .142 (.081)     White men × Change in functional limitationsa .298*** (.112) .258* (.112) Basic control measures     Age −.001 (.004) −.002 (.004) −.001 (.004)     Divorcedb .081 (.064) .090 (.063) .107 (.064)     Widowedb .012 (.051) .013 (.050) .025 (.051)     Never marriedb .112 (.101) .124 (.101) .135 (.101) Health measures     Previous or recent smoker −.004 (.056)     Body mass index −.003 (.004)     Baseline diseases .019 (.017)     New diseases .037* (.017) Constant .817 .780 .807 .814 R 2 .325 .382 .395 .409 Notes: Data are presented as unstandardized regression coefficients with standardized coefficients in parentheses. Models show regression of change in depression on baseline levels of depression (1); race–gender groups, functional limitations, and basic controls (2); interactions (3); and other health statuses and conditions (4). aCompared to African American men. bCompared to currently married. *p <.05; **p <.01; ***p <.001 (two-tailed). Table 3. Regression of Change in Depression Among Individuals in the High-SES Group (n = 457). Variable Model 1 Model 2 Model 3 Model 4 Focal associations     Baseline depression −.398*** (.045) −.496*** (.045) −.499*** (.045) −.511*** (.046)     African American womena −.040 (.068) −.039 (.069) −.034* (.068)     White womena .021 (.057) .036 (.057) .029 (.058)     White mena −.017 (.051) −.013 (.051) −.016 (.051)     Baseline functional limitations .174*** (.033) .173*** (.033) .172*** (.035)     Change in functional limitations .212*** (.040) .295*** (.070) .280*** (.071)     African American women × Change in functional limitationsa −.028 (.156) −.016 (.158)     White women × Change in functional limitationsa −.259* (.106) −.259*(.107)     White men × Change in functional limitationsa −.060 (.091) −.069 (.091) Basic control measures     Age .005 (.004) .005 (.003) .005 (.004)     Divorcedb .198** (.069) .213** (.069) .202** (.070)     Widowedb .012 (.054) .005 (.054) .004 (.054)     Never marriedb .004 (.083) .008 (.083) −.001 (.083) Health measures     Previous or recent smoker .039 (.068)     Body mass index −.002 (.005)     Baseline diseases −.016 (.015)     New diseases .039* (.016) Constant .570 .731 .730 .794 R 2 .146 .266 .277 .288 Notes: Data are presented as unstandardized regression coefficients with standardized coefficients in parentheses. Models show regression of change in depression on baseline levels of depression (1); race–gender groups, functional limitations, and basic controls (2); interactions (3); and other health statuses and conditions (4). aCompared to African American men. bCompared to currently married persons. *p <.05; **p <.01; ***p <.001 (two-tailed). Appendix Survey Items. Item Wording Response Categories Depression Index     Lack enthusiasm for doing anything No days (1)     Feel bored or have little interest in things 1 or 2 days (2)     Cry easily or feel like crying 3 or 4 days (3)     Feel downhearted or blue 5 or more days (4)     Feel slowed down or low in energy     Blame yourself for everything that goes wrong     Have your feelings hurt easily Functional Limitations Index     Level of difficulty in:         Dressing and undressing Without difficulty (1)         Getting in and out of bed With difficulty, but without help (2)         Taking a bath or shower With a little help from someone (3)         Getting to and using the toilet Unable to do this without complete help from someone or special equipment (4)         Climbing up stairs         Keeping balance while walking         Going food shopping         Getting from your home to where you need to go         Figuring out your own monthly bills     Let's suppose that you had to reach over your head to lower a bag of sugar. What is the heaviest bag of sugar you could lower? 15 or more pound bag (1) 10 pound bag (2) 5 pound bag (3) 1 pound bag (4) Not at all able to lower the bag (5)     Let's suppose that you had to stand, without help, in a long line. About how long could you stand? 1 hr or more (1) 45 min (2) 30 min (3) 15 min (4) 5 min (5) Not at all (6)     How long are you able to walk without stopping to rest? 1 hr or more (1) 45 min (2) 30 min (3) 15 min (4) 5 min (5) Not at all (6) Disease Index     Asthma or emphysema No (0)     Arthritis Yes (1)     Diabetes     High blood pressure     Heart disease and/or heart attack     Stomach disorders     Stroke     Cancer of any kind     Osteoporosis     High cholesterol     Painful joints     Cataracts, glaucoma, detached retina, or any other condition of the retina An NIA grant award AG17461 (Leonard I. Pearlin, P.I.) supports this work. Address correspondence to Scott Schieman, PhD, University of Toronto, Department of Sociology, 725 Spadina Avenue, Toronto, Ontario M5S 2J4, Canada. E-mail: scott.schieman@utoronto.ca. References American Psychiatric Association. (1994). Diagnostic and statistical manual of mental disorders (4th ed.). Washington, DC: Author. Centers for Disease Control. (2002). United States life tables, 2000. National Vital Statistics Reports, 51(3). Clark, D. O., Maddox, G. L. (1992). Racial and social correlates of age-related changes in functioning. Journal of Gerontology: Social Sciences, 47B,S222-S232. Dannefer, D. (2003). Cumulative advantage/disadvantage and the life course: Cross-fertilizing age and social science theory. Journal of Gerontology: Social Sciences, 58B,S327-S357. Derogatis, L. R., Lipman, R., Rickels, K., Uhlenhuth, E. H., Covi, L. (1974). The Hopkins Symptom Checklist (HSCL): A self-report symptom inventory. Behavioural Science, 19,1-15. Dowd, J. J., Bengtson, V. L. (1978). 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New York: Oxford University Press. McLeod, J. D., Nonnemaker, J. M. (1999). Social stratification and inequality. In C. S. Aneshensel & J. C. Phelan (Eds.), Handbook of the sociology of mental health (pp. 321–344). New York: Kluwer Academic/Plenum. Mirowsky, J., Ross, C. E. (2001). Age and the effect of economic hardship on depression. Journal of Health and Social Behavior, 42,132-150. O'Rand, A. M. (1996). The precious and the precocious: Understanding cumulative disadvantage and cumulative advantage over the life course. The Gerontologist, 36,230-238. Pampel, F. C., Rogers, R. G. (2004). Socioeconomic status, smoking, and health: A test of competing theories of cumulative advantage. Journal of Health and Social Behavior, 45,306-321. Pearlin, L. I., Skaff, M. M. (1996). Stress and the life course: A paradigmatic alliance. The Gerontologist, 36,239-247. Rieker, P. P., Bird, C. E. (2005). 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Journal of Aging and Health, 17,263-292. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AB8A2C93FA5FA6C516B97EED516962D819FB8FA.txt b/test/dataset/in/resources/corpus/Clean_0AB8A2C93FA5FA6C516B97EED516962D819FB8FA.txt new file mode 100644 index 0000000..dc2a223 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AB8A2C93FA5FA6C516B97EED516962D819FB8FA.txt @@ -0,0 +1 @@ +]>NBA5543S0197-4580(01)00218-410.1016/S0197-4580(01)00218-4Elsevier Science Inc.Fig. 1Fig. 1 represents the electrophoretic pattern of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) (SWISS-PROT number P09543). Frontal cortex, control sample.Fig. 2Fig. 2 represents the electrophoretic pattern of carbonic anhydrase II (CA II) (SWISS-PROT number P00918). Frontal cortex, control sample.Table 1Quantification of CNPase in human brain samples (arbitrary units)legend legend legendBrain regionControl mean ± SD (N)Down syndrome mean ± SD (N)Alzheimer’s disease mean ± SD (N)Frontal6.10 ± 2.35 (6)∗3.38 ± 1.64 (6)∗2.70 ± 0.95 (6)Temporal5.19 ± 2.02 (9)∗∗3.30 ± 1.28 (9)3.69 ± 1.94 (9)Occipital4.37 ± 0.98 (11)5.19 ± 1.85 (8)4.79 ± 1.86 (9)Parietal4.33 ± 2.30 (7)4.38 ± 2.78 (8)2.59 ± 1.53 (7)Cerebellum2.75 ± 0.47 (5)3.65 ± 1.24 (6)3.78 ± 1.84 (9)Thalamus5.41 ± 1.96 (7)5.06 ± 1.48 (7)4.74 ± 2.82 (5)Caudate nucleus4.02 ± 1.35 (6)6.04 ± 1.73 (7)5.53 ± 1.91 (8)legendData represent means ± SD (N = number of samples used from overall experimental group).legendMann-Whitney U-test was used to test differences between DS or AD and control group.legendStatistical significance∗p = 0.04,∗∗p = 0.02.Table 2Quantification of CA II in human brain samples (arbitrary units)legendBrain regionControl mean ± SD (N)Down syndrome mean ± SD (N)Alzheimer’s disease mean ± SD (N)Frontal11.16 ± 3.87 (9)16.70 ± 5.65 (5)9.74 ± 3.69 (7)Temporal10.54 ± 5.25 (10)13.73 ± 8.27 (8)13.55 ± 7.79 (10)Occipital11.16 ± 2.98 (9)16.25 ± 10.12 (8)12.03 ± 5.74 (10)Parietal13.86 ± 6.53 (7)10.66 ± 3.76 (8)14.14 ± 2.32 (5)Cerebellum8.87 ± 3.39 (11)7.15 ± 1.66 (7)9.50 ± 7.20 (9)Thalamus11.55 ± 2.93 (10)14.70 ± 3.41 (6)12.78 ± 3.38 (7)Caudate nucleus15.72 ± 8.73 (6)13.16 ± 3.35 (7)9.95 ± 2.41 (7)legendData represent mean ± SD (N = number of samples used from overall experimental group)Decreased brain levels of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in Down syndrome and Alzheimer’s diseaseVlkolinskyRomanVlkolinskýa1NigelCairnsbMichaelFountoulakiscGertLubeca*Gert.Lubec@akh-wien.ac.ataUniversity of Vienna, Department of Pediatrics, Waehringer Guertel 18, A 1090 Vienna, AustriabInstitute of Psychiatry, Brain Bank, King’s College, De Crespigny Park London SE5 8AF, UKcPharmaceutical Research, Genomic Technologies, F. Hoffmann-La Roche, Ltd., CH-4070 Basel, Switzerland*Corresponding author. Tel.: +43-1-40400-32151The author is on leave from Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovak Republic.AbstractIn Down syndrome (DS) as well as in Alzheimer’s disease (AD) oligodendroglial and myelin alterations have been reported. 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) and carbonic anhydrase II (CA II) are widely accepted as markers for oligodendroglia and myelin. However, only data on CNPase activity have been available in AD and DS brains so far. In our study we determined the protein levels of CNPase and CA II in DS, AD and in control post mortem brain samples in order to assess oligodendroglia and myelin alterations in both diseases. We used two dimensional electrophoresis to separate brain proteins that were subsequently identified by matrix assisted laser desorption and ionization mass-spectroscopy (MALDI-MS). Seven brain areas were investigated (frontal, temporal, occipital and parietal cortex, cerebellum, thalamus and caudate nucleus). In comparison to control brains we detected significantly decreased CNPase protein levels in frontal and temporal cortex of DS patients. The level of CA II protein in DS was unchanged in comparison to controls. In AD brains levels of CNPase were decreased in frontal cortex only. The level of CA II in all brain areas in AD group was comparable to controls. Changes of CNPase protein levels in DS and AD are in agreement with the previous finding of decreased CNPase activity in DS and AD brain. They probably reflect decreased oligodendroglial density and/or reduced myelination. These can be secondary to disturbances in axon/oligodendroglial communication due to neuronal loss present in both diseases. Alternatively, reduced CNPase levels in DS brains may be caused by impairment of glucose metabolism and/or alterations of thyroid functions.KeywordsOligodendrogliaNeurodegenerationMALDI-MSCarbonic anhydrase IIMyelin1IntroductionIn Down syndrome (DS), Alzheimer’s type of neurodegeneration develops invariably in later postnatal life, typically over the age 30. The neurodegenerative process in DS is sharing many pathological findings with Alzheimer’s disease (AD), the typical hallmarks being amyloid deposition, formation of neurofibrillary tangles, congophilic angiopathy and extensive neuronal cell loss [17,23]. In addition to neuronal involvement, glial cells also markedly participate in DS and AD pathology. For instance, persistent reactive astrogliosis has been clearly demonstrated in DS and AD brains [61,45].It was proposed that oligodendroglia, the principle myelin forming cells within CNS is also significantly affected in both diseases. In patients with DS and AD markers for oligodendroglial cells and myelin formation e.g. the activity of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), amount of myelin proteins and cerebroside were shown to be decreased [4,52]. Accordingly, myelination seems to be delayed in young DS patients as revealed by histology [65,66] and magnetic resonance imaging [28]. On the other hand, the proliferation of oligodendroglia by amyloid P component has been described in DS [1].Although evidence for the involvement of oligodendroglia in DS and AD pathology is accumulating, oligodendroglial markers such as CNPase and carbonic anhydrase II (CA II) activities but not their protein levels have been quantified so far [4,6,52]. Measuring enzyme activity may be misleading however, as this parameter can be influenced by different factors such as substrate availability/concentration, protein phosphorylation, etc. [49]. Moreover, the CNPase activity can be altered by the extent of neuro-glial communication therefore it need not to correspond to its protein levels [37]. This communication, in the case of DS and AD, may be considerably altered due to the extensive neuronal loss being present in both diseases.The limited data on white matter involvement in both diseases made us examine two oligodendroglial markers, CNPase and CA II in several brain regions of patients with DS and AD as compared to controls at the protein level. The use of 2-dimensional gel electrophoresis with subsequent unambiguous identification and characterization of the spots by matrix assisted laser desorption and ionization mass-spectroscopy (MALDI-MS) allowed the concomitant determination of two oligodendroglial markers on one gel.2′,3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37) is a protein accounting for approximately 4% of myelin protein content and for a long time it has been considered as an index of myelin formation [for review see 52,63]. There are two isoforms of this protein, CNP1ase (∼46 kDa) and CNP2ase (∼48 kDa), with CNP2ase differing from CNP1ase by a 20 amino acid extension at its N-terminus. Developmentally, it is one of the earliest myelination-specific polypeptides, synthesized by oligodendrocytes and its synthesis persists into adulthood, suggesting a role in the synthesis and maintaince of the myelin sheath [49]. It is synthesized on free polysomes in the oligodendrocyte cell bodies from where it reaches myelin. In myelin, CNPase is excluded from compact lamellar domains but it is present within the cytoplasmic channels that transverse the myelin sheath [15]. Therefore, it can be found at cytoplasmic sites of oligodendrocyte cell bodies, and at the periphery in the elongated oligodendroglial processes [43,47,67]. The physiological role of CNPase is not understood, but it is thought to be functionally involved in oligodendrocytes surface membrane expansion and migration during the earliest stages of axonal ensheathment [67].Carbonic anhydrases (carbonate hydrolase: EC 4.2.1.1) represent a group of isozymes the basic physiological function of which is to catalyze the reversible hydration of carbon dioxide to form H2CO3 which in turn decomposes to H+ and HCO3−. CA II is the main isozyme of the CA family in the human brain. It is located mainly in oligodendroglia and less in astrocytes [10,11,12,21,30]. CA II is a monomeric protein of theoretical relative molecular weight 29 kDa and theoretical pI value of 7.5 [3,35]. It has multiple functions in the brain and it’s deficiency is associated with pathological consequences such as mental retardation and brain calcification [57,62]. CA II is providing HCO3−, which regulates membrane transport of Na+/water and contributes to cerebrospinal fluid formation. CA II has been proposed to participate in the processes of myelination. Controlling extracellular ion concentration, CA II activity is probably involved also in regulation of neuronal excitability [44] and susceptibility to seizures [54].In this report we describe decreased levels of CNPase protein in frontal and temporal cortex of DS brain samples along with unchanged levels of CA II. Similarly, we detected decreased protein levels of CNPase in frontal lobe of AD brains while CA II levels were not altered.2Methods2.1Brain samples for determination of protein levelsThe brain samples from frontal, temporal, parietal, occipital cortex, cerebellum, thalamus and caudate nucleus of caryotyped patients with DS (n = 9; 3 females, 6 males; 55.7 ± 7.48 years old), AD (n = 11, 4 females, 7 males; 59.55 ± 6.1 years old) and controls (n = 14, 4 females, 10 males 56.1 ± 9.87 years old) were used for the studies at the protein level. Post mortem brain samples were obtained from the MRC London Brain Bank of Neurodegerative Diseases, Institute of Psychiatry). The controls were brains from individuals with no history of neurological or psychiatric disorders. The major cause of death was bronchopneumonia in DS and AD and heart disease in controls. Post mortem interval of brain dissection in DS, AD and controls was 31.44 ± 19.56, 32.45 ± 27.83 and 37.67 ± 21.8 h, respectively). Tissue samples were stored at −70°C and the freezing chain was never interrupted.2.2Two-dimensional electrophoresis of brain proteinsBrain tissue was suspended in 0.5 ml of sample buffer consisting of 40 mM Tris, 5 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St Louis, MO, USA), 4% CHAPS (Sigma), 10 mM 1,4 dithioerythritol (Merck), 1 mM EDTA (Merck) and a mixture of protease inhibitors, 1 mM PMSF and 1 μg of each pepstatin A, chymostatin, leupeptin and antipain. The suspension was sonicated for approximately 30 s and centrifuged at 10000 × g for 10 min and the supernatant was centrifuged further at 150000 × g for 45 min. The protein content in the supernatant was determined by the Coomassie blue method [7].The 2-D gel electrophoresis was performed essentially as reported [36]. Samples of approximately 1.5 mg were applied on immobilized pH 3-10 nonlinear gradient strips (IPG, Amersham Pharmacia Biotechnology Uppsala, Sweden), at both, the basic and acidic ends of the strips. The proteins were focused at 300 V for 1 h, after which the voltage was gradually increased to 3500 V within 6 h. Focusing was continued at 5000 V for 48 h. The second-dimensional separation was performed on 9–16% linear gradient polyacrylamide gel (Serva, Heidelberg, Germany and Bio-Rad, Hercules, CA, USA). The gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for 48 h, destained with water and scanned in a Molecular Dynamics Personal densitometer. The images were processed using PhotoShop (Adobe) and PowerPoint (Microsoft) software. Protein spots were quantified using the ImageMaster 2D Elite software (Amersham Pharmacia Biotechnology). During the quantification the volumes of the spots were calculated as a summation of pixel intensities making up the spots. The individual spot volume was expressed as a percentage of the total volume of the spots present in the gel part considered.2.3Matrix assisted laser desorption ioization—mass spectroscopy (MALDI-MS)MALDI-MS analysis was performed as described with minor modifications [20]. Briefly, spots were excised, destained with 50% acetonitril in 0.1 M ammonium bicarbonate and dried in a speedvac evaporator. The dried gel pieces were reswollen with 3 μl of 3 mM Tris-HCL, pH 8.8, containing 50 ng trypsin (Promega, Madison, WI, USA) and after 15 min, 3 μl of water were added. One μl was applied onto the dried matrix spot. The matrix consisted of 15 mg nitrocellulose (Bio-Rad) and 20 mg (α-cyano-4-hydroxycynnamic acid (Sigma) dissolved in 1 ml acetone:isopropanol (1:1, v/v). 0.5 μl of the matrix solution was applied on the sample target. Specimen were analyzed in time-of-flight PerSeptive Biosystems mass spectrometer (Voyager Elite, Cambridge, MA, USA) equipped with a reflectron. An accelerating voltage of 20 kV was used. Calibration was internal to the samples. We used standards to correct the measured peptide masses, reducing, thus, the window of mass tolerance and increasing the confidence of identification. The peptide masses were matched with the theoretical peptide masses of all proteins from all species of the SWISS-PROT database. For protein search, monoisotopic masses were used and a mass tolerance of 0.0075% was allowed.2.4Statistical analysisResults are expressed as means ± standard deviation (SD). Following Bartlett’s test for homogeneity of variances, Mann-Whitney U-test was used to evaluate differences between control vs. DS and AD study groups. The exact p values were calculated for those comparisons where the statistical significance reached p < 0.05 (∗p = 0.04, ∗∗p = 0.02).3ResultsHuman brain protein extracts from seven brain regions were separated by 2-D gel electrophoresis and visualized using colloidal Coomassie blue staining. The pattern of separated protein spots was mostly identical to that previously published [38]. Protein spots were analyzed by MALDI-MS, following in-gel digestion as described above. The 2-D gels with proteins from corresponding brain regions of patients with DS and AD were compared with each other, in order to detect differences at the protein level.CNPase was represented by a well-defined single spot (Fig. 1). By the technique used we could not distinguish between the two known isoforms of the protein, but the protein was unambiguously identified by MALDI-MS (SWISS-PROT accession number: P09543).We detected a significant decrease of CNPase protein levels in frontal and temporal cortex of brains of DS patients. In other brain regions levels of CNPase were not significantly different from controls. In brain samples from patients with AD we detected significant decrease of this protein in frontal cortex only (Table 1). CA II (SWISS-PROT accession number: P00918) was represented by two spots that could not be clearly separated from each other in most gels (Fig. 2). The sum of both CA II spots was therefore used for statistical evaluation.Compared to control group we could not detect any significant changes in CA II protein level neither in DS nor in AD brain samples (Table 2). 4DiscussionUsing 2-dimensional electrophoresis with MALDI-MS identification we have clearly shown that CNPase and CA II are regularly expressed in brains of controls. In our study CNPase was significantly reduced in frontal and temporal cortex of DS patients and in frontal cortex in AD patients.4.1CNPase and CAII protein levels in DS brain samplesIn DS patients the reduced CNPase activity (along with other parameters of myelin such as the amount of proteolipid protein and myelin basic protein) was already reported by Banik et al. [4] suggesting reduced oligodendroglia and possibly myelination in DS. This is in agreement with our observation of decreased protein level of CNPase. Impaired myelination in DS brain starting from early childhood was confirmed histologically [9,65,66] and has been proposed by magnetic resonance imaging [28].CA II protein was not altered in any of DS brain regions, which is not a contradiction to the observed decrease of CNPase activity. Unchanged levels of CA II in DS brains of our cohort may have been resulting from the (low abundance) expression of CA II in astrocytes [26,48,50,58], proliferating in DS [45,61] thus masking a potential decrease of CA II. In a previous study, increased levels of an astroglial marker, glial fibrillary acidic protein (GFAP) have been found also in DS brains of our cohort [22].A number of pathological mechanisms may explain oligodendroglia/myelination deficits in DS. The evidence is growing that myelination and oligodendroglial maturation in CNS is strongly dependent on their close communication with neurons. This communication involves e.g. cell adhesion molecules that could convey the axonal signal to oligodendroglial cells and/or electrical activity of neurons itself that may regulate the myelination process [14,16,40]. In DS brains, however, a massive neuronal loss, starting early in postnatal life, has been reported due to either β-amyloid toxicity to neurons, altered regulation of apoptotic processes or free radical damage (for review see 46,25]. Thus, the neuro-oligodendroglial communication may be profoundly disrupted with the consequence of altered myelination.Additionally, there are several other pathological changes in DS brain metabolism that may explain our results:In DS impaired brain glucose metabolisms may be responsible for oligodendroglial loss and consequently myelin deficits. Oligodendroglial cells are more susceptible to glucose deprivation than astrocytes [41] and thus, glucose metabolism disturbances in DS would preferably affect this cell population and the oligodendrocyte-dependent myelination. In temporal and frontal lobe of DS brains of our cohort we have recently shown down-regulated expression and decreased activity of phosphoglucose isomerase, a key glucose handling enzyme [32]. This change was not found in AD, however. Moreover, the expression and the activities of phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase levels, important enzymes of glucose metabolism, were found increased in DS brains but not in AD [34,39]. These findings indicate that disturbances of glucose metabolism that possibly explain oligodendroglial pathology, are linked to DS rather than to AD.Thyroid hormone abnormalities are strongly associated with DS, the most consistent finding being elevated levels of thyroid stimulating hormone [18,24]. In our cohort we showed elevated thyroid stimulating hormone receptor protein levels in DS brains [33]. It is well established that myelin protein synthesis [2] and oligodendroglial development [28] are strongly dependent on tissue thyroid hormone levels. Early in development thyroid hormones function as an instructive signal for the generation of oligodendrocytes and enhance the proliferation of the committed precursor oligodendrocyte cells. Thyroid hormones regulate the number of oligodendrocytes generated by directly promoting their differentiation and they are required to achieve adequate oligodendrocyte numbers [53]. Finally, thyroid hormone increases morphological and functional maturation of postmitotic oligodendrocytes by stimulation of the expression of various myelin genes. The direct stimulatory influence of triiodothyronine on the activity of CNPase and its mRNA expression has also been demonstrated [5,42]. Therefore, alterations of thyroid hormones commonly encountered in DS, may have led not only to altered CNPase activity in DS patient found by Banik et al. [4] but also to the decrease on CNPase protein levels in our samples. Additionally, to show another possible link between CNPase decrease and thyroid hormone disturbances, it has been suggested that sustained increase of thyroid hormone levels in DS may promote oligodendroglial cell death by apoptosis [42]. Indeed, in DS brain samples of our cohort we detected increased levels of DNAse I transcripts, an enzyme that is most probably involved in apoptotic processes [56]. In addition, by quantitative ELISA, we detected significant increase of p53 protein in frontal and temporal cortex along with increase of CD95 protein levels in temporal cortex only [55]. The upregulation of both proteins usually precedes apoptosis in many cell types and is probably involved also in apoptosis-induced cell loss in DS. Interestingly, CD95 (APO-1/Fas) receptor that mediate receptor-dependent programmed cell death, is expressed in adult human brain mainly by oligodendroglial cells. The alteration of apoptotic markers determined in these studies may well be reflecting, apart from apoptosis of neurons, the enhanced apoptosis of oligodendroglial cells with a consequence of reduced myelination. Hence, we assume that decreased CNPase protein levels detected in DS brains of our cohort might be the consequence of enhanced apoptosis either in oligodendroglial cells, or be secondarily to neuronal loss.4.2CNPase and CAII protein levels in AD brainsThe involvement of white matter in AD pathology has been described. Alterations of myelin constituents such as myelin lipids were shown to be significantly decreased in AD brains, including semioval center [64], temporal and frontal cortices, caudate nucleus and hippocampus [60]. Additionally, imaging techniques were used to reveal brain white matter changes in AD [8,19,59].In AD information on CNPase and CA II is poor and in quantitative terms only Reinikainen et al. determined activity of CNPase reflecting oligodendroglial density and possibly myelin composition of AD brains [52]. The authors found CNPase activities to be slightly increased in temporal cortex, parietal cortex and gyrus parahippocampalis, but decreased in the hippocampus and putamen, pointing to decreased oligodendroglial density or myelination deficits in the later two regions. The relatively higher CNPase activities in former three regions were ascribed to excessive loss of neurons without substantial change in oligodendroglia. However, as stated above, results from CNPase activity and protein levels may well be divergent. Hence, measuring the CNPase protein levels may be a more reliable parameter of oligodendroglial density.We revealed significantly decreased CNPase protein level in frontal cortex of AD. Although a tendency of decreased CNPase protein level could be detected also in temporal and parietal cortex of AD patients, the changes did not reach statistical significance. Nevertheless, the finding confirms the already detected pathological myelin alterations commonly found in AD [8,19,59]. We assume, however, that the myelin changes in AD are rather secondary to neuronal/axonal degeneration common in AD than the consequence of above mentioned glucose metabolism disturbances or imbalance of thyroid functions present in DS.Unaltered levels of CA II in AD brains may be explained similarly to the finding observed in DS brains by reactive astrogliosis which may mask potential decrease of CA II [51]. Indeed, using 2-D electrophoresis technique an elevated level of GFAP was found in AD brains of our cohort pointing to astroglial proliferation [22].4.3Regional differences of CNPase protein levels in DS and ADThe reasons for the uneven distribution of white matter damage, noticeable in DS in frontal and temporal lobes and in AD in frontal lobe only, are presently not clear. The regional differences might be simply explained by well known selective vulnerability of different neuronal populations to various noxious stimuli being present in both diseases and selective neuronal death with the consequence of decreased myelination in corresponding areas. The frontal and temporal lobes, where the decrease of CNPase protein reached statistical significance, are typically underdeveloped in young DS patients [9,13] and these are the brain regions, where thyroid-receptor derangement was detected [33]. Similarly, in AD the frontal lobe is usually among most affected regions based on measurements by volumetric neuroimaging [31].The differential vulnerability of individual neurons in different brain regions is well known. Studies on apoptosis in a comparable cohort of DS brain brains showed that temporal but not frontal lobes presented with deranged CD95, a hallmark of apoptosis [55]. In a comparable panel we found synaptosomal loss in temporal cortex of AD and DS patients but not in frontal cortex [68]. Similarly, regional and disease-specific changes of neuroendocrine-specific protein (NSP-C), a marker of neuronal differentiation, were decreased in frontal and temporal lobes in DS but sparing temporal cortex in AD [68]. These findings may help to explain different CNPase levels in individual brain regions but also indicate the problem to find a primary cause for myelination deficits.4.4Concluding remarksIn conclusion, we report decreased CNPase in frontal cortex of patients with DS and AD and in temporal cortex of patients with DS as compared to controls with the tentative biological meaning of decreased oligodendroglia. The methodology used permitted unambiguous identification of this marker protein for oligodendroglia and most probably for myelination. This finding is in agreement with decreased myelination in DS and AD as revealed by histological and imaging methods. Although in agreement with and confirming previous findings of decreased CNPase activity in DS brain [4], decreased CNPase protein determined by 2-dimensional electrophoresis with MALDI-MS identification may be more reliable and suitable than evaluating enzyme activity. 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Sci USA80198327522756[58]D.S.SnyderT.R.ZimmermanJrM.FarooqW.T.NortonW.CammerCarbonic anhydrase, 5′-nucleotidase, and 2′,3′-cyclic nucleotide-3′-phosphodiesterase activities in oligodendrocytes, astrocytes, and neurons isolated from the brains of developing ratsJ Neurochem401983120127[59]A.SteingartV.C.HachinskiC.LauA.J.FoxH.FoxD.LeeD.InzitariH.MerskeyCognitive and neurologic findings in demented patients with diffuse white matter lucencies on computed tomographic scan (leuko-araiosis)Arch Neurol4419873639[60]L.SvennerholmC.G.GottfriesMembrane lipids, selectively diminished in Alzheimer brains, suggest synapse loss as a primary event in early-onset form (type I) and demyelination in late-onset form (type II)J Neurochem62199410391047[61]S.TakashimaL.E.BeckerBasal ganglia calcification in Down’s syndromeJ Neurol Neurosurg Psychiatry4819856164[62]R.E.TashianD.Hewett-EmmettS.J.DodgsonR.E.ForsterW.S.SlyThe value of inherited deficiencies of human carbonic anhydrase isozymes in understanding their cellular rolesAnn N Y Acad Sci4291984263275[63]U.S.VogelR.J.ThompsonMolecular structure, localization, and possible functions of the myelin-associated enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesteraseJ Neurochem50198816671677[64]A.WallinC.G.GottfriesI.KarlssonL.SvennerholmDecreased myelin lipids in Alzheimer’s disease and vascular dementiaActa Neurol Scand801989319323[65]K.E.WisniewskiDown syndrome children often have brain with maturation delay, retardation of growth, and cortical dysgenesisAm J Med Genet Suppl71990274281[66]K.E.WisniewskiB.Schmidt-SidorPostnatal delay of myelin formation in brains from Down syndrome infants and childrenClin Neuropathol819895562[67]X.YinJ.PetersonM.GravelP.E.BraunB.D.TrappCNP overexpression induces aberrant oligodendrocyte membranes and inhibits MBP accumulation and myelin compactionJ Neurosci Res501997238247[68]B.C.YooN.CairnsM.FountoulakisG.LubecSynaptosomal proteins beta-soluble N-ethylmaleimide-sensitive factor attachment protein (beta-SNAP), gamma-SNAP and synaptotagmin I in brain of patients with Down syndrome and Alzheimer’s diseaseDement Geriatr Cogn Disord7202000[In Process Citation] \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AB9A63CD039EF49090F95B7E790CAFF27D3BCFE.txt b/test/dataset/in/resources/corpus/Clean_0AB9A63CD039EF49090F95B7E790CAFF27D3BCFE.txt new file mode 100644 index 0000000..e0e2799 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AB9A63CD039EF49090F95B7E790CAFF27D3BCFE.txt @@ -0,0 +1 @@ +eorthoeorthoThe European Journal of Orthodontics1460-22100141-5387Oxford University Press10.1093/ejo/cjp118ArticlesEffects of long-term storage and thermocycling on bond strength of two self-etching primer adhesive systemsYuasaToshihiro*IijimaMasahiro*ItoShuichi**MugurumaTakeshi*SaitoTakashi**MizoguchiItaru**Division of Orthodontics and Dentofacial Orthopedics, Department of Oral Growth and Development**Division of Cariology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-tobetsu, JapanAddress for correspondence Masahiro Iijima , Division of Orthodontics and Dentofacial Orthopedics , Department of Oral Growth and Development , School of Dentistry , Health Sciences University of Hokkaido , Kanazawa 1757 , Ishikari-tobetsu , Hokkaido 061-0293 , Japan. E-mail: iijima@hoku-iryo-u.ac.jp06201006122009323285290© The Author 2009. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.2010The effects of 2 years of storage and 6000 thermocycles on the shear bond strength (SBS) of two self-etching adhesive systems were studied. Two self-etching primer (SEP) systems (Transbond Plus and Beauty Ortho Bond) and one etch and rinse system (Transbond XT) were used to bond brackets to 126 human premolars that were then stored in artificial saliva for 24 hours or 2 years and thermocycled in distilled water before SBS testing with a universal testing machine. The adhesive remnant index (ARI) scores were calculated. Data were compared by two-way analysis of variance and chi-square analysis. Enamel/adhesive interfaces were examined by scanning electron microscopy.There was no significant difference in the mean SBS for the bonding materials among the three conditions. ARI scores showed that Transbond XT and Beauty Ortho Bond had less adhesive remaining on the teeth after ageing compared with storage for 24 hours. Specimens bonded with Beauty Ortho Bond showed leakage between the resin adhesive and enamel after ageing. Both SEP systems produced adequate SBS even after 2 years or 6000 times thermocycling. Thermocycling is an appropriate technique for determining the durability of orthodontic bracket bonding materials.IntroductionAfter the concept of acid etching was introduced by Buonocore (1955), the direct bonding of orthodontic appliances to enamel with composite resin was introduced by Newman (1965) and is now widely accepted by most orthodontists (Eliades and Eliades, 2001; Eliades et al., 2001). Bonding practices based on a self-etching primer (SEP), which combine etching and priming into a single step, are now being used in clinical orthodontics (Cehreli et al., 2005; Arhun et al., 2006; Bishara et al., 2006; Faltermeier et al., 2007; Scougall Vilchis et al., 2007; Iijima et al., 2008). In addition to saving time and reducing procedural errors, their lower etching ability, due to their higher pH compared with phosphoric acid, might minimize the potential for iatrogenic damage to enamel (Pashley and Tay, 2001; Zeppieri et al., 2003).Bracket-bonding failure sometimes occurs during the later stages of treatment due to heavy forces produced by an archwire or occlusal force. Bracket bond failure is not only frustrating for the practitioner but can also significantly affect treatment efficiency and have an economic impact on a practice (Northrup et al., 2007). Although it has been demonstrated that the shear bond strengths (SBSs) of brackets bonded with SEP adhesive systems were similar to those with a conventional etch and rinse adhesive system (Scougall Vilchis et al., 2007; Iijima et al., 2008), most of these bonding studies measured short-term adhesive bond strength and did not extend the study period to encompass the duration of normal orthodontic treatment. Recently, Oesterle and Shellhart (2008) studied the effect of composite ageing for two conventional etch and rinse adhesive systems on SBS during a normal 24 month orthodontic treatment period and concluded that the SBS of orthodontic brackets increases from 30 minutes to 24 hours and then tends to decrease over the next 24 months.The most commonly used artificial ageing technique is long-term water storage. Another widely used ageing technique is thermocycling. The International Organization for Standardization (ISO) TR 11450 standard (1994) indicates that a thermocycling regimen comprising 500 cycles in water between 5 and 55°C is an appropriate artificial ageing test, and many studies have been carried out following the ISO standard. However, this number of cycles is probably too low to achieve a realistic ageing effect (Gale and Darvell, 1999). Recent studies in orthodontics have used various number of thermocycles: approximately 1500 cycles between 10 and 50°C after 3 months of storage (Trites et al., 2004), 500 cycles between 5 and 55°C (Bishara et al., 2007), and 6000 cycles between 5 and 55°C (Faltermeier et al., 2007).The purpose of this study was to investigate the effects of long-term storage (2 years) and thermocycling (6000 iterations) on the SBS of two SEP adhesive systems. The null hypothesis tested was that the SBS of the self-etching adhesive systems would decrease with long-term storage and thermocycling.Materials and MethodsOne hundred and twenty-six non-carious human maxillary premolars were used in this study. The teeth, which had been extracted for orthodontic reasons, were randomly divided into nine groups of 14 specimens for measurement of SBS. Selection criteria included the absence of any visible decalcification or cracking of the enamel surface under a stereomicroscope (SEM; SMZ 1500, Nikon, Tokyo, Japan) at a magnification of ×10. The extracted teeth were stored in a 0.5 per cent chloramine solution at approximately 4°C. The buccal surfaces of all teeth were cleaned using non-fluoridated pumice. The teeth were also polished using a rubber cup, thoroughly washed, and dried using a moisture-free air source.Group 1: Transbond XT etch and rinse adhesive system. The enamel surfaces were treated with 35 per cent phosphoric acid etching gel (Transbond XT Etching Gel, 3M Unitek) for 15 seconds, washed for 20 seconds, and dried with oil-free air stream. Table 1 lists the bonding materials used in the present study and Figure 1 is a flow chart of the bracket-bonding instructions. Transbond XT primer was applied to the etched surface, and metal upper premolar brackets (Victory Series, 3M Unitek), with a base area of 10.0 mm2, were bonded with Transbond XT composite (3M Unitek).Table 1Materials and instruction employed in present study.MaterialManufacturerComponents (lot no.)CompositionpHaInstructionsTransbond XT3M Unitek, Monrovia, California, USAEtching gel: (6GN); primer: (5CL); paste: (6TG)35% phosphoric acid, tetraethyleneglycol dimethacrylate (TEGDMA), bisphenol-A-diglycidel methacrylate (Bis-GMA); Bis-GMA, TEGDMA, silane-treated quartz, amorphous silica, camphorquinone1.39Etch enamel 15 seconds; rinse and air-dry; apply thin coat primer; apply adhesive to bracket; 20 seconds light curingTransbond Plus self-etching system3M UnitekSelf-etching primer: (237956E); paste: (6TG)Water, methacrylated phosphoric acid, esters, amino benzoate, camphorquinone, Bis-GMA, TEGDMA, silane treated quartz, amorphous silica, camphorquinone1.85Apply primer 3 seconds; gentle air-dry; apply adhesive to bracket; 20 seconds light curingBeauty Ortho Bond self-etching systemShofu, Kyoto, JapanPrimer A: (030602); primer B: (030602); paste: (120503)Water, acetone, others, phosphoric acid monomer, ethanol, TEGDMA, surface pre-reacted glass-ionomer, filler, Bis-GMA, camphorquinone2.20Apply primer 3 seconds; gentle air-dry; apply adhesive to bracket; 20 seconds light curingaPublished values.Figure 1Flow chart of bracket bonding.Group 2: Transbond Plus SEP adhesive system. Transbond Plus SEP (3M Unitek) was applied and rubbed on the enamel surfaces for approximately 3 seconds. An air jet was lightly applied to the enamel, and the brackets were bonded with Transbond XT composite.Group 3: Beauty Ortho Bond SEP adhesive system. Beauty Ortho Bond primers A and B (Shofu) were mixed. The solution was then rubbed onto the enamel surfaces for approximately 3 seconds. An air jet was briefly applied to the enamel, and the brackets were bonded with Beauty Ortho Bond Paste (composite).Each bonding procedure was performed by the same operator (MI). The excess bonding material was removed with a small scaler. All samples were light cured for 20 seconds (Jetlite 3000, J.Morita USA Inc., Irvine, California, USA) (10 seconds from each proximal side).After bonding, the specimens were stored in artificial saliva at 37°C for 24 hours (T1) or 2 years (T2). A third group (T3) was thermocycled between 5 and 55°C for 6000 cycles after 24 hours of storage at 37°C in distilled water. SBS was then measured. The specimens were fixed to a custom-fabricated acrylic resin block using Model Repair II (Densply-Sankin, Tokyo, Japan) and the block was fixed to a universal testing machine (EZ Test, Shimadzu, Kyoto, Japan). A knife-edged shearing blade was secured to the crosshead with the direction of force parallel to the buccal surface and the bracket base. Force was applied directly to the bracket–tooth interface. The brackets were debonded at a crosshead speed of 0.5 mm/minute.After bond failure, the bracket bases and enamel surfaces were examined with a SEM at a magnification of ×10. The adhesive remnant index (ARI) scores were used to assess the amount of adhesive left on the enamel surface (Årtun and Bergland, 1984).The interface morphology between the adhesive resin and the intact enamel was evaluated under a SEM (SSX-550, Shimadzu). After the SBS was determined, the specimens were cut with a slow-speed water-cooled diamond saw (Isomet, Buehler, Lake Bluff, Illinois, USA), so that they were divided into occlusal and cervical halves; one half was encapsulated for observation of the adhesive interface. The specimens were then polished using a series of abrasives, finishing with a 1 μm diamond paste to obtain a suitable polished surface. The encapsulated specimens were immersed in 6 M hydrochloric acid for 40 seconds and then dehydrated in a graded series of ethanol and water up to 100 per cent ethanol. All specimens were sputter coated with gold (SC-701AT, Sanyu Electron, Tokyo, Japan) and examined under a SEM operating at 15 kV.Statistical analysisStatistical analysis was performed using the Statistical Package for Social Sciences (version 14.0J for Windows, SPSS Inc., Chicago, Illinois, USA). The mean SBSs, along with the standard deviation (n = 14), for the groups of bonding materials were compared by two-way analysis of variance (ANOVA). The two factors for ANOVA were bonding material (Transbond XT etch and rinse, Transbond Plus SEP, Beauty Ortho Bond SEP) and storage method (24 hours, 2 years, thermocycling). Chi-square analysis was used to test the significance of differences in the distribution of ARI scores. The level of statistical significance was set at P < 0.05.ResultsThe SBS results are shown in Figure 2. Two-way ANOVA showed that bonding material (Transbond XT etch and rinse adhesive system, Transbond Plus SEP adhesive system, Beauty Ortho Bond SEP adhesive system; P = 0.000) was a statistically significant factor. The storage method (24 hours, 2 years, thermocycling; P = 0.408) was not a statistically significant factor. Specimens bonded with Beauty Ortho Bond produced a significantly lower mean SBS (7.4 MPa) than those bonded with Transbond XT etch and rinse (9.8 MPa) or Transbond Plus SEP (9.1 MPa). There was no significant difference in the mean SBS among the three different ageing methods (T1, T2, and T3) for any of the bonding materials.Figure 2Shear bond strength (MPa) for (a) Transbond XT, (b) Transbond Plus, and (c) Beauty Ortho Bond. Horizontal bars in boxes are medians; 50 per cent of all values are within boxes. The horizontal bars represent the complete range of values. Small boxes within the boxes are average.Chi-square analysis comparing the ARI scores for the three adhesives revealed a significant difference in the distribution of frequencies among the ARI categories for the three adhesive groups at each storage interval (Table 2). Transbond XT etch and rinse and Transbond Plus SEP had a greater frequency of ARI = 1 and 2, except for the Transbond Plus SEP with thermocycling (T3). On the other hand, the Beauty Ortho Bond SEP had a greater frequency of ARI = 2 and 3 for all three storage methods. Both of ageing methods (T2 and T3) for Transbond XT and Beauty Ortho Bond SEP showed less adhesive remaining on the teeth than the 24 hour storage group (T1).Table 2Frequency distribution of adhesive remnant index (ARI) scores.ARI scoresMean12345Transbond XT24 Hours (T1)1031——1.42 Years (T2)49—1—1.9Thermocycling (T3)—113——2.2Transbond Plus24 Hours (T1)464——2.02 Years (T2)662——1.7Thermocycling (T3)1831—2.4Beauty Ortho Bond24 Hours (T1)482——1.92 Years (T2)1382—2.8Thermocycling (T3)—563—2.1ARI scores: 1, indicates all of the composite, with an impression of the bracket base, remains on the tooth surface; 2, more than 90% of the composite remains on the tooth surface; 3, more than 10% but less than 90% of the composite remains on the tooth surface; 4, less than 10% of composite remains on the tooth surface; 5, no composite remains on the tooth surface.Figure 3 shows the representative interfaces between the adhesive resin and the intact enamel after SBS testing for T1, T2, and T3, respectively. For Transbond XT, comparatively thick hybrid layers (Nakabayashi and Pashley, 1998), consisting of primer penetrating the surface enamel, were observed with tags ranging from 2 to 10 μm (Figure 3a). On the other hand, a distinct border was seen between the adhesive resin and the enamel for both self-etching adhesive systems (Transbond Plus and Beauty Ortho Bond) and the hybrid layers were less than 2 μm (Figure 3b and 3c). Tight contact between the adhesive resin and the enamel was observed for all specimens after 24 hours of storage (T1) and for the Transbond XT and the Transbond Plus SEP after 2 years of storage (T2) (Figure 3d and 3e) and thermocycling (T3) (Figure 3g and 3h). However, some of the Beauty Ortho Bond specimens showed leakage between the resin adhesive and enamel after 2 years of storage and thermocycling (Figure 3f and 3i).Figure 3Scanning electron photomicrographs of the adhesive interface between the adhesive resin and the enamel after the shear bond strength (SBS) testing. The specimens were stored in artificial saliva at 37°C for 24 hours (a, Transbond XT; b, Transbond Plus; c, Beauty Ortho Bond), artificial saliva at 37°C for 2 years (d, Transbond XT; e, Transbond Plus; f, Beauty Ortho Bond), or thermocycled between 5 and 55°C for 6000 cycles after 24 hours of storage at 37°C in distilled water (g, Transbond XT; h, Transbond Plus; i, Beauty Ortho Bond) before SBS testing. BR, bonding resin; E, enamel. Magnification ×3000, bar = 10 μm.DiscussionThe direct bonding of orthodontic appliances to enamel with acid etching, originally introduced by Newman (1965), has significantly improved the effectiveness of clinical orthodontics. Although acid etching of enamel may remove approximately 10–20 μm of enamel (Shinchi et al., 2000; Powers and Messersmith, 2001), most clinicians accept acid etching of the enamel surface as a routine technique which may cause some iatrogenic effects such as the risk of enamel fracture, surface stains from increased surface porosity, discolouration by resin tags retained in the enamel, and loss of the outer enamel surface (Powers and Messersmith, 2001). Over the past decade, progress has been made in bonding enamel with resin-modified glass ionomers (Cehreli et al., 2005) and SEPs (Cehreli et al., 2005; Bishara et al., 2006), and their lower etching ability might minimize the potential for iatrogenic damage to enamel.The durability of SEP and resin-modified glass ionomers in clinical use must be evaluated. The most commonly used artificial ageing technique, especially in restorative dentistry, is long-term water storage. Thermocycling is another widely used ageing technique. Bishara et al. (2007) studied the effect of ISO standard thermocycling (500 × 5°C/55°C) on SBS for a resin-modified glass ionomer (Fuji Ortho LC) and a SEP adhesive system (Transbond Plus); their mean SBSs after thermocycling were at clinically acceptable levels (6.4 and 6.1 MPa, respectively). However, the number of cycles used in the ISO standard is probably too low to simulate the ageing effect during long-term orthodontic treatment. Faltermeier et al. (2007) compared SBS after thermocycling (6000 × 5°C/55°C) among one-, two- (self-etching systems), and three-component (etch and rinse system) adhesive systems and concluded that there was no significant difference in SBS between the two- and three-component adhesive systems, while the one-component adhesive had a lower bond strength. Most studies have used various types of thermocycling as an artificial ageing technique to understand the durability of bracket bonding but have not used long-term water storage. A decrease in bonding effectiveness is believed to be caused by degradation of the interface components by hydrolysis (Munck et al., 2005). In addition, water can also infiltrate and weaken the mechanical properties of the polymer matrix (Ferracane et al., 1998; Ito et al., 2005). Although it is unclear whether the effect of thermocycling on bond strength is equal to that of long-term storage, limited information is available on the relative effects of thermocycling and long-term storage on bracket bonding. The present study measured SBS after 2 years, which almost corresponds to the duration of orthodontic treatment with fixed appliances and compared the results to bond strength after thermocycling (6000 × 5°C/55°C). There was no significant difference in the mean SBS for any of the bonding materials among the three different storage methods (T1, 24 hours storage; T2, 2 years storage; T3, thermocycling between 5 and 55°C for 6000 cycles). These results confirm that thermocycling is an appropriate method for understanding the durability of orthodontic bracket-bonding materials. All the bonding materials used in the present study with both ageing methods had clinically acceptable levels of SBS.The ARI scores in the present study showed that Transbond XT and Beauty Ortho Bond SEP adhesive systems had less adhesive remaining on the teeth with T2 and T3 than for the 24 hour storage group (T1). In SEM observation, some of specimens bonded with Beauty Ortho Bond showed leakage between the resin adhesive and enamel after artificial ageing. This may have been due to degradation of the interface components by hydrolysis since mild etching was carried out with a primer with a higher pH. As considerable chair time is needed to remove residual adhesive from the enamel surface, if brackets fail at the enamel–adhesive interface, there would be less residual adhesive, and this might be advantageous if the specimens still had an adequate bond strength.ConclusionsUnder the present study conditions, the following conclusions can be drawn:Thermocycling is an appropriate method for understanding the durability of orthodontic bracket-bonding materials.Both SEP adhesive systems, Transbond Plus and Beauty Ortho Bond, produced adequate SBS even after 2 years of storage and thermocycling between 5 and 55°C for 6000 cycles.ArhunNArmanASesenCKarabulutEKorkmazYGokalpSShear bond strength of orthodontic brackets with 3 self-etch adhesivesAmerican Journal of Orthodontics and Dentofacial Orthopedics2006129547550ÅrtunJBerglandSClinical trials with crystal growth conditioning as an alternative to acid-etch enamel pretreatmentAmerican Journal of Orthodontics198485333340BisharaSEAjlouniRLaffoonJFWarrenJJComparison of shear bond strength of two self-etch primer/adhesive systemsAngle Orthodontist200676123126BisharaSEOstbyAWLaffoonJFWarrenJShear bond strength comparison of two adhesive systems following thermocyclingAngle Orthodontist200777337341BuonocoreMGA simple method of increasing the adhesion of acrylic filling materials to enamel surfaceJournal of Dental Research195534849850CehreliZCKecikDKocadereliLEffect of self-etching primer and adhesive formations on the shear bond strength of orthodontic bracketsAmerican Journal of Orthodontics and Dentofacial Orthopedics2005127573579EliadesTEliadesGBrantleyWAEliadesTOrthodontic adhesive resinsOrthodontic materials: scientific and clinical aspects2001StuttgartThieme201219EliadesTKakabouraAEliadesGBradleyTGComparison of enamel colour changes associated with orthodontic bonding using two different adhesivesEuropean Journal of Orthodontist2001238590FaltermeierABehrMMüssigDA comparative evaluation of bracket bonding with 1-, 2-, and 3-component adhesive systemsAmerican Journal of Orthodontics and Dentofacial Orthopedics2007132144.e1-5FerracaneJLBergeHXCondonJRIn vitro aging of dental composites in water–effect of degree of conversion, filler volume, and filler/matrix couplingJournal of Biomedical Materials Research199842465472GaleMSDarvellBWThermal cycling procedures for laboratory testing of dental restorationsJournal of Dentistry1999278999IijimaMItoSYuasaTMugurumaTSaitoTMizoguchiIBond strength comparison and scanning microscopic evaluation of three orthodontic bonding systemsDental Materials Journal200827392399ItoSEffects of resin hydrophilicity on water sorption and changes in modulus of elasticityBiomaterials20052664496459MunckJDA critical review of the durability of adhesion of tooth tissue: methods and resultsJournal of Dentistry200584118132NakabayashiNPashleyDHHybridization of dental hard tissue1998TokyoQuintessence Publishing Co.67NewmanGVEpoxy adhesives for orthodontic attachments: progress reportAmerican Journal of Orthodontics196551901912NorthrupRGBerzinsDWBradleyTGSchuckitWShear bond strength comparison between two orthodontic adhesives and self-ligating and conventional bracketsAngle Orthodontist200777701706OesterleLJShellhartWCEffect of aging on the shear bond strength of orthodontic bracketsAmerican Journal of Orthodontics and Dentofacial Orthopedics2008133716720PashleyDHTayFRAggressiveness of contemporary self-etching adhesives. Part II: etching effects on unground enamelDental Materials200117430444PowersJMMessersmithMLBrantleyWAEliadesTEnamel etching and bond strengthOrthodontic materials: scientific and clinical aspects2001StuttgartThieme105122Scougall VilchisRJYamamotoSKitaiNHottaMShear bond strength of a new fluoride-releasing adhesiveDental Materials Journal2007264551ShinchiMJSomaKNakabayashiNThe effect of phosphoric acid concentration on resin tag length and bond strength of a photo-cured resin to acid-etched enamelDental Materials200016324329TritesBFoleyTFBantingDBond strength comparison of 2 self-etching primers over a 30 month storage periodAmerican Journal of Orthodontics and Dentofacial Orthopedics2004126709716ZeppieriILChungC-HManteFKEffect of saliva on shear bond strength of an orthodontic adhesive used with moisture-insensitive and self-etching primersAmerican Journal of Orthodontics and Dentofacial Orthopedics2003124414419 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AC3CD85FF23C83D9CD5B61ECC61D5C88A33D5E6.txt b/test/dataset/in/resources/corpus/Clean_0AC3CD85FF23C83D9CD5B61ECC61D5C88A33D5E6.txt new file mode 100644 index 0000000..192a24e --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AC3CD85FF23C83D9CD5B61ECC61D5C88A33D5E6.txt @@ -0,0 +1 @@ +]>NBA5521S0197-4580(00)00248-710.1016/S0197-4580(00)00248-7Elsevier Science Inc.Fig. 1Purification and identification of the TG-3 antigen. (A) Purification and Western blotting of the TG-3 reactive 105 kDa protein. HEp-2 cells were synchronized with nocodazole, detergent extracted, and fractionated by sequential DEAE and Heparin chromatography. TG-3 immunoreactivity eluted in a single fraction from both columns with 0.4 M and 0.5 M NaCl respectively. (B) Western blot analysis (lanes 1–5) and immunoprecipitations with anti-nucleolin MS3 antibody (lanes 6, 7). Lysates of synchronized (lanes 4–7) and asynchronous (lanes 1–3) HEp-2 cells were analyzed with MS3 (lanes 1, 4) or TG-3 (lanes 2, 3, 5). Lane 3, cells collected by the mitotic shake technique (see methods).Fig. 2Identification of the TG-3 antigen as mitotic nucleolin by chemical cleavage. The TG-3 antigen was partially purified from synchronized cells as in Fig. 1, separated by SDS-PAGE, and subjected to partial chemical digestion with N-chlorosuccinimide (A) and hydroxylamine (B). The cleavage products of each digest were separated by SDS-PAGE and transferred onto two membranes. Each membrane was probed with the TG-3 antibody (lanes 1,2), stripped, tested by ECL, and reprobed with the MS3 antibody (lanes 3,4). Controls contained no cleaving reagent (lanes 1, 3).Fig. 3Phosphorylation of nucleolin by cdc2 confers TG-3 immunoreactivity. Nucleolin partially purified from asynchronous cells was phosphorylated by Cdc2/cyclinB in the presence of 32P labeled ATP. The phosphorylated nucleolin was further immunoprecipitated with anti-nucleolin monoclonal antibody MS3 and Western-blotted with either MS3 (panel B) or TG-3 (panel C) antibodies. (A) Autoradiograph of samples separated by SDS-PAGE (Lanes 1 and 2). Partially purified preparations were incubated with [32P]ATP in the presence (lane 1) and the absence (lane 2) of the cdc2/cyclinB complex. The 105 kDa TG-3 immunoreactive band appears only in the sample where Cdc2 was present (lane 1, panel C). (Lanes 3–6) Immunoprecipitations of nucleolin with MS3 from samples prepared as in lanes 1 and 2. 32P is incorporated into nucleolin both in the presence (lane 3, panel A) and absence (lane 4, panel A) of Cdc2. The 105 kDa, TG-3 immunoreactive protein is immunoprecipitated only in the sample where cdc2 was present (lane 3, panel C). No staining was detected when no nucleolin (lane 5) or no primary antibody (lane 6) was used in the experiment. (+) and (-) above lanes indicate presence or absence of the reagent.Fig. 4Distribution of TG-3 immunoreactive nucleolin throughout mitosis. Laser confocal micrograph of asynchronous HEp-2 cells dual labeled with neurofibrillary tangle monoclonal antibody, which recognizes nucleolin phosphorylated by Cdc2: TG-3 in red, monoclonal antibody to β-tubulin in green (A, D, G), and monoclonal antibodies to histone proteins in green (J). Colocalization is shown in yellow (C, F, I, L). Note the appearance of TG-3 immunoreactivity (B). Only the cells in mitosis are immunoreactive with TG-3 (B, H, K) and the immunoreactivity is localized to the nucleus and to the segregating centrosomes (A-arrow, B, C). During late prometaphase, TG-3 immunoreactivity becomes dispersed throughout the mitotic cytosol (E, F). The centrosomes retain TG-3 immunoreactivity (F). In metaphase, the aligned chromosomes are immunoreactive with TG-3 (H) and chromosomal TG-3 staining colocalizes with histone staining (K). The centrosomes lose their TG-3 immunoreactivity. In anaphase TG-3 immunoreactivity begins to decrease (H-cell 3). A marked difference in intensity of TG-3 immunoreactivity is observed between metaphase (K-cell 1) and telophase (K-cell 2).Fig. 5Distribution of TG-3 immunoreactive and non-reactive nucleolin in the early stages of mitosis. Laser confocal micrographs of asynchronous HEp-2 cells dual labeled with monoclonal antibody to nucleolin: MS3 (green) and TG-3 (red). A nuclear distribution of nucleolin is observed in interphase cells. In the early stages of mitosis, nucleoli are intensely immunoreactive with both TG-3 and MS3. Note that the overlap in the mitotic cells is partial (A, B), suggesting that not all nucleolin is phosphorylated by Cdc2 in these cells. DAPI insert indicates cell in prometaphase (D-arrow)Fig. 6Distribution of nucleolin in control and AD brains. Light and immunofluorescent micrographs of control (B-E) and AD (G-J) hippocampal sections immunostained with monoclonal antibody MS3. Control (A) and AD (F) brain sections immunostained with TG-3 were used as a reference for illustrative value. CA3-CA4 (B, C): Nucleolin is present in large neuronal nuclei and neuronal cytoplasm, and in small glial nuclei (B, bar = 0.05 mm; C, bar = 0.1 mm). Nucleolin is primarily nuclear in some neurons, primarily cytoplasmic in others and was detected in both compartments in still other neurons (B). In CA1 (D, F), note the intense immunoreactivity in the apical dendrites (D, bar = 0.05 mm). Note the cytoplasmic distribution of nucleolin and presence of nucleolin in early NFT in the control brain (E, bar = 0.05 mm). A decrease in nucleolin immunoreactivity in all regions of the AD hippocampus was consistently observed (G-J). In CA3-CA4 note infrequently observed immunoreactive plaque-like structures (G, arrows, bar = 0.2 mm), and the granular appearance of nucleolin staining (H, bar = 0.2 mm). CA1 (I, J) - note the abundance of nucleolin positive NFT. Type 1, 2, and 3 NFT were observed (bar = 0.05 mm).Fig. 7TG-3 reactive nucleolin in the AD brain. (A) Laser confocal micrograph of AD affected hippocampus dual labeled with antibody TG-3 (red) and anti-nucleolin mAb MS3 (green) (bar = 2.5 □). Green nuclei indicate TG-3 nonreactive nucleolin in neurons. Yellow NFT indicate overlap. TG-3 recognizes microtubule associated protein tau in neurites (red). (B, C) TG-3 recognition of phosphorylated Thr 231 on tau was blocked by preincubation with anti-tau monoclonal antibody AT-180 which recognizes phosphorylated Thr231 on tau, but not mitotic nucleolin. Blocked and control sections from the same region of the same brain were dual labeled with TG-3 and anti-nucleolin antiserum. Note the complete disappearance of TG-3 staining from compartments where no nucleolin was present (B). The amount of total TG-3 immunoreactivity and the ratio of TG-3/nucleolin overlap were quantitated and compared in blocked (+AT180) and control (-AT180) sections (C). Blocking tau from recognition by TG-3 decreased the amount of total TG-3 staining, but did not decrease the amount of TG-3/nucleolin overlap, suggesting that TG-3 recognizes nucleolin in tangles. The data are presented as the ratio of the amount of TG-3/nucleolin overlap to total TG-3.☆This paper is part of the special issue ‘Cell Cycle’ (Guest Editor Inez Vincent).Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer’s disease pathologyAlexDranovskyabInezVincentcLuisaGregoribAlexanderSchwarzmanbDavidColfleshdJanEnghildeWarrenStrittmatterePeterDaviescDmitryGoldgaberb*dgoldgaber@mail.psychiatry.sunysb.eduaMedical Scientist Training Program, State University of New York, Stony Brook, NY 11794, USAbDepartment of Psychiatry and Behavioral Science, State University of New York, Stony Brook, NY 11794, USAcDepartment of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USAdUniversity Microscopy Imaging Center, State University of New York, Stony Brook, NY 11794, USAeDepartment of Neurology, Duke University, Durham, NC 27710, USA*Corresponding author. Tel.: +1-631-444-1369; fax: +1-631-444-7534AbstractNucleolin is a major multifunctional nuclear phosphoprotein that is phosphorylated by Cdc2 kinase in mitosis and that participates in a number of cellular processes. The monoclonal antibody TG-3 generated against neurofibrillary tangles (NFT) found in Alzheimer’s disease (AD) is highly specific for mitotic cells in culture. We here demonstrate that phosphorylation of nucleolin by Cdc2 kinase generates the TG-3 epitope. The unique pool of TG-3 immunoreactive nucleolin appears abruptly during the prophase. It is associated with chromosomes through the metaphase and it gradually disappears during separation of chromosomes and exit from mitosis. In the brain, nucleolin was localized not only to nuclei but also to neuronal cytoplasm, and it is a marker for early NFT. In patients with AD, Cdc2 phosphorylated nucleolin was present in NFT. These findings suggest that phosphorylation of nucleolin by Cdc2 kinase is a critical event and the point of convergence of two distinct pathways, mitosis and neurodegeneration.KeywordsNucleolinMitosisNeurodegenerationAlzheimer’s disease1IntroductionSeveral recent reports suggest that reactivation of certain cell cycle events in postmitotic neurons may underlie neurofibrillary degeneration in AD [6,25,28,29,43,44]. Evidence for involvement of cell cycle specific proteins in neurodegeneration stems from studies on the role of phosphorylation in the formation of neurofibrillary tangles (NFT), one of the neuropathological hallmarks of AD. NFT consist of paired helical filaments (PHF) and straight filaments. Phosphorylation by proline directed serine-threonine kinases results in PHF properties in tau (a structural component of PHF) [31]. Experiments in vitro have shown that many proline directed kinases phosphorylate recombinant tau [1,27,31,46]. However, the biologic relevance of such experiments is not clear due to the substrate promiscuity of these kinases [30] and the ability of tau to form PHF in the absence of phosphorylation in vitro [48].In vivo support for tau phosphorylation by mitotic proline directed kinases was provided when a kinase related to the major mitotic kinase Cdc2 was immunolocalized to NFT in the AD brain [26,50]. Moreover, Vincent and colleagues recently demonstrated that Cdc2 kinase was activated in AD neurons [43]. Furthermore, immunoreactivity of other cell cycle markers was detected in the brains of patients affected by AD [6,28–29]. Aberrant activation of mitotic mechanisms in postmitotic neurons may facilitate cellular events leading to NFT formation and neurodegeneration. Evidence was obtained for the involvement of mitotic mechanisms in NFT formation when monoclonal antibodies (TG/MC series) raised against immunopurified NFT showed specificity for both the AD brain in immunohistochemical studies, and for mitotic cells in culture [44]. The same study provided provocative evidence that monoclonal antibody MPM-2, which was raised against mitotic cells [10], was highly immunoreactive with AD, but not with control brains. Moreover, MPM-2 recognized NFT, the neuritic components of plaques, and morphologically healthy neurons in the AD brain, thus suggesting that it is a marker for the early stages of neurodegeneration. Similar to the TG/MC series, MPM-2 recognized mitotic phosphoepitopes [10,16,39]. The presence of mitotic phosphoepitopes in degenerating neurons suggests that mitotic and neurodegenerative pathways converge in AD. Therefore, antibodies that demonstrate high specificity for both AD pathology and mitotic events can serve as ideal tools for identifying common molecules within the two pathways.Of all the antibodies reported by Vincent and colleagues, TG-3 was the most specific for AD neurofibrillary pathology demonstrating no detectable immunoreactivity in control brains. Therefore, identifying and characterizing the TG-3 antigen or antigens are essential to our understanding of the convergence of neurofibrillary degeneration and mitosis. The first TG-3 immunoreactive protein in the brains of patients with AD was recently identified as an abnormally phosphorylated microtubule associated protein tau [20]. Other potentially biologically relevant TG-3 antigens in the brain remain unknown. However, in cultured cells synchronized in mitosis, TG-3 recognizes not tau but a 105 kDa protein [44]. We here report that the cellular TG-3 antigen is the major nucleolar phosphoprotein nucleolin after phosphorylation by Cdc2 kinase. We also report that in the human brain, nucleolin is present in neuronal cytoplasm and colocalizes with NFT in AD.2Materials and methods2.1AntibodiesMonoclonal antibody TG-3 (hybridoma medium) was described in a previous publication [44] and used at a 1:10 dilution for Western blotting. Anti-nucleolin monoclonal antibody MS3 (hybridoma medium) was kindly provided by Drs. H. Busch and L. Perlaky from Baylor College of Medicine, and used at a dilution of 1:10 for immunocytochemistry and at a 1:1000 dilution for Western blotting [42]. MS3 ascites fluid was used for immunoprecipitations at a 1:10 dilution. Monoclonal antibody D3 (hybridoma medium), kindly provided by Dr. J.-S. Deng from the University of Pittsburgh, was used at a 1:10 dilution for immunocytochemistry [11]. Anti-nucleolin monoclonal antibody CC98 (hybridoma medium), kindly provided by Dr. N.H. Yeh, National Yang Ming College, Taiwan, was used at a 1:5 dilution for immunocytochemistry [8]. Anti-nucleolin monoclonal antibody 4E2 (hybridoma medium) from Research Diagnostics (Flanders, NJ) was used at a 1:5 dilution for immunocytochemistry. Rabbit polyclonal antisera W15 generated against human nucleolin and nucleolin-maltose-binding protein fusion protein were generously provided by Dr. N. Maizels, Yale University [17]. Each polyclonal antiserum was used at a 1:1000 dilution. Non-immune rabbit serum was used as a control. Monoclonal antibody AT180 (Innogenetics, Belgium), which recognizes phospho-threonine 231 (Thr231) of tau [15], was used at a 1:10 dilution for the blocking experiments. Monoclonal antibodies against β-tubulin (Sigma, St. Louis, MO) and histone protein mAb052 (Chemicon, Temecula, CA) were used at a 1:200 final dilution. The specificity of each primary monoclonal and polyclonal antibody was characterized by the authors who generated the antibody and was described in the papers cited above. Secondary goat anti-mouse IgG1a and goat anti-mouse IgM antibodies conjugated to fluorescein and rhodamine (Jackson Immunochemicals, West Grove, PA) were used at a 1:200 final dilution for immunocytochemistry. Secondary antibodies conjugated to biotin along with streptavidin conjugated to CY3 and CY5 fluorochromes (Southern Biotech, Birmingham, AL) were all used at a 1:500 dilution for the immunohistochemistry of brain sections. Secondary antibodies conjugated to horseradish peroxidase (Amersham, Oakville, Ontario) were used at a 1:3000 final dilution for Western blotting.2.2Cell culture and immunocytochemistryHEp-2 human epithelial cells (ATCC, Rockville, MD) were cultured in DME containing 10% bovine calf serum, and penicillin-streptomycin (Gibco, Burlington, Ontario) was cultured at 37°C in 5% CO2. Cells were washed with PBS, harvested by trypsinization, counted and plated at 1.25 × 104 - 2.5 × 104 cells/cm2 on 8 well chambered coverslips (VWR, Piscataway, NJ). On the following day, cells were fixed in 2% paraformaldehyde/0.1% glutaraldehyde for 40 min, permeabilized with 0.5% Triton X-100 for 10 min and stored in PBS at 4°C for immunocytochemistry. Prior to immunocytochemistry, subconfluent monolayers were blocked with 5% bovine serum albumin (BSA) in PBS for 30 min and rinsed in 3 volumes of PBS 3 times followed by three 10 min washes in PBS. Immunostaining of monolayers was performed in the chambered coverslips. Primary antibodies were applied for 2 h at room temperature or overnight at 4°C in a high humidity incubation chamber. The monolayers were washed as described above and incubated with secondary antibodies in the presence of DAPI (Sigma, St. Louis, MO) for 30 min. The monolayers were washed as described above and stored in the ANTI-FADE reagent (Molecular Probes, Eugene, OR) at 4°C. Rodamine. Fluorescein was visualized by the Noran Confocal Odyssey system through a Nikon inverted Diaphot microscope, and images were captured using Image One software.2.3Protein purificationHEp-2 cells were grown in suspension according to the conditions described above. Five liters of logarithmically growing cells were synchronized in mitosis by treatment with 2 mg/ml of nocodazole (Sigma, St. Louis, MO) overnight. Nocodazole was diluted from a DMSO solution so that the final DMSO concentration was 0.05%. Cells were harvested by centrifugation at 250 × g for 2 min, rinsed with PBS, centrifuged, and cell pellets were stored at −80°C. All extraction and purification procedures were performed at 4°C. Cells were extracted in 5 volumes of buffer A for 15 min, gently vortexed, and centrifuged at 400 × g for 10 min. Buffer A contained 0.015 M Tris-Cl pH 7.4, 0.08 M KCl, 2 mM EDTA-KOH, 1% Nonidet P-40, 0.2 mM spermine, 0.5 mM spermidine, 1 mM phenylmethane-sulphonyl fluoride, 10 nM Microsystin-LR, 1 mM Na-o-vanadate (all from Sigma), and protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). The supernatant was chromatographed on DEAE-Sepharose (Pharmacia, Piscataway, NJ) preequilibrated with buffer A, washed, and eluted with a 0.1–0.5 M NaCl step gradient in buffer A using 0.1 M increments. TG-3 reactive fractions were identified by Western blotting using the ECL detection system (Amersham, Oakville Ontario). The TG-3 reactive fraction was diluted in 15 mM Tris, 2 mM EDTA, pH 7.4 (buffer B), chromatographed on HiTrap-Heparin mini column (Pharmacia, Piscataway, NJ) preequilibrated in buffer B, washed, and eluted with a 0.1–0.5 M NaCl step gradient using 0.1 M increments. TG-3 immunoreactivity was determined by Western blotting. The TG-3 reactive fraction was separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The purified TG-3 reactive 105 kDa protein was subjected to N-terminal sequencing and the obtained sequence was compared by BLAST search against the Genbank database.2.4ImmunoprecipitationNocodazole-treated HEp-2 cells from 50 ml of culture media were collected by centrifugation and washed twice with PBS. The following procedures were performed at 4°C. Cells were lysed with 1 ml of RIPA buffer [18] and centrifuged at 8000 × g for 30 s. 30 μl of supernatant were mixed with 3 μl of MS3 ascites fluid. The reaction mixtures were incubated with rotation for 1 h, and 70 μl of anti-mouse IgG agarose (Sigma, St. Louis, MO) were added for an additional 30 min. The resin was washed three times with 1 ml of RIPA buffer, and the immunoprecipitated proteins were eluted by the addition of SDS-PAGE sample buffer containing 2-mercaptoethanol. The sample was divided into two equal parts and subjected to SDS-PAGE, transferred onto PVDF and probed using either MS3 antibodies or TG-3 antibodies.2.5“Mitotic shake”HEp-2 cells were grown in 175 mm2 flasks as described above. Mitotic cells were mechanically dislodged from the rest of the monolayer by dropping the flasks from three feet. The dislodged mitotic cells were harvested by centrifugation of the supernatant at 1000 × g for 5 min. The cell pellet was lysed and analyzed by Western blotting with TG-3 as described above.2.6Chemical cleavageTG-3 positive nucleolin was purified from nocodazole treated HEp-2 cells and subjected to SDS-PAGE. The TG-3 positive band was cut out of the gel, and the gel slice was subjected to chemical digestion with N-chlorosuccinimide, which specifically cleaves at tryptophane (positions 481 and 641 in nucleolin), or with hydroxylamine, which specifically cleaves between asparagine and glycine (positions 136, 137 and 519, 520 in nucleolin). N-chlorosuccinimide was used at 10 ml of 0.1 M in 2 M urea, and 50% acetic acid for 30 min at room temperature with rotation.Hydroxylamine was used at 2 M in 6 M guanidine-HCl at pH 9.0 for 12 h at room temperature with rotation. Gel slices were washed with 15 mM Tris, pH 6.8, 0.1% SDS, and 2-mercaptoethanol for 20 min. After chemical cleavage, the gel slice was placed on top of a second SS-gel and electrophoresed in a direction perpendicular with respect to the first gel. The separated cleavage products were transferred onto PVDF membrane and analyzed by Western blotting. The membranes were first immunostained with TG-3, then stripped with SDS and 2β- mercaptoethanol [18] and reprobed with MS3 to detect nucleolin. In the control experiments the cleaving reagent was omitted.2.7ImmunohistochemistryAll human subjects used in this study were evaluated both clinically and neuropathologically at the Albert Einstein College of Medicine as described elsewhere [44]. For neuropathological evaluation, tissue was immersion-fixed in 4% paraformaldehyde and independently evaluated with antibody Alz-50 and Thioflavin S. Brains from patients with clinical symptoms of AD showed marked neuropathological lesions. No clinical diagnosis of AD was noted in the control cases. Immunostaining was performed essentially as described elsewhere by free floating 50 μm vibratome sections in 24 well culture dishes [44]. For the blocking experiment the tissue was incubated overnight at 4°C with monoclonal antibody AT180 which recognized phosphorylated threonine 231 (Thr231) on tau and does not recognize nucleolin. The tissue was then washed as described elsewhere [44], incubated with unlabeled anti-mouse IgG1 overnight at 4°C, washed again, incubated with TG-3 and nucleolin rabbit antisera W15 overnight at 4°C, and washed. TG-3 was detected with TRITC conjugated anti-mouse IgM, while W15 was detected with biotinylated anti-rabbit antibodies followed by streptavidin conjugated CY5. All secondary incubations were performed as described above. Fluorochromes were visualized by the Bio-Rad Confocal system through a Nikon inverted Diaphot microscope.3Results3.1The TG-3 antigen in mitotic cells is nucleolinTG-3 consistently recognized a major 105 kDa band in extracts from HEp-2 cells that were synchronized in mitosis by treatment with nocodazole (Fig. 1 B, lane 5). This band was also observed in extracts from synchronized MSN neuroblastoma [44] and HeLa cells (data not shown). The appearance of this band was not an artifact of nocodazole treatment because an identical band was observed in extracts from mitotic HEp-2 cells that were enriched in mitotic cells by mechanical dislodging from the flask (Fig. 1B, lane 3). The TG-3 antigen was thus purified from nocodazole synchronized HEp-2 cells by sequential column chromatography and subjected to N-terminal sequencing (Fig. 1A). The sequence of the first 18 amino acids of the TG-3 antigen was identical to the N-terminal sequence of human nucleolin, a protein with a molecular mass of 105 kDa [37].To exclude the possibility that a minor protein of a similar size, copurifying with nucleolin, could account for the TG-3 immunoreactivity, we immunoprecipitated cell lysates with anti-nucleolin monoclonal antibody MS3 and analyzed the immunoprecipitate with TG-3. A band with the molecular weight of 105 kDa was immunoprecipitated by the MS3 antibody and detected by the TG-3 antibody (Fig. 1B, lane 7). These results support our initial finding that both MS3 and TG-3 antibodies recognize nucleolin in mitotic cells.As a further confirmation, the protein purified from nocodazole synchronized cells was subjected to chemical cleavage with either N-chlorosuccinimide or hydroxylamine (Fig. 2). Human nucleolin has two recognition sites for each reagent, distributed in such a way that cleavage generates three fragments, two of which were predicted to be detectable with the antibodies TG-3 and MS3 (see methods). Major bands migrating at 92 kDa and 74 kDa were observed after cleavage with N-chlorosuccinimide (Fig. 2A) and hydroxylamine (Fig. 2B), respectively. It should be noted that although nucleolin contains two cleavage sites for each reagent, under our experimental conditions we detected the product of only one major cleavage event. After each cleavage reaction, both MS3 and TG-3 stained identical bands, suggesting that they recognized the same protein. Thus, the results from the chemical cleavage experiments, in conjunction with the sequencing and immunoprecipitation experiments, proved that the TG-3 antibody recognized nucleolin in mitotic cells.3.2TG-3 reactive nucleolin is phosphorylated by Cdc2Nucleolin is a major cellular phosphoprotein, which has nine putative phosphorylation sites for the major mitotic kinase Cdc2 and has been shown to be phosphorylated by Cdc2 both in vitro and in vivo [2]. Previous studies demonstrated that TG-3 recognizes phosphoepitopes [44,20].Since TG-3 recognizes mitotic and not interphase cells, the following experiments were performed in order to test if TG-3 immunoreactivity is the result of phosphorylation of nucleolin by Cdc2. Interphase nucleolin, which is not reactive with TG-3, was partially purified from asynchronous HEp-2 cells. Nucleolin enriched samples were phosphorylated with [32P]ATP in the presence or absence of Cdc2 kinase. After phosphorylation, nucleolin was immunoprecipitated with the MS3 antibody, separated by SDS-PAGE, and analyzed by autoradiography and immunoblotting with either MS3 or TG-3 antibodies (Fig. 3). Autoradiography revealed 32P incorporation into numerous bands suggesting that the nucleolin enriched fraction contained many proteins that could be phosphorylated by Cdc2 kinase (Fig. 3A, lane 1). A significant incorporation of 32P into the protein immunoprecipitable with MS3 in the absence of Cdc2 suggested that nucleolin was also phosphorylated by copurified kinases (Fig. 3A, lane 4).While many bands showed Cdc2 dependent incorporation of 32P, only a single band migrating at 105 kDa was immunoreactive with TG-3 (Fig. 3C, lane 1). The appearance of the single TG-3 immunoreactive band at 105 kDa after phosphorylation with Cdc2 kinase demonstrated the antigenic specificity of the TG-3 phosphoepitope-specific antibody for the nucleolin protein in the relatively crude preparation. Furthermore, immunoprecipitation of nucleolin from the enriched fraction with non-phosphospecific MS3 (see Valdez et al., 1995 for epitope mapping) [42] revealed that nucleolin became immunoreactive with TG-3 only after phosphorylation by Cdc2 (Fig. 3C, lane 3). Phosphorylation reactions carried out in the absence of Cdc2 did not produce epitopes recognizable by TG-3 (Fig. 3C, lanes 2 and 4). Therefore, TG-3 is specific for phosphoepitopes produced by Cdc2 and not by copurifying endogenous kinases. Finally, phosphorylation was time and kinase concentration dependent, and TG-3 immunoreactivity was proportional to the amount of 32P incorporation (data not shown). Together, these data demonstrate the specificity of the TG-3 antibody for nucleolin phosphorylated by Cdc2.3.3TG-3 reactive nucleolin undergoes temporal and spatial changes during mitosis in cultureTG-3 immunoreactivity was examined in an asynchronous population of HEp-2 (Fig. 4 ) and HeLa cells (data not shown) by laser confocal microscopy. Dual labeling with monoclonal antibodies to either □-tubulin or to histone proteins was used to identify cells in different stages of mitosis. TG-3 reactive nucleolin (TG-3 nucleolin) appeared abruptly in cells entering mitosis. Interphase cells were not reactive with TG-3 (Fig. 4). TG-3 nucleolin was localized to the nuclei in cells in late prophase and early prometaphase at the beginning of centrosomal segregation (Figs. 4A–C). TG-3 nucleolin was also localized to the mitotic centrosomes starting from the earliest stages of polar migration (Figs. 4C and 4F). TG-3 nucleolin became dispersed throughout the mitotic cytosol as the prometaphase progressed (Figs. 4E and 4F). Optical sectioning revealed that the condensed chromosomes were immunoreactive with TG-3 as judged by the overlap with immunostaining that was detected by anti-histone antibody mAb053 (Fig. 4L).The chromosomal localization of TG-3 nucleolin became most apparent in metaphase when the majority of the immunoreactivity was localized to the aligned chromosomes (Figs. 4H and 4K). While chromosomal TG-3 immunoreactivity was most intense, some also localized to the mitotic cytosol. The intensity of cytosolic TG-3 immunoreactivity varied between the metaphase cells of the different cell lines but was always lower than immunoreactivity associated with chromosomes (data not shown). TG-3 immunoreactivity began to decrease at the earliest stages of chromosomal segregation, which was determined by histone specific mAb053 (Figs. 4J and 4L) and DAPI staining (data not shown). Whereas the intensity of TG-3 nucleolin immunoreactivity proceeded to decrease from metaphase to telophase, its spatial distribution did not change and the chromosomes persisted to be the predominant TG-3-immunoreactive structures (Figs. 4H, cell 3; Fig. 4K, cell 2). Cells in cytokinesis and interphase showed only background levels of immunostaining.3.4TG-3 reactive and non-reactive nucleolin are differentially distributed during mitosisAsynchronous HEp-2 cells were dual-labeled with phosphoepitope specific TG-3 and with MS3 which is specific for the region of nucleolin that is devoid of phosphorylation sites [42]. Whereas TG-3 was highly specific for nucleolin in cells in mitosis, MS3 was immunoreactive with nucleolin in all phases of the cell cycle (Fig. 5). As expected, the most prominent MS3 immunoreactive structures in interphase cells were nuclei and nucleoli. MS3 immunoreactivity was more dispersed throughout the nucleus in the early stages of mitosis. In prophase, nuclear TG-3 immunoreactivity only partially colocalized with the MS3 immunoreactivity (Fig. 5A). In metaphase, MS3 immunoreactivity was distributed throughout the mitotic cell. MS3 and TG-3 immunoreactivity colocalized primarily around the chromosomes since chromosomal TG-3 immunoreactivity was most pronounced. MS3 immunoreactivity of cytosol persisted in anaphase, telophase, and cytokinesis, while TG-3 immunoreactivity gradually became undetectable (Fig. 5C). The partial overlap of MS3 and TG-3 immunoreactivities in all stages of mitosis suggests that TG3 reactive nucleolin represents a unique subpopulation of mitotic nucleolin.3.5Differential distribution of nucleolin in the normal human brain and in ADThe functions of nucleolin have been extensively studied in cultured cells and in vitro [14,41]. Nucleolin has been immunocytochemically localized primarily to the nucleolus and the nucleus. However, the distribution and the role of nucleolin in human brain cells remain unknown. Therefore, the distribution of nucleolin in the brain was examined in four individuals affected by AD and in eight age-matched controls. Identical immunolocalization was obtained with all examined anti-nucleolin monoclonal and polyclonal antibodies, strongly suggesting that the distribution of immunoreactivity reflected the true distribution of nucleolin and that it was not the result of cross reactivity with other proteins. The images on panels of Fig. 6 and 7 were obtained with anti-nucleolin monoclonal antibody MS3 and illustrate a typical distribution of nucleolin in the hippocampus. The specificity of TG-3 for AD brains has been previously described [44], and it is here presented for illustrative value (Fig. 6A and 6F).Cells corresponding to all major areas of the hippocampal formation, including the dentate gyrus, were positive for nucleolin. Nucleolin was detected in both large neuronal nuclei and smaller glial nuclei (Figs. 6C and 6D). The distribution of nucleolin in glia appeared primarily nuclear, while neuronal nucleolin was readily detected in both cytoplasm and nuclei. Moreover, some neurons displayed mostly cytoplasmic distribution of nucleolin, while in other neurons, nucleolin was primarily nuclear (Fig. 6B). In addition to staining the neuronal cell bodies, nucleolin was detected in the apical dendrites of the pyramidal cells (Fig. 6D). Neurons with predominantly nuclear, and neurons with predominantly cytoplasmic nucleolin were observed in CA3 and CA4, whereas more cytoplasmic and robust dendritic staining was observed in CA2 and CA1 (Figs. 6C and 6D). The variability in the distribution of nucleolin between cells is indicative of a dynamic function for the protein in neurons. Occasionally a small number of NFT were observed in the hippocampus from individuals who showed no cognitive impairments prior to their death. The appearance of these early pathological changes in the brain prior to development of clinical symptoms is believed to characterize AD progression. CA1 is the earliest region in the hippocampus to be affected by neurodegenerative changes [5]. Interestingly, an intense nucleolin immunoreactivity in densely packed material typical of NFT within some pyramidal cells in the CA 1 region was observed in four of our control cases (Fig. 6E, representative example).To further characterize the association of nucleolin with AD pathology, brains from 3 individuals affected by AD were immunostained with mAb MS3 (Figs. 6G,H,I, and J). The distribution of nucleolin in the AD brains paralleled the distribution of nucleolin in the control brains. However, the neurons in the AD brains exhibited a marked decrease in nucleolin immunoreactivity throughout the hippocampal formation (Fig. 6G and 6H). Nucleolin immunoreactivity, shown in red, labeled small granular structures in many of the neurons in the CA3 and CA4 regions (Fig. 6H). Nucleolin localized to NFT in the CA1 and CA2 regions of the hippocampal formation (Figs. 6I and 6J). Using the classification of NFT proposed by Wischik (1989) [49], we found that the NFT immunolabeled with MS3 had the appearance of type 1 (early intracellular), type 2 (late intracellular) and type 3 (extracellular) NFT. Type 1 NFT were observed as intensely fluorescent, densely packed material traversing the pyramidal neuron and surrounding the nucleus. Large swollen pyramidal neurons with intensely labeled NFT and a nucleus that was positioned near the plasma membrane resembled type 2 NFT. Finally, intense filament-like staining of cell remnants with no noticeable nucleus was consistent with type 3 NFT.3.6TG-3 reactive nucleolin is present in NFTA recent study demonstrated that in AD brains, TG-3 antibody recognized microtubule associated protein tau phosphorylated on Thr231 [20]. It has not been established, however, if TG-3 recognizes other proteins in AD brains. After we established that TG-3 is highly specific for mitotic nucleolin in cell cultures, it became important to determine if TG-3 immunreactive nucleolin was present in the brains of patients with AD and, if yes, to differentiate it from TG-3 immunoreactive tau.In order to identify a population of NFTs with TG-3 reactive nucleolin, dual-label confocal microscopy was first performed with TG-3 and MS3 antibodies on AD cases that showed abundant MS3 immunoreactive NFT (Fig. 7A). There were many TG-3 immunoreactive NFT and an intense neuritic staining by the TG-3 antibody. MS3 stained NFT and nuclei, but there was no staining of neurites. Thus, there were many NFT that were positive with both antibodies (Fig. 7A).We then took advantage of another monoclonal antibody, AT180, which is also specific for tau phosphorylated on Thr231 [15]. However, AT180 did not recognize nucleolin in mitotic cells in cell cultures by immunocytochemistry or immunoblotting (data not shown). In AD brains all cellular structures recognized by TG-3 were also recognized by AT180, indicating that the phosphorylated Thr231 on tau was accessible to both antibodies in the same cellular compartments (data not shown). Since AT180 recognizes phosphorylated tau, but not mitotic nucleolin, AD brain tissue sections were preincubated with AT180 to block phosphorylated Thr231 on tau from being recognized by TG-3. The tissue sections were subsequently dual labeled with TG-3 (to detect the TG-3 immunoreactive protein which was not tau) and anti-nucleolin polyclonal antisera (to detect how much of the residual TG-3 immunoreactivity overlapped nucleolin) (Figs. 7B and 7C). After preincubation with AT180, TG-3 immunoreactivity disappeared preferentially from areas where no nucleolin immunoreactivity was present. TG-3 immunoreactivity was completely blocked in dystrophic neurites and neuritic components of plaques (Fig. 7B). The laser confocal fluorescence was quantified, and the proportion of total TG-3 immunoreactivity represented by the overlap of TG-3 and nucleolin was computed (Fig. 7C). A six-fold increase in the overlap ratio was seen in the sample blocked by AT180 compared to the control sample, demonstrating that AT180 blocked TG-3 reactive tau, but not TG-3 reactivity colocalized with nucleolin. The overlapping of residual TG-3 with nucleolin indicated that TG-3 reactive nucleolin was present in the NFT.4Discussion4.1Nucleolin phosphorylated by Cdc2 is the TG-3 antigenNucleolin is a major multifunctional nuclear phosphoprotein [14,41] and was one of the first identified nuclear-cytoplasmic shuttling proteins [3]. It is thought to be intricately involved in ribosomal biogenesis and it is a marker for cellular proliferation [13,22]. Nucleolin is a physiological substrate for Cdc2 kinase [2,32], and its biologic functions are highly regulated by phosphorylation in a cell cycle specific manner [21,35]. Here we report the identification of the cellular antigen for the AD specific antibody TG-3 as a form of nucleolin that is phosphorylated by Cdc2 in mitosis.Nucleolin has 9 proline directed consensus sequences for Cdc2 recognition. Phosphorylation of nucleolin by Cdc2 and casein kinase II (CKII) has been reported in vitro and in vivo [2,32,24]. The incorporation of 32P in the absence of Cdc2 shown in the present study, complements the previous observation that CKII copurifies with and phosphorylates nucleolin [24]. However, our data indicate that in mitotic cells, TG-3 recognizes nucleolin phosphorylated by Cdc2 and not other kinases, and TG-3 does not recognize other phosphoproteins in cultured cells. These results firmly establish the TG-3 antibody as a novel and useful tool for studying nucleolin in mitosis.4.2Cdc2 phosphorylated nucleolin and mitosisThe abrupt appearance of TG-3 reactive nucleolin in early mitosis corresponds to activation of Cdc2, thus supporting the in vitro phosphorylation results discussed above. A recent report indicates that nucleolin is also phosphorylated by cell cycle kinases that are active prior to mitosis [33]. The specificity of TG-3 for cells in mitosis presents in situ evidence that TG-3 is specific for a phosphoepitope generated by Cdc2 and not for phosphoepitopes generated by other kinases. TG-3 immunoreactivity appears in the nucleus at the onset of mitosis. Here we report the translocation of nucleolin phosphorylated by Cdc2 to the cytoplasm during late prophase and early prometaphase. Schwab and Dreyer (1997) [35] recently demonstrated that Cdc2 phosphorylation localizes microinjected X.laevis nucleolin to the cytoplasm. The redistribution of Cdc2 phosphorylated nucleolin to the cytoplasm in late prophase provides functional in situ support for the above result and raises the possibility that phosphorylation may play a role in targeting nucleolin outside the nucleus at this stage of the cell cycle. Interestingly, the in situ detection of nucleolin in the cytoplasm has been elusive, suggesting that under normal conditions the amount of nucleolin in the cytoplasm of cultured cells is below the level detectable by immunocytochemical methods. However, in polio-virus infected cells [47] and in rat intestinal epithelial cells grown on a glass surface [51], nucleolin was found in the cytoplasm.The localization of Cdc2 phosphorylated nucleolin to the chromosomes is consistent with previously reported observations [12]. Moreover, colocalization of MS3 and TG-3 immunoreactivity in the perichromosomal region suggested that Cdc2 phosphorylation of nucleolin could be important for its chromosomal interaction. It has been hypothesized that interaction of Cdc2 phosphorylated nucleolin with histone H1 plays a role in chromatin condensation in mitosis [21].While most of the nucleolin is located in the mitotic cytosol, most of the TG-3 reactive nucleolin is localized to mitotic chromosomes. In every stage of mitosis, MS3 immunoreactivity only partially overlapped the TG-3 immunoreactivity, suggesting heterogeneity of mitotic nucleolin. This partial overlap also establishes that TG-3 reactive nucleolin represents a unique subpopulation of mitotic nucleolin. Functional studies with nucleolin mutated in its Cdc2 phosphorylation domains will help to elucidate the biologic role of phosphorylation in the interaction of nucleolin with chromosomes and in the cytoplasmic targeting of nucleolin.We observed a decrease in TG-3 immunoreactivity starting at anaphase, and it became undetectable in cytokinesis. The decrease in TG-3 immunoreactivity during mitotic exit may be due to dephosphorylation or degradation of nucleolin. Recent reports suggest that nucleolin is more susceptible to degradation in nonproliferating cells [our unpublished observations; 8]. Our observation that TG-3 immunoreactivity decreases in late mitosis while MS3 immunoreactivity remains unchanged suggests the possibility that TG-3 reactive nucleolin is first dephosphorylated and then degraded. Interestingly, the Saccharomyces pombe nucleolin homolog gar2 is essential for cytokinesis and phosphorylated by Cdc2 [23].Perhaps dephosphorylation and/or degradation of chromosome associated nucleolin has functional significance for chromosomal segregation or cell division. Activation of phosphatase Cdc14 triggers mitotic exit in budding yeast [36,45]. Similar events in mammalian cells may explain the disappearance of TG-3 phosphoepitope, which parallels the separation of chromosomes and mitotic exit. The recently demonstrated participation of nucleolin in special cases of recombination and replication may provide clues to the role of Cdc2 phosphorylated TG-3 immunoreactive nucleolin in mitosis [17,40,4,9]. Thus, our finding of the dynamic association of TG-3 reactive nucleolin with mitotic chromosomes may represent the long sought but elusive direct link between nucleolus and cell cycle regulation in higher organisms [7].4.3Nucleolin in the brainThis study represents the first characterization of the distribution of nucleolin in the human brain. The presented data were highly reproducible when several polyclonal antisera and monoclonal antibodies to nucleolin were used on sections of human brain tissues obtained from both archival biopsy and autopsy material.Three novel observations were made. First, nucleolin was detected in neuronal cytoplasm. Second, nucleolin was present in NFTs, including early tangles, and NFT-associated nucleolin was TG3-positive, suggesting that it was phosphorylated by Cdc2 kinase. Third, in AD, the overall level of nucleolin was dramatically decreased in the temporal cortex as well as in the CA1 and CA2 regions of the hippocampus. As reported by others in cultured cells [41,14,38] and as described here, nucleolin was found in nuclei and nucleoli of neurons and glia cells in the brain. Surprisingly, nucleolin was also present in the cytoplasm of granular neurons of the dentate gyrus and pyramidal neurons of the hippocampus and of the lower neocortical layers. Moreover, nucleolin was identified in the apical dendrites.The finding of nucleolin in the cytoplasm was surprising because it is generally believed that nucleolin, as the name indicates, is present in the nucleolus and that it can also be detected in the nucleus. Nucleolin has been found in the cytoplasm and plasma membrane by biochemical methods [41,14,38]. However, immunocytochemical detection visualized nucleolin normally in the nucleus and nucleolus of cultured cells suggesting that the quantity of nucleolin outside the nucleus is insufficient for its detection under normal conditions. Nucleolin can be detected in cytoplasm of cultured cells by immunocytochemical methods only under certain conditions [51,47]. Yu et al. [51] detected nucleolin in the cytoplasm of rat intestinal epithelial IEC-6 cells grown on glass slides. However, in cells grown on laminin, nucleolin was found in the usual location of the nucleus and nucleolus. Waggoner and Sarnow (1998) [47] reported a massive nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells. Recently a nucleolin-related protein, probably nucleolin, was detected in the cytoplasm of bovine photoreceptor cells. These studies suggest that in response to certain as yet unidentified signals, nucleolin may relocalize to the cytoplasm. Thus, it is possible to speculate that in the brain, nucleolin is localized to the neuronal cytoplam in response to signals that are absent in cultured cells.The association of nucleolin with neuronal degeneration stems from the localization of nucleolin to the NFT. It is important to stress that brain tissue from some of the so called control age-matched cases, where no clinical symptoms of AD were apparent, had a small number of nucleolin positive, early (type 1) NFT in the CA1. Since CA1 is thought to be the earliest hippocampal region affected by NFT during the course of AD progression [5], nucleolin was inferred to be a morphological marker for early neurofibrillary changes.Dual labeling experiments with antibodies to nucleolin and TG-3 yielded partial colocalization. Recognition of hyperphosphorylated tau by TG-3 serves to explain the TG-3 immunoreactivity that does not overlap with nucleolin immunoreactivity. Blocking of tau from being recognized by TG-3 by preincubation with an excess of monoclonal antibody AT180 led to the disappearance of TG-3 immunoreactivity in dystrophic neurites and neuritic components of plaques, thus confirming tau presence in these structures. Persistence of residual TG-3 immunoreactivity in NFT suggests that in NFT, TG-3 primarily recognized proteins other than tau. Inefficient blocking of Thr231 on tau in NFT is not likely since AT180 is intensely immunoreactive with NFT. The colocalization of nucleolin immunoreactivity with the residual TG-3 immunoreactivity strongly suggests that Cdc2 phosphorylated nucleolin is one of the major TG-3 immunoreactive proteins in NFT.Detection of TG-3 immunoreactive nucleolin in NFT marks the first identification of a normal cellular substrate for mitotic kinases, which appears to undergo mitotic modification in AD. Several recent reports indicating that mitotic kinases are active in the AD brain contribute important information to our understanding of mitotic mechanisms that are reactivated in AD pathogenesis. However, in order to understand the mechanisms of the resulting neuronal degeneration, substrates for mitotic kinases in AD must be identified. It is important to reemphasize that nucleolin is a marker for early (pre-clinical) neurofibrillary changes and thus its mitotic modification may prove important in the early development of neuronal degeneration associated with AD.The possibility that nucleolin is involved in both chromosomal segregation during mitosis as well as AD may help to explain the observed co-occurrence of Down syndrome (DS) and AD within certain families [19,34]. The majority of DS cases are the result of non-dysjunction of maternal chromosome 21 in miosis, or a defect in chromosomal segregation. Therefore, the co-occurrence of DS and AD within specific families suggests a converging cellular mechanism between chromosomal segregation and neuronal degeneration. Specifically, proteins and pathways involved in chromosomal segregation and cell division in general may serve as signals for, or intermediates in, the degeneration of post-mitotic neurons in AD. The possible involvement of nucleolin in mitosis and AD makes it a candidate for such a protein.AcknowledgementsThis work was supported by NIA, Alzheimer’s Association, and Long Island Alzheimer Foundation (LIAF) to DG, AG/OD 12721 to IV and MSTP GM08444, AFAR, and LIAF fellowships to AD. The authors thank M. Cammer for assistance with confocal images; G. Jicha for assistance with a phosphorylation experiment; Drs. C. Dingwall, and S. Strickland for critical reading of the manuscript; Drs. E. Bromet, P. Fisher, W. Quitschke, S. Simon, and W. Van Nostrand for helpful comments on the manuscript.References[1]Baumann, K., E.M. Mandelkow, J. Biernat, H. Piwnica-Worms, and E. Mandelkow. Abnormal Alzheimer’-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5. FEBS Lett. 1993;336:417–424.[2]Belenguer, P., M. Caizergues-Ferrer, J.C. Labbe, M. Doree, and F. Amalric. Mitosis-specific phosphorylation of nucleolin by p34Cdc2 protein kinase. 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Comm. 247:186–192;1998. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AC888AEAD1C845FBF18E51951AF4D79F62A1ED8.txt b/test/dataset/in/resources/corpus/Clean_0AC888AEAD1C845FBF18E51951AF4D79F62A1ED8.txt new file mode 100644 index 0000000..5407b5a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AC888AEAD1C845FBF18E51951AF4D79F62A1ED8.txt @@ -0,0 +1 @@ +annoncannoncAnnals of Oncology1569-80410923-7534Oxford University Press10.1093/annonc/mdp110original articleshematologic malignanciesFavorable impact of the interleukin-4 receptor allelic variant I75 on the survival of diffuse large B-cell lymphoma patients demonstrated in a large prospective clinical trialSchoofN.1*von BoninF.1ZeynalovaS.2ZiepertM.2JungW.1LoefflerM.2PfreundschuhM.3TrümperL.1KubeD.11Department of Hematology and Oncology, Medical Center of the Georg-August-University of Göttingen, Göttingen2Department of Statistics and Epidemiology, Institute of Medical Informatics, University of Leipzig, Leipzig3Department of Internal Medicine I, Saarland University, Homburg/Saar, Germany for the DSHNHL (German High-Grade Non-Hodgkin's Lymphoma Study Group)*Correspondence to: Dr Nils Schoof, Universitätsmedizin der Georg-August-Universität Göttingen, Zentrum für Innere Medizin, Abteilung Hämatologie und Onkologie, 37099 Göttingen, Germany. Tel: +49-0-551-39-8572; Fax: +49-0-551-39-10497; E-mail: nschoof@gwdg.de9200910620092091548155427122008222009© The Author 2009. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org2009Background: Recently published data indicate that host germline variations in immune genes can influence the outcome of lymphoma patients. Interleukin (IL)-4 and IL13 are crucial immune factors and may influence the course of the disease. Both cytokines signal through the interleukin-4 receptor (IL4R). Therefore, we investigated whether polymorphisms of IL4, IL13 and IL4R genes could predict the outcome of diffuse large B-cell lymphoma (DLBCL) patients.Methods: In 228 DLBCL samples of the German High-Grade Non-Hodgkin's Lymphoma Study Group, the polymorphisms of IL4 (-524CT, rs2243250), IL13 (-1069CT, rs1800925) and IL4R (I75V, rs1805010; S503P, rs1805015; Q576R, rs1801275) were analyzed and the soluble interleukin-4 receptor (sIL4R) serum level was measured before the start of chemotherapy.Results: Patients harboring IL4R V75 (IL4RI75V-AG and IL4RI75V-GG) had shorter overall survival (OS) (P = 0.044) and event-free survival (EFS) (P = 0.056) periods compared with I75 carriers (IL4RI75V-AA). Multivariate analysis adjusted to the International Prognostic Index revealed a relative risk of 1.9 for carriers of the IL4R V75 (P = 0.011) in relation to OS. DLBCL patients homozygous for the IL4R I75 and low sIL4R serum levels have the most favorable OS and EFS.Conclusions: These data support the role for host germline gene variations of immunologically important factors like the IL4R I75V gene variation to predict the survival in DLBCL patients.cytokinesDLBCLIL4RpolymorphismsIL4RintroductionDiffuse large B-cell lymphoma (DLBCL) is the major subtype of non-Hodgkin's lymphoma (NHL), accounting for approximately one-third of all lymphoma cases in the western world. Although cure rates are high in DLBCL, many patients do not achieve complete remission or relapse after induction treatment. The introduction of the International Prognostic Index (IPI) has improved risk stratification of NHL patients but is still insufficient for outcome prediction, mainly for the group of the nonresponders [1]. Therefore, factors not included in the IPI have to be further validated in order to better predict patients’ outcome, to stratify patients’ treatment strategies as well as to understand the underlying pathological mechanisms of lymphoma. Cytokine gene variations have been shown to influence the susceptibility to NHL as well as the clinical course of lymphoma [2–6]. In a cohort of 500 NHL patients from the NHL-B study of the German High-Grade Non-Hodgkin's Lymphoma Study Group (DSHNHL), we recently found an association of distal IL10 gene variations to the clinical course of high-grade NHL [2]. However, in this study, the subgroup of DLBCL patients showed neither association of the analyzed IL10 gene variations to the clinical outcome nor the susceptibility of DLBCL, leading us to question the influence of other genetic variations in immune genes in DLBCL patients [2].Cytokines and their receptors play an essential role in regulation of the host immunity and could act as potential autocrine or paracrine factors for lymphoma initiation or progression. The expression of cytokines and their respective receptors on different cell types regulate the communication between the components of immune system and could be responsible for the host–lymphoma interactions as well. Interleukin-4 (IL4) and IL13 are crucial cytokines to polarize naïve CD4 T lymphocytes to Th2 cells and also to further trigger the humoral Th2 immune responses. Both cytokines use the interleukin-4 receptor (IL4R) chain to signal through different receptor heterodimers. IL4 signals by the use of the common gamma chain together with the IL4R chain (type I receptor) and both IL4 and IL13 can signal through the dimer of the IL13RA1 chain in combination with the IL4R (type II receptor). Moreover, a soluble form of the IL4R chain (sIL4R) has been described [7]. The expression of soluble cytokine receptors is believed to alter the properties of the respective cytokine signaling cascade. Serum levels of soluble cytokine receptors have been reported to be of prognostic significance in patients with NHL [8, 9].In this exploratory study, we analyzed the association of IL4, IL13 and IL4R gene polymorphisms and the respective sIL4R serum levels to the clinical outcome in a cohort of 228 DLBCL patients of the NHL-B1/B2 study of the DSHNHL. Our results, based on clinical data of a large prospective clinical trial, indicate for the first time an association of the IL4R I75V gene polymorphism with the treatment outcome of DLBCL patients.methodsstudy designThe study was conducted in accordance with the Declaration of Helsinki. This polymorphism analysis was approved by the Ethics Committee of the University of Göttingen (4 November 2001). All patients gave written informed consent to the inclusion into the NHL-B trial. Patients were eligible for inclusion if they had previously untreated, biopsy-confirmed DLBCL according to the Revised European–American Lymphoma Classification (translated into the World Health Organization classification). Within the NHL-B1/B2 trials of the DSHNHL 1399 high-grade NHL patients were included, thereof 913 patients with DLBCL [10, 11]. Data and samples from 228 DLBCL patients were selected from the DLBCL NHL-B1/B2 study population based on the availability of samples and representativeness for all DLBCL patients of the trials, as described recently [2, 3, 10, 11]. The selected patients were equally distributed across the different study arms of the NHL-B1/B2 trials. Clinical characteristics and histology of the patients eligible for this study are shown in Table 1 in comparison with all DLBCL patients of the NHL-B1/B2 studies. The respective histology is based on a review by a panel of expert hematopathologists as described before [10, 11].Table 1.Clinical characteristics and histolopathology of study patients with DLBCLAll DLBCL patients NHL-B1/B2 trials (n = 913)DLBCL patients analyzed for gene variations (n = 228)Patient's characteristics    Sex        Male507 (55.5)126 (55.3)        Female406 (44.5)102 (44.7)    Age, median (minimum; maximum), years61 (18; 75)60.5 (22; 75)    Serum LDH >N225 (24.6)49 (21.5)    Age >60 years489 (53.6)114 (50.0)    Performance status ECOG >1110 (12.0)28 (12.3)    Ann Arbor stages III, IV371 (40.6)91 (39.9)    No. of extranodal sites >1166 (18.2)38 (16.7)    IPIa        Low (IPI = 0, 1)531 (58.2)140 (61.4)        Low intermediate (IPI = 2)164 (18.0)44 (19.3)        High intermediate (IPI = 3)131 (14.3)24 (10.5)        High (IPI = 4, 5)87 (9.5)20 (8.8)    Bulky tumor (≥7.5 cm)296 (32.4)75 (32.9)    B symptoms247 (27.1)54 (23.7)    Extranodal involvement448 (49.1)109 (47.8)Histology    DLBCL913 (100.0)228 (100.0)        Centroblastic diffuse687 (75.2)165 (72.4)            Monomorphic83 (9.1)25 (11.0)            Multilobulated57 (6.2)11 (4.8)            Polymorphic349 (38.2)86 (37.7)            Not otherwise specified198 (21.7)43 (18.9)        Immunoblastic114 (12.5)32 (14.0)        T-cell rich B-cell24 (2.6)7 (3.1)    Anaplastic28 (3.1)11 (4.8)    Not otherwise specified60 (6.6)13 (5.7)The total numbers of patients and (in parentheses) respective percentages are presented.aLDH >N, age >60 years, ECOG >1, stage III, IV and no. of extranodal sites ≥2.DLBCL, diffuse large B-cell lymphoma; NHL, non-Hodgkin's lymphoma; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; IPI, International Prognostic Index.genotyping analysesGenomic DNA from lymphoma patients was isolated as described previously [2, 3, 12]. The polymorphisms of IL4 (-524CT, rs2243250), IL13 (-1069CT, rs1800925) and IL4R (I75V, rs1805010; S503P, rs1805015; and Q576R, rs1801275) genes were analyzed. For the analysis of the genetic polymorphisms (listed in Table 3 and supplemental Table 1, available at Annals of Oncology online), an Applied Biosystems ABI 7900HT instrument was used based on the sequence data and assay conditions of the National Cancer Institute SNP500 database (http://snp500cancer.nci.nih.gov/).serum analysisSera were collected and stored frozen at the trial office biomaterials storage facility center at −70°C until measurement of serum factors. Serum concentrations of sIL4R were determined with Quantikine ELISA Kits (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) according to the manufacturer's instruction. All samples and standards were run in duplicates; 115 pg/ml of sIL4R were set to discriminate high and low sIL4R levels based on upper quartile value of the whole cohort.statistical analysisStatistical analysis was carried out as described before [2]. Event-free survival (EFS) was defined as the time from first day of treatment to progressive disease under therapy, or failure to achieve a complete remission or complete remission unconfirmed (i.e. no change or partial remission associated with additional salvage therapy), additional therapy in excess of that prescribed in the protocol, relapse or death from any cause, whatever came first. Overall survival (OS) was defined as the time from first day of treatment to death from any cause. Patients without an event in EFS or OS were censored at the last day with valid information for the respective end point. EFS and OS were estimated according to Kaplan—Meier test and compared by log-rank test. For univariate analyses, P values < 0.05 were considered significant. Estimators at 3 years are given with 95% confidence intervals. Multivariate analyses were carried out with the use of Cox proportional hazard models to estimate hazard ratios for evolving an event. All factors with P < 0.1 were included in the multivariate analysis. We are aware of the problem of multiple comparisons between different combinations of heterozygous and homozygous groups within each genotype and have chosen to extract the most prominent aspect; therefore, this study is labeled as an exploratory study. Statistical analyses were carried out with SPSS 15.0.resultsIL4R I75V polymorphism is associated with survival of DLBCL patientsWithin this cohort of DLBCL patients, we analyzed polymorphisms of IL4 (-524CT), IL13 (-1069CT) and IL4R (I75V, S503P and Q576R) genes. Genotype distribution and Hardy–Weinberg equilibrium of the analyzed polymorphisms are shown in Table 2 and supplemental Table 1 (available at Annals of Oncology online). Subsequently, the association of the polymorphisms with the OS and EFS of DLBCL patients was analyzed. Univariate analysis revealed for the IL4R I75V variation differences in the survival of DLBCL patients. Individuals with at least one V75 variant (represented by IL4RI75V-GG and IL4RI75V-AG genotypes) had decreased OS (P = 0.044) and EFS (P = 0.056) periods compared with homozygous A-allele carrier representing the I75 variant (IL4RI75V-AA) (Figure 1A and B). The 3-year survival rate of patients with I75 variant (IL4RI75V-AA) was 80.0% (OS) and 71.4% (EFS) compared with 66.8% (OS) and 58.2% (EFS) in patients with V75 variants (IL4RI75V-AG and IL4RI75V-GG) (Table 3). The two other IL4R polymorphisms (S503P and Q576R) showed also shorter survival periods for homozygous carriers of the respective minor alleles. However, this was not significant probably due to their low frequency (Table 3). No relation of the promoter polymorphisms of IL4 (-524CT) and IL13 (-1069CT) to the survival of DLBCL patients was found (supplemental Table 2, available at Annals of Oncology online).Table 2.IL4R SNPs, genotype distribution and HWESNP nameRef. SNP IDGenotypeAA variantNo. of patients% of patientsHWE P value (χ2)IL4R I75Vrs1805010AAI75/I757030.70.265AGI75/V7510546.1GGV75/V755323.2IL4R S503Prs1805015TTS503/S50315668.40.603TCS503/P5036628.9CCP503/P50362.6IL4R Q576Rrs1801275AAQ576/Q57614061.40.754AGQ576/R5767934.6GGR576/R57693.9IL4R, interleukin-4 receptor; SNP, single-nucleotide polymorphisms; HWE, Hardy–Weinberg equilibrium.Table 3.Association between IL4R nonsynonymous coding SNPs and OS and EFS of DLBCL patientsGenotype3-year rate (95% CI)P3-year rate (95% CI)PGenotype3-year rateP3-year ratePOSEFSOSEFSIL4R I75V    AA n = 70I75/I7580.0 (70.6–89.4)0.12171,4 (60.0–82.0)0.157AA n = 7080.00.04471.40.056    AG n = 105I75/V7567.4 (58.4–76.4)59.0 (49.6–68.4)AG and GG n = 15866.858.2    GG n = 53V75/V7565.6 (52.7–78.5;)56.5 (43.2–69.8)AA and AG n = 17572.40.66264.00.604GG n = 5365.656.5IL4R S503P    TT n = 156S503/S50371.1 (64.0–78.2)0.25062.8 (55.2–70.4)0.534TT n = 15671.10.61862.80.832    TC n = 66S503/P50372.4 (61.6–83.2)62.1 (50.3–73.9)TC and CC n = 7270.561.1    CC n = 6P503/P50350.0 (10.0–90.0)50.0 (10.0–90.0)TT and TC n = 22271.40.15062.60.264CC n = 650.050.0IL4R Q576R    AA n = 140Q576/Q57670.6 (63.0–78.2)0.33062.1 (54.1–70.1)0.508AA n = 14070.60.41462.10.899    AG n = 79Q576/R57673.1 (63.3–82.9)63.3 (52.7–73.9)AG and GG n = 18871.362.5    GG n = 9R576/R57655.6 (23.1–88.1)55.6 (23.1–88.1)AA and AG n = 21971.50.31262.50.279GG n = 955.655.6Patients characterized by the IL4R genotypes I75/V75 and V75/V75 had a poorer prognosis comparing them with homozygous I75/I75 genotypes. Significant P values of the IL4R I75V gene variation are shown in italics. For continuative description of significant results, see also Figure 1.IL4R, interleukin-4 receptor; SNP, single nucleotide polymorphisms; OS, overall survival; EFS, event-free survival; DLBCL, diffuse large B-cell lymphoma; CI, confidence interval.Figure 1. Overall (A) and event-free survival (B) of DLBCL patients in relation to IL4R gene variation I75V. Comparison of I75V genotypes reveals differences of homozygous I75/I75 carriers (black lines) toward the other genotypes with regards to OS (A) (P = 0.044) and EFS (B) (P = 0.056) of DLBCL patients. Patients with I75/V75 (gray lines) or V75/V75 (light gray lines) variations have a poor prognosis compared with patients with I75/I75 (black lines) genotype. For 3-year rates and multivariate analysis, see Table 2 and Table 3, respectively.multivariate analysis of IL4R I75V according to IPI factorsIn a multivariate analysis adjusting for the IPI, we investigated the effect of the IL4R I75V gene variation on the survival of DLBCL patients. The presence of IL4R V75 variants (IL4RI75V-AG and IL4RI75V-GG) is a statistically independent predictor of OS [relative risk (RR) 1.9, P = 0.011] and a relevant, though not significant, predictor for EFS (RR 1.5, P = 0.076) (Table 4). These effects are also consistent when adding the treatment results of the NHL-B1/B2 trials to the multivariate analysis. These results indicate a strong association of this gene variation with the outcome of DLBCL patients.Table 4.Multivariate analysis of IL4R I75V gene variation IL4R V75 versus I75 (IL4RI75V-AG and IL4RI75V-GG versus IL4RI75V-AA) adjusted to the IPI (Cox model) in relation to OS and EFSFactorOSEFSRR (95% CI)PRR (95% CI)PSerum LDH >N2.1 (1.3–3.6)0.0031.7 (1.1–2.8)0.024Age >60 years2.6 (1.5–4.5)0.0012.1 (1.3–3.3)0.002Performance status ECOG >12.5 (1.5–4.1)0.0012.6 (1.6–4.2)<0.001Ann Arbor stage III/IV1.4 (0.9–2.3)0.1231.5 (1.0–2.2)0.054Extranodal involvement >11.6 (1.0–2.7)0.0641.3 (0.8–2.0)0.321IL4R V75 versus I751.9 (1.2–3.2)0.0111.5 (1.0–2.3)0.076Significant P values of the IL4R I75V gene variation are shown in italics. IL4R, interleukin-4 receptor; IPI, International Prognostic Index; OS, overall survival; EFS, event-free survival; RR, relative risk; CI, confidence interval; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group.impact of IL4R I75V in combination with sIL4R serum level on survival of DLBCL patientsCytokine and respective soluble cytokine receptor serum levels are parameters that could to some extent reflect the actual host–lymphoma interaction and thereby influence the prognosis for DLBCL patients. We therefore measured sIL4R serum levels at the time of admission to the hospital before the first treatment and combined sIL4R serum levels with IL4R I75V genotypes in the survival analysis because we found that an elevation of sIL4R serum levels [high (>115 pg/ml) versus low (≤115 pg/ml)] showed adverse effects on the OS (P < 0.001) and EFS (P < 0.001) of all 228 DLBCL patients. Univariate analysis of DLBCL patients with I75 (IL4RI75V-AA) genotype and sIL4R levels ≤115 pg/ml showed the most favorable OS (Figure 2A) and EFS (Figure 2B) outcome. However, when they had serum levels >115 pg/ml, their OS and EFS were less favorable. The 3-year rates of patients with I75 variant (IL4RI75V-AA) with low sIL4R levels compared with I75 variant patients with high sIL4R levels are 84.3% versus 68.4% (OS, P = 0.003) and 74.5% versus 63.2% (EFS, P = 0.023). DLBCL patients with IL4R V75 variants (IL4RI75V-AG and IL4RI75V-GG) and high serum levels of sIL4R have a worse clinical course compared with those with low sIL4R levels (Figure 2). The 3-year rates of patients with IL4R V75 variants (IL4RI75V-AG and IL4RI75V-GG) with low sIL4R levels compared with V75 variant patients with high sIL4R levels are 71.9% versus 49.7% (OS, P = 0.001) and 63.9% versus 38.9% (EFS, P = 0.002). All genotypes of the IL4R I75V variation showed significant differences in the survival of DLBCL patients taking sIL4R serum level into account (Table 5). Our data indicate a strong association of the IL4R I75V polymorphism to the survival of DLBCL patients and show that joint effects of this gene variation with serum levels of sIL4R are observed. These may further subdivide patients into groups with slower or faster disease progression. Including sIL4R serum levels (≤115 pg/ml versus >115 pg/ml) into the multivariate analysis adjusted to the IPI factors, the effect of the IL4R I75V gene variation remains unchanged (OS: RR = 1.9, P = 0.012 and EFS: RR = 1.5, P = 0.077) whereas the sIL4R serum level (>115 pg/ml) is not an independent factor (OS: RR 1.1, P = 0.722 and EFS: RR 1.0, P = 0.998). This analysis strongly supports the hypothesis that, besides clinical and molecular lymphoma characteristics of the lymphoma itself, the genetic background may be an important additional factor predicting the outcome of DLBCL patients.Table 5.OS and EFS periods of DLBCL patients in relation to IL4R I75V gene polymorphism combined with sIL4R serum levelGenotype3-year rate (95% CI)3-year rate (95% CI)IL4R I75VAA variantsIL4R serum level (pg/ml)No. of patientsOSEFSAAI75/I75≤1155184.3 (74.3–94.3)74.5 (62.5–86.5)AGI75/V75≤1157973.1 (63.3–82.9)64.6 (54.0–75.2GGV75/V75≤1154369.6 (55.9–83.3)62.7 (48.2–77.2)AAI75/I75>1151968.4 (47.4–89.4)63.2 (41.4–85.0AGI75/V75>1152650.0 (30.8–69.2)42.3 (23.3–61.3)GGV75/V75>1151048.0 (15.9–80.1)30.0 (1.6–58.4)Combining IL4R genotypes with serum level of sIL4R reveals detailed differences in survival of DLBCL patients (global P < 0.001). Carriers of IL4R V75 (I75V-AG and -GG) variants with high serum levels of sIL4R have lowest survival periods. IL4R I75/I75 carriers with low sIL4R levels have the highest survival periods.OS, overall survival; EFS, event-free survival; DLBCL, diffuse large B-cell lymphoma; sIL4R, soluble interleukin-4 receptor; CI, confidence interval.Figure 2.Overall (A) and event-free survival (B) of DLBCL patients in relation to IL4R gene variation I75V genotypes separated in high and low serum levels of sIL4R. Patients with high levels of sIL4R and the IL4R I75/V75 or V75/V75 (dotted gray lines) variants have lowest survival rates, whereas homozygous carriers of the I75/I75 variant and low sIL4R serum levels (black lines) have the best survival rates. Patients with high sIL4R serum levels and I75/I75 genotype (gray lines) range together with patients with low sIL4R levels and the unfavorable genotypes I75/V75 and V75/V75 (black dotted lines).discussionRecent evidence indicates that gene variations of cytokines and their receptors are associated with the risk of developing hematological malignancies [3–6, 13]. However, only a limited number of studies have analyzed the clinical course and treatment outcome of lymphoma in association with inherited gene polymorphisms. This is mainly due to the fact that a sufficient number of samples from patients included in controlled prospective clinical trials are difficult to obtain. Therefore, the DSHNHL collection of patient data and pretreatment samples provides a unique opportunity to assess these correlations.For the IL10 gene, different promoter gene variations have been associated with the clinical course of lymphoma patients [2, 5]. We could recently show an association of far-distal IL10 promoter variations with the OS of lymphoma patients, however not in the subgroup of DLBCL patients [2]. Due to the absence of an association with IL10 polymorphisms in DLBCL patients, we focused here on other host immune factors with relation to DLBCL. During our study, our candidate gene approach was supported by observations made by other groups that polymorphisms within the chosen genes are associated with the risk of NHL development but may also reflect treatment response [4].To our knowledge, this study is the first analysis of nonsynonymous coding polymorphisms of the IL4R gene in DLBCL patients within a prospective multicenter clinical trial. We show that the I75V genetic variant of IL4R is associated with the survival in this cohort of DLBCL patients. Homozygous A-allele carriers reassembling the I75 variation have a better prognosis compared with the other genotypes. The multivariate analysis of the IL4R I75V variation revealed a 1.9-fold increased risk for carriers of V75 variant to have a poor prognosis (OS). This observed RR is comparable to those estimated for clinical parameters included in the IPI, the current standard risk stratification model for patients with NHL. As this model is based on clinical factors only, its power to describe the biological heterogeneity of DLBCL is limited. A refinement of this prognostic model will be achieved by the inclusion of biological risk factors. Other variations, such as IL4R S503P, IL4R Q576R, IL4 -524CT and IL13 -1069CT gene variations, had no impact on the survival of DLBCL patients in this exploratory study.The sIL4R serum level study shows a strong negative impact of levels >115 pg/ml. Therefore, we were interested in combining the sIL4R serum level in our analysis with IL4R I75V gene variations in order to further distinguish DLBCL patients. Patients with high levels of sIL4R and the unfavorable IL4R V75 variants had worse survival rates, whereas homozygous carriers of the I75 variant and low sIL4R serum levels had the best survival rates. With this combined analysis of serum sIL4R and IL4R I75V, for ∼22% of DLBCL patients (IL4R I75 and sIL4R ≤115 pg/ml), a good clinical outcome could be revealed, whereas for ∼16% of the patients (IL4R V75 and sIL4R >115 pg/ml), a significantly worse outcome is observed. However, in a multivariate IPI risk factor analysis, the effect of the IL4R I75V gene variation remained unchanged in contrast to the sIL4R serum level which is not an independent factor.Recently, Habermann et al. [14] provided evidence that the IL4R regulatory single nucleotide polymorphisms (SNP) (rs2057768) could be a predictor of long-time survival in DLBCL patients as part of a risk score combination of four cytokine SNPs. The T allele of this SNP is in linkage disequilibrium to the IL4R I75 variant (IL4RI75V-AA), both associated with better survival of the DLBCL patients [15]. This further supports our hypothesis that variations in the IL4R gene could be an important prognostic factor predicting the outcome of DLBCL patients. Nevertheless, the functional consequences of these genetic variations are not understood yet. Previous studies indicate that the nonsynonymous coding polymorphisms are likely to change IL4R functions in some in vitro assays, but the in vivo influence on the immune system has been controversially discussed [16–19]. The IL4R I75 variant seems to have a higher signal transduction capacity toward the V75 variant and was therefore referred to as ‘gain-of-function’ variant [16, 19]. Recently, a higher expression of the IL4R in the germinal center B-cell-like DLBCL subgroup was shown and distinct functions of IL4 in the DLBCL subgroups were proposed [20, 21]. Two publications based on gene expression profiling of DLBCL patients raised the possibility that an active immune response is favorable for DLBCL patients [22, 23]. Thus, the favorable impact of the IL4R I75 variant on DLBCL outcome may be related to a more efficient immune response capacity against the lymphoma cells by enhanced IL4R expression or signal transduction capacities of the IL4R.Only few studies have so far analyzed IL4R polymorphisms in patients with malignant diseases other than NHL. In a Japanese study on sporadic renal cell carcinoma, the IL4R I75V polymorphism was described as a useful genetic marker for assessing the risk of the tumor development and progression [24]. No increased risk for colorectal cancer but an increased risk for cervical cancer was found for the IL4R V75 allele [25, 26]. Cervical cancer is related to human papillomavirus infection and Th1 immune responses are essential for viral clearance. The IL4R I75V polymorphism was also related to other viral diseases like acquired immunodeficiency syndrome or Rous sarcoma virus bronchiolitis and thus may alter the Th1/Th2 balance [27, 28]. An altered host–tumor interaction by an imbalance of Th1/Th2 cytokine expression or an altered response to these cytokines may lead to harmful processes in DLBCL patients and thereby affect the clinical course in the way shown here for the IL4R I75V gene variation.In summary, we report here for the first time that the nonsynonymous IL4R gene variation I75V is associated with the treatment outcome of DLBCL patients. Additional studies will have to prove if the IL4R I75V gene variation will indeed become a prognostic factor for DLBCL outcome. The I75 variation showed a significant favorable survival prognosis compared with V75 variants. The incorporation of the serum level of sIL4R together with the IL4R I75V genotypes will give further insights into the clinical pathophysiology of DLBCL. 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profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory responseBlood20051051851186124.NakamuraEMegumiYKobayashiTGenetic polymorphisms of the interleukin-4 receptor alpha gene are associated with an increasing risk and a poor prognosis of sporadic renal cell carcinoma in a Japanese populationClin Cancer Res200282620262525.IvanssonELGustavssonIMMagnussonJJVariants of chemokine receptor 2 and interleukin 4 receptor, but not interleukin 10 or Fas ligand, increase risk of cervical cancerInt J Cancer20071212451245726.LandiSBottariFGemignaniFInterleukin-4 and interleukin-4 receptor polymorphisms and colorectal cancer riskEur J Cancer20074376276827.HoebeeBRietveldEBontLAssociation of severe respiratory syncytial virus bronchiolitis with interleukin-4 and interleukin-4 receptor alpha polymorphismsJ Infect Dis200318721128.SorianoALozanoFOlivaHPolymorphisms in the interleukin-4 receptor alpha chain gene influence susceptibility to HIV-1 infection and its progression to AIDSImmunogenetics20055764465429.HummelMBentinkSBergerHA biologic definition of Burkitt's lymphoma from transcriptional and genomic profilingN Engl J Med20063542419243030.PfreundschuhMSchubertJZiepertMSix versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60)Lancet Oncol20089105116 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0ACC48625EADFA36EF1D512306E2D0F99973F312.txt b/test/dataset/in/resources/corpus/Clean_0ACC48625EADFA36EF1D512306E2D0F99973F312.txt new file mode 100644 index 0000000..fcb7b4d --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0ACC48625EADFA36EF1D512306E2D0F99973F312.txt @@ -0,0 +1 @@ +]>MAD1973S0047-6374(97)00115-210.1016/S0047-6374(97)00115-2Elsevier Science Ireland LtdFig. 1The effect of EGF on [3H]thymidine incorporation. The serum-starved subconfluent cell monolayers in Falcon 24-well plates were exposed for 24 h to indicated concentrations of EGF in the presence of 2.5% FCS. The cells were labeled with [3H]thymidine for the last 6 h of culture and the radioactivity and DNA content determined as described in Section 2. The vertical columns are the means of four cultures and vertical bars indicate one standard deviation. All results are highly significant (Student `t' test) comparing experimental and the corresponding control (no EGF) culture.Fig. 2The effect of FCS, or FCS plus EGF on [3H]thymidine incorporation. The experimental conditions were as in the legend of Fig. 1. The [3H]thymidine incorporation and DNA content were determined 24 h after the cells have been exposed to the indicated concentrations of FCS or FCS plus 2.5 ng/ml EGF. Vertical columns are the means of four cultures and vertical bars indicate one standard deviation. The figures within columns are the ratios of the means: EGF-treated over the corresponding EGF-untreated cultures.Fig. 3The effect of FCS or FCS plus EGF on cell proliferation. The experimetal conditions were as in the legend of Fig. 1. The DNA content was measured 5 days after exposure of cells to the indicated concentrations of FCS or FCS plus 2.5 ng/ml EGF. Vertical columns are the means of six cultures and vertical bars indicate one standard deviation. The figures within columns are ratios of the means: EGF-treated over the corresponding EGF-untreated cultures.Fig. 4The time-course of the effect of EGF on [3H]thymidine incorporation. The experimental conditions were as in the legend of Fig. 1. The serum-starved cells were incubated for the indicated periods with 2.5% FCS, or FCS plus 2.5 ng/ml EGF and labelled with [3H]thymidine for the last 2 h in culture. The results are the means of four cultures. Standard deviations are not indicated for the sake of clarity, but the differencies found for EGF-treated and EGF-untreated cultures are significant (Student `t' test) at 15 h and later times of incubation.Fig. 5Time course of [125I]EGF binding at room temperature. 90% confluent monolayers were incubated for 24 h in medium containing 0.2% FCS, then for 1 h at 37°C with PBS containin 0.2% BSA. The binding was performed at room temperature in the above binding buffer containing [125I]hrEGF (60 000 cpm in 1 ml) for the indicated times. After careful rinsing, the cells were lysed and the total bound radioactivity was determined with an LKB gamma counter. The results are means of 4 cultures. The standard deviations are not indicated for the sake of clarity.Fig. 6Receptor saturation curve and Scatchard analysis. Cells were incubated for 2 h with increasing concentrations of [125I]EGF as described in the legend of Fig. 5. The cells were carefully rinsed, and the total bound radioactivity was determined. Specific binding was determined by subtracting the binding measured in the presence of a 200-fold excess of unlabelled EGF from the total binding values. Inset shows the Scatchard plots of the binding data for AC in 1- and 14-month old rats.Fig. 7Autoradiographs of RT-PCR products amplified with oligonucleotide primers for EGF, EGF-receptor and GAPDH. Amplified PCR products were separated and analyzed as described in Section 2. They were hybridized with the corresponding oligonucleotide probe labelled with γ32P-ATP. The hybrids were revealed by autoradiography and films were scanned. 1 and 2: 1-month old; 3 and 4: 8-month old; 5 and 6: 14-month old rats. Upper row shows hybrids of gene-specific cDNA coding for EGF-proteins; third row shows hybrids of gene specific cDNA coding for EGF-receptor protein. Lanes 1, 3 and 5: the cells were not stimulated with EGF prior to extraction of RNA. Lanes 2, 4 and 6: the cells were stimulated for 24 h with 2.5 ng/ml EGF prior to extraction of RNA. GAPDH was used as an internal standard for amplification protocol.Fig. 8Graphic representation of the scans in Fig. 7. Columns represent the relative density of the bands expresed as per cent of the corresponding GAPDH band density. (A) Scans of the EGF, upper row in Fig. 7. (B) Scans of the EGF-receptor, third row in Fig. 7.Table 1(125I)EGF-receptor binding data*1-month old8-month old14-month oldKd (nM)0.180.120.07Bmax (M/μg DNA)5.04×10−119.21×10−122.61×10−12No. of receptors37 80016 7501950*The data are obtained by computer analysis of the results shown in Fig. 6.Age-related decrease in the responsiveness of rat articular chondrocytes to EGF is associated with diminished number and affinity for the ligand of cell surface binding sitesDidierRibaultMessaiHabibKhatibAbdel-MajidAlainBarbaraDragoslavMitrovic*INSERM U-349, 6 rue Guy Patin, 70510 Paris, France*Corresponding author. Tel.: +33 1 49956447; fax: +33 1 49958452.AbstractThe effect of age on the responsiveness of articular chondrocytes (AC) to epidermal growth factor (EGF) was examined. Cells were isolated by digesting cartilage fragments from the humeral and femoral heads of 21-day old, 8- and 14-month old rats with collagenase. The cells were cultured under standard conditions, as monolayers. DNA synthesis was measured by [3H]thymidine incorporation and cell proliferation by the DNA content of subconfluent cultures. [125I]EGF binding and the amounts of EGF and EGF-receptor mRNAs were determined using confluent cells. DNA synthesis was decreased with age of animals. EGF stimulated DNA synthesis in cultures in 1- and 8-month old rats at low serum concentrations (<5%), and in cultures in 14-month old animals at high serum concentrations. It also increased 5-day DNA content of cultures compared to serum-treated controls but this effect was weak in cultures in 14-month old rats. The number of high affinity binding sites for [125I]EGF decreased from 37 800 in the 1-month old to 1950 in the 14-month old rat AC. The apparent dissociation constant (Kd) also decreased with age: 0.18 nmol/l in the 1-month old; 0.12 nmol/l in the 8-month old; and 0.07 nmol/l in the 14-month old cells. AC in older rats contained more EGF mRNA and less EGF-receptor mRNA. Incubation of the cells with EGF resulted in down regulation of the EGF- and upregulation of EGF-receptor mRNA expressions. These findings show the age-related quantitative and qualitative alterations in EGF and EGF-receptor which may account, at least in part, for the diminished responsiveness of senescent AC to EGF.KeywordsAgingEpidermal growth factorRat articular chondrocytesDNA synthesisEGF-receptorEGF- and EGF-receptor mRNAs expressions1IntroductionCellular senescence in culture is associated with a gradual decline in mitotic activity, in which the number of population doublings is inversely proportional to the age of a donor (Hayflick and Moorhead 1961, Hayflick 1965, Martin et al. 1970, Schneider and Mitsui 1976, Golstein et al. 1978). This is usually interpreted as an expression of cellular aging (Norwood et al., 1990). The decline in the replicative capacity of senescent cells is usually attributed to the gradual increase in the fraction of cells arrested in the nonreplicative phase, near the G1/S boundary of the cell cycle, just before the onset of DNA synthesis (Rittling et al. 1986, Cristofallo 1988, Afshari et al. 1993). The failure of senescent cells to initiate DNA synthesis and replicate is associated with a diminished responsiveness to mitogenic stimuli such as growth factors including the epidermal growth factor (EGF) (Phillips et al. 1984, Ishigami et al. 1993, Tang et al. 1994, Li et al. 1995).There is good experimental evidence indicating that cellular senescence is controlled by an active dominant genetic programme (Rittling et al. 1986, Hensler and Pereira-Smith 1995, Goldstein 1990, Ning and Pereira-Smith 1991, Goletz et al. 1993, Doggett et al. 1992, Pang and Chen 1994) and that senescent cells no longer initiate the transcription of certain growth regulatory genes such as c-fos proto-oncogene (Seshadri and Campisi, 1990). Although, changes underlying the cellular senescence are still poorly understood, a good deal of evidence points to differences in gene expression in young and senescent cells (Rittling et al. 1986, Li et al. 1995, Doggett et al. 1992, Dereventzi et al. 1996, Tahara et al. 1995).EGF is a potent mitogen for many cells including chondrocytes (Vivien et al. 1990, Kinoshita et al. 1992, Ribault et al. 1997). This 53 amino acid polypeptide with a molecular weight of 6045 Da exerts its biological effects, only after it is bound to specific cell surface receptors. Once it is bound to its receptor, it activates the receptor's intracellular tyrosine kinase domain resulting in a phosphorylation of tyrosine residues and in the generation of a signal which initiates DNA synthesis, cell cycle progression, and cellular proliferation (Carpenter and Wahl, 1990). EGF extends the replicative life span and increases the saturation density of human diploid fibroblasts in vitro (Carpenter and Cohen 1976, Kaji and Matsuo 1983), but these effects decrease with cellular ageing in vitro. The unresponsiveness of senescent cells to EGF does not seem to be associated with quantitative or qualitative changes in the EGF-receptors (Phillips et al. 1984, Ishigami et al. 1993) or to EGF-induced activation of the receptor's tyrosine kinase domain (Gerhard et al., 1991). However, Tang et al. (1994)recently reported a decrease in EGF receptor mRNA expression in fibroblasts that had attained their maximal life span.EGF has occasionally been reported to have a mitogenic action in chondrocytes (Vivien et al. 1990, Kinoshita et al. 1992, Ribault et al. 1997), and these cells bear specific high and low affinity receptors (Kinoshita et al. 1992, Ribault et al. 1997). Although, to our knowledge, the effect of EGF on chondrocytes derived from young, mature and old donors has not been studied. We have therefore examined the effects to EGF on articular chondrocytes (AC), isolated from young, mature and old rats and measured its mitogenic action, the presence of specific binding sites, and the expression of EGF- and EGF receptor-specific mRNA.2Materials and methods2.1Cell cultureRat ACs were isolated from the humeral and femoral heads of 21-day old, 8- and 14-month old male Wistar rats (Charles River, France) (Ribault et al., 1997). Briefly, cartilage slices were digested at 37°C for 10 min (young) or 5 min (old) with 0.05% trypsin-3 mM EDTA solution (Eurobio, France) with gentle shaking to remove contaminant cells and cell debris. The chondrocytes were then released by incubation for 1–2 h (1-month old rats) and 10 min (older animals) with 0.1% bacterial collagenase (Type IA, Sigma, St. Louis, MO). The released cells were sedimented and washed by centrifugation (5 min at 450×g) with DMEM nutrient medium. The cells were seeded in 25 cm2 sterile plastic flasks at 2×106 cells and cultured under standard conditions (37°C, 95% humidified atmosphere, 5% CO2) in DMEM nutrient medium containing serum and antibiotics (100 IU/ml penicillin and 50 mg/ml streptomycin). The cells in young rats were cultured in the medium containing 10% fetal calf serum (FCS), and cells in old animals in serum containing 10% FCS and 10% horse serum.The confluent cells, 4–5 days after seeding, were detached by gentle shaking for 5–10 min with trypsin-EDTA solution. The isolated cells were collected and washed by centrifugation, and the suspended cells seeded in 24-well Falcon culture plates at 2×104 cells per well, or in culture flasks at 1×104 cells per cm2. First passage cells were used in all experiments. The culture medium was changed every 3–4 days.2.2DNA synthesis and cell proliferationDNA synthesis was monitored by measuring the incorporation of [3H]thymidine into nuclear DNA, and cell proliferation by measuring the DNA content (West et al., 1985) of the cultures. The cells were seeded and cultured under standard conditions in 24-well plates; the medium was replaced with DMEM containing 0.2% FCS on the 3rd day after seeding. This medium was removed the next day and cells were incubated for 24 h in medium containing 2.5% FCS and (except controls) indicated concentrations of human recombinant EGF (Genzyme). [3H]thymidine (1 μCi/ml, NEN) was added for the last 6 h of incubation.The labelled cell monolayers were washed with PBS, fixed in cold 5% TCA (2×15 min), then in cold 80% ethanol and lysed at 37°C for 2 h in 400 μl 10 mM EDTA, pH 12.3. The alkaline lysates were neutralized with 20 μl 1 M KH2PO4 and 200 μl samples, were counted in a liquid scintillation counter (LKB Wallac 1409). The neutralized alkaline digests were also used to measure the DNA content of the cultures. The results are expressed as the means of at least four cultures±1 S.D. and were compared with controls using Student's `t' test.2.3[125]EGF bindingBinding studies were performed using 90% confluent AC cultured in 24-well plates (Ribault et al., 1997). In order to block the non-specific binding sites, serum-starved cell monolayers were pre-incubated for 1 h at 37°C degree in PBS plus 0.2% bovine serum albumin (BSA) and then 60 000 cpm of the human recombinant [125I]EGF (900 Ci/mmol, Amersham) were added per ml of the binding buffer. Preliminary experiments showed that [125I]EGF binding reached a plateau in less than 2 h at room temperature. Cells were then washed three times with the binding buffer, lysed with 1 ml 1 N NaOH, and the lysates counted in an LKB γ-counter.Saturation curves were obtained at room temperature by incubating cell monolayers for 2 h in the binding buffer containing increasing concentrations of [125I]EGF. Displacing curves were obtained similarly in the presence of [125I]EGF (60 000 cpm/ml of binding buffer) and increasing concentrations of unlabelled EGF. Non-specific binding was determined by subtracting the [125I]EGF binding obtained in the presence of 200-fold excess of unlabelled EGF from the corresponding total binding data.2.4RNA isolationTotal cellular RNA was isolated from 90% confluent rat AC monolayers subcultured in 75 cm2 flasks. The serum-starved cells were incubated for 24 h without or with 2.5 ng/ml EGF in the presence of 2.5% FCS as described above for DNA synthesis prior to RNA extraction. The cell monolayers were then rinsed with PBS and total RNA isolated using the Rapid RNA™ purification kit (Amresco, USA). RNA pellets were collected by centrifugation (10 min, 4°C, 15 000×g), air dried, and resuspended in 100 μl sterile diethylpyrocarbonate-treated water. The concentration and purity of the total cellular RNA were determined by spectrophotometry at 260, 280 and 230 nm.2.5Oligonucleotide primer design and RT-PCRThe oligonucleotide primers used for RT-PCR were designed from published cDNA sequences Saggi et al. 1992, Avivi et al. 1991, Tso et al. 1985and were synthesized by Genosis Biotechnologies (England). The sense and antisense primers in the 5′, 3′ direction were: for EGF, sense position 3033–3056 (GCTCAGACTGTCCTCCCACCTCG), antisense position 3482–3460 (CCCGTAGTCAGCATGGCGCAGC) (Saggi et al., 1992); for EGF-receptor sense position 1260–1282 (GGACCTTCACATCCTGCCAGTG), antisense position 1870–1848 (GGTGATGTTCATGGCCTGGGGC) (Avivi et al., 1991); for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense position 260–280 (TGCTGGTGCTGAGTATGTCG), anti-sense position 907–888 (ATTGAGAGCAATGCCAGCC) (Tso et al., 1985). These gave PCR products of 449 base pairs for EGF, 610 base pairs for EGF-receptor and 646 base pairs for GAPDH. Reverse transcription and PCR amplification were performed as described elsewhere (Ribault et al., 1997) with modifications. In the preliminary experiments, it was established that linear concentration-related RT-PCR amplifications were achieved with 2.5 μg RNA and 30 cycles for EGF, with 1.5 μg RNA and 25 cycles for EGF-receptor. GAPDH was amplified under the same conditions as each of the above genes. Genomic contamination was checked by monitoring amplification in the absence of MMLV-reverse transcriptase after digestion of 4 μg RNA by RNAase A (10 mg/ml) for 1 h at 37°C.2.6Analysis of RT-PCR productsThe PCR products were analyzed by agarose (2%) gel electrophoresis in Tris–borate–EDTA buffer (89.2 mM Tris, 89 mM boric acid, 0.05 mM EDTA pH 8.3) containing 0.1 μg/ml ethidium bromide, for 1 h at 20°C at 10 V/cm. The DNA bands, stained with ethidium bromide, were visualized and photographed under UV light using positive/negative instant film (Polaroid Kodak). DNA molecular weight markers (marker VI from Boehringer) were used to determine the sizes of the amplified products.All PCR products were transferred to nylon sheets and hybridized with synthetic oligonucleotide probes labelled with γ32P-ATP (10 mCi/ml, NEN) by T4 polynucleotide kinase (ATGC, USA). These probes were designed from published sequences and recognized a unique sequence located between the PCR primer templates. The sequences of oligonucleotides were: for EGF (5′GCCCATCAGCACCTGGACTGCC3′), spanning nucleotides 3227 and 3249; for EGF-receptor (5′GTGAGCAGAGGCAGGGAGTGCG3′), spanning nucleotides 1744 and1766; and for GAPDH (5′CATGGACTGTGGTCATGAGC3′), spanning nucleotides 512 and 532. The hybrids were revealed by autoradiography using Hyper film MP (Amersham). The films and membranes were exposed for 1–2 h at −80°C and then processed routinely. The radioactive bands wee analyzed by scanning densitometry.3Experimental resultsAt serum concentration of 2.5%, EGF induced a concentration-dependent increase in [3H]thymidine incorporation by AC in all three age groups of rats (Fig. 1). The AC in 1-month old animals responded much better than cells in 8-month old and 8-month old animals responded much better than cells in 14-month old animals. The effect was highly significant at 0.25 ng/ml of EGF. In all cultures maximal stimulation was attained with 1 ng/ml, but the effect decreased significantly at 10 ng/ml in cultures in 1- and 8-month old rats. The response of AC in young and old rats also differed with respect to FCS concentration (Fig. 2). AC in 1-month old rats responded to EGF maximally at low serum concentration (<2.5%). The effect decreased gradually at higher serum concentration (≥5%) and completely disappeared at 10% FCS (Fig. 2a). The cells in 8-month old animals responded better at low serum concentration and were relatively insensitive at high serum concentration (Fig. 2b). In contrast, AC in 14-month old rats were poorly stimulated by EGF at low serum concentration and responded much better to the factor at higher (≥2.5%) serum concentration (Fig. 2c). The effects of FCS or FCS plus 2.5 ng/ml EGF on cell proliferation are shown in Fig. 3. The DNA content of all cultures increased as a function of serum concentration, but much more in cultures in 1-month old animals. The exposure of cells for 5 days to 2.5 ng/ml EGF, resulted in an increase of the DNA content which was consistently higher than in cultures lacking EGF. FCS (1%) increased the DNA content of cultures in 1-month old rats by a factor of 1.83, whereas similar increase was achieved at 5% FCS in a 8-month old cultures. In the range of serum concentrations tested, DNA content of cultures in 14-month old rats was much less affected by EGF. Growth-arrested AC incubated with FCS or FCS and EGF, did not incorporate [3H]thymidine during the 12–15 h after the stimulus had been given (Fig. 4). Only a small fraction of cells initiated DNA synthesis at 15 h and a somewhat larger fraction after 18 h of stimulation. Again, a significantly larger proportion of AC in 1-month old rats incorporated [3H]thymidine at and after 15 h of stimulation compared to the cells derived from old rats.The time course of [125I]EGF binding at 20°C to cell monolayers is shown in Fig. 5. There was much less [125I]EGF binding per μg DNA by AC in 14-month old rats, although binding (plateau) was saturated earlier (45 min) than for the younger cells (120 min). An incubation time of 2 h was therefore used in subsequent binding experiments.Total, specific and non-specific bindings and the corresponding Scatchard plots are shown in Fig. 6. The specific binding was more than 80% of the total binding in all three types of culture and Scatchard plot analysis of the binding data indicated the presence of a single class of high affinity binding sites, only. Table 1 summarizes the binding data as obtained by computer analysis. As it is apparent, the binding affinity (Kd) decreased with age of cells from 0.18 nM for the 1-month old to 0.07 nM for the 14-month old cells. The maximal binding (Bmax) also decreased from 5.04×10−11–2.61×10−12 mol/μg DNA for the AC in old rats. There were 37 800, 8900 and 1950 binding sites per cell for cultures derived from 1-, 8- and 14-month old rats, respectively.The mRNAs for EGF and the EGF-receptor in unstimulated and EGF-stimulated rat AC were revealed by RT-PCR of the total extracted RNA. The amplified cDNA products were separated by agarose gel electrophoresis and stained with ethidium bromide. The products migrated in the gel at the positions predicted from the respective cDNA sequences 449 bp for EGF, and 610 bp for the EGF-receptor.The PCR products were transferred to nylon membranes and hybridized with γ32P-labelled specific oligonucleotides. cDNA fragments of 449 and 610 bp interacted with the respective probes (Fig. 7). It is apparent that AC in 8- (lanes 3 and 4) and 14- (lanes 5 and 6) month old rats contain more EGF mRNA and less EGF-receptor mRNA (lanes 1, 3 and 5). EGF stimulation also decreased the amount of mRNA transcripts specific for EGF, and increased that of EGF-receptor mRNA in all three types of cells (lanes 2, 4 and 6). Fig. 8 shows densitometric semi-quantitative analysis of the scans of the Fig. 7.4Discussion and conclusionsThese results show several age-related alterations in chondrocytes derived from the articular cartilage of adult rats. The cells from mature and aged rats have a decreased proliferative capacity and responsiveness to EGF. The concentration of EGF needed to stimulate DNA synthesis was about the same for young and old cells, but considerably less DNA was synthesized by old cells. EGF initiated DNA synthesis more effectively in young cells at a low serum concentration, but worked best at a higher serum concentration in old cells. EGF increased the DNA content of cultures at all the serum concentrations studied. The induction of DNA synthesis following growth arrest starts approximately 18 h after the cells have been challenged with FCS or FCS plus EGF. EGF does not shorten this time lag but it does increase the number of cells that initiate DNA synthesis. Assuming that each dividing cell incorporates the same amount of [3H]thymidine, the fraction of cells that enter the S phase of the cell cycle is always much greater in cultures from young animals and in those exposed to EGF.It has been reported that the responsiveness of cells to growth factors, including EGF, and the number of cell divisions in vitro diminish with the age of the cell donor (Gospodarowicz and Meschern 1977, Dominice et al. 1986, Chen et al. 1990, Colige et al. 1990, Matsuda et al. 1992, Ishigami et al. 1993, Tang et al. 1994, Li et al. 1995). The diminished proliferative capacity of senescent cells is associated with a gradual loss of cell responsiveness to growth factors like EGF. This could be ascribed to quantitative and/or qualitative changes in specific receptors, but the published findings on this point are not unanimous. Some studies found no significant age-related changes in the density or binding affinity of EGF-specific receptor (Phillips et al. 1983, Ishigami et al. 1993). However, other studies have reported fewer EGF-receptors in cells from older donors (Carlin et al. 1983, Matsuda et al. 1992, Marti 1993).Our results also indicate that a decrease in the number and binding affinity of EGF receptors can account for the lower responsiveness of senescent rat AC to EGF. The number of EGF receptors and their binding affinity were dramatically decreased in cells isolated from old rats. The number of binding sites dropped from 37 800 in cells in 1-month old to 1950 in those in 14-month old rats. The Kd value decreased similarly from 0.18 to 0.07 nmol/l.The reasons for these discrepancies are not known, but differences in the methods used may be important. It is intriguing that no evidence for receptor alteration have been reported in studies of cell senescence in vitro (Carlin et al. 1983, Gerhard et al. 1991, Ishigami et al. 1993), whereas alteration are seen in cells derived from donors of different ages (Matsuda et al. 1992, Marti 1993). However, the lower responsiveness of senescent AC to growth factors may also be associated with an increased number and unchanged affinity for the ligand of the specific binding sites as we have obtained for insulin like growth factor 1 (not shown).In agreement with the binding study results, there were also age-related differences in the EGF and EGF-receptor mRNA expressions. Decreased EGF-receptor mRNA expression in cells from older animals supports the binding data. Surprisingly, we also found that the amount of EGF mRNA is increased in old cells and that EGF modulates the expression of both EGF and EGF-receptor mRNAs. The modulation of the expression of the EGF-receptor mRNA by EGF is observed in several studies, independently of age (Clark et al., 1985). The significance of these findings is unclear. The feed-back mechanism could be put forward to explain an over expression of EGF mRNA in senescent cells where the number of receptors also decreases. Many different mechanisms could alter the amounts of receptor in senescent cells. For instance, mRNA stability, translational efficiency, rates of synthesis, post-translational modifications and the turnover of the EGF-receptor. Several other abnormalities are associated with cellular aging, in addition to the changes in the receptor density and affinity. There is transcriptional repression of several growth regulatory genes such as c-fos in senescent human fibroblasts (Seshadri and Campisi, 1990), gas-1 and gas-6 in senescent murine fibroblasts Cowled et al., 1994, a post-transcriptional block of proliferative cell nuclear antigen (PCNA) (Chang et al., 1991) and a translational defect in ornithine decarboxylase (Seshadri and Campisi, 1990). Other genes, which products inhibit cell proliferation, are in contrast expressed or over expressed in senescent cells. 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Biochem.1471985289295 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0ADBDEAB01620EB127CB75FD828702BEBC50D45E.txt b/test/dataset/in/resources/corpus/Clean_0ADBDEAB01620EB127CB75FD828702BEBC50D45E.txt new file mode 100644 index 0000000..a841373 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0ADBDEAB01620EB127CB75FD828702BEBC50D45E.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press P15210.1093/geronb/61.3.P152 Journal of Gerontology: Psychological Sciences Ambivalent Reactions in the Parent and Offspring Relationship Fingerman Karen L. Chen Pei-Chun Hay Elizabeth Cichy Kelly E. Lefkowitz Eva S. 1Child Development and Family Studies, Purdue University, West Lafayette, Indiana. 2Department of Sociology, Purdue University, West Lafayette, Indiana. 3Department of Psychology, University of Florida, Gainesville. 4Department of Human Development and Family Studies, Pennsylvania State University, University Park. 5 2006 61 3 P152 P160 19 10 2005 20 4 2005 The Gerontological Society of America 2006 Theory suggests that aging parents and their adult children experience ambivalence (conflicting emotions) as a result of unclear norms governing the tie between them. This study investigated personality differences and relationship context differences in ambivalence, as well as the reactions of parents and offspring to each other. As part of the Adult Family Study, 474 individuals from 158 family triads consisting of a mother, father, and son or daughter aged 22 to 49 years completed telephone interviews, in-person interviews, and questionnaires. Multilevel models revealed that poor parental health and neuroticism in parents and offspring were associated with greater ambivalence. Surprisingly, investment in competing roles was associated with less ambivalence. Parents also experienced greater ambivalence when offspring scored higher on neuroticism, rated the parent as less important, or were less invested in their own spousal role. Parents' characteristics were not associated with offspring's ambivalence. Parents appear to react to their children's personality and achievements even after children are grown. hwp-legacy-fpage P152 hwp-legacy-dochead RESEARCH ARTICLE IN THE United States, parents' relationships with their children under the age of 18 are guided by legal and social sanctions, but there are few norms governing relationships between adults and their parents. Recently, scholars have proposed that ambivalence, in the form of conflicting emotions and cognitions, may arise when social structures do not provide direction for family relationships (Connidis & McMullin, 2002; Luescher & Pillemer, 1998). Ambivalence can be conceptualized as (a) feeling conflicted or torn, or (b) having positive and negative sentiments toward the same object (Priester & Petty, 2001). The latter approach may be helpful in the study of intergenerational ambivalence. Prior social scientific research has tended to operationalize ambivalence as polarized feelings or cognitions (Bassili, 1996; Kaplan, 1972; Uchino, Holt-Lunstad, Uno, & Flinders, 2001; Weigert, 1991). Researchers take this approach because participants have a difficult time articulating the degree to which they feel torn or conflicted, but they can rate their actual positive and negative feelings (Luescher & Pillemer, 1998). Further, an examination of positive and negative feelings may extend researchers' knowledge of parent–offspring ties in important ways. Studies pertaining to intergenerational ties have tended to focus on either solidarity (Silverstein & Bengtson, 1997) or on conflict and tensions (Fingerman, 1996). Assessing ambivalence as positive and negative qualities may provide insights into the co-occurrence of tensions and positive feelings (see Fingerman, 2001; Willson, Shuey, & Elder, 2003). Initial theory and research pertaining to intergenerational ambivalence has been sociological in nature, focusing on how contradictory expectations that occur in status, roles, and institutions give rise to this ambivalence (Connidis & McMullin, 2002). For example, studies examining intergenerational ambivalence have focused on gender (e.g., women experience greater ambivalence; Willson et al., 2003), financial status (e.g., mothers experience more ambivalence toward financially dependent offspring; Pillemer & Suitor, 2002), and health (e.g., parents experience ambivalence over offspring's assistance; Spitze & Gallant, 2004). However, two individuals who occupy a similar status (e.g., gender) might experience varying degrees of ambivalence. Psychological factors may contribute to these individual differences because (a) these social structures may have different subjective meaning to different individuals, and (b) individuals may bring different predispositions to their relationships. In this study, we examine whether individuals' personality traits and their investment in the parent–offspring relationship contribute to intergenerational ambivalence. Further, we extend current knowledge by considering interpersonal processes and examining parents and offspring within the same family. Prior studies of ambivalence have included only the parent's reporting (Pillemer & Suitor, 2002; Spitze & Gallant, 2004) or the offspring's reporting (Willson et al., 2003) on intergenerational ambivalence. However, individuals react to features of social partners, such as their age (Erber, Szuchman, & Prager, 2001) and their personality characteristics (Gotlib & Beatty, 1985). Thus, an individual's experience of intergenerational ambivalence may also reflect characteristics of the parent or offspring partner. Subjective Investment in the Relationship The value that parents and offspring place on their relationship sets a psychological context for their emotional reactions toward one another. Role centrality theory posits that when individuals identify a particular role as self-defining, events in that role affect well-being to a greater extent than events in less salient roles (Gurin, Veroff, & Feld, 1960; Martire, Stephens, & Townsend 2000). Further, individuals who highly value a role may experience heightened positive and negative emotional reactions in that role. Appraisal theories of emotions (e.g., Lazarus, 1991) also suggest that individuals experience intense emotional reactions in situations that they consider to be personally relevant. Thus, these theories suggest that parents and offspring who greatly value one another may experience more ambivalence in their relationship. Indeed, across cultures, people report problems in their closest social ties (Akiyama, Antonucci, Takahashi, & Langfahl, 2003), and in other research we found that individuals aged 13 to 99 years were more likely to classify closer social partners as ambivalent than less close social partners (Fingerman, Hay, & Birditt, 2004). In addition, we anticipated that adults who are strongly invested in multiple roles (e.g., worker, spouse) would experience greater ambivalence toward their parents or offspring. Investment in many roles may generate conflicting feelings because it is unclear how the parent–offspring tie fits in with the other roles. For example, adults who work long hours, have small children, and value their own spouses may feel torn in their efforts to connect with parents. Personality and Ambivalence Individuals may also hold predilections to experience positive or negative emotions in their relationships as a result of their personality traits. Evidence suggests that individuals' predispositions may contribute to problems in relationships (Atkinson & Violato, 1994; Gunthert, Cohen, & Armeli, 1999). We expected higher neuroticism to be associated with increased ambivalence between parents and offspring for two reasons. First, researchers have linked neuroticism and ambivalence in a variety of studies (Carver, 1997; Emmons & Colby, 1995; Kokkonen & Pulkkinen, 2001). Some researchers have suggested that ambivalence involves dysregulation of emotion (Kokkonen & Pulkkinen). For example, certain individuals may experience ambivalence in situations with few norms (such as the parent–offspring tie), because they have a difficult time regulating their reactions to these situations. Other researchers argue that individuals bring predispositions in both personality and relationship style to their ties, and these researchers find that ambivalent relationship style and neuroticism are linked (Carver). Second, measurement issues may contribute to associations between neuroticism and ambivalence. Individuals who score higher on measures of neuroticism are more likely to report negative sentiments in relationships than are individuals who score low on neuroticism. Parents and offspring tend to rate positive qualities of their relationships high (Rossi & Rossi, 1990; Umberson, 1992), and there is little measured variability in positive sentiments. Therefore, measured variability in ambivalence is likely to reflect variability in participants' ratings of negative sentiments. Thus, we expected to find associations between neuroticism and ambivalence toward parents or offspring in this study. Family Processes in Ambivalence Prior studies of intergenerational ambivalence have relied on reports from either parents or offspring (Fingerman et al., 2004; Pillemer & Suitor, 2002; Spitze & Gallant, 2004; Willson et al., 2003), but not on reports from parents and offspring concerning the same relationship. Nonetheless, studies suggest that parents and offspring respond to characteristics of the other party. For example, Willson and colleagues found that adults' ambivalence toward their parents depended on the parents' gender and health. Likewise, Pillemer and Suitor found that mothers' ambivalence varied as a function of their perceptions of offspring's needs. This study allowed us to examine parents' and offspring's reactions to the other party's psychological characteristics. Parents and offspring may react with greater ambivalence when the other party is higher in neuroticism or is less invested in their relationship. In addition, parents' and offspring's experience of ambivalence may be associated. In studies involving dyads of mothers and daughters, some mother–daughter pairs show greater positive and negative emotionality than do other pairs (Fingerman, 2001; Lefkowitz & Fingerman, 2003; Martini, Grusec, & Bernardini, 2001). These findings suggest that mothers and daughters may respond to one another's emotionality. It is unclear whether fathers and sons also respond to their social partners' ambivalence, but this study allowed us to examine dyadic patterns of ambivalence. Finally, we examined variability in ambivalence within dyads. Across studies, parents rate positive qualities of relationships with offspring more highly than do offspring (Bengtson & Kuypers, 1971; Rossi & Rossi, 1990; Shapiro, 2004). Nevertheless, parents may experience ambivalence toward offspring, despite positive feelings. Pillemer and Suitor (2002) found that over half of the mothers in their study experienced some ambivalence toward their offspring. Similarly, our prior research revealed that nearly half of middle-aged and young-old adults classified ties to offspring as ambivalent (Fingerman, Hay, et al., 2004). Nonetheless, we examined possible generational differences in ambivalence. Other Factors Associated With Ambivalence The basic premise of this study is that the experience of intergenerational ambivalence partially reflects the value that individuals place on this relationship (relative to other roles and relationships) and the predispositions that individuals bring to their relationships. Yet the experience of intergenerational ambivalence also reflects structural factors that may generate unclear norms and that also may be associated with the value that individuals place on the relationship. These factors include gender (women are more ambivalent than men), ethnicity (African American adults are more ambivalent than European American adults), parental health (caregivers experience ambivalence), and contact (the more frequent the contact, the greater the ambivalence; see Connidis & McMullin, 2002). We considered these variables here. Age differences in ambivalence also may be evident. Socioemotional selectivity theory posits that older adults experience fewer problems in their personal relationships than do younger adults due to selection of rewarding social partners and improvements in emotion regulation (Carstensen, Isaacowitz, & Charles, 1999; Fingerman, Hay, et al., 2004). We anticipated age differences with regard to ambivalence We considered several additional variables without generating hypotheses. For example, family size may be associated with qualities of intergenerational relationships; parents with more offspring may be less invested in any given offspring (Fingerman, 2001). Further, some studies find that when offspring have partners and children of their own, their ties to their parents are stronger (Fischer, 1981, 1986; Umberson, 1992), but other studies find no such effects (Fingerman, 2000; Suitor & Pillemer, 2000). We examined offspring's number of siblings, whether or not the offspring have children of their own, and both parties' marital status as control variables. Methods Participants Data are from The Adult Family Study (Fingerman, Lefkowitz, & Hay, 2004), which examines men and women aged 22 to 49 years, their mothers, and their fathers. The total sample included 213 families who participated in telephone interviews. This study examines 158 of those families (N = 474) who participated in face-to-face interviews and completed self-report questionnaires. These families did not differ from the larger sample on background or relationship characteristics. Participants resided in the Philadelphia Primary Metropolitan Statistical Area, encompassing five counties in southeastern Pennsylvania and four counties in New Jersey (Pennsylvania Data Center, 2004). We used a stratified sampling procedure to obtain distributions by offspring's age (aged 22–33 and 34–49), gender, and ethnic group (European American or African American). We identified potential participants by using telephone lists that targeted the offspring's age range, which we purchased from Genesys Corporation. Participants who had a listed address received a prenotification letter. We supplemented this approach to recruitment by using snowball and convenience sampling. In total, 86% of the participants were recruited by means of the Genesys list, 4% by snowball sampling, and 10% by convenience sampling. Recruitment techniques were evenly distributed by offspring's age, gender, and ethnicity. To ensure participation in the face-to-face interviews, we picked those parents and offspring who resided in separate households within 50 miles (80.45 km) of each other. No parents or offspring were engaged in caregiving relationships. Table 1 includes background information for offspring and parents. Participants rated themselves as being in relatively good health on a scale of 1 (poor) to 5 (excellent; see Idler & Kasl, 1991). Participants described their ethnicity as African American or European American, except for two parents who described their ethnicity as both Hispanic and African American. For simplicity, we grouped these individuals in the African American category. Procedures Each family member completed a telephone interview. Then offspring completed in-person videotaped interviews and self-report questionnaires separately with their mother and their father (158 complete families, N = 474). We counterbalanced in-person interviews, with mother interview first for half the sample, and father interview first for the other half. We derive the data in this study from the telephone interviews and written questionnaires. Throughout the study, offspring responded to questions concerning their mothers and their fathers, and each parent responded to questions about the target offspring. For the telephone interviews we used Computer Assisted Telephone Interview software, permitting the random order of administration of sections pertaining to mother and to father across offspring. Background information Participants provided their age, gender, and ethnicity during initial telephone screening. In the telephone interviews, participants also provided their education in years, marital status, and work information, and they rated their health. Relationship characteristics Offspring identified the persons they considered to be their mother and their father during screening. Most offspring selected their biological mother (n = 153) and biological father (n = 143). Remaining participants selected stepfathers (n = 12), stepmothers (n = 2), or adoptive parents (n = 3). Importance of relationship Participants rated the importance of the parent or offspring relative to other social partners, using 6 categories: 1 (most important person in your life), 2 (among the 3 most important), 3 (among the 6 most important), 4 (among the 10 most important), 5 (among the 20 most important), and 6 (less important than that). In prior studies (Fingerman, 2001), this item was associated with emotional qualities of relationships. We reverse coded this item, so that higher numbers equal greater importance of relationship. Investment in roles Using a modified version of the role centrality measure by Martire and colleagues (2000), we had participants rate the importance of their roles as parent, romantic partner or spouse, and worker on a scale from 1 (not at all important) to 10 (very important). We examined these items for each role separately (spouse, parent, and worker), and we also combined them to obtain a total score (total roles). In this sample, 112 offspring and 259 parents rated all three roles. Some individuals who were unemployed, retired, and on leave still rated the importance of the worker role. Theoretically, parents' investment in the parenting role could overlap with their ratings of the importance of the offspring in this study. Yet the measures appeared to be distinct (r =.07). This low correlation could reflect the fact that 90% of the parents had more than one child and their ratings of the parenting role included other children. To illustrate, one parent might greatly value the parenting role, but rate the target child as only within the top 10 people in his or her life (because the target child is one of many children); another parent might rate the parenting role as important and the child as important; and still another parent might rate the role as unimportant, but the child an important social partner. Neuroticism To assess neuroticism, or the general predisposition to experience negative feelings, participants completed the widely used 12-item Revised Eysenck Personality Questionnaire (Eysenck, Eysenck, & Barrett, 1985). Example yes–no items included “Are you often fed up?” and “Are your feelings easily hurt?” Coefficient alphas were α = 0.73 for both parents and offspring. Control Variables Family variables Offspring reported number of sisters and brothers, and parents reported number of children. Offspring also indicated the number of children they had. Frequency of contact Participants reported frequency of telephone or electronic contact, using a scale from 1 (every day) to 7 (less often than once a month). They also reported face-to-face contact on a scale from 1 (every day) to 6 (less than twice a year). The use of ordinal scales to assess contact minimizes the impact of extreme values on results (Dewit, Wister, & Burch, 1988; Greenwell & Bengtson, 1997). Most participants reported frequent face-to-face contact (62% of participants reported seeing the other party once a week or more often). Thus, in analyses, we used frequency of contact by phone or e-mail. Measurement of Ambivalence We assessed positive and negative feelings by using four items from prior studies of parent–offspring ties (Umberson, 1992; Willson et al., 2003). Positive feelings reflected the average ratings of two items, “How much does he or she make you feel loved and cared for?” and “How much does he or she understand you?” These were rated 1 (not at all) to 5 (a great deal), with α = 0.69. We assessed negative feelings with the average rating of two items, “How much does he or she criticize you?” and “How much does he or she make demands on you?” Here, α = 0.68. Social psychologists have derived several formulas for calculating ambivalence scores from ratings of contradictory feelings (see, e.g., Kaplan, 1972; Thompson, Zanna, & Griffin, 1995). As in prior studies of intergenerational ties (e.g., Willson et al., 2003), we used Griffin's Similarity and Intensity of Components formula (described in Thompson et al.) to calculate ambivalence as follows:Because the calculations may result in negative numbers, we then added a constant of 1.5. For example, when both average positive and negative scores were 5, we calculated ambivalence as [(5 + 5)/2 − 0] + 1.5, generating a high ambivalence score of 6.5. When the positive score was 5 and the negative score was 1, we calculated the ambivalence score as [(5 + 1)/2 − |5 − 1|] + 1.5 = 0.5, which is a very low ambivalence. This formula has several advantages: it takes into account both the presence and intensity of coexisting positive and negative sentiments, it correlates highly with other formulas for ambivalence, and is widely used in research (Thompson et al.). Table 2 presents means and standard deviations of the independent variables and the ambivalence scores. Analysis Strategy Bivariate associations Our preliminary analyses focused on bivariate associations between independent variables. Correlations between psychological variables (neuroticism, role investment, and importance of the relationship) were all less than.12. We then considered potential covariates. The inclusion of control variables not associated with the dependent variable may generate spurious significant associations between independent and dependent variables in analyses (Rovine, von Eye, & Wood, 1988; Weisberg, 1979). Offspring's number of siblings, having children of their own, participants' work status, hours spent working, and educational attainment were not associated with ambivalence scores; therefore, we did not consider these variables further. Multilevel models Mothers and fathers responded to questions about the target offspring, and offspring responded to questions about each parent. We used PROC Mixed in SAS to estimate multilevel models to account for nonindependence of parents' and offspring's responses in analyses (Littell, Milliken, Stroup, & Wolfinger, 1996; Singer, 1998). Multilevel models allowed us to treat family as an upper level unit, with parent, offspring, and relationship characteristics as lower level units. Ambivalence scores served as the dependent variable. Lower level social structural variables included participants' age, ethnicity, generation (parent or offspring), dyad (e.g., daughter–mother, son–mother), parental health, and frequency of contact with partner as control variables. By including dyad (mother–son) and participants' generational status (parent), we implicitly included participants' gender. Lower level psychological variables included importance of the relationship, investment in roles, and neuroticism. Because members of the same family might have correlated responses, we included a random term for family effect in the model. Results We started by examining whether individuals' psychological factors are associated with their ambivalence toward parents or offspring. Multilevel models (see Table 3) revealed that individuals who scored higher in neuroticism had higher ambivalence scores. Individuals who were highly invested in a number of roles (spouse, parent, and worker) had lower ambivalence scores. Further, when parents were in poorer health, parents and offspring had higher ambivalence scores. The family variance component was significant, supporting the use of mixed models. We estimated post hoc analyses to explore these findings. Because offspring have higher neuroticism scores on average than parents, we estimated the models separately for parents and offspring; significant effects for neuroticism emerged in both models. Further, to understand the association of neuroticism with ambivalence, we examined correlations between ambivalence scores and ratings of the negative dimension of the relationship (r =.88) and the positive dimension of the relationship (r = −.48). Participants in the lowest quartile of ambivalence scores had high positive scores (M = 4.70, SD = 0.41) and low negative scores (M = 1.16, SD = 0.23). Individuals in the highest quartile of ambivalence scores showed a mixture of positive scores (M = 3.52, SD = 0.73) and negative scores (M = 2.86, SD = 0.60). Thus, low ambivalence was synonymous with high positivity and low negativity. High ambivalence was associated with moderate positive and moderate negative feelings. To understand the association of parental health with ambivalence, we examined offspring's ambivalence scores for their healthier versus their less healthy parent. Mothers' and fathers' health ratings were the same in 47 families. For the remaining families, offspring's ambivalence scores were lower for their healthier parent, paired t(110) = 2.45, p <.05. We reestimated the multilevel models, examining ratings of the three roles separately. We treated individuals who did not have one of the roles (i.e., spouse, parent, or worker) as missing cases. In this analysis, neuroticism and parental health remained significant (as in Table 3), but only investment in the parental role was significantly associated with ambivalence in a negative manner. Role as worker and parent were not significantly associated with ambivalence. Within-family patterns Next, analyses focused on patterns within families. Correlations between parents' and offspring's ambivalence scores are presented in Table 4. Within-family patterns were evident in mothers' and offspring's ambivalence scores (r =.46) and mothers' and fathers' ambivalence scores (r =.29). Not surprisingly, offspring's ambivalence scores for the two parents also were correlated (r =.37). To further understand whether parents and offspring react to one another's psychological characteristics, we estimated multilevel models examining (a) parents' ambivalence scores predicted by offspring's characteristics and (b) offspring's ambivalence scores predicted by parents' characteristics. Associations between parents' and offspring's ratings on these variables were small, ranging from r =.12 to r =.18. Our analyses focused on participants' own psychological variables as well as those of their parent or offspring. We did not include structural variables, because there were strong associations between parents' and offspring's ages (r =.79) and ethnicity. Thus, the inclusion of partner's age or ethnicity on ambivalence would be redundant with parents' or offspring's own age and ethnicity examined in prior analyses (Table 3). In addition, we estimated analyses with the partner's gender, but there were no significant effects for gender; therefore, we do not include gender in the findings presented here. Table 5 presents analyses predicting parents' ambivalence from offspring's and parents' psychological variables. Offspring's total investment in roles was associated with parents' ambivalence; when offspring were more invested in other roles, parents were less ambivalent about the relationship. As well, parents' neuroticism was significantly associated with parental ambivalence, and offspring's neuroticism tended toward association with parental ambivalence (p <.10). We also examined ratings of the three roles separately. As one can see in Table 6, parents had lower ambivalence scores when their offspring rated them as more important in their social network as well as when offspring were more invested in the spousal role; parents had higher ambivalence scores when their offspring scored higher on neuroticism. To explore these findings, we estimated post hoc tests including possible moderator effects such as frequency of contact and parental neuroticism. These tests allowed us to examine issues such as whether having frequent contact with a more neurotic son or daughter generates greater ambivalence. These models were not significant. We also examined indicators of offspring's social achievement, such as years of education, marital status (married vs not married), work status (work for pay vs not work for pay), and presence of children, on parents' ambivalence. These models, too, were not significant. Models predicting offspring's ambivalence from parental characteristics did not reveal significant associations. We do not present these findings here. Discussion There is a large literature pertaining to intergenerational ties in the field of sociology (see Bengtson, 2001, for a review). Thus, much of what we know about relationships between parents and offspring pertains to social structural variables such as age, gender, and generational status (Rossi & Rossi, 1990; Shapiro, 2004; Silverstein & Bengtson, 1997). Findings from this study suggest that social structural variables may serve as marker variables for underlying psychological processes. For example, adults who occupy several roles (parent, spouse, and worker) experience less intergenerational ambivalence if they are highly invested in these roles. Further, individuals within a given social structure (e.g., gender) may vary in their experiences of ambivalence as a result of individual differences in psychological predispositions (e.g., neuroticism). In sum, although social structural contexts can contribute to intergenerational ambivalence through unclear norms, individuals' beliefs and feelings about those contexts and relationships influence their personal experience of intergenerational ambivalence. Psychological Underpinnings of Intergenerational Ambivalence In this study, we considered individuals' investment in competing roles, the importance of the parent–offspring relationship, and the personality trait of neuroticism. Investment in competing roles and the relative importance of the relationship provide information about the context in which intergenerational ambivalence arises; these variables assess individuals' representation of the primacy of this relationship within their lives. Neuroticism represents general predispositions for negative feelings, or psychological properties that individuals may bring to their relationships. Both types of psychological variables were associated with intergenerational ambivalence. Competing roles and relative importance of the relationship Psychological perceptions of the salience of parent or offspring contributed to the experience of ambivalence, but the findings contradicted expectations. We initially predicted that individuals who valued their roles as worker, parent, and spouse would feel more ambivalent in their efforts to retain connections with their parents or offspring. Instead, we found that investment in other roles was associated with lower ambivalence scores. Several theories shed light on these findings. In the caregiving literature, researchers have posited energy expansion with multiple roles (Martire et al., 2000). According to this perspective, individuals who value multiple roles experience increased well-being because rewards across roles complement one another. Thus, individuals who are busy in a variety of contexts may feel more positive about their parents or grown offspring and focus less attention on their faults. Findings are also consistent with theories suggesting that autonomous functioning in the parent–offspring tie deters ambivalence (Fingerman, 2001; Lang & Schütze, 2002; Pillemer & Suitor, 2005). When parents and offspring value other roles, they indicate that they have outlets independent of this relationship. These roles may represent arenas in which they manifest autonomy. Neuroticism and negativity Individuals' general predispositions toward negativity (i.e., neuroticism) contributed to intergenerational ambivalence. This finding is not surprising, given the literature suggesting that neuroticism contributes to poorer quality (Atkinson & Violato, 1994; Gunthert et al., 1999) and more ambivalent relationships (Carver, 1997). Further, parents experienced greater ambivalence when their offspring scored higher on neuroticism. The directionality of these effects may be complex, however, given the long history of the parent–offspring relationship. Parents and children may influence one another's personality when children are young and reside at home. Longitudinal research might investigate associations between poor-quality relationships in childhood and subsequent neuroticism in adulthood. In addition, low ambivalence involved the presence of positive sentiments and the absence of negative sentiments. High ambivalence involved moderate to high positive and negative feelings in this study. Ambivalence is not merely the presence of negative sentiments in this tie, but rather a mixture of sentiments. Nevertheless, measurement biases give the sense that ambivalent relationships are distinguished by the presence of negative emotions. It is difficult to assess variability in positive aspects of parent–offspring ties because adults lean toward high positive ratings of this tie. Indeed, elsewhere, approximately half of parents and offspring classified their ties as solely positive, whereas the other half classified their ties as both positive and problematic (i.e., ambivalent; Fingerman, Hay, et al., 2004). Generally negative parent–offspring relationships presumably exist, but individuals in these relationships may disband their ties in adulthood or refrain from participating in studies of intergenerational ties. Future research might focus on understanding negative feelings in ambivalent parent–offspring relationships. Parents' and Offspring's Characteristics and the Other Party's Ambivalence We assessed intergenerational ambivalence from the perspective of parents and offspring within the same family. This approach allowed us to consider associations between different parties' psychological variables and ambivalence. Findings pertaining to interpersonal processes raise as many questions as they answer. Within families, mothers and their offspring experience the greatest similarity of ambivalence, more so than mothers and fathers (who responded to the same target offspring). These findings suggest that positive and negative emotional contagion or communication between mothers and offspring is greater than that between fathers and offspring in adulthood, consistent with earlier stages in the relationship (Collins & Russell, 1991; Seiffge-Krenke, 1999). Furthermore, offspring's psychological characteristics may contribute to their parents' experience of ambivalence. Findings varied when we examined parental ambivalence as a function of offspring's total investment in roles versus when we examined the same model but included investment in each role separately. For offspring who rated all three roles (worker, spouse, and parent), parents experienced less ambivalence when their offspring valued the parent–offspring relationship more, valued their own role as a romantic partner more, and scored lower in neuroticism. These variables may represent distinct facets of the relationship. Elsewhere, we found that older mothers felt better about their relationships with their daughters when the daughters valued their relationships more (Fingerman, 2001), and findings are consistent in this study. Further, parents may experience less ambivalence when their children have an easy interaction style (i.e., low in neuroticism) and have achieved normative social markers of adulthood. Prior studies have also linked parental ambivalence with perceptions of their offspring's expected social achievements (e.g., Pillemer & Suitor, 2002). Parents may worry more about a troubled or neurotic offspring (Hay, 2004). Finally, an offspring's having a strong tie to a spouse may indicate that the offspring has achieved a normative marker of adulthood, a good marriage. Alternately, the offspring's strong tie to a spouse may generate better relationships with the parents because children-in-law affect family relationships (Fingerman, 2004). Nonetheless, the fact that the mothers' and fathers' ambivalence scores were only moderately correlated suggests that offspring's traits and accomplishments are not fully responsible for parental ambivalence. If offspring's characteristics fully accounted for parental ambivalence, we might expect both parents to respond in a more similar fashion. It is unclear why parental psychological characteristics were not associated with offspring's ambivalence. Offspring might be sensitive to other parental psychological factors that we did not examine here. For example, offspring may be sensitive to parental favoritism of siblings (Suitor & Pillemer, 2000) or parents' memories of the early relationship (Shaw, Krause, Chatters, Connell, & Ingersoll-Dayton, 2004). As we discuss next, offspring were sensitive to their parents' health status. Comparisons With Other Studies As in the study by Willson and colleagues (2003), we found that poorer parental health was associated with greater ambivalence. A recent study of parent–offspring relationships when parents experience physical declines in late life revealed similar findings (Fingerman, Hay, Kamp Dush, Cichy, & Hosterman, 2005); parents and offspring were more likely to describe their relationship as growing worse if parents incurred difficulties in daily functioning. Future studies should examine whether personality and investment in the relationship explain variability in individuals' reactions to parental aging. This study also raises questions about social structural variables examined elsewhere. We did not find the gender differences in ambivalence observed by Willson and associates (2003). Regional differences may help explain the distinct findings of the studies. Willson and associates used data from a longitudinal study of rural families who resided in Iowa in the 1980s when they entered the study. Other studies using these Iowa data have noted that farm families show gender distinctions, with patrilineal preferences (e.g., King & Elder, 1995). Participants in the present study resided in the Philadelphia area. The lack of gender differences is consistent with studies of intergenerational ties in urban areas (e.g., Logan & Spitze, 1996). In contexts where families show stronger differential treatment by gender (such as rural Iowa), ambivalence may vary by gender, but in contexts where differential treatment by gender is less strong (as in urban areas), gender differences may be less evident. In sum, this study suggests that psychological aspects of parent–offspring ties contribute to intergenerational ambivalence. Intergenerational ambivalence reflects predispositions that adults and parents bring to their relationship, the importance of this relationship relative to other aspects of their lives, and interpersonal processes between the two parties. Parents, in particular, may be influenced by their offspring's achievements and views of the relationship, even after the offspring are grown. Future research should further examine the effects of psychological variables on qualities of relationships between adults and their parents. Decision Editor: Thomas M. Hess, PhD Table 1. Characteristics of the Sample. Offspring(n = 158) Parents (n = 316) Means and standard deviations     Age 34.97 (7.28) 62.13 (9.06)     Education, years 15.05 (1.97) 14.08 (2.72)     Self-reported physical healtha 3.75 (0.85) 3.31 (0.97) Proportions     Gender         Women .52 .50     Ethnicity         African American .32 .32         European American .68 .68     Marital status         Married or remarried .64 .89         Widowed .00 .00         Divorced .09 .07         Single .21 .00         Cohabitating .06 .03     Work status         Working for pay .83 .54         Unemployed .05 .03         Homemaker or caregiver .07 .07         Student .03 .00         Retired .00 .33         Disability or on leave .01 .04 Note: Due to rounding errors, not all categories sum to 1.00. aRated 1 = poor, 2 = fair, 3 = good, 4 = very good, 5 = excellent. Table 2. Descriptive Information Pertaining to Independent and Dependent Variables. Variable Total (N = 474) Offspring (n = 158) Parents (n = 316) Dependent variable     Ambivalence 2.30 (1.10) 2.29 (1.13) 2.32 (1.07) Independent variable     Importance of target relationshipa 4.40 (0.88) 4.38 (0.90) 4.41 (0.87)     Neuroticismb 3.11 (2.58) 3.75 (2.66) 2.46 (2.32)     Total rolesc 23.99 (6.01) 23.31 (6.94) 24.68 (4.82)         Spousal role 8.92 (1.57) 9.25 (1.14) 8.62 (1.84)         Parental role 9.44 (1.23) 9.48 (1.40) 9.40 (1.09)         Worker role 8.11 (1.99) 8.10 (1.97) 8.13 (2.01) Note: Standard deviations are shown in parentheses. aInvestment in relationship: 1 = less important, 2 = among 20 most important, 3 = among 10 most important, 4 = among 6 most important, 5 = among 3 most important, 6 = most important. bThe possible range of neuroticism scores is 0 to 12. cThe sums ratings of three roles—parent, spouse, and worker: 1 = not at all important, 10 = very important. Table 3. Mixed Model Predicting Ambivalence Scores From Structural and Psychological Variables. Predictor B SE B t Intercept 2.99*** 0.55 5.40 Age  0.01 0.01 −0.88 Dyada     Mother son −0.16 0.11 −1.52     Father daughter −0.15 0.14 −1.04     Father son −0.02 0.14 −0.11     Mother daughter — — — Generationb −0.29 0.20 −1.51 Ethnicityc 0.23 0.13 1.79 Neuroticism 0.05** 0.02 0.76 Importance of relationshipd 0.04 0.05 2.59 Total investment in rolese −0.02* 0.01 −2.18 Parent's healthf −0.09* 0.04 −2.02 Contactg −0.01 0.04 1.18 Family variance component .30 z = 4.87***     Residual .86 Note: Estimated parameters 14, Akaike Information Criteria = 1,839.10. aThe reference group is mother–daughter dyad. bGeneration: 0 = parent, 1 = offspring. cEthnicity: 0 = European American, 1 = African American. dInvestment in the relationship: 1 = less important, 2 = among 20 most important, 3 = among 10 most important, 4 = among 6 most important, 5 = among 3 most important, 6 = most important. eRoles of being parent, spouse, and worker: 1 = not at all important, 10 = very important. fParent's health: 1 = poor, 2 = fair, 3 = good, 4 = very good, 5 = excellent. gContact with parent or offspring: 1 = every day, 2 = several times a week, 3 = once a week, 4 = every 1 to 2 weeks, 5 = every 2 to 4 weeks, 6 = once per month; 7 = less often. *p <.05; **p <.01; ***p <.001. Table 4. Correlations of Offspring and Parent Ambivalence. 1 2 3 4 1. Offspring ambivalence for mothers — 0.37** 0.46** 0.22** 2. Offspring ambivalence for fathers — 0.12 0.24** 3. Mother ambivalence for offspring — 0.29** 4. Father ambivalence for offspring — Ambivalence score, M (SD) 2.40 (1.19) 2.19 (1.07) 2.25 (1.15) 2.39 (0.98) **p <.01. Table 5. Mixed Models Predicting Parents' Ambivalence from Offspring's and Parents' Psychological Variables and Total Investment in Roles Scores. Predictor B SE B t Intercept 3.10*** 0.57 5.40 Offspring variables     Importance of relationshipa −0.09 0.07 −1.21     Neuroticism 0.04 0.03 1.69     Total rolesb −0.02* 0.01 −2.13 Parent variables     Importance of relationshipa −0.01 0.07 −0.09     Neuroticism 0.05* 0.03 2.10     Total rolesb −0.01 0.01 −0.49 Family variance component .31 z = 3.15**     Residual .82 Note: Estimated parameters 9, Akaike Information Criteria = 924.40. aInvestment in the relationship: 1 = less important, 2 = among 20 most important, 3 = among 10 most important, 4 = among 6 most important, 5 = among 3 most important, 6 = most important. bTotal score for investment in three roles—importance of being parent, spouse, and worker: 1 = not at all important, 10 = very important. *p <.05; **p <.01; ***p <.001. Table 6. Mixed Models Predicting Parents' Ambivalence From Offspring's and Parents' Psychological Variables and Separate Investment in Role Scores. Predictor B SE B t Intercept 5.27*** 1.23 4.28 Offspring variables     Importance of relationshipa −0.24* 0.11 −2.15     Neuroticism 0.08* 0.04 2.33     Parental roleb −0.03 0.07 −0.39     Worker roleb 0.04 0.05 0.71     Spouse roleb −0.19* 0.09 −2.22 Parent variables     Importance of relationshipa −0.06 0.09 −0.63     Neuroticism 0.06 0.04 1.57     Parental role b −0.10 0.07 −1.23     Worker role b 0.00 0.04 0.06     Spouse role b 0.06 0.04 1.42 Family variance component .35 z = 2.65**     Residual .78 Note: Estimated parameters 12, Akaike Information Criteria = 550.40. aInvestment in the relationship: 1 = less important, 2 = among 20 most important, 3 = among 10 most important, 4 = among 6 most important, 5 = among 3 most important, 6 = most important. bImportance of being parent, spouse, and worker: 1 = not at all important, 10 = very important. *p <.05; **p <.01; ***p <.001. This study was supported by Grant R01AG17916 from the National Institute of Aging, “Problems Between Parents and Offspring in Adulthood.” We appreciate the efforts of Ellin Spector, Carolyn Rahe, and Ann Shinefield, who managed the field study and data collection through the Institute for Survey Research at Temple University. We are grateful to Miriam Moss and Sheryl Potashnik for assistance with recruitment. Graciela Espinosa-Hernandez and Shelley Hosterman provided invaluable assistance on all aspects of this project. Lindsay Pitzer provided assistance with analyses and tables and read drafts of the manuscript. Lauren McIntyre, Michael Rovine, and Martin Gonzalo provided support for the statistical models. References Akiyama, H., Antonucci, T., Takahashi, K., Langfahl, E. S. (2003). Negative interactions in close relationships across the life span. Journal of Gerontology: Psychological Sciences, 58B,P70-P79. Atkinson, M., Violato, C. (1994). 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Journal of Marriage and the Family, 65,1055-1072. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0ADE8D8D8D4CB9F3AD5552BC618EFFE9D6B8A6EC.txt b/test/dataset/in/resources/corpus/Clean_0ADE8D8D8D4CB9F3AD5552BC618EFFE9D6B8A6EC.txt new file mode 100644 index 0000000..3c79906 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0ADE8D8D8D4CB9F3AD5552BC618EFFE9D6B8A6EC.txt @@ -0,0 +1 @@ +]>AMGP61071S1064-7481(12)61071-910.1097/00019442-199700510-00006American Association for Geriatric PsychiatryFIGURE 1Simplified model of the interactions between stress and adjustment, with factors that might influence the relationship (adapted, with permission, from Koenig et al.2)TABLE 1Correlation matrix of independent, mediating, and dependent variables (Pearson r)Life SatisfactionPsychological DistressDepressionMasterySocial SupportReligious CommitmentFunctional status0.170.160.020.23**−0.03−0.11Life satisfaction—0 49****0.49****0.51****0.50****0.30***Psychological distress——0.72****0.46****0,41****0.18*Depression———049***0.52****0.40****Mastery————0.40****0.38****0.05

10% P < 0.0005), again providing evidence for our model that sees social support as buffering the ill effects of physical disability on distress level. Mastery contributes further to the variance in psychological distress, but at a marginally significant level (2.8% P = 0.05). Taken together, physical disability, social support, and mastery accounted for 42% of the variance in psychological distress.Our third outcome variable, depression, was surprisingly unrelated to physical disability. Nevertheless, it was strongly related to mastery, which accounted for over 28% of the variance in depression. Also, social support and religious commitment added 10% and 5%, respectively, to the variance in depressive symptoms, suggesting that these variables along with a sense of mastery, may help buffer the adverse effects of stressors on mood state. Again, these three variables together accounted for almost 44% of the variance in depression.DISCUSSIONThese data lend empirical support to our proposed model, providing evidence for the importance of internal resources (mastery), external resources (social support), and coping (religious commitment) in buffering the negative effects of physical disability on psychological adjustment in later life. The results are also consistent with a substantial body of research that shows a relationship between physical disability and emotional distress,36–38 and they demonstrate important roles for social support,7–9 mastery,5,39 and coping behavior3,4,9 in mediating the relationship between psychosocial stress and adjustment.In this analysis, the stressor and buffering factors contributed measurably, and differentially, to mental outcomes. Life satisfaction, a broad measure, was only weakly associated with physical disability, but was strongly related to mastery and social support. Religious commitment was also a weak contributor to life satisfaction. Psychological distress, on the other hand, is a more specific mental health outcome than life satisfaction. As measured in this study, it includes anxiety and somatization. It had the strongest relationship with physical disability, our biopsychosocial stressor; both social support and mastery, on the other hand, were inversely related to psychological distress, as predicted by our model.The explanatory model for depression, the most specific mental health construct, was different from either life satisfaction or psychological distress. Like life satisfaction, depression was weakly correlated with physical disability, but strongly and inversely correlated with mastery and, to a lesser degree, social support and religious commitment. Clinically, depression is marked by a sense of helplessness, the opposite of mastery. Social supports are known to be perceived as unavailable or unreliable to depressed persons, although it is unclear whether the absence of social support makes one vulnerable to depression, or whether depression causes one to perceive existing social supports as unavailable, unreliable, or undeserved.7,27,31,33The inverse relationship between religious commitment and depression, independent of social support or mastery, is noteworthy. The early (and even more recent) psychiatric literature has portrayed religious commitment as a symptom of neurosis40 or evidence of psychological disturbance.41 This study adds weight to recent findings of an inverse relationship between religiousness and depression.16,42 Of course, given the cross-sectional nature of our data, as with social support, we cannot say whether greater religious commitment causes less depression or whether depression causes less religiousness. There are both longitudinal data16 and interventional data,43 however, that support the former idea.We recognize that assigning religious commitment as primarily a coping variable is somewhat arbitrary, because it may to some extent serve as an internal and external resource. These constructs, then, may not be entirely independent of each other. First, religious commitment may act as an internal resource by enhancing mastery and making persons less psychologically vulnerable to stressors. Strong religious commitment provides a belief system that may increase mastery by giving the individual a sense that he or she has more control over their situation; that is, if he or she believes in an all-powerful God who cares about him or her and will respond to prayers, then the person will feel that he or she is not powerless, but instead can take control over a situation by influencing God to act on his or her behalf. Second, religious commitment may enhance social support (external resource) by encouraging involvement in the religious community, which may broaden one's social support network (particularly among elderly persons, whose major voluntary social participation outside family is the church); furthermore, by encouraging people to support and care for others, religious beliefs (the “social gospel”) may help form a more supportive community, which helps to reduce the adverse effects of stress.Nevertheless, in our model, we see religious commitment as primarily a coping strategy because it influences how persons perceive and respond to stressors (i.e., cope), independent of internal and external resources. Indeed, our results would suggest that although religious commitment is positively correlated with both mastery (internal resource) and social support (external resource), there is still an independent effect for religion on life satisfaction and depression. Religious belief may facilitate coping directly by altering the perception of the stressor so that consequences are not seen as disastrous. For example, poor physical health may indicate to the older person that he or she is getting closer to death; if the elder believes that there is an afterlife where he or she will be reunited with family and life will improve, then the prospect of dying is not as terror-filled. Finally, religious commitment may facilitate adaptation by providing both cognitive (trusting God, turning problems over to God) and behavioral (praying to God, singing, participating in communal worship) tools for coping with distress.There are many factors accounting for the unexplained variance in our dependent variables (life satisfaction, general distress, depression). One is measurement error. For example, psychosocial variables, like religious commitment, social support, mastery, and the like, cannot be measured with the same degree of accuracy as physiological variables, like blood pressure or heart rate. Consequently, in mental health or social science research, the explained variance in outcome is seldom greater than 50%. Second, we did not measure other important internal resources (hereditary effects or genetic vulnerability) and external resources (health problems not affecting functional status) that could have contributed substantially to our mental health outcomes. Third, there may be factors that we are not yet aware of that affect mental health (for example, until recently religion was not thought to contribute to mental health in any meaningful way).Our study group is unique in several ways. Not only does the sample represent a narrowly defined group demographically and vocationally, but also, as the data suggest, there are other distinctions. Compared with several other studies using general, community-based populations,22–24 our sample of women appears more satisfied with life and less likely to report psychological distress. This observation is all the more striking given the higher prevalence of certain mental disorders, especially depression, in women.44,45 Although only a handful of other studies have looked at mental health outcomes in Catholic religious groups, virtually all have reported similar differences between lay and Catholic religious.46–48Moore46 and Kelly47 both concluded that mental illness was less common among women religious than in the general population. Kvale and colleagues48 studied morale among retired elderly nuns, and found that on nearly all measures, the sisters had higher morale than their lay counterparts. Our study confirms these findings. Despite the lack of spouses and children, and despite being retired from active employment, these women experienced better emotional well-being than women in the general population. However the mechanism explaining the better mental health functioning of this group remains unclear. The healthier outcome could be related to some shared characteristic, such as the overall high degree of religious commitment, the predictable and reliable financial situation, or the more ready access to health care and monitoring.Perhaps women religious are not as distinctive a group as one might assume, at least in terms of the impact of stress on mental well-being. Magee,49 studying 150 retired women religious, reported that the conditions contributing to the life satisfaction of older adults in general also pertained to his study population. Furthermore, many stressors encountered by Catholic religious professionals are similar to the stressors facing many other older Americans. These stressors include the potential for alienation from society and the personal problems they must handle to deal with the changing world. For example, Kennedy and colleagues50 reported that church reforms introduced role confusion, change in occupational focus, and ideological questioning among a group of Catholic priests they studied. Stressors such as these may contribute further to poorer mental health outcomes through the same mechanisms we described previously: loss of mastery and diminished social support.Despite the homogeneity of this sample, it did generate scores on religious commitment that were normally distributed, although still clustered at the high end of the scale (mean score, 27.3 ± 5.0 out of a possible 35; range: 10–35). Perhaps development of a more complex model of religious coping would be a useful approach in future research.17This is, of course, a unique sample of unmarried, celibate, elderly women who have devoted their lives to their religious vocation. Thus, in these and perhaps other unmeasurable ways, this sample is different from the general population of older adults in America. How these differences may have biased results is difficult to predict. Many women in their later years, however, do find themselves unmarried and celibate, living near or with each other, and often congregating in church. Furthermore, the fact that we found similar relationships in this sample to those of other researchers working with nongeriatric and elderly populations helps to dispel some of these concerns. Another potential bias is the lack of information about nonresponders. If nonresponders were selectively impaired in some way that made them refuse the study, such as having poor cognition, mental illness, or physical debility, our study sample may have been a self-selected healthier group. The relatively high response rate, however, adds confidence to the study findings.SUMMARY AND CONCLUSIONSWe have developed and tested a multidimensional stress and coping model in a selected and homogenous population of elderly Catholic women religious. Conceptualizing physical disability as a biopsychosocial stressor and life satisfaction, psychological distress, and depression as mental health outcomes, we found that internal (mastery), external (social support), and coping resources (religious commitment) predicted better mental health in this population. Our internal resource (sense of mastery) was the strongest single predictor of variance in two separate mental health outcomes, life satisfaction and depression, accounting for 27% for life satisfaction and 29% for depression. Higher level of perceived social support (external resource) was also significantly related to all three mental health outcomes. Finally, religious commitment, often unexamined in models of coping and adaptation, emerged as a positive predictor of life satisfaction and lower levels of depression. These findings further support the validity of a multidimensional coping model for adaptation to biopsychosocial stressors in late life.What implications do these findings have for clinicians caring for elderly patients? First, as in other studies, we found a link between disability and emotional distress. This finding should support close attention to the emotional adjustment of older persons experiencing severe disability from health problems. Optimizing physical functioning and the capacity for independent living must be among the first goals of the treating physician. Second, in counseling disabled elders with emotional disorder or those at risk for it, clinicians should pay particular attention to the patient's sense of helplessness or powerlessness about his or her situation. Discussing with patients the different aspects of their life that they can and cannot control may help to resolve some of these feelings. Third, it is important to encourage involvement in social activities that broaden the patient's support network, particularly activities with age-matched peers who may form a support group for them. Finally, clinicians should be encouraged to inquire about and support healthy religious beliefs and activities that may help older persons adapt to chronic illness, disability, and losses over which they have little or no control.51References1DGBlazerKJordanEpidemiology of psychiatric disorders and cognitive problems in the elderlyGLKlermanMMWeissmanPSAppelbaumSocial, Epidemiologic, and Legal Psychiatry1986JB LippincottNew York2612722HGKoenigMSmileyJAPGonzalesReligion, Health, and Aging: A Review and Theoretical Integration1988Westport, CTGreenwood Press3RLazarusSFolkmanStress, Appraisal, and Coping1984SpringerNew York4SFolkmanRSLazarusCoping as a mediator of emotionJ Pers Soc Psychol5419884664755SFolkmanRSLazarusRJGruenAppraisal, coping, health status, and psychological symptomsJ Pers Soc Psychol5019865715796SFolkmanRSLazarusSPimleyAge differences in stress and coping processesPsychol Aging219871711847DWRussellCECutronaSocial support, stress, and depressive symptoms among the elderly: test of a process modelPsychol Aging619911902018PPVitalianoJRussoHMYoungPredictors of burden in spouse caregivers of individuals with Alzheimer's diseasePsychol Aging619913924029WEHaleyEGLevineSLBrownStress, appraisal, coping, and social support as predictors of adaptational outcome among dementia caregiversPsychol Aging2198732333010JSLevinReligious factors in aging, adjustment, and health: a theoretical overviewJournal of Religion and Aging4198813314611DGBlazerEPalmoreReligion and aging in a longitudinal panelGerontologist161976828512KSMarkidesJSLevinLARayReligion, aging and life satisfaction: an 8-year, three-wave longitudinal studyGerontologist27198766066513HGKoenigDOMobergJNKvaleReligious activities and attitudes of older adults in a geriatric assessment clinicJ Am Geriatr Soc36198836237414HGKoenigResearch on religion and mental health in later life: a review and commentaryJ Geriatr Psychiatry231990235315HGKoenigLKGeorgeICSieglerThe use of religion and other emotion-regulating coping strategies among older adultsGerontologist28198030331016HGKoenigHJCohenDGBlazerReligious coping and depression among elderly, hospitalized medically ill menAm J Psychiatry14919921693170017HGKoenigJNKvaleCFerrelReligion and well-being in later lifeGerontologist281988182818KASherrillDBLarsonMGreenwoldIs religion taboo in gerontology? 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Journal of Geriatric Psychiatry 29:155–184 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0ADF61F76363BFE62FEEBA003BFD2581BC290AEA.txt b/test/dataset/in/resources/corpus/Clean_0ADF61F76363BFE62FEEBA003BFD2581BC290AEA.txt new file mode 100644 index 0000000..d3fcc6b --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0ADF61F76363BFE62FEEBA003BFD2581BC290AEA.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press 22010.1093/geronb/59.4.S220 Journal of Gerontology: Social Sciences Is There Equity in the Home Health Care Market? Understanding Racial Patterns in the Use of Formal Home Health Care White-Means Shelley I. Rubin Rose M. Department of Economics, Fogelman College of Business and Economics, University of Memphis, Tennessee. Address correspondence to Dr. Shelley I. White-Means, University of Tennessee Health Science Center, 847 Monroe Avenue, Room 205N, Memphis, TN 38113. E-mail: kingram.utmem.edu 7 2004 59 4 S220 S229 17 2 2004 11 4 2003 The Gerontological Society of America 2004 Objective. This article explores whether the formal home health care (HHC) market is equitable or manifests unexplained racial disparities in use. Methods. The database is the 1994 National Long Term Care Survey. We estimate logit regression models with a race dummy variable, race interaction terms, and stratification by race. We apply the Oaxaca decomposition technique to quantify whether the observed racial gap in formal HHC use is explained by racial differences in predisposing, enabling, need, and environmental characteristics. Results. We find numerous unique racial patterns in HHC use. Blacks with diabetes and low income have higher probabilities of HHC use than their White counterparts. Black older persons have a 25% higher chance of using HHC than Whites. Our Oaxaca analysis indicates that racial differences in predisposing, enabling, need, and environmental characteristics account for the racial gap in use of HHC. Discussion. We find that the HHC market is equitable, enhancing availability, aceptability, and accessibility of care for older Black persons. Thus, the racial differences that we find are not racial disparities. hwp-legacy-fpage S220 hwp-legacy-dochead RESEARCH ARTICLE Distinct racial patterns in older persons' use of both institutional and community-based health care services are well documented (Wallace, Levy-Storms, Kington, & Andersen, 1998). In this article, we explore whether formal home health care (HHC) use among older persons varies by race, and if so, then we seek to explain why this differential use occurs. We assess the use of HHC as an integral part of community health care rather than as one component of the spectrum of long-term care, as is done by Wallace and colleagues (1995, 1998), and we delineate the use of HHC in the context of the broader literature of racial differences in community care to highlight that this market differs from other alternatives. Thus, discussion of racial patterns of use for alternative types of community health care provides important contextual background for our focus on HHC. Owing partly to limited financial access to nursing homes and cultural preferences (Falcone & Broyles, 1994), older Blacks are less likely to use nursing homes and more likely to live in the community with chronic conditions and disabilities that exceed those of older Whites (Wallace et al., 1998; White-Means, 2000). However, lower use of skilled nursing facilities by Blacks is not counterbalanced by higher use of HHC services (Cagney & Agree, 1999). Further, noninstitutionalized older disabled Blacks are less likely to use other community-based services such physician services and prescription medications (White-Means, 2000). Differential use of in-home assistive devices is also found, with Whites more likely to use home modification devices and Blacks more likely to use portable devices (Rubin & White-Means, 2001). What explains these distinct racial patterns in health services use? Racial differences in physician services use and prescription medication are not fully explained by racial differences in average income or socioeconomic or health characteristics (White-Means, 2000). Researchers (Wallace, 1990; Wallace et al., 1998; White-Means, 2000) note that racial differences are explained by three factors: availability, accessibility, and acceptability. Problems of availability arise from the lack of close geographic proximity to services and/or segregation in the use of services. Lack of knowledge about available services and limited referrals to auxiliary services lead to gaps in accessibility. Patient dissatisfaction, culturally insensitive care, and unfavorable provider–patient interactions lead to problems of acceptability. The Institute of Medicine (2002) distinguishes between racial differences and disparities. It reports that some racial differences in health care use can be expected and are acceptable, including differences due to clinical need, patient preferences, and the appropriateness of particular interventions. But if differences are due to characteristics of the health care system, provider discrimination, or incorrect or deficient patient information, then these differences are considered racial disparities in care. Thus, differences in use, reflecting such factors as stereotyping, biases, and uncertainty regarding treatment efficacy by race, limited supplies of racial/ethnic minority health care providers, and poor patient–provider communication and trust, are seen as disparities. We seek to determine whether the racial differences in HHC use are acceptable differences (and thus equitable) or actually reflect disparities, as defined by the Institute of Medicine. If the differences found are disparities, then we would expect to find divergences in availability, accessibility, and acceptability in HHC use. So, disparities are seen as differentials that are not explained by either socioeconomic or health characteristics. We define formal HHC as market-purchased services provided in the patient's home, including both Medicare-financed care associated with subacute care and community-based care that meets needs associated with activities of daily living (ADLs) and instrumental activities of daily living (IADLs). Our comparison focuses on use versus nonuse of community-based HHC and whether the comparative use is equitable by race. The dichotomous dependent variable indicates whether an older Black or White person who lives in the community uses HHC or not. Nonusers of HHC may use informal care support or various alternate formal (purchased or market-supplied) health care services or no care, as distinguished by Wallace et al. (1998), who also explore nursing home users. We hypothesize that HHC is an equitable market. If it achieves the three criteria of availability, accessibility, and acceptability, then this would suggest that HHC represents an equitable market. HHC is more available to the home bound than other community-based care because it does not require transportation (Rubin, White-Means, & Feagin, 2003). Accessibility is broadened by Medicare and by the relatively low price of HHC. Medicare is the largest single HHC payer (about 40%; National Association of Home Care [NAHC], 2000), covering HHC under Part A with no out-of-pocket charges and under Part B with a $100 deductible. Other federal programs and Medicaid also pay for some HHC for the poor or medically indigent (NAHC, 2000; Shi & Singh, 1998). Also, the culturally diverse workforce of HHC (35% are Black and 10% Hispanic) may be more acceptable to diverse groups of older persons than nonhome community-based care and may be perceived to be more culturally sensitive (Stone & Wiener, 2001). As this low-paid workforce is economically disadvantaged, cultural and class barriers tend to be minimized. Additionally, because of the relatively low start-up cost of a home health agency (approximately $150,000), many agencies are owned by ethnic minorities and are more likely to serve communities where a higher proportion of ethnic minorities reside (Brown, 1998). Data from national surveys tend to support the hypothesis of HHC as an equitable market. Indeed, Blacks are more likely to use HHC than Whites (Mui & Burnette, 1994; Wallace et al., 1998). In contrast, regression analysis provides equivocal support for the role of race in increasing home health use. Freedman (1999) reports that non-Whites are significantly more likely to use more HHC services and also to use them for longer terms. This higher intensity of use is associated with the greater likelihood that Blacks postpone use and remain in the community longer with chronic conditions (Cagney & Agree, 1999). Although the coefficients are insignificant, Picone and Wilson (1999) find higher Medicare home health agency use rates for Blacks, but they conclude that socioeconomic factors are not strong determinants compared to health status. Similarly, Miller and colleagues (1996), who do not find racial differences, find that overall health and functional status, as well as Medicaid use, are primary predictors of service use. In contrast, Wallace and colleagues (1998) note that African Americans are less likely than non-Latino Whites to use HHC, with HHC use more likely for those of advanced age, women and single women, and those with more formal education, higher ADLs, stroke or heart conditions, low income, and Medicaid. Research Questions This study makes important contributions to a growing literature on racial disparities in health care. The broad research objective is to discover whether HHC provides an equitable health care market for Blacks. We determine empirically based answers to two specific questions: (a) Are there distinct racial patterns in the use and determinants of use of HHC? (b) What is the role of racial differences in need, predisposing conditions, enabling factors, and environmental factors in explaining observed racial differences in HHC use? Methods Data The database is the 1994 National Long-Term Care Survey (NLTCS), a repeated panel survey designed to estimate chronic disability status, functional problems, and institutionalization rates of older persons and their use of community-based in-home long-term care. The NLTCS provides nationally representative data on the prevalence and patterns of functional limitations, medical conditions, and recent medical problems for Medicare-enrolled Americans aged 65 and older. It also includes data on health services use, out-of-pocket expenditures, financial resources, and sociodemographic characteristics of older persons and their families. The 1994 NLTCS sample is the fourth panel and includes living respondents from previous panels, despite constancy or changes in their disability level (Manton, Corder, & Stallard, 1997). The 1994 dataset is supplemented with additional persons meeting the impaired status screening criteria from those who were previously screened but not interviewed because at that time they did not meet the impaired status criteria or reached age 65 after the 1989 cutoff. The addition of an oversample of persons aged 95 and older and a “healthy” sample not meeting the “impaired” status criteria is unique to the 1994 NLTCS to enhance the national representativeness of the data. Detailed questions are asked if persons meet screening criteria for chronic ADL and IADL disability. However, many carried over were previously chronically disabled but in 1994 had no ADL and/or IADL disabilities so that the sample shows many people with no current ADLs or IADLs (Liu, Manton, & Aragon, 2000). In contrast to some analyses (e.g., Liu et al., 2000), we include going outside as an ADL, because it is grouped as an ADL in the NLTCS screener definitions. The NLTCS screener includes limitations in walking around inside and also going outside as ADLs; this ADL definition may result in relatively high prevalence of ADLs compared with IADLs. The NLTCS includes 5,089 noninstitutionalized older persons in the detailed community interviews, of whom 4,740 are non-Hispanic Whites and non-Hispanic Blacks; our study sample includes 3,448 non-Hispanic White and non-Hispanic Black complete reporters (participants who provided data for all variables). Similar to other survey research data, it has missing data on family income (25%) and education (5%). Typically, persons at upper and lower incomes are least likely to report income or education or both, resulting in patterns of data loss that are nonrandom or systematic. Thus, the factors explaining the likelihood of missing data are unrelated to the current study's exploration of the use of home care. With a sample of complete reporters, we lose some statistical power, but this does not lead to inaccurate parameter estimates. First, statistical tests (Kromrey & Hines, 1994; Switzer, Roth, & Switzer, 1998) indicate that in cases where no more than 30% of the data are missing (as is true for our sample), accurate parameter estimates are generated. Second, standard estimation procedures restricted to the available observations yield valid inferences when the process that generates the missing data is independent of the observed and unobserved data values (Rotnitzky & Wypij, 1994). As the data indicate that it is equally likely that Blacks and Whites have missing income data (27.19% vs 24.3%) and that home health users and nonusers have missing income data (26.81% vs 24.11%), we have further confirmation of lack of bias in the parameter estimates using the available observations. Thus, our analysis uses a sample of 3,448 persons, with 3,136 White non-Hispanic and 312 Black non-Hispanic participants, as described in Table 1. Analytic Design To examine our first research question, we use three regression strategies: one with a race dummy variable, a second with race interaction terms, and a third stratified by race. For the second research question, we apply the Oaxaca decomposition technique (Oaxaca, 1973), quantifying the percentage of the observed racial gap in HHC use explained by racial differences. This approach can contribute to our understanding because it questions whether an identified racial difference in home health use is what we should observe. As the health status of community-residing older Blacks is significantly lower than that of Whites, the Oaxaca approach can help to answer whether we should similarly find differences in home health use, with significantly higher use by Blacks, and if not, then why not? We first estimate logistic regression models of HHC use, including a race dummy variable (Model I) and also race interaction terms (Model II). Next, we estimate logistic regressions of HHC use, stratified by race. The measure of HHC use is a dichtomous variable that equals 1 if paid home services are provided by a helping organization (i.e., a home health agency or other business provider) and 0 otherwise. The NLTCS allows respondents to define a helping organization as any organization that provides in-home assistance, by describing their relationship to each in-home caregiver (or helper). If any caregiver was from a helping organization, the respondent was coded as having HHC. Our choice of explanatory variables and conceptual framework are guided by the Andersen model (1995) and previous research on HHC use. The Anderson model suggests that medical use patterns are influenced by (a) predisposing conditions, (b) enabling factors, (c) environmental factors, and (d) need. Our measures of predisposing conditions are gender, age, marital status, education, and three quantifiable behavioral characteristics (whether the respondent currently smokes, exercises or plays sports regularly, and/or drinks alcoholic beverages one to three times a month or less frequently; Cutler & Sheiner, 1994; Newman, Struyk, Wright, & Rice, 1990). Our measures of enabling factors are income, income squared, health insurance status (Medicare only, Medicare plus supplements, or Medicare and Medicaid dual eligibility), number of adults living in the home, and use of home-delivered meals. We utilize income squared because previous research (White-Means, 1997) documents a nonmonotonic relationship between home health use and income. Home-delivered meals, part of community-based nutrition programs in Title VII of the Older Americans Act and part of community-based in-home care, may enhance nutrition and forestall institutionalization (Shi & Singh, 1998). As home-delivered meals are not part of standard home care and are not covered by Medicare, we view use of home-delivered meals as an enabling factor that facilitates living in the community. Similar to previous research (Picone & Wilson, 1999), we include two proxy measures for geographic environment: urban (respondents living in cities of 50,000 or more residents or their suburbs) and South. We also include measures of (a) whether the state has a large Medicaid home- and community-based care waiver program to reflect financial support for HHC services (Sloan, Picone, & Hoerger, 1997) and (b) the number of primary care physicians per 1,000 residents by state to measure underservice by physicians. State Medicaid waiver program funding of HHC greater than the median of $142.4 million indicates a high level of funding, whereas funding less than or equal to $142.4 million indicates a low level of funding. We examine HHC need with the five most prevalent chronic health conditions in the NLTCS and the ADLs and IADLs used as screening criteria in the NLTCS. The five chronic conditions are (a) joint diseases (rheumatism, paralysis, and arthritis), (b) diabetes, (c) heart conditions, (d) hypertension, and (e) breathing disorders (bronchitis, pneumonia, flu, emphysema, and asthma). Although persons in the sample may have one or more of these conditions, we enter each condition as a separate variable in our regressions. The simultaneous impact of multiple conditions can be explored by summing the impacts of individual conditions. We include the number of ADLs and IADLs as measures of disability (Cutler & Sheiner, 1994; Newman et al., 1990; Picone & Wilson, 1999; Sloan et al., 1997). As data on severity of health conditions are unavailable in the NLTCS database, use of continuous measures of ADLs and IADLs provides an approach to a proxy for severity. The nine ADLs are limitations with eating, getting in and out of bed, getting in and out of chairs, walking around inside, going outside, dressing, bathing, toileting, and controlling bowel movement/urination; and the seven IADLs are limitations with meal preparation, laundry, light housework, grocery shopping, managing money, taking medicine, and making telephone calls. The general form of our logit regression models is as follows:where DG = a vector of demograhic and sociostructural variables (gender, age, marital status, education), PH = a vector of health behaviors (smoking, exercise, and alcohol consumption practices), EN = a vector of enabling factors (income, income squared, health insurance status, number of potential caregivers living in the home, and home-delivered meal use), EF = a vector of environmental factors (urban, South, underservice by physicians, and residence in a state with high- or low-funded Medicaid waiver programs), HS = a vector of chronic health conditions (joint diseases, diabetes, heart conditions, hypertension, and breathing disorders), ADL = number of ADLs, IADL = number of IADLs, and ε = error term. We use three regression strategies to examine our first research question. We begin with regression models that include race dummy variables (Model I) and race interaction terms (Model II), because they are most directly comparable with previous research. If the race dummy variable is statistically significant, then race has a unique and independent influence on HHC use, given the role of other regression variables. The interactive model explores whether the impact of other independent variables on HHC use varies by race. Finally, using models stratified by race, we explore differences in estimated β coefficients and marginals (to measure impact of change in the independent variable on the probability of HHC use) for Blacks and Whites. We seek to determine whether independent variables impact HHC use differently for those of different races. We explore whether factors found significant in the unstratified data are significant in the stratified models and whether they are significant for both Blacks and Whites. For our second research question, we explore the role of racial differences in need, predisposing conditions, enabling factors, and environmental factors in explaining the observed racial difference in HHC use. If the variables included in our model do not fully account for racial differences in HHC use, then other nonmeasured factors are assumed to account for these differences. A priori, we expect Blacks' relatively lower health levels to be associated with greater HHC use compared with Whites. We also predict that the magnitude of the racial gap in HHC use directly corresponds with the size of the racial gap in variables included in the model. Our methodology for the second research question is application of the Oaxaca decomposition technique. The Oaxaca technique is used to quantify the percentage of the observed racial gap in HHC use explained by racial differences in the predisposing, enabling, need, and measured environmental characteristics. This technique is a standard approach in labor economics to determine the extent of wage discrimination. It was originally applied to log-linear models of wage compensation and later to nonlinear logit, probit, and bivariate probit discrimination models (Hinks & Watson, 2001; Mohanty, 2002). With use of this technique, the total racial gap in HHC use is the difference in the probability that Whites and Blacks use HHC services: (PW − PB). PW − PB is equal to F(Xw, ßw) − F(Xb, ßb), where ßw and ßb = regression coefficients for Whites and Blacks, Xw and Xb = characteristics of Whites and Blacks, and F = the cumulative logistic probability function: 1/[1 + e−(α+βXi)]. The total gap emphasized in the Oaxaca technique can also be expressed asThe first term of the above equation measures the gap in the probability of HHC use that is explained by racial differences in characteristics, that is, acceptable differences in use. The gap explained by racial differences uses the regression coefficients for Whites and examines how the probability of HHC use varies when these regression coefficients are paired with the characteristics of Whites versus the characteristics of Blacks. The unexplained gap (i.e., disparity) is the difference in the probability of use of HHC by Blacks assuming their own characteristics (Xb) and using the regression coefficients of Blacks (ßb) and the probability of use of HHC by Blacks assuming their own characteristics and the coefficients (ßw) of Whites. If unexplained differences in service use are found, they may be due to either unmeasured demand side or supply side factors, including racial differences in the knowledge about or preferences for HHC use, barriers to formal medical care, or possibly discrimination in the provision of HHC (i.e., factors in the health delivery system that influence accessibility, availability, and acceptablity of services). If the Oaxaca technique reveals unexplained differences in services use, then an important next step of investigation would be to seek data sources that allow identification of the causal factors among the unmeasured variables. However, if application of the Oaxaca technique reveals no unexplained differences in HHC service use, then an equitable market with acceptable racial differences is indicated. An additional caveat in the use of the Oaxaca decomposition approach is that measurement errors may also generate unexplained racial variation in the use of HHC. If regression variables are systematically measured with error by race, for example, self-reported diseases, then the systematic differences would appear as components of the unexplained gap in service use. Results Sample Characteristics Table 1 presents the study sample characteristics by race. About 16.2% of Whites and 20.2% of Blacks used HHC, or Blacks had a 25% greater chance of utilizing HHC. The sample includes 91% non-Hispanic Whites and 9% non-Hispanic Blacks, with similar mean ages of 78 and gender distribution (63% female). Whereas 40% of Whites live in medium or large cities and 35% are in the South, more than half of Blacks live in medium or large cities, and two thirds live in the South. Fully 70% of Whites, but only 29% of Blacks, have Medicare plus supplemental health insurance coverage, a striking racial differential. Blacks are much more likely than Whites to have only Medicare coverage (30.7% vs 17.3%) and 3.5 times more likely than Whites to be dually eligible (36.7% vs 10.4%). These racial insurance status differences correspond to differences in education and income. The average income is slightly over $21,000 for Whites and only $13,000 for Blacks; about 70% of Whites have at least a ninth grade education compared with 39% for Blacks. Preferences for preventive health and wellness differ for the two racial groups. Whites are more likely than Blacks to report drinking one to two times a week (15% vs 5%) and also getting exercise or playing sports regularly (23% vs 14%), but Blacks are slightly more likely to smoke (13% vs 10%). The ability of older persons with chronic illnesses to remain in the community is affected by the availability of caregivers (measured by the number of other adults in the household) and nonhousehold support services (measured by use of home-delivered meals). The number of other adults living in the home differs by race, with Blacks having an average of slightly over two, while Whites average only one and a half. The data indicate differences in the use of home-delivered meals (7.3% of Blacks vs 4.8% of Whites). Blacks are more likely than Whites to live in states with high physician/population ratios (6.63 vs. 5.00), with high Medicaid waiver programs (25% vs 8%), and also with small Medicaid waiver programs (12% vs 3%). Blacks report the presence of chronic health conditions more frequently than do Whites, with the exception of breathing disorders. The presence of heart conditions is reported by Blacks and Whites with similar frequency (28%). However, Blacks report joint disease, diabetes, and hypertension more frequently. Blacks average more ADLs than Whites (1.78 vs 1.38) and report more IADLs (averaging 1.08 vs 0.81). About 56% of Blacks report four or more ADLs (compared with 47% of Whites), and 29.4% report one or more IADLs (compared with 26.5% of Whites). Note that for both Blacks and Whites, ADL prevalence is higher than IADL prevalence. Based on the NLTCS screener criteria, this is not unexpected, as suggested in the data description section above. The prevalence for difficulty in getting around inside is 33.1%, and the prevalence for difficulty in getting around outside is only 3.9% (3.9% for Whites and 3.8% for Blacks). Thus, the results might be slightly skewed by including difficulty getting around outside as an ADL rather than an IADL. Racial Patterns in Use of Formal HHC Table 2 reports two models analyzing the logistic regression results for HHC use by Blacks and Whites. Model I includes a race dummy variable, and Model II also includes race interaction terms. Chronic medicial conditions, ADLs, IADLs, and enabling, environmental, and sociodemographic factors influence HHC use. In both Model I and Model II, heart condition is the chronic condition most likely to lead to HHC use. Older persons with more ADLs and IADLs have greater HHC use, and both income and income squared are statistically significant. Interestingly, there is a nonmonotonic relationship between income and HHC use. First, HHC use decreases as income increases; then, for those with higher incomes, increases in income are associated with increases in the probability of HHC use. It appears that HHC use is U-shaped with respect to income, as has been found for nursing home care (Headen, 1993; Morrow-Howell & Proctor, 1994). Those participating in meals programs are more likely to use HHC. However, as the number of persons living in the household increases, the probability of HHC use decreases. Dual eligibility increases the likelihood of HHC use, a likelihood that varies with the extent of state funding available for Medicaid community waiver programs. Women and persons who are older and have more formal years of education are more likely to use HHC, but those who are married are less likely to use HHC. The regression indicates that the variable of race is statistically insignificant in influencing HHC use. The role of race is further explored in Model II with race interaction terms. Each variable that was significant in Model I remains significant in the full interaction model, and the variables interacting race and diabetes and race and income are also significant. Thus, if Blacks have diabetes, they are more likely to use HHC than White diabetic individuals and also more likely than Blacks who have other major chronic conditions. Further, when we examine Blacks and Whites who have the same income, Blacks are more likely than Whites to use HHC. Table 3 presents the results of our stratified logistic regressions of the use of formal care by race and the marginals for significant coefficients. The influence of race on some independent variables, as reported in Table 2, allows us to estimate the regressions separately, although a Chow test indicates that race does not affect the impact of all independent variables on the dependent variable. With the exception of the marginals for the income and income-squared terms, in the reported logistic regressions, the marginal is exp(−α − βx)*bi/[1 + exp(−α − βx)]2, where bi = the regression coefficient for the variable for which a marginal is reported. Thus, Table 3 reports the marginal effects of the significant independent variables on the probabilities of HHC use, that is, the percentage point differences in the probability of HHC use when the regression variable changes by 1 unit. For regression variables that are dichotomous, the marginal is the percentage point difference in the probability of HHC use when the regression variable has a value of 1. Computing the marginals for the income and income-squared terms is complicated because we use an interactive term (income squared) in a nonlinear regression model, that is, logistic regression (Ai & Norton, 2003; Norton, Wang, & Ai, 2003). Thus, the marginal for income is (b2 + 2b4x2) * exp(−α − βx)/[1 + exp(−α − βx)]2, where b2 = the estimated coefficient of the income term, b4 = the coefficient of the squared income term, and x2 = the mean of the income variable. Marginals are comparable with reporting odds ratios, but they are more straightforwardly comprehended. Whereas odds ratios provide data on the relative increased probability of use, marginals provide data on the absolute increased probability of use. Odds ratios indicate the effect of a unit increase of the independent variable on the odds of an event [(probability of HHC use)/(1 − probability of HHC use)], but marginals report the effect of a unit increase in the independent variable on the probability of HHC use. The results in Table 3 indicate that despite the difference in sample size for the Black and White samples, there are unique racial patterns in HHC use. As suggested in the interactive model, among Blacks, diabetes increases the probability of using HHC by 10%; among Whites, heart conditions are associated with higher probabilities (4.5%) of HHC use. ADL disability affects HHC use for both Blacks and Whites. The direct effect of each additional ADL is to increase the probability of HHC use by 4.3% for Whites and by 4.6% for Blacks. For both racial groups, participation in delivered meals programs is associated with a greater likelihood of using HHC, with greater probability of 24% for Blacks and 12% for Whites. Several impacts are unique among Whites. More IADLs are associated with greater HHC use, with each additional IADL increasing the probability of HHC use by slightly less than 1%; each year of age increases the probability of HHC use by 0.25%; women have a slightly (3%) higher probability of HHC use; more education increases the likelihood of HHC use by 3%; and married status reduces the likelihood of HHC use by 6%. Three enabling/environmental characteristics also influence HHC use among Whites. Dual eligibility increases the probability of use by 8% in states with high Medicaid community waiver programs and by 7% in states with low Medicaid community waiver programs. There is a nonmonotonic relationship between income and HHC use. At lower income levels, each $1,000 increase in income decreases the probability of HHC use by 1.7%, but higher income (above $49,500) increases the probability of using HHC. For each person living in the house, the probability of HHC use decreases 3.5%. Explained and Unexplained Racial Patterns in the Use of HHC We apply the Oaxaca technique to determine how much of the racial differential in the probability of using HHC is explained by racial differences in medical and socioeconomic characteristics. We calculate the difference between the probability that Whites would use HHC assuming their own actual characteristics (0.1554) and the probability that Whites would use HHC assuming they have the characteristics of Blacks (0.1984), as measured by [F(Xw, ßw) − F(Xb, ßw)]. This calculated differential probability is (0.1554 − 0.1984) or −0.0430, which is approximately the actual racial gap in HHC use (0.162 − 0.202). Thus, the Oaxaca decomposition suggests that differences solely in the medical, socioeconomic, enabling, and environmental characteristics of Blacks and Whites should lead to the observed racial gap in HHC use. Expressed differently, we find that HHC is indeed an equitable health care market. Discussion Consistent with our prediction that HHC provides an equitable community-based health care market, the raw data indicate that in 1994, Blacks were more likely to use HHC than Whites. In this study, we find that predisposing, enabling, environmental, and need factors influence the use of HHC by both Blacks and Whites. Blacks have a 25% higher probability of using HHC than Whites. We find that racial differences in chronic conditions and socioeconomic factors fully predict this greater likelihood of HHC use among Blacks. This finding supports our hypothesis that HHC use is equitable by race. Older Black patients who live in the community with health conditions that are more extensive than those of White patients (possibly due to lower use of nursing home care and earlier-than-average release from hospitals) are absorbed by the HHC market, which presents few barriers to access. What do these results mean when other research (e.g., Wallace and colleagues 1998) finds that racial differences persist? The highlight of our findings is that HHC provides a uniquely equitable market. Similar to other research, our descriptive statistics (see Table 1) note a racial difference in HHC use, with Blacks having greater use. However, using our regression model and an expanded set of variables relative to those of Wallace and colleagues (1998), we find that racial differences in these sample characteristics explain racial differences in use of HHC among community-based older persons. Thus, the racial differences we identify in HHC are not racial disparities (Institute of Medicine, 2002) and do not reflect divergences in availability, accessibility, and acceptability. As described in previous research (Hodgson & Cohen, 1999; Wallace et al., 1998), chronic conditions (e.g., need) significantly influence HHC use, and those with the most impact vary by race. Diabetes is most reported by black HHC users, whereas a heart condition is most reported by Whites. These findings indicate that health policies or policy changes that differentially affect HHC use by chronic condition may also have unintended differential racial impacts. For example, the 1997 Balanced Budget Act, disallowing venipuncture as a sole qualifier for HHC coverage by Medicare, possibly imposed a greater burden on Blacks than Whites, because venipuncture patients are primarily diabetic. Research indicates that loss of HHC among disqualified venipuncture patients reduced their bathing and dressing assistance, requiring them to do more for themselves (Rubin et al., 2003). Thus, an unintended effect of the Balanced Budget Act, decreasing access to a service more heavily used by Blacks, possibly reduced the racial differential in HHC use. This legislation may even have reversed the prior trend of relatively higher HHC use among Blacks, and its impact is worthy of further investigation. ADL limitations increase HHC use for Blacks and Whites, although by different percentages, which is consistent with previous research (Kemper, 1992; Wallace et al., 1998; White-Means, 1997) noting the central role of disability level in affecting HHC use. The unique role that meals programs play in increasing use of HHC needs further exploration. A remaining question is why HHC differs from other health care services for older persons. Examining the differential impacts of race and chronic conditions on older persons' use of assistive devices, Rubin and White-Means (2001) find a 50% unexplained underuse gap for Blacks. White-Means (2000) finds unexplained underutilization of medical services and prescription drugs for Blacks. For HHC, we find higher use among Blacks and no unexplained use differential. It is important to note that whereas the NLTCS involves a complex sampling method, it is nonetheless nationally representative. The NLTCS has only a minimal research design effect of sampling (maybe 10%). Therefore, use of SUDAAN in regression analysis to adjust standard errors for underestimation of the true variation is considered unnecessary. Further, putting the SUDAAN model on top of the regression would possibly portray an inaccurate degree of precision (K. Manton, personal communication, 2004). This is in contrast to the procedures necessary for numerous other complex survey design data sources where the sampling technique leads to substantive correlation among observations, necessitating the use of weights. We predict use of HHC at one point in time and show that this cross-sectional view of HHC use indicates equity. Analyzing HHC use within a dynamic modeling framework remains an interesting area for future research. For example, it is unclear whether Blacks are more likely than Whites to use HHC for extended periods or with more or less continuity than Whites. These are also additional components of equity of care that are beyond the scope of this article. Another area for future research is the importance of including detailed measures of variation in support at the community level, comparable with the enabling variables that Bradley and colleagues (2002) label “availability of support.” We are unable to incorporate these variables analytically, because although we have individual data, the NLTCS lacks detailed community-level variables, giving our research imperfect measures of the community variables of Bradley and colleagues. Inclusion of these community support variables would address significant differences at the small area level. Although our data suggest there are not unexplained racial differences in HHC use, we are unable to determine whether there are racial differences in the types and quality of HHC received, because different types of HHC are not delineated in the data. Thus, further research is still warranted to understand whether there is equity in terms of quality of care provided by different types of helping organizations. In addition, questions of how racial differences in household and family formation affect HHC use and whether the actual intrahousehold relationships are important deserve future investigation. Our results indicate that being married and also the number of other adults in the household are significant for Whites but not for Blacks. The relationship between HHC use and cultural differences is another area for prospective research. Culture, expressed in group values and beliefs, may affect the selection among long-term care options and between institutional and noninstitutional choices (Thornton & White-Means, 2000; Wallace et. al., 1998). Thus, measured racial differences may embody cultural influences that are implicit rather than explicit. Explicit measures of knowledge and cultural preferences for receipt of HHC versus other health care services, personal experiences of discriminaton when interacting with the health care system or with the HHC services market, and charactersistics of patients' community health care systems are further issues needing investigation. These analyses would provide input for effective public policy measures to address racial disparities in use of health care services and enable replication of the equity in use that our data indicate is provided by the HHC market. Decision Editor: Charles F. Longino Jr., PHD Table 1. Characteristics of the Sample. Total Non-Hispanic White Non-Hispanic Black Sample Characteristics % or Mean No. SD % or Mean No. SD % or Mean No. SD N, sample size 100% 3,448 90.4% 3,136 9.6% 312 Home health care usea 16.6% 571 0.37178 16.2% 508 0.3685 20.2% 63 0.40208 Av. age (mean), yr 77.8 7.3624 77.8 7.2659 78 8.28181 Gender     Male 36.4% 1,255 36.4% 1,142 36.7% 115     Female 63.6% 2,192 0.48113 63.6% 1,994 0.48109 63.3% 198 0.4823 Marital status     Marriedb 46.1% 1,590 0.49851 47.6% 1,493 0.49949 31.0% 97 0.46228     Nonmarried 53.9% 1,858 52.4% 1,643 69.0% 216 Location     Medium or large cityb 41.2% 1,420 0.49229 39.9% 1,251 0.48942 54.0% 169 0.49906     Small city, town or rural 58.8% 2,027 60.1% 1,885 46.0% 144 Region     Southb 37.2% 1,282 0.4834 34.7% 1,088 0.47597 62.9% 197 0.48406     Non-South 62.8% 2,165 65.3% 2,048 37.1% 116 Insurance status     Medicare only 18.5% 638 17.3% 543 30.7% 96     Medicare plus supplementb 66.7% 2,300 0.4712 70.4% 2,208 0.45638 29.4% 92 0.45672     Medicare and Medicaid 12.7% 438 10.4% 326 36.7% 115 Education     9th grade and aboveb 66.9% 2,307 0.47082 69.6% 2,183 0.46001 39.1% 122 0.48876     K through 8th grade 33.1% 1,141 30.4% 953 60.9% 190 Income (mean $)b $20,451 18,422.8 $21,182 18,821.7 $13,096 11,448.1 Other adults (mean)b 1.59 1.12943 1.55 1.06706 2.06 1.55599 Delivered meals     Yes 5.0% 172 4.8% 151 7.4% 23     No 95.0% 3,276 0.21893 95.2% 2,985 0.21412 92.6% 289 0.26173 Behavioral characteristics     Smokinga 10.3% 355 0.30395 10.0% 314 0.29979 13.5% 42 0.34186     Drinkingb 14.1% 486 0.34772 15.0% 470 0.35669 5.1% 16 0.22093     Exerciseb 21.9% 755 0.41361 22.7% 712 0.41899 13.8% 43 0.34527 Chronic health conditions     Joint diseasesb 69.1% 2,383 0.46198 68.2% 2,139 0.46549 77.9% 243 0.41569     Diabetesb 16.4% 565 0.37047 15.5% 486 0.36194 25.6% 80 0.43735     Heart conditions 27.7% 955 0.44757 27.7% 869 0.44764 27.6% 86 0.44755     Hypertensionb 43.6% 1,503 0.49591 42.3% 1,327 0.49409 56.4% 176 0.49667     Breathing disordersb 30.3% 1,045 0.45953 30.7% 963 0.46149 25.6% 80 0.43735 ADL frequency distribution     0 ADLs 51.8% 1,786 0.49973 52.6% 1,649 0.49941 44.2% 138 0.49746     1 or more ADLsb 48.2% 1,662 47.4% 1,487 55.8% 174 ADL (mean)b 1.41 1.92609 1.38 1.91197 1.75 2.03585 IADL frequency distribution     0 IADLs 73.3% 2,527 0.44267 73.5% 2,306 0.44123 70.5% 220 0.45672     1 or more LADLsb 26.7% 921 26.5% 830 29.5% 92 IADL (mean)b 0.84 1.77436 0.81 1.74772 1.08 2.00949 Underserved by physician (mean) 5.15 2.7202 5.00 2.58481 6.63 3.49789 High Medicaid waiverb 9.3% 321 0.29102 7.8% 245 0.26791 25.0% 78 0.43371 Low Medicaid waiverb 3.4% 117 0.18108 2.6% 81 0.15865 11.5% 36 0.32 Notes: ADL = activity of daily living; IADL = instrumental activity of daily living. Source: 1994 National Long-Term Care Study, Center for Demographic Studies, Duke University. aIndicates racial difference significant for variable at.10 or better, based on t test for proportions. bIndicates racial difference significant for variable at.05 or better, based on t test for proportions. Table 2. Results of Logistic Regressions of the Use of Formal Care by Race for Non-Hispanic Black and White Older Persons. Model I Model II With Dummy Race Variable With Interactive Termsa Variables Coefficient t Statistic Coefficient t Statistic Intercept −4.747 −6.70764*** −4.656 −6.0807*** Joint diseases 0.0799 0.6277 0.1508 1.1187 Diabetes 0.0895 0.6303 −0.00669 −0.0430 Heart condition 0.4267 3.7496*** 0.4555 3.7738*** Hypertension −0.0728 −0.6535 −0.1012 −0.8584 Breathing disorders 0.1098 0.9498 0.1571 1.2941 ADL sum 0.4298 15.1873*** 0.439 14.5364*** IADL sum 0.0802 3.0037*** 0.0967 3.3693*** Underserved 0.000251 0.0111 −0.00561 −0.2262 South 0.0709 0.5645 0.0356 0.2681 Gender 0.3057 2.2882** 0.3088 2.1746** Age 0.0262 3.4026*** 0.0256 3.0955*** Married −0.564 −4.0286*** −0.5959 −3.9993*** Education 9th grade+ 0.3204 2.6155*** 0.3331 2.5235** Urban −0.0707 −0.6284 −0.1099 −0.9235 Medicare plus −0.00896 −0.0634 0.0493 0.3237 Dual eligible/high waiver 0.7514 4.0182*** 0.8195 3.8950*** Dual eligible/low waiver 0.5834 2.1599** 0.7364 2.4057** Alcohol 0.0939 0.4798 0.1278 0.6330 Smokes 0.1224 0.6405 0.0901 0.4458 Exercises −0.094 −0.6196 −0.0475 −0.3006 Income ($10,000 units) −0.2 −2.1423** −0.03 −3.0374*** Income squared 0.0000026 3.1257*** 0.000003 3.4788*** Other adults −0.2983 −4.9800*** −0.3545 −5.0861*** Delivered meals 1.2842 7.0599*** 1.1943 6.1689*** Black −0.0487 −0.2542 −1.3254 −0.5915 B Joints −0.6644 −1.4397 B Diabetes 0.7688 1.7946* B Heart condition −0.0963 −0.2402 B Hypertension 0.3958 0.9633 B Breathing disorders −0.6389 −1.4141 B ADL sum −0.0773 −0.7761 B IADL sum −0.1134 −1.3065 B Underserved 0.0694 1.0206 B South 0.4699 1.0021 B Gender −0.2683 −0.5492 B Age 0.00607 0.2409 B Married −0.1268 −0.2498 B Education 0.2349 0.5696 B Urban 0.4882 1.1231 B Medicare plus −0.5015 −0.9734 B Dual eligible/high waiver −0.1561 −0.3228 B Dual eligible/low waiver −0.7839 −1.1313 B Alcohol −0.6826 −0.7285 B Smokes 0.213 0.3208 B Exercise −0.3984 −0.6171 B Income ($10,000 units) 0.97 1.6724* B Income squared −0.000015 −1.299 B Other adult 0.2093 1.3788 B Delivered meals 0.6548 1.0868 Sample size (N) 3,448 3,448 −2 log L 2,368.28 2,340.741 χ2 726.962*** 754.498*** Notes: ADL = activity of daily living; IADL = instrumental activity of daily living. aRace interaction terms begin with “B” preceding a variable name. *Significant at p =.10; **Significant at p =.05; ***Significant at p =.01. Table 3. Results of Logistic Regressions of the Use of Formal Care Stratified by Race. Non-Hispanic White Older Persons Non-Hispanic Black Older Persons Variables Coefficient t Statistic Marginals Coefficient t Statistic Marginals Intercept −4.656 −6.08071*** −5.9814 −2.84017*** Joint diseases 0.1508 1.11869 −0.5136 −1.16383 Diabetes −0.00669 −0.04299 0.7621 1.90955* 0.0975 Heart condition 0.4555 3.77382*** 0.0450231 0.3592 0.93958 Hypertension −0.1012 −0.85835 0.2946 0.74848 Breathing disorders 0.1571 1.29407 −0.4818 −1.10708 Underserved −0.00561 −0.22621 0.0638 1.0079 ADL sum 0.439 14.5364*** 0.0433922 0.3617 3.81138*** 0.04627 IADL sum 0.0967 3.36934*** 0.0095581 −0.0168 −0.20513 South 0.0356 0.26807 0.5056 1.12431 Gender 0.3088 2.17465** 0.0305228 0.0405 0.08665 Age 0.0256 3.09553*** 0.0025304 0.0317 1.33193 Married −0.5959 −3.99933*** −0.058901 −0.7227 −1.48949 Education 9th grade+ 0.3331 2.52348** 0.0329247 0.568 1.4538 Urban −0.1099 −0.92353 0.3783 0.90481 Medicare plus 0.0493 0.3237 −0.4522 −0.91873 Dual eligible/high waiver 0.8195 3.89496*** 0.0810021 0.6634 1.52366 Dual eligible/low waiver 0.7364 2.40575** 0.0727882 −0.0475 −0.07642 Alcohol 0.1278 0.63299 0.5549 0.60645 Smokes 0.0901 0.44582 0.303 0.47913 Exercises −0.0475 −0.30063 −0.4459 −0.7123 Income ($10,000 units) −0.3 −3.03736*** −0.017 0.71 1.2456 Income squared 0.000003 3.47876*** 0.051 −0.00001 −1.0392 Other adults −0.3545 −5.08608*** −0.03504 −0.1452 −1.07715 Delivered meals 1.1943 6.1689*** 0.1180486 1.8492 3.24137*** 0.23658 Sample size (N) 3,136 312 −2 log L 2,109.02 231.722 χ2 669.194*** 82.185*** Notes: ADL = activity of daily living; IADL = instrumental activity of daily living. *Significant at p =.10; **Significant at p =.05; ***Significant at p =.01. 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I. (2000). Racial patterns in disabled elderly persons' use of medical services. Journal of Gerontology: Social Sciences, 55B,S1-S14. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AE1EC5958BBE547537B2D57668C9B9FD6BD7723.txt b/test/dataset/in/resources/corpus/Clean_0AE1EC5958BBE547537B2D57668C9B9FD6BD7723.txt new file mode 100644 index 0000000..b2cf652 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AE1EC5958BBE547537B2D57668C9B9FD6BD7723.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 10410.1093/gerona/60.1.104 Journal of Gerontology: Medical Sciences Nighttime Oxygen Desaturation and Symptoms of Sleep-Disordered Breathing in Long-Stay Nursing Home Residents Martin Jennifer L. 1 Mory Aaron K. 2 Alessi Cathy A. 1 1University of California, Los Angeles, Multicampus Program in Geriatric Medicine and Gerontology, and VA Greater Los Angeles Healthcare System, Geriatric Research Education and Clinical Center, Los Angeles, California. 2Jefferson Medical College, Philadelphia, Pennsylvania. Address correspondence to Jennifer L. Martin, PhD, VA Medical Center, GRECC (11E), 16111 Plummer Street, North Hills, CA 91343. E-mail: jemartin@ucla.edu 1 2005 60 1 104 108 20 8 2003 17 7 2003 The Gerontological Society of America 2005 Background. Sleep-disordered breathing (SDB) is common in older adults and has been implicated as a cause of decreased quality of life and even death. Sparse data exist on SDB in the nursing home setting. The authors evaluated SDB (using attended nocturnal pulse oximetry) in nursing home residents with daytime sleepiness and nighttime sleep disturbance. Methods. Pulse oximetry was used to estimate the prevalence of nighttime oxygen desaturation in 109 long-stay nursing home residents (mean [standard deviation] age = 86.2 [9.2] years; 74% women). Pulse oximetry findings were compared to a structured observational measurement of symptoms of SDB, the Observational Sleep Assessment Instrument. Seventy-one participants had concurrent wrist actigraphy to estimate total sleep time during oximetry recording. Results. Using the oxygen desaturation index (ODI; average number of oxygen desaturations 4% or more below the baseline level per hour), the authors found that 40% of the residents had abnormal ODI (ODI more than 5, which is suggestive of SDB). Of all observational variables assessed, only loud breathing during sleep was significantly correlated with ODI (r =.284; p =.003). When ODI was adjusted for estimated total sleep time, higher adjusted ODI was associated with higher body mass index (kg/m2). Conclusions. Abnormal ODI is common in nursing home residents. Observed loud breathing at night and high body mass index may suggest that further assessment of SDB is indicated. Future research should determine the importance of SDB and abnormal nocturnal oxygen desaturation on functioning and quality of life in nursing home residents. hwp-legacy-fpage 104 hwp-legacy-dochead RESEARCH ARTICLE AGE-RELATED changes in sleep patterns and sleep structure have been described in older adults. Increased daytime sleepiness, taking longer to fall asleep, and more episodes of nighttime awakening are accompanied by decreased sleep efficiency (i.e., time asleep/time in bed) and shorter periods of deep sleep in older adults (1). These changes are both common and more severe in nursing home residents than in community-dwelling older adults. Sleep disruption in nursing home residents likely results from multiple factors, including increased time spent in bed, reduced daytime bright light exposure, lack of structured daytime activities, and few regular social and environmental cues to regulate sleeping and waking patterns (2). In addition to age-related changes and environmental factors, certain sleep disorders, such as sleep-disordered breathing (SDB), are more common in older persons (1). SDB is defined as repeated pauses in breathing during sleep lasting 10 seconds or more (3,4). These respiratory events often lead to decreased blood oxygen saturation levels, which can precipitate awakenings followed by resumption of breathing. In addition, SDB contributes to daytime sleepiness and nighttime sleep fragmentation, which can impair daytime functioning. Several adverse outcomes in older adults have been associated with SDB, including cardiovascular consequences (hypertension, cardiac arrhythmias, myocardial infarction, and stroke) and cognitive impairment (memory problems, attention difficulties, and difficulty concentrating) (1,5–8). Some studies have shown that older adults with severe SDB have higher mortality rates compared with those without SDB (9). Associations between SDB and increased age, higher body mass index (BMI), male sex, and dementia have been reported (4,10,11). An association between SDB and increased risk for mortality has been reported in both community-dwelling older adults (9) and female nursing home residents (12). Traditionally, SDB is studied with polysomnography in a sleep laboratory. To definitively diagnose SDB, polysomnography uses multiple channels including the electroencephalogram, electrooculogram, electromyogram, electrocardiogram, pulse oximetry, chest movement, and airflow (3). Persons undergoing polysomnography are then assigned a respiratory disturbance index (mean respiratory events per hour of sleep). A respiratory disturbance index less than 5 is typically considered normal, whereas a respiratory disturbance index of 15 or more suggests moderate to severe SDB worthy of treatment (4,13). Some persons cannot be examined in the traditional sleep laboratory setting. Less intensive ambulatory methods can be useful in detecting sleep disorders in such persons. Pulse oximetry alone has been investigated as an alternative way to screen for SDB because it is simple, allows the patient to sleep in their usual environment, and is less expensive than traditional polysomnography. Pulse oximetry is particularly attractive for use in nursing home residents with dementia because it is more easily tolerated than multichannel polysomnography. Pulse oximetry alone yields an oxygen desaturation index (ODI) rather than a respiratory disturbance index. The ODI is the average number of oxygen desaturations at least 4% below baseline level per hour. Several studies have tried to determine the sensitivity and specificity of this method, with results ranging from sensitivity of 31% to 98% and specificity of 41% to 100%, depending on the specific devices and ODI cutoffs used (14,15). Using a cutoff of ODI of 5 or more appears to optimize the sensitivity of identification of patients with SDB compared with patients without SDB in sleep clinic samples (15); nonetheless, pulse oximetry has been shown to be more specific than sensitive in most studies. As a result, pulse oximetry is unlikely to falsely identify a person as having SDB, but it is more likely to fail to identify some persons who, in fact, have SDB (14,16). To date, no validation studies have been conducted in nursing home settings. One caveat against using pulse oximetry alone is that the indices can be computed based only on the duration of the recording, whereas in the laboratory setting, the indices are computed based on the total amount of time the patient is asleep (i.e., time awake is excluded). This may lead to an underestimation of SDB severity when pulse oximetry is used alone. Estimation of total sleep time (e.g., with wrist actigraphy) may improve the accuracy of pulse oximetry-based estimation of SDB severity. The cardinal symptoms of SDB are snoring and excessive daytime sleepiness. When both symptoms are observed together, SDB is often suspected and further evaluation is indicated. Structured observations of nighttime sleep and symptoms of SDB have been studied in the nursing home setting (17,18), but only limited objective data exist on nighttime oxygen desaturation among nursing home residents. In this study, we assessed the frequency of nocturnal oxygen desaturation in nursing home residents with daytime sleepiness and nighttime sleep disruption. We hypothesized that observations of disturbed breathing during sleep would be related to ODI. We also hypothesized that persons with more cognitive impairment would have higher ODI and more observed breathing disturbances during sleep. We also expected to confirm findings of previous studies showing that male sex and BMI were correlated with SDB severity. Methods Participants Participants were residents in four Los Angeles-area community nursing homes with daytime sleepiness and nighttime sleep disruption enrolled in a randomized controlled trial of nonpharmacologic interventions to improve sleep. Pulse oximetry data for the current study were collected under usual-care conditions. Daytime sleepiness was assessed using behavioral observations of sleep versus wakefulness performed by research staff every 15 minutes from 9:00 am to 5:00 pm for 2 days for all residents at each nursing home. Only those who were bed-bound, in contact isolation, or who left the facility before screening were excluded. Daytime sleepiness was defined as being asleep on 15% or more of observations, where “sleep” was defined as “eyes closed with no purposeful movement.” Residents who met criteria for observed daytime sleepiness and who gave consent to participate then had 2 nights of wrist actigraphy. Those who were scored asleep 80% or less of the time between 10:00 pm and 6:00 am were enrolled in the study. Apparatus Enrolled participants had 1 night (approximately 9:00 pm to 6:00 am) of attended pulse oximetry monitoring with a fingertip sensor connected via cable to an Ohmeda Biox 3700 pulse oximeter (Louisville, CO), which recorded oxygen saturation, heart rate, and time continuously on a laptop computer. Observations during sleep were performed simultaneously by research staff using the Observational Sleep Assessment Instrument (OSAI). The OSAI was developed to assess symptoms of SDB in nursing home residents (18). This instrument involves hourly 3-minute observations during which snoring, breathing rate, loudness and continuity, and chest movements are recorded. To estimate total sleep time during oximetry recordings, nighttime wrist actigraphy was also performed using a Mini-motionlogger (Ambulatory Monitoring, Inc., Ardsley, NY). The actigraph was placed on the dominant wrist, and activity levels were recorded in 60-second epochs. Sleep/awake was scored using a validated algorithm within the ActionW software (Ambulatory Monitoring, Inc.). Wrist actigraphy has been shown to accurately reflect total sleep time in nursing home residents (19). The Mini-Mental Status Examination (MMSE) was used to measure cognitive functioning (20). A measure of comorbidity, the Cumulative Illness Rating Scale–Geriatrics was completed by a study physician using data from a structured medical record review and a brief physical examination (21). In our previous work, the Cumulative Illness Rating Scale–Geriatrics predicted acute illness episodes and death in long-stay nursing home residents (22). Procedures Participants underwent comprehensive screening including the MMSE and Cumulative Illness Rating Scale–Geriatrics. Demographic and medical record information (i.e., diagnoses and medications received during the study) were noted. Pulse oximetry recordings, OSAI observations, and wrist actigraphy were collected or performed while residents slept in their own rooms receiving usual care. Data Analysis For each participant, baseline oxygen saturation was determined using the mean oxygen saturation rate during the first 30 minutes of recording or the first 30-minute period with sufficient data to assign a baseline. An oxygen desaturation event was defined as a decrease to 4% or more below the baseline level. Oxygen desaturation indices were computed for each participant with at least 3 hours of pulse oximetry (mean [SD] recording length = 7.4 [1.2] hours). The ODI was defined as the average number of oxygen desaturations per hour of recording. We used actigraphically estimated total sleep time to calculate the number of desaturations per hour of sleep for 71 participants with acceptable actigraphy recordings. Although there were no differences in ODI between patients with (n = 71) and without (n = 38) wrist actigraphy recordings (t107 = −.319, p =.75), patients with wrist actigraph recordings had lower MMSE scores (9.6 vs 14.5; p =.003), fewer medical diagnoses (9.3 vs 11.0; p =.040), and took fewer routine (8.3 vs 10.6, p =.025) and as-needed (1.5 vs 2.6, p =.002) medications compared with participants without actigraphic recordings. As a result of these differences, we report analyses based on calculations of ODI with and without adjustment for total sleep time. We used Student's t tests and regression analyses to test for associations between ODI and descriptive and OSAI variables. We anticipated that higher ODI would be associated with older age, higher BMI, lower MMSE scores, more medical diagnoses, use of more medications, and higher (more severe) Cumulative Illness Rating Scale–Geriatrics scores. For variables that were highly skewed, descriptives are presented as raw values and we used a normal scores transformation for statistical analyses (23). Two-tailed probability values less than.05 were considered significant. Results Participant Screening Of the 492 nursing home residents screened with daytime behavioral observations, 339 met criteria for daytime sleepiness. Of those, 194 gave informed consent to participate and 133 met criteria for nighttime sleep disturbance. One hundred twenty-one nursing home residents were randomized into the larger controlled trial of which 109 had 3 or more hours of pulse oximetry and were included in the current study. Table 1<--?1--><--?2-->shows descriptive characteristics. No participants had a documented diagnosis of SDB in medical records. Of those participants with complete oximetry data, 77 had concurrent wrist actigraphy recordings during oximetry testing. Actigraphy recordings were considered inadequate if the patient was not asleep for at least 1 hour during the recording (n = 6). Analyses were based on the 109 participants with pulse oximetry and were repeated for the 71 participants with adequate concurrent actigraphic recordings. Table 2 shows the results of the simultaneous pulse oximetry, OSAI, and wrist actigraphy. The mean ODI for the group was 7.0 (SD, 9.3). Forty percent of patients had ODI ≥5, 23% had ODI ≥10, and 13% had ODI ≥15 (Figure 1). The ODI was not significantly correlated with age, sex, ethnicity, BMI, MMSE score, number of diagnoses, or number of medications taken. Loudness of breathing was the only OSAI variable significantly correlated with ODI (r =.28, p =.003; Table 3). Louder breathing was also related to higher BMI (r =.28 p =.004). When BMI was included in the regression model, loudness of breathing remained a significant predictor of ODI (F2,104 = 5.23, p =.007). Oxygen Desaturation Index Based on Actigraphically Estimated Total Sleep Time The mean total sleep time during the oximetry recording period for patients with acceptable actigraphy recordings (n = 71) was 4.3 hours. Patients with higher ODI had shorter total sleep time (r = −.52, p <.0005). When the ODI was computed based on total sleep time, the severity of SDB appeared substantially worse (mean [SD] adjusted ODI = 20.6 [40.6]). Fifty-one percent of patients had adjusted ODI ≥5, 62% had adjusted ODI ≥10, and 25% had adjusted ODI ≥15. The association between the standard and adjusted ODI was strong (r =.94, p <.0005). The relationship between adjusted ODI and BMI was significant such that patients with higher BMI had higher adjusted ODI (r =.30, p =.012). Adjusted ODI was not significantly associated with age, sex, ethnicity, MMSE, number of diagnoses, or number of medications, and adjusted ODI was not related to OSAI variables (i.e., snoring, breathing rate, continuity of breathing, loudness of breathing, or continuity of chest movements). Discussion We found that at least 40% of long-stay nursing home residents with evidence of daytime sleepiness and nighttime sleep disturbance had an abnormal ODI (≥5), and 13% had moderate-to-severe oxygen desaturation (ODI ≥15). When ODI was adjusted for actigraphically estimated total sleep time, the prevalence of severe oxygen desaturation appeared even higher (25% of participants had adjusted ODI ≥15). The ODI was not associated with age, sex, or comorbidity. Although we found that observed loud breathing was significantly related to oxygen desaturation, we did not find a relationship between other observed symptoms of SDB such as snoring or discontinuous chest movement. It is possible that these events are simply more difficult to detect with nighttime observations than is loud breathing. One previous study of SDB in a nursing home population using thoracic and abdominal effort sensors plus wrist actigraphy (12) found 43% of residents had at least five apneas per hour of sleep with no statistical difference between men and women. This is comparable to our results showing that 40% of nursing home residents with daytime sleepiness had ODI ≥5, with no statistical difference between men and women, although our methods of measurement were different. Although BMI was not significantly associated with ODI (based on total recording time), it was related to the adjusted ODI (based on total sleep time). Given that patients with actigraphy recordings were medically healthier (i.e., took fewer medications and had fewer diagnoses), the relationship between ODI and BMI may be stronger in these persons. Nursing home residents with multiple medical comorbid conditions and who take more medications may have SDB as the result of these factors rather than because of high BMI. When BMI and loudness of breathing were evaluated simultaneously, both were related to ODI. Although BMI is strongly predictive of SDB in community-based populations, obesity by itself may not be sufficient to predict SDB in the nursing home setting. Further study is needed to determine whether the combination of high BMI and loud breathing during sleep are predictive of SDB. Although previous studies have shown a link between cognitive impairment and SDB, we did not find an association between MMSE score and ODI. Twenty-nine participants (27%) received a score of 0 out of 30 on the MMSE, suggesting that a floor effect may have influenced our findings. Nursing home residents with intact cognitive abilities but poor physical health may have more SDB than residents who are cognitively impaired but physically healthy. The main limitation of this study is that pulse oximetry is an indirect method of estimating SDB because it only detects respiratory events sufficient to cause oxygen desaturation, and the true number of events occurring during sleep cannot be precisely determined. We tried to address this concern with concurrent wrist actigraphy; however, we could not obtain actigraph recordings on all 109 participants. The participants with actigraphic recordings tended to be more cognitively impaired but more medically healthy. Although full polysomnography would have been more accurate, many nursing home residents will not tolerate such cumbersome recording equipment, and sleep electroencephalography can be difficult to interpret in persons with dementia. The strength of this study is that we were able to complete pulse oximetry recordings for 90% of enrolled participants with daytime sleepiness and nighttime sleep disturbance, which enhances the generalizability of our findings. In addition, based on validation studies conducted in sleep clinic samples, pulse oximetry has been shown to be more specific than sensitive in the identification of patients with SDB (14), and we may have underestimated the true prevalence of SDB in our study sample as a result of using pulse oximetry rather than complete polysomnography. Conclusion We found a high prevalence of abnormal oxygen desaturation among nursing home residents with daytime sleepiness and nighttime sleep disruption. The clinical significance of this desaturation is unclear. Future research should focus on potential implications of oxygen desaturation and SDB in this setting. Decision Editor: John E. Morley, MB, BCh Figure 1. The distribution of oxygen desaturation index (ODI) scores (unadjusted for total sleep time) is shown for all participants. Sixty percent of participants fell within the normal range (ODI <5), whereas the remaining 40% had ODI of 5 or more, suggesting at least mild sleep-disordered breathing Table 1. Descriptive Characteristics (N = 109) and Associations With Oxygen Desaturation Index (ODI)*. Mean (SD) or % r p value Age 86.2 (9.2) −0.044 .65 Years in nursing home 2.9 (2.9) −.042 .68 Body mass index, Kg/m2 25.3 (5.6) 0.186 .053 MMSE score< 11.3 (9.6) −0.089 .36 No. of medical diagnoses 9.9 (3.9) −0.088 .37 No. of routine medications 9.1 (5.2) −0.042 .67 No. of PRN medications 1.9 (1.8) −0.144 .14 CIRS-G score 24.6 (5.0) 0.063 .52 t p value Gender, % female 74.3% −0.62 .54 Ethnicity, % non-Hispanic white 92.7% 1.36 .18 Sedative medications, % taking* 17.4% 0.30 .76 Cardiac disease, % of patients with† 10.1% 0.67 .52 Pulmonary disease, % patients with† 56.9% 0.40 .69 Neurologic disease, % patients with† 62.4% −0.35 .73 * SD = standard deviation; MMSE = Mini Mental Status Examination; PRN = as needed; CIRS-G = Cumulative Illness Rating Scale-Geriatrics. † Includes sedating antipsychotics, benzodiazepines, and benzodiazepine-like agents. ‡ Based on CIRS-G ratings. Table 2. Summary of Nighttime Variables From Simultaneous Pulse Oximetry, Observational Sleep Assessment Instrument (OSAI), and Wrist Actigraphy. Mean (SD) Range Pulse oximetry (N = 109)     Total recording time (minutes) 445.4 (71.87) 182.7–610.9     Baseline oxygen saturation (%) 93.7% (2.3%) 84.8%–97.8%     Mean heart rate (beats per minute) 67.8 (9.8) 31.2–96.2     Mean oxygen desaturation index (ODI) 7.0 (9.3) 0–52.9 OSAI (N = 109)     Number of observations per night* 6.5 (1.7) 2–12     Discontinuity of breathing† 0.27 (0.47) 0–1.83     Discontinuity of chest movements† 0.27 (0.46) 0–2.4     Loudness of breathing‡ 1.4 (0.51) 1.0–3.0     % of observations with snoring 13.3% (25.1%) 0%–100%     Breathing rate (per minute) 17.6 (3.5) 11.1–26.0 Wrist actigraphy (N = 71)     Total sleep time (TST; in hours) 4.3 (2.1) 1.1–9.0     Percent sleep (TST/total monitoring time) 47.9% (25.4) 5%–100%     Number of awakenings during recording 18.2 (8.4) 0–43 * Observations completed only when patient was asleep. † Total number of events during the 3-minute observation period, reported as mean number per hour. ‡ Scored as 1 (low) to 3 (high). Table 3. Relationship Between Observed Symptoms of Sleep Apnea (Observational Sleep Assessment Instrument) and Oxygen Desaturation Index (ODI). Correlation p value Number of observations per night* 0.036 .708 Discontinuity of breathing† −0.007§ .939 Discontinuity of chest movement† 0.140§ .148 Loudness of breathing‡ 0.284 .003 % of observations with snoring 0.126 .191 Breathing rate (per minute) −0.011 .914 * Observations completed only when patient asleep. † Total number of events during the 3-minute observation period, mean per hour. ‡ Scored as 1 (low) to 3 (high). § Non-parametric correlation coefficient (Spearman's rho). 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Chicago: SPSS, 2000. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AE88CADBDCDFCE37B0DEF85CBC138F76052ADB1.txt b/test/dataset/in/resources/corpus/Clean_0AE88CADBDCDFCE37B0DEF85CBC138F76052ADB1.txt new file mode 100644 index 0000000..6c8e7b6 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AE88CADBDCDFCE37B0DEF85CBC138F76052ADB1.txt @@ -0,0 +1 @@ +frenchfrenchFrench History1477-45420269-1191Oxford University Press10.1093/fh/crp072ArticlesThe Parlement of Paris and the Ordinances of Blois (1579)DaubresseSylvie**Sylvie Daubresse is ingénieur de recherche at the CNRS in Paris. She may be contacted at sylvie.daubresse@culture.gouv.fr. This article has been translated by Mark Greengrass12200927102009234446466© The Author 2009. Published by Oxford University Press on behalf of the Society for the Study of French History. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org2009The Parlement of Paris invested considerable time and energy in detailed consideration of the substantial reforming legislation that had emerged from the first Estates General of Blois (1576–77) and which formed the eventual Ordinances of Blois (1579). Using the registers of the Parlement of Paris and hitherto unexamined copies of associated remonstrances, this article assesses why they did so, focusing on the issues of ecclesiastical and judicial reform. By placing their intervention in the context of the response from the Parisian magistrates to the holding of other Estates General in the sixteenth and early seventeenth century, it concludes that the sovereign court sought as much to uphold its exalted view of the ‘law of the realm’ and its own conception of reform, as to assert its independence from the Estates General or become a decisive intermediary in the dialogue between the crown and its subjects.Could the Parlement of Paris ever collaborate with the Estates General in the framing of reform legislation?1 On 23 November 1560, when two Parisian échevins asked the Parlement to despatch councillors to the Estates General shortly to be held at Orléans ‘pour adviser à deputer ceux qui seront et debvront estre envoyez de la part de lad. Ville’, the court replied in no uncertain terms that ‘[elle] n'en deputera point pour ce qu'il pourroit advenir que lad. Court congnoistroit par appel ou aultrement des choses appartenans aux doleances desd. Estats’. Sixteen years later, the Parlement continued to stand by its view and declared that ‘la forme ancienne accoustumée [doibt] estre gardée pour le faict des Estatz’.2 It refused to send deputies to the Estates on the grounds that it could not act as both judge and plaintiff.3On 2 August 1561, the Parlement was asked to validate the cahier that contained remonstrances from the Estates General held at Orléans from 13 December 1560 to 31 January 1561.4 This was the first time that doléances had been submitted for registration by the sovereign court. Where did such an initiative originate? It is clear that it arose within the Estates General itself, whose deputies thus subjected their acquiescence in royal demands for finance to the registration without modifications of the Ordinance of Orléans.5 They sought the Parlement's approbation for its measures, its juridical guarantee, not its opinion.The gens du roi, that is, the king's law officers, did nothing to hide their astonishment at this unusual way of proceeding. The written conclusions of the procureur général were drafted around the conviction that the judges of the Parlement had nothing to do with the Estates General and that they were ‘à part par consentement du roi’.6 This was also the view of Chancellor Michel de L'Hospital who, in his opening speech to the Estates of Orléans on 13 November 1560, established a clear distinction between the Parlements, which judge the private suits of individuals, and the Estates, which deliberate on the ‘plaintes generales qui concernoyent l’universel’.7 On 9 August 1561, Antoine de Navarre came before the court to explain that it was not required to consider the proposed ordinances article by article since they had already been deliberated upon by the Privy Council and by the Estates, of which the Parlement was a part. The judges were only required to ‘voir’ the proposed ordinances and ‘l'expedier’ rapidly.8 The premier president, Gilles Le Maistre, replied icily that the Parlement of Paris was not part of the Estates General, either as a corporate body or in any particular. Although two présidents and ten to twelve conseillers had been required to attend the Estates General of Tours in 1467 and 1484, its outcomes had not been forwarded to the Parlement of Paris.9 The premier président reminded Navarre that they would deliberate on the current Ordinances and agree their opinions on them in ‘la maniere accoustumee’. There would doubtless be some articles that would cause them no difficulty, but there might be others which ‘pourront ester trouver scismatiques et grandement doubteux’. The premier président Le Maistre was banned from exercising his office for four months as a reward for his pains, a highly unusual event in the history of the court.10 So the leading sovereign court of the realm was not prepared to rubber-stamp the ordinances, but expected to deliberate upon them and express its views. Herein lay the origins of the misunderstandings between the Estates General, the royal government and the Parlement of Paris, which would be replicated during the Estates General at Blois.Almost four years after the massacre of Saint Bartholomew, the edict of Beaulieu brought the fifth war of religion to a close, embodying the concessions obtained by the Malcontents, a coalition of princes and protestants under the leadership of Henri III's brother, François d'Alençon. Article 58 of the edict confirmed the summoning of the Estates General within six months. They were duly convened at Blois, meeting from 6 December 1576 to 5 March 1577, resulting in an ordinance whose articles were submitted to the Parlement for verification and registration two months after the Estates had been dismissed.11 From 28 February to 26 March 1579 the Parlement devoted seventeen sessions to examining the cahiers of Blois, and then a whole month to the drafting of remonstrances on those articles they regarded as controversial. The debate between the Parlement and the king was marked by various remonstances, reinforced by speeches from distinguished members of the court. The whole process of verification lasted almost twelve months despite all the efforts of the royal council to smooth over the various difficulties.12 Indeed, their bitter deliberations continued for several months after eventual registration in January 1580.Jacques-Auguste de Thou, a contemporary witness, was less than forthcoming, however, on what had transpired.13 Edouard Maugis, historian, furnishes no details whatsoever of these discussions.14 However, in contrast to the situation following the Estates General of Orléans of 1560, when there was next to no discussion because the ordinances were submitted so late, the registers of the Parlement of Paris enable us to follow those for the ordinances of Blois in detail.15 Even though we do not know precisely the text of the initial draft legislation submitted to the Parlement, this article demonstrates how the premier sovereign court of the realm attempted to intervene in the legislative process, following the efforts of the great estates of the kingdom to grapple with all the major issues of the day. These were so numerous, however, that we shall concentrate our attention upon the articles regarding the Church and those concerning judicial affairs.IOn the last day of February 1579, the officers of the king presented Henri III's replies to the cahiers of the Estates General of Blois before the Paris magistrates. The chambers assembled in joint session for the first reading of the edict and the nomination of a rapporteur. On 2 March, the first clause concerning the affairs of the Church was considered and, the following day (as was customary) the greffier Jean Du Tillet presented the resolutions of the court at the time of the verification of the ordinances of Orléans in 1561 and entrusted them to the rapporteur, conseiller Jacques Viole. Previous deliberations such as these always served as points of reference and the starting-point for subsequent discussion, constituting a kind of ‘juridical’ dossier upon which the magistrates could rely for precedents so that they could retain a degree of consistency of approach. On 26 March 1579, they decided to prepare remonstrances on certain articles, which were read out in draft on 6 May in the Grand'Chambre of the Parlement.16 The président, de Harlay, and three conseillers were nominated to present them to the king. They excluded comment on the proposed regulations concerning the substituts of the procureur général in article 157 of the draft Ordinances, because these were aimed exclusively at gens du roi.17These remonstrances of May 1579 exist in two extant copies.18 The first is a copy of the draft of the initial deliberations and it includes a note of the views expressed in the Parlement from which it is possible to reconstruct the spectrum of opinion.19 These varied from straightforward approval (noted with ‘bon’) for almost 60 per cent of the articles to various forms of reserve such as the petition (‘le roi sera supplié de …’), the explication (‘sera fait entendre au roi …’), or the request for further explanation or precision.20 The Parlement could propose to enlarge the scope of an article, limit it, or even completely redraft it.21 Or, again, it could decide upon formal remonstrances. Sometimes, it maintained a hard-line position, especially when it regarded a proposed article as impossible to carry out, in which case it expressed its view in a tone that brooked no dissent: ‘les anciennes ordonnances seront gardees’. The second document, also dated May 1579, is apparently a fair copy of the first, but with some notable differences in the opinions recorded.22 These manuscripts are entirely exceptional because the Parlement of Paris was normally scrupulous about maintaining the secrecy of its deliberations.23Throughout April 1579, the Parlement discussed articles concerning the Church.24 Whilst avoiding overt criticism of the Concordat of Bologna, it presented the Pragmatic Sanction of Bourges as a means of appeasing ‘the wrath of God’, rooting out heresies and bringing the troubles to a close.25 From the onset of the civil wars, the Parlement had tenaciously pressed for the residence of bishops in their dioceses and the confiscation of absentees’ revenues in favour of the poor.26 The court opposed the appointment of lay abbots and priors, and condemned the practice of laymen using custodi nos or prête-noms to acquire the revenues of benefices. Their position was, however, in contradiction with their own privilege of an indult by which they could become the titular holders of certain benefices and enjoy the proceeds.27 The magistrates of the Parlement equally pressed for ecclesiastical benefices to be denied to foreigners, fearing that the revenues from ecclesiastical offices would leave the kingdom.28 To reinforce the reestablishment of discipline in monasteries, the court sought to abolish their possibility of an appeal to the king in litigation involving regular clergy.29 The Parlement also targeted the suppression of économats.30 It set the minimum age for taking monastic vows at twenty years old and required female vows to be monitored to ensure that they involved no improper pressure.31 Finally, the judges wanted churchwardens to have to present their accounts before their parishioners and in the presence of the priest in charge.32 The Parlement clearly accepted the case for a measure of reform in the French Church.33The sovereign court was sympathetic to the idea of a general law prohibiting clandestine marriages and explicitly asked that there should be no further dispensations from publishing bans of marriage—something which had also figured in the canons of the Council of Trent. It associated itself, therefore, with one of the key decision of the Ordinances of Blois, by which marriages became public acts, to be celebrated following the publication of bans and in the presence of a priest in charge and four witnesses, upon pain of being declared null and void. The Parlement continued, however, to oppose the disinheritance of children who entered into marriage without the consent of their parents.On judicial affairs, the cahiers de doléances incessantly repeated the same message: justice was in poor shape, too slow, too expensive and open to improper influence among judges with family connections. Not altogether consonant with reality, this image allowed the king to introduce reforms into the judicial sphere, and especially into its procedures. It was also a good opportunity to emphasize the ideal of the king as source of justice. In reality, the first article on justice projected the image of a king ready to hold court in public and dispense justice in person, without judicial representation or procedures.34 The priority of the Parlement, for its part, was to safeguard the authority of its decisions. It did not take kindly to the fact that members of the royal council ‘prennent connaissance par dessus lesdicts arrests’. The basis for the rescinding (‘cassation’) or withdrawal (‘retractation’) of the court's decisions should follow the normal legal procedures, that is, by means of a civil suit and a legal error.35 Only in the second draft remonstrances, and then for the first time, did the Parlement refer to its decisions having been ‘cassez et retractez’, no doubt a reflection of a certain amount of irritation among judges regarding a practice which they deplored.36 Article 92 of the Ordinances of Blois sought to give them satisfaction on this issue and strictly limited the recourse to extraordinary legal appeal, over and above the ordinary civil suits and legal error.The cahiers of the three orders had been unanimous on the issue of cases removed from the jurisdiction of Parlement upon the authority of the king (évocations). They were a stain on justice.37 It allowed the king to interfere in a judicial process before a tribunal, remove it from its competence and either judge it himself or reassign it to another jurisdiction.38 The Parlement of Paris sought to prevent suitors from seeking évocations whose significance the Chancellor, René de Birague, had minimized in his speech to the Estates General of Blois, claiming that they had mostly occurred because of family connections and ‘menées’ among the magistrates themselves.39 It was in fact the case that these évocations were often connected to cases where there were legitimate grounds for suspicion that one of the parties to the case was related to the magistrates in the tribunal judging the case. An edict of François I had established a strict procedure that these évocations had to follow, according to which they were initially ‘deliberated upon’ by the Chancellor following a report from maîtres des requêtes.40 The Parlement concentrated its efforts therefore on ensuring that the Royal Council clarified its procedures and established clear rules.41 The Parlement of Paris wanted some limited participation from magistrates of the sovereign court in the sessions of the Royal Council. Those summoned to attend would only do so at the express command of the king and only to consider matters directly concerning the Parlement. If a magistrate proferred his advice to the Council he would not have the right to participate in the Parlement's deliberations on the same matter since, to do so, would make him judge and plaintiff in the same case. However, the king's law officers would not be invited to give their views before the Council. The Parlement set its face against the establishment of a corps or ‘college’ of magistrates from which the king might select those to serve on his Council, since he should be free to choose whomsoever he wished and without creating offices for them, ‘forme qui n’a jamais fin’.42 The Parlement had no desire to see its magistrates lured away from their ordinary jurisdiction on the pretext of prolonged service at court. On the contrary, they wanted magistrates to serve in their posts, a plea which fell upon deaf ears.The remonstrances of May 1579 reached the king at a date that we cannot determine precisely, but which coincided with the moment when the ordinance was ‘delivered’ by the king. On 20 June 1579, the sovereign court was still working its way through verifying the articles of the cahiers of the Estates General.43 On 23 June the king's law officers asked for remonstrances specifically in order to resolve the relationship between the Parlement and the Privy Council.44 On 27 June, the judges learnt that Henri III had reacted badly to their criticism.45 On 25 and 26 June the king had declared ‘with bitterness’ that he had no intention of surrendering the regalian right accorded him by the Concordat of Bologna to nominate to ecclesiastical benefices.46 For the deputies from the Parlement, however, royal authority was diminished rather than enhanced by the Concordat and they were utterly convinced that a return to the Pragmatic Sanction was desired by all his kingdom's subjects.47 For the king, by contrast, ‘ce ne seroit approuver les estats si l'on en parloit’.48 Henri III knew only too well that the issue of the right of clerical nomination had been debated at the Estates General of Orléans ‘pour les diversités qui estoit au royaume’, an allusion to the intense debates between Catholics and Protestants at that point on the reform of the Church.49 He refused to compromise on the right of nomination, which he did not want to sacrifice, but he offered assurances that he would not nominate anyone unworthy of the post whilst letting it be known that elections would provoke ‘menees’.50 The declaration indicates the degree to which Henri III had been caught unawares by a debate that must have seemed to him like a rearguard action from a far-off battle. The apparent collective amnesia of the Parlement had led its members to forget what had once been a fundamental reality: the long judicial battles at the Parlement between candidates provided by Rome and those elected locally, battles that masked the essence of the issue, which was to choose the best candidate.51 Such incoherence barely disguised the discomfort of the Parlement towards the Concordat of Bologna. It was difficult for the judges to accuse the monarchy of being partly responsible for the abuses in clerical nominations. Was not their unthinking advocacy of the Pragmatic Sanction in reality nothing other than the only means they could find to exhort the king to choose prelates worthy of their office?On the issue of the Privy Council, Henri III never regarded it as constituting an entirely separate ‘corps’. With notable firmness, he added that he ‘ne se vouloit brider et n'estoit a la cour de limiter sa puissance’. Throughout this bitter exchange of views, the Parlement could claim no victory, but equally it showed no sign of bringing the process of verifying the articles of the proposed ordinance to an early close. On the contrary, it sought to reinforce its stance, and on 3 July the magistrates decided to present their remonstrances once more before the king without substantial modifications.IIThe second set of remonstrances on the cahier of the Estates General, dated 6 July, was more incisive.52 If the Parlement was exigent it was because its members accorded the matter grave importance and sought to react to remarks made by the king on 26 June, to several deputies of the Parlement, to the effect that he wanted to know the names of those who stubbornly maintained views opposed to his own. The king threatened to come before the Parlement, and ‘sçauroit bien ceux qui sont en cause pour en faire ce qu'il faut’. The Parlement sought to defend the freedom of its deliberations which had always been respected by his predecessors. The sovereign court referred to an ordinance of December 1566 authorizing the Parlements to make reiterated remonstrances, a measure that suppressed article 2 of the Ordinance of Moulins (1566) which aimed at forbidding successive remonstrances. It was in utter ‘sincerity’ that the Parlement presented the king with what it thought to be for the good of his service and the reestablishment of his state ‘que l'on voit etre en mauvais ordre’.On the issue of the Church, the Parlement became more direct and precise. Its remonstrances insisted on the importance of the choice of good prelates, the only means as they saw it of staunching heresy and bringing civil war to an end. This was their justification for earnestly seeking a return to the system of clerical election, albeit admitting that the king could make recommendations in certain cases.53 The Concordat of Bologna had resulted inevitably in the triumph of favour over merit. They noted that the payment of annates to Rome, regarded as contrary to the ‘droits et constitutions canoniques et ordonnances anciennes’ and to the councils of the Church to which the pope ought to adhere, was nowhere to be found in the Concordat.54 Deploying the financial argument, they argued that this was money drained out of the kingdom.On justice, the second remonstrances returned to the respect that was owed to the decisions of the Parlement, the ‘guarantee’ of all property. The ‘law of the realm’—the term was employed by the judges for the first time in the process of verifying the articles of the cahier—required that judicial cases be heard in first instance before prévôts, baillis and seneschals, and in further instance before the Parlement, ‘and not before others’. It was impossible ‘d'attribuer par souveraineté la justice aux Parlements et par concurrence de cette souveraineté au Conseil privé du roy’.55 Such a ‘law of the realm’ could not be changed without provoking confusion and disorder in the ‘orders of justice’. The président Séguier, the Parlement's spokesman, went even further and asserted that the king had promised to maintain the old order of the judicial system at his coronation.56 Such rules could only be modified if there was a clear advantage to be derived from doing so, he added. It is tempting to interpret this as a desire to establish a rule of law over and above the king, but, in reality, it was more the application of the fruits of proven historical experience that respect for judicial hierarchy was the best guarantee of good justice. Séguier's argument was one from reason and experience.The Privy Council, which had instituted the greffiers criminels, should refuse to hear the suits of private individuals because ‘il n'y a loix, ny ordonnance en ce royaume qui donne puissance et autorité aux gens de sondit Conseil de connoitre des causes des personnes privées et entreprendre jurisdiction contentieuse sur les sujets du roy …’.57 The decisions of the Parlement were issued in the name of the king. If they were set aside, it was tantamount to the king ‘weakening himself’ the remonstrances deduced with impeccable logic. Upon the maintenance of the ‘choses jugées’ depended the ‘security’ of all the wealth in the kingdom, its peace and the tranquillity of the state. The risks from such initiatives were clear: the ruin and subversion of the state. The position was entirely coherent from a juridical and institutional point of view, although it conveniently ignored the fact that the king was the source of all justice.The remonstrances reminded the king that the Grand Conseil had been instigated by Charles VIII and that its role was to hear cases concerning the ‘limites des différends des Parlements’. Thereafter, however, its competence had increased with the result that it judged all sorts of suits upon évocations from the Parlements, as well as judicial tribunals in first instance, which resulted in intolerable travelling expenses for suitors.58 The remonstrances of 5 May 1579 asked the king to see that article 37 of the Ordinance of Orléans was upheld: ‘Les gens tenans nostre Grand Conseil ne connoistront desormais et ne pourront entreprendre la jurisdiction d'autres matieres et causes que de celles qui leur sont attribuées par leur creation et institution.’59 Furthermore, they recalled :Il n'y a eu lois, ny ordonnance concernant le pouvoir dudit Grand Conseil qui ayant été publiez et veriffiez audit Parlement et qui etoit et est requis par les anciennes ordonnances, et ne suffit la publication faitte par devant eux qui n'ont aucun teritoire, ne juridiction sinon limitte comme dit est. Toutefois il se trouve qu'aud. Grand Conseil les arrestz donnez esd. Parlemens, juges ordinaires et naturels y sont facilement cassez et renversez en quoy ils sont soutenu et appuyez de ceux qui voudroient s'ils pouvoient aneantir les jurisdictions ordinaires pour avoir juges a leurs volontez dont souvent a esté fait plainte au roy et a ses predecesseurs.60As in the earlier remonstrances of May 1579, the Parlement sought the suppression of the recently established commercial magistrates (juge-consuls), reformulating an argument that had already been deployed when they were created, to the effect that the king had commuted his justice to individuals without either the necessary knowledge or experience. In addition, this innovation would encourage gentlemen and other subjects of the king to seek their own judicial tribunals, not to mention the risks of collusion among merchants. In sum, the initiative risked bringing ‘schisme et division’ among the king's subjects. The views of the Parlement (and the third estate) on this subject were almost completely ignored since articles 239 and 241 of the Ordinances of Blois maintained consular justice in commercial towns. Elsewhere, however, commercial suits between merchants were assigned to the ordinary judicial institutions with the intention of limiting the proliferation of lawsuits.The increased authority of the prévôts des maréchaux was also called into question by the Parlement. These provosts had power and jurisdiction over certain crimes and misdemeanours in last resort and without appeal, a situation that the sovereign court magistrates regarded as a source of abuses and injustice. The provosts being charged with apprehending suspected criminals, it was dangerous that they should also be their judges without possibility of appeal. Their cases should be despatched by the ordinary local judges and, if the baillis and seneschals were in residence, then the provosts, along with the vice-baillis and lieutenants de robe courte served no purpose. Finally, the Parlement also opposed the pardons obtained by the prévôt de l'hôtel, acting again in final instance, for individuals ‘craignant la severité et rigueur des justices ordinaires’. The provost of the household had ‘pris un nom bien haut de grand prevot, qui n'a esté recue, ne publié au Parlement …’.61The Parlement approved, however, the reinstitution of the ordinances of François I, known as the Ordinances of Bourdaisière (May 1529) and Chanteloup (March 1546). These had laid down the regulations to be followed in respect of évocations, with the aim of preventing improper collaborations between relatives and clients in one and the same Parlement.62 However, article 118 in the proposed Ordinances of Blois required conseillers and présidents, who had a lawsuit involving even their distant relatives, to bring the case before another Parlement with the resulting absence from court and ‘punitive’ travelling costs. The magistrates of the sovereign court reminded the king that their privilege of commitimus (du petit scel, i.e. their right to bring actions in first instance before the chamber of requests of the Parlement) was ‘le plus ancient privilege qu'ils ayent’. If constrained to defend their case before another Parlement this would be completely contrary to their privileges.In the eyes of the Parlement, all these proposed changes risked compromising respect for justice: ‘Si tout le peuple de ce royaume pouvoir parler par une voix, il feroit cette requeste au roy que la justice fut remise en son premier etat et naturel et que les membres tirés et separés du corps fussent réunis et raportez a iceluy.’ At the end of the sixteenth century, to reform was not to innovate. This stubborn defence of sovereign justice was based on attachment to the old order in a period when justice was the preserve of the king, or his representative, the Parlement. Each jurisdiction should, here as elsewhere, keep to its limits and, as with royal power itself, the jurisdiction of the Parlement should itself be better defined. However, in highlighting the various encroachments upon its authority, the Parlement was also demonstrating its own weakness and the fact that it felt under threat. To present itself as the ‘voice’ of the people was perhaps a better means of making its voice heard in the dialogue between king and subjects.Despite these various different views, royal letters of constraint (lettres de jussion) of 15 July 1579 commanded the Parlement to register the cahiers ‘sans rien changer ny innover’. On 18 July, président Pierre I Séguier delivered a speech before the king which summarized the remonstrances of the Parlement. Remonstrances, when reinforced by the rhetoric of a président, might serve more effectively to persuade the king to change his mind.IIIPiety and Justice ‘are the twin columns which sustain the weight of your crown’, declared Séguier to Henri III. If the king maintained them both, God would be favourably disposed towards him: ‘Vous sçavez, Sire, les exemples si frequens en l'Escripture sur la plaincte et clameur des subjectz, Dieu se courrouce aux roys, mais ce ne sera pas à vous, si luy plaist, car nous tenons certain que vous avez souvenance du support et soulaigement de vostre pauvre peuple.’63 He insisted on the necessity of the church being made up of ‘gens bien suffisans’, fervent in piety and models of virtue for the king's subjects. Séguier then summarized the history of the provision of benefices, starting with the primitive Church, a period in which there had been no interference from kings or popes. It had been precisely because Charles VII had introduced the decrees of the Council of Basel (1431–49) into the realm by the ordinance known as the Pragmatic Sanction (1438) that God had favoured the king of France in his war against the English. Only a general council of the Church, according to the Parlement, was in a position to resolve the differences between Catholics and Protestants. The pope's authority was not above that of a general council of the Church, and he therefore lacked the authority to conclude a ‘concordat’ with the French king. On the question of the in commendam royal nomination to abbeys, Séguier made somewhat curious use of the canon of the Council of Trent condemning the choice of laymen as heads of monastic houses.64 It was a matter on which he invited the comments of the members of the Royal Council but, he continued, they could not change the fact that the Pragmatic Sanction had brought nothing but good to the kingdom, and the Concordat the reverse.The Parlement, taking careful note of the report of this encounter, categorically refused to publish the Ordinances and explicitly decided to lodge a copy of these remonstrances in its registers as a ‘perpetuelle memoyre à ce que la posterité congnoisse que le Parlement s'est mis en son debvoir’.65 This was its familiar way of protesting when it was urged. The remonstrances do not, in fact, feature in its registers although, as we have seen, we have the manuscript copies of them surviving from elsewhere. On 3 September 1579, Henri III (acting through the gens du roi and the présidents) renewed his demand that the Parlement publish those articles which they had already considered and approved before they broke for their annual recess. The Parlement noted, however, the large number of absentees when it reconvened to continue its deliberations on 5 September and refused to ‘divide’ the publication of the articles in that fashion. It was remarkably hard to stop the legal machinery from taking its course once it had begun, and on 7 September the sovereign court was instructed to proceed with its work of verifying the articles, even though it was close to the recess. That same day, the duc de Nevers appeared before the Parlement to confirm the king's will in the matter, the court replying that it ‘a faict ce qu'elle a peu et deu durant la sceance du Parlement …’. In the days that followed, a chambre des vacations (a nominated bench of judges to sit through a judicial recess) was constituted despite the king's orders. Whether Henri III had changed his mind or not, we do not know.Returning from its vacation in November, the Parlement went back to work verifying the cahiers of Blois. On 11 December 1579, conseiller Philippe de Lenoncourt came before the Parlement to remind the judges of the ‘dommage indicible’ caused by the delay in their publication and to read out to them articles 2 and 5 from the cahiers of the provincial estates of Normandy.66 The king's law officers added that the estates had the impression that the delay was caused by the king, but the discussion ended inconclusively: ‘la cour a vacqué sur ce qu'elle avoit a faire sur ce sujet jusqu'a la sonnerie de l'heure’. The following day, it was finally decided to conclude the reading of the cahiers and publish them ‘aprés plusieurs remontrances faites tant de vive voix que par escript’, but without signalling approval for the Concordat (of Bologna) and without accepting the encroachment on the authority of the baillis and the Parlements that the Ordinances implied.67Henri III rejected these reservations, as he explicitly made clear to the premier président de Thou when he was summoned for an audience with the king on 13 January 1580. The king accepted that the reservations be noted in the court's register, but not that they be included in the legal registration, whose publication had to be ‘pure et simple’.68 The king required the court to publish the cahiers otherwise he would be obliged to ‘faire chose dont il seroyt marry’ – in other words, to impose his will by force. The président Bellièvre intervened in order to defend the king's position, describing the objections made to the Privy Council as ‘chicanery’. The discussions continued on 16 January and, on 19 January, the king once more pressed the court to register the ordinances without qualification through the offices of président de Harlay. On 20 January, however, the Parlement decided to prepare further remonstrances.69 Then, three days later, after hearing about personal missives (lettres closes) sent to the premier président, whose content was never revealed, the sovereign court decided that the cahiers should be published without reservation on the following Monday, but that remonstrances be presented on the need to choose the right people to head monastic houses, and that their deliberations be registered along with their remonstrances and proposed modifications of some of the articles.70 Was this a purely formal disposition? Apparently resigned to an inevitable outcome, the Parlement's real concern was the judgement of posterity.71 Yet this was not the end of the matter because, at least until the measure was printed, discussions on the Ordinances of Blois continued.On 28 January 1580, Christophe de Thou was summoned to an audience with the king.72 Three royal valets waylaid him en route for the Louvre to tell him that the king was in no mood to compromise. De Thou outlined the different stages in the verification of an edict to Henri III, emphasizing the necessity of respecting its processes and deliberations. The Parlement never decided anything without considering it at great length. If the king did not want to take account of the remonstrances prepared by the Parlement, the royal letters had to include the customary formula: ‘fait sur le très commandement du roi’. The attempt by Michel de L'Hospital to curtail reiterated remonstrances had never been enacted and the Parlement was free to make as many protests as it saw fit. De Thou expressed the regret that the Chancellor de L'Hospital would have experienced in seeing the Parlement's rights of remonstrance restricted in that manner. By way of conclusion, the premier président returned to the main bone of contention between the Parlement and the king, namely the nomination to ecclesiastical benefices, and expressed the wish that those in future which did not conform to established norms be declared null and void. At this point, the king expressed his approval for de Thou's suggestion. The cahiers, de Thou concluded, contained some good measures and they needed to be put into practice. The Parlement took no pleasure in opposing the king's will. It acted in accordance with his conscience. De Thou reported back to the Parlement that the king had not interrupted him but listened without saying a word. The royal response was ‘pleine de modestie’. He took all that de Thou said in good part. His objective was to see the Ordinances which had been placed before Parlement obeyed, especially those concerning religion and justice. He would not tolerate any infringement of the jurisdictions of baillis, seneschals and parlements and he was firmly resolved to restrict as much as possible the granting of évocations. The king could afford to appear conciliatory since the act had been registered ‘pur et simple’, but he conceded nothing but vague promises by way of response to the Parlement.Thanks to the nuncio Dandino we know that, during the following month (February 1580), the king instructed the Royal Council to examine the Parlement's concerns on ecclesiastical benefices.73 On 1 March 1580, the président Prévost and several conseillers were once more despatched with further remonstrances on the cahiers of the Estates General, arising from the decision to register them on 23 January. It was a purely symbolic act: the Parlement continued to proffer its advice, even though its views were ignored. On 9 March, some further ‘modifications’ were proposed to some of the articles, even though they had just been published without reservation.74 In reality, it was a matter of yet more remonstrances in which the court returned to almost all the points it had previously raised. On the ‘regulation’ of justice, these ‘modifications’ concerned two essential points: to define the role of the Royal Council vis-à-vis the Parlement; and to ensure that the ordinances of Chanteloup and La Bourdaisière were applied and, with them, that the privileges of members of the Parlement were respected when they were in legal contention with members of their own family.75It was with considerable satisfaction, then, that magistrates of the Parlement received article 91 of the eventual Ordinances of Blois limiting the processes of évocation, returning to the Parlements appeals pending before the Royal Council and preventing it from judging such cases in contentious litigation in the future.76 In March 1581, however, they demanded still more, namely the complete annulment of all such contentious litigation still pending before the Council.77 The problem of the privileges of parlementaires who were in litigation with their relatives had clearly still not been settled by then since the court decided to draft new remonstrances about it. Article 97 of the Ordinances of Blois, which forbad actions of évocation de propre mouvement instigated by the king himself, seems to have been a response to one of the demands of the Estates General.78 In one of his letters, the lawyer Etienne Pasquier praised the king's decision on the matter.79 His view was that évocations were unknown in France before the time of Charles VI, which was when the duc de Bourgogne sought to divert some cases to satisfy his clients. Speaking from personal experience, he remembered that, at the beginning of his legal practice in 1549, someone who had the audacity to ask for the diversion of a case from the Parlement on the grounds of letters of evocation was cut short in his tracks and condemned to a fine for his pains. In the Pourparlers du Prince, Pasquier wrote that the sovereign court always reserved to itself the right to remonstrate to the king against évocations de propre mouvement, since all legal actions ‘doivent ‘s'accorder à raison’.80 Otherwise, ‘favourites’ would construct law from their own passions out of self-interest.The issue of évocations de propre mouvement did not disappear with the Ordinances of Blois and, even in the eighteenth century, their use remained one that was strictly controlled. What was the real impact of the Ordinances upon legal practice? The decisions of the Privy Council in Henri III's reign are difficult to classify but there are singularly few that rescind those of the Paris sovereign court and very large numbers which return cases to the Parlement of Paris, a sign of their sensitivity to the concerns of the Parisian senior magistrates as much as their respect for judicial procedure. This observation should, however, be qualified by the fact that there are substantial gaps in our evidence.81 In the immediate aftermath of the publication of the Ordinances various reactions emerged, including the evident disapproval of the papacy.82 In its remonstrances, the Parlement spoke up for the stability of an order willed by God in which everyone implicitly had their place. Its enduring concern was to reinforce royal authority. Since royalty and justice were one and the same thing, to defend the unity of justice was to defend royal sovereignty, it argued. The king's desire, by contrast, was to rectify the abuses of judicial style and practice by ever more technical legislation with the objective of speeding up the delivery of justice. The sovereign court wanted the king to lay down the details of legal procedure, but it also wanted to preserve its power of remonstrance.83 In a general sense, this led to a certain consensus of views.84 So, for example, the Parlement could only welcome the prohibition upon extraordinary commissions (article 98), and equally approve the articles which laid down a summary procedures in justice so that petty cases involving sums under three écus could be despatched quickly (articles 153 and 154). On 25 January, Pierre de l'Estoile noted ironically in his diary:fust publié en la Cour de Parlement l'Edit fait et arresté après longue deliberation de la Cour sur les cahiers des Estats tenus à Blois, en l'an 1577, auquel y a beaucoup de belles et bonnes ordonnances: lesquelles, s'il plaisoit à Dieu et au roy qu'elles fussent bien observées, tous les Estats et peuple de France en seroient grandement soulagés et satisfaits, mais est à craindre qu'on en die, comme de l'Edit des Estats d'Orleans et de toutes autres bonnes ordonnances faites en France: Apres trois jours, non vallables’.85For Jacques-Auguste de Thou it was ‘le malheur des tems’ that prevented numerous laws from being enacted.IVAccording to Edouard Maugis, behind a possible secret complicity between the Estates General and the Parlement to secure their ascendancy there lay an eventual victory for the Parlement, distinct from the Estates General and with a jurisdiction superior to it, as demonstrated in its right to revise and verify the ordinances that resulted from the cahiers de doléances of the Estates.86 It would be unwise, of course, to underestimate the rivalry between these bodies (permanently constituted or not), each wedded to its own specificity and role.87 But if we put to one side this notion of a perpetual confrontation, what do we find? On the one hand, the deputies of the Estates General petitioned the crown to respect the rights of verification of laws in the Parlements, but on the other to reject meddling in the cahiers that resulted from their petitions.88 In these circumstances, why did the crown ask for the advice of the Parlement when it was a body from which juridical cautiousness was only to be expected? On the other hand, the Parlement customarily proferred its opinions when asked to comment on all sorts of royal edicts. By the end of the sixteenth century, the verifying of the cahiers of the Estates by the Parlement had become commonly accepted if we accept the view of conseiller Guillaume Du Vair, who vigorously asserted that the decisions of the Estates General would not have the force of law unless they were verified by the Parlement.89 Yet, as we have seen from the preceding analysis, verification was a mere formality when it was a matter of purely and simply publishing the measure as it was presented to them. In reality, the Parlement of Paris made its voice heard, but it did not seek to intervene decisively in the dialogue between the crown and its subjects. Or, at least, it only sought to do so in the more labyrinthine secret discussions that took place between its procureur général and the king.90In 1588, during the session of a subsequent Estates General, the magistrates of the Parlement were asked to present their own particular cahier of remonstrances.91 The king wanted them to establish the main lines of discussion to be treated in the Estates. Following custom, however, the Parlement submitted its remonstrances only after the Estates General had met. No doubt Henri III wanted to use the Parlement as a means of moderating the views of the Estates General, whose deputies were in large majority members of the catholic League. Not surprisingly, the remonstrances which it drew up in August 1588 were based on those prepared for preceding assemblies of the Estates General. Then, in 1593, in a completely different political context, the Parlement solemnly refused to depute any of its members to attend the Estates General of the League.92 The Parlement verified edicts only following the king's instruction to do so. In that respect the court was a faithful instrument of royal authority. Unlike the situation in 1560–61, when his brother had been forced by financial necessity to do so, nothing required Henri III to ask his sovereign court for its views on the cahiers. In inviting the Parlement to express an opinion, was not the French monarchy providing it with the opportunity to seize the initiative? Little by little, the court was inclined to become a more independent agent in its relationships with both the Estates General and the king. So, to look ahead, to 1615, four days after the closing session of the Estates General on 23 February, the various tribunals of the Parlement met in general session along with the princes and the peers of the realm to solemnly inform the king of the wretched state of the government of the realm. To justify this bold and unprecedented initiative, the Parlement of Paris used the promise that the king had made not to respond to the cahiers of the Estates General without taking account of its views.93 The Parlement no longer wanted to be treated separately and it no longer patiently waited for the royal command before it intervened. This change in attitude represented a profound shift in its relations with royal authority.1A[rchives] N[ationales] X1A 1596, fo. 20v (23 Nov. 1560).2AN X1A 1653, fo. 239v (6 Sept. 1576).3This had been its view since 1413: G. F. Denault, ‘The legitimation of the Parlement of Paris and the Estates General of France, 1560–1614’ (PhD Thesis, Washington University, 1975), 243, cited by A. E. Bakos, ‘Meddling chaperons: the involvement of the Parlement of Paris in the Estates General of 1593’, in idem, Politics, Ideology and the Law in Early Modern Europe (Rochester, 1994), 93.4AN X1A 1598, fo. 117v (2 Aug. 1561).5On 4 Aug. 1561, the sieur de Selve, a conseiller in the Privy Council, came before the Parlement to explain that the king was awaiting a definitive response from the Estates General, currently meeting at Pontoise, to his demands for money. However, this response would only be forthcoming when the publication of the Ordinance had been completed. De Selve was charged ‘d'alléguer [to the Parlement] que comme comprinse ausd. estatz, lad. Court en deust estre’.6Lalourcé and Duval (eds), Recueil de pièces originales et authentiques concernant la tenue des Etats généraux, 9 vols (Paris, 1789), i. 330. Guillaume de Taix, clerical deputy at the Estates of Blois in 1576–7, expressed the same conviction: ibid., ii. 124.7L. Petris, La Plume et la tribune. Michel de l'Hospital et ses discours (1559–1562) (Geneva, 2002), 386. Among the jurists, opinions were more divided. Jean Papon considered that the king could not proceed to alienate the royal domain without the consent of the Estates General. If they were not in session, that duty fell to the Parlement of Paris which, unlike the Estates, sat in permanent session and regularly offered counsel to the king: Secrets du troisième et dernier notaire (Lyon, 1578), 318. Philibert Bugnyon, however, thought that ordinances which had been deliberated in plenary sessions of the Estates General did not need to be submitted to the Parlement for registration: Commentaires sur les ordonnances faictes par le roy Charles neufiesme en sa ville de Moulins au mois de fevrier mil cinq cens soixante six (Lyon, 1567), 17, cited in A. Rousselet-Pimon, Le Chancelier et la loi au XVIe siècle d'après l'œuvre d'Antoine Duprat, de Guillaume Poyet et de Michel de L'Hospital (Paris, 2005), 166.8AN X1A 1598, fo. 173r–v (9 Aug. 1561).9In 1467, the Parlement had deputed the premier président Boulanger and a dozen councillors to the Estates, but this group was sent ‘non comme un corps mais afin de donner conseil’. In 1484, following royal instructions, the Parlement elected to send a deputation of four of its number but in fact none of those chosen ever left Paris: E. Maugis, Histoire du Parlement de Paris de l'avènement des Valois à la mort d'Henri IV, 3 vols (Paris, 1913–16), i. 656.10In reality, it was less the propositions of the premier président which had attracted this censure, but rather the Parlement's delay in ratifying the cahier of the Estates General of Orléans just at the moment when the royal government was in discussion with the Estates’ representatives at Pontoise in August 1561: S. Daubresse, Le Parlement de Paris ou la voix de la Raison (Geneva, 2005), 253.11According to Maugis, the delay was part of the monarchy's strategy of playing a waiting game to obliterate the memory of the Estates General of Blois: Histoire du Parlement de Paris, i. 671. In reality, Henri III was above all preoccupied to reestablish peace, because the edict of Beaulieu had been the prelude to renewed conflict. It was only thanks to pressure from the provincial estates and efficiency of the new chancellor, Hurault de Cheverny, that the new Ordinances were eventually promulgated.12M. Greengrass, Governing Passions: Peace and Reform in the French Kingdom, 1576–1585 (Oxford, 2007), 267.13J.-A. de Thou, Histoire universelle de Jacques-Auguste de Thou, depuis 1543 jusqu'en 1607, traduite sur l'édition latine de Londres (London, 1734), vol. 7, 74–5. He devoted only a few lines to the subject.14Maugis, Histoire du Parlement de Paris, i. 671: ‘Inutile d'entrer dans le détail qui remplirait un chapitre’.15On 2 Aug. 1561, the Parlement received the text of the ordinances of Orléans. On 30 Aug. their draft remonstrances to the king were read out: AN X1A 1598, fo. 295r–v. Thereafter nothing is recorded until 13 Sept. 1561, the date when the ordinances were registered.16AN X1A 1663 – sessions of 5, 6, 7, 9, 10–14, 16, 17, 19, 24 and 26 Mar. 1579. On 2 Mar. it was decided that two hours per day would be devoted to the processes of verification.17AN X1A 1664 fos 32v and 33, (6 May 1579). On article 157, the magistrates noted: ‘Sera fait registre a part et ne sera employé en la remonstrance que l'on fera au roy’. This article required ‘a nos procureurs et avocats generaux de prendre le moindre nombre de substituts qu'il leur sera possible’. It prohibited these substituts from accepting payments from suitors in return for furnishing requêtes, informations, interrogatoires or other trial documents. Guy Coquille regarded the proposal as inconsistent since it could not be right to ask substituts to serve without any payments at all: Oeuvres de Me Guy Coquille, sieur de Romenay (Paris, 1646), 512.18The two texts also exist in copies to be found in AN U 768 fos 265–327.19B[ibliothèque] N[ationale] MS Fr 2703 fos 204v–209, ‘Arrestez de la cour sur le cahier des Estats generaux faits avant les remonstrances de l'Estat ecclesiastique en l'an 1579’. The assembly of the clergy, meeting with the king's consent at Melun, began its deliberations on 26 June 1579 with an examination of the complaints about the abuses which had made their appearance in the French church.20In the latter, a straightforward ‘bon’ is inscribed against 202 of the 363 articles. We should note that a third of these concerned judicial affairs, and that there is a mismatch between the number of articles that figure in the remonstrances and those that appear in the eventual ordinances, which makes following the remonstrances somewhat difficult. This was because six articles were eventually struck out at the king's command and removed from the final published legislation.21Concerning article 97: ‘au lieu de l'article ce qui ensuit sera mis: Nous voulons ….’: ibid., fos 210r–v.).22BN MS Fr 4398 fos 337–339v, ‘Remonstrances faictes au roy par sa cour de Parlement sur l’édit fait sur leurs cahiers généraux du 5 may 1579’. Concerning article 5, the court ‘persiste aux remonstrances qu’elle a faictes sur le premier et deuxieme article ….’ (my emphasis). In other respects, this copy begins with a more developed and formal preamble in praise of the king's bountiful goodness and his desire to reform corruptions and abuses, and includes a reference to the imperative necessity of securing obedience to the law. This would be the version of the remonstrances presented to the king: ibid., fo. 337v.23The deliberations of the Parlement are never transcribed into its parchment registers: F. Hildesheimer, ‘Exemplaire Parlement’, in ‘Fabrique des archives, fabrique de l’histoire’, Revue de Synthèse, 125 (2004), 49–51. The deliberations of governmental organizations and courts of justice always took place in secret.24The papal nuncio Dandino, abreast of the latest news, told the Cardinal de Côme in a letter of 12 Apr. 1579 that the Parlement was in the course of deliberating on its responses to the clauses from the Estates of Blois on ecclesiastical matters: I. Cloulas (ed.), Correspondance du nonce en France, Anselmo Dandino, 1579–1581 (Rome, 1970), 367. From his letters we know that it was the Keeper of the Seals René de Birague who kept the nuncio informed on the discussions in the Parlement: ibid., 424, 15 June 1579.25BN MS Fr 2703 fo. 204r article 1.26Article 14 of the Ordinances of Blois enforced this obligation upon absentees on pain of their being deprived of the revenues of their sees.27The indult allowed conseillers of the Parlement to hold ecclesiastical benefices (if they were conseillers-clercs) or present a candidate of their choice (if they were laymen). Conseillers-clercs were given dispensation from residing in their benefices.28This was a demand that had already been voiced by the procureur général in his conclusions upon the cahiers of the Estates General of Jan. 1561: Lalourcé and Duval, Recueil, i. 333.29This issue was eventually dropped and did not figure in article 30 of the eventual Ordinances.30An économat was instituted in the six months of interregnum in a benefice prior to the issuing of a bull of institution to the next incumbent, during which period its revenues were handed over to an économe appointed by the king.31BN MS Fr 2703, fo. 206, article 28, in which those entering religious orders could dispose of their possessions up to three months after their final vow. The clause seems to reflect almost exactly the draft proposed by the Parlement save that the minimum age was set at sixteen years, a choice reflecting the demands of the clerical deputies. Cf. the clause in the same article concerning female vows: ‘et outre les abbesses et superieurs auparavant que faire bailler aux filles les habits de professes pour les recevoir a profession seront tenus un mois devant avertir l’evesque, son vicaire ou superieur de l’ordre pour s’enquerir par eux et informer de la volonté des dictes filles, s’il y a eu contraincte et induction de leur faire entendre la qualité du veu qu’ils vont faire auparavant qu’elles s’obligent’.32Their proposal was that they should be required to do so within three months of leaving office (article 53) but this was eventually dropped from the final version of the Ordinances.33According to Jean-Marie Le Gall, the Parlement had received numerous appeals early in the sixteenth century from those in the religious orders hostile to reform (‘déformés’), but its decisions were generally in favour of those who sought reform: Les Moines au temps des réformes, 1480–1560 (Seyssel, 2001), 116. Megan Armstrong's study indicates that the Parlement sought to limit the number of appeals from ecclesiastical to royal justice (appels comme d’abus), often pursued by those in monastic orders who regarded themselves as victims of decisions that had gone against them: ‘Spiritual reform, mendicant autonomy, and state formation: French Franciscan disputes before the Parlement of Paris, 1500–1600’, Fr Hist Stud, 25 (2002), 505–30.34See article 89 of the Ordinances of Blois. Jacques Krynen has recently drawn our attention to Jean Bodin's notable reticence about this aspect of royal justice, expressed in the Six livres de la République which had appeared three years earlier in 1576 (Book IV, ch. 6). Bodin invited the king not to seek to deprive magistrates of their authority on the grounds that he (the king) was not a jurist. Coquille, however, gave the article of the Ordinances of Blois his unqualified approval: Oeuvres, 493.35BN MS Fr 2703 fo. 209v. Suitors who had not received satisfaction from legal decisions arrived at by a parlement sought to demonstrate that the court had committed an error and to adduce the grounds for their view. If the latter were found to have weight, the king rescinded the decision of the court without questioning the substantive issues of the case. The Royal Council only considered the legal grounds for an appeal and referred the case back to the Parlement for a new hearing of the evidence. The point is an important one since the rescinding of judicial decisions on such grounds was also used by the magistrates themselves, admittedly in the exceptional circumstances of the split of the Parlement of Paris into two tribunals in the League between 1589 and 1594, when it was a device used by each court against the other: S. Daubresse, ‘De Paris à Tours, le Parlement “du roi” face au Parlement ”de la Ligue” (1589–1590)’, in Le Parlement en exil ou Histoire politique et judiciaire des translations du parlement de Paris (XVe–XVIIIe siècle), ed. S. Daubresse, M. Morgat-Bonnet and I. Storez-Brancourt (Paris, 2007), 466–70.36BN MS Fr 4398, fo. 344r.37G. M. R. Picot, Histoire des Etats Généraux considérés au point de vue de leur influence sur le gouvernement de la France de 1355 à 1614 (Paris, 1872), vol. 2, 568.38A. Rigaudière, Introduction historique à l’étude du droit et des institutions (Paris, 2006), 581. An évocation could occur following a decision by the king himself, or it could be the result of an action instigated by one of the parties to the suit.39Lalourcé and Duval , Recueil, ii. 63–4.40The report of the maîtres des requêtes was made a requirement by the edict of La Bourdaisière of May 1529. The Parlement derived the oversight of evocations back to an ordinance of Charles VI of 15 May 1389 as well as another of Louis XII of 22 Dec. 1499: BN MS Fr 2703 fo. 210v and, above all, the entry on ‘évocation’ in the Encyclopédie of Diderot and d’Alembert.41Even under the reign of Louis XIV, the Parlement was still trying to win this battle, as Albert N. Hamscher demonstrates in The Conseil Privé and the Parlements in the Age of Louis XIV: A Study in French Absolutism (Philadelphia, 1987), 80 and 107.42It is notable that the Parlement of Paris never offered a word of advice on the overall composition of the Royal Council.43According to the nuncio Dandino's correspondence with the Cardinal de Côme, their deliberations on ecclesiastical matters continued throughout May: Correspondance, 383, letter of 4 May 1579; and 386–7, letter of 10 May 1579. On 13 May, Dandino had word of the initial responses of the Parlement on ecclesiastical affairs.44AN X1A 1664 fo. 292, (23 June 1579). There were some discussions between the king and the members of the prosecutor’s office, the latter declaring that the king would enter into discussion with the papacy over the issue of papal annates.45AN X1A 1664, fos 338v–339v, (27 June 1579).46On 21 June, nuncio Dandino reported the identical view of the king, only too well aware of how much the authority to nominate to benefices mattered to him, in a letter to the Cardinal de Côme, a letter in which he totally rejected the magistrates’ views. According to the nuncio, the judges of the Parlement were seeking to poison the good relationship between the king and the papacy.47The Parlement relied on the fifteenth-century councils of the Church, which forbad the obtaining of benefices for money (‘c’estoit trafiquer’) as well as the ordinances of St Louis and Philip the Fair. We should note that the Parlement conflated the right of nomination with the problem of benefice provision. The return to election to benefices was one of the demands of the peasants studied by J.-M. Constant, ‘Le langage politique paysans en 1576: les cahiers de doléances des bailliages de Chartres et de Troyes’, in Représentation et vouloir politiques autour des Etats généraux de 1614, ed. R. Chartier and D. Richet (Paris, 1982), 31, although, as the author explains, they hoped to be able to elect their own clergy.48In their cahiers the third estate and the clergy had demanded a return to clerical elections. The nobility, however, proposed a commission of inquiry before the election of a new prelate, a suggestion that was seized upon by the Royal Council, since it incarnated the notions of the Concordat of 1516: Picot, Histoire des Etats Généraux, 393–4.49O. Christin, Les Réformes: Luther, Calvin et les protestants (Paris, 2007), 23–4.50According to nuncio Dandino, the clergy had obtained assurances from the king that he would not in future allow benefices in commendam and that he would pay no more annates to Rome: Correspondance, 469, Dandino to Côme, 8 Aug. 1579.51V. Julerot, ‘Y a un grant desordre’: élections épiscopales et schismes diocésains en France sous Charles VIII (Paris, 2006), 429–30. She notes the inefficacy of elections, the mismatch between theory and practice, and emphasizes the problem of confirming the result of a clerical election.52BN MS Fr 4398 fos 367–394v, ‘Les secondes remontrances sur les cahiers des Etats generaux, du 6 juillet 1579’, signed de Thou and Viole. The articles are not enumerated.53BN MS Fr 4398 fo. 373. Ever since the Pragmatic Sanction, the king made his recommendation to the ‘elisans’ and ‘celuy qu’il plaisoit au roi etoit ordinairement gratifié et preféré s’il etoit capable et digne’. In his reply to the cahiers of the Estates General of Orléans of 1561, the procureur général then in post had proposed that episcopal elections should involve the archbishop, the cathedral chapter and ‘douze des principaux habitans et bourgeois desdites villes qui seront élus en l’hostel d’icelle par les maire et echevins, consuls et conseillers desdites villes’. The candidate then elected would, of course, be presented to the king.54For président Pierre Séguier, annates were contrary to the word of God and human law: AN X1A 1665 fo. 13, (18 July 1579).55BN MS Fr 4398 fo. 377v. This was to create an ‘incompatible’ jurisidictional conflict and confusion, declared président Séguier on 18 July 1579: AN X1A 1665 fo. 14.56AN X1A 1665 fo. 14, (18 July 1579): ‘Sire, les loyx civilles et politicques dient qu’il ne fault poinct changer ne innover les reigles par lesquelles par le passé ont esté trouvees bonne synon que l’on trouvast au change ung bien et commodité publicq … Sire, l’ordre que dessus est l’ancien de vostre royaulme que vous avez promis a vostre sacre garder et entretenir.’57To Guy Coquille it was evident that the Privy Council was instituted to ‘connoître des affaires generales, non pour les affaires des particuliers’. It was made up of princes and other seigneurs who lacked knowledge of laws, customs and legal conventions: Oeuvres, i. 493.58The third estate equally complained that the Grand Conseil encroached upon the Parlements: Picot, Histoire des Etats Généraux, 567.59The Parlement noted that the letters patent creating offices were addressed to the Grand Conseil ‘chose directement contraire a l’institution et establissement dudit Parlement, ce qui apporte toute confusion a la justice’.60BN MS Fr 4398 fo. 388.61BN MS Fr 4398 fo. 385v, ‘Le Parlement suplie le roy puisqu’il luy plaist remettre la justice en sa splandeur et premier estat pourvoir sur ce fait que les appellations dud. Prevost de l’Hostel retourneront au Parlement.’62F. A. Isambert et al. (eds), Recueil général des anciennes lois françaises depuis 420 jusqu’à la Révolution de 1789, 29 vols (Paris, 1822–33), xii. 312–15 and 908–9.63AN X1A 1665 fos 12–15, (18 July 1579). The président also presented the king with a ‘plus ample’ memorandum from the Parlement.64AN X1A 1665 fo. 13v, ‘La commende en terme de droict commun ne vault rien, et mesme par le concile de Trente, elle est damnee et resprouvee’. The in commendam involved the collation of an ecclesiastical benefice to a cleric who was not in regular orders, or even to a layman, and the Concordat had conceded to the papacy the in commendam right: D. Richet, ‘Une famille de robe: les Séguier avant le chancelier’, in idem, De la Réforme à la Révolution: études sur la France moderne (Paris, 1991), 230.65AN X1A 1665 fo. 14v (18 July 1579).66AN X1A 1666 fo. 110v (11 Dec. 1579).67AN X1A 1666 fo. 114v–115 (12 Dec. 1579).68AN X1A 1666 fo. 237 (15 Jan. 1580). Nuncio Dandino reported the king's insistence on the matter to the Cardinal de Côme although the Parlement, he added, continued to press for the restoration of elections to ecclesiastical benefices and a return to the Pragmatic Sanction: Correspondance, 564.69AN X1A 1666 fo. 274, (20 Jan. 1580).70AN X1A 1666 fo. 281v, (23 Jan. 1580). The injunction on the registration simply reads: ‘Leues, publiees et registrees oy le procureur general du roy après plusieurs deliberations et remonstrances tres humbles faictes audict seigneur’: AN X1A 8635 fo. 184v.71As Maugis noted, Histoire du Parlement, i. 671, the Parlement was much preoccupied by ‘opinion’.72AN X1A 1666 fos 312–314v, (28 Jan. 1580).73Letter of 17 Feb. 1580, in Correspondance, 601.74BN MS Fr 4398 fos 466–471, ‘Modifications sur le cahier des estats de Blois du neuf mars mil cinq cens quatre vingt’, signed de Thou and Viole. After the Estates General of Orléans, equally, the printing of the cahier was delayed because the Parlement sought to make yet further modifications: AN X1A 1598 fo. 412, (27 Sept. 1561).75Article 117. The Parlement wanted this article modified.76Isambert et al., Recueil général, xiv. 404. According to Guy Coquille, it was an issue pursued by the Paris deputies, who ‘firent grande instance pour l’obtention de cet article’: Oeuvres, i. 493.77BN MS Fr 4398 fos 467r–v, ‘Sur le quatre vingt unziesme, le roy sera suplié de declarer toutes et chacunes les procedures qui seront faites en son Conseil nulles és causes de jurisdiction contentieuse et que les parlemens n’auront esgard a cela’.78If a case was to be evoked out of a jurisdiction, it had to be with good cause and the subject of a report prepared by the masters of requests (the Parlement also wanted oversight by the chancellor or Keeper of the Seals, assisted by the masters of requests). A signature of the secretary of state was also required by article 70 of the Ordinances of Moulins (1566).79D. Thickett (ed.), Lettres familières (Geneva, 1974), 71–5, to René Choppin, avocat at the Parlement of Paris.80E. Pasquier, Pourparlers, ed. B. Sayhi-Périgot (Geneva, 1995), 102.81F. Dumont (ed.), Inventaire des arrêts du Conseil privé (règnes de Henri III et de Henri IV) (Paris, 1969–71), vol. 2. The inventory only covers the July quarter of each year, beginning in 1579 and ending in August 1588. I am grateful to Solange Bertheau for allowing me to consult her subject catalogue before it becomes publicly available. Of the 1774 entries for the reign of Henri III in the inventory, there are three arrêts of the Parlement rescinded (‘cassés’), three others which forbad the Parlement from considering a suit, five which order the closure of proceedings in a suit, three that countermanded a decision of the court on the grounds of legal error, and only one resulting from a civil action against its decisions. There are eight evocations of suits to the Conseil privé, Grand Conseil or other jurisdictions. By contrast, seventy-six cases were sent back to the Parlement of Paris, sometimes after an attempt at an évocation (even a case sent before the Grand Conseil was returned to the Parlement: ibid., i. 34, no. 240 [12 Aug. 1580]).82Commentaries of Coquille in Oeuvres, 462–9; also ‘l’Advis de M. P. Pithou, advocat en Parlement sur l’ordonnance de Blois de 1576’, in A. Loisel, Divers opuscules des mémoires de M. Antoine Loisel, advocat en Parlement (Paris, 1652), 345–52. Cf. V. Martin, Le Gallicanisme et la Réforme catholique: essai historique sur l’introduction en France des décrets du Concile de Trente, 1563–1615 (Paris, 1919), 170. The papacy had great difficulty in accepting a measure in which the king was placed in a position of absolute superiority over bishops. It demanded its complete revision, sending a new nuncio to put its case.83L. de Carbonnières, ‘Le style du Parlement de Paris et la législation royale (XIVe–XVe siècles)’, in Modèles français: enjeux politiques et élaboration des grands textes de procédure en Europe: les enjeux politiques de la cofidication de la procédure (Paris, 2008), vol. 2, 171–93. According to Carbonnières, neither in the preamble, nor in the articles of the Ordinances of Montils-lès-Tours (1446), nor those of 1454, was there any reference made to the ‘style’ of the Parlement (i.e. the legal rules governing its procedures), not even in terms of ways of modifying it. The same was true of the Ordinances of Blois. Jean-Louis Thireau also noted that the legislative initiatives of the sixteenth century ‘fait entrer pleinement la procédure dans le domaine de la loi …’: ibid., 211.84They came up against difficulties on article 149, which forbad appeals on grounds other than procedural ‘si ce n’est pour le vuider et juger sur le champ’. In its comments on the majority of the other articles, the Parlement wanted to alter the wording, which had some juridical significance but did not change the overall legislative intent.85P. de l’Estoile, Registre-journal du règne de Henri III, ed. M. Lazard and G. Schrenck (Geneva, 1992–), vol. 3, 91–2.86Maugis, Histoire du Parlement, i. 670. Rousselet-Pimon thinks that the pretensions of the Parlement to interfere in the royal legislation, proposed in cooperation with the estates general, represent a much greater menace to royal power in the sixteenth century than that of the estates general themselves: Le Chancelier et la loi au XVIe siècle, 166.87Pierre de La Place, premier président in the Cour des Aides, thought the Parlement was not required to verify the decisions of the Estates concerning royal impositions: Commentaires de l’Estat de la Religion et Republique soubs les rois Henryy et Francois seconds, et Charles neufiesme (n.p., 1565), 157. In this respect, he was protected by the prerogatives of his own jurisdiction.88Picot, Histoire des Etats Généraux, 561–2: the third estate reminded the crown that ‘de tout temps et par l’institution de la France, nul édit ne doit être reconnu pour édit au préjudice des anciennes lois et ordonnances de France, s’il n’est premièrement vérifié par les cours souveraines’. In Nov. 1576, Blanchefort, the noble deputy expressed his desire to see the king nominate those members of his Council who would negotiate the ordinances with the Estates and then submit them to Parlement, simply for their approbation: Greengrass, Governing Passions, 90. In Dec. 1576, the deputies of the clergy wondered, in the course of a meeting with the other order, whether the reforms demanded by the three orders would be automatically adopted by the king and submitted to the Parlement of Paris as a ‘loi inviolable’: ibid., p. 92. The Parlement was here viewed above all as a guardian of the laws.89In 1593, Guillaume du Vair wrote in his Suasion: ‘veu que ce qui a accoustumé de se résoudre aux Etats généraux de la France bien et légitimement assemblez n’a force ny vigueur qu’après qu’il a esté verifié par vous séans au throsne des rois, au lict de leur justice, en la cour des pairs’, cited in Maugis, Histoire du Parlement, i. 673.90In Dec. 1576, the procureur général Jean de La Guesle presented two documents on ecclesiastical and judicial affairs before the Estates General in the name of the king. These were a project for reform whose origin had lain in a text prepared by the duc d’Anjou (the future Henri III) for the Council of Charles IX on the eve of Anjou's departure for Poland: Greengrass, Governing Passions, 93–6. Such ‘particular’ remonstrances have not been the focus of this article. In a letter of 22 Feb. 1580, nuncio Dandino reported that the responses to the Estates of Blois were in the hands of the procureur général for final modifications before being printed: Correspondance, 605. It was surely at that point that the six articles, struck out on the king's orders (four of which were about judicial matters), were removed: Isambert et al. Recueil général, xiv. 462–3). They concerned issues of legal procedures and were perhaps ‘ostés par commandement du roy’ at the request of the procureur général. They were not the object of any remonstrances by the Parlement itself.91AN X1A 1711 fos 81r–v, (9 Aug. 1588). In a letter of 6 Aug. 1588, Henri III asked the Parlement to submit a memorandum that he intended to present to the Estates General.92Maugis, Histoire du Parlement, ii. 100. Five parlementaires attended the Estates General in 1593, but that was because they were deputed from the third estate of Paris.93E. Glasson, Le Parlement de Paris, son role politique depuis Charles VII jusqu’à la Révolution (Paris, 1901), 123–4. Marie de Médicis issued an arrêt de cassation to rescind the arrêt of the Parlement which had summoned the tribunals and peers to meet in common assembly. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0AEBE99971E44E4AA329E1B8B8EF05C82EAB3BC7.txt b/test/dataset/in/resources/corpus/Clean_0AEBE99971E44E4AA329E1B8B8EF05C82EAB3BC7.txt new file mode 100644 index 0000000..56a27a9 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0AEBE99971E44E4AA329E1B8B8EF05C82EAB3BC7.txt @@ -0,0 +1 @@ +ohrohrThe Oral History Review1533-85920094-0798Oxford University Press10.1093/ohr/ohn058ARTICLESSerbian Gypsy Narrative: Between Preferred and True IdentityČvorovićJelenaJelena Čvorović (Ph.D., 2001, Anthropology, Arizona State University, AZ, US) is a long-time student of Gypsies in Serbia. During the past several years, she has authored many articles dealing with various aspects of Gypsies and their traditions. Jelena's strong interests lie in the area of human evolution and behaviorWinter-Spring200917320093614570© The Author 2009. Published by Oxford University Press on behalf of the Oral History Association. All rights reserved. For permissions, Please e-mail: journals.permissions@oxfordjournals.org2009This paper discusses the narration of a Serbian Gypsy who adopted Serbian ethnic identity. Still today in Serbia, Gypsy culture remains in the oral form. Their narratives tell as much about their present as about their past. Several themes underlying the discussion about Gypsy ethnic flexibility are explored: their position in relation to non-Gypsies, the way they perceive it; varying attitudes about ethnicity within Gypsy communities in contrast with personal experiences of a Gypsy who adopted Serbian identity; and the evolving nature of Gypsy identities.ethnicitycommunitiesoral historypreferred ethnic identitySerbian GypsiesIntroductionThis paper presents the voice of a Romanian Gypsy who declares himself to be “a true Serb.” The oral history presented comes from a Gypsy community in the village of Drenovac, in western Serbia's county of Mačva, and makes a contribution to understanding the flexibility of Gypsy ethnic identity. The account reveals that many Gypsy behaviors can be better appreciated by placing together the narration of local people with anthropological objective data.Gypsy communities in Serbia still rely on oral tradition as a source of obtaining knowledge and information on the history of their ancestors, proper kin behavior, economic life, the societies in which Gypsies live, relationships with non-Gypsies, and the everyday world.1 Many times, there are no written documents or historical records that enlarge our understanding of the Gypsy past and the choices they made. Even today, many Gypsies in Serbia are illiterate or semi-literate, but their oral tradition remains transmitted, as a form of cultural heritage, from one generation of kin to the next. Recording their oral histories provides one way of breaking this silence and allows Gypsies, otherwise marginalized on all levels, to speak for themselves about issues important to them.The following interview with a Romanian Gypsy, narratives, and interview quotes I collected in several Mačva villages provide an opportunity to explore several themes related to Gypsy ethnic flexibility: (1) the position of Gypsies in relation to non-Gypsies, and how they perceive this relationship; 2) varying attitudes about ethnicity within Gypsy communities in contrast to the personal experiences of a Gypsy who adopted a Serbian identity; and 3) the evolving nature of Gypsy identity.BackgroundRoma/Gypsies are a diverse ethnic group probably of northern Indian origin, scattered throughout Europe since arriving west of the Balkans in the fifteenth century.2 It is unclear why they left India, and there are no explanatory written documents. From linguistic influences preserved in all Romani dialects, it is most probable that the major Gypsy migration routes passed through Persia, Armenia, Greece, and the Slavic-speaking parts of the Balkans.3 A number of early European historical sources refer to the Gypsies as Egyptians, and the term “Gypsy” is thought to mirror that assumption. According to historical sources, the Gypsies themselves spread the legend about their Egyptian origin; they represented themselves as dukes, kings, and princes from Egypt.4 The inventive process of Gypsy traditions and favored identity in Europe had begun. Roma/Gypsies had begun their sojourn in Europe by taking advantage of the Christian piety of the age. Since it was a Christian obligation to help pilgrims, especially the ones with documents of recommendation from rulers, the Gypsies created letters of passage from high government officials such as King Sigismund of Hungary, representing themselves as penitents for the sins of their ancestors who had rejected Christian teachings in Egypt. As a result of the sins of their ancestors, they were required to wander the earth as pilgrims seeking charity from others.5Today linguistic and historical data, supported by new genetic studies, suggest that the European Gypsies, embracing a large number of socially different endogamous groups, may be a complex conglomerate of founder populations that originated in India.6 Genetic results suggest a limited number of related founders, compatible with a small group of migrants splitting from a distinct caste or tribal group. Individual Gypsy groups can be classified into major metagroups: the Gypsies of East European origin; the Sinti in Germany and Manouches in France and Catalonia; the Kaló in Spain, Ciganos in Portugal and Gitans of southern France; and the Romanichals of Britain.7 Out of these, the greatest variety is found in the Balkans, where numerous groups with well-defined social boundaries exist.8The present study of favored ethnic identity cannot be separated from the wider study of the history of society. In spite of their presence in Europe for centuries, Gypsy integration into European society is poor, and their history is a true story of exclusion and discrimination. Gypsy hunting and other such persecutions have occurred almost from the beginning of the Gypsy presence in Europe, with a peak during World War II.9Today the position of Gypsies in Central and Eastern Europe is more or less the same.10 In Central and Eastern Europe, the policy of socialist governments was to assimilate Gypsies and for decades they were targeted for low-skilled employment within a centrally planned economy.11 Efforts were made to help Gypsies to settle down and to improve their economic and cultural position in the postwar years. This resulted in the banning of nomadism in most of the countries in the region and the destruction of traditional Gypsy occupations.The collapse of the Eastern European socialist system in 1989 was followed by a long and still incomplete period of transition in Serbia. In Serbia, the transition started at a time when the economy was near or at the point of collapse, and as a result impoverishment and unemployment rose significantly.12 Protected to some extent by the socialist regime, Gypsies have been perhaps the most affected by the transition to a market economy because of their lack of a qualified labor force. Thus they have high levels of unemployment, substandard housing, a lack of education and skills, and a deepening dependence on state benefits and services.13 The general situation in Serbia contributed to the present day Gypsy condition. Serbia's economy has been degraded by a decade of war, isolations, and sanctions. On the other hand, due to the assimilation processes and manipulation of their ethnicity by the Roma themselves, many Roma today deny their Roma ancestry.Firstly, not all Roma people refer to themselves as Roma. Some prefer the European term cigani or zigeuner. Secondly, some European groups/tribes of Roma reject altogether the connection with Roma people in general, like the Sinti and Manouches.14 This is perhaps particularly true for Serbian Roma, where many Roma refer to themselves as “smoked Serbs” or simply “Serbs.”Today, Gypsies are perhaps the most segregated ethnic group in Europe. The same is probably true for Serbia.15 The first written document referring to Gypsies in Serbia dates from 1348.16 In Serbia, as in other South Slavic countries under Turkish rule, Gypsies constituted a separate ethnic group; they lived apart in mahalas, in towns or in isolated village areas. In the Balkans, through centuries of Turkish rule, Gypsies were strictly endogamous: even the best men at their weddings were always fellow Gypsies. In the past the Gypsy social unit was the extended family and the occupational niches they filled contributed to the Serbian economy. The traditional Gypsy occupations include making crafts such as troughs, baskets, spoons, blacksmithing, ironsmithing, and music. Despite their contribution to the agriculture-based Serbian economy, Gypsies were despised by the rural Serbian population.17 In Serbia, Gypsies form a complex mixture of groups.18 In fact, there are quite a few subgroups of Gypsies in Serbia.Some of them lost the Romani language and their mother tongue is now Serbian. There are different forms of Romani depending on which group the Gypsy belongs to. Interaction between different groups is limited, and the form of Romani spoken has become an important means of distinguishing between groups. Furthermore, Gypsies have always come under several appellations and names. Each group represents a historical and, for the most part, originally localized entity. Many times Gypsies adopted their hosts’ culture in response to the different requirements of their social and environmental surroundings. The result is a great diversity of Gypsy tribes and a lack of identity as an integrated ethnic group. Therefore, Gypsy culture in general is extremely diverse and difficult to pinpoint. Their ethnicity is also a disputed and complex issue due to the fact that most Gypsies do not consider themselves members of a unified group but identify instead with the subgroup to which they belong.19 Within these subgroups, language and religion also remain diverse; the religion which a Gypsy tribe or ethnicity might hold on to depends on location and circumstances: they adopt whatever religion is prevalent in the local area. A characteristic of Gypsies as a group is their adaptability to religious and political changes. Their religious and political standings depended always on the current political climate. For example, some contemporary Christian Gypsies are ex-Muslims. Until very recently, the characteristic of all groups was that they did not mix with each other—there appeared to be a strongly emphasized antagonism among the groups. A system that divides these groups, much like a caste system, still exists in some parts of Serbia today. In the past, with respect to other Gypsies, allowable marriage choices were largely restricted. Females in particular were expected to marry someone within their particular tribe and most obeyed the rule by marrying within their group.Another characteristic of Gypsy ethnic identity in Serbia is the so-called “ethnic mimicry” or “favored identity” by which Gypsies declare themselves to be members of a majority community. According to a 2005 census, there were 114 000 Roma/Gypsy in Serbia, which is less than 1.4%. However, an official estimate of Serbia's true Gypsies range between 360 000 and 500 000 compared to 7 478 820 Serbs.20Yet another variant of the Gypsy “favored identity” is the construction of the new ethnic identities and new traditions. Among Gypsies, these new identities and traditions assume to restore an ancient origin in spite of the complexity to attest its legality and even obvious contradictions to historical records. A notorious example of the invented tradition and change in the identity of the ethnic group usually called Gypsies is the emergence of the Egyptian community in Serbia and Kosovo.21Alternatively, some Gypsies always retained a strong ethnic attitude. For example, the president of one local Gypsy party in the village of Macvanski Pricinovici claims, “We just got a status of national minority … Some asked for it, and got it. I haven't. We were Cigani before here, now we are Roma. I don't care. I'm a Serbian Ciganin and I'll stay Ciganin forever …”22 One informant from the same village, who declares himself as Roma, argues, “The word Rom means a man, but for me it actually means that you should not be ashamed to say that you are a Rom/cigani. On the other hand, we say that we are Roma, but we live together without peasants [Serbs], we have the same customs and the same religion, and the only thing that differentiates us is our face color!” Another male informant expressed it this way: “What makes us the Roma here? We still have our tradition, that we are Roma, and our name. It's our blood that makes us Roma: if my mother and father are Roma, I'm Roma too. We've stayed ethnically pure because we've held on to our tradition and managed to maintain it and we still stick to it.” These varying attitudes about ethnicity within Gypsy communities themselves reveal distinctions between the groups.FieldworkFieldwork and the interview were conducted in the village of Drenovac, the second largest village in Mačva. Approximately 10 000 Gypsies inhabit Mačva, an agricultural area in western Serbia. There are at least seven tribes/subgroups of Gypsies in Mačva; most tribes were Muslims in the past, except for Christian Romanian Gypsies.23 The Gypsy permanent settlement in Mačva was established in two stages, during the late nineteenth century and the ‘50s, when their sedentarism was enforced by the Serbian government. Usually Gypsy villages in Mačva are relatively poor, and typically there is no running water or a sewage system. The majority of the Gypsy population in Mačva is semieducated, averaging 5.36 years of schooling; few own land and most make a living as hired field workers. Many also work in a black-market economy, engaging in trades of various sorts. Marriages are made at an early age, and having a large number of children is desirable.Drenovac is inhabited by Karavlax Gypsies (Black Vlax), or Romanian Gypsies, who arrived in Serbia during the eighteenth and nineteenth centuries.24 These Gypsies consider themselves to be “natives” of Serbia, rationalizing this statement by the fact that their ancestors were born and raised in Drenovac. They identify themselves as Lejaši, from the Romanian word laiesi. The majority of Gypsies in Romania had been slaves in the middle ages25 and were divided into groups, serving the Crown, noblemen, or households. The laiesi were the slave group, comprised mostly of musicians. After the abolition of slavery in 1864, many migrated to Serbia.Approximately 10% of the houses in Drenovac are occupied by Gypsies who are bilingual. They speak Serbian as their mother tongue, and Romanian as a second language, and Romani, or Gypsy language, is not spoken at all. They belong to the Orthodox Christian Church and all celebrate ancient, traditional Serbian family holidays. Music is their traditional occupation and they were never employed, as were other Gypsy groups, as craftsmen. Although they came from Romania to Serbia as part of the larger Karavlax Gypsies tribe, they deny any connection with Karavlaxs, based on the difference in occupation: Karavlax Gypsies in the area used to be spoon makers. They have a somewhat darker skin color than the rest of the Gypsies.26 During the 1970s and 1980s, the Lejaši traveled throughout former Yugoslavia playing their music. Today they cannot travel so easily due to the political isolation of Serbia born out of the postwar (1991–99) consequences and a strict visa regime. At present, most males work in a village factory owned by native Serbs, while the majority of females labor as housewives.The Lejaši regard themselves as more sophisticated than other Gypsies in the area due to the fact that they do not marry or form social relationships with other groups. Lasting social relationships are created with Serbs of their village. All enter into mutual godparenting relationships with Serbs, and all have traditional Serbian names which indicate Serbian ethnic origin. Most consider themselves to be “true Serbs.” Census data from 1948 to 1991 reveals that no Gypsy in Drenovac has ever declared that he/she was a Roma/Gypsy.Dragan Vasiljkovic, the president of the Roma Association of Western Serbia and a member of the National Roma Council, is a native of Drenovac and exceptional in many ways. He is one of the rare Gypsies who managed to finish his university education (only 2% of Gypsies in Serbia have higher education according to Statistics of Serbia, 2002). He lives in Sabac, the main town in Mačva, with his wife and two children who are both college students. Mr. Vasiljkovic works in a general hospital as a dentist and also operates a successful private practice. He credits his success to his wife's efforts and his upbringing, which was no different than “any Serbian child.” On the other hand, he fully accepts his Gypsy/Roma background and sees the Gypsy ethnic mimicry as a separatism that weakens the already loose unity of their national minority:This is a calculated manipulation in order to avoid and deny their Roma roots, combined with the personal interests. Even today, the Roma from Drenovac don't mix with other Roma groups. They never marry Roma from other groups and this is common to all Roma here, on Mačva territory. The data indicate that the first Roma in Drenovac were Roma immigrants from Romania. In the beginning, in the nineteenth century, there were only a few tribes living in the territory of Drenovac; they were named Jankovic, Vasiljkovic, Stankovic, and Jovanovic. They spoke only Romanian, not Gypsy Romany and they had strong solidarity and cooperation within the group. The solidarity was reflected in helping each other and they got along well. Their main characteristic was that they didn't mix with the other Roma tribes which surrounded the village of Drenovac, such as the Gypsies from Priciniovici, Sevarice and so on. It appears that language was the barrier which prevented them from mixing; and we also know that the other Roma groups didn't accept them as their own. However, there is no proof that they represented a different ethnic entity.27A totally different opinion is expressed by Dragan Buric, a 46-year-old musician, who states that he is a “true” Serb. Dragan Buric remained one of few who managed to engage in the Lejaši's traditional occupation as a source of living. Dragan has a band in which he plays with his son, while his wife sings. Hence, music remains their primary source of income. He has a comfortable house, a car, and a small bar that he operates with his wife, all of which separates him from the rest of his community. The family speaks Serbian as their first language, and Dragan does not know Romanian, or Romani, the Gypsy language. He says that he always felt as a Serb and has declared himself as such. Dragan's interview was conducted in his home during the summer months of 2002. His family was present most of the time and occasionally they would perform some of their music. Interviewer and interviewee engaged in “sharing food”—“social eating” and drinking local brandy, with frequent toasting. The story that Dragan tells testifies to the difficulty of establishing Gypsy identity and his attempt to rationalize the present social order. It provides important data for our understanding of the flexibility of Gypsy ethnic identity. Dragan's account is presented in four parts: childhood; occupation/music; ethnicity; and marriage and family. Through his narrative we can trace how he pursued a strategy worked out by his ancestors to negotiate ethnic and social identity.This is his story:ChildhoodI was born in Skela, near Obrenovac. We lived there until 1978; then we came here, to Drenovac, and we live here since then. My mom was born and raised in Drenovac; she was only married in Skela, to my father Sreten. My childhood was a happy one although our family was very poor. My mom and dad had three children: my sister, my brother and me. We are all approximately two years apart. My parents got along very well; they stayed together until death took them apart. They had what I called “an ideal” marriage; although, I have to say, if it weren't for my mom we wouldn't do anything with our lives. My mom, Nadezda, stayed with us most of the time and taught us how to behave and taught us to love school. My father was away most of the day, working. At night, he performed music. My old man was a musician, he played violin, and actually, my grandfather and his brothers were musicians, too. It runs in the family—I am a musician; I play violin, and my son inherited the talent for music, too. This is our tradition, and not only that, but we all have a good ear for music. At that time, when I was born, playing music didn't pay very well. I remember that we used to live in a poor house made of mud, 4x4m. We had only two beds. I slept with my mom because I was her youngest and her favorite child.My mom was a great mother to us and a hard worker. She used to go around the village and do all kinds of jobs—cleaning, washing, cooking. She would also go out to the fields from time to time. But she never begged, ever. Gypsy women do that—beg for food or money, but my mom never did that. She earned every dinar. She would go out into the fields to work for a whole day. Landlords usually gave workers some food, a breakfast, or something, a piece of meat … but my mom would never eat that. No, she brought home every piece of food for us kids to share. That's how she was. She also never allowed me to accompany her when she worked in the fields—I remember that I cried every time she would go out, but she never let me, for it was too hot for a child to sit in the sun on the open field the whole day.My father, also, used to take every job he could find, for he couldn't support us only with his violin. He was a strong, big, healthy man, and knew how to do a lot of things. He died two years ago, and my mom passed away in 1995. I am very glad that I was able to give them a different life—they sacrificed their whole lives for us kids, and when we moved here and I started to play around Yugoslavia and the neighboring countries—I provided for them, took care of them financially. Both of my parents deserved that, after what they'd been through, when we were little kids.I was born in 1957. The late ‘50s and 1960s were very difficult times. There was never enough food no matter how hard you worked or tried. Both of my parents worked, but I still remember the poverty around us, and that little house where we used to live. On the other hand, maybe those difficult life conditions made us all very attached to each other. My brother and sister and myself, we were always very close. We still can't live without each other: my brother lives in this same house, just has the entrance on the other side, and my sister is married into a house only a 100 meters away from us.We respected our parents—that's how we are brought up, not like the kids today. You give them one little finger, and that's not enough, no, they want the whole hand! We, the kids, all went to school, even if we were so poor. My mom insisted on our education. The nearest school was about 7 kilometers away from the place where we lived, and I had to take a bus every day to get to school. There was one part of the road that I needed to travel by foot, by some forest, and my mom would always wait for me there—during winter especially, to make sure I was safe. We also didn't have appropriate clothes—no winter shoes or gloves. But we were very fortunate, we had the best schoolteachers. My schoolteacher was especially fond of me. She didn't make any distinction between me and other kids—like I'm a Gypsy kid or something. On the contrary, she did all she could so we could enroll in school like every other kid. I'm still very grateful to her. I was a very good student, always. I never needed any special help to learn school things. I know there is a huge problem with Roma children today, in schools, but that's due to the Roma mentality; a lot depends on parents, on their attitudes, and how they advise their children. That's a real pity, a shame, for all those Roma children today, but that's the irresponsibility of their parents, nothing else. I realize that we are not the same, and we don't have the same capabilities. I know now that I have Roma origin, but I have always tried to behave properly. I raised my children, especially my daughter, like God said we all should. I tried to give my kids everything and to bring them up to be good people. I thank God that they didn't have to follow my footsteps and suffer.I have the primary education, I think it's like eight grades of elementary school. I am very sorry, even today, that I couldn't continue my schooling further. I still regret that today. But, after my eighth grade, I started to play with my father. When I was ten years old, he bought me my first harmonica; after six months, I mastered it completely, that's how talented I was for music and playing. That was a very different time than it is today. It was expected from me and my brother to learn how to play and to become musicians. After all, that's what our family, our grandfathers used to do. I have to say that I loved music from a very early age. My father taught me how to play, and I loved it. I was particularly good in playing kolo, our national [Serb] dance: I would play it and the whole room would get up to dance! That was exciting, especially since I was just a little boy. Some guests at weddings used to lift me up on their shoulders and I would play like that. I loved the attention I was getting and I knew I was good. On the other hand, I should have pursued schooling, but the circumstances were the way they were, and I neglected further schooling and became a musician. Who knows what could have happened if I continued my school? I was very good in school, always good with numbers and especially drawing. I could have been an architect if I had different life circumstances. Later on, when I grew up, I bought myself some private music lessons in Belgrade, in a music school, to specialize. I learned a lot, and today there is no melody or a song that I cannot pick up and play. Music is still what I make a living off today. Maybe I'm lucky in that sense—I did and still do what I like the most. There is something about the rhythm that can carry you away, and that's what has happened to me. My brother also dropped out of school, to play, although he wasn't such a talent as I was. My sister, on the other hand, sang like a nightingale; it was such a joy to listen to her! Well, the saddest story of my childhood is connected with her. I was always very attached to her; even though I was her younger brother, I felt a need to protect her, always, for she was the only girl in our family. That's how brothers are toward their sisters. And she was a good person, too. I remember this as if it happened yesterday. It was 1968. I was ten and a half years old, my sister was around thirteen. My father's brother took her with him to a shitty little place called Potoci, in Bosnia, to sing with his band. That place was behind God's back [remote, far away and forgotten by people], with one little furniture factory and a train that goes around once a week. And nothing else. When my uncle took her away, that broke my heart. I can still feel the pain of that separation today. She was just a little girl at the time; she even looked younger than her age. I was a kind of a sensation at ten, playing harmonica so good, and with my sister it was the same. I guess that's why my uncle took her. People would pay you more. He is my uncle, but I dislike what he did—he was using her. I don't know why my parents allowed that, maybe because we were always short on money, don't know. I think we were very good kids, respecting our parents and school and our schoolteacher. We had to obey, that's the key. I think that everything depends on where you were born; if I was born in Belgrade, who knows what I would become. I felt always that I could do whatever I wanted—no trouble in school at all, so talented and how, for music … it was just the life's conditions that determined which path to take, nothing else.My sister came home after three months; she didn't say much about the place, and the restaurant where she had a job, but she told us that she managed to arrange a job for all of us—my father, my brother and me, to come back with her and perform in that same place. It turned out that the landlady, the owner of that restaurant, came to like my sister very much, and when she heard that her whole family was into music she made the arrangement for all of us to come. That meant we were all going to make some money, thanks to my little sister! My father decided we should go, and bought us two new harmonicas, one for me, and one for my brother. Actually now when we were all back together, I was very excited about this trip. This was supposed to be my first trip, my first visit anywhere outside my village, and God knows what I expected. I had great expectations. I was just a ten-year-old kid with a harmonica, but also a musician who's going to make some serious money! I have to admit that it felt good, especially that my sister was back with us.So we left. We only had money to get to that damned place, nothing more, not even for a piece of bread. We traveled by train the whole day and my god it was slow! It had those old-fashioned narrow rail tracks and I thought we would never get to our destination. We had to take a connecting train in Mliniste, around 100 kilometers away from our final destination. It was already dark and getting cold. I remember I was very hungry and I started asking for some food, at least some bread. But my father explained that we would have to wait until we got to that restaurant where we were supposed to play—maybe the landlady would give us some food. He didn't have any money, not even a dinar. So, we were sitting in a waiting room at the station and it felt like we were beggars or something, and I couldn't handle that. I cried loud, asking for my mom and some food. That railway station was a horrible place: dirty, with some dim lights, and somehow everything looked old and beat up. Then my poor darling sister took off her sock, and there, in her elastics, she had two dinars that she had saved for “rainy days”, just like this one was. She bought us one kilogram of bread and I was very grateful; her gesture touched the bottom of my soul. She used that coin to tie/twist the elastics of her sock, and without it her sock kept falling down. My darling sis! I tell this story to my kids all the time. They can learn a lot from my life experience; don't know if they will ever use it.Finally we reached the place. I didn't like it. Who knows what I had expected to find, but this was one lousy, dirty place to be. At least our family was together. I have to say, the owner, the lady who liked my sister, she was nice. She was really nice to us. Her little restaurant was very primitive—no electric power, no heating, very modest all in all. She had put one little kerosene light on each table, and polyvinyl tablecloths. The guests were mostly factory workers; at that time, people were still uncivilized, and a restaurant with a live band was not a good place to be. But we were there and we started to perform every night. I also accompanied my sister and sang with her—my voice still hadn't changed so I had a voice like a little girl and our duets became, so to say, famous. People gathered to listen to us—and my old man was also very good. The thing that I didn't like at all was that my sister was exposed to all kinds of indecency. All female singers go through that sooner or later. Some guests like to touch them or to call them names or to behave too familiar with them. And my sister was just a little girl and very embarrassed. The first time a male guest touched her—I think he placed his hand on her—I started to cry from anger and shame. And I was supposed to be a “real musician”, but this was my sister and it was too difficult to handle. Later on I learned to deal with all kinds of situations and with people; that's what this profession is all about. We managed to earn some money and after a month we got back home, to our mother.Until the day he died, my father was always working on something. Even when he was old he wanted to help. He died in my son's arms, literally. He died from throat cancer, in a hospital. He was 67 years old when he died. I used to tell him that he should slow down, and enjoy his retirement days, but he couldn't stop. He was just very diligent and couldn't sit still. Always a fighter—that's what he was. He fought for us, to provide for us the best he could. I think he passed that down to me because I'm the same with my kids. I would do anything for them.MusicThe life of a musician can be very difficult. For the past 30 years, I go to bed in the morning, around five or seven in the morning, for I play the whole night. I sleep during the day—I get up around noon or so. The basic thing is, you depend on other people, your audience. Whatever they say or want, you learn that they have the right, always. That's the only way to survive in this profession. It took me a while to get that, but I mastered it and now I'm fine. I first played with my father, for years. Then I got a band of my own, something more modern. The band is called “Demons”; don't ask me why, my son named it. We had to adjust to current fashion, or to learn fashionable songs, always. My career was mostly good, in the sense that I don't remember that people harassed me because of my color—because I look like Roma. Maybe just a couple of times but it wasn't personal … people would say cigani to us or something. It was a different time than it is today, people didn't pay so much attention to nationalities then. Now it's different. Luckily, even when there was a potential conflict, like guests would get drunk and offend our female singer or us, I always managed to calm things down. I know how to behave and how to soothe things. But sometimes I would get personally offended when guests called me ciganin/Gypsy—then my soul hurt and I would think: Why am I not a Hungarian? Why call me a Gypsy? People love to say: “Play, Gypsies!” Or they would curse our Gypsy mother. That always hurt me the most. I don't know if there is such a thing as a “Gypsy man”—for me it looks like just one bad, ugly word. I don't like that word, and I don't like to be called one. It's a pejorative term—anybody, even an American or a German could be called a Gypsy if that person behaves bad. I feel very bad when I hear that word. I managed to stay calm, always, and learn to put up with different people, even if they are mafia. If I had behaved differently I would have never survived as a musician.I've played for politicians, for mafia, and for the greatest gentlemen. For one job, I would make around 15,000 German marks. I used to fish in Plitvicka Jezera [Lakes].My music took me to Austria, Linc, in 1989. We had a very good job over there. That was still a very good time for making money, for all of us. However, when the war started in 1991 I came back home. I still don't know why I came back, why I brought my family here. Now we can't travel—if we go to Belgrade to play, that's something. I wish that I could go around the country and play the way I used to. But that's impossible now. I was in Bosnia last year, for two months, to play. They also got it bad. Today it is very difficult to survive from playing music. It's especially difficult for me since my children are used to a good life, and now I can't give them everything the way they are used to. This is a great burden for me. I still feel responsible for them—even though my son is 21 years old; my daughter is 15 now. I don't know what will happen, I'm very concerned for the future. I have some 20 years of service as a musician; I paid for my retirement fund always, but that's not going to come up with very much when I retire. I am a member of the Independent Artists Organization of Serbia, but that's still no help. Before the war this organization used to arrange jobs for us, musicians, in Yugoslavia and abroad. Now they won't do anything; they just ask for more money and more money and we don't have an audience to play anymore. I think that our profession is dying out and I'm sad because of it. This was a nice tradition and a good life, especially when you are young.I keep a few chickens and a pig today so my children won't be hungry. After all, I live in a village and it is normal to have animals.EthnicityI am a Serb. I am a 100% original Serb. My father and his father and my great-grand father were all born in Serbia. They always behaved like Serbs. I'm the same way. I love my country very much. This is where I was born. I don't know where my ancestors came from, but I feel I belong here. Never in my life have I felt like a Gypsy, not even for a moment. I have the same soul [like the Serbs], the same blood, the same upbringing, the same everything. That's how I was raised. We were always Orthodox, always. My saint day, my slava is my Saint Arhandjel Mihajlo, we celebrate that. We have an icon, too. I always declared myself as a Serb, on all censuses. The same is true for my family; we all feel the same. My son, for example, he is a Serb too. We don't speak the Gypsy language, we don't even know one word of it. We only speak Serbian, and we know some Romanian, but it's a dialect with around 30% of Serbian words. We are different than the people in Macvancki Pricinovic, for example. They all speak the Gypsy language and we don't understand them. They are Roma, Gurbeti. The others are Roma, not us. They say they are Gypsies, always did. I'm always afraid that I will offend someone by calling him Gurbet or Roma; I almost apologize every time I have to say, “You, from Gurbeti or Roma tribe.” That's funny; they apologize to me sometimes, too, when they address me.We always had very good relations with our neighbors; my next door neighbor [a Serb] is my godparent. The godparenting runs in our families for about 60 years. His grandfather baptized my mother, and his son did the same with me, and now his children are godparents to my kids. My son now started a new godparenting, with his best friend [a Serb], but I respected our family tradition—my godfather is over 70 years old today. My godfather is very important to me; he is like my father, in the sense that I always respected him and he took care of me the best he could. His wife recently died and he's very old, but he managed to be at my grandson's baptism, and he was the most welcomed person there. That's just our tradition. It is a great honor to be a godfather. The funny thing is, they are all named the same. My godfather is named Sreten, just like my father and my son, and his wife's name was Natasa, or Nadezda, just like my mom's and my daughter's. They gave us their family name and we accepted it. My children are named after our godparents, but sometimes a confusion arises when there is something official to sing or to do since we live next to each other and they are all named the same.My family has very good relations with almost everybody in this village. On the other hand, I can see today some difficulties arising—these modern kids are different than we were. Kids in the village are a little insolent today. Before, we never had any trouble at all. My mom and her family are from Drenovac—that's why we all came back here. When I first moved here, some 25 years ago, everybody greeted me very warmly. There was no difference between me and my next door neighbor at all. I think we all had a different upbringing in those times. My first neighbor is my closest family, and we love each other still. Sometimes he was closer to me than my own brother or a father. I respect my neighbor very much; he is the first one to help me when I need help, and I'm the same way to him and his whole family. As kids, when I used to come to visit my grandparents here in Drenovac we played Cowboys and Indians—I was of course, the Indian. We go a long way back, like a true family. My son grew up with his [the neighbor's] kids; they are still best friends today, and tied with godparenting.On the other hand, there is a kind of trouble in the air lately. This all started with the beginning of the war [1991]—people became nervous or something, started paying attention to nationalities. And we had a lot of refugees coming here from Bosnia. They stayed in my village, in some empty houses, or people just took them in. Anyway, a year and a half ago the worst incident happened: one Gypsy boy got severely beaten by ten or more villagers [Serbs]. I'm very sorry that such a thing could have happened here. It happened in our village center, where young people go out. In situations like this, I always think “Why am I not a Hungarian; why they call me a Gypsy when I'm not?” Then my soul hurts; from where did they get that I'm a Gypsy? Why call me that? I'm not a Gypsy. I never stole anything. I would help everybody. I'm brought up that way; my kids are the same. We all act like our dear God told us to—by God's laws. We don't steal, we don't kill. We just play our music, that's all. That poor Gypsy boy didn't deserve what happened to him. My son, on the other hand, never had any trouble, ever. That is because he behaves well. My son was involved in only one incident. He was out with his girlfriend, now his wife, and some female friends in a restaurant here in the village. They were still teenagers. And a couple of older men started to say things to them, to the girls. They even threatened that they will move out all Gypsies from this village. They cursed them: “I'll screw your Gypsy mother.” When I think of it, it was always like that. Most arguments and fights were about girls. Usually everything ended up with some bad words and humiliation. But before it was I against that one person; now it's like we are taking opposite sides, the whole village. I don't see anything good coming out of it.These bad events, humiliations I had to take, didn't prevent me from feeling like a true Serb. I love my country; this is where I was born. If I was born in Zanzibar, I would feel the same. I don't hate anybody. I don't like to be humiliated. Why me? So what if I have dark skin? I have the same soul as anybody. Maybe I have greater Serbian soul than some Serbs. My kids were always an example in school: good students, always dressed well, better maybe than some people's kids who had more money. My children always had good pocket-money. They were clean; their mother always looked after that. I've always tried hard. My son is a Serb too; he is born here. I don't want to see him humiliated, ever, because someone calls him names. I sent him to high school in Sabac; he was a good student always. He wanted to become an economist. His third day, kids tried to harm him with a screwdriver because he is like a Gypsy. They tried to take his leather jacket. We came to visit him, my wife and daughter and myself, to see his school and how he's doing. I was very proud of him. We waited for him in the school yard. Some kids were near us, saying very bad, vulgar things about us. My daughter was there; I didn't want her to hear any more of that crap. I pulled my son out of that school.Maybe it's a mistake that we are here, in Drenovac; so nonviolent, but that's our characteristic. Roma, in Macvancki Pricinovic, or even worse, in Draginje, they don't get humiliated or beaten up. No, they fight back. If something like the event with that poor boy had happened over there, only an ambulance crew could pick up parts from Serbs. They are Gurbeti, much more violent. On the other hand, we do the only way we know. We have a different mentality than the Roma. We are much more softer, and we never sold our girls. This is maybe not a nice thing to say, but we do have more culture. We don't have differences with peasants, not at all. Whatever they do, we do it, whatever they have, we have it. Actually, we understand ourselves as Serbs. Gypsies in Macvancki Pricinovic, they declare themselves to be Roma, always; we never do. That's because we feel and see ourselves as Serbs. Gurbeti would speak their Gypsy language even in front of Tito. We all speak Serbian. I can understand some Romanian, but I can't speak it. We never used Romanian in this house. Not even in the village.I was never into politics. However, I think that it would have been much better for all of us from ex-Yugoslavia if we made a split right at the beginning. Serbia is Serbia, and it should stand alone. But God didn't let us split without any incident. In 1991, many males [Gypsy] from this village got drafted, and sent to the Croatian war line. I could have stayed in Austria; we were there just at the outset of war. At that time it was still relatively easy to get “papers” to stay and work there. But I couldn't do it; I could have applied to get an asylum status, but I just couldn't do it; that was kind of shameful for me. Maybe I made a huge mistake; if we did that, my children would be much better off today. Instead, I packed my family and we went home, to Serbia. We traveled by car and passed nearby Karlovci just one day before the Croatian's HDZ slaughtered those 12 poor soldiers. This story followed us on our way home. But we were heading back home and I thought that if it is my destiny to die then I will. If God decided that we shall all be killed, that's it. Now I think I did the foolish thing. I regret the most because of my kids; if we stayed, both of my kids would have a secure future. I'm very emotional when it comes to my home and my hearth. I never approved of Milosevic's politics. In school I was taught to love Tito and partisans, and to hate cetnik formations. Maybe that was wrong too. I'm not afraid in front of God. If only God would give us another Tito! If only God gave us another politician of that caliber, so my son can build his life! People say this and that about Tito, but that's beside the point—whatever he was, he was a master of politics, and I respect him for that. Although I was never a member of a Communist Party. I didn't care about politics. We lived like in paradise when he ruled. He was the authority for the whole world, in those black African countries, everybody [foreign politicians] kneeled before him—I don't care if he was a hooligan. When he was alive I was free to travel everywhere, to stop my car in the middle of the night, in the middle of nowhere, in Bosnia's mountains, among Muslims, and I was safe. I only wish that my son can live through a time like that—to be and feel safe in his own country. In Tito's time, and with our Yugoslav passport, there were no restrictions or visas for all of us. I was so proud to have a Yugoslav passport. That is so totally different than today. In 1990, I took a job in Austria; I was in front of one gas station, loading my car, when an elderly gentleman asked me where was I from. When I said, “Serbia, Yugoslavia” he made a facial expression and said “Serbische Dreck” [Serbian shit/worthless Serb]. That was terrible; it made me feel ashamed. I was also very much ashamed and humiliated when Milosevic surrendered Kosovo. My son served in the Army, in Prizren. We used to go and visit him many times. I'm very sorry about Kosovo; that's the old Serbia, our land. But I think that we should blame not only our politicians, but America, the most. If it weren't for the Americans we would still live together, side by side, in our beautiful Yugoslavia. I'm sorry that my children won't be able to live and travel the way we used to. Whatever happens in the future, it will never be the same.Marriage and familyWe didn't have mixed marriages with Roma. In the past it was a big deal. Now things are mixed up all together. Lately, a few peasant women got married to Romanians. They are all young couples, got married during the war years. Actually it's still a big deal, not among young people so much but their parents don't approve most of the time. They always looked at us like we are at some low position. I never minded. I would approve my son's marriage to any girl, as long as she is nice and from a good family. Also, a few males married Muslim women, the refugees from Bosnia. There are now six or seven sisters, all Muslims that are married into this village. After 1991, when the war started, many young people got married, including my son. I thought that he was too young, and he was; he was 17 at that time. But that was a special time, a war, a crisis. When the refugees started to come, first from Petrinje, Croatia, then from Bosnia, it was scary. Then I thought, “These people [the refugees] lost everything; many of their relatives are killed. Who knows what will happen tomorrow?” My son got married in 1999, during the bombing. We had his wedding while the bombs were falling. He actually got married because his girlfriend got pregnant. And in a time like that a baby seemed like a blessing. We didn't know where the war zone would end, and if we would survive.In this village, people marry late—I got married when I was 25 years of age. I'm married to Biljana. She's a great wife and a good mother to my kids. She was my first true love. My wife is from Tabanovic, another Romanian village. My aunt is married there. I met my future wife when she was just a kid. I'm older than she is, five years. I dated many girls from her village and she knew about it. I remember her—she had nice braids, and I used to pull her by her hair. She was very beautiful; even today she has the most wonderful green eyes. But she was too young for me at that time. We had some kind of a sympathy for each other, but I didn't want to get serious until I served in the Army. I was 20 when I went into the Army, in 1977. After that, when I came to visit my aunt and saw Biljana, I knew she was the one. However I was still not good financially at that time and I didn't want to drag her with me into poverty. I first wanted to create something so I could provide a decent life for my wife. Also, I wanted to become known as a good musician, as a good, respectable and stable man before I got married. So we dated for about five years. Finally, I was able to marry her. We got married on January 8th. She had a good voice, and we started to perform together. I took her to Italy on our honeymoon. We played there at some Roma wedding. Those were the Cergari Roma who escaped from Yugoslavia before 1941 to Italy. They were very rich. I don't know what they did, but they had a lot of money. These Gypsies were from the old kind—even their tzar appeared. He was their judge and their tzar. That old man, he was around 70 years of age, was so respected; I've never seen something like that before. He was really the authority for all of them. That's their Roma tradition. We earned so much money then. I remember I bought a car in Italy. Those were the days! Actually, I ended up in an Italian hospital after that wedding. I played for three consecutive days; harmonica can be heavy after a while, and my foot capillary broke. The hospital was great—they gave us a menu every day to choose from like five meals! I enjoyed their pasta very much. We traveled to Italy, Germany, Austria and all over former Yugoslavia. I used to make 4000-5000 Germans marks per night! In 1980, I bought my first Mercedes. But those days are gone now.Now we all live here in Drenovac. My son is married and recently got a little baby boy. I'm very proud to be a grandfather. My daughter is still in school. My brother is still in Austria; he used to work with his two sons in a plaster shop. Actually, my godparent employed him at first. But he got very sick: a brain cancer, but he is doing better now, thank God. My brother and I were like twins, never apart, always there for each other. Our godparent lent us money, 500 German marks, but I don't have to give it back to him. That's how close we are. So my brother and I succeeded in getting out of the poverty of our childhood and built a nice life. Most important, we managed to raise our children well and to keep our family together. The only thing I'm sorry about is that my mother died. I wish she could see us today; she would enjoy her grandchildren and great-grandson. She would enjoy a better life.DiscussionDragan's account tells us about his personal experience. Often he mentions historical events and particular crises in either his life or the life of his community. Mostly, his story is not about how things really were but about how he preferred to remember the past. Jay Ruby28 has argued that what people say about themselves is data to be interpreted, not a simple factual truth. Dragan favors “the old days” of former Yugoslavia because Gypsies, among other things, were able to benefit from Tito's communism in full: guaranteed income in spite of there being little or no work. These are typical Gypsy feelings in Serbia today, as one Gypsy informant explained: “In other countries, if you don't work, you don't have anything to eat, but in [former] Yugoslavia, you can eat and drink and don't have to work at all!”29 Even under communism, Gypsy circumstances were not bright: those employed Gypsy workers were in unskilled jobs that required no, or minimal, skills. In the former Yugoslavia, in 1986, 80% of Gypsy children did not complete primary school and the employment rate among Gypsy workers stood at 80%;30 in 2000, just 20% of the Gypsy labor force held regular jobs, and only 5% of these workers were employed in state companies. Some 58% of Gypsy men and 89% of women have no profession, either traditional or modern.31 The low education rates are a result of both the apathy of the Gypsy community and parents’ disinterest in the education of their children.32 Also, the two main reasons for the high unemployment rate are the Gypsies’ lack of interest in long-term, institutional employment combined with their traditional distrust of authority and their lack of professional skills.33At the same time, the former communist regime in Yugoslavia propagated the idea of a “classless” and ethnicity-less society for the past 60 years, which supposedly promoted general acceptance of all ethnic groups. With his Yugoslav passport, Dragan, like so many Gypsies at that time, was able to travel without restrictions in search for work. One of the preferred destinations for many Mačva Gypsies was Austria, where they have established some sort of residence and benefited from the Austrian welfare system.34Although Dragan has tried hard to trade his “gypsyness” for success in life, unlike some other Gypsies with a strong sense of ethnicity, his account implies a sense of acceptance of the Gypsy low social position and even stereotypes, no matter how much he dislikes it. In fact, it appears that Dragan himself associates Gypsy identity with theft and dubious honesty when he says: “… I'm not a Gypsy. I never stole anything. I would help everybody …” Throughout Europe, the common stereotypes associated Gypsies with tricks and deceiving non-Gypsies: theft, lock-picking, purse-stealing, horse-stealing, casting spells, general witchcraft, and trickery.35 Because of these stereotypes, Dragan seems to be engaged in the task of creating a space of exception for himself and his family, constantly defining himself in explicit opposition to anything with Gypsy connotations. Due to their distinctive phenotype and widespread stereotypes Gypsies often have less agency in choosing/creating identity than other ethnic groups. It is a great irony then, that Dragan, in all of his efforts to define himself in opposition to Gypsy ethnic stereotypes, is faced by another common ethnic stereotype, “Serbian shit,” aimed as a slur for Serbs.In Dragan's oral account, the recurring theme is one of connection—to his family, his “people,” including his neighbors, the Serbs, and his village. These connections are explored through ties of kinship and his attachment to land/place emphasizes his sense of belonging. Both kinship and land offer more than a mere background for Dragan's narrative. They actually structure and shape his story. Like so many Gypsies, Dragan married endogamously, within his own tribe, just as his parents did and, recently, his son. In Serbia, Gypsies traditionally encouraged endogamy: so cooperative in trade, they restrained from marriage with outsiders and even with different Gypsy groups, so maintaining their group identity and separation.36 The Gypsy endogamy has helped their sons get wives—it was always difficult for a Gypsy to marry into a Serbian family—so it was self-maintained as much as it was imposed. In a case of a mixed marriage, not only would a Gypsy's status improve, but his or her potential children would acquire wider kinship ties and better cooperation with Serbs. Especially in Serbian rural areas kinship still remains a very important foundation for cooperation and a lot of what passes for ethnicity at the local, micro level is, in fact, kinship. In the absence of marriage kinship ties, Dragan emphasizes instead traditional godparenting that created good cooperation with the Serbs from his village for generations.When Dragan uses traditional elements of his culture to speak about the past, his life history becomes visible cultural practice and behavior rather than merely a complementary ethnographic account.37 The social stratification and limited marriage choices of rural Gypsies in Serbia preserved their local, village traditions, including their occupations. A key socializing mechanism for Gypsies that promoted their survival and reproduction in the past was an emphasis on the particular occupations employed by various Gypsy tribes. Like his ancestors before him, Dragan chose music/entertainment as his life occupation, and his son followed the family tradition. The behavior Dragan and his family employed for many decades, and the “preferred ethnicity” is better understood by comparing his narration to available historical and anthropological data. Throughout their history, Gypsies have chosen only particular occupations in which they were self-employed and which did not require education.38 Even with modernization, when they abandoned old occupations in favor of new, they did not compromise their freedom. In light of local history and the social environment, Dragan's specific life course and decisions become more recognizable.At the time when Dragan's ancestors first appeared in Serbia, during the second half of the nineteenth century, Mačva was largely uninhabited territory due to the centuries of wars, famine, and disease. Serbia was liberated from the Turkish rule but its territories were almost deserted. At that time, the village of Drenovac had only a few inhabitants, and later on a few villages fused into the present day village.39 Since the Serbian government supported immigrants from Herzegovina, the majority of inhabitants in present day Drenovac are descendants of those immigrants. Reports relate that the natives, especially in the beginning, refused to accept the newcomers even though Mačva was largely uninhabited. Conflict was mitigated not only by various orders of the Serbian government but by the fact that the newcomers from Herzegovina belonged to the same ethnic entity. They were Orthodox Serbs who carried the same cultural models. The government also supported the colonization of various Gypsies. Workers and craftsmen were needed in rural Serbia, and the demand for their goods led to their acceptance in the villages. There are documents noting Gypsy presence in Mačva in 1718 and 1834, but the majority of Romanian Gypsies came after the abolition of slavery in Romania, after 1864. The Lejaši Gypsies, Dragan's ancestors, however, were never craftsmen like other Gypsies. They were musicians, and musicians were never needed by the rural Serbian economy. Certainly, the Lejaši music was very much prized,40 and they were the only group to perform professionally, but they could make a living from their music only occasionally, at weddings, village fairs, and special events. Their payment was in the form of a tip, which made them dependant on the kindness and mood of their audience. Unlike the Serbs from Herzegovina, Gypsies also had highly visible and diverse phenotypes like skin color, hair texture, dress, and a way of life which surely made them appear as outsiders and intruders by the natives. All these factors probably posed a potential danger of ethnic conflict in the form of territorial defense. Gypsy success in surviving has always depended on a favorable social climate. In their effort to survive and become more accepted, many Lejaši must have made a calculated decision to adopt Serbian identity and culture, as did Dragan's family. This required losing their own traditions, language, dress, and wider kinship ties to other Roma in the area. Through godparenting ties, they encouraged behavior that usually occurs between close relatives to be directed towards non-kin. The nineteenth-century Serbian government was interested in forming larger villages that would have more stability, and cooperation was greatly encouraged among ethnic groups. Therefore, the decision of Dragan's ancestors to become “true Serbs” seems justified, especially so since the Lejaši were not liked by other Gypsy groups in the area due to the different language and religion. By transmitting a particular successful behavior to their own descendants, Dragan's ancestors may not have only increased their number of descendants but also sanctified particular behavior.41 These behavioral models influenced the choices that individual Gypsies were forced to make in the context of external circumstances. The preferred identity so became the end product of their life experience, producing patterns of emotional and behavioral traits.ConclusionFor narrators from marginalized groups, oral history presents a chance to tell their own story, in their own words. Their narrative can deepen our understanding of the cultural behavior of individuals by revealing personal versions of events and individuals’ understanding of the past.Dragan's oral history reveals his love of his family and his nostalgia for the past, when he was able to travel freely and enjoy the fruits of Tito's communism, especially acceptance of all ethnic groups. From his account, we learn about his personal experiences, hopes, setbacks, and his family tradition. He remains proud of himself and of his family heritage. They managed to succeed in life despite many obstacles, including their initial poverty and background. Dragan is especially pleased by his godparenting and friendship ties with the Serbs in his village and emphasizes many times that there are no true differences between them. At the same time, he is also aware of his background and the position of Gypsies in the society. Yet, throughout his life, like his parents before him and their parents before them, he has consciously traded his ethnicity for social success in life. The local life-strategy of “the preferred ethnic” identity adopted by his ancestors has created an ongoing dialogue with the social environment and political circumstances.When I came back to the village, in the winter of 2006, many things had changed: many of the Karavlax “true Serbs” now declared themselves “true Roma,” emphasizing their “ethnic revival” and the distinctiveness of the Roma culture. This change was brought about by the general rise of nationalism in Serbia and, among other things relevant for Roma in the area, by the presence of several non-governmental, humanitarian organizations that operate at the territory of western Serbia, and whose main task is to provide humanitarian and material help for the Mačva Roma. The brick factory where the Lejaši males used to work has been sold as part of the privatization process in the transition to a market economy. For most villagers of Drenovac, the only source of income today is social help/welfare and humanitarian help. In order to become recipients of humanitarian help, these “true” Serbs had to declare themselves as Roma. The Gypsies with whom I spoke said that they had to “convert” in order to survive, but also, according to their own words, they finally see their chance to obtain their own “culture and identity,” and the fact that they are sponsored and paid in order to do so helps a lot. With the help of these organizations, the Roma from Drenovac have opened a “Gypsy club” in the village where they gather daily to discuss local news, politics, and their activities. Many of these Roma are now members of the local Roma political parties, financed by the same sources, and they claim to fight for Roma rights and social dignity.Dragan's life changed too: his wife got sick and is unable to perform in the band so he stays home most of the time trying to breed pigs and chickens. He complains that he cannot make enough money for his family anymore. On the other hand, he stays committed to his “preferred ethnicity,” although he clearly sees the need of the Roma people to “finally do something for themselves” as he says, even if that means to reject the previous identity. He supports his fellow villagers in their efforts, but warns that this change has already provoked many sparks among the local villagers and that verbal and sometimes even physical conflicts with the local Serbs are on the rise. He did not apply for any sort of humanitarian or social help.It is too early to conclude how this ethnicity change will affect the Roma from Drenovac. Gypsies always depended on the needs and contacts with their host countries as a source of their livelihood. The Lejaši may possibly lose their relatively good cooperation with their Serbian neighbors but gain more on the other side, in their alliances with the other Roma and political parties from the area. In Serbia, Gypsy ethnic flexibility comes from their readiness to adjust their behavior in order to create the most favorable strategy for making ends meet. Gypsy culture, as well as identity, responds in an ongoing dialogue with their social environment, and so far they have managed to survive because of the social strategies they used, trading their objective identity for the preferred one. Humans do not passively accept models of behavior from their social network. They accept the models that work, change those that do not, and share and transmit those changes back into their social group in a continually evolving creative process.42 Such processes are open-ended and able to strategize adaptively in response to novel environmental situations.431Jelena Čvorović, “Gypsies Drown In Shallow Water: Oral Narratives among Mačva Gypsies,” Journal of Folklore Research 43, no. 2 (2006): 129–48.2Angus Fraser, The Gypsies (Cambridge: Blackwell, 1992).3Ian Hancock, The Pariah Syndrome: An Account of Gypsy Slavery (Ann Arbor, MI: Karoma Publishers, 1987).4Fraser, The Gypsies.5Fraser, The Gypsies; Čvorović, “Gypsies Drown In Shallow Water”; Tihomir Djordjevic, Naš narodni zivot I običaji (Beograd: Knjizevna Zadruga, 1932).6Luba Kalaydjieva, Francesc Calafell, Mark A. Jobling, Dora Angelicheva, Peter de Knijff, Zoë H. Rosser, Matthew E. Hurles, Peter Underhill, Ivailo Tournev, Elena Marushiakova, and Vesselin Popov, “Patterns of Inter- and Intra-group Genetic Diversity in the Vlax Roma as Revealed by Y Chromosome and Mitochondrial DNA Lineages,” European Journal of Human Genetics 9 (2001): 97–104.7Fraser, The Gypsies.8Elena Marushiakova and Veselin Popov, Gypsies (Roma) in Bulgaria (Peter Lang: Frankfurt am Main, 1997); Jelena Čvorović, “Caste Behaviors among Serbian Gypsies,” Collection of Papers EI Serbian Academy of Sciences and Arts 23 (2007): 151–68.9Hancock, The Pariah Syndrome.10Anzej Mirga and Mruz Leh, Romi, razlike I tolerancije (Beograd: Akarit, 1997).11David M. Crowe, A History of the Gypsies of Eastern Europe and Russia (New York: St Martin's Press, 1996).12United Nations Development Programme, Srbija I Crna Gora: izvestaj 2002 (Beograd: UNDP, 2002).13Katherine Pinnock, Denied A Future? The Right to Education of Roma/Gypsy and Traveler Children in Europe (United Kingdom: Save the Children Fund, 2001).14Mirga and Leh, Romi, razlike I tolerancije.15Aleksandra Mitrović, Na dnu. Romi na granicama siromaštva (Beograd: Naučna Knjiga, 1990); Jelena Čvorović, “Sexual and Reproductive Strategies among Serbian Gypsies,” Population & Environment 25 (2004): 217–42.16Tatomir Vukanovic, Romi (Cigani) u Jugoslaviji (Vranje: Nova Jugoslavija, 1983).17Tihomir Djordjevic, Iz Srbije Kneza Miloša. Stanovništvo-naselja (Beograd: Geca Kon, 1924).18Vukanovic, Romi (Cigani) u Jugoslaviji; Čvorović, “Caste Behaviors among Serbian Gypsies.”19Jelena Čvorović, “The Making of Gypsies: Invention of Tradition,” Issues of Ethnology and Anthropology 1, no. 1 (2006): 47–59.20Pinnock, Denied A Future?21Čvorović, “The Making of Gypsies.”22Jelena Čvorović, Gypsy Narratives: From Poverty to Culture (Belgrade: The Institute of Ethnography, Serbian Academy of Sciences and Arts, 2004).23Čvorović, “Gypsies Drown In Shallow Water.”24Čvorović, “Sexual and Reproductive Strategies among Serbian Gypsies.”25Hancock, The Pariah Syndrome.26Čvorović, “Sexual and Reproductive Strategies among Serbian Gypsies.”27Dragan Vasiljkovic, interviewed by Jelena Čvorović, May 10, 2003.28Jay Ruby, “Speaking for, Speaking about, Speaking with, or Speaking Alongside: An Anthropological and Documentary Dilemma,” Visual Anthropology Review 7, no. 2 (1991): 50–67.29Jelena Čvorović, “Gypsy Oral History in Serbia,” Oral History Journal 33, no. 1 (2005): 57–68.30Children of Minorities: Gypsies, ed. Sandro Costarelli (Florence: United Nations Children's Fund, International Child Development Centre, 1993).31Pinnock, Denied A Future?32Jelena Čvorović, “Gypsy Ethnic Socialization in Serbia,” Bulletin of the Institute of Ethnography Serbian Academy of Sciences and Arts 53 (2005): 35–48.33Children of Minorities: Gypsies, 44–7.34Čvorović, Gypsy Narratives.35Fraser, The Gypsies, 62.36Čvorović, “Caste Behaviors among Serbian Gypsies.”37Julie Cruikshank, Life Lived Like a Story (Lincoln and London: University of Nebraska Press, 1992).38Fraser, The Gypsies, 62; Jelena Čvorović, “A Gypsy, Butterfly and Gadje: Narrative as Instruction for Behaviour,” Folklore 119, no. 1 (2008): 29–44.39Tihomir Djordjevic, Iz Srbije Kneza Miloša. Stanovništvo-naselja (Beograd: Geca Kon, 1924).40Djordjevic, Naš narodni zivot I običaji.41Lyle Steadman and Craig Palmer, “Myths as Instructions from Ancestors: The Example of Oedipus,” Zygon 32 (1997): 341–50.42Paul Di Maggio, “Culture and Cognition,” Annual Review of Sociology 23 (1997): 263–87.43Kevin B. MacDonald, Preface to the First Paperback Edition, Separation and Its Discontents: Toward an Evolutionary Theory of Anti-Semitism (Bloomington, IN: Firstbooks Library, 1998/2003). \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B08BB7BF94AD90CF939841DDA5DA30FD14EDA7C.txt b/test/dataset/in/resources/corpus/Clean_0B08BB7BF94AD90CF939841DDA5DA30FD14EDA7C.txt new file mode 100644 index 0000000..89a3cbb --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B08BB7BF94AD90CF939841DDA5DA30FD14EDA7C.txt @@ -0,0 +1 @@ +]>EXG5145S0531-5565(98)00048-510.1016/S0531-5565(98)00048-5Elsevier Science Inc.Fig. 1(A) Discovery site of the Ice Man from the Hauslabjoch in the Ötztaler Alps, September 19, 1991. (B) Preservation unit containing the Ice Man. (C) Face of the Ice Man. Note the high degree of dental abrasion and the diastema between the upper central incisors. (D) Three-dimensional polyacrylic model of the osseous skull (stereolithographic reconstruction). (E) Set of tattoos (vertical lines) in the lumbar region. (F) CT-plane of the thorax showing the lungs and heart for computer assisted endoscopic navigation, investigation, and sample withdrawal. (G) Endoscopic inspection of the lower middle mediastinum. The heart (covered by pericardium) and left lung can be seen. The coronary vessels are also visible. (H) Endoscopic view of the surface of the left lung covered by the pleura. Anthracotic (coal-like) pigment is clearly visible in the lung tissue. (I) Osteon method for determining the age at death of the Ice Man.Original Articles“‘Man from the HauslabjochOthmarGabera*othmar.gaber@uibk.ac.atKunzelKarl-HeinzKünzelaaInstitute für Anatomie, Universitas Leopoldino-Franciscea Oenipontana, Müllerstasse 59, A-6010 Innsbruck, Austria*Correspondence to: O. Gaber; Fax +43-512-507-3061Abstract—On September 19, 1991, at an altitude of 3,200 m (10,498 feet), the remains of a well-preserved, freeze-dried mummified body of a 3,000 bc, Late Stone Age man, was found on the Austrian–Italian border. Studies conducted at the University of Innsbruck have revealed that these are the remains of a 45–46-year-old male, 45 kg (99 lbs) (living weight) and 160 cm (5 feet, 2 inches) tall. Morphometric, pathophysiologic, paleobotanical, photogrammetric, anthropological, computer tomographic, and other studies have been conducted to determine the living conditions and possible causes of death of this Late Stone Age man.KeywordsHauslabjochTyrolmorphometrypathophysiologypaleobotanyphotogrammetryanthropologycomputer tomographyIntroductionOn September 19, 1991, a body emerging from the surrounding ice was observed by two German mountain hikers at an altitude of 3,200 m (10,498 feet), within a glacier area at the border between Austria (Tyrol) and Italy (South Tyrol) (Fig. 1A). After reporting the find, it was quickly realized that these remains were not of some unfortunate contemporary climber, but that of an individual of far greater antiquity. Within a week of the discovery, the news was known worldwide.InvestigationThe find consisted of a fairly well-preserved, freeze-dried, mummified body of a man who also carried with him a number of implements. Dating (14C) of both soft tissues and bone revealed that the individual was approximately 5,300 years old, i.e., from 3,000 bc or the Late Stone Age.To preserve the remains and allow for thorough preservation and investigation, the corpse was transferred to the Institute for Forensic Medicine, Institute for Anatomy at the University of Innsbruck in Austria.Since his discovery, the “Ice Man” has been stored within a specially constructed cooling and conservation system (maintained at −6°C, with a relative humidity of 98%). These parameters are constantly monitored by electronic sensing and warning devices. Anatomical scientists have been in charge of the “Ice Man” since he was placed in this glacier-like environment (Fig. 1B) (Gaber et al., 1990).Sixty-four scientific teams in the fields of archaeology and natural sciences (e.g., botanists, glacerologists, geologists, metallurgists, and physicians) have compiled data to determine the conditions under which the “Ice Man” lived and eventually died.A few facts regarding this Late Stone Age Man: Names: Ice Man; Man from the Hauslabjoch; Man from the Tisenjoch; Similaun Man; Homo tirolensis; Frozen Fritz, or “Ötzi.”Sex: Male.Body height: 160 cm (5 feet, 2 inches).Body weight: 13.3 kg (29.3 lbs) [living weight: 45 kg (99 lbs)].Age: 45–46 years.Historical age: 5,300 years (determined by modern dating [14C] techniques).Genetic ancestry: European (DNA determined from bone and muscle fragments).Special features: high degree of dental abrasion, lack of wisdom teeth, diastema between upper central incisors (Fig. 1C), lack of the 12 set of ribs, rib fracture on left side (healed), rib fracture on the right (recent injury), deep lesion in the left hip, gluteal–femoral region, and tattoos (stripes and crosses) at strategic locations on the body.Nationality: unfortunately, no passport was found with the Man from the Hauslabjoch, but if he was alive today he would be considered Italian. Based upon resurvey of the border between Austria and Italy (Bolzano Province), the site of the discovery was determined to lie 92.56 m (303.7 feet) within Italy. By contractural agreement, all initial investigations were conducted in Innsbruck.Items that the ice man carriedAround the Ice Man were found many objects that were transferred to the Römisch-Germanisches Zentralmuseum in Mainz for preservation and restoration. The items consisted of: 14 arrows—partly feathered and equipped with flint heads; a leather pouch containing objects of flint, sinew, bone, and pitch that appear as some sort of “repair kit;” a flint stone dagger made up of an oakwood handle covered by a braided material; an ax with a blade of nearly pure copper; pierced stone beads to which were attached a number of leather straps; a birch-bark container; two lumps of tree fungi with a leather strap; a backpack frame made of hazelwood; and clothing—footwear leggings, leather loin cloth, bear skin cap, and grass wrapping material.FindingsAnthropological and paleobotanical findingsUpon further investigation, the conditions under which the Ice Man lived were reconstructed. Although his origin and destination are forever lost in history, we have learned a great deal about his living conditions.The anthropological data and the parallel skeletal finds from other Neolithic graves led us to assign the Ice Man to the so-called “Remedello group,” which had settled at the southern part of the Lake Garda region in Northern Italy. Moreover, the paleobotanical investigations, based upon different types of grasses, and remains of grain and pollen all support the theory that the Ice Man’s origin was south of the Alps. Additional support of this theory is that the flints that he carried could have been found or mined from the Trentino region of Italy.Computer-tomographic numerical dataThese data have allowed us to reconstruct the skull three dimensionally, and a plastic model was produced using stereolithography. Once completed, further anthropological measurements and other anatomical investigations could be conducted without transferring the Ice Man from the cooling unit (Fig. 1D) (Künzel et al., 1992; Zur Nedden and Wicke, 1992).PhotogrammetryThis geographical screening method has been used to achieve a reconstruction of the Ice Man’s body surface. Within days of the discovery, some dark-gray alterations of the skin were observed (Fig. 1E) (Sjovold et al., 1995; Van D Velden et al., 1995). Other markings became visible, these consisted of stripes and crosses. Further inspection revealed more cutaneous markings or “tattoos” on both calves.Infrared photographyThis technique allowed additional visualization of the tattoos. The newly discovered tattoos, not visible to the unaided eye, revealed 57 stripes arranged in 16 groups, and in addition, one large and small cross-like marking. These tattoos were localized in certain regions where, due to physical stress, degenerative changes occur that cause chronic pain. Upon further x-ray investigation, discrete bone damage beneath these skin areas was observed.Because of its biomechanical, static, and dynamic functions, the lumbar region of the spine carries the greatest structural weight of the axial skeleton. Manifestations of joint and skeletal stress, with resulting symptoms and signs, are well known to be found in muscle origins and in inserts as well as in the ligaments apparatus and in the vertebral joints. Hence, the markings previously described appear to be localized above these sensitive areas.The position of the tattoos in both lower legs is striking because the marks project towards the functionally important muscle–tendon–ligament structures. The above markings, on both the lumbar spine and lower legs, appear to reflect the results of strain, probably due to moving on rough ground, and would identify areas where pain would originate. Another example of such a marking is the positioning of a large cross on the medial side of the right knee where the marking projects exactly to the medial capsular ligament structures and the medial meniscus, another well-known site of pain resulting from chronic stress.Hence, the tattoos could have been either a form of medical treatment (e.g., acupuncture) or could have been the result of a religious or tribal ceremony. However, the findings above lead us to hypothesize that these markings or tattoos denoted areas of pain due to overuse.Bacteriological evaluationThese studies detected 20 eggs of the parasite (Trichirus trichiura, or whip worm) in the small intestine (Aspöck et al., 1996). The colon also contained a large amount of quartz-like sand, signifying the eating of milled grains. This abrasive material also helps explain the high abrasions found on the Ice Man’s dentition.EndoscopyComputerized tomography is the basis for drawing specimens from intracorporeal regions (Fig. 1F). However, in contrast to living material, and through the process of mummification, all internal organs of the Ice Man had dried out and become much smaller, so newer methods were required for accurate sampling. Hence, a new device to enable minimal invasive endoscopical sampling was developed. This newer technique was a computer-supported “virtual reality,” so exact placement on specific organs could be obtained (Gunkel et al., 1997).The endoscopic precision probes were composed of pure titanium, which prevents any tissue contamination of trace elements that might be detected in future experiments (Mersdorf and Tessadri, 1995).As of this writing, samples have been obtained from the upper respiratory passage, the lungs, liver, spleen, rib, cartilage, as well as bone, muscle, and tissue from the diaphragm, intercostal muscles, brain, and peripheral nerves. These 100 specimens were transferred to 80 team for further investigation. These samples are to be tested for histopathology, DNA testing and determination, parasitology, and trace elements (Fig. 1G and H) (Hess et al., in press, Platzer et al., in press).Recent discoveriesThe age of the Ice Man had been considered to be between 25 and 35 years at the time of his death. However, we used two different methods of age determination based on the physiological changes in the development and transformation of bone tissue during aging. By screening a thin section, the number and the areas claimed by secondary osteons and osteon fragments were recorded, and the definitive age was determined by regression analysis and micromorphological methods. Applying macromorphological methods, we used spongious structure and its changes during aging, at the proximal ends of the humerus and femur. Combination of these two methods yielded results that now age the Ice Man to 45 to 46 years (Fig. 1I) (Gaber, 1998)(Hess et al., 1989)ConclusionThe Ice Man provides a unique view into the past, and new techniques are allowing us to reconstruct the life of an individual in the Late Stone Age. 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(Editors), pp. 287–290, Springer, New York, 1995Platzer et al in pressPlatzer, W., Künzel, K.H., Gaber, O., and Maurer, H.,Endoskopische Untersuchungstechnik und Probenentnahmen beim Eismann. Radiological and Anatomic Investigations on the Iceman. Veröfentlichungen des Forschungsinstituts für Alpine Vorzeit der Universität Innsbruck, The Man in the Ice 6, Springer, New York (in press).Sjovold et al 1995Sjovold, T., Bernhard, W., Gaber, O., Künzel, K.H., Platzer, W., and Unterdorfer, H., Verteilung und Größe der Tätowierungen am Eismann vom Hauslabjoch. Der Mann im Eis 2, Neue Funde und Eregebnisse. Veröffentlichungen des Forschungsinstitutes für Alpine Vorzeit der Universität Innsbruck, The Man in the Ice, Vol. 2, Spindler K., Rastbichler-Zissernig, Wilfling, H., Nedden, D.Z., Nothdurfter, H. (Editors), pp. 279–286, Springer, New York, 1995Van D Velden et al 1995Van D. Velden, E., Den Dulk, L., Leenders, H., Dingemans, K., Van D. Bergh Weerman, M., Van D. Putte, S., Vuzevski, V., and Naafs, B., The decorated body of the man from Hauslabjoch. Der Mann im Eis 2, Neue Funde und Eregebnisse. Veröffentlichungen des Forschungsinstitutes für Alpine Vorzeit der Universität Innsbruck, The Man in the Ice, Vol. 2, Spindler, K., Rastbichler-Zissernig, E., Wilfling H., Nedden, D.Z., and Nothdurfter, H. (Editors), pp. 275–278, Springer, New York, 1995Zur Nedden and Wicke 1992Zur Nedden, D. and Wicke, K., Der Eismann aus der Sicht der radiologischen und computertomographischen Daten. Der Mann im Eis 1, Veröffentlichungen der Universität Innsbruck, vol. 187, Höpfel, R., Platzer, W., and Spindler, K. (Editors), pp. 131–148, Eigenverlag, Innsbruck, 1992 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B08F0CB648E93125EAA550E394D6B967D877DAB.txt b/test/dataset/in/resources/corpus/Clean_0B08F0CB648E93125EAA550E394D6B967D877DAB.txt new file mode 100644 index 0000000..0934784 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B08F0CB648E93125EAA550E394D6B967D877DAB.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 9310.1093/gerona/62.1.93 Journal of Gerontology: Medical Sciences Smoking Is a Risk Factor for Decreased Physical Performance in Elderly Women Rapuri Prema B. Gallagher J. Christopher Smith Lynette M. 1Bone Metabolism Unit, Creighton University, School of Medicine, Omaha, Nebraska. 2Department of Preventive and Societal Medicine, University of Nebraska Medical Center, Omaha. Address correspondence to Prema B. Rapuri, PhD. E-mail: premarapuri@yahoo.com 1 2007 62 1 93 99 1 5 2006 18 8 2005 Copyright 2007 by The Gerontological Society of America 2007 Background. In 487 elderly women aged 65–77 years, we examined the association of smoking with physical performance measures of muscle function and whether the effect of smoking on physical performance measures is mediated through its effect on vitamin D or estrogen metabolism. Methods. Timed rise, timed walk at normal and fast speed, grip strength, and serum biochemical measurements were compared between smokers, past smokers, and nonsmokers. Analysis of covariance was used to compare physical performance variables while adjusting for confounding variables. Results. Compared to nonsmokers and past smokers, current smokers were significantly (p <.05) slower on timed rise and timed walk tests and had decreased grip strength. From multivariate analysis, smoking, age, total body fat, and serum 1,25(OH)2D examined as quartiles were predictors of physical performance measures. The effect of current smoking on physical performance was equivalent to a normal age-related decline in physical performance tests of 7–11 years depending on the test. Conclusions. The results of this study suggest that current smoking is a risk factor for decreased muscle strength leading to decreased physical performance in elderly women. The effect of smoking on physical performance is in part mediated by its effect on 1,25(OH)2D metabolism. Smoking may also have an independent effect on physical performance possibly by a direct effect on muscle or through an effect on vascular function. hwp-legacy-fpage 93 hwp-legacy-dochead RESEARCH ARTICLE A decline in muscle mass and function is a well known consequence of aging. Between the ages of 20 and 80 years, humans lose approximately 20%–30% of their skeletal muscle mass. The effects of age-related changes in muscle are declining physical function and loss of muscle strength, leading to increased incidence of falls in elderly people (1). The loss of muscle mass and function with age is the result of complex multifactorial processes, and the risk factors are multiple. Evidence suggests that increased muscle weakness and decreased functional ability with aging can be attributed in part to decreased vitamin D levels, and it has been shown that vitamin D supplementation improves physical performance in older people (2–5). Increased muscle strength has also been suggested to be associated with higher estrogen levels or estrogen therapy in some studies (6,7) but not all (8,9). The health consequences of smoking are well established. There is ample evidence indicating that cigarette smoking contributes to a number of diseases, including cancer, vascular diseases, respiratory diseases, and osteoporosis, among others (10). Smoking adversely affects the vitamin D-parathyroid hormone (PTH) system (11) and has anti-estrogenic effect in women (12). Smoking is associated with lower total body fat mass, which limits the production of estradiol in postmenopausal women (12). Increased metabolism of estradiol by smoking is another possible reason proposed for decreased bioavailability of estradiol (12). Limited cross-sectional and longitudinal studies suggest that smoking also contributes to physical function decline (13–15). In a cross-sectional study of elderly postmenopausal women, we examined the association between current smoking and physical performance measures. In addition, we also explored whether the effect of smoking on physical performance measures in these women could be mediated through its effect on vitamin D and/or estrogen metabolism. Materials and Methods The study population comprised 489 women aged 65–77 years who entered a multicenter osteoporosis intervention trial (STOP IT: Sites Testing Osteoporosis Prevention/or Intervention). The baseline data of this study population were used for the analysis. The study participants and the study design have been described in detail in an earlier publication (16). In brief, participants between the ages of 65 and 77 years were recruited through advertisements in local newspapers or by mass mailing of letters inviting them to participate in a 3-year study. The letter was followed-up with a phone call. About 8005 women were contacted by mail, and 1905 women agreed to come for a preliminary screening. Exclusion criteria have been described in an earlier osteoporosis publication (16); in general, women with any disease known to affect calcium metabolism were excluded from the study. To complete eligibility, femoral neck density had to be within the normal range (± 2 standard deviations [SD]). All participants had normal liver and kidney function for entry into the study. Four hundred eighty-nine women satisfied the eligibility criteria and were enrolled into the study. Participants using estrogen were put on a 6-month washout period before the start of the study. The volunteers recruited for the study were free living, in good health, ambulatory, and willing to give written informed consent. One participant with suspected Paget's disease and one participant with undetermined smoking status were excluded from this analysis. The analysis was performed on the remaining 487 women. The study was approved by Creighton University Institutional Review Board. All participants signed an informed consent form. Smoking and Alcohol and Dietary Intake At baseline recruitment, participants were provided with a questionnaire to report their smoking and alcohol history. They were asked to furnish information on smoking status (current, past, and/or never), number of years smoked, and number of years stopped smoking. Using the above information, we categorized the women into current smokers, past smokers, or nonsmokers. Furthermore, the smokers were classified into light (<1 pack/day) or heavy (>1 pack/day) smokers. The number of pack-years smoked was also calculated for smokers and past smokers (packs/day × number of years smoked). Comparisons were made between nonsmokers, past smokers, and current smokers and between nonsmokers, past smokers, light smokers, and heavy smokers. Dietary intake data were collected using 7-day food dairies. The average daily calcium, vitamin D, and caffeine intakes were calculated by a dietician, using the Food Processor II Plus nutrition and diet analysis system (version 5.1; ESHA Research, Salem, OR). The information on alcohol consumption was also obtained by a self-administered questionnaire. Drinking and nondrinking status was noted. Reproductive history, medical history, and present use of medications and vitamin and mineral supplements were also assessed by a questionnaire. Biochemical Analysis Fasting blood was collected from each participant. Blood samples were allowed to clot and were then centrifuged at 4°C for 15 minutes at 2056 × g to separate serum. All samples were stored frozen at −70°C until analysis. Serum 25-hydroxyvitamin D (calcidiol, 25OHD) was assayed by a competitive protein binding assay (17). The procedure involves initial chromatographic extraction and purification of serum on Sep-Pak cartridges (Waters Associates, Milford, MA) (18). The limit of detection was 12.5 mmol/L (5 ng/mL), and the interassay variation was 5%. Serum 1,25(OH)2 D was measured by a nonequilibrium radioreceptor assay (Incstar Corp., Stillwater, MN) using the calf thymus receptor, after extraction and purification of the serum on nonpolar C18OH octadecysilanol silica cartridge as described (11,16). The limit of detection for the assay was 12 pmol/L, and our interassay variation was 10%. Serum intact PTH was measured with the Allegro immunoradiometric assay (Nichols Institute, San Juan Capistrano, CA) (19). The limit of detection for the assay was 1 ng/L, and our interassay variation was 3.5%. Serum total estradiol, testosterone, and sex hormone binding globulin (SHBG) were measured in fasting baseline serum samples by radioimmunoassay kits obtained from Diagnostic Systems Laboratories (Webster, TX). Serum total estradiol was measured using an ultrasensitive assay. The minimum detection limit for these assays was 2.2 pg/mL for serum estradiol, 0.05 ng/mL for serum testosterone, and 3 nmol/L for serum SHBG. The intraassay coefficient was less than 5% for these assays. The interassay coefficient for the last 10 assays of serum total estradiol for the lowest standard used (5 pg/mL) was 8.4%, and the interassay coefficient for the in-house serum control (mean ± SD, 11.8 ± 1.35 pg/mL) used was 11.4%. The interassay coefficient for the low kit control (mean ± SD, 0.55 ± 0.03 ng/mL) and in-house control (mean ± SD, 0.33 ± 0.029 ng/mL) tested for serum total testosterone were 6% and 9%, respectively. The interassay coefficient for the lowest kit control tested for SHBG was 8.5%. The bioavailable (non-SHBG-bound) serum estradiol and serum testosterone were measured as described by Khosla and colleagues (20). Briefly, tracer amounts of 3H-estradiol or 3H-testosterone were added to aliquots of serum (200 μL), made to 500 μL with saline. To this, an equal volume of saturated solution of ammonium sulfate (final concentration, 50%) was added to precipitate the SHBG with its bound steroid. The SHBG-bound and -unbound steroid were separated by centrifugation at 1100 × g for 30 minutes at 4°C. The percentage of labeled estradiol or testosterone remaining in the supernatant (free and albumin-bound fractions) was then calculated. The bioavailable estradiol or testosterone concentration was obtained by multiplying the total estradiol or testosterone concentrations, as determined by radioimmunoassay, by the fraction that was non-SHBG-bound. All women except one had measurable levels of serum estradiol. Serum testosterone levels were undetectable in about 22 women. Physical Performance Measurements Physical performance measures of muscle function were evaluated by tests of muscle strength (grip strength, right hand), agility and coordination (timed rising), and gait and balance (timed walk, normal and fast). These tests measure physical tasks important for performing the common activities of daily living, and are easily performed in a clinical setting with simple equipment. Grip strength was measured in the right hand by a handheld Jamar dynamometer (Jackson, MI) as described, with some modification (21) and used by others (22). The participant was asked to stand with the right arm flexed at 90° (anterior–posterior plane) and parallel to the floor. The elbow was fully extended, and the wrist and forearm were in neutral anatomical position. In this position, the participants were instructed to squeeze the dynamometer (setting 2) as hard as possible. The average of three trials was recorded (measured in kilograms). Timed walk at normal and brisk pace was tested as described, with some modifications (23,24). Walking speed was assessed as the time taken to walk a 15-foot walkway at the participant's normal pace and then as quickly as possible. Timed rise test involved the time taken by the participant to rise from a chair 3 times as quickly as possible, following the protocol described, with some changes (24,25). The participants sat on a chair with no arm rests. They were asked to stand up and sit down 3 times and were timed with a stopwatch. In addition to these measurements, Physical Activity Scale for the Elderly (PASE) score was calculated based on questionnaire items assessing various domains of physical activity, including walking, sitting activities, light sport and recreational activities, moderate sport and recreational activities, strenuous sport and recreational activities, exercise, and light housework (26,27). Total Body Fat and Lean Muscle Mass Total body fat and lean muscle mass (defined as the weight of the muscle and organs and excluding total body fat and bone weight) were obtained from total body dual-energy absorptiometry scans of the study participants. Statistical Analysis All analyses were performed by the SAS statistical package (version 9.1; SAS Inc., Chicago, IL). Baseline patient characteristics (age; height; weight; dietary calcium, caffeine, and vitamin D intake; alcohol use; total body fat; lean muscle mass; medication use; and comorbid conditions) were compared by smoking status (nonsmokers vs past smokers vs current smokers) with one-way ANOVA for continuous variables and chi-square tests for categorical variables. The biochemical variables in Table 1 were compared by smoking status with ANCOVA while adjusting for significant baseline characteristics. If the overall F tests from the ANOVA or ANCOVA models were significant at a.05 level, then pair-wise comparisons were conducted and Tukey's method was used to adjust p values for multiple comparisons. Univariate comparisons of the physical performance variables were conducted with ANOVA. Multivariate analyses of the physical performance variables were conducted by ANCOVA, examining the effect of smoking status on timed rise, timed walk (normal), timed walk (fast), and grip strength, while adjusting for significant confounding variables including age, body mass index, calcium intake, caffeine intake, and total body fat. Backward selection was used to select significant confounders to adjust for while retaining smoking status in the model. An alpha level of 0.05 was used for model selection. The biochemical variables [serum 25OHD, 1,25(OH)2D, total estradiol, bioavailable estradiol, testosterone, and SHBG] were examined in the ANCOVA models to see if the effect of smoking is mediated through these biochemical variables. The biochemical variable was considered a mediator when it was a significant predictor of the physical performance tests and affected the association of smoking and physical performance measures (β coefficient and significance of smoking). The interaction of smoking status with serum 25OHD, 1,25(OH)2D, total estradiol, bioavailable estradiol, testosterone, and SHBG was also examined. An age related decline was calculated for each of the physical performance variables, using the slope of the regression coefficient for age (β) in Table 2, a 5-year increase in age corresponds to a 5 × β change in physical performance. This age-related decline was then compared to the decline in physical performance seen in smokers (as determined by the regression coefficient for smoking status) as a percentage change, using the change in physical performance tests of nonsmokers as the reference value. Results Characteristics of Study Population A comparison of the characteristics among nonsmokers, past smokers, and current smokers is given in Table 1. Twelve percent of the study population were current smokers, 33% were past smokers, and 55% constituted the nonsmokers. The median pack-years for smokers was 25.9 years (0.15–174 years), and for past smokers was 12 years (0.02–84.0 years). The median time that past smokers stopped smoking was 16 years (0.5–55 years). The median time that current smokers smoked was 50 years (1–64 years), and the median time since past smokers smoked was 20 years (0.6–55 years). On average, smokers weighed 8 kg less than nonsmokers (p <.001) and had lower dietary calcium intake compared to both nonsmokers and past smokers. Smokers had significantly (p <.001) lower total body fat compared to nonsmokers and past smokers. Amount of lean muscle mass was not significantly different between the groups. Past and current smokers had significantly higher caffeine intakes (p <.001) compared to nonsmokers. The percentage of alcohol consumers was slightly higher in smokers compared to nonsmokers (Table 1). Smokers were similar to nonsmokers with respect to age, height, and dietary vitamin D intake. The occurrence of comorbidities classified as 0–2, 3–4, 5–6, and ≥7 was not significantly different between the nonsmokers, past smokers, and current smokers. Medication use in the study population classified as 0, 1, and > 1 was not different between nonsmokers, past smokers, and current smokers (Table 1). Smoking and Biochemical Variables As seen in Table 1, serum 25OHD, 1,25(OH)2D, PTH, total estradiol, and bioavailable estradiol were lower in current smokers than in nonsmokers or past smokers, although the differences were not statistically significant. When the data were examined according to quartiles of 1,25(OH)2D, the percentage of smokers was marginally higher in the lowest quartile (Q1 = 16%) compared to higher quartiles (Q2 = 7%, Q3 = 12%, and Q4 = 13%). In univariate analyses, serum bioavailable estradiol was significantly lower (p =.034) in smokers than in nonsmokers and past smokers; adjustment for total body fat made the differences nonsignificant. Serum total testosterone levels were significantly higher in current smokers than in nonsmokers and past smokers. Serum bioavailable testosterone levels were lower in smokers than in nonsmokers and past smokers. Serum SHBG levels were significantly higher in current smokers than in nonsmokers or past smokers. Smoking and Physical Performance Measures As shown in Table 1 and Figure 1, on average, current smokers were slower on timed rise and timed walk tests and had decreased grip strength compared to past and nonsmokers after adjusting for significant confounders: age, body mass index, calcium intake, caffeine intake, and total body fat. Mean PASE scores (± standard error [SE]) were not different between the current (116.3 ± 7.4), past (110.9 ± 4.1), and nonsmokers (117.7 ± 3.5). From multivariate analysis models, the effect of current smoking on physical performance measures was much higher than that observed with a 1-year age-related decline in physical function in nonsmokers (Table 2). The effect of smoking (smokers vs nonsmokers) compared to a 5-year increase in age was higher by 39% on the timed rise test, 136% on the timed walk (normal) test, 95% on the timed walk (fast) test, and 90% on the grip strength test. Past smokers had physical performance measures similar to those of nonsmokers (Table 1 and Figure 1), except in those participants with a long pack-year history who were slower in the timed walk test (data not given). In contrast, no such effect was seen when the physical performance measures were compared according to pack-years of smoking in current smokers (data not given). There was no significant difference in physical performance measures between the light (<1 pack/day) and heavy (>1 pack/day) smokers (data not given). The interactions of smoking status with serum 25OHD, serum 1,25(OH)2D, serum SHBG, serum total estradiol, and serum bioavailable estradiol on timed rising, timed walk (normal), timed walk (fast), and grip strength were not statistically significant (all p >.05). Serum 25OHD, 1, 25(OH)2D, total estradiol, bioavailable estradiol, testosterone, and SHBG were examined in the ANCOVA models with smoking status while adjusting for age and total body fat (for serum total and bioavailable estradiol, the models were examined without total body fat) to see if the effect of smoking is mediated through these biochemical variables. When serum 1,25(OH)2D was categorized into quartiles, it was a significant predictor of grip strength (p =.026) and a marginally significant predictor of timed rise (p =.06) and timed walk normal (p =.07) (Table 2). The effect of smoking was still significant in models with serum 1,25(OH)2D, though slightly lower than that seen in models without 1,25(OH)2D. Serum 25OHD, total estradiol, bioavailable estradiol (Table 2), and SHBG were not significant predictors of physical performance measures. However, serum total testosterone was found to be a significant predictor of grip strength. Discussion The results presented in this cross-sectional study demonstrate that smoking reduces physical performance in elderly postmenopausal women. Current smokers were slower on timed rise and timed walk tests, and had decreased grip strength. The effect of smoking is equivalent to the normal age-related decline in physical performance tests of 7–11 years. The grip strength measurement in nonsmokers (65–77 years) in the present study (25.2 ± 4.41 kg) is close to the normative data reported in the literature for women between the ages of 60–79 years (24.5 ± 5.0 kg) (28). In the present study, past smokers had physical performance measures similar to those of nonsmokers, apart from the timed walk test, which was slower in those women with more pack-years of smoking. There was no significant association between the quantity of smoking and physical performance measures. There are few studies in the literature that report that smoking is associated with a decrease in physical performance. The results of the present study are in agreement with those of Nelson and colleagues (13), who demonstrated that current smoking is associated with impaired neuromuscular performance. Also, Guralnik and Kaplan (14) reported a higher physical function in nonsmokers than in smokers after 19 years of follow-up. In the elderly and disabled Medicare population, Arday and colleagues (29) reported that current smokers have worse physical and mental functional status than do never smokers. Stovring and colleagues (30) reported that the cumulative effect of smoking from age 50–70 years adversely affects the functional ability at age 75 years. None of these studies suggested the mechanism of how smoking affects physical function. Changes in vitamin D metabolism may play a role in decreased physical performance. 1,25 dihydroxyvitamin D receptor (VDR) is expressed in skeletal muscle tissue, and evidence indicates that 1,25(OH)2D plays a key role in muscle contractility, cell calcium and phosphate transport, and protein and phospholipid synthesis in muscle cells (31). VDR in muscle tissue has been reported to decrease with age (32). Several cross-sectional studies have shown that low serum 1,25(OH)2D and low serum 25OHD levels are related to lower muscle strength and falls in older men and women (2,3). In healthy elderly women, we have shown decreased physical performance in the lower quartiles of serum 1,25(OH)2D and 25OHD compared to the corresponding higher quartiles (33). Vitamin D and calcium supplementation has been demonstrated to improve musculoskeletal function and decrease fall incidence in the elderly population (5,34). In the present study, smokers have marginally lower serum 25OHD and serum 1,25(OH)2D levels. In multivariate analysis, serum 1,25(OH)2D examined as quartiles was a predictor of physical performance measures and lessened the effect of smoking, indicating that some of the effect of smoking is mediated through changes in serum 1,25(OH)2D. Although serum 25OHD levels were lower in smokers, adjustment for serum 25OHD did not affect the association of smoking and physical performance measures. In the present study, smokers had lower serum total and bioavailable estradiol, lower serum bioavailable testosterone, and higher serum SHBG levels. In univariate analyses, serum bioavailable estradiol was significantly lower in smokers compared to nonsmokers and past smokers. It is known that adipose tissue produces estrogen by aromatase conversion of androstenedione in postmenopausal women (12), so the lower body weight in smokers is a probable cause of lower circulating serum estradiol; smoking has also been suggested to increase the metabolism of estrogen (12). However, after adjustment for total body fat, the significance of estradiol was lost. Furthermore, in multivariate analyses, total estradiol and bioavailable estradiol (in absence of total body fat in the models) were not significant predictors of physical performance tests, thus the effect of smoking may not be mediated through changes in estrogen metabolism. Total body fat was a significant independent predictor of physical performance tests other than grip strength; heavier participants were slower on timed walking and chair rise tests. There was no difference in lean muscle mass in smokers compared to nonsmokers, so the loss of muscle mass is not a factor in decreased physical performance. The reason for an effect of smoking on total and bioavailable testosterone is not very clear, but it has been suggested that a decrease in adrenal production of androgens may be responsible (12). Serum total testosterone was a significant predictor of grip strength in the present study, but the mechanism of how it affects physical performance is not very clear. Decreased testosterone levels in men were reported to be associated with lower appendicular skeletal muscle mass and increased risk of falls, impairment of balance, and impaired ability to perform the tandem walk (35). Testosterone supplementation in elderly men has been reported to improve muscle mass and strength (35). Testosterone administration in men was associated with a dose-dependent increase in leg-press strength and leg power, which correlated with testosterone dose and circulating testosterone concentrations (36). However, Snyder and colleagues (37) reported no effect of testosterone on muscle strength in older men. In one controlled study of low weight HIV-infected young women, testosterone supplementation was associated with increase in shoulder flexion, elbow flexion, knee extension, and knee flexion, but not in grip strength (38); however, no corresponding studies have been reported in elderly women. In the present study, the interactions of smoking with serum 25OHD, 1,25(OH)2D, total estradiol, bioavailable estradiol, testosterone, and SHBG on physical performance measures were not significant. This could be because our study was not adequately powered to assess these interactions. The present study has some limitations. The study is cross-sectional in nature. The women enrolled into the study were healthy volunteers, and our findings may not apply to the general population. The smoking status of women in the present study is based on self-report and possibly underestimates the true use. Overall, smoking affects physical performance in part through its effect on 1,25(OH)2D status. However, an independent effect of smoking on physical performance by affecting vascular function (39) or by a direct effect on muscle (40,41) is a possibility. In summary, our results suggest that current smoking is a risk factor for decreased muscle strength leading to decreased physical performance in elderly women. Decision Editor: Luigi Ferrucci, MD, PhD Figure 1. Mean baseline physical performance measures according to smoking status. The values are unadjusted mean ± standard error of the mean. Means were compared by analysis of covariance adjusting for significant confounding variables that were significant in the models and included the covariates given in Table 2. Significance was derived from adjusted data using Tukey's post hoc multiple comparison test. *p <.05 compared to nonsmokers; †p <.05 compared to past smokers Table 1. Characteristics and Biochemical Variables According to Smoking Status. Parameter Nonsmokers Past Smokers Current Smokers N 268 163 56 Age, y 71.7 ± 0.22 71.4 ± 0.29 70.6 ± 0.42 Height, cm 159.2 ± 0.40 159.2 ± 0.53 159.5 ± 0.67 Weight, kg 69.5 ± 0.74 69.5 ± 1.02 61.3 ± 1.60* Total body fat, kg 29.0 ± 5.55 28.9 ± 7.47 22.2 ± 11.3*† Lean muscle mass, kg 37.4 ± 2.54 37.3 ± 3.34 36.2 ± 6.02 Dietary calcium intake, mg/d 755.1 ± 17.3 727.9 ± 25.7 703.5 ± 49.8 Dietary caffeine intake, mg/d 225.4 ± 10.6 294.9 ± 15.4* 420.6 ± 54.6* Dietary vitamin D intake, μg/d 3.68 ± 0.13 3.30 ± 0.16 3.08 ± 0.31 Alcohol drinkers, N (%) 66 (25) 73 (45) 24 (43) Medication use     0 108 (40%) 60 (37%) 19 (34%)     1 78 (29%) 52 (32%) 21 (38%)     >1 82 (31%) 51 (31%) 16 (29%) Comorbidities     0–2 44 (16%) 25 (15%) 8 (14%)     3–4 103 (38%) 62 (38%) 26 (46%)     5–6 80 (30%) 45 (28%) 11 (20%)     ≥ 7 41 (15%) 31 (19%) 11 (20%) Serum 25OHD, ng/mL 31.7 ± 0.61 31.6 ± 0.75 28.6 ± 1.91 Serum 1,25(OH)2D, pg/mL 34.8 ± 0.51 34.0 ± 0.59 33.9 ± 1.05 Serum PTH, pg/mL 37.9 ± 0.94 36.2 ± 1.09 35.8 ± 1.71 Serum total estradiol, pg/mL 12.34 ± 0.34 11.63 ± 0.46 10.96 ± 0.65 Serum bioavailable estradiol, pg/mL 3.71 ± 0.13 3.50 ± 0.20 2.82 ± 0.21 Serum SHBG, nmol/L 139.4 ± 3.9 143.0 ± 5.8 197.4 ± 12.2*† Serum total testosterone,ng/mL 0.24 ± 0.007 0.23 ± 0.011 0.27 ± 0.018† Serum bioavailable testosterone, pg/mL 27.08 ± 1.03 26.45 ± 1.83 24.88 ± 1.94 Notes: Values are unadjusted means and standard error of means. Characteristics were compared by ANOVA with Tukey's method for post hoc analysis. Categorical variables were compared by chi-square tests. Biochemical variables were compared by ANCOVA adjusting for significant confounding variables including age, body mass index, calcium intake, and total body fat. Significance was derived from adjusted data using Tukey's post hoc multiple comparison test. *p <.05 compared to nonsmokers. †p <.05 compared to past smokers. Table 2. Multivariate ANCOVA Models With Physical Performance Measure Outcomes, Looking at Possible Mediator Variables. Model 1 Model 2 Model 3 Model 4 Outcome Predictor Variable β ± SE p Value β ± SE p Value β ± SE p Value β ± SE p Value Timed rise, s Serum 1,25(OH)2D Quartile 1 NI 0.37 ± 0.43 .062 NI NI Quartile 2 –0.45 ± 0.43 Quartile 3 –0.70 ± 0.43 Quartile 4 Ref Serum total estradiol (1 pg/mL increase) NI 0.003 ± 0.031 .92 NI Serum bioavailable estradiol (1 pg/mL increase) NI NI –0.008 ± 0.076 .92 Total body fat (1 kg increase) 0.000051 ± 0.000017 .0011 0.000056 ± 0.000017 .0008 NI NI Age (1 y increase) 0.20 ± 0.042 <.0001 0.19 ± 0.042 <.0001 0.16 ± 0.044 .0005 0.16 ± 0.044 .0005 Smoking status Nonsmoker Ref Ref Ref Ref Past smoker –0.047 ± 0.33 .016 –0.082 ± 0.33 .025 –0.001 ± 0.35 .3 –0.005 ± 0.35 .32 Smoker 1.39 ± 0.50* 1.30 ± 0.50* 0.80 ± 0.53 0.78 ± 0.54 Timed walk (normal), s Serum 1,25(OH)2D Quartile 1 NI 0.035 ± 0.13 .072 NI NI Quartile 2 –0.060 ± 0.13 Quartile 3 −0.29 ± 0.13 Quartile 4 Ref Serum total estradiol (1 pg/mL increase) NI –0.005 ± 0.010 .63 NI Serum bioavailable estradiol (1 pg/mL increase) NI NI –0.019 ± 0.025 .46 Total body fat (1 kg increase) 0.000021 ± 0.0000052 <.0001 0.000021 ± 0.0000052 <.0001 NI NI Age (1 y increase) 0.055 ± 0.013 <.0001 0.055 ± 0.013 <.0001 0.049 ± 0.015 .0008 0.049 ± 0.015 .001 Smoking status Nonsmoker Ref Ref Ref Ref Past smoker 0.075 ± 0.10 .0002 0.063 ± 0.10 .0003 0.069 ± 0.11 .037 0.069 ± 0.11 .045 Smoker 0.65 ± 0.16** 0.64 ± 0.16** 0.45 ± 0.17* 0.44 ± 0.18* Timed walk (fast), s Serum 1,25(OH)2D Quartile 1 NI 0.13 ± 0.10 .17 NI NI Quartile 2 0.083 ± 0.10 Quartile 3 –0.085 ± 0.10 Quartile 4 Ref Serum total estradiol (1 pg/mL increase) NI NI 0.0024 ± 0.008 .75 NI Serum bioavailable estradiol (1 pg/mL increase) NI NI NI –0.010 ± 0.019 .6 Total body fat (1 kg increase) 0.000011 ± 0.0000040 .0058 0.000011 ± 0.0000040 .0071 NI NI Age (1 y increase) 0.039 ± 0.01 .0002 0.039 ± 0.01 .0002 0.036 ± 0.011 .0012 0.036 ± 0.011 .0013 Smoking status Nonsmoker Ref Ref Ref Ref Past smoker 0.077 ± 0.080 .0076 0.067 ± 0.080 .011 0.098 ± 0.086 .066 0.095 ± 0.086 .084 Smoker 0.38 ± 0.12* 0.37 ± 0.12* 0.30 ± 0.09 0.28 ± 0.13 Grip strength, kg Serum 1,25(OH)2D* Quartile 1 NI –1.20 ± 0.60 .026 NI NI Quartile 2 –0.30 ± 0.60 Quartile 3 0.57 ± 0.59 Quartile 4 Ref Serum total estradiol (1 pg/mL increase) NI NI 0.012 ± 0.048 .81 NI Serum bioavailable estradiol (1 pg/mL increase) NI NI NI 0.095 ± 0.12 .42 Age (1 y increase) –0.20 ± 0.060 .0008 –0.20 ± 0.060 .0007 –0.20 ± 0.060 .001 –0.20 ± 0.060 .0011 Serum testosterone (1 ng/mL increase) 5.39 ± 1.87 .0042 5.11 ± 1.87 .0066 5.57 ± 2.18 .011 5.03 ± 2.15 .02 Smoking status Nonsmoker Ref Ref Ref Ref Past smoker 0.50 ± 0.47 .0046 0.51 ± 0.46 .0073 0.54 ± 0.47 .0045 0.54 ± 0.47 .0067 Smoker –1.99 ± 0.71* –1.87 ± 0.71* –2.0 ± 0.73* –1.92 ± 0.73* Notes: β coefficient values are from multivariate models including significant predictors out of age, body mass index, total calcium intake, caffeine intake, total body fat, sex hormone binding globulin (SHBG), serum 25 hydroxy vitamin D (25OHD), serum 1,25 –dihydroxyvitamin D (1,25(OH)2D), serum total estradiol, serum bioavailable estradiol, serum total and bioavailable testosterone, and smoking status (smoker vs nonsmoker or past smoker). Model 1 looks at only significant predictors; Model 2 looks at significant predictors + serum 1,25(OH)2D; Model 3 looks at significant predictors + serum total estradiol without total body fat; Model 4 looks at significant predictors + serum bioavailable estradiol without total body fat. * p <.01 compared to nonsmokers. ** p <.0001 compared to nonsmokers. ANCOVA = Analysis of covariance; SE = standard error; NI = Not included in the model. Table 3. Co-morbidities in the Study Population. Non-smokers (n = 268) Past Smokers (n = 163) Smokers (n = 56) p value Hypertension 107 (40%) 52 (32%) 14 (25%) 0.052 Anxiety 13 (5%) 10 (6%) 6 (11%) 0.21 Depression 9 (3%) 8 (5%) 1 (2%) 0.61 Glaucoma 17 (6%) 7 (4%) 1 (2%) 0.4 Asthma 6 (2%) 6 (4%) 1 (2%) 0.61 Headaches 16 (6%) 8 (5%) 2 (4%) 0.88 Osteoporosis 17 (6%) 20 (13%) 5 (9%) 0.078 Scoliosis 11 (4%) 8 (5%) 3 (6%) 0.86 Diabetes 13 (5%) 11 (7%) 1 (2%) 0.38 Hypothyroid 50 (19%) 30 (18%) 9 (16%) 0.94 Cancer 10 (4%) 10 (6%) 6 (11%) 0.095 Joint Disease (Arthritis) 101 (38%) 53 (33%) 15 (27%) 0.24 Chronic GI Disease 50 (19%) 36 (22%) 13 (24%) 0.54 Heart Disease 46 (17%) 38 (24%) 11 (20%) 0.25 Lung Disease 11 (4%) 17 (11%) 5 (9%) 0.026 Hip fracture 4 (2%) 6 (4%) 3 (5%) 0.14 The values given are absolute numbers with the percentages in the parentheses. Chi-square test was used to determine the differences between the groups. Table 4. Medication use in Study Population. Non-smoker (n = 268) Past Smoker (n = 163) Smoker (n = 56) p value Thiazide Diuretics 42 (16%) 18 (11%) 5 (9%) 0.26 Loop Diuretics 41 (15%) 20 (12%) 5 (9%) 0.42 Thyroid Preparations 47 (18%) 28 (17%) 7 (13%) 0.72 Sedatives/Anticonvulsants/Narcotics 10 (4%) 8 (5%) 7 (12%) 0.14 Antidepressants 9 (3%) 10 (6%) — 0.099 Tranquilizer 6 (2%) 5 (3%) 4 (7%) 0.13 NSAIDs 45 (17%) 21 (13%) 6 (11%) 0.39 Aspirin 49 (18%) 35 (21%) 10 (18%) 0.7 Antihistamine 14 (5%) 10 (6%) 7 (13%) 0.14 Cholesterol Med 23 (9%) 14 (9%) 1 (2%) 0.2 The values given are absolute numbers with the percentages in the parentheses. Chi-square test was used to determine the differences between the groups. This work was supported by National Institutes of Health Research Grants UO1-AG10373 and RO1-AG10358. We thank Karen A. Rafferty for her help in food diary data collection and analysis. We also thank Kurt E. Balhorn for the laboratory analysis. This work was presented as an abstract at 27th Annual Meeting of the American Society for Bone and Mineral Research, Nashville, Tennessee, September 23–27, 2005. References 1 Moreland JD, Richardson JA, Goldsmith CH, Clase CM. Muscle weakness and falls in older adults: a systematic review and meta-analysis. J Am Geriatr Soc.2004;52:1121-1129. 2 Bischoff HA, Stahelin HB, Urscheler N, et al. Muscle strength in the elderly: its relation to vitamin D metabolites. Arch Phys Med Rehabil.1999;80:54-58. 3 Dhesi JK, Bearne LM, Moniz C, et al. Neuromuscular and psychomotor function in elderly subjects who fall and the relationship with vitamin D status. J Bone Miner Res.2002;17:891-897. 4 Dhesi JK, Jackson SH, Bearne LM, et al. Vitamin D supplementation improves neuromuscular function in older people who fall. Age Ageing.2004;33:589-595. 5 Pfeifer M, Begerow B, Minne HW, Abrams C, Nachtigall D, Hansen C. 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J Clin Endocrinol Metab.1998;83:2266-2274. 21 Fess EE. Grip Strength. Clinical Assesment Recommendations. Chicago, IL: American Society of Hand Therapists; 1992:41–45. 22 Lindsey C, Brownbill RA, Bohannon RA, Ilich JZ. Association of physical performance measures with bone mineral density in postmenopausal women. Arch Phys Med Rehabil.2005;86:1102-1107. 23 Bohannon RW. Comfortable and maximum walking speed of adults aged 20-79 years: reference values and determinants. Age Ageing.1997;26:15-19. 24 Judge JO, Lindsey C, Underwood M, Winsemius D. Balance improvements in older women: effects of exercise training. Phys Ther.1993;73:254-262. 25 Bohannon RW. Sit-to-stand test for measuring performance of lower extremity muscles. Percept Mot Skills.1995;80:163-166. 26 Washburn RA, Smith KW, Jette AM, Janney CA. The Physical Activity Scale for the Elderly (PASE): development and evaluation. J Clin Epidemiol.1993;46:153-162. 27 Washburn RA, McAuley E, Katula J, Mihalko SL, Boileau RA. The physical activity scale for the elderly (PASE): evidence for validity. J Clin Epidemiol.1999;52:643-651. 28 Desrosiers J, Bravo G, Hebert R, Dutil E. Normative data for grip strength of elderly men and women. Am J Occup Ther.1995;49:637-644. 29 Arday DR, Milton MH, Husten CG, et al. Smoking and functional status among Medicare managed care enrollees. Am J Prev Med.2003;24:234-241. 30 Stovring N, Avlund K, Schultz-Larsen K, Schroll M. The cumulative effect of smoking at age 50, 60, and 70 on functional ability at age 75. Scand J Public Health.2004;32:296-302. 31 Boland R, de Boland AR, Marinissen MJ, Santillan G, Vazquez G, Zanello S. Avian muscle cells as targets for the secosteroid hormone 1,25-dihydroxy-vitamin D3. Mol Cell Endocrinol.1995;114:1-8. 32 Bischoff-Ferrari HA, Borchers M, Gudat F, Durmuller U, Stahelin HB, Dick W. Vitamin D receptor expression in human muscle tissue decreases with age. J Bone Miner Res.2004;19:265-269. 33 Rapuri PB, Gallagher JC, Smith LM. Higher serum 1,25-dihydroxy vitamin D and 25-hydroxy vitamin D levels are associated with better physical performance in elderly postmenopausal women (abstract). J Bone Miner Res.2005;20:(Suppl 1): S386. 34 Bischoff HA, Stahelin HB, Dick W, et al. Effects of vitamin D and calcium supplementation on falls: a randomized controlled trial. J Bone Miner Res.2003;18:343-351. 35 Herbst KL, Bhasin S. Testosterone action on skeletal muscle. Curr Opin Clin Nutr Metab Care.2004;7:271-277. 36 Storer TW, Magliano L, Woodhouse L, et al. Testosterone dose-dependently increases maximal voluntary strength and leg power, but does not affect fatigability or specific tension. J Clin Endocrinol Metab.2003;88:1478-1485. 37 Snyder PJ, Peachey H, Hannoush P, et al. Effect of testosterone treatment on body composition and muscle strength in men over 65 years of age. J Clin Endocrinol Metab.1999;84:2647-2653. 38 Dolan S, Wilkie S, Aliabadi N, et al. Effects of testosterone administration in human immunodeficiency virus-infected women with low weight: a randomized placebo-controlled study. Arch Intern Med.2004;164:897-904. 39 Kilaru S, Frangos SG, Chen AH, et al. Nicotine: a review of its role in atherosclerosis. J Am Coll Surg.2001;193:538-546. 40 Larsson L, Orlander J. Skeletal muscle morphology, metabolism and function in smokers and non-smokers. A study on smoking-discordant monozygous twins. Acta Physiol Scand.1984;120:343-352. 41 Orlander J, Kiessling KH, Larsson L. Skeletal muscle metabolism, morphology and function in sedentary smokers and nonsmokers. Acta Physiol Scand.1979;107:39-46. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B09EA51EB71D2482A6296AB120DD0363B40F44F.txt b/test/dataset/in/resources/corpus/Clean_0B09EA51EB71D2482A6296AB120DD0363B40F44F.txt new file mode 100644 index 0000000..f5a1719 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B09EA51EB71D2482A6296AB120DD0363B40F44F.txt @@ -0,0 +1 @@ +amjepidajeAmerican Journal of Epidemiology1476-62560002-9262Oxford University Press10.1093/aje/kwp032ORIGINAL CONTRIBUTIONSAre the Short-term Effects of Air Pollution Restricted to Cardiorespiratory Diseases?LarrieuSophieLefrancAgnèsGaultGaëlleChatignouxEdouardCouvyFranckJouvesBernardFilleulLaurentCorrespondence to Dr. Sophie Larrieu, French Institute of Public Health Surveillance (InVS), Cire Aquitaine, Espace Rodesse, 103 bis rue Belleville, BP952, 33063 Bordeaux Cedex, France (e-mail: sophie.larrieu@sante.gouv.fr).1552009242009169101201120818720082612009American Journal of Epidemiology © The Author 2009. Published by the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2009Short-term effects of air pollution on common morbidity are largely unknown. The authors explored links between daily levels of air pollution (nitrogen dioxide, ozone, and particulate matter less than 10 μm in diameter (PM10)) and medical home visits made for diverse reasons in Bordeaux, France, during 2000–2006. Daily numbers of visits were obtained from a network of general practitioners. The excess relative risk (ERR) of a visit for each indicator associated with increased pollutant levels was estimated by fitting a Poisson regression model, controlling for well-known confounding factors and temporal trends. Positive and significant associations were found between air pollution and most health indicators. A 10-μg/m3 increase in PM10 levels was associated with increases in visits for upper and lower respiratory diseases (ERRs were 1.5% (95% confidence interval (CI): 0.3, 2.7) and 2.5% (95% CI: 0.5, 4.4), respectively), headache and asthenia (ERR = 3.5%, 95% CI: 1.3, 5.9), and skin rash and conjunctivitis (ERR = 3.2%, 95% CI: −0.2, 6.8). Significant associations were also found between nitrogen dioxide and ozone and several health indicators. Distributed-lag models showed no harvesting effect, and some effects persisted up to 15 days after exposure increased. These results suggest that considering only the most severe effects of air pollution leads to underestimation of its impact on public health.air pollutionastheniaconjunctivitisexanthemaheadacheprimary health carerespiratory tract diseasesThe health effects of air pollution have been subject to intense epidemiologic and experimental study during the past 2 decades. It is now well-documented that day-to-day variations in air pollutant levels are associated with cardiorespiratory morbidity and mortality (1), and several biologic hypotheses have been proposed to explain air pollutant effects, mainly involving acute pulmonary inflammation and oxidative stress (2). These effects have been widely studied for particulate air pollution, but there is also substantial evidence concerning gaseous pollutants such as ozone and nitrogen dioxide. The exposure-risk relations observed are usually described as linear without any threshold (3, 4), or results suggest that the threshold above which no effect would be observed is far below the concentrations observed in urban areas (5). Furthermore, the severity of these effects is widely variable, and they can affect more or fewer people; indeed, short-term effects of air pollution are often represented as a pyramid, with the mildest but not uncommon effects at the bottom and the least common but more severe effects at the top (Figure 1).Figure 1.Pyramidal representation of the hypothesized short-term effects of air pollution on health.Most epidemiologic studies conducted to date have focused on severe events such as mortality, hospitalizations, and emergency room visits. On the other hand, more common effects have been less well described, mainly because of the unavailability of reliable indicators of primary-care setting, symptoms, or medication use. A few studies showed a significant association between air pollutant levels and general practitioner home visits in Paris, France (6), London, United Kingdom (7–9), and Hong Kong, China (10), but those studies focused on respiratory diseases; other symptoms have rarely been investigated.In Bordeaux, a mid-sized metropolitan area in southwestern France, a network of 60 general practitioners has been collecting data on the practitioners’ activities for many years, allowing us to link their activity with air pollutant levels measured over the Bordeaux area. Therefore, our aim in this study was to explore the links between daily levels of air pollution indicators (nitrogen dioxide, ozone, and particulate matter less than 10 μm in diameter (PM10)) and daily numbers of medical home visits made for nonsevere reasons, some of which are known to be associated with air pollution (respiratory diseases) and others of which have a less well described association with air pollution (headache, asthenia, conjunctivitis, and skin rash) in this area.MATERIALS AND METHODSStudy population and periodThis study was conducted during the period 2000–2006 in the area of Bordeaux, France. It included Bordeaux and 21 other neighboring cities where urbanization and background air pollutant levels could be considered homogeneous, representing a total population of approximately 600,000 inhabitants.Estimates of pollution exposureThe local air quality monitoring network, AIRAQ (http://www.airaq.asso.fr/), provided data on daily levels of ambient air pollutants measured within the study area: nitrogen dioxide, PM10, and ozone. We used measurements from the 4 background monitoring stations available in the study area, because they are not influenced by occasional sources of pollution, since they are located some distance away from major roads and industries.For each air pollutant, we calculated correlation coefficients for correlations between the different stations in order to ensure that they all measured the same type of pollution (r > 0.60); the daily level of each air pollution indicator was then computed as the arithmetic mean of the daily levels recorded by the stations (for PM10 and nitrogen dioxide) or the arithmetic mean of the daily maximum of 8-hour moving averages (for ozone).Health outcomesSOS Médecins is the primary emergency and health-care network in France, providing home medical visits by general practitioners in response to private house calls 24 hours a day, 7 days a week. In the urban area of Bordeaux, SOS Médecins comprises 60 general practitioners who make more than 400 visits per day, operating in an area of approximately 800,000 inhabitants. Characteristics concerning each visit are logged into a local database; all symptoms and/or complaints reported by the patients are coded and recorded according to the International Classification of Primary Care, Second Edition (11), as well as the final diagnosis. From this database, we extracted daily numbers of visits made to people living in the study area for the following reasons: upper and lower respiratory diseases, asthma, asthenia, headache, conjunctivitis, and skin rash (Table 1). In order to evaluate any bias in the analysis, we also extracted and modeled daily numbers of visits made for lumbago, which is unrelated to air pollution a priori, according to the same method.Table 1.Health Indicators and Corresponding ICPC-2 Codes Used in a Study of Air Pollutant Effects, Bordeaux, France, 2000–2006Health IndicatorCorresponding Syndrome(s) and/or Symptom(s)ICPC-2 Code(s)Upper respiratory diseasesTonsillitis, sinusitis, rhinitis, nasopharyngitis, pharyngitis, laryngitis, tracheitisR75, R76, R77, R83, R97Lower respiratory diseasesBronchitis, bronchiolitis, chronic obstructive pulmonary disease, coughR78, R79, R95, R05AsthmaAsthmaR96AstheniaAstheniaA04HeadacheHeadacheN01, N89ConjunctivitisConjunctivitisF70, F71Skin rashDermatitis, eczema, urticaria, skin rashS07, S87, S98Abbreviation: ICPC-2, International Classification of Primary Care, Second Edition (11).Potentially confounding factorsData on the following potentially confounding factors were collected: ambient temperature measured at the Bordeaux meteorologic station, obtained from the national meteorologic institute (Météo-France); periods of influenza epidemics, obtained from the SOS Médecins database (according to a local influenza epidemic threshold that usually allows monitoring of the disease in the area); pollen counts measured at the Bordeaux pollen monitoring station, obtained from the French surveillance system for pollen counts (Réseau National de Surveillance Aérobiologique); and dates of holidays, obtained from the French Ministry of Education.Statistical analysisDaily numbers of medical home visits were analyzed with time-series methods, using generalized additive Poisson regression models allowing for overdispersion (12). Each air pollution indicator was included in the model as a linear term, and different lags were tested: pollutant levels on the current day and up to 3 days before (lags of 0, 1, 2, and 3 days) and mean levels during the current day and the previous 1, 2, or 3 days (lags of 0–1, 0–2, and 0–3 days). The lag that minimized Akaike's Information Criterion (13) was retained. Adjustments were made for possible confounders—including long-term trends, seasonality, days of the week, holidays, minimum temperature on the current day and maximum temperature on the previous day, and influenza epidemics—following the methods of the APHEA-2 [Air Pollution And Health: A European Approach] Study (14). Long-term trends and seasonality were modeled using penalized cubic regression splines. The degree of smoothing of the spline function was chosen to remove seasonal and long-term temporal trends by minimizing the autocorrelation in the residuals. Dummy variables for days of the week and holidays were included as other independent variables. Temperature and influenza epidemics were modeled using natural splines with 3 degrees of freedom for each. The lack of residual autocorrelation was checked through the partial autocorrelation, and the Bartlett test was used to check that white noise was obtained.A term for interaction between ozone and season was introduced into the regression models to specifically assess the effect of ozone during the warmer months (April–September).All results are presented as the excess relative risk (ERR) of a medical home visit (expressed as a percentage) associated with a 10-μg/m3 increase in air pollution indicator level. To determine whether there were any effects in subgroups of the population, we also performed analyses on specific age groups, when the number of events was large enough, notably to estimate specific ERRs for frail populations such as children under age 15 years (15) and people aged 65 years or more (16).Since the health effects of air pollution may persist for several days after exposure or, on the contrary, potential morbidity displacement may exist (17), we also used distributed-lag models to better describe pollutant effects that were delayed by up to 15 days, as follows:with γ being constrained to follow a cubic polynomial function. As in the generalized additive Poisson regression models, the number of degrees of freedom was chosen to minimize residual autocorrelation.This model could not be used for ozone, because the term for interaction between ozone and season led to many breaks in the pollutant series.RESULTSThe study area comprised 22 cities and more than 600,000 inhabitants, of whom 15.5% were less than 15 years of age and 15.7% were aged 65 years or older. Descriptive results for daily air pollutant levels and health indicators are shown in Table 2.Table 2.Daily Air Pollutant Levels and Daily Numbers of General Practitioners’ Visits Made for Different Health Indicators, Bordeaux, France, 2000–2006Air Pollution or Health IndicatorMeanMinimum5th Percentile50th Percentile95th PercentileMaximumPollutant Level, μg/m3Nitrogen dioxide21.93.88.020.341.380.3Ozone69.32.732.568.2111.8142.7Ozone in spring and summera84.837.548.781.5117.1142.7PM1021.15.010.318.838.588.2No. of VisitsUpper respiratory diseases57.68255696149Lower respiratory diseases16.404143877Asthma3.5003819Asthenia2.2002511Headache4.1014818Conjunctivitis0.600028Skin rash3.5003815Lumbago (control)5.70251120Abbreviation: PM10, particulate matter less than 10 μm in diameter.aApril 1–September 30.Bordeaux is a moderately polluted area, with annual mean levels of nitrogen dioxide and PM10 that are largely below the current French annual guidelines (40 μg/m3) (18), but PM10 levels are slightly above the 2005 World Health Organization guidelines (20 μg/m3) (19). Daily maximum levels of 8-hour moving averages of ozone overtook the threshold for health protection (120 μg/m3) during less than 5% of the study period.During the 7-year study period, a total of 895,710 medical home visits were made by SOS Médecins Bordeaux, corresponding to a daily mean of 350 visits. Visits for respiratory diseases represented approximately 20% of the activity of the general practitioners. Since the numbers of visits for the 4 other indicators were quite low, we grouped together the indicators asthenia and headache, which correspond to a general health impairment without another diagnosed disease, and conjunctivitis and skin rash, which can be related to irritation and atopy.As Figure 2 shows, the general practitioners’ activity showed important day-of-the-week and seasonal variations. The daily number of visits increased on weekends and during the winter.Figure 2.Seasonal (main graph) and daily (inset) numbers of medical home visits (gray line) made to persons in the Bordeaux metropolitan area and 7-day moving averages (black line), Bordeaux, France, 2000–2006.We constructed specific models for each health indicator and each air pollution indicator. The mean of the lag 0–3 days led to the best model for all indicators except headache/asthenia, for which a lag of 0 was considered for every air pollution indicator. Table 3 presents the ERR of a medical home visit associated with a 10-μg/m3 increase in air pollutant levels for each health indicator.Table 3.Excess Relative Risk (%) of a Medical Home Visit Associated With a 10-μg/m3 Increase in Air Pollutant Levels, Bordeaux, France, 2000–2006DiagnosisNitrogen DioxidePM10OzoneERR95% CIERR95% CIERR95% CIUpper respiratory diseasesa0.8−0.7, 2.31.50.3, 2.7−0.6−1.7, 0.5Lower respiratory diseasesa2.60.2, 4.92.50.5, 4.4−0.4−2.5, 1.7Asthmaa1.1−3.0, 5.20.5−3.1, 4.1−0.8−3.9, 2.3Headache or astheniab2.80.4, 5.33.51.3, 5.91.70.2, 3.3Skin rash or conjunctivitisa0.3−3.3, 4.23.2−0.2, 6.83.00.4, 5.7Lumbago (control)a−0.3−3.4, 2.90.5−2.3, 3.50.5−1.6, 2.6Abbreviations: CI, confidence interval; ERR, excess relative risk; PM10, particulate matter less than 10 μm in diameter.aLag of 0–3 days.bLag of 0 days.The risk of a visit for upper respiratory diseases was significantly increased by 1.5% (95% confidence interval (CI): 0.3, 2.7) during the 3 days following a 10-μg/m3 increase in PM10 levels; a similar but nonsignificant trend was also observed for an increase in nitrogen dioxide levels. Similarly, the risks of consulting a general practitioner for lower respiratory diseases increased by 2.6% (95% CI: 0.2, 4.9) and 2.5% (95% CI: 0.5, 4.4) following 10-μg/m3 increases in nitrogen dioxide and PM10 levels, respectively. Asthma was not associated with any of the indicators considered, and no trend was found, nor was an association found between visits made for respiratory diseases and ozone.The daily number of visits made for headache or asthenia was significantly associated with the 3 air pollution indicators considered; same-day ERRs were 2.8% (95% CI: 0.4, 5.3), 3.5% (95% CI: 1.3, 5.9), and 1.7% (95% CI: 0.2, 3.3) for 10-μg/m3 increases in nitrogen dioxide, PM10, and ozone levels, respectively. The risk of a visit for skin rash or conjunctivitis was also increased during the 3 days following increases in PM10 (ERR = 3.2%, 95% CI: −0.2, 6.8 (close to significance)) and ozone (ERR = 3.0%, 95% CI: 0.4, 5.7) levels.Lastly, the daily number of visits made for lumbago, which we chose as the control outcome in order to evaluate any bias in the analyses, was not associated with any of the pollutant indicators considered.Results from complementary analyses of specific age groups are illustrated in Figure 3. Although the confidence intervals overlapped, central estimates of ERR were much higher in the elderly for respiratory diseases (upper and lower), regardless of the type of pollutant, suggesting a higher effect in this subgroup. This difference was particularly important for upper respiratory diseases, with ERRs of 12.3% (95% CI: 4.9, 19.7) and 8.3% (95% CI: 2.0, 14.7) being associated with 10-μg/m3 increases in nitrogen dioxide and PM10 levels, respectively, in this subgroup. In the other age groups, effects were close to each other.Figure 3.Excess relative risk (%) of a medical home visit for upper and lower respiratory diseases associated with a 10-μg/m3 increase in air pollutants, Bordeaux, France, 2000–2006. NO2, nitrogen dioxide; O3, ozone; PM10, particulate matter less than 10 μm in diameter. Bars, 95% confidence interval.For asthma, in addition to the whole population, no association was observed in children.When distributed-lag models were used, no harvesting effect was observed, regardless of which health indicator was considered. Figure 4 compares the results from initial models and from distributed-lag models, in which up to 15 days of lag time between the exposure and health effects were considered. Taking into account lags over 15 days did not lead to larger associations between PM10 and numbers of home visits, whatever the diagnosis considered, whereas estimates for nitrogen dioxide were much higher when the effects of lags of up to 15 days on upper respiratory diseases (global ERR = 5.7%, 95% CI: 2.7, 8.8) and lower respiratory diseases (global ERR = 9.4%, 95% CI: 4.7, 14.3) were considered. It is also noteworthy that there was a much higher effect of nitrogen dioxide on visits for skin rash and conjunctivitis when delayed effects were considered, even though the excess risk was not significant because of a large confidence interval.Figure 4.Comparison of excess relative risks (%) of a medical home visit obtained using a lag time of 0–3 days with excess relative risks obtained using distributed-lag models (15-day cumulative effect), Bordeaux, France, 2000–2006. LRD, lower respiratory diseases; NO2, nitrogen dioxide; PM10, particulate matter less than 10 μm in diameter; URD, upper respiratory diseases. Bars, 95% confidence interval.The persistence of nitrogen dioxide effects over 15 days is illustrated in Figure 5, which represents the ERR of a visit for upper respiratory diseases estimated for each lag up to 14 days through distributed-lag models. This ERR started to decrease progressively about 10 days after exposure and was significant up to a lag of 11 days.Figure 5.Excess relative risk (%) of a medical home visit for upper respiratory diseases associated with a 10-μg/m3 increase in nitrogen dioxide levels, by lag time, Bordeaux, France, 2000–2006. Bars, 95% confidence interval.DISCUSSIONThis study is one of the few to have investigated short-term relations between air pollution and morbidity through diagnoses made by general practitioners. It underlines that the health effects of air pollution are not restricted to the cardiorespiratory system but can also increase the risks of other conditions that have rarely been described as being associated with air pollution, such as headache, asthenia, conjunctivitis, and skin rash.The main strength of this study was the availability of reliable health data reflecting the activity of general practitioners in the general population, which is usually hard to monitor. Indeed, anyone in need of a medical visit can call SOS Médecins, which therefore makes visits in the general population by definition. The medical diagnoses are recorded by the physicians themselves after every visit, using standardized codes for each disease. This is not the case for similar organizations in France, which have only been collecting information on the complaints, symptoms, and infections reported by the patients. That is the reason for the main limitation of this study: It was a unicentric study because of the lack of similar available data from other cities covering the past several years. The population served by SOS Médecins might have changed during the study period, which was quite long, and it might also have changed according to season, since the study area has a high level of tourism. However, the adjustment for season and long-term trends allowed us to take into account those variations, as well as variations in the general practitioners’ activity. Concerning exposure, all of the monitoring stations were selected in accordance with local experts who considered them representative of the background exposure within the study area. Although levels measured by these stations do not accurately reflect individual levels of exposure, published research has shown that their day-to-day variations are well correlated with variations in individual exposure (20).Our results for respiratory diseases are globally in agreement with those of the few studies that have investigated relations between air pollution and general practitioner activity, and are supported by biologic hypotheses. Indeed, almost all of the studies that investigated the association between PM10 and general practitioners’ visits for upper (6, 9, 10) and/or lower (6, 21) respiratory diseases observed such a relation; the exception was 1 study focusing on children, for which the study period was very short (22). Concerning nitrogen dioxide, most of the studies found an association between this indicator and consultations for respiratory diseases (10, 21–23); this was not found in the latest period of a study based on visits made by SOS Médecins in the Greater Paris area (6). The associations between PM10 and nitrogen dioxide, on the one hand, and respiratory diseases, on the other hand, were higher in the elderly. It is probable that air pollution exacerbates preexisting conditions that are more likely to affect elderly people. Furthermore, aging is characterized by a decrease in antioxidant defenses, and the elderly may constitute a group at high risk of suffering from oxidation phenomena induced by air pollution (24). Like other investigators (6, 21), we did not find any association between respiratory diseases and ozone, although this pollutant is well-known for its oxidative properties and was significantly associated with respiratory medical visits in London and Hong Kong (8, 10).The most innovative results of this study concern the association between air pollution and conjunctivitis and skin rash, in relation to a potential atopic etiology of these 2 diseases, as well as the association between air pollution and headache and asthenia, which reflects general health impairment without a clearly diagnosed disease. To our knowledge, no experimental study has shown such an association, because these symptoms cannot be objectively measured and therefore are diagnosed on a declarative basis. However, a link between air pollution and physicians’ visits for headache was observed in the Evaluation des Risques de la Pollution Urbaine sur la Santé (ERPURS) program (23), with ERRs slightly lower than but in the same range as those in the current study. Szyszkowicz (25) also found a link between several air pollutant indicators and emergency visits for headache in Montreal, Canada; several lag times were tested, and the highest ERRs associated with pollutant level increases were observed for the same day, just as in the present study, and were within the same range. Thus, air pollutants could have a very short-term effect on unspecific syndromes like asthenia and/or headache—conditions which are not very severe but can be painful and affect many people.Conjunctivitis and skin rash were also significantly associated with PM10 and ozone levels, confirming the potential link between air pollution and allergic diseases other than respiratory diseases. Indeed, exposure to air pollutants is able to trigger an immunoglobulin E response and to induce oxidative protein damage in the stratum corneum, leading to the disruption of barrier function and exacerbation of atopic dermatitis (26). In the ERPURS program (22), eye conditions were found to be exclusively related to ozone levels; the concordance of results from both our study and the ERPURS study strongly favors an association between eye diseases and ozone, which could be explained by its well-known irritant and oxidant properties. Ocular inflammation and dryness were also shown to be related to high concentrations of atmospheric pollutants in patients suffering from eye discomfort syndrome (27). Lastly, 2 other studies comparing prevalences of atopic eczema (28) or atopic dermatitis and allergic rhinoconjunctivitis (29) showed that prevalences were higher in the most polluted areas. Our results are therefore in accordance with those of the few published studies available, which all suggest the existence of a link between air pollutant exposure and dermatitis and/or eye diseases. Furthermore, results found for more commonly studied indicators are totally in accordance with results from the literature, which also favors a robustness of our analyses with regard to these innovative health indicators.Also favoring the robustness of our results is the lack of any significant relation between air pollution and visits made for lumbago, the outcome chosen as the control health indicator, since it is not related a priori to air pollution.In addition, we used distributed-lag models which have been recently used in environmental epidemiology in order to quantify the so-called “mortality displacement effect” (17). In our study, distributed-lag models produced effect estimates similar to or higher than those of models using 3-day moving averages, suggesting that 1) the effect of air pollution is not simply advanced by a few days, since no obvious harvesting effect is observed, and 2) effects associated with increases in nitrogen dioxide levels persist for 2 weeks after exposure for both upper and lower respiratory diseases. The fact that the use of distributed-lag models led to higher effect estimates for nitrogen dioxide and several health indicators (respiratory diseases, conjunctivitis, and skin rash) but not for other pollution and health indicators suggests that different pollutants can have more or less delayed effects. However, it is difficult to know whether these different types of effects are really linked to the pollutant itself or whether the pollutant acts as a surrogate for other exposures.The lack of association between air pollution and asthma can be considered surprising in the context of results existing in the literature (30–32). However, studies using data on general practitioner activity are more controversial: In the London study, visits for asthma were associated with several air pollution indicators (7); in the ERPURS program in Paris, this was also the case during the 1991–1995 period (23), but such an association was no longer observed during 2000–2003 (6). Thus, the lack of association could be explained, on the one hand, by the lower number of daily events in comparison with the previous period (representing only 1% of all visits) and, on the other hand, by the fact that asthma treatment has changed, and the context of crisis that motivates general practitioners’ visits might have been consequently modified. Indeed, asthma patients often treat themselves if a crisis occurs and might go directly to the emergency room (without calling a general practitioner) if the crisis cannot be controlled using their usual medication.In conclusion, we found evidence of an association between air pollutant levels and daily numbers of general practitioners’ visits for various syndromes. This study proves the relevance of such data for epidemiologic research in the field of environmental health. Furthermore, the results of this study will sensitize doctors to the importance of giving as precise a diagnosis as possible, and this will probably help to further increase data quality; better precision in diagnosis will allow us to obtain more specific data on health indicators in the future. The links observed with conjunctivitis, skin rash, headache, and asthenia are very innovative, since few studies have shown relations with them because of the lack of suitable data. They strongly suggest that cardiorespiratory diseases are not the only conditions that can be induced or exacerbated by air pollution and show that focusing on very severe events leads to underestimation of air pollution effects. In terms of public health, this study suggests that a large number of medical visits are attributable to air pollution in Bordeaux, where current levels of air pollutants are globally close to European air quality guidelines for health protection. This is one more convincing argument for promoting all measures aimed at reducing pollutant emissions, on both the individual and collective levels, even in moderately polluted areas.AbbreviationsCIconfidence intervalERPURSEvaluation des Risques de la Pollution Urbaine sur la SantéERRexcess relative riskPM10particulate matter less than 10 μm in diameterAuthor affiliations: French Institute of Public Health Surveillance, Bordeaux, France (Sophie Larrieu, Gaëlle Gault, Laurent Filleul); French Institute of Public Health Surveillance, Saint Maurice, France (Agnès Lefranc); SOS Médecins, Bordeaux, France (Franck Couvy, Bernard Jouves); and Regional Observatory of Health Ile-de-France, Paris, France (Edouard Chatignoux).The authors thank SOS Médecins Bordeaux and AIRAQ for their collaboration in providing data and their very useful participation.Conflict of interest: none declared.1.BrunekreefBHolgateSTAir pollution and healthLancet20023609341123312422.DanielsMJDominiciFZegerSLThe National Morbidity, Mortality, and Air Pollution Study. 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Second Edition1998New York, NYOxford University Press12.WoodSNGeneralized Additive Models: An Introduction With R2006Boca Raton, FLChapman & Hall/CRC13.AkaikeHPetrovVNCsakiFInformation theory and an extension of the maximum likelihood principleSecond International Symposium on Information Theory1973Budapest, HungaryAkailseoniai-Kiudo26728114.TouloumiGAtkinsonRLe TertreAAnalysis of health outcome time series data in epidemiological studiesEnvironmetrics200415210111715.SunHLChouMCLueKHThe relationship of air pollution to ED visits for asthma differ between children and adultsAm J Emerg Med200624670971316.SunyerJBallesterFTertreALThe association of daily sulfur dioxide air pollution levels with hospital admissions for cardiovascular diseases in Europe (the Aphea-II study)Eur Heart J200324875276017.ZanobettiAWandMPSchwartzJGeneralized additive distributed lag models: quantifying mortality displacementBiostatistics20001327929218.Council of the European UnionDirective 1999/30/CE du Conseil du 22 Avril 1999 Relative à la Fixation de Valeurs Limites pour l'Anhydride Sulfureux, le Dioxyde d'Azote et les Oxydes d'Azote, les Particules et le Plomb dans l'Air Ambiant1999Brussels, BelgiumEuropean Union19.World Health Organization Regional Office for EuropeWHO Air Quality Guidelines for Particulate Matter, Ozone, Nitrogen Dioxide and Sulfur Dioxide. Global Update 2005. Summary of Risk Assessment2006Geneva, SwitzerlandWorld Health Organization20.JanssenNAHoekGBrunekreefBPersonal sampling of particles in adults: relation among personal, indoor, and outdoor air concentrationsAm J Epidemiol1998147653754721.HwangJSChanCCEffects of air pollution on daily clinic visits for lower respiratory tract illnessAm J Epidemiol2002155111022.KeidingLMRindelAKKronborgDRespiratory illnesses in children and air pollution in CopenhagenArch Environ Health199550320020623.MedinaSLe TertreAQuénelPAir pollution and doctors’ house calls: results from the ERPURS system for monitoring the effects of air pollution on public health in Greater Paris, France, 1991–1995Environ Res.1997751738424.KellyFJDunsterCMudwayIAir pollution and the elderly: oxidant/antioxidant issues worth considerationEur Respir J Suppl.20034070s75s25.SzyszkowiczMAir pollution and daily emergency department visits for headache in Montreal, CanadaHeadache200848341742326.NiwaYSumiHKawahiraKProtein oxidative damage in the stratum corneum: evidence for a link between environmental oxidants and the changing prevalence and nature of atopic dermatitis in JapanBr J Dermatol2003149224825427.VersuraPProfazioVCelliniMEye discomfort and air pollutionOphthalmologica1999213210310928.Martin Fernandez-MayoralasDMartin CaballeroJMGarcia-MarcosALPrevalence of atopic dermatitis in schoolchildren from Cartagena (Spain) and relationship with sex and pollution [in Spanish]An Pediatr (Barc)200460655556029.DotterudLKOdlandJØFalkESAtopic dermatitis and respiratory symptoms in Russian and northern Norwegian school children: a comparison study in two arctic areas and the impact of environmental factorsJ Eur Acad Dermatol Venereol200418213113630.AtkinsonRWAndersonHRSunyerJAcute effects of particulate air pollution on respiratory admissions: results from APHEA 2 projectAm J Respir Crit Care Med2001164101860186631.VilleneuvePJChenLRoweBHOutdoor air pollution and emergency department visits for asthma among children and adults: a case-crossover study in northern Alberta, Canada [electronic article]Environ Health200764032.BarnettAGWilliamsGMSchwartzJAir pollution and child respiratory health: a case-crossover study in Australia and New ZealandAm J Respir Crit Care Med20051711112721278 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B15075F176E5D810C9C1627B6698F6727D80CC4.txt b/test/dataset/in/resources/corpus/Clean_0B15075F176E5D810C9C1627B6698F6727D80CC4.txt new file mode 100644 index 0000000..e9eccdb --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B15075F176E5D810C9C1627B6698F6727D80CC4.txt @@ -0,0 +1 @@ +MAD2033S0047-6374(97)00174-710.1016/S0047-6374(97)00174-7Elsevier Science Ireland LtdTable 1Analysis of the expected distribution based on the total number of events found in 21 younger DS individuals between 0 to 25 years old (105 lesions), number of identifiable bands per haploid set (400) and number of analyzed metaphases (1050), according to Mariani (1989)ha (h)E(h)p (h)F (h)00.7691264307.6506140010.201895780.758270.230873692.3494322.649881E-0210.599522.897792E-0211.5911632.318645E-030.92745822.47911E-030.991633441.52161 lE-046.0864445E-021.604646E-046.417519E-0257.988458E-063.195383E-038.303454E-063.310744E-03**63.49495E-071.39798E-043.149962E-071.153606E-04***Column h, events per band; Column a (h), probability of finding h events at a given band; Column E (h), expected number of bands showing h events; Column p (h), probability of finding h or more events at a given band; Column F (h), expected number of bands that would show h or more events.*Significant (P<0.05); **Highly significant (P<0.01); ***Very highly significant (P<0.001).Table 2Analysis of the expected distribution based on the total number of events found in 17 older DS individuals between 29 to 48 years old (81 lesions), number of identifiable bands per haploid set (400) and number of analyzed metaphases (850), according to Mariani (1989)ha (h)E (h)p (h)F (h)00.8166865326.6746140010.16537966.15160.183313573.3254121.674462E-026.697851.793447E-027.17380531.130262E-030.45210481.189847E-030.475955545.721952E-052.288781E-025.958439E-052.385065E-02*52.317391E-069.269562E-042.364868E-069.628423E-04**Column h, events per band; Column a (h), probability of finding h events at a given band; Column E (h), expected number of bands showing h events; Column p (h), probability of finding h or more events at a given band; Column F (h), expected number of bands that would show h or more events.*Significant (P<0.05); **Highly significant (P<0.01); ***Very highly significant (P<0.001).Table 3Frequency of lesions in bands characterized as fragile sites, minimum expected number of lesions according to Poisson distribution (Mariani, 1989) and total number of lesions in lymphocytes from younger DS individuals and older DS individualsGroups of individualsNumber of individualsAge/years (±S.D.)Total number of analyzed cellsTotal number of lesionsMinimum expected no. of lesions (Mariani, 1989)Frequency (%) of lesions in bands characterized as fragile sites2q113p145q316p219q12Younger (DS)2117.99 (±6.32)1050105≥54.87.6———Older (DS)1736.39 (±10.15)85081≥44.912.346.27.46.2Down’s syndrome, ageing and fragile sitesMariliade Arruda Cardoso Smith*BiancaBorsattoDepartamento de Morfologia, Disciplina de Genética, UNIFESP Escola Paulista de Medicina, Rua Botucatu, No. 740, 04023 900 CEP São Paulo, Brazil*Corresponding author. Fax: +55 011 5492127/5708378; e-mail: macsmith.morf@epm.brAbstractFragile sites have been interesting for mapping chromosomal regions involved in disease and ageing. The chromosomal fragile site expression from 38 Down’s syndrome (DS) individuals aged 0–48 years was investigated in blood peripheral lymphocytes. Fragile sites were statistically characterized as the minimum expected number of lesions per band based on a Poisson distribution. The results showed that the fragile site 2q11 was associated with the DS condition and fragile sites 5q31, 6p21 and 9q12 with ageing in DS subjects. Fragility in 6p21 has also been associated with Alzheimer’s disease patients.KeywordsDown’s syndromeAgeingFragile sites1IntroductionDown’s syndrome (DS) or trisomy of chromosome 21 is the most common congenital chromosomal aberration in human beings. The majority of individuals (95%) with trisomy 21 have three free copies of this chromosome and in a small percentage of patients (5%), one copy is translocated to another acrocentric chromosome or is present in mosaic form. DS in marked by particular phenotypes that include mental retardation, characteristic physical features and reduced life expectancy. This syndrome generally originates in a meiotic nondisjunction, with an estimated frequency of 1:600–1:800 births. In adults with DS, most of the clinical signs associated with normal ageing occur prematurely. These individuals show an early cognitive decline with age and generally develop the neuropathological changes of Alzheimer’s disease (AD) (Wisniewski et al., 1992). Fragile sites are points in the chromosomes where they preferentially show lesions such as breaks or gaps, either spontaneously or after exposure to specific tissue culture conditions (Sutherland 1979, 1985). The fragile sites can be used as cytogenetic markers in association and linkage studies (Sutherland 1988, Sutherland and Hecht 1995). The use of chromosomal fragile sites has been useful for mapping chromosomal regions of the genome that contain genetic loci important for the causation of diseases and/or ageing. In our laboratory, we observed the chromosomal fragile site 6p21 associated with elderly healthy people and AD patients (Kormann-Bortolotto et al., 1992). Mariani (1989)proposed a characterization of fragile sites according to a minimum expected frequency of lesions (gaps, breaks and rearrangements) per band based on a Poisson distribution. Those sites in which the probability of chance recurrence was less than 5% are considered fragile sites. Up to now no approaches similar to ours have been described which investigated the occurrence of ‘spontaneous’ chromosome lesions in DS subjects. In the present study we investigated the expression of fragile sites under low folate culture conditions in Down’s syndrome patients in order to verify whether some fragile sites can be correlated with the clinical condition of the patients or with their ageing process.2Material and methods2.1Selection of subjectsThe sample of individuals with DS was made up of 38 patients, of whom 21 were between 0 and 25 years old and 17 between 29 and 48 years old. The individuals were selected and supervised within a co-operative program between the Universidade Federal de São Paulo-Escola Paulista de Medicina (UNIFESP-EPM) and Associação de Pais e Amigos dos Excepcionais (APAE-São Paulo). The average age (±S.D.) of younger DS individuals was 17.99 (±6.32) and the average age (±S.D.) of older individuals was 36.39 (±10.15). All the DS patients had free trisomy. The younger group was made up of 11 females and ten males. The older group had one female and 16 males. The DS patient groups were divided arbitrarily based on evidence that persons with DS develop neuropathological changes similar to those of AD after the 4th decade of life (Malamud, 1972). The older group was almost entirely composed of people in the 4th and 5th decade of life. The younger group was almost entirely composed of people in the 2nd and 3rd decade of life.2.2Cytogenetic analysisThe cytogenetic analysis was done on the phytohaemagglutinin-stimulated lymphocytes from these individuals. These cells were cultured for 72 h in TC 199 medium, which is a low folic acid medium, known to enhance the appearance of folate sensitive fragile sites, with 7% fetal calf serum. The investigation of fragile sites was carried out in a blind test using at least 50 cells (all containing 47 chromosomes) per individual. All cells were initially analyzed by conventional staining. Chromosomal lesions (gaps, breaks or rearrangements) were recorded and the slides were destained with fixative solution (methanol:acetic acid 3:1) and restained with G-banding technique for analysis of the same metaphases afterwards. The number of lesions in each band site were computed and only those sites were considered fragile in which the probability of random recurrence was less than 5% (Mariani, 1989)3Results and discussionThere was a total of 105 lesions in the younger DS individuals group and there were 81 among older DS individuals group. These differences were not statistically significant according to the Mann–Whitney test (zcalc=0.75 and zcrit=1.64). Thus, ageing in DS did not increase the number of chromosomal lesions. The older DS sample is almost formed by men, however some findings from the literature have not shown differences between sexes related to the incidence of autosomal fragile sites (Sutherland 1982). A band was considered a fragile site based on a Poisson distribution that indicated the minimum expected frequency of events (lesions) per band (Mariani, 1989). Table 1Table 2Table 3 show the frequency of bands considered fragile sites in younger DS individuals group and older DS individuals group. Fragile site 3pl4 was common to both groups. The occurrence of this fragility was similar in younger and older DS groups. Band 3pl4 is widely cited as the most common fragile site in humans (Simonic and Gericke, 1996). Recently a gene called FHIT has been reported that is located in this chromosomal region. This gene is either completely or partially missing in many neoplasias such as colon, breast and lung cancer which suggests it may be a tumour suppressor gene. Fragile site 6p21 was observed only in the older DS group. The gene of a microtubule associated tau like protein (MTBT2) is located in this band (Kidd et al., 1989). Thus, this gene may be related to chromosomal alterations which involve defects at the level of microtubules and may also be a primary mechanism of the ageing process of DS syndrome. Fragile site 2q11 was observed both in older DS group and younger DS group. This chromosomal region has been described as an unstable secondary constriction on the long arm of this chromosome in patients with different clinical features (Lejeune et al., 1968). Other cases with this secondary constriction show that clinical features can vary considerably, with phenotypes that include mental retardation, congenital heart malformation and growth retardation, as well as some healthy individuals (Ferrante et al., 1968Buhler et al., 1970Williams and Howell, 1976). Whereas this chromosomal alteration might be considered a normal variant, Anneren and Gutstavson (1981)suggest that cell lines with it might have a pathological effect if they occur at critical stages in the organogenesis. The fragility in chromosome 9ql2 was exclusively observed in older DS patients. Fragile site 9ql2 is a chromosomal region formed by a constitutive heterochromatin, with a still unclear biological function. Previous studies have also shown a possible increase of chromosome 9 pericentric inversion among DS subjects and it has been suggested that this inv(9)(qh) predisposes the carrier parent to nondisjunction. Cytogenetic studies show an interaction between the heterochromatin and the acrocentric chromosomes during the cell division process that can interfere in disjunction and chromosome distribution (Natarajan and Ahnstrom, 1969Gagné et al., 1974Miklos and John, 1979Hultén et al., 1987). Fragile site 5q31 was observed only in older DS individuals. In this region, for instance the gene for a lymphokine, interleukin-13, is located which is expressed in activated human T-lymphocytes. Minty et al. (1993)suggested that interleukin-13 may be critical in regulating inflammatory and immune responses. It is well known that DS individuals exhibit an increased frequency of infections and malignancies, especially those involving the head, neck and respiratory tracts. Finally, fragile site 2q11 has been observed in both DS groups whereas has not been observed in young and/or elderly control groups previously analyzed in our laboratory (Kormann-Bortolotto et al., 1992). Thus, we believe that this fragile site is associated with DS condition. Fragile site 6p21 was observed in the older DS syndrome group, in the elderly control group and in AD analyzed previously in our laboratory (Kormann-Bortolotto et al., 1992). Thus, we believe that fragile site 6p21 is associated with the elderly condition.Some studies have suggested that the fragile site could be a cytogenetic representation of gene expression (Yunis et al., 1987Hecht, 1988). Austin et al. (1992)have shown a clustering of spontaneous aberrations at fragile sites that may indicate that these regions are physiologically active during the S-phase of the cell cycle. Fragile sites FRAXA, FRAXE, FRAXF and FRA16A are formed by the (CGG)n trinucleotide repeat unit expansion with subsequent methylation of a adjacent CpG island that can introduce a recognition site for de novo imprinting (Nancarrow et al., 1994). Therefore fragile sites could represent adjacent gene expression. Finally, our findings have shown fragile site 2q11 associated with DS condition and fragile sites 5q31, 6p21 and 9q12 associated with older DS individuals. These fragile sites could indicate chromosomal regions or genes to be further investigated in DS ageing or in this syndrome.AcknowledgementsThe authors are grateful to Prof. Eduardo Katchburian, Prof. Antonio dos Santos Clemente and Dr Elisa Maria Doceu Moreira Garcez and to Associação de Pais e Amigos dos Excepcionais (APAE-São Paulo) for allowing this co-operative program. We would also like to thank Elenice Rosana Salas for technical assistance and Dr Tulio Mariani who provided the statistical computer program. This work was supported by Sandoz Foundation of Gerontological Research, Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP), Coordenação de Aperfeiçoamento de Nı́vel Superior, Brazil (CAPES) and Conselho Nacional de Desenvolvimento Cientı́fco e Tecnológico, Brazil (CNPq).ReferencesAnneren and Gutstavson, 1981G.AnnerenK.GustavsonA fragile secondary constriction on chromosome 2 in five patients with different clinical featuresHereditas9519816367Austin et al., 1992M.J.F.AustinJ.M.CollinsL.A.CoreyW.E.NanceM.C.NealeR.M.SchiekenJ.A.BrownAphidicolin-inducible common fragile sites expression: results from a population survey of twinsAm. J. Hum. Genet.5019927683Buhler et al., 1970E.M.BuhlerU.LuchsingerU.K.BuhlerK.MéhesG.R.StalderNoncondensation of one segment of a chromosome no 2 in a male with otherwise normal karyotype (and severe hypospadias)Humangenetik9197097104Ferrante et al., 1968E.FerranteL.BruniS.GrimaldiRottura paracentromerica del braccio lungo di un chromosome 2 in una bambina di 9 mesi affetta da cistationuriaritardo psicomotorio e cataratta.. Riv. Clin. Ped.811968612619Gagné et al., 1974R.GagneJ.M.LucianiM.Devictoc-VuilletA.StahlC9 heterochromatin during the first meiotic prophase in human fetal oocyteExp. Cell. Res.851974111116Hecht, 1988F.HechtFragile sites, cancer, chromosome break points and oncogenes all cluster in light G-bandsCancer Genet. Cytogenet.331988110Hultén et al., 1987M.HulténN.SaadallahJ.BatanianMeiotic chromosome pairing in the human male: Experience from surface spread synaptonemal complexesChromosomes Today91987218229Kidd et al., 1989K.K.KiddA.M.BowcockJ.SchmidtkeReport of the DNA committee and catalogs of cloned and mapped genes and DNA polymorphismsCytogenet. Cell Genet.511989622947Kormann-Bortolotto et al., 1992M.H.Kormann-BortolottoM.de A.C.SmithJ.Toniolo NetoFragile sites, Alzheimer’s disease and ageingMech. Ageing Dev.651992915Lejeune et al., 1968J.LejeuneB.DutrillauxJ.LafourcadeR.BergerD.AbonylM.O.RethoreEndoreduplication selective du bras long du chromosome 2 chez une femme et sa filleAcad. Sci. Paris26619682426Malamud, 1972Malamud, D.N., 1972. Ageing and the Brain, Gaitz, C.M., Plenum, New York.Mariani, 1989T.MarianiFragile sites and statisticsHum. Genet.811989319322Miklos and John, 1979G.L.G.MiklosB.JohnHeterochromatin and satellite DNA in man: properties and prospectsAm. J. Hum. Genet.311979264280Minty et al., 1993A.MintyP.ChalonJ.M.DerocqX.DumontInterleukin-13 is a new human lymphokine regulating inflammatory and immune responsesNature3621993248250Nancarrow et al., 1994J.K.NancarrowE.KremerK.HolmanH.EyreN.A.DoggetD.Le PaslierD.F.CallenG.R.SutherlandR.I.RichardsImplications of FRAl6A structure for the mechanism of chromosomal fragile site genesisScience264199419381941Natarajan and Ahnstrom, 1969A.T.NatarajanG.AhnstromHeterocromatin and chromosome aberrationsChromosoma2819694861Simonic and Gericke, 1996I.SimonicG.S.GerickeThe enigma of common fragile sitesHum. Genet.971996524531Sutherland, 1979G.R.SutherlandHeritable fragile sites on human chromosomes I Factors affecting expression in lymphocyte cultureAm. J. Hum. Genet.311979125135Sutherland, 1982G.R.SutherlandHeritable fragile sites on human chromosomes VIII, Preliminary population cytogenetic data on the folic-acid-sensitive fragile sitesAm. J. Hum. Genet.341982452458Sutherland, 1985G.R.SutherlandHeritable fragile sites on human chromosomes XII Population CytogeneticsAnnu. Hum. Genet.491985153161Sutherland, 1988G.R.SutherlandThe role of nucleotides in human fragile site expressionMutat. Res.2001988207213Sutherland and Hecht, 1995Sutherland, G.R., Hecht, F., 1995. Fragile Sites on Human Chromosomes, Oxford University Press, New York.Williams and Howell, 1976A.J.WilliamsR.T.HowellA fragile secondary constriction on chromosome 2 in a severely mentally retarded patientJ. Ment. Defic. Res.211976227230Wisniewski et al., 1992Wisniewski, K.E., Hill, A.L., Wisniewski, H.M., 1992. Down’s Syndrome-Advances in Medical Care, Willey-Liss, New York.Yunis et al., 1987J.J.YunisA.L.SorengA.E.BoweFragile sites are targets of diverse mutagens and carcinogensOncogene11987248250 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B15F4F7680CE01F26C1996D1F74278719AEE02C.txt b/test/dataset/in/resources/corpus/Clean_0B15F4F7680CE01F26C1996D1F74278719AEE02C.txt new file mode 100644 index 0000000..698bd57 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B15F4F7680CE01F26C1996D1F74278719AEE02C.txt @@ -0,0 +1 @@ +geronageronaJournals of Gerontology Series A: Biomedical Sciences and Medical Sciences1758-535X1079-5006Oxford University Press10.1093/gerona/glp105Journal of Gerontology: Medical SciencesRapid CommunicationCardiac Autonomic Dysfunction Is Associated With White Matter Lesions in Patients With Mild Cognitive ImpairmentGalluzziSamantha1NicosiaFranco2GeroldiCristina13AlicandriAlberto2BonettiMatteo4RomanelliGiuseppe2ZulliRoberto2FrisoniGiovanni B.1351Laboratory of Epidemiology, Neuroimaging and Telemedicine (LENITEM), IRCCS San Giovanni di Dio-Fatebenefratelli, Brescia, Italy2Institute of Internal Medicine, Department of Medical Sciences, University of Brescia, Italy3Psychogeriatric Unit, IRCCS San Giovanni di Dio-Fatebenefratelli, Brescia, Italy4Service of Neuroradiology, Istituto Clinico Città di Brescia, Italy5Associazione Fatebenefratelli per la Ricerca (AFaR), Rome, ItalyAddress correspondence to Giovanni B. Frisoni, MD, Laboratory of Epidemiology Neuroimaging & Telemedicine (LENITEM), IRCCS San Giovanni di Dio FBF—The National Center for Research and Care of Alzheimer’s Disease, Via Pilastroni 4, 25125, Brescia, Italy. Email: gfrisoni@fatebenefratelli.itDecision Editor: Luigi Ferrucci, MD, PhD122009307200964A121312131529820082032009© The Author 2009. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2009BackgroundCardiac autonomic dysfunction has been associated with cognitive impairment, but the underlying pathogenesis is complex and cerebral white matter lesions (WMLs) might be implicated.MethodsTime and frequency heart rate variability (HRV) and visual rating of WMLs were carried out in 42 patients with mild cognitive impairment.ResultsAfter adjustment for relevant demographic and clinical characteristics, including left ventricular mass, reduced HRV indices of parasympathetic (root mean square of successive difference of RR intervals, RMSSD) and sympathetic modulation (low-frequency [LF] power) were associated with increased WML score (RMSSD: B −0.30, 95% CI −0.52 to −0.08, p = .01; LF: B −0.24, 95% CI −0.46 to −0.02, p = .05). In a multiple-adjusted model, RMSSD was the major independent predictor of WMLs (B −0.35, 95% CI −0.57 to −0.13, p = .002).ConclusionThe evidence for an independent association of cardiac autonomic dysfunction with WMLs might suggest its role in the pathogenesis of WMLs.Heart rate variabilityCardiac autonomic dysfunctionWhite matter lesionsMild cognitive impairmentHEART rate variability (HRV) is a marker of cardiovascular autonomic function (1). Reduced HRV has recently gained attention in the dementia research field because of its association with cerebrovascular risk factors (2) and Alzheimer’s disease (3). Moreover, in a sample of older disabled women from the community, root mean square of successive difference of RR intervals (RMSSD) and high-frequency (HF) power, indicators of parasympathetic-mediated short-term variations in heart rate, were predictors of cognitive impairment development, being associated with 3.4 and 6.7 times greater odds of cognitive impairment, respectively (4).Understanding pathophysiological mechanisms leading to cognitive impairment is of great interest to develop preventive therapies. However, the pathway underlying the association between reduced HRV and cognitive impairment is unclear. Cerebral white matter lesions (WMLs) might have a mediating role (4), but brain imaging studies are lacking.The aim of this study was to evaluate the independent association of HRV with WMLs in patients with mild cognitive impairment (MCI). Given the higher prevalence of WMLs in MCI patients relative to cognitively normal elders, we focused this study on the former group in order to improve the power to detect an association with HRV.METHODSMCI PatientsPatients were recruited from a prospective study on the natural history of MCI (“Mild Cognitive Impairment in Brescia” study) carried out in the outpatients’ section of the Alzheimer’s Unit, IRCCS San Giovanni di Dio-Fatebenefratelli, Brescia, Italy. Inclusion criteria included the presence of subjective and objective memory or other cognitive domain disturbance in the absence of functional impairment. More details were published elsewhere (5).One hundred thirty-six patients were enrolled from April 2002 to March 2005. For the present study, only those 94 patients who performed both 24-hour electrocardiogram (ECG) monitoring and magnetic resonance (MR) imaging were selected. Twelve patients were excluded because of inadequate quality of the exams. Those 82 patients finally included in the study were not different from 54 excluded as education, sex, and vascular risk factors but were younger (69.3 + 7.3 vs 72.5 + 8.8 years, p = .03).Clinical AssessmentGlobal cognition and depressive symptoms were assessed using the Mini-Mental State Examination (6) and the depression subscale of the Brief Symptom Inventory (7), respectively, the latter ranging from 0 to 24 (higher scores indicated poorer depressive symptoms). Hypertension and diabetes mellitus and heart diseases (coronary artery disease, primary arrhythmias, and heart failure) were clinically diagnosed and defined present if currently treated. Echocardiography was made in 67 of 82 patients. The remaining 15 patients refused the examination because of logistic difficulties. The left ventricular (LV) mass was calculated from Devereux’s formula and indexed by height.HRV MeasurementTwenty-four-hour ECG recording (Accuplus 363; Del Mar Avionics, Irvine, CA) was used to analyze HRV using implemented software. In the time domain, standard deviation of the RR intervals, standard deviation of the 5-minute mean values of RRs for each 5-minute interval, average of standard deviations of RR for each 5-minute interval, and the RMSSD were calculated. In the frequency domain, low frequency (LF) 0.04–0.15 Hz, HF 0.15–0.40 Hz, and low- or high-frequency ratio were calculated. A detailed description of ECG recording procedures was available on http://www.centroalzheimer.it/public/Methods_HRV.MR ImagingMR images were acquired at the Service of Neuroradiology, Istituto Clinico Città di Brescia, Brescia, using a 1.0 Tesla Philips Gyroscan. Axial T2 weighted, proton density, and fluid-attenuated inversion recovery images were acquired. WMLs were assessed using the rating scale for age-related white matter changes (ARWMC) (8). Total score (range 0–30) was the sum of subscores for frontal, parieto-occipital, temporal, infratentorial areas, and basal ganglia areas (range 0–3) in the left and right hemispheres. In the present study, WMLs were considered as present when ARWMC scale total score was 4 or more or when beginning confluence of lesions (subscore 2) was observed in at least one area. Based on this criteria, patients were divided into 32 with and 50 without WMLs.Statistical AnalysisThe data were analyzed using SPSS version 13.0 (SPSS, Chicago, IL). Sociodemographic and clinical differences between MCI groups with and without WMLs were assessed with t test for normally distributed continuous variables, Mann–Whitney test for nonnormally distributed continuous variables, and chi-square for categorical variables. In the whole-MCI group, the association between each HRV index and WMLs (i.e., the ARWMC scale total score) was assessed with linear regression models and the power of HRV indices to predict the extent of WMLs with a stepwise multiple regression model. Confounders were chosen among the main demographic and clinical characteristics that, based on current knowledge, have been associated with WMLs (age, diabetes, heart disease, hypertension) or have been showed to affect cardiac autonomic function (beta-blockers use, LV mass).RESULTSTable 1 shows that MCI patients with WMLs were older than those without WMLs while cognition and depression were similar. All the vascular disease and risk factors were more prevalent in MCI with WMLs, only hypertension reaching statistical significance. Among HRV indices, the RMSSD and LF were reduced in MCI patients with WMLs relative to those without WMLs (unadjusted p < .001).Table 1.Demographic and Clinical Characteristics of 82 Patients With MCI by Presence of WMLsCharacteristicsWMLs Absent (n = 50)WMLs Present (n = 32)p*Demographics    Age67.6 ± 7.672.2 ± 5.9.005    Gender, female31 (62%)20 (63%).96    Education7.9 ± 4.56.6 ± 2.9.28Cognition and mood    Mini-Mental State Examination27.0 ± 1.527.3 ± 1.5.43    Depression, Brief Symptom Inventory4.1 ± 4.83.4 ± 4.2.72Vascular diseases and risk factors    Hypertension12 (24%)17 (53%).007    Heart disease8 (16%)7 (22%).50    Diabetes mellitus4 (8%)4 (13%).50    Beta-blocker use7 (14%)9 (28%).12    Left ventricular mass†105.1 ± 31.4116.1 ± 31.0.16Heart rate variability features    SD of the RR intervals, ms118.9 ± 36.8111.7 ± 36.9.39    SD of the 5-minute mean values of RR   for each 5-minute interval, ms68.3 ± 18.262.6 ± 16.9.16    Average of SDs of RR for each   5-minute interval, ms95.0 ± 38.589.0 ± 35.9.49    Root mean square of successive   difference of RR intervals, ms26.5 ± 8.720.2 ± 5.5<.001    Low-frequency power, ms2212.1 ± 152.5151.1 ± 118.8.05    High-frequency power, ms288.0 ± 65.570.4 ± 73.7.06Notes: *Significance on t test or Mann–Whitney test for normally or nonnormally distributed continuous variables and chi-square for categorical variables.†Available in 41 and 26 MCI patients, respectively. Values denote mean ± SD or number (%). MCI = mild cognitive impairment; WMLs = white matter lesions.Table 2 shows that in the whole-MCI group, the RMSSD, LF, and HF were inversely associated with the extent of WMLs in the unadjusted models (p < .001), whereas only the association of decreased RMSSD with increased WML score retained significance in the adjusted models (p = .01). When also the confounding effect of LV mass was taken in account, the association was even significant for RMSSD (p = .01) and reached significance also for LF (p = .05).Table 2.Association of HRV Indices With White Matter Lesions in 82 Patients With Mild Cognitive ImpairmentHRV IndexUnadjustedAdjusted for Age, Diabetes Mellitus, Heart Disease, Hypertension, Beta-blockersAdjusted for the Previous Plus LV MassB95% CIpB95% CIpB95% CIpSDNN−0.08−0.30 to 0.14.480.10−0.12 to 0.32.350.06−0.16 to 0.28.61SDANN−0.18−0.40 to 0.04.11−0.03−0.25 to 0.19.78−0.08−0.30 to 0.14.51SDNN idx−0.05−0.27 to 0.17.680.13−0.09 to 0.35.230.10−0.12 to 0.32.43RMSSD−0.37−0.59 to −0.15.001−0.27−0.49 to −0.05.01−0.30−0.52 to −0.08.01LF−0.30−0.52 to −0.08.006−0.15−0.37 to 0.07.15−0.24−0.46 to −0.02.05HF−0.23−0.45 to −0.01.04−0.11−0.33 to 0.11.31−0.18−0.40 to 0.04.14Notes: Regression coefficients were computed in 18 separate linear regression models and denote the increase of the dependent for a 1-Unit increase of the independent variable. Models adjusted for LV mass included 67 patients.HF = high-frequency power; HRV = heart rate variability; LF = low-frequency power; LV = left ventricular; RMSSD = root mean square of successive difference of RR intervals; SDANN = standard deviation of the 5-minute mean values of RR for each 5-minute interval; SDNN = standard deviation of the RR intervals; SDNN idx = average of standard deviations of RR for each 5-minute interval.Stepwise multiple regression analysis including HRV indices significantly associated with WMLs in univariate models (RMSSD, LF, and HF) and confounders showed that the RMSSD and age were significant predictors of WMLs (B −0.30, 95% CI −0.52 to −0.08, p = .002; B 0.38, 95% CI 0.16–0.60, p < .0005, respectively), also in the model adjusted for LV mass (RMSSD: B −0.35, 95% CI −0.47 to −0.22, p = .002).DISCUSSIONThis study showed that in MCI patients, reduced RMSSD, an index of parasympathetic-mediated short-term component of HRV, was associated with extent of WMLs, suggesting a possible role of cardiac autonomic dysfunction in the WML pathogenesis.Studies assessing the relationship between HRV and WMLs are scanty and have used different methods to measure HRV, making results difficult to compare (9–11). To our best knowledge, the only study assessing HRV using power spectral analysis analyzed only frequency-domain measures and found that LF was significantly lower in hypertensive patients with lacunar infarctions than in those without lacunar infarctions (12).In the present study, RMSSD was the only HRV index to demonstrate an association with WMLs in multivariate models. This was in line with previous finding that proved RMSSD to be more closely associated with cognitive impairment than indicators of long-term variations in heart rate (4). The pathological mechanisms possibly linking cardiac autonomic dysfunction to WMLs might include abnormalities in heart rate control and vascular dynamics (13) or the role of intermediate factors (neurally mediated syncope, orthostatic hypotension, or increased blood pressure variability) that were found to be associated with both HRV (4,13,14) and WML development (15).A possible alternative explanation is that reduced HRV is a consequence and not a cause of WMLs. Although cerebrovascular diseases frequently cause disturbances to cardiovascular autonomic functions, this has been reported only in patients with clinically overt stroke due to large-vessel disease (16,17). Only longitudinal studies assessing causality might clarify this issue.There were some limitations to this study. First, this was a cross-sectional association study that prevents to determine the causal relationships and definitively indicate pathophysiological pathways leading from reduced HRV to WMLs. Second, the possible confounding role of neurodegeneration that, in addition to WMLs, might contribute to cardiac autonomic dysfunction and of vascular risk factors, such as dyslipidemia, has not been taken into account. Lastly, the clinical setting where the study has been implemented prevents generalizability before the results are replicated in a large population-based sample of older persons.In conclusion, we showed that reduced short-term component of HRV was associated with WMLs in patients with MCI. Further studies in larger cohorts of patients are warranted to confirm the role of HRV in the WML pathogenesis.FUNDINGThis study was funded in part by an ad hoc grant of the Italian Ministry of Health “Ricerca Finalizzata RA 00. 6A”.1.Circulation199693104310652.SinghJPLarsonMGTsujiHEvansJCO’DonnellCJLevyDReduced heart rate variability and new-onset hypertension: insights into pathogenesis of hypertension: the Framingham Heart StudyHypertension1998322932973.ZulliRNicosiaFBorroniBQT dispersion and heart rate variability abnormalities in Alzheimer’s disease and in mild cognitive impairmentJ Am Geriatr Soc200553213521394.KimDHLipsitzLAFerrucciLAssociation between reduced heart rate variability and cognitive impairment in older disabled women in the community: Women’s Health and Aging Study IJ Am Geriatr Soc200654175117575.GeroldiCRossiRCalvagnaCMedial temporal atrophy but not memory deficit predicts progression to dementia in patients with mild cognitive impairmentJ Neurol Neurosurg Psychiatry200677121912226.FolsteinMFFolsteinSEMcHughPR“Mini-mental State”. A practical method for grading the cognitive state of patients for the clinicianJ Psychiatr Res1975121891987.De LeoDFrisoniGBRozziniRTrabucchiMItalian community norms for the Brief Symptom Inventory in the elderlyBr J Clin Psychol1993322092138.WahlundLOBarkhofFFazekasFA new rating scale for age-related white matter changes applicable to MRI and CTStroke200132131813229.GoldsteinIBBartzokisGHanceDBShapiroDRelationship between blood pressure and subcortical lesions in healthy elderly peopleStroke19982976577210.SierraCde La SierraAMercaderJGómez-AngelatsEUrbano-MárquezACocaASilent cerebral white matter lesions in middle-aged essential hypertensive patientsJ Hypertens20022051952411.ToyryJPNiskanenLKLansimiesEAPartanenKPUusitupaMIAutonomic neuropathy predicts the development of stroke in patients with non-insulin-dependent diabetes mellitusStroke1996271316131812.KarioKMotaiKMitsuhashiTAutonomic nervous system dysfunction in elderly hypertensive patients with abnormal diurnal blood pressure variation: relation to silent cerebrovascular diseaseHypertension1997301504151013.VinikAIMaserREMitchellBDFreemanRDiabetic autonomic neuropathyDiabetes Care2003261553157914.GrimmWWirthsAHoffmannJMenzVMaischBHeart rate variability during head-up tilt testing in patients with suspected neurally mediated syncopePacing Clin Electrophysiol1998212411241515.BallardCO’BrienJBarberBNeurocardiovascular instability, hypotensive episodes, and MRI lesions in neurodegenerative dementiaAnn N Y Acad Sci200090344244516.KorpelainenJTSotaniemiKAMäkikallioAHuikuriHVMyllyläVVDynamic behavior of heart rate in ischemic strokeStroke1999301008101317.BassiAColivicchiFSantiniMCaltagironeCCardiac autonomic dysfunction and functional outcome after ischaemic strokeEur J Neurol200714917922 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B160E385BFB1BF9AFB7B2E9EFC39E1661E58F77.txt b/test/dataset/in/resources/corpus/Clean_0B160E385BFB1BF9AFB7B2E9EFC39E1661E58F77.txt new file mode 100644 index 0000000..6482ac8 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B160E385BFB1BF9AFB7B2E9EFC39E1661E58F77.txt @@ -0,0 +1 @@ +geronbgeronbJournals of Gerontology Series B: Psychological Sciences and Social Sciences1758-53681079-5014Oxford University Press10.1093/geronb/gbq036Journal of Gerontology: Psychological SciencesArticlesDual-Task Performance Reveals Increased Involvement of Executive Control in Fine Motor Sequencing in Healthy AgingFraserSarah A.LiKaren Z. H.PenhuneVirginia B.Center for Research in Human Development, Department of Psychology, Concordia University, Montreal, Quebec, CanadaAddress correspondence to Sarah Fraser, PhD, Department of Psychology, UQAM et le Centre de recherche institut universitaire de gériatrie de Montréal, 4545 Queen Mary, Montréal, Québec, Canada H3W 1W4. Email: sfraser@live.concordia.ca.Decision Editor: Elizabeth Stine-Morrow, PhD0920101705201065B5526535211020092042010© The Author 2010. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2010The purpose of the current study was to examine the role of executive control in fine motor sequencing using a motor-cognitive dual-task paradigm. Younger and older adults performed a sequential tapping task separately and concurrently with a semantic judgment task (Experiment 1) and a mental arithmetic task (Experiment 2). Experiment 1 established that under low cognitive load, older adults were slower and less accurate in sequential tapping than younger adults. Load was manipulated in Experiment 2, and across mental arithmetic difficulty levels, older adults were less accurate in sequential tapping when performing mental arithmetic than younger adults. At the highest difficulty level, both groups suffered performance costs. In line with gross motor research, these findings suggest a role for executive functions in fine motor performance in old age.Dual taskExecutive FunctionFine motor sequencingHEALTHY aging is associated with declines in both motor (Ketcham & Stelmach, 2001; Krampe, 2002) and cognitive control functions (Kramer & Madden, 2008; Verhaeghen & Cerella, 2002). In addition, motor and cognitive functions appear to become more strongly correlated with increasing age, suggesting an increased interdependence between the two domains (Baltes & Lindenberger, 1997; Li & Lindenberger, 2002). Other evidence of this interdependence comes from motor-cognitive dual-task research. The majority of such evidence involves simultaneous gross motor and cognitive task performance, showing in many cases greater dual-task costs (DTCs) for older adults compared with younger adults (for review, Woollacott & Shumway-Cook, 2002). This pattern has been interpreted to mean that motor performance requires more cognitive resources in old age. The coordination of simultaneous task performance has been considered a component of the executive system (Baddeley, 2002), which shows age-related decline (Kramer & Madden). The involvement of executive control in gross motor performance has recently been shown using measures of gait and balance (Mendelson, Redfern, Nebes, & Jennings, 2010; Yogev-Seligmann, Hausdorff, & Giladi, 2008). Fewer studies have explored the possibility of age-related increases in executive involvement during fine motor performance (e.g., Albinet, Tomporowski, & Beasman, 2006). Therefore, our goal was to examine the role of executive control in fine motor performance using a motor-cognitive dual-task paradigm.In both gross and fine motor dual-task research, several factors have been suggested to account for age differences in dual-task performance (Krampe, 2002; Woollacott & Shumway-Cook, 2002). Some of the factors that have been implicated include a general slowing, declines in executive function, type of tasks combined, and physiological arousal. In the case of executive function, it is well documented that executive control processes may be invoked during motor tasks when adaptive online control is needed (Ble et al., 2005; Krampe; Woollacott & Shumway-Cook; Yogev-Seligmann et al., 2008). Kahneman (1973) maintained that all individuals have a limited capacity to process information and that they should be able to process two tasks at once as long as the two tasks do not exceed the individual’s limited capacity or processing resources. If the tasks demands exceed an individual’s capacity, then performance on one or both tasks can deteriorate (Kahneman). Given what is known about declines in executive and motor processes, it is not surprising that age differences are predicted in cognitive-motor dual tasks.Despite this prediction, a growing number of walking and postural control studies have found that results vary depending on the tasks combined and the cognitive load of the component tasks (i.e., Huxhold, Li, Schmiedek, & Lindenberger, 2006; Li, Lindenberger, Freund, & Baltes, 2001; Lövdén, Schäefer, Pohlmeyer, & Lindenberger, 2008). For example, in a study of mildly challenging dual-task treadmill walking, younger and older adults showed cognitive dual-task facilitation and motor DTCs, which were more pronounced in older adults (Fraser, Li, DeMont, & Penhune, 2007). A follow-up experiment that included a cognitive load manipulation demonstrated that both age groups incurred costs in both domains and were negatively affected by the increase of cognitive difficulty (Li, DeMont, Penhune, Fraser, & Abbud, 2008). Interestingly, the younger adults were able to adjust their stride length to accommodate the increase in cognitive demands (Abbud, Li, & DeMont, 2009). The changing pattern of DTCs across experiments suggests that the choice of tasks and the cognitive load of the tasks chosen can have a large impact on the resulting pattern of performance. An added dimension of walking dual-task research is the influence of postural threat (Brown, Shumway-Cook, & Woollacott, 1999). It has been argued that older adults might adopt a “posture-first” principle, prioritizing walking and balance above all other tasks in order to avoid a fall (Woollacott & Shumway-Cook, 2002).The potential confound of postural threat influencing age differences in dual-task performances is removed in fine motor dual-task research. In addition, motor measures (particularly fine and complex motor measures) have been shown to be as accurate as standard cognitive measures in delineating cognitively normal versus cognitively impaired older adults (mild cognitive impairment and mild Alzheimer’s disease; Kluger et al., 1997). This close relationship between cognitive tasks and fine motor tasks in aging has been explored with the dual-task paradigm (Crossley & Hiscock, 1992). Using a within-subjects manipulation of cognitive load, Crossley and Hiscock compared young, middle-aged, and older adults on their performance of a simple tapping task with a concurrent cognitive load. At the highest level of cognitive difficulty, there were no age differences in cognitive performance, but older adults had larger decrements in simple tapping rates in comparison with younger and middle-aged adults. This simple tapping study demonstrates age differences in fine motor dual-task performance that increase with cognitive load. Would the same be true in the dual-task performance that involves a fine motor sequence? Or would the increased complexity of sequential tapping increase the overall cognitive load and increase age differences? One study that directly contrasted simple and sequential tapping with a cognitive load (speech production) found age group differences in DTCs only for sequential tapping (Kemper, Herman, & Lian, 2003). This finding suggests that sequential tapping places an added load on older adults in comparison with simple tapping.The few published studies on aging and dual-task fine motor performance suggest that increasing the complexity of the motor task is more detrimental to older adults than young. However, the literature does not indicate if a similar pattern will emerge when cognitive complexity is varied. The current study was designed to address this gap in the literature. Our approach was to manipulate cognitive complexity in cognitive-sequential tapping task pairings in order to examine the possibility of increasing executive involvement in fine motor performance. Given the evidence for increased age-related involvement of executive functions in the gross motor literature, we began with the prediction that there would be age-related increases in motor DTCs. Such a finding would extend the existing body of research on the increasing role of cognition in motor performance.EXPERIMENT 1MethodParticipants.—Twenty younger adults (20–31 years) and 21 older adults (60–75 years) participated in the experiment. Younger adults were recruited through Concordia Psychology’s undergraduate participant pool and the older adults were recruited from a preexisting participant database. Younger adults received class credits for their participation, and older adults received a small honorarium. All participants were right handed, fluent in English, had normal or corrected vision, had never suffered a stroke, and were screened for medical conditions (e.g., Parkinson’s disease, severe arthritis) and medications that would affect their movement. Individuals who reported hearing difficulties or who wore a hearing aid were excluded. The Forward Digit Span and the Digit Symbol Substitution Test of Wechsler Adult Intelligence Scale III (WAIS; Weschler, 1997), as well as the Trail Making Test (A & B; Spreen & Strauss, 1998), were administered to assess short-term memory, processing speed, and task switching, respectively. All participants were within a normal range for their age on these tests. Descriptive statistics for each group are presented in Table 1. All procedures were approved by the Concordia University Human Research Ethics Committee.Table 1.Descriptive Statistics of the SamplesExperiment 1Experiment 2YoungerOlderYoungerOlderAge23.10 (3.16)67.67 (4.33)21.10 (2.15)70.37 (4.96)Years of Education15.95 (2.09)14.81 (4.18)14.95 (0.89)15.11 (3.26)Digit Symbol88.60 (13.82)*73.85 (18.23)*69.90 (19.10)*56.42 (14.19)*Trails B-A24.42 (12.68)*59.81 (32.92)*27.50 (15.81)*46.47 (30.75)*Digits Forward7.35 (1.04)*6.48 (1.08)*7.15 (1.09)6.68 (1.11)ERVT——7.93 (4.46)*13.03 (4.94)*WAIS math-raw——13.30 (2.92)13.95 (2.90)WAIS math-scaled——10.35 (2.08)10.63 (2.81)Notes: Mean values and standard deviations (in brackets) presented. Years of education = total number of years of formal education; Digit Symbol value based on the total number of symbols correctly completed in 120 s; Trails B−A = time to complete Trails test A minus the time to complete Trails test B; Digits forward value based on the total number of items recalled. ERVT and WAIS math subtest were administered in Experiment 2. ERVT = extended range vocabulary test; WAIS = Wechsler Adult Intelligence Scale III.*p < .05 for age group comparisons.Materials.—Fine motor task.The fine motor task was a modified version of the multifinger sequence task (MFST) used in Fraser, Li, and Penhune (2009). The MFST is a serial reaction time (RT) task, in which a visual stimulus presented in one of four squares on a computer screen and participants tap in response to the stimulus with the four fingers of their right hand on four keys of a piano-like keyboard. The visual stimuli were presented repetitively in fixed 10-tap sequence (4-1-3-4-2-3-1-2-4-3) or in random 10-tap sequences. For the purposes of the current dual-task experiment, only the repeating sequence type was used. For each tap in the repeating sequence, the intertap interval was set at 1,000 ms, in which the stimulus stayed on the screen for 600 ms and disappeared for 400 ms. Therefore, the duration of a motor trial was 10 s. In the previous experiment (Fraser et al., 2009), age equivalence in the performance of the sequence was achieved after 10 presentations of the sequence; therefore, for the current experiment, 14 trials were presented during practice to ensure age equivalence prior to the test phase. Thirty trials were presented in each of the four test runs. For both the practice and the test sessions, participants completed half of the motor trials in isolation (single-task block) and half with the semantic task (dual-task block). An example of each trial type (single motor, single cognitive, and dual task) is presented in Figure 1.Figure 1.Graphic of the trials: single motor, single cognitive, and dual task. Dashed lines represent taps. Numbers under the dashed lines represent the key the participant had to tap. The fingers that corresponded to the keys were index = 1, middle = 2, ring = 3, and pinkie = 4. The solid line represents the time line of each trial (10 s). Arrows represent the word stimuli that were presented auditorially (i.e., mother, tractor, hammer). Note. Word stimuli were presented at random intervals during the trial and a trial could contain one, two, or three words.The visual stimulus in the sequence consisted of a 4.5-cm2 cartoon animal (i.e., “Rolly the Hamster”) that was programmed in C-Sharp and shown on a 19-inch Dell desktop monitor. Each stimulus was displayed in one of four horizontally presented colored 5-cm2 frames that stayed on the screen for the total duration of each trial. The participants responded to the stimuli on an M-Audio O2 Midi Controller piano keyboard. Participants were instructed to “catch the animal” by placing the four fingers of their right hand (i.e., index, middle, ring, and pinkie) on four marked keys, and the keyboard recorded the accuracy and RT of each key press.Cognitive task: semantic judgments.For this task, participants were presented auditorially with word stimuli at random time intervals and they were asked to judge if the word they heard was living (e.g., mother) or nonliving (e.g., chair). Word stimuli used in the current experiment were the same as those presented in Fraser and colleagues (2007). The trial time structure mimicked the motor trials, such that each trial lasted 10 s (see Figure 1). Furthermore, all participants had a practice session in which they judged 30 words and four test sessions that contained 60 words each. Half of the words were presented in isolation (single-task block) and half were presented with the fine motor task (dual-task block). Each list included an equal number of living and nonliving words to judge. The digitized words consisted of two-syllable high-frequency distinct nouns (written frequency less than or equal to one word per million; Kuçera & Francis, 1967) and were spoken in a female voice. To minimize the predictability of the presentation of the words, a trial could contain one, two, or three words. The minimum interstimulus interval (ISI) for each word presentation was 1,500 ms and the maximum was 7,000 ms. An algorithm programmed with Matlab software (The MathWorks, Inc., Natick, MA) produced ISIs that would result an equal distribution of the words across each 10-s trial (equal numbers of words presented at the beginning, middle, or end of the trial). The words presented in the practice lists were not reused in the test lists. All test words were presented twice with a minimum separation of two lists. The word stimuli were randomly ordered within each list and presented with customized software, C-Sharp, through a Dell Inspiron 1300 laptop. Participants heard the words through a Plantronics (Santa Cruz, CA) DSP-300 headset that also recorded vocal RTs. Speech recognition software (Microsoft Speech API) identified participants’ responses (“yes” for living words or “no” for nonliving words), and they were subsequently scored as correct or incorrect with Matlab software.Procedure.—The testing took place in the Adult Development and Aging laboratory at Concordia University. After informed consent, all participants underwent a task familiarization session. For the motor task, participants imitated simple forward (1-2-3-4-1-2-3-4-1-2-3-4) or backward (4-3-2-1-4-3-2-1-4-3-2-1) 12-element sequences to familiarize them with the keyboard and visual stimuli. For the semantic task, participants performed the word repetition baseline where they had to repeat 30 words that were presented auditorially. To ensure adequate hearing for the test phase, participants needed to score 90% or more on the word repetition baseline. All participants met this criterion.Participants then had practice in each of the conditions: single task (semantic), single task (motor), and dual task (semantic and motor). They completed seven trials per condition. Once they practiced the tasks, they completed four counterbalanced test runs of single motor, single semantic, and dual task. For each test run, there were 15 trials per condition. For both the practice and the test sessions, participants completed half of the motor trials in isolation (single-task block) and half with the semantic task (dual-task block). For both the practice and the test runs, participants were instructed that both tasks were equally important and that they should try to respond quickly and accurately. After the test session, participants completed the Digit Symbol, the Trail Making tests, the Digits Forward test, and a demographics questionnaire. Participants were debriefed and received course credit (younger) or an honorarium (older) for their time. The entire session lasted approximately 90 min.Statistical analyses.—Four dependent variables were calculated: accuracy and RT for the cognitive task and accuracy and RT for the motor task. For both the cognitive and the motor data, the mean correct RT (ms) for each trial type was calculated for each participant. The time window for valid motor responses had a 1,000-ms duration, which started 100 ms prior to the presentation of each stimulus, to allow for anticipated responses. Any correct tap within this time window was considered part of the mean RT. For the vocal RT data, RTs were calculated from the offset of the verbal stimuli and responses were excluded if they were ±3 SD from an individual’s overall mean RT. Only a small proportion of the responses were considered outliers (MOlder = 0.02, SE = 0.001; MYounger = 0.01, SE = 0.001). Accuracy for the cognitive task and the motor task were based on the number of correct responses (i.e., correct semantic judgments, correct taps) in all possible responses for each trial type (single and dual). For the cognitive accuracy, motor accuracy, and motor RT, the data were checked for outliers based on the group mean. No such outliers were found.DTCs were calculated for each of the four dependent variables. In the case of RT, dual-task RTs were subtracted from single-task RTs for each individual. For accuracy, single-task accuracy was subtracted from dual-task accuracy on an individual basis. The resulting difference scores represent four DTC scores: DTC motor accuracy, DTC motor RT, DTC semantic accuracy, and DTC semantic RT. For each variable, planned contrasts (α = .05) were conducted to assess age differences in DTCs. All post hoc analyses used a Bonferroni corrected p value (p = .025).Results and DiscussionMean values for single- and dual-task performances are reported in Table 2.Table 2.Mean Single- and Dual-Task Performance Values for Younger and Older AdultsExperiment 1Experiment 2Cognitive taskSemantic judgmentsMinus-1Minus-7YoungerOlderYoungerOlderYoungerOlderAccuracy    Single89.2592.9099.5099.7475.0882.37    Dual91.0093.1498.3399.0467.5869.47Reaction times    Single662.42762.63567.09600.641584.961462.89    Dual690.52786.61665.46641.201647.081439.82Fine motor taskSequential tappingYoungerOlderYoungerOlderYoungerOlderAccuracy    Single96.9094.7697.8293.1396.9791.30    Dual96.3589.9097.4386.8684.2869.96Reaction times    Single281.89405.48290.54413.03294.92391.59    Dual307.28466.98313.59447.24377.82486.62Notes: Accuracy values = percentage points (a value of 100 = all responses correct). Reaction time values in milliseconds. Although single-task sequential tapping requires only a response to the visual stimulus, results for Minus-1 and Minus-7 are reported separately in this table because they were presented separately in a Minus-1 or Minus-7 test run. There were no significant differences in single-task sequential tapping accuracy between Minus-1 and Minus-7, t(38) = 1.59, p = .120.Fine motor: MFST.—Accuracy.The t test revealed significant age differences in motor accuracy DTCs, t(39) = 2.23, p = .032, such that older adults had higher motor accuracy DTCs (M = 4.71%, SE = 1.73) than younger adults (M = 0.60%, SE = 0.53). Only older adults’ accuracy DTCs were significantly different from zero, t(20) = 2.72, p = .013.Reaction times.—The t test for motor RT DTCs resulted in a significant age difference, t(39) = 2.39, p = .022. Again, older adults had higher motor RT DTCs (M = 61.50 ms, SE = 11.47) than younger adults (M = 25.39 ms, SE = 10.20). After Bonferroni correction, both younger, t(19) = 2.49, p = .022, and older adults’, t(20) = 5.36, p < .001, motor RT DTCs were significantly different from zero.Cognitive: semantic judgment task.—Accuracy.The t test comparing younger and older adults on their cognitive accuracy DTCs was nonsignificant, t(39) = 0.92, p = .362. An additional analysis on the full sample revealed that the DTCs in accuracy were not significantly different from zero, t(40) = 1.24, p = .222. When split by age, neither younger, t(19) = 1.28, p = .215, nor older adults’, t(20) = 0.31, p = .760, accuracy DTCs were significantly different from zero.Reaction times.—In line with the accuracy results, the t test comparing younger and older adults’ vocal RTs DTCs was nonsignificant, t(39) = 0.35, p = .728. In this case, the t test comparing vocal RT DTCs to zero was significant for the whole sample, t(40) = 2.65, p = .012. However, when split by age, neither younger, t(19) = 1.96, p = .065, nor older, t(20) = 1.73, p = .098, adults’ vocal RT DTCs were significantly different from zero.Testing for trade-offs: within and across domains.—Within each domain (cognitive and motor), bivariate correlations between mean dual-task accuracy and speed (reciprocal of RT: 1/RT) were computed to test for speed-accuracy trade-offs. A negative correlation between speed and accuracy measures would be expected if participants were slowing to maintain accuracy levels or making more mistakes to maintain speed. For younger adults, the correlation between vocal accuracy and speed, r(18) = −.01, p = .973, was nonsignificant. The correlation between and motor accuracy and speed, r(18) = −.46, p = .044, was significant for younger adults; however, a close examination of the scatterplot for this correlation revealed that one participant was driving this finding. When this participant was removed, the negative correlation between motor accuracy and speed was no longer significant, r(17) = .05, p = .831. Older adults had significant positive correlations between motor accuracy and speed, r(19) = .53, p = .015, and vocal accuracy and speed, r(19) = .43, p = .050. This positive correlation suggests a relationship between speed and accuracy such that older individuals who were quick to respond also had high-accuracy scores and those who were slower to respond had lower accuracy scores.To rule out cross-domain trade-offs, bivariate correlations were conducted between motor and cognitive accuracy DTCs, as well as motor and cognitive RT DTCs. A negative correlation between these DTCs would suggest that lower costs in one domain (i.e., cognitive) are associated with greater costs in the other domain (i.e., motor). For correlations between domains in accuracy, no significant trade-offs were found for younger, r(18) = −.07, p = .773, or older adults, r(19) = −.03, p = .910. Similarly, there were no significant correlations between domains in RT for younger, r(18) = .22, p = .353, or older adults, r(19) = −.11, p = .648.Summary.—The results of Experiment 1 replicate the general findings of Crossley and Hiscock (1992) using a sequential tapping task. The lack of an age difference in DTCs in combination with the age differences in motor DTCs aligns well with the notion of an increasing role for cognition in fine motor performance. Beyond age differences in fine motor performances, Crossley and Hiscock demonstrated that these age differences increased when cognitive load increased. In the case of sequential tapping, additional support for age-related cognitive-motor interdependence should be found in conditions with greater cognitive load. In keeping with previous findings, we hypothesized that in Experiment 2, a high concurrent cognitive load would produce greater costs to sequential tapping than a lower cognitive load (for both age groups) and that this difficulty manipulation would have a greater impact on the older adults’ dual-task performances than the young.EXPERIMENT 2MethodParticipants.—Twenty younger adults (18–27 years) and 20 older adults (60–78 years) participated in the experiment. Recruitment and exclusion criteria were the same as in Experiment 1. In addition to the standardized tests administered in Experiment 1, all participants completed the Extended Range Vocabulary Test (ERVT; Educational Testing Service, 1976), and the Math subtest of the WAIS III, to assess vocabulary and math abilities, respectively. Descriptive statistics of the sample are presented in Table 1. All procedures were approved by the Concordia University Human Research Ethics Committee.Materials.—Fine motor task.The motor task was identical to that used in Experiment 1.Cognitive task: mental arithmetic.The cognitive task in this experiment had two levels of difficulty. For the Minus-1 level, participants subtracted one from randomly ordered two-digit numbers presented over headphones. For the Minus-7 level, participants subtracted seven from each stimulus. Stimuli consisted of two-digit numbers ranging from 11 to 99, not including numbers ending with seven (e.g., 17, 27, 37 …) or zero (e.g., 10, 20, 30 …). Two lists composed of 30 stimuli were used during the practice session. Sixty new stimuli were randomly arranged into four lists to be used in the four conditions (single Minus-1, single Minus-7, dual Minus-1, and dual Minus-7). The ISI range used in the current study (ISIs: minimum 2,300 ms and maximum 5,500 ms) was based on the average response times found in Abbud and colleagues (2009) for Minus-7. As compared with Experiment 1, the ISIs were lengthened here to accommodate the more complex cognitive tasks. In all other respects, the delivery of cognitive stimuli was the same as in Experiment 1.Procedure.—The testing took place in the Adult Development and Aging laboratory at Concordia University. After informed consent, all participants underwent the motor familiarization session described in Experiment 1. After the motor familiarization, all participants completed two practice blocks (15 trials each). In the first block, they completed a fixed order of Minus-1, single-task motor, and dual Minus-1; in the second block, they completed a fixed order of Minus-7, single-task motor, and dual Minus-7. Participants were instructed that both tasks were equally important and that they should try to respond quickly and accurately. Prior to the test runs, participants were asked to complete the Digit Symbol test.Once they had practiced the component tasks, they completed four test runs of the single motor, single cognitive, and dual-task trials. Runs 1 and 2 were always the Minus-1 difficulty level and Runs 3 and 4 were always the Minus-7 difficulty level. Within each run, the order was fixed: for Runs 1 and 3, single cognitive was always presented first, and for Runs 2 and 4, single motor was always presented first. The dual-task condition was always at the end of a run. These four runs were counterbalanced (i.e., 1-2-3-4, 4-1-2-3, 3-4-1-2, etc.) so that the difficulty manipulation was evenly distributed across the test session (i.e., with some participants having Minus-1, Minus-1, Minus-7, Minus-7; others Minus-7, Minus-1, Minus-1, Minus-7, etc.). For each of the four test sessions, there were 30 fine motor trials, 15 performed alone (single-task motor) and 15 performed concurrently with mental arithmetic (sequential tapping & Minus-sequential tapping & Minus-7). After the first two test runs, participants completed the Trail Making Test (A & B) and the ERVT, followed by the two remaining test runs. Finally, the participants completed the Digits Forward and the arithmetic subtest of the WAIS. Participants were debriefed and received course credit (younger) or an honorarium (older) for their time. The entire session lasted 90–120 min.Statistical analyses.—Accuracy and RTs were derived in the same way as Experiment 1. DTCs were calculated for each dependent variable in each domain (motor and cognitive) and difficulty level (Minus-1 and Minus-7). For the vocal RT data, responses were excluded if they were ±3 SD from each individual’s overall mean RT. Only a small proportion of the responses were considered outliers (MOlder = 0.01, SE = 0.001; MYounger = 0.01, SE = 0.002). For cognitive accuracy, motor accuracy, and motor RT, the data were checked for outliers ±3 SD from the group mean (younger and older) on single-task performances. One older adult was removed based on this criterion. Consequently, analyses were conducted on 20 younger and 19 older adults. Mixed factorial analyses of variance (ANOVAs; α = .05) were carried out using the four dependent variables (DTCs) with difficulty level (Minus-1 and Minus-7) as the within-subjects factor and age group (younger and older) as the between-subjects factor. All post hoc analyses used a Bonferroni corrected p value (.025).Results and DiscussionMean values for single- and dual-task performances are reported in Table 2, and DTCs for each domain are presented in Figure 2.Figure 2.Experiment 2: (A) Mean dual-task costs (DTCs) in motor accuracy by difficulty level (Minus-1 and Minus-7). (B) Mean DTCs in motor reaction times (RTs) by difficulty level. (C) Mean DTCs in cognitive accuracy by difficulty level. (D) Mean DTCs in cognitive RTs by difficulty level. Error bars are ±1 SE of the mean. Note. * = significant age difference in DTCs; + = DTCs are significantly greater than zero.Fine motor: MFST.—Accuracy.Figure 2A depicts the motor accuracy DTCs for both difficulty levels. The analysis revealed a main effect of difficulty level, F(1,37) = 44.33, p < .001, η2 = .545, such that the Minus-7 had higher costs (M = 17.00%, SE = 2.20) than Minus-1 (M = 3.30%, SE = 1.00). In addition, there was a main effect of age group, F(1,37) = 7.11, p = .011, η2 = .161, where older adults had higher DTCs in motor accuracy (M = 13.80%, SE = 2.00) than younger adults (M = 6.50%, SE = 1.90). The interaction was not significant, F(1,37) = 0.46, p = .503. The younger adults’ motor accuracy DTCs were not significantly different from zero, t(19) = 0.55, p = .590, for Minus-1 but were significantly different from zero for Minus-7, t(19) = 4.93, p < .001. For both difficulty levels, older adults’ motor accuracy DTCs were significantly different from zero, Minus-1: t(18) = 3.41, p = .003; Minus-7: t(18) = 5.85, p < .001.Reaction times.Figure 2B displays the motor RT DTCs. The ANOVA for motor RT DTCs resulted in a significant main effect of difficulty, F(1,37) = 39.48, p < .001, η2 = .516, where Minus-7 resulted in higher DTCs (M = 88.96 ms, SE = 9.55) than Minus-1 (M = 28.63 ms, SE = 5.56). The main effect of age, F(1,37) = 0.89, p = .351, and the interaction, F(1,37) = 0.01, p = .960, were nonsignificant. Analyses of the RT DTCs for the full sample confirmed that the DTCs for both difficulty levels were significantly different from zero, Minus-1: t(38) = 5.13, p < .001; Minus-7: t(38) = 9.38, p < .001.Cognitive: mental arithmetic.—Accuracy.Figure 2C depicts the cognitive DTCs in accuracy for both difficulty levels. The mixed factorial ANOVA revealed a main effect of difficulty, F(1,37) = 23.33, p < .001, η2 = .387, on the accuracy DTCs, such that DTCs were higher on Minus-7 trials (M = 10.20%, SE = 1.90) than on Minus-1 trials (M = 0.94%, SE = 0.40). Both the main effect of age, F(1,37) = 1.49, p = .230, and the interaction of difficulty and group, F(1,37) = 2.33, p = .135, were nonsignificant. Additional analyses on the full sample revealed that the DTCs in accuracy were significantly different from zero for both the Minus-1, t(38) = 2.13, p = .039, and the Minus-7, t(38) = 5.22, p < .001, conditions. When split by age, younger adults’ DTCs in Minus-1 were not significantly different from zero, t(19)= 1.47, p = .158, but they were significantly different from zero in Minus-7, t(19) = 3.10, p = .006. Similarly, the older adults’ DTCs were not significantly different from zero in the Minus-1 condition, t(18) = 1.91, p = .072, but were significantly different from zero in the Minus-7 condition, t(18) = 4.30, p < .001.Reaction times.Figure 2D depicts the cognitive DTCs in RTs for both difficulty levels. There were no significant effects in the cognitive RT data. The DTCs in vocal RTs did not differ by difficulty level, F(1,37) = 0.73, p = .398, nor by age group, F(1,37) = 1.32, p = .258, and the interaction between difficulty level and group did not reach significance, F(1,37) = 0.06, p = .816. Pooling together both age groups, the DTCs in the Minus-1 condition were significantly different from zero, t(38) = 3.42, p = .002, but the DTCs in the Minus-7 condition were not, t(38) = 0.37, p = .717. When split by age, only younger adults’ DTCs were significantly different from zero in the Minus-1 condition, t(19) = 3.97, p = .001, and neither age group had DTCs that were different from zero in the Minus-7 condition, Younger: t(19) = 1.77, p = .092; Older: t(18) = 0.21, p = .837.Testing for trade-offs: within and across domains.—At each level of difficulty, the mean dual-task scores within each domain were tested for a speed-accuracy trade-off. A negative correlation between speed and accuracy measures would indicate a trade-off. Correlations between mean dual-task motor accuracy and speed in the Minus-1 condition were nonsignificant for both age groups, Younger: r(18) = −.04, p = .884; Older: r(17) = .39, p = .104. Both younger and older adults had a significant positive correlation between dual-task motor accuracy and speed in the Minus-7 condition, Younger: r(18) = .52, p = .018; Older: r(17) = .62, p = .005. For both age groups, the cognitive dual-task correlations between accuracy and speed were nonsignificant for all conditions, Minus-1: r(37) = .26, p = .105; Minus-7: r(37) = .21, p = .202. Across both age groups, the lack of a significant negative correlation indicates that there was no speed-accuracy trade-off within domain.Using DTCs, cross-domain trade-offs (i.e., responding quickly in motor task but slowing in cognitive) were tested with bivariate correlations between the cognitive and motor DTCs. For correlations between motor and cognitive accuracy DTCs, neither younger, Minus-1: r(18) = .15, p = .540; Minus-7: r(18) = .20, p = .398, nor older adults, Minus-1: r(19) = .03, p = .913; Minus-7: r(19) = −.24, p = .334, demonstrated any significant cross-domain trade-offs. Similarly, there were no significant cross-domain trade-offs for correlations between motor and cognitive RTs for younger, Minus-1: r(18) = −.22, p = .349; Minus-7: r(18) = .22, p = .354, or older adults, Minus-1: r(19) = .05, p = .846; Minus-7: r(19) = −.07, p = .774.Summary.—Similar to the simple tapping findings of Crossley and Hiscock (1992) and the sequential tapping findings of Kemper and colleagues (2003), tapping sequentially while performing a cognitive task had a greater impact on older adults’ motor performances than younger adults. The younger adults were able to maintain their motor accuracy in the Minus-1 condition, whereas older adults demonstrated significant accuracy costs in both difficulty levels. Both groups slowed when sequentially tapping with a cognitive task but there was no age difference in the degree of slowing. In the cognitive measures, the pattern of results is similar for younger and older adults with the only exception being significant cognitive RT DTCs in the Minus-1 condition for the younger adults. The lack of speed-accuracy trade-offs and cross-domain trade-offs suggests that younger adults were not slowing to maintain performance on another measure. In the Minus-7 condition, the lack of significant cognitive RT DTCs in combination with significant motor DTCs for both age groups in both measures might indicate a prioritization of cognitive task under the highest cognitive load.GENERAL DISCUSSIONThe primary goal of this study was to examine the role of executive control in fine motor performance using a motor-cognitive dual-task paradigm. This study extends previous work on dual-task simple tapping (Crossley & Hiscock, 1992) and complex tapping (Kemper et al., 2003) with age differences found primarily in fine motor performances. The first experiment combined a low-load semantic judgment task with sequential tapping and older adults were slower and less accurate than younger adults on the sequential tapping task. In the second experiment, in which cognitive load was manipulated, there were age differences in motor accuracy, with older adults demonstrating costs in both difficulty levels, whereas motor accuracy costs only emerged in the harder condition for younger adults. Because older adults demonstrate costs in sequential tapping even in the conditions of lowest load and these costs reliably emerge in motor accuracy performance, we propose that older adults require greater executive control processes in order to perform the sequential tapping task.In both experiments, there was an asymmetry in the pattern of results, such that DTCs occurred mainly in the motor domain. This occurred despite differences in temporal predictability across the cognitive and motor tasks. Indeed, one might hypothesize that the less predictable cognitive task (that occurred at different time points during the trial) would be more affected by dual-task interference than the motor task that was presented in a more predictable fashion (one stimulus each second). Ultimately, across the different levels of cognitive load, the cognitive tasks interfered with the sequential nature of the tapping task and older adults were more affected by this interference than younger adults. The interference from the cognitive task affected the older adults’ fine motor performance even in the easiest condition, and younger adults only faltered when task demands were too great. With the exception of the Minus-1 condition RT measure (cognitive and motor), all performance costs for younger adults were found in the harder Minus-7 condition. Perhaps mild cognitive loads taxed younger adults’ coordinative processes (i.e., coordinating the performance of the two tasks). Whereas for older adults, all cognitive loads were sufficiently challenging that key press accuracy or response selection in the motor task was affected.Given that the sequence we presented was repeated throughout the each block of trials, younger and older adults may have encoded the sequence of key presses into a single action plan (Tubau, Hommel, & Moliner, 2007). Findings with younger adults have demonstrated that execution of an action plan can be disrupted by visual and auditory verbal distracters. In addition, sequence learning and action plans have both been shown to involve the prefrontal cortex (Tubau et al.). The prefrontal cortex and the executive control processes it subserves are known to decline with normative aging (Verhaeghen & Cerella, 2002). Therefore, in the current experiment, because older adults relied more heavily on executive control functions for sequential tapping, they demonstrated greater performance costs than their younger counterparts. In support of this proposal, existing sequence learning research (Aizenstein et al., 2006) has found age differences in frontal activity during concurrent sequence learning, such that older adults show greater activity than younger adults in the left dorsolateral prefrontal cortex.The results of the current experiment are also consistent with our previous findings in dual-task walking experiments, which used the same cognitive tasks (Fraser et al., 2007; Li et al., 2008). In particular, Fraser and colleagues (2007) found age differences only in walking performance when performing the semantic task, and Li and colleagues (2008) demonstrated maintenance of walking performance during the Minus-1 condition for younger adults but costs similar to older adults in the harder condition. Similarly, in Experiment 1, there was age equivalence in performance of the semantic task, and age differences emerged in sequential tapping. Furthermore, in Experiment 2, although younger adults slowed their sequential tapping in the Minus-1 condition, they maintained their accuracy when older adults demonstrated accuracy costs and both groups had similar costs in the Minus-7 condition. These similarities suggest that gait and sequential tapping may draw on similar executive control functions. The age-related dual-task effects reported by Crossley and Hiscock (1992) may have been a reflection of age-related reductions in general dual-task coordination processes rather than an indication that simple tapping requires executive control. Indeed, previous research suggests that simple tapping does not rely on executive functions (Hausdorff, Yogev, Springer, Simon, & Giladi, 2005). The similarity of the current pattern of results with that of previous walking research (Fraser et al., 2007; Li et al., 2008) suggests that executive functions might play a role in both walking and fine motor DTCs and that the previous walking findings were not primarily driven by postural threat. Although it is clear that postural threat influences attentional allocation (Brown et al., 1999), it has also been found that different degrees or levels of difficulty of postural threat (Lajoie, Teasdale, Bard, & Fleury, 1996) can modulate DTCs in older adults.CONCLUSIONSTaken together, the findings extend the research on aging and dual-task fine motor performance in demonstrating that concurrent sequential tapping costs are greater in older adults due to the disruption of a planned execution of taps at the executive processing level. Under low cognitive load, younger adults have a more proceduralized or automatic approach to the sequential tapping task that does not require executive control. In contrast, older adults demonstrate costs at every load level demonstrating the cognitive penetration of motor task performance (Teasdale, Bard, LaRue, & Fleury, 1993).FUNDINGThis project was funded by a Natural Sciences and Engineering Research Council of Canada PhD scholarship awarded to S.A.F. and a Canadian Institutes of Health Research operating grant awarded to K.Z.H.L. and V.B.P. (MOP-67757, 68807).Many thanks to Alejandro Endo, Ricco Boma, Madeleine Ward, Laura Fontil, Stephanie Torok, Monique Leblanc, Hilary Greenstone, Simon Desmarais-Zalob, and Vanessa Raccio who all helped with this project.AbbudGACLiKZHDeMontRGAttentional requirements of walking according to the gait phase and onset of auditory stimuliGait and Posture200930227232AizensteinHJButtersMAClarkKAFigurskiJLStengerVANebesRDReynoldsCFIIICarterCSPrefrontal and striatal activation in elderly subjects during concurrent implicit and explicit sequence learningNeurobiology of Aging200627741751AlbinetCTomporowskiPDBeasmanKAging and concurrent task performance: Cognitive demand and motor controlEducational Gerontology200632689706BaddeleyAStussDTKnightRTFractionating the central executivePrinciples of frontal lobe function2002New YorkOxford University Press246277BaltesPBLindenbergerUEmergence of a powerful connection between sensory and cognitive functions across the adult lifespan: A new window to the study of cognitive aging?Psychology and Aging1997121221BleAVolpatoSZulianiGGuralnikJMBandinelliSLauretaniFBartaliBMaraldiCFellinRFerrucciLExecutive function correlates with walking speed in older persons: The InCHIANTI studyJournal of the American Geriatrics Society200553410415BrownLAShumway-CookAWoollacottMHAttentional demands and postural recovery: The effects of agingJournal of Gerontology: Series A: Biological and Medical Sciences199954M165M171CrossleyMHiscockMAge-related differences in concurrent-task performance of normal adults: Evidence for a decline in processing resourcesPsychology and Aging19927499506Educational Testing ServiceExtended Range Vocabulary Test: Kit of factor-referenced cognitive tests1976Princeton, NJAuthorFraserSALiKZHDeMontRGPenhuneVBEffects of balance status and age on muscle activation while walking under divided attentionJournal of Gerontology: Psychological Sciences200762B171178FraserSALiKZHPenhuneVBA comparison of motor skill learning and retention in younger and older adultsExperimental Brain Research2009195419427HausdorffJMYogevGSpringerSSimonESGiladiNWalking is more like catching than tapping: Gait in the elderly as a complex cognitive taskExperimental Brain Research2005164541548HuxholdOLiSSchmiedekFLindenbergerUDual-tasking postural control: Aging and the effects of cognitive demand in conjunction with focus of attentionBrain Research Bulletin200669294305KahnemanDAttention and effort1973Englewood Cliffs, NJPrentice-HallKemperSHermanRELianCHTThe costs of doing two things at once for young and older adults: Talking while walking, finger tapping, and ignoring speech or noisePsychology and Aging200318181192KetchamCJStelmachGEBirrenJSchaieKWAge-related declines in motor controlHandbook of psychology of aging20015th ed.San Diego, CAAcademic Press313348KlugerAGianutsosJGGolombJFerrisSHGeorgeAEFranssenEReisbergBPatterns of motor impairment in normal aging, mild cognitive decline, and early Alzheimer’s diseaseJournal of Gerontology: Series B: Psychological Sciences and Social Sciences199752BP28P39KramerAFMaddenDJCraikFIMSalthouseTAAttentionThe handbook of aging and cognition20083rd ed.New YorkPsychology Press189249KrampeRTAging, expertise and fine motor movementNeuroscience and Behavioral Reviews200226769776KuçeraHFrancisWNComputational analysis of present day American English1967Providence, RIBrown University PressLajoieYTeasdaleNBardCFleuryMUpright standing and gait: Are there changes in attentional requirements related to normal aging?Experimental Aging Research199622185198LiKZHDeMontRPenhuneVFraserSAAbbudGThe dynamic relationship between cognition and walking under dual-task conditions in healthy agingInternational Journal of Psychology200843366LiKZHLindenbergerURelations between aging sensory/sensorimotor and cognitive functionsNeuroscience and Biobehavioral Reviews200226777783LiKZHLindenbergerUFreundAMBaltesPBWalking while memorizing: Age-related differences in compensatory behaviorPsychological Science200112230237LövdénMSchäeferSPohlmeyerAELindenbergerUWalking variability and working memory load in aging: A dual process account relating cognitive control to motor performanceJournal of Gerontology: Psychological Sciences200863BP121P128MendelsonDNRedfernMSNebesRDJenningsJRInhibitory processes relate differently to balance/reaction time dual tasks in young and older adultsAging, Neuropsychology, and Cognition201017118SpreenOStraussEA compendium of neuropsychological tests: Administration, norms, and commentary1998New YorkOxford University PressTeasdaleNBardCLaRueJFleuryMOn the cognitive penetrability of postural controlExperimental Aging Research199319113TubauEHommelBMolinerJLModes of executive control in sequence learning: From stimulus-based to plan-based controlJournal of Experimental Psychology: General20071364363VerhaeghenPCerellaJAging, executive control, and attention: A review of meta-analysesNeuroscience & Biobehavioral Reviews200226849857WechslerDManual of the Wechsler Adult Intelligence Scale—III1997New YorkPsychological CorporationWoollacottMShumway-CookAAttention and the control of posture and gait: A review of an emerging area of researchGait and Posture200216114Yogev-SeligmannGHausdorffJMGiladiNThe role of executive function and attention in gaitMovement Disorders200823329342 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B19D36BBF10A2648F2CD63FC5EF4968BE5075C9.txt b/test/dataset/in/resources/corpus/Clean_0B19D36BBF10A2648F2CD63FC5EF4968BE5075C9.txt new file mode 100644 index 0000000..520bd48 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B19D36BBF10A2648F2CD63FC5EF4968BE5075C9.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 33510.1093/geront/43.3.335 USE OF TECHNOLOGY A Comparison of Assistive Technology and Personal Care in Alleviating Disability and Unmet Need Agree Emily M. PhD 1 Freedman Vicki A. PhD 2 Address correspondence to Emily M. Agree, PhD, Department of Population and Family Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Room E4014, Baltimore, MD 21205. E-mail: eagree@jhsph.edu 6 2003 43 3 335 344 16 9 2002 25 4 2002 The Gerontological Society of America 2003 Purpose:The authors examine differences in reports of residual disability and unmet need by type of long-term care arrangement (assistive technology or personal care).Design and Methods:This study compares three specific dimensions of residual difficulty (pain, fatigue, and time intensity) and reports of unmet need across care arrangements. Samples from the U. S. 1994–1995 National Health Interview Survey Phase 2 Disability Supplements include adults with limitations in bathing, transferring, walking, and getting outside.Results:Even when differences in underlying disability are accounted for, assistive technology (AT) confers no additional benefit in the three dimensions of residual difficulty analyzed here. AT users equally or more often report that tasks are tiring, time consuming, or painful, even when they use assistance. Though this would appear to indicate unmet needs for care, fewer AT users report a desire for hands-on personal care.Implications:Though disability alleviation by technology is no better on specific dimensions of difficulty, technology users report less unmet need for personal care. Designing appropriate and cost-effective home care for adults with disabilities requires a better understanding of the ways in which technology users may differ from others and the circumstances under which technology can be most effective. Long-term care Effectiveness Activities of daily living hwp-legacy-fpage 335 hwp-legacy-dochead RESEARCH ARTICLE Designing appropriate and cost-effective home care for adults with disabilities is a priority in aging societies. The number of older adults with disabilities is large and continues to grow, despite reductions in disability rates over the past two decades (Schoeni, Freedman, & Wallace, 2001). Currently, approximately 15% of the adult U. S. population, or 40 million people, are limited in activities as a result of a chronic health condition (Kaye, LaPlante, Carlson, & Wenger, 1996). The vast majority of adults with disabilities live in the community, and facilitating their ability to live independently is an increasingly important public health concern. More than 75% of older adults with disabilities use some kind of assistive device, most often to assist with mobility (Russell, Hendershot, LeClere, Howie, & Adler, 1997), and the number has been growing over time (Manton, Corder, & Stallard, 1993; Russell et al., 1997). Devices are used both independently and in conjunction with personal caregiving services. However, the relative advantages of assistive technology and personal care are not well understood, in part because most studies evaluate one or the other, but rarely the two together. Most research on assistive technology evaluates the efficacy and costs of specific devices (e.g., Chen, Mann, Tomita, & Burford, 1998; Gitlin & Levine, 1992; Kohn, LeBlanc, & Mortola, 1994; Mann, Hurren, Charvat, & Tomita, 1996; Sanford, Arch, & Megrew, 1995; Steinfeld & Shea, 1995), or it broadly examines the use of any assistive devices (Manton et al., 1993; Norburn et al., 1995; Zimmer & Chappell, 1994) or the number of devices used (Hartke, Prohaska, & Furner, 1998; Heide et al., 1993; Mann, Hurren, & Tomita, 1993). Studies of assistive technology emphasize the extent to which the acceptability and effectiveness of home-based technologies vary, depending on the training provided (Gitlin & Levine, 1992), the amount of stigma perceived by the user (Arras, 1995), and how much the introduction of assistive technologies transforms the home from a personal space to a health care delivery location (Tamm, 1999). Similarly, population-based efforts to understand the effectiveness of personal care arrangements often altogether exclude a consideration of self-care through assistive technology (Allen & Mor, 1997; Desai, Lentzner, & Weeks, 2001; Tennstedt, McKinlay, & Kasten, 1994). These studies instead focus on the trade-offs between formal and informal care or on the extent to which formal care can defer institutionalization (Soldo & Freedman, 1994). Attempts to measure outcomes that reflect the effectiveness of interventions for disability are relatively new and differ depending on whether the goal is to evaluate rehabilitation services specifically or more general effects on the ability to live independently (Andresen, Lollar, & Meyers, 2000). A recent national study examined the effectiveness of assistive technology in terms of its impact on hours of personal care for older adults with disabilities, assuming that the decision to use devices is independent of the acquisition of caregivers (Allen, Foster, & Berg, 2001). This study found that those who use assistive technology also use fewer hours of personal care. Two additional small-scale experiments have evaluated the effectiveness of interventions to increase the use of assistive devices (Mann, Ottenbacher, Fraas, Tomita, & Granger, 1999) and environmental modifications for dementia (Gitlin, Corcoran, Winter, Boyce, & Hauck, 2001). Although sample sizes were small, both studies suggested that increased use of technology may slow functional declines, lower health care costs, and increase efficacy among some caregivers. However, none of these studies directly compare the effect of assistive technology and personal care on functional health. Two other nationally representative studies have evaluated the relative effectiveness of assistive technology and personal care in terms of reported reduction of disability (Agree, 1999; Verbrugge, Rennert, & Madans, 1997). Both studies found that the use of assistive technology was associated with greater reduction of overall levels of difficulty in either activities of daily living (ADLs) or mobility impairments when compared with personal care. Though each used distinct measures and different data sets, these studies both relied on broad judgments about levels of difficulty with tasks to determine effectiveness. In sum, much of the prior research on this topic either has failed to directly compare outcomes related to assistive technology and personal care, begging the question as to when and whether each is most appropriately used (Allen et al., 2001; Gitlin et al., 2001; Mann et al., 1999), or has relied on global assessments of residual difficulty that do not offer insight into the mechanisms by which different care arrangements alleviate needs (Agree, 1999; Verbrugge et al., 1997). The purpose of this study is to further investigate the relative effectiveness of assistive technology and personal care in alleviating disability among adults with difficulty in ADLs. Using nationally representative disability survey data, we compare three dimensions of disability for which both underlying and residual difficulty can be assessed—pain, fatigue, and time intensity—across different combinations of assistive technology and personal care use. We also examine reports of the need for hands-on assistance across care arrangements. Framework The conceptual framework of the disablement process (e.g., Pope & Tarlov, 1991; Verbrugge & Jette, 1994) gives us some foundation for understanding how different care arrangements may alleviate different dimensions of disability. In this framework, disability is socially defined, being the product of the individual's functional capacity, the demands of the physical and social environment, and his or her own expectations about daily life. Verbrugge and Jette (1994) make the additional important distinction between underlying difficulty—the level of difficulty without help or special equipment—and residual difficulty—that is, with whatever assistance is generally used. Both underlying and residual disability encompass various levels of difficulty. At one extreme, an individual may be completely unable to carry out an activity without assistance; alternatively, an individual may be able to carry out an activity without assistance but with some or a lot of difficulty. The most commonly reported dimensions of difficulty in this context are pain and fatigue (Albrecht & Devlieger, 1999); tasks that are time consuming to carry out (or more time consuming than under usual or optimally functioning circumstances) may also be identified as being difficult. Closely related to residual difficulty is the concept of unmet need, that is, whether an individual thinks he or she requires more assistance. The disabling effects of an impairment can be reduced or potentially eliminated in a number of ways (see Figure 1). Individuals may compensate for their disability by using personal care, they may alter their ability to carry out the task (through rehabilitation or device use), or they may alter the demands of the environment (through, e.g., a home modification). Each of these approaches may be adopted in isolation or may be combined into a more complex care arrangement. Compensation, because it involves the cooperation of one or more helpers, creates a state of dependency. In contrast, assistive technologies and related environmental modifications enhance independence either by expanding the capacity of the individual or reducing the demands of the environment. In this article we hypothesize that different approaches to bridging disability may alleviate difficulty in different ways. We expect that assistive devices may alleviate difficulty by minimizing the pain associated with a task, whereas personal care may lessen the time and energy necessary to accomplish a task. Because devices increase an individual's capacity for self-care, we expect devices to be more effective than personal care in reducing unmet need. Methods Data Data are from the 1994 and 1995 Phase 2 Disability Supplements to the U. S. National Health Interview Survey, hereafter referred to as the NHIS-D2 (National Center for Health Statistics, 1998a, 1998b). These data provide detailed information on the use of assistive devices and environmental modifications for limitations in ADLs. The U. S. NHIS is a nationally representative health survey of approximately 48,000 households (122,000 persons) conducted annually by the U. S. National Center for Health Statistics. The survey consists of a core questionnaire that collects health information and demographic background on every person in the household. Each year supplemental surveys on specific health topics are included. In 1994 and 1995, a supplement on disability was administered in two phases over approximately 3 years. Phase 1, administered at the same time as the NHIS core interview, collected basic measures of impairment and functional health from a designated individual about all household members. Household members who were identified as disabled in Phase 1, using a broad definition of disability (the presence of any impairment, functional limitation, or disability), were interviewed in person 7–17 months later to obtain more detailed information (Phase 2). Separate questionnaires were administered to children (under 18) and adults (age 18 and older). Response rates were approximately 95% for the core and 87% for the supplements. Sample Selection To examine the effectiveness of care arrangements in alleviating disability, we focus on the adult population with underlying limitations in ADLs—those who report that they have difficulty carrying out an activity by themselves and without special equipment. This definition includes both persons currently using some form of care as well as a substantial number who report difficulty but make use of no personal care or assistive devices. Because assistive devices tend to be task specific in response to disabling conditions (Agree & Freedman, 2000), analyses are conducted separately for samples reporting underlying difficulty with each of four daily activities: bathing or showering (n = 3,493), getting in or out of bed or chairs (n = 3,834), walking (n = 7,051), and getting outside (n = 3,542). Although respondents also were asked about difficulty with dressing, eating, and getting to or using the toilet, sample sizes were too small, and the use of assistive technology too rare in the case of dressing and eating, to analyze care arrangements for these activities. Respondents with multiple ADL difficulties may be in more than one sample. Measures of Assistive Technology and Personal Care Questions are asked about assistive technology in the ADL assessment section of the NHIS-D2 instrument. For each ADL, participants who answer that they use a special device or piece of equipment to perform a task are asked to name the type of technology that they use. Interviewers use a checklist to simplify the recording of answers. For the four activities we analyze here, the specific equipment includes hand bars or rails, and bath stools, seats, or chairs (bathing); canes, walkers, special cushions, lift chairs, a hospital bed, and a trapeze or sling (transferring); and canes, walkers, crutches, and wheelchairs (getting around indoors and going outside). An analysis of specific items was not possible because devices other than canes and walkers were not mentioned frequently enough to allow separate analyses. Grouping all assistive technologies for a given activity together also allowed us to include sample persons who reported using special equipment without providing a specific type. Respondents also were asked in the same section whether they received hands-on help from another person with that activity. For each ADL, information was obtained on the type and amount of personal care provided to the respondent. On the basis of this information, we classified respondents into four groups: no care, assistive technology only, hands-on personal care only, and both personal care and assistive technology. Level of Underlying Difficulty Comparing the effectiveness of care arrangements requires some ability to control for the differential selection of these arrangements based on the severity of underlying disability. For each activity, respondents were classified as having some difficulty, a lot of difficulty, or being completely unable to carry out the activity without assistance. We also present analyses for all respondents and for the subgroup reporting a lot of underlying difficulty with a given task. Measures of Effectiveness The NHIS-D2 provides task-specific measures of residual difficulty and unmet need. The survey included information on three explicit dimensions of residual difficulty. For each activity, respondents (with the exception of those who were completely unable to perform the task) were asked to assess whether the activity is “very tiring,” “takes a long time,” or is “very painful” under two circumstances: first, if it is performed without personal care or assistive technology, and second, when it is performed with the assistance of personal care or devices (if used). This information was used to classify whether respondents' difficulty with an activity was “eliminated” when using personal care or assistive technology or whether they report residual difficulty even when using assistance. In addition, all respondents who reported underlying difficulty with a given task were asked whether they needed (more) hands-on help with that activity, which was used to derive a measure of unmet need. Limitations of the Data Although the NHIS-D is a rich source of information on the health and care arrangements of persons with disabilities in the United States, there are some limitations of the data that merit attention here. First, as already noted, the NHIS-D2 excludes the most severely disabled (those who report that they are unable to perform a task without assistance) from the questions about specific dimensions of underlying and residual difficulty, thus limiting our conclusions to a more moderately disabled population for those particular analyses. Second, the sensitivity of a disability measure to clinically detectable differences in functional status is an important factor in comparing across types of care (Cohen & Marino, 2000). In this study, the dichotomous indicators for each specific dimension of difficulty made it possible to examine effectiveness only in terms of complete elimination of difficulty. We are therefore limited in our ability to examine more refined gradations of effectiveness in disability reduction as has been done in previous studies (Agree, 1999; Verbrugge et al., 1997). Finally, the cross-sectional nature of the data make it impossible to assess the role that the timing of acquisition of assistive technology and personal care plays in the effectiveness of such arrangements. As already noted, prior research has indicated that assistive technologies may be more effective than personal care in alleviating disability. Consequently, we expect in the following analyses to find that, when we control for the level of underlying need, those who use assistive devices are less likely than those using personal care to report (a) residual difficulty on all dimensions of disability and (b) unmet need for personal care. Methods Because the NHIS-D2 is not a simple random sample but instead has a complex design with geographic clustering, statistical tests have been adjusted to take into account the complex survey design. All estimates are weighted with analytic weights normed to the appropriate sample size. Results discussed in the text are statistically significant at the p <.05 level unless otherwise noted. Results Assistive device use is quite common among persons who have underlying difficulty with the four activities considered here. Between 16% (of persons with underlying difficulty transferring) and 39% (of those who have underlying difficulty walking) use assistive technology alone to accommodate their difficulty (see Table 1). An additional 13–33% use assistive technology in combination with personal care. Taken together, 29–64% use one or more devices, with devices dominating most clearly in basic mobility activities (walking and going outside). Those with underlying difficulty bathing or getting outside are slightly older on average (67 vs. 63 years). However, the distributions by sex, minority status, and education are similar across the samples, with a majority of people being female; approximately 16–17% are non-White, approximately 7% are Hispanic, and the majority have at least completed high school (∼55%). Table 2 shows the distribution of the severity of underlying disability stratified by the type of care used for each of the four ADLs. This table confirms that the combination of care adopted is clearly related to the overall amount of underlying difficulty with the task. Those who use no care of any kind are the least disabled, with between 66% and 79% reporting some underlying difficulty and 1–7% reporting they are completely unable to carry out the activity without assistance (11–20% report some difficulty and 48–60% report being completely unable to carry out the activity). Among those who do use some type of assistance, those who use assistive devices alone report more moderate levels of underlying difficulty compared with those who use hands-on help (alone or in combination with assistive technology). Only 13–21% of those who use technology by itself state that they would be unable to perform the task without assistance, compared with 26–43% of those depending on personal care alone. As already noted, those who rely on both personal care and assistive technology report the greatest amount of difficulty, with 51–59% reporting that they would be completely unable to perform the task without assistance. Figure 2 shows for those with underlying difficulty in each of the four activities the proportion who also report underlying difficulty on three specific dimensions: whether the activity is very tiring, very painful, or takes a long time. The top panel shows estimates for all levels of difficulty, and the bottom panel is limited to those reporting a lot of underlying difficulty. For example, 63% of those with difficulty bathing say that they find the activity to be tiring when they do not have help or equipment. We find that substantial proportions of persons who report underlying difficulty with a given task find the activity very tiring, time consuming, or very painful when they do not have hands-on help or use equipment (47–76%, depending on the specific task). Not surprisingly, the proportions reporting underlying difficulty on each of the specific dimensions are consistently higher among those who report a lot of underlying difficulty on the task, ranging 59–88% on average for all activities. We also investigated (not shown) whether the proportion who reported underlying difficulty on each of the three specific dimensions varied across care arrangements. We found that those using both personal care and assistive technology were generally more likely than those using either one alone to report that a task is very tiring, takes a long time, or is very painful when carried out without assistance. When we stratified by the level of underlying difficulty, we found a much weaker relationship between care arrangement and specific dimensions of underlying difficulty. We next examine reports of residual difficulty, using the same three dimensions (whether the task is tiring, time consuming, or painful) and controlling again for the level of underlying difficulty. As shown in the top panel of Table 3, among persons with underlying difficulty, those who use assistive technology alone report residual difficulty more often than those using personal care (with or without devices). This relationship is significant, however, only for two dimensions (whether the task is tiring or time consuming) and three of the tasks (bathing, transferring, and going outside). For example, among those who report underlying difficulty with bathing, 75% of assistive technology users report that the task is very tiring when they use their equipment, compared with only 55% of those who use personal care alone (p <.05). Pain varies the least across care arrangements and is not significantly related to the type of care for any of the activities. The same general pattern is evident when the investigation is restricted to those with more severe activity limitations (bottom panel of Table 3), although the differences are statistically significant only for bathing. For many activities and dimensions of difficulty, especially pain, care arrangements are not significantly related to residual difficulty. However, in those cases in which a significant association is found, it is opposite to the expected direction—assistive technology users more often report that a task is tiring or takes a long time even when they use assistance. Figure 3 shows the proportion of people reporting a need for hands-on help by activity and level of underlying severity of disability. The proportions shown here are consistent with those of other studies (e.g., Desai et al., 2001) and generally low in comparison with the reports of residual difficulty shown in Table 3. The proportions reporting a need for (more) hands-on help are consistent across levels of underlying disability and types of care (the highest levels are 12–18% reported by those with a lot of underlying difficulty and who use personal care alone). However, those using assistive technology alone are consistently less likely to state that they need any hands-on help, at levels as low or lower than for those who use no care. For bathing, levels of unmet need are highest, but the differences across types of care are minimal and not significant (significance tests not shown). For the three mobility-related ADLs, the differences between those who use personal care (either alone or in combination with assistive technology) and those who do not (using either assistive devices alone or no care at all) are substantial and consistently significant across levels of disability. Discussion Facilitating the ability to live and work independently in the community is a central goal for gerontologists, policymakers, and public health practitioners. Although it is difficult to measure optimal care arrangements for adults with disabilities (Jette & Keysor, 2002), the results presented here further our understanding of the relative effectiveness of assistive technology and personal care in alleviating difficulty with daily activities. They also provide insights into the conceptualization and measurement of unmet need. Consistent with previous studies (Agree & Freedman, 2000; Manton et al., 1993; Verbrugge et al., 1997), we find that assistive devices are the most common means of managing day-to-day tasks for older adults and that the extent to which assistive technology and personal care are used in combination is closely related to the amount of difficulty reported. Unlike previous researchers, however, we compare across care arrangements reports of residual disability on specific dimensions such as fatigue, duration, and pain. We find that although adults with disabilities using only assistive technology tend to be less disabled than users of personal care, they are more likely to report that a task is tiring or time consuming when they use their equipment. We also find that users of assistive technology are less likely to report a need for any hands-on help than those already using personal care. Our analysis is limited in several ways. We were unable to compare the effectiveness of care arrangements on specific dimensions for the most severely disabled; thus we cannot draw any conclusions about the relative efficacy of care arrangements for those unable to perform a task without assistance. Moreover, we were able to explore only the complete elimination—rather than the reduction—of disability. There are many reasons that devices, which require some physical and cognitive effort to use, might reduce overall difficulty more easily than personal care, but not completely eliminate it. Future research should consider the relative effectiveness of personal care and assistive technology both on specific dimensions and in finer gradations. Finally, we were unable to explore with these cross-sectional data the more dynamic acquisition process underlying the disablement process. For example, a long-term user of assistive technology may become more proficient and consequently more satisfied than one who has newly acquired the device. Surely this is a fruitful area for further exploration. Despite these limitations, our analyses provide several new insights into the relative effectiveness of personal care and technology. First, the present study suggests a need to probe more deeply into the salient dimensions of disability that assistive technology and personal care alleviate. Using global measures of difficulty, prior research found that assistive technology appeared to be more effective than personal care in reducing or eliminating disability (Agree, 1999; Verbrugge et al., 1997). We find instead that users of such technology report similar or greater amounts of residual difficulty on the three specific dimensions of residual difficulty with ADL tasks in these data. These findings do not contradict earlier work, but rather inform it, by expanding our definition of effectiveness. Qualitative research may help identify the most important dimensions related to the effectiveness of different care arrangements. In one of the few studies to address this question, Albrecht and Devlieger (1999) report that pain and fatigue are among the most common problems reported, but further work illuminating the nature and meaning of these dimensions is warranted. Second, we find that persons with more severe disabilities are more likely to use personal care and less likely to use assistive technology exclusively. At the same time, like Agree (1999), we show the relative effectiveness of personal care and assistive technology on specific dimensions appears to diminish at higher levels of disability. These findings suggest that selection according to underlying disability is critical to take into account in any comparisons of effectiveness across care arrangements. In prior work this selection may have artificially increased the benefit attributed to assistive technology relative to personal care. In this study, we find the disadvantage of such technology on specific dimensions of disability does not appear to be explained by the underlying severity of disability, which is lower among assistive technology users. Third, the finding that assistive technology users less often report a need for hands-on help despite greater residual difficulty illustrates the likelihood that factors other than the severity of underlying disability influence the choice of care arrangements and must be attended to in both research and clinical practice. In particular, the use of assistive technology may be related to psychological factors such as receptivity, self-efficacy, and motivation, all of which have been shown to be related to the success of rehabilitative efforts (Arnstein, 2000; Grahn, Ekdahl, & Borquist, 2000; Zimmer & Chappell, 1999). Values related to autonomy and privacy also are important aspects of the acceptance and use of technology in rehabilitation and home care (Tamm, 1999). Our findings also have implications for the conceptualization and measurement of unmet needs for care. Studies of unmet need have traditionally focused on personal care (Allen et al., 2001; Gitlin et al., 2001; Mann et al., 1999), finding, as we did here, quite low reported levels of unmet need, even among adults with severe disabilities. Such research may be missing a substantial amount of unmet need for assistance, were the concept more broadly defined to include needs for and use of assistive technology. Not only may there be unmet needs that can be better fulfilled by technology rather than personal care, but the findings from this study suggest that some proportion of the disabled population may have unmet needs but would not report a desire for personal care. Mann, Hurren, and Tomita (1995) reported that persons with arthritis expressed a need for additional devices, even describing new technologies to be invented. Including specific questions about the need for additional devices to patient assessments and survey instruments would contribute a great deal to our understanding of and interventions to reduce unmet needs. In sum, indicators such as residual disability and unmet need do not in and of themselves allow us to identify the reasons that a particular care arrangement is more or less satisfactory. They do, however, make it possible to uncover circumstances in which types of care are less than optimal (Eldar, 2000). To meet the goal of facilitating the ability of adults to live and work independently in the community, it is essential for future research to explore the role of self-care more thoroughly. In particular, greater attention should be paid to identification of the different dimensions by which assistive technology and personal care alleviate difficulty and enhance independence. Prior versions of this article were presented in Syracuse, NY, at the Center for Policy Research Seminar Series in Aging, Labor, and Public Finance in October 2000 and in Washington, DC, at the Annual Meetings of the Population Association of America in March 2001. We acknowledge the able assistance of Jonas Marainen and Hakan Aykan in preparing the data and the tables, and Mary Alice Ernish for bibliographic assistance. We also thank Donna Strobino, Douglas Wolf, and Timothy Smeeding for their helpful comments. This work was supported by Grant R01-AG15135 from The National Institute on Aging. 1 Department of Population and Family Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD. 2 Polisher Research Institute, Madlyn and Leonard Abramson Center for Jewish Life (formerly Philadelphia Geriatric Center), North Wales, PA. Decision Editor: Laurence G. Branch, PhD Figure 1. Alleviating underlying disability through personal care and assistive technology Figure 2. Percentage of those with difficulty on each activity of daily living who report that a task is tiring, time consuming, or painful without assistance (those who are completely unable to perform an activity are not included) Figure 3. Percentage of those reporting an unmet need for hands-on care by type of assistance and activity. AT = assistive technology. Table 1. Care Arrangements and Sample Characteristics. Characteristic Bathing (n = 3,493) Transferring (n = 3,834) Walking (n = 7,051) Getting Outside (n = 3,542) Care arrangement (%)     No care 25.8 50.1 40.0 23.3     AT only 20.5 15.9 39.2 31.0     Personal care only 24.6 20.6 6.3 13.1     Personal care and AT 29.2 13.4 14.5 32.7     Total 100.0 100.0 100.0 100.0 Age (mean; years) 67.7 62.6 63.8 66.9     % Female 63.5 61.2 60.4 64.2     % Non-White 16.5 16.2 16.7 17.9     % Hispanic 6.7 7.1 6.7 6.8 Education (%)     Primary only (0–7) 17.5 14.5 14.7 17.1     Some HS (8–11) 27.6 27.4 28.6 28.6     HS graduate (12) 32.6 33.5 33.4 32.5     Some college (13+) 22.3 24.7 23.2 21.8     Total 100.0 100.0 100.0 100.0 Note: AT = assistive technology; HS = high school. Table 2. Underlying Global Difficulty by Type of Assistance and Activity. Type of Assistance Activity None AT Only PC Only PC and AT Bathing (n = 3,493)*     Some difficulty 71.1 46.9 34.3 17.0     A lot of difficulty 23.4 32.4 22.5 23.8     Completely unable 5.6 20.7 43.2 59.2     Total 100.0 100.0 100.0 100.0 Transferring (n = 3,834)*     Some difficulty 78.8 50.0 48.8 20.1     A lot of difficulty 19.9 37.1 24.4 29.2     Completely unable 1.3 13.0 26.8 50.7     Total 100.0 100.0 100.0 100.0 Walking (n = 7,051)*     Some difficulty 66.0 39.7 39.3 15.7     A lot of difficulty 29.7 41.3 34.7 36.7     Completely unable 4.2 19.1 26.0 47.6     Total 100.0 100.0 100.0 100.0 Getting outside (n = 3,542)*     Some difficulty 66.0 37.5 28.1 11.3     A lot of difficulty 27.1 37.1 30.7 30.8     Completely unable 6.9 25.4 41.3 58.0     Total 100.0 100.0 100.0 100.0 Notes: Denominators are in parentheses. AT = assistive technology; PC = personal care. *χ2 test significant at p <.05. Table 3. Residual Difficulty on Specific Dimensions by Global Level of Underlying Difficulty and Type of Assistance. Type of Assistance Activity n AT Only PC Only PC and AT All With Underlying Difficulty Bathing     Very tiring*     (695) 74.9 55.1 66.0     Long time* (709) 83.0 49.4 62.8     Very painful, ns (490) 70.2 62.5 67.0 Transferring     Very tiring* (694) 70.1 56.0 59.3     Long time* (785) 76.2 57.0 57.3     Very painful, ns (801) 82.4 79.1 76.9 Walking     Very tiring, ns (1877) 80.1 77.0 76.6     Long time, ns (1892) 83.0 78.9 78.5     Very painful, ns (1531) 82.1 79.7 83.0 Getting outside     Very tiring* (760) 86.7 78.2 77.2     Long time* (766) 88.9 76.0 79.3     Very painful, ns (617) 86.9 85.7 79.7 Those With a Lot of Underlying Difficulty Bathing     Very tiring* (317) 76.3 60.2 70.3     Long time* (332) 85.7 60.9 64.0     Very painful, ns (222) 70.7 71.7 68.8 Transferring     Very tiring, ns (326) 75.1 68.6 66.5     Long time, ns (350) 78.7 72.1 63.7     Very painful, ns (336) 87.6 88.4 84.9 Walking     Very tiring, ns (1009) 85.4 87.1 82.7     Long time, ns (1029) 87.2 86.6 85.8     Very painful, ns (870) 85.1 87.0 86.1 Getting outside     Very tiring, ns (410) 90.7 87.2 84.3     Long time, ns (419) 87.5 85.3 85.1     Very painful, ns (346) 86.3 85.6 83.5 Notes: Reported information is for those who use some type of assistance. 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The Gerontologist,. 33:168-176. National Center for Health Statistics., 1998;. 1994 National Health Interview Survey on Disability, Phase I and II [CD-ROM Series 10, No. 8A]. Hyattsville, MD: U. S. Department of Health and Human Services. National Center for Health Statistics., 1998;. 1995 National Health Interview Survey on Disability, Phase I and II [CD-ROM Series 10, No. 10A]. Hyattsville, MD: U. S. Department of Health and Human Services. Norburn, J. E. K., Bernard, S., Konrad, T., Woomert, A., DeFriese, G., Kalsbeek, W., et al 1995;. Self-care and assistance from others in coping with functional status limitations among a national sample of older adults. Journal of Gerontology: Social Sciences,. 50B:S101-S109. Pope, A. M., Tarlov, A., (Eds.) 1991;. Disability in America: Toward a national agenda for prevention. Washington, DC: National Academy Press. Russell, J. N., Hendershot, G. E., LeClere, F., Howie, L. J., Adler, M., 1997;. Trends and differential use of assistive technology devices: United States, 1994 (Advance Data No. 292). Washington, DC: National Center for Health Statistics. Sanford, J. A., Arch, M., Megrew, M. B., 1995;. An evaluation of grab bars to meet the needs of elderly people. Assistive Technology,. 7:36-47. Schoeni, R., Freedman, V. A., Wallace, R., 2001;. Persistent, consistent, widespread, and robust? Another look at recent trends in old-age disability. Journal of Gerontology: Social Sciences,. 56B:S206-S218. Soldo, B. J., Freedman, V. A., 1994;. Care of the elderly: Division of labor among the family, the market, and the state. In S. Preston & L. G. Martin (Eds.), The demography of aging. Washington, DC: National Academy Press. Steinfeld, E., Shea, S. M., 1995;. Enabling home environments: Strategies for aging in place. Paper presented at the 48th Annual Scientific Meeting of The Gerontological Society of America, Los Angeles, CA. Tamm, M., 1999;. What does a home mean and when does it cease to be a home? Home as a setting for rehabilitation and care. Disability and Rehabilitation,. 21:49-55. Tennstedt, S., McKinlay, J., Kasten, L., 1994;. Unmet need among disabled elders: A problem in access to community long term care? Social Science and Medicine,. 38:915-924. Verbrugge, L. M., Jette, A. M., 1994;. The disablement process. Social Science and Medicine,. 38:1-14. Verbrugge, L., Rennert, C., Madans, J., 1997;. The great efficacy of personal and equipment assistance in reducing disability. American Journal of Public Health,. 87:384-392. Zimmer, Z., Chappell, N., 1994;. Mobility restriction and the use of devices among seniors. Journal of Aging and Health,. 6:(2), 185-208. Zimmer, Z., Chappell, N., 1999;. Receptivity to new technology among older adults. Disability and Rehabilitation,. 21:(5/6), 222-230. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B1F3500F68FAC8AFFECC0A2A4E4F4E5326FEED7.txt b/test/dataset/in/resources/corpus/Clean_0B1F3500F68FAC8AFFECC0A2A4E4F4E5326FEED7.txt new file mode 100644 index 0000000..0789cc3 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B1F3500F68FAC8AFFECC0A2A4E4F4E5326FEED7.txt @@ -0,0 +1 @@ +]>EXG5379S0531-5565(00)00189-310.1016/S0531-5565(00)00189-3Elsevier Science Inc.Fig. 1Net percent gain in muscle weights of young and old rats after 4weeks of remobilisation. Data are expressed as means ±SD (n=5). ∗p<0.05 for muscles of old rats versus young rats.Fig. 2Net percent change of biochemical parameters in gastrocnemius muscles of young and old rats after 4weeks of remobilisation. CPK, creatine phosphokinase; ACP, acid phosphatase; LPX, lipid peroxidation. Data are expressed as means ±SD (n=5). ∗p<0.05 for biochemical data of old versus young muscles.Fig. 3Transverse sections of gastrocnemius muscle (frozen sections stained Hematoxylin and Eosin). (a) left control (contralateral) leg of young (6months old) rat; (b) left control (contralateral) leg of old (26months old) rat; (c) right immobilised leg of old rat (4weeks of external fixation); (d) right leg of old rat (4weeks of external fixation, 4weeks of remobilisation). The external fixation causes major atrophic changes in myofibres, which only manage partial recovery on remobilisation.Fig. 4Details of myofibres of gastrocnemius muscle of old rats after 4weeks of external fixation (a, b); and after 4weeks of external fixation followed by 4weeks of remobilisation (c, d). (a) Transverse section showing myofibre degeneration. Many myofibres develop small vacuoles. (b) Ultrastructural details of myofibre showing sarcomere disruption and loss of myofilaments. (c) Myopathic damage is still clearly evident and there is only limited recovery 4weeks after removal of the external fixation. (d) In some cases lysosome-like bodies are prominent in myofibers, which in many cases remain vacuolated. (a, c, d, 1μm epon sections stained alkaline toluidine blue; b, transmission electron micrograph)Table 1The mean (±SD) body-weight of old rats before and after 4weeks of external fixation and before and after an additional 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationBeforeAfterBeforeAfterBody-weight (g)398±69.8373.8±62.7428.3±63.3450±57Mean difference (g)−2521.795% confidence interval (CI) of difference−127.76 to 71.76−66.15 to 109.55Change (%)−6.1%5.14%p ValueNSNSTable 2The mean (±SD) change in the weight (mg) of the hindlimb muscles in old rats after 4weeks of external fixation and 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationMuscleContralateral legImmobilised legMean difference95% CI of differenceChange (%)Contralateral legImmobilised legMean difference95% CI of differenceChange (%)Gastrocnemius1556±204914±129−642−891 to −393−41.27*1510±1671073±118−437−648 to −226−28.9*Quadriceps2172±1001221±88−951−1088 to −814−43.77**2314±1261541±139−773−996 to −579−33.4*Plantaris231±11121±11−110−126 to −94−46.6**250±17144±9>−106−126 to −86−42.3*Soleus171±16103±20−6.8−94 to −42−39.5*166±15104±1−62−77 to −46−37.3**p<0.001.**p<0.01, contralateral control v immobilised legs.Table 3Mean (±SD) values for the activity of ACP, CPK and lipid peroxidation in the gastrocnemius muscle of old rats after 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationMuscleContralateral legImmobilised legMean difference95% CI of difference (%)ChangeContralateral legImmobilised legMean difference95% CI of differenceChange (%)ACP activity (mU/mg)a28.6±1.9538.1±69.472.96 to 15.97+33.229.5±1.8732.6±1.763.10.45 to 5.75+10.7p valuep<0.02p<0.02CPK activity (U/mg)a4.5±0.793.73±0.78−0.77−1.91 to 0.37−17.24.17±0.413.63±0.31−0.54−1.07 to −0.01−12.6p valuep<0.01Lipid peroxidation (nmol MDA/mg protein)b4.13±16.61±0.352.481.38 to 3.57+60.04.04±1.75.26±1.71.22−1.26 to 3.70+30p valuep<0.02NSaActivity of enzymes is expressed per mg of soluble proteins in the supernatant.bMDA, malondialdehyde.Table 4Comparison between young and old rats in their capacities for recovery of hindlimb muscle weights after 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationYounga % weight lossOld % weight lossYounga % weight lossOld % weight lossYoung net % gainOld net % gainYoung/Old relative % gainGastrocnemius−58−41.27−28.9−28.929.912.42.41Quadriceps−62−43.77−42.7−33.419.310.31.87Plantaris−46.5−46.6−35.7−42.310.94.32.53Soleus−46.6−39.5−41.0−37.35.62.22.54aThe data for the young animals are taken from a recent publication (Zarzhevsky et al., 1999).Table 5Comparison between young and old rats in their capacities for recovery of biochemical parameters (% change) after 4weeks of external fixation followed by 4weeks of remobilisation (n=5)External fixationExternal fixation plus remobilisationYoungaOldYoungaOldYoung net % gainOld net % gainYoung/Old relative % gainACP activity+82.4+33.2−7.3−10.789.745.91.95CPK−36.5−17.2+2.9−12.639.44.68.56Lipid peroxidation+132.5+60.0+32.4+30.0100.130.03.33aThe data for the young animals are taken from a recent publication (Zarzhevsky et al., 1999).Recovery of muscles of old rats after hindlimb immobilisation by external fixation is impaired compared with those of young ratsNZarzhevskyaECarmelibDFuchscRColemanaHSteindA.ZReznicka*reznick@tx.technion.ac.ilaDepartment of Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096, IsraelbPhysical Therapy Program, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, IsraelcDepartment of Orthopaedics, Bnai Zion Medical Center, Haifa 31408, IsraeldDeparment of Orthopaedics A, Rambam Medical Center, Haifa 31096, Israel*Corresponding author. Tel.: +972-4-829-5388; fax: +972-4-829-5392AbstractThe right hindlimbs of 24-month-old female Wistar rats were immobilised for 4weeks using external fixation of the knee joint. In a further group, after the external fixation was removed, the rats were allowed to remobilise for an additional 4weeks. Hindlimb immobilisation for 4weeks caused a 32–42% reduction in wet weights of the hindlimb muscles of the rats as compared to those of the contralateral non-immobilised legs. After 4weeks of remobilisation the hindlimb muscles had not returned to the “control” weights. Biochemical changes in the gastrocnemius muscle resulting from the external fixation showed greatly elevated acid phosphatase activities (33.2%) and markedly reduced creatine phosphokinase activities (17.2%), which did not recover to preimmobilisation values after 4weeks of remobilisation. Light and transmission electron microscopy showed that remobilisation for 4weeks (after external fixation) resulted in only partial morphological restoration of the damage to the muscles in these aged rats. A comparison of similar hindlimb external fixation and remobilisation in young (6months old) rats showed that remobilisation caused a substantial recovery in biochemical parameters in both age groups, with the muscles of the young group (but not the old group) often reaching almost complete recovery accompanied by morphological restoration. We conclude that the net gain in the recovery period of biochemical and morphological parameters is significantly greater in the young rats compared to the old rats indicating that muscle metabolism and capacity for recovery from disuse atrophy is impaired with ageing.KeywordsDisuse atrophySkeletal muscleExternal fixationRemobilisationAgeingRats1IntroductionAs early as 1944, the cause-and-effect relationship of limb immobilisation and disuse atrophy was recognised (Eccles, 1944; Thomsen and Luco, 1944). Inactivity, bed rest, non-weight bearing, external casts or external fixation all result in loss of muscle mass or muscular atrophy. Atrophy is associated with a net loss of muscle protein synthesis initiated within hours of immobilisation (Goldspink, 1977; Booth and Seider, 1979). The initial atrophy is very rapid, especially within the first week of immobilisation (Booth, 1977; Maxwell et al., 1992; Appell, 1990). Recently a comparison of two different modes of hindlimb immobilisation (Plaster of Paris casts and external fixation) was reported (Reznick et al., 1995). In these studies several biochemical and morphological parameters were investigated as a measure of damage to muscles of immobilised limbs. It was shown that external fixation, with its more rigid immobilisation, resulted in more drastic changes and damage to the hindlimb muscles (Reznick et al., 1995).The capacity for recovery after immobilisation has been little investigated, though it is of the utmost clinical importance as the orthopaedic surgeon needs to decide at what stage the cast or external fixation should be removed in order to achieve the best outcome of the operation. Common clinical experience reveals that muscle recovery following limb immobilisation is more rapid and complete in young individuals rather than in the elderly. Relatively little is known about the effects of muscle hypoactivity in old age (Ansved, 1995). Our working hypothesis was that muscles of old animals will be affected more severely by immobilisation and will be slower to recover compared with muscles of young animals. We have recently reported experimental studies on the capacity for recovery of hindlimb muscles of young (6month old) rats after external fixation (Zarzhevsky et al., 1999). In these studies, 4weeks of remobilisation resulted in the restoration of biochemical and morphological parameters to preimmobilisation values, whereas muscle mass was still lower than preimmobilisation values. In the present study we report similar experiments conducted in aged rats (24months old) and evaluate various biochemical and morphological parameters as indicators of the capacity for recovery from disuse atrophy in these old animals.2Materials and methods2.1Animals15 female Wistar rats (24months old) were housed in standard plastic breeding cages, in groups of three, at 20°C with a 12-h light–dark cycle. They were fed a standard diet and water ad libitum. The animals were maintained in conformity with the Guiding Principles in the Care and Use of Animals of the American Physiological Society and the experimental protocol received approval by the Animal Welfare and Ethics Committee of the Technion Faculty of Medicine.The animals were divided into three groups each of five rats. Group 1 consisted of rats in which the right knee was immobilised by external fixation in a position of 40–50° flexion for 4weeks (Reznick et al., 1995). Group 2 had similar hindlimb immobilisation for 4weeks after which the external fixation was removed and the limb remobilised for a further 4weeks. Group 3 consisted of untreated age-matched animals.2.2Immobilisation by external fixation (Reznick et al., 1995)Rigid immobilisation was achieved by the insertion of two Kirschner wires (0.8mm diameter) driven in pairs through the lateral plane of the femur and tibia. Then, two threaded brass rods fitted with nuts, connected them in order to construct a rigid frame. The brass rods were 4.8mm diameter and 33mm long. Each rod was cut longitudinally from both ends to an equal length of 13mm. These cuts were 1.0–1.2mm in width in order to contain the Kirschner wires. The overall weight of the above device was 12g.2.3Biochemical studiesAnimals were sacrificed by ether anesthesia at 4weeks for Group 1 and 8weeks for Group 2. Group 3 (untreated controls) were sacrificed at 24months of age prior to the start of the experimental hindlimb immobilisation. Muscle specimens for biochemical studies (200mg each) were taken from the belly of the gastrocnemius and quadriceps muscles of the right (immobilised) and left (contralateral) hindlimbs. (The soleus and plantaris muscles weigh less than 200mg and are insufficient for biochemical analysis). The muscles were immersed in 1.4ml of 50mM tris(hydroxymethyl) aminomethane buffer, pH 7.4. The mixture was homogenised in a Polytron homogeniser (Kinematica GmbH, Lucerne, Switzerland) three times for 15s. The homogenate was centrifuged for 30min at 14,000g. The supernatant was separated and utilised for enzymatic assays. Acid phosphatase activities were determined as previously described (Reznick et al., 1995). Creatine phosphokinase assay was performed according to the method of Tanzar and Gilvard (1959) using 0.05M glycine buffer, pH 7.4. Lipid peroxidation measurements were performed using thiobarbituric acid (TBA) according to the procedure of Ohkawa et al. (1979). Protein concentration in muscle extracts was determined according to the method of Lowry et al. (1951).2.4Light and electron microscopyAt sacrifice, the gastrocnemius muscle was rapidly removed. The belly of the gastrocnemius muscle was rapidly frozen in isobutane cooled by liquid nitrogen. 6μm cryostat transverse sections were stained with Hematoxylin and Eosin (H and E). For higher resolution sections, a 2mm-thick transverse slice of the fresh gastrocnemius muscle was cut with a razor blade from a site approximately 3mm distant from the tendon of origin and immersion fixed in 3% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4 containing 0.01% calcium chloride at room temperature. Blocks of tissue approximately 1mm3 were cut and left in the fixative for 3h prior to rinsing and storage overnight in 0.1M sodium cacodylate buffer containing 7.5% (wt/vol.) sucrose. This was followed by post-fixation in 1% unbuffered aqueous osmium tetroxide for 2h, dehydration in ascending ethanols, treatment with propylene oxide and epoxy embedding in Pelco Eponate 12 (Pelco International, Redding, CA, USA). After heat polymerisation (60°C for 18h), 1μm thick sections were cut on an ultramicrotome and stained with 0.1% toluidine blue in 1% borax for light microscopy. Sections (60–90nm thick) were cut with a diamond knife on the ultramicrotome, collected on uncoated copper grids, and contrast-stained with 1% uranyl acetate in 70% ethanol followed by 1% lead citrate prior to examination in a JEOL 100SX transmission electron microscope at 80kV.2.5Statistical analysisStatistical analysis was performed using either paired or independent Student's t-test. Statistical significance was set at p<0.05. Results are reported as ±SD.3Results3.1Body weightsThe body weights of animals with hindlimb immobilisation by external fixation for 4weeks and remobilisation are shown in Table 1. After 4weeks of immobilisation (Group 1), the old animals lost 6.1%, which was not significant. On remobilisation for 4weeks (Group 2) the rats had made a complete recovery of body weights and even showed a slight increase (5.14%). The differences between body weights at the start of the experiment and following external fixation and remobilisation were not significant.3.2Muscle weightsTable 2 shows the weights of the four muscles studied in the old animals after 4weeks of external fixation and after further 4weeks of remobilisation. The weights of the gastrocnemius, quadriceps, plantaris and soleus muscles were reduced by 41.2, 43.7, 46.6 and 39.5%, respectively. Following remobilisation the weights of these muscles were still much reduced (28.0, 33.4, 42.3 and 37.3%, respectively) compared with those of the contralateral legs, which was still statistically significant (p<0.001).3.3Biochemical studiesThe changes in enzymatic activities of acid phosphatase (ACP), creatine phosphokinase (CPK) and lipid peroxidation of gastrocnemius muscle in old rats after external fixation and remobilisation are shown in Table 3. Immobilisation for 4weeks caused a 33.2% increase in acid phosphatase activities, whereas there was a decrease of 17.2% in CPK activities. Lipid peroxidation increased by 60% as measured by the TBA assay. These changes were significant for acid phosphatase (p<0.02), lipid peroxidation (p<0.02) and CPK activities (p<0.01). On remobilisation for 4weeks, activities of acid phosphatase and CPK remained significantly lower than the control values indicating an incomplete recovery of enzymatic activities after 4weeks of remobilisation. Lipid peroxidation values recovered on remobilisation and were no longer significantly different from the contralateral control values.3.4Comparative age-associated studiesTable 4 presents a comparison of changes in hindlimb muscle weights of young and aged rats after external fixation and remobilisation. As a result of hindlimb immobilisation in the young rats the larger muscles, quadriceps and gastrocnemius, lose about 60% of their muscle weight, but in the aged rats these muscles lose only 41–43%. The weight loss in the smaller muscles, plantaris and soleus, was similar in both age groups. After 4weeks of remobilisation, none of the muscles of both age groups recovered to preimmobilisation weights. If values are expressed as net % gain (the difference between maximum loss in % after 4weeks of EF and the percentage weight loss after 4weeks of remobilisation) the values for the young animals are much higher than those of the aged animals. The relative % gain as calculated from the ratio young: old in the net % gain for the gastrocnemius, quadriceps, plantaris and soleus muscles was 2.41, 1.87, 2.53 and 2.54, respectively. The net % gain of both young and old animals is illustrated graphically in Fig. 1.Table 5 presents a comparison of the capacity of enzyme activities (acid phosphatase, creatine phosphokinase) and lipid peroxidation in the gastrocnemius muscle of young and old rats to recover after hindlimb immobilisation and remobilisation. After 4weeks of remobilisation in young rats the % change of acid phosphatase, CPK and lipid peroxidation was −7.3, +2.9 and +32.4%, respectively, which was not significantly different from that of the contralateral control leg. The % change for these same parameters in the old rats was −10.7, −12.6 and +30.0, respectively, which was significant for both acid phosphatase and CPK.Hence, it was possible to conclude that the net % change for young animals was much more pronounced for all the biochemical parameters studied compared with the values for the old animals. The net % change in biochemical parameters is illustrated graphically in Fig. 2. It is clear that in all categories of muscles, net % weight gain and biochemical differences, the changes of muscles of the young animals were much more pronounced than in the muscle of old animals.3.5Morphological studiesThe morphological studies were performed on gastrocnemius muscle of the aged rats. Frozen sections (Fig. 3a and b) show that the morphological appearance of myofibres is fairly similar in gastrocnemius muscles of control legs in both the young (6months old) and old (26months old) rats. Fig. 3c shows that 4weeks of external fixation in the old rats causes marked myopathic changes with the myofibres becoming markedly shrunken and distorted. On remobilisation after external fixation (Fig. 3d) there is only a partial structural recovery of the fibres. Fig. 4a shows greater detail of the myopathic changes after external fixation. Many myofibres show marked degeneration and numerous small vacuoles are seen in myofibres. At the ultrastructural level (Fig. 4b) the myopathic changes are clearly visible including sarcomere disruption and myofilament loss. After remobilisation (Fig. 4c and d), myopathic damage is still clearly evident and there is only limited recovery of myofibre structure. Many myofibres remain swollen and distorted. Sarcomeres in many myofibres remain irregular and distorted. Many myofibres still are highly vacuolated and others contain large numbers of lipid droplets. Some myofibres develop large numbers of lysosome-like bodies (Fig. 4d). Rounded cells with large regular nuclei and pronounced nucleoli (possibly activated satellite cells), as well as inflammatory cells and adipose cells are common in the connective tissue between the myofibres.4DiscussionExternal fixation is a highly effective and widely used method for bone and joint immobilisation that requires the invasive use of pins through bone and muscle. In the present study we showed that the body weights of aged rats subjected to unilateral external fixation of a hindlimb were not changed significantly as a result of the limb immobilisation or after subsequent remobilisation. We also demonstrated that none of the muscles studied from the immobilised limb were capable of returning to preimmobilisation weights or to the weights of the non-immobilised contralateral muscles after 4weeks of remobilisation. This interesting observation confirms that of Maeda et al. (1993), who wrote: “Both muscle and bone require a time period longer than the period of immobilisation in order to make a complete recovery from temporary deterioration”. Similar observations were reached by Booth (1978), who showed that gastrocnemius muscle of rats immobilised in plaster-of-Paris casts for 28days returned to preimmobilisation weight only 50days following the cast removal. However, in another study of 90 day immobilisation of rat hindlimbs, the soleus muscle weight was shown to return to control values 14days after the start of the recovery period (Booth and Seider, 1979). It should be noted that the loss of muscle weight due to immobilisation does not occur as a result of changes in muscle water content, as it was previously shown that the ratio of muscle dry weight to muscle wet weight remains the same for immobilised and control legs (Reznick et al., 1995).Alkaline phosphatase, acid phosphatase and CPK activities have been shown to change considerably after limb immobilisation (Witzmann et al., 1982). Using acid phosphatase and CPK activities as criteria for normal levels of muscle function and metabolism, we have shown that hindlimb immobilisation in old rats altered the level of activities of these enzymes in gastrocnemius muscles, however, after 4weeks of remobilisation, the levels of acid phosphatase and CPK have still not returned to pre-immobilisation levels. Lipid peroxidation was shown to increase considerably after 4weeks of limb immobilisation but on remobilisation recovered by 30% to values that were not statistically significantly different from the contralateral controls.In comparing the recovery responses of young rats to aged rats after external fixation of hindlimbs we have shown that the relative % change of muscle weights and of biochemical parameters is by far greater in the young animals. This may indicate that the capacity for active metabolism and turnover of muscle protein is indeed far greater in young animals than in the aged animals. It would appear that the older animals need much longer times to recover from the stress and disuse atrophy of limb immobilisation than the younger animals and this reflects common clinical experience on traumatic healing of muscle and bone in the elderly.These conclusions are confirmed by the morphological studies. We recently showed that the gastrocnemius muscle of young rats subjected to hindlimb immobilisation by external fixation shows an excellent structural recovery after 4weeks of remobilisation (Zarzhevsky et al., 1999). In contrast, the present study has shown that under similar experimental circumstances, the gastrocnemius muscle of old rats shows only a very limited structural recovery in this time period. We are led to conclude that skeletal muscles of hindlimbs of aged rats show very limited recovery processes after disuse atrophy compared with their younger counterparts.As can be seen from Table 4, % weight loss of gastrocnemius and quadriceps muscles of immobilised hindlimbs was higher in young animals compared to old ones. However, the % weight loss was similar for plantaris and soleus muscles for both age groups (Table 4). Indeed it appears that the larger muscles, such as the gastrocnemius and quadriceps, are undergoing a faster rate of catabolism in young animals compared to old ones. This phenomenon of reduction in the rate of protein breakdown in tissues of old animals compared to young ones has been observed in the past. A recent paper has shown using 3-methylhistidine excretion, that whole body muscle protein catabolism is slower in elderly humans compared to younger ones, by as much as 30–40% (Morais et al., 1997). Similarly, various subcellular fractions of proteins, as well as total proteins, are degraded more slowly in livers of old mice compared to livers of young mice (Lavie et al., 1982). A very recent study has shown that chaperone-mediated autophagy of lysosomes is reduced in livers of ageing rats as compared with young rats (Cuervo and Dice, 2000). Thus it is not unique that muscles of old animals in our experimental model were degraded to a lesser extent that those of the younger animals.The capacity of muscle recovery from muscle degeneration following bupivacaine injection in aging rats was studied by Marsh et al (1997a,b). The mass of tibialis anterior muscle was restored to control values within 21days in young rats (3months old), whereas in adult (18months old) and old rats (31months old) it remained 40% less than that of the contralateral controls at 21 and 28days of recovery. Furthermore, expression of myogenin and myoD mRNA levels increased in regenerated muscles of young, adult and old rats at 5 and 14days, respectively. Whereas in the young (3months old) rats mRNA levels returned to preinjection control values by 21days, in old animals they remained elevated even at 28days of recovery. It appears that either old animals have diminished capacities to down-regulate myogenin and MyoD mRNAs, or possibly their reduced ability for myogenic effect includes elevated levels of these mRNAs (Marsh et al., 1997a). Similar results were also obtained with IGF-1 mRNA, where impaired regeneration of the tibialis anterior was associated with a prolonged elevation of IGF-1 mRNA expression in the old muscle (Marsh et al., 1997b). Since the turnover of muscle proteins (synthesis and degradation) appears to slow down with age, it will be of great interest in future studies to investigate the molecular events behind the differences between young and old animals. Understanding these mechanisms in old animals may provide some clues as to the general phenomenon of sarcopenia of old age, and ways to alleviate some of the associated clinical problems.AcknowledgementsThis research was supported in part by a grant from the Jan M. and Eugenia Krol Foundation, Lakewood, NJ, USA, and by grant #181-883 from the Technion, Vice-Provost for Research. We would also like to acknowledge the excellent technical help of Mrs Pessiah Shenzer.ReferencesAnsved, 1995T.AnsvedEffects of immobilization on the rat soleus muscle in relation to ageActa Physiol. Scand.1541995291302Appell, 1990H.J.AppellMuscular atrophy following immobilizationSports Med.1019904252Booth, 1977F.W.BoothTime course of muscular atrophy during immobilization of hindlimbs in ratsJ. Appl. Physiol.431977656661Booth, 1978F.W.BoothRegrowth of atrophied skeletal muscle in adult rats after ending immobilizationJ. Appl. Physiol.441978225230Booth and Seider, 1979F.W.BoothM.J.SeiderRecovery of skeletal muscle after 3months of hindlimb immobilization in ratsJ. Appl. Physiol.471979435439Cuervo and Dice, 2000A.M.CuervoJ.F.DiceAge-related decline in chaperone-mediated autophagyJ. Biol. Chem.2000(in press)Eccles, 1944J.C.EcclesInvestigations on muscle atrophies arising from disuse and tenotomyJ. Physiol. (Lond)1031944253256Goldspink, 1977D.F.GoldspinkThe influence of immobilization and stretch on protein turnover of rat skeletal muscleJ. Physiol. (Lond)2641977267282Lavie et al., 1982L.LavieA.Z.ReznickD.GershonDecreased protein and puromycinyl-peptide degradation in livers of senescent miceBiochem. J.20219824751Lowry et al., 1951O.H.LowryN.J.RosebroughA.L.FarrR.J.RandallProtein measurement with the Folin phenol reagentJ. Biol. Chem.1931951265275Maeda et al., 1993H.MaedaD.B.KimmelD.M.RaabN.E.LaneMusculoskeletal recovery following hindlimb immobilization in adult female ratsBone141993153159Marsh et al., 1997aD.R.MarshD.S.CriswellJ.A.CarsonF.W.BoothMyogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old ratsJ. Appl. Physiol.83199712701275Marsh et al., 1997bD.R.MarshD.S.CriswellM.T.HamiltonF.W.BoothAssociation of insulin-like growth factor mRNA expressions with muscle regeneration in young, adult, and old ratsAm. J. Physiol.2731997R353R358Maxwell et al., 1992L.C.MaxwellM.R.MoodyC.S.EnwemekaMuscle atrophy continues after early cast removal following tendon repairAnat. Rec.2331992376386Morais et al., 1997J.A.MoraisR.GougeonP.B.PencharzP.J.H.JonesR.RossE.B.MarlissAm. J. Clin. Nutr.661997880889Ohkawa et al., 1979H.OhkawaN.OhishiK.YagiAssay for lipid peroxides in animal tissues by thiobarbituric acid reactionAnal. Biochem.951979351358Reznick et al., 1995A.Z.ReznickG.VolpinH.Ben-AriM.SilbermannH.SteinBiochemical and morphological studies on rat skeletal muscles following prolonged immobilization of the knee joint by external fixation and plaster cast: a comparative studyEur. J. Exp. Musculosketal Res.419956976Tanzar and Gilvard, 1959M.L.TanzarC.GilvardCreatine kinase measurementJ. Biol. Chem.234195932013204Thomsen and Luco, 1944P.ThomsenJ.V.LucoChanges of weight and neuromuscular transmission in muscles of immobilized jointJ. Neurophysiol.71944241245Witzmann et al., 1982F.A.WitzmannA.J.TroupR.H.FittsAcid phosphatase and protease activities in immobilized rat skeletal musclesCan. J. Physiol. Pharmacol.60198217321736Zarzhevsky et al., 1999N.ZarzhevskyR.ColemanG.VolpinD.FuchsH.SteinA.Z.ReznickMuscle recovery after immobilisation by external fixationJ. Bone Jt. Surg. Br.81-B1999896901 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B2A2F56C3B4690B89D311653C95EAD549C2829B.txt b/test/dataset/in/resources/corpus/Clean_0B2A2F56C3B4690B89D311653C95EAD549C2829B.txt new file mode 100644 index 0000000..0bbea45 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B2A2F56C3B4690B89D311653C95EAD549C2829B.txt @@ -0,0 +1 @@ +]>MAD2077S0047-6374(98)00042-610.1016/S0047-6374(98)00042-6Elsevier Science Ireland LtdFig. 1The inhibition kinetics of DNP-SG transport elicited by various inhibitors and effects of increasing concentrations of inhibitors on CumOOH-induced MDA formation in erythrocytes. Suspensions of erythrocytes from aged (n=5) or young (n=5) subjects were treated with increasing concentrations of Tween 80 (A), fluoride (B) or vanadate (C) as described in Section 2. After centrifugation the supernatants were assayed for DNP-SG transport. MDA levels of erythrocytes treated with 0.1 mmol/l CumOOH after pretreatment with inhibitors were also determined. Percent inhibition was expressed as the percentage decrease of DNP-SG efflux from erythrocytes. The values are means of duplicate analyses from 10 experiments.Fig. 2Effects of various inhibitors of the glutathione conjugate pump on CumOOH-induced lipid peroxidation in erythrocytes from healthy aging (n=18) and young (n=18) adults. DNP-SG inhibitors, Tween 80 (1/1200), fluoride (30 mmol/l) or o-vanadate (0.2 mmol/l) were added before the erythrocyte suspensions were incubated with 0.1 mmol/l CumOOH for 15 min at 37°C. MDA values shown are means with standard deviations.Fig. 3(See left) Effects of the addition of CumOOH to the reaction medium following preincubation with various inhibitors of glutathione conjugate pump on the changes in chemiluminescence. (A) Chemiluminescence response of erythrocytes treated with 0.1 mmol/l CumOOH following the inhibition of DNP-SG transport of erythrocytes from aging group. (B) Comparison of the chemiluminescence response in erythrocytes from aging and young adults. DNP-SG transport inhibitors, Tween 80, fluoride or o-vanadate were added to erythrocyte suspensions before mixing with 0.1 mmol/l CumOOH and chemiluminescence was recorded at indicated time. Data represent the mean of 16 determinations in the elderly group and 15 determinations in the young group.Table 1Transport of 2,4-dinitrophenyl-S-glutathione in erythrocytes from aging and young subjectsTime (h)Erythrocyte efflux (nmol DNP-SG/ml erythrocytes)Aging group (n=18)Young group (n=18)1410±77390±422685±117674±713971±128933±11441174±1651118±157All data expressed as mean±S.D.Transport of glutathione conjugate in erythrocytes from aged subjects and susceptibility to oxidative stress following inhibition of the glutathione S-conjugate pumpİlhanOnarana*AhmetÖzaydinaMustafaGültepebGönülSultuybekaaDivision of Biomedical Sciences, Cerrahpaşa Medical Faculty, Istanbul University, Istanbul, TurkeybGATA, Military Educational Hospital, Department of Biochemistry, Istanbul, Turkey*Corresponding author. Present address: Ortaklar Cd. Bütan Sk., Hyzal Apt. No:2 D:3, Mecidiyeköy 80290, İstanbul, Turkey. Fax: +90 212 5299433.AbstractThe aim of the present study was to investigate the effect of donor aging on the glutathione conjugate transport in erythrocytes and whether it plays a role in the resistance to oxidative stress of the erythrocytes of aging subjects. In our comparative study on intact erythrocytes of healthy aging and young adults, in which 2,4-dinitrophenyl-S-glutathione (DNP-SG) was used as model glutathione S-conjugate, we found that the efflux of DNP-SG remained unchanged in the aged subjects. This result suggests that the detoxification function is maintained against the chemical stress employed in erythrocytes of aging subjects. In the assay conditions used, which were optimized to obtain maximal inhibition of glutathione S-conjugate transport, our results also indicated that the susceptibility of erythrocytes to in vitro lipid peroxidation generated by cumene hydroperoxide was enhanced by pretreatment with DNP-SG inhibitors in both age groups. However, the difference in susceptibility was not a function of aging. Further, the results suggested that inhibition of glutathione S-conjugate pump may impair cellular protection of the erythrocytes against oxidative damage.KeywordsAgingErythrocyteGlutathione S-conjugateGlutathioneTransportOxidative stress1IntroductionTransport of glutathione S-conjugates is an important element of xenobiotic detoxification. Glutathione is enzymatically conjugated to many electrophilic compounds in a reaction catalyzed by glutathione S-transferases (GST). The glutathione S-conjugates formed in these reactions are afterwards exported out of cells by a specific adenosine 5′-triphosphate (ATP)-dependent glutathione S-conjugate pump (for review see Zimniak and Awasthi, 1993). The active transport systems for glutathione S-conjugates have been described for a number of human tissues and this system has also been identified in human erythrocyte membranes (Awasthi et al. 1983, Ishikawa and Sies 1984, La Belle et al. 1986, Kunst et al. 1989). The glutathione S-conjugate pump also plays a significant role in cellular defence under oxidative stress conditions since it is able to transport glutathione conjugates of lipid peroxidation products and extrudes oxidized glutathione (GSSG) (Ishikawa 1989, Grune et al. 1991, Akerboom et al. 1992). Erythrocytes are a good model system for the explanation of such processes and the molecular nature of the defect in the transport of glutathione conjugates since their lack of γ-glutamyltranspeptidase precludes further metabolizing of formed conjugates. Although the transport system exhibited a broad substrate specificity towards different types of glutathione S-conjugate (Ishikawa, 1989), the compound most widely used in such studies is 2,4-dinitrophenyl-S-glutathione (DNP-SG) formed from 1-chloro-2,4-dinitrobenzene (CDNB) in a reaction catalyzed by GST.Different detoxification mechanisms such as glutathione, GST, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase but not glutathione S-conjugate transport have been tested for aging process (Stohs et al. 1984, Jozwiak and Jasnowska 1985, Stohs and Lawson 1986, Al-Turk et al. 1987, Farooqui et al. 1987, Foldes et al. 1988, Matsubara and Machado 1991, Picot et al. 1992). Results from a number of studies have suggested that a glutathione-deficiency state is a general phenomenon in aging cells, since aging-specific decrease in glutathione levels was observed in all tissues including erythrocytes (Stohs et al. 1984, Stohs and Lawson 1986, Al-Turk et al. 1987, Farooqui et al. 1987, Matsubara and Machado 1991). Furthermore, different results showing decreased or unchanged GST activity in elderly subjects when compared with young subjects were also reported (Stohs et al. 1984, Al-Turk et al. 1987, Picot et al. 1992). On the other hand, results of our previous study indicated that the susceptibility of intact erythrocytes to in vitro oxidative stress by cumene hydroperoxide (CumOOH) in aging subjects is not much greater than young subjects (Onaran et al., 1997). The above observations prompted us to study how the transport of glutathione conjugate in erythrocytes, generated through the reactions of glutathione and GST, is affected by the aging process. We also wished to examine whether glutathione S-conjugate transport has any effect on the resistance to oxidative damage of the intact erythrocytes of aging subjects.2Materials and methods2.1SubjectsSubjects were young (aged 18–31) or elderly (aged 67–85) normal volunteers. All subjects were non-smokers and had normal blood counts, normal blood levels of urea, glucose, creatinine, albumin, alkaline phosphatase, lactate dehydrogenase and bilirubin, and no history of hematologic abnormality, recent infectious disease or significant medical illness. The participants were instructed not to take aspirin-like drugs for 2 weeks prior to blood sampling.2.2ReagentsAll reagents were of analytical quality whenever possible, obtained mainly from Sigma (St. Louis, MO) and Merck (Darmstadt, Germany).2.3Preparation of erythrocytesFresh heparinized fasting blood sample was centrifuged at 1500×g for 10 min. Erythrocytes were separated from plasma and buffy coat and then washed three times with 10 vol of phosphate buffered saline (PBS, pH 7.4). Erythrocytes containing chronologically young population rich with reticulocytes were removed by discontinuous Percoll density gradient essentially as described by Rennie et al. (1979). The gradient was built up in two layers containing Percoll with specific density values between 1.100 and 1.124 g/ml. At the end of centrifugation while the cells were collected at the interface of the Percoll above 1.124 g/ml, enriched fraction of reticulocytes above low density were removed. The erythrocytes were then washed twice with PBS.Hemoglobin (Hb) concentrations in the samples were determined with Drabkin's reagent as described by Beutler (1984). Reticulocyte count in the fraction was performed on glass slides after staining with 0.1% brilliant cresyl blue saline. The number of reticulocytes in the high density cell fractions studied did not show significant differences between elderly and young control groups.2.4Reaction systemsFor inhibition of glutathione conjugate transport, erythrocytes were incubated at hematocrit 10% in PBS (with 8 mmol/l glucose) containing one of the following reagents (15 min at 37°C with continuous shaking at 120 cycles/min): 5–30 mmol/l sodium fluoride; 0.025–0.2 mmol/l sodium o-vanadate; diluted 1/4000–1/1200 Tween 80 (polyoxyethylenesorbitan-monooleate). Controls were incubated with PBS alone. To check whether the glutathione conjugate transport was inhibited in erythrocyte suspensions the export of DNP-SG was measured. Hemolysis was also checked in samples and it was always below 1–2%. After preincubation with inhibitors, lipid peroxidation was induced by addition of freshly prepared CumOOH (final concentration, 0.1 mmol/l). Immediately after CumOOH addition, the tubes were sealed and incubated at 37°C with continuous shaking at 120 cycles/min. In parallel, control samples were incubated under the same conditions but without CumOOH. After 15 min, cell suspensions were centrifuged at 7000×g for 1 min to sediment the erythrocytes. Both pellet and supernatant were used for malondialdehyde (MDA) determination.2.5Measurement of the transport of glutathione S-conjugateTransport of DNP-SG was measured according to the procedure of Board (1981). Washed erythrocytes were resuspended in PBS and incubated with 1 mmol/l CDNB for 15 min at 37°C to form DNP-SG. Then the cells were washed of excess CDNB at 4°C and suspended at a hematocrit of 20% in the PBS (pH 7.4) containing 1 mmol/l MgCl2 and 8 mmol/l glucose. The cell suspensions were then incubated at 37°C and the export of DNP-SG was quantified by withdrawal of aliquots of the cell suspensions after 1, 2, 3, and 4 h. After centrifugation, DNP-SG was detected in the supernatant by spectrophotometric determination at 340 nm using an mmol/l extinction coefficient of 9.6.2.6Malondialdehyde (MDA) measurementThe concentration of MDA in the samples was measured with fluorometric determination improved by synchronous fluorescence (Conti et al. 1991, Onaran et al. 1997). For erythrocytes, 0.2 ml of packed cells were suspended in 0.8 ml PBS, 0.025 ml of 4% (w/v) butylated hydroxytoluene and 0.050 ml of 0.025 mmol/l disodium-ethylenediaminetetraacetic acid, and 0.5 ml of 30% trichloroacetic acid was then added. Tubes were vortexed and allowed to stand in ice for 2 h. After centrifugation, assay conditions were the same in both the supernatant from this step and the supernatant fraction from the erythrocyte suspension (Onaran et al., 1997). The level of MDA was expressed as total mmol MDA/g Hb.2.7Chemiluminescence assayChemiluminescence measurements were carried out in a TriCarb 1500 liquid scintillation analyzer in the single-photon count mode (Videla et al., 1984). Briefly, erythrocytes were resuspended in 3 ml of PBS containing 8 mmol/l glucose and preincubated with indicated concentrations of fluoride, vanadate or Tween 80 for 15 min at 37°C. The erythrocytes were transferred to scintillation vials. After the addition of CumOOH (final concentration, 0.1 mmol/l) the chemiluminescence measurements were taken every 10 min for 60 min. The zero time is the time of CumOOH addition.2.8Glutathione content and glutathione S-transferase (GST) activityThe concentration of glutathione was determined by the method of Tietze (1969). Total GST activity with CDNB as substrate was measured according to Habig et al. (1974).2.9Statistical analysisAll tests were performed in duplicate or triplicate samples, and the results were expressed as means±S.D. Statistical difference was determined by Student's t-test, with significance defined as P<0.05.3ResultsThe aging group (n=18) and young controls (n=18) used in this study showed no difference in their erythrocyte GST activity, 2.1±0.8 and 2.1±0.6 mmol of CDNB conjugated/min per g Hb, respectively (P>0.05). However, we found statistically significant lower glutathione levels in older group. The mean±S.D. values of glutathione were 5.24±0.69 and 6.59±0.71 mmol/g Hb for the elderly and controls, respectively (P<0.05).On incubation with CDNB at 37°C, glutathione is completely depleted in erythrocytes of the two age groups. Under these experimental conditions, the rate of DNP-SG efflux of the erythrocytes from aging subjects during 4 h did not differ from those of the young controls (P>0.05). The rate of transport of DNP-SG in both age groups was linear for a period of at least 4 h (Table 1).In order to find out whether glutathione S-conjugate transport has any effect on the resistance to oxidative stress of the erythrocytes of aging subjects, transport of erythrocytes was inhibited by incubation with various inhibitors, including sodium fluoride, vanadate or Tween 80. The transport process was, more markedly, inhibited as a function of the concentration of these inhibitors. Despite the differences in inhibition profiles of the transport rate of DNP-SG elicited by these agents, the inhibition kinetics of erythrocytes from aged subjects were not different from that of young control subjects. When the erythrocytes were exposed to 0.1 mmol/l CumOOH after preincubation with inhibitors, increasing relative concentration of inhibitors resulted in an enhanced MDA formation. The production of MDA elicited by CumOOH corresponded generally to the degree of inhibition in DNP-SG transport caused by different inhibitors. As shown in Fig. 1, significant differences in MDA formation as a function of the concentrations of inhibitors were not observed in erythrocytes of aged subjects in comparison to that of young subjects. Therefore, the concentrations corresponding to 30 mmol/l fluoride, 0.2 mmol/l vanadate and 1/1200 Tween 80 which were effectively able to inhibit the DNP-SG transport were selected for further experiments. In these experiments, MDA levels of erythrocytes treated with inhibitors of DNP-SG transport were not significantly different from the levels of erythrocytes treated with CumOOH alone (Fig. 2). MDA levels also remained unchanged within each group when compared to basal conditions (data not shown). However, MDA levels of erythrocytes treated with CumOOH after preincubation with inhibitors were significantly different from those treated with CumOOH without the inhibitors (P<0.05) (about 1.6–2-fold increase over controls with CumOOH alone). Also in this case, no differences in MDA overproduction were observed between the aging and the young groups, i.e., the ratios between values of the samples treated with CumOOH alone and values of the samples treated with CumOOH after pretreatment with inhibitors were statistically unchanged (data not shown). The MDA production in erythrocytes which were kept under oxidative stress was very similar for Tween 80 and vanadate. Inhibition by fluoride caused slightly less MDA production than other inhibitors (Fig. 2).Chemiluminescence intensity of erythrocytes treated with inhibitors of DNP-SG transport were not significantly different from the intensity of erythrocytes treated with CumOOH alone or from those in their basal conditions (Fig. 3A); during 60 min, light output in erythrocytes remained constant. Addition of 0.1 mmol/l CumOOH to erythrocytes pretreated with inhibitors enhances chemiluminescence signal in erythrocytes of both age groups. However, no differences in spectral distributions of chemiluminescence were observed between the aging and the young groups (Fig. 3B), i.e., the ratios of increase in chemiluminescence between treated and control (erythrocytes treated with CumOOH alone) values were statistically unchanged (data not shown). In addition, in the erythrocytes treated with Tween 80 or vanadate, onset and maximal chemiluminescence were slightly higher than those of fluoride.4DiscussionIn the present study, we employed human erythrocytes to investigate the effect of aging on the glutathione S-conjugate transport by determining the DNP-SG transport. We used erythrocytes other than the chronologically young population rich with reticulocytes, the reasons for which were explained in our previous article (Onaran et al., 1997). In experimental conditions, in which erythrocyte glutathione is completely depleted by incubating the erythrocytes with CDNB, we did not observed a significant difference in the efflux of DNP-SG in erythrocytes from aged subjects when compared with young subjects. The result indicates that the detoxification function is maintained against the chemical stress employed in their erthyrocytes.It is known that the cells transport the glutathione S-conjugates outward through ATP-dependent efflux process. The transport of glutathione S-conjugate in erythrocytes was first reported by Board (1981)and it was shown that the addition of CDNB to erythrocytes results in the efflux of DNP-SG with rapid irreversible depletion of glutathione. The transport of this conjugate is inhibited by the depletion of intracellular ATP or by known organic anion transport inhibitors such as vanadate, an inhibitor of P type ATPases (La Belle et al., 1986), and fluoride (interaction with the transporter molecule versus cellular energy depletion) (Awasthi et al., 1983). We also observed pronounced inhibition of DNP-SG transport by these inhibitors.Although the effects of aging on different detoxification mechanisms such as glutathione and GST in erythrocytes have been reported (Stohs et al. 1984, Jozwiak and Jasnowska 1985, Stohs and Lawson 1986, Al-Turk et al. 1987, Foldes et al. 1988, Matsubara and Machado 1991, Picot et al. 1992), to our knowledge no data on the effect of donor age on transport of glutathione conjugates of membrane is available. It has been shown that there is an age-related decrease in glutathione content of the erythrocytes (Stohs et al. 1984, Al-Turk et al. 1987, Matsubara and Machado 1991). However, some evidence pointed out that glutathione status, physical health and longevity of life were closely interrelated (Foldes et al. 1988, Calvin et al. 1992). Furthermore contradictory results showing decreased or unchanged GST activity in erythrocytes with donor aging were also reported (Stohs et al. 1984, Al-Turk et al. 1987, Picot et al. 1992). In addition, it has been shown that changes in glutathione concentration in mammalian cells have various effects, influencing cellular radiosensitivity and the cytotoxicity of several chemotherapeutic agents (Bump et al. 1982, Li and Kaminskas 1984, Fernandes and Cotter 1994). In the aging group that provided erythrocytes for our study, glutathione levels were lower for about 20% and GST activities remained unchanged. The present results thus reveal that cellular glutathione status of the aging subject has no adverse effect on glutathione S-conjugate transport and the present amount of glutathione in their erythrocytes might be sufficient to detoxify the employed chemical stress. However, we do not know how the transport of glutathione S-conjugate is affected by different levels of intracellular glutathione.It has been previously reported that transport systems for glutathione S-conjugates play significant roles in the biotransformation of toxic products arising from oxidative stress induced lipid peroxidation (Ishikawa et al. 1986, Grune et al. 1991, Akerboom et al. 1992). On the other hand, results of our previous study indicated that the intact erythrocytes from aged subjects were not more susceptible to oxidative stress than those of young subjects (Onaran et al., 1997). In the second part of our study we therefore decided to determine how glutathione S-conjugate transport plays a role in the resistance to oxidative stress of the erythrocytes of the aged subjects.To investigate this, DNP-SG transport was inhibited by various agents, i.e., fluoride, vanadate or Tween 80. These agents were used for inhibitory action in the studied system as their diverse effects on glutathione conjugate pump have been explained previously (Akerboom et al. 1992, Pulaski and Bartosz 1995). In preliminary studies we observed that under experimental conditions employed the concentrations of the inhibitors did not affect the glutathione and MDA contents, chemiluminescence formation, rate of hemolysis and GST activities in the individual's erythrocytes. The erythrocytes were then treated with 0.1 mmol/l CumOOH in order to induce oxidative stress and then MDA levels, a common end product of oxidative damage, were measured to asses whether the inhibition of glutathione transport is subjected to increased oxidative damage in erythrocytes. Analyses of the effects of inhibitors on DNP-SG transport as a function of the inhibitor concentrations under the in vitro standard oxidative stress conditions, indicate that increasing the relative concentrations of inhibitors to erythrocytes resulted in an enhanced MDA formation and that enhancement of sensitivity caused by each of the concentrations used was unchanged as a function of aging. In addition, the degree of the DNP-SG transport inhibition for each of inhibitors used was very similar in young and aged subjects. Because of these results it was decided that further experiments could be performed in the inhibition conditions which were able effectively to prevent the conjugate transport, in order to obtain an insight into the effects of glutathione S-conjugate transport on the resistance to oxidative stress. At this stage of the study the extent of oxidative damage was evaluated by measuring the chemiluminescence formation as well as MDA formation. The present study reports that following stimulation by CumOOH after the inhibition of DNP-SG, peroxidation- dependent changes of MDA and chemiluminescence formation took place at the same level in aging and young adults. This indicates that the erythrocytes from the elderly are equally capable of withstanding the oxidative stress following the inhibition of glutathione conjugate pump and oxidative effect of lipid peroxidation is not much greater in aging individuals with diminished glutathione levels but normal GST activity than young adults. Furthermore, these results also indicate that the erythrocytes with inhibited DNP-SG transport were shown to be more susceptible to oxidative stress generated by CumOOH than those which were not inhibited. We concluded, therefore, that this result indicates that inhibition of glutathione conjugate transport may impair cellular protection to oxidant stress. Although we are at present not able to explain the mechanisms governing these changes, several possible explanations of the effects observed may be considered. As inhibitory effects of glutathione S-conjugates on GSTs as well as glutathione reductase (Ishikawa et al. 1986, Ishikawa 1989) and the transport of GSSG in various tissues including erythrocytes (Bilzer et al. 1984, Akerboom et al. 1991) have been reported (Akerboom et al. 1982, 1991, Bilzer et al. 1984, Ishikawa et al. 1986, Ishikawa 1989), accumulation of the S-conjugates resulting from radical induced lipid peroxidation in the cell may be crucial for detectable oxidative damage. It is known that GST and glutathione reductase reduce organic hydroperoxides such as CumOOH (Prohaska and Ganther 1977, Awasthi et al. 1980), and it was also shown that the transport of GSSG is an important process for the cell to avoid highly oxidative stress (Adams et al., 1983). Even though such an effect is not known, inhibition of glutathione conjugate transport may also directly compromise the oxidant suppression of the cells by inhibiting key enzymes. However, further investigation is obviously necessary to understand the function of glutathione S-conjugate transport responsible for the protection from oxidative damage.In conclusion, this study indicates that the transport of DNP-SG in erythrocytes of healthy elderly with low glutathione is unaffected and their erythrocytes are not more susceptible to oxidative stress than those of young subjects after in vitro pretreatment with inhibitors which inhibit glutathione conjugate pump. Our findings also suggest that the ability of the erythrocytes to withstand oxidative damage may be diminished with inhibition of glutathione conjugate transport.ReferencesAdams et al., 1983J.DAdamsB.HLauterburgJ.RMitchellPlasma glutathione disulfide as an index of oxidant stress in vivo: effects of carbon tetrachloride, dimethylnitrosamine, nitrofurantoin, metronidazole, doxorubicin and diquatPharmacol. Exp. Ther.2271983749754Akerboom et al., 1982T.P.MAkerboomMBilzerHSiesCompetition between of glutathione disulfide (GSSG) and glutathione S-conjugates from perfused rat liver into bileFEBS Lett.14019827376Akerboom et al., 1991T.P.MAkerboomVNarayanaswamiMKunstHSiesATP-dependent S-(2,4-dinitrophenyl) glutathione transport in canalicular plasma membrane vesicles from rat liverJ. Biol. Chem.26619911314713152Akerboom et al., 1992T.P.MAkerboomGBartoszHSiesLow and high-Km transport of dinitrophenyl glutathione in inside out vesicles from human erythrocytesBiochim. Biophys. 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Biochem.1381984373378Board, 1981P.GBoardTransport of glutathione S-conjugates from human erythrocytesFEBS Lett.1241981163165Bump et al., 1982E.ABumpN.YYuJ.MBrownRadiosensitization of hypoxic tumor cells by depletion of intracellular glutathioneScience2171982544545Calvin et al., 1992A.LCalvinSNaryshkinD.LSchneiderB.JMillsR.DLindemanLow blood glutathione levels in healthy aging adultJ. Lab. Clin. Med.1201992720725Conti et al., 1991MContiP.CMorandPLevillainALemonnierImproved fluorometric determination of malondialdehydeClin. Chem.37199112731275Farooqui et al., 1987M.Y.HFarooquiW.WDayD.MZamoranoGlutathione and lipid peroxidation in the aging ratComp. Biochem. Physiol.88B1987177180Fernandes and Cotter, 1994R.SFernandesT.GCotterApoptosis or necrosis: intracellular levels of glutathione influence mode of cell deathBiochem. Pharmacol.481994675681Foldes et al., 1988JFoldesT.MAllweisJMenczelOShalevErythrocyte hexose monophosphate shunt is intact in healthy aged humansClin. Physiol. Biochem.619886467Grune et al., 1991TGruneWSiemsJKowalewskiHZollnerHEsterbauerIdentification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by enterocytes of rat small intestineBiochem. Int.251991963971Habig et al., 1974W.HHabigM.JPabsW.BJakobGlutathione S-transferasesJ. Biol. Chem.249197471307139Ishikawa, 1989TIshikawaATP/Mg2+-dependent cardiac transport system for glutathione S-conjugatesJ. Biol. Chem.26419891734317348Ishikawa and Sies, 1984TIshikawaHSiesCardiac transport of glutathione disulfide and S-conjugateJ. Biol. Chem.259198438383843Ishikawa et al., 1986TIshikawaHEsterbauerHSiesRole of cardiac glutathione transferase and of the glutathione S-conjugate export system in biotransformation of 4-hydroxynonenal in the heartJ. Biol. Chem.261198615761581Jozwiak and Jasnowska, 1985ZJozwiakBJasnowskaChanges in oxygen-metabolizing enzymes and lipid peroxidation in human erythrocytes as a function of age of donorMech. Ageing Dev.3219857783Kunst et al., 1989MKunstHSiesT.P.MAkerboomS-(4-Azidophenacyl) [35S] glutathione photoaffinity labeling of rat liver plasma membrane-associated proteinsBiochim. Biophys. Acta98219891523La Belle et al., 1986E.FLa BelleS.VSinghS.KSrivastavaY.CAwasthiEvidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytesBiochim. Biophys. Acta1391986538544Li and Kaminskas, 1984JLiEKaminskasAccumulation of DNA strand breaks and methotrexate cytotoxicityProc. Natl. Acad. Sci. USA81198456945698Matsubara and Machado, 1991L.SMatsubaraP.E.AMachadoAge-related of glutathione content, glutathione reductase and glutathione peroxidase activity of human erythrocytesBraz. J. Med. Biol. Res.241991449454Onaran et al., 1997YOnaranA.SYalçynGSultuybekEffect of donor age on the susceptibility of erythrocytes and erythrocytes membranes to cumene hydroperoxide-induced oxidative stressMech. 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Elsevier, Amsterdam, pp. 59–70.Stohs et al., 1984S.JStohsF.HEl-RashidyTLawsonR.HKobayashiB.GWulfJ.FPotterChanges in glutathione and glutathione metabolizing enzymes in human erythrocytes and lymphocytes as a function of age of donorAge7198437Tietze, 1969FTietzeEnzymic method for quantitative determination of nanogram amounts of total and oxidized glutathioneAnal. Biochem.271969502522Videla et al., 1984L.AVidelaM.IVillenaGDonosoJ.DFuenteELissiVisible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensionsBiochem. Int.81984821830Zimniak and Awasthi, 1993PZimniakY.CAwasthiATP-dependent transport systems for organic anionsHepatology171993330339 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B2D0D17A02E9D3F4810ED4B2C7748AAF937E04E.txt b/test/dataset/in/resources/corpus/Clean_0B2D0D17A02E9D3F4810ED4B2C7748AAF937E04E.txt new file mode 100644 index 0000000..73a4a2f --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B2D0D17A02E9D3F4810ED4B2C7748AAF937E04E.txt @@ -0,0 +1 @@ +fampractfamprjFamily Practice1460-22290263-2136Oxford University Press10.1093/fampra/cmm012Primary Care EpidemiologyPatterns of pain and consulting behaviour in patients with musculoskeletal disorders in rural Crete, GreeceAntonopoulouMariaabAntonakisNacHadjipavlouAdLionisCaaClinic of Social and Family Medicine, Department of Social Medicine, School of Medicine, University of Crete, Crete, GreecebSpili Health CentrecAnogeia Health Centre, Regional Health and Welfare System of Crete, Crete, GreecedDepartment of Orthopaedics and Traumatology, School of Medicine, University of Crete, Crete, GreeceCorrespondence to: Maria Antonopoulou, Clinic of Social and Family Medicine, University of Crete, PO Box 2208, Heraklion 71003, Crete, Greece; Email: antonopm@uoc.grAntonopoulou M, Antonakis N, Hadjipavlou A and Lionis C. Patterns of pain and consulting behaviour in patients with musculoskeletal disorders in rural Crete, Greece. Family Practice 2007; 24: 209–216.6200715520072432092163620063220071832007© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2007Background. Musculoskeletal disorders (MSDs) account as a reason for frequent consultations in primary care. However, the magnitude of the problem at the GP's clinic, the patterns of pain and the consulting behaviour has not been sufficiently explored.Objectives. The aim of this study was to report on patterns of pain relevant to MSDs and explore the co-morbidities and consulting behaviour in rural primary care settings in Crete.Methods. Three primary care centres (PCCs) of Crete were selected for a study period of 2 weeks. Every visitor, aged 20–75 years, regardless of the reason for visiting the facility was invited to participate. The Greek version of the general Nordic questionnaire for the analysis of musculoskeletal disorders (NMQ) was used for data collection.Results. A total of 455 subjects answered the NMQ. Three hundred and seventy-six (82.6%) of the study population reported having one or more symptoms during the previous year. Low back (56.9%), neck (34.1%), shoulder (29.9%) and knee (27.9%) were the commonest sites of pain. In almost half cases (48.6%), the complaints about pain were accompanied by activity restrictions. Multivariate statistical analysis showed significant correlations with increasing age and female gender (P < 0.05). Common chronic conditions were associated with co-morbidities from the musculoskeletal system. Only one-third of those who reported MSDs had consulted their GPs for the same problems within the previous year.Conclusions. MSDs are highly prevalent among rural population in Crete but fewer patients seek care than those who report symptoms.Greece, musculoskeletal disorderspain patternsprimary careruralIntroductionMusculoskeletal disorders (MSDs) comprise a major health problem for the general population, affecting their quality of life, demanding increased health care and organization.1 According to reports from Canada and the Netherlands,2,3 the prevalence of musculoskeletal problems range from 29% to 74.5%, respectively. However, the annual consultation rate by health care professionals of musculoskeletal problems is about 20%.4 MSDs often cause pain and significant disability, especially in the elderly population, signifying a heavy community burden.5 Furthermore, working conditions correlate well with MSDs.6 In family practice settings, MSDs often co-exist with multiple medical conditions, addressing special care needs.7In Greece, although MSDs have been considered as common reasons for patients' visits to GPs in rural areas,8,9 issues of pain patterns and consulting behaviour are still unexplored. This paper reports on the magnitude of musculoskeletal problems within the primary care setting with emphasis on co-morbidities and consulting behaviour and intends to discuss its implications for GPs in a southern European country.MethodsSettingData were gathered from three PCCs in rural Crete. These PCCs provide around the clock outpatient care for a rural district, serving a population of approximately 37 000 inhabitants10 with most of them being farmers [International Standard Classification of Occupations, 88 (ISCO-COM 6) and craft and related trades sectors (ISCO-COM 7)]. People are also employed in restaurant services and travel-related services (ISCO-COM 5), since a lot of tourists are visiting these areas. The unemployment rate is nearly 12%, slightly higher, as compared to 10% in the rest of the country. The educational status is low with more than 50% of the general population being either illiterate or with primary education only [International Standard Classification of Education 1997 (ISCED 0 + 1)].SamplingAll consecutive visitors (20–75 years of age) in the three PCCs during a period of 10 working days in November 2002 participated in the study. A written consent was obtained and the study protocol was applied by health professionals. The month of November was selected because most agricultural tasks in the fields take place during this period throughout the catchment area of the PCCs. During that period, farmers are engaged in intensive harvesting of grapes and olives, demanding hard manual work with the spine in a bend over position for a prolonged period of time. Other awkward positions during farming can also extract musculoskeletal strain. Moreover, at this time there are no tourists visiting the PCCs, so the study population comes from the permanent inhabitants served by these PCCs. The sample collected was further scrutinized for clinical co-morbidities and the consulting behaviour, using the medical records of one PCC.MethodsThe Greek version of the general form of the standardized Nordic questionnaire for the analysis of musculoskeletal symptoms (NMQ) was used for data collection. NMQ had been previously translated and validated in the Greek language.11 Apart from a few general demographic questions, it contains a picture of the back aspect of the human body divided in nine anatomical areas (neck, shoulders, elbows, wrists/hands, upper back, low back, hips/thighs, knees and ankles/feet) (Fig. 1). The rest of the questions are distributed in three columns. There are nine screening questions in the first column. For example, ‘have you at any time during the last 12 months had trouble i.e. ache, pain or discomfort in this area?’. In case of a positive answer, the respondents answer whether or not they had been prevented from doing their normal work at home or away from home, during the same period because of that ailment. They are also asked if they had experienced that symptom during the previous 7 days (questions in the second and the third column). Therefore, the questionnaire's structure investigates the prevalence of different MSDs and examines which of the particular problems are the more debilitating (i.e. prohibited patients from engaging in their normal work routine within the previous 12 months). Even though, NMQ is self-administered, because of the special features of the study population (mean age of 51.9 years and education level between 0 and 6 years), trained health professionals assisted the subjects with the completion of the questionnaire.FIGURE 1The back aspect of the human body as it appears in the NMQAdditional data concerning age, sex, body mass index (BMI) and working records were extracted by the NMQ. BMI was classified into four categories: underweight (BMI < 20 kg/m2), normal (BMI ≥ 20 and < 25 kg/m2), overweight (BMI ≥ 25 and < 30 kg/m2) and obese (BMI ≥ 30 kg/m2).12 To determine the extent to which NMQ identified musculoskeletal symptoms during primary care visits, we carried out a medical audit in one of the participating PCCs and all medical records of the subjects were reviewed. Co-morbidities (according to International Classification for Primary Care 2), education level (according to ISCED 1997), type of occupation (according to ISCO-COM 88), cohabitation status (any kind of cohabitation or living alone) and consultation patterns were also collected using a pre-tested questionnaire.Statistical analysesStatistical analyses were performed using SPSS version 12.0. A P-value of P < 0.05 was accepted as significant. For the nine anatomical areas, the 1-year period prevalence, the prevalence of daily limitations due to reported symptoms and the point prevalence (musculoskeletal symptoms during last week) were calculated. Those were the dependent variables. Independent variables were age, gender, BMI, working time at present work and working hours per week. Adjusted odds ratios (ORs) were calculated. Multiple logistic regression analysis was performed to evaluate the association between each exposure variable and musculoskeletal symptoms. This method was applied stepwise in an enter approach. The association between education level, type of occupation, cohabitation status, co-morbidities and the patterns of pain was also measured. To study the effects of different occupations to limitations in daily activities, the total score of positive answers for each patient and for each question was calculated. Wilcoxon paired and unpaired (Mann–Whitney) tests were used to compare these scores into the same and among different occupational groups. Chi-square test was used to compare those who had consulted their physicians for MSDs with those who did not.ResultsSample characteristicsOverall, 455 subjects completed the questionnaire at the three PCCs with participation rate at 95.2%. Table 1 shows the general demographic characteristics. Two hundred and sixty (57.2%) of the respondents were females and the mean age was 51.9 [95% confidence interval (CI): 50.3–53.7] years.Table 1Sample characteristicsn = 455, %GenderMen42.8Women57.2Age group20–3929.340–5928.260–7542.5BMIUnderweight (BMI < 20)0.8Acceptable (20 ≤ BMI < 25)20.2Overweight (25 ≤ BMI < 30)55.4Obese (BMI ≥ 30)21.1No records available2.4Residence areaAnogeia15.4Perama45.9Spili38.7Years at present work<1–412.15–1420.415–2413.025–3412.335–4411.645+4.8No records available25.7Weekly working hours5–144.215–245.325–342.935–4417.145–5425.155+17.1No records available28.3Patterns of painThree hundred and seventy-six subjects (82.6%) reported at least one musculoskeletal problem during the previous year, while 219 (48.1%) subjects reported limitations of activities due to their symptoms during the same period of time. Low back pain was the commonest complaint (56.9%), followed by neck (34.1%), shoulder (29.9%) and knee (27.9%) problems. Two musculoskeletal co-morbidities were present in 24% of patients (n =  109); one in 23.3% (n =  105), while 3.3% (n =  15) reported widespread pain in all nine regions of the body. Low back pain was also the most debilitating symptom reported (28.6%), causing limitations in daily activities. Low back and neck pain was the commonest combination reported (n =  124, 27.5%), resulting in the higher disability rate (11.0%). Although the co-existence of more than two symptoms was not frequent, this seemed to be associated with more disability but without statistical significance. The reported patterns of pain by age groups and gender are presented in Table 2.Table 2Patterns of pain by age and genderPain patterns/age groupsPrevalence of pain during the last year/genderMalesFemalesLevel of significancean%n%Neck4121.011443.8<0.0001    20–39 years31.5166.20.025    40–64 years157.74918.80.001    65+ years2211.34818.50.048Shoulder4523.19135.00.008    20–39 years73.6197.30.139    40–64 years157.73413.10.092    65+ years2211.33714.20.441Elbow189.23915.00.087    20–39 years00.031.20.336    40–64 years73.6197.30.139    65+ years105.1176.50.670Wrist/hand2110.86725.8<0.0001    20–39 years31.593.50.307    40–64 years63.13513.50.0002    65+ years126.2238.80.393Upper back2010.37729.6<0.0001    20–39 years31.5114.20.166    40–64 years63.1186.90.113    65+ years115.62911.20.054Low back8644.117366.5<0.0001    20–39 years2412.33513.50.813    40–64 years2914.97328.10.001    65+ years3216.46324.20.056Hip2010.37729.6<0.0001    20–39 years21.083.10.234    40–64 years42.13212.30.0001    65+ years136.73613.80.023Knees4322.18432.30.024    20–39 years63.1114.20.716    40–64 years115.62710.40.096    65+ years2713.83613.80.890Foot/ankle3316.95220.00.926    20–39 years42.193.50.549    40–64 years84.1228.50.093    65+ years2010.3218.10.519a Statistical significant values are printed in italic.The prevalence of the reported symptoms was statistically related to increasing age (P < 0.05) for neck, elbow, low back, hip and knee pain. Multiple musculoskeletal symptoms were also related to aging. Patients with shoulder and low back pain reported also more disability in terms of limitation in their daily activities.Morbidity patterns also differed between women and men. While low back pain was the commonest symptom in both genders, female patients reported higher prevalence for every symptom (P < 0.05) except for elbow and foot (Table 2). Women reported more pain conditions than men 11.9/10.7 (P < 0.05) and more disability.In the questions about working schedules, the majority of the sample reported 20 (95% CI: 18.6–21.8) years of work for 45.6 (95% CI: 43.6–47.6) hours per week. Men worked longer hours but this tended to decrease, as they grew older. Women worked mainly at home or at the farms, fewer hours per week than men (men/women ratio: 1.1). Working for a period of more that 15 years was related to higher morbidity mainly from shoulder and elbow pain (Table 3). Limitations in daily activities due to neck, shoulder, low back and foot complaints were related to longer working schedule per week. Multiple regression analysis for the most frequent patterns of pain is presented in Table 3.Table 3Logistic regression analysis of most frequent patterns of pain by age, gender, BMI and working timeVariableBSEP-valueExp. B95% CILow back pain    Age0.0230.0120.0461.0241.000–1.047    Gender0.8580.2390.00032.3591.477–3.768    BMI0.0330.0310.2941.0330.972–1.098    Years in the present work0.0010.0120.9561.0010.977–1.025    Hours per week0.0050.9840.4580.9950.981–1.009Neck pain    Age0.0540.0130.000031.0561.029–1.083    Gender1.3230.2740.0000013.7562.193–6.431    BMI0.0090.0330.7920.9910.930–1.057    Years in the present work0.0070.0130.5820.9930.968–1.018    Hours per week0.0100.0080.1910.9900.975–1.005Shoulder pain    Age0.0140.0130.2851.0140.989–1.040    Gender0.6310.2670.0181.8791.114–3.171    BMI0.0290.0320.3731.0290.966–1.096    Years in the present work0.0280.0130.0311.0281.033–1.055    Hours per week0.0040.0080.5961.0040.989–1.019Knee pain    Age0.0330.0130.0111.0341.008–1.061    Gender1.0060.2800.0032.7351.578–4.738    BMI0.0330.0330.3211.0330.969–1.102    Years in the present work0.0150.0130.2581.0150.989–1.041    Hours per week0.0070.0080.3961.0070.991–1.022Further analysis from the sample in one PCC showed that occupation did not constitute a statistically significant risk factor for the presence of MSDs. Thirty-one per cent of the sample were farmers (ISCO-COM 6), 11% were employed in restaurant services and travel-related services (ISCO-COM 5) and 27%—almost half of all the women—were housewives. Comparing the total scores in the three questions of the NMQ among various occupational groups, it came out that the majority kept their usual everyday activities during the previous year, even though they reported a great number of musculoskeletal symptoms (P < 0.001). Different occupations did not statistically affect this result.Cohabitation and educational status did not significantly differ in the patterns of reported symptoms.Co-morbiditiesHalf of the study population (55.4%) for both sexes were overweight (BMI > 25 kg/m2) with mean BMI = 29.0 (95% CI: 28.6–29.9), especially women in advanced age (BMI > 30 kg/m2 in 24.6% of females) (Pearson R correlation, Rp = 0.328, P < 0.001 of correlation between overweight women and age). Overweight (BMI > 25) and obese (BMI > 30) patients had a tendency to report more musculoskeletal symptoms but not of statistical significance. However, they reported more activity limitations as a result of shoulder, elbow, wrist and foot pain.Review of medical records in one PCC revealed that the most common diagnoses co-existing with MSDs were hypertension (64/176, 36%), osteoarthritis (OA) of hip and/or knee (56/176, 32%), coronary artery disease (CAD) (21/176, 12%) and mental disorders (20/176, 11%). The number of co-morbidities was increasing with age without statistical significance. Mental disorders, mainly depression, ranked as the medical conditions with the highest proportion of musculoskeletal symptoms (95.0%), followed by chronic obstructive pulmonary disease (COPD) (92.8%), CAD (90.4%), OA (84.0%), diabetes (83.3%) and hypertension (82.8%). The multiple logistic regression analysis of the most common co-morbidities in correlation to the reported symptoms highlighted strong correlations, especially with arthritic conditions. Shoulder pain was statistically associated with COPD (adjusted OR = 8.94, 95% CI 1.47–54.17, P = 0.017) and OA (adjusted OR = 4.25, 95% CI 1.67–10.81, P = 0.002), whereas wrist pain was more frequent in subjects with diabetes (adjusted OR = 3.87, 95% CI 1.09–13.68, P = 0.035). The prevalence of elbow pain was correlated to CAD (adjusted OR =5.01, 95% CI 1.01–24.87, P = 0.048). Patients with OA also reported statistically significant activity restriction (P < 0.05) due to neck, shoulder, upper back, hip, knee and foot symptoms, unlike diabetic patients who did not complain for many daily restrictions. Cancer of any form, mental disorders or stomach problems were not statistically correlated to MSDs.Consulting behaviourData from one PCC showed that although the prevalence of reported MSD was measured at 71.4% (125 out of 176), only 56 subjects (32%) had reported the same symptoms to their GP during previous consultations. These symptoms originated mainly from low back, neck, knee and hip areas. Patients with multiple co-morbidities were more likely to have previously consulted their GPs for those problems (adjusted OR = 2.21, 95% CI 1.6–2.9, P < 0.0001). Seventy-four of the participants (41.7%) reported symptoms during the last week (question in the third column of the NMQ), but only 13 (17.5%) visited their GP complaining for musculoskeletal symptoms. When comparing those visiting GPs with those who did not (chi-square test) during the previous year, we found that younger patients of both genders with limited education did not consult their physician due to MSDs. A patient with knee or shoulder pain was seeking care more often regardless of age. Older patients and patients suffering from OA or hypertension reported significantly more consultations. Other factors like gender, BMI, working time or occupation were not found to have statistical significance.DiscussionSummary of resultsThere are several interesting points revealed by this study such as the patterns and distribution of pain, the differences between genders and the consulting behaviour. Low back pain was the commonest reported site among all age groups. Low back pain, followed by neck and shoulder pain, was also the main symptom for daily living limitations. This finding is in accordance with the results of a recent systematic review in general practice of work-related diseases.13 Age was found to be a risk factor for many musculoskeletal symptoms. Younger patients (20–39 years) reported fewer symptoms and less frequently, as compared with older patients (66.2% prevalence of any MSDs versus 90% for the older group over 60 years of age, P < 0.05).Pain patterns also differed between genders. In our study, women reported on average more symptoms and more disability. Women to a greater extent than men blamed musculoskeletal pain in neck, wrist, low back, hip or knee for restrictions in daily activities. The tendency for MSDs to affect more women than men has been shown in other epidemiological studies.3,14 Reasons for gender differences, apart from the apparent biological differences (genetics, physiology, hormones, etc.), include different psychosocial aspects of symptoms and care. Women also seem to visit more promptly health care facilities and recall health problems to a greater extent than men.15 However, in our study, such gender preference was not revealed.In our study, overweight or obesity, a common public health problem in contemporary Crete,16 showed significant correlations only with the reported disabilities due to pain of upper limb and foot. According to other studies, obesity has been related to hip and low back pain in middle-aged men,17 whereas in rural communities, it causes more disability to patients with knee OA.18Working for many years was correlated to shoulder and elbow pain. More comprehensive surveys that included physical and psychosocial work factors, such as work intensity or working postures, have previously shown associations with MSDs,6 but these were not explored in our study.Unlike other epidemiological evidence which relates MSDs to physically demanding professions like farming,19 occupation did not statistically relate to any MSD. However, analysing the positive answers for all occupational groups, we found that the majority of participants carried on with their daily functions, independently of their job, despite the occurrence of multiple musculoskeletal problems. Cultural or local social network variables, not included in this study, could play a role in the perceived limitations of activity for this population.20Other social factors, such as living alone or low education, were not found to affect the reported patterns of pain, which comes in agreement with other studies.21The clinical co-morbidity rate was high. Patients with OA, COPD, diabetes, hypertension or CAD tended to report more musculoskeletal symptoms. Co-morbidities with MSDs were also shown to prevent their daily activities. Co-morbidity for OA in general practice has also been found to be extensive with musculoskeletal as well as non-musculoskeletal conditions in the UK.22 Patterns of co-morbidity in the Dutch population have shown that MSDs were most likely to coincide with lung and heart diseases, neurological disorders, diabetes and cancer.23 The explanation for the perceived co-morbidity lies beyond the present study in shared pathology of diseases or the impact which one condition might have in the occurrence of another. However, the recognition of this large extent of co-morbidity has implications for the way in which primary care should be organized. The burden is on primary care physicians to provide the majority of care, not only for the target condition but also for all co-morbid conditions.Another interesting point was that only 13 out of 74 who reported musculoskeletal symptoms were actually seeking care at the time of the study. On an annual basis, 32% of this sample had contacted a primary care physician because of musculoskeletal pain which can be considered high in comparison with other studies,4 although it corresponds to less than half of the same population reporting MSDs in the NMQ. The more concurrent chronic conditions they had, the more likely they were to have consulted their GP and complain for MSDs, too. Those were mainly older patients with higher education suffering from OA and hypertension that came complaining for knee or shoulder pain. Similarly, another study in a rural Swedish population showed that only one-fifth of those reporting current neck and/or low back pain had a primary care consultation.24 Possible explanations could be either the poor registration rates of primary care physicians who underestimate the symptoms or that symptoms are not severe enough to be reported. A recent report on primary care consultations due to indigestion problems in the same district showed under diagnosis of functional gastrointestinal disorders by physicians.25 However, a recent European study involving eight countries regarding musculoskeletal pain showed that up to 27% of people with pain do not seek medical care in spite of constant or daily pain.26 The disbelief or ignorance of effective treatments for MSDs could prevent patients from consulting a health professional.23 Even when they do seek help, this is often limited to prescription for non-steroidal anti-inflammatory drugs (NSAIDS), which result in non-compliance to their treatment.26 In fact, according to a previous study conducted in the same district, prescriptions for painkillers and NSAIDS ranked second accounting for 19% of prescriptions during 1-year period.27 To overcome the doctors' difficulty in identifying those diseases, a change in doctor–patient communication with an emphasis on patients' perceptions and needs seems as a promising option.26Strengths and weaknessSeveral concerns should be raised when discussing study findings. Overestimating prevalence due to sampling method is one issue: our sample was probably overrepresented by patients with multiple morbidities waiting to see their physicians. High prevalence of MSDs has been documented in many studies, especially when self-administered questionnaires are being used for data collection.28 People tend to recall the most recent or most debilitating painful conditions when asked to complete questionnaires. Whether or not the reported symptoms were major or minor cannot be explored through the NMQ.Although the NMQ has been used in a great number of studies in different countries,29 it still has several limitations. Since NMQ is counting symptoms throughout the human body, the estimated prevalence is expected to be high. Most general population studies focus on special anatomic sites, while others deal with specific occupational groups.30,31 The used questionnaire seems to be appropriate for general screening in occupational groups, without permitting further discrimination. For example, the pain in the hips, considering the area marked as ‘hip/thighs’ in the picture of NMQ is unclear if it is actually a hip problem or originates from the low back area and reflects at the hips. Moreover, the use of a picture of the back aspect of the body may not allow problems in the frontal aspect to be revealed.The timing of data collection did not seem to have an effect on the measured prevalence. Data from one PCC that was collected during 10 working days in spring did not show a seasonal variation for the reported MSDs.Although the sample size reflected the underlying population i.e. the general population of rural primary care in Crete (95% confidence level and 95% CI 7%), limitations should be taken into account interpreting the results for co-morbidities and consulting behaviour since the data came only from one PCC (n  =  175).ConclusionsIn conclusion, this study showed that musculoskeletal pain is very common in the rural population of Crete. GPs need to be alert and well skilled in order to prevent or identify early the musculoskeletal problems. The observed patterns of co-morbidity highlight the need for primary care oriented towards patients' overall health care. Patients, especially women and elderly, who suffer from hypertension or diabetes, could also suffer from musculoskeletal problems, even if this does not come up as their major complain, and they should be treated accordingly. Further evaluation of the consequences of MSDs to this population in terms of disability or effect in the quality of life should contribute towards that effort.DeclarationFunding: None.Ethical approval: Approval of a specific scientific committee of the University of Crete.Conflicts of interest: None.1WoolfADPflegerBBurden of major musculoskeletal conditionsBull World Health Organ2003816466562BadleyEMWebsterGKRasoolyIThe impact of musculoskeletal disorders in the population: are they just aches and pains? Findings from the 1990 Ontario Health SurveyJ Rheumatol1995227337393PicavetHSJSchoutenJSAGMusculoskeletal pain in the Netherlands: prevalences, consequences and risk groups, the DMC3-studyPain20031021671784BadleyEMRasoolyIWebsterGKRelative importance of musculoskeletal disorders as a cause of chronic health problems, disability and health care utilization: findings from the 1990 Ontario Health SurveyJ Rheumatol1994215055145MakelaMHeliovaaraMSieversKKnektPMaatelaJAromaaAMusculoskeletal disorders as determinants of disability in Finns aged 30 years or moreJ Clin Epidemiol1993465495596ColeDCIbrahimSAShannonHSScottFEylesJWork correlates of back problems and activity restriction due to musculoskeletal disorders in the Canadian national population health survey (NPHS) 1994–5 dataOccup Environ Med2001587287347MartinFBravoGHudonCVanasseALapointeLPrevalence of multimorbidity among adults seen in family practiceAnn Fam Med200532232288TzimisLKatsantonisNLeledakiAVasilomanolakisKKafatosAAntibiotics prescription for indigent patients in primary careJ Clin Pharm Ther1997222272359KoutisADIsacssonALionisCLindholmLHSvenningerKFioretosMDifferences in the diagnose panorama in health care in Dalby, Sweden and Spili, CreteScand J Soc Med199321515810General Secretariat of National Statistical Service of Greece, 2001http://www.statistics.gr (accessed on 6 January 2005)11AntonopoulouMEkdahlCSgantzosMAntonakisNLionisCTranslation and validation into Greek of the standardised general Nordic questionnaire for the musculoskeletal symptomsEur J Gen Pract200410353612Measuring Obesity: Classification and Description of Anthropometric Data. Report on a WHO Consultation on the Epidemiology of Obesity, Warsaw, 21–23 October, 19871988CopenhagenWHO Regional Office for Europe, (document EUR/ICP/NUT 125)13WeeversHJvan der BeekAJAnemaJRvan der WalGvan MechelenWWork-related disease in general practice: a systematic reviewFam Pract20052219720414LerouxIDionneCEBourbonnaisRBrissonCPrevalence of musculoskeletal pain and associated factors in the Quebec working populationInt Arch Occup Environ Health20057837938615FillingimREdwardsRPowellTSex-dependent effects of reported familial pain history on recent pain complaints and experimental pain responsesPain200086879416MamalakisGKafatosAPrevalence of obesity in GreeceInt J Obes Relat Metab Disord19962048849217BergeruddHNillsonBThe prevalence of locomotor complaints in middle age and their relationship to health and socioeconomic factorsClin Orthop199430826427018JordanJMLutaGRennerJBSelf-reported functional status in osteoarthritis of the knee in a rural southern community: the role of sociodemographic factors, obesity, and knee painArthritis Care Res1996927327819HolmbergSStiernstromELThelinASvardsuddKMusculoskeletal symptoms among farmers and non-farmers: a population-based studyInt J Occup Environ Health2002833934520PincusTBurtonAKVogelSFieldAPA systematic review of psychosocial factors as predictors of chronicity/disability in prospective cohorts of low back painSpine200227E109E12021SaastamoinenPLeino-ArjasPLaaksonenMLahelmaESocio-economic differences in the prevalence of acute, chronic and disabling chronic pain among ageing employeesPain200511436437122KadamUTJordanKCroftPRClinical comorbidity in patients with osteoarthritis: a case control study of general practice consulters in England and WalesAnn Rheum Dis20046340841423WestertGPSatarianoWASchellevisFGVan de BosGAPatterns of comorbidity and the use of health services in the Dutch populationEur J Public Health20011136537224HolmbergSAThelinAGPrimary care consultation, hospital admission, sick leave and disability pension owing to neck and low back pain: a 12-year prospective cohort study in a rural populationBMC Musculoskelet Disord200676625LionisCOlsen-FaresjoAAnastasiouFWallanderMAJohanssonSFaresjoTMeasuring the frequency of functional gastrointestinal disorders in rural Crete: a need for improving primary care physicians' diagnostic skillsRural Remote Health2005540926WoolfADZeidlerHHaglundUMusculoskeletal pain in Europe: its impact and a comparison of population and medical perceptions of treatment in eight European countriesAnn Rheum Dis20046334234727AntonakisNTsoulouSPeiosDSimeonidisPLyrarakiELionisCDrug prescribing in primary health. Part II: drugs for cardiovascular, musculoskeletal and central nervous system diseasesArch Hell Med2001185057(in Greek)28PicavetHSHazesJMPrevalence of self reported musculoskeletal disease is highAnn Rheum Dis20036264465029BaoSWinkelJShahnavazHPrevalence of musculoskeletal disorders at workplaces in the People's Republic of ChinaInt J Occup Saf Ergon2000655757430MansfieldNJMarshallJMSymptoms of musculoskeletal disorders in stage rally drivers and co-driversBr J Sports Med20013531432031HagenKBMagnusPVetlesenKNeck/shoulder and low-back disorders in the forestry industry: relationship to work tasks and perceived psychosocial job stressErgonomics19984115101518 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B2E51E5CC19F61CCD947B081C04BF3592E1CC86.txt b/test/dataset/in/resources/corpus/Clean_0B2E51E5CC19F61CCD947B081C04BF3592E1CC86.txt new file mode 100644 index 0000000..de2be13 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B2E51E5CC19F61CCD947B081C04BF3592E1CC86.txt @@ -0,0 +1 @@ +annoncannoncAnnals of Oncology1569-80410923-7534Oxford University Press10.1093/annonc/mdn179original articlespalliative care/quality of lifeUse of palifermin for the prevention of high-dose methotrexate-induced oral mucositisSchmidtE.ThoennissenN. H.RudatA.BiekerR.SchliemannC.MestersR. M.ZühlsdorfM.Müller-TidowC.BerdelW. E.*Department of Medicine, Hematology and Oncology, University Hospital Muenster, Muenster, Germany*Correspondence to: Dr W. E. Berdel, Universitätsklinikum Muenster, Medizinische Klinik, Albert-Schweitzer-Strasse 33, D-48149 Muenster, Germany. Tel: +49-251-8347587; Fax: +49-251-8347588; E-mail: berdel@uni-muenster.de92008252008199164416492811200726320083132008© The Author 2008. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org2008Background: Oral mucositis is a frequent problem after high-dose methotrexate (HD-MTX), impairing patient's quality of life, leading to higher rates of infections and delaying subsequent chemotherapy. This report describes the effect of palifermin in patients treated within the GMALL-B-ALL 2002 protocol containing HD-MTX who developed a severe mucositis in cycle A1/B1.Patients and methods: Ten patients, all with World Health Organization grades III–IV oral mucositis in cycles A1/B1, obtained palifermin with subsequent similar or identical cycles to reduce mucositis. Thus, patients serve as their own control for efficacy of palifermin.Results: All 10 patients developed grades III–IV mucositis in cycles A1/B1 without palifermin, whereas only two of 10 developed grades III–IV mucositis in corresponding cycles A2/B2 with palifermin. Only four of 10 patients showed infections in the cycles with palifermin compared with 10 of 10 patients without palifermin. The duration of mucositits in patients who acquired a higher grade mucositis despite treatment with palifermin could be reduced from 12.9 days (median) without to 11 days with palifermin. The amount of i.v. opioid analgetics could be significantly reduced.Conclusion: Palifermin might reduce the incidence, severeness and duration of oral mucositis in HD-MTX-based chemotherapy and may influence clinical sequelae such as infection and quality of life.methotrexateoral mucositispaliferminintroductionOral mucositis is a severe and frequent adverse effect of high-dose methotrexate (HD-MTX)-based chemotherapy. It is caused by damage of the mucosal lining of the gastrointestinal tract (GIT) as a result of a dynamic series of biological events involving different cellular and tissue compartments of the GIT mucosa. Understanding of the pathobiology has increased rapidly during the last years [1–5]. Mucosal barrier injury is based on a network of interactions involving the endothelium, extracellular matrix, metalloproteinases, submucosal reactions and connective tissue [1–3]. Sonis and coworkers developed a pathophysiological model dividing the dynamical process of mucositis in five phases: initiation, up-regulation, signal amplification, ulceration and healing [2, 3].Oral mucositis and myelosuppression represent dose-limiting toxicity effects of HD-MTX. Mucositis is associated with an increased risk of life-threatening infections [6, 7], with the need for total parenteral nutrition, i.v. analgetic therapy, and may lengthen hospital stay with increased economic burden and consumption of health care resources [8–11]. Oral mucositis impairs the patient's quality of life by pain, the inability to eat, swallow and talk. For many patients with grades III–IV mucositis, the subsequent cycle of chemotherapy is delayed. A grade IV mucositis is an emergency situation in oncology, ethically demanding to relieve the patient's situation. The recent evidence-based management guidelines from the Multinational Association of Supportive Care in Cancer [12] make several recommendations to provide a better patient comfort including good oral hygiene, oral decontamination with antibacterial and antifungal mouthwash and topical and systemic pain management.Numerous substances have been used in addition to treat oral mucositis such as ice chips [13, 14], antioxidants as glutamine, N-acetylcysteine, benzydamine hydrochloride and antiinflammatory agents like prostaglandine E1 and E2 [15]. Until now none of them has proven an unequivocal clinical benefit.Palifermin is a recombinant human keratinocyte growth factor (KGF) which is known to stimulate growth of epithelial cells in a wide variety of tissues. In murine models, a beneficial effect on mucositis could be shown [16]. In December 2004, the Food and Drug Administration approved palifermin (recombinant KGF from Escherichia coli, Amgen, Thousand Oaks, CA, USA) on the basis of randomized data showing a decrease of incidence and duration of severe oral mucositis in patients with hematologic malignancies receiving myelotoxic therapy requiring hematopoietic stem-cell support [17]. Therefore, the updated guidelines of the Multinational Association of Supportive Care in Cancer recommend the use of palifermin in patients who are receiving high-dose chemotherapy and total body irradiation with autologous stem-cell transplantation (level 1, grade A recommendation) [12].KGF receptors are present on epithelial cells in many tissues including the tongue, buccal mucosa, salivary gland, esophagus, stomach, intestine, lung, liver, pancreas, kidney, bladder, mammary gland and eye lens as well as on tumor cells of different histology [18]. Receptors could not be found on lymphoma and lymphoblastic leukemia cell lines [18].The mechanism of action of palifermin in mucositis is not completely elucidated. It leads to a down-regulation of proinflammatory cytokines [19] and increases antiinflammatory cytokines such as IL-13 [20, 21]. Palifermin protects the epithelium against reactive oxygen molecules by modifying the expression of detoxifying transcription factors and enzymes [22].In this series, we observed patients who were treated in the GMALL-B-ALL/B-NHL 2002 protocol, which is based on HD-MTX and is used for certain aggressive B-cell lymphomas and lymphoblastic leukemias. The incidence of grades III–IV (World Health Organization, WHO, oral toxicity scale, Miller et al. [23]) mucositis upon HD-MTX within this protocol ranges from 56% (cycle A1) to 32% (cycle B1) as described by Hoelzer et al. [24]. Another study shows a mucositis rate of 42% [25]. The data of the B-NHL90 trial (GMALL study group) with 3 g MTX/m2 (>55 years 500 mg MTX/m2) showed a grade III (Donelly scale) mucositis rate of 41%–54% in cycle A1 and 61%–75% in cycle B1. In previous trials (B-NHL 83, B-NHL 86), mucositis grades I–IV were observed in 50%–53% of chemotherapy cycles [26].All patients reported here developed a severe mucositis in cycle A1 or B1. In order to prevent additional mucositis, these patients received palifermin for the corresponding following cycles of similar or identical chemotherapy. Thus, patients in this retrospective series can serve as their own control for efficacy of palifermin in preventing mucositis.patients and methodspatientsWe report on a retrospective series of 10 patients with B-cell acute lymphoblastic leukemia (B-ALL) and aggressive B-cell lymphoma classified according to the WHO classification. Patients' characteristics are given in Table 1. We have included only patients into this report who developed severe mucositis in initial treatment cycle A1 or B1 without palifermin and were then given palifermin in the subsequent treatment cycles.Table 1.Patients treated in the GMALL-B-ALL/B-NHL 2002 protocol (severe infection is defined as body temperature >38.5°C and use of systemic antibiotics)Patient ID No.DiagnosisMaximum grade of mucositis (WHO)Maximum grade of mucositis (WHO)Dosage of paliferminWithout paliferminWith palifermin1B-ALLGrade IV, A1, 12 days; Grade III, B1, 8 days; infection A1, B1Grade 1 A2–C2 10 daysA2 60 μg/kg bodyweight (BW); B2 and C2 30 μg/kg BW2Burkitt-like lymphomaGrade IV, A1, 15 days; grade III, C1, 14 days; infection A1, C1Grade II, B1, 9 days; grade II, B2, 7 daysA2–C2 60 μg/kg BW; B1 30 μg/kg BW3Mediastinal B-NHLGrade IV B1, 15 days; infection B1Grade II C1, 4 daysA2–C2 60 μg/kg BW; C1 30 μg/kg BW d−3 to −1; 60 μg/kg BW d1–3 after chemotherapy4Mediastinal B-NHLGrade IV A1, 10 days; grade III B1, 13 days; infection A1–C1Grade I A2A2–C2 30 μg/kg BW5B-ALLGrade IV A1, 13 days; grade III, B1 7 days; infection A1Grade II cycle B2, 5 daysA2–C2 30 μg/kg BW6Burkitt lymphomaGrade II, A1, 4 days; grade IV, B1 18 days; grade III, C1, 18 days; infection A1, C1Grade I, A2, 6 days; grade I, B2, 7 days; infection A2, B2A2 ∼40 μg/kg BW; B2–C2 ∼50 μg/kg BW7Atypical Burkitt lymphomaGrade IV B1, 12 days; grade II A1, 5 days; infection A1, B1, C1Grade III, A2, 10 days; grade IV, B2, 12 days; infection A2A2-B2, 60 μg/kg BW; protocol could not be finished because of infectious complications8B-ALL, elderly protocolGrade IV A1, 16 days; infection A1Grade II B1/B2, 3 days; infection B2∼40 μg/kg BW B1–B39Burkitt lymphomaGrade IV B1, 12 days; infection B1Grade III C1 13 days; grade III A2, 10 days60 μg/kg BW10B-ALLGrade IV A1, 14 days; grade IV B1, 8 days; infection A1, B1Grade I A2-C2, 7 days; grade III C1, 10 days; infection C160 μg/kg BW C1–C2treatment protocolThe patients were treated from September 2004 to March 2007 on protocol GM-ALL-B-ALL/B-NHL 2002 (upon approval by ethical board and written informed consent) and developed severe mucositis. The protocol outline with age-related dose modifications is depicted in Figure 1. Two of 10 patients were treated in the protocol for patients >55 years of age with a slightly reduced MTX dosage (500 mg/m2 compared with 1500 mg/m2). One patient was treated in the ‘elderly protocol’.Figure 1.Prephase: cyclophosphamide 200 mg/m2 d1–5, prednisone 60 mg/m2 d1–5. Cycle A1/A2: rituximab 375 mg/m2 d7 (A2 d77); dexamethasone 10 mg/m2 d8–12 (A2 d78–82); vincristine 2 mg d8 (A2 d78); MTX 1500 mg/m2 (500 mg/m2 > 55 years) d8 (A2 d78); ifosfamide 800 mg d8–12 (A2 d78–82); cytarabine 2 × 150 mg/m2 d11–12 (A2 d81–82); VP16 100 mg/m2 d11–12 (A2 d81–82); cytarabine 40 mg i.th. d8, 12 (A2 d78, 82); MTX 15 mg i.th. d8, 12 (A2 d78, 82); dexamethasone 4 mg i.th. d8, 12 (A2 d78, 82). Cycle B1/B2: rituximab 375 mg/m2 d28 (B2 d98); dexamethasone 10 mg/m2 p.o. d29–33 (B2 99–103); vincristine 2 mg d29 (B2 d99); MTX 1500 mg/m2 (500 mg/m2 > 55 years) d29 (B2 d99); cyclophosphamide 200 mg/m2 d29–33 (B2 99–103); adriamycine 25 mg/m2 d32–33 (B2 d102–103); cytarabine 40 mg i.th. d29, 33 (B2 d99, 103); MTX 15 mg i.th. d29, 33 (B2 d99, 103); dexamethasone 4 mg i.th. d29, 33 (B2 d99, 103). Cycle C1/C2: rituximab 375 mg/m2 d49 (C2 d119); dexamethasone 10 mg/m2 p.o. d50–54 (C2 120–124); vindesine 3 mg/m2 maximum 5 mg d50 (C2 120); MTX 1500 mg/m2 (500 mg/m2 > 55 years) d50 (C2 d120); VP16 250 mg/m2 d53–54 (C2 d123–124); cytarabine 2 × 2 g/m2 (2 × 1 g/m2 > 55 years) d54 (C2 d124). Protocol for elderly patients: Prephase: cyclophosphamide 200 mg/m2 d1–5, prednisone 60 mg/m2 d1–5. Cycle A1/A2/A3: rituximab 375 mg/m2 d7 (A2 d49, A3 d98); dexamethasone 10 mg/m2 d8–12 (A2 d50–54, A3 99–103); MTX 500 mg/m2 d8 (A2 d50, A3 99); ifosfamide 400 mg d8–12 (A2 d50–54, A3 99–103); cytarabine 2 × 60 mg/m2 d11–12 (A2 d53–54, A3 d102–103); VP16 60 mg/m2 d11–12 (A2 d53–54, A3 d102–103); MTX 12 mg i.th. d8 (A2 d50, A3 99). Cycle B1/B2/B3: rituximab 375 mg/m2 d28 (B2 d77, B3 d119); dexamethasone 10 mg/m2 p.o. d29–33 B2 78–82, B3 d120–124); vincristine 1 mg d29 (B2 d78, B3 d120); MTX 500 mg/m2 d29 (B2 d78, B3 d120); cyclophosphamide 200 mg/m2 d29–33 (B2 78–82, B3 120–124); adriamycine 25 mg/m2 d32–33 (B2 d81–82, B3 123–124); MTX 12 mg i.th. d29 (B2 d78, B3 120).Eight of 10 patients received a ‘run-in’ therapy (prephase) of 5 days with prednisone 60 mg/m2 and cyclophosphamide 200 mg/m2 before the cycle A1. Two patients with mediastinal B-cell non-Hodgkin's lymphoma (B-NHL) did not receive any prephase therapy.Patients with mucositis in cycle A1 or B1 received prophylaxis and supportive treatment including morphine, tetracaine-, panthenole and camomile-containing mouthwashes, oral hygiene and amphotericin B suspensions to prevent intestinal candidosis. All patients received recombinant granulocyte colony-stimulating factor (G-CSF) to shorten the time of grade IV neutropenia. Prophylactic oral hygiene, amphotericin B and G-CSF were not changed during the subsequent cycles.grading of mucositisMucositis grade was judged daily by the four-point WHO scale [23]. The observation was carried out by the authors and, to ensure uniformity of judgment, by the first and the last author during the time of mucositis maximum. All these patients developed severe WHO grades III–IV oral mucositis in the cycle A1 (seven of 10) or B1 (three of 10). One of these patients (number 9, Table 1) did not get MTX in cycle A1 because of a surgical bowel intervention.palifermin applicationBecause of their mucositis, all 10 patients obtained palifermin (Kepivance™, Amgen) for 3 days before and another 3 days after the following corresponding chemotherapy cycle (A2 or B2) to reduce the risk of further higher grade mucositis. Prior written informed consent was obligatory. Palifermin dose was 60 μg/kg/day, which is the full dose recommended or a lower daily dose as outlined in Table 1.resultsFigure 2A and B depicts the maximum grade of mucositis observed in chemotherapy cycles A1/B1 without palifermin versus A2/B2 with palifermin support. Evaluated together, there were 16 episodes of severe grade IV and one grade III mucositis in the chemotherapy cycles without palifermin support. In contrast, we have observed only one episode of grade IV, four episodes of grade III and four episodes of grade II mucositis during the chemotherapy cycles with palifermin support (difference P < 0.05, Mann–Whitney–Wilcoxon test).Figure 2.(A) Maximum grade mucositis with and without palifermin in cycles A1 versus A2 (P < 0.05, Mann–Whitney–Wilcoxon test). Numbers in circles refer to patients as given in Table 1. Patient 9 did not receive MTX during cycle A1. (B) Maximum grade mucositis with and without palifermin in cycles B1 versus B2 (P < 0.05, Mann–Whitney–Wilcoxon test). Numbers in circles refer to patients as given in Table 1. Patient 2 received palifermin in cycle B1.The amount of i.v. opioid analgetics could be reduced from a total of 2403 mg morphine in cycle A1 (3380 mg cycle B1) without palifermin to 640 mg in cycle A2 (480 mg cycle B2) with palifermin (difference P < 0.05, Mann–Whitney–Wilcoxon test). The intraindividual dose changes are presented in Figure 3A and B.Figure 3.(A) Total amount of i.v. given morphine (in milligram) with and without palifermin in cycles A1 versus A2 (P < 0.05, Mann–Whitney–Wilcoxon test). Numbers in diamonds refer to patients as given in Table 1. Patient 9 did not receive MTX during cycle A1. (B) Total amount of i.v. given morphine (in milligram) with and without palifermin in cycles B1 versus B2 (P < 0.05, Mann–Whitney–Wilcoxon test). Numbers in diamonds refer to patients as given in Table 1. Patient 2 received palifermin during cycle B1 and developed no higher grade mucositis.Furthermore, the duration of mucositis in patients who acquired a higher grade mucositis despite treatment with palifermin could be slightly reduced. The median duration of grade III/IV mucositis without palifermin was 12.9 days (16 cycles with mucositis) with a range from 7 to 18 days. The median duration of grades III–IV mucositis with palifermin was 11 days with a range from 10 to 13 days (not significant, Mann–Whitney–Wilcoxon test). In this evaluation, patients not developing severe mucositis under palifermin protection were not included.Ten of 10 patients developed a severe infection in cycle A1 or B1 without palifermin. Severe infection is defined as fever (>38.5°C) in neutropenia which had to be treated with empiric systemic antibiotic therapy. In the palifermin-supported chemotherapy cycles, only four of 10 patients showed a severe infection. Interestingly, infection is associated with a higher grade mucositis.Since cycles A1 and A2 contain identical (with the exception of the run-in phase of corticosteroids and cyclophosphamide given to eight of 10 patients before A1) and cycles B1 and B2 contain identical chemotherapy, patients can serve as their own controls for the antimucositis efficacy of palifermin. Figure 4 documents the maximum appearance of mucositis in three patients at identical times following chemotherapy cycles without (part A, C and E) or with palifermin (part B, D and F) support. Only three individual cycle comparisons for two patients (numbers 7 and 9, Figure 2, Table 1) did not show an improvement of mucositis severity by palifermin. Patient number 10 did receive palifermin in cycle C1, but showed a grade III mucositis. However, in the corresponding cycle C2, a mucositis of only grade I was observed.Figure 4.(A) Grade IV mucositis at day 13 after cycle B1 without palifermin support (patient 3, Table 1). (B) Grade I mucositis and hyperkeratosis at day 13 after cycle B2 with palifermin support (patient 3, Table 1). (C) Grade III mucositis at day 11 after cycle B1 without palifermin support (patient 10, Table 1). (D) Grade I mucositis at day 12 after cycle B2 with palifermin support (patient 10, Table 1). (E) Grade IV mucositis at day 11 after cycle B1 without palifermin support (patient 6, Table 1). (F) Grade I mucositis at day 10 after cycle B2 with palifermin support (patient 6, Table 1).As we report on an individual patient series, there was some variation in the course of supportive palifermin treatment concerning individual cases and these are depicted in Table 1.discussionWe report descriptive observation data from 10 patients who were treated in the trial protocol GM-ALL-B-ALL/NHL 2002 or the respective protocol for elderly patients (one patient) and developed severe mucositis. In summary, we observed a considerable effect of palifermin reducing the dosage of i.v. morphine, severity, duration and clinical sequelae (such as infection) of mucositis when similar (A1/A2) or identical (B1/B2) chemotherapy cycles without palifermin support were compared with those with palifermin support in identical patients.Recently, palifermin has been described to reduce the prevalence and severity of oral mucositis in patients with hematologic malignancies undergoing high-dose therapy with autologous peripheral blood stem-cell transplantation [17]. However, there are also standard chemotherapy regimens not requiring stem-cell support, which can be associated with severe mucositis. In a placebo-controlled phase II study published recently, it was demonstrated that palifermin is effective in reducing the incidence of oral mucositis in patients with metastatic colorectal cancer undergoing 5-fluorouracil/calcium folinate chemotherapy [27]. In contrast to these data, a recently published Cochrane review about ‘Interventions for preventing oral mucositis for patients with cancer receiving treatment’ by Worthington et al. [28] on the basis of three randomized trials [17, 29, 30] shows that there is no statistically significant difference between palifermin/repifermin and placebo. It was concluded that recombinant KGF could not be supported or refuted as more or less effective than placebo due to insufficient evidence. However, HD-MTX-based chemotherapy is well known to be associated with high mucositis rates and MTX-based chemotherapy regimen was not included in the Cochrane review [28]. This opens the possibility that there might be a stronger mucositis-preventive effect of palifermin in MTX-based chemotherapy.The cases reported here receiving HD-MTX provide evidence that palifermin might reduce the risk of oral mucositis and its severe side-effects such as infections. Our data lead to the suggestion that the effect is due to palifermin. In cycle A1, the incidence of mucositis could be increased by the application of the run-in therapy. Therefore, it cannot be completely excluded that the decrease in mucositis rate is partly caused by the absence of the run-in therapy in the subsequent cycles. This, however, is not the case in cycles B1 and B2, where treatment is identical. Furthermore, it seems theoretically possible that single patients may tolerate subsequent cycles of chemotherapy with a less severe mucositis than the first cycle. This, however, is not in line with our experience treating patients in the time before accessability of palifermin. With two exceptions (both obtaining the run-in treatment in the first cycles), every patient had a benefit with a reduction in mucositis grade and also shown by the amount of opioid analgetics given during the mucositis period. Having used palifermin for a while in our department, patients started to actively ask for off-label use of this specific drug for mucositis prevention and refused HD-MTX-containing chemotherapy without. Furthermore, our observations suggest the possibility of a dose reduction for palifermin which should be further studied.However, this is a report on a case series with retrospective evaluation and cannot replace prospective, controlled studies which should be carried out before the use of palifermin in HD-MTX regimen can be recommended as standard of care for mucositis prevention in HD-MTX chemotherapy.1.SonisSTMucositis as a biological process: a new hypothesis for the development of chemotherapy-induced stomatotoxicityOral Oncol19983439432.SonisSTThe pathobiology of mucositisNat Rev Cancer200442772843.SonisSTEltingLSKeefeDPerspectives on cancer therapy-induced mucosal injury: pathogenesis, measurement, epidemiology, and consequences for patientsCancer20041009 Suppl199520254.SonisSTThe biological role of nuclear factor—kappaB in disease and its potential involvement in mucosal injury associated with antineoplastic therapyCrit Rev Oral Biol Med2002133803895.ScullyCSonisSDizPDOral mucositisOral Dis2006122292416.RuescherTJSodeifiAScrivaniSJThe impact of mucositis on alpha-hemolytic streptococcal infection in patients undergoing autologous bone marrow transplantation for hematologic malignanciesCancer199882227522817.CostaSFMiceliMHAnaissieEJMucosa or skin as source of coagulase-negative staphylococcal bacteraemia?Lancet Infect Dis200442782868.SezerOEuckerJMetznerBMucositis is associated with increased rate of documented infections and treatment related mortality after high-dose therapy and autologous peripheral stem-cell transplantationProc Am Soc Clin Oncol20001956a(Abstr 216)9.SonisSTOsterGFuchsHOral mucositis and the clinical and economic outcomes of hematopoietic stem-cell transplantationJ Clin Oncol2001192201220510.EltingLSShihYTStiffPJEconomic impact of palifermin on the costs of hospitalization for autologous hematopoetic stem-cell transplant: analysis of phase 3 trial resultsBiol Blood Marrow Transplant20071380681311.FanningSRRybickiLKalaycioMSevere mucositis is associated with reduced survival after autologous stem cell transplantation for lymphoid malignanciesBr J Haematol200613537438112.KeefeDMSchubertMMEltingLSUpdated clinical practice guidelines for the prevention and treatment of mucositisCancer200710982083113.DumontetCSonnetABastionYPrevention of high-dose L-PAM-induced mucositis by cryotherapyBone Marrow Transplant19941449249414.EdelmanMJGandaraDPerezEAPhase I trial of edatrexate plus carboplatin in advanced solid tumors: amelioration of dose-limiting mucositis by ice chip cryotherapyInvest New Drugs199816697515.KuhrerIKuzmitsRLinkeschWTopical PGE2 enhances healing of chemotherapy-associated mucosal lesionsLancet1986162316.FarrellCLRexKLChenJNThe effects of keratinocyte growth factor in preclinical models of mucositisCell Prolif200235Suppl 1S78S8517.SpielbergerRStiffPBensingerWPalifermin for oral mucositis after intensive therapy for hematologic cancersN Eng J Med2004351252590259818.OelmannEHaghguSKulimovaEInfluence of keratinocyte growth factor on clonal growth of epithelial tumor cells, lymphoma and leukemia cells and on sensitivity of tumor cells towards 5-flourouracil in vitroInt J Oncol2004251001101219.KrijanovskiOLHillGRCookeKRKeratinocyte growth factor separates graft-versus-leukemia effects from graft-versus-host diseaseBlood19999482583120.EllisonCANatulikSAFischerJMEffect of recombinant human keratinocyte growth factor (rHuKGF) on the immunopathogenesis of intestinal graft-vs.-host disease induced without preconditioning regimenJ Clin Immunol20042419721121.BlijlevensNSonisSPalifermin (recombinant keratinocyte growth factor-1): a pleiotropic growth factor with multiple biological activities in preventing chemotherapy- and radiotherapy-induced mucositisAnn Oncol20071881782622.BraunSHanselmannCGassmannMGNrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin woundMol Cell Biol2002225492550523.MillerABHoogstratenBStaquetMReporting results of cancer treatmentCancer19814720721424.HoelzerDBaurKHGiagounidisAShort intensive chemotherapy with rituximab seems successful in Burkitt NHL, mature B-ALL and other high grade B-NHLBlood2003102(Abstr 236)25.ReiterASchrappeMZimmermannMA 4-hour i.v. infusion of methotrexate 5 g/sqm is not as efficacious for treatment of advanced B-cell neoplasms of childhood and adolescence than 24 hour i.v. infusion although less toxic. Interim results of trial NHL-BFM 95Blood200198(11 Part 1)26.HölzerDLudwigWDThielEImproved outcome in adult B-cell lymphoblastic leukemiaBlood19968749550827.RosenLSAbdiEDavisIDPalifermin reduces the incidence of oral mucositis in patients with metastatic colorectal cancer treated with fluorouracil-based chemotherapyJ Clin Oncol2006245194520028.WorthingtonHVClarksonJEEdenOBInterventions for preventing oral mucositis for patients with cancer receiving treatmentThe Cochrane Library2007429.FreytesCORatanatharathornVTaylorCPhase I/II randomized trial evaluating the safety and clinical effects of repifermin administered to reduce mucositis in patients undergoing autologous stem cell transplantationClin Cancer Res200410248318832430.MerepolNJSomerRAGutheilJRandomized phase I trial of recombinant human keratinocyte growth factor plus chemotherapy: potential role as mucosal protectantJ Clin Oncol200321814521458 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B3AAA7EFC7FFCAD2D500C2EE40EEC027617B835.txt b/test/dataset/in/resources/corpus/Clean_0B3AAA7EFC7FFCAD2D500C2EE40EEC027617B835.txt new file mode 100644 index 0000000..44a8a22 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B3AAA7EFC7FFCAD2D500C2EE40EEC027617B835.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 0012710.1093/gerona/56.1.M14 Journal of Gerontology: Medical Sciences Prevalence Rate of Urinary Incontinence in Community-Dwelling Elderly Individuals The Veneto Study Maggi Stefania a Minicuci Nadia a Langlois Jean b Pavan Mara a Enzi Giuliano a Crepaldi Gaetano a aCenter on Aging, National Research Council, Padova, Italy bNational Center for Injury Prevention and Control, Centers for Disease Control, Atlanta, Georgia 1 1 2001 56 1 M14 M18 26 6 2000 20 6 2000 The Gerontological Society of America 2001 Background. Urinary incontinence (UI) is a common problem in elderly people, due mainly to functional impairments and concurrent medical diseases. Few studies, however, have assessed the prevalence of UI in noninstitutionalized individuals. The objectives of the present work were to estimate the prevalence of UI in a community-based population of elderly Italians and to determine the associated physical, social, and psychological factors. Methods. A random sample of noninstitutionalized men (n = 867) and women (n = 1531), aged 65 years and older, from the Veneto region of northeastern Italy, were interviewed at home, using an extensive multidisciplinary questionnaire, to assess their quality of life and social, biological, and psychological correlates. Results. The prevalence rate of UI was of 11.2% among men and of 21.6% among women. Among those reporting the condition, approximately 53% of women and 59% of men reported experiencing incontinence daily or weekly. Association of UI was found for participants older than 70 years in both men (odds ratio [OR] 2.49, 95% confidence interval [CI] 1.45–4.28) and women (OR 1.49, 95% CI 1.11–2.02). Three of the medical conditions investigated were associated with increases in the odds in women, namely chronic obstructive pulmonary disease (OR 1.53, 95% CI 1.11–2.12), Parkinsonism (OR 2.27, 95% CI 1.14–4.54), and hip fracture (OR 1.38, 95% CI 1.02–1.88), whereas chronic diarrhea was the only condition associated with UI in men (OR 6.92, 95% CI 2.22–21.5). Participants with a physical disability were two times more likely to report incontinence, and the odds were increased by 50% in women who had sleep disturbances. Conclusions. Incontinence is highly prevalent in the Italian elderly population, and several common chronic conditions are significantly associated with it. Moreover, very few people with incontinence seek health care or are aware of potential treatments. hwp-legacy-fpage M14 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: John E. Morley, MB, BCh URINARY incontinence (UI) is a common problem in elderly people, due mainly to functional impairments and concurrent medical diseases (1)(2). Several studies have determined the prevalence of UI in nursing homes (3)(4) and in the community (5)(6). The reported prevalence rates approximate 50% for people in nursing homes and range between 2% and 55% for adults living in the community, depending on the definition of incontinence, the population characteristics, and the methodological approach (7). The burden of UI on elderly people is evidenced not only by the economic costs but also by other adverse effects on health and quality of life. UI has been estimated to account for 2% of health care costs in the United States and Sweden (8) and is one of the main reasons for the permanent institutionalization of elderly people (9). In Italy, cost estimates are available for patients with dementia and UI: An average of 1000 US$ is spent each year for pads alone, the most commonly used remedy for incontinence in elderly persons (10). The physical complications may be serious and include an increased risk of urinary tract infections, pressure sores and other skin problems, and sleep disturbance. People with UI also report significant and often long-lasting social and psychological effects such as decreased social interaction and greater feelings of depression and isolation (11)(12)(13)(14)(15)(16)(17). The purpose of this cross-sectional study was to estimate the prevalence of UI in a community-dwelling population of elderly Italians and to determine the associated physical, social, and psychological factors. Methods Study Population The details of the methods have been described elsewhere (18). Briefly, the population for the Veneto Study consisted of a random sample of 2700 noninstitutionalized individuals aged 65 years and older, residing in the community on May 1, 1989 in five rural and four urban defined geographic areas of the Veneto region of northeastern Italy. Names and addresses of eligible individuals were obtained from the resident lists maintained by the municipalities, in which all births and deaths are registered within one week of their occurrence. A random sample from each of five age strata (65–69, 70–74, 75–79, 80–84, and 85+ years) was taken, with an over-sampling of those aged 85 years and older to obtain 20% of the total sample in this age category. Approximately 8.4% of the total elderly population of the Veneto Region was aged 85 years and older in 1989 (19). Eighty-nine percent (n = 2402) of people identified as eligible participated in the study. The 298 nonrespondents included those who refused to participate, those who were not found at home after three attempts on different days, or those for whom a proxy refused their participation due to severe dementia or terminal illness (approximately 2%). Only basic demographic data for nonrespondents were collected from a proxy; all data presented herein were provided by the respondents themselves. After exclusion of four people with missing or incorrect demographic data, the final number available for analysis was 2398. Assessment of Participants All participants were administered a comprehensive interview and a brief physical examination in the home. The interview obtained information on sociodemographic characteristics, living arrangements, family composition, social support, income (including family contributions), participation in social activities, self-reported history of medical conditions, health status, physical functioning (including activities of daily living [ADLs] (20) and instrumental activities of daily living) (21), health behaviors, self-rated health, and use of health and social services. Low mental status score was defined as a score of <0.8 for the ratio of the number of questions answered correctly to the number of possible answers (i.e., less than 24 correct answers out of 30, if all questions were applicable), or an adjusted score (number of questions answered correctly divided by the number answered, if some of the questions were not applicable) on the Mini-Mental State Examination (22). Participants whose scores on the Center for Epidemiologic Studies–Depression scale (CES-D) (23) were in the upper quintile (CES-D score >8.8 for men and >24.5 for women) were categorized as having depressive symptomatology. The interviewers were physicians enrolled in a postgraduate geriatric medicine program, trained to administer performance measures in a standardized manner and certified by a scientific training committee. The examination included assessments of height, weight, vision, hearing, and a qualitative assessment of physical performance. Assessment of Urinary Incontinence Participants were asked if they had UI problems and how often they occurred: rarely, 1 to 2 times per month, at least once per week, or every day. Participants were asked whether the incontinence occurred only under strain (i.e., during coughing or sneezing), if they used any remedies, and if they were aware that there are remedies. Health Status Indicators and Medical Conditions Participants were classified as having a disability in the basic ADLs if they reported that they needed help from another person or that they were unable to perform one or more of the following activities: bathing, dressing, getting out of bed, or eating. Mobility disability was classified as “difficult” or “impossible” to climb stairs, to walk 800 m without resting, or as the need for help from another person or the inability to walk across a room. Participants were asked how often they (i) have trouble falling asleep, (ii) have trouble with waking up during the night, (iii) have trouble with waking up too early and not being able to fall asleep again, and (iv) feel really rested when they wake up in the morning. Night awakeners were defined as those who reported often or always waking up during the night. History of arthritis, diabetes, hypertension, chronic respiratory disease, stroke, heart disease, Parkinsonism, and chronic diarrhea was ascertained by asking participants if a doctor had ever told them that they had the condition. Data Analysis To account for the over-sampling of persons aged 85 years and older, the prevalence of urine incontinence was calculated using weights determined by the ratio between the population fraction relative to the 1991 census population and the sampling fraction relative to the age stratum. All statistical analyses were conducted using the SAS statistical analysis package (SAS, Inc, Cary, NC) (24). The association of UI with the sociodemographic, behavioral, health, and other factors was assessed in logistic regression models with incontinence as the dependent variable. Results Table 1 shows the distribution of our sample by the main demographic and health-related characteristics. The final sample consisted of 867 men (mean age 75.2 ± 7.1 years) and 1531 women (mean age 76.9 ± 7.6 years). Women had a significantly lower education level and higher prevalence rates of mental and physical health disorders and obesity than males. A greater percentage of women reported fair or poor self-rated health and used sleep medications more often than men. Conversely, more men than women reported drinking alcohol and cigarette smoking. Data regarding the prevalence rates and the frequency of UI are presented in Table 2 and Table 3 . The prevalence rate was 11.5% among men and ranged from 4.6% for those aged 65 to 69 years to 23.6% for participants aged 85 years and older. In women, the prevalence rate was of 21.6%, ranging from 16.4% to 34.7% for the same age groups. Among the 415 participants with this condition, 36.8% reported that they experienced incontinence every day, and 17.7% reported that they experienced incontinence every week. Approximately 59% of men and 53% of women reported that they experienced UI frequently (every week or day). Although the percentages showed a trend with age, the association was not statistically significant (p = .06). Nevertheless, more than 50% of the affected people aged 65 to 69 years faced this problem frequently versus 67% of subjects aged 85 years and older. Approximately 58% of women and 28.5% of men (data not shown) stated that they experienced incontinence only under stress. Table 4 shows the ORs for the association of all sociodemographic and health-related factors with incontinence by sex. After adjustment for other factors, age 70 years and older, ADL disability, and mobility disability were significantly associated with incontinence in both elderly men and women. Diarrhea showed the strongest association in men but was not significant in women. Chronic obstructive pulmonary disease (COPD), Parkinsonism, hip fracture, and night awakening were significant in women but not in men. Self-rated health was borderline significant in men only. Discussion UI is a highly prevalent condition in elderly persons and deeply affects quality of life because of its negative effects mainly on social interactions, but also on physical health. The prevalence rates of UI in our study are high, similar to those reported in previous studies (25)(26)(27), but seem to be higher compared with those reported in a recent study conducted in Italy (28). However, the different methodologies could easily explain the discrepancies. The study by Bortolotti and colleagues (28) was carried out using a telephone interview, and we believe that it might exclude sicker people and therefore those at higher risk for incontinence and other chronic conditions. Moreover, our survey was carried out by physicians, who left the most sensitive questions, such as those related to incontinence, toward the end of the personal interview, when a more relaxed interaction with the participants was established, which would enhance the reliability of their answers. We confirmed a higher prevalence of UI among women than men and noted that it represents a severe problem for most of them, given that about half of those reporting incontinence experience it every day or more than once a week. The increasing prevalence with age has been also found in other studies (7), and this could be associated with age-related conditions, such as prostatic hyperplasia among men (29). Data on the presence of diseases were self-reported by the participants, and therefore we did not ask information about the prevalence of prostatic hyperplasia because of the high probability of underreporting for that condition. Age, parity, and menopausal status with the consequent incompetence of the internal urethral sphincter could be determinants of stress incontinence among elderly women (30), and, indeed, its frequency was twice as much as in men. Physical disability was a positive risk factor for functional incontinence in this population, and problems in mobility could easily explain this association. We also found a strong association with Parkinsonism and hip fracture, easily explained both in terms of the neurological impairments and mobility problems of these patients (31). The higher risk of incontinence in women with COPD could be due to the effect of increased abdominal pressure when coughing and the consequent stress incontinence, as already described in previous reports (32). Night awakening was associated with incontinence in women and not in men. This was surprising, given that prostatic problems and need to void urine usually lead to frequent night awakening and to nocturia among elderly men. It is possible that the higher prevalence of physical disability among women was responsible for their functional incontinence. A recent study (5) has underlined that few community-dwelling elderly persons seek health care for UI in spite of its high prevalence in this group. Given that symptomatic improvement or a cure would be possible in many cases, a better evaluation of this condition is needed. This would require increasing the sensitivity and knowledge of the population about these problems and about potential medical and behavioral interventions. It is certain that most people, particularly women, tend to use pads as the only solution to the problem, ignoring completely that other more appropriate and definite approaches could be very effective. Of course, treatment of incontinence requires attention to the general health status of an individual (1)(33). Treatment of urinary infections or estrogen deficiency or prostatic problems should be the first step when these conditions are present. Pelvic floor exercise could be very effective for patients with stress incontinence, while behavioral interventions, such as decreasing fluid intake, particularly caffeine-rich drinks, could be general measures applied to all patients (34)(35). This study has some limitations. First, the use of cross-sectional data does not allow for the assessment of the cause-and-effect relationship between incontinence and health conditions. Second, information on medical conditions and physical function was self-reported. However, several studies support the reliability of information on health status reported by the elderly population (36)(37). Moreover, the prevalence of diseases investigated was comparable to that reported in other studies (38), and the association with incontinence was consistent with previously reported findings in different populations (1)(2)(3)(4)(5)(6). This study has some peculiar strengths, such as the high response rate and the representativeness of the sample, that allow the generalizability of our results to the elderly population of Italy. In conclusion, incontinence is probably more frequent than generally known among community-dwelling individuals and has a negative impact on their quality of life and general health status. Given that symptomatic improvement or treatment is possible in many cases, physicians should always ask their elderly patients about incontinence and then provide appropriate evaluation and management of it. Table 1. Sociodemographic and Health-Related Characteristics by Sex (Veneto Study, 1989) p Value Men (n = 867) Women (n = 1531) Difference Between Men and Women Age Trend for Men Age Trend for Women Demographics Mean Age, y (SD) 75.2 (7.1) 76.9 (7.6) <.001 Marital Status (%) <.001 <.001 <.001 Married 78.1 32.0 Not married 5.9 12.5 Widowed 15.1 54.1 Divorced 0.9 1.4 Education (%) <.001 .156 <.001 ≤5 y 70.6 82.5 6–8 y 14.8 10.7 9–13 y 9.7 5.7 University 4.9 1.1 Mental Health (%) <.001 <.001 <.001 Low mental status\|[dagger]\| 18.8 27.1 Depressive symptomatology (%) <.001 .522 .028 Low CES-D score\|[Dagger]\| 8.8 24.5 Physical Health (%) Disability status Mobility disability\|[sect ]\| 28.5 41.5 <.001 <.001 <.001 ADL disability\|[Verbar]\| 12.4 17.6 <.001 <.001 <.001 Body mass index\|[para ]\| <.001 <.001 <.001 ≤20 4.1 4.8 >20 and <26 46.1 38.9 ≥26 and <31 37.8 37.4 ≥31 and <35 10.5 15.0 ≥35 1.5 3.9 Self-rated health <.001 .017 0.837 Excellent/good 62.6 50.3 Fair/poor 37.4 49.7 Behavioral Factors (%) Use of sleep medications <.001 .219 .702 Sometimes 6.6 11.3 Often/always 13.9 24.2 Alcohol use <.001 .157 .412 Abstainers 3.6 16.2 Past users 10.5 15.2 Current users 86.0 68.5 Cigarette smoking <.001 <.001 <.001 Never smokers 17.6 81.1 Former smokers 57.9 10.9 Current smokers 24.4 8.0 Note: CES-D = Center for Epidemiologic Studies–Depression Scale; ADL = activities of daily living. \|[dagger]\| Mini Mental State Examination score ratio ≤0.8. \|[Dagger]\| CES-D score >80th percentile. \|[sect ]\| Reported climbing stairs or walking 800 m without resting as difficult or impossible. \|[Verbar]\| Reported needing help from another person or unable to perform one or more of the following activities: bathing, dressing, getting out of bed, eating, or walking across a room. \|[para ]\| Weight (kg)/height (m)2. Table 2. Prevalence of Urinary Incontinence by Age and Sex 65–69 y 70–74 y 75–79 y 80–84 y ≥85 y Total Men 4.6 12.6 12.3 22.2 23.6 11.5 Women 16.4 17.8 24.8 23.9 34.7 21.6 Note: Data are given as percentages. Table 3. Frequency of Urinary Incontinence by Age and Sex 65–69 y 70–74 y 75–79 y 80–84 y ≥85 y Total Men Women Rarely 34.6 44.6 24.7 33.3 20.0 31.6 26.5 33.3 1 to 2 times a mo 13.0 10.8 18.0 14.1 12.6 13.9 14.5 13.7 Every week 20.2 13.4 22.9 15.4 14.1 17.7 13.6 19.0 Every day 32.2 31.2 34.4 37.2 53.3 36.8 45.4 34.0 Note: Data are given as percentages. Table 4. Odds Ratios for the Association of Sociodemographic and Health-Related Factors With Incontinence, by Sex Factor Odds Ratio 95% CI p Value Men Age ≥70 y 2.49 1.45–4.28 .0009 Diarrhea 6.92 2.22–21.50 .0008 Comorbidity 1.52 0.89–2.61 .12 ADL disability 2.31 1.23–4.32 .009 Mobility disability 2.11 1.16–3.85 .01 Self-rated health 1.69 0.997–2.88 .05 Women Age ≥70 y 1.49 1.11–2.02 .008 COPD 1.53 1.11–2.12 .01 Parkinsonism 2.27 1.14–4.54 .02 Hip fracture 1.38 1.02–1.88 .04 ADL disability 1.75 1.22–2.52 .002 Mobility disability 1.81 1.32–2.49 .0003 Night awakening 1.48 1.11–1.97 .007 Note: CI = confidence interval; ADL = activities of daily living; COPD = chronic obstructive pulmonary disease. Address correspondence to Stefania Maggi, MD, MPH, Center on Aging, National Research Council, Clinica Medica 1°, University of Padua, Via Giustiniani, 2, 35128 Padova, Italy. 1 Sirls LT, Rashid T, 1999. Geriatric urinary incontinence. Geriatr Nephrol Urol9:87-99. 2 Koyama W, Koyanagi A, Mihara S, et al. 1998. Prevalence and conditions of urinary incontinence among the elderly. Methods Inf Med37:151-155. 3 Brandeis GH, Baumann MM, Hossain M, et al. 1997. The prevalence of potentially remediable urinary incontinence in frail older people: a study using the Minimum Data Set. J Am Geriatr Soc45:179-184. 4 Ouslander JG, Kane RL, Abrass IB, 1982. Urinary incontinence in the elderly nursing home patients. JAMA248:1194-1198. 5 Roberts RO, Jacobsen SJ, Rhodes T, et al. 1998. Urinary incontinence in a community-based cohort: prevalence and healthcare-seeking. J Am Geriatr Soc46:467-472. 6 Herzog AR, Fultz NH, 1990. Prevalence and incidence of urinary incontinence in community-dwelling population. J Am Geriatr Soc38:273-281. 7 Thom D, 1998. Variation in estimates of urinary incontinence prevalence in the community: effects of differences in definition, population characteristics, and study type. J Am Geriatr Soc46:473-480. 8 Hu TW, 1990. Impact of urinary incontinence on health care costs. J Am Geriatr Soc38:292-295. 9 Baker DI, Bice TW, 1995. The influence of urinary incontinence on publicly financed home care services to low-income elderly people. The Gerontologist35:360-369. 10 Levorato A, Rozzini R, Trabucchi M. I costi della vecchiaia. Ed. Bologna, Italy: Il Mulino;1994. 11 Dugan E, Cohen SJ, Bland DR, et al. 2000. The association of depressive symptoms and urinary incontinence among older adults. J Am Geriatr Soc48:413-416. 12 Naughton MJ, Wyman JF, 1997. Quality of life in geriatric patients with lower urinary tract dysfunction. Am J Med Sci314:219-227. 13 Ouslander JG, 1997. Aging and the lower urinary tract. Am J Med Sci314:214-218. 14 Chutka DS, Fleming KC, Evans MP, Evans JM, Andrews KL, 1996. Urinary incontinence in the elderly population. Mayo Clin Proc71:93-101. 15 Busby Whitehead JM, Johnson TM, 1998. Urinary incontinence. Clin Geriatr Med14:285-296. 16 Fiers SA, 1996. Breaking the cycle: the etiology of incontinence dermatitis and evaluating and using skin care products. Ostomy-Wound-Manage42:32-34. 17 Wyman JF, Harkins SW, Fantl JA, 1990. Psychosocial impact of urinary incontinence in the community-dwelling population. J Am Geriatr Soc38:282-288. 18 Maggi S, Bush TL, Enzi G, Crepaldi G, 1991. Quality of life among the elderly in Veneto, Italy: a cross-national study. Vital Health Stat 56:211-214. 19 National Institute of Statistics. Le regioni in cifre. Rome, Italy: National Institute of Statistics;1990:39. 20 Katz SC, Ford AB, Moskowitz RW, Jackson BA, Jaffe MW, 1963. Studies of illness in the aged. The Index of ADL: a standardized measure of biological and psychosocial function. JAMA185:914-919. 21 Lawton MP, Brody EM, 1982. Assessment of older people: self-maintaining and instrumental activities of daily living. J Gerontol37:91-99. 22 Folstein MF, Folstein SE, McHugh PR, 1975. “Mini-Mental State”: a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res12:189-198. 23 Radloff LS, 1977. The CES-D Scale: a self-report depression scale for research in the general population. Appl Psych Measur1:385-401. 24 SAS Institute1989. SAS/STAT User's Guide, Version 6 4th ed. SAS Institute, Inc, Cary, NC. 25 Temml C, Haidinger G, Schmidbauer J, Schatzl G, Madersbacher S, 2000. Urinary incontinence in both sexes: prevalence rates and impact on quality of life and sexual life. Neurourol Urodyn19:259-271. 26 Gavira Iglesias FJ, Caridad y Ocerin JM, Perez del Molino Martin J, et al. 2000. Prevalence and psychosocial impact of urinary incontinence in older people of a Spanish rural population. J Gerontol Med Sci55A:M207-M214. 27 Ushiroyama T, Ikeda A, Ueki M, 1999. Prevalence, incidence, and awareness in the treatment of menopausal urinary incontinence. Maturitas33:127-132. 28 Bortolotti A, Bernardini B, Colli E, et al. 2000. Prevalence and risk factors for urinary incontinence in Italy. Eur Urol37:30-35. 29 Johnson TM, Ouslander JG, 1999. Urinary incontinence in the older man. Med Clin North Am83:1247-1266. 30 Karram MM, Partoll L, Bilotta V, Angel O, 1997. Factors affecting detrusor contraction strength during voiding in women. Obstet Gynecol90:723-726. 31 Nakayama H, Jorgensen HS, Pedersen PM, Raaschou HO, Olsen TS, 1997. Prevalence and risk factors of incontinence after stroke: The Copenhagen Study. Stroke28:58-62. 32 DuBeau CE, 1996. Interpreting the effect of common medical conditions on voiding dysfunction in the elderly. Urol Clin North Am23:11-18. 33 Milsom I, 1996. Rational prescribing for postmenopausal urogenital complaints. Drugs Aging9:78-86. 34 Iqbal P, Castleden CM, 1997. Management of urinary incontinence in the elderly. Gerontology43:151-157. 35 Gallo ML, Fallon PJ, Staski DR, 1997. Urinary incontinence: steps to evaluation, diagnosis, and treatment. Nurse Pract22:21-24. 36 Bush TL, Miller SR, Golden A, 1989. Self-report and medical report of selected chronic conditions in the elderly. Am J Public Health79:1554-1556. 37 Kehoe R, Wu SY, Leske MC, Chylack LT, 1994. Comparing self-reported and physician-reported medical history. Am J Epidemiol139:813-818. 38 National Institute of Statistics. Indagine multiscopo sulle famiglie, IV e V ciclo. Roma, Italy;1994. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B3B0444849AA7F246C15872661128DBD7584D0E.txt b/test/dataset/in/resources/corpus/Clean_0B3B0444849AA7F246C15872661128DBD7584D0E.txt new file mode 100644 index 0000000..67c0685 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B3B0444849AA7F246C15872661128DBD7584D0E.txt @@ -0,0 +1 @@ +]>HEARES3362S0378-5955(99)00194-X10.1016/S0378-5955(99)00194-XElsevier Science B.V.Fig. 1Example of intracellular calcium concentration changes in a single type I sensory cell induced by iontophoretic applications (arrows) of glutamate (Glu), glycine (Gly), NMDA, aspartate (Asp) and NaCl. Significant, rapid and transient increases in the concentration of intracellular calcium were observed for Glu, Gly, NMDA and Asp. No significant change was induced by NaCl.Fig. 2Effect of 7-chlorokynurenate (7CK) on the calcium response to glycine. The presence of 7CK (10 μM) reduced the calcium response induced by glycine application by about 60% (A and B). APV (10 μM), a competitive antagonist of the NMDA receptor, did not affect the calcium response induced by glycine (B). The addition of 7CK (10 μM) and strychnine (50 μM) together resulted in a larger decrease in the calcium response to glycine stimulation (A).Fig. 3Effect of strychnine, a specific antagonist of the glycine Cl−- channel receptor. The presence of strychnine (50 μM) decreased the calcium response to glycine stimulation by about 60%. Strychnine caused no significant decrease in the calcium response induced by glutamate.Fig. 4Effect of Cl−-free gluconate medium. The presence of gluconate (137 mM) decreased the calcium response induced by glycine. In the same condition, there was no significant decrease in the calcium response induced by glutamate application. The addition of gluconate and 7-chlorokynurenate (7CK, 10 μM) together resulted in a larger decrease in the calcium response induced by glycine stimulation.Fig. 5Effect of chelating Ca2+ in the medium by the addition of EGTA. The calcium response induced by glycine was reduced by adding EGTA (1.5 mM) to the medium. When the cells returned to normal medium, the calcium peak level tended toward the typical calcium levels induced by glycine application.Fig. 6Schematic drawing, representing the putative presence of NMDA and glycine receptors on vestibular type I sensory cells and the hypothesis of a peripheral control of the sensory cell activity by the afferent nerve calyx. An activation of these receptors by glycine could induce changes in [Ca2+]i. The increase in [Ca2+]i could also be due to the activation of ionotropic and metabotropic glutamate receptors after release of glutamate.Table 1Analysis of calcium responses to Glu, Gly, NMDA, Asp and NaCl applications in isolated type I sensory cellsGluGlyNMDAAspNaClHair cells(n)2026557Baselineratio 340/3800.77±0.100.77±0.090.76±0.030.78±0.030.76±0.05Peakratio 340/3801.2±0.231.19±0.221.02±0.131.15±0.070.76±0.06Time to peak(s)7.36±1.219.15±2.347.00±1.418.20±1.64Regulation time(s)19.09±3.5920.58±5.0415.08±3.8719.40±3.36Increase/base(%)565534470[Ca2+]i are means of the fluorescence signal of the whole cell soma. The baseline [Ca2+]i values (mean±S.D.) and the response peak values (mean±S.D.) are expressed as the 340 nm/380 nm fluorescence ratio. The time course of the increase [Ca2+]i was rapid, and then returned to basal levels. The estimated time-to-peak and regulation time are given in seconds (mean±S.D.). The nuber of type I sensory cells (n) tested for each agent is indicated.Table 2Calcium levels following glycine stimulation in the presence of 7-chlorokynurenate (7CK, 10 μM), a selective antagonist of the glycine site on NMDA receptor and of APV (10 μM), a competitive antagonist for the glutamate site on NMDA receptorGluGlu+APVGlyGly+APVGly+7CKNMDANMDA+APVHair cells(n)7171711Baselineratio 340/3800.71±0.100.70.71±0.100.70.71±0.100.70.7Peakratio 340/3801.16±0.110.951.09±0.161.280.87±0.101.030.75Time to peak(s)8±157.14±1.0776.83±1.1755Regulation time(s)17.67±3.061019.71±2.431917.67±3.14165Increase/base(%)6333548022455Decrease/specific(%)4806090The [Ca2+]i baseline values (mean±S.D.) and peak response values (mean±S.D.) are expressed as the 340 nm/380 nm fluorescence ratio. The values are expressed as in Table 1. The percentage decrease was evaluated as the ratio of the amplitude of the increase in [Ca2+]i in the presence of the antagonist to that in the presence of glycine alone.Table 3[Ca2+]i changes following glutamate and glycine stimulations in the presence of strychnine (50 μM)GluGlu+strychn.GlyGly+strychn.Gly+strychn.+7CKHair cells(n)62664Baselineratio 340/3800.65±0.060.63±0.030.65±0.060.65±0.060.66±0.06Peakratio 340/3800.98±0.080.96±0.060.94±0.110.76±0.090.72±0.04Time to peak(s)6.33±1.157.5±0.716.83±0.756.83±1.946.25±0.83Regulation time(s)19.33±3.0621±1.4116.17±2.3217±3.5817.25±2.38Increase/base(%)5148451812Decrease/specific(%)66073The values are expressed as in Table 1. The percentage decrease in the calcium response is calculated as the ratio of the peak calcium level in the presence of strychnine to that following glutamate or glycine stimulation alone. The addition of both strychnine (50 μM) and 7CK (10 μM) resulted in a larger decrease, by about 73%, in the calcium response induced by glycine application.Table 4[Ca2+]i changes in Cl−-free gluconate mediumGluGlu+glucoGlyGly+glucoGly+gluco+7CKGly after rinseHair cells(n)336613Baselineratio 340/3800.71±0.060.71±0.060.71±0.060.71±0.060.60.68±0.03Peakratio 340/3801.17±0.100.96±0.251.31±0.160.96±0.140.81.23±0.16Time to peak(s)8±07.33±1.159.5±2.178±1.2677.67±2.08Regulation time(s)21.33±3.2118±4.5823.33±4.520.17±6.052622.33±3.21Increase/base(%)643484351373Decrease/specific(%)19588413Glutamate or glycine were applied in normal or Cl−-free gluconate medium. The values are expressed as in Table 1. After rinsing with normal medium, the peak calcium level tended to be similar to that in control after glycine stimulation. The concomitant absence of Cl− and presence of 7CK resulted in a larger decrease in the calcium response to glycine than the one obtained in the absence of Cl− alone.Table 5[Ca2+]i after glycine application in the presence of EGTA (1.5 mM), a specific calcium chelator, and after rinsing with normal mediumGlyGly+EGTAGly after rinseHair cells(n)444Baselineratio 340/3800.67±0.090.54±0.040.67±0.09Peakratio 340/3800.99±0.280.57±0.180.81±0.12Time to peak(s)7.25±0.965.50±0.716.50±2.12Regulation time(s)17.25±2.0619±1.4117.50±0.71Increase/base(%)47621The values are expressed as in Table 1.Glycine induced calcium concentration changes in vestibular type I sensory cellsGinaDevau*gdevau@univ.montp2.frINSERM U432, Université Montpellier II, place Eugène Bataillon, 34095 Montpellier Cedex 5, France*Tel.: +33 4 67 14 48 30; Fax: +33 4 67 14 36 96AbstractGlutamate is the neurotransmitter of the synapse between vestibular type I hair cells and the afferent nerve calyx. This calyx may also be involved in local feedback, which may modify sensory cell activity via N-methyl-D-aspartate (NMDA) receptors. Glycine is the co-agonist of glutamate in NMDA receptor activation. Both agents have been detected by immunocytochemistry in the nerve calyx. Glutamate and NMDA stimulations cause changes in the intracellular calcium concentration ([Ca2+]i) of isolated type I sensory cells. We investigated the effect of glycine stimulation on [Ca2+]i in guinea pig type I sensory cells by spectrofluorimetry with fura-2. Glycine application to isolated type I sensory cells induced a rapid and transient increase in [Ca2+]i. The fluorescence ratio increased by 55% above the resting level. The peak was reached in 9 s and the return to basal level took about 20 s. A specific antagonist of the glycine site on NMDA receptors, 7-chlorokynurenate (10 μM), decreased the calcium response to glycine by 60%. Glycine may activate NMDA receptors. Glycine may also activate the strychnine-sensitive glycine receptor-gated channel. Strychnine (50 μM) decreased the calcium response to glycine by 60%. Thus, glycine probably induces calcium concentration changes in type I vestibular sensory cells via NMDA receptors and/or glycine receptors.KeywordsN-Methyl-D-aspartateGlycineCalciumVestibular sensory cellGuinea pig1IntroductionIn mammals, vestibular sensory cells are mechano-sensory cells, classified as type I or type II according to morphological criteria (Wersäll, 1956; Wersäll and Bagger-Sjöback, 1974). Type I sensory cells are surrounded by an afferent nerve calyx. Type II sensory cells are contacted only by button-shaped afferent endings. Physiological and pharmacological studies have shown that glutamate is the principal excitatory neurotransmitter at the cytoneuronal synapse between sensory cells and primary afferent nerve fibers, in vestibular hair cells (Annoni et al., 1984; Drescher et al., 1987; Drescher and Drescher, 1992; Guth et al., 1988; Soto and Vega, 1988; Zucca et al., 1992) and in cochlear hair cells (Anson and Ashmore, 1994; Kataoka and Ohmori, 1996). Different glutamate receptor types are involved in vestibular neurotransmission between the sensory cells and the primary afferent neurons. Non-N-methyl-D-aspartate (NMDA) receptors have been detected mostly by electrophysiological and pharmacological studies in the vestibular system of batracians (Soto and Vega, 1988; Prigioni et al., 1990, 1994), although NMDA receptors have also been shown to be present (Zucca et al., 1993; Soto et al., 1994). In mammals, immunocytochemical and in situ hybridization studies have implicated the GluR2/R3 and GluR4 subunits of AMPA receptors (Demêmes et al., 1995; Niedzielski and Wenthold, 1995; Rabejac et al., 1997), and the NR1 and NR2A–D subunits of NMDA receptors in vestibular neurotransmission (Fujita et al., 1994; Niedzielski and Wenthold, 1995). Moreover, presynaptic glutamate receptors may also interact as autoreceptors (Valli et al., 1985; Prigioni et al., 1990; Guth et al., 1991, 1998b). The presence of NMDA receptors has been detected on isolated type I sensory cells from guinea pig by calcium spectrofluorimetry with fura-2 (Devau et al., 1993). Vestibular hair cell neurotransmission has been reviewed by Guth et al. (1998a).Type I sensory cells have the particularity that they are surrounded by an afferent nerve calyx. Various studies support the hypothesis of a local control of type I sensory cell activity by this afferent nerve calyx (Scarfone et al., 1988, 1991). In particular, the calyx contains synaptic-like microvesicles and proteins involved in the synaptic vesicle cycle (synaptophysin, synapsin I and rab3a, Dechesne et al., 1997) suggesting that neuroactive substances may be released. This local control could involve glutamate (Demêmes et al., 1990) and/or a neuropeptide, substance P (Scarfone et al., 1996).The NMDA receptors on isolated type I sensory cells may act as presynaptic receptors in the active zone of neurotransmitter release or may be involved in the local control by the afferent nerve calyx. Glutamate and glycine are the co-agonists that activate NMDA receptors (Johnson and Ascher, 1987; Langosch et al., 1988; Corsi et al., 1996). Glycine and glutamate have both been detected by immunochemistry, co-localized in the thick calyceal afferent fibers of the vestibular epithelium (Reichenberger and Dieringer, 1994; Straka et al., 1996a,b; Bäurle et al., 1997). Glycine may have a modulatory effect: possibly co-released with glutamate from the nerve calyx, glycine may activate NMDA receptors or act independently on putative glycine receptors.To test whether there was local control of sensory cells involving the afferent nerve calyx, we investigated the action of glycine on isolated type I sensory cells of guinea pig by measuring variations in intracellular calcium concentration ([Ca2+]i) by spectrofluorimetry with the sensitive dye fura-2. We show that glycine can act on at least two different sites: the glycine site of the NMDA receptor selectively antagonized by 7-chlorokynurenic acid (Kemp et al., 1988; Kemp and Leeson, 1993), and the glycine receptor, which is strychnine-sensitive and highly permeable to chloride (Van den Pol and Gorcs, 1988; Vandenberg et al., 1992; Zafra et al., 1997; Breitinger and Becker, 1998).2Materials and methods2.1Vestibular type I sensory cell preparationIsolated vestibular sensory cells were prepared as previously described (Devau et al., 1993). Briefly, young pigmented guinea pigs (200–250 g) were anesthetized and then decapitated. The labyrinths were removed, opened and the cristae ampullaris quickly dissected in Hanks’ balanced salt solution (HBSS, Sigma, France). This medium contained 137 mM NaCl, 5.4 mM KCl, 1.7 mM Ca2+, 0.4 mM MgSO4, 0.31 mM Na2HPO4, 0.46 mM KH2PO4 and 5.5 mM D-glucose (Sigma, France). HBSS solution was buffered to pH 7.4 with 5 mM HEPES and adjusted to 290 mOsm. Hair cells were dissociated enzymatically by incubating the sensory epithelium in HBSS containing collagenase (1 mg/ml, Sigma, France) for 10 min at room temperature (20°C). After three rinses in HBSS, hair cells were mechanically dissociated by gentle trituration with fine iron microelectrodes. The isolated hair cells were then transferred onto slides coated with poly-L-lysine (0.5 mg/ml, Sigma, France).Type I hair cells were identified by their characteristic amphora shape, which was preserved after isolation. An elongated neck region separated the spherical basal region (containing the nucleus) from the cuticular plate, into which the intact hair bundle was inserted. The type I sensory cells were selected on the following criteria: smooth plasmalemma and no granules within the cytoplasm. This morphological examination also verified that there was no shape abnormality, for example turgescent swelling or plasmolytic shrinking due to osmotic pressure imbalance. This preparation was used for a patch clamp study (Griguer et al., 1993).The care and the use of animals in this study were approved by the French Ministère de l’Agriculture et de la Forêt (authorization number 04889).2.2Calcium measurements[Ca2+]i was measured using the fluorescent dye fura-2. The cells were loaded with 2.5 μM fura-2/AM (Molecular Probes, Eugene, OR, USA) in HBSS containing 0.02% pluronic-DMSO for 30 min at room temperature. Experiments were performed with an inverted microscope (Axiovert 10, Zeiss, Le Pecq, France) equipped with a fast fluorescence photometer controlled by MSP 69/AIS. Excitation light was provided by a xenon lamp (XBO 75 W) and was passed through filters to select the two excitation wavelengths, 340 and 380 nm. Light at both excitation wavelengths was dimmed by about 90% by a neutral density filter (green UG11) to protect the cells from phototoxicity and bleaching. The emission signals were passed through a 510 nm narrow bandpass filter. A circular 10 μm diaphragm was used to limit fluorescence measurement to a field containing a single sensory cell and centered on the soma. Each fluorescence ratio value was determined by an 80 ms time resolution of the measurement. Changes in fluorescence ratio (free dye 340 nm/bound dye 380 nm) were used to estimate changes in free cytosolic calcium concentration as previously described (Grynkiewicz et al., 1985).2.3Pharmacological applications2.3.1Iontophoretic application of glutamate, NMDA, aspartate and glycineIsolated sensory cells were stimulated by focal iontophoretic application of various agents. Glutamate, NMDA, aspartate and glycine were applied via a micropipette with the tip situated about 2 μm from the basolateral hair cell membrane. Microelectrodes were filled with one of the following solutions: glutamate (1 M, pH 8), NMDA (0.4 M, pH 8), aspartate (1 M, pH 8) and glycine (1 M, pH 8) (Sigma, France). The tip diameter of the micropipettes was less than 1 μm and their resistance when they were filled with any of the various solutions was about 25 MΩ. Electrolytes were ejected using a negative pulse (−3 μA) lasting 5 s. Breaking currents of opposite polarity were used to prevent cation leakage from iontophoretic electrodes. Control tests of the iontophoretic procedure were performed by ejection of Cl− using a negative current or of Na+ using a positive current from microelectrodes filled with sodium chloride (NaCl, 1 M).The electric charge ejected through the pipette was estimated from the Faraday equation Q=It/F where I is the amplitude of the current applied, t the duration of the application and F the Faraday constant (96 500 C). After ejection, the diffusion of the agent depends on several factors including the viscosity of the medium and the size of the molecule.2.3.2Microperfusion of glutamate and glycine antagonistsAntagonist microperfusion was started 30 s before stimulation with glutamate or glycine. Antagonists were dissolved in HBSS at a concentration of 50 μM for strychnine (Sigma, France) and 10 μM for 7-chlorokynurenate (Sigma, France) and DL-2-amino-5-phosphonovaleric acid (APV, Sigma, France). Microperfusion of antagonist alone did not affect the resting calcium concentration.2.4Data analysisCells loaded with fura-2 responded to the application of glutamate agonists or glycine by an increase in fluorescence intensity ratio corresponding to an increase in [Ca2+]i. The fluorescence ratio is expressed in terms of molarity using the equation of Grynkiewicz et al. (1985): [Ca2+]i=(R−Rmin/Rmax−R)αKD, where Rmin=0.4; Rmax=11.3; α=11.9; KD=224 nM.Fluorescence ratio changes are presented rather than changes in absolute [Ca2+]i because many factors (e.g. viscosity of the medium, diffusion, etc.) can affect the absolute [Ca2+]i. The mean basal [Ca2+]i was calculated for all the cells tested in the different experiments. Data are expressed as the peak increase in [Ca2+]i from initial basal levels. The calculated mean amplitude and duration of the calcium responses of each hair cell were averaged for each agonist and antagonist tested. Values are expressed as means±S.D.3Results3.1Calcium concentration changes induced by glutamate, glutamate agonists and glycine in type I vestibular sensory cellsThe calcium fluorescence ratio for type I sensory cells was 0.75±0.09 (n=40) at rest, corresponding to a calcium concentration of 88.4 nM. This value is consistent with the satisfactory maintenance of viable cells in vitro. Applications of glutamate, NMDA and aspartate induced rapid and transient increases in fluorescence ratio of 56, 34 and 47% respectively (Fig. 1). Glycine application induced a similar increase of 55% (Fig. 1). The delay between application and response was about 1 s. The peak value was reached in about 9 s and the return to resting level took about 20 s, indicating that regulatory processes were occurring (Table 1). The shapes of response curves were similar for all cells tested.The specificity of the calcium response induced by iontophoretic applications was checked by ejection of a different anion (Cl−) or cation (Na+). Neither caused any change in fluorescence ratio (Fig. 1).3.2Effect of 7-chlorokynurenic acid on the calcium response to glycine in sensory cellsTwo consecutive applications of glycine, separated by a rinse of 5 min, induced similar calcium responses (Fig. 2A,B), with similar profiles and peak amplitudes.A selective antagonist of the glycine site on NMDA receptors, 7-chlorokynurenate (10 μM), reduced the calcium response evoked by glycine stimulation by about 60% (Fig. 2A,B). The presence of APV (10 μM), a competitive antagonist of the glutamate site on the NMDA receptor, had no significant effect on the calcium response evoked by glycine application (Fig. 2B), whereas the presence of APV decreased the calcium response induced by glutamate by 48% and that induced by NMDA by 90% (Table 2).3.3Effect of strychnine on the calcium response induced by glycine in type I sensory cellGlycine may also activate the ionotropic glycine receptor, which is selectively antagonized by strychnine. The calcium response evoked by glycine was therefore evaluated in the presence of strychnine (50 μM): it was inhibited by 60% (Fig. 3, Table 3). The calcium response induced by glutamate application was not affected by the presence of strychnine (Table 3).Strychnine and 7-chlorokynurenate, applied together, reduced the calcium response induced by glycine by 73% (Fig. 2, Table 3).3.4Effect of extracellular chloride ions on the calcium response induced by glycineThe ionotropic glycine receptor is selectively permeable to chloride ions. We tested whether there was a relationship between chloride influx and changes in [Ca2+]i. The chloride ions of the medium were replaced with gluconate which cannot pass through the glycine receptor channel or the plasmalemma. In the presence of sodium gluconate (137 mM), the calcium resting level was slightly lower (Fig. 4) and the calcium response evoked by glycine was 58% smaller (Fig. 4, Table 4). The addition of both gluconate and 7-chlorokynurenate decreased the calcium response to glycine by 84% (Fig. 4, Table 4).3.5Effect of extracellular calcium ions on the calcium response induced by glycineWe tested whether external calcium affects the changes in calcium concentration induced by glycine by adding a specific calcium chelator EGTA (1.5 mM) to the medium (HBSS). In the presence of EGTA, calcium resting levels in the cell decreased by about 20% and the increase in calcium variation by glycine was only 6% of the basal calcium level measured in presence of EGTA (Fig. 5, Table 5). Therefore, extracellular calcium seemed to be the major source of the calcium concentration changes.4Discussion4.1Calcium concentration changes induced by glycine in type I vestibular sensory cellVestibular sensory cells are mechano-transducers, encoding and sending sensory messages by the afferent nerve to the vestibular nuclei. In isolated type I vestibular sensory cells, the low and stable [Ca2+]i before each stimulation indicated that the cells in our preparations were not altered or excited. The [Ca2+]i in type I sensory cells was similar to that observed in other cells in vitro such as cochlear ganglion neurons (Harada et al., 1994), vestibular ganglion neurons (Rabejac et al., 1997), hippocampal neurons (Murphy and Miller, 1988), and Purkinje neurons (Hockberger et al., 1989).Glycine application induced a rapid and transient increase in [Ca2+]i. Increases in [Ca2+]i were also evoked by glutamate and by two NMDA receptor agonists, aspartate and NMDA. The response curve following glycine stimulation was similar to those obtained after glutamate and aspartate stimulation. After stimulation, [Ca2+]i returned to basal levels. The [Ca2+]i is regulated by processes involving calcium binding proteins, the Ca-ATPase pump in the plasmalemma and intracellular membrane of the endoplasmic reticulum, and the Na/Ca exchanger (Tucker and Fettiplace, 1995; Tucker et al., 1996). Intracellular calcium is a messenger in fast event cascades in various cellular processes. Electrophysiological and confocal microscopy studies have shown that [Ca2+]i increases particularly in microdomains in which the calcium concentration reaches about 100 μM. These microdomains are located near the basolateral membrane in frog vestibular sensory cells (Lenzi and Roberts, 1994; Issa and Hudspeth, 1994; Tucker and Fettiplace, 1995; Tucker et al., 1996). This site corresponds to active zones of the sensory cells characterized by the presence of voltage-activated Ca2+ channels and Ca2+-activated K+ channels (Hudspeth and Lewis, 1988) and synaptic bodies involved in neurotransmitter release, which requires the rapid regulation of Ca2+ levels (Parsons et al., 1994; Fuchs, 1996). It has also been suggested that changes in [Ca2+]i at the apex of sensory cells may affect the adaptation of the mechano-transduction channels in the hair bundle, adjusting the operating range of the transducer (Denk et al., 1995).4.2Effect of glycine on NMDA/glycine receptorsGlycine may activate a glycine site on the NMDA receptor in type I vestibular sensory cells. We found that a selective antagonist of this site, 7-chlorokynurenate, reduced the increase in [Ca2+]i induced by glycine. Glycine is a co-agonist with glutamate to activate the NMDA receptors (Moriyoshi et al., 1991; Hollmann and Heinemann, 1994; Mori and Mishina, 1995; Corsi et al., 1996; Sucher et al., 1996). However, in some NMDA receptor subunit configurations, heteromeric NMDA receptors may be activated by glycine alone. The presence of the NR1 subunit associated with NR2A or NR2B or NR2C subunits in Xenopus oocytes results in the induction of a small but clear inward current induced by 10 μM glycine alone (Meguro et al., 1992; Kutsuwada et al., 1992). In rat neocortical neurons, nerve terminals, which release cholecystokinin and somatostatin, possess NMDA receptors, the channels of which can be operated by glycine or D-serine alone with no apparent activation of the glutamatergic co-agonist site (Paudice et al., 1998). The functional properties of the NMDA receptor channel are determined by the NR2 subunit (Kutsuwada et al., 1992; Molinoff et al., 1994; Paudice et al., 1998). The composition of NMDA subunits, in the vestibular epithelium, is unknown but the NR1 subunit has been detected in vestibular sensory cells (Devau et al., 1997). Glycine may act as a co-agonist with residual glutamate released by the sensory cell near the soma before rapidly spreading (schematic drawing Fig. 6). This contamination by glutamate may account for the NMDA receptor activation. The presence of APV (Watkins and Olverman, 1987), a competitive antagonist of the glutamate site on NMDA receptors, decreased the calcium response to NMDA by 90% and that to glutamate by 48% but did not alter the calcium response to glycine. This suggests that NMDA receptors sensitive to glycine are present on vestibular sensory cell. The almost complete inhibition of NMDA by APV, and only about 50% inhibition by glutamate alone suggest that glutamate acts on both ionotropic and metabotropic receptors (Devau et al., 1993; Guth et al., 1998a,b)However, 7-chlorokynurenate, a selective antagonist of the glycine site of NMDA receptors, contributes to the partial inhibition of the calcium response induced by glycine.4.3Effect of glycine on strychnine-sensitive glycine receptorsGlycine also activates ionotropic glycine receptors, which are specifically permeable to chloride ions and sensitive to strychnine. Strychnine reduced the increase in [Ca2+]i after glycine application by 60%, but decreased by only 6% the calcium response induced by glutamate. Strychnine interacts with the nicotinic receptors of acetylcholine as a potent antagonist. In frog vestibular hair cells, strychnine interacts with nicotinic-like receptors (Guth et al., 1994; Guth and Norris, 1996). The α9 subunit of the nicotinic receptor is very sensitive to strychnine and mRNA encoding the α9 subunit has been detected in the adult rat peripheral vestibular system (Hiel et al., 1996). Acetylcholine is the principal neurotransmitter of the efferent system and cholinergic receptors are present in type II sensory cells. However, the type I sensory cells of mammals are surrounded by afferent nerve calyces and are not in contact with any efferent nerve endings. Thus, the cholinergic receptors are not responsible for the type I sensory cell response to strychnine.The stimulation of glycine receptors resulted in chloride permeability. In the hair cell, chloride ions contribute greatly to the maintenance of cell volume (Rennie et al., 1997). In low chloride medium, the outer hair cells of the cochlear epithelium first shorten, then rapidly increase in length. The cells then collapse due to an efflux of Cl− followed by K+ and water (Cecola and Bobbin, 1992). In Cl−-free gluconate-HBSS, resting calcium levels were low in isolated type I sensory cells. Transferring the cells to normal HBSS resulted in a return to normal calcium resting levels. In the medium, gluconate may be associated with sodium or calcium. In many preparations, gluconate has been used to substitute for chloride ions in experiments with isolated vestibular type I hair cells (Rennie et al., 1997). However, gluconate may chelate external calcium and partially reduce the glycine effect.In other systems, for example, at some stage of development in chick embryo ciliary ganglion cells, activation of the ionotropic glycine receptor triggers an increase in [Ca2+]i, which is inhibited by strychnine (Sorimachi et al., 1997). Activation of the embryonic ionotropic glycine receptor causes depolarization and calcium transients in other neurons such as rat dorsal horn neurons (Wang et al., 1994). In adult neurons and smooth muscle cells, calcium activates chloride channels (Marsh et al., 1997; Wang and Kotlikoff, 1997). We found that in adult vestibular type I sensory cells, activation of the strychnine-sensitive glycine receptor induced an increase in [Ca2+]i, but the biochemical steps linking these events are unknown.The addition of strychnine and 7-chlorokynurenate together strongly decreased the calcium response suggesting that there are two components involved in the calcium transient. In the presence of an agonist, the activity of a ligand-gated channel such as an NMDA or glycine receptors may be modified by desensitization, which may in turn affect cell excitability (Jones and Westbrook, 1996). After washing for 5 min, a second glycine stimulation induced a calcium response with an amplitude similar to that of the first. Thus, we found no evidence of desensitization in type I vestibular sensory cells.4.4Glycine and amino acid transportersGlycine may also directly activate glycine transporters. These transmembrane proteins have 12 transmembrane segments like other members of the transporter family, and their function depends on the cotransport of electrogenic sodium and chloride (Attwell and Mobbs, 1994; Olivares et al., 1997; Zafra et al., 1997). The detection and the cellular localization of glycine transporters in the vestibular sensory epithelium could be determined by an immunocytochemical study, with specific antibodies directed against the glial glycine transporter (GLYT1) and the neuronal glycine transporter (GLYT2). The distribution of GLYT2 is correlated with that of strychnine-sensitive glycine receptors in most of the central nervous system except cerebellum (Nelson, 1998). They may be responsible for the residual response in the presence of 7-chlorokynurenate and strychnine. The sodium and chloride flux induced by glycine uptake in vestibular sensory cells may cause a change in [Ca2+]i contributing to the overall calcium response to glycine stimulation. To measure the sodium concentration changes induced by activation of glycine transporters a specific sodium-sensitive dye, SBFI, could be used.4.5Calcium source: influx of extracellular calcium and mobilization of intracellular calcium storesThe increase in [Ca2+]i induced by glycine may result from the entry of external calcium or from release from internal stores or both. Calcium may enter through the highly sensitive calcium channel, of the NMDA receptor, or may be released from the internal calcium store following signal transduction. Activation of the NMDA receptor may also depolarize the membrane and calcium influx may be increased by opening of the L-type calcium channels present in both types of vestibular sensory cells (Boyer et al., 1998). The presence of nickel and cadmium ions (Ni2+/Cd2+), which block voltage-dependent calcium channels, decreased by 30% the calcium response induced by glycine (personal observation). Calcium channel specific blockers (such as dihydropyridines for L-type or ω-conotoxin-GVIA for N-type) could be used to determine the type of the voltage-dependent calcium channels involved in the calcium concentration changes in vestibular sensory cell after glycine application. However, only L-type calcium channels have been shown in type I vestibular sensory cell (Boyer et al., 1998). In the presence of EGTA, a specific calcium chelator, the calcium response to glycine application was very small indicating that an influx of external calcium was the major component of the response. However, a residual response persisted demonstrating that internal calcium stores were not totally empty.In conclusion, the application of glycine induced an increase in [Ca2+]i in isolated type I vestibular sensory cells from guinea pig. Changes in calcium concentration involved at least two receptors: (i) heteromeric NMDA receptors, which are highly sensitive to glycine, and (ii) the strychnine-sensitive glycine receptor. Changes in calcium concentration may affect neurotransmitter release in the basolateral region of the cell. 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Res.6319925256Zucca et al., 1993G.ZuccaG.N.AkoevA.MaracciP.ValliNMDA receptors in frog semicircular canalsNeuroReport41993403404 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B44A118F148947F01791D40DDD487B44E25650B.txt b/test/dataset/in/resources/corpus/Clean_0B44A118F148947F01791D40DDD487B44E25650B.txt new file mode 100644 index 0000000..9dd4afe --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B44A118F148947F01791D40DDD487B44E25650B.txt @@ -0,0 +1 @@ +fampractfamprjFamily Practice1460-22290263-2136Oxford University Press10.1093/fampra/cmq021Primary Care EpidemiologyA pilot study on the validity and reliability of the Patient Enablement Instrument (PEI) in a Chinese populationLamCindy LKaYuenNatalie YKa*MercerStewart WbWongWendyaaFamily Medicine Unit, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 3/F, Ap Lei Chau Clinic, 161 Main Street, Ap Lei Chau, Hong Kong, ChinabDepartment of General Practice and Primary Care, Faculty of Medicine, University of Glasgow, 1 Horselethill Road, Glasgow G12 9LX, Scotland, UK*Correspondence to Natalie YK Yuen, Family Medicine Unit, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 3/F, Ap Lei Chau Clinic, 161 Main Street, Ap Lei Chau, Hong Kong, China; Email: nykyuen@gmail.com820103042010274395403281020092003201023032010© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2010Background. The Patient Enablement Instrument (PEI) was developed to measure patients’ enablement, which is an indicator of the effectiveness of a primary care consultation; however, to date, the PEI has not been tested in Asian populations.Objectives. The purpose of this study is to test the acceptability, validity, reliability and other psychometric properties of a Chinese [Hong Kong (HK)] translation of the PEI in Chinese patients in Hong Kong and whether these properties would be affected by different timing of administration.Methods. A Chinese (HK) translation of the PEI was developed by iterative forward–backward translations and the content validity was assessed by a cognitive debriefing interview with 10 Chinese patients. It was then administered to 152 adult patients attending a government-funded primary care clinic in Hong Kong both immediately after the consultation and 2–3 weeks later by telephone. Internal construct validity was assessed by item-scale correlations and factor analysis, test–retest reliability was assessed by intraclass correlation (ICC) and sensitivity was assessed by known group comparison.Results. The Chinese (HK) PEI was semantically equivalent to the original PEI for all items. Acceptability of the PEI was high with 83.1% response and 100% completion rates. Statistical analyses showed no difference between test and retest means as well as good reproducibility (ICC 0.75). Internal reliability determined by Cronbach’s alpha was >0.8 irrespective of timing of administration. Scale construct validity was confirmed by strong (r > 0.4) item-scale correlations and resumed to a one-factor hypothesized structure. PEI scores were significantly higher in younger patients supporting sensitivity. There was no significant difference in the psychometric properties or scores between the assessment results from immediately after and 2-weeks post-consultation.Conclusions. A Chinese (HK) translation of the PEI equivalent to the original is now available for application to Chinese populations. Pilot testing supported its acceptability, validity, reliability and sensitivity. Further studies to confirm its construct validity and responsiveness will help to establish the Chinese (HK) PEI as an outcome measure of the effectiveness of primary care consultations in Chinese patients.Chinesecontent validitypatient enablementprimary carereliabilityIntroductionThe clinical encounter between patient and practitioner is the core activity of all systems of health care. As such, the practitioner–patient relationship has been extensively investigated in conventional care, particularly in the primary care setting.1–4 However, while various methods exist to measure the effectiveness of primary care consultations, few exist to measure the effectiveness of interpersonal care in primary care.5 Many primary care consultations are for problems that are self-limiting, for which a favourable biomedical outcome can be anticipated regardless of the activities of the doctor. On the other hand, a large number of consultations are for multidimensional problems, where physical, psychological and social elements as well as the need for health promotion interact in a complex way that traditional clinical outcomes are not sensitive enough to capture.6There is some evidence to suggest that there is a positive link between patient-centred consultations and health outcomes.2,3,7 Two core values of family medicine are patient-centred care and holism,8 upon which is built the concept of patient enablement, first articulated by Howie et al.9,10 ‘Enablement’ describes a consultation outcome that reflects the extent to which patients understand their health problems and feel able to cope with them as a result of the consultation. According to Wensing, patients find it very important to be able to understand the nature of their problem and manage their own illness,11 which supports the concept of enablement as a patient-centred health-related benefit resulting from the consultation.Howie’s theory of quality of care implies that greater enablement is achieved when the patient’s needs are identified, acknowledged and dealt with in the appropriate context.12 It recognizes the importance of themes of patient centeredness and empowerment and of the patient’s ability to understand and cope with their health and illness and recognized these issues as being conceptually distinct from satisfaction.5 The original version of the Patient Enablement Instrument (PEI) was developed by Howie et al.5 based on these concepts to assess the enablement of patients after a consultation with their primary care clinician. The PEI has six questions: able to cope with life, able to understand your illness, able to cope with your illness, able to keep yourself healthy, confident about your health and able to help yourself. Each question has four response options: much better/better (Questions 1–4) or much more/more (Questions 5–6) or same or less or not applicable. The PEI is intended to be a measure of the effectiveness of primary care consultations13 and has been used for this purpose in several studies in Western countries, including the UK,10,14 Croatia,8 the Netherlands11 and Poland.15Previous studies have shown that patient enablement may be increased by doctor’s empathy,14 familiarity of the patient with the practitioner,16 positive patient evaluation of the doctor’s communication,17 as well as longer consultation duration18 and appropriate doctor training.15 In turn, patient enablement results in greater compliance with therapy and improved outcomes,18,19 as well as increased well-being up to 1 month post-consultation.14 A 6-week programme enabling patients with chronic disease in Hong Kong showed that Chinese patients undergoing the programme exhibited significantly higher self-efficacy in managing their illness and felt more energetic, the results of which supported the validity and relevance of the concept of enablement in Chinese culture.20 In contrast, low enablement is associated with a lack of continuity of care, poor self-perceived health, low educational level and low quality of life.8Hong Kong’s health system is a combination of a tax-funded health model combined with a private sector, which account for 56% and 44% of health care spending respectively.21 Total expenditure on health care amounts to ∼5.6% of the gross domestic product. Hong Kong has no mandatory primary care network, which means that patients are free to choose to consult doctors of any specialty, which results in a lack of gate-keeping, increased doctor shopping and increased health care costs.Ninety-five percent of Hong Kong’s population is Chinese and for 91% of the population, the first language is Cantonese.22 The culture of consultation is traditionally seen as paternalistic, with little chance for conversation or joint decision making by patient and doctor. Chinese society often embodies collectivism and power distance23 which means that patients rarely assert individual rights during their consultations and that they often behave deferentially towards doctors. While their dissatisfaction with doctor-centred consultation may not be evident during the consultation, it is exhibited by the high rate of doctor shopping and increased consultation rate. One study found that patients in Hong Kong preferred patient-centred consultations, regardless of differences in their age, income, education, housing and sex.23There is very little research on patient-centred care or enablement in Chinese populations. The PEI would be a very useful tool for the evaluation and enhancement of patient enablement in the consultation in order to shift the doctor-dependent paradigm in this part of the world. For a patient-reported outcome measure to be used in Hong Kong, it must first be translated into Chinese and then tested for cross-cultural equivalence, validity and psychometric adequacy. The PEI has never been tested in an Asian population, and the data on its psychometric properties in any populations are rather limited.The aim of our study was to develop a Chinese [Hong Kong (HK)] equivalent translation of the PEI and to evaluate its acceptability, validity and reliability in Chinese patients. We wanted to confirm that the items of the PEI were valid indicators of the underlying construct and that they share a common principal factor in order to justify direct summation of the item scores. In addition, we wanted to find out whether the Chinese (HK) PEI results would be influenced by patient characteristics and timing of its administration. Results showing the validity and reliability of the PEI in our Chinese population would support its use in other Chinese populations and adaptation of the PEI to other Asian cultures.MethodsDevelopment of the Chinese (HK) PEI and evaluation on content validityThe Chinese (HK) translation of the PEI was developed according to an internationally recommended method.24 Two professional translators, who were native speakers of Chinese, translated the original English PEI into Chinese independently. A panel consisting of the translators and two bilingual authors (CLKL and WW) reviewed the translations to form the first draft of the Chinese (HK) PEI. Another professional translator blind to the original PEI back-translated this first draft to English. The back-translation was assessed for equivalence to the original by one of the authors who are a native English speaker familiar with the original PEI (SM). Discrepancies between the original English language PEI and the back-translation of the first draft Chinese (HK) PEI were reviewed and revised by the panel (Appendix 1). Cognitive debriefing on the second draft was performed with 10 Chinese patients to assess the clarity and interpretation of each item and response option. The cognitive debriefing subjects were a convenient sample balanced for age and sex recruited among attendees of a government primary care clinic in Hong Kong. The final version of the Chinese (HK) PEI (Appendix 2) was then used to collect data for psychometric testing.SubjectsAll patients aged 18 years or more consulting one randomly selected doctor in an average size government-funded primary care clinic in Hong Kong from 20 April to 9 May 2009 were included. The clinic is a teaching practice with three to four doctors consulting in any one session. It is situated in an urban, low socio-economic district of Hong Kong. Patients present with similar problems to other government clinics, namely predominantly chronic conditions such as hypertension and diabetes. All doctors speak Cantonese and 99% of the consultations are conducted in this dialect. To recruit patients, a random number table generated by computer was used to select one doctor (by room number) from all those available for consultation during a particular clinical session. The aim, procedures and nature of the study were explained and written consent was obtained from each subject. Patients were excluded if they were unable to communicate in Cantonese, if they were too ill to complete the interview or if they refused to take part in the study. Patients did not receive any incentives for participating in the study.Data collectionEach subject answered the Chinese (HK) PEI via interview with a trained research assistant, who was not part of usual clinic staff, after the consultation. Information was gathered on purpose of consultation (chronic disease follow-up or acute illness), age, sex, marital status (single, married, divorced and widowed), education level and occupation (according to the British Registrar General’s classification of occupation).25All subjects were followed up by telephone 2 or more weeks after the initial consultation, when the PEI was administered again by a different trained research assistant. Lam et al.26 previously showed that telephone interviews give similar results on health service utilization as those found in face-to-face surveys.Outcome measures and statistical analysesThe data of all subjects who had completed the follow-up (retest) interviews were included in the analysis of the test–retest reliability. In order to evaluate whether the psychometric properties of the PEI would be affected by the timing of administration, we split the subjects in half for this part of the data analysis. Seventy-six subjects were randomly selected from the 131 who completed the retest to form the sample for the evaluation of delayed administration, and the rest of the 152 who completed the baseline interviews were used as the sample for the evaluation of immediate administration of the Chinese (HK) PEI.The Chinese (HK) PEI score was calculated by the mean of the scores of the applicable items multiplied by six. Cases that had more than three ‘not applicable’ items were excluded. Responses of ‘much better/much more’, ‘better/more’ and ‘same or less’ were scored 2, 1 and 0, respectively, giving a summation PEI score ranging from 0 to 12. Descriptive statistics were determined, including mean, SD, percentage of floor and ceiling of scores by assessment time.Test–retest reliability was evaluated by paired t-tests on the difference in mean and also with intraclass correction coefficient (ICC) between test and retest scores. ICC is the recommended test on reproducibility of psychometric measures and the expected standard is ≥0.7.27 Internal reliability was measured by Cronbach’s alpha coefficient, for which ≥0.7 has been suggested as the standard for group comparison.28The internal construct validity of the PEI items was first evaluated by Spearman’s rho correlation, corrected for overlap, between the item and the scale (total PEI) score; an item should be substantially linearly related (r ≥ 0.4) to the underlying concept (total PEI score) being measured. Factor analysis using the varimax rotation method was then performed on the item scores to extract factors that had an eigenvalue >1. As all items should measure the same construct of patient enablement, one principal component factor was hypothesized and they should have similar factor loading and correlation. Sensitivity was also explored by known group comparison of the mean scores by age, sex and consultation reason. It has been suggested in the literature that patient enablement would be higher in patients who are younger, male, and who present for an acute problem.8,17 The sensitivity in detecting a difference between groups was evaluated by two-sample t-tests. All data analyses were conducted by SPSS 17.0. Ethics approval was given by HKU/HA HKW IRB reference number, UW 07-226.ResultsContent validity and translation equivalence of the Chinese (HK) PEIThe back-translation of the first draft Chinese (HK) PEI was equivalent to the original PEI for all questions and responses, with few exceptions. The review panel found that the literal translation of ‘cope with’ in Chinese (e.g. or ) was linguistically inappropriate when collocated with ‘illness’ (); thus, we translated it to the Chinese term that means ‘to face’ (), which is close in meaning to the original. In addition, ‘life’ was translated to ‘daily living’. The native English expert (SM) agreed that the back-translation of the Chinese (HK) PEI was equivalent in meaning to the original. The cognitive debriefing group was composed of 10 patients, 5 male and 5 female, aged 21–74 years (mean 48.4 years), who took 1–6 minutes to complete the test (mean 3.3 minutes). Each item was understood, rated as relevant, appropriate and not difficult and correctly interpreted by >80% of respondents of the cognitive interviews. A few subjects found the title of the instrument redundant and did not clearly interpret that the stem statement was asking about a change. In addition, several suggested that the separation of the response option ‘the same or worse (less)’ into two [‘the same’ and ‘worse (less)’] options. Therefore, revisions were made to simplify the title, reword the stem question as a question and to separate the response options to improve the clarity of the instrument.Acceptability and completion rateThe flow of patient response and follow-up is shown in Figure 1: 203 patients were contacted, of whom 31 patients were excluded because of cognitive or physical impairment (9), inability to speak Cantonese (7), aged <18 years (6) or not accessible by telephone for follow-up (9) and 20 refused to take part. One hundred and fifty-two (83.1% response rate among those eligible) completed the baseline survey with no missing data. One hundred and thirty-one (86% of 152) of the participants completed the telephone follow-up interviews, and the median time interval between the two assessments was 15 days (range 14–22 days). Data from two subjects at baseline and three subjects on follow-up were excluded from further analysis because there were more than three not applicable responses on their Chinese (HK) PEI.FIGURE 1Response and follow-up of patients consulting at Ap Lei Chau ClinicThe characteristics of the study subjects can be seen in Table 1. Most subjects were elderly and of low education and socio-economic class, and more consultations were for chronic disease rather than acute illnesses. Subject characteristics were similar except for the education level between those who completed and defaulted the follow-up interviews, suggesting little systematic bias in the retest results.TABLE 1Characteristics of study subjectsCharacteristicsBaseline (n = 152)Follow-up (n = 131)Default follow-up (n = 21)Mean age (SD)62.1 (12.68)62.1 (12.84)62.2 (11.92)Age group (years, %)    18–5942.142.042.9    60 or above56.657.352.4    Missing1.30.84.8Gender (%)    Male38.238.238.1    Female61.861.861.9Education level (%)a    Nil23.019.147.6    Primary36.240.59.5    Secondary35.535.138.1    Tertiary5.35.34.8Marital status (%)    Married67.167.266.7    Widower17.816.823.8    Single6.66.94.8    Divorced8.69.24.8Occupation (%)    Professional3.33.14.8    Associated professional7.27.64.8    Skilled worker19.719.819.0    Semi-skilled worker16.418.34.8    Unskilled worker27.026.033.3    Housewife23.023.719.0    Others3.31.514.3Consultation reason (%)    Chronic disease67.167.961.9    Acute illness32.932.138.1aSignificant difference between follow-up and default follow-up subjects by chi-square test (P < 0.05).Psychometric properties of the Chinese (HK) PEITable 2 shows the distribution of baseline and follow-up Chinese (HK) PEI scores. There were few subjects who had the lowest possible (floor) or highest possible (ceiling) scores at baseline, however a relatively high proportion (18.9%) scored at the floor in the follow-up assessment. The paired t-test (P < 0.124) indicated no difference between the test and retest means, and the test–retest reliability ICC was 0.75, indicating good reproducibility. The Cronbach’s alpha coefficient of internal reliability was 0.84 and 0.85 at baseline and at follow-up, respectively, further supporting the reliability of the PEI.TABLE 2Distribution and reliability of baseline and follow-up Chinese (HK) PEI scoresMean (SD) (95% CI)% Floor% CeilingCronbach’s αPaired t-test, P valueaTest–retest reliability (ICC)Baseline (n = 150)4.65 (2.76) (4.21–5.10)6.03.30.84Follow-up (n = 127)4.22 (3.37) (3.63–4.81)18.93.90.850.1240.75PEI score calculated as mean of answered items times 6, excluding cases that were not applicable or missing in more than three items, range 0–12. Two baseline and three follow-up subjects were excluded because cases answered not applicable in more than three items.aOnly 127 subjects who had valid PEI scores in both baseline and follow-up assessments were included in the test–retest analyses.Table 3 shows the item-scale correlation between each item and the total PEI score by administration time. The correlations were all strong (≥0.65) indicating good internal consistency, in support of the internal construct validity.TABLE 3Item-scale correlations of Chinese (HK) PEIBaseline (n = 76)Follow-up (n = 76)Mean (SD)Correlation with total PEI scoreaMean (SD)Correlation with total PEI scoreaAble to copy with life0.61 (0.66)0.680.55 (0.70)0.70Able to understand your illness0.80 (0.67)0.680.74 (0.76)0.77Able to copy with your illness0.77 (0.70)0.830.74 (0.79)0.78Able to keep yourself healthy0.96 (0.56)0.750.92 (0.80)0.76Confident about your health0.86 (0.71)0.840.89 (0.79)0.79Able to help yourself0.66 (0.60)0.670.76 (0.75)0.72aSpearman’s rho correlation, corrected for overlap, were all significant, P < 0.001.Only one factor with eigenvalue >1 was extracted by factor analysis, which explained >70% and 67% of the reliable variance of the item scores at baseline and follow-up, respectively. Table 4 shows the factor coefficients and correlations between principal component and the PEI items at baseline and follow-up. Factor loadings of all the items were similar, and correlation between principle component and the Chinese (HK) PEI were strong (0.69–0.8), further confirming internal construct validity.TABLE 4Factor coefficients and correlations between principal component and items of Chinese (HK) PEIBaseline (n = 76)Follow-up (n = 76)Factor coefficientsFactor correlationsFactor coefficientsFactor correlationsAble to copy with life0.1830.680.2120.73Able to understand your illness0.1930.710.2330.80Able to copy with your illness0.2310.850.2260.78Able to keep yourself healthy0.2220.820.2180.75Confident about your health0.2270.840.2250.78Able to help yourself0.2130.790.2040.70Eigenvalue3.7003.448Total variance explained (%)61.6757.46Table 5 shows the mean PEI scores by age, sex, and consultation reasons for all subjects. Older (aged 60 years or above) subjects had significantly lower mean PEI scores than younger (aged 18–59 years) subjects, both at baseline and at follow-up. There was no significant difference in mean PEI score with respect to sex or consultation reason. The PEI score on follow-up was significantly lower than that at baseline in subjects who consulted for chronic diseases, but there was no difference for those consulting for acute illness.TABLE 5Mean (SD) of PEI summary score by age, gender and consultation reasonBaseline (n = 150)Follow-up (n = 128)nMeanSDESnMeanSDESAge group (years)    18–59635.64a2.76565.20a2.89    60 or above853.84a2.470.65723.47a3.530.60Gender    Male584.662.99494.183.57    Female924.642.620.01794.273.240.03Consultation reason    Chronic disease1024.85b2.95874.31b3.41    Acute illness484.222.280.21414.083.290.07ES, effect sizeaSignificant difference in mean PEI score between age group by independent t-test (P value < 0.01).bSignificant difference in mean PEI score for chronic disease between baseline and follow-up by paired t-test (P value = 0.047).DiscussionWe believe that this is the first report in the literature on the validity, reliability and other psychometric properties of the PEI in an Asian population. Such evidence is important to support the cross-cultural application of a patient-reported outcome measure. An equivalent translation is an essential although not sufficient step for the application of an instrument to another population with a different culture and language. In our case, an equivalent Chinese translation could be made for all the PEI items and response options. The term most difficult to translate was enablement in the title as this is a relatively new concept in the collective Chinese culture. Nevertheless, our Chinese patients could understand and identify with the indicators (items) of this construct. This further supports the relevance of the concept of enablement in the Chinese culture, as found by an earlier study that enabling programmes led to beneficial outcomes for Chinese patients with chronic disease.20Our results demonstrated good acceptability of the PEI by Chinese patients with a response rate of 83%, completion rate of 100% and analysable data from 98% of the respondents. The cognitive debriefing interviews showed that all subjects, including elderly ones, could complete the instrument in a few minutes. Most of our subjects were elderly and had a low level of education, which is typical of patients attending public primary care clinics in Hong Kong. We would expect the PEI to be even more acceptable to younger better-educated Chinese patients.Our mean PEI scores were higher than those found in studies conducted in English-speaking patients (3.1) and comparable to studies for patients consulting in other languages (4.5).10 This may be explained by cultural differences in illness coping or response to questionnaires. Howie et al.10 found significantly higher enablement scores in ‘other language patients’, while Freeman et al.29 found higher enablement scores when non-English-speaking patients had consultations in their own language. It may not be valid to directly compare the absolute scores between different cultural groups because each may have different life expectations and coping needs. Qualitative exploration on the meaning of the scores from respondents would shed light on any difference in the interpretation and function of the concept of enablement between different cultures.The proportion of subjects scoring the lowest and highest scores were all <20%, which is generally regarded as the threshold for a floor or ceiling effect,30 suggesting that the Chinese (HK) PEI is potentially sensitive in detecting differences in the outcomes of consultations.The results of the item-scale correlation and factor analyses supported the internal construct validity of the PEI as a measure of the core concept of patient enablement. All items were relevant and important as shown by the high and almost equal correlations with the total score and the principal factor. The high item-score correlation indicated that not only were the original and Chinese (HK) versions of the PEI similar in language (semantics) but there were also equivalent in function.The finding of lower PEI scores in the elderly provided some support to the external construct validity of the PEI, which is consistent with existing Western literature.8 However, there was no difference in PEI score according to reason of consultation contrary to findings in a previous study.17 One would expect higher enablement scores in those presenting with acute illness, as chronic conditions entail regular follow-up, and doctors would assume that patients are stable and have acquired knowledge of their condition over time, thereby resulting in less enablement achieved at each subsequent consultation. On the other hand, doctors might see the need to help patients with chronic diseases to cope better. Pawlikowska et al.15 also did not find any effect from case mix on PEI scores.It should be noted that the findings with relation to those presenting for acute versus chronic conditions need to be interpreted with caution since the ‘acute illness’ group was relatively small. Our study patients presenting with ‘acute’ conditions were instructed to focus on their original acute problem only; however, there is a chance that they may have found it difficult to separate acute from chronic illness, had they also had chronic illnesses. In addition, it may also have been possible that they had separate consultations (other than those included in the baseline) that might have affected their responses. Nevertheless, we expected that the number of patients presenting for re-consultation in the 2 weeks of the study would have been low.There was no difference in the scores or psychometric performance of the PEI according to timing of administration (immediately post-consultation compared with delayed for 2 or more weeks). This not only supported test–retest reliability but also the feasibility of measuring patient enablement with the PEI by telephone up to a few weeks after the consultation. The PEI was originally developed to be administered immediately post-consultation.5 Our results indicate that a ‘delayed’ completion of the PEI may be used instead, with beneficial implications for its usage. For patients, they would not have to spend more time at the clinic in addition to their consultation time duration. For clinical practice and research, the option of delayed administration of the PEI over the telephone (or via other means) confers a definite logistical advantage.It was interesting to note that a higher proportion of subjects had a floor PEI score in the assessment at 2 or more weeks, which might imply the enablement perceived immediately after the consultation was lost with time. This was more apparent in those consulting for chronic diseases whose mean PEI score also decreased with time. This more likely represented a true change rather than poor reproducibility. If it were a systematic bias, then the same change should have been found in all subjects. It is not possible to ascertain whether immediate post-consultation, compared with delayed PEI administration, represented a more accurate reflection of the ‘truth’, but conceptually, it would take time before a patient could really tell whether he/she had been more enabled. In addition, some patients might feel obligated to give a more positive response when they are interviewed immediately after the consultation, especially if they have a good relationship with the doctor.LimitationsThis pilot study intended to establish the cross-cultural applicability of the PEI in a Chinese population. The results provide preliminary evidence on its internal construct validity and reliability; however, its construct validity in relation to external criteria has not been firmly established. Our findings on the differences between known groups were inconclusive due to limitations of the sample size and the study setting. There are certain factors documented in the literature to be associated with patient enablement, such as patient age and sex, doctor’s communication, length of consultation, how well the doctor and the patient know each other (proxy for continuity of care) and overall satisfaction of the visit, which were not explored due inherent difficulties in our primary care clinic. For instance, in our setting, patients are usually not seen by the same doctor, and consultation lengths are fairly standard (around 8 minutes). Further studies should be carried out to test the construct validity of the PEI and to further examine determinants of patient enablement.Conclusion(s)The Chinese (HK) version of the PEI is semantically equivalent to the original English language PEI, and our results have supported its acceptability, content and internal construct validity, reliability and sensitivity. As our subjects are of different ages and sex, education levels and social classes, we propose that the PEI is valid for use with the general Hong Kong Chinese population. As all Chinese share the same written language (be it in traditional or simplified form), we hope in the future it can be applied to other Chinese populations worldwide. Further studies should be carried out to evaluate the relationship between PEI scores and other external criteria to confirm its construct validity. Sensitivity in differentiating consultation outcomes in different settings or morbidity groups and responsiveness to changes with intervention will also need to be established.DeclarationFunding: Tung Wah Group of Hospitals.Ethical approval: HKU/HA HKW IRB reference number, UW 07-226.Conflict of interest: none.We would like to thank Professor John Howie in giving us permission to adapt the PEI to our Chinese population. Thanks also go to Mr Carlos Wong and Ms Mandy Tai for helping with the data collection and analysis.TRIAL REGISTRATION: The research was registered with the HKU Clinical Trial Centre Registry (HKCTR-477).Appendix 1. Draft Chinese (HK) PEI with back-translation used in cognitive debriefing with English back-translations in brackets1StewartMBrownJBWestonWWPatient-Centred Medicine. 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Tests of data quality, scaling assumptions and reliability across diverse patient groupsMed Care1994324066 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B48C07191826EBBDBADD0B5B2A899A764E75367.txt b/test/dataset/in/resources/corpus/Clean_0B48C07191826EBBDBADD0B5B2A899A764E75367.txt new file mode 100644 index 0000000..01a27de --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B48C07191826EBBDBADD0B5B2A899A764E75367.txt @@ -0,0 +1 @@ +carcincarcinCarcinogenesis1460-21800143-3334Oxford University Press10.1093/carcin/bgp132CARCINOGENESISDisruption of estrogen receptor signaling enhances intestinal neoplasia in ApcMin/+ miceClevelandAlicia G.†OikarinenSeija I.1†BynotéKimberly K.MarttinenMaija1RafterJoseph J.23GustafssonJan-Åke23RoyShyamal K.4PitotHenry C.5KorachKenneth S.6LubahnDennis B.78910MutanenMarja1GouldKaren A.*Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, USA1Department of Applied Chemistry and Microbiology (Nutrition), PO Box 66, University of Helsinki, Helsinki FIN-00014, Finland2Department of BioSciences and Nutrition, Karolinska Institute, NOVUM, S-14186 Huddinge, Sweden3Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA4Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198, USA5McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706, USA6Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA7Department of Biochemistry8Department of Child Health9Department of Animal Sciences10MU Center for Phytonutrient and Phytochemical Studies, University of Missouri, Columbia, MO, USA*To whom correspondence should be addressed. Tel: +1 402 559 2456; Fax: +1 402 559 7328; Email: kagould@unmc.edu†These authors contributed equally to this work.92009116200930915811590611200830420092352009© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org2009Estrogen receptors (ERs) [ERα (Esr1) and ERβ (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERα and ERβ is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERα knockout and ApcMin mouse strains, we demonstrate that ERα deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in ApcMin/+ mice. Within the normal intestinal epithelium of ApcMin/+ mice, ERα deficiency is associated with an accumulation of nuclear β-catenin, an indicator of activation of the Wnt–β-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERα deficiency is associated with activation of Wnt–β-catenin signaling, ERα deficiency in the intestinal epithelium of ApcMin/+ mice also correlated with increased expression of Wnt–β-catenin target genes. Through crosses between an ERβ knockout and ApcMin mouse strains, we observed some evidence that ERβ deficiency is associated with an increased incidence of colon tumors in ApcMin/+ mice. This effect of ERβ deficiency does not involve modulation of Wnt–β-catenin signaling. Our studies suggest that ERα and ERβ signaling modulate colorectal carcinogenesis, and ERα does so, at least in part, by regulating the activity of the Wnt–β-catenin pathway.IntroductionColorectal cancer incidence is 35% lower in women than men (1). Although the basis for this difference is unknown, gonadal steroids are considered a contributing factor. In women, high parity, early age at first pregnancy, oral contraceptive use and estrogen replacement therapy are each associated with decreased colorectal cancer risk (2–4). In the Women's Health Initiative study, hormone replacement therapy conferred a 40% reduction in colorectal cancer risk (5). Altogether, these data suggest that estrogens and/or progestins protect against colorectal cancer.Consistent with the hypothesis that estrogens protect against colorectal cancer, administration of either a synthetic estrogen or the naturally occurring 17β-estradiol (E2) inhibits carcinogen-induced gastrointestinal tumorigenesis in rats (6,7). Similarly, E2 attenuates intestinal tumorigenesis in ApcMin/+ mice (8,9), in which inheritance of the multiple intestinal neoplasia (Min) mutation in the adenomatous polyposis coli (Apc) gene predisposes carriers to spontaneous intestinal tumorigenesis (10,11). The effects of estrogens are mediated by estrogen receptors (ERs) [ERα (Esr1) and ERβ (Esr2)], which function as ligand-dependent transcription factors. Both receptors are expressed in the intestinal epithelium (12–15), but their function in this tissue is not known. Loss of ERα expression due to promoter hypermethylation occurs in colorectal cancers in both men and women (13), suggesting that ERα functions as a tumor suppressor in the colon. ERα promoter hypermethylation occurs in normal colonic epithelium during aging (13,16) and is postulated to create a microenvironment permissive to carcinogenesis. In contrast, loss of ERβ expression correlates with advanced Dukes stages (17).Some support for the hypothesis that ERα and ERβ impact intestinal tumorigenesis has been obtained from studies using ApcMin/+ mice. In one study, heterozygosity for an ERα knockout allele (Esr1Pcn1) was associated with increased incidence of colon tumors in ApcMin/+ mice (18). The impact of homozygosity for this ERα knockout allele (ERα−/−) on tumorigenesis in ApcMin/+ mice could not be evaluated because no ERα−/− mice carrying the ApcMin/+ mutation were obtained from the intercross. Although two studies have examined the impact of an ERβ knockout allele (Esr2Pcn1) on tumorigenesis in ApcMin/+ mice, the results of these studies are strikingly different. In one study, heterozygosity and homozygosity for the ERβ knockout allele were associated with increased incidence of colon tumors (18). In the second study, this same ERβ knockout allele had no impact on colon tumorigenesis (19). Rather, these investigators observed that homozygosity for the ERβ knockout was associated with an increase in small intestinal tumor number in ApcMin/+ female but not male mice (19). Altogether, these results do support the idea that disruption of ERα and ERβ signaling promotes intestinal tumorigenesis in ApcMin/+ mice. However, because a crucial genotypic class was absent in the ERα study and the two ERβ studies yielded disparate results, the nature of the impact of ERα and ERβ signaling on intestinal tumorigenesis remains largely unknown.In the present study, we sought to clarify the role of ER signaling in tumorigenesis in the ApcMin/+ mouse by crossing targeted disruptions of ERα and ERβ that are distinct from those used in the previous studies described above with the ApcMin strain. We intercrossed these ERα and ERβ knockout strains with the ApcMin strain and evaluated their impact on intestinal tumorigenesis. The impact of these knockouts on the activity of the Wnt–β-catenin pathway in ApcMin/+ mice was also evaluated. Our data indicate that loss of ERα is associated with increased adenoma multiplicity in ApcMin/+ mice, whereas loss of ERβ increases the incidence of colon tumors in ApcMin/+ mice without significantly altering tumor multiplicity. Loss of ERα but not ERβ is associated with activation of Wnt–β-catenin signaling in the intestinal epithelium of ApcMin/+ mice. These studies suggest that both ERα and ERβ modulate colorectal carcinogenesis, and ERα does so, at least in part, by regulating the Wnt–β-catenin pathway.Materials and methodsCare and treatment of miceThe ERα cross and the second ERβ cross were conducted at the University of Nebraska Medical Center. The Institutional Animal Care and Use Committee of the University of Nebraska Medical Center approved all procedures involving live animals in these studies. B6 ApcMin/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The ERα knockout strain (B6.129-Esr1tm1Ksk) (20) was provided by D.Lubahn. The ERβ knockout strain (B6.129-Esr2tm1Ksk) (21) was obtained from Taconic (Hudson, NY). Animals were housed under controlled temperature, humidity and 12 h light–12 h dark lighting conditions in a facility accredited by the American Association for Accreditation of Laboratory Animal Care and operated in accordance with the standards outlined in Guide for the Care and Use of Laboratory Animals (The National Academies Press, 1996). Mice were provided Harlan irradiated rodent diet 7904 (Harlan Teklad, Madison, WI), which contains soy, milk and meat-based protein sources and allowed to feed ad libitum.B6 females heterozygous for targeted disruption of the ERα gene (ERα+/−) were crossed to B6 ApcMin/+ males. The resulting ERα+/− ApcMin/+ males were crossed to B6 ERα+/− females to produce ERα−/−, ERα+/− and ERα+/+ ApcMin/+ mice. Similar crosses were performed with the ERβ knockout. The ERα and Apc genotypes were determined by polymerase chain reaction (PCR) as described previously (20,22). ERβ genotype was determined by PCR using neo primers (20) and primers that amplify ERβ exon 3 (forward primer 5′-AGAGACCCTGAAGAGGAAGC-3′ and reverse primer 5′-GCTTCTTTTAAAAAAGGCCT-3′) only in the absence of the neo insert.The first ERβ cross was conducted at the University of Helsinki. The Laboratory Animal Ethics Committee of the Faculty of Agriculture and Forestry, University of Helsinki, approved this study protocol. ApcMin/+ mice on a C57BL/6J (B6) genetic background were obtained from The Jackson Laboratory. Mice from the B6.129-Esr2tm1Ksk strain (21) that was heterozygous for the ERβ knockout (ERβ+/−) mice were serially backcrossed to B6 mice. Mice were genotyped at Apc and ERβ using PCR-based assays as described previously (22,23).After 10 generations of backcrossing to B6 (N10), ERβ+/− females were intercrossed with ApcMin/+ males to generate ApcMin/+ ERβ+/− mice. Male ApcMin/+ ERβ+/− mice were then bred with female ERβ+/− mice to obtain ApcMin/+ ERβ+/+, ApcMin/+ ERβ+/− and ApcMin/+ ERβ−/− offspring. All mice were fed with Teklad Global 14% Protein Rodent Maintenance Diet 2014 (Harlan Teklad, Horst, The Netherlands), which contains 14% protein, 3.5% oil, 5.5% fiber and no alfalfa or soybean meal. In this diet, the level of natural phytoestrogens is negligible. Animals were housed in plastic cages in a temperature- and humidity-controlled animal facility, with 12 h light–dark cycle. Food and water were provided ad libitum.Analysis of tumor multiplicity, size and histologyMice were euthanized by CO2 asphyxiation. Intestinal tumors were counted and measured as described previously (10,24–26) except that the small intestine was cut into four segments of equal length and the entire length of these segments, along with the colon, was examined. In Omaha crosses, segments of intestinal tissue fixed in 10% neutral buffered formalin were processed, sectioned and stained with hematoxylin and eosin. Stained sections were examined by an observer blinded with respect to the ER genotype. For intestinal tissue used for extraction of RNA or protein, adenomas were excised under the stereomicroscope at ×10 magnification. The remaining normal epithelium was scraped from the submucosal smooth muscle, frozen in liquid nitrogen and stored at −70°C.Quantitative reverse transcription–PCRTotal RNA was prepared using the Absolutely RNA miniprep kit (Stratagene Corporation, La Jolla, CA). Reverse transcription reactions were performed as described previously (27). For Cyclin D1 and c-Myc complementary DNA, PCR was performed using 900 nM each primer and 200 nM probe. The primer and probes sequences were as follows: 5′-TCCGCAAGCATGCACAGA-3′ (Cyclin D1 forward primer); 5′-CTTTGTGGCCCTCTGTGCCACAGA-3′ (Cyclin D1 probe); 5′-GGTGGGTTGGAAATGAACTTCA-3′ (Cyclin D1 reverse primer); 5′-TGAGCCCCTAGTGCTGCAT-3′ (c-Myc forward primer); 5′-AGGAGACACCGCCCACCACCAG-3′ (c-Myc probe) and 5′-CCTCATCTTCTTGCTCTTCTTCAGA-3′ (c-Myc reverse primer). PCR was performed and analyzed using the ABI 7500 Real-Time PCR System and Sequence Detection Software version 1.9 (Applied Biosystems, Foster City, CA) as described previously (27). Transcript abundance was normalized to glyceraldehyde 3′-phosphate dehydrogenase (Gapdh) (27). Neither the ApcMin mutation nor the ERα deficiency impacted Gapdh expression (data not shown).Determination of genotype at polymorphic markersOligonucleotide primers specific to simple sequence length polymorphism repeats polymorphic in length between the 129P2 and the B6 strains were used for genotyping as described previously (27,28).RadioimmunoassayE2 and progesterone in unextracted sera collected at killing were measured using a coated tube radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA) as described previously (29). Samples were assayed in duplicate. For ERα+/+ Apc+/+ and ERα+/+ ApcMin/+ mice, E2 measurements were obtained during the follicular phase of estrous and progesterone measurements were obtained during the luteal phase. Phase of estrous was determined based on ovarian histology. ERα−/− females do not cycle normally and are anovulatory; E2 measurements were made in these mice without selection for phase of estrous.ImmunoblottingMucosal scrapings were isolated in ice-cold phosphate-buffered saline supplemented with protease and phosphotase inhibitors. Nuclear and cytoplasmic extracts from fresh tissue were prepared using the Nuclear Extract kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Whole-cell extracts were prepared from frozen tissue and the same kit using manufacturer's instructions. Western blots were performed as described previously (30) except that the Li-Cor Odyssey Imaging system (Li-Cor, Lincoln, NE) was used. The antibodies used were as follows: E-5 β-catenin and MS-3 nucleolin mouse monoclonal antibodies and I-19 actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin mouse monoclonal antibody, clone B-5-1-2 (Sigma Corporation, St. Louis, MO) and secondary antibodies labeled either with Alexa fluor® 680 (Invitrogen Corporation, Carlsbad, CA) or with IRDye800™ (Rockland Immunochemicals, Gilbertsville, PA). Primary and secondary antibodies were used at a dilution of 1:1000 and 1:2000, respectively.Quantitative assay for Apc locusDNA was isolated from normal intestinal epithelium scrapings. The quantitative PCR assay for the Apc locus was performed and analyzed as described previously (31). For this assay, the Apc+ and ApcMin alleles are amplified and digested with HindIII, resulting in a 123 bp product from the Apc+ allele and a 144 bp product from the ApcMin allele. Radiolabeled products were resolved on a 5% denaturing polyacrylamide gel and quantitated using a Molecular Dynamics PhosphorImager and ImageQuaNT 5.0 software (GE Healthcare, Piscataway, NJ).Statistical analysisAverage tumor multiplicity, tumor diameter and tumor burden were compared using the Wilcoxon rank sum test. Differences in the distribution of tumors along the intestine were evaluated with the chi-square test. Tumor incidence data were compared using Fisher's exact test. Differences in gene expression were evaluated using a t-test. All statistical analyses were conducted using SPSS software, version 13 (SPSS, Chicago, IL). P ≤ 0.05 was considered to be significant.ResultsERα deficiency enhances ApcMin-induced tumorigenesisTo examine the impact of disrupting ERα in ApcMin/+ mice, we intercrossed the ERα knockout and ApcMin mouse strains. All genotypes of F2 progeny were obtained at the expected frequencies (P > 0.05; data not shown). Mice were killed at 80 days of age. Mean tumor multiplicity in ERα+/+ ApcMin/+ mice was 37.9 (Figure 1A). Mean tumor multiplicity in ERα+/− ApcMin/+ mice was 37.1 and did not differ from that in ERα+/+ ApcMin/+ mice (P > 0.05; Figure 1A). Relative to ERα+/+ ApcMin/+ and ERα+/− ApcMin/+ mice, the mean adenoma multiplicity in ERα−/− ApcMin/+ mice was increased by 50% to 56.4 (P = 0.01; Figure 1A). Gender did not impact tumor multiplicity (Figure 1B; P > 0.05). ERα genotype did not alter the distribution of adenomas along the length of the intestine (Figure 1C; P > 0.05).Fig. 1.Disruption of ERα leads to enhanced intestinal tumorigenesis in ApcMin mice. The mean adenoma multiplicity in the intestinal tract of ERα+/+ ApcMin/+ (n = 22), ERα+/− ApcMin/+ (n = 17) and ERα−/− ApcMin/+ mice (n = 16) at 80 days of age is illustrated (A). The same data are illustrated in panel (B), except that the mean multiplicity in females (F) is shown separately from that in males (M). In panel (C), the mean percentage of adenomas located within each of the four defined regions of the small intestine (SI) and the colon (C) is shown. For the SI, the regions are depicted proximal to distal, with region 1 beginning just distal of the stomach and region 4 ending immediately proximal of the cecum. The mean adenoma burden in the intestinal tract of ERα+/+ ApcMin/+ (n = 22), ERα+/− ApcMin/+ (n = 17) and ERα−/− ApcMin/+ mice (n = 16) at 80 days of age is illustrated in panel (D). The ‘1’ indicates a statistical difference from ERα+/+ ApcMin/+ and the ‘2’ indicates a statistical difference from ERα+/− ApcMin/+.To examine the effect of ERα deficiency on tumor size, we focused on small intestinal adenomas because these lesions are flat; therefore, tumor diameter and/or tumor area are used as indicators of tumor size (25). The mean tumor area in ERα+/+ ApcMin/+ mice did not differ significantly from that in ERα+/− ApcMin/+ mice (P > 0.05). In contrast, the average tumor area in ERα−/− ApcMin/+ mice was significantly larger than that in either ERα+/+ or ERα+/− mice (P ≤ 0.001). Combining tumor multiplicity and tumor size data, we calculated tumor burden. The average tumor burden in ERα+/+ ApcMin/+ mice averaged 70 ± 23 mm2. The mean tumor burden in ERα+/− ApcMin/+ mice of 81 ± 14 mm2 did not differ significantly from that in ERα+/+ ApcMin/+ mice (P > 0.05; Figure 1D). In contrast, the mean tumor burden of 143 ± 28 mm2 in ERα−/− ApcMin/+ was significantly greater than that in ERα+/+ and ER+/− mice (P < 0.005; Figure 1D). ERα genotype did not impact adenoma histology in ApcMin/+ mice (Figure 2A and B).Fig. 2.Impact of ERα deficiency on intestinal adenoma and ovarian histology in ApcMin/+ mice. Representative examples of fairly well-differentiated polypoid adenomas from ERα+/+ ApcMin/+ (A) and ERα−/− ApcMin/+ (B) mice. A healthy ovary from an 80-day-old ERα+/+ Apc+/+ female showing multiple functional corpora lutea (indicated by *) and healthy maturing follicles (indicated by arrows) are shown in panel (C). By comparison, a representative ovary from an age-matched ERα+/+ ApcMin/+ female (D) exhibits few corpora lutea (*) and those that are present show signs of diminished function and steroidogenesis. The majority of maturing follicles here show evidence of degeneration (arrows). A quantitative analysis of the mean number (±SD) of corpora lutea (E) and mean serum progesterone (F) is also shown. Corpora lutea were counted in both ovaries from Apc+/+ (n = 6) and ApcMin/+ mice (n = 12) during the luteal phase of the estrous cycle. Serum progesterone was measured by radioimmunoassay during the luteal phase. The ‘1’ indicates a statistical difference from wild-type. The ovary from an ERα−/− ApcMin/+ mouse showing portions of two large, hemorrhagic, cystic follicles is also shown (panel G). The edge of one follicle, showing what appears to be a single layer of granulosa cells and underlying ovarian stromal cells, is defined by the box with the dashed line and is shown at higher magnification in the inset. The arrow indicates the stromal cells. In panel (H), the ovary from an ERα−/− Apc+/+ mouse shows portions of four large, hemorrhagic and cystic follicles. A portion of the hypertrophied thecal layer lining one follicle, defined by the box with the dashed line, is shown at higher magnification in the inset. The arrowhead indicates the thecal layer and the arrow indicates ovarian stromal cells. The scale bar represents 100 or 44 μM (inset panels).Impact of ERα deficiency on E2 levels and ovarian histology in ApcMin/+ miceERα deficiency in female Apc+/+ mice is associated with a 10-fold elevation in serum E2 by 60–120 days of age (32). These observations raised the possibility that ERα deficiency might modulate tumorigenesis via an unknown mechanism driven by high levels of E2. Therefore, we examined serum E2 levels in ERα−/− ApcMin/+ females. We observed that in 80-day-old ERα−/− ApcMin/+ females, the mean serum E2 concentration was 32 ± 7 pg/ml. This value is at the low end of the normal physiological range of E2 in female mice, which varies from 20 to 100 pg/ml depending upon the phase of estrous. Thus, the enhanced intestinal tumorigenesis in female ERα−/− ApcMin/+ mice is not a consequence of high serum E2.We postulated that ERα−/− ApcMin/+ females might exhibit low levels of serum E2 as a result of deleterious effects of the ApcMin mutation on ovarian function. In the estrus phase, when E2 levels are highest, the mean concentration of serum E2 in 80-day-old ERα+/+ ApcMin/+ mice is <20 pg/ml, the lower limit of detection of the assay used. In contrast with ERα+/+ Apc+/+ females (Figure 2C), ERα+/+ ApcMin/+ females at 80 days of age possess ovaries exhibiting a high degree of follicular degeneration (Figure 2D) and few corpora lutea (Figure 2E; P ≤ 0.05). ERα+/+ ApcMin/+ females also exhibit low-serum progesterone levels (Figure 2F; P ≤ 0.05), indicating reduced ovarian function. Although ovaries in ERα−/− ApcMin/+ mice showed the enlarged, hemorrhagic and cystic follicles (Figure 2G) characteristic of ERα−/− Apc+/+ females [Figure 2H; (32)], these follicles in ERα−/− ApcMin/+ females did not show the hypertrophied theca, indicative of leutinizing hormone stimulation, frequently seen in older ERα−/− Apc+/+ females.Potential impact of 129-derived loci in the ERα knockout cross?We genotyped our B6.129-Esr1tm1Ksk founders using markers distributed across the genome (Table I) to identify residual 129P2 alleles even though this strain had been backcrossed for 12 generations. In these founders, the only 129P2 alleles detected were at three markers linked to ERα on proximal chromosome 10. The 129P2 alleles at these markers were segregating in the F2 intercross mice (Figure 3). Although 70% of ERα−/− ApcMin/+ mice retained the 129 allele at D10Mit152, the marker most closely linked to ERα, linkage disequilibrium dropped off sharply distal of this marker. Genotype at D10Mit51, the next marker distal of D10Mit152, exhibited poor concordance with ERα genotype. Only 10% of ERα−/− mice were homozygous for the 129P2 allele at D10Mit51, indicating that the 129P2 allele at this marker is not in disequilibrium with the 129P2-derived ERα knockout allele. All mice evaluated were homozygous for the B6 allele at markers including and distal to D10Mit87. Thus, <13 Mbp from the 129P2 genome on chromosome 10 is retained in association with the ERα knockout.Table I.Markers used in genotypic evaluation of B6.129-Esr1tm1Ksk and B6.129-Esr2tm1Ksk strainsaMarker (location in cMb; Mbpc)D1Mit236 (26; 45)D10Mit95 (51; 92)D1Mit440 (54; 91)D10Mit233 (62; 114)D1Mit159 (82; 161)D11Mit71 (1; 7)D1Mit111 (92; 171)D11Mit4 (37; 68)D1Mit206 (96; 175)D11Mit333 (66; 108)D1Mit426 (101; 182)D12Mit182 (2; 11) (ERβ)dD2Mit61 (34; 61)D12Mit60 (16; 35)D2Mit395 (70; 119)D12Mit91 (29; 73) ERβ)D2Mit148 (105; 175)D12Mit118 (45; 92) (ERβ)D3Mit203 (11; 27)D12Nds2 (59; N.D.e)D3Mi98 (40; 86)D13Mit275 (16; 37)D3Mit320 (72; 143)D13Mit13 (35; 56)D4Mit193 (7; 32)D13Mit151 (71; 116)D4Mit17 (31; 63)D14Mit60 (15; 46)D4Mit308 (57; 124)D14Mit39 (30; 68)D5Mit348 (8; 24)D15Mit252 (15; 23)D5Mit201 (45; 75)D15Mit107 (49; 84)D5Mit95 (68; 125)D15Mit161 (69; 97)D6Mit138 (1; 4)D16Mit131 (4.3;) (ERβ)D6Mit284 (38; 93)D16Mit101 (17; 24)D6Mit373 (74; 147)D16Mit139 (43; 66)D7Mit267 (11; 29)D17Mit245 (3; 12)D7Mit248 (28; N.D.e)D17Mit51 (23; 43)D7Mit323 (50; 100)D17Mit10 (20; N.D.e) [ERα]D8Mit155 (1; 5)D17Mit180 (29; 50) [ERα]D8Mit292 (19; 36)D17Mit93 (45; 74)D8Mit45 (40; 90)D18Mit177 (20; 41)D9Mit90 (9; 32)D18Mit144 (57; 86)D9Mit129 (26; 43)D19Mit96 (15; 22)D9Mit350 (61; 111)D19Mit90 (41; 42)D10Mit152f (4; 12) [ERα]gDXMit64 (31; 73)D10Mit213f (11; 20)DXMit223 (73; 163)D10Mit36 (29; 48) [ERα]aERα (Esr1) is located at 12cM and 5.7 Mbp and ERβ (Esr2) is located at 33cM and 77 Mbp (www.informatics.jax.org).bLocation of marker in centiMorgans obtained from mouse genome informatics (www.informatics.jax.org).cLocation of marker in mega base pairs obtained from mouse genome informatics (www.informatics.jax.org).d(ERβ) indicates that this marker was evaluated in mice derived from B6.129-Esr2tm1Ksk strain.eN.D., location of marker on physical map has not been determined.fThese markers were mapped proximal to Esr1 but the physical mapping places these markers distal to Esr1. Genetic and physical mapping data obtained from mouse genome informatics. 129 alleles at these markers were segregating in N12 mice from the B6.129-Esr1tm1Ksk strain.g(ERα) indicates that this marker was evaluated in mice derived from B6.129-Esr1tm1Ksk strain.Fig. 3.A small region of the 129P2 genome is retained in association with the ERα knockout allele. The genotypes of ERα−/− ApcMin/+ mice (n = 10), ERα+/− ApcMin/+ mice (n = 7) and ERα+/+ ApcMin/+ mice (n = 7) on chromosome 10 are shown. The white bars represent homozygosity for the 129P2 allele, the shaded bars represent heterozygosity for the 129P2 allele and the black bars represent homozygosity for the B6 allele. The position of each marker in Mbp is indicated in parentheses.ERα deficiency is associated with an accumulation of nuclear β-catenin and increased expression of Wnt–β-catenin target genesThe Wnt–β-catenin pathway is constitutively activated in virtually all colon cancers. Although the impact of estrogen on Wnt–β-catenin activity in the intestine is not known, estrogens do influence this pathway in other tissues (33–36). These data, together with our observations that ERα deficiency enhanced intestinal tumorigenesis, led us to postulate that ERα deficiency might activate Wnt–β-catenin signaling in the intestinal epithelium of ApcMin/+ mice. In whole-cell extracts, β-catenin expression was higher in normal intestinal epithelium of ERα−/− ApcMin/+ mice relative to ERα+/+ ApcMin/+ mice (Figure 4A). Nuclear accumulation of β-catenin, a molecular indicator of activation of Wnt–β-catenin signaling, was also evaluated. Quantitative analysis of these western blots indicated that nuclear β-catenin is increased by, on average, 60% in intestinal epithelium of ERα−/− ApcMin/+ mice relative to ERα+/+ ApcMin/+ mice (Figure 4B), consistent with an increased activation of Wnt–β-catenin signaling in the absence of ERα. Expression of Cyclin D1 and c-Myc, two transcriptional targets of the Wnt–β-catenin pathway, was increased on average 2.2-fold and 2.4-fold, respectively, in the intestinal epithelium of ERα−/− ApcMin/+ mice relative to ERα+/+ ApcMin/+ mice (Figure 4C), lending further support to the hypothesis that ERα deficiency is associated with increased Wnt–β-catenin signaling.Fig. 4.ERα deficiency activates the Wnt–β-catenin pathway in the intestinal epithelium of ApcMin/+ mice. β-Catenin expression in whole-cell extracts (A) and nuclear (N) and cytoplasmic (C) protein extracts (B) prepared from normal intestinal epithelium of 80-day-old ERα+/+ ApcMin/+ (n = 6) and ERα−/− ApcMin/+ (n = 6) mice. Actin expression was used as a positive and loading control. Nucleolin was used as a positive control for the nuclear fraction and a negative control for the cytoplasmic fraction, whereas α-tubulin was used as a positive control for the cytoplasmic fraction and a negative control for the nuclear fraction. Cyclin D1 and c-Myc expression was determined using quantitative reverse transcription–PCR (C). Both male and female mice were used. For each sample, expression of Cyclin D1 and c-Myc was normalized to Gapdh expression. The average level of normalized Cyclin D1 and c-Myc expression in the ERα+/+ ApcMin/+ mice (n = 4) was set at 1.0 expression units. The relative expression level in ERα−/− ApcMin/+ mice (n = 7) was expressed as a percentage of that in ERα+/+ ApcMin/+ mice. Each bar represents the mean gene expression ± SEM. The asterisk indicates a statistically significant difference from ERα+/+ ApcMin/+. The mean Apc+/ApcMin ratio values in normal intestinal epithelium of 80-day-old ERα+/+ ApcMin/+ (n = 6) and ERα−/− ApcMin/+ (n = 6) mice is show (D). Each bar represents the mean ratio value ± SD.To verify that the normal intestinal epithelium specimens used in these analyses did not contain microadenomas, small precursors of adenomas that have lost wild-type Apc allele (Apc+) and thus exhibit increased nuclear β-catenin (37), we used a quantitative assay to determine the relative abundance of the Apc+ and ApcMin alleles in genomic DNA isolated from these specimens. It has been reported that in histologically normal intestinal epithelium of B6 ApcMin/+ mice, the mean Apc+:ApcMin ratio value was 0.83 ± 0.18 (31). Because microadenomas and adenomas in B6 ApcMin/+ mice exhibit loss of the Apc+ allele (31), contamination of normal tissue with these lesions would reduce the Apc+:ApcMin ratio. Here, normal intestinal epithelium samples from ERα+/+ ApcMin/+ mice yielded a mean Apc+:ApcMin ratio value of 0.78 ± 0.24 (Figure 4D). A mean Apc+:ApcMin ratio value of 0.90 ± 0.21 was obtained from normal intestinal epithelium samples from ERα−/− ApcMin/+ mice (Figure 4D). The mean Apc+:ApcMin ratio values from ERα+/+ ApcMin/+ in mice did not differ significantly from the value ERα−/− ApcMin/+ mice (P > 0.05), and both means were consistent with the value reported previously (31). This result indicates that the normal epithelium samples used were not contaminated with microadenomas.Effect of deletion of ERβ on tumorigenesis in ApcMin/+ miceTo evaluate the effect of ERβ deletion on tumor formation in ApcMin/+ mice, we intercrossed ERβ+/− mice with ApcMin/+ mice. In this intercross (Helsinki intercross), tumor multiplicity was evaluated at 90 days of age. ERβ genotype did not have a significant impact on the mean number of adenomas in either the entire intestinal tract (data not shown; P ≥ 0.05) or the small intestine alone (Figure 5A; P ≥ 0.05). Because ERβ is thought to play a role during the later stages of colorectal cancer progression, a second set of intercross mice was produced and evaluated at 140 days of age. This second set of intercross mice (Omaha intercross) was performed at a separate institution. However, both intercrosses involved the same ERβ knockout (Esr2Ksk1) and ApcMin/+ alleles, both of which were extensively backcrossed to the B6 strain. Consistent with our observation in the first set of intercross mice, ERβ deficiency did not impact total tumor multiplicity (data not shown; P ≥ 0.05) or small intestinal tumor multiplicity (Figure 5B; P ≥ 0.05) in the second intercross population.Fig. 5.ERβ deficiency increases incidence of colon tumors in ApcMin mice in the Helsinki but not Omaha intercross. Adenoma multiplicity data from the small intestinal tract (A–B) and colon (C–D) are illustrated. Colon adenoma incidence data are shown in panels (E and F). The ‘1’ indicates a value significantly different from that in ERβ+/+ ApcMin/+ mice and the ‘2’ indicates a value significantly different from that in ERβ+/− ApcMin/+ mice. The number of mice from the Helsinki N10 intercross was as follows: ERβ+/+ ApcMin/+ (n = 19), ERβ+/− ApcMin/+ (n = 14) and ERβ−/− ApcMin/+ mice (n = 14). The number of mice from the Omaha N10 intercross was as follows: ERβ+/+ ApcMin/+ (n = 9), ERβ+/− ApcMin/+ (n = 18) and ERβ−/− ApcMin/+ mice (n = 17).Analysis of the tumorigenesis in the colon indicated that ERβ genotype had no significant effect on the mean number of adenomas in the colon at 90 days of age (Figure 5C; P = 0.06–0.20) or 140 days of age (Figure 5D; P ≥ 0.05). In contrast, the 100% (14/14) incidence of colon tumors in 90-day-old ApcMin/+ ERβ−/− mice was greater than the 57% (8/14) incidence in age-matched ApcMin/+ ERβ+/+ mice (Figure 5E; P < 0.01) and the 68% (13/19) incidence in age-matched ApcMin/+ ERβ+/− mice (Figure 5E; P < 0.05). ERβ had no impact on colon adenoma incidence in the Omaha intercross mice evaluated at 140 days of age (Figure 5F; P ≥ 0.05).Potential impact of 129P2-derived loci in the ERβ knockout crosses?To assess the potential impact of residual 129P2 alleles present in the ERβ intercross populations, we genotyped the B6.129-Esr2tm1Ksk mice used in each study with markers distributed across the genome (Table I) to identify 129P2-derived regions. In the Helsinki intercross, 129P2 alleles were detected at only two markers—one unlinked to ERβ (D1Mit206) and one linked to ERβ (D12Mit91) (Table I). At these two markers, ApcMin/+ ERβ+/+, ApcMin/+ ERβ+/− and ApcMin/+ ERβ−/− intercross mice were either homozygous for the B6 allele or heterozygous. Genotype at these markers had no significant impact on total adenoma number in the intercross mice (data not shown; P = 0.3). Likewise, genotype at these markers did not have a significant impact on colon adenoma number (data not shown; P > 0.05).In the Omaha intercross mice, 129P2 alleles were detected at one marker unlinked to ERβ (D3Mit203) and three markers linked to ERβ (D12Mit60, D12Mit91 and D12Mit118) (Table I). Genotype at D3Mit203 had no impact on tumor multiplicity (P > 0.05; data not shown). This analysis is straightforward because D3Mit203 and ERβ are unlinked. In contrast, evaluating the potentially confounding effects of 129P2 alleles at markers linked to the ERβ knockout was complicated by the significant linkage disequilibrium. Genotype at ERβ and the three markers proximal of ERβ on chromosome 12 showed a high degree of concordance as follows: 93% for D12Mit91, 84% for D12Mit60 and 62% for D12Mit182. Thus, as much as 77 Mbp of the 129P2 genome on chromosome 12 is retained in association with the ERβ knockout allele (Table I).Thus, both the Helsinki and the Omaha intercrosses were performed on a congenic B6 background in which only a single marker unlinked to ERβ was still segregating 129P2 alleles. This uniform genetic background facilitates the interpretation of results because the possible impact of modifier loci unlinked to the ERβ mutation can be discounted. However, there were significant differences between the ERβ knockout mice used in these two crosses with respect to markers linked to ERβ. In the Helsinki cross, the degree of linkage disequilibrium between the ERβ knockout and linked 129P2 alleles is quite small, and the potential for linked modifiers is therefore minimal. In contrast, a larger block of 129P2 alleles that was retained in association with the ERβ knockout in the Omaha cross. Although there is no direct evidence supporting the idea that a locus linked to ERβ modifies tumorigenesis in ApcMin/+ mice, our observations prevent us from discounting this possibility.ERβ deficiency does not impact the levels of β-catenin, Cyclin D1, E-cadherin or ERα in the colonic mucosaBecause ERβ deficiency increased colon tumor incidence, we examined the impact of ERβ deficiency on β-catenin expression and localization, the expression of Wnt–β-catenin target genes including Cyclin D1 and the interaction of β-catenin with the cell adhesion molecule E-cadherin. We used western blotting to analyze the levels of β-catenin, E-cadherin and Cyclin D1 in the nuclear and membrane fraction of the colonic mucosa of ApcMin/+ ERβ+/+ and ApcMin/+ ERβ−/− mice. Additionally, we analyzed the impact of ERβ deficiency on the expression of the ERα. Deletion of ERβ had no effect on the expression of the nuclear β-catenin or Cyclin D1 or membrane β-catenin or E-cadherin (supplementary Figure 1 is available at Carcinogenesis Online). Furthermore, the expression of ERα protein was unaffected (supplementary Figure 1 available at Carcinogenesis Online). Thus, we found no evidence for ERβ-dependent modulation of the Wnt pathway.DiscussionIn the present study, ERβ deficiency did not increase adenoma multiplicity in ApcMin/+ mice. This lack of effect of ERβ deficiency on total tumor multiplicity or small intestinal tumor multiplicity was confirmed in a second, independent cross involving the same ERβ knockout allele (Esr2Ksk1) and ApcMin/+ mice. This second cross was conducted at a separate institution using distinct husbandry conditions. A similar lack of effect of ERβ deficiency on small intestinal tumorigenesis in ApcMin/+ mice was also reported by Cho et al. (18) in a study using a distinct ERβ knockout allele (Esr2Pcn1) (38). These three observations differ from that of Giroux et al. (19), in which it is reported that deficiency of the Esr2Pcn1 ERβ knockout allele increased tumor multiplicity and size in female but not male ApcMin/+ mice. However, it should be noted that in the study of Giroux et al., female mice had been ovariectomized and implanted with E2 and progesterone pellets that result in continuous serum levels of these hormones in the high physiological range. In the two intercrosses we performed, as well as the study of Cho et al., ovary intact females were used. Consistent with the ovarian defects described previously in ApcMin/+ females (39), we observe that ApcMin/+ females have reduced serum levels of E2 and progesterone. Thus, there are significant differences in hormone levels between mice in these various studies. These differences may contribute to the disparate results observed.In the present study, we observed that ERβ deficiency was associated with an increased incidence of colon tumors in ApcMin/+ mice. Although our results differ from those described in Giroux et al. (19), in which the Esr2Pcn1 ERβ knockout allele was found to have no impact on colon tumorigenesis in ApcMin/+ mice, our data are consistent with the study of Cho et al. (18), which reported that homozygosity for the Esr2Pcn1 ERβ knockout allele increased colon tumor incidence in ApcMin/+ mice. In our study, we found that the increased incidence in colon tumors in ERβ-deficient ApcMin/+ mice was not associated with enhanced activation of the Wnt–β-catenin pathway. These observations are consistent with a previous report (19) suggesting that ERβ deficiency may promote intestinal tumorigenesis by modulating the transforming growth factor β pathway. Thus, our present studies using ApcMin/+ mice and a distinct ERβ knockout (Esr2Ksk1) help resolve the controversy regarding the impact of ERβ on colon tumorigenesis and provide independent support for the hypothesis that ERβ deficiency may promote tumorigenesis through a Wnt–β-catenin-independent pathway.We also observed that genetic disruption of ERα in ApcMin/+ mice was associated with an increase in intestinal adenoma multiplicity, size and burden. These data are consistent with the hypothesis that loss of ERα expression contributes to intestinal tumorigenesis. Although the ERα knockout used here is a hypomorph, resulting in a truncated receptor (40) with a deleted amino-terminus and no activation function 1 domain but with some residual ligand binding and/or function, it is clear that disruption of ERα, even if incomplete, is associated with enhanced tumorigenesis ApcMin mice. It is noteworthy that when Cho et al. (18) crossed another ERα knockout (B6.129-Esr1Pcn1), one reported to be a more complete disruption of ERα, with ApcMin/+ mice, no ERα−/− ApcMin/+ mice were recovered. One intriguing interpretation of this result is that there is a synthetic lethal interaction between the ERα allele and the ApcMin mutation. In our study, ERα−/− ApcMin/+ mice were recovered at the expected frequency.Based on data from humans implicating ERα as a tumor suppressor in colon, we conclude that ERα deficiency itself is responsible for the enhanced tumorigenesis in ERα−/− ApcMin/+ mice. However, it is impossible to completely exclude the possibility that the enhanced tumorigenesis in ERα−/− ApcMin/+ mice is due to another allele that resides within the 129P2-derived region of <13 Mbp on chromosome 10 that is retained in association with the ERα knockout allele. Less than a dozen genes map to this region, and none of these is known to modulate tumorigenesis in ApcMin/+ mice. The Scc14 locus, which modulates susceptibility to azoxymethane-induced colon cancer in a cross between the BALB/c and the CcS19/Dem strains, is linked to a marker 3.4 Mbp proximal of ERα (41). Nothing is known about the impact of the 129P2 allele of Scc14 on colon cancer. Assuming that ERα and Scc14 represent distinct genes, we cannot exclude the possibility that the 129P2 allele of Scc14 increases tumor multiplicity in our cross. However, our previous studies provide no evidence that 129 substrains carry alleles that enhance tumorigenesis in ApcMin/+ mice. Rather, we have shown that the 129S2/SvPas substrain carries alleles that reduce tumor multiplicity in ApcMin/+ mice (27). Additionally, in Oikarinen et al., in this issue of Carcinogenesis, we report the localization of Mom5, a modifier of tumorigenesis in ApcMin/+ mice; the 129P2 allele of Mom5 is associated with reduced tumor number.The impact of ERα deficiency was equivalent in ApcMin/+ males and females. These results are consistent with the fact that ERα expression is silenced in colorectal cancers in both men and women (13,42). Although ERα signaling reduces intestinal tumorigenesis in both male and female ApcMin/+ mice, it is unclear whether this effect requires endogenous estrogens or is ligand independent. ApcMin/+ females in our colony exhibit diminished ovarian function and low serum concentrations of E2 and progesterone. Although the physiological basis for the impact of the Min mutation of ovarian function is unknown, these observations, together with our observation that tumorigenesis in ApcMin/+ females is not influenced by ovariectomy (K.A.Gould, unpublished data), suggest that in our colony, ovarian estrogens do not play a role in ERα-dependent processes that attenuate tumorigenesis in ApcMin/+ mice.We report that ERα deficiency in ApcMin/+ mice is associated with an accumulation of nuclear β-catenin, suggesting that ERα deficiency activates canonical Wnt–β-catenin signaling. The level of β-catenin in normal intestinal epithelium of ERα−/− ApcMin/+ mice is less than that in ApcMin-induced intestinal adenomas (K.A.Gould, unpublished data), suggesting that further activation of Wnt–β-catenin signaling occurs during tumorigenesis, probably as a consequence of loss of Apc+, which occurs at a frequency of 100% in adenomas of B6 ApcMin/+ mice (11,31). Consistent with the hypothesis that ERα deficiency enhances Wnt–β-catenin signaling, the expression of the Wnt–β-catenin target genes Cyclin D1 and c-Myc was increased in the normal intestinal epithelium of ERα−/− ApcMin/+ mice. The expression of Cyclin D1 and c-Myc is regulated by many factors, including estrogens (43,44). Thus, it is possible that the increased expression of Cyclin D1 and c-Myc in ERα−/− ApcMin/+ epithelium is unrelated to Wnt–β-catenin activation. Nonetheless, the upregulation of these genes, which are known to play a role in colorectal cancer, indicates that ERα deficiency in ApcMin/+ mice is associated with increased expression of genes involved in intestinal tumorigenesis.Although it is known that there is cross talk between the estrogen and the Wnt pathways in estrogen-responsive tissues such as the mammary gland (33), further studies will be required to understand the molecular basis and significance of cross talk between ERα and Wnt signaling in the intestinal epithelium. In contrast to what is observed in the mammary gland, our studies suggest that Wnt and estrogen signaling have opposing effects in intestine, with Wnt signaling promoting tumorigenesis and ERα signaling inhibiting tumorigenesis.Supplementary materialSupplementary Figure 1 can be found at http://carcin.oxfordjournals.org/FundingNebraska Department of Health and Human Services; National Institutes of Health (K01-CA113413 to K.A.G.).AbbreviationsApcadenomatous polyposis coliERestrogen receptorE217β-estradiolMinmultiple intestinal neoplasiaPCRpolymerase chain reactionConflict of Interest Statement: None declared.1.American Cancer SocietyColorectal Cancer Facts and Figures Special Edition 20052005Atlanta, GAAmerican Cancer Society2.CalleEEEstrogen replacement therapy and risk of fatal colon cancer in a prospective cohort of postmenopausal womenJ. 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USA1999961121711222 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B48D236D9F55F298C43CF722F0CFC3D53AD9D5C.txt b/test/dataset/in/resources/corpus/Clean_0B48D236D9F55F298C43CF722F0CFC3D53AD9D5C.txt new file mode 100644 index 0000000..6b99d2f --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B48D236D9F55F298C43CF722F0CFC3D53AD9D5C.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 0020410.1093/geront/41.4.449 RESEARCH ARTICLE Come Talk With Me Improving Communication Between Nursing Assistants and Nursing Home Residents During Care Routines Burgio Louis D. a b Allen-Burge Rebecca a b Roth David L. c Bourgeois Michelle S. d Dijkstra Katinka d Gerstle John a Jackson Erik a Bankester Leanna a aApplied Gerontology Program, The University of Alabama, Tuscaloosa bDepartment of Psychology, The University of Alabama, Tuscaloosa cDepartment of Psychology, University of Alabama at Birmingham dDepartment of Communication Disorders, Florida State University, Tallahassee Louis D. Burgio, The University of Alabama, Department of Psychology and The Applied Gerontology Program, Box 870315, Tuscaloosa, AL 35487-0315. E-mail: lburgio@sw.ua.edu. 1 8 2001 41 4 449 460 6 4 2001 18 9 2000 The Gerontological Society of America 2001 Purpose: We examined the effects of communication skills training and the use of memory books by certified nursing assistants (CNAs) on verbal interactions between CNAs (n = 64) and nursing home residents (n = 67) during care routines. Design and Methods: CNAs were taught to use communication skills and memory books during their interactions with residents with moderate cognitive impairments and intact communication abilities. A staff motivational system was used to encourage performance and maintenance of these skills. Formal measures of treatment implementation were included. Results:Results were compared with those for participants on no-treatment control units. Trained CNAs talked more, used positive statements more frequently, and tended to increase the number of specific instructions given to residents. Changes in staff behavior did not result in an increase in total time giving care to residents. Maintenance of CNA behavior change was found 2 months after research staff exited the facility. Although an increase was found in positive verbal interactions between CNAs and residents on intervention units, other changes in resident communication were absent. Implications: Nursing staff can be trained to improve and maintain communication skills during care without increasing the amount of time delivering care. The methodological advantages of including measures to assess treatment implementation are discussed. Long-term care Staff training Dementia hwp-legacy-fpage 449 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: Laurence G. Branch, PhD The social milieu of the nursing home is a critical component of both quality of care and quality of life in this setting. In recent years, researchers have focused their efforts on understanding and improving various aspects of staff–resident and resident–resident social interactions. Direct behavioral observation of residents on a psychogeriatric ward revealed that 21% of residents' time was spent in physical care interactions (Hallberg, Norberg, and Eriksson 1990). Observation of certified nursing assistants' (CNAs') work behavior in nursing homes revealed that 53% of observation time was spent in a broader category of resident care, with 11.8% of observation time engaged in staff–resident verbal interaction (Burgio, Engel, Hawkins, McCormick, and Scheve 1990). Sadly, positive verbal interactions appear to be as rare as negative verbal interactions in nursing homes (Burgio et al. 1990), where neutral interactions appear to be the norm (Carstensen, Fisher, and Malloy 1995). These data suggest that intervention is necessary to structure staff–resident social interactions during care to positively affect resident quality of life. Communication-based interventions in nursing homes have the potential to improve resident behavioral deficits such as excess disability, low-rate social engagement, and behavioral excesses such as disruptive vocalization (Allen-Burge, Stevens, and Burgio 1999; Burgio and Stevens 1999). Through these interventions, staff are taught therapeutic strategies for approaching residents and responding to both adaptive and maladaptive behaviors. Recent small-sample studies of communication-based staff-training interventions to reduce resident problem behaviors have suggested that training staff to communicate more effectively might reduce the incidence of physical aggression (Boehm, Whall, Cosgrove, Locke, and Schlenk 1995; Hoeffner, Radar, McKenzie, Lavelle, and Stewart 1997). Many recent studies have used packaged staff interventions that include both communication skills training and the use of prosthetic memory aids, such as memory books, to compensate for residents' diminished cognitive capacity. Memory books contain images and brief, simple sentences that use the preserved automatic processing abilities of frail older adults to improve the structure and quality of communication with others, hopefully making interactions more pleasant for both older individuals and their partners in communication (Bourgeois 1990, Bourgeois 1992a, Bourgeois 1992b, Bourgeois 1993; Bourgeois and Mason 1996). Memory books can serve two functions. First, they can provide meaningful stimulation to nursing home residents in an environment that can be both physically and socially barren. Second, they can be used as a tool by staff or other residents to facilitate communication with cognitively impaired residents. Through the use of memory books, community-residing dementia patients with a wide range of cognitive deficits have increased the informativeness and accuracy of their conversations and decreased their ambiguity and repetitiveness (Bourgeois 1990, Bourgeois 1992a, Bourgeois 1992b; Bourgeois and Mason 1996). Use of these aids has decreased disruptive verbal behaviors such as repetitive questioning (Bourgeois, Burgio, Schulz, Beach, and Palmer 1997). In nursing homes, content analysis of researcher-initiated, semistructured 5-min conversational interactions between demented residents and their primary CNAs, and social validity ratings of these interactions by unfamiliar judges, revealed modest improvements in resident and CNA conversational behaviors (Hoerster, Hickey, and Bourgeois in press). Ripich, Wykle, and Niles 1995 developed and tested a didactic training program for improving communication between CNAs and residents. In addition to teaching specific verbal and nonverbal communication strategies, training modules included information on language difficulties associated with different stages of dementia, depression, and cultural–ethnic considerations. Results showed that training improved both CNA knowledge and attitudes toward residents with dementia. McCallion, Toseland, Lacey, and Banks 1999 evaluated a Nursing Assistant Communication Skills Program on various staff and resident outcomes in nursing homes. In addition to teaching verbal and nonverbal communication skills, staff were taught to use memory aids. Memory aids included placing written and graphic signs in important locations and the use of memory charts placed on resident bedroom walls. Memory charts contained photographs, brief statements, and conversation topics that were meant to encourage communication with the residents. To facilitate maintenance of therapeutic effort, the trainer made informal monthly visits to the nursing homes to verify continued implementation of the Nursing Assistant Communiation Skills Program techniques. Results showed durable effects (6 months posttreatment) on resident depressive symptomatology. However, the effects on resident disruptive behaviors were mixed and generally transient. McCallion, Toseland, and Freeman 1999 also developed a Family Visit Education Program, which focused on improving communication among residents, nursing staff, and visiting family members. Compared with a control group, the Family Visit Education Program showed benefits for residents and family members, but not for nursing staff. Specifically, family members demonstrated improved communication patterns with residents; residents showed improvements in depression, irritability, and verbal behavior. Staff also reported a reduction in agitation. The success of communication-based interventions designed to improve nursing home care depends heavily on the receptiveness of nursing staff to learn new skills and on the establishment of staff motivational systems to ensure the maintenance of these skills (Burgio and Burgio 1990; Burgio and Scilley 1994; Burgio and Stevens 1999; Stevens et al. 1998). Staff motivational systems such as behavioral supervision (Burgio and Burgio 1990) and total quality management (Schnelle, Ouslander, Osterweil, and Blumenthal 1993) have been used successfully to motivate staff in the implementation of a variety of interventions in nursing homes. Behavioral supervision is designed to motivate management and direct care staff to observe and analyze skill performance in order to identify problems and to induce supervisory staff to provide direct and specific feedback suggesting practical ways of maximizing skills (Burgio and Stevens 1999; Daniels 1994). Using behavioral supervision, Stevens et al. 1998 showed that CNAs could be trained to increase and maintain the rate of task announcements and positive statements made to residents during care over a 46-week assessment period. In a small intra-subject pilot study (N = 8), Allen-Burge, Burgio, Bourgeois, Sims, and Nunnikhoven 2001 examined the effects of communication skills training and the use of memory books. CNAs were taught to use communication skills and memory books during their interactions with residents with mild to moderate cognitive impairment but relatively intact communication abilities. An abbreviated system of behavioral supervision was attempted to encourage performance of these skills on the nursing units. Results showed that, regardless of sporadic implementation of the intervention by nursing staff, the intervention improved communication between staff and residents during care routines, increased the amount of time other residents and visitors spent talking with target residents, and increased the rate of positive statements made by the target residents and others in their immediate environment. The purpose of the present study was to examine the efficacy of communication skills training and the use of individualized memory books in improving communication between CNAs and a larger group of residents (N = 67) during care. In contrast to the pilot study, we used a more comprehensive staff motivation system (behavioral supervision). In addition, formal measures of treatment implementation were included (Lichstein, Riedel, and Grieve 1994). We hypothesized that (a) through the use of behavioral supervision, CNAs' use of therapeutic procedures would increase, compared with their baseline performance, and would maintain at a 2-month follow-up assessment; (b) trained CNAs would increase their frequency of overall verbal interaction and positive verbal interactions compared with their own baseline performance and untrained CNAs; and (c) residents on trained nursing units would show increases in overall amount of coherent verbal interaction (i.e., understandable speech) compared with residents on untrained units. Methods Settings This study was conducted in five nursing homes with an average census of 120 residents on three units. On average, 11% of the beds were Medicare, 50% were Medicaid, and the remainder were private pay. Four of the nursing homes were corporately owned facilities; one was privately owned with a religious affiliation. Special care units were not included in the study. Residents had an average length of stay of 3 years; 93% were female, 75% were white, and 25% were African American. Resident-to-CNA ratios were 9:1 during the day shift and 12:1 during the evening shift. Reported yearly rates of staff turnover were 8% among registered nurses (RNs), 18% among licensed practical nurses (LPNs), and 49% among CNAs. The American Health Care Association reported that the national turnover rate is 97% for CNAs (as cited in Harrington et al. 2000). Participants Residents Residents were entered into the study on the basis of the following criteria: (a) age of at least 55 years; (b) Mini-Mental Status Examination (MMSE; Folstein, Folstein, and McHugh 1975) total score greater than 0 or Short Portable Mental Status Questionnaire (Pfeiffer 1975) with fewer than 10 errors (our purpose for using these criteria were to exclude nonresponsive residents); (c) retention of minimal ability in verbal communication involving spontaneous speech based on criteria established by Bourgeois 1993(criteria available by writing Louis D. Burgio or Rebecca Allen-Burge); (d) absence of major sensory impairment; (e) life expectancy greater than 6 months; and (f) residence within the facility for at least 1 month. Because of the importance in this study of observing staff–resident interactions during care routines, residents who completed activities of daily living (ADLs) independently during baseline were excluded. Residents were considered independent if the care tasks were completed with no nursing assistant present for at least 5 minutes across two observational occasions during baseline. Two hundred sixty-three of 403 residents in the five nursing homes met entry criteria on the basis of nursing staff referral; 111 consented to participate, the majority through proxy. The consent rate was 42%. Nineteen were excluded during the baseline phase because the communication assessment and ADL observation indicated that they did not meet entry criteria; thus, 92 residents meeting entry criteria completed baseline. A decision was made to include in the data analyses only residents who had data available for all three assessment points (baseline, Postintervention Assessments I and II). Twenty-five residents were excluded due to incomplete data, leaving 67 residents for data analysis (33 control and 34 intervention residents). The reasons for exclusion were the following: eight residents died prior to the intervention phase, 9 moved to another nursing unit or different facility, 4 residents or sponsors withdrew consent for participation, and 4 residents refused to be observed during care. There were no significant differences between the surviving (n = 67) and the excluded (n = 25) residents on age, gender, race, dementia diagnosis, MMSE, or Functional Independence Measure (ADL) scores. Table 1 shows selected characteristics of surviving residents in the study sample at baseline. As can be seen, there were no significant differences between residents on the control and treatment units. Staff Ninety-eight CNAs were working on either the intervention or control units during baseline assessment. Observational data were available during all three assessment points for 64 CNAs; thus, 34 CNAs were dropped from the analysis. There were no differences in gender, race, education, age, or length of service between the 98 CNAs available at baseline and the 64 surviving CNAs. Table 2 shows selected characteristics of surviving CNAs in the study sample at baseline; however, demographic data were available for only 37 CNAs in the intervention group. As can be seen, there were no significant differences between CNAs on the control units (n = 25) and treatment units (n = 39; 37 providing demographic data). Design and Procedures We used a two-group comparison design with an intragroup comparison component embedded in each group. The two groups were (a) memory book intervention with a staff motivational system and (b) no-treatment control (NTC). The study was conducted sequentially across nursing homes. Upon entering a nursing home, two units were selected randomly and also randomly assigned to treatment and control conditions. Data were collected on treatment and control units concurrently. We spent the first 3 to 4 weeks in each facility, prior to baseline recording, gathering information on all consenting treatment and control residents and on regular day and evening shift nursing staff. This was followed by a 4-week (Weeks 1 to 4) baseline phase (both units) designed to assess the social environment in the nursing homes before intervention. During this and all phases, direct observational and paper-and-pencil assessments were completed (see Measures section). On treatment units, communication–memory book skill inservice workshops were conducted during a 1-week period immediately following baseline (Week 5; see Intervention Conditions section). This was followed by hands-on training on the intervention units during the next 4 weeks (Weeks 6 to 9). During this phase, CNAs were instructed in the use of communication–memory book skills by research and indigenous nursing staff. Research staff also instructed supervisory nursing staff in the use of the staff motivational system during this time. The next 8 weeks consisted of evaluation of the intervention's effectiveness using the same assessments as at baseline. This assessment period was divided into two phases: Postintervention Assessment I (PIA–I) represented staff and resident performance during the first 4 weeks (Weeks 10 to 13); Postintervention Assessment II (PIA–II) represented their performance during the second 4 weeks of this phase (Weeks 14 to 17). During the first segment of this latter phase, supervisory nursing staff continued to receive assistance from research staff on the use of the staff motivational system. By the end of the phase, indigenous staff controlled all aspects of the system and received no assistance from research staff. To enhance maintenance of the staff motivational system, consultation visits by the project manager were conducted at 3 and 6 weeks after we had departed from the facility (i.e., after PIA–II). Two months after PIA–II, probe follow-up data were collected on staff and resident performance during a 1-week period in four of the five nursing homes. The fifth nursing home declined the follow-up assessment because of a high level of administrative and nursing staff turnover. Observational data were collected using a reduced sampling schedule. Intervention Conditions Communication–Memory Book Skills and Staff Motivational System Participating residents received a personalized 12-page laminated memory book consisting of biographical, orientation, and daily schedule information. Each 5- × 5-in. page included a 6–10 word declarative sentence printed in 14- or 20-point Times New Roman type with a color or black-and-white photograph or line drawing illustrating the statement. Residents' books might contain pictures of their wedding and family, their CNA, other residents, their daily schedule, instructions on bathing, and pages targeting behavior problems such as wandering, aggression, or repetitive questioning. It was the CNA's responsibility to ensure that books were always present in the residents' living space. Residents were provided with individually tailored vests that attached the book to the resident's torso, wheelchair caddies that attached the book to the resident's wheelchair, or a stand to display the book prominently in the resident's room. Additional pages were added to the books every 4 weeks in order to maintain staff and resident interest in this aspect of the intervention. Memory books were replaced by research staff if lost. Replacements were provided for 38% of the residents. All nursing staff on intervention units were trained in the use of memory books and general communication skills. The first component of staff training consisted of a 2-hr inservice session on communication–memory book skills conducted by the project manager (a licensed clinical psychologist). Members of the nursing staff were instructed to use memory books with residents (a) to increase general communication among residents and between nursing staff and residents, (b) to increase the independent functioning of residents during targeted care routines, and (c) as a distraction to decrease resident disruptive behaviors. Inservice materials are available from Louis D. Burgio or Rebecca Allen-Burge on request. CNAs and supervisory staff then attended a 1-hr inservice to introduce the staff motivational system. An additional hour of training was provided to LPNs and RNs on the supervision of this system. The critical roles of the CNA and LPN were emphasized because these members of the nursing staff have the most direct contact with residents (Burgio et al. 1990). In addition to didactic training, active learning techniques including the use of role play, discussion of real-life examples from the nursing units, and discussion of written vignettes were used to engage staff. Notebooks were provided to each member of the nursing staff that included all information from the inservices. Staff were encouraged to refer to these notebooks while working with residents on the unit. To increase the likelihood that staff would use the newly learned skills on the units, behavioral supervision (Burgio and Burgio 1990) was taught during the inservice, implemented by research staff during the 4-week hands-on training phase and trans-ferred to indigenous nursing home staff during the 8-week postintervention assessment period. Initially, the CNAs were trained by research staff in the use of memory books, communication skills, and components of the staff motivational system. CNAs on the day and evening shifts were observed by research staff once per day while providing care to residents. Researchers and LPNs used the Communication Skills Checklist (CSC) during the care routine to record whether CNAs displayed the skills taught in the inservice; feedback was provided to CNAs regarding their use of the memory books, use of specific versus general instructions, one-step instructions, positive statements, responses to behavioral disturbances, and general distraction techniques. CNAs were provided verbal performance feedback immediately following the care routine. The project manager also completed the Observation of the LPN's Supervisory Activities (OLSA) on the LPNs during this period. Only data collected by research staff were used in outcome analysis. Thirty-nine CNAs on the intervention units were taught to monitor and record their own skill performance as a means of tracking their performance and as a daily reminder of the skills needed to be an effective communicator. CNAs were asked to use the self-monitoring form during shifts worked throughout the week. CNAs had the opportunity to use the form an average of four times (range 1–10) per week. To meet job performance goals, CNAs were asked to complete at least an average of 80% of their assigned self-monitoring forms. To reach performance criterion, they were also expected to obtain an average of 80% accuracy on the CSC completed by LPNs or research assistants. Trained CNAs received public recognition for meeting job performance criteria (i.e., 80% completion of forms and 80% accuracy) by having their names posted weekly on a CNA Honor Roll. Those CNAs whose names appeared on the honor roll received an opportunity for a performance incentive. All honor-roll CNAs listed were entered into a performance-based lottery held once each week for day and evening shifts (Reid, Parsons, and Green 1989). For each shift, the individual winning the lottery was provided with his or her choice of incentives from a list of choices determined by each nursing home, including the following: (a) free lunch in the cafeteria every day for 1 week, (b) a "goodie bag" full of inexpensive snacks, beauty products, and knick-knacks, (c) permission to arrive at work 15 min later than scheduled every day for 1 week, (d) permission to leave work 15–30 min earlier than scheduled every day for 1 week, (e) permission to leave work 2.5 hr early on Friday, or (f) 2.5 hr of extra pay. Across nursing homes, the most frequently chosen incentives were the opportunity to leave work earlier than scheduled, extra pay, and goodie bags. LPNs on the intervention unit also received public recognition for meeting job performance criteria (i.e., 80% completion of CNA monitoring and supervisory forms) by having their names posted weekly on an LPN Honor Roll. On average, LPNs were assigned 2–5 CNAs to monitor and supervise in their section on their shift. NTC CNAs and LPNs on the NTC units did not participate in any intervention-related activity. Assessments were conducted on NTC units on the same schedule as on the intervention units. CNAs and LPNs on the intervention units were occasionally pulled to the NTC units. When this occurred, they were asked not to use any of the skills learned on the intervention units. After the 2-month follow-up assessment period, staff on the NTC units were offered instructions and materials on communication–memory book skills. Measurement CNA Communication Skills Checklist The CSC is a direct observation behavioral frequency measure allowing the researcher to assess CNAs' use of specific versus general instructions, one-step instructions, positive statements, biographical statements not included in the memory book (generalization), responses to behavioral disturbances, and use of general distraction techniques (Allen-Burge et al. 2001). Research staff attempted to observe every CNA daily during hands-on training, twice during baseline and each of the two postintervention assessments, and once during follow-up. We were successful in completing this target number of observations for 83% of the CNAs. The CSC focused on communication skills demonstrated during a care interaction. To allow for an adequate sampling of CNAs' use of communication–memory book skills, at least three separate care activities (i.e., bathing, dressing, transfer) were included during the observation in order for the data to be retained. A total of 76.37 hr of observation (range 2–60 min per CSC) were completed across all study phases (i.e., 22.60 hr during baseline, 18.94 hr during PIA–I, 16.20 hr during PIA–II, and 18.63 hr during follow-up). Data were expressed as total number of occurrences of each type of statement divided by the total number of seconds in the observation period. Interobserver reliability was assessed independently among observers during 13% (80/603; 14.03 hr) of the observations across all sites and phases. Observer agreement for each behavioral category measured by the CSC was calculated by using a total occurrence agreement calculation. The average percentage of agreement for this measure across all categories was 84% (range 71%–99%). OLSA The OLSA allows the researcher to measure LPNs' accuracy in observing and recording CNA skill performance during supervision, and LPNs' skill in providing CNAs with verbal performance feedback. As part of the staff motivational system, LPNs observed each of the CNAs under their supervision once per week using a form similar to the CSC during 15-min samples of care routines with residents. During these observations, LPNs recorded the occurrence of CNAs' use of memory books, specific versus general instructions, one-step instructions, positive statements, negative or unhelpful statements, biographical statements not included in the memory book, and CNAs' responses to behavioral disturbances. LPNs' accuracy was checked against the project manager's recording of the same behaviors during the same observation. The project manager was present with LPNs observing CNAs using the CSC during the staff training period. The OLSA also allowed the project manager to rate the LPNs' use of verbal supervisory feedback. Verbal supervisory feedback is rated with regard to beginning and ending a session with a supportive statement to the CNA, the provision of accurate and specific positive and corrective feedback to the CNA, the provision of specific performance scores to the CNA with a statement indicating the CNA's training status, and the provision of an opportunity for the CNA to discuss any of the feedback given by the LPN. CNAs received positive or negative written feedback letters at the end of the hands-on training period and at the end of PIA–I. To receive a positive feedback letter, CNAs had to attain an average performance score of 80% or better in their completion of self-monitoring forms and LPN evaluations for the previous 4-week period. These letters were placed in the CNAs' personnel files, and copies were given to CNAs and to administrative nursing staff. Computer-Assisted Behavioral Observation System (CABOS): Hardware and Software As in Burgio, Scilley, Hardin, Hsu, and Yancey 1996 prior research, the Portable Computer Systems for Observational Research software programs from Communitech International (DeKalb, IL) were used for this project (Repp, Karsh, van Acker, Felce, and Harman 1989). The recording of behavior was synchronized with each computer's internal time clock and controlled through a software routine. Real-time CABOS data were generated by sampling behaviors during care interaction between residents and their primary day- and/or evening-shift CNA. All participating residents were observed twice during baseline, PIA–I and PIA–II, and once during follow-up. Care routines had to be at least 5 min in length for the data to be retained. Domains of behavior divided into mutually exclusive and exhaustive categories relevant to the hypothesized outcomes were identified, and detailed operational definitions were generated for all behavior codes within categories. One category coded the residents' activity, including the amount of ADL care. Four categories coded aspects of the residents' social environment: (a) nonverbal presence of staff or other residents, (b) verbal interaction, (c) verbal content, and (d) disruptive behaviors. Each behavioral category contained codes that allowed specification of type of behavior within the category and whether it was initiated by staff or resident (e.g., content had separate positive and negative statement codes). A total of 70.29 hr of observation (range 5–51 min per care interaction) was completed across all study phases (i.e., 24.24 hr during baseline, 20.31 hr during PIA–I, 18.90 hr during PIA–II, 6.84 hr during follow-up). Interobserver agreement was calculated using Cohen's kappa (Cohen 1968; Hays 1994). Reliability was assessed independently among four observers during 14% (14.03 hr) of the total observation time across all phases. Average kappa reliability across all categories was .79 (range .68–.95). In behavioral research, recommended lower limits for acceptable kappas range from .60 to .75 (Hartmann 1982). Memory Book Checks Research assistants recorded whether residents were in possession of memory books (i.e., memory books were prominently displayed and within arm's reach of the resident) during separate observational checks conducted twice daily, once during the morning and evening shifts. This yielded a total of 282 checks during PIA–I, PIA–II, and the 2-month follow-up assessment. Data were recorded dichotomously (yes/no) at both checks for each resident. Because NTC residents did not possess memory books, checks were conducted only on intervention units. Paper-and-Pencil Measures Assessment instruments were administered either by a clinical psychologist or by research assistants trained and supervised by the clinical psychologist to administer these measures. MMSE (Folstein et al. 1975) The MMSE, a measure of global cognitive ability, measures orientation, immediate and delayed recall for words, attention and concentration, language, and praxis (total score range 0–30). The test–retest and interevaluator reliabilities are .89 and .83, respectively. Functional Independence Measure (FIM)—REACH Version Information for completing this instrument was provided by CNAs familiar with the daily care needs of the participating resident (Hamilton, Laughlin, Fiedler, and Granger 1994; Kidd et al. 1995). The version used in this study was developed for the National Institutes of Health–funded cooperative agreement Resources for Enhancing Alzheimer's Caregiver Health (REACH; Coon, Schulz, and Ory 1999). As in the REACH project, only the motor subscale consisting of self-care, sphincter control, transfer, and locomotion items was used in this study (total score range 13–91). Reliability data indicated an intraclass correlation coefficient of .96 for the motor domain, with unweighted kappas ranging from .53 to .66 (Hamilton et al. 1994). Method of Analysis Analyses were conducted on the CSC and on CABOS data collected during care routines. CNAs were the unit of analysis for CSC data; data were expressed as mean rate of CNA statements per hour. CSC outcome data included (a) the rate of specific, one-step instructions, (b) the rate of CNA positive statements, (c) the rate of biographical statements, (d) the rate of multiple-step instructions, and (e) the total duration of care. CABOS data were expressed as mean total percentage of observation time spent in each activity or mean rate per hour of positive statements, and the unit of analysis was the resident. CABOS outcome data included (a) total percentage of time of resident coherent verbal interaction, (b) total percentage of time of staff speech directed to the resident, and (c) the rate per hour of positive statements made by either residents or CNAs. We did not discriminate between staff and resident positive statements on this variable. Positive statements are brief duration events and were expressed as the rate of statements per hour. Our analytic questions centered on (a) assessment of treatment delivery and enactment (Lichstein et al. 1994), (b) establishing an initial change in behavior for both staff and residents after implementation of the intervention, and (c) assessing the maintenance of any staff or resident behavior change 2 months later. Descriptive statistics were used to address the first question. For the second analytical question, analyses were run by calculating 2 (group) × 3 (time) mixed-factor analysis of variance (ANOVA), where the between-subjects factor Group included intervention and control and the within-subjects factor Time included baseline, PIA–I, and PIA–II phases. The primary outcome of interest in these analyses were significant Group × Time interaction effects, showing that the intervention group improved in the targeted behavioral outcomes for CNAs and residents over and above any improvements seen in the control group as a result of the passage of time or observation. For these interaction effects, Greenhouse-Geisser p values are reported throughout in order to correct for potential violations to the sphericity assumption (Winer 1971). Finally, a 2 (group) × 2 (time) mixed-factor ANOVA was run comparing PIA–II outcomes to follow-up. The primary outcome of interest in these analyses were significant main effects of Group, showing that intervention participants maintained any behavior change. Results Treatment Receipt and Enactment Memory Book Availability Observational data from research staff memory book checks indicated that residents were in possession of their memory books during 72% of the morning checks and 75% of the afternoon checks during the hands-on training phase. This figure remained steady or increased during the PIA–I (70% morning, 73% afternoon), PIA–II (78% morning, 81% afternoon), and 2-month follow-up phases (77% morning, 80% afternoon). CNA/LPN Training and Performance of the Staff Motivational System Thirty-nine CNAs with data at all three assessment times (baseline, PIA–I, PIA–II) completed the hands-on training phase on the intervention units. Of these 39 CNAs, 92% (n = 36) passed a final evaluation with a performance score of 80% or above in the use of communication skills as measured by the CSC. The average final evaluation score for CNAs was 84.77% (range 53%–97%). The average number of training sessions on the unit per CNA was 6 (range 1–13). CNAs completed an average of 64% of their self-monitoring forms during the training phase (SD = 28%; range 5%–100%). The percentage of self-monitoring forms completed increased somewhat during the postintervention phases (PIA–I M = 67%, SD = 33%, range 0%–100%; PIA–II M = 66%, SD = 36%, range 0%–100%). CNAs were not required to self-monitor during the follow-up phase. Across the five nursing homes, a total of 55 written feedback letters regarding CNAs' skill performance were distributed. Fifty-eight percent of these letters were positive; 42% were negative. The number of supervisory training sessions conducted with 20 LPNs ranged between two and four. One hundred percent of LPNs reached a criterion of 80% correct performance in their accuracy of CSC recording and the provision of verbal feedback to CNAs regarding communication skill performance by the end of the hands-on training phase. On average, during the training phase LPNs conducted 62% (SD = 36%, range 0%–100%) of their assigned observations of CNAs. This number remained relatively stable after the hands-on training phase when LPNs conducted CNA observations independently (i.e., not accompanied by the project manager). During PIA–I, LPNs conducted 57% (SD = 44%, range 0%–100%) of their assigned independent observations, and during PIA–II LPNs conducted 61% (SD = 46%, range 0%–100%). CNA Use of Communication Skills There were no baseline differences between intervention and control groups on the CSC-generated overall communication skills score, time spent giving care to residents, specific one-step instructions, multistep instructions, positive statements, or biographical statements. Results of the 2 (group) × 3 (time) ANOVA analyses are shown in Fig. 1Fig. 2Fig. 3. The mixed-factor ANOVA for the overall total correct communication skill percentage score revealed significant main effects of group, F(1,62) = 16.87, p = .0001, and time, F(2,124) = 37.30, p = .0001, and a significant Group × Time interaction, F(2,124) = 17.20, p = .0001 (see Fig. 1). CNAs in the intervention group improved their use of general communication skills with residents during care in comparison with NTC group CNAs at both PIA–I and PIA–II. More specifically, CNAs in the intervention group tended to increase their rate per hour use of specific, one-step instructions during care in comparison with control group CNAs at both PIA–I and PIA–II (see Fig. 2), as shown by a marginally significant Group × Time interaction, F(2,124) = 2.80, p = .06. Intervention CNAs significantly increased their rate of positive statements made to residents during care in comparison with control group CNAs at both PIA–I and PIA–II (see Fig. 3), as shown by a significant Group × Time interaction, F(2,124) = 6.16, p = .004. There was also a significant group effect with intervention CNAs using fewer multistep instructions, F(1,62) = 5.21, p = .03, but no Group × Time interaction. There were no differences between intervention and control group CNAs across time in use of biographical statements. Notably, there was also no difference between intervention and control group CNAs across time in the amount of time spent in daily care with residents. Forty-three residents (21 control, 22 intervention) and 29 CNAs (13 control, 16 intervention) had data available from all assessment points from baseline assessment through the 2-month follow-up (i.e., maintenance sample). Analyses indicated that there were no significant differences in the characteristics of residents or CNAs who survived to follow-up and those who completed baseline. There were also no differences between CNAs on the control and intervention units at the 2-month follow-up assessment. The only significant difference between residents on the control and intervention units at the 2-month follow-up was that residents on intervention units were more independent in self-care as measured by the FIM, F(1,41) = 4.33, p = .04. Results of the ANOVAs show significant main effects of group in overall communication skills score on the CSC, F(1,27) = 12.92, p = .001 (Fig. 1) and the use of positive statements, F(1,27) = 11.91, p = .002 (Fig. 3), indicating maintenance of change at the 2-month follow-up assessment point. CNA–Resident Interactions During Care Results of the 2 (group) × 3 (time) mixed-factor ANOVA of the CABOS care interaction data are shown in Fig. 4 and Fig. 5. There were significant main effects of group, F(1,65) = 5.37, p = .02, and time, F(2,130) = 8.57, p = .0003, and a significant Group × Time interaction, F(2,130) = 4.11, p = .02, for the total amount of staff speech directed toward residents (see Fig. 4). CNAs in the intervention group increased their verbal interaction with residents during care in comparison with control group CNAs at PIA–II. Regarding resident coherent verbal interaction, only the main effects of group, F(1,65) = 6.50, p = .01, and time, F(2,130) = 3.20, p = .05, were significant. However, the rate of positive verbal interactions between CNAs and residents during care routines increased in the intervention group across time, as shown in Fig. 5 by a significant Group × Time interaction, F(2,130) = 4.81, p = .01. Analysis of maintenance data indicated that there were significant main effects of group for total amount of staff speech, F(1,41) = 5.20, p = .03 (Fig. 4), and positive statements by either staff or resident (i.e., positive verbal interactions), F(1,41) = 5.83, p = .02 (Fig. 5). Intervention group CNAs maintained a higher rate of speech with residents during care, and there were more positive statements made during care in CNA–resident dyads in the intervention group. The occurrence of agitation during care among the 67 residents was extremely low during all phases of the study (e.g., on average less than 1% of observation time). Thus, changes in this behavior could not be assessed. Discussion The results of this study suggest that, through the use of communication–memory book skills training and a staff motivational system, CNAs can improve aspects of their communication with nursing home residents. CSC data, derived from direct observation of care interactions, showed that CNAs' overall communication skills level increased significantly as compared with an NTC group. CNAs increased their use of positive statements and showed a marginal increase (p = .06) in single, one-step instructions, which can be less confusing than multiple nonspecific instructions when interacting with individuals with cognitive impairments. Moreover, CABOS data showed a significant increase in the amount of staff speech directed toward the resident during care routines. Finally, a behavioral code measuring the amount of positive statements during CNA–resident verbal interactions also showed a significant increase. Importantly, all of the changes in staff behavior were maintained at an assessment conducted 4 months after the initiation of treatment. Thus, the current data would appear to corroborate pilot data from Allen-Burge and colleagues 2001 and the findings of McCallion and colleagues 1999. These changes in staff behavior were brought about without increasing the amount of time necessary to deliver daily care. It has been argued that even if CNAs can be taught to play a more therapeutic role in the nursing home, it would require a time commitment that might not be feasible in the current nursing home environment (Schnelle and Beck 1999). For example, Rogers and colleagues 1999 reported success in training research therapists to increase independent dressing in residents; however, their therapeutic procedure required more time for staff to complete. Our data suggest that using better communication skills and memory books during care routines does not require more staff time. It is important that researchers delineate which therapeutic routines do and do not require additional time commitments so that staffing adjustments can be made when planning therapeutic routines. Contrary to our hypothesis, residents in the intervention group did not improve their rate of coherent verbal interactions with CNAs during care routines in comparison with control group residents. As discussed above, there was a significant increase in the rate of positive statements during CNA–resident interactions after training. However, due to our definition of this behavioral code, we did not examine how much residents contributed to this increase. Moreover, we were unable to assess any positive effects of the intervention on disruptive behavior due to its extremely low rate of occurrence in this sample. In this study, residents were required to show minimal ability in verbal communication involving spontaneous speech for entry. Consequently, residents were, on average, moderately cognitively impaired (MMSE M = 13.39), and prior research has indicated that moderately cognitively impaired residents are less likely to display serious disruptive behaviors (Burgio et al. 1994; Cohen-Mansfield, Werner, and Marx 1990). A main feature of this study was the careful attention given to the assessment of treatment implementation (Lichstein et al. 1994). Although the assessment of treatment implementation has long been a standard in nongerontological psychosocial intervention research (Cook and Campbell 1979; Moncher and Prinz 1991; Sechrest, West, Phillips, Redner, and Yeaton 1979), it has seldom been used in gerontological intervention research (Burgio, Corcoran, et al. 2001). Aspects of treatment receipt (e.g., CNAs' understanding of and use of the treatment) and treatment enactment (e.g., use of the staff motivational system and memory books by CNAs and LPNs) were investigated. The methodological advantages of directly assessing the implementation of interventions in applied settings are numerous and include greater assurance in the internal and external validity of the study. Internal validity concerns whether observed changes in behavior in CNA and resident outcomes actually coincide with increased use of the communication–memory book skills by CNAs. Measurements of treatment implementation also provide guidelines for the assessment of external validity by indicating what level of enactment of therapeutic skills by indigenous nursing staff was necessary to produce the observed therapeutic gains. Our results show that both CNAs and LPNs received (i.e., learned) the intervention as we intended. Ninety-two percent of CNAs demonstrated through direct observational evaluation that they could perform communication skills at the 80% criterion. One hundred percent of the LPNs reached a criterion of 80% accuracy of completing the CSC as compared with the project manager's completion of the CSC. Demonstrating skill acquisition in no way guarantees that staff will use these skills in the clinical setting. However, our assessment of treatment enactment shows that memory books were available to staff and residents during an average of 76% of observations. Their availability remained constant after the intervention was transferred completely to indigenous staff. CNAs completed an average of 66% of their self-monitoring forms. LPNs sent CNA performance feedback memos as instructed, and they completed an average of 60% of assigned observations of CNAs with the CSC form. This also remained relatively constant throughout all phases of the study. Although the completion of CNA self-monitoring forms and LPN CSC observations were only in the 60% range, considering the workload of these personnel we consider this to indicate successful, though less than ideal, treatment enactment. There are several limitations of this study. First, the reported CNA turnover rate in the nursing homes was 49%, well below the national average of 97% (American Health Care Association, 1997, cited in Harrington et al. 2000). Large staff turnover rates present significant difficulties in implementing psychosocial interventions, and our results may not be replicable in more typical nursing homes with larger turnover rates. In this study, newly hired staff viewed videotapes of the inservice, and hands-on training was also provided. Still, it is possible that continuous training of incoming staff in nursing homes with turnover near 100% may present challenging logistical problems. In all studies using direct observational measurement, participant reaction to observation is a potential problem. Specifically, the CNAs' performance of skills during observation may be influenced by social desirability factors and may not be representative of their actual day-to-day performance. There is no completely satisfactory answer to this problem, and we do not know to what degree social desirability influenced the CNA results. However, in this and our prior studies, we have developed specific procedures for observing staff unobtrusively; thus, and presumably, limiting reactivity (see Burgio 1996, and Burgio, Scilley, Hardin, and Hsu 2001, for a more detailed discussion of this issue). Multiple outcome measures were collected to provide a comprehensive assessment of treatment effects. This was useful for broadly identifying effects on CNA behavior, resident responses, and CNA–resident interactions. However, the Type I error rate is undoubtedly inflated by the fact that no adjustments were made for each individual analysis. A Bonferroni correction might be considered, although this procedure markedly elevates the Type II error rate and decreases power. Given the relatively limited sample size available for this initial efficacy evaluation, we chose to not implement this more conservative approach. Exact probability levels are reported instead so that the reader can make any interpretive adjustments deemed necessary. Another potential limitation was our choice to randomize nursing units within each nursing home into groups instead of randomizing entire nursing homes. Because CNAs were occasionally pulled to work on other nursing units, it is possible that trained CNAs applied communication skills on control units. This is a potential threat to the study's internal validity. However, we believe that this threat was minimized by including only nursing homes using fixed staffing and minimal pulling. The alternative of randomizing groups to nursing homes presents its own methodological problems, including the difficulty of finding nursing homes that are equivalent on all factors that might affect outcome. We have written about this dilemma in more depth elsewhere (Burgio and Stevens 1999). Because this was an evaluation of a multicomponent treatment package, we do not know the separate contribution of communication training and memory book usage to the changes in staff behavior. Perhaps more important, we do not know whether the inclusion of a staff motivational system was necessary for producing and maintaining staff behavior change. Studies that have used similar training techniques, including hands-on training, have generally shown an immediate training effect but no maintenance of behavior change (e.g., Schnelle, Newman, and Fogarty 1990). In a study currently under review (Burgio, Stevens, et al. 2001) we compared a behavior management skills training package with and without a staff motivational system. As hypothesized, results showed an immediate training effect for both groups, but maintenance of CNAs' behavior change was demonstrated more frequently at a 6-month follow-up in the group that received a staff motivational system. In conclusion, the results of this study suggest that CNAs can be trained to use improved communication skills and memory books with their residents during care interactions. With the use of a staff motivational system integrated into the training program, staff will perform these skills and will maintain this performance up to 4 months after the initiation of training. Although increases were observed in the overall amount of staff speech during interactions, use of positive statements, and positive statements during dyadic interactions between staff and residents, there is no direct evidence of changes in the resident communication behaviors targeted in this study. Future studies may want to examine other resident behaviors that may be influenced by improved CNA communication skills. One candidate for investigation is resident affect, which has been shown to be sensitive to change due to intervention in residents with dementia (Lawton, Van Haitsma, and Klapper 1996). Finally, although management of the intervention program was transferred to indigenous staff midway through the intervention phase, this was predominantly an efficacy trial. To optimize the internal validity of the study, training was conducted by a PhD-level licensed clinical psychologist, both inservice and hands-on training were used, and, as discussed above, nursing homes with relatively low rates of CNA turnover were chosen. Although it is our belief that this intervention can be manualized and implemented by typical staff development personnel in most nursing homes, only an effectiveness trial can shed light on the true feasibility of this intervention program. Practice Concepts The Forum Book Reviews Table 1. Surviving Resident Characteristics by Group Variable Intervention (n = 34) No-Treatment Control (n = 33) F /χ2 (df) Agea 81.78 (8.88) 82.42 (7.10) 0.10 (1,65) Mini-Mental Status Examinationa 13.50 (6.72) 12.94 (6.00) 0.13 (1,65) Functional Independence Measurea 46.88 (22.26) 37.30 (18.79) 3.61 (1,65) No. medicationsa 15.59 (9.79) 15.52 (8.49) 0.00 (1,65) Gender (women)b 73.53 75.76 0.04 (1) Race (White)b 76.47 90.91 2.54 (1) Dementiab,c 52.94 72.73 2.80 (1) a Characteristics reported as means (SD). b Characteristics reported as percentages (SD). c Dementia is listed as a diagnosis in the medical chart. Table 2. Surviving Staff Characteristics by Group Variable Intervention (n = 37)a No-Treatment Control (n = 25) F /χ2 (df) Ageb 39.30 (11.46) 35.13 (7.05) 2.43 (1,56) Years of educationb 13.57 (0.99) 14.00 (0.85) 3.01 (1,58) Months at facilityb 46.79 (50.62) 40.41 (53.71) 0.21 (1,56) Months as certified nursing assistantb 111.50 (81.26) 106.78 (64.94) 0.05 (1,57) Gender (women)c 86.49 88.00 0.03 (1) Race (Black)c 86.49 88.00 1.59 (1) a Demographic data were available for only 37 of the 39 CNAs in the intervention group. b Characteristics reported as means (SD). c Characteristics reported as percentages (SD). Figure 1. Mean total percentage correct on the certified nursing assistant Communication Skills Checklist across study phases for control (white bars) and intervention (black bars) groups. Asterisks denote significant differences between groups at p < .05. PIA–I = Postintervention Assessment I; PIA–II = Postintervention Assessment II. Figure 2. Mean rate per hour of specific, one-step instructions by certified nursing assistants across study phases for control (white bars) and intervention (black bars) groups. Asterisks denote significant differences between groups at p < .05. PIA–I = Postintervention Assessment I; PIA–II = Postintervention Assessment II. Figure 3. Mean rate per hour of positive statements by certified nursing assistants during care across study phases for control (white bars) and intervention (black bars) groups. Asterisks denote significant differences between groups at p < .05. PIA–I = Postintervention Assessment I; PIA–II = Postintervention Assessment II. Figure 4. Mean percentage of total observation time across study phases during which certified nursing assistants were speaking to residents for control (white bars) and intervention (black bars) groups. Asterisks denote significant differences between groups at p < .05. PIA–I = Postintervention Assessment I; PIA–II = Postintervention Assessment II. Figure 5. Mean rate per hour of positive statements made by certified nursing assistants or residents during care across study phases for control (white bars) and intervention (black bars) groups. Asterisks denote significant differences between groups at p < .05. PIA–I = Postintervention Assessment I; PIA–II = Postintervention Assessment II. Portions of this article were presented at the 52nd annual scientific meeting of the Gerontological Society of America, San Francisco, CA, November 1999. The research reported in this article was supported by funding from the National Institute on Aging (RO1AG13008) to M. Bourgeois and L. Burgio. We thank the nurses, certified nursing assistants, and administrative staff of Civic Center Nursing Home, Pleasant Grove Health Care Center, St. Martin's in the Pines, Montclair East Nursing Home, and Shelby Ridge Nursing Home for their support and assistance. 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McGraw-Hill, New York. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B49154E14BFAF64F435F6C799AF7E239785764F.txt b/test/dataset/in/resources/corpus/Clean_0B49154E14BFAF64F435F6C799AF7E239785764F.txt new file mode 100644 index 0000000..0b7d070 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B49154E14BFAF64F435F6C799AF7E239785764F.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 020035BS10.1093/gerona/57.11.B400 Journal of Gerontology: Biological Sciences Designer Microarrays From Soup To Nuts Wang Eugenia a Lacelle Chantale a b Xu Suying a Zhao Xuechun a Hou Michael a aDepartment of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Kentucky bDepartment of Anatomy and Cell Biology, McGill University, Montréal, Canada Eugenia Wang, Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 570 South Preston Street, Baxter Building Room 304, Louisville, KY 40292 E-mail: eugenia.wang@louisville.edu. 1 11 2002 57 11 B400 B405 7 8 2002 28 5 2002 The Gerontological Society of America 2002 The recognition that multigene mechanisms control the pathways determining the aging process renders gene screening a necessary skill for biogerontologists. In the past few years, this task has become much more accessible, with the advent of DNA chip technology. Most commercially available microarrays are designed with prefixed templates of genes of general interest, allowing investigators little freedom of choice in attempting to focus gene screening on a particular thematic pathway of interest. This report describes our “designer microarray” approach as a next generation of DNA chips, allowing individual investigators to engage in gene screening with a user friendly, do-it-yourself approach, from designing the probe templates to data mining. The end result is the ability to use microarrays as a platform for versatile gene discovery. hwp-legacy-fpage B400 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: James R. Smith, PhD OVER the past century, as a result of increasing medical knowledge, better sanitation, and improved nutrition, developed countries are experiencing an unprecedented increase in average human life span, along with a higher incidence of multifactorial diseases such as cardiovascular diseases, neurodegenerative disorders, type 2 diabetes, and cancers (1). These age-dependent diseases, plaguing people as young as their mid-50s, are products of the combined influences of genetics and environment (2). Nature and nurture together provide predispositions to cancer, cardiovascular disease, diabetes, and neurodegenerative disorders, presenting a complex picture for the development of these perils in the fast-growing middle- and old-age subpopulations of our society. Although recent advances in medical research have enabled us to diagnose several age-associated diseases, alleviate pain associated with them, and retard the onset of their acute stages, we remain largely incapable of identifying at an early stage those individuals bearing genetic predispositions to these diseases, and thus of administering preventive medicine or treatment. Because the human genome contains some 30,000 genes (3), and modern industrialized society yields increasing environmental complexity, it is an ever-greater challenge to perceive how the integration of our genes and surrounding environment creates disease-predisposed states. For example, why do certain individuals suffer lung cancer at an early age, after a few years of cigarette smoking, whereas some centenarians tolerate lifelong smoking without dying of the same disease? Such questions led to the idea of the need to identify “genetic signatures.” Once genetic signatures are secured, one may develop means to prevent and/or treat diseases in an individual manner, creating individualized medicine for prognostic, diagnostic, and therapeutic purposes. In general, large-sample gene chips, bearing perhaps 10,000 genes, are applicable only to early-stage screening and may yield voluminous lists of potential positive results; here we describe a next-generation, medium-density microarray approach embodying considerable quality control in both chip design and analysis, which results in fewer hits of enhanced accuracy and pertinence. Genetic Signatures and Microarray Technology During the 1980s and early 1990s, biologists were busy dissecting the functions of single genes, by using a reductionist approach, which, although thorough in its exact methodological analysis of genetic impact, was restricted in explaining how each particular single gene functions in the context of many homologous genes or partners to accomplish a biological task. In an attempt to shed light on these biological tasks, “biochips” were introduced in the mid-1990s (4) as a new tool for molecular medicine; they now constitute a rapidly developing field of biomedical research, which combines molecular medicine, nanotechnology, computer science, and engineering. Biochip technology was developed for high-throughput gene screening, capable of simultaneously identifying changes in the expression of hundreds or thousands of genes, and thus genetic signatures defining particular physiological states. Consequently, as a result of its power, the young field of DNA microarray technology has rapidly gathered speed and popularity within the biomedical research community. However, as universities across North America have started to establish microarray core facilities, they are realizing that the next generation of microarrays must be more versatile, user friendly, and inexpensive to ensure that these facilities will meet the divergent needs of their researchers, and ultimately provide practical answers to fundamental biological problems. Thus, 4 years ago, parallel to the development of commercial microarrays, we began devoting significant time and effort to developing pathway-specific “designer biochips”; here we provide an account of our quest for designer arrays, and we discuss some of the challenges that lie ahead for those seeking the perfect array. The Theory Behind Microarrays Unlike Northern blots, in microarrays the probes, not the targets, are immobilized to a physical platform. Microarrays consist of a platform to which are independently attached numerous nucleic acid sequences known as probes, to screen targets of prelabeled nucleic acids obtained from donors (human or animal). The quantification of probe–target complexes formed during hybridization permits measuring gene distribution and intensity, as complementary probe-labeled target sequences bind together. Although the principle behind microarrays is simple, creating and implementing microarray technology is difficult, as several parameters (discussed in the paragraphs that follow) can drastically affect the validity of the results obtained from microarrays. Furthermore, because the number of probes included on each microarray platform is great, the magnitude of results obtained from microarrays is huge, and thus requires powerful computerized image processing and statistical software to classify and analyze the data; without these, little significant gain can be obtained from using microarrays. Thus, microarray core facilities must integrate expertise in biology, computer science, engineering, and statistics. It is with this in mind that we started our quest for designer biochips. Platform and Printing Robots The first consideration when developing microarray technology is the type of platform, commonly either membrane or glass slide (5)(6)(7)(8)(9). Membrane-based microarrays use nylon or nitrocellulose membranes, and they are generally used when radioactive or colorimetric tags are used to label the targets, whereas glass slides are more suitable for microarrays using fluorescent tagging; we chose to use a membrane-based platform for our microarrays. Unlike glass slides, which must be chemically treated to permit the attachment of probes and decrease background fluorescence before use as a platform for microarrays (5)(8)(9)(10)(11)(12)(13), most membranes (whether bearing positive, negative, or neutral charge) require no such pretreatment. Because nucleic acids are negatively charged, the use of positive or neutral membranes greatly improves the signal; however, neutral membranes are generally more suitable, as positive-charged membranes yield higher background readings as well. Negatively charged membranes, although providing very little background, are usually not suitable because they yield poor signal. Thus we chose to use neutral-charged membranes for our arrays. Once a microarray platform has been chosen, probes must be attached to the platform. Two obvious methods exist: synthesis of probes directly on the platform (14)(15)(16)(17)(18), and probe-spotting by use of a contact or noncontact printing robot (4)(19)(20)(21)(22). Although leading biochip companies often synthesize oligos directly on their microarrays by using techniques such as photolithography, this method is not easily mastered, nor accessible to most laboratories. In contrast, probe-spotting can be accomplished using any of several commercially available printing robots (22). Because we use membranes attached to glass slides as our platform instead of glass slides directly, we encountered several problems, including skipped dots and uneven printing, when we first attempted to print arrays. We had to substantially modify the printing heads of the first robot we purchased, and we had to build an enclosure over it that permitted maintenance of constant humidity, to ensure even printing of the probes. Probes Almost any type of nucleic acid can be printed with a printing robot; nucleic acids obtained from complementary DNA (cDNA) libraries, as well as polymerase chain reaction (PCR) products generated by reverse transcription PCR (RT-PCR), are commonly used as probes for microarrays. The use of cDNA libraries is of considerable value for screening previously unknown genes or a great many genes with no predefined preferences for certain gene families. However, mistakes arising from mislabeling of clones or contamination can cause problems (23). We spot PCR products on our chips to generate thematic arrays of particular known genes of interest. Commercially available biochips are often restricted to specific sets of genes contained on each biochip, posing user-unfriendly conditions. In the gene discovery task, users are generally conditioned to screen according to the preset configuration of genes, without the possibility of generating thematic or pathway-specific microarrays covering genes known to belong to a specific family, as demanded by a particular research program. The use of designer biochips can circumvent this problem. The following sections describe the strategy we used to fabricate thematic arrays. Thematic Microarray Design Gene Selection and Primer Design To generate thematic microarrays bearing genes from a particular family or the same pathway, we use several public databases, including Unigene and GenBank (http://www.ncbi.nlm.nih.gov/GenBank, http://www.ncbi.nlm.nih.gov/Unigene), and conduct an extensive literature search (http://www.ncbi.nlm.nih.gov/PubMed) to obtain a repertoire of genes. Once a list of potential genes is constructed, we use GenBank and Unigene to obtain the sequences of all candidate genes. Primers for each gene sequence are designed using Primer3 software (http://www.genome.wi.mit.edu/genome_software/other/primer3.html) to generate a PCR product, or “amplicon,” with a length between 300 and 700 bp and a melting constant that ranges from 75°C to 89°C. For each gene we choose a pair of sense and antisense primers with an annealing temperature of approximately 60°C and an average length of 23 bp, for amplicon production using 96-well PCR plates. These clustered amplicon sizes and melting constants support standardized hybridization conditions for all probes across the microarray platform, including stringent washing, thus decreasing nonspecific signals while maximizing a specific signal. The length of the PCR product, as documented by Stillman and Tonkinson (6), is particularly important in maximizing a specific signal. Primer design is perhaps the most time-consuming step in our microarray production, because once a primer pair is selected, an analysis must be performed with Blast (proprietary software available on the National Center for Biotechnology Information website) to ensure that each primer pair amplifies only the gene of interest. This is crucial, because results obtained from the microarray are dependent on the specificity of the amplicons. However, in some instances, the specificity of the primer may not guarantee the specificity of the generated amplicon, when a conserved or shared domain lurks somewhere within the sequence. It is therefore highly recommended that the entire amplicon sequences themselves be Blasted to identify homologous regions, which can cause nonspecific binding. With probes obtained from cDNA libraries this may become a pitfall, especially when the spotted nucleic acid sequence is unknown; often highly homogenous sequences may result in nonspecific binding between genes of high homology. Stringent hybridization conditions and washing can generally eliminate this nonspecific binding, if the homologous region is not too long. Controls As in any biological experiment, and most importantly for microarrays, controls must be carefully selected. It is important to spot on all microarrays negative and positive controls as well as “housekeeping genes,” used in more traditional experiments such as quantitative RT-PCR, which show little or no physiological change in expression among the subjects or conditions being studied. The inclusion of housekeeping genes is useful for data normalization; for our designer microarrays targeted to mouse models, we selected six mouse genes (glyceraldehyde phosphate dehydrogenase, ribosomal S6, beta-actin, hypoxanthine-guanine phosphoribosyltransferase, phospholipase A2, and ubiquitin) commonly used in the literature as controls. In general, the validity of these controls must be determined a priori by using independent tests, such as Northern blotting assays or quantitative RT-PCR (24). For instance, EF-1α would be a poor choice for a housekeeping gene if the target nucleic acids were obtained from skeletal muscle, as it is not expressed in adult muscle cells; it would, however, be a good control when cDNA from liver is used as a target (25). The use of housekeeping genes permits the measuring of changes in gene expression against a gene whose expression does not vary significantly; in some cases this can be of great value. Negative controls should include buffer, bacterial, and viral DNA, as well as amplicons from genes known not to be expressed in target tissues. Negative controls are used to assess the level of background noise arising from nonspecific nucleic acid binding during probe–target hybridization. Positive controls such as total cDNA or genomic DNA permit the detection of suboptimal conditions of hybridization and staining, which may obscure appropriate signal intensity. Quality Control for Amplicon Production In order to avoid producing the wrong amplicon for printing as a result of contaminated PCR reactions, the use of dedicated equipment and reagents in the PCR setup and reaction areas is recommended. For each PCR reaction with a unique amplification primer pair, a negative control should be used to ensure the absence of reagent contamination, often caused by the presence of exogenous nucleic acids. This control reaction is identical to the regular reaction, except that no template is present. Agarose gel electrophoresis is used to verify the amplicons and ensure that they are of expected size. In instances where multiple bands result from the PCR, products can be resolved on an agarose gel, and the fragment of expected size excised; these amplicons can then be sequenced to confirm their identity. It is our experience that, when a primer pair is well chosen, multiple bands seldom result from the PCR reaction. Printing the Arrays Once amplicons have been produced for all genes of interest as well as housekeeping genes, arrays can be printed. To avoid positional bias, arrays should be printed in a scattered fashion, with several repeats of the same amplicon located in different regions of the chip. It is important to avoid positional bias, as uneven distribution of charges on the membrane can result in regions of increased background. A typical microarray manufactured in this fashion carries arrayed triplicates or quadruplets of amplicons from selected genes, positioned on the array among many control spots. The rationale for triplicate printing is to provide three data points for statistical analysis of significance; ideally, the three could be expanded to four or five repeats, to yield more data points for statistical analysis. This approach of scattered array printing requires considerable careful analytical software design, to enable tracking of amplicon repeats across the platform; however, it approaches an ultimate solution to resolving positional bias. Although the spots of microarrays printed onto membranes affixed to glass slides are usually colorless, it is possible to monitor quality to detect gross errors in printing, such as missing, smeared, or non-uniform spots; immediately before a batch of microarrays are printed, a colored dye can be used to print a test array of dots onto a membrane. Microscopic visual inspection of the spots enables any necessary adjustments to be made to the robot before sample printing begins. While large batches of chips are being printed, quality can be monitored by inserting poly-L-lysine-coated glass slides among the membrane platforms. Unlike membranes, the clear surface of glass slides permits the researcher to see printed spots by breathing on the slide and viewing it through a transmitted light source. Once the probes are printed on the membranes, they are cross-linked to the microarray to permit better attachment of the nucleic acids to the substratum; probes are denatured by boiling the membranes before hybridization. Target Labeling Donor nucleic acids can be labeled by adding a labeled base to the RT reaction used to generate target cDNA. Whereas most commercial arrays use fluorescence-conjugated or radiolabeled bases, we developed our microarrays based on a nonradioactive colorimetric method. The bulkiness of some of the fluorescent tags, fluorescent quenching over time, and the need of specialized scanners to read fluorescent signals dissuaded us from using the fluorescent approach (26). Similarly, we wanted to avoid using radioactive labels for safety reasons, because they often give saturated signals, and to save the time required for exposure of the labeled arrays to x-ray film. Using commercially available digoxigenin (DIG)-labeled dUTP (Roche, Palo Alto, CA) and alkaline phosphatase (AP)-conjugated anti-DIG antibody (27), we have developed a new application for DIG in microarrays (28). In our method, the cDNA to each donor RNA is synthesized with a DIG-labeled base. Following hybridization of the DIG-labeled target with the probes, positive reactions are revealed by incubating with anti-DIG antibody conjugated to AP, and subsequent staining with Nitro-blue-tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche) to detect AP (29). Taking advantage of the fact that two complementary nucleotide strands can hybridize with each other, we generate microarray results by quantifying the signal obtained from the labeled targets bound to the immobilized probes. Thus the positive loci are visible as bluish spots, easily identified as round deposits for each positive locus. The final detection is revealed as a matrix of many round dots of varying intensity of staining. Microarray Inventory Extensive records should be maintained on the microarray printing process, including logistical parameters of the physical status of the microarrayer (e.g., relative humidity of the chamber during printing, or how often the printing pins are cleaned during the run), the specifics of printing, including the printing sequence, the identity of each amplicon-containing spot, and all other applicable data or comments. To ensure that identical records are made for each printing, we record all data for our microarrays onto a standardized form that we have developed over the past several years. We are also developing a two-dimensional bar-coding system to keep detailed track of our inventory of microarrays. Image Acquisition and Data Processing The work just described supports the process of data generation; the following sections describe the mining of the resulting data. Array Normalization and Background Subtraction As our arrays are based on a colorimetric detection method, a high-resolution scanner is used to scan them into digital images. Before a normal office scanner is used, it is important to ensure that it digitizes accurately without transforming the image (26). If the image is transformed by the scanner, mathematical correction transformation should be applied to the result. Following acquisition, the digitized images can be normalized and subtracted as desired. We have developed a software program, GeneAnalyzer, which accomplishes background subtraction, array normalization, and quantification. When colorimetric microarrays are analyzed, several types of background must be considered; for instance, regional background subtraction is useful when the array shows differential intra-array background expression, whereas global background subtraction is suitable when the background value is constant within arrays but variable between arrays, as a result of experimental conditions. For interarray comparison to be supported, arrays may be normalized by several methods, including reference to housekeeping gene levels and median chip values. However, investigators should think carefully about the effects of performing such background standardization or normalization before they start analyzing their results; they should especially consider the effect of background subtraction on diminishing the signal of low-expression genes. Software for Microarray Data Acquisition In general, image acquisition and data analysis include the following processes: (a) image grabbing and digitizing; (b) image processing; and (c) data mining, including a qualitative and quantitative analysis of all digitized images, and a statistical analysis of data. We developed our software with a user-friendly interface and a limited number of preset functions, to enable researchers to analyze their own data. The main features of our program are as follows. First, we provide users a personal identification number, which allows optimal security of their data and access to the interactive functions of our web server facility. Second, users can upload their electronic images from remote sites over the Web. Third, our system processes the users' initial data to enhance the image profile, through standard computer software such as MatLab. Fourth, our system supports the users' data archiving and database organization for the next stage of data analysis. Statistical Analysis and Data Mining Although it is necessary to use statistical methodologies to analyze voluminous microarray data, it is equally important to generate adequate microarray-derived data to support statistical testing. Thus it is recommended for most studies to use at least three mice (or individuals) chosen at random from each test group (e.g., young vs old mice). RNA extracted from each donor tissue source (e.g., lung) is then subjected to three separate RT reactions, and each of these nine independently obtained cDNAs is used as targets for three different microarrays. Because on our microarray each amplicon is spotted in triplicate, we have a total of 81 (34) spots, or data points, for each gene being analyzed (RNA from three mice multiplied by three RT reactions multiplied by three microarrays multiplied by three spots on each slide). The use of at least triplicates for each step of microarray fabrication enables us to obtain statistically meaningful data. Without this replication, simple variations in the efficiency of the RT reaction, interanimal variation, or misprinting of a spot could all result in falsely perceived changes in gene expression. Because the statistical analysis of microarrays presents a challenge to many biologists, it is recommended that a statistician be consulted as necessary. Statistical consultants can be extremely helpful, not only at the final stage of data analysis, but also at the initial experimental design step; for example, they may provide answers to fundamental questions, such as how many animals are needed to establish a statistically significant data analysis, or whether or not one may pool RNA samples. Once microarray data are processed through statistical analysis, data entry points deemed of true “significant” value, that is, gene expression changes as effects of an experimental physiological change, should be subjected to the next level of data analysis, now popularly termed the data mining process (30). Many established methods have been popularized among microarray users, including principal component analysis (31)(32)(33), hierarchical clustering (34)(35)(36)(37), multidimensional scaling (38)(39)(40), and self-organizing maps (41). In general, the selection of any of these methods is dependent on the individual investigator's preference and expertise. For example, GeneSpring software, sold by Silicon Genetics, Inc. (San Carlos, CA), and Significance Analysis for Microarrays from Stanford University (42), are preferred for many gene screening data mining tasks because they can analyze data generated by several different microarray platforms. These data mining software packages enable researchers to display their data in forms suitable for publication, easily conveying the essence of the results. Following data mining, microarray data that seem to be significant should be validated by using one of two popular methods: Northern blotting or quantitative RT-PCR. In general, it is advisable that microarray data be validated by the selection of four or five randomly designated genes from each of three categories: those showing high, intermediate, and low levels of significant difference. Because we use amplicons to generate our probes, we can easily validate our results, using the same primers used to generate our amplicons by quantitative or semi-quantitative RT-PCR. During data analysis, special consideration must be given to low-expression genes, which generally exhibit the greatest variance in expression levels. On any given microarray, these genes show very weak intensities, and in some cases they are barely visible above the background value. Here, standard global normalization and thresholding are not practical, because the signals are so weak. Often we find that global thresholding is too crude, allowing in one case the gene expression to be quantified as a gain, and in another case allowing the same gene expression to be quantified as a loss. One possible solution for this problem is to use “segmental thresholding,” localized thresholding for each individual weak spot. Then the local background level is calculated against the global background level to obtain confidence level indices. The actual gene expression level for these low-abundance genes is then the “minithresholding level” divided by the confidence level. We realize that this is not a perfect solution; often we have to disregard these data points altogether. Conclusions The notion that the bioinformatics of microarray studies is still in its infancy pertains not only to studies in the aging area, but also to many other biological systems as well. The entire field of microarrays is experiencing volatile changes in methodological approaches, technological design, and bioinformatics development for data interpretation. Part of the growing pains in the use of microarray technology is the constant need for new cutting-edge technology and the reevaluation of methodology. Therefore, for any designer microarray projects, it is necessary to be vigilant for any new methodology and technology developments, to improve the design and fabrication process as well as the bioinformatic aspects of gene screening tasks. As with all technologic advances, the microarray approach is not an end in itself; it is just a beginning. Obviously, one wants to know whether the genes identified as significantly changed at the RNA level are truly manifested at the protein level. For this purpose, the recent explosion of proteomic technology is certainly a testimony to the need for follow-up to microarray data. Ultimately, gene expression microarray studies have to be followed with experiments to examine protein changes, thus permitting a comprehensive examination of gene expression changes from RNA to protein levels. This work was supported by Grant R01 AG07444 from the National Institute on Aging of the National Institutes of Health, and from the Defense Advanced Research Project Agency of the Department of Defense, to E. Wang. We express our sincere gratitude to Ms. Sherry Chen, Ms. Angel Wang, Mr. Keith Liang, Dr. Yih-Jing Tang, Dr. Nagathihalli Nagaraj, Dr. Bo Yu, and Ms. Jane Williams for their excellent technical assistance, and to Mr. Alan N. Bloch for proofreading this manuscript. 1 Hayflick L, 2000. 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Proc Natl Acad Sci USA.96:2907-2912. 42 Tusher VG, Tibshirani R, Chu G, 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA98:5116-5121. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B4A738BFF1E9CD2E041D19AFD5E6A3AD39D01D9.txt b/test/dataset/in/resources/corpus/Clean_0B4A738BFF1E9CD2E041D19AFD5E6A3AD39D01D9.txt new file mode 100644 index 0000000..fd60b00 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B4A738BFF1E9CD2E041D19AFD5E6A3AD39D01D9.txt @@ -0,0 +1 @@ +]>HEARES3104S0378-5955(98)00140-310.1016/S0378-5955(98)00140-3Elsevier Science B.V.Fig. 1An example of click waveform recorded from animal's concha in a sound-attenuating chamber.Fig. 2The responses of a PL/Type I/III AVCN unit to clicks and click pairs. Upper right panel shows the PSTH of the response of this unit to tone bursts at characteristic frequency (CF). The coefficients of variation (CV) for this unit were 0.62 and 0.57 in the 2–14.9 and 20–39.9-ms time windows (called CV2–14.9 and CV20–39.9), respectively. Responses to single click and click pairs at interclick intervals (ICI) between 1 and 16 ms at 65 and 85 dB SPL are shown in the two left columns. Lower right panel shows the second click response as a function of ICI at 45, 65 and 85 dB SPL and represents the percentage of recovery of second-click responses.Fig. 3The responses of a Chop-S/Type III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2. CV2–14.9=0.1; CV20–39.9=0.14; Ronset/Rsust.=1.0.Fig. 4The responses of a Chop-S/Type III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2 except that click levels of 105 dB SPL are included. CV2–14.9=0.27; CV20–39.9=0.23; Ronset/Rsust.=1.7.Fig. 5The responses of an On-C/Type I/III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2. CV2–14.9=0.07; there were no spikes in the 20–39.9-ms window to calculate the CV and Ronset/Rsust..Fig. 6The responses of a Chop-T AVCN unit to clicks and click pairs. Upper right panel shows the PSTH of the response of this unit to tone bursts at CF. The format of this figure is the same as Fig. 2. CV2–14.9=0.34; CV20–39.9=0.61; Ronset/Rsust.=1.3. This unit was classified as Chop-T type based the CV-vs.-time pattern even though chopping was not visible in the PSTH.Fig. 7The responses of an On-L/Type I/III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2. CV2–14.9=0.14; there were insufficient spikes in the 20–39.9-ms window to calculate the CV; Ronset/Rsust.=4.5.Fig. 8The second-click recovery function for primary-like (left column), chopper (middle column) and onset (right column) AVCN units at the four stimulus levels (45, 65, 85 and 105 dB SPL). The thick line in each panel represents the mean second-click response recovery function. The number of units represented, N, is shown in each panel. Only those units with data available for three or more ICIs are included.Fig. 9Mean second-click response±standard error of means (S.E.M.) vs. ICI for the three main PSTH types (primary-like, chopper and onset; panels a–c) and the three AVCN EIA types (Type I, Type I/III and Type III; panels d–f) at four stimulus levels (45, 65, 85, and 105 dB SPL). Mean recovery function for Type I units at 45 and 105 dB SPL are not available due to the small sample size.Fig. 10Comparison of SR distribution of AN fiber and AVCN unit. The number of units represented, N, is shown in each panel. AN data are from Kim et al. (1991).Fig. 11Mean second-click recovery functions (±S.E.M.) for two populations of AVCN neurons at four stimulus levels. AVCN units are divided into a low-SR (SR<20 spikes/s) group and a high-SR (SR>20 spikes/s) group.Fig. 12Comparison of mean second-click recovery functions among low-SR and high-SR AN fibers and AVCN neurons at four stimulus levels (45, 65, 85 and 105 dB SPL). Data from AN fibers at 105 dB SPL are not available.Fig. 13Mean spike counts (a), absolute number of spikes (b) and mean response duration (c) in response to single clicks as a function of stimulus level among low- and high-SR AN fibers and AVCN units at four stimulus levels (45, 65, 85 and 105 dB SPL).Table 1Number of AVCN units in PSTH and EI-area classification schemesPSTH TypeEI-area typeII/IIIIIIII or IVUnclassifiedTotalPercentPL6918174134Chop02223395747On073141512Unusual0410387Total64245523121Percent53537419Responses of anteroventral cochlear nucleus neurons of the unanesthetized decerebrate cat to click pairs as simulated echoesKParhamH.-BZhaoYYeD.OKim*Division of Otolaryngology, Surgical Research Center, Department of Surgery, Neuroscience Program, The University of Connecticut Health Center, Farmington, CT 06030-1110, USA*Corresponding author. Tel.: +1 (860) 679-3690; Fax: +1 (860) 679-2451; E-mail: kim@neuron.uchc.eduAbstractTo elucidate the contribution of the anteroventral cochlear nucleus (AVCN) to `echo' processing, this study documents the responses of AVCN neurons to simulated echoes and compares them to those of auditory nerve (AN) fibers. Single unit discharges were recorded from 121 units in the AVCN of 21 unanesthetized decerebrate cats in response to click pairs with inter-click intervals ranging from 1 to 32 ms between 45 and 105 dB SPL re 20 μPa. Units were classified according to the post-stimulus time histogram (PSTH) and excitatory-inhibitory response area (EI-area) schemes. Based on their spontaneous rates (SR), units were subdivided into low- (<20 spikes/s) and high- (>20 spikes/s) SR groups. A majority of the units exhibited second-click responses whose recovery time courses were similar to those of AN fibers. These units included primary-like, chopper and onset units in the PSTH scheme and Types I, I/III and III units in the EI-area scheme. A minority of the units exhibited responses that were distinct from those of AN fibers, in that they had second-click response recovery times that were either markedly reduced or prolonged. This group of units included those with primary-like, chopper and onset PSTHs and Type I/III and III EI-areas. No significant difference was found in the second-click response among various PSTH or EI-area types. High-SR AVCN units exhibited a decrease in the second-click response with increasing level. In contrast, low-SR AVCN units showed little level-dependent change in the second-click responses. This SR-based difference was similar to that previously found among AN fibers. The present results suggest that, although a majority of AVCN units exhibit similar time courses of second-click response recovery to those of AN fibers, there do exist mechanisms in the cochlear nucleus that can substantially alter this representation. Furthermore, the difference between the second-click response recovery functions of low- and high-SR AVCN units and the consistency of this finding between AVCN and AN suggest that SR represents an important dimension for signal representation in the AVCN neurons.KeywordsAnteroventral cochlear nucleusEcho processingClick pairSpontaneous rate1IntroductionIn a reverberant environment, such as a lecture hall, an acoustic signal originating from one location is typically followed by its echoes that arrive at the ears from multiple directions. Nevertheless, a listener can correctly perceive a single auditory image and its correct direction. This phenomenon of the first wavefront taking precedence over later-arriving wavefronts has been referred to as the `law of the first wavefront' or `precedence effect' (Wallach et al., 1949; Haas, 1951; Von Bekesy, 1960; Blauert, 1997). To gain an understanding of the mechanisms involved in auditory processing of the echoes, it would be useful to study this phenomenon across multiple structures within the ascending auditory pathway.We recently reported on echo processing features of the auditory nerve (AN) fibers in the unanesthetized decerebrate cat (Parham et al., 1996). A main finding was that the recovery of the response to the trailing click of a click pair among AN fibers not only depended on stimulus parameters (i.e., interclick interval and click level) but also on fiber spontaneous rate (SR). High-SR fibers exhibited longer recovery times than their low-SR counterparts.The cochlear nucleus (CN) serves as the obligatory termination site of AN fibers. Anatomically, it can be subdivided into three main divisions: the anterior and posterior ventral cochlear nuclei (AVCN and PVCN, respectively) and the dorsal cochlear nucleus (DCN). Each subdivision is composed of populations of neurons that, by virtue of different combinations of intrinsic membrane properties and synaptic inputs, can express diverse responses to sound. As a result, the CN consists of an array of neurons whose responses fall on a continuum from a faithful preservation to a substantial transformation of the incoming afferent signals. These responses are conveyed to higher auditory centers along parallel pathways arising from the CN subdivisions.Based on their responses to sounds, CN neurons have been characterized according to two schemes: temporal discharge patterns reflected in the post-stimulus time histogram (PSTH) scheme (e.g., Pfeiffer, 1966) and the expressions of excitatory and inhibitory regions and responsiveness to broad-band noise reflected in the excitatory-inhibitory area (EI-area) scheme (e.g., Young, 1984). The response types within the two schemes appear to be distributed differentially within CN subdivisions and are correlated with different morphological classes of neurons in the CN. In the present context, the anatomical and physiological diversity of CN neurons gives rise to three important questions: (i) Is echo representation among CN neurons different from that among AN fibers? (ii) Do various physiological classes of CN neurons differ from one another with respect to echo representation? (iii) Are SR-based differences in echo processing of AN fibers also present in the CN?Recently, Wickesberg (1996)reported that ventral cochlear nucleus (VCN) units showed rapid recovery of the second-click response in the anesthetized chinchilla. Units characterized by primary-like and chopper PSTHs showed 50% recovery by 2 ms interclick interval and a nearly complete recovery by 4 ms. Wickesberg also reported some variability in the responses of VCN neurons, in that a subset exhibited little to no reduction or enhanced response to the second click.In this study we attempt to elucidate the contribution of AVCN to echo processing. We systematically investigated the behaviors of AVCN neurons regarding their response recovery functions under a click-pair stimulus paradigm in decerebrate unanesthetized cats. In this assessment of AVCN behavior in response to click pairs, neurons were characterized according to both PSTH and EI-area schemes and SR-based differences were investigated. Early results of this study were presented at a meeting of the Association for Research in Otolaryngology (Kim et al., 1992).2MethodsThe care and use of animals reported on in this study were approved by the University of Connecticut Health Center Animal Care Committee (protocol title: `Physiology of the Cochlear Nucleus'; protocol number: 91-014-97-2). The details of Section 2are described elsewhere (Parham et al., 1996). Briefly, we used unanesthetized decerebrate cats. We opened the left posterior fossa, aspirated a portion of the cerebellum, and exposed the CN. A micropipette was positioned over the AVCN which was visualized through an operating microscope. Single unit spike discharges were recorded using micropipettes filled with 3 M sodium chloride or potassium acetate that had resistances of 20–50 MΩ. In some penetrations the recording location was marked by iontophoretic injection of markers, 10% biotinylated dextran amine or 10% horseradish peroxidase in 0.5 M potassium acetate in Tris buffer. As the electrode was advanced, we applied a tonal search stimulus whose frequency was swept with a triangular modulating waveform over 0–50 kHz with a modulation repetition period of 2 or 4 s. The level of the swept tone was in the range of 40–60 dB SPL (re 20 μPa). For each AVCN unit encountered, we recorded the following: (i) spontaneous discharge rate based on a 10- or 20-s sample without acoustic stimulation; (ii) response area curve representing discharges induced by 200-ms tone bursts (two repetitions) at various frequencies spaced at 50 frequencies per decade on a logarithmic scale and at a constant dB SPL, typically 10–30 dB SPL; from the response area we estimated the characteristic frequency (CF) (the frequency to which a unit is most sensitive) of the unit; (iii) responses to 60-dB SPL CF tone bursts (50 ms duration, 500 ms repetition interval, 5 ms rise/fall time, 60 repetitions) from which a post-stimulus time histogram was constructed; (iv) responses to 200-ms noise bursts (three repetitions, 5 ms rise/fall; 1000 ms repetition interval) in 5-dB steps between 20–70 dB SPL (measured as rms level with A-weighting); and (v) responses to single rarefaction clicks and equilevel click pairs with inter-click intervals (ICI) ranging from 1 to 32 ms; repetition period of 500 ms, 40 repetitions, at 45, 65, 85 and 105 dB SPL peak. The rarefaction clicks were produced by applying 30-μs pulses to the earphone. The acoustic waveform of a single click recorded in the concha of the cat is shown in Fig. 1.Event times for spike discharges and stimulus markers were stored with 20 μs resolution in the data files such that subsequent `off-line' analyses would have access to full information about spike discharge and stimulus events. Single units were classified according to the PSTH and EI-area schemes. Discharges of all units were examined for regularity based on the behavior of the coefficent of variation (CV) of spike discharges as a function of time (e.g., Young et al., 1988; Parham and Kim, 1992). Chopper units were subdivided into three subtypes: Chop-S (CV<0.35); Chop-T (CV increased from <0.35 to >0.35 within the first 15 ms of the response) and Chop-O (CV>0.35). Based on PSTH of the responses to pure tone at CF, onset units were identified based on a quantitative criterion that the ratio of the discharge rate over the first 5 ms of the response (Ronset) to the last 40 ms of the response (Rsust.) be greater than 2.5.The procedure for analysis of single unit responses to click pairs is described in detail elsewhere (Parham et al., 1996). Briefly, we examined the response of each AVCN unit to single clicks and click pairs in the form of PSTHs with binwidths of 0.08 ms. For graphical illustrations binwidths of 0.2 or 0.4 ms were used. To quantify the results, we defined the time window that included all of the evoked spike discharges to the single click as W1. W1 was defined for each unit and at each click level because response latency and duration varied among units and as a function of stimulus level. W1 served as the reference time window for subsequent computations of the responses to a click pair. The time window of the response to the second click of a pair (W2) was defined as a window identical to W1 except for a delay by the amount of ICI. The second-click response (SCR) was expressed as percentage of the response to the first click. At short ICIs, the quantification of the SCR took into account the tail end of W1 which overlaps with W2 (for details see Parham et al., 1996).At each ICI the level dependent differences between SCRs of various unit types were statistically evaluated using analyses of variance (ANOVAs). Significant interactions were followed up by lower order ANOVAs and significant main effects were further examined with Tukey HSD tests of mean comparisons.3ResultsThis study is based on the responses of 121 AVCN units recorded in 21 cats. Table 1 shows the number of AVCN units subdivided according to the PSTH and EI-area schemes. In the PSTH scheme, the majority of the units (93%) exhibited either primary-like (PL, 34%), chopper (Chop, 47%) or onset (On, 12%) PSTHs. The remaining units (7%) had unusual or non-descript PSTH appearances. The PL group (N=41) included primary-like (98%) and primary-like-with-notch (2%) PSTH types. The Chop group (N=57) was further subdivided into sustained (Chop-S, 33%), transient (Chop-T, 53%) and other (Chop-O, 14%) chopper subtypes. The On group (N=15) consisted of units exhibiting onset without sustained discharges (On-I, 13%), onset chopping (On-C, 47%), onset with low level of sustained activity (On-L, 33%) and onset followed by inhibition of spontaneous activity (On-inh, 7%). In the EI-area scheme, the majority of the units (77%) were characterized as Type I (5%), I/III (35%) or III (37%) with a minority falling into other categories (Type II, 3%; Type IV, 1%). For the remaining 19% of the units, response to broadband noise was unavailable and these units were not classified in the EI-area scheme. Among the present PL units, 18 out of 41 units were EI-area Type III. This is a much greater tendency than a previous report (Shofner and Young, 1985). The reason for this difference is not clear.Fig. 2 shows the responses of a low-SR PL/Type I/III unit to single clicks and click pairs. Responses of this unit to single clicks at 65 and 85 dB SPL peak are shown in the top PSTHs of the two left-most columns. The response was characterized by a large peak followed by a gradual decay of the response over 5–6 ms. Responses to click pairs at short ICIs (1–3 ms), shown in the remaining rows of the two columns, tended to be slightly more prolonged than the response to single clicks. At larger ICIs the response to the second click was evident. As the ICI increased, the second-click response gradually increased in strength such that by 16-ms ICI, the second-click response was similar to the first-click response. The lower right panel provides the magnitude of the second-click response as a function of ICI at 45, 65 and 85 dB SPL. The second-click response gradually recovered from near 25% at 1-ms ICI to above 50% by 4-ms ICI and to near a complete recovery (80–100%) by 16-ms ICI. This PL/Type I/III unit exhibited a slower recovery of the second-click response as click level was increased from 45 to 85 dB SPL.The behavior of the sample PL/Type I/III unit shown in Fig. 2 was typical of a majority of the units recorded from the AVCN in this study (to be shown further below) and resembled the behavior of AN fibers recorded under similar conditions (Parham et al., 1996). However, a minority of the units exhibited responses to click pairs that were distinct from the typical behavior resembling AN fibers. The next five figures (Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7) illustrate units characterized by sustained and transient responses to pure tones that exhibited click pair responses that were distinct from that of the AN fibers.The three examples shown in Figs. 3–5 exhibited faster recovery of the second-click responses than AN fibers. The responses of a Chop-S/Type III AVCN unit to single clicks and click pairs is shown in the two left-most panels of Fig. 3. This unit's responses preserved the representation of both clicks of a pair at low and moderate stimulus levels (45 and 65 dB SPL), even at ICIs as short as 1 ms. At 45 dB SPL, the PSTH of the response of this unit to single clicks consisted of a single narrow peak. At 1-ms ICI the response to the second click is distinct and nearly equal to that of the first click. At 85 dB SPL, the single click response consisted of two peaks, separated by an interval (1.8 ms) which is approximately equal to the interpeak interval of the PSTH of the response to the CF tone (upper right panel). The response to click pairs at 1-ms ICI was similar to that of the single-click response except that the second peak of the PSTH was narrower and taller (i.e., distributed across fewer bins). A distinct response to the second click was present at 2-ms ICI which gradually recovered as ICI was increased to 32 ms. The lower right panel shows the second-click response recovery functions of this unit. At 45 and 65 dB SPL the second-click response remained near 100% for all ICIs, but at 85 dB SPL the second-click response recovered from 0% at 1-ms ICI to 100% at 32 ms.The responses of another Chop-S/Type III AVCN unit are shown in Fig. 4. This unit exhibited a poor recovery of the second-click response at a low stimulus level for ICIs even up to 32-ms ICI, but strong second-click response at high stimulus level even at 1-ms ICI. At 45 dB SPL a weak response to the second click was present by 2-ms ICI. Unlike previous examples, however, second-click response was completely absent by 8-ms ICI and remained below 50% for ICIs up to 32 ms. In contrast, at 105 dB SPL the second-click response was present at 1-ms ICI in the form of a broad peak. By 2-ms ICI the second-click response was nearly the same as that of the first. The second-click recovery functions of this unit are shown in the lower right panel. At 45 dB SPL the second-click response remained below 50%, but at 105 dB SPL the second-click response was recovered to 85–100% across all ICIs examined. At intermediate levels of 65 and 85 dB SPL the second-click response exhibited more rapid recovery than that expected from AN-like recoveries.Fig. 5 shows the responses of an On-C/Type I/III unit. At 65 dB SPL the second-click response was strong (>75%) across all ICIs. At other click levels the recovery of the second-click response was more gradual, although somewhat faster than those exhibited by AN fibers. For example, at 85 dB SPL the response to the second click was greater than 75% at ICIs beyond 3 ms.The next two examples shown in Figs. 6 and 7 exhibited slower recovery of the second-click response recovery function than AN fibers. The responses of a Chop-T AVCN unit is shown in Fig. 6. The second-click response of this unit at 45 dB SPL exhibited a strong response at 1-ms ICI, but weak responses at 3–8-ms ICIs. The second-click response at 65 dB SPL was similar to that of 45 dB SPL (lower right panel). At 85 dB SPL this unit exhibited a more gradual recovery (middle column and lower right panel).The responses of an On-L/Type I/III AVCN unit are shown in Fig. 7. This unit was characterized by a weak second-click response up to 32-ms ICI for all stimulus levels tested. Interestingly, the second-click response increased between 2- and 8-ms ICIs, but decreased for longer ICIs.Fig. 8 shows recovery functions for populations of primary-like (left column), chopper (middle column) and onset (right column) units at 45–105 dB SPL. Individual curves in Fig. 8 represent separate units, while the thick line in each panel represents the mean second-click response recovery function. In general, the majority of the units exhibited recovery functions qualitatively similar to those observed at the level of the AN. However, a small number of non-AN-like recovery functions were observed in all three populations. Examples of fast recovery to the second click can be seen in primary-like (panel d), chopper (panels b, e and k) and onset (panels c and f) units. Examples of weak response to the second click can also be seen in primary-like (panel g), chopper (panels b, e, h and k) and onset (panels f, i and l) units.The mean recovery functions of the three main AVCN PSTH types are shown in Fig. 9a–c. Primary-like units showed little change in the mean second-click response with the click level (Fig. 9a). Only at 16- and 32-ms ICIs were the second-click responses to 45 and 65 dB SPL significantly greater than those at 105 dB SPL stimuli (Tukey HSD, P<0.05). Chopper units exhibited an orderly weakening of the mean second-click response as click level was increased from 45 to 85 dB SPL for 1–3-ms ICIs (Fig. 9b). However, at 105 dB SPL the chopper units showed the strongest mean second-click responses for 1–3-ms ICIs. The mean second-click response of the onset units also decreased with increasing stimulus level (Fig. 9c). However, the level-dependent differences among chopper and onset units were not statistically significant (P>0.05).The mean second-click recovery functions of the three unit types were similar at 65 and 85 dB SPL. At 45 dB SPL the onset units exhibited more rapid recovery of the second-click to above 75% than the primary-like and chopper units (i.e., 3 vs. 4 ms, respectively). However, at 105 dB SPL the onset units exhibited slower recovery of the second-click response than the primary-like and chopper units (75% SCR: 7 vs. 6 ms, respectively). At 45 and 105 dB SPL the mean second-click recovery functions of the primary-like units were intermediate to those of the chopper and onset units. The differences between the three PSTH types were not statistically significant (P>0.05).The mean second-click recovery functions for the three EI-area types are shown in Fig. 9d–f. The mean second-click response of the Type I group increased as the stimulus level increased from 65 to 85 dB SPL (Fig. 9d). Because of the small sample size for Type I units mean second-click response recovery functions are not available at 45 and 105 dB SPL. The mean second-click response decreased from 45 to 85 dB SPL for Type I/III and III units (Fig. 9e,f). However, at 105 dB SPL Type I/III and III units showed an increase in the mean second-click responses relative to those at 85 dB SPL (Fig. 9e,f). The mean second-click response recovery functions of the three EIA types differed little when compared at each click level.Fig. 10 shows the SR distributions for AVCN units recorded in this study and a sample of AN fibers recorded in a previous study (Kim et al., 1991). The AVCN units were divided into low- (<20 spikes/s, N=76, 63%) and high-SR (>20 spikes/s, N=45, 37%) groups, as was previously done for AN fibers (Parham et al., 1996). The mean second-click response recovery functions of these two groups are shown in Fig. 11. Low-SR AVCN units showed little level-dependent changes in the second-click response recovery functions between 45 and 85 dB SPL. Interestingly, at 105 dB SPL the low-SR AVCN units exhibited stronger second-click responses than at lower stimulus levels. One-way ANOVAs at each ICI indicated a significant main effect of level only at the 2-ms ICI (F(3,124)=3.42, P=0.02). In contrast, the high-SR AVCN units exhibited a more noticeable level-dependent decrease in the second-click response between 45 and 105 dB SPL. This decrease was statistically significant at 3-, 4- and 6-ms ICIs (P<0.02). The mean recovery functions of the two AVCN SR groups are compared to one another and their AN counterparts in Fig. 12. At 45 and 65 dB SPL, the four populations exhibited similar second-click response recovery functions. One-way ANOVAs at 45 and 65 dB SPL showed no significant differences in mean second-click responses of the two SR groups. At 85 dB SPL, the high-SR AVCN and AN fibers exhibited weaker second-click response recoveries than their low-SR counterparts. Two-way ANOVAs at each ICI at 85 dB SPL revealed main effects of SR at all ICIs less than 32 ms (P<0.05) without any significant interaction with location (i.e., AN vs. CN).At 105 dB SPL differences in second-click response between the low- and high-SR AVCN units are much more evident, with the high-SR AVCN units having weaker second-click response for ICIs less than 32 ms. The AN data for 105 dB SPL are not available. ANOVAs indicated that the second-click responses of the high-SR AVCN units were significantly weaker than their low-SR counterparts at all ICIs less than 8 ms (P<0.05).Since the second-click response recovery functions presented above are a relative measure, they do not convey information about the absolute neural activities. The latter information can be inferred by combining the relative recovery functions with an absolute measure of neural activities for various stimulus conditions. For this purpose, we examined the number of spikes elicited by single clicks at various levels, as shown in Fig. 13. For all groups, the mean number of spikes elicited by single clicks increased as the stimulus level increased (Fig. 13a). However, this increase was substantially larger for the high-SR AVCN and AN units. Consequently, the low-SR AVCN units and AN fibers consistently exhibited fewer spikes per click than their high-SR counterparts across all levels. The duration of the single-click response is shown in Fig. 13c. Although the response duration increased as stimulus level increased for all four groups, the response durations of high-SR AVCN units and AN fibers were consistently longer than those of their low-SR counterparts.4DiscussionA majority of AVCN units recorded in this study exhibited second-click response recovery time courses that were similar to those of AN fibers. These units had primary-like, chopper and onset PSTHs and Types I, I/III and III EI-areas. A minority of AVCN units, however, exhibited second-click responses whose recovery time courses were markedly reduced or prolonged relative to those of AN fibers. The latter group of units had primary-like, chopper and onset PSTHs and Type I/III and III EI-areas. The mean second-click response recovery functions showed little difference among the three main PSTH and EI-area types. When AVCN units were subdivided into low- and high-SR groups, however, a clear difference in the second-click response recovery functions was found between the two SR groups.In this study, the responses of many AVCN neurons to single click stimuli were often prolonged. For example, multipeaked PSTHs of click responses were relatively common in low-CF (i.e., below 2 kHz) primary-like units. Similar to previous observations in the AN, the PSTH of the responses of these low-CF primary-like units to clicks had a multi-peaked appearance, with the interpeak interval being equal to the 1/CF interval. Some high-CF CN units also had PSTHs of the response to click stimuli that were multi-peaked (e.g., Figs. 3, 5 and 6). However, as previously noted by Kiang (1965), the inter-peak intervals in these high-CF units did not correspond to 1/CF. Kiang's report did not classify CN units according to the PSTH type. In the current study, multi-peaked PSTHs among high-CF units were associated with chopper and onset-chopper type units. The inter-peak intervals in the PSTHs of the response to the clicks in these units approximated those in the PSTHs of the responses to pure-tone bursts at their CFs.The level-dependent weakening of the second-click responses of high-SR AVCN units and their slower recovery relative to low-SR AVCN units are analogous to our earlier observations in the AN (Parham et al., 1996). Spontaneous activities of AVCN neurons appear to be mainly dependent on their AN inputs. Koerber et al. (1966)observed that, after cochlear destruction, spontaneous activity of AVCN neurons was abolished suggesting that SR of AVCN neurons mainly arises from their AN afferent inputs. Therefore, the differences between the two SR groups in the AVCN for the most part appear to be a reflection of their AN afferent inputs.The results presented in this report offer a possible reason for the difference in the dependence of the second-click response on the level between the two SR populations of the AN and AVCN, i.e., neuronal refractoriness. Gaumond et al. (1982)characterized refractoriness of AN fibers in terms of a conditional discharge probability conditioned on the time since the last spike, called tau, for a steady stimulus. The conditional discharge probability of an AN fiber typically was zero (i.e., completely refractory) for tau<0.75 ms, rapidly increased from zero to about 60% of a fully recovered value at tau=4 ms, and more gradually increased to the fully recovered value (i.e., zero refractoriness) between 4 and about 40 ms. Gaumond et al., 1982(Figs. 12 and 13) also observed that, when there were successive short (e.g., <10 ms) interspike intervals, the refractoriness of AN fibers was accumulated, i.e., the conditional discharge probability further decreased. The fact that the time courses (0 to about 40 ms) and general shapes of the two measures, i.e., second-click response vs. inter-click interval and refractoriness vs. time since the last spike, are quite comparable to each other, supports the possibility that neuronal refractoriness is the main mechanism underlying the reduction and a gradual recovery of the second-click response being described in the present report and in Parham et al. (1996). In Fig. 13a, it is seen that, as the stimulus level was increased from 45 to 105 dB SPL, the mean absolute number of spikes increased from about 1 to 3 spikes per click for high-SR AN and AVCN units whereas they changed little for low-SR AN and AVCN units. It is also seen in Fig. 13c that the multiple spikes at high stimulus levels occurred over a period of about 4–10 ms. This corresponds to interspike intervals of about 2–4 ms.In view of the Gaumond et al. study discussed above, the refractoriness of a high-SR unit should be substantially greater during a period following the clicks presented at high levels than that at low levels because additional spikes were discharged with short interspike intervals at high stimulus levels. This expected change in the amount of refractoriness of high-SR units with stimulus level is consistent with the decrease of second-click response observed in high-SR AVCN units (Fig. 11) and AN fibers (Parham et al., 1996, Fig. 7). Likewise, this reasoning as applied to low-SR units regarding their absolute number of spikes per click being nearly independent of stimulus level (Fig. 13a) predicts that their second-click response recovery functions should be nearly independent of stimulus level. Low-SR AVCN units (Fig. 11) and AN fibers (Parham et al., 1996, Fig. 7) indeed exhibited much less dependence of their second-click recovery functions on stimulus level than their high-SR counterparts, largely consistent with the prediction. There is one discrepancy between the refractoriness-based prediction and the observation in this regard. That is, the second-click response of low-SR AVCN units increased somewhat at 105 dB SPL compared with those at lower levels (Fig. 11, left panel) whereas, based on Fig. 13, one predicts that it should remain nearly constant with a slight decrease. The reason for this discrepancy is not clear.When one considers that the discharge behavior of an AVCN neuron is affected by neuronal refractoriness, two components of refractoriness can be considered: (i) refractoriness of AN fibers acting as an input to the AVCN neuron, and (ii) refractoriness of the AVCN neuron. The present observations (Fig. 12) indicate that the second-click response recovery functions are rather similar between the AN fibers and AVCN neurons. In interpreting the second-click response recovery functions in terms of neuronal refractoriness, this similarity implies that the combined total refractoriness observed in AVCN units is similar to that in AN fibers. To our knowledge, direct examinations of neuronal refractoriness (analogous to the Gaumond et al. study) for AVCN neurons have not been reported. We believe that such a study should be useful.The faster recovery of the second-click response of low-SR AN fibers and AVCN neurons than their high-SR counterparts contrasts with findings of forward masking studies using tone bursts at the level of the AN (Relkin and Pelli, 1987; Relkin and Doucet, 1991) and CN (Boettcher et al., 1990; Shore, 1995). Forward masking studies noted that low-SR units required longer times for recovery of responses to tone bursts than high-SR units, opposite of the present observation of the second-click response. Previously, we considered that possible reasons for the discrepancy might include longer signal duration (tone burst) and a large level difference between the masker and probe in the forward masking studies compared with click pairs of the present studies (Parham et al., 1996). Even when the masker and probe levels were nearly equal in the CN forward masking studies, low-SR neurons showed slower recovery than high-SR neurons (Boettcher et al., 1990; Shore, 1995). Therefore, the difference between masker and probe levels does not explain the discrepancy between click-pair and forward masking studies. Different mechanisms may underlie the two phenomena. Adaptation at the inner hair cell and its afferent synapse and suppression of cochlear responses mediated by the olivocochlear efferent system (Bonfils and Puel, 1987) may mainly underlie the forward masking phenomenon whereas neuronal refractoriness may mainly underlie the second-click response reduction phenomenon. We do not expect that the second-click response recovery function is mainly mediated by the efferent system because the click stimulus had a brief duration (Fig. 1) presented with a long (500 ms) repetition period, whereas efferent-mediated effect required a sufficiently long (greater than 35 ms) stimulus (Bonfils and Puel, 1987).The similarity of the recovery rates of low- and high-SR AVCN neurons to their AN counterparts supports the notion that SR may be an important axis in signal representation at the level of the CN. This conclusion is consistent with that of Shore (1995)in her study of forward masking in the VCN. The suggestion of an SR-based segregation of AN fiber projections to the CN, first made by anatomical studies (Leake and Snyder, 1989; Kawase and Liberman, 1992; Liberman, 1991), has received additional neurophysiological support. Recent recordings from the AVCN showed that neurons in the marginal shell were predominantly low SR, whereas the central core included high- and low-SR neurons (Ghoshal and Kim, 1996Ghoshal and Kim, 1997).Since a majority of the AVCN units display AN-like behavior with respect to second-click responses, these neurons do not modify the AN afferent inputs but act to relay AN signals to higher auditory centers. In a forward masking study of CN neurons, Boettcher et al. (1990)arrived at a similar conclusion. A minority of AVCN units, however, showed echo processing behaviors that were distinct from those of the AN fibers. These units had primary-like, chopper and onset PSTH types. Forward masking studies in the CN have also identified neurons whose recovery from short-term adaptation is distinct from those of AN fibers (Boettcher et al., 1990; Shore, 1995). In vivo intracellular recording and labeling experiments have shown that PSTH types tend to be associated with morphological cell types in the AVCN (Rhode et al., 1983; Rouiller and Ryugo, 1984; Smith and Rhode 1987, 1989; Ostapoff et al., 1994). For example, the primary-like responses are generally associated with bushy cells, whereas chopper responses are generally associated with multipolar cells. Since the unusual second-click recovery behaviors of some AVCN neurons were not associated with a distinct response type in the AVCN, the present observation suggests that these novel echo processing strategies may not be associated with a unique cell type in the AVCN.The unusual echo processing patterns of AVCN neurons consisted of neurons with either enhanced or suppressed representation of the echo relative to that of the AN fibers. The enhancement of the second-click response was observed at short ICIs (i.e., less than 4 ms) where individual AN fibers show typically weak responses (less than 50%; Parham et al., 1996). The present study is the first to demonstrate the existence of neurons with very fast second-click response recovery times in the AVCN. One possible way to extract adequate information regarding the temporal position of an echo, is for some AVCN neurons to sample and integrate the responses of many AN fibers. In other words, convergence of several AN fiber inputs onto an AVCN neuron could lead to summation of temporally coincident excitatory post-synaptic potentials thus eliciting an action potential. This is analogous to previously proposed mechanisms for certain VCN neurons (e.g., Smith and Rhode, 1986; Winter and Palmer, 1995; Palmer et al., 1996). Responses of spherical bushy cells in the AVCN are dominated by their AN fiber inputs which are believed to be form one or two calyx-type endings on one bushy cell (e.g., Sento and Ryugo, 1989). Therefore, the spherical bushy cells are a poor candidate for this type of integration. More likely candidates are multipolar cells receiving bouton-type endings from the AN which are distributed over their soma and proximal dendrites (Cant, 1981).The suppression of the second-click responses beyond that exhibited by the AN fibers is likely mediated by circuits releasing inhibitory neurotransmitters such as glycine and/or GABA in some CN neurons (Backoff et al., 1997). Suppression of the second-click response of AVCN neurons could arise partly from intranuclear and/or descending inhibitory projections to AVCN neurons. The intranuclear mechanisms that may underlie such suppression have been discussed in detail elsewhere (Wickesberg and Oertel, 1990; Wickesberg, 1996). Briefly, one hypothesized source is the tuberculoventral neurons of the DCN which provide glycinergic input to the AVCN. In vitro excitation of these neurons through stimulation of AN fibers which project to tuberculoventral neurons, as well as AVCN neurons, showed inhibitory postsynaptic potentials in AVCN neurons about 2 ms after AN induced excitatory postsynaptic potentials. Wickesberg (1996)provided further support for this circuit in a pharmacological perturbation study. Wickesberg showed that, following application of lidocaine in the DCN, responses of 19% of the VCN units (primary-like and chopper type) to the trailing click increased. Such inputs could account for the trough-like recovery functions observed in this study (e.g., Fig. 6). For 62% of the VCN units, however, Wickesberg found that the response to the first click decreased after lidocaine administration. The reason for the latter unexpected finding is not clear.In the AVCN, ICI at 50% recovery of the second-click response ranged from 1.8 ms at 45 dB SPL to 3 ms at 85 dB SPL without any substantial differences between the two AVCN SR groups (Fig. 12). This compares with 1.3–3 ms over the same stimulus level range in the AN (Parham et al., 1996). Thus, there was only a very slight increase in the average 50% recovery time in AVCN relative to the AN. The recovery functions of AVCN units are similar to those reported for units in the SOC by Fitzpatrick et al. (1995). Monaural units in the SOC exhibited second-click recovery functions that were analogous to those of AVCN neurons. The average 50% recovery time of monaural units in the SOC was ∼2 ms (Fitzpatrick et al., 1995). The fact that recovery functions in the SOC are comparable to those in the AVCN with the average 50% recovery times being similar in the two nuclei suggests that no significant modification of the echo processing occurs between these two structures. Substantial alterations apparently occur higher up at the level of the IC as the average 50% recovery time increases to ∼6–20 ms (Yin, 1994; Fitzpatrick et al., 1995). Additional suppression mechanisms are interpreted to be present in the cortex, since in the auditory cortex the average 50% recovery times ranged up to 300–500 ms (Fitzpatrick et al. 1997, 1998).Some of the auditory cortical neurons were observed to exhibit rapid recovery times of <2 ms (Fitzpatrick et al., 1997). Considering that the AVCN also contains neurons exhibiting comparably rapid recovery times (e.g., Figs. 3–5), the projections of such AVCN neurons could form the beginning of a channel that may convey information about the presence and nature of echoes. Such information may underlie the quality of the sound heard, i.e., its timbre (e.g., Blauert, 1997; Harris et al., 1963; Sayers and Toole, 1964). The projections from the majority of AVCN neurons, whose second-click responses recovery times are 2–10 ms, may represent the beginning of a separate channel that may underlie the perception of the sound location, thus mediating the precedence effect which has a time window of about 2–10 ms (Wallach et al., 1949). In addition, certain neurons in the AVCN (present study) and higher auditory centers (e.g., Fitzpatrick et al. 1995, 1997), whose second-click recovery times are in the range of about ten to a few hundred milliseconds, may mediate a time window (`echo window') where a lagging sound (i.e., echo) is heard but its perception is still affected by a leading sound (Fitzpatrick et al., 1998).Based on the findings of our studies in the AN and AVCN, it is important in future studies to keep in mind two important variables: SR and stimulus level. In both AN and AVCN, the 50% recovery time of the second-click response increased from 1.3 and 1.8 ms, respectively, to 3 ms between 45 and 85 dB SPL. At 105 dB SPL, however, the 50% recovery time for low-SR AVCN neurons was less than 1.5 ms, while that of high-SR AVCN neurons was ∼5 ms. Results for the AN at 105 dB SPL are not available for comparison. Given that both SR and stimulus level exert a substantial influence on echo processing, greater attention needs to be paid to these variables when making comparisons across structures.Regarding psychophysical studies of precedence effect which examined the influence of the overall stimulus level, we are aware of only one, Shinn-Cunningham et al. (1993). Using two stimulus levels (80 and 110 dB SPL 1-ms noise bursts), they found: (i) that the stimulus level had relatively small influence on the strength of the precedence effect; (ii) that the strength of the precedence effect was little affected by the stimulus level for 1-ms ICI (or `lag' of Shinn-Cunningham et al.); and (iii) that the strength of the precedence effect tended to decrease somewhat at the higher stimulus level for 10-ms ICI. This psychophysical observation of a decrease of the precedence effect strength with increasing stimulus level for 10-ms ICI appears to be in opposite direction to what one may anticipate from the neurophysiological observations of an increase of the recovery time with increasing stimulus level (Fig. 11 of the present paper; Figs. 7 and 11 of Parham et al., 1996). The reason for this apparent discrepancy is not clear.To summarize, a survey of the available results throughout the auditory pathway suggests that the response to a trailing sound or echo can be either weakened (a characteristic of the AN fibers), enhanced or suppressed. As we ascend the auditory pathway, suppression appears to become more robust or prolonged, but an enhanced representation of a simulated echo continues to persist. These results suggest that, with respect to echo processing, the CN may be the origin of several parallel channels within which the AN signal is either relayed or altered on its way to higher centers.AcknowledgementsThis study was supported in part by the National Institute on Deafness and Other Communication Disorders, NIH, Grant P01-DC-01366. We thank Drs. S. Kuwada, D. Fitzpatrick, and A. Palmer for their helpful comments on this manuscript.ReferencesBackoff et al., 1997P.M.BackoffP.S.PalombiD.M.CasparyGlycinergic and GABAergic inputs affect short-term suppression in the cochlear nucleusHear. Res.1101997155163Von Bekesy, 1960Von Bekesy, G., 1960. Experiments in Hearing. McGraw-Hill, New York, NY.Blauert, 1997Blauert, J., 1997. Spatial Hearing: The Psychophysics of Human Sound Localization. MIT Press, Cambridge, MA.Boettcher et al., 1990F.A.BoettcherR.J.SalviS.S.SaundersRecovery from short-term adaptation in single neurons in the cochlear nucleusHear. 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Neurophysiol.601988129 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B4C81E27458178FDFF60AA067D04A887C57B142.txt b/test/dataset/in/resources/corpus/Clean_0B4C81E27458178FDFF60AA067D04A887C57B142.txt new file mode 100644 index 0000000..c8d76d9 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B4C81E27458178FDFF60AA067D04A887C57B142.txt @@ -0,0 +1 @@ +]>HEARES2986S0378-5955(98)00018-510.1016/S0378-5955(98)00018-5Elsevier Science B.V.Fig. 1Drawing of a stage 52 larva of the newt Cynops pyrrhogaster, including a three-dimensional reconstruction of the inner ear and endolymphatic system, which are shown at higher magnification below. Abbreviations: UTO, utricular otolith; ES, endolymphatic sac; EO, endolymphatic otolith; ED, endolymphatic duct; SO, saccular otolith.Fig. 2Light micrographs of gross changes in the endolymphatic sac, otic vesicle, midbrain, during development and maturation in the newt Cynops pyrrhogaster. The sections were cut transversely through the middle portion of the endolymphatic sac (1 μm thickness) and stained with 1% toluidine blue. Bar=50 μm. a: Stage 39, an oval shaped endolymphatic sac first appears at the dorsomedial tip of the otic vesicle. The endolymphatic sac is adjacent to the midbrain. b: Stage 41, several tiny vacuoles (arrow in enlarged insert) appear in the sac, indicating that the endolymphatic sac is starting to expand. Scale bar in magnified insert is 10 μm. c: Stage 46, the endolymphatic sac elongates significantly. A slit-like lumen starts forming in the middle portion of the saccule. The endolymphatic duct opens into the otic vesicle. The epithelial lining becomes distinguishable. d: Stage 48, the lumen of the endolymphatic sac expands, and is clearly distinguished from the outer epithelial lining. This section shows that the proximal portion of the endolymphatic sac has extended to the upper end of the medial wall of the otic vesicle. e: Stage 50, the endolymphatic sac lumen enlarges and a number of bright otoconia are present. f: Stage 54, the endolymphatic sac expands and extends markedly forming a large lumen. The lumen of the endolymphatic sac accumulates numerous otoconia nearest the duct entering the otic vesicle (OC and arrow in enlarged insert, scale bar=10 μm). Abbreviations: OT, otic vesicle; MB, midbrain; LM, lumen; EL, epithelial lining; OC, otoconia. Other abbreviations as in Fig. 1.Fig. 3TEM of epithelial cells of the ES at different developmental stages. Bar=1 μm. a: Stage 39, the epithelial cell is of polymorphous type and contains a minimum of cytoplasm. Note the intercellular space is narrow and has a smooth appearance. b: Stage 44, the epithelial cell has a columnar shape. The cytoplasm is not dense with only a few mitochondria seen. The intercellular space contains several vacuoles and primitive microvilli on its surface. The basement membrane blends with the fibrils of the connective tissue (unlabeled arrow). c: Stage 48, the epithelial cell contains dense cytoplasm in which mitochondria are evident. The dilated intercellular space has an irregular surface with finger-like projections where cell junctions form. d: Stage 54, the epithelial cell contains a very dense cytoplasmic matrix and has a fibrous appearance. Abundant cell organelles such as mitochondria, smooth endoplasmic reticulum and Golgi complexes are seen in the cytoplasm. Note many granules and vesicles are also located in the apical and lateral cytoplasm. The intercellular space is loose and has a labyrinthine appearance. Abbreviations: EP, epithelial cell; IS, intercellular space; LV, lipid vesicle; VC, vacuole; M, mitochondria; MV, microvilli; N, nucleus; CJ, cell junction; GR, granules; V, vesicles. Other abbreviations as in Fig. 2.Fig. 4TEM of the lumen of the ES at different stages during development. Bar=5 μm. a: Stage 46, a slit-like lumen goes longitudinally through the middle part of the sac. The intercellular spaces are perpendicular to the surface of the lumen. The vacuoles in the space are fused, which makes the space have a wave-like surface. Note that there is a widened intercellular space between and beneath the epithelial cells. b: Stage 48, the ES lumen expands. This shows an uneven lining formed by microvilli. Floccular material appears in the lumen. c: Stage 50, the lumen dilates markedly. Otoconia are present in the lumen. The spaces where the otoconia were appear as holes, indicating artifacts of processing. The floccular material becomes more dense nearest to the otoconia. The intercellular spaces have a honeycomb appearance. d: Stage 54, the lumen expands its width and length significantly with dense floccular material inside. The relatively thick and long microvilli arise from the luminal surface of the flattened epithelial cells. Abbreviations: FM, floccular material. Other abbreviations as in Figs. 2 and 3.Fig. 5TEM of two morphologies of the ES appearing at stage 54. a: High resolution TEM of a matured microvillus on the luminal surface of an epithelial cell. Note that the lipid bilayer is visible. A small vesicle is also found in the cytoplasm. Bar=0.1 μm. b: TEM of a free floating cell. The cytoplasm of this cell is relatively light with numerous vacuoles and empty mitochondria inside. Long microvilli are present around all the free surfaces of the cell. Bar=1 μm. Abbreviations: LB, lipid bilayer; FF, free floating cell. Other abbreviations as in Figs. 2–4.Fig. 6TEM of the endolymphatic duct during development and maturation. The sections were cut longitudinally through the duct. a: Stage 46, the primitive endolymphatic duct with a narrow lumen within the medial wall of the otic vesicle. Bar=5 μm. b: Stage 50, the lumen of the endolymphatic duct expands at the intermediate portion. Some floccular material is present in the lumen. Note that the luminal surface is relatively smooth with only a few microvilli seen. Bar=2 μm. c: Stage 54, the intermediate portion of the endolymphatic duct. Note that the epithelial cells contain dense cytoplasm. The luminal surface bears numerous microvilli that display an intermeshing appearance. Also note that there are fibrils in the basement membrane. Bar=1 μm. Abbreviations: GC, Golgi complex; F, fibrils. Other abbreviations as in Figs. 2–4.Fig. 7TEM of junctional complex between the epithelial cells of the ES during development. a: Stage 46, tight junctions are formed close to the lumen. The outer layers of the adjacent plasma membrane fuse in the wall of the junction. Bar=0.1 μm. b: Stage 50, a tight junction and an adherent junction are present between the epithelial cells, indicating that junctional complex has developed. Note some granular substance is adhering along the outer wall of the tight junction and cytoplasmic filaments are associated with the adherent junction. Bar=0.2 μm. c: Stage 54, the walls of the tight junction are dense and parallel with a narrow interspace. The cytoplasmic filaments crossing the adherent junction are longer than those at stage 50. Below the junctional complex, both of the cell membranes are closely apposed at some parts. Bar=0.2 μm. Abbreviations: AJ, adherent junction; TJ, tight junction; CF, cytoplasmic filaments. Other abbreviations as in Figs. 2–4.Table 1Abbreviations used in figuresAJadherens junctionFMfloccular materialNnucleusCFcytoplasmic filamentsGCGolgi complexOCotoconiaCJcell junctionGRgranuleOTotic vesicleEDendolymphatic ductISintercellular spaceSOsaccular otolithELepithelial liningLBlipid bilayerTJtight junctionEOendolymphatic otolithLMlumenUTOutricular otolithEPepithelial cellLVlipid vesicleVvesicleESendolymphatic sacMmitochondriaVCvacuoleFfibrilsMBmidbrainFFfree floating cellMVmicrovilliTable 2Quantitative and qualitative characteristics of the developing ESStageDSizeType of cellCytoplasm and cell organellesMicrovilli (μm)Intercellular spaceLumen (μm)Material in lumen391165×25×15polymorphousminimumnarrow, smooth4415columnarnot dense, only a few Mvacuoles4618120×30×25cylindricalincrease in M0.3–0.5fused vacuoles, expanded, wave-like39×3.3×44820150×35×40cylindrical, cuboidalrelatively dense M, scattered R and G0.4–0.6dilated, finger-like protrusions, expandedfloccular material5026160×35×50cuboidaldense, packed M, R, and G vesicles0.5–0.7honeycomb, intercellular digitations120×20×20floccular materialotoconia5448190×40×55cuboidal, flatvery dense with fibrous appearance; abundant M, R, and G vesicles and granules1.0labyrinthine, intercellular digitations, floccular material140×30×25floccular materialotoconiacell debrisfloating cellsD, days after eggs are laid; M, mitochondria; R, ribosome; G, Golgi complex.Table 3Quantitative and qualitative characteristics of the developing EDStageLength (μm)Diameter of lumen (intermediate) (μm)Diameter of lumen (low end) (μm)Cell typeMicrovilli (intermediate)Microvilli (lower end)Material in lumen39250046450.50.8duct-likea few501000.81.0lower cuboidalonly a fewa fewfloccular material542001.22.0low cuboidal and flatintermeshinga fewfloccular materialotoconiaDevelopment of the endolymphatic sac and duct in the Japanese red-bellied newt, Cynops pyrrhogasterWenyuanGaoMichael LWiederhold*Jeffery LHarrisonDepartment of Otolaryngology-Head and Neck Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7777, USA*Corresponding author. Tel.: +1 (210) 567 5655; Fax: +1 (210) 567 3617.AbstractThe development and maturation of the endolymphatic sac (ES) and duct (ED) were studied in the newt Cynops pyrrhogaster. The ES first appears as an oval capsule at the dorsal-medial tip of the otic vesicle at stage 39, about 11 days after oviposition. The ES consists of polymorphous epithelial cells with a minimum of cytoplasm. The intercellular space (IS) between the epithelial cells is narrow and has a smooth surface. At stage 44, the size of the ES increases as many vacuoles in the IS become filled. At stage 46, 18 days after oviposition, the ES elongates markedly and a slit-like lumen is found in the ES. The epithelium contains a few cell organelles which are scattered in the cytoplasm. The vacuoles in the IS are fused, which expands the IS. Two days later (stage 48), floccular material (endolymph) is present in the expanded lumen. The IS dilates and has a wide and irregular appearance. At stage 50, approximately 26 days after oviposition, the ES extends and expands significantly and crystals (otoconia) can now be seen in the widened lumen of the ES. The cytoplasm of the cuboidal epithelial cells contains an abundance of vesicles surrounded by ribosomes and Golgi complexes. Intercellular digitations are formed in the expanded IS. At stage 54, the ES forms a large bellow-like pouch. Numerous otoconia accumulate in the lumen. Free floating cells and cell debris can be seen in the lumen at this stage. The epithelial cells contain numerous cytoplasmic organelles which are evenly distributed in the cytoplasm. Granules are found in the apical and lateral cytoplasm. The IS is loose and displays a labyrinthine appearance. The primitive ED first appears as a connection between the ES and the saccule but no lumen is present inside at stage 39. At stage 46, a narrow lumen is formed in the ED, which corresponds to the formation of the ES lumen. At stage 50, as the ED extends, floccular material is seen in the lumen. At stage 54, the ED bears numerous microvilli on its luminal surface. Otoconia and endolymph are present in the ED. Tight junctions between the epithelial cells are formed at stage 46. A fully developed intercellular junctional complex is produced at stage 54. Based on the development of the ES and ED, the maturation of function of the ES and ED are discussed.KeywordsEndolymphatic sac and ductDevelopmentOtoconiaIntercellular space1IntroductionThe endolymphatic sac (ES) and duct (ED), which are part of the membranous labyrinth, exist in most vertebrates. The biological function of this portion of the inner ear has received much attention in mammals. Absorption and endocytosis of endolymph (Fukazawa et al. 1990, 1991, 1995; Hoshikawa et al., 1994), secretion of endolymph and other substances (Friberg et al., 1986; Barbara, 1989; Rask-Andersen et al., 1991), pressure regulation of the endolymphatic compartment (Bagger-Sjöbäck and Rask-Andersen, 1986; Friberg et al., 1986; Barbara et al., 1987; Takumida et al., 1988) and immunoreactive function in the inner ear (Rask-Andersen and Stahle, 1980; Tomiyama and Harris, 1986) have been suggested for the entire endolymphatic system. In amphibians, however, the only function of the ES which is known so far is to store calcium. This stored calcium is used to make otoconia and later to mineralize the skeleton and in egg production (Guardabassi, 1960; Marmo et al., 1986).One way to obtain further knowledge about the ES and ED is to study the embryology of the system. Bast and Anson (1949)found that the human ES forms early in a 7-mm embryo by an anterior-posterior crease in the otic vesicle. Watske and Bast (1950)showed changes in size and position of the ES during its maturation. Apart from the studies by Hultcrantz et al. (1987, 1988)in which the ES and ED were investigated at the light and electron microscopic level in CBA/CBA mouse, few studies have been devoted to the development of this system in mammals. In amphibians, only a few early studies of the embryology of the ED and ES have been reported. Whiteside (1922)demonstrated in Rana temporaria linne, that the ED is a well-differentiated canal, situated at the medial side of the otic vesicle; its upper end expands into a small vesicle to form the ES during the first stage (4 mm long). Dempster (1930)reported that the formation of the ES starts as a dorsal evagination of the closed otic vesicle in 15 mm long cryptobranchidae alleganiensis. Unfortunately, these studies were based on light microscopic observations and did not describe the whole developmental process.The Japanese newt Cynops pyrrhogaster is a favorable species in which to study development, since its vestibular system is similar to those of mammals but develops much more rapidly. The development of the otolith organs and semicircular canals, the gross appearance of the ES and ED and the morphogenic features of otoconia in the utricle and saccule have been investigated in this laboratory (Wiederhold et al. 1992, 1995, 1997; Steyger et al., 1995). However, there is a lack of detailed information concerning the development of the ES and ED. The present study was performed to elucidate the pattern and sequence of events during organogenesis and cytodifferentiation of the ES and ED at the light and electron microscope level. It is hoped that this study will shed light on the function of this system in amphibians.2Materials and methodsNewt embryos of the desired developmental stage were obtained as reported previously (Wiederhold et al., 1995; Steyger et al., 1995). Development stages were determined by observation through a dissecting microscope and comparison with the sequence and stage description reported by Okada and Ichikawa (1947)and Okada (1989).Three embryos or larvae at each stage (from stage 25 to 56) were used as subjects. The specimens were fixed by immersion in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h. After a triple-rinse in 0.1 M cacodylate buffer, the specimens were post-fixed in 1% osmium tetroxide (pH 7.4) for 1 h, and again rinsed in buffer. Specimens were dehydrated with increasing concentrations of ethanol (35% to 100%, 15 min each). A transition fluid of propylene oxide was used (45 min) before infiltration and embedding in Epon. The polymerization process required three days in an oven (37, 45, 60°C for 24 h each).Serial transverse sections (1 μm thickness) were cut through the ES and ED. The sections were stained with 1% toluidine blue, mounted on slides and cover slipped. The sections were examined under an Olympus BH-2 light microscope. Ultrathin sections (90 nm) were cut transversely at the middle portion of the ES and ED. The sections were stained with uranyl acetate and Reynold's lead citrate (Reynolds, 1963), and examined with a Philips 301 electron microscope.The care and use of the animals reported in this study were approved by the University of Texas Health Science Center at San Antonio's Institutional Animal Care and Use Committee.3Results3.1Development of the endolymphatic sac (ES)3.1.1Gross structureThe endolymphatic system is much larger, relatively, in adult and developing amphibians, than in mammals. Fig. 1 (in Table 1 are the abbreviations used in the figures) shows a drawing of a stage 52 Cynops larva and a three-dimensional reconstruction of the inner ear and ES and ED. Stage 52 is approximately 28 days after oviposition and 16 days after the larvae usually hatch from the egg. At this stage, the two ESs are separate but represent large projections from the dorsomedial aspect of the saccule. As early as stage 31, the precursor of the ES can be recognized as a slight extension of the otic vesicle (see Wiederhold et al., 1995) and both the ES and ED expand greatly from stages 50 to 56. In an adult newt, the ES from the two sides join to form a continuum across the top of the brainstem (see Koike et al., 1995).Before stage 38, serial sections did not reveal an ES. At stage 39, the ES first appears as an oval capsule at the dorsomedial tip of the otic vesicle (Fig. 2a). The ES is about 65 μm long, 25 μm wide and 15 μm thick. Serial sections did not show a lumen inside the ES. At stage 41, several tiny vacuoles (arrow in insert of Fig. 2b) appear in the sac, indicating the ES is ready to expand. At stage 46, the ES elongates markedly, to approximately 120 μm long and 30 μm wide. The capsule boundary appears in 25 serial sections. Its proximal portion extends to the upper part of the medial wall of the otic vesicle. A narrow slit-like lumen can barely be seen in the middle of the ES (LM in Fig. 2c). At stage 48, the ES increases in size with a long and short diameter of 150 μm and 35 μm, respectively. The whole ES continued to display its outline in about 40 serial sections with the expanded lumen clearly distinguished from the wall of the ES (LM in Fig. 2d). At stage 50 the ES is 160 μm long, 35 μm wide and 50 μm thick. The lumen becomes much wider than that at stage 48. In the lumen, a number of crystals (otoconia) can now be seen (OC in Fig. 2e). The birefringence of these otoconia display the same colors in polarized light microscopy as do the saccular and utricular otoconia, indicating that they are, indeed, crystalline. At stage 54, the ES forms a large bellow-like pouch, which is 190 μm long, 40 μm wide and 55 μm thick. The sac extends from the middle portion of the medial wall of the otic vesicle to the dural membrane. The large lumen cavity contains numerous otoconia piled together (arrow in magnified insert in Fig. 2f).3.1.2Epithelial cell and intercellular spaceAt stage 39, the epithelial cells are of a polymorphous type and contain a minimum of cytoplasm with very few organelles (Fig. 3a). At stage 44, the epithelial cells have a columnar shape. The cell cytoplasm has increased in size but is not dense, with only a few mitochondria inside (Fig. 3b). At stage 46, the epithelium of the ES contains a single layer of cylindrical cells. The content of mitochondria appears increased. A few microvilli appear on the epithelial cell luminal surface. At stage 48, the epithelial cells are of both cuboidal and cylindrical types. The epithelial cells contain relatively dense cytoplasm in which many mitochondria, as well as scattered ribosomes and Golgi complexes can be found (Fig. 3c). At stage 50, the epithelial cells are cuboidal, with a height of about 8 μm and a width of 7 μm. The dense cytoplasm contains an abundance of vesicles surrounded by Golgi complexes and ribosomes. Mitochondria are packed together in the cytoplasm of some epithelial cells (see Fig. 4c). At stage 54, cells are cuboidal at the proximal portion but more flattened at the intermediate and distal portion. The cytoplasm is of high density with a fibrous appearance. Abundant cytoplasmic organelles such as mitochondria, ribosomes, Golgi complexes, endoplasmic reticulum are distributed in the cytoplasm. Numerous granules are found in the apical and lateral cytoplasm (GR in Fig. 3d) at this stage. These granules, 0.2–0.4 μm in diameter, are usually round or oval and surrounded by a limiting membrane.The epithelial cells are separated by intercellular spaces (ISs). At stage 39, these spaces are narrow and have a smooth surface, with nothing inside (Fig. 3a). At stage 44, the ISs are radially distributed from the center of the ES with some vacuoles filled in the space (see Fig. 3b). Primitive microvilli are occasionally found on the lateral surface of the intercellular spaces (MV in Fig. 3b). At stage 46, the vacuoles in the IS are fused, which makes the space expand and have a wave-like surface (see Fig. 4a). At stage 48, the ISs dilate irregularly, in which finger-like protrusions arise from adjacent surfaces (CJ in Fig. 3c). The ISs expand markedly and have a honeycomb-like appearance at stage 50 (see Fig. 4c). Numerous protrusions arise from the surface of the space to form intercellular digitations. At stage 54, floccular material is found in the widened irregular IS (not illustrated). The IS is loose and contains many digitating projections, which display a labyrinthine appearance (Fig. 3d).3.1.3LumenIn Fig. 4a, the slit-like lumen of the ES lies longitudinally in the middle of the ES at stage 46. The lumen is approximately 39 μm long and 3.3 μm wide and displays its outline in only four serial sections. The luminal surfaces of the epithelial cells bear a few short microvilli, which are about 0.3–0.5 μm long. At stage 48, the ES lumen becomes wider and longer than before. The epithelial surface is uneven with several short protrusions and microvilli in the lumen (MV in Fig. 4b). The microvilli are longer than before (0.4–0.6 μm). Floccular material (endolymph) appears in the lumen (FM in Fig. 4b). This material is not dense and is evenly distributed within the lumen. Crystals were not found in the lumen in serial sections at this stage. At stage 50, the ES lumen dilates markedly and is about 120 μm long, 20 μm wide and 20 μm thick. The luminal surface is irregular with several protrusions into the lumen (not illustrated). The microvilli on the luminal surface are relatively long (0.5–0.7 μm) and curved. Otoconia are first found in the lumen at stage 50. The spaces where the crystals were appear bright under TEM, indicating that these spaces are ghosts of dislodged or dissolved otoconia. The endolymph becomes dense at places adjacent to the otoconia. At stage 54, the ES lumen forms a large cavity (Fig. 4d) that is about 140 μm long, 30 μm wide and 25 μm thick. In the lumen, numerous otoconia pile together. The size of individual otoconia is similar to that at stage 50. The floccular material is dense and contains occasional cell debris. Thick and long microvilli (1 μm long and 0.1–0.2 μm wide) arise from the luminal surface of the epithelial cell. At high magnification, these microvilli have a triple layered unit membrane with the middle layer more dense, indicating a lipid bilayer (LB in Fig. 5a). The floccular material close to the microvilli and the otoconia sometimes becomes very condensed as seen in Fig. 5a. Free floating cells first appear in the ES lumen at this stage. The cytoplasm of this cell is relatively light with numerous vacuoles inside. Long microvilli (2 μm) are present around all the free surface of the cell (Fig. 5b).3.2Development of the endolymphatic duct (ED)Before stage 38, no ED was found in serial sections. At stage 39, a primitive ED is present as a connection, about 25 μm long, between the ES capsule and the medial side of the otic vesicle (ES in Fig. 2a). Serial sections did not show a lumen inside this connection. At stage 41, the ED extends and expands, but still no lumen was found. A true short and thin tube (ED lumen), which joins the ES lumen and passes into the otic vesicle lumen at the upper end of the saccule (SC), is present at stage 46 (LM in Fig. 2c,Fig. 6a). The ED is about 55 μm long and has a lumen diameter of 0.5 μm. The ED is lined with a single layer of duct-like epithelial cells which rest on a smooth basement membrane. A large nucleus with its long axis parallel to the duct can be seen in the cytoplasm. Intercellular spaces are present between the epithelial cells. At the low end of the ED, near the saccule, the duct dilates to 0.8 μm in diameter. Microvilli are seen only on the luminal surface of this dilated end of the ED (ED in Fig. 6a).At stage 50, the ED extends its length to about 100 μm in length with a lumen diameter of 0.8 μm. At the low end of the ED, the diameter of the lumen extends to 1.0 μm. The lower cubical epithelial cells of the ED contain more cytoplasm than before. The nucleus usually lies at the base. Some floccular material (FM in Fig. 6b) can now be seen in the lumen. The luminal surface of the ED is relatively smooth and bears only a few microvilli (Fig. 6b).At stage 54, the ED is about 200 μm long with a lumen diameter of 1.2 μm. At the low end of the ED, close to the saccule, the diameter of the lumen extends to 2 μm. In the low portion of the ED, the epithelial cells bear a few short microvilli which rise perpendicularly from the apical surface. In the intermediate and transitional portion, the epithelial cells bear numerous relatively long and thick microvilli. These microvilli protrude into the lumen at different directions and touch or overlap each other, which makes the lumen have an intermeshing appearance (MV in Fig. 6c). The epithelial cell becomes low cubical. The cytoplasm is rather dense and has many mitochondria, Golgi complexes, vesicles and vacuoles. The nucleus is irregular in shape with condensed nucleoplasm inside. Floccular material and otoconia are occasionally found in the lumen of the ED. A well-defined and dilated IS can be seen between the epithelial cells.3.3Development of junctional complexes between the epithelial cellsBefore stage 45, there is no sign of junctions between the adjacent epithelial cells in the ES and ED. The epithelial cells are connected to each other with a smooth or rugose IS. At stage 46 (Fig. 7a), tight junctions first appear in the lateral space between the epithelial cells close to the luminal surface of the ES. The outer layers of the adjacent plasma membrane fuse in the wall of the junction. The junction is rather short (0.2 μm long). Very fine filamentous material fills the interspace of the junction. Below the junction, the IS expands and has an irregular surface.At stage 50 (Fig. 7b), the length of the tight junction increases (0.4 μm). In the cytoplasm of the adjacent cells close to the junction, some granular substance adheres to the outer wall of the junction. Below the tight junction, an adherent junction 0.15 μm long is formed, indicating that a junctional complex has developed. The adherent junction consists of two dark cytoplasmic plaques very close to the innermost layer of the plasma membrane (AJ in Fig. 7b). Several parallel filaments traverse across the junction and extend to the cytoplasm of both cells (CF in Fig. 7b). Below the junctional complex, cellular digitations can be seen in the lateral intercellular space.At stage 54 (Fig. 7c), the cell walls of the tight junction are parallel with a narrow intercellular space of about 200 Å. In the adhering junction, there are two oval plaques of dense material in the cytoplasm close to the inner layer of the plasma membrane. The crossing parallel filaments become longer and have a microtubule-like appearance (CF in Fig. 7c). Below the junctional complex, both of the cell membranes are closely apposed at some point (TJ in Fig. 7c) indicating that multiple cell junctional complexes are formed.4DiscussionThe membranous labyrinth of the inner ear derives from ectoderm. In C. pyrrhogaster, a small protuberance of the otic vesicle appears as a precursor of the ES, on the dorsal-medial aspect of the saccule at stage 31, about 7 days after the eggs are laid (Wiederhold et al., 1995). In the present study, a true egg-like capsule, which is composed of epithelial cells of the ES and a connection between the capsule and the saccule, which forms the primitive ED, are seen at stage 39, approximately 11 days after oviposition. It is believed that the protuberance of the otic vesicle extends to produce the ED and its upper end expands to form the ES during development.Before the 1980's, investigations on the development of the inner ear only briefly mentioned or failed to describe the process of organogenesis of the ES and ED in mammals (Ruben, 1967; Sher, 1971). In amphibians, earlier descriptions of the development of C. pyrrhogaster showing histological sections through the brain of embryos up to stage 42 did not mention the ES and ED (e.g. Okada and Ichikawa, 1947). In this study, the whole sequence of maturation of the ES and ED has been demonstrated, both concerning organogenesis and epithelial cell cytogenesis (Table 2Table 3).From Tables 2 and 3, it is known that a slit-like lumen of the ES is formed at stage 46. At the same time, a true lumen of the ED, which joins the ES lumen at its upper end and the lumen of the otic vesicle at the low end, first appears. Several days later (stages 48 and 50), floccular material (endolymph) is found in the expanded ES lumen and the ED. It is believed that the entire endolymphatic system is in communication with the other parts of the inner ear at this period. The floccular material flows into the ES from the saccule, or out to the otic vesicle lumen from the ES, by passing through the ED at stage 46. Apparently, in Cynops, the ES and ED play their biological function only from this stage.The cytodifferentiation of the epithelial cells in Cynops occurs from stage 39 to stage 54. During the process, the height of the cell reduces but the cytoplasm becomes larger with increasing number of cytoplasmic organelles. This indicates that the metabolic activity within the epithelial cells of the ES increases during development. At stage 54, the cytoplasm of the epithelial cells is of high density with a fibrous appearance. Abundant cytoplasmic organelles such as mitochondria, ribosomes, Golgi complexes and endoplasmic reticulum are distributed in the cytoplasm. The fine structure of the epithelial cells at stage 54 is similar to that of the epithelial cells at stages 56 and 58 (a nearly adult newt). Thus, it is concluded that the epithelial cells in the newt C. pyrrhogaster have reached maturation at stage 54.In the present study, the cytoplasm of the epithelial cells of the ES contains numerous vesicles at stage 50 and many granules at stage 54. These vesicles and granules are surrounded by a limiting membrane and located close to the apical cell membrane. Salamat et al. (1980)have also observed vesicles in the cells of the labyrinthine wall of the rat. They believed that the content within the vesicles would be expelled into the labyrinthine lumen. In amphibians, Kawamata et al. (1987)found that most epithelial cells of the ES in the tree frog, Hyla arborea japonica, contain granules. These granules, varying in size, are surrounded by a limiting membrane and apposed to the apical membrane of the cell. Kawamata suggested that these granules are released into the lumen by the epithelial cells. In a histological and ultrastructural study, Dahlmann and Düring (1995) found many ribosomes in some epithelial cells of the ES of the rat. They suggested that supranuclear accumulation of organelles, especially ribosomes, Golgi complexes and vesicles in the cytoplasm are interpreted as signs of secretory activity. In this study, numerous ribosomes and Golgi complexes are richly distributed around the vesicles and granules in the apical cytoplasm of the epithelial cells at stage 50. These findings strongly suggest that, in the newt, the epithelial cells of the ES are secretory after the animals reach stage 50.The intercellular spaces (ISs) between epithelial cells of the ES have received much attention (Friberg et al., 1985; Takumida et al., 1988). This study demonstrates the developmental process of this structure. At stage 39, the primitive IS is narrow and has a smooth surface. Vacuoles are found in the IS at stage 41. At stage 46, about 18 days after the eggs are laid, the IS starts to expand. The intercellular spaces dilate evidently and have an irregular appearance at stage 48. After stage 50, intercellular digitations arise from the surface of the IS. The IS is honeycomb-like between the epithelial cells. At stage 54, the IS contains many intercellular digitations and displays a loose labyrinth appearance.The function of the intercellular spaces of the ES is still not known. Many morphological and experimental studies indicate that the lining of the ES and ED may represent a fluid transporting epithelium with an ability to transfer water and solutes to and from the ES and ED (Lundquist, 1965; Adlington, 1967; Rask-Andersen et al., 1981; Bagger-Sjöbäck and Rask-Andersen, 1986). Friberg et al. (1985)and Takumida et al. (1988)suggested that the intercellular spaces of the ES may form a pathway for transepithelial water flow from the ES to blood vessels. According to their assumption, the epithelial cells may actively transfer salt from the ES lumen to the IS, and then water is drawn through local osmotic force from the lumen to the intercellular space. Based on the `longitudinal theory' (Guild, 1927), endolymph flows from the vestibule out to the ED and ES. Friberg et al. (1985)confirmed that cessation of longitudinal flow to the ES by surgery would make the intercellular spaces collapse. They considered that this cessation of flow also arrests transepithelial movement of fluid. In the present study, the intercellular spaces are not dilated before stage 45, when the lumen of the ED and ES have not yet been formed. After the lumen of the ED and ES have developed at stage 46, the intercellular spaces start to expand. The IS dilates evidently at stage 48 when abundant floccular material appears in the lumen. This finding suggests that fluid transport across the sac epithelium may be initiated at the same time the IS starts to dilate.Tight junctions first appear in the epithelium at stage 46, about the same time the lumen is formed. At stage 50, adherent junctions are present below the tight junctions, indicating that a junctional complex has developed. At stage 54, fully developed multi-junctional complexes are present between the epithelial cells. Tight junctions act as a permeability barrier to fluids and ions (Claude and Goodenough, 1973; Bagger-Sjöbäck and Anniko, 1984). Anniko and Bagger-Sjöbäck (1982) reported that the tight junctions of the mouse have developed before formation of the high potassium concentration in the endolymph. In the present study, tight junctions are formed at stage 46, at which time a slit lumen of the ES and very narrow ED have just developed. Apparently, formation of the tight junctions occurs before endolymph is available in the ES lumen. These tight junctions probably play an important role in maintaining ion gradients between the endolymphatic lumen and lateral and extralabyrinthine space, and the elevated Ca2+ concentration in endolymph which is necessary for formation of otoconia, a major function of the ES in amphibians.An interesting finding in this study is that so many microvilli touch and overlap each other, which gives an intermeshing appearance to the lumen when the ED reaches maturity. The functional significance of this organization of the microvilli is not known, but would suggest an active secretory function.AcknowledgementsThese studies were supported by the NASA Space Biology Program (Grants NAG 2-952 and NAG 10-0180) and the National Science Foundation (Grant BIN-95-29136). 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Bull.1019979196 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B55479AB153C8E2AA83528EEF0D29CD88601242.txt b/test/dataset/in/resources/corpus/Clean_0B55479AB153C8E2AA83528EEF0D29CD88601242.txt new file mode 100644 index 0000000..a3b0d63 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B55479AB153C8E2AA83528EEF0D29CD88601242.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press P4110.1093/geronb/63.1.P41 Journal of Gerontology: Psychological Sciences Spousal Social Activity Trajectories in the Australian Longitudinal Study of Ageing in the Context of Cognitive, Physical, and Affective Resources Hoppmann Christiane A. Gerstorf Denis Luszcz Mary 1School of Psychology, Georgia Institute of Technology, Atlanta. 2Department of Human Development and Family Studies, The Pennsylvania State University, University Park. 3Centre for Ageing Studies, Flinders University, Adelaide, Australia. Address correspondence to Christiane A. Hoppmann, Georgia Institute of Technology, School of Psychology, 654 Cherry Street, Atlanta, GA 30332. E-mail: ch295@mail.gatech.edu 1 2008 63 1 P41 P50 6 9 2007 21 12 2006 Copyright 2008 by The Gerontological Society of America 2008 We examined the dyadic interdependence of spousal social activity trajectories over 11 years by using longitudinal data on 565 couples from the Australian Longitudinal Study of Ageing (Mage = 76 years at Time 1). Social activity trajectories were interrelated in elderly couples, and they depended not only on individual but also on spousal cognitive, physical, and affective resources at baseline. Most associations examined were similar in husbands and wives. However, wives performed more social activities and displayed different depression–social activity associations than did husbands. We found stronger within-couple associations in the domain of social activities than for cognition. Our findings illustrate the important role of social relationships for late-life development and suggest that the mechanisms involved in dyadic interdependencies may be domain and gender specific. Successful aging Social activities Couples Growth curve modeling hwp-legacy-fpage P41 hwp-legacy-dochead RESEARCH ARTICLE ENGAGEMENT in social activities is key to successful aging and has attracted considerable interest regarding its association with cognition, physical functioning, and affect (Andrews, Clark, & Luszcz, 2002; Bath & Deeg, 2005; Ghisletta, Bickel, & Lövdén, 2006; Hultsch, Hertzog, Small, & Dixon, 1999; Lang & Baltes, 1997; Lövdén, Ghisletta, & Lindenberger, 2005; Rowe & Kahn, 1997; Ryff & Singer, 2000). Social activities comprise a person's activities that are performed for the purpose of direct interaction with others (Herzog, Ofstedal, & Wheeler, 2002). An important benefit of social activities is that they can be implemented by older adults despite physical limitations that may restrict other activities. Hence, engagement in social activities may continue to contribute to a person's sense of competence in a way not afforded by other types of activities (Herzog et al.). Studies on social activities have primarily been based on unrelated people. Because social activities inherently depend on others, marriage partners are prime social targets, particularly in late life. Couples comprise a unique social unit in old age that is often characterized by a long relational history, frequent interaction, and considerable closeness (Antonucci & Akiyama, 1991; M. M. Baltes & Carstensen, 1998; Dixon, 1999; Lang, 2001). Simply being married may be socially advantageous, given the high rates of bereavement in old age. However, past research shows that the positive potential of marriage has to be qualified by spousal behaviors (Coombs, 1991; Hagedoorn et al., 2006). Investigating social activities in couples therefore addresses one specific process that occurs within marital contexts. Changes in, and entrainment of, social activity trajectories over 11 years in couples from the Australian Longitudinal Study of Ageing (ALSA; Luszcz, 1998) is the central focus of this article. Spousal development is interdependent in many central life domains (Carstensen, Gottman, & Levenson, 1995; Gruber-Baldini, Schaie, & Willis, 1995; Townsend, Miller, & Guo, 2001). The social activities of elderly couples may be interrelated for at least two reasons. First, social networks are dominated by joint kin and become smaller but increasingly interdependent throughout adulthood and into old age (M. M. Baltes & Carstensen, 1998; Kahn & Antonucci, 1980; Lang, 2001; Milardo & Helms-Erikson, 2000). Second, elderly couples typically have long marital histories with many shared experiences. Because each partner has developed an in-depth knowledge of the other's strengths and weaknesses (Dixon, 1999; Meegan & Berg, 2002), each partner's expertise and skills can be invoked for the sake of both partners. For example, if one spouse initiates a social activity (e.g., inviting friends for dinner), the other spouse also will have a social engagement opportunity. We therefore expect social activity trajectories of elderly couples to be closely interrelated. Our second major objective is to examine associations among social engagement by spouses and resources in other domains, namely cognitive, physical, and affective resources. Associations between social activities and cognitive resources may in part be due to the complex nature of social activities that place high demands on cognitive skills, thereby potentially protecting against cognitive decline (Fratiglioni, Paillard-Borg, & Winblad, 2004; Ghisletta et al., 2006; Lövdén et al., 2005; Pushkar et al., 1999). Alternatively, cognitive fitness may be a prerequisite for social activities (Hultsch et al., 1999). We also expect social activities to be positively associated with physical resources. Social activities modulate stress reactivity and promote psychobiological recovery processes that are associated with age-related illnesses (Seeman & McEwen, 1996). Finally, social activities may be linked to affective resources (e.g., resistance to depression) because they fulfill an inner need for affiliation (Cantor & Sanderson, 1999; Herzog et al., 2002). We therefore expect social activities to be positively associated with cognitive, physical, and affective resources. Some of these links with resources may be shared with other activity categories. Cognitive resources in particular are likely to shape a broad range of activities, for example, crossword puzzling (Ghisletta et al., 2006). In contrast to crossword puzzling, we expect that additional resource domains impinge on social activity. This makes social activities particularly interesting for researchers interested in successful aging, and it also highlights the special role of cognitive resources for activities. Hence we also explore dyadic associations in longitudinal trajectories of cognitive functioning (cf. Anstey, Hofer & Luszcz, 2003). This enables us to investigate the domain specificity of entrainment of partner's functioning and to assess whether social activity and cognitive trajectories wax and wane together. Thoroughly examining the relative contributions of different categories of activities or domains of functioning is beyond the scope of this article; rather, we target entrainment and change in social activities as particularly salient in the context of long-term marriage. One feature of this study is the capacity to identify associations of social activities to functioning in other domains, not only at the individual level but also at the couple level. For example, if one spouse experiences serious cognitive decline, resources of the partner may compensate, at the expense of social activities (Clark & Bond, 2000; Tremont, Davis, & Bishop, 2006). Hence, we assume that one spouse's cognition, physical functioning, and affect not only relate to his or her own but also to spousal social activities. Following the terminology of the dyadic literature (Cook & Kenny, 2005), we refer to associations of an individual's characteristics with his or her own score on a given variable as actor effects and with the partner's score on a given variable as partner effects. Applying this nomenclature, we hypothesize that a spouse's cognitive, physical, or affective resources relate not only to his or her own social activities (actor effects) but also to those of the other spouse (partner effects). Finally, the expected relationships between social activities and cognitive, physical, and affective resources may not be symmetric in husbands and wives. A large body of literature indicates that women take a more active part in their social networks than do men, playing an important role in initiating and organizing social activities (Antonucci, 1994, 2001; Moen, 2001). Additionally, relationship maintenance is often seen as obligatory by women whereas men's social activities are more voluntary (Antonucci, 1994). Hence, husbands may not enter all social engagement opportunities that are provided by their wives. We therefore expect wives to report more social activities than husbands. Being more socially invested than men (Moen, 2001) may have both positive and negative implications for wives, because they may not only benefit more from their social activities but also be more vulnerable when circumstances necessitate a change in them (Rook, 1998). We therefore expect that social activities are more strongly linked with both wives' own and their spouses' resources. Hence, wives' social activities are expected to be more closely related to both actor and partner resources than husbands' social activities are. In summary, we examine three major questions by using 11-year longitudinal couple data. First, we examine dyadic interdependencies in level and overall change in social activities in couples. Second, we examine how social activity trajectories are related to individual and spousal resources. We further explore dyadic interdependencies of longitudinal changes in social activities and cognition. Third, we investigate potential gender-based (a) symmetries of the associations examined. Methods The ALSA is a broad biopsychosocial and behavioral panel study. On the advice of the third author (M. Luszcz), we selected variables on an a priori basis as the best available markers of the constructs targeted for investigation. Detailed descriptions of variables and procedures are published elsewhere (Andrews et al., 2002; Anstey et al., 2003; Luszcz, Bryan, & Kent, 1997). Participants and Procedure The baseline ALSA sample (N = 2,087) was stratified by age and sex into four cohorts (70–74 years, 75–79 years, 80–84 years, and 85+ years); men and those over the age of 85 years were oversampled. The sample (55% response rate) was obtained from the South Australian Electoral Roll that identified households with residents over 70 years of age (Hugo, Healy, & Luszcz, 1987). Randomly sampled individuals within these households were invited to volunteer for ALSA; spouses over age 65 were also invited to participate. We included all couples with valid data on the variables of interest (N = 565 or 1,130 people). Average marriage duration was 46.33 years (SD = 11.23; range = 0–70 years); number of children was 2.7 (SD = 1.63). Husbands were about 3 years older than their wives, at 77.7 versus 74.3 years; F (1, 1128) = 101.1, p <.001. Responding to a 5-point Likert-scale, couples were “extremely” to “very” satisfied with their marriage (M = 1.69, SD = 0.78) and family life (M = 1.97, SD = 0.81), but slightly less satisfied with their friendships (M = 2.37, SD = 0.76). As compared with the remaining 957 participants from the total ALSA sample, participants in the ALSA couple subsample did not differ in number of social activities, education, or health constraints (all p >.10). However, they were younger [M = 76.66, SD = 5.87 vs M = 80.72, SD = 6.70; F (1, 2085) = 295.6, p <.001], reported fewer depressive symptoms [M = 7.27, SD = 6.90 vs M = 9.36, SD = 7.85; F (1, 1991) = 40.0, p <.001], and performed better on the Digit Symbol Substitution task [M = 30.59, SD = 10.89 vs M = 27.20, SD = 11.20; F (1, 1241) = 28.6, p <.001]. Data were collected in two 1.5- to 2-hour sessions at participants' home (98.4% private household or 1.6% institution). Session 1 assessed demographics, social activities, depression, and health. The Session 2 functional assessment occurred approximately 2 weeks later and included tests of perceptual speed for a subsample of, on average, 70% of Session 1 participants (see Table 1). We use four waves of longitudinal data spanning 11 years: baseline in 1992–1993 (Time 1 or T1; n = 1,130), 1994–1995 (Time 3 or T3; n = 929), 2000–2001 (Time 6 or T6; n = 487), and 2003–2004 (Time 7 or T7; n = 315). Assessments at Times 2, 4, and 5 did not provide data on the targeted domains; we excluded them. On average, T3 occurred 1.53 years (SD = 0.50), T6 occurred 7.43 years (SD = 0.50), and T7 occurred 10.57 years (SD = 0.50), respectively, after T1. By T3, T6, and T7, 9.9%, 38.4%, and 51.7%, respectively, of participants had become deceased (this corresponds closely to the incidence of widowhood, i.e., 10%, 37%, and 52%); whereas in the residual sample of 987 participants, there were 12.9%, 52.9%, and 65.1% of the sample who became deceased across the same period. Measures Social activities We selected four items from the Adelaide Activity Profile (Clark & Bond, 1995) that specifically assessed social-life aspects in which both partners can potentially participate. These concerned the frequency of (a) having invited other people to one's home, (b) making phone calls to friends or family, (c) attending social activities at a center such as a club, a church, or a community center, and (d) participating in outdoor social activities. Participants responded by means of a 4-point Likert-scale, with higher scores indicating more activity. A structural model at T1 indicated that all factor loadings for husbands and wives were above.60 (M =.79), suggesting a reliable social activity factor. Covariates We considered education, T1 health constraints and depression, and processing speed at T1 and over time. We dichotomized years of education by contrasting participants who left school at age 15 plus (higher education) with those who left under age 15. Health constraints were self-reports of the number of chronic medical conditions from a comprehensive list of 61 (e.g., stroke, diabetes, arthritis). For depressive symptoms, we used the Center for Epidemiological Studies–Depression scale (Radloff, 1977); 20 items asked participants how often over the past week they felt symptoms such as lack of energy or sad feelings. For a parsimonious indicator of cognition, we selected perceptual speed because it is highly reliable and sensitive to change throughout adulthood (Anstey et al., 2003; Salthouse & Madden, in press), and, as a cognitive primitive (Luszcz & Bryan, 1999), is conceptually closer to a resource than other abilities. In contrast, decline in verbal ability, for example, occurs very late in life (Anstey et al., 2003), when it may be too late to engage in compensatory behaviors (Ghisletta et al., 2006), and memory change is fundamentally limited by change in perceptual speed. We assessed perceptual speed by using the Digit Symbol Substitution subscale of the revised Wechsler Adult Intelligence Scale (Wechsler, 1981; for details, see Luszcz et al., 1997), at T1, T3, T6, and T7. Participants substituted symbols corresponding to the numbers 1–9 as rapidly as possible into a randomly ordered array of 93 digits. Symbols were presented throughout the task. We used the number of correct substitutions in 90 seconds. Data Preparation We standardized social activities and perceptual speed to a T metric (M = 50; SD = 10), with the T1 ALSA couples providing the reference. This transformation ensured a common metric while maintaining the psychometric properties of the scores and the longitudinal changes in means and variances. Missing data for social activities amounted to 7% (206 out of 2,861 attainable data points over four occasions). Missing data for perceptual speed amounted to 6% of Session 2 participants and were primarily due to poor vision.1 No data imputation procedure was applied. The average longitudinal observation interval was 3.68 years (SD = 3.63). Table 1 presents, separately for husbands and wives, the age at assessment as well as means and standard deviations for the variables under study. Participants were tested, on average, in their late seventies and early eighties. Husbands and wives did not differ in education or health problems (p >.10), but wives reported more depressive symptoms than did husbands, F (1, 1084) = 4.5, p <.05. The effects of social activities and perceptual speed were our main focus and are presented in the Results section. Statistical Procedures We applied two-variable and four-variable extensions of the latent growth model over time (McArdle, 1988) that estimates fixed effects (average level and change) and random effects (interindividual differences in level and change). The two-variable growth curve is a straightforward extension of a univariate growth curve. Specifically, we consider the couple as the unit of analysis and model separate growth processes for social activities of husbands and wives. A graphical representation of the model is given in Figure 1. The diagram shows observed variables as squares, latent variables as circles, and the required constant as a triangle as well as fixed model parameters as one-headed arrows and random parameters as two-headed arrows. Unlabeled paths are fixed to 1. The four repeated measures of X (husband) and Y (wife) have three sources: the latent intercept, X0, Y0, with unit loadings; the latent slope, Xs, Ys, with linear loadings (0, 1.5, 7.4, 10.6); and the time-specific residual, ex[t], ey[t]. Intercepts and slopes are estimated at the population level and are allowed to vary and to covary. Time-specific residuals ex[t], ey[t] have a mean of zero, a single variance (σ2ex, σ2ey), and are allowed to covary with each other within occasion (σ2ex,ey). Because of the age-heterogeneous sample, all models reported include husbands' and wives' ages as covariates to adjust otherwise inflated covariances among variables that are due to their common association with chronological age. In Figure 1, covariates are represented by variables Zx and Zy with their own mean (μZx, μZy), variance (σ2Zx, σ2Zy), and covariance (σ2Zx, Zy), as well as regression effects on intercepts and slopes both as actor effects (βZx,X0, βZx,Xs, βZy,Y0, βZy,Ys) and partner effects (βZx,Y0, βZx,Ys, βZy,X0, βZy,Xs). We used the full-information maximum likelihood estimation algorithm and included all data points available, which allowed us to accommodate for unbalanced data structures and incomplete data under the missing-at-random assumption (McArdle, 1994). We estimated the models by using the Mplus program, Version 4 (Muthén & Muthén, 1998–2006). We carried out three sets of analyses. First, we examined level and overall change of social activities among husbands and wives as well as associations among these factors at the couple level. Second, we investigated whether level and overall change in social activities were related to T1 measures of education, perceptual speed, health constraints, and depression, both at the individual and couple level. We did this by including additional Z variables for each covariate in the model shown in Figure 1, allowing for intercorrelations among all covariates. In follow-up analyses, we explored covariations among longitudinal changes in social activities and perceptual speed, both at the individual and couple level. To do so, we extended the two-variable growth model shown in Figure 1 to a four-variable model that also included linear and quadratic slopes of perceptual speed for both partners. Third, we evaluated the (a) symmetry of covariate–growth factor associations between spouses by means of statistically nested model comparisons of models that either freely estimated the respective parameters or set it invariant across spouses. Results Level and Change of Social Activities in Older Couples Results for spousal social activity trajectories (Table 2) indicate that the model fit the data well (e.g., root mean square error of approximation or RMSEA =.023). Consistent with our expectation, fixed effects for the levels indicate that wives reported more activities than did husbands, which was corroborated by a substantial loss of model fit when we set the levels invariant (Δχ2/df = 34.1/1, p <.001). Fixed effects for both slopes were not significantly different from zero, suggesting that social activities did not show much change over time on average. Significant estimates for all random effects among husbands as well as wives revealed, however, that individuals reliably differed from one another in both their activity levels and change over time. We also note the significant and negative level–slope covariance among wives, but not among husbands, indicating that wives who initially reported more social activities tended to show an overall subsequent decline. Of particular interest were the three significant partner effects. Social activities between partners were strongly correlated, and more activities among wives were associated with activity decline for husbands. In addition, associations between the time-specific residuals indicate that, after we accounted for individual activity changes, activities of both partners were still related with one another. In line with our expectations, these findings suggest that social activity trajectories were interrelated not only at the individual level but also at the couple level. Relations of Level and Change in Social Activities to Individual and Spousal Correlates To examine associations between level and change in social activities with individual and spousal correlates (Table 3), we introduced husbands' and wives' T1 measures of education, perceptual speed, health constraints, and depressive symptoms as covariates. Regarding actor effects, husbands' activity levels were negatively associated with husbands' age and health constraints and positively with husbands' perceptual speed. Similarly, wives' activity levels related negatively to wives' age and depression and positively to wives' education and perceptual speed. The only significant correlate of change in activity was husbands' depression, and the negative sign indicates that more baseline depression related to more activity decline. Again, partner effects were of primary interest. The only statistically significant association at the partner level was that depression of wives related positively to husbands' activity change, suggesting that husbands of depressed wives showed an increase in activities. In follow-up analyses, we examined a four-variable growth model of longitudinal changes in social activities and perceptual speed among husbands and wives.2 This model again provided reasonable fit to the data (e.g., RMSEA =.040). Fixed effects for social activities were very similar to the aforementioned two-variable model (e.g., wives reported more activities than husbands); for perceptual speed, we found no spousal differences at any time, but both husbands and wives showed significant decline over time. The fixed effects from this model are shown in Figure 2. <--CO?1-->What is most important for our question is that both husbands and wives showed significant interindividual differences in their linear change components on social activities and perceptual speed (whereas no interindividual differences were found in the quadratic change component on perceptual speed). This allowed for a close inspection of the covariances across level and linear slope factors at the individual and couple level (Table 4). These intercorrelations did not provide evidence that activity changes either at the individual or couple level were accompanied by cognitive changes, but showed three significant actor effects. Analogous to the two-variable model, level–slope intercorrelations for social activities were negative among wives, but not among husbands. Linking domains of functioning, we found that levels of activities and perceptual speed were associated among both husbands and wives. We also found four significant partner effects: Levels of activities and perceptual speed were positively related, as were husbands' activity levels and wives' level of perceptual speed. In addition, level of activity for wives was strongly associated with activity decline for husbands. Finally, examining asymmetrical effects in the four-variable model revealed that activity levels showed stronger associations among both husbands and wives (r =.59) than did levels of perceptual speed (r =.13; Δχ2/df = 5.4/1, p <.01). This suggests that mutual engagement in activity is stronger than entrainment of cognitive capacity. In sum, our results suggest that social activity trajectories were not only associated with one's own resources but also with the resources of one's partner.3 Asymmetry of Associations Between Husbands and Wives Finally, we conducted a series of statistically nested model comparisons to evaluate whether social activity trajectories show differential associations among husbands and wives. To begin with, the nominal difference in level–slope associations among spouses (−.26 vs −.41; see Table 2) was not statistically different, given that a model with these parameters set invariant across spouses did not result in loss of model fit (Δχ2/df = 0.2/1, p >.10). Regarding asymmetrical actor effects in covariate–growth factor associations (see Table 3), we found no effects for age, education, perceptual speed, and health (all p >.10). However, level and slope effects of depression differed between spouses, suggesting that depression was associated with fewer activities among wives but not among husbands (Δχ2/df = 6.5/1, p <.01); and in husbands, but not wives, depression related to activity decline (Δχ2/df = 5.7/1, p <.01). Asymmetrical partner effects indicated that the effects of wives' depression on husbands' activity slope were not mirrored in the effects of husbands' depression on wives' activity slope (Δχ2/df = 3.2/1, p <.05).4 In sum, affective resources of husbands and wives are differentially associated with social activity trajectories of the spouse. Discussion On the basis of 11-year longitudinal couple data, we demonstrated that level of, and change in, social activities are closely interrelated among elderly spouses and that wives perform more social activities than do husbands. We also showed that spousal social activity trajectories were related to both individual cognitive, physical, and affective resources, and to spousal resources. Specifically, significant partner effects emerged linking depression with social activity changes and levels of cognition and social activities. Longitudinally, significant actor effects concerning cognition–social activity associations emerged. Partner effects were stronger in social activities than perceptual speed. Finally, several social activity–depression associations were gender specific. Level and Change of Social Activities in Older Couples Our findings concerning dyadic interdependencies in social activities show that developmental trajectories of spouses are closely intertwined. As expected (Antonucci, 2001; Moen, 2001), wives reported more social activities than husbands. We can only speculate as to the mechanisms linking social activity trajectories in couples. In keeping with socioemotional selectivity theory, our findings may reflect an increased focus on emotionally meaningful relationships in old age as manifested in high mutual engagement in social activities among spouses (Carstensen, 1995). More work is needed, however, to differentiate whether such interdependencies are due to similar background characteristics, overlapping networks, or a reciprocal activity involvement (Milardo & Helms-Erikson, 2000). Relations of Level and Change in Social Activities to Individual and Spousal Resources Our findings that social activity trajectories are related to individual cognitive, physical, and affective resources are in line with past research on unrelated individuals. Within our sample, poorer physical functioning at baseline was related to fewer social activities (Zunzunegui et al., 2005), and spouses with more educational and cognitive resources reported more social activities (Hultsch et al., 1999). Although we found correlation coefficients of different sizes across spouses (e.g., linking physical functioning and social activities), we note that statistically nested model comparisons indicated that these relationships were similar among husbands and wives. Over and above these actor affects, we also found partner effects linking depression and social activities. These findings are discussed in the context of gender-specific associations. We also explored longitudinal associations between social activities and perceptual speed. In line with previous research, participants living socially active lifestyles showed better cognitive performance than did socially inactive participants (Ghisletta et al., 2006; Lövdén et al., 2005). We also found partner effects, and these were much stronger in social activities relative to cognition, thereby highlighting the important role of spouses for social lifestyles. This difference in relative strength of dyadic associations illustrates the increased need for cultural support at advanced ages (P. B. Baltes, 1997). Culture is likely to be more efficacious in the social domain than in the cognitive domain, because spouses can provide input that directly helps their partner to remain socially engaged. Perceptual speed is a fluid ability and more reliant on biological propensities than on cultural resources. Hence, older adults are likely to encounter loss-based cognitive limitations that may be harder for marriage partners to influence. When conducting analyses of cognition and social activities including both partners' age, education, health, and depression as covariates, we found that only partner effects in social activities remained. In sum, our findings suggest that taking into account long-term relationships as an important contextual factor may help researchers to better understand the processes underlying successful aging. Asymmetry of Associations Between Husbands and Wives Our findings of gender-specific actor and partner effects in depression–social activity associations illustrate that husbands and wives are both vulnerable to the effects of depression on social activities. However, the underlying processes may arise at different times. Whereas for wives the negative depression–social activity associations may result from processes that occurred earlier in life, husbands' negative social activity slopes suggest that higher depression scores are followed by social activity decline over the study period. This interpretation is in line with research showing that women report more depression than men, but the relative difference is larger in midlife than in old age (Anstey, van Sanden, Cox, & Luszcz, in press; Barefoot, Mortensen, Helms, Avlund, & Schroll, 2001). Hence, the study period may coincide with a point in time when husbands' depression-related processes unfold, whereas those processes for wives have already emerged. Over and above these actor effects, we also found evidence that wives' depressive symptoms were associated with an increase in husbands' social activities. These results can be interpreted in the context of substantial positive correlations between spousal depression scores (Townsend et al., 2001) and suggest that husbands assume responsibility for social activities when their wives are emotionally challenged. These findings demonstrate that associations of depression and social activity trajectories in elderly couples are best studied in dyads, rather than unrelated individuals. Strengths and Limitations We examined a unique longitudinal couple data set that allowed us to explore actor and partner effects in the relationship between social activities and cognitive, physical, and affective resources. Despite the longitudinal data, most findings refer to mean level results. Like other studies (Ghisletta et al., 2006; Hultsch et al., 1999; but see Lövdén et al., 2005), our study indicated that social activities showed relatively little average change over time (but reliable individual differences therein), which may impose limits on detecting relationships of longitudinal changes to other individual and partner variables. One promising route for future research could thus be to explore whether other (social) activity indicators that are more sensitive to normative change (e.g., loss of bridge partners or closure of a favorite social club) reveal different patterns of longitudinal associations. In addition, our focus on only genuinely social activities, such as inviting friends, contrasts with previous reports from the ALSA (Luszcz et al., 1997; Newson & Kemps, 2005) and other studies (Ghisletta et al., 2006; Hultsch et al., 1999; Lövdén et al., 2005; Pushkar et al., 1999), which used much broader activity measures covering a wide range of physical, leisure, and work activities. We selected social activities that both partners could potentially participate in, but we cannot differentiate whether social activities were performed jointly or individually. Follow-up analyses found that accounting for variability in marriage duration did not change our results, but we could not determine how other relationship characteristics such as mutual affection shape social activities. Together, our results highlight the necessity to distinguish the nature of “activity” in order to accurately understand interrelationships between activities and resources. We acknowledge that our growth curve models produced estimates of average within-person change independent of whether or not an individual (or couple) stayed in the sample over time (i.e., missing-at-random assumption). Nonetheless, we corroborated our main results in a restricted sample of longitudinal participants.<5 Furthermore, these models consider concurrent associations between level and overall change on a given set of variables but not their causal ordering, such as whether one spouse's social activities affect subsequent change in the other spouse's perceptual speed (for an overview of alternative approaches, see Ghisletta et al., 2006). Future research may apply “dynamic” modeling techniques such as bivariate dual change score models (McArdle & Hamagami, 2001) to examine potential lead–lag associations. Conclusions We showed that social activity trajectories are interrelated in elderly married couples and associated with not only individual, but also spousal, characteristics. This attests to the importance of social relationships for late-life development and some domain specificity in underlying dynamics. Together, our results suggest that although physical and cognitive resources provide an environment that is conducive to social activities, the affective resources of one marital partner are crucial to variations in both level and change in social activities of the other and hence to that of the couple as a unit. Our findings are heuristic in posing new questions about the dynamics of long-term spousal relationships for successful aging. For example, is the codependence in social activities due to a priori similarities or does it develop over time (Luo & Klohnen, 2005)? Does the codependence originate in shared environmental characteristics or is it similar across different environmental contexts? Is the relationship between social activities and resources driven by social activity performance or by social embeddedness? Future research may substantiate these findings by going beyond individual differences variables and addressing the underlying dyadic processes. This may get at issues concerning social reserve capacities and vulnerabilities in late life. Decision Editor: Thomas M. Hess, PhD 1Because of the specific study design, sample attrition between Session 1 and Session 2 over the 2-week period was substantial. As discussed extensively by >nstey and Luszcz (2002), attrition primarily reflected self-selection as a result of poor health or physical or cognitive functioning. Using the sample in Session 1 as a reference, we found that missing data for the Digit Symbol task amounted to 31%. 2Results from a preliminary two-variable latent growth model for perceptual speed revealed that both husbands and wives showed significant decline over time (husbands, −0.74, SE = 0.09; wives, −0.55, SE = 0.09; both p < .001) as well as interindividual differences in decline (husbands, 0.25, SE = 0.08, p < .01; wives, 0.21, SE = 0.09, p < .05). 3To examine whether or not our main results concerning partner effects for social activity trajectories were contingent upon physical activities, we included a covariate in our models that comprised a unit-weighted composite of the following items: doing heavy housework, light and heavy gardening, or household or car maintenance; engaging in sports; and walking outside for more than 15 minutes. These analyses revealed substantively identical results to those reported here. In addition, we examined the relationship between physical activities and spousal cognitive, physical, and affective resources. Results showed that the pattern of relationships concerning physical activities differs from the reported findings concerning social activities. In addition, we found no association between physical activity performances of both spouses. Hence, our findings appear to be specific to social activities and suggest that social activities do not represent a proxy for activities in general. 4When introducing the covariates, we found that the reported individual (actor) and spousal (partner) covariances among level and change factors of social activities remained virtually the same. 5For our major findings at the couple level (husband's activity decline relates to wives' level of activities and depression at T1; correlated residuals), analyses of a restricted sample of those who remained in the study up to T6 or longer (n = 164) revealed substantively identical results to those reported here. Figure 1. Graphical representation of the two-variable latent growth curve model (McArdle, 1988) as applied in the present study. Observed variables are represented by squares, latent variables by circles, regression weights by one-headed arrows, and variances and covariances by two-headed arrows; the triangle represents a constant indicating means and intercepts. All unlabeled paths are set to 1. There are four repeated measures of social activities for husbands (X) and wives (Y), and covariates are represented by variables Zx (husbands) and Zy (wives) Figure 2. Model-implied means over time in study from a four-variable latent growth curve model of social activities (solid lines) and perceptual speed (dashed lines) among husbands and wives. We standardized scores to the T metric by using the Time 1 Australian Longitudinal Study of Ageing couple sample (N = 1,130), M = 50, SD = 10 Table 1. Age at Assessment and Descriptive Statistics for Measures Used in the Present Study. Husbands Wives Measure n Age M SD n Age M SD Social activities     T1 563 77.71 48.37 9.88 561 74.30 51.63 9.86     T3 415 78.79 49.11 8.89 452 75.88 52.89 9.19     T6 164 83.38 50.00 9.89 242 80.70 52.87 9.20     T7 96 85.08 52.50 9.71 170 82.85 55.01 9.72 Perceptual speed     T1 371 77.23 49.40 9.79 357 74.03 50.64 10.19     T3 339 78.45 49.69 10.06 350 75.55 52.43 10.03     T6 128 82.60 48.88 9.70 180 79.92 51.45 9.20     T7 85 84.94 47.17 9.66 158 82.60 50.01 9.66 Health constraints     T1 508 77.44 5.43 3.00 508 74.11 5.33 3.13 Depression     T1 489 77.39 6.83 6.51 497 74.05 7.71 7.25 Percent Percent High education     T1 242 77.85 43% 267 74.35 47% Note: T1, T3, T6, and T7 = Times 1, 3, 6, and 7, respectively. We standardized scores of social activities and perceptual speed to the T metric by using the T1 Australian Longitudinal Study of Ageing couple sample (N = 1,130), M = 50, SD = 10. We collected data on perceptual speed in Session 2, which was attended by two thirds to three fourths of Session 1 participants. The average age of Session 1 and Session 2 participants is highly similar. High education signifies that the participants were 15 years of age or older when they left school. For depression, scores >16 correspond to the cutoff for depression according to the Center for Epidemiological Studies–Depression scale. Table 2. Estimates of a Two-Variable Latent Growth Model for Social Activities of Husbands and Wives. Actor Effect Husbands Wives Partner Effect Social Activities Estimate SE Estimate SE Estimate Fixed effects     Level μ0 49.12*** 0.44 51.54*** 0.45     Slope μs 0.01 0.10 0.03 0.08 Random effects     Variance of level σ20 39.02*** 4.71 44.14*** 4.69     Variance of slope σ2s 0.28* 0.13 0.21* 0.09     Correlation level σ20, slope σ2s −0.26 −.41*     Residual variance σ2e 47.33*** 3.01 46.35*** 2.62 Correlations     Husband level σ20 − Wife level σ20 .60***     Husband slope σ2s − Wife slope σ2s .14     Husband level σ20 − Wife slope σ2s −.23     Wife level σ20 − Husband slope σ2s −.43*     Residual variance σ2e Husband − Wife .26*** Notes: All noncorrelation estimates are unstandardized. The significance tests assigned to the correlations refer to the corresponding covariances. Model includes age of husbands and wives as covariates. We standardized social activities to the T metric by using the Time 1 Australian Longitudinal Study of Ageing couple sample (N = 1,130), M = 50, SD = 10. Model fit statistics: χ2 (df) = 45.0 (35); Comparative Fit Index, CFI = 0.988; and root mean square error of approximation, RMSEA = 0.023. * p <.05, **p <.01, ***p <.001. Table 3. Estimates of a Two-Variable Latent Growth Model for Social Activities of Husbands and Wives, Including the Effects of Individual Difference Variables Assessed at Time 1. Husbands Wives Parameter or Covariate Estimate SE Actor Effect Partner Effect Estimate SE Actor Effect Partner Effect Parameter     Level μ0 48.51*** 0.59 51.44*** 0.60     Slope μs −0.10 0.13 −0.07 0.11     Variance of level σ20 35.45*** 4.52 38.69*** 4.43     Variance of slope σ2s 0.12 0.12 0.19 0.09     Residual variance σ2e 47.51*** 3.00 46.06*** 2.60 Covariate     Age −.23** .01 −.15* −.06     Age × Slope −.30 −.22 −.16 −.12     Education .04 .08 .14* −.09     Education × Slope −.18 .22 −.15 .24     Perceptual speeda .14 .06 .21** −.02     Perceptual Speed × Slope −.02 .04 −.16 −.17     Health constraints −.18** −.07 −.06 −.06     Health Constraints × Slope .11 .27 −.11 .11     Depression .11 −.04 −.16* .13     Depression × Slope −.50** .39* .07 −.05 Notes: All noncorrelation estimates are unstandardized. The significance tests assigned to the correlations refer to the corresponding covariances. We standardized social activities to the T metric by using the Time 1 Australian Longitudinal Study of Ageing couple sample (N = 1,130), M = 50, SD = 10. Age, health constraints, and depression were centered at zero. Education: 0 = 0–14 years of age when the participant left school; 1 = 15 years of age or older when the participant left school. Model fit statistics: χ2 (df) = 84.1 (67); Comparative Fit Index, CFI = 0.981; root mean square error of approximation, RMSEA = 0.021. Variance in social activities accounted for level of husbands (R2 =.170), linear slope of husbands (R2 =.601), level of wives (R2 =.190), and linear slope of wives (R2 =.131). a For perceptual speed, p =.06. * p <.05, **p <.01, ***p <.001. Table 4. Intercorrelations Among Level and Slope Factors of Social Activities and Perceptual Speed Among Husbands and Wives (Obtained From a Four-Variable Latent Growth Model). 1 2 3 4 5 6 7 Husbands Actor effect     1. Activity level σ20 —     2. Activity slope σ2s −.25 —     3. Speed level σ20 .16** −.03 —     4. Speed slope σ2s −.06 .39 −.10 — Wives Partner effect Actor effect     5. Activity level σ20 .59*** −.43** .00 .07 —     6. Activity slope σ2s −.22 .15 −.07 −.16 −.39* —     7. Speed level σ20 .16* −.07 .13* −.07 .28*** −.14 —     8. Speed slope σ2s .17 −.09 −.02 −.11 .01 .36 −.22 Notes: Model includes age of husbands and wives as covariates. The significance tests assigned to the correlations refer to the corresponding covariances. Model fit statistics: χ2 (df) = 226.5 (116); Comparative Fit Index, CFI = 0.940; root mean square error of approximation, RMSEA = 0.041. * p <.05, **p <.01, ***p <.001. Preparation for this manuscript was supported by a Research Fellowship awarded by the German Research Foundation (DFG) to Christiane Hoppmann. Large parts of this article were prepared while Denis Gerstorf was at the Department of Psychology, University of Virginia on a Research Fellowship awarded by the German Research Foundation (DFG). The ALSA was conducted by the Centre for Ageing Studies, Flinders University, Adelaide, Australia. ALSA was financially supported by grants from the U.S. National Institute on Aging (Grant AG08523), the South Australian Health Commission, Australian Rotary Health Research Fund, National Health and Medical Research Council (Grant 229922), the Australian Research Council, the South Australian Department of Human Services, Flinders Medical Centre Foundation, Elderly Citizens Homes PL (ECH), and the Flinders Research Grants Scheme. We thank the ALSA participants, who have given their time over many years, and without whom the present study would not have been possible. 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European Journal of Ageing, 2,40-47. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B55C433ABD4232EAEDE4027FD5FA0733BAF644A.txt b/test/dataset/in/resources/corpus/Clean_0B55C433ABD4232EAEDE4027FD5FA0733BAF644A.txt new file mode 100644 index 0000000..2ed1804 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B55C433ABD4232EAEDE4027FD5FA0733BAF644A.txt @@ -0,0 +1 @@ +eltjeltjELT Journal1477-45260951-0893Oxford University Press10.1093/elt/ccn068ArticlesThe collaborative development of teacher training skillsStillwellChristopherChristopher Stillwell has conducted workshops on teacher observation and collaborative professional development throughout Asia and the USA, and has contributed several chapters to TESOL's Classroom Practices series. Currently a senior lecturer and research coordinator at Kanda University of International Studies in Chiba, Japan, he has also worked as a teacher trainer at Teachers College Columbia University, where he received his MAEmail: stillwellc@aol.comFinal revised version received June 200810200926112008634353362© The Author 2008. Published by Oxford University Press; all rights reserved.2009This paper describes ‘mentor development’, a means of collaborative professional development through peer observation that was initiated by the author with 18 peers, all native English speaker EFL teachers at Kanda University of International Studies in Chiba, Japan. It shows how such a programme allows teachers to learn from one another through classroom observations and peer mentoring, where observers practise teacher-educator skills by taking on the role of ‘mentor’ in post-observation conferences. A third colleague attends the post-observation conference with the aim of helping both the mentor and observed teacher reflect on and learn from their interaction during the conference, and to explore the implications these discoveries may have for effective teaching and mentoring.Professional development through peer observationPeer observation is a powerful means by which language teachers can become aware of a broad range of possibilities for conducting classes effectively. While many language teaching professionals will already have experienced peer observation in their training, a recent review of the state-of-the-art in ELT professional development indicates that the practice continues beyond graduate school as an important component of such undertakings as lesson study, team teaching, and peer coaching (Mann 2005). Of these three, peer coaching makes perhaps the greatest use of peer observation as colleagues visit one another's classrooms to help refine practices, gather information related to persistent problems, or get and give feedback on the implementation of new teaching methods (Benedetti 1997).A great deal of the literature on peer coaching, and teacher observation in general, focuses on the post-observation conversation, going to great lengths to warn participants of the risks involved with providing evaluative feedback (Bailey, Curtis, and Nunan 2001; Richards and Farrell 2005). In ‘An etiquette for nonsupervisory observation of L2 classrooms’, Murphy (1992: 225) includes the statement that ‘even well-intentioned feedback to classroom teachers often misfires’. For peer coaching, Showers and Joyce (1996: 15) therefore state that it is ‘necessary and important to omit verbal feedback as a coaching component’. Sheal (1989: 93) notes that feedback issues extend to ‘official’ observations by supervisors and administrators as well, for much observation ‘… is unsystematic and subjective. Administrators and teachers generally have not been trained in observation … Consequently they tend to use themselves as a standard, and they observe impressionistically’.Still, many of these same writers concede that teachers typically ask their observers for feedback, and Cosh (1999) notes that teachers are unlikely to get the full reflective value of being observed if they do not receive any comments. Therefore, it seems unrealistic to expect participants in peer observation to refrain from sharing their opinions on what they have seen. If, on the contrary, the opportunity to exchange insights is embraced as a chance to develop skill and experience with feedback, participants can gain heightened awareness of the power of their positive and negative comments, finding optimal ways to offer guidance that inspires rather than disheartens. This article describes mentor development, an approach to peer observation that taps into teachers’ ability to teach themselves, as well as their potential to be valuable resources to one another, by providing frameworks through which colleagues can work together to train themselves in the best practices of observation and mentoring.Mentor developmentThe inspiration for mentor development came from a desire to help teachers enhance their skills and potentially make a transition to teacher training by providing opportunities for colleagues to practise training techniques with, and on, one another. The first participants began by taking part in a number of reading discussions on teacher observation literature, coming to consensus on ground rules for visits to peers’ classrooms and the conferences that follow. In ensuing years these reading discussions have been replaced by workshops that give participants hands-on experience with various observation and conferencing techniques recommended by the literature. Once the participants have had some exposure to the key issues involved in peer observation, they are divided into groups of three to begin the actual process.First, the colleagues meet in pairs to make the necessary preparations for a series of three classroom visits, in which Teacher A observes Teacher B, B observes C, and C observes A (Figure 1).FIGURE 1The first cycle of observationsAfter this set of classroom visits has been completed, all three colleagues come together for a post-observation conference that takes place in three two-part stages, with each of the participants playing a different role at each stage (Table 1). Each stage focuses on a different classroom observation, with the teacher and classroom observer (‘mentor’) discussing predetermined areas of interest regarding the execution of the lesson while the third party silently listens in as the conference observer. At the end of the discussion, this third party develops teacher training skills by guiding the other two teachers through a conference review in which they reflect and share their perspectives on the post-observation conference, identifying those mentoring behaviours that promoted reflection as well as those that had the potential to have negative consequences (Table 2). Through this debriefing process, all parties gain deeper understanding of the challenges involved with giving feedback and develop greater self-awareness with regard to their own skill at sharing and receiving it.TABLE 1Roles during post-observation conferenceFirst stageSecond stageThird stageTeacher ATeacherThird partyMentorTeacher BMentorTeacherThird partyTeacher CThird partyMentorTeacherTABLE 2Three-stage sequence of post-observation conferenceFirst stageSecond stageThird stageTime0:00–20:0020:00–30:0030:00–50:0050:00–60:0060:00–80:0080:00–90:00FocusConferenceReviewConferenceReviewConferenceReviewRoleTeacher (A) and Mentor (B) discuss the observed lesson. Third party (C) listens.Third party (C) guides Teacher (A) and Mentor's (B) reflection on the conference.Teacher (B) and Mentor (C) discuss the observed lesson. Third party (A) listens.Third party (A) guides Teacher (B) and Mentor's (C) reflection on the conference.Teacher (C) and Mentor (A) discuss the observed lesson. Third party (B) listens.Third party (B) guides Teacher (C) and Mentor's (A) reflection on the conference.At the start of each new stage, a different classroom observation becomes the focus, and all participants change roles accordingly. In this fashion, the three stages of mentor development afford every participant experience giving and receiving feedback at the levels of teacher, mentor, and even as a mentor educator (‘third party’). The cyclical nature of the process and the focus on reflection can lead to great insight, with additional self-awareness and sensitivity resulting from the fact that every feedback giver will have a recent or impending experience in the ‘hot seat’ to consider.Ground rules for peer observationThe execution of the pre-observation and observation stages of mentor development closely resemble other typical practices of peer observation. Key principles include the following:The pre-observation conference1 The observed teacher should set the agenda, determining what the goals of the observation are (Mann 2005). The more specific the observed teacher can be, the more the mentor is likely to provide useful information (Sheal op. cit.).2 The mentor can gain valuable insight and promote better preparation prior to the observation by asking such questions as:▪ What are the goals of this lesson?▪ How does this material fit with previous learning?▪ What problems might be anticipated? (Zuck 1984).3 The more time that is invested in this meeting, the more likely the mentor and teacher are to have a fruitful post-observation discussion. As a frame of reference, Richards and Lockhart (1991) state that in their work with peer coaching, the meetings typically last no more than an hour.During the observation1 The observer is often an invasive presence in the classroom. It is up to the observer to take whatever measures possible to minimize the potential ill-effects of the visit (Master 1983).2 The teacher can put the students at ease by telling them that the observer is not there to watch them but rather to help the teacher develop more skills.3 At the end, the observer should thank the teacher for the opportunity to visit, but should not make any substantive comments about the class. Comments of that nature can provoke premature discussion and circumvent the teacher's own reflection process.The post-observation conferenceOnce each teacher has observed one other, the three come together for the post-observation conference, where each participant takes a turn as a mentor holding a conference with the observed teacher while a third party watches and prepares for the conference review. The following section outlines best practices of mentor development post-observation conferences, by supporting ideas from relevant literature with data collected from teachers from the USA, Canada, the UK, Australia, and New Zealand who chose to participate in the mentor development programme at Kanda University of International Studies (KUIS), an EFL-focused university in Chiba, Japan. The participants had between 2 and 14 years of classroom teaching experience and all hold Masters degrees. At the time of the data collection, the length of their participation in the mentor development programme ranged from one semester to two years or more. Data were collected in two forms:1 anonymous responses given by 16 of the 18 participants to a short online survey;2 excerpts from personal teaching portfolios and journals that were volunteered by six participants.A few technical points were clarified in the survey data. For one thing, it is important to schedule the post-observation conference shortly after the classroom observations. For those nine participants who held their conferences within a week of the observations, only two reported difficulties sharing feedback as a result of delay, while four others reported that the delay was actually helpful for organizing notes and fostering reflection. On the other hand, four of the seven who put off their conference for a week or more found the delay problematic.To make sure that everyone gets equal attention and practice at the conference, time allotments for each part of the discussion can be agreed upon at the outset. KUIS participants settled on 20 minutes for each mentor–teacher discussion and ten minutes for each third party-guided conference review, with a full session thus taking 90 minutes in total. While many of the teachers initially expressed doubt that the sessions should be so long, survey responses show that 75 per cent of the participants ultimately found this amount of time was either ‘adequate’ or even ‘too short’.Mentor and observed teacherZuck (op. cit.: 340) states that it is not advisable for a mentor to begin a post-observation conference by telling a teacher that a class was good or bad because it ‘denies the teacher's role as self-evaluator and gets the discussion off to a bad start’. In order for any conclusions to be internalized by the teacher, the work of the mentor and teacher needs to be fully collaborative. The mentor ‘will need to listen to the concerns of the teacher and signal this to the teacher’ (Randall and Thornton 2005: 87). Indeed, for most cases, it is the teacher who is uniquely capable of accounting for why things went the way they did (Nunan 1996).If the teacher is given a large degree of control over the direction that the conversation takes, the door will be open for the teacher to ‘own’ the discoveries that are made. KUIS participants reported a variety of techniques for making this happen. A mentor may begin by inviting the teacher to share his/her own perspective on the lesson, perhaps by talking through the lesson from beginning to end. Typically, the result of this start is that ‘the points that the observer planned to raise are brought up first by the teacher’. Another reported approach was to encourage reflection on the positive aspects of the class:I knew if I launched into ‘So tell me about your lesson’, she would outline negatives only … I decided to start with a question for her to find one of the positives of her lesson. This was commented on by the other group members, as they said it set the tone for the peer observation feedback.As the conversation proceeds, a mentor's sensitivity can create a safe atmosphere:Throughout the interview, I wanted to make sure I phrased questions that didn't ask ‘why?’ as they can often sound like one is asking the other to justify their rationale for an activity or so on.Allowing the teacher control over the direction of the conversation may at times mean that certain issues remain overlooked, but this should not be taken as an indication that the process is a failure, for it must be remembered that participants in mentor development are peers voluntarily engaging in collaborative professional development. Mentors should resist the urge to stamp out any and all imperfect teaching behaviours, for this approach will only put a teacher on the defensive and demoralize. Nor does this mean that participants should contain themselves to positive feedback, for then ‘the whole exercise becomes a pointless act of mutual back-patting’ (Cosh op. cit.: 24). On this point, some KUIS teachers agreed, saying ‘You don't just want positive feedback—you do it for the negative feedback’ and ‘It can be frustrating for a teacher who wants some honest feedback to hear that everything was “great”’.Generally speaking, it is when the teacher's need to be heard and understood is satisfied that the path is best laid for the teacher to comfortably invite feedback. At that point, the mentor can make appropriate substantive comments within the context of what the teacher has already shared, ideally keeping in mind Gottesman's (2000: 8) advice that feedback should be… specific in nature, about items the teacher can control, … descriptive rather than evaluative, tactful, well-timed, checked for clarity and simplicity, dealing with behaviours rather than personalities (of either teacher or students), … and well organized.Criteria such as these can also be useful to guide the participants’ discussion in the following conference review, when the third party shifts the focus to the appropriateness and effectiveness of the feedback given.The conference reviewThe conference review:… enables us (all in turn) to explore the extremely important aspect of how we provide feedback that is affirming, non-threatening, and, at the same time, effective. Asking a colleague into the ‘sacred space’ of our classroom is a potentially threatening proposition, as is soliciting feedback. It is therefore important that we monitor ourselves (both givers and receivers of feedback) so that we are getting feedback that we want without needing to have a crying session afterwards. (Survey response)The inclusion of a conference review guided by a third party allows for exploration of the way teachers talk to one another and the sort of feedback they view as acceptable, and how this varies across cultures. Collaboration between two colleagues alone would of course be simpler in many ways, but survey responses showed that▪ 69 per cent of the participants would not have preferred to work in a pair▪ 75 per cent felt that they had benefited from taking the role of third party▪ 81 per cent felt that they had benefited from having a third party present when they were giving and/or receiving feedback about teaching.The third party faces a challenging but rewarding dance, having to both lead and demonstrate a discussion on good mentoring behaviours—proverbially ‘walking the walk’ while ‘talking the talk’. Although third parties have little or no time to prepare for the discussion immediately following the mentor–teacher conference, they may have the most dynamic material to work with of all, having the opportunity to generate instantaneous reflection on a mentoring situation witnessed from point blank. One simple way to begin is to follow the template of a typical mentor–teacher discussion by putting the observee in control of the discussion, having the mentor talk through his/her perspective of how each stage of the conference went. Throughout this post-conference discussion, the third party should ask probing questions to promote deeper reflection, making sure that the conversation does not dry up at a superficial level. For instance, the mentor may be asked how the conference did or did not go according to plan and how things might be handled differently if they come up again in the future.At times, the observed teacher's presence can complicate the dynamics of the discussion, making it difficult for the mentor to be completely open about challenges that arose. This can be especially troublesome for mentors who tend to make long lists of faulty teaching behaviours to ‘correct’. Mentors who instead concentrate on helping others to reflect and come to conclusions for themselves will likely have more fulfilling post-conference discussions, as they can more candidly explore the successes and failures of the various techniques employed. Though the presence of the observed teacher may still lend an air of artificiality to the proceedings at times, a touch of awkwardness is not necessarily a bad thing, as it can heighten the senses, making everyone more keenly aware of how well the impact of their words matches their intentions. When the time feels right, the observed teacher can also be brought into the conversation to provide the perspective of what it was like to be mentored in this instance and how the mentor's intended messages were received. It can be instructive to consider whether the teacher felt the mentor's language came across as neutral or judgemental, how much of the mentor's feedback fell into the areas the teacher had previously requested, and how the mentor made the teacher feel safe to reflect candidly on the lesson and come to conclusions for himself/herself.The third party's lack of prior information about the observed class can facilitate complete focus on teacher training issues such as the mentor's techniques for setting the tone and creating a safe environment, asking questions effectively and helping the teacher reflect, and responding appropriately to what the teacher shares. Still, a bit of context can be of great help. Just as a pre-observation conference can lay the groundwork for a good post-observation discussion, the mentor and third party can benefit from a conversation prior to the conference as well. In this meeting, the mentor can make choices about what sort of feedback would be desirable from the third party. A mentor may tell the third party that he/she plans to use a particular style of mentoring or that he/she wants to talk as little as possible during the discussion (much like a teacher of a student-centred class may choose to do) or simply to avoid closed questions, and thus the third party will be invited to comment on the mentor's successfulness in these particular areas. The mentor may even choose to share a ‘conference plan’ (the mentoring equivalent of a lesson plan) with the third party to further guide the observation of the conference.At the end of the conference review, the mentor and observed teacher should take a moment to share reflections on the mentor educator's work as well, closing the circle and taking full advantage of opportunities to increase awareness of the way feedback is given and received. This process is not fully completed until a second cycle is carried out in the opposite direction, so everyone can once again have the experience of the shoe being on the other foot. In other words, instead of Teacher A visiting Teacher B, this time Teacher A will visit Teacher C, B will visit A, and C will visit B (Figure 2), and this will once again be followed by a round of post-observation conferences and conference reviews. Scheduling can be tricky: working in these trios for two full cycles of observation and conference typically occupied KUIS participants for as long as a semester or more. After that point, the group members can decide whether to disperse into new groups to gain further experience with new colleagues in different classrooms or to repeat the process with the same partners so as to reap the benefits of the increased level of trust that will have developed over the course of the two rounds of observation and conference.FIGURE 2The second cycle of observationsFuture directionsThere is much that could be learnt from additional research into mentor development in practice, particularly from a conversation analysis of post-observation conferences to see exactly what sort of feedback tends to be shared, in what manner, and the effects various techniques tend to have. It would also be valuable to have concrete information about the adaptations that groups make as they tailor the model to their own needs and interests. Recordings are now being collected, and it is hoped that additional research can be shared in the future.In the meantime, some ideas for adapting the practices outlined in this article to meet various teaching circumstances and interests may be of use. In cases where it is not possible to find three colleagues to collaborate, pairs of teachers can work together and do the ‘policing’ for themselves. A video camera can serve as a substitute for the third party, and the pair can later watch the video (together or apart) to observe and reflect on their performances as mentors. An additional layer of reflection and feedback can be brought into the process if the participants write journals on the experience, which they can then exchange and respond to, and a distant third party can even participate asynchronously if the tape and journals are shared electronically. A completely different option practised by some KUIS participants was to diminish the focus on the feedback and instead turn the post-observation conference into a sort of collective brainstorming session on solutions to typical classroom problems. In this approach, the participation of a third party in the conversation is welcome but not absolutely essential.Teachers are notoriously busy people, so the idea of setting aside 90 minutes for a post-observation conference may be unrealistic for some. Some teachers at KUIS dealt with this problem by instead having short sessions after each observation, perhaps over lunch. Others simply agreed to tighter time limits, squeezing the whole process into 45 minutes or an hour. Another approach is to make the third-party conference review an optional step that is only taken when the participants feel there is a need.The multilayered practices of mentor development can lead to a variety of rewards. Insofar as it is a form of collaborative professional development, it brings peers together to talk shop and tap into one another's experience, breaking down barriers and giving novice teachers a chance to learn from the pros, and vice versa. The peer observation component adds the opportunity for participants to become more self-aware as they recognize their own behaviours in the practices undertaken by the observed teacher. At times, these observations can lead to renewed enthusiasm for teaching: peers may get new ideas for things to try out, or they may take comfort in the knowledge that they are not alone in facing certain challenges. Students ultimately stand to benefit from mentor development as their teachers find fresh approaches to instruction and improve their skill at delivering feedback. And for those interested in making a career transition into teacher education, mentor development provides practical hands-on experience and training.BaileyKCurtisANunanDPursuing Professional Development: The Self as Source2001Boston, MAHeinle and HeinleBenedettiT‘Enhancing teaching and teacher education with peer coaching’TESOL Journal19977/1412CoshJ‘Peer observation: a reflective model’ELT Journal199953/1227GottesmanBPeer Coaching for Educators2000Second EditionLondonScarecrow PressMannS‘The language teacher's development’Language Teaching20053810318MasterP‘The etiquette of observing’TESOL Quarterly198317/3497501MurphyJ‘An etiquette for the nonsupervisory observation of L2 classrooms’Foreign Language Annals199225/321525NunanDBaileyKNunanD‘Hidden voices: insiders’ perspectives on classroom interaction’Voices from the Language Classroom1996CambridgeCambridge University PressRandallMThorntonBAdvising and Supporting Teachers2005CambridgeCambridge University PressRichardsJFarrellTProfessional Development for Language Teachers: Strategies for Teacher Learning2005CambridgeCambridge University PressRichardsJLockhartC‘Teacher development through peer observation’TESOL Journal19911/2710ShealP‘Classroom observation: training the observers’ELT Journal198943/292103ShowersBJoyceB‘The evolution of peer coaching’Educational Leadership199653/61216ZuckJ‘The dynamics of classroom observation: evening the odds through information’TESOL Quarterly198418/233741 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B5A24388E2BC52A6A3774D010A856296E90D769.txt b/test/dataset/in/resources/corpus/Clean_0B5A24388E2BC52A6A3774D010A856296E90D769.txt new file mode 100644 index 0000000..bc43d6e --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B5A24388E2BC52A6A3774D010A856296E90D769.txt @@ -0,0 +1 @@ +jexbotexbotjJournal of Experimental Botany1460-24310022-0957Oxford University Press10.1093/jxb/ern137Research PapersOver-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formationPineda RodóAlbert1BrugièreNorbert2VankovaRadomira3MalbeckJiri3OlsonJaleh M.1HainesSara C.1MartinRuth C.4HabbenJeffrey E.2MokDavid W. S.1MokMachteld C.1*1Department of Horticulture and Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331-7304, USA2Agronomic Traits/Discovery Group, Pioneer Hi-Bred International, Inc., Johnston, IA 50131-1004, USA3Institute of Experimental Botany v.v.i., Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic4United States Department of Agriculture, Agricultural Research Service, National Forage Seed Production Research Center, Corvallis, OR 97331, USA*To whom correspondence should be addressed. E-mail: mokm@hort.oregonstate.edu72008315200859102673268610120083132008742008© 2008 The Author(s).2008This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots.Corncytokininplant developmenttasselseedZea mayszeatin O-glucosyltransferaseIntroductionCytokinins are plant hormones that are essential for cell division and plant development (Mok and Mok, 1994). Natural cytokinins are adenine derivatives, generally with an isoprenoid side chain at the N6 position, although cytokinins with aromatic side chains are also known to occur (Strnad, 1997). Zeatin is considered to be an essential cytokinin in higher plants due to its ubiquitous nature and high activity. Other free bases with cytokinin activity, cis-zeatin, dihydrozeatin, and N6-(Δ2-isopentenyl) adenine, are also present in most plant tissues. Derivatives of these bases include the corresponding ribosides and nucleotides, as well as glucosides with the sugar moiety at the O of the side chain or at the N7, N9, or N3 of the adenine ring.Until recently, the true activities of the various cytokinin metabolites were difficult to assess. The activity of natural as well as synthetic compounds have been traditionally determined by bioassays such as the tobacco callus, bean callus, or radish cotyledon bioassays (Murashige and Skoog, 1962; Letham, 1971; Mok et al., 1978). The results of these bioassays indicated that free bases and ribosides had high cytokinin activity. However, apparent cytokinin activities may not necessarily reflect their true activities in planta due to rapid metabolism or catabolism. For instance, while the N7 and N9 glucosides of zeatin are mostly inactive in bioassays (Letham et al., 1983), the O-glucoside is very active in bioassays, sometimes more than zeatin itself (Mok et al., 1992). The high activity of O-glucosylzeatin was assumed to result from conversion to the aglycone since the side chain far exceeds the optimal molecule size for cytokinin activity (Skoog and Armstrong, 1970).The identification of cytokinin receptors provided an alternative evaluation of cytokinin activity. For example, studies with the CRE1/AHK4 and AHK3 receptors of Arabidopsis showed that indeed, zeatin O-glucoside is not an active cytokinin (Spíchal et al., 2004). In general, these studies indicate that the highest cytokinin activity is conferred by free bases. Relatively lower activity was displayed by ribosides, while no activity was detected with glucosides (Spíchal et al., 2004; Yonekura-Sakakibara et al., 2004). There are, however, differences between receptors in cytokinin recognition as illustrated by the binding of only the trans isomer of zeatin by the Arabidopsis CRE1/AHK4 receptor, but both the cis and trans isomers by the maize ZmHK1 receptor (Yonekura-Sakakibara et al., 2004; Mok et al., 2005).Cytokinin O-glucosides are generally assumed to be storage products. While they lack activity per se, they can be activated through hydrolysis by β-glucosidases. Although conversion of zeatin to its O-glucoside by O-glucosyltransferases temporarily inactivates zeatin, this process also protects zeatin from cytokinin oxidases/dehydrogenases, which can degrade zeatin but not its O-glucoside (McGaw and Horgan, 1983; Armstrong, 1994). Complicating this picture is the distribution of these metabolites in the cell, resulting possibly in differential localization of substrates and enzymes. For instance, dihydrozeatin O-glucoside and the glucosides of a number of other plant growth regulators were found in the vacuoles (Garcia-Martinez et al., 1981; Schmitt and Sandermann, 1982; Bray and Zeevaart, 1985; Lehmann and Glund, 1986; Fusseder and Ziegler, 1988; Dean et al., 2003) whereas the maize β-glucosidase is targeted to plastids (Kristoffersen et al., 2000). Thus, while the high activity of O-glucosylzeatin in bioassays indicates that it can be converted to zeatin, the extent of this conversion in plants is unclear. It is possible that it is limited to certain tissues or particular stages of plant development or that it only occurs under specific conditions.Enzymes and genes involved in zeatin glycosylation and glucoside hydrolysis have been identified. The first zeatin O-glucosyltransferase (EC 2.4.1.203) was isolated from immature P. lunatus seeds (Dixon et al., 1989) while a variant of this enzyme, a zeatin O-xylosyltransferase (EC 2.4.2.40), was obtained from those of P. vulgaris (Turner et al., 1987). Subsequently, the genes encoding these enzymes, ZOG1 and ZOX1, were cloned (Martin et al., 1999a, b). In addition, two genes encoding O-glucosyltransferases with a preference for cis-zeatin were isolated from maize (Martin et al., 2001b; Veach et al., 2003). Other zeatin O-glucosyltransferase genes as well as two cytokinin N-glucosyltransferase (EC 2.4.1.118) genes were identified in the Arabidopsis genome (Hou et al., 2004). These enzymes all belong to Family 1 of the UDP-sugar requiring glycosyltransferases (http://www.cazy.org) and contain the signature sequence for this family in the C-proximal portion.An enzyme converting zeatin O-glucoside to zeatin was first identified in maize (Brzobohatý et al, 1993). This β-glucosidase (EC 3.2.1.21) has somewhat broader substrate specificity than the O-glucosyltransferases since it cleaves kinetin-N3-glucoside as well as some other substrates. The corresponding gene was cloned (Zm-p60.1) and found to be highly expressed in root meristems (Brzobohatý et al., 1993). A similar gene was isolated from Brassica napus (Falk and Rask, 1995).We are interested in the effects of over-expressing as well as repressing ZOG-type genes to determine the regulatory properties of O-glucosylation on plant development. The developmental modifications of maize transformants harbouring ZOG1 driven by the constitutive ubiquitin (Ubi) promoter is reported here. Our data indicate that zeatin O-glucosylation clearly affects root formation, leaf development, chlorophyll content, senescence, and male flower differentiation. Most interesting are the effects of the ZOG1 transgene on tassel development, with the formation of tasselseed in the homozygous transformants.Materials and methodsGeneration and selection of transgenic linesTransgenic Zea mays plants were obtained using an Agrobacterium-mediated transformation procedure of GS3×HC69 hybrid embryos as previously described (Zhao et al., 1998). The construct that was used contained the Ubi (ubiquitin) promoter (Christensen et al., 1992) upstream of the ZOG1 gene (accession no. AF101972) and a bialaphos resistance gene (Block et al., 1987) as the selectable marker. For further details on the construct see Fig. S1 in Supplementary data at JXB online. T0 plants were backcrossed four times to HC69.Five replications of four BC4 lines (P1, P3, P6, and P7) were planted from February through April and grown in the greenhouse at 25/20 °C (day/night) under natural light. Plants were identified as bialaphos-resistant or bialaphos-sensitive by painting part of the lower leaf with 200 mg l−1 bialaphos. For each replication, data were obtained from four plants per BC4 line.Crosses between sibs of the BC4 lines produced progeny equivalent to F2s. These progenies segregated at the expected 3:1 ratio of bialaphos-resistant to bialaphos-sensitive plants. Three repeats of two F2 lines (P3 and P7) with 30 plants per line were planted in early October to study the F2 generation. The photoperiod was gradually extended by using high-pressure sodium lamps (Hortilux LU1000B/Ht1/En) to simulate daylength conditions in spring and summer (April planting). Two additional 30 plant replications of P3 and P7 were planted in the spring and grown under natural light.Distribution of materialsNovel materials described in this publication may be available for non-commercial research purposes upon acceptance and signing of a material transfer agreement. In some cases such materials may contain or be derived from materials obtained from a third party. In such cases, distribution of material will be subject to the requisite permission from any third-party owners, licensors or controllers of all or parts of the material. Obtaining any permission will be the sole responsibility of the requestor. Plant germplasm and transgenic material will not be made available except at the discretion of the owner and then only in accordance with all applicable governmental regulations.PCRDNA was isolated from 100 mg tissue samples by sequential treatment with 500 μl extraction buffer (200 mM TRIS-HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), 500 μl saturated phenol, and 500 μl chloroform, followed by precipitation with 400 μl isopropanol. Internal primers for ZOG1, ZOG411F (5′-CATCTCAAATGTTGAA-AACTAC-3′) and ZOG930B (5′-CTTCACTTCCGGCAAAGATGTC-3′), were used for PCR. An initial denaturation cycle at 95 °C for 4 min was followed by 30 cycles of 95 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min. After an additional cycle at 72 °C for 10 min, samples were stored at 4 °C.RT-PCRTotal RNA was isolated from leaves, roots, and tassels at various stages using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Polysaccharide-rich samples were treated with 20% polyvinylpyrrolidine (PVP) and 8 M lithium chloride and incubated overnight at –20 °C. Synthesis of cDNA was achieved using the SuperScript™ II reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA product was subjected to semi-quantitative PCR using actin to normalize samples. Primers for actin PCR were mzACTIN-32F (5′-GTGACAATGGCACTGGAATG-3′) and mzACTIN-741B (5′-GACCTGACCATCAGGCATCTC-3′). The internal primers ZOG411F and ZOG930B were used to amplify ZOG1. The following conditions were used for PCR: initial denaturation at 94 °C for 4 min followed by 30 cycles of 94 °C for 1 min, 57 °C for 45 s, and 72 °C for 1 min. The last cycle was followed by incubation at 72 °C for 10 min.Western analysesProtein was extracted from leaves with buffer containing 55 mM TRIS, 50 μM EDTA, 5 mM DTT, pH 7.4 (800 μl extraction buffer for 200 mg leaf tissue) followed by 30–75% NH4SO4 fractionation. Samples were desalted and then purified by Blue Sepharose 6B affinity chromatography (Dixon et al., 1989). A fraction equivalent to 7 mg fresh leaf tissue was separated on a 12% SDS polyacrylamide gel and blotted to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA). Western blots were developed as described in Martin et al. (1990).Cytokinin analysesCytokinin levels were determined in roots and leaves of 28-d-old and in leaves of 110-d-old non-transformed and hemizygous plants. For extraction and purification of cytokinins as well as determination of cytokinin levels, the procedures described by Veach et al. (2003) were followed. The HPLC/MS system consisted of a HTS-Pal auto-sampler with a cooled sample stack (CTC Analytics, Zwingen, Switzerland), quaternary HPLC pump Rheos 2200 (Flux Instruments, Basel, Switzerland), Delta Chrom CTC 100 Column oven (Watrex, Prague, Czech Republic) and TSQ Quantum Ultra AM triple-quad high resolution mass spectrometer (Thermo Electron, San Jose, USA). For HPLC a Synergi Hydro column (Phenomenex, Torrance, USA) was used. Data were obtained from three samples per genotype, with two HPLC injections and analyses per sample.Phenotypic characterizationsPhenotypic characterization of transformed lines included measurements on vegetative growth rate, plant height, root length, chlorophyll content, leaf width, stomatal distribution, meristem structure, development of reproductive organs (time of appearance, size), pollen viability, and kernel development.Foliar data presented were taken from the 10th leaf, which was found to be representative of the whole plant by measuring all leaves from fewer plants (data not shown). Foliar chlorophyll levels were monitored with a chlorophyll content meter CCM-200 (Optisciences Inc., Tyngsboro, MA, USA) over a 175-day period. Four measurement points were taken from each leaf. To convert these measurements to chlorophyll units per leaf fresh weight, chlorophyll was extracted by incubating leaf tissues in N,N′-dimethylformamide (7% leaf weight/solvent volume) at 4 °C for 48 h, as described in Inskeep and Bloom (1985). Sample absorbances were measured at 647 nm and 664.5 nm (maximum for chlorophyll b and a, respectively) and absolute chlorophyll contents calculated according to the equation:Optimeter CCI units were then transformed into μg of chlorophyll per mg fresh weight with restriction curves. Random leaf samples were examined with a light microscope to determine stomatal cell distribution patterns.Apical meristem samples were collected from 30-d-old plants, fixed in FAA (50% ethanol, 10% acetic acid, 5% formalin, by vol.), dehydrated in a graded ethanol series, and embedded in Technovit 7100 plastic (Heraeus Kulzer, Wehrheim, Germany). Sections were cut on an AO 820 rotary microtome to obtain 46-μm-thick serial sections. Samples were stained with Toluidine Blue-O, and analysed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).Pollen viability was tested prior to pollination assays. Fresh pollen grains were collected at 09.00 h and kept for 2 h on liquid germination media containing (l−1) 236.2 mg Ca(NO3)2, 24.8 mg H3BO3, and 30% (w/v) of polyethyleneglycol (PEG). Silk filaments from the female flower were added to enhance pollen tube growth. A pollen cryo-preservation protocol was optimized to ensure a constant supply of viable pollen during pollination assays. Pollen grains were collected, dried at 20 °C and 20–40% humidity for 2 h and shock-frozen by immersion in liquid nitrogen as described by Barnabas (1994). A saturated MgCl2 solution was used to stabilize the humidity in the desiccator. Prior to use, pollen was thawed in a water bath at 40 °C for 2 min. The combination of fresh and cryopreserved pollen allowed uniform pollination coverage during the receptive period of the maize ear.The experimental design for all phenotypic traits was fully randomized. Data were analysed statistically by multivariate analysis of variance (MANOVA) with repeat, line, and type as the main sources of variation, followed by a Tukey test at 5% significance level. The analyses aimed to identify the interactions among the independent variables as well as their effect on the response variables. For those traits where no relevant variable interactions were observed, two-way and one-way ANOVA were performed.ResultsTransgenic Ubi:ZOG1 maize lines were generated using Agrobacterium-mediated transformation. Bialaphos resistance was the selectable marker and initial transformants were backcrossed to the parental inbred HC69 for four generations to generate transformants with genetic backgrounds similar to this inbred. Four BC4 lines (P1, P3, P6, and P7), derived from independent transformation events and containing a single insert each, were selected for further study. The BC4 lines segregated into bialaphos-resistant hemizygous (P+) and bialaphos-sensitive non-transformed (NT) plants (1:1). To obtain homozygous transformants (P++), hemizygous BC4 plants were selfed. For simplicity, the progeny obtained after selfing the hemizygous BC4 is referred to as F2.Bialaphos-resistant plants transcribe and translate ZOG1RT-PCR (Fig. 1a) and western (Fig. 1b) blots of BC4 plants indicated that transcription and translation of Ubi:ZOG1 occurs in transgenic maize tissues. Tissue samples from bialaphos-resistant plants of all four lines produced clear RT-PCR and protein bands, but those from bialaphos-sensitive plants did not.Fig. 1.Ubi::ZOG1 expression. (a) RT-PCR products with ZOG1-specific primers and actin as a control showing expression of the transgene in roots, leaves, and tassels. P1+, P3+, P6+, and P7+ are independent hemizygous lines. NT is a non-transformed control. Plasmid I-1124 contains the Ubi::ZOG1 insert. (b) Western blot of ZOG1 product in leaves (4 mg equivalent) from hemizygous transformants and non-transformed sibs, developed with monoclonal antibodies specific to ZOG1.Over-expression of ZOG1 leads to large increases in zeatin O-glucosideCytokinins were analysed in roots and leaves of young plants and in leaves of mature plants (Fig. 2). As expected, the zeatin O-glucoside levels were increased significantly in the hemizygous transformants: 100-fold in roots, and 38-fold and 88-fold higher in leaves of young and older plants, respectively (Fig. 2a, d, g). By contrast, the cis-zeatin O-glucoside levels differed only up to 2-fold and glucosylation of ribosides was low. These levels are both in agreement with the substrate preference of the ZOG1 enzyme (Martin et al., 1999a). Overall, O-glucosides constituted the main pool of cytokinin metabolites, even in the non-transformed plants. The levels of N-glucosides were negligible compared with those of O-glucosides (data not shown).Fig. 2.Cytokinin concentrations in roots and leaves. (a, b, c) Cytokinin concentrations in roots of 28-d-old non-transformed (NT) and hemizygous Ubi::ZOG1 (+) maize plants. (d, e, f) Cytokinin concentrations in leaves of 28-d-old maize plants. (g, h, i) Cytokinin concentrations in leaves of 95-d-old maize plants. (a, d, g) O-glucosides; (b, e, h) free bases; (c, f, i) ribosides and nucleotides. Z, trans-zeatin; cZ, cis-zeatin; iP, N6-(Δ2-ispentenyl)adenine; ZR, trans-zeatin riboside; cZR, cis-zeatin riboside; iPR, N6-(Δ2-ispentenyl)adenosine; ZOG, trans-zeatin O-glucoside; cZOG, cis-zeatin O-glucoside. Values are means (±SE) of three samples with two HPLC analyses for each. Significant differences between transformants and controls are indicated by *P <0.05, **P <0.01, or ***P <0.001.In roots, levels of free bases were extremely low, both in transformed and control plants (Fig. 2b). Root riboside levels were higher than free bases but did not differ between transformants and controls (Fig. 2c). In leaves of both young and old plants, the levels of zeatin were higher in transformants than in controls (Fig. 2e, h). Although this increase in zeatin could be accounted for by hydrolysis of only 1% of the zeatin O-glucosides in transformants during storage and extraction, the ribosides and nucleotides were also elevated in transformants (Fig. 2f, i), which could not stem from glycoside hydrolysis. The levels of dihydrozeatin and its derivatives were generally below detection levels. Altogether, the data indicate that the levels of active cytokinins (free bases and ribosides) are slightly lower in transformed roots but higher in transformed leaves.Increased cytokinin conjugation leads to pronounced changes in maize plant architecture and leaf widthThe hemizygous Ubi:ZOG1 BC4 plants were morphologically distinguishable from non-transformed sibs. They grew slower, were shorter, and had narrower leaves. There were three types of F2 plants: bialaphos-sensitive non-transformed plants, plants resembling the hemizygous BC4 transformants, and a class of extremely small plants (Fig. 3a). The segregation ratios were 16:36:10 and 15:27:22 in the P3 and P7 lines, respectively, fitting a 1:2:1 ratio and thus supporting the hypothesis that the very small plants were homozygous for the transgene. There were significantly fewer leaves on the hemizygous transformants than on the non-transformed plants and even fewer leaves on the homozygous transformants (Fig. 3b). At the time of tassel emergence, plant height was reduced by 22–41% in the hemizygous and by 48–60% in the homozygous Ubi:ZOG1 plants compared with non-transformed plants (Fig. 3c). There was a greater reduction in plant height than in the number of leaves, indicating that internodes were shortened in response to transgene activity. Stems were slender in hemizygous and very slender in homozygous Ubi:ZOG1 plants. The leaves of hemizygous plants were significantly narrower than those of non-transformed sibs (Fig. 3d, e), but their length was the same (data not shown). The leaves of homozygous transformants were extremely narrow, even at plant maturity (Fig. 3e), and also shorter than control and hemizygous plants. The thinner stems and leaves of transformants suggested smaller meristematic regions and microtome sections confirmed that hemizygous and homozygous meristems were indeed significantly smaller (Fig. 3f-i). These characteristics were not influenced by environmental fluctuations (light intensity or day length).Fig. 3.Vegetative development. (a) Non-transformed (left), hemizygous (centre) and homozygous (right) 97-d-old plants. (b) Number of leaves over time. P+ and P++ represent hemizygous and homozygous lines, respectively. (c) Plant height at tassel emergence. (d) Non-transformed (top), hemizygous (centre) and homozygous (bottom) leaves. (e) Average width of the tenth leaf. (f) Shoot meristem size of 40-d-old plants. (g) Non-transformed meristem. (h) Hemizygous meristem. (i) Homozygous meristem. Values are means (±SE), obtained from two experiments with a total of 60 plants each. Values for (f) are means (±SD) of 10 meristems.Chlorophyll formation and distribution of stomates are altered in transformantsDifferences in foliar chlorophyll content were noticeable as early as 23 d after germination. The transformed plants were darker green than the non-transformed ones. Chlorophyll levels in transformants were elevated throughout their life span and ultimately resulted in the delay of senescence which kept transformed plants green weeks after control sibs had senesced (Fig. 4a). This is interesting since increased chlorophyll formation and delay of senescence are traits usually associated with an increase in cytokinins (Richmond and Lang, 1957; Gan and Amasino, 1995) whereas the plant architecture of Ubi::ZOG1 transformants is generally indicative of a decrease in active cytokinins (see Discussion). However, these findings are supported by the cytokinin analyses, which showed increases in active cytokinins in transformant leaves (Fig. 2). The delay of senescence was influenced by the environment since it occurred in the summer but not in the winter under artificial light (Fig. 4b), even though the daylength was adjusted to mimic summer conditions. It is most likely that the difference in light intensity and/or quality was a major factor in this seasonal effect.Fig. 4.Leaf characteristics. (a, b) Chlorophyll levels in the 10th leaf through senescence. Plants grown in the spring/summer under natural long-day conditions (a) and in the autumn under artificial lighting (b). (c) Paired chain of stomata from a non-transformed leaf. (d) Single chain of stomata from Ubi::ZOG1 leaf. (e) Number of paired stomata mm−2 (f) Total number of stomata mm−2. Values for (a) and (b) are means (±SE) of two experiments with four plants per line and six measurements per leaf. Values for (e) and (f) are means (±SD) of cross-sections of 20 random mm2 fields.Closer examination of the leaves revealed that the number of major veins was the same in non-transformed plants and hemizygous transformants but that the distance between the veins was smaller in the latter. On normal maize leaves, the stomata usually occur in rows, both in double files with stomata at alternate positions (Fig. 4c; Hernandez et al., 1999) and single files (Fig. 4d). Transformants had fewer double file stomata (Fig. 4e) but more single files. The overall density of the stomata in the leaves did not differ between the two groups (Fig. 4f).Root mass and branching is increased in Ubi:ZOG1 transformantsRoot morphology differed between non-transformed plants and hemizygous transformants. Ubi:ZOG1 roots were thicker, more branched (Fig. 5a) and longer (Fig. 5b). As a result, the total root weight was much greater in the transformants (Fig. 5c).Fig. 5.Root development. (a) Non-transformed (two at left) and hemizygous (two at right) roots 3 weeks after planting. (b) Root fresh weight. (c) Root length. Values for (b) and (c) are means (±SD) of 10 plants.Ubi:ZOG1 causes reduction of tassel size and feminization of floretsHemizygous Ubi:ZOG1 plants showed delayed tassel initiation (Fig. 6a) and, more strikingly, a drastic reduction in tassel size, branching, and spikelet production compared with the non-transformed control (Fig. 6b, c, d). The weight of transformed tassels was about 75% less than that of non-transformed tassels (Fig. 6b), due to the decreased number and smaller size of branches. Moreover, the tassels were abnormal. Normal maize tassels have many florets up to the tip of each branch (Fig. 6e), but the hemizygous Ubi:ZOG1 tassels had functional spikelets only at the lower end of the tassels while the tips of the branches were devoid of most floral structures (Fig. 6f). Sterile spikelets consisted of two external glumes with no florets, lemma or stamens. The normal florets underwent anthesis and produced functional pollen grains as demonstrated by the ability of the pollen to germinate (Fig. 6g). Pollen viability was further supported by the roughly equal numbers of transformed and untransformed kernels in the BC4 populations, which were generated using BC3 transformants as male parents.Fig. 6.Tassel development. (a) Age of plant at tassel emergence. (b) Tassel fresh weight at maturity. (c) Non-transformed tassel. (d) Hemizygous Ubi::ZOG1 tassel. (e) Control lateral rachis with fully developed fertile spikelets. (f) Hemizygous lateral rachis with empty spikelets. (g) Viable Ubi::ZOG1 pollen in liquid germination medium. (h) Homozygous tassels with various degrees of floret feminization. Values for (a) and (b) are means (±SE) of five experiments with four plants each.Homozygous F2 tassels were phenotypically even more extreme, showing a dosage effect of the ZOG1 gene. They were very small, had no or very few branches, and most interestingly, showed various degrees of floret feminization (Fig. 6h). Fertilization of these female florets resulted in formation of complete kernels (Fig. 6h). The feminization varied in the homozygous population, from a few female florets and seeds to almost complete silking of the tassel (Fig. 6h, left to right). Normal tassel floret development is initially bisexual, after which florets become unisexual through gynoecium abortion (Cheng and Pareddy, 1994). This programmed gynoecium abortion was inhibited by ZOG1 over-expression in some of the lower florets since fertile ovules and elongated silk were formed. The few male florets on the apical portion of the homozygous tassels yielded a small amount of viable pollen. The phenotype varied depending on the light source during plant growth. Plants grown in the winter under artificial light always showed some degree of feminization while plants grown in the spring and summer under ambient light conditions had only a few tasselseeds.Ubi:ZOG1 reduces seed weightEar development on hemizygous Ubi:ZOG1 plants lagged behind that of non-transformed plants but the ears looked normal (Fig. 7a). Hemizygous ears had only partial seed set, in contrast to fully filled non-transformed ears under the same greenhouse conditions; however, when the ears were artificially pollinated, the hemizygous ears were also completely filled, indicating that pollen availability (not viability) was the limiting factor. When the Ubi:ZOG1 plants were used as the male parent, plants produced kernels with a lower average weight than those pollinated with non-transformed pollen (Fig. 7b). To confirm this initial observation, hemizygous plants were used to pollinate non-transformed plants and the resulting kernels were weighed and classified as transgene positive or negative (using PCR with ZOG1-specific primers). The data indicate that kernels on the same ear resulting from pollination with Ubi:ZOG1 pollen grains were significantly smaller than those resulting from pollination with non-transformed pollen (Fig. 7c).Fig. 7.Ear and kernel development. (a) Non-transformed (left) and hemizygous (right) ears from plants of the same age before pollination. (b) Weight of kernels from non-transformed ears pollinated with pollen from non-transformed (NT) or hemizygous Ubi::ZOG1 (P+) plants. (c) Weight of kernels from non-transformed ears pollinated with pollen from hemizygous transformants. Kernels were taken from the same ear and classified as P–×P– or P–×P+ by PCR with ZOG1-specific primers. Values for (b) are means (±SE) of all kernels of four ears. Values for (c) are means (±SD) of 15±2 kernels.DiscussionRelevance of cytokinin determinationsConstitutive over-expression of ZOG1 resulted in elevated levels of O-glucosides, as expected. There was, however, no concomitant decrease in active cytokinins (bases and ribosides) in roots and an increase in overall active cytokinins in leaves. This indicates that the initial decrease in zeatin led to an increase in cytokinin biosynthesis. It should be noted though that cytokinin analyses can only determine the cytokinin composition in a large amount of tissue, whereas the distribution of cytokinins within specific tissues and cells may be most critical. As a consequence of ZOG1 over-expression, there are probably changes in the inter- and intracellular distribution of various active cytokinins and their metabolites due to differential expression of cytokinin biosynthetic genes. Thus it is possible that cytokinins are elevated at or near biosynthetic sites but not at cytokinin receptor sites.Root development and plant architecture of Ubi:ZOG1 transformants are characteristic of cytokinin deficiencyRoot development was enhanced in the Ubi:ZOG1 maize plants similar to that observed in Arabidopsis and Lotus japonicus transformed with maize cytokinin oxidase/dehydrogenase genes (Werner et al., 2001, 2003; Lohar et al., 2004; Kopečný et al., 2006). This suggests that the decrease in physiologically active cytokinins caused by increased glucosylation stimulated initiation and growth of root tips. Both the increased number of root tips and possible increased cytokinin biosynthesis may have led to the adjustment in free cytokinins to levels of non-transformed maize.While root development was stimulated by the presence of the transgene, the opposite was the case with shoot development. The hemizygous Ubi:ZOG1 transformants were shorter and less robust than the controls and this size reduction was more severe in the homozygous plants, indicating dosage effects of the transgene. The phenotypes were similar to those of maize plants having a CKX transgene (N Brugière, unpublished results). Slower growth rates and reduced plant stature were also observed in dicots having decreased cytokinin levels or reduced cytokinin sensitivity due to increased expression of oxidases/dehydrogenases (Werner et al., 2003; Kopécný et al., 2006), deficiencies in biosynthesis (Miyawaki et al., 2006), or mutations in receptor genes (Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006).Observations regarding cytokinin effects on monocot leaf development are limited. Classical experiments involving exogenous cytokinin applications conducted with dicots demonstrated that cytokinins stimulated leaf expansion (Kuraishi and Okumura, 1956). However, transformants and mutants of Arabidopsis with either reduced cytokinin levels or deficient in cytokinin perception have smaller leaves, but the ratio between the length and width remains constant (Riefler et al., 2006; Werner et al., 2001, 2003). The leaf width of Ubi:ZOG1 hemizygous maize plants was significantly reduced by over-expression of ZOG1, while both the leaf width and length were decreased in Ubi:ZOG1 homozygous plants. Maize genetic mutants with smaller leaves usually have smaller meristems (Scanlon et al., 1996). This is also the case with the Ubi:ZOG1 transformants. The meristems of the homozygous Ubi:ZOG1 plants were slightly smaller than those of the hemizygous Ubi:ZOG1 plants which were smaller than those of the non-transformed control plants. The meristems of Arabidopsis plants over-expressing cytokinin oxidase/dehydrogenase genes were also smaller those of control plants (Werner et al., 2003; Kopečný et al., 2006). Another notable difference between the Ubi:ZOG1 transformants and control plants was the arrangement of leaf stomata. There were more single file stomata and fewer double file stomata in the transformants, which may be related to the reduced number of cell files between veins.Chlorophyll formation and leaf senescence reflect higher levels of active cytokininsChlorophyll levels were increased and senescence was delayed in Ubi:ZOG1 transformant leaves. These characteristics are indicative of an increase in active cytokinins since exogenous cytokinins and up-regulation of cytokinin biosynthesis produce similar phenotypic changes (Richmond and Lang, 1957; Smart et al., 1991; Li et al., 1992; Gan and Amasino, 1995; Robson et al., 2004). The cytokinin analyses confirmed that active cytokinins (free bases and ribosides) are increased in leaves. Although it is possible that accumulation of extremely high amounts of O-glucosides can eventually lead to increased free bases due to the action of β-glucosidases, higher chlorophyll and delayed senescence were also reported for tobacco over-expressing the AtCKX2 gene of Arabidopsis (Mýtnová et al., 2006). A factor common to both the transgenic maize and tobacco is the generally slower development due to cytokinin O-glucosylation and degradation, respectively, thus prolonging the growth period. However, if this slower development is delaying maturation to a significant extent, time of flowering would also be greatly delayed. This was not the case since tassels emerged only about 2 d later in transformants than in untransformed controls, whereas leaf senescence showed a much longer delay (Fig. 4a). Both the ZOG1 and AtCKX2 leaves may respond to the resulting cytokinin deficiency by increasing cytokinin biosynthesis. Since biosynthesis takes place in the plastids (Kasahara et al., 2004; Sakakibara, 2006), there may be a localized increase of cytokinins in plastids. Whether higher cytokinin levels in chloroplasts could bring about an increase in chlorophyll is an interesting question. It would assume direct action of cytokinins in chloroplasts, which has never been demonstrated.Indirect effects contributing to the higher chlorophyll in the transformed maize leaves can not be excluded. Non-transformed maize leaves expand very rapidly under natural summer conditions while those of the transformants remain much narrower, which could result in differential chlorophyll accumulation. Furthermore, the ratio between root and shoot growth is much higher in the transformants, possibly leading to higher accumulation of nutrients (including N) in the leaves. If this is the case, the much higher cytokinin levels in the mature transformant leaves may not be the cause, but rather a consequence, of the delayed senescence.Increased cytokinin conjugation leads to defective tassel development and feminization of the lower floretsThe drastic reduction in tassel size of the Ubi:ZOG1 transformants points to the importance of cytokinins in maize reproductive development. Normal tassels have 10–50 lateral branches which contain paired spikelets (Cheng and Pereddy, 1994). By contrast, the hemizygous Ubi:ZOG1 transformants had fewer and shorter lateral branches. Terminal florets were often missing. Maize plants transformed with the CKX1 gene driven by a pollen-specific promoter also had smaller tassels even though CKX1 expression was targeted to the pollen (Huang et al., 2003). A rice cultivar with reduced expression of a cytokinin oxidase/dehydrogenase gene and increased cytokinins had more reproductive organs (Ashikari et al., 2005). Although reproductive programmes of maize and rice are different in a number of aspects, these studies suggest that cytokinins have a positive effect on terminal flower development, most probably through its effects on meristem size.Most intriguing was the occurrence of female florets at the lower end of the homozygous Ubi:ZOG1 tassels which formed seed when pollinated. Maize florets begin as complete bisexual flowers containing pistil and anther initials, but later the pistils abort (Cheng and Pareddy, 1994). The process of pistil abortion must have been inhibited in the homozygous Ubi:ZOG1 plants. Tasselseeds were found on all four transformed lines examined indicating that they were not a consequence of accidental insertion in one of the tasselseed (TS) loci but rather as the result of altered cytokinin levels or composition. Tasselseed in maize is a known phenomenon and several genetic mutations causing feminization of the tassel have been described (Irish and Nelson, 1989; Dellaporta and Calderon-Urrea, 1994). The most prominent phenotypes occur in recessive mutants ts1/ts1 and ts2/ts2, which display complete reversion from male to female inflorescences, with the failure of pistil abortion and the induction of stamen abortion (Emerson, 1920; Irish et al., 1994; DeLong et al., 1993). Somewhat less extreme is the dominant Ts5 mutant, which shows positional effects, with female florets occurring at the basal portion of the tassel (Nickerson and Dale, 1955), similar to the Ubi:ZOG1 transformants. The ts4/ts4 and ts6/ts6 mutants also show partial reversions (Dellaporta and Calderon-Urea, 1994). The TS2 gene was cloned and found to have homology to short chain alcohol dehydrogenases (DeLong et al., 1993). The maize transformants described here establish the first link between tasselseed and cytokinins.Previously, cytokinins have been implicated in sex expression of a number of plants (Durand and Durand, 1994), but to our knowledge they have not been researched in connection with sex expression in maize tassels. The occurrence of tasselseed on the homozygous Ubi:ZOG1 transformants indicates that increased zeatin O-glucosylation and the associated disturbance in cytokinin homeostasis result in feminization. Whether this is due to a decrease in active cytokinins (as shown by the retarded shoot development and smaller meristem size) is difficult to assess. However, tasselseed formation was also observed on maize transformants over-expressing a cytokinin oxidase/dehydrogenase gene under the control of the Ubi promoter (N Brugière, unpublished results). In most plant species (although primarily dicots), cytokinins are feminizing, but exceptions are known (Durand and Durand, 1994). Interestingly, only the lower inflorescences showed this feminization whereas the more apical florets had the usual male characteristics. Thus there may be a gradient of active cytokinins or some polarity in signal distribution, causing female flowers to develop at the basal end of the tassel.A possible alternative explanation for the tasselseed characteristic could reside in the changed composition of cytokinins. Not much is known about the influence of particular cytokinin metabolites on sex expression. In the most extensively studied system, Mercurialis, where a number of genes control sex expression, specific cytokinins have been linked with sex differentiation and male sterility. For instance, occurrence of trans-zeatin in apices was correlated with femaleness, while its riboside and nucleotide were more abundant in males (Durand and Durand, 1994). The presence of cis-zeatin and its riboside were associated with male sterility. Thus the changes in the ratios between cis and trans isomers in the Ubi::ZOG1 homozygotes, as a result of the preference of ZOG1 for trans-zeatin, may also be contributing to the abnormal tassel phenotypes. However, this is difficult to assess since any changes in cytokinin levels and composition could be the cause, but also merely the consequence, of altered development.The influence of light intensity or quality on the tasselseed trait in Ubi:ZOG1 transformants indicates an interaction between the cytokinin and light signalling pathways. Such interactions have been previously observed. For instance, exogenous cytokinin caused de-etiolation of dark-grown Arabidopsis seedlings (Chory et al., 1994). Furthermore, Arabidopsis transformants and mutants with altered expression of the cytokinin type A response regulator ARR4 showed altered red light sensitivity (Sweere et al., 2001; To et al., 2004).Pollen grains of hemizygous Ubi:ZOG1 plants appeared normal and germinated on artificial medium. Even the few anthers on homozygous transformants contained normal pollen. Also Arabidopsis transformants with constitutive over-expression of CKX genes produced fewer but still functional pollen grains (Werner et al., 2003); however, maize plants over-expressing CKX1 via a pollen-specific promoter (pZtap) showed pollen sterility (Huang et al., 2003).Female inflorescence development is normal but seeds are smaller in Ubi:ZOG1 plantsThe female inflorescences are not altered in Ubi:ZOG1 maize plants indicating that the changes in cytokinin levels have stronger effects on male flower development. Also, no obvious abnormalities in the flowers were observed in transgenic tobacco over-expressing ZOG1 (Martin et al., 2001a) even though tobacco flowers are derived from the terminal bud. Flower abnormalities were also absent from Arabidopsis plants constitutively over-expressing CKX genes (Werner et al., 2003). However, when cytokinin levels were increased through senescence-induced expression of the Agrobacterium IPT gene, the pistil was retained in the lower floret and fused kernels were formed (Young et al., 2004).Until recently, very few studies have addressed the effects of cytokinin on seed size. Transgenic Arabidopsis with decreased cytokinin levels or sensitivity produced fewer but larger seeds (Werner et al., 2003; Kopečný et al., 2006; Riefler et al., 2006). However, the maize seeds with increased cytokinin conjugation were smaller than non-transformed seeds from the same ears when control plants were pollinated with pollen from hemizygous Ubi:ZOG1 transformants. It should be noted though that the increased seed size in Arabidopsis was associated with a decrease in the number of seeds, while seed fill of the maize cobs was complete. Moreover, maize and Arabidopsis seed development can not be compared due to the fact that maize kernels are largely endosperm while Arabidopsis seeds consist mainly of embryos.Implications of high levels of cis isomersIn the present study, the predominant cytokinin in non-transformed maize leaves was the O-glucoside of cis-zeatin. In a previous study, cis-zeatin and its derivatives were found to be the major components in maize roots and stems (Veach et al., 2003). Levels of cis isomers were also very high in kernels although lower than the trans counterparts (Veach et al., 2003). Maize has a cytokinin glucosyltransferase with a preference for cis-zeatin over trans-zeatin (Martin et al., 2001b). In addition, a maize cytokinin receptor, ZmHK1, with high affinity for cis-zeatin and trans-zeatin has been identified (Yonekura-Sakakibara et al., 2004). These findings suggest that cis-zeatin is an active cytokinin in tissues where ZmHK1 is expressed.A related issue is the possible origin of cis-zeatin. The presence of a hydroxylase for the synthesis of cis-zeatin, similar to the hydroxylase found in Arabidopsis for the formation of trans-zeatin (Takei et al., 2004), is a distinct possibility. Recent findings in Arabidopsis (Miyawaki et al., 2006) favoured tRNA degradation rather than de novo synthesis as the source of cis-zeatin. Maize may be different from Arabidopsis in that the cis-zeatin level is too high to be accounted for solely by tRNA breakdown. In addition, the presence in maize of a receptor responsive to cis-zeatin and a cis-specific glucosyltransferase indicates species-specific pathways, supporting direct synthesis of cis-zeatin in monocots.ConclusionsThe changes in plant architecture associated with ZOG1 over-expression are consistent with the reduction of active cytokinins, but chlorophyll levels and retention seem to reflect increases in cytokinins, as supported also by the cytokinin analyses. The most interesting effect of ZOG1 over-expression was the feminization of tassel floret development. This novel observation provides a link between cytokinins and sex-specific floral development in maize.Supplementary dataA supplementary figure with specifics of the construct used for transformation is available at JXB online.We thank K Cook for her assistance with microtome sectioning. We thank M Rossman and C Pereira for their help with the statistical analyses. 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Differential ligand preferences and response to cis-zeatinPlant Physiology200413416541661YoungTEGiesler-LeeJGallieDRSenescence-induced expression of cytokinin reverses pistil abortion during maize flower developmentThe Plant Journal200438910922ZhaoZGuWCaiTTaglianiLAHondredDBondDKrellSRudertMLBruceWBPierceDAMolecular analysis of T0 plants transformed by Agrobacterium and comparison of Agrobacterium-mediated transformation with bombardment transformation in maizeMaize Genetics Cooperative Newsletter1998723437 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B5F8CAA2C191B84BE158AB0DAA18E6A754871DF.txt b/test/dataset/in/resources/corpus/Clean_0B5F8CAA2C191B84BE158AB0DAA18E6A754871DF.txt new file mode 100644 index 0000000..2eae479 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B5F8CAA2C191B84BE158AB0DAA18E6A754871DF.txt @@ -0,0 +1 @@ +amjepidajeAmerican Journal of Epidemiology1476-62560002-9262Oxford University Press10.1093/aje/kwq044ORIGINAL CONTRIBUTIONSTrajectories of Neighborhood Poverty and Associations With Subclinical Atherosclerosis and Associated Risk FactorsThe Multi-Ethnic Study of AtherosclerosisMurrayEmily T.*Diez RouxAna V.CarnethonMercedesLutseyPamela L.NiHanyuO'MearaEllen S.*Correspondence to Dr. Emily T. Murray, Laboratory of Epidemiology, Demography, and Biometry, Gateway Building, 3C309, 7201 Wisconsin Avenue, Bethesda, MD 20814 (e-mail: emily.lemelin@nih.gov).15520102742010171101099110827720091522010American Journal of Epidemiology © The Author 2010. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2010The authors used data from the Multi-Ethnic Study of Atherosclerosis and latent trajectory class modeling to determine patterns of neighborhood poverty over 20 years (1980–2000 residential history questionnaires were geocoded and linked to US Census data). Using these patterns, the authors examined 1) whether trajectories of neighborhood poverty were associated with differences in the amount of subclinical atherosclerosis (common carotid intimal-media thickness) and 2) associated risk factors (body mass index, hypertension, diabetes, current smoking) at baseline (January 2000–August 2002). The authors found evidence of 5 stable trajectory groups with differing levels of neighborhood poverty (∼6%, 12%, 20%, 30%, and 45%) and 1 group with 29% poverty in 1980 and approximately 11% in 2000. Mostly for women, higher cumulative neighborhood poverty was generally significantly associated with worse cardiovascular outcomes. Trends generally persisted after adjustment for adulthood socioeconomic position and race/ethnicity, although they were no longer statistically significant. Among women who had moved during the 20 years, the long-term measure had stronger associations with outcomes (except smoking) than a single, contemporaneous measure. Results indicate that cumulative 20-year exposure to neighborhood poverty is associated with greater cardiovascular risk for women. In residentially mobile populations, single-point-in-time measures underestimate long-term effects.body mass indexcarotid artery, internaldiabetes mellitushypertensionmodels, statisticalresidential mobilityretrospective studiessmokingA number of studies have reported associations of neighborhood socioeconomic characteristics with cardiovascular-related outcomes that persist after statistical adjustment for individual characteristics. For example, neighborhood disadvantage has been linked to coronary heart disease prevalence, incidence, and mortality (1–6); subclinical coronary heart disease (7); and cardiovascular risk factors (3, 4, 8–11). However, almost all of these studies are based on the measurement of neighborhood characteristics at a single point in time.Very few studies have documented what trajectories of neighborhood socioeconomics people experience in adulthood and how they are related to health outcomes (including cardiovascular outcomes) later in life. Because atherosclerosis develops over long periods, long-term neighborhood exposures may be more relevant to investigate than single-point-in-time exposures. Prior work has researched cumulative effects of neighborhood exposures over the life course by summing or averaging neighborhood exposures over a specified period of time (12, 13) or has estimated “independent” effects of exposures for different life epochs.If all persons in a population stay in the same neighborhood or move between neighborhoods of a similar socioeconomic level, then an average measure and any single measure for the specified time period would show the same association with the outcome. However, if the accumulation of neighborhood effects over the life course is affecting cardiovascular disease risk, then the use of a single-point-in-time measure may result in misestimates of area effects. Additionally, socioeconomic mobility may itself have an independent effect on cardiovascular disease health (14, 15). To test whether this is the case, cardiovascular disease health outcomes need to be compared in populations of upward- or downward-moving neighborhood poverty with what would be expected under a purely cumulative model. Prior approaches do not allow for these comparisons.We used data from the Multi-Ethnic Study of Atherosclerosis (MESA), and latent trajectory class modeling (16, 17), to determine patterns of neighborhood poverty over a 20 year period during mid- to late adulthood. We then examined how these patterns are related to the amount of subclinical atherosclerosis as well as associated risk factors later in life. We also assessed effects of mobility by comparing cardiovascular outcomes for groups with decreased (upwardly mobile) neighborhood census tract poverty over time with groups with equivalent stable cumulative poverty. In addition, we investigated the added advantage of using a cumulative measure of neighborhood poverty compared with a single measure in adulthood.MATERIALS AND METHODSStudy populationMESA was initiated to investigate the prevalence, correlates, and progression of subclinical cardiovascular disease. Participants were 45–84 years of age, from 6 US communities (Baltimore, Maryland; Chicago, Illinois; Forsyth County, North Carolina; Los Angeles County, California; northern Manhattan, New York; and St. Paul, Minnesota), clinically free of cardiovascular disease at baseline, and sampled to be ethnically diverse (∼38% white, 28% African American, 23% Hispanic, and 11% Chinese). The methods used for sampling and study design have been reported elsewhere (18).Assessment of neighborhoodsDuring one study clinic visit, participants were asked by trained interviewers to complete a 20-year residential history questionnaire (January 1980–date of visit). Each address was geocoded and assigned latitude and longitude coordinates. Census tract codes from the 1980, 1990, and 2000 US Censuses were coded to each address. For intercensal years, we interpolated the value for that year based on the 2 closest censuses. The percentage of residents living below the poverty level (i.e., neighborhood census tract poverty) for each location was obtained from the Neighborhood Change Database, which enables comparison across various census years by recalculating and normalizing past census years to 2000 US Census tract boundaries (19).We selected neighborhood census tract poverty as our primary key neighborhood variable for the following reasons: it is often used in sociologic work to characterize neighborhood conditions (20), it was measured in a standardized manner for all the US Censuses relevant to the period of study, and its definition is modified over time by the US Census to account for changes in the cost of living. Using this approach, we created a database that contains a measure of census tract poverty for each month between January 1980 and the date of the MESA baseline examination for each study participant. Only participants with complete address history information for the entire 20 years were included in the analyses.Measures of long-term patterns of neighborhood povertyTrajectory classes of neighborhood poverty over the 20 years prior to the MESA examination were identified by using hierarchical latent growth curve modeling. Individuals were assigned to trajectory classes based on their pattern of repeated measurements of annual average neighborhood poverty for each year between 1980 and 1999 (16, 17). The data trajectory for each subject consisted of repeated measurements of neighborhood census tract poverty over T time periods, Yi = (Yi1, …, YiT), that were independent given the group Ci. The trajectory was modeled aswhere μijk represents the ith response of person j in group K, β0jk is the estimated mean neighborhood poverty value for group k, where Yearij = 0 (January 1980) and Yearij denotes the number of years since January 1980 at subject j’s time i. Up to a third-order polynomial in year was included to flexibly model the effects of time. K numbers of groups were assigned so that the likelihood of observing the data trajectory for subject j, given that he or she belonged to group K, was estimated by using a censored normal model.We investigated models for 2–8 trajectory groups. The number of groups selected was based on which model as a whole had the best overall Bayesian Information Criterion (BIC) value. Group membership for a given person was assigned based on which K group had the highest likelihood of observing the data trajectory observed, given that he or she belonged to group k.In addition to categories based on trajectories of neighborhood poverty, we also investigated a measure of neighborhood percent poverty at the time of the baseline examination (referred to as the contemporaneous measure) as well as an average measure for the whole 20-year period. The average measure was the area under a continuous line fitted to each participant's data, divided by the number of months from January 1980 to the date of the baseline examination (January 2000–August 2002) (mean = 0.14, range = 0.01–0.71).Outcome variablesCommon carotid intimal-media thickness (IMT) was the measure of subclinical atherosclerosis examined. IMT is a relatively simple, inexpensive, precise, reproducible, and valid measure of early atherosclerotic changes in the carotid artery. IMT has been shown to be associated with coronary heart disease risk factors, prevalent coronary heart disease, and subsequent coronary heart disease events (21, 22). Common carotid IMT was measured noninvasively with high-resolution B-mode ultrasonography (Logia 700 ultrasound machine; General Electric Medical Systems, Little Chalfont, Buckinghamshire, United Kingdom). Common carotid IMT reflects the mean of all available maximum wall thicknesses across all scans, across both left and right sides, and across the near and far walls. Central reading of IMT was performed at the Tufts-New England Medical Center (Boston, Massachusetts) (18).We studied the following cardiovascular risk factors: body mass index (BMI), hypertension, diabetes, and current smoking. Low density lipoprotein cholesterol and high density lipoprotein cholesterol were also examined, but no associations were apparent for either gender, so results are not shown in this paper. BMI was calculated as weight (kg)/height (m)2. Resting blood pressure was measured 3 times with a Dinamap PRO 100 automated oscillometric device (Critikon Inc., Tampa, Florida). The average of the final 2 blood pressure readings was used for this analysis. Hypertension was defined by the 6th report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (23) criteria of self-reported treatment of hypertension or systolic blood pressure of ≥140 mm Hg or diastolic blood pressure of ≥90 mm Hg. Diabetes mellitus was defined by the 2003 American Diabetes Association criteria of fasting glucose ≥126 mg/dL, use of insulin or oral hypoglycemic agents, or self-reported physician diagnosis. Cigarette smoking was based on self-report and was classified into 1 of 3 categories: current, former, or never (18).CovariatesAge, gender, adult socioeconomic position (SEP), and race/ethnicity were obtained from the baseline examination interview. As in prior work (13), adult SEP was created by combining information on income, education, and wealth. Participants were asked to select their total gross family income in the past 12 months from 13 categories (collapsed into 4 for this analysis: <$25,000, $25,000–$39,999, $40,000–$74,999, or ≥$75,000). Highest educational level completed was collapsed into 4 categories: ≤high school, some college but no degree/technical school certificate, associate's or bachelor's degree, or graduate/professional degree. Wealth was assessed with the following 4 items: 1) whether the participant, or his or her family, had investments such as stocks, bonds, mutual funds, retirement investments, or other investments (yes/no); 2) whether the participant owned the home (yes/no); 3) whether the participant owned a car (yes/no); and 4) whether the participant owned land or another property that was not his or her primary residence (yes/no). A summary adult SEP score was created by summing scores for income (0–3, from lowest to highest category) and education (0–3, from lowest to highest) and adding one point for each wealth indicator present. Thus, the range of values for adult SEP was 0 to 10, with higher values indicating greater adult SEP. Race/ethnicity was classified as 1 of 4 categories: white non-Hispanic, African American non-Hispanic, Chinese, or Hispanic.Statistical analysisSociodemographic characteristics and mean common carotid IMT for the chosen categories of neighborhood poverty trajectory group were compared by using analysis of variance (continuous variables) and the chi-square statistic (categorical variables). For each analysis, linear regression was used to assess mean differences in common carotid IMT and BMI, and a generalized linear model with a binomial distribution (24) was used to estimate the relative prevalence of hypertension, diabetes, and current smoking associated with the neighborhood poverty measures. These regression models were used to estimate associations of trajectory classes with the outcomes by including trajectory classes as dummy variables in the regressions. For those in stable trajectory classes, tests for trend were conducted to examine whether higher levels of neighborhood poverty were associated with worse cardiovascular outcomes.In addition, in these same stable trajectory classes, we also contrasted associations of contemporaneous (each participant's last known address at the MESA baseline examination (January 2000–August 2002)) and average poverty with the outcomes by fitting models with each of these exposures separately and estimating the adjusted mean difference or adjusted relative prevalence of the outcome for the 90th versus the 10th percentile of each measure. The nonstable trajectory group was excluded from these analyses; because of the changing nature of poverty over time in this group, comparison of the contemporaneous and average measures is not very meaningful.Because race/ethnicity and adult SEP may be partly confounding associations of neighborhood poverty with common carotid IMT, and associated risk factors, models were fit before and after adjustment for adult SEP and race/ethnicity. Analyses were stratified by gender because of potential differences in associations of SEP with atherosclerosis between men and women.RESULTSOf the total 6,814 men and women, 5,871 (86.2%) completed a residential history questionnaire at baseline (January 2000–August 2002). Of these participants, 929 (16%) were excluded because 1) one or more addresses could not be geocoded (n = 797) or 2) poverty values were not available for one or more of the tracts in which they had lived since January 1980 (n = 132); thus, data on 4,942 participants were available for analysis. Compared with persons included in the analysis, persons excluded were significantly more likely to be younger; be Chinese or Hispanic; be in the lower income or educational categories; have lower common carotid IMT, BMI, and high density lipoprotein cholesterol; and have a lower prevalence of hypertension, although differences were generally not large.The mean age of the sample was 62.4 years; 47.6% were male; 43% were white, 30% were African American, 7% were Chinese, and 20% were Hispanic; mean BMI was 28.7 (standard deviation, 5.4); 46% were hypertensive; 14% were diabetic; and 13% were current smokers. The fit of the trajectory class models, as assessed by BIC value, improved from 2 classes (BIC = 110,809) through 6 classes (BIC = 148,513), but it worsened with 7 classes (BIC = 143,758). The model with 8 classes was a slightly better fit (BIC = 151,448), but zero participants were assigned to 2 of the classes, suggesting that the 6-class model was the best fit for the data (Table 1).Table 1.Mean Neighborhood Poverty Trajectory Values for 1980, 1985, 1990, 1995, and 2000 for the 6 Neighborhood Trajectory Groups, Multi-Ethnic Study of Atherosclerosis, United StatesNo.%19801985199019952000Medium high–low2044.10.290.270.140.100.11High stable1783.60.440.470.490.450.42Medium-high stable4709.50.310.330.340.330.31Medium stable80616.20.200.210.220.240.24Low-medium stable1,11622.70.120.130.150.150.15Low stable2,16843.90.060.060.050.060.07Participants in the low stable poverty group were significantly more likely to be male, white, and US born. These participants had the most favorable risk factor profiles of all of the groups: the lowest mean BMI and the lowest prevalence of hypertension, diabetes, and current smoking. Moving from the lowest stable group to the highest stable group, the groups were younger, included more females, included fewer whites or Chinese, included more blacks and Hispanics (with a decrease in Hispanics and an increase in blacks in the highest poverty group), were less likely to be US born, and had a higher mean BMI and a higher prevalence of hypertension, diabetes, and current smoking. The percentage of persons who moved over the 20 years decreased monotonically from a high of 56% in the low stable poverty group to a low of 42% in the high stable poverty group (Table 2).Table 2.Sociodemographic Characteristics by Neighborhood Trajectory Class, Multi-Ethnic Study of Atherosclerosis, United States, 2000–2002aLow Stable (n = 2,168)Low-Medium Stable (n = 1,116)Medium Stable (n = 806)Medium-High Stable (n = 470)High Stable (n = 178)Medium High–Low (n = 204)Mean (SD)%Mean (SD)%Mean (SD)%Mean (SD)%Mean (SD)%Mean (SD)%Baseline neighborhood povertyb,c0.06 (0.04)0.12 (0.05)0.20 (0.07)0.31 (0.08)0.44 (0.10)0.29 (0.11)20-Year average neighborhood povertyb,c0.06 (0.02)0.14 (0.02)0.22 (0.03)0.33 (0.03)0.45 (0.06)0.18 (0.05)Age, yearsb62.8 (9.7)61.9 (10.2)63.5 (10.3)62.4 (10.4)60.1 (9.0)59.1 (10.3)Maleb504746454241Moved during the last 20 yearsb565246403796No. of movesb2.1 (1.7)2.1 (1.5)1.9 (1.4)1.7 (1.2)1.6 (1.0)3.1 (1.5)Raceb    White non-Hispanic6344217524    Chinese8563012    African-American non-Hispanic193143476239    Hispanic101931433325US bornb,d858271555769Married at baselineb695751464851Body mass index, kg/m2b28.1 (5.2)28.5 (5.3)29.4 (5.7)29.6 (6.0)30.0 (5.5)29.2 (5.5)LDL cholesterol, mg/dL117.4 (29.9)117.1 (32.0)117.7 (33.5)116.5 (31.7)116.2 (31.0)118.7 (33.1)HDL cholesterol, mg/dL51.5 (15.1)51.6 (15.0)50.2 (14.9)51.1 (13.5)51.1 (15.3)49.4 (13.1)CIMT, μm872.5 (195.2)870.9 (189.6)893.2 (183.6)885.7 (199.5)879.2 (193.5)833.2 (166.7)Hypertensionb444451525446Diabetesb91315172411Current cigarette smokerb101415161917Abbreviations: CIMT, common carotid intimal-media thickness; HDL, high density lipoprotein; LDL, low density lipoprotein; SD, standard deviation.aNeighborhood poverty from January 1980 to 2000: ∼6% all = low stable; ∼12% all = low-medium stable; ∼20% all = medium stable; ∼31% all = medium-high stable; ∼45% all = high stable; ∼30% to ∼10% = medium-high–low.bTest for trend significant at the 0.05 level.cThe value 0.06, for example, indicates that the mean number of households in that census block living below the poverty line is 6/100.dAll participants have been in the United States since January 1980.Only one group with a changing (i.e., not stable) neighborhood poverty trajectory was identified. This group (labeled medium high–low) began the period at a medium-high neighborhood poverty level (mean, 29%) and experienced a decline in neighborhood poverty over time (mean, 11%) (Table 1). The medium high–low group was the youngest and most female. The racial/ethnic distribution was similar to that of the medium stable group except for a larger representation of Chinese. This group, compared with the other groups, also had the lowest mean common carotid IMT, a relatively low prevalence of diabetes, and a high prevalence of current smokers. Virtually all (96%) persons in this group moved at some point over the 20-year period (Table 2).Among women, there was clear evidence that, after age adjustment, groups experiencing higher stable neighborhood census tract poverty over time had a higher common carotid IMT and BMI and more diabetes and hypertension than those in the low stable poverty group. For men, this finding was true for only diabetes—with less clear patterns. For both genders, after adjustment for race and adult SEP, only the medium-high stable group for BMI and the medium-high and high stable groups for diabetes in women had mean outcomes significantly higher than those in the low stable group. However, point estimates showed that a general trend of greater common carotid IMT, greater BMI (except for the highest poverty category), and greater prevalence of diabetes and hypertension associated with higher levels of neighborhood poverty tended to persist after adjustment, although the trends were no longer statistically significant at the 0.05 level (Figures 1–4). For men and women, stable poverty trajectory groups above the low stable group had a higher prevalence of current smoking, but a dose-response relation was not apparent (Figure 5). When we adjusted for adult SEP, results were similar when the individual SEP components of the composite score were adjusted for rather than the composite score (data not shown).Figure 1.Adjusted mean differences in common carotid intimal-media thickness (CIMT) (μm), and 95% confidence intervals, for neighborhood trajectory classes compared with the low stable group for A) males and B) females. Solid vertical line: age adjusted; dotted vertical line: age–adult socioeconomic position and race adjusted. P for trend (stable groups only)—age adjusted: A) 0.2362, B) 0.0002; age–adult socioeconomic position and race adjusted: A) 0.3451, B) 0.2568.Figure 2.Adjusted mean differences in body mass index (BMI), and 95% confidence intervals, for neighborhood trajectory classes compared with the low stable group for A) males and B) females. Solid vertical line: age adjusted; dotted vertical line: age–adult socioeconomic position and race adjusted. P for trend (stable groups only)—age adjusted: A) 0.6932, B) <0.001; age–adult socioeconomic position and race adjusted: A) 0.0022, B) 0.1043.Figure 3.Adjusted relative prevalences of hypertension, and 95% confidence intervals, for neighborhood trajectory classes compared with the low stable group for A) males and B) females. Solid vertical line: age adjusted; dotted vertical line: age–adult socioeconomic position and race adjusted. P for trend (stable groups only)—age adjusted: A) 0.0962, B) <0.001; age–adult socioeconomic position and race adjusted: A) 0.1925, B) 0.5045.Figure 4.Adjusted relative prevalences, and 95% confidence intervals, of diabetes for neighborhood trajectory classes compared with the low stable group for A) males and B) females. Solid vertical line: age adjusted; dotted vertical line: age–adult socioeconomic position and race adjusted. P for trend (stable groups only)—age adjusted: A) 0.0055, B) <0.0001; age–adult socioeconomic position and race adjusted: A) 0.4063, B) 0.0012.Figure 5.Adjusted relative prevalences, and 95% confidence intervals, of current smoking for neighborhood trajectory classes compared with the low stable group for A) males and B) females. Solid vertical line: age adjusted; dotted vertical line: age–adult socioeconomic position and race adjusted. P for trend (stable groups only)—age adjusted: A) <0.0001, B) 0.0008; age–adult socioeconomic position and race adjusted: A) 0.1904, B) 0.2246.With few exceptions, cardiovascular outcomes for the medium high–low trajectory group (category shown to the right of the stable groups in each figure) were somewhere in between those for the low-medium stable and medium stable groups (which averaged 12% and 20% neighborhood poverty levels, respectively, comparable to the medium high–low group's average of 18.5%). For men, mean common carotid IMT was lower (Figure 1) and the prevalence of hypertension was nonsignificantly higher (Figure 3) in the medium high–low group than in any of the other trajectory groups. Residential mobility over the 20 years was not associated with any of the outcomes after adjustment for age, cumulative neighborhood poverty, and race/ethnicity (data not shown).For all outcomes except smoking, associations using the contemporaneous or average poverty measure were similar for women reporting 1 address. However, for women reporting 2 or more addresses, associations were consistently stronger when the average poverty measure was used. In contrast, for current smoking, among women with 2 or more addresses, associations were stronger for the contemporaneous measure than for the average measure. There was no clear pattern for men. These analyses were restricted to the stable trajectory groups (Table 3).Table 3.Mean Differences in CIMT and Body Mass Index, and Relative Prevalences of Hypertension, Diabetes, and Current Smoking, for the 90th vs. 10th Percentile of Contemporaneousa and Average Neighborhood Poverty During a 20-Year Period (1980–2000) for Women With Stable Trajectories (n = 2,592), Multi-Ethnic Study of Atherosclerosis, United StatesOutcome and AdjustmentWomen Reporting Only 1 Address (n = 1,245)Women Reporting ≥2 Addresses (n = 1,347)ContemporaneousAverageContemporaneousAverageMean Difference95% CIMean Difference95% CIMean Difference95% CIMean Difference95% CICIMT (n = 2,561)    Age31.811.0, 52.528.98.2, 49.618.5−2.3, 39.335.412.5, 58.4    Age and adult SEP32.310.4, 54.229.17.3, 50.98.1−13.9, 30.125.31.0, 49.7    Age, adult SEP, and race/ethnicity28.74.5, 52.925.01.0, 48.9−14.2−37.3, 8.9−2.6−29.1, 23.9Body mass index (n = 2,592)    Age2.41.7, 3.22.31.6, 3.12.51.7, 3.33.42.5, 4.2    Age and adult SEP2.11.3, 2.92.01.2, 2.81.81.0, 2.72.71.7, 3.6    Age, adult SEP, and race/ethnicity0.8−0.1, 1.60.7−0.2, 1.50.1−0.8, 0.90.4−0.6, 1.4Relative Prevalence95% CIRelative Prevalence95% CIRelative Prevalence95% CIRelative Prevalence95% CIHypertension (n = 2,592)    Age1.71.5, 2.01.61.4, 1.91.51.2, 1.82.01.6, 2.3    Age and adult SEP1.71.4, 2.01.61.3, 1.91.31.0, 1.61.71.4, 2.0    Age, adult SEP, and race/ethnicity1.20.9, 1.51.10.8, 1.40.90.5, 1.21.00.7, 1.4Diabetes (n = 2,582)    Age2.21.8, 2.62.42.0, 2.82.72.3, 3.13.73.3, 4.1    Age and adult SEP1.91.5, 2.32.11.7, 2.52.11.7, 2.52.92.4, 3.3    Age, adult SEP, adult and race/ethnicity1.20.7, 1.61.30.9, 1.81.51.0, 1.91.91.4, 2.3Current smoking (n = 2,584)    Age2.31.7, 2.72.11.7, 2.51.81.4, 2.11.41.0, 1.8    Age and adult SEP2.11.7, 2.51.91.5, 2.31.51.1, 1.91.20.7, 1.6    Age, adult SEP, and race/ethnicity2.21.7, 2.72.01.5, 2.41.30.9, 1.70.90.4, 1.4Abbreviations: CI, confidence interval; CIMT, common carotid intimal-media thickness; SEP socioeconomic position.aContemporaneous refers to each participant's address at the baseline Multi-Ethnic Study of Atherosclerosis examination (January 2000–August 2002).DISCUSSIONWe found evidence of several different subpopulations of neighborhood trajectory groups, with most groups experiencing approximately stable neighborhood poverty over the entire time period but differing in level of neighborhood poverty. For women, higher cumulative neighborhood poverty was generally significantly associated with worse cardiovascular outcomes in age-adjusted models, and, whereas the general trends (as indicated by the point estimates) tended to persist after adjustment for adult SEP and race/ethnicity, confidence intervals for many of the estimates were wide and trends were no longer statistically significant. No consistent association of neighborhood poverty with the outcomes was observed for men. In general, the medium high–low neighborhood poverty mobility group had outcomes comparable to those for stable groups with similar cumulative exposures. In addition, for women who moved, the long-term average measure of poverty was more strongly associated with outcomes than a single, contemporaneous measure. In contrast, the opposite was true for current smoking.Few prior studies have investigated trajectories of neighborhood poverty over time. In our sample, the relative stability of residence over the study period (47% did not move during the entire 20 years), coupled with very few changes in poverty within census tracts over time, resulted in stable trajectory groups. However, almost 50% of the stable groups had moved at least once, and correlations between poverty levels in subsequent neighborhoods were positive although not very high (Pearson's correlations of neighborhood poverty between each pre- and postmove neighborhood, 0.36). In general, our sample was less mobile than the US population of the same age (percentage who moved at least once between 1995 and 2000: US Census, 33.1%; MESA, 16.7%). The peak age for mobility is 20–29 years, with mobility decreasing with age (25). Consequently, the relative residential stability of our sample may be attributable to its age (41% were older than age 65 years) or to selection effects related to participation in a long-term study such as MESA.These results are consistent with prior analyses of other cohorts showing that cumulative neighborhood SEP is associated with higher mean common carotid IMT in white women (12), with patterns still apparent but nonsignificant after adjustment for individual SEP and race/ethnicity (12, 13). The measures of neighborhood SEP used in previous work were summaries or average measures of neighborhood socioeconomic status. In contrast, we examined distinct trajectories of neighborhood poverty over time. Potential mechanisms linking long-term neighborhood conditions to cardiovascular disease include access to resources and services that promote healthy lifestyles, as well as social features such as social norms, social support, and features of neighborhoods (such as violence or disorder) that could be stressful (2). To the extent that neighborhood poverty is a reasonable proxy for these conditions, our results may reflect cumulative effects of these exposures.Few data exist on how fluctuations in neighborhood poverty over time could translate into better or worse cardiovascular disease risk factors and outcomes for residents. It is plausible that changes in neighborhood poverty are associated with changes in specific environmental features linked to cardiovascular disease. For instance, some studies have found that lower income neighborhoods have fewer supermarkets and more fast-food restaurants than wealthier neighborhoods do (26). Persons moving from higher to lower income neighborhoods or experiencing a change in the socioeconomic features of their neighborhoods over time could also be exposed to changes in food environments related to cardiovascular disease. Measuring neighborhood poverty at a single point in time would give an incomplete assessment of what neighborhood exposures an individual had experienced over that time period and could therefore lead to underestimates of neighborhood effects.Consistent with our results, previous literature has documented a stronger effect of neighborhood deprivation on the incidence and prevalence of coronary heart disease in women than men (1, 3, 27). A number of processes could explain the stronger associations among women, including greater exposure to neighborhood conditions or greater reliance on neighborhood resources for women than for men (13). Further study is needed to examine mechanisms related to heterogeneity of neighborhood effects by gender. We found that most associations of neighborhood poverty with the cardiovascular disease risk factors were attenuated after adjustment for adult SEP and race/ethnicity. General patterns tended to remain, although trend tests were no longer statistically significant. The strong association of trajectories of neighborhood poverty with adult SEP and race/ethnicity in these data makes it difficult to isolate their “independent” contributions. In addition, adult SEP may be partly influenced by a history of exposure to neighborhood poverty. Statistical controls for race/ethnicity and adult SEP may therefore result in underestimates of neighborhood effects.One group, the medium high–low group, displayed a substantial change in neighborhood poverty over the period (dropping from 29% to 11%). This change was largely attributable to residential mobility (96% had moved over the period, and Pearson's correlations between census years for all census tracts in the Neighborhood Change Database were very high (1980–2000: 0.73, 1980–1990: 0.76, 1990–2000: 0.85). Our results with respect to cardiovascular outcomes in this group support an accumulation model of neighborhood exposure to poverty with little evidence of an independent effect of social mobility (15, 28). However, our ability to examine this trajectory group was limited by small sample size (n = 204 or 4.1% of the sample). Further study is needed to examine whether residential mobility patterns in MESA are representative of patterns of moving during mid- to late adulthood.The finding that 96% of the sample was classified into a “stable” trajectory group implies that a contemporaneous single-point-in-time measure of neighborhood poverty may accurately represent past neighborhood exposure. However, we found that, even in the “stable” trajectory groups, when women had moved during the historical period of interest, use of the long-term average measure of exposure rather than a contemporaneously measured exposure resulted in stronger associations with health outcomes that develop over long periods (such as subclinical atherosclerosis). In contrast, a current behavior such as smoking was more strongly related to current conditions than to past conditions. These results suggest that, when estimating long-term exposures in the presence of residential mobility, reliance on a single-point-in-time measure could lead to underestimates of the effects of cumulative long-term exposures to neighborhood conditions.Limitations of our study include limited trajectory groups in which neighborhood poverty changed substantially over time and retrospective collection of residential history information, which could have resulted in important misclassification of poverty exposures. The exposure to neighborhood was measured with only a single indicator, neighborhood census tract poverty, and captured only 20 years of exposure in late adulthood. Census tracts may not be the most relevant geographic unit, 20 years may not fully capture the historical time frame relevant to the outcomes we studied, and the 20 years might be capturing a different period in adulthood depending on the participant's age when he or she entered the study.Our results indicate that a summary of 20-year exposure to neighborhood poverty is associated with greater cardiovascular risk for women. They also suggest that, in many cases, single-point-in-time measures may be reasonable proxies for historical exposures, although they may underestimate neighborhood effects in the presence of important residential mobility and for outcomes that develop over long periods.AbbreviationsBICBayesian Information CriterionBMIbody mass indexIMTintimal-media thicknessMESAMulti-Ethnic Study of AtherosclerosisSEPsocioeconomic positionAuthor affiliations: Department of Epidemiology, University of Michigan, Ann Arbor, Michigan (Emily T. Murray, Ana V. Diez Roux); Department of Preventive Medicine, Northwestern University, Chicago, Illinois (Mercedes Carnethon); Department of Epidemiology and Community Health, University of Minnesota, Minneapolis, Minnesota (Pamela L. Lutsey); Epidemiology Branch, Division of Prevention and Population Services, National Heart, Lung, and Blood Institute/National Institutes of Health, Bethesda, Maryland (Hanyu Ni); and Department of Biostatistics, University of Washington, Seattle, Washington (Ellen S. O’Meara).This study was supported by grant 2R01-HL071759 from the National Heart, Lung, and Blood Institute (A. D. R., Principal Investigator). The Multi-Ethnic Study of Atherosclerosis is supported by contracts N01-HC-95159 through N01-HC-95165 and N01-HC-95169 from the National Heart, Lung, and Blood Institute.The authors thank the MESA investigators and staff for their valuable contributions.A full list of participating MESA investigators and institutions can be found at the following website: http://www.mesa-nhlbi.org.Conflict of interest: none declared.1.Diez-RouxAVNietoFJMuntanerCNeighborhood environments and coronary heart disease: a multilevel analysisAm J Epidemiol1997146148632.Diez RouxAVMerkinSSArnettDNeighborhood of residence and incidence of coronary heart diseaseN Engl J Med20013452991063.SundquistKMalmströmMJohanssonSENeighbourhood deprivation and incidence of coronary heart disease: a multilevel study of 2.6 million women and men in SwedenJ Epidemiol Community Health200458171774.SundquistKWinklebyMAhlénHNeighborhood socioeconomic environment and incidence of coronary heart disease: a follow-up study of 25,319 women and men in SwedenAm J Epidemiol200415976556625.CubbinCWinklebyMAProtective and harmful effects of neighborhood-level deprivation on individual-level health knowledge, behavior changes, and risk of coronary heart diseaseAm J Epidemiol200516265595686.StjärneMKFritzellJDe LeonAPNeighborhood socioeconomic context, individual income and myocardial infarctionEpidemiology200617114237.NordstromCKDiez RouxAVJacksonSAThe association of personal and neighborhood socioeconomic indicators with subclinical cardiovascular disease in an elderly cohort. The Cardiovascular Health StudySoc Sci Med20045910213921478.HartCEcobRDavey Smith G. People, places and coronary heart disease risk factors: a multilevel analysis of the Scottish heart health study archiveSoc Sci Med19974568939029.SmithGDHartCWattGIndividual social class, area-based deprivation, cardiovascular disease risk factors, and mortality: the Renfrew and Paisley StudyJ Epidemiol Community Health199852639940510.CubbinCHaddenWCWinklebyMANeighborhood context and cardiovascular disease risk factors: the contribution of material deprivationEthn Dis200111468770011.CubbinCSundquistKAhlénHNeighborhood deprivation and cardiovascular disease risk factors: protective and harmful effectsScand J Public Health200634322823712.CarsonAPRoseKMCatellierDJCumulative socioeconomic status across the life course and subclinical atherosclerosisAnn Epidemiol200717429630313.LemelinETDiez RouxAVFranklinTGLife-course socioeconomic positions and subclinical atherosclerosis in the Multi-Ethnic Study of AtherosclerosisSoc Sci Med200968344145114.PollittRARoseKMKaufmanJSEvaluating the evidence for models of life course socioeconomic factors and cardiovascular outcomes: a systematic reviewBMC Public Health20055715.HallqvistJLynchJBartleyMCan we disentangle life course processes of accumulation, critical period and social mobility? An analysis of disadvantaged socio-economic positions and myocardial infarction in the Stockholm Heart Epidemiology ProgramSoc Sci Med20045881555156216.NaginDSAnalyzing developmental trajectories: a semi parametric group-based approachPsychol Methods19994213915717.JonesBLNaginDSRoederKA SAS procedure based on mixture models for estimating developmental trajectoriesSociol Methods Res.200129337439318.BildDEBluemkeDABurkeGLMulti-Ethnic Study of Atherosclerosis: objectives and designAm J Epidemiol2002156987188119.Normalized data—neighborhood change database [NCDB] tract data from 1970–20002006East Brunswick, NJGeolytics Inc(http://www.geolytics.com/USCensus, Neighborhood-Change-Database-1970-2000, Products.asp). (Accessed May 19, 2009)20.KriegerNWilliamsDRMossNEMeasuring social class in US public health research: concepts, methodologies, and guidelinesAnnu Rev Public Health19971834137821.BotsMLCarotid intima-media thickness as a surrogate marker for cardiovascular disease in intervention studiesCurr Med Res Opin200622112181219022.PoredosPIntima-media thickness: indicator of cardiovascular risk and measure of the extent of atherosclerosisVasc Med200491465423.The sixth report of the Joint National Committee on PreventionDetection, Evaluation, and Treatment of High Blood PressureArch Intern Med1997157212413244624.SpiegelmanDHertzmarkEEasy SAS calculations for risk or prevalence ratios and differencesAm J Epidemiol2005162319920025.PlaneDAHenrieCJPerryMJMigration up and down the urban hierarchy and across the life courseProc Natl Acad Sci U S A200510243153131531826.MooreLVDiez RouxAVAssociations of neighborhood characteristics with the location and type of food storesAm J Public Health200696232533127.WinklebyMSundquistKCubbinCInequities in CHD incidence and case fatality by neighborhood deprivationAm J Prev Med20073229710628.Davey SmithGLynchJWKuhDBen-ShlomoYSocioeconomic differentialsA Lifecourse Approach to Chronic Disease Epidemiology2003Vol 2Oxford, United KingdomOxford University Press \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B60CE69C89F751133FE358CED2307D9E9B90004.txt b/test/dataset/in/resources/corpus/Clean_0B60CE69C89F751133FE358CED2307D9E9B90004.txt new file mode 100644 index 0000000..b8e569d --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B60CE69C89F751133FE358CED2307D9E9B90004.txt @@ -0,0 +1 @@ +jmpjmpJournal of Medicine and Philosophy1744-50190360-5310Oxford University Press10.1093/jmp/jhq048ArticlesA Transhumanist Fault Line Around Disability: Morphological Freedom and the Obligation to EnhanceBradshawHeather G.*University of Bristol, Bristol, UKTer MeulenRuudUniversity of Bristol, Bristol, UK*Address correspondence to: Heather G. Bradshaw, Centre for Ethics in Medicine, University of Bristol, 3rd Floor Hampton House, Cotham Hill, Bristol BS6 6AU, UK. E-mail: heather.bradshaw@bristol.ac.uk12201012112010356Bioethics and Transhumanism670684© The Author 2010. Published by Oxford University Press, on behalf of the Journal of Medicine and Philosophy Inc. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org2010The transhumanist literature encompasses diverse nonnovel positions on questions of disability and obligation reflecting long-running political philosophical debates on freedom and value choice, complicated by the difficulty of projecting values to enhanced beings. These older questions take on a more concrete form given transhumanist uses of biotechnologies. This paper will contrast the views of Hughes and Sandberg on the obligations persons with “disabilities” have to enhance and suggest a new model. The paper will finish by introducing a distinction between the responsibility society has in respect of the presence of impairments and the responsibility society has not to abandon disadvantaged members, concluding that questions of freedom and responsibility have renewed political importance in the context of enhancement technologies.disabilityhuman enhancementmorphological freedomnegative libertytranshumanistI.INTRODUCTIONEnhancement in the context of disability draws out areas of continuing debate and development in transhumanist ethics and political philosophy. We will introduce the fault line by contrasting the words of people with disabilities with two strands of thought in the transhumanist literature. Section II explains the fault line, following Isaiah Berlin (2002/1958), via two models of freedom. In Section III, the transhumanist conception of freedom is developed and critiqued. Sections IV and V each address objections to our preferred model of freedom in transhumanist work referring to disability. The conclusion summarizes the contextual threats of the rejected model of freedom. We turn now to the fault line.James Hughes (2004, 147) writes that we have “a duty and right to provide children with the healthiest and most able bodies we can.” He calls this “a really basic idea.”But there exist persons living with differences or disabilities who do not think they do have such an obligation to enhance themselves or their children.1 For example, Louisa, a congenitally deaf academic and linguist who is also a childhood cochlear implantee (CI) said: “[I]f for example you get a deaf baby, there is enormous pressure from doctors and society etc to take CI, because ‘it is there’ . . . whilst I think it is not because it is there, that it is automatically ‘good’ or matching the particular situation/environment of the person.”“Healthiest” and “most able” can be seen as situationally dependent (Savulescu, 2006, 324) and thus less basic than Hughes’ use suggests. This partially explains why Louisa and others living in the Deaf community (Savulescu, 2002; Atkinson, 2006; Murphy, 2008) are uncomfortable with and do not see themselves as bound by what is expressed in Hughes’ “basic idea.”2 Of course, there are others living with disabilities of various types who do want the opportunity to enhance themselves, for example, a young congenitally partially sighted man interviewed in the same research project as Louisa: “It's just … ah you know … just … just be able to see in my case.”A view from the transhumanist literature that contrasts with Hughes’ acknowledges this complexity:There clearly exist many people who deeply wish to be cured from various disabilities. But there are also many people who over time have become used to them and instead integrated them into their self-image. The investment of personal growth and determination necessary to accept, circumvent or overcome a disability is enormous. Suggesting a cure to them implies a change to themselves on a far deeper level than just “fixing” a broken tool, and quite often is experienced as an attack on their human dignity. (Sandberg, 2001, S6 Why do we need morphological freedom?, Para 9)Sandberg develops the concept of “morphological freedom” to express one transhumanist attitude to morphology or body configuration:3 “The desirability to [of] many of the possibilities allowed by morphological freedom also helps support the right to not change, as people see that they are two sides of the same coin” (Sandberg, 2001, S6, Para 4). Hughes (2004, 138) similarly writes: “People should have a right to control their own genomes and have children without permission from the government”; however, he goes on:But if eugenics includes believing that individuals, free of state coercion, should have the right to change their own genes and then have children, then the advocates of human enhancement and germinal choice are indeed eugenicists. If eugenics also includes the belief that parents and society have an obligation to give our children and the next generation the healthiest bodies and brains possible, then most people are eugenicists. (Hughes, 2004, 131)It may be that most people do believe that parents and society have such an obligation (Hughes does not give evidence) but “most” leaves out perhaps significant minorities and most people can be ethically mistaken, collectively as well as individually.Strikingly, Hughes adds with reference to Deaf people having this same “right to control their own genomes and have children”: “Physicians should refuse to accede to such a request, and public and private insurance should refuse to pay for it. When the deaf child reaches maturity they should be able to sue their parents for damages . . .” (Hughes, 2004, 140).4Sandberg, in contrast to Hughes, makes an in-principle defense of the right not to change, including the right to stay disabled or choose disability (or Deafness). He is concerned with defending radical technologies against the serious charges that they might be misused “in a coercive manner, enforcing cultural norms of normality or desirability” (Sandberg, 2001, S6, Para 3). He proposes that this extension of the concept of freedom to the control of the form of one's body is required to defend against the threat of coercive normalization from radical technologies.If it is widely accepted that we have the right to control how our bodies are changed both in the positive sense (using available tools for self-transformation) and in the negative sense of being free to not change, then it becomes harder to argue for a compulsory change (Sandberg, 2001, S6, Para 4).Sandberg and Hughes represent two diverse streams of transhumanist thought that are not yet always clearly delineated. One symptom of their divergence is the different weight they place on countering the risk that enhancement technologies and the knowledge underpinning them will be used in tyrannical or exploitative ways. That is, the weight they place on protecting freedom.5 These contentious differences about how the risks should be addressed are especially prominent in transhumanist attempts to come to terms with political, social, ethical, and economic questions involving human diversity, especially, impairment and disability. As differences over freedom are at the epicenter of the fault line, we turn now to Berlin's analysis of freedom.II.ABANDONMENT AND OPPRESSION ARE THE RISKS OF FREEDOMIn our view, the fissure in transhumanist positions (and politics) is a modern continuation of older political debates. Isaiah Berlin wrote of (but does not claim to have created the distinction between) two sorts of political or social risk arising from an institution or society's attempt to meet citizens’ psychological need for control of their own actions, freedom. Two sorts of risk will be outlined in this section then related to transhumanist through a dilemma. A potential solution and objection will take us to Section III.Abandonment is the first type of risk. Abandonment occurs when freedom is produced by society withdrawing from individual lives. We shall call this the absence model of freedom. This has been experienced in recent human history as “politically and socially destructive policies which armed the strong, the brutal and the unscrupulous against the humane and the weak, the able and ruthless against the less gifted and the less fortunate. Freedom for the wolves has often meant death to the sheep” (Berlin, 2002/1969, 38). Berlin wrote early in Europe's welfare state era. He excuses his lack of critique of the absence model by alluding to the voluminous contemporaneous literature on the harms pure capitalism causes to all but a few (Berlin, 2002/1969, 38).Oppression is the second type of political or social risk. Oppression's relation to freedom is complex. With regard to the involvement of society in individual lives in the form of the welfare state or socialism, “the case for intervention” (Berlin, 2002/1969, 38) to counter abandonment is “overwhelmingly strong” (Berlin, 2002/1969, 38). Yet in his view, the case for intervention still does not justify any model of freedom that carries the risk of oppression. Models in which your control of your actions is accepted as only partial invite others to help you increase your control. Oppression follows because, in Berlin's slippery slope argument:This renders it easy for me to conceive of myself as coercing others for their own sake, in their, not my, interest. I am then claiming that I know what they truly need better than they know it themselves. What, at most, this entails, is that they would not resist me if they were rational and as wise as I and understood their interests as I do. But I may go on to claim a good deal more than this. I may declare that they are actually aiming at what in their benighted state they consciously resist, […] Once I take this view, I am in a position to ignore the actual wishes of men or societies, to bully, oppress, torture them in the name, and on behalf, of their “real” selves […]. (Berlin, 2002/1958, 180)Such models of freedom we will call partial models (Berlin's “positive” liberty; Berlin, 2002/1969, 4). We explain their relevance to transhumanist via respect for “the person-as-they-are.” By summarizing the argument so far as a dilemma, we introduce John Christman's “autonomy” resolution and Takala's liberal objection.Berlin is clear that no matter how obvious it might appear from the outside that certain people might do better, in some sense, if they were different, they are actually not other than they are. One has to start from the person as they present themselves. To do otherwise is to oppress. One cannot start from the person one hopes another will become. To do this is to fail to recognize and respect the person-as-they-are as a morally whole person, as one who has his or her own ends in mind. It is to place one's own mind in place of theirs. “This monstrous impersonation, which consists in equating what X would choose if he were something he is not, or at least not yet, with what X actually seeks and chooses, is at the heart of all theories of political self-realisation” (Berlin, 2002/1958, 180).6The dilemma abandonment and oppression pose is emotive in disability and the transhumanist human enhancement context. The absence model risks abandoning to involuntary suffering even those who are suffering from a difference, impairment, disability or not being enhanced.7 Partial models risk oppression for those who have a difference, impairment, disability, or are not enhanced. Abandoned or oppressed? A poor choice for those already perceived as at a disadvantage!John Christman (2005) attempts a solution to the version of the dilemma posed in medical care and especially relevant in mental health care (Gutridge, 2010) by choosing a partial model but attempting to provide a barrier on the slippery slope. He argues that thinking about having “idealized,” complete, control over our actions, or “effective agency” is useful (Christman, 2005, 86, 80). We have thus discovered that autonomy is partial because… Autonomy is defined in various ways, but most conceptions stress the capacity for critical self-reflection in the development of value systems and plans of action. Such capacities do not merely emerge naturally, but must be developed through various processes involving educational, social, and personal resources. (Christman, 2005, 87)But autonomy is beneficial because it is self-limiting. Unlike other models of self-realization, in autonomy the only goal that can be imposed on one by others is the goal of being able to choose one's own goals. So even coercion in the name of autonomy can never lead to oppression and Berlin's “monstrous impersonation.”But Christman's model, as he admits all defenses of self-realization are, is based on what he calls a “cognitivist” understanding of justification: an action is justified by being the right action, not by being an action that was chosen (Christman, 2005, 84). Voluntarists, in contrast, claim that justification requires only that the action was freely chosen (Christman, 2005, 84). The catch with Christman's “cognitivist” justification is that we require some way of knowing which actions are right independently of whether people choose them or not. But how can the rightness of the action be thus abstracted from the circumstances in which it is made? In particular, how can it be separated from the nature, background, and subjective judgments of the agent that makes it?Takala (2007) develops this objection to Christman's abandonment/oppression dilemma solution. She reminds us that societies like those of the United States and Britain today do not have ways of agreeing on the right action in every ethically contentious case. This is because of cultural and value divergence:If the account of autonomy is grounded on a positive notion of liberty, it annihilates the value neutrality that respect for autonomy was supposed to protect. In multicultural societies there is no justification to build the notion of autonomy on an account of liberty that presupposes a particular value system. (Takala, 2007, 228)Christman argues that any clarification of freedom must be normative, thus taking some value as prior. In contrast, Takala argues, liberally, for separation of freedom from morality. Freedom is constituted by the space to follow and develop one's own conscience, perhaps in opposition to the ways of the surrounding society. In the transhumanist future, this question of a preset guiding value versus value-free space is exceedingly difficult due to the potentially greatly increased diversity and consequent threat to social coherence from the absence model of freedom in this context. Freedom in transhumanist thought adds a further complication: variety in the sources of impediments to freedom is greater than in Berlin's work. In Section III, both Berlin's human source of impediment and transhumanist natural sources of impediment will be discussed in order to complete and criticize the transhumanist model of freedom.III.TRANSHUMANIST FREEDOM: THE HUMAN SOURCE RESTRICTION AND THE DISPOSITIONAL ACCOUNT OF VALUESHere we discuss the human source restriction's relevance to transhumanist freedom and introduce objections from resource scarcity and obligation scope. We counter by appeal to subjective, not universal, enhancement valuations, arguing that universalization requires particularly problematic empirical evidence in the enhancement context. Here the dispositional theory of value is subject to Berlin's objections from oppression. In contrast to Bostrom, we see the absence model as providing part of the solution to the evidence deficit. We conclude by returning to disability through Sandberg's work.Christman (2005) and Garnett (2007) emphasize Berlin's “human source restriction” (Christman, 2005, 82). Berlin wrote: “Helvetius made this point very clearly: ‘The free man is the man who is not in irons, not imprisoned in a goal, nor terrorised like a slave by the fear of punishment.’ It is not lack of freedom to fly like an eagle or swim like a whale” (Berlin, 2002/1958 FN, 169). So, on Berlin's view we are not unfree when subject to traditional and contemporary human limitations, for example, lacking wings. But transhumanist is “a belief that the human race can evolve beyond its current limitations, especially by the use of science and technology” (Oxford English Dictionary, 2009), and the Transhumanist FAQ version 2.1, which summarizes the movement's values, describes it thus:(1) The intellectual and cultural movement that affirms the possibility and desirability of fundamentally improving the human condition through applied reason, especially by developing and making widely available technologies to eliminate aging and to greatly enhance human intellectual, physical, and psychological capacities.(2) The study of the ramifications, promises, and potential dangers of technologies that will enable us to overcome fundamental human limitations, and the related study of the ethical matters involved in developing and using such technologies. (Bostrom, 2003a, 1, my italics)So a transhumanist may hold that if it would be possible given available or near future technology and desirable; according to her understanding of “‘enhance,” for her to fly like an eagle, then anyone preventing her from accessing, developing, or encouraging the development of such technology is interfering with her freedom to spend her money and time and her freedom to change herself—body, brain, and mind—in accordance with her ends. Sandberg writes: “As a negative right, morphological freedom implies that nobody may force us to change in a way we do not desire or prevent our change” (Sandberg, 2001, S2, Para 11, my italics). That contradicts the human source restriction.An objection is society's lack of resources to address these new limitations to freedom as well as traditional human oppression. In reply we disagree that Sandberg's position entails a universal obligation to fund, buy, or use eagle technology. Universalization would require the significant further step that others ought to come to share that particular transhumanist's value system. A universal social obligation to enhance would require the enhancement to be likely to improve anyone's well-being, not just one's own (for individual welfarist definitions of enhancement, see Savulescu, 2006, Savulescu and Kahane, 2008; Kahane and Savulescu, 2009) as well as taking one beyond one's present state. But why should others share this value system, and how could we know if they did? Transhumanists who talk of universal enhancement obligations face a normativity problem similar to Christmans’—their justification is not value neutral and so risks Berlin's oppression rather than abandonment. In effect, they are using a partial model of freedom not an absence model.To generate a universal obligation to support an enhancement, the easiest way around our reply is to start with an enhancement which it can be assumed everyone already does value, such as reducing the incidence of the diseases of old age, as demonstrated by the Transhumanist FAQ. John Harris (2007, 36) uses a version of this strategy and defines an enhancement as such a widely valued ability.We respond first that the original objection of oppression is not addressed by seeking a universal value because it leaves no room for dissent. Second, it is still an assumption in need of evidence that these technologies are valued universally by the sorts of beings who will invest in developing them. It is not enough in this context of oppression avoidance that they ought to be so valued if the arguments supporting that ought are themselves based on a partial model of freedom or are otherwise oppressive. Third, it is a different question whether beings who actually had such technologies would still value them. And this question leads to its own set of problems, which we shall call the Chimpanzee Challenge.Not even the transhumanists have conducted large-scale surveys to determine what technological changes to the human organism are actually desired by the members of all human populations currently existing, let alone what the members of all human populations think they might desire if they were different beings, that is, beings who actually had these technologies.8 As Bostrom (2003b, 3) acknowledges, this would be difficult because “Just as chimpanzees lack the brainpower to understand what it is like to be human, so too do we lack the practical ability to form a realistic understanding of what it would be like to be posthuman”. Research with differently abled people (Bradshaw, forthcoming) shows that the change does not have to be large, or in the direction of increased ability, for a being, even a human, embodied in one way to “lack the practical ability to form a realistic understanding of what it would be like to be” differently embodied, that is, to have a different morphology. Sighted teachers lack a realistic understanding of partially sighted pupils’ phenomenological experiences; even technically well educated, oral, and hearing-friendly deaf persons may lack a realistic understanding of what life with a CI will be like. Hearing, orally communicating people generally lack a realistic understanding of what it is to be a Deaf parent.9 Most of us humans, like the chimpanzee, are just not very good at projecting what different circumstances will feel like for us let alone how these will change our values. If we were, there would be little point to literature and film.So the human “chimpanzee” seeking posthuman enlightenment will, phenomenologically, tread some very foreign roads, and visit some quite fantastic towns, and may well change value direction at each of them, seeking a destination that always eludes and having forgotten after a while where he or she came from. But none of this should stop him or her from setting out on the journey if they so wish and as long as they are aware of there being risks, including the risk of losing any sense of value direction.Even the nature of the more specific, personal, risks must be largely, though not entirely, unknown. “Here be serpents, Eve.” Not every road will lead to Utopia, and if some claim they have found it and we should follow them, how are we to know they have not just been taken in by some beguiling detour? (Bostrom, 2008; Miah, 2008, Letters from and to Utopia).But this does not constitute an objection to voluntary individual enhancement or morphological variation, for instance choosing to remain deaf, as supported by morphological freedom. Indeed, it is an argument for morphological freedom. This is because none of these risks, or the chimpanzee's necessary ignorance, should detract from the value of the journey itself, for how can we learn to navigate such waters if none of the various willing volunteers are allowed to explore them?But, to return us yet again to the question of there being a social obligation to enhance, Bostrom (2003b) goes further than the case of what one may choose for oneself and writes:Additionally, we may favour future people being posthuman rather than human, if the posthumans would lead lives more worthwhile than the alternative humans would. Any reasons stemming from such considerations would not depend on the assumption that we ourselves could become posthuman beings.We agree that the ethical force of this need not depend on the posthumans being in some way continuous with the original humans, but if there is no such connection, and the posthumans are, for example, other people's children, then the poor chimpanzee's problems are multiplied.This is because, although it is clear that I can favor, or wish, that my future life, whether as the person I am now, with my present identity and values, or with a new identity and values, will be posthuman rather than human, without risk of oppressing others (assuming the means I take to this end do not themselves harm others), it is not clear that I could favor this for someone else without risking oppressing them. There are various possibilities, such as altruism, and hope in despair, but we will show that they are not adequate.Altruistically we can wish that others’ lives will be better than our own. The hope that their children's lives will be better than their own appears to have carried many parents through dark times. To support and wish for technology that will benefit others, even if it arrives too late to help oneself, is admirable and may provide something to hope for when there is no personal hope remaining (see Bradshaw, forthcoming, for an example of this).Our objection to these two options is that they depend on a certainty that what the technology has to offer will be better for that other person. But this certainty is unavailable because of the Other Chimpanzee Challenge: If an individual cannot predict his/her own future well-being, then how can we predict well-being on behalf of another? And without certainty we have to take account of the risk of loss. If it is that the technologically modified being will have known nothing else (e.g., new morphology congenitally present), then at least they will not suffer a loss. So consider if we chose to alter a child's morphology and our imagined “better” turns out worse for them than what we can see in hindsight the original morphology would have done. Then the child will have lost nothing, in the sense of having experienced loss.10 But equally they will not experience a gain from the technology either.Such a risk of loss would, however, be present for any existing being who takes our destination as his or her own, for better or, in this case, worse. Because of this risk of loss due to the difficulty of predicting future well-being, it is important that he or she chooses of his or her own accord, and not because of anyone else's preferences so as to maximize the accuracy of the well-being forecast, to clearly assign responsibility for the consequences and to preserve agency. And for the choice to be fully theirs, it must be a real option for them to refuse the technology.This sort of refusal is, we think, what Sandberg means when he says that the opportunity to enhance needs to be countered by the “right not to change” to form morphological freedom. A true enhancement, rather than just a change, will be supported by many people's uncoerced, and retrospectively valued, choices over time. But any hint of coercion will detract from the information content of such changes as well as their psychological acceptance. Furthermore, as the proportions of people choosing one way or the other varies, so will the value of either choice. Staying the same when everyone else changes is itself to explore a subspace different from staying the same when everybody else also stays the same. And this requirement of free choice is precisely what Bostrom's formulation puts in doubt, especially when interpreted with his references to Lewis’ dispositional theory of value in mind. Bostrom writes:The conjecture that there are greater values than we can currently fathom does not imply that values are not defined in terms of our current dispositions. […] According to Lewis’ theory, something is a value for you if and only if you would want to want it if you were perfectly acquainted with it and you were thinking and deliberating as clearly as possible about it. On this view, there may be values that we do not currently want, and that we do not even currently want to want …. (Bostrom, 2003b, 4)This may be quite consistent but in terms of political philosophy it is reminiscent of Berlin's passages on the risks of positive or partial models of freedom (Berlin, 2002/1958, 180). For Lewis and Bostrom are here suggesting that a person's set of present values can be split into the values they are aware of and the values they are unaware of. Berlin claims such partial consciousness entails only that if the subject were (i) rational, (ii) “as wise as I,” and (iii) “understood their interests as I do,” then they “would not resist me.” It does not imply that present resistance is not significant! Nor that such people “are actually aiming at what in their benighted state they consciously resist” (Berlin, 2002/1958, 180). Berlin sees a great risk of oppression in attributing such currently unwanted or occluded values to those who are obviously not presently in the ideal state. “Once I take this view,” he writes, “I am in a position to ignore the actual wishes of men or societies, to bully, oppress, torture them in the name, and on behalf, of their ‘real’ selves” (Berlin, 2002/1958, 180). And that, when connected with the sorts of technologies in question, is more than enough to generate, and realize, the fears Sandberg refers to that “technologies such as genetic modifications would be used in a coercive manner, enforcing cultural norms of normality or desirability” (Sandberg, 2001, S6, Para 3).Sandberg connects his idea of morphological freedom as a defense against such risks with the experience of those living with disabilities and facing present technological choices. As they are the group for whom the obligation to enhance is often thought to be strongest, but rest lightest, it is a good test case. He writes:A simple ban of coercive medical procedures would not be enough, even if it is better than nothing. The reason is that it does not imply any right to have an alternative body or protect differently bodied people. The official [a bureaucrat considering that being disabled is a very expensive lifestyle] could encourage “normal” bodies through various means, including officially pronouncing disabled people who did not change as irresponsible and wasting public resources. Without any protection of the right to have a different body, both in the legal sense to prevent discrimination and in the ethical sense as a part of public ethics guiding acceptance and tolerance, the disabled would be in a very disagreeable situation. It should be noted that the disability movement have been strong supporters of right to determine ones body just for this reason. (Sandberg, 2001, S6, Para 11)Transhumanist pushes the boundaries of the human source constraint by attributing to human actions or omissions one's bodily inability to perform posthuman actions. Thus, transhumanist reiterates in a particularly concrete form the questions of freedom and oppression wrestled with by Berlin.IV.MATERIALIZATION OF THE SELF AND TWO RESPONSIBILITIES: A NEW DISTINCTIONThis transhuman materialization precipitates the hitherto abstract “self” to be realized into a material, though malleable, body or brain. Then the suffering of the “sheep” becomes not an act of fate beyond human power (as it was considered in the early lassaiz-faire period) but a human responsibility because of our matter manipulating technology. This apparently strengthens the argument against the absence model of freedom under which not only would the disadvantaged be abandoned but also now their disadvantage as well as their abandonment could be directly attributed to others’ omissions.11But now there are two responsibilities: the disadvantage responsibility and the non-abandonment responsibility. We may reason that if we can ameliorate disadvantage, we should, to fulfill our disadvantage responsibility. But exactly this benevolent (though self-serving) intent also leads to coercion and oppression. Consider instead who is relieved of this responsibility when the person concerned voluntarily accepts it, for example, when a Deaf person freely chooses to remain Deaf even when speech reception technology is available to them. This transfer of responsibility is how morphological freedom manages disadvantage. Society has no further obligation to “cure,” enhance, or encourage self-enhancement for those disadvantaged by freely chosen morphologies.We do retain a strong obligation to continue with enhancement research for the benefit of others with the same disadvantage who, voluntarily, do not choose to retain it. Our non-abandonment responsibility is unaffected by either choice and implies support for the ends of those who choose to retain a disadvantage just as we support the ends of other members of society within the constraints of just resource allocation.In our view, there is also a further set of constraints on morphological freedom. They can be derived from consideration of a model society within which morphologically homogenous, economically independent subgroups interact in a structured way supported by a neutral state. In this model, there is no single “basic cooperative framework” (Buchanan et al., 2000, 20, 288–303). Instead, multiple cooperative frameworks, each representing different combinations of physical, social, and morphological factors, are coordinated through collectively financed state institutions.In this model, the efficiencies of morphological similarity are exploited within the subgroups, but technology and new political institutions ensure that the benefits of morphological diversity are available, collectively to the whole society and personally to individual members wishing to explore and take up other morphologies or lifestyles. Freedom to found new subgroups helps the society to explore a variety of morphology-environment fits. It should be noted that constraints on acceptable morphologies will still be present in this model, but they will be far fewer than at present in our single basic cooperative framework societies. Such multiple cooperative framework societies seem capable of supporting much greater levels of morphological freedom while minimizing the risks of both abandonment and tyranny.V.CONCLUSIONThrough the lens of transhumanist's attempts to address disability, we have glimpsed one of the schisms fundamental to the movement's history and future. The fault line lies between freedom as the absence of interference with people's existing ends—a pluralist, voluntarist, liberal conception, compatible with disability studies’ models of disability—and the less compatible, partial—monistic, rationalist, objectivist—conception of freedom. This conceptual gap has not grown with the advent of greater understanding of human psychology, neuroscience, or biotechnology; it has just become more material.Commercialization of recent technological innovations may make the political importance of this schism greater than at any time since Berlin wrote. Transhumanist literature such as Hughes (2004), Stock (2003), and Naam (2005) demonstrates that sociopolitical understanding is still stymied by humans’ inability to imagine how morphological fluidity (MacKenzie, 2008, 399) will affect their identities and value systems (Scully, 2008). Subjective experience, by analogy or experimentation, may be the only way out of the impasse. Meanwhile, reducing the risks posed by oppression, tyranny, despotism, abandonment, and the use of force against discontent requires more diligent study of existing reports, theory, and history of value change. Attention to available data may yet enable technological, political, and social progress to continue without war's destruction, fueled by perceived oppression, and conducted with the full force of our crude, present or near future, levels of technological understanding.AtkinsonRI hoped our baby would be deafThe Guardian2006BaumanH.-D. LDesigning deaf babies and the question of disabilityJournal of Deaf Studies and Deaf Education2005103115BerlinINHardyHTwo concepts of libertyIsaiah Berlin Liberty1958Oxford, UKOxford University Press166217———HardyHIntroduction to five essays on libertyIsaiah Berlin Liberty1969Oxford, UKOxford University Press355BostromNTranshumanist FAQ version 2.1 [On-line]2003World Transhumanist AssociationAvailable: http://www.transhumanist.org/resources/FAQv21.pdf (Accessed December 29, 2008)———Human genetic enhancements: A transhumanist perspectiveJournal of Value Enquiry200337493506———Letter from UtopiaStudies in Ethics, Law, and Technology200827BradshawHGDefining enhancement, disability and therapy: how technology affects identity and the ethical implications of thisPhD Dissertation, University of Bristol, forthcomingBuchananABrockDDanielsNWiklerDEFrom chance to choice: Genetics and justice2000CambridgeCambridge University PressChristmanJSaving positive freedomPolitical Theory: An International Journal of Political Philosophy2005337988GarnettMIgnorance, incompetence and the concept of libertyJournal of Political Philosophy20071542846GutridgeKSafer self-injury or assisted self-harm?Theoretical Medicine and Bioethics2010317992HarrisJEnhancing evolution: The ethical case for making better people2007Princeton, NJPrinceton University PressHughesJCitizen cyborg: Why democratic societies must respond to the redesigned human of the future2004Cambridge, MAWest View Press, Perseus Books GroupKahaneGSavulescuJBrownleeKCuretonAThe welfarist account of disabilityDisability and disadvantage2009Oxford, UKOxford University Press1452MacKenzieRSomatechnics of medico-legal taxonomies, elective amputation, transableism and functional somatic syndromesMedical Law Review200816123MiahALetter to Utopia: A reply to BostromStudies in Ethics, Law and Technology200827MurphyFDraft letter to Professor Marcus Pembrey2008 [On-line]. Available: http://www.grumpyoldeafies.com/2007/11/hfeb_bda_draft_letter_to_profe.html (Accessed October 8, 2009)NaamRMore than human: Embracing the promise of biological enhancement2005New YorkBroadway BooksOxford English Dictionary onlineOxford, UKOxford University PressAvailable: http://www.oed.com/ (Accessed October 7, 2009)ParfitDReasons and persons1984Oxford, UKOxford University PressSandbergAMorphological freedom—Why we not just want it, but need it2001[On-line]. Available: http://www.nada.kth.se/∼asa/Texts/MorphologicalFreedom.htm (Accessed February 26, 2009)SavulescuJDeaf lesbians, “designer disability” and the future of medicineBritish Medical Journal20023257713Sims BainbridgeWRoccoM———Justice, fairness and enhancement Progress in convergenceAnnals of the New York Academy of Science2006109332138SavulescuJBostromNHuman enhancement2009Oxford, UKOxford University PressSavulescuJKahaneGThe moral obligation to create children with the best chance of the best lifeBioethics20082327490ScullyJ. LDisability bioethics: Moral bodies, moral differences2008Lanhan, MDRowman and LittlefieldStockGRedesigning humans: Choosing our genes, changing our future2003Boston, MAMariner BooksTakalaTConcepts of “Person” and “Liberty,” and their implications to our fading notions of autonomyJournal of Medical Ethics20073322581For the purposes of this paper, we will consider enhancement to include any intervention that takes an individual beyond the level they have previously experienced for that ability. Therapy, in contrast, returns an individual to an ability level they had previously experienced. In the case of children, for the purposes of this paper we will take enhancement to be any intervention on the part of the parents to deliberately produce offspring who have greater abilities than either of the parents do or to increase the probability with which the offspring are likely to carry a particular trait that at least one of the parents has experience of. We would consider it unethical to deliberately cause offspring to carry traits considered to be detrimental to life success by the parents and which neither parent has personal experience of. This leaves open the risk that some traits considered by the parents to be enhancing will prove to be detrimental. It also allows parents who live with a trait considered undesirable by those without it to deliberately pass this trait on to their children.2And enshrined in UK law at present in the form of the illegality of implanting embryos known to have an “abnormality” which involves a “significant risk” of the development of a “serious” “disability,” “illness,” or “any other serious medical condition.” (Human Fertilisation and Embryology Act 2008 c.22. Clause 14 Subsection (9)). People identifying as Deaf do not consider Deafness to be a medical condition of any sort and certainly not a disability or illness.3Sandberg was the main author of Version 1 of the Transhumanist FAQ. See S7, Acknowledgments and Document History, within Bostrom (2003a).4Later the same page he acknowledges that the benefits of respect for reproductive freedom would allow us to ignore the few such cases of “bizarre” choices because the resulting harm done would be small in comparison to the overall benefits of reproductive freedom. This seems ad hoc and in need of empirical confirmation though we agree the numbers involved may be quite small.5Neither they nor we feel that these fears and risks justify preventing the development of technologies with such potential for ameliorating involuntary suffering and increasing achievement. Indeed, we would all argue strongly for more support for research in these areas.6Transhumanist is often seen as a theory of physical self-realization.7Despite common speech not everyone affected suffers from such differences.8Some such work has been done. See Savulescu and Bostrom (2009, 8).9We use Deaf where a clear allegiance to the Deaf community is known to be present and deaf where such an allegiance cannot be assumed. We also differentiate between hard of hearing but orally oriented and deaf and/or Deaf in the sense of nonorally oriented persons who may feel a greater or lesser allegiance to the Deaf community.10This is a version of Derek Parfit's nonidentity problem. Parfit (1984, 351–90).11We are using “disadvantage” here to refer to those whose competitive advantage may be considered low in their society, for example, those with body morphologies today considered “impaired” or those who are not “enhanced.” That is, those with a smaller range of abilities or a lower level of key abilities than is usual in their society. We would like to contrast this sense of “disadvantaged” with what is implied by “disabled” in the social model of disability—the negative effects of an impairment that are due not to the impairment itself but to the social circumstances in which the impaired individual finds themselves. The latter have always been within society's power to change and thus have always been the responsibility of society to ameliorate. But the impairment was until now often accepted as being beyond human power to alter. That is no longer the case. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B6423DC51059B6040DE8A5A383991C474FAB3B6.txt b/test/dataset/in/resources/corpus/Clean_0B6423DC51059B6040DE8A5A383991C474FAB3B6.txt new file mode 100644 index 0000000..d2d1017 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B6423DC51059B6040DE8A5A383991C474FAB3B6.txt @@ -0,0 +1 @@ +]>MAD1887S0047-6374(97)01887-310.1016/S0047-6374(97)01887-3Elsevier Science Ireland LtdFig. 1Proliferative responses of PBMC from 14 centenarians, 10 middle-aged and 16 young controls to soluble anti-CD3 mAb (25 ng/ml) or PMA (5 ng/ml).Fig. 2Proliferative responses of purified T cells from 14 centenarians, 10 middle-aged and 16 young controls to PMA (5 ng/ml); PMA added to immobilized anti-CD28 mAb, immobilized anti-CD3 mAb or to coimmobilized anti-CD3 plus anti-CD28 mAb.Table 1Cytofluorimetric analysis of T lymphocytes from centenarian, middle-aged and young subjectsCentenariansMiddle agedYoung adultsMean age and number of subjects101.1±0.4 (10)63.5±1.9 (17)28.8±0.9 (24)% of CD28+ among T cells50.5±3.5**77.3±3.8**90.25±1.4Number of CD28+ T cells per μl583±47**1138±70**1558±47CD28 median fluorescence channel in linear scale385±13.9358±21372±18Data are reported as mean values±S.E.M.PBMC were double stained with FITC-conjugated mAb anti-CD3 and PE-conjugated mAb anti-CD28 as described in Section 2.Analysis of CD28 expression was obtained by Lysis II® software.Statistical analysis was done by using the ANOVA Fischer test.**P<0.01 vs. either of the two other groups.T lymphocyte proliferative capability to defined stimuli and costimulatory CD28 pathway is not impaired in healthy centenariansPaoloSansonia*FrancescoFagnoniaRosannaVescoviniaMarcoMazzolaaVincenzoBriantiaGiovanniBolognaaEnricaNigroaGiampaoloLavagettoaAndreaCossarizzabDanielaMontibClaudioFranceschibcMarioPasseriaaIstituto di Clinica Medica Generale e Terapia Medica, University of Parma, via Gramsci 14, Parma 43 100, ItalybDipartimento di Scienze Biomediche, Sezione di Patologia Generale, University of Modena, via Campi 287, Modena 41 100, ItalycINRCA, via Birarelli, 8, 60 100 Ancona, Italy*Corresponding author. Tel.: +39 521 290783/4; fax: +39 521 290776.AbstractIt is generally assumed that T cell proliferation is impaired in aged individuals. We report data on the proliferative capability of peripheral blood mononuclear cells (PBMC) and T lymphocytes from 40 healthy people of different ages, (19–107 years), including 14 centenarians, to defined mitogenic stimuli. We observed no age-related proliferative impairment both in PBMC and in purified T cells stimulated by anti-CD3 mAb or phorbol myristate acetate (PMA). Furthermore, T cells stimulated by anti-CD3 mAb or PMA and costimulated by CD28 mAb did not proliferate differently among young, middle aged subjects and centenarians. Thus, short term T cell proliferation is not affected even at extreme age when well defined stimuli are used on cells deriving from carefully selected healthy subjects.KeywordsCentenariansT cellsCD28Proliferation1IntroductionAn old tenet in medicine is that aging is accompanied by a deterioration of the immune system that could contribute to increased risk of morbidity and mortality [1–4]However, the study of carefully selected healthy aged subjects showed that most immune functions are well preserved even in advanced age [5, 6]. We have shown that several immune responses are well preserved in healthy centenarians in comparison to middle-aged and young subjects[7, 8].A very important function which has been suggested to deteriorate with age and to play a major role in the aging process is the capability of cells from aged subjects to respond to mitogenic stimuli and, consequently, to undergo cell proliferation [9]. A defect in this function is very important for immune cells, whose capability to undergo clonal expansion is critical to set up an effective response to antigenic stimuli [10]. Accordingly, we thought it worthwhile to assess the proliferative capability of peripheral blood mononuclear cells (PBMC) and purified T lymphocytes from healthy centenarians in comparison to middle-aged and young subjects. Our results demonstrate that this function is well preserved when defined stimuli, such as anti-CD3 mAb or phorbol 12-myristate acetate (PMA), are used. This conclusion is further supported by experiments showing that the costimulatory pathway via the CD28 molecule is well preserved in centenarians.2Materials and methods2.1Subjects.We studied a total of 14 centenarians with a mean age of 100.8±0.3 years (range, 100–107 years), 3 males and 11 females. All subjects were in relatively good clinical condition without relevant acute or chronic disease affecting the immune system and mentally competent to give informed consent. In particular, none of the subjects had cancer, were suffering from serious cardiac, brain or kidney disease or taking drugs known to affect the immune system. Along with a complete social and medical history, we performed a physical examination. As control groups we studied 10 healthy middle-aged subjects with a mean age of 58.2±2.1 years (range, 48–68 years) and 16 healthy young donors with a mean age of 28.1±0.6 years (range, 19–36 years). All these control subjects were selected according to the SENIEUR protocol [5, 6].2.2Monoclonal antibodies.MAb to CD3 (OKT3) was generously provided by Dr E. Engleman, Stanford University (Stanford, CA); anti-CD8 conjugated with fluorescein (FITC), anti-CD28 conjugated with phycoerythrin (PE) and isotype-matched control mAb were purchased from Becton Dickinson (San José, CA).2.3Cell preparation and enrichment of T lymphocytes.PBMC were obtained by Ficoll-Hypaque gradient centrifugation from freshly drawn venous blood collected around 08:00 from a centenarian, a middle-aged and a young control on the same day. After washings with PBS, the cells were suspended in RPMI-1640 with 10% AB serum, 2mM l-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, hereafter referred to as complete medium. PBMC were fractionated into T and non-T cells by a single step rosetting method [11]. T cells were separated from sheep red blood cells by hypotonic lysis of the latter. T cells were further depleted of monocytes by adherence (1 h) to plastic. The purity of T cells preparation was always >95% as assessed by cytofluorimetric analysis.2.4Proliferation assays.All proliferation assays were performed in round-bottomed microtitre wells in a final volume of 0.2 ml complete medium. Stimulation of 1×105 PBMC with soluble anti-CD3 mAb (25 ng/ml) or 5 ng/ml PMA was carried out for 3 days at 37°C in 6% carbon dioxide–air. Purified T cells were stimulated with immobilized mAb as follows: 100 μl anti-CD3 (0.1 μg/ml) and/or 100 μl anti-CD28 (5 μg/ml), diluted in PBS, were placed in 96 wells microtiter plates and incubated at room temperature overnight and then washed with PBS. T cells were challenged with several stimuli or a combination of them: immobilized anti-CD3; immobilized anti-CD3+immobilized anti-CD28; 5 ng/ml PMA; PMA+immobilized anti-CD28 as specified in the figure legends. Assays were performed in triplicate and 0.5 μCi [3H]thymidine ([3H]TdR) was added to each well 6 h before cell harvesting on glass fiber filter paper. Uptake of [3H]TdR was measured in a liquid scintillation counter and the results expressed as the mean counts per min±S.E.M. (cpm±S.E.M.).2.5Cytofluorimetric analysis.Cytofluorimetric analysis was performed on PBMC following standard methods [12]. Briefly, 1×106 PBMC were incubated with 1 μg anti-CD8 FITC and anti-CD28 PE for 20 min at 4°C, washed with PBS and suspended in 200 μl PBS until analysis. Two colors FACS analysis was performed on a FACScan cytofluorimeter (Becton-Dickinson, CA) as previously described [12]. The expression per cell of CD28 molecule was calculated by flow cytometry using PE-conjugated anti-CD28 mAb, considering the mean fluorescence channel of each histogram, as described [13].2.6Statistical analysis.Statistical analysis was performed by ANOVA and Fisher test by SPSS for Windows® software. P<0.05 was considered significant.3Results3.1Proliferative responsiveness of PBMC and purified T cells.When PBMC were stimulated by defined mitogenic stimuli, such as PMA or anti-CD3 mAb, no significant difference concerning the proliferative capability was observed among cells from young, middle-aged and centenarians (Fig. 1). PBMC are mainly composed of T and B lymphocytes, NK cells and accessory cells such as monocytes. It is well known that optimal T cell responsiveness requires a primary and a costimulatory signal that usually is delivered by accessory antigen presenting cells through the B7 molecule which is capable of interacting with the CD28 molecule on the T cell membrane [14]. Accordingly, purified T cells from young, middle aged and centenarians were stimulated by the above mentioned primary stimuli (PMA or anti-CD3 mAb) and costimulated by anti-CD28 mAb in order to activate the most important costimulatory pathway in T cells [15]. Fig. 2 shows that costimulation via anti-CD28 mAb significantly increased T cell responsiveness to PMA or anti-CD3 mAb in cells from all groups including centenarians and again no significant age-related difference was found.3.2CD28 expression.Since the target of anti-CD28 mAb is the CD28 molecule on the T cell membrane we performed a cytofluorimetric analysis of resting lympocytes in order to study the expression of this molecule on T lymphocytes from centenarians, middle-aged and young controls.The results indicate that the costimulatory capability of anti-CD28 mAb is not likely to be related to the percentage of CD28+ cells in culture. Indeed Table 1 shows that the percentage of CD28+ cells is significantly reduced in centenarians, whose cells, however, responded well to costimulation with anti-CD28 in the presence of PMA or anti-CD3 mAb (Fig. 2). Table 1 also shows that, notwithstanding the decrease in the percentage of CD28+ lymphocytes, the expression of CD28 molecules per cell was, however, unchanged.4DiscussionPrevious studies indicate that a profound remodelling of the immune system occurs with age [7]. In particular we have reported that, in the peripheral blood of aged people including centenarians, there is a progressive increase in serum level of IgA, IgG1, IgG2, IgG3 but not of IgM or IgG4 [16], a decrease in absolute number of T lymphocytes (CD3+) involving both CD4+ and CD8+ subsets, a marked decrease of B lymphocytes and an increase of cells with natural killer (NK) markers [8, 17]. We have also found that healthy elderly and centenarians are almost free of organ-specific autoantibodies [18, 19]and equipped with very well preserved cytotoxic activities (NK, anti-CD16 and anti-CD3 redirected killing activities) [8, 17, 20].An important and extensively studied immune function is lymphocyte capability to undergo cell proliferation. Several reports indicate that this function is impaired with age [1, 2]. The reason for this defect has been variably referred to among T cell subsets, an imbalance [21], a reduced proliferation of CD8+ T cells [22], a failure to produce IL-2 [23]or to a decreased number of cells capable of completeing the S phase of the cell cycle [24]. However, most of the previous studies refer to the capability of PBMC to proliferate when stimulated with PHA, a mitogen used to assess such a function since 1960 but whose fine mechanism is not completely understood [25]. The available data indicate that PHA induces activation of T cells in a complex way through cross-linking of several surface structures, including CD2, CD3, CD5 molecules [26, 27]and perhaps others. Thus, we thought it worthwhile to study the proliferative capability of either PBMC or purified T cells from people of different ages using more defined stimuli acting at different levels of the signal transduction cell machinery, i.e. plasma-membrane (anti-CD3) or transmembrane (PMA). When this approach was used no proliferative defect was evident with both stimuli in cells from any group, including people who are close to the maximum life span such as subjects over 100 years of age. These data are in accord with earlier reports in unseparated PBMC from subjects aged between 70 and 82 years [28, 29].We tried to further dissect the pathways known to be involved in lymphocyte proliferation and particularly the main costimulatory T-cell pathway via the CD28 molecule. To this end we used anti-CD28 mAb to act as surrogate for this most important contribution by accessory cells to T cell activation [14]. This system has the advantage of optimizing the lymphocyte proliferative conditions, indeed the ligation of CD28 molecules increases the expression of IL-2 receptors and IL-2 availability [15, 30, 31]which, in turn, are thought to be defective in lymphocyte cultures from elderly subjects [23, 32]. Under these conditions, costimulation via CD28 significantly increased T lymphocyte proliferation in middle-aged and young controls, as well as in centenarians, and this phenomenon was of the same extent in the three groups.According to the two signal theory, T lymphocyte activation derives from MHC-peptide specific engagement of TCR-CD3 and contestual crosslinking of non antigen-specific accessory molecules such as CD28 [33, 34]. Our data indicates that both CD3 and CD28 pathways are well preserved in T cells from aged people including centenarians. Our results suggest that the cell machinery responsible for the transduction of primary and costimulatory signals is functioning well even in cells from these subjects of very advanced age. The results presented are in apparent contrast with the data in the literature indicating that lymphocytes from elderly people show a proliferative defect when complex stimuli such as PHA [2]or alloantigens are used [35]. The defective response to PHA could be related to the complexity of this stimulus (proliferation of different T cell subsets? Activation-induced cell death? Induction of cell nonresponsiveness?).The results shown here suggest that, when cells from carefully selected aged subjects are used and stimulated with well defined stimuli T lymphocyte proliferation is not impaired as previously thought and that some important activation pathways, such as those involving CD3, are well preserved into the last decades of life. Furthermore, the efficient costimulation through CD28 molecules in PMA and in anti-CD3 stimulated cultures indicate that the costimulation through the CD28 pathway is not impaired in centenarians despite the reduced number of CD28 positive cells with age.About 1/3 of centenarians, despite their very advanced age, are still in good mental and physical condition. These subjects are the best example of successful aging, being free of the major age-related diseases. Therefore, healthy centenarians are a model of human longevity and constitute a very select group of exceptional individuals. The current observation of a well preserved T cell proliferative capability involved in adaptive immune responses, together with previous observations concerning well preserved innate immunity, suggests that such a well equipped immune system may contribute to longevity in human and, on the other hand, indicate that the immune system of healthy centenarians is still apparently capable of coping with infectious diseases.In summary, we suggest that a generalized age-related defect in the cell proliferative machinery can be excluded and that important activation pathways are intact in lymphocytes even in the last decade of life. The data presented here fit the hypothesis that aging is not characterized by unidirectional deterioration but by a complex remodelling of immune functions [7, 36].AcknowledgementsThis work was supported by the National Research Council (CNR) target project `Ageing', M.U.R.S.T. 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Immunol., 12 (1995) 57–74. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B69B273F75DCBF65BAAB6506108A3F83D71D665.txt b/test/dataset/in/resources/corpus/Clean_0B69B273F75DCBF65BAAB6506108A3F83D71D665.txt new file mode 100644 index 0000000..1304f7b --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B69B273F75DCBF65BAAB6506108A3F83D71D665.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 358 10.1093/geront/48.3.358 PHYSICAL ACTIVITY Long-Term Effects of a Stage-Based Intervention for Changing Exercise Intentions and Behavior in Older Adults Greaney Mary L. PhD 1 Riebe Deborah PhD 2 Ewing Garber Carol PhD 3 Rossi Joseph S. PhD 8 4 Lees Faith D. MS 5 Burbank Patricia A. DNSc, RN 6 Nigg Claudio R. PhD 7 Ferrone Christine L. MS 8 Clark Phillip G. ScD 5 Address correspondence to Mary L. Greaney, PhD, Public Health Nutrition, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115. E-mail: mgreaney@hsph.harvard.edu 6 2008 48 3 358 367 12 9 2007 5 1 2007 Copyright 2008 by The Gerontological Society of America 2008 Purpose: We examined the efficacy of an intervention tailored to the individual's stage of change for exercise adoption on exercise stage of change, physical activity, and physical function in community-dwelling older adults. Design and Methods: We randomized participants to a print and telephone intervention or a contact comparison group. Through the use of longitudinal analyses we examined the intervention's effectiveness in promoting stage progression, altering decisional balance and the processes of change, increasing self-efficacy and physical activity, and improving physical function among older adults who completed the 24-month study (N = 966). We conducted similar analyses that excluded individuals who were in maintenance at baseline and 24 months. Results:  At the end of the study, there were no differences in stage progression, self-efficacy, decisional balance, the processes of change, physical activity, or physical function by intervention assignment. When the analyses excluded those participants (n = 358) who were in the maintenance stage for exercise throughout the intervention, we found that, compared with the comparison group, a greater proportion of individuals who received the exercise intervention progressed in stage by 24 months. Conversely, more individuals in the comparison group remained stable or regressed in stage compared with the intervention group. Implications: Results indicate that a tailored intervention is effective in increasing motivational readiness for exercise in individuals who were in stages of change other than maintenance. Behavior change Exercise Health promotion Intervention Transtheoretical model hwp-legacy-fpage 358 hwp-legacy-dochead RESEARCH ARTICLE Regular physical activity is associated with a decreased risk of functional limitation among older adults, a delay or decrease in the incidence of chronic health problems, an improved quality of life, and a reduced risk of falling (Miller, Rejeski, Reboussin, Ten Have, & Ettinger, 2000). Despite the known physiological and psychological benefits of a physically active lifestyle, physical inactivity remains a major health problem among older adults in the United States, with a majority of older adults failing to reach the minimum recommended levels of physical activity (Kamimoto, Easton, Maurice, Husten, & Macera, 1999; Kruger, Carlson, & Buchner, 2007). Physical activity interventions that include older adults and use an assortment of behavioral and educational strategies have been somewhat effective in increasing the adoption of physical activity (Hillsdon, Foster, & Thorogood, 2005; Marcus et al., 2006). Many intervention studies with older adults have provided organized, center-based exercise classes for a small number of participants (e.g., DiBrezzo, Shadden, Raybon, & Powers, 2005), but several studies of middle-aged and older adults have shown that most of these individuals prefer to do their physical activity outside a formal setting, and home-based exercise has higher adherence rates (Ashworth, Chad, Harrison, Reeder, & Marshall, 2005; Juneau et al., 1987; King et al., 2000; King, Haskell, Taylor, Kraemer, & DeBusk, 1991). Several theories of behavior change have been used to develop interventions to promote physical activity, including the transtheoretical model of health behavior change (TTM). The TTM is an integrative theoretical model that uses individual decision-making processes to explain intentional health behavior change (Prochaska & Velicer, 1997). It has been used to tailor interventions to increase physical activity and exercise in diverse populations (Jones et al., 2001; Marcus et al., 1998), and it has been validated for use in community-dwelling older adults (Barké & Nicholas, 1990; Riebe et al., 2005). Although it has been demonstrated that interventions based on the TTM are effective in increasing exercise under some circumstances, it has not been applied to a large-scale community-based intervention in older adults. Our purpose in this study was to determine whether an exercise intervention tailored to an individual's stage of change for exercise would be effective in progressing stage of change, increasing utilization of TTM constructs (self-efficacy, decisional balance, and the processes of change), increasing physical activity behavior, and improving physical function in community-dwelling older adults. Methods Participants The study participants were volunteers for the Study of Exercise and Nutrition in Older Rhode Islanders (SENIOR) Project, a community-based health-promotion study designed to increase fruit and vegetable consumption and exercise among community-dwelling older adults (Clark et al., 2005). We recruited participants into the study over a period of 14 months by using a variety of methods, including direct mailings, advertisements, flyers, and presentations given at senior housing sites (Greaney, Lees, Nigg, Saunders, & Clark, in press). Study entry criteria included being 60 years of age or older and residing in the greater East Providence, Rhode Island area. We excluded those individuals who were not living independently in the community. Participants completed the Physical Activity Readiness Questionnaire as modified for older adults, which identifies participants who should seek out medical consultation before engaging in a new program of exercise (Thomas, Reading, & Shephard, 1992). Participants who answered “yes” to any of the questions on the Physical Activity Readiness Questionnaire were strongly urged to check with their health care provider prior to entering the study, but medical clearance was not required. The University of Rhode Island's Institutional Review Board approved the study, and all participants provided informed consent. Design The SENIOR Project consisted of a 12-month intervention followed by a 12-month nonintervention observational period (Clark et al., 2005). We randomly assigned participants to one of four interventions: Group 1 increased fruit and vegetable consumption; Group 2 increased exercise; Group 3 increased both exercise and fruit and vegetable consumption; and Group 4 was a contact-comparison group. In this article we examine the effects of the intervention designed to increase exercise, so we combined the four groups to form two: an intervention group consisting of all participants who received the intervention for increasing exercise (Groups 2 and 3) and a comparison group consisting of participants who did not receive the intervention for increasing exercise (Groups 1 and 4). Data reported here were collected at baseline, 12 months, and 24 months. Intervention The SENIOR Project's intervention components have been discussed in detail elsewhere (Clark et al., 2005). Briefly, we developed the interventions on the basis of the TTM, and they included a series of written materials and counselor calls designed to encourage movement through the stages of change to increase exercise, fruit and vegetable consumption, or both. The tailored materials included behavior-specific manuals, newsletters, expert systems reports, and coaching calls. We contacted participants monthly by means of print or telephone contact throughout the 12-month intervention period. For those participants who received the exercise intervention, we strongly encouraged them to adopt a habitual pattern of intentional physical activity (exercise) performed at home or in formal exercise settings consistent with the recommendations for apparently healthy adults by the American College of Sports Medicine (1998). This guideline recommends several sessions per week of aerobic (cardiorespiratory), flexibility, and muscle-strengthening exercises. We chose these criteria for physical activity because this level of activity has been demonstrated to improve physical fitness, physical function, and general health, which are particularly important in older adults (American College of Sports Medicine). In the intervention's print materials and during the coaching calls, we encouraged participants to engage in aerobic exercise of moderate to vigorous intensity, such as brisk walking, cycling, swimming, and exercising in aerobic exercise classes or on exercise machines, for 3 to 5 days per week for a minimum of 20 minutes. We recommended flexibility exercises, such as stretching, yoga, Pilates, and tai chi, for at least 2 days per week, and we encouraged muscle-strengthening exercises such as weight lifting, calisthenics, and resistance equipment for 2 to 3 days per week. We gave participants information on how to do these exercises at home and we encouraged them to use exercise facilities and to join formal exercise programs available within the community. Details of the intervention components follow. Manual At the start of the intervention, each participant received an intervention-specific manual that was organized by stages of change and that incorporated stage-specific behavioral strategies commonly employed to advance to the next stage. The manual also included information about the benefits of physical activity, what types of exercise are recommended, how to safely engage in exercise, and a listing of community resources for exercise, including formal exercise programs. Individuals not receiving the exercise intervention received either a manual about fruit and vegetable consumption or a fall-prevention manual; neither of these manuals included any information about physical activity or exercise. Newsletters We sent intervention-specific newsletters to participants on a monthly basis, except for Months 4, 8, and 12, when they received an expert system report (subsequently described). Newsletters were stage specific, and participants received the newsletter designed for their stage of change as determined during the evaluations conducted at baseline and at 4 and 8 months. The newsletters included stories about older adults, practical tips about exercise, suggested physical activities, interactive sections, and local resources for exercise. The newsletters addressed stage-appropriate processes of change, and they included material to effect positive changes in self-efficacy and decisional balance. Expert System Assessments and Reports During the 12-month intervention, we collected data on TTM constructs and other behavioral and health information at baseline and at 4 and 8 months. On the basis of the participants' responses, a computer-based expert system generated a 4- to 5-page report that was mailed directly to each participant's home. Reports provided normative (compared with peers in the same stage of change, or SOC) and ipsative (based on current and previous responses from the individual) feedback on the individual's physical activity behavior to encourage continued positive behavior change. Coaching Calls Participants received three 15-minute coaching calls from trained counselors during the 12-month intervention period, approximately 4 to 6 weeks after they received their expert system reports, which the counselors used to guide calls. Counselors used a standardized protocol incorporating motivational interviewing strategies in a stage-matched manner (Rollnick, Heather, & Bell, 1992). Demographics and Overall Health Participants provided demographic information, including gender, age, race or ethnicity, and educational level. Participants also completed the Medical Outcomes Study 36-item Short Form, and the answer to the first question on this form; we used the General Health subscale (general health perception) to assess perceived health status (McHorney, Ware, and Raczek, 1993). The first measure is categorical and we used it for descriptive purposes; we used the latter measure as a covariate in the statistical analyses. Outcome Measures Participants completed a baseline assessment in person at their home or in the project office. We had the assessment administered by trained project staff members who were of similar age and ethnicity as the participants and from the intervention community. The project staff members administered the assessment before randomization (baseline), at the end of the 12-month intervention period, and 12 months after the completion of the intervention (24 months), during which no intervention was delivered. The present study includes measures obtained at baseline and at 12 and 24 months; they are described in the paragraphs that follow. TTM constructs Measuring SOC for exercise is ideal for gaining a better understanding of physical activity and exercise behavior, because it focuses on intention and perception of behavior rather than actual behavior (Nigg & Riebe, 2002). We measured all TTM constructs (SOC, self-efficacy, decisional balance, and processes of change) by using instruments validated in community-dwelling older adults, and details of these instruments follow here. SOC for Exercise We determined SOC for regular aerobic exercise by using a validated questionnaire and scoring algorithm (Nigg & Riebe, 2002). The criterion used for SOC for exercise was the volume of exercise recommended by the American College of Sports Medicine (1998), which recommends aerobic exercise of moderate to vigorous intensity, lasting at least 10 minutes, for a total of 20 to 60 minutes per day, 3 to 5 days per week. This criterion is consistent with the exercise message used in the intervention and with the U.S. Surgeon General's recommendations for physical activity (U.S. Department of Health and Human Services, 1996), which emphasizes moderate to vigorous physical activity on most days of the week. On the basis of their responses to the questionnaire, we classified participants into one of five stages. Participants classified in the precontemplation stage had no intention to begin exercising in the next 6 months, whereas those in the contemplation stage intended to begin exercising within the next 6 months. Individuals in the preparation stage intended to change within the next month. Those in action had been exercising regularly over the past 6 months, and participants in the maintenance stage had been exercising regularly for 6 months or more (Nigg & Riebe, 2002). Self-Efficacy The six-item exercise self-efficacy scale measured a participant's confidence in his or her ability to exercise despite adverse or challenging situations (Benisovich, Rossi, Norman, & Nigg, 1998). Self-efficacy scores range from 1 to 5, with higher scores indicating greater self-efficacy. Decisional Balance (Pros and Cons) This two factor, ten-item Likert-format scale measures the pros (advantages) and cons (disadvantages) of adopting or maintaining exercise (Nigg, Rossi, Norman, & Benisovich, 1998). Scores range from 1 to 5, with higher scores demonstrating more exercise pros or exercise cons. Processes of Change This questionnaire includes 30 statements that participants are asked to rate in terms of frequency of occurrence over the past month on a 5-point Likert scale ranging from 1 = never to 5 = repeatedly (Nigg & Riebe, 2002). The questionnaire contains three items for each of the 10 processes of change and provides individual scores (ranging from 1 to 5) for each process. For analyses, we divided the processes into two groupings—experiential and behavioral processes. Scores range from 15 to 75, with higher scores indicating greater use. Physical Activity Behavior We measured physical activity by using the Yale Physical Activity Survey (YPAS). The YPAS is a validated survey for older adults that is designed to assess exercise, household, and recreational physical activity during a typical week in the previous month (DiPietro, 2001). We calculated five activity dimensions: vigorous activity, leisurely walking, moving on feet, standing, and sitting. We then summed the results to calculate the YPAS summary score. A higher score indicates higher levels of physical activity. Physical Function We measured physical function by using the Timed Up-and-Go (TUG) test. The TUG test is a practical, reliable measure of functional mobility in older adults (Podsiadlo & Richardson, 1991). The TUG test measures, in seconds, the time taken by an individual to stand up from a standard armchair, walk a distance of 3 m, turn, walk back to the chair, and sit down again. TUG scores have been able to distinguish between older adults who need assistance in activities of daily living, those who are independent, and those with somewhat impaired mobility (Van der Bij, Laurant, & Wensing, 2002). TUG scores of less than 12 seconds reflect normal physical function in community-dwelling adults (Bischoff et al., 2003). Analysis We conducted all analyses by using SPSS version 15.0 (SPSS, Chicago, IL). We calculated means, standard deviations, and frequencies for demographic characteristics, health status, SOC, physical activity, and physical function variables at baseline by using SPSS descriptives. We set significance a priori at p <.05. The purpose of the analyses was to evaluate the intervention's effect on progressing SOC, promoting use of TTM constructs, increasing physical activity, and improving physical function. To examine SOC progression, we conducted chi-square analyses using three stage movement groups (progress, regress, stable) to test the hypothesis that stage progression differed by exercise intervention assignment at 12 and 24 months. We defined stage progression as an increase of one or more stages from baseline; we defined stage regression as a decrease of one or more stages from baseline. We categorized participants who remained in the same stage as being in the stable stage. Because there was a large proportion of participants who remained in a maintenance mode for exercise throughout the intervention who would not be expected to progress in stage, we also evaluated SOC progression while excluding participants who remained in maintenance at baseline and 24 months. We used a series of repeated measures analysis of covariance to answer these research questions: First, do self-efficacy, decisional balance, and processes of change scores differ in participants who received the intervention? Second, did the intervention have an effect on physical activity and physical functioning? We included the following covariates in all models: gender, age at baseline, and perceived health status at baseline. Time was the within-subject factor and intervention assignment was the between-subject factor. We also evaluated these questions while excluding participants who remained in maintenance at both time points (baseline and 24 months). Results Participants The results presented in this article include study participants who completed the 24-month evaluation (N = 966). This includes 21 people who completed the 24-month assessment but did not complete the 12-month assessment. Table 1 shows the characteristics of these people at baseline. Baseline Equivalence of Intervention Groups As shown in Table 1, there were no significant differences between individuals in the intervention and comparison groups based on age, education, gender, race, perceived health status, SOC, YPAS summary score, or TUG score. Overall, 58.2% (n = 562) of the total sample was in action or maintenance stages for exercise at baseline (56.4%, n = 265, of the intervention group and 59.9%, n = 297, of the comparison group). Adherence Of the 1,274 people enrolled in the study at baseline, 80.5% (n = 1,026) completed the 12-month follow-up, and 75.8% (n = 966) completed the 24-month evaluation. Analyses published elsewhere have examined differences between participants who withdrew and those who completed the study, and there were no differences in attrition by race–ethnicity or intervention assignment (Greaney et al., in press). Compared with participants who withdrew, participants completing the study were more likely to be female (77.8% vs 70.8%, p =.01), in the action or maintenance stages for exercise at baseline (80.1% vs 70.3%, p <.01), younger (76.2, SD = 6.8, vs 78.5, SD = 6.8; p <.001), and have more years of education (12.87 years, SD = 2.2, vs 12.11 years, SD = 3.4; p <.01; see Greaney et al.). Participants who dropped out of the study also had lower self-efficacy than did participants who completed the 24-month study (3.22, SD =.95, vs 3.37, SD =.90; p =.02). Program Outcomes With the exception of participants in the precontemplation stage who progressed in stage at 24 months, participants who received the intervention were no more likely to progress in stage, regress in stage, or remain stable in stage than were participants who did not receive the intervention. In addition, there were no differences in self-efficacy, decisional balance, and the processes of change by intervention assignment. Furthermore, there were no differences in physical activity behavior and physical function, as measured by the YPAS and TUG, respectively, by intervention assignment over the course of the study. Additional details about the program's outcomes are provided in the following paragraphs. Progress in Stage of Change Table 2 shows the proportion of participants in the intervention and comparison groups who progressed, regressed, or maintained SOC from baseline to 12 months and from baseline to 24 months. At 12 months, χ2(2, N = 941) = 2.02, p =.36, and 24 months, χ2(2, N = 966) = 5.61, p =.06, there were no differences between the intervention group and comparison group with respect to the proportion of individuals who progressed, regressed, or maintained stage. When we excluded participants who were in the maintenance stage at baseline and 12 months from the analysis (n = 378), the results remained unchanged, χ2(2, n = 563) = 2.01, p =.37. However, when we excluded participants (n = 358) who were in the maintenance stage throughout the 24-month period from this analysis, we found that a greater proportion of individuals who received the exercise intervention progressed in stage by 24 months compared with individuals in the comparison group. Consistent with this finding, more people in the comparison group than people in the intervention group remained stable or regressed in stage, χ2(2, N = 608) = 6.30, p =.04. A description of stage progress according to the baseline SOC by intervention assignment is shown in Table 3. Although not significantly different, a greater percentage of participants in the intervention group who were in contemplation, preparation, and action stages at baseline had progressed in stage at 12 months, compared with the comparison group. Overall, 22.3% of the intervention group progressed in stage by 12 months compared with 19.4% of the comparison group (p =.36). We found similar results at 24 months (27.2% vs 20.7%, p =.06), and this time individuals receiving the intervention who were in the precontemplation stage at baseline were more likely to progress in stage than individuals in the comparison group who were in precontemplation stage at baseline, χ2(2, n = 177) = 4.11, p = 04. TTM Constructs The means of the TTM constructs are shown in Table 4. With the exception of exercise pros at 24 months, there was no difference in the use of these constructs by intervention assignment over the course of the study. When we excluded individuals who were in the maintenance stage at baseline and 24 months from the analysis (n = 358), the results remained unchanged (data not shown). Behavioral Outcomes The adjusted means of the YPAS summary score and the TUG score at 12 and 24 months by intervention assignment are shown in Table 5. There were no significant differences between the intervention and comparison groups with respect to physical activity (YPAS summary score) or physical function (TUG score) at 12 or 24 months. When we excluded the individuals who remained in the maintenance stage at baseline and 24 months from the analysis (n = 358), the results remained unchanged (data not shown). Discussion There were no significant differences between the intervention group and the comparison group in the percentage of individuals in each of the SOCs for exercise at 12 months, at the end of the intervention period, or a long-term effect at 24 months. There were limited differences in stage progression between the intervention and comparison groups (individuals in the precontemplation stage who received the intervention were more likely to progress in stage than were individuals in the comparison group). When we excluded the large number of participants who remained in the maintenance stage for the entire study period from the analysis, there was a long-term effect at 24 months, so that individuals who received the exercise intervention were more likely to progress in stage and individuals in the comparison group were more likely to regress in stage or remain stable in stage. Concurrently, with the exception of exercise pros at 24 months, there were no differences in use of the TTM constructs, or any changes in physical activity or physical function throughout the intervention. It is intriguing that individuals in the precontemplation stage at baseline who received the intervention had significantly more stage progression than did similar individuals in the comparison group. This suggests that the intervention was more effective in individuals who were not considering becoming physically active at the start of the study. This could have important public health implications if confirmed in subsequent studies, as it suggests that the most sedentary older adults could benefit from a low cost, primarily print-based intervention. Sedentary individuals (in the precontemplation, contemplation, or preparation stages) were more likely to drop out of the study, although the drop-out rate among sedentary individuals was similar between the intervention group and the comparison group. Participants who dropped out of the study had lower exercise self-efficacy than did those who completed the study. Some studies of exercise adherence have reported that low levels of self-efficacy and readiness to change are associated with drop out from the exercise program (Young, King, Sheehan, & Stefanick, 2002), whereas higher levels of self-efficacy predict adoption and maintenance of exercise (Oman & King, 1998). Overall, individuals completing the study were younger and had more education than those who dropped out of the study; each of these variables is associated with higher levels of physical activity and better health (Bish, et al., 2005; U.S. Department of Health and Human Services, 1996). There were no differences between the intervention and comparison groups in YPAS summary score and TUG scores at 12 or 24 months, suggesting that the intervention was not effective in improving self-reported physical activity or physical function. The lack of effect of the intervention on these variables remained when individuals in maintenance at each time point were excluded from the analyses. The lack of effect on physical activity behavior and function is not surprising, because the primary outcome of this study was SOC, which reflects intentions for and perception of exercise behavior and does not necessarily reflect actual exercise behavior. For example, an individual who moved from precontemplation to contemplation or who remained in the maintenance stage, as a large portion of our participants did, would not be expected to change physical activity behavior and consequently physical function. Advancing age is associated with declines in physical activity, physical fitness, and physical function (American College of Sports Medicine, 2004). Interestingly, neither physical activity nor physical function declined significantly in our sample, as evidenced by the stable YPAS summary scores and TUG scores over 24 months. The mean scores on the TUG test are well below the 12-second cutpoint for normal physical function in community-dwelling older adults, demonstrating that our participants were particularly vigorous. We do not know if those who dropped out of the study were more likely to experience declines in physical functions and physical activity or be institutionalized, but this is certainly a possibility. The modest results found in our study of younger and older adults are consistent with the findings of previous studies in younger and older adults that used similar methodologies (Banks-Wallace & Conn, 2002; Conn, Valentine, & Cooper, 2002; Eakin, Glasgow, & Riley, 2000; Hillsdon et al., 2005; Kahn et al., 2002; Van der Bij, et al., 2002). A recent Cochrane review concluded that physical activity interventions have moderate to little effect (Hillsdon et al.). It is believed that physical activity interventions have had equivocal results in part as a result of methodological problems, including incomplete or inconsistent application of behavioral theory (Baranowski, Anderson, & Carmack, 1998; Epstein, 1998; Prochaska & Velicer, 1997). The SENIOR Project's intervention was designed with one theoretical model, the TTM, and it maintained theoretical integrity in both the development of the intervention and the outcome measurements. Nevertheless, the results were still modest, indicating the difficulty in understanding and altering this complex behavior. The main focus of our intervention was at the intrapersonal (individual) level; the intervention did not incorporate interpersonal and environmental factors that are thought to mediate physical activity and exercise behavior (King, 2001). In a very small subsample of our participants, we have shown that a number of personal and physical environmental factors affected physical activity behavior (King et al., 2006), but we are unable to evaluate this possibility in the context of the intervention. Print interventions have generally had a small effect on physical activity behavior (Marshall, Owen, & Bauman, 2004). The use of tailoring and adding other methods such as counseling in combination with print materials, similar to the methods used in the SENIOR Project, have improved the impact of print and other types of physical activity interventions (Kahn et al., 2002; Van der Bij et al., 2002). One difficulty with print interventions, such as the SENIOR project, is that it is difficult to assess the dose of the intervention that the particpants received; we were unable to assess the amount of time spent using the materials or ensure that people actually read and understood the materials. We did not screen for literacy or cognitive function, although it is known that cognitive decline is common in aging adults (Craik & Bialystok, 2006; Raz, Rodrigue, Kennedy, & Acker, 2007). A high proportion of our sample was in action or maintenance stages at the start of the study, which may have affected our ability to assess the impact of our intervention, because the participants perceived that they were already engaging in adequate amounts of exercise. Over 70% of the individuals in maintenance for exercise at baseline remained in maintenance at 24 months. These results are not unexpected, as a recent study of long-term maintenance of regular exercise (McAuley et al., 2007) reported that regular exercise behavior was a strong predictor of maintaining exercise over the long term. The high proportion of people in the maintenance stage is consistent with other studies of adults of all ages obtained in statewide samples in Rhode Island (Garber, Allsworth, Hesser, & Marcus, 2006; Laforge et al., 1999). When we excluded participants who were in the maintenance stage at both time points (baseline and 12 months, baseline and 24 months), we found that individuals in all SOCs in the exercise intervention group were more likely to advance in stage by 24 months. This was not an immediate effect of the intervention: At 12 months there were no differences between the intervention and comparison groups with respect to the proportion who progressed, regressed, or remained stable in stage. These results are similar to results found in studies of physical activity (McAuley et al., 1999) and smoking cessation (Prochaska et al., 2001) that have found that individuals who received behaviorally based interventions continued to improve their behavior after completion of the intervention, indicating that the effects of the treatment continued long after the end of the intervention. The consistency between the SOC and self-reported physical activity and independently assessed physical function provides further validation for the SOC for exercise, extending the results of previous studies (Schumann, Estabrooks, Nigg, & Hill, 2003; Schumann et al., 2002; Spencer, Adams, Malone, Roy, & Yost, 2006). Use of TTM Constructs There were no differences in the scores of the TTM constructs by intervention assignment. We were unable to detect differences between the intervention and comparison groups with respect to exercise self-efficacy, decisional balance, experiential processes, and behavioral processes at 12 and 24 months, even when we excluded the large number of participants who remained in maintenance throughout the intervention. These results are in contrast to other studies of exercise adoption and maintenance in older and younger adults, in which self-efficacy and the use of cognitive and behavioral strategies predict exercise behavior (Bock et al., 1997; Brassington, Atienza, Perczek, DiLorenzo, & King, 2002; Cheung et al., 2007; Kosma, Cardinal, & McCubbin, 2004; McAuley et al., 2007; Oman & King, 1998; Velicer, Norman, Fava, & Prochaska, 1999). However, it is possible that the number of participants who progressed or regressed in stage was inadequate for us to detect differences in the use of these behavioral processes. Study Limitations This study had several limitations, the greatest being the potential for sampling (volunteer) bias, whereby persons in the preaction stages may have been less likely to volunteer as participants in this study, whereas persons in maintenance were more likely to volunteer. There is also the possibility of drop-out bias because individuals who were sedentary and with lower levels of self-efficacy at baseline were more likely to withdraw than participants who were regular exercisers. The drop-out pattern was similar between the exercise intervention and the comparison groups, so there is no differential group effect of the dropouts. Lastly, all of the measures, except physical function, were measured by self-report, which can reduce the sensitivity and specificity of the instruments as a result of report and response biases. However, TUG scores are an objective measure of physical function, and the TUG scores and self-report of physical activity mirrored each other, suggesting that the self-reported physical activity data were valid. Conclusions The SENIOR exercise intervention demonstrated that a community-based intervention to promote exercise that is grounded by behavioral theory can be modestly effective in promoting stage progression over the long term. Nevertheless, the intervention did not increase physical activity behavior or improve physical function. Future research evaluating the intrapersonal, interpersonal, and environmental mediator and moderators variables affecting the adoption and maintenance of exercise behavior is needed to provide important insights into factors that can be addressed in future behavior-based interventions to improve outcomes. This research was supported by Grant 1R01AG16588 from the National Institute on Aging, National Institutes of Health. Preliminary results were presented in a symposium at the 57th Annual Scientific Meeting of the Gerontological Society of America, Washington, DC, November 19–23, 2004, and at the 26th Annual Meeting of the Society of Behavioral Medicine, Boston, MA, April 13–16, 2005. We thank all participants of the Study of Exercise and Nutrition in Older Rhode Islanders (SENIOR) Project and acknowledge the contributions of all the members of the SENIOR Project Research Team, including the following individuals: Bryan Blissmer, PhD; Robert Dufresne, PhD; Catherine English, PhD, RD; Geoffrey W. Greene, PhD, RD, RN; Andrea Luisi, PharmD; Norma Owens, PharmD; Cynthia Padula, PhD, RN; James Prochaska, PhD; Susan Rossi, PhD, RN; Sandra Saunders, MS, MPH; Laurie Ruggiero, PhD; Kira Stillwell, MS; and Nancy Fey-Yensan, PhD, RD. 1 Public Health Nutrition, Harvard School of Public Health, Boston, MA. 2 Department of Kinesiology, University of Rhode Island, Kingston. 3 Department of Biobehavioral Sciences, Columbia University, New York, NY. 4 Department of Psychology, University of Rhode Island, Kingston. 5 Program in Gerontology and Rhode Island Geriatric Education Center, University of Rhode Island, Kingston. 6 School of Nursing, University of Rhode Island, Kingston. 7 Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu. 8 Cancer Prevention Research Center, University of Rhode Island, Kingston. Decision Editor: William J. McAuley, PhD Table 1. Characteristics of 966 Participants Completing the SENIOR Project by Intervention Assignment. Intervention Assignment<--CO?1--> Intervention Group Comparison Group Characteristic n (M) % (SD) n (M) % (SD) p Baseline age: years (75.2) (6.7) (74.7) (6.6) .28 Education: years (12.9) (2.7) (12.9) (3.1) .59 YPAS summary score (44.9) (26.4) (45.7) (31.4) .66 TUG: seconds (8.7) (4.1) (8.8) (5.7) .74 Gender     Male 128 27.2 146 29.6     Female 342 72.8 348 70.4 .43 Race or ethnicity     White 362 78.4 379 77.0     Black 10 2.2 12 2.4     Portuguese–Cape Verdean 60 13.0 71 14.4     Other 30 6.5 30 6.1 .91 Perceived health status     Excellent–very good 221 47.4 244 49.3     Good 188 40.3 192 38.8     Fair–poor 57 12.2 59 11.9 .65 SOC for exercise at baseline     Precontemplation 89 18.9 88 17.7     Contemplation 32 6.8 29 5.8     Preparation 84 17.9 82 16.5     Action 22 4.7 30 6.0     Maintenance 243 51.7 267 53.8 .77 Note: The intervention and comparison groups are n = 470 and n = 496, respectively; some data are missing, so the sample size for all variables may not equal 966. Values given in parentheses refer to means or standard deviations, as shown. SENIOR = Study of Exercise and Nutrition in Older Rhode Islanders; YPAS = Yale Physical Activity Survey; TUG = Timed Up-and-Go test; SOC = state of change; SD = standard deviation. Table 2. SOC Movement from Baseline by Intervention Assignment. Intervention Assignment<--CO?2--> Intervention Group Comparison Group Stage Movement n % n % 12 months     Progress 102 22.3 94 19.4     Regress 102 22.3 124 25.6     Stable 253 55.4 266 55.0 24 months     Progress 128 27.2 103 20.8     Regress 117 24.9 138 27.8     Stable 225 47.9 255 51.4 Note: Intervention and comparison groups are n = 470 and n = 496, respectively; some data are missing, so the sample size for all variables may not equal 966. SOC = stage of change. Table 3. SOC Movement from Baseline by Stage and Intervention Assignment. Progress<--CO?3-->: %<--CO?4--> (n) Regress: % (n) Stable: % (n) Baseline Stage Intervention Comparison Intervention Comparison Intervention Comparison 12 Months     Precontemplation 37.1 (33) 37.6 (32) 62.9 (56) 62.4 (53)     Contemplation 59.4 (19) 48.3 (14) 37.5 (12) 44.8 (13) 3.1 (1) 6.9 (2)     Preparation 45.6 (36) 38.3 (31) 41.8 (33) 40.7 (33) 12.7 (10) 21.0 (17)     Action 77.8 (14) 58.6 (17) 16.7 (3) 37.9 (11) 5.6 (1) 3.4 (1)     Maintenance 22.6 (54) 25.8 (67) 77.4 (185) 74.2 (193) 24 Months     Precontemplation* 58.4 (52) 43.2 (38) 41.6 (37) 6.8 (50)     Contemplation 62.5 (20) 51.7 (15) 31.3 (10) 31.0 (9) 6.3 (2) 17.2 (5)     Preparation 51.2 (43) 46.3 (38) 33.3 (28) 40.2 (33) 15.5 (13) 13.4 (11)     Action 59.1 (13) 40.0 (12) 36.4 (8) 50.0 (15) 4.5 (1) 10.0 (3)     Maintenance 29.2 (71) 30.3 (81) 70.8 (172) 69.7 (186) Notes: Intervention and comparison groups are n = 470 and n = 496, respectively; some data are missing, so the sample size for all variables may not equal 966. SOC = stage of change. *Significant difference (p <.05) in stage progression by intervention assignment. Table 4. Adjusted Means of the TTM Constructs by Intervention Assignment. Intervention <--CO?5-->Group Comparison Group Construct M SE M SE p Self-efficacy     Baseline 3.41 .04 3.37 .04 .57     12 months 3.50 .05 3.41 .04 .10     24 months 3.52 .05 3.41 .05 .11 Pros     Baseline 3.51 .05 3.47 .05 .52     12 months 3.64 .05 3.65 .04 .98     24 months 3.81 .05 3.67 .05 .03 Cons     Baseline 1.22 .02 1.24 .02 .31     12 months 1.16 .02 1.49 .02 .20     24 months 1.15 .02 1.16 .02 .80 Experiential processes     Baseline 50.87 .57 50.52 .55 .63     12 months 53.32 .52 52.46 .51 .21     24 months 54.83 .58 52.83 .56 .10 Behavioral processes     Baseline 45.04 .55 43.61 .53 .05     12 months 46.40 .53 45.51 .51 .20     24 months 46.13 .56 45.13 .54 .17 Note: Intervention and comparison groups are n = 470 and n = 496, respectively; some data are missing, so the sample size for all variables may not equal 966. Adjusted means indicates that the analyses controlled for gender, age, and perceived health status at baseline. TTM = transtheoretical model of behavior change; SE = standard error. Table 5. Adjusted Means of the YPAS Summary Score and TUG Scores by Intervention Assignment. Intervention Group Comparison Group Scores M SE M SE p YPAS summary score     Baseline 46 1.4 46 1.3 .96     12 months 46 1.2 47 1.1 .81     24 months 47 1.3 47 1.2 .92 TUG score (seconds)     Baseline 8.4 0.2 8.6 .2 .51     12 months 8.6 0.2 8.7 .2 .50     24 months 8.9 0.3 8.9 .3 .93 Note: Intervention and comparison groups are n = 470 and n = 496, respectively; some data are missing, so the sample size for all variables may not equal 966. Adjusted means indicates that the analyses controlled for gender, age at baseline, and perceived health status at baseline. YPAS = Yale Physical Activity Survey; TUG = Timed Up-and-Go test; SE = standard error. References American College of Sports Medicine. (1998). American College of Sports Medicine Position Stand. 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R., King, A. C., Sheehan, M., & Stefanick, M. L., (2002). Stage of motivational readiness: Predictive ability for exercise behavior. American Journal of Health Behavior, 26, 331-341. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B6C0887F02B47C2DBB922E056BE4C67FB660CB8.txt b/test/dataset/in/resources/corpus/Clean_0B6C0887F02B47C2DBB922E056BE4C67FB660CB8.txt new file mode 100644 index 0000000..0a054bd --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B6C0887F02B47C2DBB922E056BE4C67FB660CB8.txt @@ -0,0 +1 @@ +cercorcercorCerebral Cortex1460-21991047-3211Oxford University Press10.1093/cercor/bhl036ArticlesExtrahippocampal Contributions to Age Differences in Human Spatial NavigationMoffatScott D.12KennedyKristen M.12RodrigueKaren M.12RazNaftali121Institute of Gerontology2Department of Psychology, Wayne State University, Detroit, MI 48202, USAAddress correspondence to email: moffat@wayne.edu.62007207200617612741282© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org2007The hippocampus (HC) and associated neural structures are hypothesized to contribute to individual differences in human spatial navigation. However, functional imaging studies and theoretical models underscore the importance of extrahippocampal regions as well. The purpose of the present study was to examine age differences in virtual environment navigation and to assess possible relationships between navigation and structural integrity of hippocampal and extrahippocampal brain regions. Healthy adult volunteers completed a virtual navigation task and underwent magnetic resonance imaging to assess volumes of the caudate nucleus (CN), cerebellum, HC, prefrontal, and primary visual cortices. Results demonstrated robust age-related differences in place learning. Moreover, individual differences in regional brain volumes as well as performance on the tests of memory and executive functions contributed to age differences in human place learning. High performance in a virtual navigation task was associated with larger volume of the CN and prefrontal gray and white matter. Larger hippocampal volume was associated with improved performance in the young but not old participants. We conclude that human navigation requires both hippocampal and extrahippocampal brain systems and draws on executive resources for successful performance.agingAlzheimer's diseaseexecutive functionhippocampusplace learningwanderingIntroductionAge-related deficits in spatial maze learning are evident in spatial navigation tasks across several mammalian species (Barnes 1979; Ingram 1988; McLay and others 1999). In particular, these deficits are observed in performance on the Morris water task (MWT) (Morris and others 1982) that has been used extensively in studying cognitive aging in animals. Briefly, in the MWT, an animal is required to search for a platform hidden beneath the surface of a circular pool. Because the platform cannot be seen directly, the animal must locate the platform position with reference to external cues throughout the environment and remember its location across a series of trials. Multiple studies of MWT performance have yielded a plethora of data, and age-related deficits on the MWT have been well characterized (Gallagher and Pelleymounter 1988; Lindner 1997; Lukoyanov and others 1999; Begega and others 2001). One of the reasons for popularity of MWT in studies of cognitive aging is its sensitivity to hippocampal lesions and its functional dependence on the hippocampal formation (Morris and others 1982).The investigation of age-related differences in human spatial navigation is a topic of considerable import. Nondemented elderly experience substantial deficits in spatial navigation (Kirasic 1991; Kirasic and others 1992; Burns 1999; Wilkniss and others 1997; Newman and Kaszniak 2000), and impairment in navigational skills is often apparent in the early stages of dementia (Passini and others 1995; Klein and others 1999). Recently, virtual environment (VE) spatial learning tasks have been successfully applied to the study of age differences in spatial navigation among healthy elderly (Moffat and others 2001, 2006; Moffat and Resnick 2002; Driscoll and others 2005). In a virtual MWT, elderly individuals traveled a longer linear distance in locating the hidden goal, spent less time searching in the vicinity of the goal on a retention trial, and were impaired in constructing a cognitive map of the environment in comparison to their younger counterparts (Moffat and Resnick 2002).Functional neuroimaging and lesion studies have identified a complex network of structures that are involved in spatial navigation. The proposed network includes the hippocampus (HC), parahippocampal gyrus, cerebellum (CB), parietal cortex, posterior cingulate gyrus, prefrontal cortex, retrosplenial cortex, and other cortical and subcortical regions (Aguirre and others 1996; Barrash 1998; Maguire and others 1998; Katayama and others 1999; Gron and others 2000). In a recent functional neuroimaging study, we found that in performing a virtual navigation task, elderly participants showed reduced activation in some of the same neural systems that young subjects use to navigate successfully. The cerebral regions identified in that study included the HC, parahippocampal gyrus, and retrosplenial cortex. Thus, reduced activation in the HC and extrahippocampal regions may underlie the observed age deficits in performance (Moffat and others 2006).Most attention in research on human navigation has focused on the role of the HC and associated structures. This focus may be justified in light of the prominent place of the HC in models of human spatial and episodic memory and in animal models of spatial navigation. However, a closer examination of the executive and strategic demands of human navigation and their neural underpinnings is overdue. Successful navigation requires the selection of an appropriate search strategy and also depends on appropriate behavioral monitoring and alterations of searching behavior if the selected strategy proves unsuccessful. Such reliance on executive skills during acquisition is common in many tasks, including those that in their developed states are highly proceduralized. On other procedural tasks, shrinkage of the prefrontal cortex, neostriatum, and CB have been shown to contribute to age-related differences in these skills (Raz and others 2000; Kennedy and Raz 2005). Thus, it is plausible that age-related deficits in acquiring and maintaining navigational skills may reflect, at least in part, differential shrinkage of multiple regions rather than strictly hippocampal atrophy.The age-related deficits observed in the virtual analog of MWT cannot be fully accounted for by models of performance restricted to spatial memory. In particular, we have observed that healthy elderly perform more poorly than their younger counterparts even on the first trial, which does not depend on memory for platform location (Moffat and Resnick 2002; see Driscoll and others 2005, for similar results). According to strictly spatial memory models of performance, there should be no age difference on trial 1 performance because the search for the hidden platform on the first trial should be random. Importantly, our analyses indicated that this trial 1 deficit was not attributable to psychomotor impairment in the elderly. We hypothesize that the deficits observed among elderly participants in navigational behavior may be partially attributed to impaired executive and strategic functions that play a significant role in skill acquisition (Anderson 1983). In particular, our observations indicate that elderly individuals are reluctant to disengage from searching in locations that have been adequately explored, suggesting a perseverative component to their behavior or, more generally, the selection of inefficient spatial search strategies. This behavioral effect is illustrated in Figure 1. We therefore hypothesize that the deficits observed among elderly subjects in navigational behavior may be partially attributed to impaired executive and strategic functions and shrinkage of the neural systems that support executive control.Figure 1.Top view of virtual MWT search paths (trial 1) in 5 representative young and old subjects in a previous study (Moffat and Resnick 2002). Older subjects took longer to find the platform and traveled a longer linear distance in their initial search. Inefficient search strategies and a reluctance to disengage from nongoal locations may contribute to this age difference on trial 1.In the present study, we quantified navigational behavior in a sample of men and women whose regional brain volumes were assessed via structural magnetic resonance imaging (MRI). In addition to an extensive cognitive test battery, the individuals in the present study performed a VE place navigation task modeled after the MWT. The purpose of the study was 3-fold: 1) to investigate age differences on a virtual Morris water task (vMWT), regional brain volumes, and other cognitive tests; 2) to investigate associations between regional brain volumes and vMWT performance; and 3) to investigate associations between cognitive tasks and vMWT performance. We hypothesized age-related deficits in spatial navigation as assessed by the vMWT. We hypothesized that a significant portion of variance in vMWT performance would be explained by hippocampal, prefrontal, and striatal volume differences, whereas individual differences in the volume of a control region (primary visual cortex [VC]) would evidence no significant association with navigation performance. In addition, both hippocampal dependent measures of memory as well as frontal lobe executive measures would be positively associated with vMWT performance.Materials and MethodsParticipantsIn this study, we examined cross-sectional differences in the normal aging brain using a sample of healthy volunteers who participate in an ongoing study of brain and cognitive aging. Participants in the present study (18 years and older) were recruited from a pool of more than 140 subjects who underwent MRI scanning and cognitive testing in our laboratory. These participants, who live in the Detroit metropolitan area, were contacted and agreed to return to complete the virtual MWT. Sixty-eight participants who had undergone MRI scanning agreed to participate in the study and met all study qualifications. These participants were divided into 2 groups, hereafter referred to as young and elderly. Persons who reported history of cardiovascular (except treated essential hypertension), neurological, or psychiatric illness, diabetes, head trauma with loss of consciousness for more than 5 min, thyroid problems, treatment for drug and alcohol problems, or a habit of taking 3 or more alcoholic drinks per day were excluded from the study as were participants who use antiseizure medication, anxiolytics, or antidepressants. Persons who experience emotional distress that could interfere with cognitive performance were screened using a geriatric depression questionnaire (Radloff 1977). In addition, participants in our study were classified into those with current or history of hypertension. This was done to investigate the effects on performance of hypertension that is increasingly acknowledged as an important moderator of cognitive and brain aging (Raz and others 2003; Korf and others 2004; Wiseman and others 2004). General cognitive status was assessed by Mini Mental State Examination (Folstein and others 1975). All participants were strongly right handed as screened by the Edinburgh handedness questionnaire (Oldfield 1971). Table 1 presents demographic data for the subject sample. Mean delay between MRI and cognitive testing was 3.8 months (standard deviation [SD] = 2.7), and there were no differences in delay between the young and the old participants t = 0.01, not significant (NS). Before cognitive testing, all participants were interviewed to ensure that they had not experienced any health changes in the interim period. Because we performed a cross-sectional study comparing a group of older participants with a group of younger participants, any small cognitive or brain changes over the 3.8-month interval are unlikely to affect our cross sectional comparisons that average 44 years difference between old and young participants.Table 1Demographic characteristics of participantsNPercent femaleAge (mean ± SE)Education (mean ± SE)Percent hypertensiveYoung327524.5 (0.91)15.16 (0.32)0Old366768.5 (0.92)15.22 (0.52)47.22Note: SE, standard error.ProceduresStructural MRI and Regional VolumetryImage acquisition and processing.Participants in the existing project underwent MRI scanning at Children's Hospital of Michigan on a GE Signa 1.5-T scanner. Regional brain volumes were measured on images acquired with T1-weighted 3-dimensional spoiled gradient recalled (SPGR) sequence with 124 contiguous axial slices, echo time = 5 ms, time repetition = 24 ms, field of view = 22 cm, acquisition matrix 256 × 192, slice thickness = 1.3 mm, and flip angle = 30°. The images were reformatted to correct for undesirable effects of head tilt, pitch, and rotation with BrainImage 2.3.3 software. The procedure is described in detail elsewhere (Raz and others 2004).Volumetry: region selection, demarcation, and tracing.The regions of interest (ROIs), selected on the basis of the hypotheses to be tested in this study, included HC, lateral prefrontal cortex gray matter (LPFC), prefrontal white matter (PFW), caudate nucleus (CN), and the CB. Each of these selected structures has been implicated as contributing to spatial navigation in human neuroimaging or animal studies. Primary VC was selected as a comparison (control) region for which no correlations were expected. To control statistically for individual differences in body size, especially for sexual dimorphism, we used intracranial volume (ICV). All regional brain volumes were corrected for total ICV using a regression analysis of covariance approach. Interrater reliability of all measures exceeded an intraclass correlation of 0.90.The rules for demarcation and tracing of brain regions as well as the estimates of interrater reliability of volume estimates obtained with National Institutes of Health Image software (Version 1.62) are described in detail in previous publications (Raz and others 1997, 2004) and will be presented here in condensed form. Unless otherwise noted, we used the average of left and right hemisphere volumes in statistical analyses.Caudate nucleus.The volume of the head and the body of the CN were estimated from 15 to 20 coronal slices. The most rostral slice was the one on which the CN first appeared, usually lateral to the lateral ventricles. The CN was traced on every other slice (interslice distance 3 mm) until no longer visible.Cerebellum.The cerebellar hemispheres were measured on 32–40 coronal slices. The vermis, the cerebellar peduncles, and the forth ventricle were excluded, whereas the hemispheric gray matter, the cerebellar tonsils, the vellum, and the corpus medullare were included in the tracing of each cerebellar hemisphere. The rostral border was defined as the first slice on which cerebellar gray matter became visible and distinguishable from the cerebellar peduncles, and the caudal border ended when the hemispheres were no longer distinguishable.Hippocampus.HC volume was measured on continuous slices aligned perpendicular to the long axis of the right HC between the mammillary bodies and slice showing the fornices rising from the fimbria. The HC included sectors CA1–CA4, the dentate gyrus, and the subiculum.Intracranial volume.The ICV was estimated from the coronal sections. The operator traced ICV on every eighth slice (a total of 11–12 slices) between the first slice following the orbits and last slice on which brain tissue was visible.Lateral prefrontal cortex.Eight to 12 coronal slices located within 40% of the distance between the genu of the corpus callosum and the frontal pole are included in the prefrontal ROI.Prefrontal cortex white matter.The range is identical to LPFC above and also includes orbitofrontal cortex. The volume includes all white matter on the coronal slice, excluding ventricles and other cerebrospinal fluid spaces.Primary VC (calcarine).The volume of the VC is calculated as the volume of the cortical ribbon lining the calcarine sulcus on the anterior 50% of the coronal slices between the midvermis slice and the occipital pole.Spatial Navigation Assessments: Virtual Water MazeFor the behavioral navigation testing, all subjects completed a vMWT that has been developed and validated previously (Moffat and Resnick 2002). Before beginning the navigation task, practice trials were allowed to familiarize subjects with movement through the environment and to be certain that participants were comfortable with the computer-administered task. The task environment was a circular arena surrounded by several cues, which could be used to guide navigation. Hidden beneath the surface of the arena was a platform, and the task required the participant to locate the platform as quickly as possible. Because the platform was not visible to the participant, it must be located with reference to its position relative to the external objects and cues. When the participant passed over the platform, the platform became visible, and a tune notified the participant that he or she had located the platform. Participants were informed that the platform remained in the same location on each trial and that they should try to remember its location. On each of 6 learning trials, participants were “placed” into one of the 3 quadrants of the pool, which did not contain the platform and facing a different orientation on each trial. The primary dependent measure was the distance traveled on each trial. Following the 6 learning trials, a “probe” trial was performed in which participants explored the same VE with the exception that the platform had been removed. Participants were given 1 min of search time in the probe trial. Individuals who have properly encoded platform location will spend a greater proportion of their time and distance searching in the vicinity of the platform. Thus, the dependent measures were the percentage of distance spent in the goal quadrant and the number of platform intersections (number of times a participant's path would have passed over the platform had it been present).Control TestsPretest training and assessment of joystick visuomotor control.Prior to virtual MWT testing, extensive pretraining was provided to familiarize participants with the VE and with the use of a joystick for movement. This was accomplished with an initial period of experimenter instruction, followed by a period of free exploration of a VE using the joystick. After participants were comfortable with the joystick and had satisfactorily demonstrated their ability to guide themselves to targets designated by the experimenter, the participants underwent a joystick control speed test. During the speed test, participants were required to navigate a long winding corridor as quickly and accurately as possible until they reached a trophy at the goal point. Participants were required to demonstrate their competency with the joystick by completing the corridor in less than 120 s.Visible platform performance.As a further experimental control, a trial was performed in which the platform was visible on the surface of the pool. Subjects were instructed to move onto the platform from the starting location as quickly as possible. This served as an additional control for possible age differences in visual and motor skills. The magnitude of any age differences observed on this task should be considerably smaller than those observed in the vMWT trials and probe trials. The dependent measure was the total distance traveled to the platform.Cognitive TestsCognitive tests were selected based on theoretical and empirical considerations. In particular, we selected tests that measure cognitive skills that have been empirically demonstrated to be related to spatial navigation in previous studies (Moffat and others 1998, 2001). In addition, we selected traditional neuropsychological measures that are sensitive to frontal lobe function to assess the contribution of prefrontal cortex to spatial navigation.“Computation span” (Salthouse 1989) was obtained as a measure of verbal working memory, and the absolute span was used as the index of performance, calculated by summing the number of correct items across all trials.A modified “size-judgment span” (Cherry 1993) was administered as a measure of nonverbal working memory. In this task, participants are orally presented with a list of objects and animals and asked to repeat them in order of size from smallest to largest. Index of performance is total number of correct trials.“Letter comparison” and “pattern comparison” tests (Salthouse and Meinz 1995) were indices of perceptual speed.A computerized version of the “Wisconsin card sorting test” (WCST; Psychological Assessment Resources, Odessa, FL) was administered. Total number of perseverative errors was the index of perseveration.“Vocabulary” (V-3) is a paper-and-pencil test from Educational Testing Service Factor-Referenced Test Kit (Ekstrom 1976) and was used as a measure of word knowledge.“Spatial recall” test was modified from a task described previously (Salthouse 1994). In this task, participants were instructed to memorize the locations of a stimulus in 1 of 7 target locations within a 5 × 5 matrix and then asked to place an X in the location of the target in a blank matrix. Average number of correct items across the 25 trials was the index of performance.“Buildings memory” test is a paper-and-pencil test from the Educational Testing Service Factor-Referenced Test Kit (Ekstrom 1976). After studying a fictitious map for 4 min, participants were shown a blank map and tested on their memory for the location of buildings on the map studied in a multiple-choice format. The index of performance was the number correct minus the number incorrect.Data ReductionTo reduce the data, we analyzed cognitive performance by 4 construct domains: speed of processing, working memory, spatial memory and executive control. Each construct, except executive control, was measured by 2 tests; executive control was assessed by WCST. The working memory domain was assessed by computation span and size judgment span. The spatial memory construct was measured by spatial recall and the buildings memory test. The construct of perceptual speed was measured by letter and pattern comparison tests. Although the domains were determined on the basis of theoretical considerations, we performed principal component analysis on our data in order to verify the postulated factor structure. We conducted principal component analysis followed by varimax rotation on the young and older participants separately. For 32 young participants, the analysis yielded 3 factors that accounted for 78% of the score variance. The first factor was speed (letter comparison and pattern comparison loadings both 0.87, 29% of the variance accounted), the second factor was spatial memory (building memory 0.79, spatial recall 0.88, 25% of the variance), and the third factor was working memory (size judgment 0.87, computation span 0.86, 24% of the variance).In the older age group, a different factor structure was obtained, with the first factor (letter and pattern comparison tests, spatial recall and building memory, loadings ranging between 0.74 and 0.91) accounting for 59% of the variance and the second factor (size judgment and computation span measures, loadings 0.87 and 0.88) accounting for additional 16%. The difference illustrates an often observed tendency for dedifferentiation of cognitive abilities with age (Ghisletta and Lindenberger 2004). For statistical analyses, we converted all raw scores from cognitive tests to standardized (Z) scores and computed composites consisting of the average of test performance in common cognitive domains as described above.Statistical Approach and AnalysisAs noted earlier, there were 3 overarching aims of the study: 1) to investigate basic age differences in vMWT performance, cognitive performance, and regional brain volumes; 2) to investigate correlations between vMWT performance and regional brain volumes; and 3) to investigate correlations between cognitive measures and vMWT performance. For all 3 aims, our hypotheses were tested within the framework of the general linear model with age and sex as independent variables and appropriate dependent variables. The primary dependent variable on the vMWT was the total distance traveled on each trial. Because the distribution of that index was positively skewed, a logarithmic transformation was applied. Following the evaluation of hypothesis 1, which established basic age effects on vMWT, cognitive tests and ROI's, analyses were performed incorporating the regional brain volumes into the identical models, separately for each brain region with distance traveled on the 6 trials of vMWT as dependent variable. This allowed us to evaluate the contribution of each brain region to vMWT performance (hypothesis 2). Lastly, we deleted brain volumes from our statistical models and incorporated cognitive test scores allowing us to assess the correlation between performance on vMWT and other cognitive domains (hypothesis 3). To assess the specificity of possible associations between vMWT performance and specific cognitive domains, we also evaluated correlations with vocabulary scores as a control measure for which we expected no significant positive correlations.ResultsAge and Sex Effects on Brain, vMWT, and Cognitive MeasuresRegional Brain VolumesThe effects of age and sex on regional brain volumes were evaluated in the framework of a general linear model with age group and sex as independent variables and regional volumes as a 6-level repeated measure. Before being entered into the equations, the volumes of all brain regions were adjusted for individual differences in ICV. Overall, there was a significant main effect of age (F1,65 = 34.06, P < 0.001; η2 = 0.34) and no significant sex differences (all main effects and interactions F values < 1). The significant age × ROI interaction, F5,325 = 10.09, P < 0.001, η2 = 0.13, indicated that the magnitude of the age differences varied across ROIs. There was a significant effect of hemisphere (F1,65 = 6.19, P < 0.05, η2 = 0.09), with right hemisphere volumes, on average, larger than left but no interactions involving hemisphere were significant (but see below for the analysis of simple effects).Analysis of simple effects with univariate F-tests indicated that the differences between the age groups were significant in LPFC (F1,65 = 46.38, P < 0.001), the HC (F1,65 = 25.34, P < 0.001), the caudate (F1,65 = 21.07, P < 0.001), the CB (F = 11.37, P < 0.001), and PFW (F1,65=6.21, P < 0.05) but not in the primary VC (F1,65 = 2.82, P = 0.10). Comparison of effect sizes revealed that the magnitude of age differences in the prefrontal volume was significantly greater than that for the pericalcarine volume. Other regional differences fell between the 2 values and did not differ significantly from each other (see Table 2).No lateral differences were found in LPFC (F < 1), VC (F < 1), the caudate (F1,65 = 1.71, NS), or the CB (F1,65 = 2.57, NS). Right PFW was significantly larger than left, F1,65 = 13.87, P < 0.001. Left HC was larger than the right but only in the young (F1,30 = 25.74, P < 0.001) not in the older participants (F < 1).We examined the possible influence of hypertension in the older age group on the observed volume differences. Only the older age group could be considered in these analyses as there were no young subjects with a diagnosis of hypertension. Separate analyses of the older participants revealed a trend for the diagnosis of hypertension to be associated with overall smaller regional brain volumes (F1,32 = 3.53, P < 0.07, η2 = 0.09).Virtual Water Maze: LearningThere was a significant main effect of age, F1,65 = 84.34, P < 0.001, η2 = 0.56, with younger subjects outperforming older subjects and a main effect of trial, F5,325 = 9.11, P < 0.001, η2 = 0.12, in which performance improved across trials. There was a nonsignificant trend for an age × trial interaction: F5,325 =1.97, P < 0.09, η2 = 0.03. The linear component of the trial effect was significant, F1,65 = 23.29, P < 0.001, η2 = 0.26, as was the quadratic component for an age × trial interaction: F1,65 = 5.99, P < 0.05 η2 = 0.08. Changes in performance across trials for both age groups are depicted in Figure 2. As seen in Figure 2, there was an unexpected increase in distance traveled on trial 4 in both the younger and older groups. This effect was an artifact of the random placement procedure across the 6 learning trials. On trial 4, participants were placed at the maximum possible distance and facing a direction opposite from the platform resulting in the increase in distance traveled. We performed all analyses again deleting trial 4 from our repeated measure and obtained virtually identical results. In particular, the task resulted in the same robust age and trial effects. In all remaining analyses, all 6 trials are used as dependent measure.Figure 2.Total distance traveled in the vMWT as a function of age and trial. Performance improved across trials and older participants traversed a longer path in locating the hidden platform on all 6 trials.The effects of hypertension on vMWT performance were investigated in the older participants. Within the older group, there was a trend for normotensive participants to perform better than hypertensive participants: F1,32 = 3.94, P = 0.056, η2 = 0.11.Virtual Water Maze: Probe TrialIn this analysis, the new dependent variable was the distance traveled in the quadrant of the pool that formerly contained the platform. The results indicated that younger (Mean = 52.50, SD = 22.16) individuals spent more of their distance searching in the goal quadrant than the older (Mean = 28.17, SD = 20.06) participants: main effect of age, F1,64 = 6.49, P = 0.013. Similarly, there was a trend for younger subjects (Mean = 2.94, SD = 1.56) to have more frequent platform intersections than their older (Mean = 1.97, SD = 1.34) counterparts: F1,64 = 3.69, P = 0.059. We observed neither a main effect of sex nor significant interactions.Cognitive TestsThe descriptive statistics and the effect size indices (Cohen's d) for all cognitive tests by age group are presented in Table 3. The distribution of perseverative errors was skewed and was entered into the analysis after a logarithmic transformation.Table 2Descriptive statistics (mean ± standard error of mean) and effect sizes for regional brain volumes (cm3) in the 2 age groupsLPFCPFWHCCBCNPVCYoung20.94 (0.40)43.51 (1.07)7.39 (0.13)133.06 (1.98)10.15 (0.17)5.36 (0.14)Old16.30 (0.38)37.59 (1.01)6.36 (0.12)123.12 (1.87)8.77 (0.16)5.23 (0.13)Effect size (d)2.010.971.440.881.410.37P value<0.0010.001<0.0010.001<0.0010.12Note: Lateral prefontal cortex white matter (PFW); Primary visual cortex (PVC).Table 3Descriptive statistics (mean ± standard error of mean) and effect sizes (d) for cognitive test scores in the 2 age groupsWCSTaSpatialVocabBuildSizeCSPANPatcompLetcompYoung6.75 (0.64)5.33 (0.10)18.25 (1.14)7.86 (0.52)9.84 (0.31)26.94 (3.07)41.21 (1.26)24.87 (0.72)Old19.23 (2.08)4.22 (0.13)26.52 (1.25)4.26 (0.56)8.50 (0.32)18.06 (1.90)32.19 (0.97)18.26 (0.86)Effect size (d)1.311.41−1.161.120.720.611.381.40P value<0.001<0.001<0.001<0.0010.0040.014<0.001<0.001Note: Wisconsin card sorting test (WCST) perseverative errors; spatial recall (Spatial); vocabulary (Vocab); buildings memory (Build.); size judgment (Size); computation span (CSPAN); pattern completion (Patcomp); letter completion (Letcomp). One older subject had missing data on Letcomp, making for that test the total N = 67.aScoring scale is reversed. Higher scores reflect poorer performance.The results of the analysis revealed a significant main effect of age (F1,64 = 10.87, P < 0.01, η2 = 0.15) and no sex differences (F1,64 = 1.56, NS, η2 = 0.02). However, the magnitude of age differences varied across the tested domains, as indicated by a significant age × test interaction, F7,448 = 15.35, P < 0.001, with Hyunh-Feldt correction, η2 = 0.19. Univariate analyses showed that the age differences (favoring the younger participants) were the smallest in the working memory domain (F1,64 = 6.05, P < 0.05 for computation span and F1,64 = 9.49, P < 0.01 for size judgment span) in comparison to the other domains: speed (F1,64 = 33.64, P < 0.001 for pattern comparison and F1,64 =32.92, P < 0.001 for letter comparison); spatial memory (F1,64 =33.95, P < 0.001 for spatial recall and F1,64 = 20.64, P < 0.001 for building memory); and perseveration: F1,64 =38.37, P < 0.001. The effect of age on vocabulary was significant (F1,64 = 23.70, P < 0.001) but in the opposite direction (favoring the older participants).Regional Brain Volumetry and Virtual MWT PerformanceBecause high correlations among the regional volumes would result in multicollinearity if all entered in the same model, we performed a separate analysis for each ROI. Before being entered into the equations, the volumes of all brain regions were adjusted for individual differences in ICV. In the interest of brevity, main effects of age and trial on vMWT performance that were reported above are not reported in these analyses. All zero-order correlations between ROI's and vMWT performances on each trial are presented in Table 4.Table 4Correlations between regional brain volumes and distance traveled on each trial of the vMWTTrial 1aTrial 2aTrial 3aTrial 4aTrial 5aTrial 6aLPFC−0.28**−0.47**−0.43**−0.40**−0.40**−0.37**PFW−0.25*−0.40**−0.41**−0.31**−0.23−0.27*HC−0.17−0.33**−0.38**−0.38**−0.30**−0.36**CB−0.10−0.19−0.34**−0.34**−0.31*−0.27*CN−0.16−0.43**−0.39**−0.50**−0.47**−0.46**PVC−0.11−0.39**−0.15−0.100.03−0.10Note: Lateral prefrontal cortex white matter (PFW); Hippocampus (HC); Primary visual cortex (PVC). Correlations were essentially the same for right and left hemisphere volumes of each structure (data not shown).aScoring scale is reversed. Higher scores reflect poorer performance.*P < 0.05; ** P < 0.01.The inclusion of hippocampal volume as an independent variable revealed a complex pattern of differences in performance reflected in a significant interaction between age, trial, and HC volume: F5,300 = 2.37, P = 0.04, η2 = 0.04, although the main effect of HC on vMWT performance was NS (F1,60 = 1.18, NS, η2 = 0.02). Analysis of simple effects showed that the association between HC volume and maze performance was significant only among the younger participants and only on the first trial with young adults with larger HC accumulating shorter distances in their initial search for the virtual platform (r = −−0.38, P < 0.05).The analyses of the influence of other regional volumes revealed a less complex pattern of results. Larger volumes of prefrontal gray matter and PFW were linked to better vMWT performance: F1,60 = 3.89, P = 0.05, η2 = 0.06 and F1,60 = 7.28, P = 0.009, η2 = 0.11, respectively. A similar association between better vMWT performance and larger volumes was observed for the CN: F1,60 = 11.07, P < 0.001, η2 = 0.16. A trend in the same direction was observed for the CB volume: F1,60 = 2.89, P = 0.09, η2 = 0.05. Consistent with our hypothesis, VC, designated as a control region, showed no significant association with vMWT performance: main effect F < 1. In none of the models were there significant interactions between the regional volumes and age, sex, or trial.In the models that included brain regional volumes, the effect of hypertension was significant only in conjunction with the caudate and HC volumes: F1,31 = 7.43, P = 0.01, η2 = 0.19 and F1,31 = 4.48, P < 0.05, η2 = 0.13, respectively. For all other ROI models, the trends for hypertension effect on performance were in the same direction but failed to reach the 0.05 level of significance. The probability levels of the trends ranged from P = 0.056 to P = 0.20, all NS.Cognitive Test Performance and Virtual MWT PerformanceAll zero-order correlations between cognitive composites and vMWT performance on each trial are presented in Table 5. The analyses revealed that performance on executive function tests predicted vMWT performance: participants with better working memory (F1,64 = 3.97, P = 0.05) or fewer perseverative errors on WCST (F1,64 = 7.98, P = 0.006) attained higher vMWT scores. There were no significant interactions involving age, sex, or trial.Table 5Correlations between cognitive domains and distance traveled on each trial of the vMWTTrial 1aTrial 2aTrial 3aTrial 4aTrial 5aTrial 6aWorking memory0.00−0.28*−0.39**−0.28*−0.24*−0.33**Spatial memory−0.11−0.41**−0.47**−0.62**−0.38**−0.57**Perseverationa0.170.38**0.42**0.50**0.34**0.55**Perceptual speed−0.13−0.46**−0.48**−0.52**−0.25*−0.53**Vocabulary0.090.160.120.33**0.22−0.09aScoring scale is reversed. Higher scores reflect poorer performance.*P < 0.05, **P < 0.01.Better spatial memory was associated with better performance on vMWT as indicated by the significant main effect of spatial memory: F1,64 = 17.68, P < 0.001, η2 = 0.22. A trend for trial by spatial memory interaction (F5,320 = 2.17, P = 0.057, η2 = 0.03) suggested that spatial memory may be linked to performance on each trial except the first (see Table 5). A similar pattern of results was observed for perceptual speed: main effect F1,63 = 7.15, P = 0.01, η2= 0.10. Individuals who were faster on the perceptual speed tests performed better on the vMWT. Moreover, a significant interaction between perceptual speed and trial (F5,315 = 2.52, P = 0.03, η2 = 0.04) indicated that perceptual speed was positively associated with performance on each trial but the first (see Table 5).When vocabulary scores, intended as a measure of general crystallized abilities, were introduced into the model, we observed an unexpected significant main effect on vMWT performance: F1,64 = 5.77, P = 0.02, η2 = 0.08. Individuals with higher vocabulary scores performed more poorly on the vMWT. There were no significant interactions.DiscussionThe results of the present study replicate extant findings of age differences in spatial navigation (Moffat and others 2001; Moffat and Resnick 2002; Driscoll and others 2005) and contribute new evidence toward understanding of their neural substrates. We found that age effects on VE place learning are indeed substantial; older individuals traversed a longer linear distance in solving the learning trials of the vMWT. On a probe trial, a measure of retention of spatial location, younger individuals traveled a greater distance in the vicinity of the platform and had more frequent platform intersections relative to older subjects. These findings indicate that younger individuals learned and retained more accurate knowledge of platform location than their older counterparts. In search of neural correlates of the observed behavioral differences, we found that larger LPFC, PFW, and CN volumes were positively associated with navigational skill. However, individual differences in HC volume did not explain the age-related deficit in navigation performance. In addition, we found that success in spatial navigation was associated with superior executive skills, spatial memory, and speed of processing. Notably, the observed relationships between regional brain volumes, cognitive skills, and navigation performance were specific and dissociable from the control brain region (primary VC) and control cognitive variables (vocabulary).In addition, our study replicated our earlier findings demonstrating that the age difference in vMWT performance emerges on the very first trial (see Figs 1 and 2) arguing against a strictly hippocampal/episodic memory explanation of the age difference. This behavioral difference may reflect planning, strategy selection or perseveration (i.e., persistence in searching of the same region despite ample opportunity to determine that a particular pool location has been searched adequately), cognitive skills that are dependent on prefrontal executive resources. As well, it is important to note that within a trial, participants must remember preceding moves and locations and continually update this information to avoid returning to the same (incorrect) locations. Although clearly memory related, such within-trial immediate recall might best be characterized as requiring working memory, an age-sensitive ability (Salthouse and Meinz 1995) that has also been shown to be dependent on the prefrontal regions (Courtney and others 1998; Smith and Jonides 1999; D'Esposito and others 2000).The most important evidence supporting our view that vMWT performance is influenced by frontal systems is the fact that both the gray and white matter volumes of the prefrontal cortex were positively associated with vMWT performance. In addition, 2 cognitive measures of executive/frontal function (the number of perseverative errors and working memory span) were both associated with performance on the vMWT. This strongly suggests that successful navigation in humans require substantial contribution from prefrontal circuits and associated cognitive systems. It should be noted that frontal lobe contributions to vMWT performance may not be restricted to the first trial but may extend across several trials, as evidenced by the significant correlations between frontal lobe volumes and vMWT performance across all 6 trials (see Table 4). However, it should also be acknowledged that our primary measure of frontal lobe executive resources (perseveration on the WCST) predicted performance on trials 2–6 but not trial 1 (see Table 5). Although this may be attributable to chance variation in a single study, it is also possible that other frontal lobe measures (or other cognitive domains) may better capture individual differences in trial 1 performance.Studies in nonhuman species confirm important contributions from prefrontal systems in solving the MWT. The respective roles of the frontal cortex and hippocampal system have been delineated by adopting both the place and response versions of the MWT. In the former, the platform remains in the same position while the release site varies, thus requiring a place strategy for successful solution. In the latter, both the platform and release site vary from trial to trial with the spatial relationship between the release site and platform location held constant (e.g., a fixed distance to the right). Several studies have reported double dissociations with hippocampal and or fimbria/fornix lesions impairing place learning in the place version of the MWT and frontal cortex lesions impairing response learning in the MWT (de Bruin and others 1997, 2001). It is noteworthy that we did not specifically assess response learning in our vMWT. However, as we outlined above, even in the place MWT, there are likely substantial executive demands required and at least one study observed impairments in the place version of the MWT following temporary inactivation of the orbital frontal cortex by tetrodotoxin (Vafaei and Rashidy-Pour 2004). Interestingly, consistent with the age differences observed in our study, this impairment was evident on the very first block of trials, suggesting that frontal dysfunction may impair MWT place learning early in the acquisition phase before there are significant mnemonic demands. Although separate tasks may be designed to investigate place and response strategies, respectively, animals (Packard and McGaugh 1996) and humans likely employ both strategies to some extent concurrently or successively across trials.In addition to positive associations with frontal cortex measures, we also observed a positive correlation between CN volume and navigation performance. These findings are compatible with previous studies in humans and other mammals. The CN plays an important role in learning and spatial memory and is often activated in young subjects during virtual navigation tasks in functional imaging studies (Maguire and others 1998; Moffat and others 2006). Because of its extensive connections with the LPFC and HC (Alexander and others 1986), the CN is a part of distributed frontostriatal and striato-hippocampal systems, which are vulnerable to aging (Raz 2000; Raz 2003). Studies investigating contributions of the CN to human spatial navigation suggest that the CN may work in concert with hippocampal systems (Voermans and others 2004) and may play a role in nonspatial or procedural response components of spatial behavior (Hartley and others 2003; Iaria and others 2003). As the MRI methods used in this study were not geared to assessment of the integrity of corticocortical and corticosubcortical connections, the intriguing question of the joint effects of multiple systems on navigation remains open.Somewhat unexpectedly, we observed that hippocampal volume was associated with vMWT performance in the young but not in the elderly participants. The extant literature on HC memory relations in human aging consists primarily of studies that relied on verbal and visual memory tasks with positive associations between HC volume and episodic memory reported in some samples or specific groups of participants (Soininen and others 1994; Raz and others 1998; Jack and others 2000; Petersen and others 2000; but c.f. Van Petten 2004). A notable exception to that focus is a recent study that assessed the effects of age and HC volume and navigation performance in the framework of a virtual MWT similar to the one used in the current study (Driscoll and others 2003). In that investigation, HC volume was positively correlated with vMWT performance across all subjects. However, when these researchers incorporated age into their model as we did in this study, the relationship between HC volume and vMWT performance was reduced to nonsignificant. Thus, to our knowledge, the only studies to investigate the contributions of HC volume to age differences in navigation have produced similar results.Although, the lack of a relationship between HC volume and vMWT performance in the elderly group should be interpreted with caution, a possible explanation for this finding may be found in the animal literature. It is known that animals have an initial preference for the use of a place strategy in the MWT (de Bruin and others 1997). Thus, when confronted with the response version of the MWT, animals first adopt a place strategy, which when unsuccessful is replaced by a response strategy. Importantly, Barnes has demonstrated that the preference for initial selection of a place strategy is age dependent with younger animals adopting a place (hippocampal) strategy, whereas older animals adopt a response (extrahippocampal) strategy. If a similar phenomenon is at work in humans, then it could be argued that our younger and elderly participants were adopting different solution strategies that were differentially dependent on the HC with younger participants adopting a place (hippocampal) strategy and older participants adopting a reference (extrahippocampal strategy), resulting in positive and null associations with hippocampal structure, respectively. Conceivably, after a reference strategy proved unsuccessful, elderly participants may switch to a place strategy. However, the number of trials employed in the present study was not sufficient to test this hypothesis. In the future, the design of distinct place and response versions of the vMWT incorporating more learning trials may help to address this question.It is also important to note that we assessed only the volume of the HC and not other important neurochemical and/or functional measures of HC integrity. An alternative conceptualization is that a variety of “functional” changes in the HC that were not reflected in our volume measurement may play a more important role in cognitive aging (Driscoll and others 2003; Grady and others 2003; Moffat and others 2006).In the framework of a cross-sectional study, it is impossible to discern the influence of longstanding individual differences in brain and cognitive variables from true longitudinal declines. It is possible that smaller frontal and striatal volumes that predict poorer performance in the young and the elderly alike do so for age-specific reasons. For the younger participants, the frontal executive links may reflect developmental or even inborn differences in the size of the prefrontal cortex and white matter. In the older subgroup, better performance may be due to preservation of these age-vulnerable regions.One of the factors that could have affected performance and the relationships between brain variables and navigation is hypertension, a commonly acknowledged negative modifier of age trajectories (Breteler and others 1994; Raz and Rodrigue 2006). An interesting trend was observed for the influence of hypertension on brain and cognitive performance. The diagnosis of hypertension is usually acquired in older age, and the current sample of older adults was relatively small to allow a powerful test of that hypothesis. However, our results indicated that hypertensive participants tended to show poorer navigation skills than their normotensive counterparts. Thus, hypertension and possibly other vascular risk factors need to be taken into account in future evaluation of age differences in cognitive performance.In summary, the present study confirms robust age-related deficits in navigational skill. 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Aging199712372375WisemanRMSaxbyBKBurtonEJBarberRFordGAO'BrienJTHippocampal atrophy, whole brain volume, and white matter lesions in older hypertensive subjectsNeurology20046318921897 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B716D778D468DEFC32EE4B62F70AA93A8601B17.txt b/test/dataset/in/resources/corpus/Clean_0B716D778D468DEFC32EE4B62F70AA93A8601B17.txt new file mode 100644 index 0000000..2f3c18a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B716D778D468DEFC32EE4B62F70AA93A8601B17.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 66710.1093/geront/45.5.667 EXERCISE Characteristics of Physical Activity Programs for Older Adults: Results of a Multisite Survey Hughes Susan L. DSW 1 Williams Barbara PhD 2 Molina Lourdes C. MPH 1 Bayles Constance PhD 3 Bryant Lucinda L. PhD, MHSA 4 Harris Jeffrey R. MD, MPH 2 Hunter Rebecca MEd 5 Ivey Susan MD, MHSA 6 Watkins Ken PhD 7 Address correspondence to Susan L. Hughes, DSW, Center for Research on Health and Aging, University of Illinois at Chicago, 1747 W. Roosevelt Road, Room 558, M/C 275, Chicago, IL 60608. E-mail: shughes@uic.edu 10 2005 45 5 667 675 8 2 2005 23 8 2004 The Gerontological Society of America 2005 Purpose: Although increased participation in physical activity by older adults is a major public health goal, little is known about the supply and use of physical activity programs in the United States. Design and Methods: Seven academic centers in diverse geographic areas surveyed physical activity programs for older adults. Five sites conducted surveys by mail with telephone follow-up, and two administered surveys primarily by telephone. Reported program attendance rates were compared with local census data to assess unmet needs. Results: Of the 2,110 targeted facilities, 77% responded. Aerobic programs were offered by 73%, flexibility by 47%, and strength training by 26%. Commercial gyms or YMCAs, senior centers, park or recreation centers, and senior-housing facilities offered 90% of available programs. The 2000 Census enumerated 1,123,401 total older adults across the seven sites. Facilities reported 69,634 individuals as current weekly program participants, equaling 6% of the sites' total older-adult population. This percentage varied from 3% in Pittsburgh to 28% in Colorado. Implications: Based on conservative estimates of demand, the number of physical activity programs would have to increase substantially (by 78%) to meet the needs of older adults. The data also indicate the need to develop more strength-training programs and to engage a higher percentage of older adults in these programs. There is a clear need to stimulate demand for programs through health promotion. Exercise Needs and demand Providers Facilities hwp-legacy-fpage 667 hwp-legacy-dochead RESEARCH ARTICLE Current estimates predict that the number of adults over the age of 65 years (older adults) will increase from 13% of the U.S. population in 2000 to 20% by 2030, with the most rapid expansion occurring among those aged 85 and older (Federal Interagency Forum on Aging-Related Statistics, 2000). Although 17% of younger adults aged 18 to 64 have disabilities, disability prevalence increases to 50% in older adults (Centers for Disease Control and Prevention [CDC], 2001). Physical inactivity, a known modifiable risk factor for future disability, also increases with age (Huang et al., 1998; Hubert, Bloch, & Fries, 1993; LaCroix, Guralnik, Berkman, Wallace, & Satterfield, 1993; Seeman et al., 1995; Strawbridge, Cohen, Shema, & Kaplan, 1996). Physical activity interventions have demonstrated multiple benefits among older adults, including improved functioning (Fiatarone et al., 1994; Singh, 2002), improved health-related quality of life, and decreased levels of depression (CDC, 1996). Physical activity also has been shown to benefit older adults with specific chronic conditions, including arthritis, heart disease, and diabetes (Stahle, Nordlander, & Bergfeldt, 1999). Specifically, studies show that regular physical activity reduces the risk of dying prematurely and of developing diabetes, high blood pressure, and colon cancer; reduces feelings of depression and anxiety; helps control weight and maintains bone mineral density; and promotes psychological well-being (Binder, Birge, & Kohrt, 1996; Blumenthal et al., 1991; Fried et al., 1998; Preisinger et al., 1996; Singh et al., 1999). Despite these documented benefits, estimates suggest that 33% of men and 50% of women over the age of 75 engage in no physical activity (CDC, 2004). The prevalence of inactivity varies by racial and ethnic group and by gender, from 47% in White women aged 75 and older to 59% in older Black men and 61% in older Black women (Rejeski, Brawley, McAuley, & Rapp, 2000). Healthy People 2010 national health objectives recommend an increase in the proportion of adults who engage in regular, moderate physical activity for 30 min or more per day or vigorous physical activity 3 or more days per week for 20 min or more per occasion (U.S. Department of Health and Human Services, 2000). Current estimates suggest that, of older adults who engage in any physical activity, only 25% aged 65 to 74 and 15% aged 75 and older meet these recommendations for vigorous or moderate physical activity (CDC, 2002). A Robert Wood Johnson Foundation (2001) report identified the removal of barriers to increased physical activity among older adults as a major current national public health need. However, a potential key barrier to engaging in physical activity—the available supply of affordable physical activity programs for older adults—has not been studied to date. In this article we address this important issue by presenting findings from a multisite survey of physical activity programs for older adults in seven diverse geographic locations across the United States. Members of the Healthy Aging Research Network (HAN) conducted the survey between January and July 2003 with support from a CDC program—the Prevention Research Centers (PRCs). The goal of the PRC program is to support the development of academic and community partnerships to conduct prevention research, and each PRC conducts research and demonstration projects to address the most pertinent public health problems (Doll & Kreuter, 2001). The HAN survey built on the methods and findings of an earlier survey conducted by the Senior Health Alliance Promoting Exercise (SHAPE) in Cook County, IL, which compared the existing supply of physical activity programs to potential demand among older adults. The survey found that approximately 4% of the potential demand, defined as persons older than 65 in Cook County, based on the 2000 Census, could be met by the current supply (Hughes & Molina, 2002). The purposes of the current survey were to (a) obtain new information regarding the types of facilities that provide programs for older adults and the types of activities provided; (b) undertake an initial effort to estimate the supply of these programs in a variety of geographic areas across the nation; and (c) compare capacity data for the specific geographic areas studied to estimate the potential demand for programming by using U.S. census data. To our knowledge, this is the first study of the extent of physical activity programming for older adults at multiple sites across the United States. Methods HAN Sites The seven selected participating HAN academic centers target areas for study that are diverse with respect to geographic and demographic characteristics. Table 1 describes the population of older adults in each target area, including the size of each target area, its population density, the percentage of ethnic minorities, and the percentage of the population living below poverty level. The target-area size ranged from 26 square miles (67.34 km2) in southeast Seattle, WA, to 8,194 square miles (21,222.46 km2) in San Luis Valley, CO. The population of persons aged 65 or older ranged from 5,921 in San Luis Valley to 630,265 in Cook County, IL. In addition, the population density of persons aged 65 or older varied across target areas, from fewer than 1 older adult per square mile in San Luis Valley to almost 700 older adults per square mile in Cook County. Differences also existed with respect to the percentage of persons aged 65 or older who are minorities or non-Hispanic Whites, ranging from 9.1% in Allegheny County, PA, to 57.6% in southeast Seattle. The percentage of older adults living below poverty level ranged across target areas from 8.1% in Alameda County to 14.6% in the San Luis Valley, with an average of 10.7%. Survey Instrument The HAN survey evolved from a previous survey by the SHAPE, administered in 2001 to more than 1,000 facilities in Cook County, IL (Hughes & Molina, 2002). Revisions to the SHAPE survey included more detailed questions on program capacity, accessibility, features, participation barriers, and reasons why facilities did not provide programs for older adults. The survey asked about programming designed for older adults (those over the age of 65) and also sought information on programs older adults used that were not necessarily designed for them. The eight-page survey included an activity grid (e.g., type of activity offered, frequency), yes–no questions, open-ended questions, and checklists. Copies of the instrument are available from the corresponding author. In addition to collecting data on existing physical activity programs for older adults, the survey collected information about reasons that some organizations do not provide programs. The first survey questions asked respondents if they provided programming specifically for older adults or if older adults participated in any of their programs (e.g., 1. Do you provide physical activity programs designed specifically for older adults? 2. Do you have physical activity programs that younger as well as older adults attend?). If respondents answered “no” to both questions, they were asked why they did not provide programming for seniors. If respondents answered “yes” to either question, the instructions asked respondents to complete the entire questionnaire. Several survey items addressed program capacity. They included questions on the maximum capacity of older adults (aged 65+) per week, the actual number of older-adult participants per week, whether there were waiting lists for activities, and the estimated unduplicated number of older-adult participants in the past year. Organizations that provide physical activity programs for older adults pilot tested the survey twice. The National Council on the Aging conducted the first pilot test in 14 senior centers across the country. HAN members at the University of North Carolina conducted the second pilot test at 10 community sites. Survey items were revised and clarified based on the pilot findings. Sampling Frame All seven participating HAN academic centers agreed to participate in the study, but each site chose its own geographic target area to study. The Seattle site, for example, wanted to do an in-depth assessment of programming availability within a precisely defined, underserved minority community. In contrast, the Chicago site had previously surveyed Cook County, had already developed an initial sampling frame, and wanted to update its findings on the same population of respondents. As previously noted, the size of the geographic target area included in the surveys varied across sites from 26 square miles (67.34 km2) in southwest Seattle to 8,194 square miles (21,222.46 km2) in San Luis Valley. Despite this variation in size, each participating site attempted to assemble as exhaustive a list of potential physical activity providers as possible within its geographic area, given available resources. To err on the side of inclusiveness, each site initially included a broad spectrum of community organizations thought to provide physical activity programs for older adults. Thus, the initial sampling frames included a wide array of potential providers, including senior centers, community centers, YMCAs or YWCAs, commercial gyms, county and city parks and recreation facilities, churches, schools, hospitals, private or public housing for seniors, and residential facilities for able-bodied older adults (e.g., independent-living facilities). These sampling frames included most of the organizations that might have had contracts with Area Agencies on Aging and all known senior centers. Information gathered during the survey process led to refinement of the sampling frames and elimination of initially suggested facilities or programs that no longer existed, had moved from the area, or did not provide physical activity programming for persons of any age. Survey Administration Each HAN site acquired approval for the study protocol from its human subject research institutional review board. All HAN sites began survey administration at the same time and followed similar protocols. Five sites conducted surveys initially by mail and followed up by telephone or in person. The remaining two sites administered the survey primarily by telephone. One of the seven sites also made the survey accessible on the Internet. In most cases, sites used a combination of methods to maximize response. Initial contact by an introductory letter or phone call gave recipients information about the HAN, the survey's history and purpose, benefits of the survey to the organization (specifically, future publication of directories of available programs and facilities), HAN site-specific contact information, and expected survey timing. This introduction also requested confirmation of the appropriate contact person in the facility or organization to receive the survey. Most HAN sites waited 2 weeks after the introduction letter or call to mail the survey. For the HAN site that conducted the survey by telephone, the initial call also served as the first attempt to complete the survey. If the first mailing or phone calls did not generate responses, all sites made follow-up calls or on-site visits to nonrespondents. The follow-up process spanned several months. During follow-up, sites offered organizations the choice of responding to the survey by telephone or in person, and having the survey re-sent or faxed. Some sites with a comparatively small number of providers in their target areas were able to achieve very high response rates by calling repeatedly until they achieved a response. Others with larger numbers of providers in their target areas had lower response rates, but still used multiple call backs to all potential respondents. The use of a combination of administration methods yielded higher response rates than any single method and offered the opportunity to clarify respondents' questions about the survey. Each HAN site recorded survey activities on an Excel tracking sheet that was separate from data received from programs and facilities. This tracking sheet listed the organization name or identification number, contact information, date of initial mailing, follow-up activity, response dates, and any specific data-collection issues that arose. The University of Washington HAN site's Health Promotion Research Center (UWHPRC) served as the central data-collection site. UWHPRC created a universal Microsoft Access database and provided each HAN site with detailed instructions for data entry. Submitted data excluded all personal identifiers (institutional or individual). UWHPRC conducted a database reliability check 1 month after each HAN site began using the database. HAN sites sent all survey data to UWHPRC for analysis when data collection and entry were completed. Table 2 provides sampling frame sizes for each HAN site (adjusted for initially incorrect or incomplete information as described earlier), response rates, and the number of facilities in each sample that offered programs. Respondents included organizations that offered physical activity programs for older adults and completed the entire survey, organizations that did not offer physical activity programs for older adults and completed the abbreviated version of the survey, and organizations that responded orally that they did not offer physical activity programs for older adults and did not complete any version of the survey. Results Although the number of facilities surveyed by each site varied more than 20-fold, ranging from 29 organizations in Colorado to 737 in Chicago, response rates were good for all seven sites (see Table 2). Response rates ranged from 67% in Alameda County, CA, to 100% in San Luis Valley, CO, with an average response rate of 77% across sites. Among the 1,168 responding facilities, 675 (58%) reported that they offered programs for older adults. Of 326 facilities that stated they did not offer programs and provided a reason for lack of programs, 161 (50%) identified the most common reason as a perceived lack of interest from older adults, followed by lack of funding (46%), lack of staff interest (44%), lack of staff knowledge regarding frail adults (34%), staff shortage (34%), lack of staff training regarding older adults (24%), and concerns about liability (23%). Respondents could give multiple reasons for not offering programs. Of 675 facilities that provided programming designed for or used by older adults, 652 (97%) provided information regarding the specific types of programs offered (Table 3). Overall, aerobic programs were offered most frequently (73%), in contrast to flexibility (47%) and strength training (26%), and 31% of facilities offered multicomponent programs. Among facility types, senior centers most frequently offered aerobic programs, and hospitals and clinics most frequently offered strength-training and flexibility programs. Most facilities surveyed provided more than one program. Specifically, the 652 facilities surveyed provided 2,546 programs to 69,634 older adults weekly (Table 4). This amounts to an average of four programs per facility. The most commonly offered and best-attended programs were aerobics (47% of programs, 53% of attendance), followed by flexibility (24% of programs, 19% of attendance). Although strength training was offered by 26% of facilities, it represented only 10% of total programs and 11% of attendance. The most popular aerobic programs were aerobic exercise (unspecified), stationary equipment, chair-based activities, walking, and dance. Four types of facilities accounted for 90% of programs offered (Table 5). Although we originally tracked commercial gyms and YMCAs separately, we combined them into one type of respondent in Table 5 because calls to both types of facilities showed that their fee structures were similar in terms of both initial membership and monthly fees. An analysis of program offerings by facility type showed that commercial gyms and YMCAs offered 27% (685 programs), senior centers offered 24% (604), park and recreation centers or community centers offered 23% (582), and senior housing facilities offered 16% (415). These same facility types have the largest number of older adults participating in programs per week, constituting 88% of attendance. Across facility types, the percentage of programs offered closely reflects the percentage of older-adult participants per week, with the exception of park and recreation centers or community centers, which have a greater percent of programs offered than attended. It is important to note, however, that facility types with the greatest number of programs might also have the largest capacity to serve older adults. Several items on the survey addressed issues of access. First, with respect to populations served, facilities reported that they served the following specific, nonexclusive subpopulations of older adults: sedentary (52% of all older adults served), low income (47%), frail (43%), and non-English speaking (25%). Second, regarding physical access to programs, of 675 respondents, 88% reported having parking available on site, 66% were within one fourth of a mile (0.40 km) of public transportation, and 33% had senior transportation or shuttles that conveyed participants to the facility. The survey also inquired about program fees with respect to financial access, but it found too much variation across respondents in terms of monthly memberships versus daily fees versus class fees to be able to report meaningful information in a consistent way. We also report findings regarding two measures of demand. The first measure estimates demand as a function of the existence of waiting lists to gauge demand for physical activity programming in each geographic area (Table 6). Only 4% of programs reported that they had waiting lists—a consistent finding across all sites. The second measure compares the U.S. census population at each site with reported participation in programs. Of 1,123,401 total older adults that the 2000 Census enumerated across the seven sites combined, the facilities identified 69,634 individuals as current weekly program participants. This number of participants equals 6% of the total older-adult population across the sites. The participation percentage varied from 3% in Pittsburgh to 28% in Colorado and was generally higher in areas with the fewest numbers of programs. It is important to note that these percentages, although low, may overestimate participation because they likely include individuals who participated in more than one activity. Facilities also estimated their maximum capacity to serve older adults seeking programs. The total estimated maximum capacity across all sites, 207,328, would meet the needs of only 18% of older adults residing in the survey sites. Finally, responses to several survey items indicate the presence of program-management issues. First, nonresponses and follow-up calls to a number of facilities regarding the aforementioned capacity items revealed that several respondents had difficulty documenting the number of persons served per year or the number of persons attending programs during a given year and had a particularly difficult time estimating the number of persons who could be served by equipment as opposed to classes. Other responses concerning program-management issues show that, for the 675 facilities offering programs, 49% conducted program evaluations, 74% tracked attendance, 47% tracked participant progress, and 56% trained instructors. Senior centers and hospitals, more than other facilities, performed program evaluations (62% and 70%, respectively) and tracked attendance (83% and 89%, respectively), whereas churches were least likely to conduct program evaluations (38%) or track attendance (64%). Hospitals also were most likely to train instructors (81%). Forty-one percent of organizations indicated an interest in obtaining assistance with programming for older adults. Discussion Evidence regarding the benefits of physical activity to older adults is strong and compelling, especially when coupled with the prevention imperative posed by the rapidly growing aging population and costs known to be associated with inactivity. Equipped with this knowledge, the public health, personal health, and aging communities increasingly call for older adults to take to the trails, dance floor, or pool. Although these calls may actively stimulate demand, we know remarkably little about the available supply of organizations that provide physical activity programming for older adults. Is supply adequate to meet current demand? Is there room for growth? How can we build increased capacity among active organizations or potential program providers? These questions become increasingly urgent as the older population grows, and we continue to encourage physical activity as sound prevention. We know that many older adults seek physical activity opportunities independently, but others need structured programs (King et al., 2000). Structured programs may be particularly helpful for sedentary older adults who need instruction and support in getting started and integrating behavioral change into their lifestyles (King, Haskell, Taylor, Kraemer, & DeBusk, 1991). The HAN physical activity program capacity survey provides new information regarding the types of organizations that provide programs to older adults and the types of activities provided. The data also yield an estimate of the supply of these programs in a variety of settings across the nation, and they provide crude estimates of the existing supply compared with the potential demand for programming based on U.S. census data for the specific geographic areas studied. With respect to those facilities that do not provide programs, the most commonly reported reason cited was a perceived lack of interest from older adults (50%), followed by lack of funding, lack of staff interest, and lack of staff knowledge regarding frail older adults. This finding indicates that substantial effort has to be invested in educating older adults about the benefits of physical activity, in increasing funding for programs, and conducting staff training regarding the exercise needs and capacities of older adults. The overall low percentage of total population served (6% across study sites) is another noteworthy finding of the survey. The fact that study sites with the highest response rates reported greater attendance on average tempers our confidence in this finding. Improved response rates could result in an upward adjustment of attendance. In contrast, the data may overestimate participation because some individuals participate in more than one physical activity program in a given week. For example, they may attend an aerobics class and also participate in a senior golf league. Despite these cautionary notes, this indicator of program participation causes concern regarding the engagement of older adults in physical activity. Even if we take into account individuals in institutions and the portion of the older adult population that prefers individual physical activity to structured programs, a significant gap remains to be filled. For example, consider Cook County, which, according to data from the 2000 Census, has 630,265 persons aged 65 and older (U.S. Census Bureau, 2000). CDC (2003) data indicate that nationally about 4% of the elderly population resides permanently in nursing homes, reducing the size of the relevant Cook County population to roughly 600,000. If we further estimate, using national averages on participation in leisure-time physical activity, that 33% of the population is currently sedentary, that reduces the number of persons in Cook County needing programs to 198,000 (CDC, 2001). If we further assume, on the basis of prior reports in the literature, that 28% of the remaining population prefer group as opposed to individual or home-based exercise, then we estimate that 55,440 persons not currently involved in facility-based programming in Cook County might participate in and benefit from programming if it were available (Mills, Stewart, Sepsis, & King, 1997). Because the findings presented in this article indicate that 31,171 older adults already participate in programs, program capacity in Cook County would have to increase by 78% to meet the needs of this group, assuming that we can motivate them to become involved. Findings regarding attendance per facility type are also of interest. A majority of participants (57%) attend programs that are not geared specifically to older adults, and 43% attend programs tailored for older adults. Across all programs offered, 55% are not geared to older adults, and 45% of programs target only older adults. These data underscore the important role of general physical activity programs in addressing the needs of both older and younger adults. Not all older adults need specialized or senior-only programs. These data demonstrate the key contribution that private-sector organizations make to physical activity programming. Accordingly, as we move to build capacity, we must look both to the private sector and to traditional senior-service organizations for growth in size and program scope. Additional need for programming documented by survey results is also of interest. First, findings indicate that only 31% of 163 facilities that serve non-English-speaking populations tailor programs for non-English-speaking participants. This finding demonstrates a need for additional focus on non-English-speaking populations. Second, given that attendance largely parallels program offerings and that 73% of facilities offer some form of aerobic activity, it is not surprising that aerobic programs are best attended. However, this finding also raises the question of whether facilities provide what consumers request or whether consumers use what is available. Another consideration relates to consumer and organization perceptions about what constitutes appropriate and safe activity for older adults. For example, flexibility programs are widely believed to be safe and low risk, whereas strength training is less familiar to older adults, and both older adults and organizations that serve them may perceive it as a more risky undertaking. Given the strong evidence concerning the benefits of strength-training activity for older adults (CDC, 2004), these data point clearly to the need to develop more strength-training programs and to engage a higher percentage of older adults in these programs. A need to create demand for such programs through public education also may be indicated. It is likely that strength training requires technical assistance for providers to support sound program development. This study has limitations that merit discussion. The communities surveyed have diverse ethnic and geographic characteristics but constitute a convenience sample and do not represent the nation as a whole. Although the actual data collected were consistent across sites, administration methods varied (e.g., telephone, mail, and in-person interviews). For example, South Carolina used a telephone survey as the primary administration method because the site believed it would be better received, particularly at churches in its sampling frame. This variability can affect response rates. Moreover, some sampling-frame differences existed across sites. For example, in some areas, churches generally did not offer programs; in other communities, faith-based organizations represented a key provider type. In those cases, sites chose to include or exclude specific types of organizations. Other types of organizations like Federally Qualified Health Centers also were not included. In some instances, responses indicated possible problems among responding organizations relative to the estimation of capacity. For example, some organizations could not provide data pertaining to the number of persons served over the course of a year or the number of persons in attendance during a week. Organizations had a particularly difficult time estimating the number of persons who could be served by using equipment as opposed to attending classes. Similarly, organizations serving both older and younger adults had to estimate participants' ages (older or younger than age 65), and they found estimating use by older adults to be challenging. Other program-management issues noted by survey respondents included limited attention to evaluating programs, tracking participant progress, and training instructors. These management issues also may be appropriate foci for technical assistance, especially because 41% of the surveyed organizations indicated an interest in obtaining assistance with their programming for older adults. Despite these limitations, findings from this survey suggest that the current supply of physical activity programs designed for older adults in the target communities does not adequately meet potential demand for programs by older adults. More energy should be focused on increasing demand by raising awareness of the importance of physical activity among older adults and reducing barriers to exercise. The barriers perceived by providers indicate a substantial need for health-promotion campaigns for users and providers, as well as increased funding for programs. Future research should examine ways to refine the survey sampling methodology, streamline the survey instrument, and replicate this survey in other communities. These efforts are vital if we are to obtain valid data on the existing supply of physical activity programs and be empowered to make valid assessments of the fit between supply and demand in the future. This research was supported by grants from the Centers for Disease Control and Prevention (CDC) Prevention Research Centers Program (Grant U48/CCU0009654) and the Health Care and Aging Studies Branch, and from the National Council on the Aging and the Robert Wood Johnson Foundation. We acknowledge the following people for their contributions to survey development, data collection, or manuscript review: Melissa Kealey, University of California, Berkeley; Jennifer I. McLean, University of Colorado; Thomas Prohaska, Ella Fermin, Megan Renehan, University of Illinois at Chicago; members of the Cook County Senior Health Alliance Promoting Exercise; Christen Sible, Cindy Schrauder, Michael Randall, Carol Giuliani, Franzi Zabolitizki, Victor Marshall, Mary Altpeter, and Tiffany Small, University of North Carolina; Jane Schall, University of Pittsburgh; Harriet Williams, Sara Wilcox, Bridget Kane, Larissa Oberrecht, Jill Maxwell, and Joey Vrazel, University of South Carolina; Gwen Moni and James LoGerfo, University of Washington; and Nancy Whitelaw at the National Council on the Aging. 1 Center for Research on Health and Aging, University of Illinois at Chicago. 2 Health Promotion Research Center, University of Washington, Seattle. 3 Center for Healthy Aging, University of Pittsburgh, PA. 4 Division of Health Care Policy and Research, University of Colorado Health Sciences Center, Denver, CO. 5 Chapel Hill School of Medicine Program on Aging, University of North Carolina. 6 Berkeley School of Public Health, University of California. 7 Department of Health Promotion, Education, and Behavior, University of South Carolina, Columbia. Decision Editor: Linda S. Noelker, PhD Table 1. Description of Target Areas. City or County State Area (square miles) 65+b Densityc Minority (%)d Below Poverty (%) Alameda CA 737 147,591 200.2 43.1 8.1 San Luis Valleya CO 8,194 5,921 0.7 39.7 14.6 Cook IL 945 630,265 666.9 30.3 10.3 Durham, Henderson, and Northampton NC 1,200 44,755 37.3 20.9 11.4 Allegheny PA 730 228,416 312.9 9.1 9.0 Richland and Lexington SC 1,457 53,459 36.7 22.4 10.9 Southeast Seattlea WA 26 12,994 500.0 57.6 10.8 aSan Luis Valley is Alamosa, Conejos, Costilla, Mineral, Rio Grande, and Saguache counties; Southeast Seattle is King County census tracts 93–95, 99–104, 107–114, and 117–119. bPopulation aged 65 and older (U.S. Census 2000 table P12I; available at http://www.census.gov). cDensity = population aged 65 and older per square mile. dPopulation aged 65 and older who reported other than non-Hispanic White race or ethnicity (http://www.census.gov). Table 2. Response Rates by Site. City or County State No. of Facilities in Initial Sampling Framea No. of Eligible Facilitiesb No. of Facilities That Responded (%) No. of Facilities That Offer Programsc Alameda CA 289 251 168 (67) 88 (52) San Luis Valley CO 36 29 29 (100) 29 (100) Cook IL 804 737 529 (72) 273 (52) Durham, Henderson, and Northampton NC 617 179 143 (80) 58 (41) Allegheny PA 70 70 60 (86) 57 (95) Richland and Lexington SC 237 197 191 (97) 138 (72) Southeast Seattle WA 57 49 48 (98) 32 (67) aNumber of facilities believed to have the potential to offer physical activity programs in the target area and were mailed a questionnaire. bNumber of eligible facilities included facilities that did not offer physical activity programs but completed the survey. Nonrespondents, except churches, were assumed to have physical activity programs and were included. Nonrespondent churches were assumed to not have physical activity programs and were excluded (see text). Facilities that indicated they did not offer physical activity programs and did not fill out the survey were removed. cNumber of facilities that responded positively to at least one of the two screening questions. Table 3. Percent of Facilities Offering Various Types of Physical Activity Programs, for Facilities With Any Programs. Type of Facility Total No. of Facilities Types of Programs (% of Facilities) Aerobic Strength Training Flexibility Multicomponent Recreational Other Commercial gym or YMCA 184 70 22 45 35 25 15 Senior center 150 81 21 55 29 15 13 Park or recreation and community center 146 73 32 40 18 32 10 Housing 103 71 30 53 41 11 6 Church 39 51 13 28 38 18 8 Hospital or clinic 26 77 46 62 35 15 23 School 4 100 50 75 0 50 0 Total 652 73 26 47 31 22 12 Table 4. Program Attendance by Physical Activity Program Subtype. Program Type Programs Offered Attendance per Week N % of Total N a % of Total Aerobic 1,184 47 37,041 53     Aerobic exercise 290 25 8,952 24     Stationary equipment 168 14 8,057 22     Walk 178 15 4,660 13     Chair based 181 15 4,591 12     Dance 154 13 4,436 12     Water aerobics 106 9 3,253 9     Swimming 58 5 1,988 5     Other 49 4 1,104 3 Flexibility 607 24 13,281 19 Strength training 266 10 7,356 11 Multicomponentb 205 8 4,949 7 Recreational 188 7 4,649 7 Otherc 96 4 2,358 3 Total 2,546 69,634 100 aAttendance is the sum of actual number of older adults reported as participants in all programs or activities (except those that were educational only). If an actual number is missing, then attendance is computed as the median value for that activity or as the mean proportion of maximum capacity (whichever is less). Because adults may participate in more than one activity per week, the sum may not be an unduplicated number. bMulticomponent program example: aerobic and free weights. Multicomponent programs are counted as one program, assuming that same older adults attend both components. If two different attendance numbers are listed, then take maximum of the two (and assume that some older adults left before other component). cOther does not include programs that were only educational. Table 5. Program Attendance by Facility Type. Facility Type Programs Offered Attendance per Weeka N % N % Commercial gym or YMCA 685 27 20,917 30 Senior centerb 604 24 16,830 24 Park or recreation and community center 582 23 13,379 19 Housing 415 16 10,813 15 Hospital or clinic 146 6 5,970 9 Church 78 3 1,165 2 School 36 1 560 1 Total 2,546 100 69,634 100 aSum of actual number of older adults for all programs or activities (Q7–Q34, but not Q32). If missing actual number, we used the median value for that activity or the mean proportion of maximum capacity (whichever is less). Because adults may participate in more than one activity per week, sum is an not unduplicated number. bSenior center includes any center or day program geared to older adults. Table 6. Waiting Lists and Weekly Attendance by Site as Percentage of Total Population Aged 65 and Older. City or County State Total No. of Programsa Programs With Waiting List Attendance per Week N % of Programs N b % of Total Population Aged 65+c Alameda CA 305 12 4 11,338 8 San Luis Valley CO 78 0 0 1,643 28 Cook IL 1079 43 4 31,171 5 Durham, Henderson, and Northampton NC 236 12 5 9,710 22 Allegheny PA 286 10 3 7,601 3 Richland and Lexington SC 305 4 1 5,384 10 Southeast Seattle WA 97 3 3 2,787 21 Total 2,386 84 4 69,634 6 aTotal number of programs is not the same as those in Table 4 because some programs in Table 5 are missing waiting list information and are not included. bSum of actual number of older adults (65+) per week for all activities reported by responder, aerobic exercise to other, not including educational materials (Q32). If missing actual number, we used the median value for that activity or the mean proportion of maximum capacity (whichever is less). 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American Journal of Epidemiology, 144, 135-141. U.S. Census Bureau. (2000). Summary File 1 and Summary File 3—Census 2000 demographic profile highlights for Cook County, IL. Washington, DC: U.S. Government Printing Office. U.S. Department of Health and Human Services. (2000). Healthy people 2010: Understanding and improving health and objectives for improving health (2nd ed.). Washington, DC: U.S. Government Printing Office. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B7707F0F4E4D9DD633419401936BBA794C59402.txt b/test/dataset/in/resources/corpus/Clean_0B7707F0F4E4D9DD633419401936BBA794C59402.txt new file mode 100644 index 0000000..af8c840 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B7707F0F4E4D9DD633419401936BBA794C59402.txt @@ -0,0 +1 @@ +JAGP61041S1064-7481(12)61041-010.1097/00019442-199800640-00003American Association for Geriatric PsychiatryTABLE 1Prevalence of psychiatric symptoms in patients with and without hoarding behavior, n (%)CharacteristicsHoarding(n = 30)Nonhoarding (n = 103)χ2[1]PDelusions21 (70.0)67 (65.0)0.2540.614 Theft16 (53.3)56 (54.4)0.0100.920 Persecutory5 (16.7)30 (291)1.8600.173 Jealousy3 (10.0)18 (17.5)0.9770.405Hallucinations9 (30.0)37 (35.9)0.3600.548 Auditory5 (16.7)19 (18.4)0.0500.823 Visual7 (23.3)27 (26.2)0.1010.750Misidentifications14 (46.7)30 (291)3.2290.072 Phantom boarders symptoma8 (26.7)15 (14.6)2.3800.123 Residence is not home7 (23.3)15 (14.6)1.2940.271 Mirror signb3 (10.0)5 (4.9)1.0800.379 Caregiver is impostor2 (6.7)11 (10.7)0.4240.732Other disturbances Repetitive behaviors24 (80.0)55 (53.4)6.8170.009 Day/night disturbance8 (26.7)28 (27.2)0.0030.955 Aggressiveness18 (60.0)48 (46.6)1.6680.196 Agitation18 (60.0)49 (47.6)1.4350.231 Wandering15 (50.0)44 (42.7)0.4990.480 Hyperphagia18 (60.0)24 (23.3)14.482<0.001 Pilfering15 (50.0)26 (25.2)6.6780.010athe belief that other (imaginary) people are in the house.bthe inability to recognize one's own mirror reflection (or statements such as “Someone else is in the mirror.”)Received March 24, 1997; revised August 10, 1997, December 24, 1997; accepted January 15, 1998.The authors are indebted to the geropsychiatric staff at Veterans General Hospital–Taipei for their assistance in this study.This study was supported by Veterans General Hospital–Taipei, Grant VGH-85-150.Regular ArticlesHoarding Behavior in Dementia: A Preliminary ReportJen-PingHwangM.D.Shih-JenTsaiM.D.*Chen-HongYangM.D.King-MingLiuM.D.Jiing-FengLirngM.D.Department of Psychiatry, Veterans General Hospital-Taipei, Republic of China*Address correspondence to Dr. Tsai, Department of Psychiatry, Veterans General Hospital-Taipei, No. 201, Shih-Pai Road Sec. 2, Taipei, Taiwan, Republic of ChinaHoarding behavior has been reported in several mental disorders and is occasionally reported by the caregivers of dementia patients. Such behavior may have adverse effects on the patients and increase the burden of the caregivers. This study was conducted to investigate the prevalence of hoarding behavior in patients with dementia and identify the characteristics and psychiatric symptoms associated with it. The sample was 133 dementia patients admitted to a geropsychiatric ward. Of the 133 dementia patients, 30 (22.6%) showed hoarding. Hoarding was found in various types of dementia. Patients with hoarding had a higher prevalence of repetitive behaviors, hyperphagia, and pilfering. Results suggested that hoarding behavior is a common symptom in dementia patients and a complex phenomenon. Better understanding of the underlying pathogenesis may highlight specific pharmacological or behavioral methods for treatment of the behavior.Hoarding—collecting a large number of unneeded objects—is commonly found in the general population1 and in a variety of mental disorders, including schizophrenia,2–4 dementia,2 obsessive-compulsive disorder,2 and eating disorders.5 Greenberg et al.2 proposed that when hoarding is the main symptom, it is the final common pathway for a spectrum of different processes. At one end of the spectrum is obsessive-compulsive disorder; at the center, paranoid disorders, and at the other end, organic mental disorders.Hoarding behavior has long been recognized as a feature of dementia2 and is frequently noted by the caregivers of dementia patients. It can interfere with the hygienic management and health of patients, and patients may become extremely agitated and even violent when family members threaten to discard their possessions.2,6 Also, many nurses view hoarding as negative and assume that the patient is a bit “strange” or psychotic.7 A search of the MEDLINE database revealed only one case report concerning hoarding in dementia patients.2 Our study was designed to investigate the prevalence of hoarding behavior in dementia patients admitted to the geropsychiatric ward of a general hospital. We also made an attempt to identify the characteristics and psychiatric symptoms that are associated with hoarding behavior.METHODSThe sample population consisted of 133 consecutive dementia patients admitted to the geropsychiatric ward of Veterans General Hospital–Taipei between August 1989 and February 1996. Most of the patients were admitted because of violence, inappropriate behaviors, or emotional problems. All patients met the criteria for dementia set by the Diagnostic and Statistical Manual of Mental Disorders (DSM-III-R).8 Those with Dementia of the Alzheimer's type (DAT) met DSM-III-R criteria for primary degenerative dementia as well as the diagnostic criteria developed by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA).9 All patients with multi-infarct dementia met the DSM-III-R criteria for multi-infarct dementia. The other nonspecific dementias were defined on the basis of their etiologies, according to the DSM-III-R criteria. Diagnosis was based on a complete medical and neuropsychiatric examination, including history, physical examination, ECG, EEG, blood count, biochemistry (electrolytes, liver and renal function, vitamin B12, folic acid), thyroid function tests, and a serological test for syphilis. Computed tomographic scans of the brain were available for all patients.Patients were divided into two groups, based on the presence or absence of hoarding. Hoarding was defined as repeatedly collecting mostly useless or unneeded objects during some time from the onset of the illness. Such behaviors were indiscriminate and developed after the onset of dementia.All demographic data were supplied by caregivers. Psychotic symptoms and behavioral problems were assessed by a checklist adapted from the Behavioral Pathology in Alzheimer's Disease Rating Scale (BEHAVE-AD) by Reisberg et al.,10 with some modifications. In each case, the patient, as well as at least one caregiver familiar with the behaviors of the patient, was interviewed by a geriatric psychiatrist. Symptoms were evaluated and classified as dichotomous variables. Dementia severity was assessed on admission by use of the Folstein Mini-Mental State Exam (MMSE); scores ranged from 0 to 30.11Data were analyzed by use of SPSS for Windows. Two-tailed t tests were used to compare the differences between the means of continuous variables. The chi-square test was used to compare differences in dichotomous variables. A P value less than 0.05 was considered to be statistically significant.RESULTSThe mean age of these patients was 74.1±6.3 years (range: 65–91), and the mean MMSE score was 11.6±6.2 (median: 11). There were 98 men and 35 women; the large male predominance is characteristic of a veterans' hospital. Of the 133 dementia patients, 30 (22.6%) showed hoarding. Patients with hoarding were found in various types of dementia classifications: Alzheimer's dementia (24 of 75), multi-infarct dementia (4 of 40), and dementia not otherwise specified (NOS; 2 of 18). The items patients hoarded included daily necessities (10), food (7), garbage (6), newspapers or magazines (6), broken umbrellas or electrical items (6), plastic bags (4), old clothes (4), and cigarette butts (1). Patients in the hoarding group stored their objects around their apartment, in closets, in drawers, under pillows or bedclothes, under trash cans, under the bed, or in boxes, or carried the items with them. Reasons given by patients for hoarding included “It will come in handy or can be sold.”, “This object belongs to me.”, “It will be stolen.”, “It is too good to throw away.”, “I don't want to be caught without a needed item.”, “Even though the food is outdated, it is still edible.”, and “I usually feel hungry.”There were no significant differences between the two groups in regard to age, age at onset, gender, educational level, or MMSE score. Table 1 summarizes the frequency of psychiatric symptoms in the two groups. The prevalence of repetitive behaviors was significantly higher in the hoarding group. Also, patients with hoarding were more likely to have hyperphagia and to pilfer other people's possessions.DISCUSSIONThe prevalence of hoarding (22.6%) in dementia in this study was high. In a study of behavioral complications of dementia, the prevalence of hoarding was 1.9%.12 The discrepancy in these two studies may stem from the differences in the definition of hoarding and the sample source. In the previous study, the patients were in the community, and their disease severity varied widely. Behavior was assessed for only one week. The sample in our study was patients with moderate-to-severe dementia admitted to a geropsychiatric ward. The duration being assessed was from the onset of illness. Our study found that hoarding occurred in Alzheimer's dementia, multi-infarct dementia, and dementia NOS; this finding suggests that hoarding is a universal problem in various kinds of dementia.It was believed that older women hoard more than older men,7 but our study showed no sex differences. We also did not find a difference in the MMSE score between groups. This finding may be because our study group was more homogenous, with most patients having moderate-to-severe dementia. Homma et al.12 reported that hoarding was found in 5.3% of patients with moderate dementia but was not found in those with mild dementia. Persons suffering from cognitive impairment may be unable to determine the relative importance of articles, so they tend to save everything, resulting in “conditions of hoarding and filth.” Some patients in this study tended to save everything they considered valuable even before disease onset. Initially, they stored their collection well, but it eventually became disorganized as their dementia progressed.Analysis of the psychiatric symptoms showed that patients who hoarded had a higher prevalence of repetitive behaviors, hyperphagia, and pilfering. Hoarding has been classified with repetitive behaviors and has been postulated to involve hippocampus dysfunction.3,4 In a recent report, magnetic resonance imaging (MRI) showed some atrophy of the hippocampus in both Alzheimer's dementia and multi-infarct dementia patients.13 Alteration of eating habits has been found in dementia patients, and one study demonstrated that 26% of patients had eaten significantly more at some stage since the onset of dementia.14 One patient in this study stored spoiled food, which caused gastrointestinal problems. He insisted that he was hungry and the food was still edible. Some patients with hyperorality stored food around their bedside during hospitalization. During hospitalization, some hoarders took objects from the ward or from other patients. Often these patients failed to discriminate and claimed that these objects belonged to them. Newspapers, magazines, food, soap, and toilet paper were stored under the bed, in the bedclothes, or in the closet. Greenberg et al.2 also reported this phenomenon.One study reported that some patients carried or stored their hoarded items out of fear of theft.2 We also found this to be true in our patients, but in this study, the delusion of theft was not significantly different in the two groups. It is possible that the delusion of theft is common in dementia and explains hoarding behavior in only some patients.Hoarding sometimes is a safety hazard and can damage health. Patients may collect dangerous objects, newspapers may cause a fire, and outdated food spoils. Intervention is needed in these circumstances. However, the causes of hoarding vary from individual to individual. Before there is intervention, the underlying etiology must be explored. For example, dementia patients with hyperphagia who store food may benefit from drugs such as fluvoxamine.15 Phenylpropanolamine, which has been reported to inhibit feeding and hoarding in rats, may also help these patients.16 Patients who exhibit hoarding because of delusion of theft may respond to antipsychotic treatment. Also, Hogstel7 has proposed some nonpharmacological interventions for hoarding in elderly patients, such as distraction, limiting opportunities, behavioral therapy, group discussion, and removing potentially dangerous objects.7This is a preliminary report of hoarding behavior in dementia patients. There were two limitations in this study. First, because of the protean expression of hoarding behavior, it is difficult to clearly define such behavior. Second, the population in this study consisted of inpatients with moderate-to-severe dementia. Further study is needed to identify hoarding behavior in a community-based population. Also, it would be of interest to compare transnational and cross-cultural differences in hoarding behavior among dementia patients.CONCLUSIONHoarding is common in patients with dementia of various etiologies, particularly in those who exhibit repetitive behaviors, hyperphagia, and pilfering. The pathogenesis of hoarding is complex. The underlying etiologies must be clarified before there is intervention.References1LWWarrenJCOstromPack rats: world-class saversPsychology Today22198858622DGreenbergEWitztumALevyHoarding as a psychiatric symptomJ Clin Psychiatry5119904174213DJLuchinsA possible role of hippocampal dysfunction in schizophrenic symptomatologyBiol Psychiatry28199087914DJLuchinsMBGoldmanMLiebRepetitive behaviors in chronically institutionalized schizophrenic patientsSchizophr Res819921191235FRFrankenburgHoarding in anorexia nervosaBr J Med Psychol57198457606DGreenbergCompulsive hoardingAm J Psychother4119874094167MOHogstelUnderstanding hoarding behaviors in the elderlyAm J Nurs93199342458American Psychiatric AssociationDiagnostic and Statistical Manual of Mental Disorders3rd Edition1987American Psychiatric AssociationWashington, DCRevised9GMcKhannDDrachmanMFolsteinClinical diagnosis of Alzheimer's disease: reports of the NINCDS-ADRDA Work Group under the auspices of the Department of Health and Human Services Task Force on Alzheimer's DiseaseNeurology34198493994410BReisbergJBorensteinSPSalobBehavioral symptoms in Alzheimer's disease: phenomenology and treatmentJ Clin Psychiatry48suppl198791511MFFolsteinSEFolsteinPRMcHughMini-Mental State: a practical method for grading the cognitive state of patients for the clinicianJ Psychiatr Res12197518919812AHommaTIshiiRNiinaRelationship of behavioral complications and severity of dementia in Japanese elderly personsAlzheimer Dis Assoc Disord81994465313TMatsuzawaMTHishinumaHMatsuiSevere atrophy of amygdala and hippocampus in both Alzheimer's disease and multi-infarct dementiaScientific Reports of the Research Institute, Tohoku Univ. (Med.)371990232514CHMorrisRAHopeCGFaiburnEating habits in dementia: a descriptive studyBr J Psychiatry154198980180615RHopePAllmanHyperphagia in dementia: fluvoxamine takes the biscuitJ Neurol Neurosurg Psychiatry5419918816PJWellmanALevyInhibition of feeding and hoarding behaviors by phenylpropanolamine in the adult ratPharmacol Biochem Behav2919887981 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B78A3E78B5029ADA30D3B9CB9FF382C6880564F.txt b/test/dataset/in/resources/corpus/Clean_0B78A3E78B5029ADA30D3B9CB9FF382C6880564F.txt new file mode 100644 index 0000000..bca53dc --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B78A3E78B5029ADA30D3B9CB9FF382C6880564F.txt @@ -0,0 +1 @@ +gerontgerontThe Gerontologist1758-53410016-9013Oxford University Press10.1093/geront/gnp170FUNCTIONAssistive Device Use as a Dynamic Acquisition Process in Later LifePresslerKaris A.MA12FerraroKenneth F.PhD22Center on Aging and the Life Course and Department of Sociology, Purdue University, West Lafayette, Indiana1Address correspondence to Karis A. Pressler, MA, Center on Aging and the Life Course, Purdue University, Ernest C. Young Hall, 155 South Grant Street, West Lafayette, IN 47907-2114. E-mail: kpressler@purdue.eduDecision Editor: William J. McAuley, PhD620102712010503371381281020098122009© The Author 2009. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2010Purpose: This study identifies risk factors, including incident disability, for the use of assistive devices (ADs) among older people. Design and Methods: Three waves of data from the National Long-Term Care Survey (NLTCS) are used to examine whether upper and lower body disability lead to use of ADs (both number of devices used and number of activities of daily living domains for which ADs are used). Predictors of AD use include demographic variables, body mass index, and disability (both initial and incident). Relationships are estimated with negative binomial regression models. Results: Lower body disability, advanced age, and obesity are consistent predictors of the number of ADs used. An interaction between age and incident disability revealed the highest rate of adoption among the younger respondents who experienced increases in disability. Implications: Many older adults use ADs in response to the disablement process. In addition to need driven by rising disability, obese older adults use more ADs. Results from this study clarify who and why ADs are adopted by older persons and should facilitate effective intervention by health care personnel and caregivers.Assistive devicesDisabilityDisablement processAssistive technologyObesityAssistive devices (ADs), also referred to as assistive technology, are “items frequently used by people with functional deficits as alternative ways of performing actions, tasks, and activities” (LaPlante, Hendershot, & Moss, 1992, p. 2). Examples of ADs include walkers, wheelchairs, raised toilet seats, and grab bars. It is now widely recognized that ADs aid functional independence, enabling older adults to remain active when facing disability in activities of daily living (ADL; Agree & Freedman, 2003; Cornman, Freedman, & Agree, 2005). The use of ADs has risen substantially in the past 20 years, and current estimates reveal that approximately one third of all adults aged 65 years or older use at least one device (Freedman, Agree, Martin, & Cornman, 2006; Schoeni, Freedman, & Martin, 2008; Spillman, 2005). From another viewpoint, more than half of all AD users are older adults, and demand is expected to increase due to population aging and growing sales and acceptance of such aids (Centers for Disease Control and Prevention and The Merck Company Foundation, 2007).ADs can be an important resource for older adults, but timely adoption can mean the difference between maintaining and losing one’s functional independence. We conceptualize the adoption of ADs as a response to the disablement process, but find that many previous studies of the topic rely on interpretations from cross-sectional analyses or point estimates of disability. The present study makes use of 10 years of data from a longitudinal panel study to identify incident disability and whether it, in turn, increases the likelihood of using one or more ADs.Disability Dynamics and Use of ADsThe disablement process can be an abrupt transition for some people, such as for stroke victims (Gitlin, Luborsky, & Schemm, 1998), but it is a long and somewhat circuitous process for others. Research during the past decade has revealed that many functional limitations emerge during late middle age and that many older people experience spurts, plateaus, and declines in disability (Manton, 2008; Manton, Gu, & Lamb, 2006; Schoeni et al., 2008). Despite predictions of a compression of morbidity (and disability), most people spend years adjusting to growing disability, seeking help through environmental modifications, health care, and activity accommodations to manage their conditions (Verbrugge & Jette, 1994). ADs are one such resource that many people use effectively in the struggle to maintain functional independence (Verbrugge, Rennert, & Madans, 1997).Broadly speaking, previous research focused on ADs has had one of two main aims. First, several studies identify trends over time in the use of ADs, and most such studies use data from cross-sectional studies, including repeated cross sections (Freedman et al., 2006; Spillman, 2005). The overall trend is straightforward—increasing use of ADs—but especially for devices to aid walking, bathing, and toileting (Freedman et al., 2006; Russell, Hendershot, LeClere, Howie, & Adler, 1997).Second, a number of studies have been conducted to identify which older adults are most likely to adopt ADs to help maintain their functional independence. Findings from these studies generally concur that advanced age and disability are the most consistent predictors of use of ADs by older adults (Agree, Freedman, Cornman, Wolf, & Marcotte, 2005; Agree, Freedman, & Sengupta, 2004; Cornman et al., 2005; Hartke, Prohaska, & Furner, 1998; Tomita, Mann, Fraas, & Stanton, 2004). At the same time, there is some evidence of a nonlinear relationship between disability and AD use: a rise in AD use across low-to-moderate levels of disability but less so thereafter suggesting a threshold effect (Mathieson, Kronenfeld, & Keith, 2002). Research on predictors of AD use has also addressed the related question of whether such devices supplement or substitute for personal care. Most studies of these processes provide support for a process of supplementation (Agree, 1999; Hoenig, Taylor, & Sloan, 2003; Manton, Corder, & Stallard, 1993), although ADs may substitute for informal care on specific tasks (Agree & Freedman, 2000; Agree et al., 2005).Previous research has contributed much to our understanding of the magnitude of recent increases in AD use and who is likely to adopt them, but we identify four limitations of prior research in order to advance our knowledge and improve interventions. First, most studies examine overall disability, but Verbrugge and colleagues (1997) showed that lower body disability is more consequential to AD use. Accordingly, we differentiate upper and lower body disability as predictors of ADs.Second, beyond age and disability, there is little agreement in the literature as to other factors that predict AD use. For instance, some studies show that persons with less education are more likely to use ADs (e.g., Kaye, Kang, & LaPlante, 2000), whereas other studies show the opposite (e.g., Cornman et al., 2005). Similarly, there is inconsistency in reports of racial differences in AD use (cf. Cornman & Freedman, 2008; Kaye, Yeager, & Reed, 2008). Moreover, some variables that are theoretically relevant have received scant attention. One example is obesity, which many studies show is related to disability (Ferraro & Kelley-Moore, 2003; Guralnik, Fried, & Salive, 1996), but few studies actually examine whether obesity increases AD use (Cornman & Freedman; Verbrugge & Sevak, 2002). The current study considers these and other covariates while prospectively modeling use of ADs over time.Third, there is some disagreement as to what actually constitutes AD use. Some studies (including the present one) identify ADs used specifically for physical disability (Hoenig et al., 2003; Jette, 1994; Manton et al., 1993), but other studies use a more expansive definition that includes visual, cognitive, and hearing devices (Mann, Ottenbacher, Hurren, & Tomita, 1995; Mann et al., 2008; Tomita, Mann, Fraas, & Burns, 1997). Of course, with the more inclusive definition, nearly all older adults would be AD users. In addition, Cornman and colleagues (2005) show quite convincingly that some surveys underestimate AD use by posing the question to only those respondents who report difficulty with daily activities. This decision may contribute to inconsistency in findings on the topic and understate the importance of AD use in the daily lives of older people.Fourth, if AD use is presumed to be a resource that is activated in response to growing disability, longitudinal panel studies are needed to uncover how people respond to the disability episodes. Cross-sectional analyses might even give the appearance of less disability if AD use is part of the question tapping performance of activities. The extant literature is replete with cross-sectional analyses as well as calls for panel studies (e.g., Agree et al., 2004). Nevertheless, we were able to identify only one study that used a prospective two-wave panel design on a national sample (Cornman & Freedman, 2008) and a few others that are experiments, testing the effect of AD use on a select sample over time (Gosman-Hedström, Claesson, & Blomstrand, 2002; Mann, Ottenbacher, Fraas, Tomita, & Granger, 1999; Mann et al., 2008).There are two primary reasons why longitudinal panel designs make sense for research on ADs. First, illness episodes are the likely turning points in AD adoption. During the early stages of a disease, a person may be able to manage their condition without an AD, but the advanced stages of a disease may mean that the person needs to do more to maintain the same functional level. Tracking this over time is important. Indeed, Agree and colleagues (2004, p. 267) called for incorporating change in disability into the study of the “dynamic acquisition process” whereby older people adopt an AD—and this is precisely the aim of the present analysis.Second, as disability increases, it may diffuse to other domains of functioning (Ferraro & Shippee, 2009). Do most people use multiple devices within one domain of functioning? Or might the disablement process lead to AD use in multiple domains of functioning? To answer these questions, it would be helpful if research could not only identify who uses ADs—and how many—but also the number of functional domains affected. As the number of domains rises for which a person uses one or more ADs, this is likely an indicator of overall vulnerability.Three main research questions guide the present research. First, does incident disability lead to the use of one or more ADs? Second, if yes, do both upper and lower body disability increase the likelihood of adopting ADs? Third, does rising disability also lead to domain diffusion in the adoption of ADs? Answers to these questions are vital to effective intervention by health care personnel and caregivers who assist older adults with ADLs. Our aim is to capture important transitions in the disability process as it unfolds and test the effect that this change has on subsequent use of ADs.MethodsSampleData for this study come from the National Long Term Care Survey (NLTCS), a nationally representative sample of those aged 65 years and older in the United States, drawn from the Medicare beneficiary list, accounting for 97% of U.S. older adults (Manton, Corder, & Stallard, 1997). Even though the NLTCS began in 1984, the current study focuses on the three most recent waves of data: 1994, 1999, and 2004. The 1994 survey will be referred to as W1 (Wave 1), 1999 as W2, and 2004 as W3. Although the NLTCS tracks respondents who enter institutions, measures needed for this study vary across the institutional and community surveys. Therefore, only community-dwelling older adults were included here.A detailed questionnaire was administered during 1994 to 4,126 chronically disabled persons (i.e., difficulty performing an ADL or independent activity of daily living for at least 3 months) and a sample of 963 nondisabled older persons, resulting in 5,089 respondents. The inclusion of initially nondisabled persons is important given our aim to capture incident disability as it occurs naturally.Among the 5,089 W1 respondents, 1,706 (33.5%) died by W2; 320 (6.3%) were institutionalized; and 600 (11.8%) were nonrespondents. Among the 2,463 interviewed at both W1 and W2, 957 (38.9%) died by W3, 131 (5.3%) were institutionalized, and 328 (13.3%) were nonrespondents. Analyses proceeded with 2,436 respondents between W1 and W2 and 1,047 respondents across three waves.To account for sample attrition, multivariate analyses are adjusted with selection bias modeling (Heckman, 1979). A probit model predicting death was estimated with the following predictors: age, gender, lives alone, body mass index (BMI), personal care, upper body disability, lower body disability, incident lower body disability, self-rated health, smoker, and whether a proxy provided responses to the cognitive questions. A selection factor based on the inverse Mills ratio of the probit results was used to adjust for attrition in the final equations.MeasuresTwo dependent variables were created to assess use of ADs. (Of note, we follow Agree & Freedman’s, 2000, use of the term “assistive device” as opposed to “assistive technology” because of our focus on ADL disability; see also Cornman et al., 2005). The first is an overall count of ADs that respondents reported currently using, referred to as number of ADs used. When respondents indicated they used devices for both getting into or out of bed and getting around indoors, each device was counted only once. Respondents could report using as many as 29 ADs, but 88% reported using 5 or fewer ADs in 2004. A complete list of ADs used in the analyses is found in Table 1 (note a).Table 1.Descriptive Statistics of NLTCS Respondents Present at W1, W2, and W3 Community SurveysSurvey yearW1W2W3VariableRange199419992004Number of ADs useda0–161.26b,c (2.01)d1.64 (2.47)2.05 (2.83)Number of ADLs with ADse0–60.94 (1.31)1.04 (1.37)1.23 (1.44)Age65–98f78.67 (7.66)Male0–134.09%White0–187.74%Education1–73.35 (1.60)Lives alone0–137.91%42.27%Underweight (BMI <18.5)0–17.28%4.90%Normal weight (BMI 18.5–24.9)0–144.20%43.07%Overweight (BMI 25–29.9)0–131.49%34.02%Obese (BMI >30)0–117.03%18.01%Personal care0–124.37%21.84%Upper body disability (Nagi)0–121.93 (2.86)1.78 (2.87)Lower body disability (Nagi)0–123.76 (3.65)3.39 (3.60)Incident upper body disability, W1–W20–60.55 (1.32)Incident lower body disability, W1–W20–60.74 (1.37)Number of cases5,0892,4631,047Note: AD = assistive device; ADL = activity of daily living; BMI = body mass index (kg/m2); NLTCS = National Long Term Care Survey.aNumber of ADs used included the following, which are ranked in order from most to least frequently used at W3 among respondents who participated at all three waves: Shower seat/tub stool, grab bars/handle bars for bathing, walker, cane, raised toilet seat, rubber mat for bathing, hand-held shower, rail/grab bar for toileting, wheelchair, portable toilet/bedside commode, special underwear/diapers, indoor railing, furniture/walls to get around indoors, bedside railing, bedpan or urinal, lift to get in/out of bed, oxygen/respirator, orthopedic shoes, elevator/escalator, brace (leg or back), crutch, prosthesis, and chairlift on stairs. Respondents also indicated whether they used “any” AD for eating and dressing and were asked whether they used any “other” ADs for each of the ADL domains—both of these items were included in count of ADs at W2 and W3.bMean or percentage.cFor respondents available at all three waves, the mean values for number of ADs used were 0.47 at W1 and 0.89 at W2.dStandard deviation.eADLs include eating, dressing, getting in/out of bed, getting around indoors, bathing, and toileting.fAge at W1 (1994).The second dependent variable is the number of ADL domains for which respondents reported using one or more ADs. This measure, number of ADL domains with AD, taps AD use across domains of ADL (i.e., eating, dressing, getting in/out of bed, getting around indoors, bathing, and toileting). Use of these two outcomes enables us to compare the robustness of predictors by differentiating between people who use many devices within a single ADL domain and those who use few devices spread across several ADL domains.Disability was measured with the Nagi items and differentiated as upper and lower body (Nagi, 1976). Lower body disability questions asked respondents the degree of difficulty experienced climbing one flight of stairs, walking to the end of the room and back, bending to put on socks or stockings, and lifting a 10-pound package and holding it for a few minutes. Upper body disability captured the degree of difficulty respondents experienced reaching above their head, combing and brushing their hair, washing their hair, and using fingers to grasp and handle small objects (no Nagi items were used for both disability variables). Responses for each disability variable were captured on a 4-point scale (0 = not difficult, 1 = somewhat difficult, 2 = very difficult, and 3 = cannot do the task at all). Each disability index has an alpha reliability coefficient greater than .80 at each wave. To capture incident disability, persons who experienced an increase in disability were assigned the difference between waves (top-coded so that 6 = 6 or more). Persons who were followed at all three waves and had no change between W1 and W2 (n = 411) or experienced a disability decline (n = 266) were assigned a value of 0.Personal care was measured with a binary variable if the respondent reported someone helping them to perform at least one ADL during the previous week. To assess the potential influence of BMI, we used a categorical form of kilograms per meters squared. Binary variables were created for the categories outlined by the National Institutes of Health, National Heart, Lung, and Blood Institute (1998): underweight, BMI less than 18.5; normal weight, BMI of 18.5–24.9; overweight, BMI of 25.0–29.9; obese, BMI greater than or equal to 30. Control variables included age, gender (male), race (White or non-White), education, and lives alone. Several additional variables were included in preliminary multivariate analyses but omitted from the final analyses because they were nonsignificant in various specifications. These variables included marital status, comorbidity, and social isolation.Analytic PlanGiven the overdispersion of the outcome variables, negative binomial regression was selected as the statistical estimator (Long, 1997). All analyses were conducted with Stata 10 (StataCorp, 2007).The analysis was divided into two main stages. First, equations predicting number of ADs used at W2 and the number of ADL domains with ADs at W2 were estimated; two models were estimated for each outcome, where the W1 variable of the outcome was excluded and then included. The first model provides point estimates at W2 and the second reveals change in the outcome. Second, negative binomial regression models were estimated to examine change in the outcomes by W3. We capitalized on the three waves of data to isolate the influence of both initial disability (W1) and incident disability (between W1 and W2, using lagged models). In both stages, we tested for potential statistical interactions (e.g., age and disability) and polynomial terms for independent variables (e.g., disability).ResultsDescriptive statistics for the sample are presented in Table 1. AD use increased substantially over the decade, from 1.26 at W1 to 2.05 at W3 (p < .001). The number of ADL domains with AD also increased over time (p < .001). At W1, the majority of respondents were either normal weight (44%) or overweight (31%); about 17% were obese. Approximately one quarter of respondents reported receiving personal care at W1. Lower body disability was greater than upper body disability, with W2 respondents reporting a value of about 2 for upper body disability and about 4 for lower body disability.Table 2 displays the results of the negative binomial regression analyses for the two W2 dependent variables, and two models are presented for each outcome. Model 1 provides estimates of overall AD use at W2; Model 2 adds AD use at W1 in order to examine the adoption of ADs since W1.Table 2.Negative Binomial Regression Predicting Number of ADs Used at W2 (1999)Number of ADs usedNumber of ADLs with ADsW1 independent variablesModel 1Model 2Model 1Model 2Age0.065a,*** (0.010)b0.057*** (0.010)0.059*** (0.008)0.053*** (0.008)Male−0.073 (0.104)−0.128 (0.101)−0.033 (0.083)−0.054 (0.082)White−0.004 (0.108)−0.021 (0.105)−0.063 (0.085)−0.080 (0.083)Education0.087*** (0.022)0.061** (0.022)0.059** (0.018)0.041* (0.018)Lives alone0.064 (0.074)0.000 (0.073)0.078 (0.059)0.047 (0.059)Underweight (BMI <18.5)c0.133 (0.256)0.076 (0.246)0.066 (0.198)0.058 (0.195)Overweight (BMI 25–29.9)c−0.003 (0.088)0.032 (0.087)0.004 (0.072)0.030 (0.071)Obese (BMI >30)c0.343** (0.106)0.382*** (0.104)0.259** (0.084)0.267** (0.083)Personal care0.407** (0.130)0.145 (0.130)0.328** (0.099)0.138 (0.101)Upper body disability0.019 (0.019)0.022 (0.019)0.008 (0.014)0.009 (0.014)Lower body disability0.181*** (0.019)0.122*** (0.020)0.155*** (0.015)0.116*** (0.016)Number of ADs used, W10.190*** (0.023)0.127*** (0.017)Mortality λ−0.346 (0.300)−0.179 (0.290)−0.305 (0.221)−0.193 (0.218)Observations2,1612,1612,1612,161Pseudo R20.0600.0710.0870.096Likelihood ratio χ2437.45512.42529.60587.62Note: AD = assistive device; ADL = activity of daily living; BMI = body mass index (kg/m2).aUnstandardized coefficient.bStandard error.cCompared with those with a normal BMI (18.5–24.9).*p < .05. **p < .01. ***p < .001.Findings from Model 1 of Table 2 are quite similar across the W2 two outcomes. The number of ADs used and the number of ADL domains with ADs were higher for persons who were older, had more education, obese, received personal care, and had more lower body disability. When AD use at W1 was added in Model 2—predicting change in AD use from W1 to W2—most results are consistent with Model 1, except that personal care is no longer significant. Across all four equations, lower body disability predicts AD use, but upper body disability is not independently related to the outcomes.Supplementary analyses reveal that 42% of respondents present at W1 reported using ADs for bathing and/or indoor mobility difficulty at W2, which are devices that are more useful to those with compromised lower body function and require a certain degree of upper body strength and dexterity to operate. The percentage of respondents using bathing and/or mobility devices increased to 50% by W3.The results displayed in Table 3 carry the analyses forward to W3 and take advantage of data from the prior two waves. Both Models 1 and 2 include the W2 measure of AD use in order to focus on change in use of ADs. Model 1 for number of ADs reveals that age, obesity, and lower body disability at W2 are associated with an increase in AD use by W3. Again, upper body disability was not a significant predictor of AD use. In addition to initial level of lower body disability (W1), Model 1 also includes a variable for incident lower body disability (between W1 and W2), which is associated with the adoption of additional ADs (p < .01). (Incident upper body disability was not included in the equation due to the W2 level of upper body disability having no significant influence on AD use in W3.)Table 3.Negative Binomial Regression Predicting ADs Used at W3 (2004), NLTCSNumber of ADs usedNumber of ADLs with ADsIndependent variablesModel 1Model 2Model 1Model 2Number of ADs used at W20.154a,*** (0.031)b0.133*** (0.032)0.099*** (0.020)0.078*** (0.021)Age0.059*** (0.016)0.068*** (0.016)0.054*** (0.012)0.058*** (0.012)Male−0.219 (0.133)−0.261* (0.123)−0.138 (0.104)−0.179 (0.097)White0.147 (0.153)0.155 (0.152)0.082 (0.119)0.061 (0.119)Education0.026 (0.032)0.036 (0.032)0.034 (0.025)0.041 (0.025)Lives alone0.096 (0.097)0.103 (0.097)0.126 (0.075)0.138 (0.075)Underweight (BMI <18.5)c−0.433 (0.353)−0.506 (0.345)−0.437 (0.275)−0.534* (0.272)Overweight (BMI 25–29.9)c0.184 (0.131)0.261 * (0.126)0.082 (0.100)0.149 (0.096)Obese (BMI >30)c0.539*** (0.145)0.594*** (0.140)0.344** (0.109)0.394*** (0.105)Personal care0.011 (0.206)−0.098 (0.213)0.083 (0.143)−0.015 (0.148)Upper body disability at W20.004 (0.029)0.012 (0.029)−0.005 (0.020)−0.003 (0.020)Lower body disability at W10.067* (0.030)0.060* (0.026)0.079** (0.023)0.070*** (0.020)Incident lower body disability, W1–W20.135** (0.048)1.630** (0.488)0.111** (0.035)1.150*** (0.329)Interaction between age and incident lower body disability between W1–W2−0.019** (0.006)−0.013** (0.004)Mortality λ0.422 (0.387)0.659 * (0.326)0.263 (0.272)0.518 * (0.230)Observations933933933933Pseudo R20.0680.0710.1020.106Likelihood ratio χ2232.74244.07287.94300.84Note: AD = assistive device; ADL = activity of daily living; BMI = body mass index (kg/m2); NLTCS = National Long Term Care Survey.aUnstandardized coefficient.bStandard error.cCompared with those with a normal BMI (18.5–24.9).*p < .05. **p < .01. ***p < .001.Several interactions between incident lower body disability and predictors were tested in supplementary analyses, and the product term of incident disability and age was significant as shown in Model 2 of Table 3. The effect of incident lower body disability on AD use varies across age groups. Incident lower body disability increased AD use, but this relationship was moderated somewhat for the oldest respondents. Note also that with the interaction specified in Model 2, overweight emerged as a significant predictor of using more ADs by W3, and the mortality selection factor (λ) was also significant.The last two equations (columns) in Table 3 display similar models for the second outcome. Findings for the number of number of ADL domains with ADs are quite similar. The pattern of significant relationships for Model 1 is similar to the equation for number of ADs used, but the effect due to initial lower body disability is slightly stronger for number of ADLs with ADs.The findings from Model 2, which include the interaction between age and incident lower body disability, are similar to those for the first outcome, albeit overweight is not significant. The product term in Model 2 reveals the same pattern: Incident lower body disability increases AD use but this relationship is stronger for the younger members of the sample.To better understand the interaction between age and incident lower body disability that was observed for both outcomes, we provide a graphical representation for two different levels of AD use at W3 while controlling for the demographic, BMI, and disability variables. Using a logistic regression model, Figure 1 displays the predicted probability of using one or more ADs at W3 across four age groups while accounting for incident lower body disability between W1 and W2. As shown in Figure 1, the probability of using one or more ADs by W3 varies considerably by age even if there was no new disability between W1 and W2. Incident lower body disability, however, raised the likelihood of using ADs for all age groups, and the slope for persons aged 65–69 years was fairly linear. By contrast, for the oldest respondents, the slope rose faster across incident disability and then tapered off at the highest levels approaching 100% saturation.Figure 1.Predicted probability of using 1+ assistive devices (ADs) by W3 (2004) by incident lower body disability and age (N = 933).Figure 2 predicts the probability of a less frequent scenario—when a person used three or more ADs at W3. Again, there are age differences even with no incident disability. The slopes for the middling age categories are fairly linear. By contrast, the probability of using three or more ADs rose more slowly across incident disability for the youngest age group and rose faster for the oldest age group before tapering off at about 80%.Figure 2.Predicted probability of using 3+ assistive devices (ADs) by W3 (2004) by incident lower body disability and age (N = 933).DiscussionSeveral years ago, Agree and colleagues (2004, p. 267) called for research to examine use of ADs as a “dynamic acquisition process, with attention to age and disability severity.” We sought to answer that call by using three waves of panel data from a nationally representative sample of older adults to examine how ADs are adopted by persons facing ADL disability. The current study examines not only who is more likely to use ADs but also how incident disability and prior AD use influence number of ADs used and the number of ADL domains for which ADs are used.Three research questions were examined in the present study. First, we sought to determine whether incident disability leads to the use of one or more ADs. Although many gerontologists no doubt conceptualize AD use as a consequence of the disablement process, we are unaware of any nationally representative studies that used three or more waves of data to isolate whether incident disability accelerates the adoption of ADs. The present study found consistent evidence across two different outcome variables that incident disability is the precursor of AD adoption. This is not to say that baseline levels are unimportant; they are and result in AD adoption. Nevertheless, incident disability leads to additional use of ADs. For practice, therefore, it may be helpful to identify persons in the early stages of incident disability to optimize the effectiveness of ADs.Second, we sought to determine if both upper and lower body disability increase the likelihood of adopting ADs. Although some studies do not differentiate types of disability, findings from the current study are consistent with other investigations showing that lower body disability is the engine of AD adoption (Cornman & Freedman, 2008; Spillman, 2005; Verbrugge et al., 1997). In addition, the present research builds upon this finding by demonstrating that AD use is not only driven by baseline levels of lower body disability but also by incident lower body disability as well. We believe that there is now ample evidence to call for distinguishing between these two forms of disability in order to identify persons who are likely adopters of ADs.Third, we asked whether increasing disability also leads to domain diffusion in the adoption of ADs. This is an important question for intervention. Are older people in need of additional devices for one domain of ADL functioning or will the need spill over into other domains? Our results point to the latter. When incident lower body disability occurs, it leads not only to additional AD use within one domain, but across multiple domains. Underlying physical capacity is important for the performance of many ADLs. Persons who have difficulty with transfers to the toilet are probably also at risk for transfers involving getting in/out of bed. Future research could confirm this by investigating the sequence of AD adoption among older adults. These results from the NLTCS clarify that incident lower body disability diffuses to AD use across ADL domains.While answering these three research questions, two additional conclusions emerged as noteworthy. First, as suggested by Agree and colleagues (2004), age merits special attention. Based on this national sample of older adults, AD use varied by age in a nonlinear way. Although there are age differences in the likelihood of AD use—such that persons of advanced age (80+ years at W1) used more devices—we also observed a tapering of the rising probability of AD adoption for the oldest age groups (perhaps reflecting a saturation effect for the oldest respondents). Small changes in incident lower body disability were associated with rising AD use among the youngest respondents, an important finding for intervention efforts.Second, we were struck by the consistent predictive ability of obesity as a risk factor for AD use. Across all models, regardless of the outcome considered, obesity was a consistent, significant, and positive predictor. Obesity is clearly a risk factor for disability, but few previous studies have included it as a predictor of AD use. Of those that did, the results revealed either no influence on AD use (Verbrugge & Sevak, 2002) or few findings that merited discussion (Cornman & Freedman, 2008). The current study reveals the lasting impact that obesity has on the use of ADs: Those who were obese at either W1 or W2 were more likely to use a greater number of ADs at W2 and W3, respectively. Given the increased prevalence of adult obesity during recent decades, it should be recognized as a critical risk factor for both disability and AD use.This study’s contributions must be weighed against three limitations. First, although we devised an analytic plan to take advantage of the sequence of events observed in these data, the NLTCS 5-year time frame is fairly coarse when attempting to measure both incident disability and AD use (see also Freedman, Martin, & Schoeni, 2002; Spillman, 2005). Whereas the precise date of AD adoption was not known, we know only that it occurred during the 5-year period between surveys. Accordingly, we used a lagged analytic strategy to guard against conclusions that could be due to reverse causality (because both incident disability and AD adoption can occur within either of the 5-year periods). This strategy enabled us to isolate the influence of incident disability on adoption of ADs, but the 5-year periods are fairly long for studying these dynamic processes. There is clearly a need for research with shorter time frames to detect early stages of incident disability.Second, although we theorized that incident disability is a risk factor for AD use, there are other factors that merit attention, including illness episodes and hospitalizations (Wolff, Agree, & Kasper, 2005). Hospitalizations linked to specific illness episodes such as a stroke serve as a gateway to both incident disability and AD use (Gitlin et al., 1998). Now that we have documented that AD adoption is linked to incident disability, there is a need for future research to identify these “upstream” factors that may lead to both rising disability and AD use.Third, although we know that AD adoption does not occur in a social vacuum, the NLTCS does not provide information on which social network members facilitate the process. We envision caregivers as pivotal agents in the decision to use ADs, but this proposition merits systematic examination in future research.Implications for Policy and PracticeOur findings have implications for caregivers, clinicians, and policy makers, and we articulate two that are especially important. First, according to several studies, poor lower body function is a strong indicator of additional functional decline among older adults (e.g., Guralnik et al., 2000). We found that incident lower body disability triggers a response of increased reliance on ADs—and across multiple ADL domains—to counteract the slippery slope associated with functional decline. Moreover, those who begin experiencing this decline after age 70 are especially sensitive to respond to this change by using more ADs. Therefore, clinicians and caregivers should take note of increases in lower body disability among those benefitting from their care and be prepared to respond by suggesting the use of ADs to effectively bridge the gap between impairment and function. Medicare’s durable medical equipment benefit has helped millions of Americans obtain ADs, but many remain unaware of this benefit (Wolff et al., 2005).Second, obesity is more prevalent among the baby boom generation (born between 1946 and 1965) compared with those born in the prior decades (Leveille, Wee, & Iezzoni, 2005). Based on our finding that obese individuals are more likely to use more ADs—and for a wider number of ADLs—it should be anticipated that AD use will climb as the baby boom population ages. Beyond their sheer numbers, the higher prevalence of obesity among baby boomers and their higher levels of education will make ADs even more widely used in the future.We appreciate the comments on an earlier version of the manuscript by Kathy Abrahamson, Neale R. Chumbler, Ann Howell, Markus H. Schafer, Tetyana P. 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Retrieved October 13, 2009, from http://www.urban.org/url.cfm?ID=1001277StataCorpStata Statistical Software: Release 102007College Station, TXStata CorporationTomitaMRMannWCFraasLFBurnsLLRacial differences of frail elders in assistive technologyAssistive Technology19979140151TomitaMRMannWCFraasLFStantonKMPredictors of the use of assistive devices that address physical impairments among community-based frail eldersJournal of Applied Gerontology200423141155VerbruggeLMJetteAMThe disablement processSocial Science & Medicine199438114VerbruggeLMRennertCMadansJHThe great efficacy of personal and equipment assistance in reducing disabilityAmerican Journal of Public Health199787384392VerbruggeLMSevakPUse, type, and efficacy of assistance for disabilityJournal of Gerontology: Social Sciences200257BS366S379WolffJLAgreeEMKasperJDWheelchairs, walkers, and canes: What does Medicare pay for, and who benefits?Health Affairs20052411401149 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B84673E2A77F8873ADE3BF869C67FECB1786C32.txt b/test/dataset/in/resources/corpus/Clean_0B84673E2A77F8873ADE3BF869C67FECB1786C32.txt new file mode 100644 index 0000000..5a8c676 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B84673E2A77F8873ADE3BF869C67FECB1786C32.txt @@ -0,0 +1 @@ +cercorcercorCerebral Cortex1460-21991047-3211Oxford University Press10.1093/cercor/bhp250ArticlesPrefrontal Cortical Contribution to Risk-Based Decision MakingSt. OngeJennifer R.FlorescoStan B.Department of Psychology and Brain Research Center, University of British Columbia, Vancouver, BC, Canada V6T 1Z4Address correspondence to Stan B. Floresco, PhD, Department of Psychology and Brain Research Center, University of British Columbia, 2136 West Mall, Vancouver BC V6T 1Z4, Canada. Email: floresco@psych.ubc.ca.82010511200920818161828© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org2010Damage to various regions of the prefrontal cortex (PFC) impairs decision making involving evaluations about risks and rewards. However, the specific contributions that different PFC subregions make to risk-based decision making are unclear. We investigated the effects of reversible inactivation of 4 subregions of the rat PFC (prelimbic medial PFC, orbitofrontal cortex [OFC], anterior cingulate, and insular cortex) on probabilistic (or risk) discounting. Rats were well trained to choose between either a “Small/Certain” lever that always delivered 1 food pellet, or another, “Large/Risky” lever, which delivered 4 pellets, but the probability of receiving reward decreased across 4 trial blocks (100%, 50%, 25%, and 12.5%). Infusions of gama-aminobutyric acid agonists muscimol/baclofen into the medial PFC increased risky choice. However, similar medial PFC inactivations decreased risky choice when the Large/Risky reward probability increased over a session. OFC inactivation increased response latencies in the latter trial blocks without affecting choice. Anterior cingulate or insular inactivations were without effect. The effects of prelimbic inactivations were not attributable to disruptions in response flexibility or judgments about the relative value of probabilistic rewards. Thus, the prelimbic, but not other PFC regions, plays a critical role in risk discounting, integrating information about changing reward probabilities to update value representations that facilitate efficient decision making.anterior cingulateorbitofrontal cortexprelimbic cortexreversible inactivationratIntroductionDecision making entailing cost–benefit evaluations about risks and rewards recruits numerous regions of the prefrontal cortex (PFC). Initial work by Damasio, Bechara, and colleagues, employing what is now referred to as the Iowa Gambling task, revealed that patients with damage to the ventromedial and orbital regions of the PFC make risky, disadvantageous choices compared with control subjects (Bechara et al. 1994, 1999; Damasio 1994). Subsequent research, using a variety of different tasks, patient populations, as well as neuroimaging in healthy subjects, has implicated multiple interconnected regions of the PFC in mediating these forms of decisions. These include the dorsolateral PFC (Ernst et al. 2002; Manes et al. 2002; Brand et al. 2004; Fellows and Farah 2005; Labudda et al. 2008), the medial and lateral regions of the orbitofrontal cortex (OFC; Rogers, Everitt, et al. 1999; Rogers, Owen, et al. 1999; Manes et al. 2002; Clark et al. 2003, 2008; Ernst et al. 2004; Fukui et al. 2005), the dorsal (Brodmann's Area 24) and ventral (Area 32) regions of the anterior cingulate (Ernst et al. 2002; Labudda et al. 2008; Lawrence et al. 2009), and the insular cortex (Ernst et al. 2002; Bar-On et al. 2003; Clark et al. 2008; Smith et al. 2009).Although multiple frontal lobe regions have been implicated in facilitating risk/reward decisions, the specific contribution that different PFC regions make to risk-based decision making remains unclear. For example, impaired decision making on the Iowa Gambling task displayed by patients with ventromedial PFC damage may reflect a failure to learn the win–lose contingencies associated with the different choice options, a preference for high-risk options regardless of potential loss or deficits in strategy acquisition and maintenance (Clark et al. 2004). Likewise, damage to either the OFC or dorsolateral PFC has been associated with impaired decision making in some studies (Bechara et al. 1994, 1999; Rogers, Owen, et al. 1999; Brand et al. 2004; Fellows and Farah 2005) but not in others (Bechara et al. 1998; Manes et al. 2002; Clark et al. 2003). Different regions of the anterior cingulate have been implicated in multiple aspects of decision making, depending on the type of task used. These include monitoring increasing potential or received gain (Rogers et al. 2004; Tom et al. 2007), loss or negative feedback (Blair et al. 2006; Marsh et al. 2007; Cohen et al. 2008), or changes in reward contingencies that require modifications in behavior (Bush et al. 2002; Cohen et al. 2008). These discrepant findings may be due in part to differences in the specific loci and laterality of damage that may vary considerably between subjects and may also encompass multiple PFC regions. Thus, studies that afford greater experimental control, as may be achieved with animal models, may help clarify how different PFC regions contribute to aspects of risk-based decision making.One component of decision making that can be assessed in rodents is the evaluation of certain costs associated with different actions relative to the potential reward that may be obtained by those actions. In these studies, rats typically choose between smaller rewards associated with a nominal cost, and larger, yet more costly rewards (Floresco, St Onge, et al. 2008). Dissociable regions of the rat PFC appear to mediate different forms of these cost–benefit judgments. The OFC appears to be a critical component of the circuitry underlying delay discounting, where animals choose between small, immediate rewards and larger, delayed rewards (Mobini et al. 2002; Winstanley et al. 2004; Rudebeck et al. 2006). On the other hand, the dorsal anterior cingulate has been implicated in mediating decisions related to effort costs associated with larger rewards (Walton et al. 2003; Rudebeck et al. 2006; Floresco and Ghods-Sharifi 2007).Interestingly, there have been relatively few studies on the involvement of different regions of the rat PFC in risk-based decision making. Of these, the OFC has received the most attention. Mobini et al. (2002) reported that large lesions of the OFC (encompassing portions of the medial PFC) increased preference for a small, but certain, food reward relative to a larger reward delivered in a probabilistic manner. Another study used a procedure modeled after the Iowa Gambling task, where rats learned contingencies of decision alternatives in 1 session and adjusted choice based on evaluations of previous outcomes (Pais-Vieira et al. 2007). Rats with OFC lesions developed a preference for the high-risk/large reward (LR) over the low-risk/small reward (SR) option, even though the expected value of the 2 options was equal. In both of these studies, lesions were induced prior to training on the task, indicating that the OFC may play a role in the initial learning of risk/reward contingencies. In contrast, the contribution of the rat OFC to risk/reward judgments after animals have learned the relative value of different response options is unknown. Moreover, to our knowledge, there have been no studies on the contribution of the rodent medial PFC or insular cortex to risk-based decision making.To address this issue, the present study investigated the effects of reversible inactivation of different subregions of the rat PFC on risk-based decision making, using a probabilistic discounting task conducted in an operant chamber. Rats chose between responses that yielded a small/certain food reward and a larger reward delivered in a probabilistic manner, where the odds of receiving the larger reward decreased over a session. Previous studies using similar procedures have implicated the nucleus accumbens (Cardinal and Howes 2005), the basolateral amygdala (Ghods-Sharifi et al. 2009), and the dopamine system (St Onge and Floresco 2009) in biasing choice toward larger, probabilistic rewards. Each of these systems shares connections with different regions of the PFC (Beckstead et al. 1979; McDonald 1991). In the present study, rats were well trained on this task prior to receiving reversible inactivations of 1 of 4 different PFC subregions. Each of these regions sends projections to the nucleus accumbens core (Brog et al. 1993), which has been shown previously to mediate to risk discounting (Cardinal and Howes 2005). Risk discounting was altered selectively by inactivation of the prelimbic region of the medial PFC, a region that is anatomically homologous to Area 32 of the human anterior cingulate (Uylings and van Eden 1990) but also shares functional homology to the dorsolateral PFC (Brown and Bowman 2002; Uylings et al. 2003). Accordingly, subsequent experiments were conducted to clarify the specific nature of the disruption in risk-based decision making induced by medial PFC inactivation.Experiment 1: The Effects of Inactivations of the Medial PFC, OFC, Anterior Cingulate, and Insular Cortex on Risk DiscountingMaterials and MethodsAnimalsMale Long Evans rats (Charles River Laboratories, Montreal, Canada) weighing 275–300 g at the beginning of behavioral training were used for the experiment. On arrival, rats were given 1 week to acclimatize to the colony and food restricted to 85–90% of their free-feeding weight for an additional 1 week before behavioral training. Rats were given ad libitum access to water for the duration of the experiment. Feeding occurred in the rats’ home cages at the end of the experimental day, and body weights were monitored daily to ensure a steady weight loss during food restriction and maintenance or weight gain for the rest of the experiment. All testing was in accordance with the Canadian Council of Animal Care and the Animal Care Committee of the University of British Columbia.ApparatusBehavioral testing for all experiments described here was conducted in 12 operant chambers (30.5 × 24 × 21 cm; Med-Associates, St Albans, VT) enclosed in sound-attenuating boxes. The boxes were equipped with a fan to provide ventilation and to mask extraneous noise. Each chamber was fitted with 2 retractable levers, one located on each side of a central food receptacle where food reinforcement (45 mg; Bioserv, Frenchtown, NJ) was delivered via a pellet dispenser. The chambers were illuminated by a single 100-mA houselight located in the top center of the wall opposite the levers. Four infrared photobeams were mounted on the sides of each chamber. Locomotor activity was indexed by the number of photobeam breaks that occurred during a session. All experimental data were recorded by an IBM personal computer connected to the chambers via an interface.Lever-Pressing TrainingOur initial training protocols have been described previously (Floresco, Tse, et al. 2008; St Onge and Floresco 2009). On the day prior to their first exposure to the chambers, rats were given approximately 25 sugar reward pellets in their home cage. On the first day of training, 2–3 pellets were delivered into the food cup, and crushed pellets were placed on a lever before the animal was placed in the chamber. Rats were first trained under a fixed-ratio 1 schedule to a criterion of 60 presses in 30 min, first for one lever, and then repeated for the other lever (counterbalanced left/right between subjects). Rats were then trained on a simplified version of the full task. These 90 trial sessions began with the levers retracted and the operant chamber in darkness. Every 40 s, a trial was initiated with the illumination of the houselight and the insertion of 1 of the 2 levers into the chamber. If the rat failed to respond on the lever within 10 s, the lever was retracted, the chamber darkened, and the trial was scored as an omission. If the rat responded within 10 s, the lever retracted, and a single pellet was delivered with 50% probability. This procedure was used to familiarize the rats with the probabilistic nature of the full task. In every pair of trials, the left or right lever was presented once, and the order within the pair of trials was random. Rats were trained for approximately 5–6 days to a criterion of 80 or more successful trials (i.e., ≤10 omissions).Risk-Discounting TaskThe task was modified from Cardinal and Howes’ (2005) procedure, which we have previously used to assess the role of dopamine and the basolateral amygdala in risk-based decision making (Ghods-Sharifi et al. 2009; St Onge and Floresco 2009). Rats received daily sessions consisting of 72 trials, separated into 4 blocks of 18 trials. The entire session took 48 min to complete, and animals were trained 6–7 days per week. A session began in darkness with both levers retracted (the intertrial state). A trial began every 40 s with the illumination of the houselight and, 3 s later, insertion of one or both levers into the chamber (the format of a single trial is shown in Fig. 1). One lever was designated the Large/Risky lever, the other the Small/Certain lever, which remained consistent throughout training (counterbalanced left/right). If the rat did not respond by pressing a lever within 10 s of lever presentation, the chamber was reset to the intertrial state until the next trial (omission). When a lever was chosen, both levers retracted. Choice of the Small/Certain lever always delivered one pellet with 100% probability; choice of the Large/Risky lever delivered 4 pellets but with a particular probability (see below). When food was delivered, the houselight remained on for another 4 s after a response was made, after which the chamber reverted to the intertrial state. Multiple pellets were delivered 0.5 s apart. The 4 blocks were comprised of 8 forced-choice trials where only one lever was presented (4 trials for each lever, randomized in pairs) permitting animals to learn the amount of food associated with each lever press and the respective probability of receiving reinforcement over each block. This was followed by 10 free-choice trials, where both levers were presented and the animal chose either the Small/Certain or the Large/Risky lever. The probability of obtaining 4 pellets after pressing the Large/Risky lever varied across blocks: It was initially 100%, then 50%, 25%, and 12.5%, respectively, for each successive block. For each session and trial block, the probability of receiving the LR was drawn from a set probability distribution. Therefore, on any given day, the probabilities in each block may vary, but on average across many training days, the actual probability experienced by the rat will approximate the set value. Using these probabilities, selection of the Large/Risky lever would be advantageous in the first 2 blocks, and disadvantageous in the last block, whereas rats could obtain an equivalent number of food pellets after responding on either lever during the 25% block. Therefore, in the last 3 trial blocks of this task, selection of the larger reward option carried with it an inherent “risk” of not obtaining any reward on a given trial. Latencies to initiate a choice and overall locomotor activity (photobeam breaks) were also recorded.Figure 1.Task design. Cost–benefit contingencies associated with responding on either lever (A) and format of a single free-choice trial (B) of the risk-discounting task.A priori we determined that if inactivation of a particular PFC region resulted in an increased preference for the Large/Risky lever, a separate group of animals would be trained on a similar discounting task, in which the probabilities associated with the Large/Risky lever increased, rather than decreased across the 4 blocks (i.e., 12.5%, 25%, 50%, and 100%). The purpose of this was to determine if an increased preference for the Large/Risky lever following PFC inactivation was due specifically to a general increase in risky choice or to alterations in other cognitive processes that facilitate risk discounting. All other aspects of the training procedures were identical to those described above.Training Procedure, Surgery, and Microinfusion ProtocolRats were trained on the task until as a group they 1) chose the Large/Risky lever during the first trial block (100% probability) on at least 80% of successful trials and 2) demonstrated stable baseline levels of choice. Brain inactivations were conducted once a group of rats displayed stable patterns of choice for 3 consecutive days, assessed using a procedure similar to that described by Winstanley et al. (2005), Floresco, Tse, et al. (2008), and St Onge and Floresco (2009). In brief, data from 3 consecutive sessions were analyzed with a repeated-measures analysis of variance (ANOVA) with 2 within-subjects factors (Day and Trial Block). If the effect of Block was significant at the P < 0.05 level but there was no main effect of Day or Day × Trial Bock interaction (at P > 0.1 level), animals were judged to have achieved stable baseline levels of choice behavior. After the stability criterion was achieved, rats were provided food ad libitum and 2 days later were subjected to surgery.Rats were anesthetized with 100 mg/kg ketamine hydrochloride and 7 mg/kg xylazine and implanted with bilateral 23-gauge stainless steel guide cannulae into one of the following regions: the medial PFC (prelimbic cortex; anteroposterior [AP] = +3.4 mm; medial–lateral [ML] = ± 0.7 mm from bregma; and dorsoventral [DV] = −2.8 mm from dura); lateral OFC ([AP] = +3.9 mm; [ML] = ± 2.6 mm from bregma; [DV] = −2.9 mm from dura); anterior cingulate ([AP] = +2.0 mm; [ML] = ± 0.7 mm from bregma; and [DV] = −1.0 mm from dura); and agranular insular cortex ([AP] = +2.7 mm; [ML] = ± 3.8 mm from bregma; and [DV] = −3.8 mm from dura). For all surgical preparations, the mouthbar was set to −3.3 mm (flat skull). Thirty-gauge obdurators flush with the end of guide cannulae remained in place until the infusions were made. Rats were given at least 7 days to recover from surgery before testing. During this recovery period, animals were handled at least 5 min each day and food restricted to 85% of their free-feeding weight. Body weights were continued to be monitored daily to ensure a steady weight loss during this recovery period. Rats were subsequently retrained on the task for at least 5 days and until, as a group, they displayed stable levels of choice behavior. For 3 days before the first microinfusion test day, obdurators were removed, and a mock infusion procedure was conducted. Stainless steel injectors were placed in the guide cannulae for 2 min, but no infusion was administered. This procedure habituated rats to the routine of infusions in order to reduce stress on subsequent test days. The day after displaying stable discounting, the group received its first microinfusion test day.A within-subjects design was used for all experiments. Inactivation of PFC regions was achieved by infusion of a drug solution containing the GABAA agonist muscimol (Sigma–Aldrich Canada, Oakville, Ontario, Canada) and the GABAB agonist baclofen (Sigma–Aldrich). Both drugs were dissolved separately in physiological saline at a concentration of 500 ng/μL and then combined in equal volumes, with the final concentration of each compound in solution being 250 ng/μL. Intracranial microinfusions used a volume of 0.5 μL so that the final dose of both baclofen and muscimol was 125 ng per side. These doses were chosen because we have found them to be effective at altering risk discounting when infused in other brain regions (Ghods-Sharifi et al. 2009). In addition, infusions of comparable doses of baclofen and muscimol in the OFC (Takahashi et al. 2009) or muscimol alone into the anterior cingulate (Ragozzino and Rozman 2007) have been reported to disrupt cognition. Note that these doses are substantially higher than doses used in previous experiments that have also reported behavioral effects (e.g., 10 ng; Corrigall et al. 2001). Infusions of gama-aminobutyric acid (GABA) agonists or saline were administered bilaterally into one of the PFC regions via 30-gauge injection cannulae that protruded 0.8 mm past the end of the guide cannulae, at a rate of 0.5 μL/75 s by a microsyringe pump. Injection cannulae were left in place for an additional 1 min to allow for diffusion. Each rat remained in its home cage for another 10-min period before behavioral testing. On the first infusion test day, half of the rats in each group received saline infusions and the other half received muscimol/baclofen. The next day, they received another training day with no infusion. If, for any individual rat, choice of the Large/Risky lever deviated by more than 15% from its preinfusion baseline, it received an additional day of training prior to the second infusion test. On the following day, rats received a second counterbalanced infusion of either saline or muscimol/baclofen.HistologyAfter completion of all behavioral testing, rats were sacrificed in a carbon dioxide chamber. Brains were removed and fixed in a 4% formalin solution. The brains were frozen and sliced in 50-μm sections before being mounted and stained with Cresyl Violet. Placements were verified with reference to the neuroanatomical atlas of Paxinos and Watson (1998). The locations of acceptable infusions for rats used in Experiment 1 are presented in the right panels of Figure 2.Figure 2.Effects of inactivation of different PFC regions on risk discounting. The panels of each figure plots percentage choice of the Large/Risky lever (left) and response latencies (middle) as a function of the trial block (x-axis) following infusions of saline (white circles) or muscimol/baclofen (inactivation, black squares) into each of the 4 PFC subregions: (A) Medial PFC; (B) OFC; (C) anterior cingulate; and (D) insular cortex. Symbols represent mean + standard error of the mean. Black stars denote a significant (P < 0.05) main effect of treatment. White stars denote significant (P < 0.05) differences between treatments during a specific block. Right panels are schematics of coronal sections of the rat brain showing the location of acceptable infusion placements through the rostral–caudal extent of each of the 4 PFC regions.Data AnalysisThe primary dependent measure of interest was the percentage of choices directed toward the Large/Risky lever for each block of free-choice trials factoring in trial omissions. For each block, this was calculated by dividing the number of choices of the Large/Risky lever by the total number of successful trials. The choice data were analyzed using 2-way within-subjects ANOVAs, with Treatment and Trial Block as the within-subjects factors. The main effect of block for the choice data was significant in all experiments (P < 0.05) indicating that rats discounted choice of the Large/Risky lever as the probability of the LR changed across the 4 blocks. This effect will not be mentioned further. Locomotor activity (photobeam breaks) and the number of trial omissions were analyzed with 1-way within-subjects ANOVAs. Response latencies were analyzed using either 2-way or 1-way within-subjects ANOVAs.ResultsMedial PFC (Prelimbic)Initially, 16 rats were trained for this experiment. Two animals died during surgery, and the data from another rat was eliminated due to inaccurate placement. The remaining rats (n = 13) required an average of 35 days of training on the risk-discounting task prior to receiving counterbalanced infusions of saline or muscimol/baclofen into the medial PFC. Analysis of choice behavior following bilateral infusions of muscimol/baclofen or saline into the medial PFC revealed a significant main effect of treatment (F(1,12) = 6.20, P < 0.05; Fig. 2A, left panel). Medial PFC inactivation caused a significant increase in the proportion of choices directed toward the Large/Risky lever relative to saline infusions. Interestingly, inactivation of the medial PFC also significantly increased response latencies but only in the 25% and 12.5% probability blocks, where the relative long-term value of the Large/Risky option was comparable or less than that of the Small/Certain option (treatment × block interaction: F(3,36) = 3.85, P < 0.05, Dunnett's, P < 0.05; Fig. 2A, middle panel). However, this effect was only apparent during free-choice trials, as analysis of the response latency data collected during forced-choice trials revealed no differences between treatment conditions (F(1,12) = 1.89, n.s.). There were no differences in the number of trial omissions or locomotor activity between saline and muscimol/baclofen treatments (all F's < 0.89, n.s.). Thus, under these conditions, inactivation of the medial PFC reduced risk discounting, leading to an apparent increase in risky choice.OFCA total of 16 rats were initially trained for this experiment. Five of these animals had placements that extended ventral to the OFC, and their data were excluded from the analyses. The remaining rats (n = 11) required an average of 28 days of training on the risk-discounting task prior to receiving counterbalanced infusions of saline or muscimol/baclofen into the OFC. Analysis of the choice data revealed no main effect of treatment (F(1,10) = 0.38, n.s) or treatment x block interaction (F(3,30) = 0.43, n.s.; Fig. 2B, left panel). Although bilateral inactivation of the OFC had no effect on choice behavior, this manipulation did alter response latencies, as indicated by a significant treatment × block interaction (F(3,30) = 4.26, P < 0.05; Dunnett's, P < 0.05; Fig. 2B, middle panel). Simple main effects analyses revealed that OFC inactivation did not affect response latencies during the 100% and 50% probability blocks, but significantly increased deliberation times in the latter 2 blocks, relative to saline infusions. As was observed from the medial PFC group, OFC inactivation did not affect response latencies during forced-choice trials (F(1,10) = 3.48, n.s.). There was also a slight decrease in locomotion following OFC inactivation (1580 ± 229) relative to saline treatment (1712 ± 253), but this difference failed to reach significance (F(1,10) = 4.27, P = 0.07). There was no effect of OFC inactivation on trial omissions (F(1,10) = 1.79, n.s.). Thus, inactivation of the OFC did not affect risky choice under conditions where rats were familiar with the relative risk/reward contingencies associated with different response options and changes in these contingencies that occurred over the course of a session.Anterior CingulateThirteen rats were trained for this experiment. The data from 2 rats were excluded due to inaccurate placements and another rat died during surgery. The remaining 10 rats took an average of 33 days of training prior to receiving counterbalanced infusions of saline or muscimol/baclofen into the anterior cingulate. Inactivation of this region caused no discernable change in choice of the Large/Risky lever relative to saline treatment. This observation was confirmed by the lack of a significant main effect of treatment (F(1,9) = 0.00, n.s.) and treatment × block interaction (F(3,27) = 0.31, n.s; Fig. 2C, left panel). Likewise, response latencies during free-choice trials did not differ between treatment conditions (all F's < 1.0, n.s.; Fig. 2C, middle panel). However, during forced-choice trials, rats displayed slightly longer response latencies following anterior cingulate inactivation (1.3 ± 0.1 s), compared with saline infusions (1.0 ± 0.1 s; F(1,9) = 11.26, P < 0.01). Locomotion and trial omissions were not altered significantly by anterior cingulate inactivations (all F's < 1.0, n.s.).Insular CortexThirteen rats were trained for this experiment, but data for 6 rats were excluded due to inaccurate placements. The remaining 7 rats took an average of 28 days of training on the risk-discounting task prior to surgery and receiving counterbalanced infusions of saline or muscimol/baclofen into the agranular insular cortex. Again, we observed no change in choice behavior following muscimol/baclofen infusions, as indicated by the lack of a significant main effect of treatment (F(1,12) = 0.16, n.s), or treatment × block interaction (F(3,18) = 1.67, n.s; Fig. 2D, left panel). Insular inactivation did not significantly affect response latencies during either free-choice (Fig. 2D, middle panel) or forced-choice trials nor did it alter locomotion or trial omissions (all F's < 2.51, n.s.).Medial PFC Inactivation (Ascending Probabilities)Medial PFC inactivation increased selection of the Large/Risky lever when the probabilities of obtaining the larger reward decreased over the course of a risk discounting session. In a subsequent experiment, a separate group of 12 rats were trained for 25 days on a variant of this task, where the probability of obtaining the LR “increased” over the session, prior to receiving counterbalanced infusions of saline or muscimol/baclofen into the medial PFC. In contrast to the effects on choice behavior reported above, inactivation of the medial PFC induced the opposite effect on risk discounting under these conditions, with rats displaying a decreased preference for the Large/Risky lever over the 4 trial blocks. Analysis of these data revealed a significant main effect of treatment (F(1,11) = 12.28, P < 0.05; Fig. 3A). Rats chose the Large/Risky lever less often following medial PFC inactivations relative to saline infusions. There was no significant effect of inactivation on trial omissions or locomotion (F's < 2.5, n.s.). However, there was a significant increase in average response latencies during free-choice trials across all the blocks after medial PFC inactivation (F(1,11) = 8.82, P < 0.05; Fig. 3B). Again, this effect was selective to trials where rats were required to choose between the 2 levers, as response latencies during forced-choice trials did not differ between treatments (F(1,11) = 2.87, n.s.).Figure 3.Effects of medial PFC inactivation on risk discounting with ascending probabilities. (A) Percentage of choice of the Large/Risky lever during free-choice trials following infusions of saline or muscimol/baclofen into the medial PFC for all 12 rats used in this experiment. Inactivation of the medial PFC decreased risky choice. Inset displays data from a subset of rats (n = 9) that demonstrated comparable levels of choice of the Large/Risky lever after both saline and inactivation treatments during the initial, 12.5% probability block (i.e., matching for performance). (B) Response latencies displayed as a function of trial block. Black stars denote a significant (P < 0.05) main effect of treatment. (C) Location of acceptable infusion placements for rats used in this experiment.Inspection of Figure 3A reveals that the decreased preference for the Large/Risky lever after inactivation of the medial PFC was apparent during the first trial block (12.5%) and persisted over the remainder of the session. However, the decreased choice of the Large/Risky lever during the first block was primarily attributable to 3 rats. We conducted a supplemental analysis on the data from the 9 remaining rats that displayed a comparable preference for the Large/Risky lever during the first trial block (i.e., matching for performance) after inactivation and saline treatments. In this instance, the analysis of these data again revealed a significant overall decrease in choice of the Large/Risky lever (F(1,8) = 7.39, P < 0.05; Fig. 3A, inset). Thus, in this subset of rats, even though medial PFC inactivation did not reduce preference for the Large/Risky lever during the first block, these animals were still slower to adjust their choice behavior as the probability of obtaining the larger reward increased over subsequent blocks. This suggests that the medial PFC may facilitate adjustments in preference for the Large/Risky option in response to increases in the probability of obtaining the larger reward.Experiment 2: Effects of Medial PFC Inactivation on Performance of a Within-Session ReversalInactivation of the medial PFC (but not other frontal lobe regions) induced differential effects on risk discounting depending on the manner in which the probabilities of obtaining the larger reward changed over a session. When the odds of the Large/Risky reward were initially 100% and then decreased, medial PFC inactivation increased risky choice, whereas when the probabilities were initially low and subsequently increased, similar inactivations reduced preference for the Large/Risky lever. Both variants of this task require a shift in the direction of responding, whereby rats choose the Large/Risky lever less or more often as the probabilities decrease or increase over the session, respectively. Thus, one potential interpretation of the effects of medial PFC inactivation on risk discounting may be that these manipulations impaired behavioral flexibility, such that rats were perseverating on the lever that they preferred during the first block (Large/Risky or Small/Certain). Notably, similar inactivations of the medial PFC do not impair performance of standard or probabilistic reversal tasks, although they do impair shifting between discrimination strategies (Ragozzino et al. 1999; Block et al. 2007; Ragozzino 2007; Floresco, Block, and Tse 2008). Nevertheless, we conducted a subsequent experiment to determine whether alterations in risk-based decision making induced by medial PFC inactivations were due to perturbations in response flexibility. Rats were trained on a modified within-session reversal task that was similar to the risk-discounting procedure in a number of respects.Materials and MethodsWithin-Session Reversal TaskRats underwent initial lever-pressing training in a manner identical to that described in Experiment 1. They then received daily training sessions consisting of 56 trials, separated into 2 blocks of 28 trials. The 2 blocks were each comprised of 8 forced-choice trials where only one lever was presented (4 trials for each lever, randomized in pairs) permitting animals to learn the amount of food associated with each lever. This was followed by 20 free-choice trials, where both levers were presented (i.e., 40 free-choice trials in total, as in the risk-discounting task). Each 38-min session began in darkness with both levers retracted, and trials began every 40 s with the illumination of the houselight and insertion of one or both levers into the chamber 3 s later. One lever was designated the LR lever, the other the SR lever (counterbalanced left/right). When a lever was chosen, both levers retracted. During the initial block, a press on the LR lever delivered 4 pellets, whereas the SR lever delivered 1 pellet, both with 100% probability, similar to the first block of the risk discounting task. However, during the second, reversal block, selection of the LR resulted in the immediate retraction of both levers, extinguishing of the houselight, and no pellets were delivered. During this block, responses on the SR lever continued to deliver 1 pellet. All other aspects of the task were identical to those described in Experiment 1. Rats were trained with these same contingencies until as a group, they chose the LR lever on at least 80% of successful trials during the first block, less than 20% of successful trials during the reversal block, and the group displayed stable patterns of choice for 3 consecutive days. Subsequently, they were subjected to surgery, and then retrained on the task for at least 5 days prior to receiving intracranial microinfusions.Surgery, Microinfusions, and HistologyRats were implanted with bilateral guide cannulae into the medial PFC as described in Experiment 1. All other procedures were identical to those described in Experiment 1.ResultsEight animals were trained for 18 days prior to receiving counterbalanced infusions of muscimol/baclofen into the medial PFC. One rat had 2 infusions located in 1 hemisphere of the PFC, and its data were excluded from the analyses. Figure 4A displays choice data for the remaining rats (n = 7). After saline infusions, rats showed a strong preference for the LR lever during the initial block when it delivered 4 pellets, compared with the SR lever associated with 1 pellet. During the reversal block, rats shifted their choice away from the LR lever that now delivered 0 pellets, instead choosing the SR lever on the vast majority of free-choice trials, which continued to deliver 1 pellet. This pattern of choice was not altered by inactivation of the medial PFC. Analysis of the choice data revealed a significant main effect of block (F(1,6) = 103.16, P < 0.001) but no significant main effect of treatment (F(1,6) = 0.84, n.s.) or treatment x block interaction (F(1,6) = 2.75, n.s.; Fig. 4A). Interestingly, in this experiment, there were no effects of medial PFC inactivations on response latencies (F(1,6) = 0.01, n.s.). Likewise, locomotion and trial omissions did not differ between treatment conditions (all F's < 0.9, n.s.). The location of acceptable medial PFC infusion placements in this experiment is displayed in Figure 4B. Thus, medial PFC inactivation did not impair the ability to perform a within-session reversal in response to changes in reinforcement contingencies. This suggests that alterations in risk discounting induced by medial PFC inactivation cannot be easily attributed to a general impairment in response flexibility.Figure 4.Medial PFC inactivation does not affect performance of a within-session reversal. Data are plotted as percentage choice of the LR lever over 2 blocks of 20 free-choice trials (initial and reversal). The LR lever was associated with a 4-pellet reward during the initial block, but did not deliver any reward (0 pellets) during the reversal block. Responding on the SR lever delivered 1 pellet during both blocks. Rats were able to shift responding away from the LR lever during the reversal block after both saline and muscimol/baclofen treatment. (B) Location of acceptable infusion placements for rats used in this experiment.Experiment 3: Effects of Inactivation of the Medial PFC on Risk Discounting with Fixed ProbabilitiesAnother potential explanation for the alterations in risk discounting observed in Experiment 1 may be that medial PFC inactivation disrupted monitoring of “changes” in the probability of obtaining the larger reward that occurred within a session. Alternatively, this manipulation may have induced a more fundamental disruption in calculating the relative value of the Large/Risky option within each trial block. To assess this latter hypothesis, a separate group of rats were trained on a simplified version of the task, in which the probability of obtaining the larger reward remained constant throughout a session. Initially, the probability was fixed at 40%, making the relative long-term value of the Large/Risky option (4 pellets @ 40%) higher than the Small/Certain option (1 pellet @ 100%). Rats were then retrained and tested under conditions where the probability of obtaining 4 pellets was set at 10%. In this instance, selection of the Small/Certain option would yield a greater amount of reward in the long term.Materials and MethodsRisk Discounting with Fixed ProbabilitiesEight rats underwent initial lever-pressing training in a manner identical to that described in Experiment 1. They were then trained on the modified risk discounting procedure, consisting of 40 trials, broken down into 20 forced-choice, followed by 20 free-choice trials. Each session took 26 min to complete. All other aspects of the task were identical to the risk-discounting task used in Experiment 1. On each trial, selection of the Large/Risky lever delivered 4 pellets with a 40% probability, whereas pressing the Small/Certain lever delivered 1 pellet with 100% probability. The 40% probability was chosen to maximize the possibility of detecting either increases or decreases in the preference for the Large/Risky lever, given our observation in Experiment 1 that rats tend to select the Large/Risky lever on ∼90% of trials when the probability of obtaining 4 pellets was higher (i.e., 50%; e.g., see Fig. 2A). Rats were trained 6–7 days a week until, as a group, they chose the Large/Risky lever on at least 60% of successful trials and demonstrated stable performance for 3 consecutive days. They were then subjected to surgery, retraining, and their first sequence of intracranial microinfusions.Following the first set of microinfusion test days, rats were retrained on the same task for another 11 days but with the probability of obtaining the larger reward now set to 10%. This probability was chosen because it biases rats to use a risk-aversive strategy but was large enough to ensure that, over 20 forced-choice trials, rats will experience at least some reward so that the probability would not be inferred to be 0%. By the end of this training period, rats as a group chose the Large/Risky lever on less than 40% of the free-choice trials and demonstrated stable performance for 3 consecutive days. They then received a second series of counterbalanced infusions into the medial PFC.Surgery, Microinfusions, and HistologyRats were implanted with bilateral guide cannulae into the medial PFC as described in Experiment 1. All other procedures were identical to those described in Experiment 1.ResultsRats were trained for 24 days prior to receiving the first sequence of counterbalanced infusions of muscimol/baclofen or saline into the medial PFC. Of the initial 8 rats, one died after surgery, and the infusion placement for another was outside the boundary of the prelimbic cortex. Its data were excluded from the analyses. Following saline infusions, the remaining 6 rats displayed a slight preference for the Large/Risky option when the probability of obtaining 4 pellets was 40%, selecting this lever on ∼60% of free-choice trials (Fig. 5A, left panel). Importantly, inactivation of the medial PFC did not alter this preference (F(1,5) = 0.40, n.s.; Fig. 5A left panel). There were also no differences between treatment conditions in terms of locomotion, omissions, or response latencies (all F's < 2.0, n.s.).Figure 5.Inactivation of the medial PFC does not affect risky choice using fixed probabilities associated with larger reward. Bars represent percentage choice of the Large/Risky lever following saline (white) or muscimol/baclofen (black) infusions into the medial PFC. The left panel displays data from infusion tests days when the Large/Risky reward probability was fixed at 40%. The right panel displays data from infusions test days following 11 days of retraining with the Large/Risky probability at 10%. The inset diagrams the significant reduction in preference for the Large/Risky lever averaged across the last 3 days of training prior to the first (40%) and second infusion tests (10%). The star denotes a significant (P < 0.05) difference between reward probability conditions. (B) Location of acceptable infusion placements for rats used in this experiment.Rats were subsequently retrained for another 11 days with the probability of obtaining the Large/Risky reward set at 10%. By the end of this retraining period, rats were selecting the Large/Risky lever less often, compared with their performance when the probability was set at 40% (Fig. 5, inset). Analysis of the choice data obtained from the last 3 baseline days prior to the first (40% chance of the Large/Risky reward) and second (10% chance) infusion tests days confirmed that rats significantly discounted the Large/Risky lever as the probability of reward was reduced (F(1,5) = 7.82, P < 0.05). Again, inactivation of the medial PFC failed to alter choice behavior (F(1,5) = 3.91, n.s.; Fig. 5A right panel), locomotion, omissions, or response latencies relative to saline infusions (all F's<1.9, n.s.). The location of acceptable medial PFC infusion placements in this experiment is displayed in Figure 5B. Thus, inactivation of the medial PFC does not alter choice between small/certain and larger/uncertain rewards when the relative value of larger, probabilistic rewards remains constant during a session.DiscussionThe major finding of the present study is that inactivation of the prelimbic region of the medial PFC induces dramatic effects on risky choice assessed with a probabilistic discounting task. However, the specific effect of medial PFC inactivation was dependent on the manner in which the reward probabilities changed over a session. When the probabilities associated with the Large/Risky option were initially high (100%) and subsequently decreased, medial PFC inactivations increased risky choice. Yet, when the 4-pellet option was initially disadvantageous (12.5%) and then increased in value, similar inactivations had the opposite effect. Subsequent experiments indicated that these effects were not easily attributable to general disruptions in response flexibility or to evaluating the relative value of larger/uncertain versus smaller/certain rewards. In contrast, inactivation of the OFC did not affect risk discounting but did increase response latencies during the latter portion of the session. Similar inactivations of the anterior cingulate or insular cortex did not reliably alter choice behavior. Collectively, these findings indicate that the medial prelimbic PFC plays a critical role in guiding choice between certain and uncertain rewards of different magnitudes in response to changes in reward probability.Involvement of the Rat Medial PFC in Risk DiscountingAt first glance, the increase in risky choice induced by medial PFC inactivation in Experiment 1 may have indicated that this region normally biases choice toward small, certain rewards. However, similar inactivations had the opposite effect when the probabilities of obtaining the Large/Risky reward were initially low and then increased, suggesting that this relatively simple explanation is insufficient to account for our findings. Alternatively, it is possible that these treatments may have induced a general impairment in response flexibility, given that in both experiments, rats persisted in selecting the lever they displayed a bias for during the first probability block. It is notable that following medial PFC inactivations, rats were able to adjust choice of the Large/Risky lever in response to changes in reward probability, albeit at a slower rate than that displayed after saline treatments. Nevertheless, we conducted a subsequent experiment where rats were required to perform a within-session reversal, patterned after the risk-discounting task. Responding on 1 of 2 levers initially delivered a large 4-pellet (LR) or smaller 1-pellet (SR) reward. In the latter part of the session, the LR lever no longer delivered food, but the SR lever continued to deliver 1 pellet. If medial PFC inactivations induced a general tendency to perseverate toward the lever they initially displayed a bias for at the beginning of a session, we would have expected to observe an impaired ability to shift choice between levers after the reversal. However, this effect was not observed, arguing against the notion that the results of Experiment 1 were attributable to impairments in response flexibility. These results are in keeping with numerous other studies showing that inactivation of the prelimbic region of the medial PFC does not affect reversal learning (Ragozzino et al. 1999; Birrell and Brown 2000; Floresco, Block, and Tse 2008).The prelimbic PFC has been implicated in guiding goal-directed behavior in response to changes in the value of rewards linked to particular actions (Balleine and Dickinson 1998; Killcross and Coutureau 2003). Experiment 3 examined whether medial PFC inactivations interfered with choice behavior when the relative long-term value of larger, probabilistic rewards remained constant throughout a session. Here, the probability of obtaining 4 pellets was fixed so that over 20 free-choice trials, this option either had a greater (40% = ∼32 pellets) or lesser (10% = ∼8 pellets) long-term value than the certain, 1 pellet option (20 pellets). Following saline infusions, rats displayed an appropriate bias toward the Large/Risky or Small/Certain lever when the odds of obtaining the larger reward were 40% or 10%, respectively. Importantly, inactivation of medial PFC did not alter choice behavior under either condition. This suggests that the medial PFC is not critical for basic estimations of reward probabilities when animals must evaluate the relative long-term value associated with smaller, certain versus larger, uncertain rewards. Moreover, this lack of effect on choice indicates that the decreased choice of the Large/Risky option following medial PFC inactivations observed in Experiment 1 (i.e., risk discounting with ascending probabilities) cannot be attributed to a general reduction in preference for larger, probabilistic rewards. Rather, it appears that this region plays a more specialized role in biasing choice in response to changes in the probabilities of obtaining larger, uncertain rewards.Further insight into the contribution of the medial PFC to risk-based decision making comes from a discussion of how rats may identify changes in reward probabilities to facilitate risk discounting. Over repeated training, animals learn that changes in the probabilities associated with the Large/Risky reward are signaled by intermittent blocks of forced-choice trials that precede each set of 10 free-choice trials. Thus, rats must remember the outcomes of previous trials and use this information to update their representation of the relative value associated with the Large/Risky lever as a session proceeds. Rats may also use internal temporal cues to estimate the relative value of the Large/Risky option, as they are trained that the likelihood of obtaining 4 pellets early in a session is substantially different from that later in a session (Catania 1970). There is some suggestion that damage to the medial PFC impairs aspects of time perception (Dietrich and Allen 1998; Thorpe et al. 2002) and induces insensitivity to within-session shifts in delays in a delay-discounting task (Cardinal et al. 2001). Thus, in this context, the medial PFC may subserve an “updating function,” whereby mnemonic and temporal information used to identify changes in reward probabilities is integrated to adjust the direction of behavior to maximize reward. Following inactivation of the medial PFC, rats were impaired at using within-session cues to update their choice according to the reward contingencies of the current trial block. Therefore, dysfunction in this region may limit the ability to use internally generated information to update representations of expected reward probabilities based on recently experienced events.The rat medial PFC displays anatomical connectivity homologous to the ventral portion of the anterior cingulate (Area 32) in primates (Uylings and van Eden 1990) and recent studies suggest that this region plays an important role in mediating risk-based decisions in humans. General increases in activity in this region are observed when healthy subjects perform common cost–benefit decision-making tasks (Ernst et al. 2002; Labudda et al. 2008; Lawrence et al. 2009), particularly when they make risky relative to safe choices on the Iowa gambling task (Lawrence et al. 2009). Similarly, using a task where the risk associated with the different choices was “not winning” a given reward (as in the task used in the present study), Smith et al. (2009) found greater activation in the ventral anterior cingulate (Area 32) during risky versus safe choices, as well as during low-probability versus high-probability choices. Moreover, patients with ventromedial PFC lesions that include damage to Area 32 show impaired decision making on the Cambridge Gamble Task (Clark et al. 2008). Yet, the rat medial PFC has also been proposed to share “functional” homology to the dorsolateral PFC of primates (Brown and Bowman 2002; Uylings et al. 2003). For example, the medial and lateral PFCs have been implicated in working memory in rats (Seamans et al. 1995; Floresco et al. 1999), and humans (Owen et al. 1990; Stuss and Alexander 2000), respectively. In humans, dorsolateral PFC activity has been linked to changes in contexts associated with different gains and losses of reward, particularly when outcomes are the opposite of expectations (Akitsuki et al. 2003). Activity in this region is also increased during performance of the Iowa gambling task (Ernst et al. 2002), particularly in the decision phase (Labudda et al. 2008). Likewise, damage to the dorsolateral PFC has been associated with impairments in different forms of risk-based decision making (Manes et al. 2002; Brand et al. 2004; Fellows and Farah 2005). These findings, viewed in light of the present data, suggest that with respect to risk-based decision making, the medial PFC of the rat may share functions that are similar to those mediated by both the ventral anterior cingulate cortex and dorsolateral PFC in humans.The Role of Other PFC Regions in Cost–Benefit Decision MakingInactivation of the OFC did not affect risk discounting, although these manipulations dramatically increased response latencies in a manner similar to that induced by medial PFC inactivation, whereby deliberation times were increased only in the last 2 trial blocks. This effect on response latencies was only apparent during free-choice trials when rats had to choose 1 of the 2 response options. Notably, it is during these blocks that the relative long-term value of the Large/Risky option switched from having a greater value (100% and 50%) to roughly equal (25%) or lesser (12.5%) value than the Small/Certain option. This suggests that although the OFC may not be necessary for making advantageous choices involving risks and rewards under these conditions, neural activity in this region may aid speed of processing about the cost–benefit contingencies associated with each decision following shifts in the relative long-term values of probabilistic rewards. Consistent with these findings, patients with OFC damage also display increased deliberation times when performing certain risk-based decision making tasks (Rogers, Everitt, et al. 1999; Manes et al. 2002; Clark et al. 2008).Our finding that inactivation of the OFC did not alter risk discounting was somewhat surprising, considering previous studies with both rodents and humans that have implicated this region in cost–benefit decision making tasks about risks and rewards (Rogers, Everitt, et al. 1999; Rogers, Owen, et al. 1999; Manes et al. 2002; Clark et al. 2003, 2008; Ernst et al. 2004; Fukui et al. 2005). Lesions of the rat OFC induced prior to training led to a reduced preference for larger probabilistic rewards (2 pellets) relative to a smaller certain reward (1 pellet; Mobini et al. 2002). In that study, the larger reward probability remained constant over a daily session and was decreased systematically every 20–25 days. The “risk-averse” tendencies displayed by OFC lesioned rats were most prominent when the relative long-term value of the larger, probabilistic option (32% or 20%) was lesser than that of the small certain option. This suggests that OFC lesions may impair learning about the relative value of larger, probabilistic rewards. This notion is supported by findings from human neuroimaging studies, where lateral OFC activity is associated with learning risk/reward contingencies of the Iowa Gambling task over a session (Lawrence et al. 2009). In contrast, rats in the present study were trained for 3–4 weeks until they demonstrated stable patterns of risk discounting, implying that they had learned to adjust their choice behavior in response to within-session changes in reward probabilities. Thus, the lateral aspects of the OFC may facilitate the initial learning of risk/reward contingencies, but neural activity in this region may not be required to guide decision making once these contingencies have been acquired.It is notable that patients with OFC damage also display impaired decision making on tasks where subjects are explicitly informed of reward probabilities prior to making a choice (Clark et al. 2008), similar to the task used in the present study, where rats were familiarized with changes in reward probability over many training days. However, in that study, the patient group that showed impairments included those with damage to the medial portions of the OFC. In fact, a lesion control group that included individuals with lateral OFC damage performed similar to healthy controls. Thus, it may be that the medial, rather than the lateral OFC, plays a more prominent role in facilitating risk-based decisions in situations where subjects have prior knowledge about reward probabilities.Damage to the insular cortex in humans has been associated with alterations in risk-related judgments (Bar-On et al. 2003; Clark et al. 2008). These patients are able to make advantageous decisions (i.e., they correctly choose options yielding the most reward), but do not adjust their betting according to gain–loss probability, suggesting that they are less “risk sensitive” than healthy controls. In a similar vein, imaging studies in healthy controls suggest that the insular cortex may have a specific role in using negative outcomes (i.e., punishment) to bias choice behavior to maximize potential reward (O'Doherty et al. 2003; Cohen et al. 2008; Preuschoff et al. 2008). In contrast, we did not observe significant alterations in risk discounting following insular cortex inactivation. Note that the task used in the present study measured whether rats were able to choose the most advantageous option based on changing reward probabilities. However, our procedures did not employ any explicit punishments per se; the risk was a lost opportunity to obtain a reward, which may explain the lack of effect on risk discounting following inactivation of this region. Thus, it would be of particular interest to investigate the role of the rat insular cortex in guiding decision making when animals evaluate potential aversive consequences associated with different options.Inactivation of the dorsal anterior cingulate cortex also did not affect risk discounting, even though infusions of GABA agonists into the adjacent prelimbic cortex did affect choice and response latencies. These 2 regions of the medial PFC receive dissociable patterns of innervation from the mediodorsal thalamus (Conde et al. 1990) and also exhibit different afferent and efferent connectivity with other cortical and subcortical structures (Sesack et al. 1989; McDonald 1991; Heidbreder and Groenewegen 2003). The present findings suggest that despite their close proximity, these 2 cortical regions have dissociable functions. This notion is in keeping with numerous studies demonstrating that lesions or inactivation of the prelimbic versus anterior cingulate cortex induce differential effects on cognitive, executive, and decision-making functions (Seamans et al. 1995; Bussey et al. 1997; Walton et al. 2003).It is interesting to compare the present findings with previous studies on the dissociable contribution of rat PFC subregions to other forms of cost–benefit decision making, such as delay or effort discounting. The OFC appears to play a role in delay discounting, guiding choice between larger, delayed versus smaller, immediate rewards, whereas lesions of the anterior cingulate do not interfere with this form of decision making (Cardinal et al. 2001; Mobini et al. 2002; Winstanley et al. 2004; Rudebeck et al. 2006). Conversely, the anterior cingulate, but not the OFC, is a component of the circuitry underlying effort-related decisions, but not delay discounting (Walton et al. 2003; Rudebeck et al. 2006; Floresco and Ghods-Sharifi 2007; but see Schweimer and Hauber 2005). Notably, medial PFC lesions do not significantly alter cost–benefit judgments involving either delay- or effort-related costs (Cardinal et al. 2001; Walton et al. 2003). Our findings that the prelimbic PFC (but not the OFC or anterior cingulate) contributes to guiding choice between certain and probabilistic rewards further supports the notion that different response costs (e.g., delays, effort, and uncertainty) are processed by separate frontal lobe regions (Rudebeck et al. 2006; Floresco, St Onge, et al. 2008). It is important to highlight that with delay or effort discounting procedures, animals typically obtain “some” reward after each choice, receiving immediate feedback about the costs associated with larger rewards. Imposition of these costs may reduce the perceived value of larger rewards, biasing choice toward smaller but more easily obtainable rewards. On the other hand, risk discounting requires monitoring of rewarded and nonrewarded outcomes, as well as integration of this information over many trials to estimate the long-term value of small/certain versus large/uncertain rewards. In turn, this information may be incorporated in computations about the probability of obtaining the larger reward under conditions where reward probabilities change. Thus, the seemingly selective involvement of the medial PFC in this form of discounting may be linked to its role in the temporal organization of behavior (Fuster 2000), whereby internally generated information is used to keep track of changes in reward probabilities and update value representations that, in turn, may facilitate efficient decision making.FundingCanadian Institutes of Health Research (MOP-89861 to S.B.F., a Michael Smith Foundation for Health Research Senior Scholar); Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foundation for Health Research (to J.R.S.O.).We are grateful to Ying Zhang for her assistance with behavioral testing and surgical procedures and Colin Stopper for his helpful comments on the manuscript. 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projections of the medial prefrontal cortex in the rat: an anterograde tract-tracing study with Phaseolus vulgaris leucoagglutininJ Comp Neurol1989290213242SmithBWMitchellDGHardinMGJazbecSFridbergDBlairRJErnstMNeural substrates of reward magnitude, probability, and risk during a wheel of fortune decision-making taskNeuroimage200944600609St OngeJRFlorescoSBDopaminergic modulation of risk-based decision makingNeuropsychopharmacology200934681697StussDTAlexanderMPExecutive functions and the frontal lobes: a conceptual viewPsychol Res200063289298TakahashiYKRoeschMRStalnakerTAHaneyRZCaluDJTaylorARBurkeKASchoenbaumGThe orbitofrontal cortex and ventral tegmental area are necessary for learning from unexpected outcomesNeuron200962269280ThorpeCFlorescoSCarrJWilkieDAlterations in time-place learning induced by lesions to the rat medial prefrontal cortexBehav Proc20025987100TomSMFoxCRTrepelCPoldrackRAThe neural basis of loss aversion in decision-making under riskScience2007315515518UylingsHBGroenewegenHJKolbBDo rats have a prefrontal cortex?Behav Brain Res2003146317UylingsHBvan EdenCGQualitative and quantitative comparison of the prefrontal cortex in rat and in primates, including humansProg Brain Res1990853162WaltonMEBannermanDMAlterescuKRushworthMFFunctional specialization within medial frontal cortex of the anterior cingulate for evaluating effort-related decisionsJ Neurosci20032364756479WinstanleyCATheobaldDECardinalRNRobbinsTWContrasting roles of basolateral amygdala and orbitofrontal cortex in impulsive choiceJ Neurosci20042447184722WinstanleyCATheobaldDEDalleyJWRobbinsTWInteractions between serotonin and dopamine in the control of impulsive choice in rats: therapeutic implications for impulse control disordersNeuropsychopharmacology200530669682 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B853D5B870F3D88CB012A22899484E2ECE632AD.txt b/test/dataset/in/resources/corpus/Clean_0B853D5B870F3D88CB012A22899484E2ECE632AD.txt new file mode 100644 index 0000000..73c998a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B853D5B870F3D88CB012A22899484E2ECE632AD.txt @@ -0,0 +1 @@ +]>NBA5261S0197-4580(99)00016-010.1016/S0197-4580(99)00016-0Elsevier Science Inc.Fig. 1Mean (SEM) body weight values. ∗Significant age effect for data combined across group (ANOVA, p < 0.0001). Tukey pairwise notations for combined data: 25-month and 17-month significantly differ; 317-month and 27-month significantly differ. Sample sizes are listed in parentheses.Fig. 2Morris swim maze performance. The first group of mice received 20 trials (5 days) of hidden platform training (A). The second group received 28 trials (7 days) of hidden platform training (B). ∗Significant age effect (ANOVA, p < 0.05). The 90-s probe trial was subdivided into six 15-s epochs for the first group (C), and for the second group (D). +Significantly different from chance (25%) for data combined across age (one-sample t, p < 0.05). Mean (SEM) cued platform distance for the second group collapsed across 5 trials (1 day) of training (E), and for each training trial (F). Tukey pairwise notations: 15-month and 27-month significantly differ; 25-month and 17-month significantly differ; 317-month and 27-month significantly differ. Sample sizes are listed in parentheses.Fig. 3Psychomotor battery performance. Mean (SEM) tightrope suspension time (A), crossings (B), and transformed score (C) collapsed across three trials. ∗Significant age effect for data combined across group (Brown–Forsythe, p < 0.05). Mean (SEM) square open field activity during a 10-min exposure (D). ∗Significant age effect for data combined across group (ANOVA, p < 0.05). Tukey pairwise notations for combined data: 15-month and 27-month significantly differ; 25-month and 17-month significantly differ; 317-month and 27-month significantly differ. Sample sizes are listed in parentheses.Fig. 4Eye pathology correlation with Morris swim maze performance of 17- and 27-month 129/SvJ mice. Spearman’s rank correlation was not significant for mean distance to find the hidden platform (A) nor mean probe trial performance (B). However, this correlation was slightly significant (p < 0.05) for mean distance traveled to the cued platform (C). Gross eye pathology was ranked on a four-point scale ranging from 0 (no eye pathology) to 3 (severe eye pathology; see text).Table 1Two groups, each comprising three counter-balanced age cohorts of 129/SvJ mice, were subjected to both a psychomotor battery and Morris swim maze. The schedule of behavioral tests is listed along with the day of the test for each groupBehavioral TestGroup 1 (Weeks 1–2)Group 2 (Weeks 8–9)Swim MazePretrainingN/ADay 1 (5 trials)Hidden PlatformDays 1–5 (20 trials)Days 2–8 (28 trials)Probe TrialDay 5 (1 trial 6 epochs)Day 8 (1 trial 6 epochs)Cued PlatformN/ADay 9 (5 trials)Psychomotor BatteryTightropeDay 10 (3 trials)Day 10 (3 trials)Open FieldDay 16 (10-min exposure)Day 11 (10-min exposure)ArticlesAge-related psychomotor and spatial learning deficits in 129/SvJ miceJohn MHengemihleaJeffrey MLongaJenniferBetkeybMathiasJuckercDonald KIngrama*doni@vax.grc.nia.nih.govaLaboratory of Cellular and Molecular Biology, Molecular Physiology and Genetics Section, National Institute on Aging, Intramural Research Program, Gerontology Research Center, Baltimore, MD 21224, USAbDenison University, Granville, OH, USAcDepartment of Neuropathology, Institute of Pathology, University of Basel, Schonbeinstr. 40, CH-4003 Basel, Switzerland*Corresponding author. Tel.: +1-410-558-8180; fax: +1-410-558-8323AbstractThe 129 mouse strain has been widely used to construct mutations that model behavioral aging in humans. The current study found significant age-related declines in both psychomotor and swim maze performance of 5-, 17-, and 27-month-old 129/SvJ mice. However, the age differences in swim maze acquisition were inconsistent with poor performance in the probe trial which assesses spatial memory. This inconsistency may result from the high degree of genetic polymorphisms and age-related visual pathology which afflicts this mouse strain. Therefore, we concluded that 129/SvJ mice present a problematic model of mammalian cognitive aging and involve a risk for behavioral contamination in studies involving mutant mice derived from this strain.KeywordsAgingAnimal behaviorEye pathologyGeneticsMaze learningTransgenic mice1IntroductionThe use of transgenic and knockout murine models has expanded opportunities for examining complex behavioral processes and may be clinically relevant in establishing models of neurodegenerative disease states. Homologous recombination in embryonic stem (ES) cells has allowed the development of chimeric mice with stable, germ line transmission of heritable targeted mutations [28]. Homozygous F2 offspring carrying the mutation can be backcrossed for several generations to create a congenic line on a fixed genetic background suitable for behavioral testing. The behavioral phenotype of these genetically engineered mutant mice is influenced by the mutation but also reflects the interaction of background genes [4], compensatory developmental mechanisms [18], and epistatic interactions between genes [15]. Therefore, before conclusions can be reached about the effect of a targeted mutation on polygenic learning and memory processes, it is important to understand the endogenous behavioral phenotype of the host strain. Because complex behaviors are polygenic, the genetic background of the host strain may affect the behavioral expression of a single gene mutation [28].ES cells isolated from the 129 mouse strain show the highest success rate for germ line integration [22,23]. Consequently this inbred mouse strain has been widely used in the construction of mutant mice, including murine models of Alzheimer’s disease [19,31]. However, relatively little has been published about age-related behavioral changes in this strain that would be useful for assessing the impact of mutant genes. It is possible that the behavioral abnormalities seen in mutant mice derived from 129 ES cells arise from polymorphisms in the mutant 129 genetic background rather than from any exogenous genetic manipulation [4]. Because of potential behavioral contamination from these background or hitchhiker genes [3,5,7,14], there is clearly a need for improved behavioral characterization of wild type 129 mice.To the best of our knowledge, this is the first study of aging in wild type 129/SvJ mice. We attempt to determine the suitability of this inbred strain to studies on behavioral aging. This is important because most previous studies on behavioral aging in mice have used the C57BL/6 strain [9,10,13]. In the current study, 129/SvJ mice of three ages underwent a motor and cognitive assessment, specifically locomotor activity in an open field, strength and coordination in a tightrope test, and spatial learning ability in a Morris water maze. Because previous studies had indicated relatively poor performance of 129/SvJ mice in the water maze [20], we examined whether extended acquisition training (28 versus 20 trials) could enhance performance in this task. We also attempted to ascertain the influence of age-related gross eye pathology on water maze performance of this albino mouse strain [24,25]. Previous studies have also indicated that 129 mice may be especially vulnerable to handling stress [17]. Therefore, we attempted to habituate our mice to handling and thereby overcome some of the known stress related activity confounds of the swim maze, such as passive floating [30] and jumping.2Methods2.1AnimalsExperimentally naive male 129/SvJ mice were obtained from the Jackson Laboratory, Bar Harbor, ME at 5-weeks of age (JAX® Stock Number 000691; now designated as 129XI/SvJ). Over a period of 22 months, three cohorts of mice were acquired and maintained at the Gerontology Research Center (GRC). At the time of behavioral testing, mice were either 5, 17 or 27 months old. Mice were housed in groups of five in a 30 × 19 × 13 cm plastic cage with corncob bedding and had access to food (NIH formula 07) and filtered water ad libitum. Mice were maintained on a 12:12-h light:dark cycle. Lights were turned on at 0700. The room temperature of the vivarium was about 22°C and the relative humidity was about 48%.2.2Behavior testing2.2.1HandlingFor logistical reasons, mice were divided into two counterbalanced groups and behaviorally tested 8 weeks apart. Before testing, mice were systematically handled in an attempt to minimize the previously observed passive floating behavior of this strain in the Morris swim maze (unpublished observation). Each mouse received 10-min of daily handling by the same experimenter. This consisted of picking up each mouse and continuously grooming its fur with brushes of various textures. This was done each day for a period of 2 weeks before behavior testing. To facilitate identification, a sterile AVID® microchip (AVID Inc., Norco, CA USA) was aseptically placed subcutaneously (s.c.) into each mouse before behavior testing.2.2.2Apparatus and procedureThe testing area consisted of a black rectangular table (112 cm × 104 cm) elevated 64 cm from the floor. This table was surrounded by white curtains (183 cm long) and was surveyed by a video camera mounted 122 cm overhead. The area was illuminated with overhead fluorescent lights that provided adequate contrast for the camera. Locomotor activity was monitored using a Videomex® tracking system (Columbus Instruments, Columbus, OH USA) which measured the amount of distance traveled by the mouse during the test period. Before testing, mice were transferred from the vivarium to the testing room 30 min before testing to allow for acclimation. All testing was conducted in the same testing room by the same experimenter between 0730 and 1500 h in the order described in Table 1. 2.2.3Maze trainingThe Morris swim maze is a spatial learning task in which mice must learn to escape by swimming to a concealed, submersed platform within a tank. The maze was a black, circular plastic pool, 80 cm in diameter and 50 cm in height, filled with clear (i.e. non-opaque) tap water to a depth of 25 cm. Water was maintained at 21–22°C. A transparent circular platform, 7 cm in diameter, was fixed in the center of one quadrant of the maze, 1 cm below the water surface. The swim maze was always maintained in the same position in the room as were all visible extra-maze cues. A white cloth curtain was draped around all sides of the maze to allow for adequate video camera tracking of mice within the maze. Previous studies conducted in our laboratory determined that C57BL/6J mice learn the Morris swim maze significantly faster in the presence of multiple three-dimensional cues in close proximity to the maze (unpublished data). It was assumed that these same cues would also benefit 129/SvJ mice. Therefore, seven three-dimensional objects (soda can, toy mouse, floppy disk box, nondescript foam rubber cutout, origami crane, light bulb, rubber stopper) were anchored equidistantly around the top inside wall of the maze and served as cues in this study. The cues varied in shape, size, color, and were no larger than 15 × 15 cm. Because a curtain surrounded the tank, the mice were unable to view the experimenter at any time. Distance traveled (cm) to reach the platform was the dependent variable.As shown in Table 1, the second group of mice (but not the first) were administered five pretraining trials to familiarize them with the presence of a submersed goal and to minimize any potentially confounding effect of nonspatial kinesthetic strategies that the mice may employ to solve the task. During pretraining, all cues were removed, and a Plexiglas® insert was placed into the tank forming a 7-cm wide alley leading to the submerged platform. Each mouse in the second group was given a total of five 90-s trials to locate the platform starting at the opposite end of the alley.For the hidden platform trials, the cues were replaced, and a trial was begun by placing the mouse into a counterbalanced starting quadrant (either N, S, E, or W) facing the wall of the pool. The computer then began recording the distance traveled. Each mouse had a maximum of 90-s to swim to the platform, at which point activity tracking was terminated. When the mouse reached the platform, it was permitted to remain there for 30 s before being removed from the maze. If the mouse failed to find the platform within 90 s, it was placed onto the platform by the experimenter and was removed after 30 s. Following a trial, the mouse was returned to its home cage with free access to food and water between trials. Following completion of a trial for all mice, a 70-min inter-trial interval (ITI) was provided before the start of a new trial. All mice dried themselves off during this rest interval and did not seem to be fatigued. Each mouse received four trials per day; 1 trial per day from each of the four counterbalanced starting quadrants. The platform’s location remained constant throughout training for each mouse. As shown in Table 1, the first group of mice received 20 trials (5 days) of hidden platform training, whereas the second group received 28 trials (7 days). Acquisition was measured as mean distance traveled each day, i.e. each block of four trials, for each group of mice. Thus, a decrease in distance traveled over hidden platform trials implies an improvement in reference memory spatial learning processes.A probe trial was given to all mice following their final trial on the last day of training (see Table 1). The probe trial started from a position directly in the center of the tank and was identical to previously described trials, except that the goal platform was removed. The percent of total distance traveled in the training quadrant (the quadrant where the platform had been located throughout the experiment) was calculated for the entire 90-s trial. The probe trial was divided into six contiguous 15-s epochs, and the percent training quadrant distance was calculated for each epoch in an attempt to access extinction. The probe trial was used to determine the amount of spatial bias or habit strength that mice developed for the location of the platform. An increased spatial bias in the probe trial indicated that the mice had developed a memory for the spatial location of the platform and was an additional indicator of the degree of spatial learning improvement with training.As shown in Table 1, on the day following the probe trial, the second group of mice received five cued platform trials with a 70-min ITI. For this block of trials, the peripheral cues described above were removed. The submerged platform was restored to a quadrant 180° diagonal to its original location and was prominently cued by the insertion of a dowel stick with a green Styrofoam® ball (10-cm diameter) attached 15 cm above the water. The 129/SvJ strain is albino, and therefore subject to visual impairments that may confound a pure spatial learning interpretation of the swim maze task [27]. The cued platform trials can address this problem by serving to identify gross impairments in visual discrimination ability in addition to motoric and motivational impairments all of which are potential confounds of the swim maze.2.2.4TightropeStrength and motor coordination of each mouse was tested by its ability to grasp and to remain suspended from a rope (2 mm in diameter and 50 cm in length). The rope was stretched taut inside a white polyethylene tank (50 cm diameter × 30 cm high) which was filled with water (at 21–22°C) to a depth of 10 cm. During testing, each mouse was kept in a dry holding cage that did not contain any bedding material. Before the test began, the mouse was introduced to the water by holding it by the tail in the water for 5 s. For each of three subsequent trials, the mouse was raised by its tail above the rope and then lowered slowly until it grasped the center of the rope with both front paws. The body of the mouse was then slowly lowered below the rope and released so that the animal had to support its body weight by its grip or fall into the water 20 cm below. The suspension time until a fall into the water was recorded (60-s maximum); the clock was stopped when the mouse entered the water (not when it lost its grip). In addition, the rope had pen markings at 5 cm intervals along its length to measure the amount of horizontal movement (crossings) which occurred while the mouse was suspended (a crossing was counted when the animals leading front paw touched the marking). When the mouse fell into the water, it was immediately removed to the holding cage and allowed to rest for 30-s before the next trial. The suspension time and number of crossings were averaged across three trials. A transformed score was calculated for each mouse: Transformed Score = (mean suspension time) + 10 (mean number of crossings). Thus, an elevated transformed score indicates superior performance either in terms of grip strength or motor coordination or both.2.2.5Open fieldSpontaneous locomotor activity in a novel environment was assessed in this test. A black Plexiglas® square (40 cm wide × 17 cm high) with wire mesh (1.5-cm square openings) attached to one side was used to monitor total distance traveled during a 10-min exposure. The device was elevated 14 cm from the surface of a black table to allow for passage of waste. The apparatus was illuminated from above with fluorescent lights such that the center of the square received approximately 9 foot-candles whereas the corners received 5 foot-candles of light. Mice were allowed to acclimate to the test room for 30 min before testing. Ten seconds after the mouse was placed at the center of the square, distance tracking began and was measured for 10 min. The apparatus was cleaned with 100% ethanol after each mouse was tested to remove both odors and boli. All alcohol vapors were thoroughly removed before the next mouse was tested. Two mice (5 and 17 months old) from the first group died following tightrope testing; open field data were not collected for these subjects.2.3Eye pathologyAt the conclusion of behavior testing, the second group of mice was examined for blepharoconjunctivitis. This ophthalmic condition can affect humans and is observed in several strains of mice, A/HeJ, BALB/cJ, CBA/J, and 129/J [24,25]. It is characterized either by a flaking hyperkeratosis of the eyelids or by excessive production of sebum-like material from the meibomian glands, and the incidence increases with advancing age. To assess the degree of pathology, we examined the eyeball presentation of each mouse and ranked the observations on a four-point scale as follows: 0 = no detectable eye involvement; 1 = mildly swollen eyelid; 2 = moderate or bulging eye; 3 = severe periorbital abscess. No attempt was made to determine the etiology of the ocular pathology nor the relationship between this pathology and visual acuity.2.4Statistical analysisBody weight, tightrope and open field data were first analyzed using a 2(Group) by 3(Age) analysis of variance (ANOVA; BMDP 7D) to determine the appropriateness of combining the two separately tested groups of mice for statistical analysis. If the main effect of group was not significant, data were collapsed across group, and a 3(Age) ANOVA was calculated. If the main effect of age was significant, the Tukey Studentized Range method (with harmonic mean adjustment for unequal sample size) was used to determine individual differences between age groups. If Levene’s test revealed heterogeneity of variance between the groups, the Brown–Forsythe equality of means test [1] was used in lieu of ANOVA because it does not assume that the group variances are equal. Critical values for the BrownForsythe test were obtained from the F distribution with a loss of degrees of freedom to allow for unequal group variances. To determine the degree to which body weight affected tightrope performance, the Pearson correlation coefficient (r) was calculated.Swim maze data for each group were analyzed separately. Hidden platform data for the first group of mice was analyzed using a 3(Age) by 5(Block) ANOVA with repeated measures (ANOVA-RM; BMDP 2V) on the last factor—mean swim distance for each block of four trials. Likewise, hidden platform data for the second group of mice was analyzed using a 3(Age) by 7(Block) ANOVA-RM. Probe trial data for each group of mice were analyzed using a 3(Age) by 6(Epoch) ANOVA-RM on the last factor—percent total distance in the training quadrant for each 15-s epoch. Probe trial data for each group were also analyzed using a one-sample t-test to determine if the observed spatial bias was significantly above chance level (25%). In addition, probe trial data for each quadrant were separately analyzed using a 3(Age) ANOVA collapsed across group. This analysis was conducted to verify selective searching in the training quadrant. Cued platform data for the second group was analyzed using a 3(Age) by 5(Trial) ANOVA-RM on the last factor—swim distance for each cued platform trial. In all cases, significant F tests were followed by pairwise comparisons using the Tukey method. If Levene’s test was significant for any repeated measure, a variance stabilizing square root transformation was conducted.The relation between eye pathology and swim maze performance was determined using the Spearman rank-order correlation (rs; BMDP 3D). This coefficient was selected because exact numerical values could not be assigned to gross eye pathology rankings.3Results3.1Body weightThe body weight of each mouse was taken before the start of behavior testing. Data were collapsed across the two separately tested groups of mice and a 3(Age) ANOVA was computed. As shown in Fig. 1, this analysis revealed a significant main effect of age (F(2,70) = 13.41, p < 0.0001). Pairwise mean comparisons using the Tukey method showed that 17-month-old mice weighed significantly more than either 5- or 27-month-old mice (p < 0.01).3.2Morris swim mazeThe hidden platform data for each group of mice was analyzed separately to ascertain the influence of extended training (see Table 1). Because Levene’s test was significant, a variance stabilizing square root transformation was conducted on both groups of hidden platform data. Fig. 2A presents 20 trials of data for the first group of mice. A 3(Age) by 5(Block) ANOVA-RM showed a significant main effect of age (F(2,33) = 6.56, p < 0.01). Likewise, Fig. 2B presents 28 trials of data for the second group of mice. A 3(Age) by 7(Block) ANOVA-RM revealed a significant main effect of age (F(2,34) = 7.35, p < 0.01). Tukey pairwise mean comparisons were used to access age differences at different time points (see Fig. 2). Both groups of mice demonstrated appreciable learning across trials as evidenced by the significant main effect of block (Group 1: F(4,132) = 15.26, p < 0.0001; Group 2: F(6,204) = 6.99, p < 0.0001). The significant interaction between block and age in each group reflects impaired learning among the 27-month-old mice compared to the 5- and 17-month-old mice in each group (Group 1: F(8,132) = 2.87, p < 0.01; Group 2: F(12,204) = 2.70, p < 0.01). When examining the data collected during extended training provided to the second group of mice, two salient points emerged. First, no age group improved their performance when training was extended from 4 to 7 days. Second, the 27-month-old mice showed significant deterioration in performance during extended training.On their last day of hidden platform training, both groups of mice were administered a 90-s probe trial consisting of six 15-s epochs. Fig. 2C presents probe trial data (spatial bias for training quadrant) for the first group of mice which received 20 trials of hidden platform training; Fig. 2D presents probe trial data for the second group of mice which received 28 trials of hidden platform training. A 3(Age) by 6(Epoch) ANOVA-RM did not reveal a significant main effect of age, epoch nor interaction for neither group. One-sample t-tests revealed that when data were collapsed across age, the first group of mice performed at or slightly below chance level (25%) for each 15-s epoch (see Fig. 2C). However, the same analysis revealed that the second group of mice, which received extended hidden platform training, performed significantly above chance level in all but the first epoch of the probe trial (see Fig. 2D). Thus, despite not showing improved acquisition learning with extended training trials as measured by swim distance, probe trial memory for the location of the platform was improved with extended training, although no significant age differences were evident using percent quadrant distance as the measure of memory. Moreover, the relative probe trial performance was not great considering that levels of 35% to 40% would be clear evidence of a spatial bias. Based on an analysis of distance swam in each quadrant in the combined groups, no significant spatial bias was observed in any maze quadrant. In addition, there were no significant age differences observed in any of the other quadrants. The percent distance scores for other (i.e. non-platform) quadrants were as follows: 5-month group (23.5%, 25.3%, 22.7%), 17-month group (24.2%, 24.4%, 24.8%), 27-month group (25.2%, 23.4%, 27.1%).The second group of mice was given five cued platform trials on the day following the probe trial (see Table 1). As shown in Fig. 2E, when the data were collapsed across all five cued trials, a 3(Age) ANOVA was not significant. As shown in Fig. 2F, when the data were analyzed as a function of training trials, a 3(Age) by 5(Trial) ANOVA-RM did not reveal any significant main effects nor interaction. There was an apparent age-related increase in distance to find the cued platform; however, this trend was not significant.3.3Psychomotor batteryBoth groups of mice were tested on the tightrope task following maze training (see Table 1). Data from the two separately tested groups were combined. Because Levene’s test revealed heterogeneity of variance between groups, the Brown–Forsythe statistic (F∗) was used. As shown in Fig. 3A, a 3(Age) Brown–Forsythe test on the tightrope suspension time did not show a significant main effect of age. However, as shown in Fig. 3B, a 3(Age) Brown–Forsythe test on tightrope crossings revealed a significant main effect of age (F∗ (2,55) = 15.51, p < 0.0001). Pairwise mean comparisons using the Tukey method showed that 5-month-old mice made significantly more crossings than either 17- or 27-month-old mice (p < 0.01). Fig. 3C presents transformed data from the tightrope task for which a 3(Age) Brown–Forsythe test revealed a significant main effect of age (F∗ (2,55) = 4.43, p < 0.05). Tukey pairwise comparisons showed that 5-month-old mice performed significantly better than 17-month-old mice (p < 0.05).Interpretation of the age differences in the tightrope task were complicated to some degree by differences in body weight. Previous studies in our laboratory have shown that heavier mice tend to perform somewhat more poorly on the tightrope task [8,12]. As evidence of possible extraneous variation in the present study, the correlation between body weight and the transformed score for all age groups was significant, r (71) = −0.27, p < 0.05. However, this correlation was significant only within the 27-month-old group, r (16) = −0.48, p < 0.05. The significant decline in the tightrope transformed score between 5- and 17-month-old groups could be related to the heavier body weight of the 17-month-old group. However, as shown in Fig. 1, there was no significant difference in body weight between the 5- and 27-month groups to account for the decline in performance of this task.Both groups of mice were tested in the square open field (see Table 1). Data from the two groups were combined. As shown in Fig. 3D, a 3(Age) ANOVA revealed a significant main effect of age (F(2,68) = 5.50, p < 0.01). Pairwise Tukey comparisons showed that 27-month-old mice were significantly less active than either 17- (p < 0.05) or 5-month-old mice (p < 0.01).3.4Eye pathologyThe second group of mice was examined for gross eye pathology at the completion of behavior testing. Only 6% (1:18) of the 5-month-old mice were observed to have moderate to severe eye pathology suggesting that this cohort was relatively healthy. In contrast, 46% (6:13) of the 17- and 67% (4:6) of the 27-month-old mice were observed to have an eye pathology ranking of 2 or 3. Because the 5-month-old cohort was relatively healthy, their data were culled from the eye pathology correlation; only data from the 17- and 27-month-old cohorts were included because these mice presented most of the overt eye pathology. As shown in Fig. 4A and B, the Spearman rank correlation with eye pathology was not significant for hidden platform distance (mean of first 20 trials) nor probe trial performance (mean of 6 epochs). However, as shown in Fig. 4C, a significant Spearman correlation was found between eye pathology and distance traveled to the cued platform (mean of 5 trials; rs (17) = 0.46, p < 0.05).4DiscussionEvidence of behavioral aging in male 129/SvJ mice was observed in tests of psychomotor performance and spatial learning in this cross-sectional study. Specifically, age was associated with diminished strength and motor coordination as measured by tightrope performance and decreased spontaneous locomotor activity measured in the open field. Significant age-related deficits in the water maze were evident in this strain of mice during the acquisition trials measured as distance to platform; however, there were no significant age differences in probe trial performance. The later observation would confound any conclusions about age differences in spatial memory abilities, despite the age differences in learning in this task.The behavioral phenotype of 129 mice is continuing to emerge in the literature. Previous studies have reported hypoactivity of 129/SvJ mice in the open field [15,17,18]. However, this robust finding was not reproduced in the current study. When compared to unpublished observations in 6-month-old male C57BL/6J mice from our own laboratory, the 5-month-old male 129/SvJ mice of the current study were found to be significantly (18%) more active in the open field. One previous study [18] using the tightrope task found no difference between C57BL/6J and 129/Sv mice. In contrast, the 5-month-old male 129/SvJ mice from the current study were found to demonstrate a significantly (36%) lower transformed score in the tightrope task when compared to 6-month-old male C57BL/6J mice (unpublished data). Methodological differences, such as the handling, may account for these discrepancies. However, it is more likely that the mixed genetic background of the 129/SvJ strain [26] accounts for many of these discrepancies with the previous psychomotor literature on 129/SvJ mice.The Morris swim maze is heavily dependent upon visual, motor, and cognitive abilities. As such, interpretation of a cognitive deficit in the current study is problematic from several perspectives. First, the observed age-related decline in psychomotor performance of this strain may confound a pure spatial deficit interpretation of the swim maze results. Second, although learning was evident in the two youngest groups, the aged group showed no significant improvement across sessions. Third, the level of performance achieved over 20 trials in 129/SvJ mice was substantially less than we have observed in young C57BL/6NIA [11] and C57BL/6J [2] mice as measured by distance traveled. Fourth, performance in the probe trial was poor for all three age groups and did not differ significantly. There was no evidence of spatial bias to indicate use of a spatial strategy based on utilization of visual cues. Although no significant age effect was found in the cued platform test, it was clear that many older mice seemed impaired as shown by the great variability. This variability was correlated with the degree of eye pathology. Over 52% (10:19) of the 17- and 27-month-old mice presented with overt eye symptoms, suggestive of blepharoconjunctivitis [24,25]. It is possible that the high incidence of age-related visual pathology in these mice may cause a visual acuity deficit resulting in a potentially confounding measurement error [9]. As the severity of the pathology increased, performance in the cued trials diminished (Fig. 4C). However, no such correlation was observed in the hidden platform task (Fig. 4A) or in the probe trial (Fig. 4B). These latter observations would indicate that the mice were not utilizing available visual cues to direct their performance in the swim maze. In summary, interpreting the observed age-related differences in spatial learning ability is very problematic to the extent that these learning deficits may not be a function of normal aging in 129/SvJ mice [9].As noted previously, the 5-month-old 129/SvJ mice in the current study presented a very low incidence of overt visual pathology. Therefore, their swim maze behavior was not likely to be influenced by the measurement errors discussed above. As such, the current study supports previous findings which showed young 129/SvJ mice achieved an inferior level of asymptotic performance in the swim maze relative to pigmented strains of mice [20]. Although 5-month-old male 129/SvJ mice from the current study were able to consistently find the hidden platform, this asymptote was only achieved with a highly significant (98%) increase in distance traveled compared to 7-month-old male C57BL/6NIA mice from our laboratory [11]. The current study demonstrated that without extended hidden platform training, 129/SvJ mice lack development of appreciable spatial bias as measured in the probe trial (see Fig. 2C); this finding replicates negative probe trial results of a previous study in 129/SvJ mice [20]. It is possible that the percent training quadrant distance measure frequently used in the probe trial is not a sensitive measure of spatial bias in 129/SvJ mice. Annulus crossings seems to be a better choice because another study [18] was able to demonstrate significant probe trial spatial bias in 129/Sv mice using that measure. The current study reports two manipulations which taken together were partially successful in improving probe trial performance. First, extended hidden platform training was able to improve spatial bias above chance performance (Fig. 2D). Second, our handling manipulation was apparently successful in reducing stress in that neither passive floating nor jumping behaviors were observed in any of our mice in the swim maze. This observation is significant because previous studies have shown that handling reduces stress and thereby improves spatial learning in mice [6]. Despite these relative improvements in spatial bias, however, probe trail performance did not vary significantly across age.Previous studies using aged rats have shown that a shortened ITI can impair water maze performance due to fatigue [21]. Therefore, we were careful to space the trials out in an attempt to avoid fatigue in our aged subjects. Absence of fatigue was evidenced by the fact that all the mice completely groomed themselves to dryness between trials and did not shiver. Moreover, the distance measure used in the current study to assess acquisition is more robust to fatigue-related activity confounds than the latency measure used in previous studies [18,20]. These previous studies have relied on distal room cues for navigation, however, in the current study proximal cues were placed inside the rim of tank. Because it has been suggested that mice are less adapted to swimming tasks than other rodent species [29], it is possible that this proximal location of cues favors the development of a spatial rather than kinesthetic strategy to solve the task.In conclusion, we feel that the high degree of documented genetic polymorphisms [26] and possible ancestral contamination [22] makes the 129/SvJ mouse strain generally unsuitable to studies of behavioral aging. Although we feel that the 129/SvJ strain should be avoided in experiments assessing age-related effects on behavior, we appreciate the value of this strain in the production of mutant mice. The strategy of maintaining the targeted mutation heterozygous (by backcrossing chimeras into a common inbred strain such as C57BL/6J) and then testing the homozygous mutant in an F1 cross is sound. Because F1s are used, specific deficits in the 129 strain influenced by deleterious homozygous genes will be attenuated. In addition, the current study reports visual pathology in aged 129/SvJ mice severe enough to potentially confound swim maze studies of aged mutant mice derived from 129/SvJ ES cells. Future studies using conditional mutants may avoid potential behavioral problems resulting from genetic variation and contamination [16].AcknowledgementsThe Gerontology Research Center (NIH-NIA-IRP) is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. 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Louis Pasteur s/n, E-29071 Málaga, Spain2Department of Renal Diseases and Hypertension, School of Medicine, University of Colorado Health Sciences Center, 4200 E. 9th Av. Denver, CO 80262, USA3Department of Pathology, School of Medicine, University of Colorado Health Sciences Center, 4200 E. 9th Av. Denver, CO 80262, USA*To whom correspondence should be addressed: E-mail: segovia@uma.es320096038158284720082011200824112008© 2009 The Author(s).2009This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago.Aponecrosis-likeapoptosis-likeautophagic/vacuolar cell deathcaspase-likecell deathDEVDase activityDunaliellaenvironmental stressmicroalgaenecrosis-likeparaptosis-likephytoplanktonTUNELIntroductionWhether cell death is beneficial or detrimental for the organisms depends on the biological circumstances in which this happens and on the cell type or species. For both, the question arises concerning the significance of programmed cell death (PCD) in the biology of multi- and unicellular organisms. The origin of the capacity for self-destruction may be as ancient as the origin of the very first cell (Ameisen, 1998). Thus, if effectors of the cell survival machinery can also be effectors of the self-destruction of the cell in which they operate, then the requirement for coupling cell survival to the prevention of self-destruction may be as old as the origin of the first cell, and cell suicide is therefore an unavoidable consequence of self-organization. PCD is ubiquitous in multicellular systems and is essential for normal growth and development (Leist and Nicotera, 1997). The PCD type known as apoptosis, is characterized by very specific morphological and biochemical requirements such as chromatin condensation and margination, ordered DNA cleavage while the cytoplasm and organelles remain unchanged, and the participation of a family of cysteine proteases named caspases, as central regulators (Kerr et al., 1972). Apoptosis is clearly different from necrosis which is characterized by a loss of membrane integrity, cell swelling, and lyses. However, the classical necrotic nature described as passive, unprogrammed cell death, can be questioned. There are examples of cells presenting necrotic morphologies that are subjected to an active cell death programme called ‘necrotic-like’-PCD that can be either caspase-dependent or -independent (Edelstein et al., 1999; Kitanaka and Kuchino, 1999). Consistently, it is considered a type of PCD due to the presence of underlying regulatory mechanisms. However, other types of PCDs, which do not completely fulfil either the typical apoptotic features or the necrotic ones, have also been reported (Clarke, 1990; Sperandio et al., 2000; Leist and Jäätelä, 2001; Papucci et al., 2004; Elmore, 2007). In this regard, a cell death type named ‘paraptosis’, which is fundamentally different from apoptosis, was discovered. It involves cytoplasmic vacuolization, mitochondrial swelling, and the absence of caspase activation or typical nuclear changes, including pyknosis and DNA fragmentation, and is mediated by mitogen-activated protein kinases (MAPKs) (Sperandio et al., 2000, 2004). Other cells have been shown to exhibit features of both apoptosis and necrosis and the term ‘aponecrosis’ was coined (Formigli et al., 2000; Papucci et al., 2004). These authors suggested that apoptosis and necrosis represent two extremes of a wide range of aponecrotic responses that operate under caspase activation. One distinct model is ‘autophagic/vacuolar’-like cell death. The typical hallmark of this form of cell death is the consumption of the cytoplasm in the absence of leakage of the intracellular content and intact cell membranes. This type of cell death may display nuclear degradation and pyknosis, depending on the nature of the cell (Jones, 2000) and it has been shown to be a lysosomal degradation pathway, since final evidence for lysosome-like organelles in plants has been reported (Swanson et al., 1998).Among all the cell death classes, apoptosis has been mainly studied in metazoans, but vascular plants, as well as some unicellular eukaryotic organisms, show characteristic apoptotic-like features. Apoptosis-like phenomena occur in vascular plants (Greenberg, 1996; Pennell and Lamb, 1997; Lam and del Pozo, 2000; Lam et al., 2001) and they have also been portrayed in unicellular organisms, including chlorophytes (Berges and Falkowski, 1998; Segovia et al., 2003; Segovia and Berges, 2005), dinoflagellates (Vardi et al., 1999; Dunn et al., 2004; Franklin and Berges, 2004; Segovia, 2007), diatoms (Casotti et al., 2005), yeast (Frohlich and Madeo, 2000), kinetoplastids and slime moulds (Cornillon et al., 1994), and bacteria (Lewis, 2000), including cyanobacteria (Berman-Frank et al., 2004). Berges and Falkowski (1998) described a form of autocatalysed cell death in the single cell algae D. tertiolecta. When this chlorophyte was deprived of light, it underwent apoptotic cell death (Segovia et al., 2003). During the process of cell dismantling in D. tertiolecta, the DNA suffered fragmentation and the nucleus disintegrated while the cytoplasm and organelles remained intact. In parallel, both caspase-like activity and expression increased. Despite the identification of metacaspases in vascular plants and protists their roles are not clear yet (Vercammen et al., 2007; Deponte, 2008) and neither is the nature of caspase-like activities in vascular plants (Bonneau et al., 2008). In the majority of cases, measurements of these activities in unicellular species have been carried out by using the classical aspartate-containing caspase substrates. Consequently, the activities measured must be ‘caspase-like’ activities. Although data are scarce, modes of cell death different from apoptosis are adopted by other phytoplanktonic species under stress conditions (Dunn et al., 2002, 2004; Franklin and Berges, 2004). The factors that cause cell death in unicells, their roles, and the details of the apoptotic process remain unclear and, nowadays, the existence and evolution of PCD in unicellular organisms is still controversial (Deponte, 2008). Hence evidence that these alternative, non-apoptotic, PCDs exist has important implications for understanding the fate of unicellular organisms.Microalgae from the genus Dunaliella are among the most ubiquitous eukaryotic organisms in hypersaline environments, and often the major primary producers in salt lakes and in the evaporation ponds of salt works (Borowitzka, 1981). As an adaptation to the strong environmental seasonal changes operating in these systems, they show a remarkable degree of acclimation to salinity, temperature, nitrogen, and irradiance (Ginzburg, 1987). These features make this species a perfect candidate as a model organism.Exposure of D. viridis to environmental stresses that impair cell division such as hyperosmotic shock, UV radiation, heat shock, and nutrient starvation, causes a marked decrease in the phosphorylated form of an extracellular signal-regulated kinase (ERK), known to be involved in cell proliferation and differentiation in mammalian cells, through protein kinase cascades (Jimenez et al., 2007). The authors had formerly demonstrated that ERK phosphorylation was critical for cell division in D. viridis (Jiménez et al., 2004). Cell numbers and viability of D. viridis cultures under these conditions were tested and suffered no changes when exposed to sub-lethal stress conditions.In the present study, evidence is presented that D. viridis has the capacity to undergo different modes of cell death depending on the stress factor and on its intensity. The existence of several biochemical and morphological features typical of each cell death-like morphotype in this microalga is further demonstrated. In all the cases, cell death in D. viridis was linked to an increase in the caspase-like activity DEVDase that matched their accumulation.Materials and methodsCulture conditionsDunaliella viridis Teodoresco was grown in Johnson et al. (1968) medium, at 2 M NaCl (Jiménez et al., 2004) and 25 °C under continuous photosynthetic active radiation (PAR) (400–700 nm) at 150 μmol quanta m−2 s−1, while maintaining gentle stirring and bubbling with filtered air. When cultures reached mid log-phase they were submitted to the following environmental stress treatments: (i) osmotic shock, (ii) UV radiation, (iii) heat shock, (iv) nitrogen starvation, and (v) cells were left to reach late stationary phase (senescence). All experiments were performed in triplicate and each sample was measured by triplicate.Stress treatmentsOsmotic shock:Osmotic stress consisted of an increase in the osmotic pressure of the medium by the addition of NaCl. In acute hypertonic stress experiments, a final concentration of 4 M was considered non-lethal while 5.5 M NaCl was lethal. Cells were sampled at 0, 0.5, 1, and 2 h after the shock.UV radiation (UVR):Cultures were placed in 14 cm diameter Petri dishes transparent to UVR and exposed to either 40 (non-lethal) or 70 mJ cm−2 (lethal) of UVR in the range 200–400 nm using a GS Gene Linker UV chamber (Bio-Rad). The UVR spectral band is divided into three sub-bands corresponding to UV-C (<280 nm), UV-B (280–315 nm), and UV-A (315–400 nm). After UV exposure, cultures were maintained with continuous orbital shaking and 150 μmol m−2 s−1 PAR. Cultures were sampled at 0, 2, 4, and 6 h after UV radiation.Heat shock:For thermal stress, cultures were transferred to 50 ml conical tubes and submerged in a water bath at either 35 °C (non-lethal) or 40 °C (lethal). Continuous light was provided at the same irradiance as the PAR used during culture growth. Cultures were sampled at 0, 1, 2, 3, and 4 h after the heat shock.Nitrogen starvation:Cells were harvested by centrifuging the cultures at 1500 g for 10 min and resuspending them in nitrogen-free growth medium for 7 d. Control cultures were maintained with normal nitrogen-containing medium under the same conditions. Cultures were sampled at 0, 2, 5, and 7 d.Senescence:Cells were left to grow under continuous PAR, at the same irradiance as above, for 12 d until they reached late stationary phase. Sampling took place at 0, 2, 5, 7, 9, and 12 d after inoculation.Transmission electron microscopy (TEM)Cell pellets of the last point of the time-course for each treatment were used for morphological analysis. For that, such pellets were fixed in 2% glutaraldehyde for 1 h, washed and resuspended in 1 ml of 0.01 M phosphate buffer (pH 8). The pellets were post-fixed in 1% buffered osmium tetroxide followed by dehydration in a graded series of ethanol and embedded in epoxy resin. Ultra-thin sections were viewed and photographed on a Philips EM 201 electron microscope. TEM images of all the treatments were examined at ×3750, ×11 750, and ×25 000. Representative pictures (×25 000) under the different stress conditions are presented. Quantification of the cells under the TEM is always difficult, therefore counting of cells showing each different morphotype was carried out by analysing three fields of view (FOV) for each treatment under the smallest magnification.Dead cells stainingEvans Blue is an acidic dye which has the inverse staining properties of a vital stain when determining the survival of plant and planktonic cells. Cells with intact semi-permeable membranes exclude the dye, whereas the dye penetrates and stains dead cells. Therefore, Evans Blue is referred as a mortal stain rather than a vital stain (Crippen and Perrier, 1974). Since this method does not give an indication of the mode of cell death, it should only be used in conjunction with other techniques. Accordingly, the method described by Crippen and Perrier (1974) was applied for marine plankton. Samples were measured in triplicate independent cultures for each treatment. Two aliquots of 1 ml of each culture were taken. One of them was preserved in Lugols iodine for counting the total number of cells. The other, was used for counting the number of Evans Blue-stained cells, by using a final concentration of 1:2000 (w/v) of Evans Blue stock 1% (w/v). Samples were counted under the microscope using a haemocytometer.Confocal laser microscopy (CLM)DAPI is a popular nuclear counterstain for use in multicolour fluorescent techniques. Its blue fluorescence stands out in vivid contrast to green, yellow, or red fluorescent probes of other structures and stains nuclei specifically, with little or no cytoplasmic labelling. DAPI (Invitrogen) was added to a concentration of 1–10 μM and incubated for 5 min at room temperature. A small droplet of algae suspension was placed on a pre-cleaned glass slide (VWR, Aurora, CO) and trapped under a coverslip. Algae were imaged on an inverted laser scanning confocal microscope (LSM 510, Carl Zeiss Inc., Thornwood, NY) through a ×40 1.2 N.A. water immersion objective. DAPI and endogenous chlorophyll fluorescence were simultaneously excited using two-photon excitation at 780 nm from a Ti: sapphire laser (Coherent Inc., Santa Clara, CA). A dichroic filter (HFT KP 700/543) was used to split the fluorescence emission into two channels. DAPI fluorescence was observed through a 435–485 nm band pass filter and chlorophyll fluorescence through a 650–710 nm bandpass filter. More than 100 cells from each condition were examined under a ×40 objective and representative pictures were taken.TUNEL stainingNuclear DNA fragmentation was identified in situ by TUNEL labelling (Gavrieli et al., 1992). Basically, cells were fixed with 0.1% glutaraldehyde, centrifuged for 5 min at 14 000 g at 4 °C. The pellet containing cells was permeabilized by the addition of 0.1% Triton-X100 for 15 min, washed with PBS, and labelled following the manufacturers’ instructions (Apoptag Direct Kit, Chemicon). Samples were then resuspended in PBS, and green fluorescence was observed using an epifluorescence microscope (Nikon, Eclipse E 800, Japan) (excitation 490 nm, emission of 525 nm). Samples were also analysed by using a DAKO cytomation flow cytometer (MoFlo, Beckman Coulter, Fullerton, CA, USA). Counts were triggered using forward scatter (FSC) signals. Positive controls consisted of cells pretreated with 10 μg ml−1 of DNAse I (nickase); for negative controls, distilled water was substituted for the terminal deoxynucleotidyl transferase.DEVDase activityFor estimating the protein concentration of samples, 50 ml of culture were centrifuged at 1500 g for 10 min. The pellets were resuspended in 1 ml of lysis buffer and kept at 4 °C for 1 h. The lysis buffer contained 25 mM Na+ HEPES, 2 mM dithiothreitol (DTT), 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS), 10% sucrose, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1 μM pepstatin A, pH 7.2. Then the homogenate was sonicated and centrifuged at 4 °C at 100 000 g in a Beckman ultracentrifuge using a Ti70 rotor for 1 h. The resultant supernatants were immediately frozen in liquid N2 and stored at –80 °C until use. Lysate protein concentration was measured by the bicinchoninic acid method (Pierce, Rockford, IL), with bovine serum albumin as standard.The DEVDase activity was determined by use of fluorescent substrates as previously described (Edelstein et al., 1999). The DEVDase assay was performed using 20–50 μl of the supernatant obtained as described above containing 50 μg total protein. A caspase assay buffer was added to the supernatant to achieve a total sample volume of 190 μl. The assay buffer consisted of 250 mM K+ HEPES, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, and 0.1% CHAPS, pH 7.4. The solution was pre-incubated for 10 min at 30 °C before the addition of the caspase substrate (Ac-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin (Ac-DEVD-AMC) in 10% DMSO) (Thornberry et al., 1997). Ten μl of the substrate (25 μM final concentration) were added to make a final assay volume of 200 μl. Peptide cleavage was measured over 1 h at 25 °C using a Cytofluor 4000 series fluorescent plate reader (Perseptive Biosystems) at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. An AMC standard curve was determined for each experiment. Caspase activity was expressed in nmol AMC released min−1 of incubation time mg−1 of lysate protein. To check whether or not these activities were real DEVDase, increasing concentrations of the irreversible caspase-3 inhibitor Z-DEVD-FMK (Calbiochem) were added to the homogenates according to Segovia et al. (2003) and samples were pre-incubated for 60 min before running reactions.ImmunodetectionSDS-PAGE electrophoresis in 12% gels run in Tricine buffer, as well as immunedetection and band analysis were carried out according to Capasso et al. (2001). Antibodies against the full-length precursor form of caspase-3 of human origin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). This antibody is highly specific and reacts with the active form and the full-length precursor of caspase-3. Jurkat (human T-cell leukemia) whole cell lysates were used (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as positive controls.Statistical analysisMultiple group comparisons were performed using a one-way analysis of variance (ANOVA). Where significant differences were detected, post-hoc multiple comparisons were made by using the Tukey tests. A P value of <0.05 was considered statistically significant. To quantify the relationship between the variables, the Pearson product moment correlations were performed (considering P <0.05 as significant). Values are expressed as means ±standard deviation. The statistical analyses were carried out by using the STATISTICA 7 statistical package (StatSoft Inc. Tulsa, Oklahoma, USA).ResultsCellular morphologyTEM micrographs of control cells actively growing in light (Fig. 1A) showed that D. viridis has one big cup-shaped chloroplast that occupies more than 50% of the cell volume with the pyrenoid in the centre containing starch granules. The nucleus is located in the apical part of the cell surrounded by a well-defined nuclear membrane. Mitochondria and Golgi apparatus are easily recognized. The flagellar insertion point can also be seen in the cellular apex. Ninety-eight per cent of the cells presented this morphology (Table 1). When cells were injured with sub-lethal doses of the environmental stressors, they did not die and were able to cope with the damage (essentially identical to that described by Jimenez et al., 2007). However, exposure of cells to sudden hyperosmotic shock (5.5 M NaCl) (Fig. 1B) revealed that 55% of the cells had swollen and showed chromatin condensation but also extensive cytoplasmic vacuolation (arrow 1) in the absence of nuclear fragmentation and cellular blebbing (arrow 2). This morphology seems to resemble the paraptotic cell death phenotype. Seventy-six per cent of cells exposed to lethal UV radiation (Fig. 1C) presented symptoms of morphological changes typical of necrosis such as cell swelling, disruption of organelle membranes, condensation of mitochondria, and the formation of cytoplasmic blebs (arrow 1). Nevertheless, other features clearly indicated some apoptotic characteristics such as an intact cell membrane and neat membrane blebbing (arrow 2). This appearance gives the impression of an intermediate cell-death phenotype combining both apoptotic and necrotic features, or aponecrosis. However, chromatin condensation was not clear. Fig. 1D corresponds to cells exposed to acute heat shock (40 °C). Under these conditions, 96% of the cells displayed swelling and nuclear oedema. Mitochondrial rupture and disrupted organelle membranes were also observed (arrow 1). Eventually, altered plasma membranes typical of necrosis were also apparent. However, since some chromatin clusters can be identified as spots within the nucleus, it is suggested that the morphology might be necrotic-like (arrow 2). In nutrient-starvation experiments, cells were deprived of nitrogen for 7 d (Fig. 1E) and 77% of the cells demonstrated margination of the nucleus in the cell, pyknosis, clumping and condensation in the nucleus (arrow 1), and intact cell membrane with blebbing (arrow 2) and the absence of leakage of the intracellular content. However, cytoplasmic consumption was observed as indicated by the disappearance of chloroplast as well as other organelles (arrow 3). This morphotype is very difficult to interpret as it seems to be coincident with autophagic/vacuolar cell death. When control cultures of Dunaliella are allowed to reach a late stationary phase of growth or senescence (Fig. 1F), cell death started to happen and cell density declined sharply. In this case, chromatin aggregation and a certain level of karyorrhexis can be seen (arrow 1) as well as membrane blebs (arrow 2). Cytoplasmic disassembling suggests secondary necrosis after apoptosis. Eighty-three per cent of the cells were in this state.Table 1.Percentage of cells showing each different morphotype (Fig. 1) counted for each environmental stress under the TEM with the smallest magnification used (×3750)Cell morphotype% of cells(A) (normal)98 (8.76)B (paraptotic-like)65 (11.23)C (aponecrotic-like)76 (10.57)D (necrotic-like)96 (2.23)E (autophagic-like)77 (6.16)F (apoptotic-like)83 (5.44)Standard deviation in brackets. (A) Control cells growing in PAR. (B) Cells exposed to sudden hyperosmotic shock (5.5 M NaCl). (C) Cells exposed to lethal UV radiation. (D) Cells submitted to acute heat shock. (E) Cells under nitrogen deprivation. (F) Cells left to reach the late stationary phase of growth, i.e. senescence. n=200–250 (cells treatment−1).Fig. 1.Representative transmission electron micrographs showing the morphological changes in Dunaliella viridis subjected to different lethal stress treatments. C, chloroplast; Fi, flagellar insertion; G, Golgi, M, mitochondria; Mb, plasmalemma; N, nucleus; P, pyrenoid; S, starch. Arrows indicate the alterations indicated in the text. (A) Normal vegetative cells grown in PAR. (B) Cells After 3 h of lethal hyperosmotic shock (5.5 M NaCl). Note chromatin condensation together with extensive cytoplasmic swelling and vacuolation (arrow 1) in the absence of nuclear fragmentation and cellular blebbing (arrow 2). (C) Cells after 4 h of UV radiation. Characteristic cell swelling, organelle membranes are disrupted, mitochondria are condensed and cytoplasmic blebs appear (arrow 1). Cell membrane is intact and shows blebbing (Arrow 2). (D) Cells after 2 h of heat shock. Cells experience nuclear edema and swelling, mitochondrial rupture, disrupted organelle membranes and eventually altered plasma membrane (arrow 1), some chromatin clusters are observed (arrow 2). (E) Cells after 7 d of nitrogen starvation. Note pyknosis (arrow 1) but not margination of chromatin, cell membrane with blebbing (arrow 2), and cytoplasmic consumption (arrow 3). (F) Senescent cells after 12 d. Nucleus suffers margination in the cell, chromatin is condensed (arrow 1), clumped and marginated in the nucleus, while cell membrane is intact with blebbing (arrow 2) and undamaged organelles. Picture augmentation was ×25 000.Dead cellsCounting of Evans Blue-stained cells revealed that under the non-lethal stress the cells did not incorporate the dye. After the first 30 min following osmotic shock around 35% of the cells were already dead. One and 2 h later, more than half of the population was dead (Fig. 2A). The response was different after UV radiation and thermal shock. After the UV stress, the cells also died during the first 4 h of treatment. However, the amount of dead cells increased 2-fold between 4 h and 6 h, reaching almost 80% of the total number of cells (Fig. 2B). The number of dead cells rose to about 3-fold, 2 h after the heat shock treatment and remained constant during the whole period of time (Fig. 2C). Sixty per cent of the cells died suddenly after day 7 under nitrogen deprivation whilst during the previous days only 5% or 10% (days 2 and 5, respectively) of the cells showed blue staining (Fig. 2D). Senescent cells started to die during day 7 and by the end of the sampling period (day 12), around 90% of the population was already dead (Fig. 2E).Fig. 2.Cell death judged by Evans Blue mortal staining of cells under light microscopy with lethal stress treatments. The dashed horizontal line represents live control cells in PAR at t=0. (A) Cells after osmotic shock. (B) Cells after UV radiation. (C) Cells after heat shock. (D) Cells under nitrogen starvation. (E) Culture senescence.DNA condensationD. viridis cells were also analysed by means of CLM using DAPI nuclear staining. Normal cells growing in PAR (Fig. 3A) showed homogeneous DAPI staining well confined to the nuclear area. Two hours after the onset of a lethal osmotic shock (5.5 M NaCl) (Fig. 3B) cells presented irregular DAPI staining and a slight degree of chromatin clumping could be observed. Fig. 3C represents cells exposed for 2 h to lethal UV irradiation. In this case, chromatin aggregation was not only restricted to the nuclear area, blue-stained granules were also observed in the centre of the cell, therefore suggesting some degree of karyolysis. After 2 h of acute heat shock (Fig. 3D) cells demonstrated chromatin aggregation but again, not strictly restricted to the nuclear area. Figure 3E corresponds to cells subjected to 7 d of nitrogen starvation in which well-defined DNA condensation is observed, appearing only in the nuclear zone. Senescent cells also showed clear chromatin condensation in the nucleus (Fig. 3F).Fig. 3.DNA condensation in Dunaliella viridis revealed by DAPI staining and confocal laser microscopy. (A) Control cells in PAR. (B) Cells after osmotic shock (5.5 M NaCl). (C) Cells after 4 h of UV radiation. (D) Cells after 2 h of heat shock. (E) Cells after 7 d of nitrogen starvation. (F) Culture senescence. Horizontal bar is 1 μm. (This figure is available in colour at JXB online.)DNA strand breaksWhile the morphological changes mentioned above occurred, nuclear DNA was concurrently degraded in some of the stress treatments. Free 3′ OH ends of DNA, generated by activation of endonuclease activity in dying cells, were fluorescently labelled with a conventional TUNEL assay (Gavrieli et al., 1992). No labelling was observed either in cells growing in PAR (Fig. 4A) or in the the non-lethal conditions. In the FACS chart, quadrant R1 contain cells with positive TUNEL labelling, and quadrant R3 corresponds to the absence of TUNEL green fluorescence. Red chlorophyll fluorescence comprised both quadrants. After osmotic shock (Fig. 4B) only about 5% of the cells presented green fluorescence and such labelling was very faint. Around 68% of cells showed labelling after UV radiation (Fig. 4C) and a similar pattern, was observed after 3 h of heat shock (Fig. 4D). Results similar to those obtained under osmotic shock were also found under the nitrogen-starvation treatment (Fig. 4E), in which the number of green fluorescent cells was about 28%. A greater number of cells (93%) presented green fluorescence under the senescence treatment (Fig. 4F) after 12 d. Some of the counts in R1 correspond to cellular debris (for instance the dots at 100 both for FITC and red chlorophyll). Positive controls, consisting of cells treated with DNAse showed strong staining, indicative of DNA degradation. Negative controls were analysed by using 9 d senescent cells and by substituting MilliQ water for the TdT enzyme. These cells did not stain (controls data not shown).Fig. 4.DNA fragmentation in Dunaliella tertiolecta after different environmental stresses revealed by TUNEL staining. (A) Control cells in PAR. (B) Cells after 3 h of osmotic shock. (C) Cells after 4 h of UV radiation. (D) Cells after 3 h of heat shock. (E) Cells after 7 d of nitrogen starvation. (F) Senescence after 12 d. Horizontal bar is 20 μm. (This figure is available in colour at JXB online.)DEVDase activityDEVDase activity was detected in D. viridis cell extracts, in response to the different stress conditions tested in this work (Fig. 5). Exposure of cultures of D. viridis to either lethal or sub-lethal hyperosmotic stress by the addition of NaCl (5.5 M and 4 M final concentrations, respectively) produced different results (Fig. 5A). While non-lethal hyperosmotic stress did not result in significant increases in activity, cells exposed to 5.5 M NaCl exhibited a rapid increase in DEVDase activity within 0.5 h after the shock, and resulted in values of 337±67 nmol min−1 mg−1 protein after 2 h (about a 17-fold increase). An increase in activity was also determined for cultures subjected to either lethal doses of UV radiation (70 mJ cm−2; Fig. 5B) or temperature (40 °C; Fig. 5C). In these cases a similar increase in DEVDase activity (100–120 nmol min−1 mg−1 protein) was noted under both treatments in the first few hours with a significant drop in activity at later time points most likely representing cell death. The activity of the enzymes was around half of that obtained under osmotic shock. Unlike hyperosmotic stress, sub-lethal UV and temperature shock did not induce a significant increase of DEVDase activity.Fig. 5.DEVDase activity in Dunaliella viridis following exposure to various stresses. Changes in enzymatic activity were measured as hydrolysis of 7-amino-4-fluoromethyl coumarin-labelled specific substrate DEVD, in all stress treatments. (A) Osmotic shock. (B) UV radiation. (C) Heat shock. Lethal conditions of stress factors are represented by black bars and sub-lethal conditions by white bars. (D) Nitrogen starvation. (E) Senescent cultures. (F) Inhibition of the enzymatic activity by using the irreversible caspase-3-inhibitor Z-DEVD-FMK after 4 h of UV lethal stress. The highest point of DEVDase activity for the rest of the treatments was inhibited with the highest concentration of the inhibitor (200 μM) and any of them showed activity. Statistical differences (P <0.05) during the time-course for the lethal stress are marked with letters. Same letter means no differences.Nitrogen starvation (Fig. 5D) is a much slower process leading to cell death, and a peak of maximal DEVDase activity (17-fold increase compared with control cells) was found 5 d after nitrogen removal from the medium. The maximum DEVDase activity in nitrogen-starved cells at 5 d was more than double compared to treatment with UV or temperature and similar to that caused by osmotic stress (Fig. 5B, C, respectively), reaching average values of 250 nmol min−1 mg−1 protein.Having demonstrated that DEVDase activity was induced in cells exposed to different forms of lethal stress, the influence of natural cell deterioration due to culture ageing was studied. Fig. 5E depicts a dramatic increase in DEVDase activity in senescent cultures of D. viridis at 12 d following initial inoculation (late stationary phase of growth). The activity reached values of more than 350 nmol min−1 mg−1 protein, indicating that activation of this particular caspase-like activity naturally occurs in ageing cultures of D. viridis. DEVDase activity dropped 50% after the addition of 100 μM of the irreversible caspase 3 inhibitor Z-DEVD-FMK (Fig. 5F), and was not inhibited at all by 1 mM PMSF or 1 μM pepstatin A, well-known inhibitors of serine proteases and acid proteases (aspartyl peptidases), respectively.Immunoblot analysisA mammalian anti-caspase 3 antibody was used to detect the caspase-like protein in D. viridis in response to lethal environmental stress (Fig. 6). Such protein was essentially absent in normal steady-state cells; however, a rapid increase in the intensity of the active 32 kDa caspase-like band was detected after the shock treatments. According to the apparent molecular weight and to the migration of the caspase-3 positive control, the band is similar to the caspase-3 of human origin. The presence of the caspase-like band was only slightly detected after the lethal hyperosmotic shock (Fig. 6A) and no bands appeared under the sub-lethal condition. Under UV radiation (Fig. 6B), maximal band intensity occurred 4–6 h after the treatment, while 3–4 h was needed following heat shock (Fig. 6C). It started to accumulate 4 d after the transfer of cells to nitrogen-free medium, reaching maximal band intensity after 7 d (Fig. 6D). Senescence of cultures of D. viridis also induced the appearance of the caspase-like enzyme (Fig. 6E) 9 d after inoculation, showing maximal band intensity after 12 d (late stationary phase of growth). Thus, there was a clear induction of caspase-like enzyme synthesis in response to lethal stress, irrespective of the cell death morphology presented.Fig. 6.Western blot showing cross-reactions of protein extracts from Dunaliella viridis with antibodies raised against human caspase 3. Lanes correspond to the hours or days exposed to each particular stress treatment. (A) Osmotic shock. (B) UV radiation. (C) Heat shock. (D) Nitrogen starvation. (E) Culture senescence.DiscussionApoptosis is an active form of cell death by which individual cells commit suicide. It is a highly controlled and organized process characterized by well-defined morphological changes. The possible role of programmed cell death (PCD) in unicellular organisms has received much attention recently (Cornillon et al., 1994; Ameisen, 1996; Vardi et al., 1999; Frolich and Madeo, 2000; Lewis, 2000; Ning et al., 2002; Segovia et al., 2003). The presence of key components of cell death pathways in some of the earliest-evolved organisms (Berman-Frank et al., 2004) suggests that their origins are truly ancient, and it has been speculated that they may be the result of viral-eukaryote genomic mixing during ancient evolutionary history (Berges and Falkowski, 1998; Segovia et al., 2003; Bidle and Falkowski, 2004). However, the existence and evolution of PCD in unicellular organisms is controversial (Deponte, 2008) and obviously confusing because unlike multicellular organisms, it results in complete loss of the organism. However, apoptosis is not the only way by which cells may die. Evidence shows that other alternative forms of non-apoptotic PCD exist in parallel or in an independent manner with apoptosis (Golstein and Kroemer, 2005; Bredesen, 2007), with important implications for understanding the type of PCD that occurs in unicellular microalgae and how they operate. It is now commonly thought that subtle or dramatic changes in the cell-death phenotype are a direct result of the relative degree of the injury to which the cells are exposed. Supporting this contention, there are data that environmental stimuli can produce different types of cell death depending on the intensity of the stimulus and on ATP availability within the cell, and that classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell death (Papucci et al., 2004).In the present work, both morphological and biochemical approaches have been used to study the forms of cell death in D. viridis. Only senescent cultures showed unambiguous features of apoptosis, followed by secondary necrosis; in turn, the other stress factors provoked different cell-death phenotypes under lethal conditions. In the case of heat shock, necrosis-like was the single cell-death process, whilst UV radiation produced an intermediate cell-death morphotype combining both apoptotic and necrotic features, with aponecrotic-like characteristics. A different pattern was observed under hyperosmotic shock, where cells highly resembled the paraptotic-like phenomenon. Finally, algae exposed to nitrogen starvation showed a different death morphotype, that, acording to the results obtained, suggest that it is a similar mechanism to that of autophagic/vacuolar-like cell death. The ends of the death chain would then correspond to necrosis and apoptosis (Aigner, 2002; Papucci et al., 2004), fitting within the cell-death phenotypes found in D. viridis under heat shock and senescence, respectively. The necrotic-like morphology observed after heat stress implies active cell death (Kitanaka and Kuchino, 1999). Chromatin condensation was not observed, but the cells presented chromatin clusters as well as disorganized spots accompanied by cytoplasmic swelling, also described by Leist and Jäätelä (2001), and TUNEL labelling was positive. This is opposed to the necrotic event per se, which is passive and unprogrammed. Cell disintegration under heat stress seems to be programmed and organized in Chlorella pyrenoidosa (Leu and Hsu, 2004) and shows apoptotic morphology as well as intermediate morphotypes in Chlorella saccharopila (Zuppini et al., 2007). Interestingly, the temperature used in both species was higher than the temperature used for D. viridis, which showed a necrotic-like pattern. In the same context, Symbiodinium sp., the symbiotic dinoflagellate of the sea anemone Aiptasia sp., presented PCD characteristics under experimental bleaching, that shifted between both necrosis and apoptosis, depending on the extent of the stress (Dunn et al., 2002, 2004). When D. viridis cells were left to reach late stationary phase, they died apoptotically-like. The cell-death phenotype shown by these cells was totally coincident with that described for D. tertiolecta under light deprivation (Segovia et al., 2003). This is not surprising as ageing has been widely reported to be one of the conditions by which apoptosis takes place in vascular plants (Fukuda, 1994; Buchanan-Wollaston et al., 2003) and by which phytoplankton blooms disappear (Berges and Falkowski, 1998; Vardi et al., 1999; Ross and Sharples, 2007). In fact, dead cells in natural phytoplankton populations closely resemble senescent cultured cells (Veldhuis et al., 2001).Intermediate features of both apoptosis and necrosis morphotypes or aponecrosis (Formigli et al., 2000) were exhibited when D. viridis was irradiated with UV, including a high degree of TUNEL labelling. UV has been reported to cause apoptotic-like cell death in the unicellular alga Chlamydomonas reinhardtii subjected to high doses of UV (100 J m−2) (Moharikar et al., 2006). However, the difference between D. viridis showing an aponecrotic-like appearance and C. reinhardtii with apoptotic-like morphology might reside in the UV doses received, i.e. in the level of the injury caused to the cells. So, depending on the damage infringed, the cells seem to ‘choose’ how to die.Completely distinct responses took place under nitrogen starvation and hyperosmotic shock. Under nitrogen starvation, cells may die by means of a mechanism similar to autophagic/vacuolar-like cell death as indicated by the disappearance of clear cytoplasm. Nevertheless, the nucleus was not degraded and chromatin was slightly clumped and condensed. Strikingly, a small percentage of cells showed TUNEL positive labelling. Several reports support that autophagic cell death goes through without the generation of DNA strand breaks (Kissova et al., 2006; Bassham, 2007). Nuclear degradation and pyknosis seem not to be universal in all cells undergoing autophagic death (Jones 2000), and this would rather depend on the cell type. Although the positive TUNEL assay was not statistically significant, the reason lying beyond the chromatin cluttering observed with DAPI and TEM, deserves further studies. Yet, the most important fact is that autophagic/vacuolar cell death occurs in nutrient-starved cells, serving as a cellular protective mechanism up-regulated by nutrient starvation (Yue et al., 2003; Shimizu et al., 2004; Levine and Yuan, 2005). In vascular plants, autophagy has been known for some time to be important for nutrient remobilization during sugar and nitrogen starvation and leaf senescence, and recent reports focus on its role in housekeeping functions related to oxidative stress (Bassham, 2007).Finally, when cells were subjected to hyperosmotic shock they showed an analogous appearance to paraptotic-like morphology, i.e. chromatin condensation and also cytoplasmic swelling and vacuolation. Like apoptosis, it seems to be an active process requiring transcription and de novo protein synthesis (Aigner, 2002). Paraptosis has been reported to occur in the unicellular dinoflagellate A. carterae when cultured in darkness and during culture senescence (Franklin and Berges, 2004) concurring with rapid vacuolization, loss of internal structure, intact membranes, and lack of DNA fragmentation. Paraptosis is mediated by mitogen-activated protein kinases (MAPKs) in human cells (Sperandio et al., 2004). Coincidently, authors have demonstrated the presence of mitogen-activated protein (MAP) kinase signalling pathways in D. viridis, and that operation of the p38 and the c-Jun N-terminal kinase (JNK) cascades are crucial for adaptation and survival of this microalga upon hyperosmotic stress (Jiménez et al., 2004), the very stress treatment described in this work under which the cells seem to undertake a paraptotic-like demise. Moreover, hyperosmotic shock, nitrogen starvation, and UV irradiation, impaired cell division and caused a marked decrease in the phospho-ERK levels in D. viridis (Jiménez et al., 2007). These data suggest that, indeed, D. viridis might undergo a paraptotic-like event.Hence, it is now generally accepted that multiple forms of programmed cell death exist and that some of them do not require the activation of caspases (Leist and Jäätelä, 2001; Clarke, 2002). Thus, the term apoptosis in most cases, but not always, is exclusively used for caspase-dependent cell death (Blagosklonny, 2000; Leist and Jäätelä, 2001). On the other hand, while necrosis does not require caspase activation, necrosis-like, being active cell death might or might not require caspase activation (Kitanaka and Kuchino, 1999), and the same is true for aponecrosis (Papucci et al., 2004). However, paraptosis and autophagic/vacuolar cell death traditionally do not call for the participation of caspases (Sperandio, 2000; Jones, 2000; Wyllie and Golstein, 2001; Leist and Jäätelä, 2001). A highly significant increase of DEVDase activity and activation in cell extracts was found after the induction of cell death by means of the environmentally relevant stresses. Caspase-like enzymatic activities and immunodetection were reported for the first time in the unicellular cholorophyte D. tertiolecta (Segovia et al., 2003), other reports regarding metacaspase activity in several microalgal species have also been described (Berman-Frank et al., 2004; Moharikar et al., 2006; Zuppini et al., 2007) and analyses of completed genome sequences of prokaryotic and eukaryotic phytoplankton have revealed the widespread presence of metacaspases in some cyanobacteria, and in unicellular eukaryotic microalgae (Bidle and Falkowski, 2004). However, despite the fact that metacaspases have been reported to operate in an analogous manner to caspases, they are distinct in terms of target site specificity from caspases, at least in vascular plants [their target substrate contains either lysine or arginine at the P1 position, whilst caspase-like enzymes seem to have specificity for aspartate (Vercammen et al., 2004., 2007; Watanabe and Lam, 2005)], and their roles in protists are not obvious yet (Deponte, 2008). DEVDase was not apparent in steady-state D. viridis cells; however, a rapid increase in the intensity of the 32 kDa band was detected after shock treatments. Our data suggest that these activities must be DEVDases because PMSF (an inhibitor of serine proteases) and pepstatin A (an inhibitor of aspartyl peptidases) did not inhibit the activity, whilst the irreversible caspase-3 inhibitor Z-DEVD-FMK inhibited them completely. In this context, it seems clear that environmental treatments induced the activation and the increase in DEVDase activity in unicellular organisms. Yet, the nature of caspase-like proteins in plants is diverse (Bonneau et al., 2008) and a more thorough inhibitor analyses is necessary. Similar results were obtained in other unicellular chlorophytes exposed to UV (Moharikar et al., 2006) and to heat shock (Zuppini et al., 2007). In both cases, antibodies against a mammalian caspase-3 from human origin cross-reacted with a protein of 28–32 kDa and its pattern of expression correlated with the onset of cell death. DEVDase activity and accumulation of the 32 kDa band was observed in cells undergoing all the different cell-death types described in this work, despite the fact that some of those cell-death events, as for example autophagic-like or paraptotic-like deaths, do not necessarily concur with caspase activation. A non-apoptotic role for proapoptotic caspases was previously proposed (Zeuner et al., 1999), indicating that high caspase-3 activity is not exclusive of apoptotic cell death, and in phytoplankton they have been reported to be constitutive and having housekeeping functions (Segovia and Berges, 2005). Thus, growing evidence suggests the participation of caspases in other cellular processes such as development, cell cycle, cell proliferation, and receptor internalization (Algeciras-Schimnich et al., 2002), in addition to their well-characterized role in apoptosis, helping to understand apoptosis-dependent and apoptosis-independent functions of caspases.PCD is difficult to explain as it is a mechanism that offers negative selective pressure. In spite of this, cells have managed to use PCD for several ecologically relevant purposes. One theory suggests that PCD in unicellular organisms would provide for evolutionary advantage for their genome to survive harsh conditions, optimize adaptation of cell numbers to environmental conditions (i.e. nutrient availability), maintain tight regulation of the cell cycle and differentiation (i.e. formation of resistant forms: cysts), enhance defence against pathogens (i.e. viral infection), and to promote and maintain clonality within the population (Welburn et al., 1997; Ameisen, 2000). In this sense, PCD in unicellular organisms may be considered as a safety mechanism for the population. The evidence that alternative, non-apoptotic PCD exists in unicellular organisms has important implications for understanding cell dynamics. That environmental stimuli can produce different types of cell death depending on the intensity of the stimulus, and that classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell death, is also applicable to unicellular organisms. If PCD in phytoplankton is truly the result of a programme activated by environmental factors, then it is important to understand how activation happens and what occurs within the cell in response. The existence of genetically driven cell-death phenomena in phytoplankton indicates that the regulation mechanisms responsible for the dichotomy cell survival/cell death were already present in single-celled organisms 1.2–1.6 billion years ago and preceeded multicellularity.It has been demonstrated in this work, by using morphological and biochemical approaches, that the presence of different cell-death programmes, mediated by DEVDase activity, can occur in a single-celled organism such as the unicellular chlorophyte D. viridis, and that cell death may vary from apoptosis to necrosis, going through different intermediate morphotypes such as aponecrosis, or adopt autophagic/vacuolar or paraptotic-like appearance, depending on the stimulus received. New advances in the elucidation of the molecular pathways leading to cell death in phytoplankton will decipher the genes involved in the responses to different stress factors.This research was supported by grants from the Ministry of Science and Innovation (MICINN) (Spain) to C Jimenez (CGL05-01071 partially funded by FEDER) and to M Segovia (CTM06-09710), and grants from the National Institutes of Health (NIH) to CL Edelstein (DK-56851). 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b/test/dataset/in/resources/corpus/Clean_0B8AA39E3E4530D655DC168C153C6510FEA35523.txt new file mode 100644 index 0000000..b47662c --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B8AA39E3E4530D655DC168C153C6510FEA35523.txt @@ -0,0 +1 @@ + geront Gerontologistgeront The Gerontologist The Gerontologist 0016-9013 1758-5341 Oxford University Press 90810.1093/geront/43.6.908 PRACTICE CONCEPTS The Savvy Caregiver Program: Developing and Testing a Transportable Dementia Family Caregiver Training Program Hepburn Kenneth W. PhD 1 Lewis Marsha PhD, RN 1 Sherman Carey Wexler MA 2 Tornatore Jane PhD 3 Address correspondence to Kenneth W. Hepburn, PhD, School of Nursing, 6-169 Weaver-Densford Hall, University of Minnesota, 308 Harvard St. SE, Minneapolis, MN 55455. E-mail: hepbu001@umn.edu 12 2003 43 6 908 915 10 10 2002 23 7 2002 The Gerontological Society of America 2003 Purpose: This article reports on the development and field testing of the Savvy Caregiver Program, the transformation of a successful, academic-based caregiver psychoeducational program into a self-contained program that can be adopted in other locations.Design and Methods: Program development began with a prototype of a 12-hr course with the aims of introducing family caregivers to the caregiving role, providing them with the knowledge, skills, and attitudes needed to carry out that role, and alerting them to self-care issues. Results from initial field trials dictated a substantial revision of the workshop materials. The next version was field tested in multiple sites in southern rural Minnesota, Colorado, and Alaska. In this expanded testing, participants evaluated the program, and cross-group comparisons were conducted by use of well-established caregiver well-being scales.Results: Virtually all respondents reported increased skill, knowledge, and confidence, and all would recommend the program to others. A preintervention versus postintervention analysis indicates that caregivers' reaction to the overall behavior of the persons for whom they provide care (i.e., “total reaction”), their self-reported burden, and their beliefs about caregiving (emotional enmeshment) changed significantly in directions indicating better caregiver well-being. Implications:Results suggest that it is feasible to translate a research-based caregiver intervention into a packaged program that can be adopted in other settings without the direct involvement of the program initiators. Alzheimer's disease Dissemination Community intervention Translational research hwp-legacy-fpage 908 hwp-legacy-dochead RESEARCH ARTICLE Family members provide the bulk of community-based care for persons with dementia and often do so for long periods of time (National Alliance for Caregiving, 1997; Stone, Cafferata, & Sangl, 1987). This practice provides a substantial cost benefit to society (Arno, Levine, & Memmott, 1999; Max, Webber, & Fox, 1995), but it can exact a heavy toll on caregivers in the form of depression, social isolation, family stress, and other adverse outcomes (Alspaugh, Zarit, Stephens, Townsend, & Greene, 1999; Schulz & Beach, 1999; Schulz, O'Brien, Bookwala, & Fleissner, 1995). The most frequently identified sources of caregiver burden and distress are issues associated with care receiver behavior and efforts to manage that behavior (Gaugler, Davey, Pearlin, & Zarit, 2000; George & Gwyther, 1986; Teri, 1997). Effectively dealing with these issues while maintaining one's own well-being is particularly challenging for family caregivers, given their close emotional ties to the care receiver, the changing nature of their relationship with the ill family member, and their need to take on new caregiving-related tasks for which they are typically unprepared. Recent research suggests that caregivers can be trained to succeed in their new role with less detriment to themselves. In a National Institute for Nursing Research (NINR)-supported randomized controlled trial, we demonstrated that the Minnesota Family Workshop, a 14-hr psychoeducation program that drew on stress mediation theory (Lazarus & Folkman, 1984), was effective in reducing caregiver burden, depression, and reaction to behavior and in helping caregivers achieve a less emotionally enmeshed attitude (Hepburn, Tornatore, Center, & Ostwald, 2001; Ostwald, Hepburn, Caron, Burns, & Mantell, 1999). A number of other psychoeducational and counseling interventions have demonstrated benefits for caregiver well-being, delay of institutionalization of the care recipient, and even care recipient mortality (Brodaty, McGilchrist, Harris, & Peters, 1993; Buckwalter et al., 1999; Gallagher-Thompson & DeVries, 1994; Gendron, Poitras, Dastoor, & Perodeau, 1996; Mittelman, Ferris, Shulman, Steinberg, & Levin, 1996). For large numbers of family caregivers to benefit from successful research-based training programs, the programs must be translatable to other locations and auspices. One might expect to see widespread translation efforts, given the anticipated increase in the number of persons with dementing disorders (Brookmeyer, Gray, & Kawas, 1998). However, reports of such efforts are notably absent from the literature. In this article, we report on the development and testing of an innovative “practice-translation” project, the Savvy Caregiver Program (SCP). Modeled on the Minnesota Family Workshop (MFW), the SCP was designed to offer organizations a turn-key means of implementing our successful, academic-based caregiver psychoeducational program—without the direct involvement of the program initiators. We describe developing the SCP and field testing it with 140 caregivers in rural Minnesota; Denver, Colorado; and Anchorage, Alaska. Challenges to Creating a Transportable Intervention The original MFW program provided 7 weekly 2-hr training sessions to dementia caregivers and other family members. The sessions were designed and conducted by university faculty with disciplinary backgrounds in nursing, family therapy, education, and occupational therapy. A hallmark of the program was its emphasis on training caregivers for the unfamiliar work role into which they had been thrust. The program's curriculum was designed to help caregivers objectively appraise their situation (e.g., assess the progression of the disease in the care receiver and identify feasible caregiving goals) while providing them with the knowledge, skills, and attitudes needed to mediate their stress situation and more effectively carry out the caregiving role they have assumed. We recognized four major challenges in developing a transportable version of the MFW. The first was translating expertise. Teaching activities in the MFW curriculum drew on individual faculty knowledge and skills in the provision of tasks and exercises. The problem we faced was how to codify the teaching activities in a way that did not render them sterile and canned and yet did not require trainers and workshop leaders in other sites to have the same knowledge and skill set as the expert faculty. Moreover, because the program touches on sensitive issues, caregivers often sought additional information, causing segments to run longer than expected. In such circumstances, our faculty were able to draw on their well-developed sense of the program's content and pace to bring closure to the segment and to abridge future segments so as to fit all critical content into the session. Inherent in this problem was the recognition that providing individualized training to SCP workshop leaders would not be feasible. The second challenge was in transporting content. The MFW information base, presented in talks and exercises by four faculty members, had not formally been written. Although these instructors used overheads, they did not have verbatim texts of their talks. MFW participants were given manuals that contained some of the materials covered in the talks, but these also were not comprehensive. The third challenge was in maintaining program integrity. The MFW curriculum and order of presentation remained constant in the randomized trial. The final version had been refined over time in a series of previous field tests and beta versions. In developing the transportable SCP, we sought to preserve the program intact and particularly to ensure that the stress-mediation theoretical framework (Lazarus & Folkman, 1984) would be maintained. We also wanted to ensure that the workshop did not serve as a platform for any idiosyncratic permutations a local trainer might wish to use. The fourth challenge was in obtaining care recipient assessment. During the MFW, a parallel activity program for care recipients offered the opportunity for an occupational therapist to assess the cognitive performance status of the care recipients, using a modified Allen scale (Allen, 1988; Burns, Mortimer, & Merchak, 1994). A key part of the learning activity in the MFW involved participants' viewing a videotape of their care recipient being assessed and then learning the results of the assessment from the occupational therapist. For the SCP, we needed to find a way to teach caregivers to estimate their care recipient's cognitive performance abilities, short of having potential SCP sites hire an occupational therapist to conduct such assessments. Development and Piloting of SCP Curriculum and Materials In 1998, working with a staff member from the Metro Lakes chapter of the Alzheimer's Association of Minnesota, we developed a prototype version of the SCP, designed to overcome the challenges to transportability. The prototype was a 12-hr, self-contained dementia caregiver training program that could be offered in the community by governmental, educational, medical, or social service provider organizations and led by volunteer professionals from the sponsors. Table 1 outlines the key learning objectives of this SCP curriculum. The prototype version of the SCP included the following materials. Trainer's Manual This manual orients the workshop facilitator to the SCP and helps him or her to conduct the program. The manual introduces the facilitator to the intent of the program, emphasizing the tone he or she should strive to maintain (training, in contrast to a support group). The manual specifies the order of talks and exercises in each of six 2-hr sessions. It provides specific learning objectives for each segment of each session, describes how the segment fits into the session and the overall program, and gives detailed directions for conducting each segment. The manual provides the slides and handouts the instructor needs for each segment of the program (also given to the participants). Finally, the manual specifies the “homework” the instructor should encourage participants to engage in after each session. Caregiver's Manual The caregiver's manual provides a written version of the material conveyed in the workshop. Initially, the manual was designed to follow along with the instructional sequence of the program, so the sections were divided by where the material fell in the curriculum. As we discuss in what follows, the structure of this manual changed in the field trial. CD-ROM In a separate project, a CD-ROM, Alzheimer's Caregiving Strategies, was developed at the Minneapolis Veterans Affairs Medical Center (Adelson, Burns, Hepburn, Maddux, & Smith, 1999). This CD-ROM involved faculty from the MFW and drew on its curriculum for content. Equally, it drew on material that was developed at the Minneapolis Geriatric Research, Education, and Clinical Center program. Although the content of the CD-ROM parallels that of the MFW and the SCP, of particular interest is the cognitive performance estimation procedure that is embedded in the program. Using video clips of persons with dementia who are being tested in the performance of an everyday task (making toast), the CD-ROM introduces caregivers to the performance staging system we use and encourages them to estimate where their care recipient falls along the performance stages. Once the prototype material had been developed, two of the authors (K. Hepburn and C. Sherman) led a workshop for five family caregivers, using the materials and following the scripts. The participating caregivers provided detailed and extensive commentary on the program itself and the materials used in it. This pilot resulted in a revision of the curriculum (primarily a reshuffling, but in some cases a revising or a deletion, of materials and segments). It also resulted in an expansion of both the caregiver's and the trainer's manuals. Field Testing Local Field Testing The Metro Lakes chapter of the Alzheimer's Association recruited volunteers to serve as trainers for SCP workshops in their home organizations. These persons were from a variety of backgrounds such as public health nursing, social work, and therapeutic recreation. All had direct service experience with dementia patients and their caregivers. They received approximately 6 hr of training or orientation for the role to familiarize them with the pace and structure of the SCP, to show how certain exercises worked, and to illustrate the linkages between the trainer's and the caregiver's manuals. From the spring through the fall of 1999, three of these volunteer trainers conducted a total of five SCP workshops throughout the metropolitan area of Minneapolis–St. Paul, Minnesota. The volunteers recruited participants for the workshops and found community locations in which to offer the workshops. We observed the trainers conducting three complete workshops and portions of two others, providing us with firsthand experience in seeing trainers who were not invested in the original program attempt to teach from the materials we provided. By agreement with the volunteers, we did not take part in these workshops but observed in silence. We kept notes on what worked and what did not work in the program, particularly assessing the extent to which (a) the trainers appropriately conveyed the intent and meaning of each segment; (b) which segments of the program were effective; (c) the segment achieved our intended or expected result; and (d) the content was (could be) presented in the time available. At the end of each session and then again at the end of each of these programs, we asked program participants to complete evaluation forms. These forms sought the family caregivers' reactions to the individual segments within each session, to the teaching materials (slides and handouts), to the reading and homework assigned for the session, and, where appropriate, to the segments of the CD-ROM assigned for the session. We also sought more general responses and judgments about the trainers' style and delivery and about the value of the segments, sessions, and the program as a whole. The results of this initial field trial informed a substantial revision of the SCP materials. Overall, we tightened and removed elements from the curriculum program to sharpen the program's focus on caregiver role mastery. Acquiring the skills, knowledge, and attitudes important for success in the caregiver role became the central design feature of the program. Because the trainers, typically, did not get through all of the material in the sessions, we eliminated content we determined to be less than central to the purpose of the program. For example, we eliminated an exercise that sought to have participants work through ways of improving family interaction related to caregiving. We retained this content in the manual and the homework assignments, but it was no longer an in-class exercise. We also eliminated or simplified some of the handout materials and slides. Although the caregiver's manual remained essentially intact, we added sections on communication and difficult behaviors and carried out minor revisions to simplify the language and remove jargon from the text. We added text to the trainer's manual to better convey the intent of the segments and to provide fuller direction for their conduct. As a means of ensuring the inclusion of key material, we added directions for how much time training should spend on each segment in each session. Our observations reinforced the importance of using a specified amount of unstructured “debriefing time” at the beginning of each session to address issues and questions raised by the previous session or the homework and to review the results of experiential assignments. Expanded Field Test Next we initiated a field test of the SCP in settings outside the Twin Cities. One site, in southern Minnesota, provided linkages to a number of projects and agencies (including the regional Area Agency on Aging) with whose missions the SCP aligned. The second site, in Denver, Colorado, involved the Centura Health System Senior Clinic and the Rocky Mountain chapter of the Alzheimer's Association, both participants in a National Chronic Care Consortium/Alzheimer's Disease project seeking to implement a caregiver training program as part of the demonstration. The authors provided brief on-site training to the leadership of each site and have since provided problem-solving consultation by phone. The first implementation of this round of field testing of the SCP began in the fall of 2000. In the spring of 2001 we added a third field test site, the Alzheimer's Resource Center of Alaska in Anchorage. This site initiated programs without the use of any face-to-face training; the program leader read through the materials and began programs after only a brief phone consultation with one of the authors (K. Hepburn). This larger, three-site field trial was conducted in the framework of a quasi-experimental design, following review by the University of Minnesota Institutional Review Board. Each participating site recruited participants for SCPs in their area. Participants were informed that the organizations were taking part in a formative study of the program and were asked if they would consent to be part of the study. The organizations sent a list of these participants (and their signed consent forms) to our study center. We then sent participants a brief questionnaire, which we asked them to complete and return (using a stamped envelope we provided) prior to the beginning of the SCP at their site. In addition to basic background questions about the caregiver and care recipient (e.g., age, gender, relationship to care recipient, living situation, education, and income), the questionnaire included five brief, well-established scales. First, the Revised Memory and Behavior Problem Checklist (Teri et al.,1992) includes a caregiver rating of the extent and severity of care recipient behaviors in four domains (memory, behavior, paranoia, and depression); it also includes a caregiver self-assessment of the extent to which she or he is bothered by each of these behaviors. Second, the Caregiver Burden Scale (Zarit, Orr, & Zarit, 1985) seeks to assess the extent to which various facets of caregiving are problematic for caregivers. Third, we used the Center for Epidemiologic Studies-Depression scale (Radloff, 1977) to measure one element of the emotional aspect of caregiving. Fourth, we used a Mastery scale (Pearlin, Mullan, Semple, & Skaff, 1990) to record caregiver self-assessment of caregiving competence. Fifth and finally, we used the Beliefs About Caregiving Scale (Phillips, Rempusheski, & Morrison, 1989) to assess the emotional enmeshment of the caregiver. Approximately 3–4 months after the beginning of each program, we again mailed the same questionnaire to caregivers and asked them to complete and return it. At the end of each SCP workshop, the site leaders distributed evaluation questionnaires that sought caregivers' ratings of the benefits of the program (e.g., improvements in knowledge, confidence, or attitude), their ratings of the effectiveness of the program and its materials, and their suggestions for ways to improve the program. Participants were asked to complete the evaluations and mail them back to the study center (using stamped envelopes we provided). We did no phone or mail follow-up to increase our survey response rate, so we report on considerably fewer participants than total entrants into the program. All data were coded and managed at our study center. Expanded Field Test Results Participant Demographics In the year and a half of field trials held in Alaska, Colorado, and rural Minnesota, 22 separate SCP programs were offered at these sites by a wide variety of professionals—educators, nurses, social workers, recreational therapists, and even geriatricians. A total of 140 family caregivers took part; 40% were spouses of the care recipient, and 47% were adult children; 55% lived with the care recipient. Several paid caregivers (e.g., nursing assistants) also took part, but their data were not included in the analyses reported here, nor were those of participants in the earlier Twin Cities prototype development groups (local field test participants). Participants in the larger field trial were relatively well educated (approximately 70% had more than a high school education) and affluent (modal household income was $30,000–39,000). Almost all (95.7%) were non-Hispanic White. Evaluation Findings As the data in Table 2 indicate, participant response to the program, its materials, and the quality of the workshop trainers was very favorable. Virtually all respondents reported increased skill, knowledge, and confidence as a result of participation, and all would recommend the program to others in similar situations. The caregiver manual was also judged favorably. We note that, although they were still quite positive, the relevance items in Table 2 were scored lower than the other items, particularly in the “strongly agree” category. A closer examination of the data revealed that caregiving daughters tended to rate the program as slightly less relevant to them than did caregiving spouses. Of particular importance, the participants reported a high degree of linkage and concordance between the text in the caregiver manual and the material presented by the trainers. This gives us reassurance that the trainers were faithful to the curriculum of the program, as specified in the trainer's manual. Table 2 also provides caregivers' reports on the effectiveness of the trainers and the training process. These results suggest that the program can be taught effectively by trainers from a wide variety of professional backgrounds. Preintervention Versus Postintervention Findings Table 3 reports on the results of a preintervention versus postintervention t-test analysis comparison of caregivers on the small set of measures we used (taken prior to and 3–4 months after participation in the SCP). It is of note that on three important dimensions—caregivers' reaction to the overall behavior of the persons for whom they provide care (i.e., “total reaction”), their self-reported burden, and their beliefs about caregiving (emotional enmeshment)—significant changes occurred in directions that indicate better caregiver well-being. Discussion The principal finding of this effort is that it is feasible to “translate” a research-based caregiver intervention into a packaged program that can be adopted in other settings. The experience of dissemination suggests that the SCP is flexible enough to be facilitated by a variety of leaders with different areas of expertise. Results indicate that we met the four primary challenges we faced. Participant assessment of strong concordance between classroom activities and material in the caregiver manual indicates that the program is kept intact when other leaders implement it. The other evaluation data indicate that participation yields benefits that are similar to those found in the parent program. This encourages us to conclude that expertise was transported and program integrity was maintained. The participants' evaluation of the caregiver manual leads us to conclude that the transportation of content was successful. Finally, when caregivers used the CD-ROM-based assessment of their care recipient's performance stage, they judged it to be helpful and put the information to use. However, caregivers' access to computers limited the utility of the CD-ROM, a situation we have sought to resolve (see the paragraphs that follow). Thus far, we have learned three main lessons regarding implementation of the SCP. First, leaders have to be committed to the program and willing to lead within the parameters of a training paradigm. Given the SCP's training emphasis, workshop leaders have to be willing to refer caregivers to other resources to address their other needs (e.g., for information or support) and curtail impulses to transform the SCP into a counseling program. Second, leaders have to prepare to lead the program. It takes time to read through both manuals and to understand the flow and timing of the curriculum. It does not work well to try to teach directly from the manual during the sessions. The manual can cue the leaders about the exercises and talks during the sessions, but leaders have to be in possession of the material beforehand. Most facilitators report that the first time through can be challenging but that they gain mastery through this experience. Third, the SCP benefits from strong organizational support. Those leaders working in organizations in which aiding caregivers fits well and centrally with the mission found it easier to recruit caregivers and to offer the SCP as an integral part of their work. Thus, in Denver, where a natural linkage existed with a large clinical operation, the leaders found that caregivers were readily identified by clinicians and referred into the SCP. Since our field testing, SCP has continued to evolve. Using feedback from the field trial trainers, we completely revised the caregiver's manual. The book was restructured so it could be read more as a stand-alone text. The language, reading level, and printing format were carefully examined; the resulting text is less complex, easier to read, visually less dense, and more reader friendly. Although the CD-ROM was well received, it was not used by everyone. Lack of equipment (a CD-ROM-equipped computer) or computer skills made it inaccessible to some participants. At the same time, we heard from trainers that a component of the training related to decision-making skills was difficult to teach. To remedy both problems, we produced a video that incorporates key elements of the CD-ROM (particularly the segment on care recipient assessment), provides a guided lecture on decision making by one of the authors (M. Lewis), and includes a segment illustrating the care recipient involvement strategies that SCP teaches. The new training video has helped to make the program even more portable. Lastly, we found that a greater proportion of caregiving daughters took part in the SCP than in either of our NINR-supported trials. The evaluation responses suggest that we need to attend better to their situation, both in the curriculum and in the program materials. Thus, we are in the process of developing additional material for this caregiver group. Several limitations of this report should be acknowledged. Approximately 37% of those taking part in the SCP trials did not provide follow-up data. Our examination of the nonrespondent demographic data does not suggest any differences between the two groups. The positive findings we are able to report stem from a within-group, preintervention versus postintervention analysis. A randomized trial of the SCP is currently underway in sites in Alaska, Colorado, and Mississippi; this trial will provide a more rigorous test of SCP effectiveness. Despite these limitations, the SCP has demonstrated that it can offer a wide range of organizations the opportunity to provide dementia caregivers with training from which the caregivers themselves claim to experience the same kinds of benefits as those demonstrated in the randomized trial of the MFW. The SCP offers organizations a turn-key means to add to the repertoire of assistance they can provide to this important and growing group of family caregivers. This research was supported by grants from the UCare Minnesota Foundation (1009-98), the National Alzheimer's Association (IIRG-99-1591), and the National Institute for Nursing Research (NR02981). 1 School of Nursing, University of Minnesota, Minneapolis. 2 Department of Family Practice and Community Health, University of Minnesota, Minneapolis. 3 Screen, Inc., 8017½ 8th Avenue S., Seattle, WA. Decision Editor: Eleanor S. McConnell, RN, PhD Table 1. Caregiving Learning Objectives in the Savvy Caregiver Curriculum. Objective Description Acknowledge the disease Understand and come to grips with the disease that is affecting the person. Recognize it is not personal—it is the disease and not the person that is causing difficulties. Make the cognitive shift Develop a strategic sense of what cognitive losses are occurring and how caregiver behavior has to adapt to these as they take place and progress. Develop emotional tolerance Recognize the central role of confusion in dementia and how it contributes to troubling behaviors (e.g., shadowing or repetitive questioning). Appreciate the care recipient's need for emotional stability in the face of confusion. Accept the caregiver's role of providing calm and stability. Take control Understand that dementia gradually erodes autonomy. Appreciate the social and emotional difficulties involved in taking control of another adult (thinking of the other as somehow not equal). Recognize the need to take control and be willing and able to do so. Establish a realistic care goal Accept that striving to ensure as good a quality of life as possible is the most realistic goal for caregiving. Recognize that rehabilitation, restoration, or retardation of dis-ease progress are not feasible goals and let go of them, when applicable. Gauge the care recipient's capabilities Be able, using an occupational-therapy-based staging system, to estimate the care recipient's capacity for involvement in tasks and activities (kind, complexity, duration, etc.). Design opportunities for satisfying occupation Be able, based on the estimate of capability, to design tasks or activities in which the person can become engaged and which she or he will enjoy. Be able to communicate effectively with the person to promote and maintain involvement. Become a sleuth Adopt a problem-solving approach to caregiving, one that involves being able to stand back from the situation (emotionally), create hypotheses about causes of behavior, formulate and try out responses, observe and learn from results, and repeat the sequence as needed. Table 2. Results of Participant Evaluation of the Savvy Caregiver Program. Evaluation Response Category Strongly Agree (5) Agree (4) Total (4 + 5) Overall response to SCP (%) More knowledgeable as a caregiver 86.4 11.4 97.8 Learned useful caregiving strategies 85.2 11.4 95.6 Feel more confident as a caregiver 68.2 27.3 95.5 Have more caregiving skills 75.0 19.3 94.3 Program content relevant to caregiving 72.7 18.2 90.9 Would recommend to other caregivers 95.5 4.5 100.0 Evaluation of SCP caregiver manual (%) Clear and easy to understand 75.0 19.3 94.3 Information relevant to caregiving 68.2 22.7 90.9 Information helps in caregiving 75.0 18.2 93.2 Information ties in with sessions 84.1 12.5 96.6 Expect to use manual in the future 68.2 22.7 90.9 Evaluation of workshop trainers (%) Information (talks or exercises) clear 86.4 9.1 95.5 Trainers presented clearly and well 85.2 11.4 96.6 Trainers encouraged discussion 92.0 6.8 98.8 Class time used effectively 78.4 15.9 94.3 Trainers made information relevant 88.5 10.3 98.8 Note: For all evaluation response categories, N = 88. Table 3. t-Test Comparison of Caregiver Measures Taken Before and After Participation in the SCP. Measurea Score p value n Before After Reaction to care recipient behavior (−) 8.56 7.29 .075 56 Reaction to care recipient paranoia (−) 4.62 3.65 .104 56 Reaction to care recipient depression (−) 9.71 7.47 .023 57 Total reaction (−) 31.02 26.12 .020 56 Zarit burden (−) 40.40 38.13 .047 63 CES–Depression scale (−) 16.33 15.10 .186 63 Master scale—caregiving competence (+) 2.48 2.47 .941 62 Beliefs about caregiving scale (+) 47.75 49.85 .003 60 Notes: Time after participation in the program was 3–4 months. SCP = Savvy Caregiver Program; CES = Center for Epidemiologic Studies. aThe plus or minus sign following each measure indicates the direction of change sought through the intervention, thus a decrease in the reaction to behavior score and an increase in the mastery score would both be good outcomes. References Adelson, R., Burns, T., Hepburn, K., Maddux, M., Smith, S, 1999;. Alzheimer's caregiving strategies. Minneapolis, MN: Veterans Affairs Education Service. Allen, C. K., 1988;. Occupational therapy: Functional assessment of the severity of mental disorders. Hospital and Community Psychiatry,. 39:140-142. Alspaugh, M. E. L., Zarit, S. H., Stephens, M. A. P., Townsend, A. L., Greene, R., 1999;. Longitudinal patterns of risk for depression in dementia caregivers: Objective and subjective primary stress as predictors. Psychology and Aging,. 14:34-43. Arno, P. S., Levine, C., Memmott, M. M., 1999;. The economic value of informal caregiving. Health Affairs,. 18:182-188. Brodaty, H., McGilchrist, C., Harris, L., Peters, K. E., 1993;. Time until institutionalization and death in patients with dementia: Role of caregiver training and risk factors. Archives of Neurology,. 50:643-650. Brookmeyer, R., Gray, S., Kawas, C., ;. Projections of Alzheimer's disease in the United States and the public health impact of delaying disease onset. American Journal of Public Health,. 88:1337-1342. Buckwalter, K. C., Gerdner, L., Kohout, F., Hall, G. R., Kelly, A., Richards, B., et al 1999;. Nursing intervention to decrease depression in family caregivers of persons with dementia. Archives of Psychiatric Nursing,. 13:80-88. Burns, T., Mortimer, J. A., Merchak, P., 1994;. Cognitive performance test: A new approach to functional assessment in Alzheimer's disease. Journal of Geriatric Psychiatry and Neurology,. 7:46-54. Gallagher-Thompson, D., DeVries, H. M., 1994;. “Coping with frustration” classes: Development and preliminary outcomes with women who care for relatives with dementia. The Gerontologist,. 34:548-552. Gaugler, J. E., Davey, A., Pearlin, L. I., Zarit, S. H., 2000;. Modeling caregiver adaptation over time: The longitudinal impact of behavior problems. Psychology and Aging,. 15:437-450. Gendron, C., Poitras, L., Dastoor, D. P., Perodeau, G., 1996;. Cognitive-behavior group intervention for spousal caregivers: Findings and clinical considerations. Clinical Gerontologist,. 17:3-19. George, L. K., Gwyther, L. P., 1986;. Caregiver well-being: A multidimensional examination of family caregivers of demented adults. The Gerontologist,. 26:253-259. Hepburn, K., Tornatore, J., Center, B., Ostwald, S. W., 2001;. Dementia family caregiver training: Affecting beliefs about caregiving and caregiver outcomes. Journal of the American Geriatrics Society,. 49:450-457. Lazarus, R. S., Folkman, S., 1984;. Coping and adaptation. In W. D. Gentry (Ed.), The handbook of behavioral medicine (pp. 282–325). New York: Guilford. Max, W., Webber, P., Fox, P., 1995;. Alzheimer's disease: The unpaid burden of caring. Journal of Aging and Health,. 7:179-199. Mittelman, M. S., Ferris, S. H., Shulman, E., Steinberg, G., Levin, B., 1996;. A family intervention to delay nursing home placement of patients with Alzheimer disease: A randomized controlled trial. Journal of the American Medical Association,. 276:1725-1731. National Alliance for Caregiving, American Association of Retired Persons., 1997;. Family caregiving in the U.S.: Findings from a national survey (Final Report). Bethesda, MD: Author. Ostwald, S. K., Hepburn, K. W., Caron, W., Burns, T., Mantell, R., 1999;. Reducing caregiver burden: A randomized psychoeducational intervention for caregivers of persons with dementia. The Gerontologist,. 39:299-309. Pearlin, L. I., Mullan, J. T, Semple, S. J., Skaff, M. M., 1990;. Caregiving and the stress process: An overview of concepts and their measures. The Gerontologist,. 30:583-594. Phillips, L. R., Rempusheski, V. F., Morrison, E., 1989;. Developing and testing the Beliefs about Caregiving Scale. Research in Nursing and Health,. 12:207-220. Radloff, L. S., 1977;. The CES-D scale: A self-report depression scale for research in the general population. Applied Psychological Measurement,. 1:385-401. Schulz, R., Beach, S. R., 1999;. Caregiving as a risk factor for mortality: The caregiver health effects study. Journal of the American Medical Association,. 282:2215-2219. Schulz, R., O'Brien, A. T., Bookwala, J., Fleissner, K., 1995;. Psychiatric and physical morbidity effects of dementia caregiving: Prevalence, correlates, and causes. The Gerontologist,. 35:771-791. Stone, R., Cafferata, G. L., Sangl, J., 1987;. Caregivers of the frail elderly: A national profile. The Gerontologist,. 27:616-626. Teri, L., 1997;. Behavior and caregiver burden: Behavioral problems in patients with Alzheimer disease and its association with caregiver distress. Alzheimer Disease and Associated Disorders,. 11:S35-S38. Teri, L., Truax, P., Logsdon, R., Uomoto, J., Zarit, S., Vitaliano, P. O., 1992;. Assessment of behavioral problems in dementia: The revised memory and behavior problems checklist. Psychology and Aging,. 7:622-631. Zarit, S., Orr, N., Zarit, J., 1985;. The hidden victims of Alzheimer's disease: Families under stress. New York: New York University Press. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B8CE3F28C7ECF202C365FB46B0D7DF0825FD78C.txt b/test/dataset/in/resources/corpus/Clean_0B8CE3F28C7ECF202C365FB46B0D7DF0825FD78C.txt new file mode 100644 index 0000000..94fa14f --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B8CE3F28C7ECF202C365FB46B0D7DF0825FD78C.txt @@ -0,0 +1 @@ +]>AGG997S0167-4943(99)00048-510.1016/S0167-4943(99)00048-5Elsevier Science Ireland LtdFig. 1(a) The structure of the hip protector. (b) The appearance of the patient with hip protector.Fig. 2Profile of fall (orientation and time).Table 1Summary table of falls and fractures dataControlTreatmentCombinedFracture groupFalls10119129232Residents3140718aAverage falls per resident3.264.784.114Fractures639aOne resident had two fractures (one on each side).Effectiveness and acceptability of a newly designed hip protector: a pilot studyDaniel KChana*d.chan@unsw.edu.auGaryHillierbMichelleCoorebRosemaryCookebRebeccaMonkbJanetteMillscWai THungdaDepartment of Geriatric Medicine, Prince of Wales Hospital, High Street, Randwick 2031, NSW, AustraliabAged Care Assessment Team, Community Health Services, Orange, NSW, AustraliacDepartment of Orthopaedics, Prince of Wales Hospital, High St., Randwick 2031, NSW, AustraliadFAR, School of Mathematical Sciences, University of Technology, Broadway 2007, NSW, Australia*Corresponding author. Tel.: +61-2-93824242; fax: +61-2-93824241AbstractHip fracture has a significant economic and personal cost, involving hospital admission and functional impairment for elderly people. To assess the benefit of using a newly designed hip protector (new material and new design) to prevent fracture in a realistic setting, a randomised intervention-control design was used to trial the effectiveness of pads worn by high falls risk residents (n=71) in nursing home for 9 months. 40 residents were in the intervention group and 31 were in the control group. A profile of falls, including time of day, and orientation was obtained to demonstrate the potential effectiveness of the protectors for injury prevention. Acceptance of the hip protector was also surveyed amongst nursing home staff and residents. One hundred and one falls and six fractures occurred in the control group. In contrast, one hundred and ninety one falls and three fractures occurred in the hip protector (pads) group. The three fractures in the protector wearing group occurred when pads were not in place. This was extrapolated as 1 in every 16.8 falls and 1 in every 63.7 falls resulting in fracture in the two groups, respectively. The relative risk of fracture was 0.264 (95% CI=0.073–0.959) when the fracture incidence rate in the intervention group (three fractures per 191 falls) was compared to the control group (six fractures per 101 falls). This is a statistically significant result and implies that this newly designed hip protector is effective in preventing hip fracture. The majority of falls occurred during the day, which was when protectors were worn in this study, but the data on orientation was incomplete, with direction unknown in 74% of falls. Compliance was an issue, which was interpreted as only 50.3% of falls recorded with protectors in place. Dementia was identified as the explanation for this as the pads were often removed by these residents who comprised the majority of participants. Perception of low risk was the primary barrier to residents accepting the intervention. Comfort of protectors was not a significant concern for staff or residents, and only staff described appearance as an issue. In conclusion, the newly designed hip protector is protective against fractures in a realistic setting. Compliance and acceptance of the protectors will ultimately determine the viability of this prophylaxis.KeywordsNovel hip protectorHip fractureDementia1IntroductionFracture of the hip has been recognised as a significant problem in the elderly population from both an economic perspective, and in terms of the personal impact on the individual experiencing the injury. The impairment in function associated with the fracture, and surgical intervention alters the lives of individuals, increasing the likelihood of permanent institutionalisation five times for previously community dwelling elderly (Cumming et al., 1996). Pre-fracture levels of physical functioning are not regained by more than half the injured patients, and fractured neck of femur increases mortality in the elderly significantly (Butler et al., 1996; Fox et al., 1996).In the majority (60–90%) of fractures direct trauma is the aetiology (Cummings and Nevitt, 1989), which indicates the importance of addressing falls in the elderly. However, this is a complex issue as most falls are multifactorial in cause, including, lower limb weakness, gait instability, vision, cognitive and functional impairments as well as the effects of medication (Rubenstein et al., 1994). External factors have been found to be unreliable determinants of risk, as multiple hazards have been implicated in fractures (Clemson et al., 1996). Therefore, the value of a secondary prophylaxis is suggested as a fracture prevention, to mediate the impact of the fall, when it inevitably occurs.Hip fracture risk is known to be reduced by a larger body mass index as increased soft tissue appears to absorb the impact of a fall and protect the femur (Hayes et al., 1991; Robinovitch et al., 1995). Even in a direct fall onto the hip in the elderly, research has shown that natural padding is insufficient to attenuate the force, and other mechanisms such as reflex contraction of the quadriceps protect the hip fracture (Robinovitch et al., 1995).As a solution to the prevention of fracture, several clinical trials have demonstrated that external hip protector is effective in protecting the hip, as no fractures occurred in subjects while wearing the appliances (Lauritzen et al., 1993; Tracey et al., 1998). However, the compliance rate is only about 50% (Lauritzen et al., 1993; Tracey et al., 1998) and it drops to about 30% (Tracey et al., 1998) in the long term. The design of these hip protectors are such that they have to be worn with underpants to cover a small area over the greater trochanter. Any movement of the protector may mean that the covering position may be shifted. Therefore, the hip protectors are quite tightly fitted and this may explain a significant proportion of people feeling the discomfort (Tracey et al., 1998). Furthermore, the polypropylene material used is quite hard which may add to the discomfort.We therefore designed a new hip protector using softer material, which is fitted to the inner surfaces of trousers or tracksuit pants. The hip protectors also cover the greater trochanters but have the propensity to allow for some movement, and hence may cause less discomfort. Our aims are to find out both the effectiveness and acceptability of this newly designed hip protector.2Patient and method2.1ParticipantsSubjects were drawn from nine nursing homes and the criteria for inclusion was that the nursing home staff identified the residents as high falls risk. This was not on the basis of particular diagnosis, or any formal evaluation of falls risk, but the perception of the staff themselves. The subjects or the person responsible was then asked to sign consent to participate. Therefore, the purposes of the study were clear to participants and/or the guardians, and involvement was voluntary. Random assignment of subjects was achieved in most nursing homes with some participants designated as control and some to wear the protectors. The current results are based on 71 participants (control=31, treatment=40) on whom data has been collected after 9 months.2.2Hip protectorThe hip protectors were designed to absorb the impact in the upper femur, hip region, in particular to cover the greater trochanter in a fall. They are made of pads as shown in Fig. 1. The pads are worn in pockets sewn into the inner surfaces of tracksuit pants or trousers. Pads are 2×3 rows of cube shapes with dimensions 6 (width)×7 (length)×2.5 (depth) cm in each cube. The material is made from EVA foam. This material is waterproof and the shock absorbency is demonstrated through the successful use in Tai Kwan Do matting. The mould used is also jointed allowing flexibility and comfort. Therefore, this new hip protector is different from that by Lauritzen (Lauritzen et al., 1993) in both material and design.2.3Measurement2.3.1Effectiveness of protectorsA simple form was designed for inclusion in residents files which staff were asked to complete for every fall. Important information, which would indicate the effectiveness of the protector, was injuries, orientation of the fall, and the time of day, as protectors were not worn at night in bed in this current study.2.3.2Acceptance of protectorsCompliance was extrapolated from the percentage of falls recorded for which protectors were worn.Separate surveys were designed for staff and residents, with closed responses to enhance acceptability to nursing staff in terms of time constraints. However, the survey was administered verbally and response options were not designed to quantify responses, but facilitate those actually using the protectors to articulate their opinion of their acceptability.2.4Data analysisThe calculation of the fracture incidence rate ratio (or relative risk) was as follows:three fractures per 191 falls (hip pad group)/six fractures per 101 falls (control group).This is based on the test-based method by Sahai and Khurshid (Sahai and Khurshid, 1996).3Results3.1Effectiveness of protectorsFifty of the 71 subjects had fallen with a total 292 falls recorded. Nine fractures occurred with none of the eight residents wearing protectors at the time of injury (one resident fractured both hips 1 month apart). 101 of the falls and six of the fractures were recorded in the control group. This may be interpreted as a risk of 1 in every 16.8 falls resulting in a hip fracture. The protector group in comparison accounted for three of the fractures and 191 of the falls with an extrapolated fracture risk of one in every 63.7 falls. The relative risk of fractures in the hip protector group as compared with the control group was 0.264 (95% CI=0.073–0.959). The average falls for control, protector, total and fracture groups is shown in Table 1.The orientation of falls for those resulting in fractures was that three were unknown, one was backwards, and five were sideways. All occurred during the day.In the total study group 81.8% of falls occurred during the day (6:00–22:00 h) and 9.6% were sideways. As 74% of falls were of unknown orientation, it is possible that the protectors may have been more useful in mediating impact than indicated. A profile of falls (orientation and time) is shown in Fig. 2.3.2Acceptance of protectors3.2.1ComplianceIn this study, compliance was defined as the percentage of falls recorded for which hip protectors were worn in the treatment group. The compliance of the participants was 50.3%.In the staff survey dementia was indicated as the reason for non-compliance. 64.8% of the total 71 participants have been identified as having dementia.3.2.2Perceptions of protectorsThe surveys were administered to seven staff and four residents. As most participants were diagnosed with dementia this limits the reliable resident sample. Three of the residents surveyed had been nominated as suitable for the study, but declined to participate. Comments from staff and residents noted informally have also been documented.3.2.3ComfortOnly one staff member noted concern about the comfort suggesting the seams on adapted underwear had rubbed. Also only one resident had serious concerns regarding the protectors highlighting the bulkiness and awkwardness, especially when continence pads are already worn. The tracksuit pants were also an issue for hot weather, so alternative clothing fitted with the protectors was preferred.3.2.4CostSince a pair of protectors cost only $10.00 AUD ($30 with tracksuit pants), no one was unable to afford it.3.2.5AppearanceFifty seven percent of staff described this as concern. However, no residents indicated it was an issue. The obvious bulkiness of the protectors was noted, and one staff member commented public dignity was compromised, such as at a doctors appointment.3.2.6Barriers to acceptanceTwo of the staff indicated they would have serious concerns regarding the use of protectors in resident management. The reasons suggested included, hygiene and the inconvenience of putting them on, especially when resident non-compliance was an issue. Enthusiasm for the protectors appeared to have a direct relationship with perceived effectiveness, as one staff member was sufficiently convinced by their own observation of the protectors, that they would like to continue their use beyond the study. Another respondent indicated that implementation of the protectors would ultimately depend on resident feelings.Residents explanations for not using the protectors centred on a perceived lack of personal risk. This included the belief in the two respondents already post fracture, simply that as they had experienced one fractured hip, they were now safe, and an indication from the doctor that the first fracture was pathological, therefore the resident felt external protection would be of no benefit in preventing fracture. Another resident noted that she had ridden horses for years, and would have needed them more then, as would other people in higher risk occupations, than simply being in danger of falling. Independence to manage their own risk was also an issue as one resident felt she was now too old to care, and another asserted the right to refuse intervention and experience the consequences of their own decision. The one resident surveyed who was wearing the protectors queried the position of the protectors for effectiveness, but was otherwise satisfied with the intervention. His daughter actually commented on the peace of mind provided by knowing he was wearing the protectors.4DiscussionUltimately the aim of this study was to implement protectors in a realistic setting and record data as to their effectiveness. This data has supported the value of pads worn to prevent fracture of the hip as no fractures occurred while they were in place. Furthermore, the reduction of relative risk of hip fracture in this new hip protector (RR=0.264) is comparable or marginally better than the old design (RR=0.44) by Lauritzen (Lauritzen et al., 1993). As most falls occurred during the day, this suggested that current wearing regime should be sufficient, it is the actual compliance with wearing the protectors that is problemsome. 50.3% is a low compliance, even allowing for the minority of falls occurring at night when the protectors were not worn (i.e. compliance rate is actually higher if night time falls are excluded from the calculation). However, this compliance rate is consistent with other designs of hip protectors (Lauritzen et al., 1993; Tracey et al., 1998). Dementia is the most significant factor in this study regarding compliance, which is an area that perhaps should be addressed in the design of the pads so they are not able to be removed easily and are less obvious to the person as their presence appears to irritate these residents.Unfortunately the low compliance could also reduce the strength of the fracture risk findings comparing the treatment and control groups. Although compliance was related to dementia in this research, the surveys regarding acceptance have gauged the importance of attitudes to the protectors in actually implementing them as an intervention tool.As for the perception and acceptance survey, the sample used was small, especially of residents. However, the diversity of responses even in this group suggests the difficulty of assessing attitudes and marketing the protectors to the consumer. In terms of staff it is interesting that they accorded more concern regarding the appearance of the protectors than the residents. It is the actual perception and understanding of fracture risk that appears to be the central barrier to acceptance amongst residents. This supports the value of education to the elderly who are high risk, such as frequent fallers or post fracture clients, in understanding the risk not simply of falling, but of fracture (Cameron and Quine, 1994). Therefore, the preferred management of the problem of fractured neck of femur would include not just falls prevention but fracture protection.The findings of this study were limited to a nursing home population where protectors have had been implemented and monitored by staff. Therefore, issues such as ease of use for the client have not been explored which could be a significant difficulty for community living elderly with mobility impairments. A similar study using community living individuals would be appropriate, not to validate the actual effectiveness of protectors but their practicality.In terms of the participant group selected as suitable for the research the variables were not monitored by the researcher, it was based on the subjective opinion of staff. However, the comparison in the average falls per year per bed of 1.5 in institutions to the 4.11 recorded for this group in a 9-month period, suggests that staff are accurate in determining high falls risk residents. Another point of note is the higher rate of fall (4.78 falls per bed per 9 months) in treatment group compared to controls (3.26) may be due to willingness of staff to allow patients to ambulate after they are put on hip protectors (rather than restricting them).The hip fracture per fall rate in the control group is 5.94% (six fractures in 101 falls). This is considerably higher than fracture rates of elderly in the community but is in accord with findings from Butler et al. (Butler et al., 1996) (the risk of hip fracture in institutions is 101.5 times those living in private homes).Since a lot of the fallers are demented people whose rehabilitation and functional outcome after a fracture hip is poor, this raises the ethical question as to their rights to choose or deny wearing the hip protectors. As our study actually includes proxy consent from carers or guardians, this raises the important question as to whether the important information about hip protectors should be focused on carers or guardians, not just elderly fallers.The price of a pair of protectors is only $10.00 AUD, making it possible to be affordable to nearly all pensioners. Cost was a concern in some other protectors (Cameron and Quine, 1994).5ConclusionThe descriptive data about falls and fractures in a realistic trial in nursing homes indicates the viability of protectors as a valid instrument in the prevention of fractured hips as none occurred while the device was in place. However, further information regarding the profile of falls, especially direction of fall, would increase the evidence for the potential effectiveness of this device. Potential barriers to implementation include compliance, and attitudes to perceived risk.AcknowledgementsThe authors express their gratitude and thanks to Calare Nursing Home, Orange; Cudal Memorial Hospital; Eugowra Memorial Hospital; Mater Misericordiae Nursing Home, Forbes; Moyne Eventide Home, Canowindra; Niola Nursing Home, Parkes; Wontama Nursing Home, Orange; Sir Joseph Banks Nursing Home, Botany; Camelot Nursing Home, Maroubra for their co-operation and help.ReferencesButler et al., 1996M.ButlerR.NortonL.TrevorA.ChengA.J.CampbellThe risk of hip fracture in older people from nursing homes and institutionsAge Ageing251996381385Cameron and Quine, 1994I.CameronS.QuineExternal hip protectors: likely non-compliance among high risk elderly people living in the communityArch. Gerentol. 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Soc.161991139Lauritzen et al., 1993J.B.LauritzenM.M.PetersonB.LundEffect of external hip protectors on hip fracturesLancet34119931113Robinovitch et al., 1995S.N.RobinovitchT.A.McMahonW.C.HayesForce attenuation in trochanteric soft tissues during impact from a fallJ. Orthop. Res.1319955662Rubenstein et al., 1994L.Z.RubensteinK.R.JosephsonA.S.RobbinsFalls in the nursing homeAnn. Intern. Med.1211994442451Sahai and Khurshid, 1996H.SahaiA.KhurshidStatistics in Epidemiology: Methods, Techniques and Applications1996CRC PressBoca Raton, FL171174Tracey et al., 1998M.TraceyA.VillarP.HillH.InskipP.ThompsonC.CooperWill elderly rest home residents wear hip protectorsAge Ageing271998195198 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B937F9461CCAEEA575B6A276E7894E53C3759CC.txt b/test/dataset/in/resources/corpus/Clean_0B937F9461CCAEEA575B6A276E7894E53C3759CC.txt new file mode 100644 index 0000000..255757c --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B937F9461CCAEEA575B6A276E7894E53C3759CC.txt @@ -0,0 +1 @@ +gerontgerontThe Gerontologist1758-53410016-9013Oxford University Press10.1093/geront/gnp031INEQUALITY AND AGEISMManaging Age Discrimination: An Examination of the Techniques Used When Seeking EmploymentBergerEllie D.PhD122Department of Sociology, Nipissing University, North Bay, Ontario, Canada1Address correspondence to Ellie D. Berger, PhD, Department of Sociology, Nipissing University, 100 College Drive, North Bay, Ontario, Canada P1B 8L7. E-mail: ellieb@nipissingu.caDecision Editor: Nancy Schoenberg, PhD62009174200949331733221120071382008© The Author 2009. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2009Purpose: This article examines the age-related management techniques used by older workers in their search for employment. Design and Methods: Data are drawn from interviews with individuals aged 45–65 years (N = 30). Results: Findings indicate that participants develop “counteractions” and “concealments” to manage perceived age discrimination. Individuals counteract employers’ ageist stereotypes by maintaining their skills and changing their work-related expectations and conceal age by altering their résumés, physical appearance, and language used. Implications: This research suggests that there is a need to reexamine the hiring practices of employers and to improve legislation in relation to their accountability.AgeismHiring practicesManagement techniquesOlder workersUnemploymentIt is widely recognized that the Canadian population is aging, and as a result, there are a wide number of social and economic changes expected to occur in the coming years. In particular, changes in the domain of work will be considerable as individuals aged 45–65 years are estimated to represent close to half of the working-age population by the year 2015 (Forum of Labour Market Ministers, 2002). Mandatory retirement was eliminated in Ontario in 2006, and thus, increasing attention has been paid to the aging of the workforce by both lay and academic audiences. In fact, the Canadian government has been trying to encourage older workers to remain in the labor force longer through various policy changes (Policy Research Initiative, 2005). Despite these changes, the average age of retirement in Canada has been decreasing from 64.9 years in 1976 to 61.4 years in 2005 (Conference Board of Canada, 2006).Further, industries in Canada, such as manufacturing, have had large layoffs in recent years and switched their focus to more contract and part-time work, which leaves older workers with little protection under employment standards law and employment insurance (McMullin, Cooke, & Downie, 2004). In fact, it has been recognized that recent changes in the Canadian economy have led to the need for workers to be geographically, occupationally, and industrially mobile as well as invest more heavily in job searches, training, and education (Secretariat for the Expert Panel on Older Workers, 2007). Thus, displaced older workers face the most challenges due to their lack of mobility, education, and training and due to the existence of age discrimination. These factors cause older Canadian workers to have a reduced chance of finding reemployment once unemployed. Further, older workers take considerably longer than younger ones to find work once unemployed due in part to the existence of age discrimination (Ontario Human Rights Commission, 2000; Walker, 2002). In fact, as age increases, so does the average duration of unemployment for working-age Canadians. Individuals aged 15–24 years spend an average of 7.9 weeks unemployed, those aged 25–54 years spend 17.9 weeks unemployed, and 55- to 64-year-olds spend an average of 22.8 weeks unemployed (Statistics Canada, 2007). This finding is similar to that in the United States, where individuals aged 16–19 years spend an average of 11.2 weeks unemployed and those aged 20–24 years spend 14.4 weeks unemployed on average (U.S. Bureau of Labor Statistics: Division of Labor Force Statistics, 2007). This pattern continues, where number of weeks spent unemployed increases as age increases, and 45- to 54-year-olds spend an average of 21.2 weeks unemployed and 55- to 64-year-olds spend an average of 21.9 weeks unemployed.Therefore, despite the growing number of older individuals and policy changes that encourage them to remain in the workforce longer, labor shortages will still occur due to their tendency to retire earlier (both voluntarily and involuntarily due to discrimination) and to their poor retention and recruitment. Although an increasing amount of research has been focused on policy changes related to the aging workforce and quantitative studies examining employers’ attitudes toward older workers (reviewed subsequently), there is a lack of research on how various policies and practices (both positive and negative) affect older workers. Specifically, little is known about the hiring practices of employers from the older worker's perspective and how these practices influence older workers when searching for employment. Therefore, the purpose of this article is to examine older workers’ perceptions of employers’ attitudes toward them and their management of these (mostly) negative attitudes.Literature ReviewEmployers' Attitudes Toward Older WorkersEmployers’ first impressions of older workers are often based on stereotypes—“mental pictures of people based on their membership in a group” (American Association for Retired Persons [AARP], 1994, p. 8). Although research suggests positive attitudes toward older workers on certain dimensions (knowledge, experience, honesty, dependability, work ethic, mentoring, and stability; Berger, 1999; Marshall, 1996; Walker & Taylor, 1998), they are perceived negatively in many other areas. For example, studies indicate that employers believe older workers are less flexible, in poorer health, less creative, less interested in technological change, and less trainable than are younger workers (Chiu, Chan, Snape, & Redman, 2001; Guillemard & Walker, 1994). In addition, older workers are seen as being more prone to accidents and staying with the company for a shorter period of time as compared with younger workers. Older workers are also viewed by employers as less productive and less able to engage in physically demanding work than younger workers (Henkens, 2005).Although most of these stereotypes have been disproved in the literature, employers continue to believe many of them and often make their hiring decisions accordingly (Berger, 1999, 2004). The key reasons that employers cite for not hiring older workers relate to beliefs concerning their lack of computer literacy, followed by lack of other appropriate skills, lack of appropriate qualifications, and difficulty adapting to varying work settings (Marshall, 1996). Employers’ stereotypical attitudes have also been linked to poor opportunities for training, promotion, and retention of older workers (Chiu et al., 2001). Another study that specifically examined the relationship between employers’ attitudes and their behaviors toward older workers also found a direct association between the two in relation to hiring, training, and advancement opportunities (Taylor & Walker, 1998). Further, Taylor and Urwin (2001) found that older workers participate less than younger workers in employer training programs due in large part to an age bias in employers’ decision making. In addition, stereotypical attitudes of employers have been tied to negative views concerning the retirement or retention of their older employees (Henkens, 2005). In fact, employers who have less personal contact with older workers have more negative attitudes toward them than employers who have more personal contact with older workers. This could suggest that although employers may be positive toward older workers already employed by their organization, they are negative toward the recruitment of new older employees. Employers’ attitudes toward older workers also vary significantly according to company size, employers’ age, and employers’ gender, with older female employers from smaller companies displaying the most positive attitudes (Berger, 1999).Although there is a growing body of literature on employers’ attitudes toward older workers, there is a lack of research that examines older workers’ perceptions regarding ageism. However, according to a national American work and career survey of more than 2,500 individuals aged 45–74 years, 80% of individuals who are searching for employment feel that age discrimination exists in the workplace (AARP, 2002, p. 66). Also, 15% of older individuals feel that they were not hired for a particular job because of age (AARP, 2002, p. 13). Findings from the survey speculate that older workers’ perceptions about age-related barriers to reemployment are a reality due to the increasing number of complaints about age discrimination made to the Equal Employment Opportunity Commission. In fact, between 2000 and 2002, complaints increased by 25% (AARP, 2002, p. 14).Managing Attitudes and BehaviorsConcepts that are taken from the symbolic interactionist framework are used in this article to gain a meaningful understanding of individuals’ motives, behaviors, and interactions (Blumer, 1969). In this perspective, individuals are seen as having agency and as engaging in mindful interaction and self-reflexive behavior. Therefore, individuals act, they do not simply respond, and they make choices within the constraints of the broader social context. Under the symbolic interactionist umbrella, Goffman (1959, 1963) has demonstrated the importance of stigma, identity, self-presentation, and impression management through his dramaturgic perspective. The concept of stigma refers to “an attribute that is deeply discrediting” (Goffman, 1963, p. 3) and results in discriminatory attitudes and behaviors being directed toward the stigmatized individual. More recently, the conceptualization of stigma has been redefined as existing “when elements of labeling, stereotyping, separating, status loss, and discrimination co-occur in a power situation that allows these processes to unfold” (Link & Phelan, 2001, p. 382).In relation to identity, Goffman (1963) described three types of identity that are used in the developmental process: “felt identity,” the meaning attributed to one's own identity; “presented identity,” the view of oneself that is projected to others; and “social identity,” the meaning that others attribute to the individual. He argued that one's presented identity can be altered to meet the needs of any given situation. By using certain techniques, individuals actively control the image they are projecting to others (presented identity), to obtain a desirable social identity. Goffman's (1959, 1963) use of the term “impression management” explains how individuals present themselves to others by conveying certain images that they consider appropriate in a given situation. In this way, individuals can manipulate the presentation of themselves displayed to others and create a distinct “definition of the situation” (Thomas, 1972) to sway others’ perceptions of them in a positive direction. For example, in their sociological study of the process of becoming doctors, Haas and Shaffir (1991) discussed medical students’ “constant need to create and manage the image of a competent self through the process of impression management” (p. 74). Thus, the importance of managing self-presentations is a well-developed concept in many realms of the sociological literature. With respect to aging, research on older widows has shown that to avoid being stigmatized and maintain a positive identity, the women “suppressed” evidence of age (Matthews, 1979, pp. 74–75). “People have time allotted to tend to physical and mental needs . . . to cover potential age stigma signs, such as wrinkles or gray hair, by surgical intervention or hair dyes” (Luken, 1987, p. 185). Thus, research suggests that individuals use certain management strategies to avoid being classified as “old” and being stigmatized by others.Studies indicate that impression management tactics are often used in the context of a job interview (Delery & Kacmar, 1998; Rosenfeld, 1997). More specifically, Delery and Kacmar showed three types of impression management techniques that are useful in an interview setting: (a) entitlements (taking credit for a prior work-related success), (b) enhancements (making statements reflecting one's own positive attributes), and (c) self-promotion (highlighting strengths in relation to those required by the ideal applicant). Their research considered the importance of applicants’ age in an interview setting but only included participants with a maximum age of 38 years and considered age to be an advantage in relation to having more experience with the interview process itself. Other research indicates that individuals use impression management strategies when they perceive that discrepancies exist between the feedback received in an organizational setting and their desired social identity (Bozeman & Kacmar, 1997). When discrepancies do occur, individuals acquire alternative techniques to use and then wait for additional feedback, which determines the future interaction between the participants. Accounts (Scott & Lyman, 1968) and disclaimers (Hewitt & Stokes, 1975) are also used in the employment context (Bozeman & Kacmar), to avoid negative stigmatization and negotiate identity in relation to past and future behaviors. Thus, although previous research has not focused on older workers seeking employment, it does suggest that certain strategies are particularly useful for enabling individuals to focus the interview on their strengths and positive attributes.Despite these findings, there is a lack of research that seeks to understand older workers’ experiences during the job search process. This article is part of a larger research project, which examined the meaning and import of age in the job search process (Berger, 2004). The majority of participants in this project experienced ageism when looking for work. The present article examines how these participants manage this ageism when searching for employment. To do this, I draw on data from 30 semistructured interviews with older unemployed workers. More specifically, this qualitative study addresses the following research questions: Once older job hunters perceive they have been discriminated against by potential employers, do they develop specific management techniques that they feel help them in the job search process? Are these techniques similar to the ones used by younger workers or are there specific age-related techniques that develop during the job search process?MethodsThis study used a qualitative methodological approach that allowed for a direct examination of older workers’ experiences in the job search process. The use of qualitative methodologies allowed for the subjective meanings and interpretations of my participants’ experiences to be revealed in a way that is often not possible through quantitative methodologies (George, 1990). This study also used the grounded theory approach (Glaser & Strauss, 1967) in an attempt to let the data generate the theory, and thus, initial research questions were reformulated as the study progressed to better reflect the emerging data.The data for this study were drawn from semistructured interviews with 30 unemployed individuals aged 45–65 years. Individuals became unemployed for a variety of reasons, including corporate downsizing, layoffs, or desire to leave their previous place of employment. I also conducted 35 hrs of participant observation in three older worker programs in the greater Toronto area to help provide a context for older workers’ experiences. Human Resources Development Canada (1999) created these programs to assist older workers in their job search process by providing them with specific workshops to meet their needs. These workshops covered topics such as interviewing skills, personality assessments, computer training, résumé writing, and specific sessions on understanding the myths and realities of being an older worker. I recruited most of my respondents by attending these workshops (n = 20) and posting recruitment advertisements in older worker programs (n = 4) and in employment agencies geared to all age groups (n = 0). To obtain participants who may not have sought out specific employment assistance (anticipating that this may lead to distinct results), recruitment advertisements were also placed in community centers (n = 1) and libraries (n = 0) in a major Canadian city, in a monthly newspaper aimed at individuals aged 55 years and older (n = 3), on a university Web site (n = 0), and through personal referral techniques (n = 2). To be included in my study, participants had to be between the ages of 45 and 65 years (in congruence with the definition of older workers given in the majority of literature), actively searching for work, and unemployed for 3 months or longer. (The stipulation that individuals must be unemployed for 3 months or longer was used because individuals unemployed for less than this may still be highly distressed from losing their jobs and may not have had the opportunity to develop age-related management techniques for their job search.)The interviews conducted in this study lasted from 45 min to 2 hrs. The first 8 interviews were conducted in 1999. An additional 22 interviews were done in 2002 to expand the initial research project. (As the unemployment rates were similar in 1999 and 2002 [7.6% and 7.7%, respectively], I do not believe that the economic conditions differentially affected participants’ ability to secure employment in the two time periods [Statistics Canada, 2004a, 2004b]. Further, due to the qualitative nature of this project and the related focus on individuals’ meanings that were attributed to their experiences, the fact that data were collected at two different time points is not perceived to have altered the findings of my study.) Prior to conducting the interviews, the study was approved by the McMaster University Research Ethics Board. Interviews were held at a variety of locations (including older worker programs, community centers, libraries, and coffee shops), depending on individuals’ personal preferences. A semistructured interview format was used to guide the interview along several key areas, as well as to provide enough flexibility for the participants to discuss issues freely that arose during the course of the interview. The interview guide included questions on demographics, employment background, perceptions of age discrimination, feelings related to these perceptions, and strategies used to gain employment. With consent from the participants, interviews were tape-recorded and later transcribed verbatim.The sample for this study included individuals who varied by age, gender, marital status, ethnic or religious background, country of origin, education, income, and length of time unemployed. Just over half of the participants were aged 45–54 years (n = 16) and the remaining ones were 55–65 years (n = 14). There was an equal number of men and women in this study and the majority of participants were married (n = 16). The remaining participants were divorced (n = 6), never married (n = 6), or widowed (n = 2). In terms of ethnic or religious background, the majority of participants indicated that they were atheist or preferred not to answer this question (n = 16). The remaining respondents varied in their ethnic or religious background. The sample was quite well educated, as the majority of the participants had a college or university degree (n = 19). In terms of participants’ personal income before they became unemployed, individuals who were comfortable disclosing their income were quite dispersed among the income categories provided to them in the interview, aside from the lowest income category ($19,999 or less). Participants were also asked their personal and family income level since being unemployed; however, only a small number of participants were willing to provide this information. The length of time that individuals were unemployed also varied substantially, ranging from 3 months to 6 years. The most common length of time unemployed for the participants was between 6 and 8 months. The occupational backgrounds of the participants interviewed were quite varied. Occupational categories represented in this study included administrative, construction, consulting, education, engineering, executive, finance, food and service, health care, human resources, law, management, sales, marketing, and television and film.Data AnalysisThe data were coded according to several key themes, which were broken down further into various subthemes and subcategories (Taylor & Bogdan, 1998, p. 142). I used three stages to analyze my data based on the grounded theory approach (Strauss & Corbin, 1988). First, I used open coding and explored preliminary themes that emerged from the data. I then used axial coding, which allowed me to create a framework involving these themes and helped me to identify and link more specific subthemes and subcategories. Finally, I used selective coding, which involved confirming and refining my initial set of codes.This article is part of a larger research study containing six main research themes. “Management techniques,” the focus of this article, was one of my six key themes, which was broken down further into three subthemes and nine subcategories. After reading through the data and recognizing similar statements from various participants in terms of a pattern beginning to present itself, I created a theme, subtheme, or subcategory. For example, looking at the theme “management techniques” as a whole, I began to notice some common strategies that participants were using in an effort to find employment. I divided these strategies into three subthemes based on “preparation for the interview,” “impression management during the interview,” and “alteration of employment.” I then more closely examined these subthemes and found that within the subtheme “preparation for the interview,” various patterns began emerging, such as “physical preparations” (related to appearance), “training,” “résumé modification,” and “mental preparations.” These became the subcategories. Two subcategories also presented themselves with respect to “impression management during the interview,” which I grouped into “clothing for the interview” and “youth-oriented language during the interview.” Finally, the third subtheme, “alteration of employment,” was broken down into the subcategories “alteration of employment goals,” “alteration of employment type,” and “alteration of geographic location for employment.” I developed codes (e.g., 1.1, 1.2) and recorded them on the transcripts by hand and then I loaded the data into the qualitative analytical software program QSR NUD*IST (Non-Numerical Unstructured Data Indexing Searching and Theorizing). This program allows researchers to code large amounts of text, which can then be stored according to themes and accessed easily in the future. Thus, once the transcripts were loaded into QSR NUD*IST, I entered the codes I had created by hand so that when I was ready to write about a particular subtheme or subcategory, this software would highlight these previously coded quotations.To ensure trustworthiness of the data (Lincoln & Guba, 1985) or accuracy of its interpretation, several techniques were used. First, interviews were transcribed verbatim by a research assistant, and I then checked them independently for errors. Second, although I coded the data myself, themes that emerged were shared with my peers and only finalized once this consultation took place. At that point, a consensus was reached that these themes accurately represented the data they were describing. Transcripts were also reexamined on several occasions to confirm accuracy of the themes that initially presented themselves. Finally, I continued to recruit participants until I felt that no new themes were emerging and data saturation was achieved (Patton, 2002). It is anticipated that by using the methods previously described, the transferability of my findings (similar to external validity in quantitative studies) to other research contexts will be heightened (Lincoln & Guba). In addition, member checks, a technique that involves the corroboration of preliminary findings with research participants, were used early on in my study to ensure the credibility (similar to internal validity in quantitative studies) of my findings. These member checks involved follow-up telephone conversations with several participants. During these conversations, I suggested specific themes that were emerging from the data and asked respondents if these themes accurately reflected their job search experiences.Results: Managing Age DiscriminationIn the job-seeking process, participants felt that prospective employers used various mechanisms to discriminate against them that reflected negative stereotypes with respect to skills, training, adaptability or flexibility, and financial costs. Further, participants felt that employers examined their résumés in a discriminatory fashion (i.e., choosing candidates to interview based on the year their degree was received or the number of years of experience they had), that the job interview was a mechanism for employers to assess the age of job candidates, and that ageist language was used during the hiring process. (For more detail on participants’ perceptions concerning age discrimination in the hiring process, see Berger [2004].) As a result of these negative experiences, the vast majority of individuals in this study developed specific strategies that they felt helped them avoid being stigmatized as old when searching for employment. Although it is recognized that most individuals, regardless of age, use various management techniques during their job searches (e.g., wearing appropriate attire, preparing mentally for an interview), the strategies that I refer to in this section are management techniques that are related to age. I have grouped these techniques into two categories. The first type of technique used by respondents I refer to as “counteractions”—tactics used to offset negative stereotypes. These techniques involve participants attempting to counteract employers’ ageist stereotypes by maintaining their skills and changing their work-related expectations. The second type of technique used by participants was “concealments”—strategies used to hide specific information (i.e., age). These techniques, developed to conceal age, included participants altering their résumés, physical appearance, and language used.CounteractionsSkill Maintenance.—The first age-related management technique used by respondents to counteract employers’ negative attitudes toward them involved keeping up-to-date with training. This technique was used by the majority of participants, slightly more often by women as compared with men. Overall, they felt that employers did not want to invest the money and time in training older workers:They look at a person and—this has been told to me point-blank by human resources people—they look at people as a monetary investment. They invest a certain amount of time and effort in training a new recruit—the higher level of the recruit, the more expensive the time in training. And they expect to get back ten times their investment, or else it isn't worth it to them to hire them. So if they spend a year acclimatizing me, training me, getting me integrated into the system, then they would expect to get ten years of profitable time out of me. Now, if I am 50 years old, they look at me as a poor prospect. Even at 46, they were looking at me as a very poor prospect. (man aged 52)Respondents kept their training current to avoid being classified as “out of touch” by potential employers:Any training going on, I signed up for it, and I did it. You know, when it came to computer training, I was one of the older ones. Everyone realized it, but I'm computer literate, so that's not an issue. … I have also gone for all the training on how to job search. They have my résumé, a follow-up letter, and the whole bit—doing it professionally. (woman aged 60)I went to the University of Toronto to take some courses to update my knowledge. I also go when different organizations offer specific workshops that I am interested in. (woman aged 56)Despite the recognized importance of keeping training and skills up-to-date, many of the participants were very frustrated with their current inability to pay for the training needed to obtain employment and described a type of “catch-22” situation during our interview:I need the qualifications, but I need money to get the qualifications, but I need the qualifications in the first place to get the job to have the money. It's kind of like a catch-22. It's very similar to the 17-year-old fresh out of high school who can't get a job because he doesn't have any experience and doesn't have any experience because he can't get a job. It is the same kind of catch-22, only 35 years later. (woman aged 53b)If you have an education here [Canada] you can reach whatever you want. But I have no money to take courses to upgrade my education from [my previous country of residence]. It is not a nice feeling. It is unpleasant and very difficult. … I am planning to learn more and get a Canadian license but the problem for me is money. If I can get a job and start to work, I will be able to save money and get a license. (woman aged 45b)There were three groups of women and four groups of men who shared the same age in my study. To distinguish between participants, I have used the letters “a,” “b,” and “c” after their ages. A few of the participants mentioned that they were doing volunteer work, in the hopes that this would help them gain reentry into their desired occupation:We both [she and her husband] are volunteering and always keeping busy. This allows us to improve our knowledge. … And I am reading every day on the computer to improve my professional skills. (woman aged 45b)I have worked for two years as a volunteer. … I am probably too chicken to get a [paid] job because I haven't worked in a while. (woman aged 56)Volunteering was viewed as a temporary (and undesirable) solution to prolonged unemployment; yet, participants felt that this would help them update their skills or keep them current and hoped that it would give them the chance to get a foot back in the door.Another way that participants were able to update their skills was by attending workshops held by older worker programs:I never really used the Internet very much where I worked ‘cause you know, you were busy with your other things. … They [older worker program] spent a lot of time on computer skills, like using Word and getting on the Internet. (man aged 62)I was advised to go for that [older worker] program. … I needed some updating in my computer skills because the courses I had in computers was from more than 20 years ago. (man aged 57b)I have changed majorly since I took the training [provided through the older worker program]. … There were a lot of books available there. There was a lot of advice given. … I went to the centre and every day I learned something new. (woman aged 50a)Interestingly, what was previously considered an advantage by participants—having job security by remaining in one job for a long period of time—was now something that they believed was hindering their job search success. To overcome this newfound limitation, participants felt that they not only needed to update their skills but they also needed to mentally alter their expectations to find work. Participants’ changing expectations will now be discussed.Changing Expectations.—Another way that participants attempted to counteract employers’ ageist stereotypes was in relation to their work-related expectations. Most respondents (men and women equally) changed their employment expectations with respect to altering their original employment goals, the type of employment being sought, or the geographical location of the place of employment. First, a third of participants indicated that they altered their original employment goals. Second, a majority of participants changed the type of employment they were seeking (i.e., monetary remuneration, career change, employment status). Finally, one third of respondents also considered geographical relocation to secure employment. Although these mental strategies may be applicable to all individuals regardless of age, older workers have had to use these management techniques to heightened degrees due to the experience and higher salaries that are often associated with advancing age. Thus, once older workers perceived that their age was a barrier to reemployment, they soon realized that they could no longer have the same expectations they once had if their main goal was to secure employment.In terms of employment goals, although monetary rewards were viewed as important to a degree, satisfaction and fulfillment with work were seen as the primary motivators in seeking employment by some of the participants in higher-income brackets. For example, according to one respondent:You know, I'm not an entry-level person, and I don't expect an entry-level kind of remuneration. Not that money is the most important thing to me—satisfaction to me is as important or more important at this stage of my life than money. (man aged 52)This tendency may assist older individuals in their job-seeking process, as employers often cite higher financial costs (associated with employee turnover, salaries, and benefits) as a reason for not hiring older employees. Respondents described how they came to this realization:The end result for me is to find a position. I'm not finding a position. The end result of a company is to make money. They think they are going to make money off younger people better than they are going to make money off an older person because they think that the younger person will be with the company longer and is going to be able to produce or benefit revenue for a longer period of time. It's not true. An older person doesn't make the mistakes of the less experienced. So, the learning curve is a lot shorter. But they don't look at that. They don't look at the result's line. (man aged 52)I think salary is a barrier because employers think we expect more money. (woman aged 45b)It follows that many of the respondents realized that receiving the same level of financial compensation as they had prior to becoming unemployed was not necessarily feasible and thus lowered their monetary expectations in relation to the type of employment being sought. For example:I know that I will take a lower [pay] rate to come in [to the labor force]. … You just have to keep going, you know. Lower your expectations, unfortunately, and just keep going. … It seems the longer you're unemployed, the more you can rationalize it. You don't feel any better, but you just sort of, like I said, you lower your expectations just basically to get back into the job market. (woman aged 58)Thus, job advertisements stating lower salaries, a mechanism that participants believed employers used to discriminate against them, would no longer be considered a barrier to these respondents.Furthermore, after long periods of unemployment, many individuals begin to consider a change in career or industry to gain reentry into the workforce:The frustration is immense because you keep sending your résumés back out to the same people. … I've been about 18 months out of work, and a couple of months ago I decided I've got to switch this trade. That's when I considered bartending. … Then I decided on this building maintenance course. . . . But [it is hard] knowing that no matter what industry I go into now, I'll never make that kind of money again. (man aged 50c)I am trying now to shift my focus. … I am trying to diversify. … It has been very difficult to get interviews, so now I am trying to look into other areas. (woman aged 50b)Although a change in career was not their preference, many respondents realized that this sacrifice had to be made at some point in their job search process. Some individuals even deliberated between returning to their previous careers with lowered salary expectations and changing careers in the hopes of achieving any level of financial stability. This decision was not an easy one, as many participants soon realized that once they left their original career, they would have great difficulty reentering it at a later date. For example, one, woman who was a dental assistant explained her current predicament:I am looking into—I mean I'm not really sure that I would be just as good doing other things and I'm thinking why should I waste 25 years of training. … I have set up a deadline for myself that at least by the end of January if I do not get a job as a dental assistant—I never thought I could think along the lines and I'm still not happy doing that—but I just might consider other jobs. … But then that's where the problem lies, that if you go into other things, it…s that much harder to go back. If you go for an interview and the doctor…s asking you what you are doing and you say, “I’m in a bookstore” or something like that—“So why are you applying for the job? I can have 20 assistants that are actually assistants.” So that’s the dilemma there. (woman aged 50a)Expectations may also change in relation to the status of employment that is being sought. Many older individuals decide (voluntarily or not) to work on a contract or part-time basis with various organizations. For example:I would accept part-time work, and at the moment I'm doing some consulting work for a boutique; it gets me out of the house. (woman aged 60)There are some jobs that admittedly the salary is not what I want, but right now, would I take a part-time job? Yes. … Right now I'm willing to listen to any offers that come around. (man aged 62)Although the security of full-time employment was not present for these individuals, participants felt that they were able to display their strengths to employers, which may ultimately lead to more stable employment for them in the future. Many participants felt that changing their own “definition of the situation” (Thomas, 1972) from unemployed to semiretired helped them accept a possible change in the type of employment that was being sought. For example:I sort of call myself semi-retired ‘cause that means that I could tolerate, say, a lesser job than I had before. Like part-time work or something. … When I first looked [for a job], that's what I told people—that I wasn't anxiously looking. Like when I say people, I mean the neighbor I bump into on the street, or something, wondering why I was home during the day. (man aged 60)You know, I almost made up my mind that this is semi-retirement, but I'm not ready for it, I'll tell you. (woman aged 60)Individuals reconceptualized their status from unemployed to semiretired to provide a new and more acceptable social meaning to their current situation, similar to findings reported in the literature on displaced workers (McMullin & Marshall, 1999).Despite making drastic changes in monetary, career, and employment status expectations, participants were still unable to secure employment:I'd like to have full-time but if like I could only get part-time, well, I would do that, you know, in the interim. … I'd take another job. Like I even applied at different places like IGA and Blockbuster, you know, I would take something like that, you know, just to be working and doing something. (woman aged 53c)I'm willing to try anything and I'm not asking 30 dollars an hour, what I used to make before, I'm not asking that. I'm going to go for—how much is minimum wage—six eighty-five? I’m willing to start with that if the job is normal and I’m capable of doing it. I’m not picky. I’m not asking this kind of wages, minimum wage is going to do it. I’m going to be there. I’m going to try. (man aged 56)I’ve junk-mailed every employer out there with my résumé—even survival jobs where I went to a car dealer and wanted to be a lot person—you know, park cars, take them in and get them washed, vacuum them out, and that kind of stuff—even to get minimum wage. And I can’t even land that ‘cause they look at you and go—“What is your problem?” You know, it’s just tough. (man aged 46b)Making these extreme changes in their expectations without success (i.e., securing employment) was obviously quite disturbing to many of the respondents. In fact, many of the comments made by the participants conveyed both a sense of desperation and a sense of adaptableness. For example:I’m willing to do practically anything to work rather than not work. (woman aged 64)Many of the individuals in this study were also quite willing to move to another geographical location to find work:Don't just depend on Toronto, Mississauga, Brampton, or York, because there is nothing out there. And you gotta have your mind made up to want to leave your home and to be traveling wherever the job is. …You have to try all different angles. (woman aged 59)When I started my job search.… I placed that restriction—my job search parameters—and then realized it. I used to say, “I want only full-time and I want it downtown.” Then I said, “I want full-time but I’m willing to stay anywhere in greater Toronto or contract in greater Toronto.” So now it's full-time, part-time, or contract anywhere in Ontario.… And this month I've also begun sending résumés off to the [United] States.… So now I've widened my search.… So we’ll see. (man aged 62)However, several participants noted that although they would personally be willing to relocate to secure employment, they felt that the needs of their families would best be met by remaining in their current geographical location. For example, despite the hardships that they had encountered since immigrating to Canada, some of the participants felt that there would be better long-term opportunities for their families if they remained in Canada due to the educational system or a general feeling of safety and security:I would have, you know, left the country [if] I didn't have the problem of my daughter studying here. It's only for her that I continue to stay here. (man aged 57b)We came to Canada because of the dangers of living in [my previous country of residence]. …My husband was a big company director and I was a medical doctor and now we are both unemployed and on welfare. … We can't find work here. We have been unemployed for five years—since we have been in Canada. (woman aged 45b)The changes in mental expectations described in this section were deemed necessary by the participants to try to overcome some of the stereotypes associated with being an older worker, such as higher salary expectations. Unfortunately, making these mental adjustments did not help participants secure employment, so many of them felt that more drastic measures were needed to conceal their ages to potential employers. The analysis now turns to these types of management techniques.ConcealmentsRésumé Modification.—It should be recognized that stereotypes can also be positive, and some of the participants felt that they possessed certain positive characteristics that are associated with being older. For example, the experience that comes with age was recognized by one respondent when describing her previous work situation:I mean, there is no substitute for experience. … It does make the work that much more efficient; that much more professional. Honestly, I think a lot of people—say, my age—when they go to an office and see a person my age, they would say “Okay, this person knows what she or he is doing. It seems that they’ve been doing this for a long enough time.” (woman aged 50a)While some participants tried to emphasize positive stereotypes associated with older workers, such as loyalty and experience, many were not able to do this due to a variety of reasons. For example, assuming that age and experience go hand in hand reflects a gendered bias in that most of the women in my sample had less paid work experience than did the men, due mainly to raising their children. Thus, they were not able to highlight to employers one of the few positive stereotypes associated with older workers in the same way that men could do so. See McMullin and Berger (2006) for more details on gender differences in the route to reemployment for older workers.However, having experience can also be a negative thing:To just look at my résumé, they know how many years I’ve been in the business and they can sort of deduct that I’m not 35 or 40. (woman aged 60)You don’t know if they're looking at your résumé and looking at … like, figuring you got all that experience so obviously you got to be a certain age, right? (woman aged 53c)In the aforementioned examples, respondents described how their extensive experience was being used by employers as a way to determine age from their résumés and subsequently identify and select younger candidates to interview for job openings. Therefore, most respondents felt that they had to conceal age on their résumés by eliminating some of their work experience, number of jobs held, or the year a degree was received:I try to hide it [my experience]. Sometimes I don’t mention my degree and that sort of thing, and I only show the last ten years of work experience. (man aged 60)I changed my résumé somewhat to exclude the number of years I’ve worked and said, “I have extensive [experience].” … Even though—I guess now everybody who uses “extensive,” they [employers] say, “Oh. This guy's got 20 plus years.” (man aged 62)Participants also indicated that they switched the style of their résumés from chronological ones to “functional” ones, where the importance of skills rather than specific jobs or years in the labor force is highlighted:I changed my résumé around. I was using a chronological résumé at first, but for the last four months I have been using a functional one. (man aged 50a)I use a particular style of résumé that emphasizes skills more than the number of jobs or length of time that I have been in certain jobs. (man aged 50b)Responses from the participants made it increasingly clear that “de-emphasizing age” was a strategy taught by personnel at the older worker programs:At the [older worker program] they helped me modify my résumé—to de-emphasize age. … They would tell us don't give them [employers] any more information, basically, than is necessary. Like sort of just give them your last 10 years of work, let them see you versus, you know, putting down everywhere you've worked for the last 30 years or 40 years. (woman aged 58)I was taught [at the older worker program] that when you send a résumé, you don't put the date you graduated from school … or stuff like that. So they really don’t know how old I am when I send them the résumé. (woman aged 55)Thus, although some individuals may have eventually decided to alter their résumés on their own using some of the techniques previously described, it was clear that the overt encouragement at the older worker programs was the reason that the majority of adjustments were made.“Improving” Appearances.—Another way respondents felt that employers discriminated against them related to the interview process. These individuals believed that after employers had the opportunity to see them during an initial interview, they could immediately estimate how old they were and only request second interviews from the younger applicants. Several participants provided justification for this belief:What is quite interesting is that a friend of mine who is 45 years old, about the same age as me and who just got her degree, is getting more interviews than me. She is only getting the first interview though. I am not sure if it is because the employers assume that she is younger because of her recent degree, but once she gets in there and has an interview, she is never asked back for a second interview. I am not sure if it is age per se, but that is just my feeling. (woman aged 45a)We [she and other older workers] get interviews but not jobs. … When they [employers] look at you, they decide. (woman aged 53a)Thus, another type of concealment used by the majority of participants—by women far more often than men—was to portray a “youthful” appearance to potential employers. Several of the participants referred to physical preparations for the job interview. For example, many men and women acknowledged dying their hair prior to an important interview:When I actually get an interview, … I dye my hair. (man aged 50b)When you look around you and you see the faces getting younger and younger, you know you've got to keep it up. Even if you can't afford it, that's the one thing you need is your bottle of dye. (woman aged 58)Several men also considered using a toupee if they were balding and shaving their beards to appear younger, despite the fact that they did not necessarily agree with this or acknowledge that this was their idea in the first place:They want to see me have a full head of dark brown hair. Maybe I should go to Honest Ed's [discount department store] and buy a nylon toupee. That would improve my appearance, don't you think? (man aged 52)People say to me, because I'm bald, “Why don’t you get one of these wigs?” or whatever you call them, or “Why don’t you shave your beard?” I have had my beard all of my life—since I was 20. It was never a problem, so why is it a problem now? (man aged 50a)Similarly, several respondents mentioned that after going to job interviews, they recognized the importance of “fitting in” to an employer’s organizational culture, which essentially meant giving the impression of being younger:I think that the organizational culture in an organization is very important. You need to fit into the company’s culture. Many companies have youth-oriented cultures and they may only hire someone who fits into this culture. (man aged 50b)Therefore, he explained how he would dress a certain way for an interview in an attempt to “fit in” with what he believed was a young organizational culture:There are certain techniques that I use [in an interview] to seem younger. … I always wear young-looking clothes. … I dress in certain clothes that fit in with their company's culture. (man aged 50b)Another strategy that respondents used to maintain youthful appearances was to make a concerted effort to maintain their health. Thus, although many of the participants encountered changes in their mental health following experiences with age discrimination and prolonged unemployment (see Berger [2006] for more detail on the psychosocial consequences of perceived age discrimination), the majority of participants were in good physical health prior to becoming unemployed and remained this way following unemployment. In fact, several of the respondents felt that their physical health had improved since they became unemployed. For example, when asked to describe his health since he had become unemployed, one individual explained:My health has improved quite a bit because I've got back into a running program. … My previous job, you know, they wanted the 60-hour weeks. … So physically I'm doing a lot better and my eating habits have improved. (man aged 46a)Other respondents expressed how they felt fortunate that they did not physically look “old” due to their good health and thus felt they did not have to alter their physical appearance to avoid experiencing age discrimination from employers:I guess [in relation to] how I look—I really haven’t had that kind of age discrimination. I think that when I go to a [work-related] event I always look good, so I think that also helps a lot. (woman aged 50b)Appearance-wise I find I’m okay … to be able to say, “yes, I'm still capable of working; I don’t have a cane yet.” (woman aged 58)Finally, it should be noted that not all the participants agreed with modifying their physical appearance to obtain employment. Some individuals actually were quite against this active age concealment behavior. For example:I have grey hair and I refuse on general principle to dye my hair. … I don't want to cater to this. … I can see the reaction of people to somebody who has dyed hair—you know they dye their hair blond or whatever—they tend to think of them as younger and prettier and so on and I refuse to do it. I’m stubborn that way. It’s costing me I know but I don’t believe in catering to that. I don’t believe the color of your hair should be an influence on how people treat you. (woman aged 53b)It is clear that this woman felt very strongly about not wanting to conform to society’s expectations regarding her physical appearance; yet, she also recognized that this conviction may be harming her job search success.The “Right” Age Talk.—The last way that many participants attempted to conceal their age was by managing their outward portrayal during job interviews through language. This was done by either using certain discourse to reframe otherwise undesirable conversations or using “youth-oriented” language. Turning first to reframing undesirable age-related conversations, one woman said:I was told that [I was too qualified for a position] but I turned it around because I had so much experience or knowledge in finance that—I was applying for a job in mutual funds and I said “You are getting kids out of school who don’t even own them so how do you expect them to sell them.” I turned it around. (woman aged 53a)Many of the respondents explained how they managed to avoid or deflect discussions in which their age could become known to employers and explained the importance of anticipating age-related questions during job interviews to mentally rehearse desirable answers:I’m not saying falsify an image [during an interview]. Be yourself and be honest. But, again, depending on the questions, you know—“How old are you?”—rather than respond—“That’s none of your business”—I would have said something like—“Well, I’m old enough to have 25 years of experience in this business,” which gives them an age bracket and it doesn’t affect me. “As you can see by my résumé, I have 25 years [experience], so you know I’m not 35.” (man aged 50c)I try to verbalize what I’m going to say to them [employers] because they seem to do this constantly …. I guess I just sort of prepare myself mentally for these, I call them, stupid questions—“What are your goals, let’s say ten years down the road?” Well, I don’t have ten years left, you know. So you have to verbalize what you are going to say. (woman aged 58)Several individuals also engaged in what they considered to be youthful language. For example, one participant explained the lengths he went through to avoid being defined as “out of touch” more generally in terms of current societal trends:What I talk about is really youth-oriented. I make sure I discuss very physically active kinds of sports and I mention various social groups that I have joined. (man aged 50b)Participants also realized the importance of using certain “buzzwords” (e.g., in the sales field, what was once referred to as a “forecast” is now called a “pipeline”) during a job interview to illustrate to employers that they were up to date with the language being used in their field. For example:The high-tech industry has so many different skills and languages. Like, you have to be an expert. … You’ve got to really move quickly and learn new stuff. (man aged 46a)I think the lingo has changed considerably. … There are a whole slew of buzzwords that I am sure are being used today by corporations that perhaps a person that was trained 20, 25 years ago understands but is not comfortable using because it’s not in their everyday vocabulary. They were trained with a different sets of words. … I think it's a barrier. … As soon as you go into an interview and you use your own terminology, you date yourself—automatically, subconsciously. (man aged 57a)This participant went on to describe a whole list of buzzwords that he felt were being used by individuals in his field and in human resources more generally:We were just discussing a whole list of words yesterday [at the older worker program]. And they were words that I personally had not seen, let’s say 20 years ago. Such as “telecommuting” and “motor-skilling” and “life-long learning” and “career planners” and “dejobbing.” This is just on the human resources side. … The same thing occurred in the marketing industry in a different way. For example, you know, having “new enhancements.” That’s one way people would refer to things. In the old days you would just call it, you know, “increased sales.” … I do believe that it would be useful to create, I don’t know, a whole set of words, buzzwords, that are currently in use in an industry that show involvement and progression—at least of terminologies. (man aged 57a)Another individual used some other buzzwords to explain his difficulties in an interview situation:Let's say they [employers] describe a particular position you have to do. … You have to meet “revenue targets” and you have to “integrate pricing and portfolio managements” and “database marketing” and you have to work “retention strategies.”. … Unless you are able to repeat these words that they put in their job description, you may sound outdated. You may sound like you will not be able to fit because you don’t understand their terminology, which is not true. You are [just] more comfortable working with established business terms. (man aged 45b)Thus, some of the respondents felt they were at a disadvantage in relation to the language being used in their field after realizing that the lingo they were trained with in the past was already outdated; being unemployed for a lengthy period of time complicated the matter. They found this quite frustrating, similar to the catch-22 situation that they found themselves in in relation to their skills and training. Thus, individuals felt that the only way to maintain a current lingo in their field was to be employed in it, and this was not happening for them at the time of these interviews.Discussion and ConclusionsThis study explored how older individuals’ perceptions of age discrimination in the job search process led to the development of age-related management techniques. Participants’ feedback on seeking employment illustrated how age became a discrediting attribute or stigma (Goffman, 1963), which resulted in discriminatory attitudes and behaviors by potential employers. Using Goffman’s three notions of identity, individuals began to feel old (felt identity) when the image they were projecting to employers (presented identity) was perceived to be that of an “older” worker (social identity). Because presented identity can be modified depending on social context, by using the management techniques described in this article, participants were actively controlling the image they were projecting to others to present a more advantageous social identity.Similar to findings discussed previously in relation to impression management techniques in general (Goffman, 1959, 1963) and their use in the organizational behavior literature (Bozeman & Kacmar, 1997), once individuals perceived that a discrepancy existed between the image they wanted to convey to potential employers and the feedback they received (perceived age discrimination), the vast majority of them developed alternative management techniques that were age related to project a positive presented identity (Goffman, 1959) and avoid being stigmatized by potential employers. For example, most respondents altered their physical appearance to portray a more youthful image in a job interview setting to manipulate their self-presentations and sway potential employers to hire them.Overall, counteractions were used by participants more often than concealments. Looking at all five types of management techniques, “changing expectations” was used most often, followed by “skill maintenance,” “the ‘right’ age talk,” “résumé modification,” and “‘improving’ appearances.” With respect to gender differences, in descending order of use, women were most likely to “‘improve’ appearances,” equally used “changing expectations” and “skill maintenance,” and then used “the ‘right’ age talk” and “résumé modification.” For men, the pattern of usage was slightly different. They predominately used “changing expectations,” followed equally by “résumé modification” and the “right age talk,” “skill maintenance,” and “improving appearances.” I was surprised that there were not larger gender differences in terms of the management techniques used overall, but it is interesting to note that the biggest gender difference appeared with respect to the subcategory “improving” appearances. Aged bodies are more commonly perceived to be a barrier to finding employment for women than for men because youth is more often equated with attractiveness for women (McMullin & Berger, 2006). As many of the women in my sample had come to this realization over the course of their job search, most felt the need to alter their appearance before a job interview.Management techniques were developed by the participants to combat employers’ negative attitudes in relation to their skills, training, adaptability or flexibility, and higher perceived monetary costs by staying up-to-date with their skills and training and changing their work-related expectations. In addition, participants felt that employers examined their résumés and chose candidates to interview in a discriminatory fashion. After coming to this realization, individuals began to use concealment tactics on their résumés that involved eliminating the year that they received their degree or reducing the amount of experience (i.e., number of jobs or number of years in a job) displayed on their résumés to gain a more competitive advantage in the job market. Similarly, respondents felt that the job interview and language used by employers were key mechanisms for discrimination. By altering their appearance or using specific language to mentally prepare for age-related concerns, they felt a more youthful image was being conveyed to potential employers.This study contributes theoretically and substantively to the literature by applying concepts related to management techniques and the aging process to older individuals who feel stigmatized in their job search process. More specifically, in relation to management techniques, previous researchers have argued that the following types of impression management techniques have been used in an interview setting: (a) entitlements (claiming responsibility for a positive event that occurred in the past), (b) enhancements (making statements that reflect one’s positive attributes), and (c) self-promotion (highlighting strengths in relation to those required by the ideal applicant (Delery & Kacmar, 1998). (Although this was not the focus of my current investigation, I found evidence that participants in my study were using all three of the impression management techniques described by Delery and Kacmar. Many of them made statements to me that highlighted their past accomplishments, emphasized their positive attributes, and stressed how their work-related experience would benefit them in a job situation. Further, they explained that they would make similar comments to employers in an interview setting.) Findings from my study expand on the impression management techniques described by Delery and Kacmar. My research demonstrated that older workers use these three techniques in a similar fashion to younger workers. However, they also use two additional techniques in an attempt to combat age discrimination. The first technique was counteractions—tactics used to offset negative stereotypes. The second technique used by participants was concealments—strategies used to hide specific information (i.e., age). The notion of concealment has been examined in the literature on aging in relation to older widows who suppressed evidence of age (Matthews, 1979, pp. 74–75). However, the use of these management techniques has not been applied specifically to age discrimination in the hiring process prior to this study. I anticipate that this contribution to knowledge can also be applied to a broader area relating to impression management and aging more generally. Both counteractions and concealments can relate to behaviors that attempt to offset any type of stereotypes or hide any type of stigma, although further research is needed to demonstrate the utility of these concepts beyond their applicability to older workers and the hiring process.The findings from my research suggest that more needs to be done to assist older individuals in their search for reemployment; thus, I believe that additional interviews with older workers, employment counselors, and employers themselves will greatly enrich our understanding of the job search process. These findings would help expand those presented in this article, which could then be investigated on a larger scale. A quantitative study that tests the frequency that these management techniques are used would expand our knowledge regarding structural barriers in the job search process. Examining these strategies on a larger scale in future research would help quantify the extent that counteractions and concealments are being used by older workers. This would highlight the magnitude of this issue, which is becoming of growing importance due to the aging of the workforce. This suggestion highlights the utility of combined research methods or triangulation (Creswell, 1994). I suspect that the insights into the lived experiences of the participants could only be obtained from qualitative interviews; yet, to illustrate the applicability of the themes I created beyond the participants in my study, a larger-scale investigation is warranted. Although gender was examined to some extent in this article, the ways in which age combines with other social structural factors such as race, ethnicity or country of origin, and occupation in the job search process need to be examined.Another area worthy of future research relates to conducting interviews with individuals who found work immediately following unemployment or those older than 65 years who are working past the “typical” retirement age. This research would provide insights into whether these individuals have used the specific age-related techniques described in this article. This could lead to an exploration of lessons learned from “success stories” of older workers who have succeeded in finding and retaining employment, which could help unemployed older individuals avoid or manage encounters with ageist employers. Further, it would be interesting to discover if these individuals are located in certain industries or occupations (e.g., those with less physically demanding jobs).Next, longitudinal research is needed to follow older individuals who experience prolonged periods of unemployment. Interviewing individuals several times over an extended period would provide greater insight into the job-seeking process and advance knowledge in relation to the amount of time spent in each stage of the job search process. Further, both physical and mental health could be assessed at various points in time to observe specific changes and relate them to their coinciding experiences with their search for employment. Thus, both long- and short-term health of older workers could be assessed in relation to experiences with age discrimination and unemployment.Further research is also needed in relation to the newfound contradiction that has surfaced during my research. On the one hand, prior quantitative analyses have demonstrated that employers’ attitudes toward older workers have become more positive in recent years; yet, on the other hand, my interviews with older workers suggest that age discrimination currently exists in the hiring process. Although quantitative studies that document employers’ attitudes toward older workers are growing, there is little research that examines employers’ accounts of the hiring process. Therefore, qualitative interviews with employers would allow for greater insight into the meanings that they give to various experiences with older job candidates and provide a greater understanding of their attitudes toward older workers. It would also provide clarification in relation to whether employers’ attitudes lead to the development of age-related hiring and retention practices and policies.In summary, research indicates that as a result of prolonged periods of unemployment, older workers are often considered to be “peripheral and marginal to the workforce” (Henkens, Sprengers, & Tazelaar, 1996, p. 575). This is due in large part to the existence of age discrimination in employers’ hiring processes. Thus, the current societal norms that favor youth have created structural barriers for older workers seeking reemployment. However, despite the existence of these structural barriers, findings from this study suggest that participants actively negotiate age-related strategies and thus clearly have agency in the job search process. Although the findings from this study cannot be generalized to all older workers due to the small nonrepresentative sample, the sociological insights gained contribute to knowledge of older workers’ job search process and provide a foundation for further research into this area on a broader scale. This research suggests that there is a need to reexamine the hiring practices of employers and to improve legislation in relation to their accountability. The Conference Board of Canada (2006) has suggested that policy makers work with employers to make age part of their diversity strategies; yet, this is still not largely occurring. It is only by combating age discrimination on a broader societal level that the age-related management strategies used by the participants will no longer be necessary.FundingThe research reported in this article was funded by a Social and Economic Dimensions of an Aging Population fellowship, supported by the Social Sciences and Humanities Research Council of Canada, Byron Spencer, principal investigator.I thank Dr. Carolyn Rosenthal, Dr. Margaret Denton, and Dr. William Shaffir for comments on various drafts of this article as well as the three anonymous reviewers for their helpful feedback. 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(a) Anterior is to the right. (b) Anterior is to the left. The saccular and lagenar maculae and nerve branchelets are indicated for orientation purposes. AC, anterior semicircular canal; HC, horizontal semicircular canal; L, lagena; LM, lagenar macula; LO, lagenar otolith (sagitta); N, vestibular and auditory branches of the eighth nerve; PC, posterior semicircular canal; S, saccule; SM, saccular macula; SO, saccular otolith; U, utricle; UM, utricular macula; UO, utricular otolith.Fig. 2Saccular maculae from (a) B. splendens (1.6 g, 39 mm), (b) M. opercularis (1.87 g, 39 mm) and (c) T. vittata (2.00 g, 44 mm). In each case, the long dashed lines separate groups of ciliary bundles oriented in different directions while short dashed lines in the saccular epithelia separate the anterior portion of the macula from the ribbon-shaped portion. The arrows point in the direction of the kinocilia on the cells in a particular group.Fig. 3Scanning electron micrograph showing the posterior saccular epithelium of M. opercularis. Cells shown are divided into two groups that differ in their orientation (separated by a dashed line). All cells in these two groups are oriented in the same direction and have their kinocilia pointed in the same way (arrows). Scale bar=3 μm.Fig. 4Lagenar maculae from (a) B. splendens, (b) M. opercularis and (c) T. vittata. See Fig. 2 for further explanations.Fig. 5Utricular maculae from (a) B. splendens, (b) M. opercularis and (c) T. vittata. See Fig. 2 for further explanations. Lc, lacinia; St, striola.Fig. 6Scanning electron micrographs of the saccular sensory epithelia of (a) B. splendens, (b) M. opercularis and (c) T. vittata. These SEMs show F1 and F2 ciliary bundles at the ventral margin on the posterior part of the maculae. The bundles are oriented ventrally. The arrows point in the direction of the kinocilia. Note that the kinocilia (K) of the F2 bundles are several times longer than the kinocilia of F1 bundles compared to stereocilia. Scale bar=5 μm.Fig. 7Scanning electron micrograph of the ventral saccular sensory epithelium of T. vittata. Arrowheads point to the separated row of F2 ciliary bundles at the ventral margin. Scale bar=10 μm.Fig. 8Scanning electron micrographs of the lagenar and utricular maculae of T. vittata showing ciliary bundle types. (a) Caudal margin of the lagenar macula. (b) Lateral margin of the striola of the utricle near the lacinia. Scale bar=5 μm.Table 1Mean (±S.E.M.) body weights, standard lengths (SL) and length of the saccular maculae in the rostral caudal direction in the three species examinedB. splendensM. opercularisT. vittataANOVASignificanceWeight (g)1.67±0.12.06±0.21.03±0.3F2,15=1.55P=0.01SL (mm)39.0±1.340.4±1.634.2±3.3F2,14=1.25P=0.14Macula (mm)1.05±0.011.04±0.010.98±0.09F2,14=0.63P=0.55Significances of the differences between species were tested using one-way ANOVA.Table 2Mean (±S.E.M.) ciliary bundle densities per 1000 μm2 in the rostral, middle and caudal area of the saccular maculae in the three species examinedB. splendensM. opercularisT. vittataANOVASignificanceRostral53.1±7.450.9±2.148.4±4.7F2,13=0.25P=0.78Middle49.5±3.754.0±1.451.6±5.8F2,15=0.42P=0.67Caudal51.1±2.156.3±2.360.5±2.4F2,14=3.70P=0.051Significances of the differences between species were tested using one-way ANOVA.Comparison of the inner ear ultrastructure between teleost fishes using different channels for communicationFriedrichLadicha*friedrich.ladich@univie.ac.atArthur NPopperbaInstitute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, AustriabDepartment of Biology, University of Maryland, College Park, MD 20742, USA*Corresponding author. Tel.: +43 (1) 4277 54227; Fax: +43 (1) 4277 9544AbstractThe anatomy and ultrastructure of the inner ear of three species of gouramis which differ widely in acoustic behavior were studied using scanning electron microscopy. Of the three species, Trichopsis possess a pectoral sound-producing mechanism while Macropodus and Betta lack sonic organs. The general structure of the inner ear and the shapes of the sensory epithelia are very similar, although they do differ on the posterior part of the saccular macula which is more S-shaped in Trichopsis and Macropodus than in Betta. The maculae on the three species do not differ either in ciliary bundle type (cells with long kinocilia on the periphery of the maculae and cells with short kinocilia in the central region) or in hair cell orientation pattern. Quantitative measurements of hair cell densities and the size of the sensory epithelia of the saccule did not show significant differences between species. Data presented correlate with physiological results from other investigators showing similar auditory sensitivity in Trichopsis and Macropodus. The similarity in structure and function of the inner ears of gouramis on one hand, and the occurrence of sound-generating organs in just one genus, suggests that hearing evolved prior to vocalization and thus acoustic communication in this taxon.KeywordsSound productionAcoustic communicationHearing specialistHair cellAnabantoidEvolutionAbbreviationsAC, anterior semicircular canalF1, F2, ciliary bundle typesHC, horizontal semicircular canalK, kinociliaL, lagenaLc, laciniaLM, lagenar maculaLO, lagenar otolithN, vestibular and auditory branches of the eighth nervePC, posterior semicircular canalS, sacculusSBC, suprabranchial chamberSL, standard lengthSM, saccular maculaSO, saccular otolithSt, striolaU, utriculusUM, utricular maculaUO, utricular otolith1IntroductionTeleost fishes have evolved a diverse array of sound-generating organs, and many species regularly vocalize during social interactions (Myrberg, 1981; Ladich, 1997; Ladich and Bass, 1998; Zelick et al., 1999). Interestingly, only a few groups of fishes are known in which sonic organs are taxonomic characteristics. One such a group is the ‘arioids’, a suborder of siluriforms which includes five families such as the African mochokids (upside down catfishes) and the neotropical doradids (thorny catfishes). This group is characterized by having an elastic spring mechanism that is used in sound production (Lundberg, 1993). In many other fish groups, such as gadids (cods and relatives) and pomacentrids (damselfishes), some genera are known to be vocal while others are not (Hawkins and Rasmussen, 1978; Hawkins and Myrberg, 1983). Furthermore, within mormyrids (elephant-nose fishes), sound generation is only described in the genus Pollimyrus (Crawford et al., 1997; Crawford and Huang, 1999), and within anabantoids (gouramis) in the genera Trichopsis and Colisa (Kratochvil, 1985; Schuster, 1986; Ladich et al., 1992; Bischof, 1996). Within the family Cyprinidae (minnows and carps), a group which comprises about 2000 species, only representatives of a few genera such as Cyprinella, Gobio and Pimephales are known sound producers (Winn and Stout, 1960; Ladich, 1988; Johnston and Johnson, 2000).This limitation of sound production to certain genera of particular families has several behavioral and physiological implications which are poorly understood. Non-vocal species may primarily utilize other channels for intraspecific communication such as vision or chemoreception. Nelissen (1978) analyzed acoustical behavior of cichlid species and found that the number of sounds produced is inversely correlated to the number of color patterns shown. Fish which do not display visually or acoustically during social interactions might communicate chemically via pheromones (goldfish, Sörensen et al., 1989), via the lateral line (salmon, Satou et al., 1994), or through use of electric signals (knifefishes, Hagedorn and Heiligenberg, 1985).Does the occurrence of sound-generating mechanisms and acoustical communication correlate with the structure and function of the inner ear? Correlations between the region of the greatest hearing sensitivity and sound energy spectra in the characid Serrasalmus nattereri, the toadfish Porichthys notatus, the pomacentrids Eupomacentrus spp., and the gourami Trichopsis vittata support this idea (Cohen and Winn, 1967; Myrberg and Spires, 1980; Stabentheiner, 1988; Ladich and Yan, 1998; McKibben and Bass, 1999). However, comparative studies which also included closely related non-vocal species failed to find vocalization-specific differences in auditory sensitivity among anabantoids and otophysans (Ladich and Yan, 1998; Ladich, 1999).In order to determine if structural differences exist in the inner ear between teleosts that use acoustical communication and those which exploit other communication channels, we examined the ear and hearing ability of several anabantoid species (gouramis or labyrinth fishes). These are a group of small perciforms from Southeast Asia and Africa which differ widely in their methods of intraspecific communication. Only representatives of the genus Trichopsis (croaking gouramis) possess an elaborate sound-producing mechanism based on modifications of the pectoral fins (Kratochvil, 1978). Both males and females of these species emit loud croaking sounds during agonistic behavior. Closely related genera, such as the Siamese fighting fish Betta splendens and the paradise fish Macropodus opercularis, lack sonic organs and do not vocalize except with pharyngeal teeth as an incidental by-product of feeding. Betta and Macropodus, however, produce intensive visual displays by erecting fins, opercula, and gill membranes and by pronounced color changes (Simpson, 1968; Bischof, 1996).Contrary to this variation in the presence of sonic organs, all of the labyrinth fishes are characterized by having a ‘labyrinth’ which is an air-filled cavity located dorsal to the gills (=suprabranchial chamber). These chambers are supplementary respiratory organs utilized for air breathing since these fishes live in poorly oxygenated waters. At the same time, the chambers are located lateral to the inner ears and thereby potentially serve to enhance the hearing ability of these fish (Schneider, 1941; Ladich and Yan, 1998; Yan, 1998). Prior comparison of the saccular sensory epithelia in the blue gourami, Trichogaster trichopterus, and the kissing gourami, Helostoma temmincki, representatives of two different anabantoid families (Belontiidae vs. Helostomatidae), revealed major differences in the size and number of sensory cells of the saccule, despite the two species having similar hearing capabilities (Saidel and Popper, 1987).The goal of the present study was to investigate the ultrastructure of the otic end organs of T. vittata, M. opercularis, and B. splendens. We have analyzed the shape of the sensory epithelia (maculae), distribution of the hair cell types, and the hair cell orientation patterns on the saccular, lagenar and utricular sensory epithelia. In addition, we compared hair cell densities and the size of the saccular maculae in all three species since the saccule is generally considered to be the main hearing end organ in most fishes (Popper and Fay, 1999).2Materials and methodsSix specimen of B. splendens (1.27–1.9 g; 34–42 mm in standard length), eight of M. opercularis (1.41–2.65 g; 34–45 mm SL), and nine of T. vittata (0.29–2.0 g; 25–44 mm SL) obtained from aquarium stores were used for investigations. Fish were kept at 28°C in aerated and filtered aquaria and fed daily until used in the study. All work was done with the approval of the Institutional Animal Care and Use Committee of the University of Maryland.2.1Scanning electron microscopyFish were deeply anesthetized with tricaine methanosulfonate (MS 222, Sigma) and the trunk of the fish, opercula, and gills were dissected away. Cold fixative (2% glutaraldehyde in 0.1 M phosphate or cacodylate buffer) was put into the opened ear capsules. The head was then placed in fixative for at least 24 h. The tissue was then rinsed twice in buffer before the ears were dissected out and otoliths removed. The ears were post-fixed in 1–2% osmium tetroxide for 1 h, washed three times in double distilled water, and dehydrated through a graded series of alcohols to 100% ethanol. Sensory epithelia were then critical point dried with liquid carbon dioxide as the intermediate fluid. The samples were mounted flat on aluminum stubs, coated with gold-palladium alloy and viewed with an Amray 1820 or a Philips XL 20 SEM operating at 20 kV.2.2Density measurements and statisticsIn order to compare the density of ciliary bundles, low power scanning electron micrographs were taken perpendicular to the surface of the maculae of six B. splendens, seven M. opercularis and five T. vittata. Three areas were sampled from each saccule, one in the rostral region of the epithelium in which the ciliary bundles on the hair cells are oriented along the fish’s horizontal axis (see below), and one each in the middle and caudal (where the macula curves ventrally) regions in which ciliary bundles are oriented along the fish’s vertical axis. Although this procedure may not give precise density values because of the shrinkage of the tissue during fixation, it does not effect a general comparison since shrinkage would be about the same in all specimens. The same regions of each end organ were sampled in all specimens. In all cases, peripheral margins of the sensory epithelia were avoided because density increases on the periphery and due to the presence of otolithic membrane which, in these species, were almost impossible to remove from the very edges of the maculae with any consistency.The density (number of hair cells/1000 μm2) was calculated from each of the three sample areas in pictures taken at 3000×. Each picture was 31×24 μm. Approximately 2000 ciliary bundles were counted. Statistical analysis between species were calculated for each area as well as for the mean using one-way ANOVA. In addition, the total length of the saccular macula in the anterior–posterior axis was determined using the scanning electron micrograph (SEM) measuring device.The hair cell orientation patterns were mapped by examining the orientation of ciliary bundles along transects of the maculae. Orientation of transects generally followed the orientation of ciliary bundles. Dorsal–ventral transects were taken from the caudal part of the saccular epithelium while anterior–posterior transects were taken from the anterior part of the saccular macula. Radially oriented transects were taken from the utricular macula, and anterior–posterior transects from the lagena. At least 10 such transects were taken from each epithelium. The orientations of ciliary bundles in each region were marked on a low power picture of the whole epithelium. Orientation patterns were determined for at least two specimens for each species.3Results3.1Gross morphologyThe gross morphology of the ear of all three species is basically similar to that found in most other teleosts. There are three semicircular canals and associated cristae (for detection of angular acceleration) and three otolithic end organs (saccule, lagena, utricle), each containing a sensory epithelium and an overlying single otolith (Fig. 1a,b). No macula neglecta was found. The saccule is the largest otic end organ and the lagena the smallest. The utricle is anterior and dorsal to the saccule and the two regions are connected by a narrow opening. The lagena is located dorso-posteriorly to the saccule. The utricular macula lies on the animal’s horizontal plane, while the saccular and lagenar maculae lie in the vertical plane.Otoliths of the end organs differ considerably in size. The sagitta and the small asterisci fill almost all of the volume of the large saccular and the lagenar chambers respectively, whereas the lapilli do not fill the whole utricular chamber (Fig. 1). Sensory epithelia are smaller than the overlying otoliths especially within the saccule where the ovoid sagittae only contact the sensory macula at the sulcus, an L-shaped shallow indentation. However, despite the otolith’s size, the dorsal parts of the rostral region of the saccular maculae are not covered by the sagittae. In this region, the otolith membrane, which lies between the sensory epithelium and otolith, is the only contact for the ciliary bundles.The otolithic end organs in all three species are innervated by four branches of the eighth cranial nerve, an anterior utricular, two saccular, and one posterior lagenar branch. The first saccular branch leaves the brain along with the utricular nerve and innervates the oval rostral region of the saccular maculae. This branch also goes to the cristae of the anterior and lateral semicircular canals. The posterior part of the saccular macula is innervated by a massive separate branch of the eighth nerve (Fig. 1b). The posterior branch innervates the lagena and the crista of the posterior semicircular canal.3.2Sensory epitheliaThe shapes of the sensory epithelia are very similar in all three species (Figs. 2, 4, 5). The saccular macula consists of two regions, the oval anterior region and a more ribbon-shaped posterior region. The posterior region differs slightly between species. It was rather stretched in B. splendens and more S-shaped in M. opercularis and T. vittata (Fig. 2). The lagenar macula is moon-shaped, while the utricular macula is almost round with a lateral finger-like projection pointing laterally (=lacinia) (Figs. 4, 5).Morphological comparisons revealed that species differed in weight but not in standard length or length of the saccular maculae in the rostro-caudal dimensions (Table 1). Saccular epithelia ranged from 0.66 mm to 1.15 mm long and the size was correlated significantly with body measures (standard length: r=0.68, n=17, P=0.003; weight: r=0.58, P=0.016).Hair cells on each macula are divided into groups, with all of the ciliary bundles in each group oriented in approximately the same direction as defined by having their kinocilia on the same side of the ciliary bundle (see Fig. 6). Saccular hair cells are divided into four orientation groups. The rostral region of the saccular macula has posteriorly oriented hair cells in front of, and directed towards, a group of cells that are oriented anteriorly. The posterior region of the saccular macula has a dorsally oriented hair cell group on the dorsal half and a ventrally oriented group on the ventral half. These cells generally change orientation to follow macula curvatures (Fig. 2). The transitions between groups of hair cells, such as that from dorsal to ventral groups, occur over one or two rows of hair cells (Fig. 3).Lagenar hair cells are oriented dorsally on the anterior side of the epithelium while the cells on the posterior side are oriented ventrally (Fig. 4). Both cell groups tend to curve as the maculae curves. The regions at which the two orientation groups overlap by a small amount is usually called the transition zone. Utricular maculae differed from the other epithelia in the size of the two orientation groups. The cells along the narrow anterior border are oriented medially while the cells of the large central groups are oriented peripherally. This pattern is also found within the lacinia (Fig. 5), a lateral extension of the anterior margin of the utricular epithelium. The actual transition occurs in a narrow region that has been called the striola (e.g. Werner, 1933). In this region, as discussed below, the lengths of the ciliary bundles are somewhat smaller than in surrounding regions (called the extrastriola region).3.3Hair cell types and densitiesSensory epithelia in all three species consist of different types of hair cell ciliary bundles. The most common ciliary bundle type is F1 (definitions according to Popper, 1977) and consists of a series of short graded stereocilia and a kinocilium that is no more than two times longer than the longest stereocilia. Type F2 bundles, on the other hand, are characterized by short graded stereocilia, and kinocilia that are approximately three times longer than the largest stereocilia. Ciliary bundle types are fairly discrete, but there are intermediary forms making it difficult to discriminate between various bundle types.F1 bundles are found in the central region of the saccular macula and F2 ciliary bundles at the periphery (Fig. 6) of all three species. A common feature of all three species is the presence of one row of isolated F2 cells at the ventral saccular epithelium. This row of ciliary bundles is only found at the ribbon-shaped posterior part of these epithelia (Fig. 7).Within the utricular macula, type F1 bundles are found in the central region of the striola and in the extrastriolar region. F2 bundles are found at the margins of the striola and within the lacinia. Similarly, the central part of the moon-shaped lagenar maculae is built up of type F1 bundles whereas the periphery consists of type F2 bundles (Fig. 8). Densities of ciliary bundles varied but are always smaller in the central region of the saccular, lagenar and striolar maculae. Particularly low densities were observed within the extrastriolar regions of the utricle.Densities of hair cells ranged from 48.5 to 60.4 bundles per 1000 μm2 (Table 2) on all three maculae in the three species. No significant differences were found between species in the mean densities or in the densities of the anterior, middle or posterior areas. Differences were close to significance in the caudal area (Table 2). In addition, densities did not differ between the rostral, middle and caudal areas within species (one-way ANOVA, F2,48=1.97, P=0.15).4Discussion4.1Gross and fine structure of anabantoid earsThe ears of B. splendens, M. opercularis and T. vittata resemble each other and the typical pattern of most teleosts other than for the otophysan fishes (goldfish and relatives) (Retzius, 1881; Popper, 1977, 1981; Popper and Coombs, 1982). The saccule is by far the largest end organ and the lagena the smallest.The acoustical coupling of the inner ears of labyrinth fishes to the lateral air-filled suprabranchial chambers (SBCs) is based on the reduction of otic bones surrounding the saccule and separating it from the SBC. According to Schneider (1941), the thin bony sheet may be replaced by a membraneous window which markedly improves hearing in some individuals of Macropodus. In essence, Schneider (1941) first proposed that this air-filled chamber could serve as an accessory hearing organ, a role that is similar to the swim bladder in fishes that have connections between this organ and the inner ear, or air bubbles that are closely associated with the ear, as in the mormyrid fishes (Stipetić, 1939).In contrast to the lagenar and utricular maculae, the saccular maculae do not follow the shape of the otolith. The ovoid sagittae are much larger than the corresponding J-shaped sensory epithelia.4.2Hair cell orientation patternsHair cell orientation patterns within the lagenar and utricular maculae are quite similar to those described in other teleosts (Popper, 1977, 1980, 1981). However, significant differences from many other teleost groups are found in the saccule. Five hair cell orientation patterns have been identified on saccular maculae in different teleosts (Popper, 1981; Popper and Coombs, 1982). Except for the otophysans (goldfish and relatives) and a few non-otophysan species, saccular maculae generally have two horizontally and two vertically oriented groups of ciliary bundles (Popper and Coombs, 1982). The anabantoids described here represent the ‘opposing’ pattern described by Popper and Coombs (1982) in that the fish have two opposing horizontally oriented ciliary bundles on the anterior saccule. Vertically oriented bundles are found on the posterior ribbon-shaped part. This pattern is encountered in other anabantoids (e.g. Popper and Hoxter, 1981) and in the unrelated deep-sea myctophids (lantern fishes) (Popper, 1977).Popper and Coombs (1982) argued that there is a close correlation between the presence of an air bubble near the ear or a connection between the swim bladder and the ear with specializations in the structure of the anterior end of the saccular macula. They also argued for a correlation between such saccular epithelial specializations and enhanced hearing capabilities as compared to fishes that have no connections to an air bubble and the ‘general’ saccular hair cell orientation pattern. The results from the three species of anabantoids reported here tend to support this hypothesis.Thus, based upon the morphological results we not only show a strong correlation between the saccular structure of these three species and the presence of the suprabranchial organ as a hearing structure, but we would predict that were hearing studies to be done on the Siamese fighting fish Betta, it could be shown that it too has a wide hearing bandwidth (ranging from 100 Hz to 5 kHz).4.3Sensory hair cellsThe ciliary bundle types of gouramis investigated are basically similar to those found in other fishes. In the central populations the ratio of kinocilium to stereocilia length is between 1:1 and 2:1 (F1 type). At the margins of the sensory epithelia cells have bundles with ratios of about 3:1 (F2 type). These cells have very short stereocilia. Platt and Popper (1981, 1984) described similar patterns in the blue gourami T. trichopterus.4.4Interspecific differences between labyrinth fishesOur results show that all three species studied have specialized saccular sensory epithelia, although the function of this type of specialization is still not understood (Popper and Fay, 1999). These results support the data in the literature (Ladich and Yan, 1998) showing that two of these species, Trichopsis and Macropodus, have an extended hearing bandwidth and increased sensitivity when compared to fishes without such specializations. Fishes without hearing specializations tend to have saccular maculae where the rostral region is not very much deeper (from dorsal to ventral margin) than the caudal region. While we have not done statistical comparisons, examination of the literature shows that the rostral end of the saccular epithelium in non-specialists is generally less than twice the depth of the caudal region (e.g. Popper, 1977, 1981). In these anabantoids, however, the rostral end of the saccular macula is three times as deep as the caudal macula and the caudally oriented cells are in front of the anteriorly oriented cells. Further supporting the hypothesis that hearing specializations are paralleled by changes in the shape and/or the hair cell orientation pattern on the rostral region of the saccular macula (Popper and Coombs, 1982) is the finding of Saidel and Popper (1987) which showed that the blue gourami T. trichopterus, a species with a saccular specialization similar to that reported here, had lower hearing thresholds than the kissing gourami H. temmincki that has a ‘general’ saccular pattern.Comparison of shape and size of sensory epithelia as well as hair cell orientation patterns did not reveal any significant differences between the species investigated. The J-shaped saccular epithelium is also found in the blue gourami (Popper and Hoxter, 1981; Saidel and Popper, 1987) and seems to be a characteristic of the family Belontiidae. Differences exist in the shape of the saccular maculae between belontiids and the kissing gourami H. temmincki, the only representative of the family Helostomatidae. The rostral portion of the saccular macula of this species is widened but lacks the characteristic ventral extension of belontiids. In addition, the total area of the saccular macula in Helostoma is approximately 40% larger as compared to similar-sized specimens of T. trichopterus.Differences in hair cell densities are minor among representatives of the belontiid family. (While we did not do a statistical analysis on cells at the very margins of the epithelia, examination of tissue from all of our animals leads to the impression that the density was similar between species.) No significant differences were found between B. splendens, M. opercularis and T. vittata and densities of ciliary bundles appear to be similar to those found in T. trichopterus. However, the densities of hair cells in T. trichopterus were slightly higher than in H. temmincki in each sample area, again indicating an interfamiliar difference between anabantoids. With the larger area and slightly higher hair cell densities, Helostoma had about 90% more hair cells than T. trichopterus (44 000 vs. 23 000, respectively – Saidel and Popper, 1987).Do these differences between families account for any difference in hearing abilities? Saidel and Popper (1987) observed that the number of hair cells in the saccules of these two anabantoid fishes inversely correlates with the saccular microphonic thresholds. The somewhat lower microphonic thresholds were almost always obtained from T. trichopterus which had fewer hair cells than Helostoma. In general, the auditory sensitivities of the fish resembled one another in that both species have very similar thresholds and bandwidth (Saidel and Popper, 1987; Yan, 1998). So far it is unknown if differences in hair cell densities result in any physiological differences such as sound localization abilities, frequency discrimination or detection of masked sound.4.5Behavioral and evolutionary considerationsOur findings, and the similarity in gross and fine structure of the inner ears of B. splendens, M. opercularis, T. vittata and T. trichopterus, suggest that no major differences in hearing sensitivities are to be expected among the belontiid family. Ladich and Yan (1998) measured auditory sensitivities by utilizing auditory brainstem response (ABR) recording techniques and observed the lowest thresholds in T. trichopterus (76 dB re 1 μPa at 800 Hz). The paradise fish and the croaking gourami were about 8 dB less sensitive and possessed higher most sensitive frequencies (MSFs) than the blue gourami but otherwise did not differ from one other. This difference in hearing capacities among belontiids might rather be attributed to the size differences of the peripheral suprabranchial chambers than to fine structural differences of the inner ears such as number of hair cells. One possibility is that smaller pharyngeal air bubbles in smaller species such as Macropodus and Trichopsis might have higher resonant frequencies than larger bubbles, but resonance properties of constrained air bubbles, such as these (and the swim bladder) are hard to predict based upon bubble size alone (Popper, 1971, 1974; Sand and Enger, 1973a,b; Clarke et al., 1975).Are differences in auditory sensitivities between the anabantoids investigated (Ladich and Yan, 1998) related to their acoustical behavior? Dominant frequencies of sounds correspond with best hearing bandwidth in T. vittata (1–2 kHz). However, B. splendens, M. opercularis and T. trichopterus which communicate primarily using visual signals, and only incidentally with sound, have similar or even better hearing sensitivities than T. vittata and did not differ in their inner ear morphology. These findings strongly suggest that enhanced sound-detecting abilities in anabantoids evolved prior to, or independently of, the evolution of pectoral sound-generating mechanisms in the genus Trichopsis. Most likely the SBC is an adaptation to waters poor in oxygen and the improvement of hearing a by-product of this process. A similar development obviously took place in otophysans. The evolution of the Weberian apparatus resulted in a pronounced improvement of hearing capacities in cypriniforms, catfishes, and characiforms. However, auditory sensitivities in vocalizing otophysans resemble those of non-vocalizing species and most sensitive frequencies lack a clear relationship to the main energies of sounds (Ladich, 1999). Interestingly, only a few representatives of cypriniforms, the most primitive otophysan group, are known to be vocal and in no case was there any vocal organ described.This again points to the fact that constraints other than optimization of acoustical communication is likely to have caused the ancestors of otophysans, and perhaps fishes in general, to develop and enhance their sound-detecting abilities. A major selective pressure might have been the analysis of the auditory scene which means separating sounds of different origins due to differences in their frequency content or temporal patterns (Bregman, 1990). This could have been a major advantage in avoiding predators and detecting prey (Popper and Fay, 1993; Ladich, 1999).If one accepts the idea that detection of the general auditory scene is of importance for fishes, and particularly for fishes that may live in dark or murky waters, the evolution of mechanisms to enhance detection of higher frequencies than found in most hearing non-specialists may have been a response to particular environments in which species ancestral to hearing specialists lived. Rogers and Cox (1988) pointed out that the transmission characteristics of sound in shallow water (up to several meters in depth) are quite different than in deeper water. In shallow waters, there is a very high attenuation rate for low frequencies that is correlated with depth. While this would not have any impact on fishes living in deeper waters, fishes living in shallow waters would only be able to detect sounds from very near-by sources, unless they have evolved hearing to higher frequencies (Schellart and Popper, 1992). We speculate that anabantoids, like otophysans, clupeids (herrings and relatives), and a number of other diverse teleost taxa may have arisen in shallow waters and evolved high frequency hearing. The fact that the specificities of the specializations are so different in various species argues for the evolution of high frequency hearing several times. For example, as the anabantoids evolved the ability to use the air bubble in the pharyngeal chamber for high frequency hearing, otophysans evolved Weberian ossicles while clupeids evolved a highly specialized utricle (e.g. Mann et al., 1997, 1998).In contrast, there are many other fishes currently living in relatively shallow waters that do not have hearing specializations. 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Springer, New York, pp. 363–411. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B980D08A04604E69176319ABDFB77351E8A67B5.txt b/test/dataset/in/resources/corpus/Clean_0B980D08A04604E69176319ABDFB77351E8A67B5.txt new file mode 100644 index 0000000..feba1ad --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B980D08A04604E69176319ABDFB77351E8A67B5.txt @@ -0,0 +1 @@ +geronbgeronbJournals of Gerontology: Series B1758-53681079-5014Oxford University Press10.1093/geronb/gbp003Journal of Gerontology: Psychological SciencesArticlesEffects of Optic Flow Speed and Lateral Flow Asymmetry on Locomotion in Younger and Older Adults: A Virtual Reality StudyChouYing-hui1WagenaarRobert C.1SaltzmanElliot1GiphartJ. Erik1YoungDaniel1DavidsdottirRosa2Cronin-GolombAlice21Department of Physical Therapy and Athletic Training, Boston University, Massachusetts2Department of Psychology, Boston University, MassachusettsAddress correspondence to Ying-hui Chou PhD, ScD, Department of Occupational Therapy, Medical College, Fu-Jen Catholic University, No. 510, Zhongzheng Road, Xinzhuang City, Taipei County 242, Taiwan. Email: yinghuichou@gmail.com32009103200964B222223182200820122008© The Author 2009. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2009The purpose of the study is to investigate whether there are age-related differences in locomotion due to changes in presence of vision, optic flow speed, and lateral flow asymmetry using virtual reality technology. Gait kinematics and heading direction were measured using a three-dimensional motion analysis system. Although older and younger adults were affected differentially by the availability of vision, a greater dependence on optic flow information in older adults during walking was not found. Linear relations were observed between walking performance and flow speed as well as heading direction and flow asymmetry. The findings suggest that the ability to integrate optic flow information into the multimodal system for assessment of walking speed and heading direction is comparable in older and younger adults.Optic flow speedVirtual realityLocomotionInterlimb coordinationHeading directionVisual dependenceTHE control of locomotion depends on the integration of information from visual, vestibular, and somatosensory systems. When information from one or more sensory systems is withdrawn or becomes less reliable, the central nervous system changes its weighting of sensory inputs to maintain appropriate postural responses (van der Kooij, Jacobs, Koopman, & van der Helm, 2001). A number of studies have reported that older adults are more dependent on the availability of vision for posture and gait than are younger adults. For example, older adults show greater sway with eyes closed during quiet standing (Choy, Brauer, & Nitz, 2003; Hay, Bard, Fleury, & Teasdale, 1996; Manchester, Woollacott, Zederbauer-Hylton, & Marin, 1989) and display diminished head stability with eyes closed during walking (Cromwell, Newton, & Forrest, 2001, 2002) compared with younger adults. A greater reliance on vision in older adults is considered a central sensory reweighting deficit (Teasdale, Stelmach, & Breunig, 1991) and/or age-related peripheral sensory loss in the vestibular and somatosensory systems. However, some studies (Anderson, Mulder, Nienhuis, & Hulstijn, 1998; Konczak, 1994; Schubert, Prokop, Brocke, & Berger, 2005) did not find significant differences in locomotion between younger and older adults due to changes in visual information, especially when optic flow parameters were involved. Additionally, research findings on age-related changes in perception of optic flow are equivocal. Billino, Bremmer, and Gegenfurtner (2008) and Atchley and Andersen (1998) found that perception of radial optic flow was not affected by age, whereas Warren, Blackwell, and Morris (1989) and Gilmore, Wenk, Naylor, and Stuve (1992) reported increased thresholds for heading detection and motion coherence, respectively, with age. Whether older adults are more dependent on optic flow information compared with younger adults during locomotion is still open to further investigation.Optic flow patterns and their changes are used to assess speed and direction of self-motion (Gibson, 1958). Previous studies demonstrate that gait parameters are inadvertently modulated by manipulations of optic flow speed. When optic flow speed decreases, walking speed (Prokop, Schubert, & Berger, 1997; Schubert et al., 2005; Varraine, Bonnard, & Pailhous, 2002) and stride length (Prokop et al., 1997; Schubert et al., 2005) increase; decreasing optic flow speed shows opposite effects. The manipulation of lateral asymmetry of optic flow speed influences heading direction in honey bees (Srinivasan, Lehrer, Kirchner, & Zhang, 1991) and young adults (Duchon & Warren, 2002). Bees and young adults steered away from the faster moving wall with larger differences of optic flow speeds, resulting in more drift. The purpose of the present study is to examine whether there are age-related changes in gait patterns and heading direction as a function of the presence of vision (eyes open vs. blindfolded vs. virtual environment), optic flow speed, and lateral flow asymmetry.In addition to walking speed and stride parameters, how limbs dynamically coordinate in response to changes in optic flow speed during locomotion remains to be determined. Wagenaar and colleagues have shown that shifts between patterns of interlimb coordination would occur when an appropriate control parameter is scaled (Donker, Beek, Wagenaar, & Mulder, 2001; Wagenaar & van Emmerik, 2000). For example, when walking speed (i.e., a control parameter) is systematically increased from 0.2 to 1.6 m/s, the arm-to-leg movement frequency ratio gradually changes from 2:1 (a less stable pattern) to 1:1 (a more stable pattern). The transition between coordination patterns is accompanied by an increased standard deviation of relative phase between arm and leg movements (Wagenaar & van Emmerik, 2000). Clinically, the degree of flexibility in interlimb coordination has been found to correlate with bradykinesia and rigidity in Parkinson's disease (Winogrodzka, Wagenaar, Booij, & Wolters, 2005) and with asymmetry in stroke (Ford, Wagenaar, & Newell, 2007). Older adults with risk for falls showed a reduced flexibility in their coordination of walking at speeds higher than 0.8 m/s compared with the walking patterns of healthy older adults (Wagenaar, Holt, Kubo, & Ho, 2003). The present study will investigate whether optic flow speed is an appropriate control parameter for changes in the coordination dynamics during walking and whether the coordination dynamics in response to changes in optic flow speed is age-related.The first hypothesis is that older adults are more dependent on the presence of visual information during walking compared with younger adults, as shown by a reduced walking speed, stride length, and stride frequency during the blindfolded condition compared with the conditions of normal vision and virtual reality. Second, we expect that older adults will modulate their walking performance and heading direction to a greater extent in response to changes in optic flow speed and lateral flow asymmetry than younger adults. Third, for both younger and older adults, we hypothesize that walking speed, stride length, and stride frequency will be modulated linearly with manipulations of optic flow speed during overground walking. For example, increasing optic flow speed will result in a reduced walking speed, stride frequency, and stride length. It is also expected that the interlimb coordination will demonstrate a change from a 1:1 to a 2:1 arm-and-leg frequency ratio with increasing optic flow speed. Finally, with regard to heading direction, it is hypothesized that participants will drift away from the wall that is moving faster, and the degree of lateral drift will be proportional to the level of lateral flow asymmetry.METHODSParticipantsThe participants consisted of two groups: 16 younger adults (8 men, 8 women; 15 right-handers, 1 left-hander; mean age 22.2 ± 3.4 years; age range 18−30 years) and 17 older adults (8 men, 9 women; 16 right-handers, 1 left-hander; mean age 60.5 ± 8.7 years, age range 46−73 years). Older adults were recruited through local newspaper advertisements and personal contacts, and younger adults were undergraduates at Boston University. Exclusion criteria included walking disability or history of leg, knee, hip impairments, serious cardiac disease, other serious chronic medical illness, history of traumatic brain injury, psychiatric or neurological diagnoses, history of alcoholism or other drug abuse, or history of eye disease or other abnormalities as noted on neuro-ophthalmological examination. Individuals whose corrected binocular acuity was poorer than 20/40 were excluded. Informed consent approved by the Institutional Review Board of Boston University and conforming to the 1964 Declaration of Helsinki was obtained prior to participation. All participants were paid for participation.MaterialThe VR system.—A virtual hallway was created using World ToolKit Release 9 (Sense8, San Francisco, CA) on an Onyx2 Reality graphics work station (Silicon Graphics, Inc., Mountain View, CA). The hallway was composed of two sidewalls of white random dots on a black background (Figure 1). The width of the hallway was 2 m, the height of the hallway 2.55 m, and the depth of the hallway 8.88 m. The middle of the virtual hallway along the anteroposterior axis was always aligned with a reference axis in the real environment. The orientations of the virtual hallway in the frontal and sagittal planes were calibrated with respect to each participant's head orientation, whereas the participant was instructed to stand upright and face forward. The visual scene was displayed on a ProView 60 head-mounted display (HMD; Kaiser Opto-Electronics, Inc., Mountain View, CA) weighting 1.75 lbs. This HMD contained two active LCD panels (640 × 480 resolution, true color, 60 Hz) and had a 60° field-of-view (diagonal) with 100% overlap to allow for true stereo viewing. Participant's field of view was restricted to the VR environment by a mask that occluded vision outside the LCD panels. Head coordinates were tracked in real time, using an IS 900 LAT system (InterSense, Burlington, MA), and the information was used to update the visual scene with a delay of four frames (67.7−83.3 ms). The movement tracking system covered the overground walking area with an approximate error of less than 4 mm root mean square for position data and 0.1° for orientation data. The optic texture of the virtual hallway's walls in the anteroposterior direction could be manipulated by a computer. The validity of this VR simulation has been demonstrated by Giphart, Chou, Kim, Bortnyk, and Wagenaar (2007).Figure 1.Display of virtual reality environment.Three-dimensional kinematics.—Three-dimensional kinematic data were collected using an Optotrak 3020 System (Northern Digital Inc., Waterloo, ON, Canada), with a spatial resolution of 0.1 mm. One position sensor was placed on each side of the walkway, and a third position sensor was located at the end of the walkway in order to provide an environmental reference plane for capturing bilateral locomotor movements for at least eight strides. Calibrations among the three position sensors were accepted when the mean error was 1.5 mm or less. Infrared light-emitting diodes (IREDs) were placed on participant's chin (lower mandible) and bilaterally on the ankle (lateral calcaneus), hip (anterior superior iliac spine), wrist (radiocarpal joint), and shoulder (humeral head). The instantaneous position of each IRED was sampled at a rate of 100 Hz and stored to disk for further analysis.Experimental ProcedureAfter intake, three experimental conditions were carried out in a fixed order, that is, Trial 1 (manipulation of vision), Trial 2 (manipulation of optic flow speed), and Trial 3 (manipulation of lateral flow asymmetry). The experimental procedure was described as follows.Intake.—Visual dependence was assessed by using a rod and frame test (Azulay, Mesure, Amblard, & Pouget, 2002). Participants were presented with a 1.5-m horizontal line that was slightly tilted at the outset of each run (initial tilt ranging from 9° to 12°) in a large screen. The angle of the line was gradually altered to become more horizontal by 1° increments. Participants were asked to indicate when the rod was horizontal. Mean deviation (degree) averaged from 10 runs was taken as the measure of the participants’ level of visual dependence. All stimuli sequences were created using Adobe Photoshop and PowerPoint for presentation.Trial 1.—Participants were trained to walk 8 m at 0.8 ± 0.05 m/s because the state of interlimb coordination at this walking speed is relatively unstable (Wagenaar & van Emmerik, 2000). Therefore, if there were any changes in coordination pattern as a result of changes in optic flow speed, it would be observed more sensitively. Three vision conditions were presented in a fixed sequence: (1) eyes opened (EO) condition—participants were instructed to walk down the middle of a walkway in the laboratory with eyes open; (2) blindfolded (BF) condition—participants were instructed to walk down the middle of the walkway in the lab blindfolded; (3) virtual reality (VR) condition—participants were instructed to walk down the middle of a virtual hallway created by the virtual reality system. In the VR condition, the optic flow speed was set by feedback control to equal −1 times the participant's walking speed in the anteroposterior direction. The minus sign indicated that the optic flow was programmed to move in a direction opposite to the participant's walking direction. Participants had the opportunity to practice walking in the virtual environment for at least 10 min, until they demonstrated that they were able to walk comfortably at 0.8 ± 0.05 m/s. For each condition, at least five runs were performed. Each participant received feedback on walking speed after each run. The average walking speed was measured in real time with two sets of infrared switches (Safe House, Fort Worth, TX) placed 6 m apart along the walkway.Trial 2.—Five different optic flow speed conditions (0, −0.4, −0.8, −1.2, and −1.6 m/s) were presented in random order across participants. Five consecutive runs were performed for each optic flow speed condition, yielding 25 runs in total for each participant. Participants were instructed to walk at 0.8 m/s throughout the experimental session. During Trials 2 and 3, the speed of optic flow was held constant within a condition and not a function of the participant's walking speed. For example, when the optic flow speed was −0.8 m/s, participants had the impression that they were walking at the trained speed through a stationary hallway; when the optic flow speed was faster/slower than −0.8 m/s, participants had the impression that they were walking more quickly/slowly than the trained speed. A special case was that when the optic flow speed was equal to 0 m/s, participants had the impression that they were not moving forward relative to the hallway, even though they were walking (i.e., a situation similar to that experienced visually during walking on a treadmill). No feedback on walking speed was provided during Trials 2 and 3.Trial 3.—Participants were trained to walk at 0.8 ± 0.05 m/s again in the VR environment for at least five runs prior to Trial 3. In Trial 3, the optic flow speed in one visual field was always equal to −0.8 m/s, whereas the flow speed in the other visual field was 0, −0.4, −0.8, −1.2, or −1.6 m/s. The 10 different optic flow speed conditions were presented in random order across participants. Five runs were performed for each condition, yielding 50 (2 × 5 × 5) runs in total for each participant.Data AnalysisFor kinematic analysis, the position data were filtered using a zero-lag, fourth-order Butterworth low-pass filter with a cutoff frequency of 5 Hz. Shoulder and wrist time series were used to compute the angular displacement of arm movements and hip and ankle time series to calculate the angular displacement of leg movements relative to vertical. Forward wrist or ankle movement resulted in a positive angle. Each stride cycle was identified by two consecutive maxima from the angular position data of leg movements. All the variables were computed for only the middle six strides in order to avoid acceleration and deceleration variations at the beginning and at the end of the distance walked. The data were processed using MatLab (The MathWorks, Inc., Natick, MA).Walking speed and stride parameters.—The average walking speed was estimated by the displacement of the chin time series in the anteroposterior axis divided by the time it took the participant to travel for six stride cycles. Stride length was calculated from both the left and right ankle time series, separately, by dividing the displacement in the anteroposterior axis divided by 6 (i.e., the number of strides). Stride frequency was calculated in strides per second (as the inverse of the time it took the participant to travel six stride cycles divided by the number of strides).Relative power index.—The angular displacement data of the arm and leg movements were used for the analysis of interlimb coordination. Figure 2a shows an example of the time series data of arm and leg movements during walking at lower speeds. The power spectral density function was used to transform the time series data into movement frequencies and corresponding spectral power for leg (Figure 2b) and arm movements (Figure 2c). The spectral power was normalized by dividing each frequency power by the mean power calculated over the 0.2- to 2.5-Hz frequency range for each run.Figure 2.Fourier transform of time series of arm and leg movements.A step is heel contact of one leg to heel contact of the other leg, a stride from heel contact to heel contact of the same leg. As in Figure 2b, the frequency with the largest power was regarded as the stride frequency (Wagenaar & van Emmerik, 2000). The step frequency was the peak at twice the stride frequency. The corresponding power at the stride and step frequencies of arm movements was identified using the stride and step frequencies from the leg movements, respectively (see Figure 2c). To quantify the frequency ratio of arm and leg movements, a relative power index (RPI) was calculated using the following Equation:(1)where P1 is the power at the stride frequency observed in the arm movements and P2 is the power at the step frequency observed in the arm movements (Figure 2c). RPI ranges from −1 to 1. RPI equal to 1 indicates a 1:1 frequency ratio between arm and leg movements. RPI equal to −1 points out a 2:1 coordination pattern in which the arms cycle twice during every stride cycle of the legs.Drift.—The average of left and right hip position data in the mediolateral axis was used as the body reference point. The drift is defined as the difference in mediolateral coordinates between the last and the first stride. A positive value indicates rightward drift and a negative value indicates leftward drift. Each participant's drift data in Trials 2 and 3 would be normalized by his or her baseline measures of drift in the VR condition during Trial 1.Statistical AnalysisVisual dependence.—The difference of visual dependency between the older and younger adults was examined using a one-way analysis of variance.Trial 1: Manipulation of visioen.—Data across the five runs were averaged for each condition. Whether the average walking speed in the older adults and the younger adults was significantly different from 0.8 m/s was evaluated by means of Wilcoxon one-sample signed-rank tests (with a two-tailed level of significance alpha = .05). In order to test whether there were significant effects of group and vision conditions, a general linear model was performed with a within-subject factor for Vision (EO, BF, and VR) and a between-subject factor for Group (younger and older adults).Trial 2: Manipulation of optic flow speed.—Data across the five runs were averaged for each condition. A general linear model was performed with a linear contrast for Optic Flow Speed (within-subject factor, five levels) and a between-subject factor for Group.Trial 3: Manipulation of lateral flow asymmetry.—Data across the five runs were averaged for each condition. A general linear model was performed with a within-group factor for Visual Field (i.e., optic flow speed changed in the left or the right visual field), a linear contrast for Optic Flow Speed (within-subject factor, five levels), and a between-subject factor for Group.Post hoc analyses were employed in all three trials in the case of a significant interaction effect or a significant main effect of a variable with more than two levels. Multiple comparisons were conducted with Bonferroni corrections. All analyses were performed using SPSS (SPSS, Inc., Chicago, IL).RESULTSThere was no statistically significant difference between younger and older adults in visual dependency as measured by the rod and frame task, F(1, 31) = 0.68, p = .42.Trial 1: Manipulation of VisionWalking speed and stride parameters.—The walking speeds in the younger adults were not significantly different from 0.8 m/s (EO: p = .86; BF: p = .15; VR: p = .80). The mean walking speed in the older adults was significantly lower in BF than 0.8 m/s (p = .03). However, no significant differences were found for EO (p = .36) and VR (p = .75). At all conditions, the 95% confidence intervals of the mean walking speed were within 0.8 ± 0.05 m/s for both groups.Analysis of walking speed yielded a significant main effect for Vision, F(2, 62) = 18.76, p = .0001, and a significant interaction effect between Vision and Group, F(2, 62) = 6.33, p = .003 (Figure 3a). Post hoc analyses revealed that a significant effect of Vision was observed in the older adults, F(2, 32) = 17.97, p = .0001, but not in the younger adults, F(2, 30) = 2.66, p = .09. Post hoc analyses of the vision effect in the older adults showed that walking speed in BF was significantly lower than that in EO (p = .0001) and VR (p = .006).Figure 3.Influence of vision condition on (a) walking speed and (b) stride length in younger and older adults. Error bars show 95% confidence intervals around the means. EO = eyes opened; BF = blindfolded; VR = virtual reality.Significant main effects for Vision, F(2, 62) = 12.38, p = .0001, and Group, F(1, 31) = 4.42, p = .05, were observed for stride frequency. Older adults had a significantly higher stride frequency (1.10 Hz) than the younger adults (1.03 Hz). Post hoc analyses revealed that stride frequency in BF (1.14 Hz) was significantly higher than that in EO (1.01 Hz, p = .001) and VR (1.05 Hz, p = .001).A significant main effect for Vision, F(2, 62) = 32.12, p = .0001, and a significant Vision × Group interaction effect, F(2, 62) = 5.51, p = .006 (Figure 3b) were found for stride length. Post hoc analyses revealed that (1) there were significant differences among vision conditions in the older adults, F(2, 32) = 22.25, p = .0001, as well as the younger adults, F(2, 30) = 11.24, p = .0001, and (2) the group effect was significant only in BF, F(1, 31) = 8.24, p = .007, indicating that the older adults had significantly shorter stride length compared with the younger adults in BF. In addition, both groups showed that the greatest stride length was observed in EO, followed by VR, and then BF.The RPI.—Analysis of RPI yielded a significant main effect for Vision, F(2, 62) = 3.88, p = .03. Post hoc analyses showed that the RPI in VR (0.74 ± 0.34) was significantly greater than that in EO (0.65 ± 0.33, p = .03).Drift.—A significant main effect for Vision, F(2, 62) = 4.51, p = .02, was found. The average drifts are 32.30 (SD = 50.44), −119.82 (SD = 395.66), and 32.37 (SD = 62.20) mm for the EO, BF, and VR conditions, respectively. Post hoc analyses showed no significant effect among the three vision conditions.Trial 2: Manipulation of Optic Flow SpeedWalking speed and stride parameters.—The linear main effects for Optic Flow Speed were significant for walking speed, F(1, 31) = 16.42, p = .0001, and stride frequency, F(1, 31) = 22.87, p = .0001, indicating that both groups decreased walking speed (see Figure 4a) and stride frequency (see Figure 5a) when the optic flow speed was increased. The main Group effects were significant for walking speed, F(1, 31) = 6.38, p = .02, and stride frequency, F(1, 31) = 9.52, p = .004, showing that older adults walked at a higher speed (0.86 ± 0.08 m/s) and stride frequency (1.09 ± 0.11 Hz) than the younger adults (0.81 ± 0.08 m/s; 1.00 ± 0.09 Hz, respectively). No significant interaction effects between Group and Optic Flow Speed were found for walking speed, F(1, 31) = 0.42, p = .52, and stride frequency, F(1, 31) = 1.43, p = .24. No significant main or interaction effects were found for stride length.Figure 4.Influence of optic flow speed on walking speed in (a) Trial 2 and (b) Trial 3. Error bars show 95% confidence intervals around the means.Figure 5.Influence of optic flow speed on stride frequency in (a) Trial 2 and (b) Trial 3. Error bars show 95% confidence intervals around the means.The RPI.—There was a significant linear effect for Optic Flow Speed, F(1, 31) = 5.73, p = .02 (see Figure 6a). The RPI in both groups decreased with the increment of optic flow speed, and the impacts of Optic Flow Speed on RPI were not significantly different between the older and the younger adults, F(1, 31) = 3.61, p = .07. As shown in Figure 6a, the mean RPIs were positive, indicating that the overall distribution of RPI was confined primarily to the 1:1 frequency ratio.Figure 6.Influence of optic flow speed on relative power index in (a) Trial 2 and (b) Trial 3. Error bars show 95% confidence intervals around the means.Drift.—No significant main or interaction effects were observed (Figure 7a).Figure 7.Influence of optic flow speed on drift in (a) Trial 2 and (b) Trial 3. Error bars show 95% confidence intervals around the means.Trial 3: Manipulation of Lateral Flow AsymmetryWalking speed and stride parameters.—The linear effects for Optic Flow Speed were significant for walking speed, F(1, 31) = 16.69, p = .0001, and stride frequency, F(1, 31) = 11.99, p = .0001, indicating that both groups decreased walking speed (see Figure 4b) and stride frequency (see Figure 5b) when the optic flow speed in unilateral visual field was increased. The Group effects were significant for walking speed, F(1, 31) = 16.96, p = .0001, and stride frequency, F(1, 31) = 14.78, p = .001, showing that older adults walked at a higher speed (0.89 ± 0.08 m/s) and stride frequency (1.11 ± 0.11 Hz) than the younger adults (0.81 ± 0.07 m/s; 0.99 ± 0.10 Hz, respectively). No significant interaction effects between Group and Optic Flow Speed were found for walking speed, F(1, 31) = 0.09, p = .76, and stride frequency, F(1, 31) = 0.35, p = .56. No significant main or interaction effects were found for stride length.The RPI.—A significant linear effect for Optic Flow Speed, F(1, 31) = 7.23, p = .01, was found. The RPI in both groups decreased with the increment of optic flow speed in left or right visual field, and the effects of Optic Flow Speed on RPI were not significantly different between the older and the younger adults, F(1, 31) = 0.99, p = .33. As shown in Figure 6b, the mean RPIs were positive, indicating that the overall distribution of RPI was confined primarily to the 1:1 coordination pattern.Drift.—A significant linear effect for Optic Flow Speed was observed, F(1, 31) = 26.37, p = .0001, indicating that both groups drifted away from the wall that was moving faster, and the degree of drift was positively related to the difference in optic flow speeds between the two walls (see Figure 7b). The effects of Optic Flow Speed manipulations were not significantly different between the older and the younger adults, F(1, 31) = 0.04, p = .85. No other significant main or interaction effects were observed.DISCUSSIONThe purpose of the study was to investigate whether older adults show different modulations in locomotion compared with younger adults in response to availability of vision, optic flow speed, and lateral flow asymmetry. The result in Trial 1 corroborates previous findings showing that, in terms of walking speed and stride length, older and younger adults were affected differentially by the availability of vision (Choy et al., 2003; Cromwell et al., 2001, 2002; Hay et al., 1996; Manchester et al., 1989). However, in Trials 2 and 3, the effects of flow speed and lateral flow asymmetry were not influenced by age. Our findings, complementing previous studies (Anderson et al., 1998; Konczak, 1994; Schubert et al., 2005), did not support a greater dependence on optic flow information in older adults during walking. Additionally, no age-related difference was found for the rod and frame test. Taken together, the findings suggest that the ability to integrate optic flow information into the multimodal system for assessment of walking speed and heading direction was comparable in older and younger adults. When vision was deprived, adapted walking performance in older adults emerged possibly due to changes in vestibular or somatosensory function or previous experiences, making them walk more cautiously. We are aware that some participants in the older group were younger than 65. Could the insignificant interaction effect between age and optic flow speed be attributed to the existence of subgroups in the older group? We, thus, divided the older group into two by a cutoff age of 65 years and ran the statistical analyses again. The findings with three groups were not different from those with two groups, indicating that this possibility can be ruled out.Confirming previous results (Prokop et al., 1997; Schubert et al., 2005; Varraine et al., 2002), changes in absolute value of optic flow speed were linearly and negatively correlated with variations in walking speed, stride frequency, and RPI. The finding can be interpreted as that optic flow speed presented in Trials 2 and 3 was compared with the flow speed perceived during training (i.e., −0.8 m/s). When the flow speed was faster/slower than −0.8 m/s, participants felt that they were walking more quickly/slowly than the trained speed, and they modulated locomotion inadvertently or intentionally to keep their walking speed as instructed. Interestingly, even when changes in flow speed occurred in the unilateral wall only, the effects of optic flow speed could still be observed. One possible explanation is that the perceived flow speed might be the average of the flow speeds from the two walls. More experiments are needed to understand how optic flow speed is perceived when flow speeds are different in two visual fields.With respect to the effects of lateral flow asymmetry, participants drifted away from the wall that was moving faster, and the degree of drift was positively related to the difference of optic flow speeds between the two walls. This agrees with data from previous studies (Duchon & Warren, 2002; Srinivasan et al., 1991), suggesting that when optic flow speeds are different in two visual fields, participants consider their trajectory as curved and would drift to a balance point at which the flow speed in both sides appears equal.In all, 30 of 33 participants retained a pattern of 1:1 arm-to-leg frequency ratio across all conditions and did not show any transitions with the manipulations of optic flow speed. This finding suggests that optic flow speed may not be an appropriate control parameter for changes in interlimb coordination during walking. However, it may also be the case that walking overground at 0.8 m/s reduced the likelihood of finding effects of optic flow speed manipulations on interlimb coordination. It appears that more stable patterns (i.e., 1:1 arm-to-leg frequency ratio) were observed during overground walking at 0.8 m/s than during treadmill walking at the same speed, and there is a possibility that the transition point during overground walking is at a lower walking speed compared with treadmill walking. Future work on interlimb coordination during overground walking with a systematic manipulation of walking speed will provide further insight into coordination dynamics.Older adults walked significantly faster than younger adults in Trials 2 and 3, but not in Trial 1. There are two possible explanations. First, the reported preferred walking speed was between 0.82 and 1.38 m/s for older adults and around 1.20 m/s for younger adults (Malatesta et al., 2004; McGibbon & Krebs, 2001; Patel et al., 2006). Because the trained walking speed might not be the preferred walking speed for all the participants, older adults who could not maintain the muscle strength needed for smooth walking at 0.8 m/s, or found the exposure to the VR environment challenging (Giphart et al., 2007), might have unintentionally walked at their preferred speed to reduce gait variability (i.e., to improve stability). Second, it has been reported that the perceived speed reduced after prolonged exposure to optic flow (Krekelberg, van Wezel, & Albright, 2006; Smith, 1985). It is possible that the perceived speed in older adults was lower than that in younger adults after adaptation, thereby inducing higher walking speeds in older adults. Further studies are needed to test these hypotheses.In the current study, we provide evidence that older adults are able to integrate optic flow information into the multimodal system to monitor their walking speed and heading direction in much the same manner as younger adults. Future research on the fall-prone elderly is needed to understand whether perception of optic flow is degraded in this population and how they use the optic flow information to guide locomotion.FUNDINGThis research was supported by the National Institute of Neurological Disease and Stroke (grant 1R21 NS043730-01 awarded to A.C.-G.) and the Dudley Allen Sargent Research Fund (awarded to Y.-H.C.).We thank Dr. Terry Ellis and Ms. Julie Starr for help with participant recruitment.AndersonGAMulderTNienhuisBHulstijnWAre older adults more dependent on visual information in regulating self-motion than younger adults?Journal of Motor Behavior199830104113AtchleyPAndersenGJThe effect of age, retinal eccentricity, and speed on the detection of optic flow componentsPsychology and Aging199813297308AzulayJPMesureSAmblardBPougetJIncreased visual dependence in Parkinson's diseasePerceptual and Motor Skills20029511061114BillinoJBremmerFGegenfurtnerKRDifferential aging of motion processing mechanisms: Evidence against general perceptual declineVision Research20084812541261ChoyNLBrauerSNitzJChanges in postural stability in women aged 20 to 80 yearsJournals of Gerontology: Series A, Biological Sciences and Medical Sciences200358525530CromwellRLNewtonRAForrestGHead stability in older adults during walking with and without visual inputJournal of Vestibular Research200111105114CromwellRLNewtonRAForrestGInfluence of vision on head stabilization strategies in older adults during walkingJournals of Gerontology: Series A, Biological Sciences and Medical Sciences200257M442M448DonkerSFBeekPJWagenaarRCMulderTCoordination between arm and leg movements during locomotionJournal of Motor Behavior20013386102DuchonAPWarrenWHJr.A visual equalization strategy for locomotor control: Of honeybees, robots, and humansPsychological Science200213272278FordMPWagenaarRCNewellKMThe effects of auditory rhythms and instruction on walking patterns in individuals post strokeGait and Posture200726150155GibsonJJVisually controlled locomotion and visual orientation in animalsBritish Journal of Psychology195849182194GilmoreGCWenkHENaylorLAStuveTAMotion perception and agingPsychology and Aging19927654660GiphartJEChouY.-H.KimDHBortnykCTWagenaarRCEffects of virtual reality immersion and walking speed on coordination of arm and leg movementsPresence: Teleoperators and Virtual Environments200716399413HayLBardCFleuryMTeasdaleNAvailability of visual and proprioceptive afferent messages and postural control in elderly adultsExperimental Brain Research1996108129139KonczakJEffects of optic flow on the kinematics of human gait: A comparison of young and older adultsJournal of Motor Behavior199426225236KrekelbergBvan WezelRJAlbrightTDAdaptation in macaque MT reduces perceived speed and improves speed discriminationJournal of Neurophysiology200695255270MalatestaDSimarDDauvilliersYCandauRBen SaadHPréfautCCaillaudCAerobic determinants of the decline in preferred walking speed in healthy, active 65- and 80-year-oldsPflugers Archiv: European Journal of Physiology2004447915921ManchesterDWoollacottMZederbauer-HyltonNMarinOVisual, vestibular and somatosensory contributions to balance control in the older adultJournal of Gerontology198944M118M127McGibbonCAKrebsDEAge-related changes in lower trunk coordination and energy transfer during gaitJournal of Neurophysiology20018519231931PatelITuranoKABromanATBandeen-RocheKMuñozBWestSKMeasures of visual function and percentage of preferred walking speed in older adults: The Salisbury Eye Evaluation ProjectInvestigative Ophthalmology and Visual Science2006476571ProkopTSchubertMBergerWVisual influence on human locomotion. Modulation to changes in optic flowExperimental Brain Research19971146370SchubertMProkopTBrockeFBergerWVisual kinesthesia and locomotion in Parkinson's diseaseMovement Disorders200520141150SmithATVelocity coding: Evidence from perceived velocity shiftsVision Research19852519691976SrinivasanMVLehrerMKirchnerWHZhangSWRange perception through apparent image speed in freely flying honeybeesVisual Neuroscience19916519535TeasdaleNStelmachGEBreunigAPostural sway characteristics of the elderly under normal and altered visual and support surface conditionsJournal of Gerontology199146B238B244van der KooijHJacobsRKoopmanBvan der HelmFAn adaptive model of sensory integration in a dynamic environment applied to human stance controlBiological Cybernetics200184103115VarraineEBonnardMPailhousJInteraction between different sensory cues in the control of human gaitExperimental Brain Research2002142374384WagenaarRCHoltKGKuboMHoCLGait risk factors for falls in older adults: A dynamic perspectiveGenerations2003262832WagenaarRCvan EmmerikREResonant frequencies of arms and legs identify different walking patternsJournal of Biomechanics200033853861WarrenWHJrBlackwellAWMorrisMWAge differences in perceiving the direction of self-motion from optical flowJournal of Gerontology198944P147P153WinogrodzkaAWagenaarRCBooijJWoltersECRigidity and bradykinesia reduce interlimb coordination in Parkinsonian gaitArchives of Physical Medicine and Rehabilitation200586183189Decision Editor: Rosemary Blieszner, PhD \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B98637223569DFC105B062206637AA93134062C.txt b/test/dataset/in/resources/corpus/Clean_0B98637223569DFC105B062206637AA93134062C.txt new file mode 100644 index 0000000..fe3a04d --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B98637223569DFC105B062206637AA93134062C.txt @@ -0,0 +1 @@ +EXG5174S0531-5565(98)00078-310.1016/S0531-5565(98)00078-3Elsevier Science Inc.Table 1Means (± standard errors) for longevity and female fecundity indices of the short-lived and long-lived bean weevils lines legendTraitShort-lived lineLong-lived linet-testnmean ± S.E.nmean ± S.E.LongevityFemales9511.58 ± 0.279719.19 ± 0.5412.59aMales9211.61 ± 0.259315.42 ± 0.437.67aFecundityEarly (1–2 days)10123.83 ± 1.1510015.60 ± 1.214.93aLate (≥9 days)1000.36 ± 0.201003.59 ± 0.754.14aTotal10049.57 ± 1.8210048.02 ± 2.341.19legendn = The number of analyzed individuals.ap < 0.001.Table 2Means (± standard errors) for activities (in units/mg proteins) of SOD and catalase in the young (one-day-old) and old (10-day-old) virgin and mated females and males of the short-lived and long-lived lineslegendEnzymeShort-lived lineLong-lived lineYoungOldYoungOldSODFemalesVirgin31.69 ± 3.4822.57 ± 1.3929.59 ± 1.5427.40 ± 2.67Mated26.86 ± 1.2427.59 ± 0.55MalesVirgin31.87 ± 1.6419.14 ± 1.1832.49 ± 2.4223.79 ± 1.65Mated19.52 ± 1.5227.99 ± 3.28Grand Mean31.78 ± 1.9122.02 ± 0.9631.04 ± 1.5126.69 ± 1.09CatalaseFemalesVirgin29.62 ± 0.4229.89 ± 2.7227.43 ± 0.8234.77 ± 3.38Mated35.33 ± 5.9343.91 ± 6.18MalesVirgin8.84 ± 0.298.37 ± 0.998.03 ± 0.507.36 ± 0.15Mated10.99 ± 0.4612.79 ± 2.33Grand Mean19.23 ± 3.6621.15 ± 3.1617.73 ± 3.4434.71 ± 3.96legendSample sizes for each mean are 5. Grand means are calculated on the pooled data of the corresponding groups.Table 3Two-way ANOVA for SOD and catalase activities in the young (one-day-old) females and males originated from the short-lived and long-lived linesSource of variationdfSOD M.S. (×10−3)FCAT M.S. (×10−3)FLine10.320.077.585.24aSex12.750.581409.48974.27bLine × sex11.140.230.130.09Error164.771.47ap < 0.05;bp < 0.001.Table 4Two-way ANOVA for SOD and catalase activities for virgin weevils of different ages (one day old and 10-days old) from short- and long-lived linesSource of variationdfSOD M.S. (×10−3)FCAT M.S. (×10−3)FFemalesLine13.960.631.150.27Age140.866.51a10.862.58Line × age113.332.1212.012.85Error166.284.21MalesLine112.583.279.922.35Age1159.4641.48b5.991.42Line × age19.762.540.000.00Error163.844.22ap < 0.05;bp < 0.001.Table 5Three-way ANOVA for SOD and catalase activities in the old (10-day-old) virgin and mated females and males from the short- and long-lived linesSource of variationdfSOD M.S. (×10−3)FCAT M.S. (×10−3)FLine (A)172.4615.59b13.590.79Sex (B)146.289.96b3070.31179.69bMating status (C)116.193.49126.507.40aA × B114.913.2115.950.93A × C10.030.007.480.43B × C10.140.0333.691.97A × B × C19.842.122.160.13Error324.6517.09ap < 0.05;bp < 0.001.Original PapersActivity of superoxide dismutase and catalase in the bean weevil (acanthoscelides obtectus) selected for postponed senescenceSeslijaDarkaŠešlijaaBlagojevicDuškoBlagojevićaSpasicMihajloSpasićaTucicNikolaTucića*tucic@opennet.orgaDepartment of Evolutionary Biology, Institute for Biological Research, 29 Novembra 142, 11000 Belgrade, Serbia, Yugoslavia*Corresponding authorAbstractRelationship of superoxide dismutase and catalase activities and aging were tested using bean weevil lines selected for postponed senescence. The beetles of different age (young and old) and mating status (virgin and mated) from the extended longevity lines were compared with their counterparts derived from the short-lived lines for activities of SOD and catalase. The old beetles from the long-lived lines had statistically significant higher activity of SOD than their controls. Although we did not find a significant effect of catalase on longevity, beetles originating from both types of lines exhibited an increased catalase activity during mating processes. In addition, we did observe an increased activity of catalase in one-day-old beetles of the short-lived lines relative to the same-aged individuals of the long-lived lines.KeywordsAcanthoscelides obtectusSuperoxide dismutaseCatalaseLongevitySelectionThe identification of underlying mechanisms of aging is regarded to be an elusive task, largely due to the complexity of aging processes. In addition, most of experimental methods approaching this problem have proven to be inadequate for delineation of the physiological mechanisms that determine rates and patterns of aging. For example, the usual methods of pursuing this goal, such as the studies of the temporal correlates of aging in defined cohorts, have not contributed much to the understanding of genetic and physiological mechanisms controlling aging (see review in Finch, 1990; Rose, 1991). The most important drawback to chronological measures of aging is that many age-related physiological changes are not the same as time-dependent changes (see, e.g., Baker, 1976). Also, experimental studies attempting to test causal mechanism of the aging processes by using a single gene mutant or different environmental factors that “accelerate the rate of aging” seem to be inappropriate because under these circumstances organisms could be dying because of novel pathologies rather than an acceleration of normal aging processes (Maynard Smith, 1966; Hutchinson & Rose, 1987). For these reasons, it has been argued (Johnson, 1987; Rose, 1991; Arking, 1995) that organisms undergoing genetically postponed aging represent the best experimental system that would allow study of the processes important in the normal aging situation.Such a model system for the analysis of underlying genetic and physiological mechanisms of aging constitutes lines with genetically postponed aging obtained from selection for survival to, and reproduction at, later ages. The important advantage to the use of these lines is that they have been obtained by the action of selection on allelic variation affecting aging at great many loci within outbred populations of Drosophila melanogaster (Luckinbill et al., 1984; Rose, 1984, Partridge & Flower, 1992) and Acanthoscelides obtectus (Tucić et al., 1996, 1997). In experiments with these holometabolous insect species in which selection on quantitative genetic variation proceeded by using either young or old adults as parents in each generation, it was found that the “old” lines had higher longevity (and delayed fertility) than the “young” ones. Besides, these experiments have successfully corroborated the basic assumption of the general evolutionary theory of aging (Medawar, 1952; Rose, 1991). As pointed out above, the obtained lines are an excellent experimental system for the study of the nonpathological processes of aging.Because short-lived and long-lived lines are derived by imposing selection on natural variants of many genes affecting aging, it is very likely a priori, that different species (e.g., Drosophila vs. Acanthoscelides) or lines of the same species originating from different laboratories have attained specific longevities by various underlying mechanisms. This expectation seems to be fulfilled with lines of D. melanogaster, with extented longevity stemmed out from different laboratories (see Arking, 1995). Accordingly, when dealing with a topic as complex as aging, it is essential to determine empirically mechanism(s) operative in the short-lived and long-lived lines of A. obtectus obtained in our laboratory.As a part of our effort to elucidate actual mechanisms affecting the aging processes in the bean weevils, in the present study we investigated the activities of two enzymes of great interest to gerontologists—superoxid dismutase (SOD), and catalase. These free radical scavenging enzymes are of interest because of SOD’s role in catalyzing the conversion of superoxide radicals to hydrogen peroxide (McCord & Fridovich, 1969), the latter then undergoing conversion to water due to the action of catalase (Deisseroth & Dounce, 1970). Because free radicals act damaging macromolecules within a cell, it has been proposed that they are important factors in aging (Harman, 1956), and that SOD and catalase could play the antiaging role. The most convincing experimental evidence that supports the free radical theory of aging was provided by Orr and Sohal (1994). In their experiments, D. melanogaster transgenic flies carrying three copies of SOD and catalase genes exhibited about a one-third extension of longevity, a lower rate of senescence, and a lower amount of protein oxidative damage compared to diploid controls. However, analyses of these enzymes in D. melanogaster lines with markedly different adult longevities led to contradictory results. For example, Bartosz et al. (1979) and Fleming et al. (1992) detected a positive correlation between longevity and antioxidant capacity, whereas Massie et al. (1975), Niedzwiecki et al. (1992), and Durusov et al. (1995) did not observe such a correlation. Moreover, a comparison of antioxidant defense in several mammalian species did not suggest a clear association between this defense and longevity (Sohal & Orr, 1992).The long-lived D. melanogaster lines selected by Luckinbill et al. (1984) and those by Rose (1984) differ substantially in their physiology, including the antioxidant defense system. The long-lived flies obtained in the former laboratory show a significant increase in the level and activity of both SOD and catalase (and some other enzymatic and nonenzymatic components of the antioxidant defense system) relative to the control flies, but their resistance to starvation and desiccation is low (Arking et al., 1993; Arking, 1995; Dudas & Arking, 1995). However, although more active allele of the SOD gene is associated with postponed aging lines of Rose (1984), it seems that this allele does not directly increase life span or later fecundity (Tyler et al., 1993). Here, however, the long-lived flies display enhanced resistance to starvation, desiccation, ethanol vapor, and heat (Service et al., 1985; Service, 1987; Graves et al., 1992). Thus, in D. melanogaster there is more than one route to extended longevity. The present work is an attempt to obtain further information on the activity of SOD and catalase in aging of Acanthoscelides obtectus, another insect species for which long-lived and short-lived lines produced by the manipulation with reproductive schedules exist.The fact that sexual activity reduces longevity complicates the interpretation of the long-term studies that use artificial manipulation of reproductive schedules. Although the hypothesis that selection for increased longevity acts on genes that regulate short-term mortality risk through the regulation of sexual behavior (Partridge, 1987) has been refuted (Service, 1989; Partridge & Flower, 1992; Tucić et al., 1996), it is possible that elevated sexual activity in the later ages of the long-lived lines is in a positive correlation with the efficient system of antioxidant defense. This could be expected because, in accordance with “rate of living theory” (Pearl, 1928), a higher sexual activity of females and males means that they are more metabolically active, which implies that these individuals produce a higher level of free radicals. To test this hypothesis, activities of the SOD and catalase were analyzed in the virgin and mated females and males originating from the short- and long-lived lines of the bean weevil.1Materials and methods1.1The short- and long-lived bean weevil linesThe lines used in the present study were derived from a population of bean weevil (Acanthoscelides obtectus) maintained in the laboratory from 1986 (Tucić et al., 1996). This population (the “base”) was synthesised by mass mating equal numbers of adults from three local populations of A. obtectus captured in the vicinity of Belgrade (Yug.). The base population was maintained at large size (about 5,000 individuals each generation) on Phaseolus vulgaris, c.v. “gradistanac” seeds for about a 40-day interval. All cultures were maintained in a dark incubator at 30°C and about 70% humidity.The lines used in this study were obtained from later generations of lines on which selection had been imposed for either early (“young” lines, Y) or late-life (“old” lines, O) reproductive success (Tucić et al., 1996, 1997). The four replicate lines per each selection regime were established. The following summarizes the selection procedures used to maintain the Y-selected and O-selected lines (details are given in Tucić et al., 1996).Beetles in the Y-selected lines were allowed to lay eggs at the ages of one to two days after emergence. In each generation about 300–500 individuals were used as the next generation breeders. This treatment should have given rise to beetles with enhanced fitness during early adult life. In the present study we have used “young” lines selected for the 69 generations.In the O-selected lines (55 generations), the beetles were reproduced from 10th day after emergence until death. Prior to that period experimental adults were kept in vials (females and males together) without bean seeds (in the absence of seeds the egg production is low).All the assays described below were done using four-way crosses within each selection regime. These crosses were obtained by crossing the F1s of different pairs of replicate lines within each selection regime, that is, (Y1 × Y2) × (Y3 × Y4), and (O1 × O2) × (O3 × O4), where the subscript numbers refer to the specific replicate lines (thereafter denoted simply as Y and O lines). The outcrossing of replicate lines should have removed any effect of inbreeding depression, and it diminished any epistatic interactions among genes originating during the long-term selection due to Wahlund’s effect or mutation pressures. Also, protocols used to construct four-way crosses (see Tucić et al., 1997 for details) ensured that selected lines passed through two generations of common conditions.1.2Enzyme assaysEnzyme assays were carried out for females and males of different age (1 and 10 days old) and mating status (virgin and mated) from each experimental group. Twenty individuals were homogenized in 0.25 M sucrose, 50 mM Tris, and 0.1 mM EDTA solution, pH 7.4, using a Janke-Kunkel Ka-Werk Ultra-Turrax homogenizer at 0–4°C Homogenates were sonicated according to Takada et al. (1982). After centrifugation (90 min, 10,500g, 4°C), protein content was determined in the supernates (Lowry et al., 1951) used also in enzyme activity assays. Measurements of the five different samples of each enzyme were made for each sex/age/mating status group.1.3Superoxide dismutaseSuperoxide dismutase (SOD, EC 1.15.1.1) activity was measured as described by Misra and Fridovich (1972). The rate of inhibition of epinephrine autoxidation at alkaline pH in the presence of SOD was spectrophotometrically estimated. One unit of SOD activity was defined as the amount of enzyme inhibiting oxidation of epinephrine by 50% under appropriate reaction conditions. The SOD specific activity was expressed as units per mg protein.1.4CatalaseCatalase (EC 1.11.1.6) activity was determined after Beutler (1982). Decomposition of H2O2 was monitored spectrophotometrically, and a unit of catalase activity was defined as 1 μM H2O2 decomposed per minute, assuming a molar extinction coefficient of 62.4 at 230 nm.2ResultsData shown in Table 1 are the mean longevities and three important fecundity indices of females that characterized lines selected for early and late reproduction for 69 and 55 generations, respectively. As may be seen, there are significant direct responses to both selection regimes; females from the Y lines exhibited significantly higher early fecundity than those from the O lines, whereas for the late fecundity, the opposite trend was observed. In addition, we have corroborated our earlier findings (Tucić et al., 1996, 1997) that selection for early and late reproductive effort does not change the total number of eggs laid by females during the whole lifespan. It is important to note that here, as in the earlier generations of selection, the effect of imposed age-specific selection on longevity, cohorts sampled from the O lines have significantly greater mean longevities relative to Y lines.Records of SOD and catalase activities in 1- and 10-day-old virgin and mated females and males originating from the short-lived and long-lived lines are listed in Table 2. To test differences in the activities of the analyzed enzymes among these groups we performed several ANOVAs (data are log transformed).A two-way ANOVA, with lines and sex of one-day-old beetles as the factors (Table 3), revealed significant differences in the catalase activities (but at the 0.05 level), both between the lines (at the 0.05 level; with the activity of the catalase significantly higher in the short-lived than in the long-lived lines) and sex (in both lines catalase activities in females exceeded more than three times those in males; Table 2).The differences observed above in catalase activities between the lines disappeared when virgins of both 1- and 10-day-old beetles were compared (Table 4). Because the data in Table 1 indicate the opposite pattern of age-dependent catalase activity between the sexes, a two-way ANOVA (with line as one factor and age of beetles as the other factor) were done separately for each sex (Table 4). Increased catalase activity in old females relative to the one-day-old females and its opposite trend in males was, however, statistically insignificant. Although the “line effect” was also absent with regard to SOD activity, virgin 10-day-old females and males had significantly lower activity of this enzyme than their one-day-old counterparts (Table 4).The results of three-way ANOVAs on the SOD and catalase activities over line, sex, and mating status of 10-day-old beetles as factors are listed in Table 5. Here we observed interesting differences between the SOD and catalase activities with respect to line and mating status effects. The old beetles sampled from the long-lived lines exhibited significantly higher SOD activity (at the 0.01 level) than the same aged beetles from the short-lived lines. At the same time, significant “line effect” was absent for catalase activity. Although both the SOD and catalase activities were somewhat higher in females and males kept together their whole life (Table 1), a significant difference between virgin and mated beetles has been observed only for catalase (but at the 0.05 level; Table 5). Also here, in contrast to the data for one-day-old beetles, both enzyme activities were significantly lower in males than in females.3DiscussionThe results presented in this study suggest that SOD might play a role in the postponing, and thus controlling, aging in Acanthoscelides obtectus. Although there are no available data on allelic differences at the SOD structural gene between the lines, pattern of differentiation in the SOD activities between short- and long-lived lines (i.e., statistically detectable difference between 10-day-old beetles and the lack of differentiation between young individuals sampled from different lines; Tables 3 and 5) could be attributed to differences in the regulation of structural genes common to both lines. In other words, in both lines we observed a significant age-specific decline in SOD activity (the “age effect” in Table 4), but this decrease was less pronounced in the long-lived lines. In this respect, our lines seem quite different from those obtained by Tyler et al. (1993), who found that more active SOD alleles were associated with postponed aging of Drosophila melanogaster laboratory lines. However, our interpretation is in accordance with the findings of Dudas and Arking (1995) and Burde and Arking (1998). Their data, obtained by gel electrophoresis, did not provide any evidence on the allelic differences at the SOD gene between the short- and long lived Drosophila lines, which, however, exhibited differences at the level of SOD activity.Orr and Sohal (1994) showed that transgenic Drosophila flies carrying extra copies of SOD and catalase exhibited lifespan extension as a consequence of a slower rate of aging (evidenced by the Gompertz parametric analysis), whereas selected long-lived Drosophila lines seemed to live long as a consequence of a decreased initial mortality rate with no change in the aging rate (Arking, 1995; Dudas & Arking, 1995). Our data, based on the Gompertz parametric analyses (Tucić et al., 1996), suggest that the “young” and “old” selection regimes in the bean weevil produce a change both in the timing of mortality (or in the initial mortality rate) and in rate of change of age-specific mortality. Which of these two important aging events in the bean weevil is possibly connected with the observed level of SOD activity remains to be shown.If, as our data suggest, the long-lived lines exhibit elevated level of the SOD activities, then a concordant increase could be expected in the activity of the catalase, which, acting together with SOD provides the primary enzymatic antioxidant defense. Elevated levels of SOD in the absence of a compensatory upregulation of catalase are thought to lead to H2O2 accumulation, which may give rise to cytotoxic effects (Fleming et al., 1992, and references therein). However, we did not detect any deterioration in the life history traits of the long-lived beetles (on the contrary; egg-to-adult viability, e.g., of the long-lived weevils, was higher than that of the short-lived individuals; Tucić et al., 1996), despite the fact that we failed to demonstrate the increase of catalase activity in these lines. However, this failure arose from the lack of a statistically detectable (in our sample sizes) increase of catalase activity in the long-lived lines but not from the entire absence of any increment in the activity of this free radical scavening enzyme. Careful inspection of the data in Table 2 indicates that grand mean (over both mating status and sexes) of 10-day-old beetles for the catalase is about 14% higher in the long-lived than in the short-lived lines. At the same time, the increase in SOD activity of the same age group in the long-lived relative to the short-lived lines was, on average, some 18%. There are two conclusions that could be drawn from these data. First, the observed increase in the SOD activity appears to provide additional protection against oxidative stress and results in an increased lifespan. Fleming et al. (1992) estimated the increase of SOD expression to be up to 35% (without any change of catalase activity), which could result in an increased longevity, and that levels of SOD above those appear to be toxic for Drosophila. Unfortunately, in the absence of transgenic individuals such an estimation is not possible for the bean weevil. Second, and more important from the standpoint of the evolutionary biologists, it seems that imposed selection regimes generate genotypes in which functionally coupled activities of the free radical scavening enzymes are elevated in a balanced way.Our data also raise the possibility that changes in catalase activity may be an important mechanism operating in the mating activities of the bean weevil. Although both the SOD and catalase activities increased in the mated females and males relative to the virgin beetles (Table 2), only in the latter was a statistically detectable increment of activity recorded (Table 5). It seems, therefore, that different aspects of reproductive activity in the bean weevils, such as courtships and other mating activities of females and males, oogenesis, and spermatogenesis, are protected by antioxidant enzymes. This is not unexpected, because neuropeptides involved in the expression of ecdysone during ovarian development (Lagaueux et al., 1977; Goltzene et al., 1978; Zhu et al., 1983) and spermatogenesis (Koolman et al., 1979) are important regulators of the mating activities, fertilization, and production of gametes in insects. Physiological effects of the ecdysone are realized through 20-hydroxyecdysone, which is regulated by the activity of the cytochrome P-450–linked enzymes which in turn, generate free radicals (Hoffman & Hetru, 1983).However, how could one explain increased level of catalase activity in the one-day-old beetles in the short-lived lines relative to the same aged individuals of the long-lived lines? The reason for such a result could be found in our experimental procedures involved in production of the short- and long-lived lines. Namely, our “young” lines (which bring about to the short-lived beetles) are leaving their progenies only during the first two days after emergence. They are, therefore, under strong selection for shortening the period between emergence and peak of reproductive activity in this insect species (about three to four days after emergence; Tucić et al., 1996). As a by-product of such a selection regime, we have genotypes characterized by elevated catalase activity coincident with the period of the “permitted” reproduction. Presently, we do not know what these genotypes are. They could be more active catalase alleles expressed in the early adult life, or the alleles of the other genes involved in the regulation of catalase activity.ReferencesArking 1995R.ArkingAntioxidant genes and other mechanisms involved in the extended longevity of DrosophilaR.G.CutlerL.PackerJ.BertramA.MoriOxidative Stress and Aging1995Birkhauser VerlagBasel123138Arking et al 1993R.ArkingS.P.DudasG.T.BakerGenetic and environmental factors regulating the expression of an extended longevity phenotype in a long lived strain of DrosophilaGenetica911993127142Baker 1976G.T.BakerInsect flight muscleMaturation and senescenceGerontology221976334361Bartosz et al 1979G.BartoszW.LeykoR.FriedSuperoxidase dismutase and life-span of Drosophila melanogasterExperientia35197911931194Beutler 1982E.BeutlerCatalaseE.BeutlerRed Cell Metabolism, A Manual of Biochemical Methods1982Grune and Straton, IncNew York105106Burde and Arking 1998H.R.BurdeR.ArkingImmunological confirmation of elevated levels of CuZn superoxide dismutase protein in an artificially selected long-lived strain of Drosophila melanogasterExp Geront331998227237Deisseroth and Dounce 1970A.DeisserothA.L.DounceCatalasePhysical and chemical properties, mechanism and catalysis and physiological rolePhysiol Rev501970319375Dudas and Arking 1995S.P.DudasR.ArkingA coordinate up-regulation of the antioxidant gene activities is associated with the delayed onset of senescence in a long-lived strain of DrosophilaJ Gerontol Biol Sci501995B117B127Durusov et al 1995M.DurusovN.DirihN.BozcukAge-related activity of catalase in different genotypes of Drosophila melanogasterExp Gerontol3019957786Finch 1990C.E.FinchLongevity, Senescence, and the Genome1990University of Chicago PressChicagoFleming et al 1992J.E.FlemingI.ReveillaudA.NiedzwieckiRole of oxidative stress in Drosophila agingMutat Res2751992267279Goltzene et al 1978F.GoltzeneM.LaugueuxM.CharletJ.A.HoffmannThe follicle cell epithelium of maturing ovaries of Locusta migratoriaA new biosynthetic tissue for ecdysoneHoppe-Seyler’s Z Physiol Chem359197814271434Graves et al 1992J.L.GravesE.C.ToolsonC.JeongL.N.VuM.R.RoseDesiccation, flight, glycogen, and postponed senescence in Drosophila melanogasterPhysiol Zool651992268286Harman 1956D.HarmanAging—A theory based on free radical and radiation chemistryJ Gerontol111956298300Hoffmann and Hetru 1983J.A.HoffmannC.HetruEcdysoneR.G.H.DownerH.LauferEndocrinology of Insects1983Alan R. 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Adult daughters (40 African American, 40 European American) completed assessments of adult attachment, instrumental and emotional caregiving, and caregiver burden. The author performed hierarchical linear regressions to examine the relationships between attachment dimensions (Security and Anxiety) and the provision of instrumental and emotional care. Both attachment dimensions were unrelated to the provision of instrumental care. In contrast, high scores on the Security dimension and low scores on the Anxiety dimension were associated with the provision of more emotional care to mothers. A final analysis revealed that high scores on the Security dimension were associated with less caregiver burden. These results suggest that practical care that daughters provide to their mothers may be independent of attachment patterns within the child–parent relationship, whereas affective, discretionary care may be promoted or hindered by attachment patterns. Moreover, the stress of caregiving may be mediated by a more secure attachment bond. The potential impact of adult attachment patterns has implications for both family intervention and public policy, given that more families are expected to participate in caregiving in coming decades. hwp-legacy-fpage P257 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: Margie E. Lachman, PhD Anumber of factors influence the degree to which adult children assist their older parents, such as obligation, affection, reciprocity, and other cultural norms (Blieszner and Hamon 1992). More concrete factors are important as well and include geographical distance, size of the family, gender of caregiver and care recipient, and socioeconomic resources and constraints (Dwyer and Coward 1991; Litvin, Albert, Brody, and Hoffman 1995; Wolf, Freedman, and Soldo 1997). In the current study I examined an additional factor, adult attachment patterns, to determine whether attachment-based emotional bonds between daughters and their older mothers were associated with (a) the amount and nature of care that daughters provide and (b) caregiving burden experienced by daughters. Basic Concepts in Attachment Theory Originating with the work of John Bowlby 1982, attachment theory describes a socioemotional behavioral system that guides how individuals manage their need for emotional security. This system is first evident early in life as children interact with their primary caregiver. When they are physically or psychologically threatened, children turn to their caregiver for comfort, and ideally their caregiver responds with immediate, positive, and consistent support. In reality, of course, caregivers do not always respond in ways that children expect. On the basis of their accumulated experiences with caregivers, children develop mental representations, or internal working models (Bowlby 1988), that reflect their beliefs about the responsiveness of caregivers and the environment more generally. Seminal work by Mary Ainsworth (Ainsworth, Blehar, Waters, and Wall 1978) identified behavioral manifestations of internal working models in the form of attachment styles, secure versus insecure attachment being the most broad differentiation. Children with a secure attachment were likely to seek and savor contact with their caregiver, to use that person as a secure base for exploration. Meanwhile, children with an insecure attachment were likely to avoid their caregiver or demonstrate anxiety in contact with him or her. A broad array of research has suggested that a child's initial attachment bond has an impact well beyond their first critical relationship and influences not only subsequent relationships but also a wide range of social and emotional outcomes later in life (Feeney and Noller 1996; Rothbard and Shaver 1994). Despite a burgeoning empirical literature on attachment dynamics, significant conceptual issues remain. For example, it is unclear whether attachment patterns represent an aspect of individuals (i.e., attachment as a trait), a facet of specific relationships (e.g., types of attachment differing within an individual's social network), or some combination of the two (Bartholomew and Shaver 1998). Likewise, there is debate about whether attachments are categorical phenomena or are best thought of as graded entities (Feeney, Noller, and Hanrahan 1994). Diversity of theory is also apparent in the variety of attachment taxonomies that exist, although a parsimonious nomenclature for attachment patterns is beginning to emerge (see Feeney et al. 1994; Griffin and Bartholomew 1994). Another issue is whether a construct originally validated with research on children is relevant to individuals at other ages. Although Bowlby first focused on the attachment dynamic between infants and their caregivers, he asserted that "attachment behaviour is held to characterize human beings from the cradle to the grave" (Bowlby 1979, p. 129), a life-span perspective shared by Ainsworth 1989. And indeed, research on attachment in childhood has been complemented by expanding attention to attachment in adults. Attachment in Adulthood Attachment patterns are hypothesized to persist across the life span through the reinforcing properties of internal working models (Bowlby 1973; Main, Kaplan, and Cassidy 1985). The first attachment relationship provides a template, a self-perpetuating schema that influences subsequent relationships. Mental representations from early attachment bonds thereby influence how individuals seek, anticipate, and interpret future interpersonal interactions (West and Sheldon-Keller 1994). Reflecting its roots, attachment theory also has emerged as a framework for understanding the relationship between adult children and their parents. Some theorists have suggested that adult children relinquish their parents as attachment figures (Weiss 1982), whereas others have amassed secondary evidence that attachments to parents are sustained (Krause and Haverkamp 1996). In a study of adult children whose parents were institutionalized, Crispi, Schiaffino, and Berman 1997 found that children's attachment style predicted aspects of their well-being. Specifically, a secure attachment style was associated with less caregiving difficulty and less psychiatric symptomatology. Another study of children whose parents were in nursing homes found that children's attachment was related to parent mood (Pruchno, Peters, Kleban, and Burant 1994). Attachments were less intense when parents were depressed and no longer able to provide the emotional support that children expected from the relationship. It may be that attachment dynamics, forged in childhood, continue to influence child–parent relationships later in the life span. But even if attachment patterns in adult children are discontinuous from their earliest manifestation, contemporary attachment patterns may still be relevant to the way in which adult children interact with their parents via children's capacity for self-reflectiveness, empathy, and their own needs for security (Crose 1994). Cicirelli 1991 has suggested that adult children provide care to their parents to forestall the dissolution of their attachment relationship. As parents age and weaken, their impermanence becomes more apparent to children. Anxious about the threatened loss of their attachment figure, children may provide support to bolster their parent and preserve the important attachment object. Cicirelli 1993 study of caregiving daughters revealed that attachment had a direct and positive relationship with the care that daughters were providing to their mothers: Stronger attachment bonds were associated with greater amounts of care, independent of mothers' level of functional dependency. Stronger attachment bonds also were associated with lower caregiver burden. One limitation of Cicirelli 1993 work is that it employed a global index of attachment rather than an assessment of specific attachment styles or dimensions. Yet just as attachment styles are associated with different behavior patterns and outcomes in other realms (e.g., Crispi et al. 1997; Hazan and Shaver 1990), they also may be associated with differences in parent care. Securely attached adult children, whose parents have been responsive and supportive, may be highly motivated to care for their parents to protect the valued attachment figure. They may continue to experience felt security (Sroufe and Waters 1977) in the attachment relationships with their parents, and they may desire to preserve that secure base as long as possible. In contrast, insecurely attached children, whose parents have been unresponsive and unsupportive, may be less eager to care for their parents because the psychological rewards for sustaining that relationship are unreliable. Their anxiety, distrust, or unease may translate into a lack of contact compared with that of peers with more secure attachments. Thus, one question I pursued in the current study was whether specific attachment patterns are related to the amount of care children provide to older parents. Another question was whether attachment patterns also are associated with the nature of parent care provided. Securely attached children may be comfortable providing a range of both emotional and practical care. Their stable, internal working models enable them to get close to their parents and offer a balance of pragmatic support as well as emotional availability. Also, because of their security and emotional flexibility they are likely to perceive caregiving as less burdensome. In contrast, insecurely attached children may be less willing to provide emotional support because of the psychological risks, although they may still provide practical assistance because of its relative emotional safety. Moreover, any care that insecurely attached children do provide is likely to be stressful because of underlying uneasiness in the dyad. In summary, my purpose in this study was to examine the relationship between attachment dimensions and the nature of care that adult daughters were providing to their older mothers. In this initial study I chose to focus on this one type of family dyad given its commonality in caregiving, but future research will need to include the full range of family relationships. I explored whether attachment dimensions contribute to the explanation of instrumental and emotional caregiving above and beyond the influence of demographic and contextual factors that may be related to parent care. The following hypotheses were tested:Securely and insecurely attached daughters provide comparable amounts of instrumental care to their older mothers.Securely attached daughters provide more emotional care to their mothers than insecurely attached daughters.Securely attached daughters experience lower caregiver burden than insecurely attached daughters. Methods Participants and Procedure Eighty adult daughters completed one semistructured interview. Participants were recruited from the greater Cleveland community via announcements in employee newsletters (29% of the sample) and local newspapers (19%), from agencies that provide social services (21%), and from recommendations made by other participants (31%). Inclusion criteria for the study were that daughters provided at least 5 hr of care each week to their mother and had been doing so for at least 2 months. In addition, mothers were required to be aged 60 and older and living alone or with their daughters. The sample size reflected a desired power of .80, an alpha level set at .05, and a medium effect size of .61 based on Cicirelli 1993 findings. Interviews were conducted by an advanced clinical psychology graduate student, lasted approximately 1–2 hr, and took place without the mother being present. After completing the interview daughters were provided with a description of the study, debriefed, and paid $10. Referrals were made to social services agencies (e.g., the Alzheimer's Association, caregiver support groups) when appropriate. The current sample expanded on Cicirelli 1993 European American sample and included 40 African Americans and 40 European Americans. Statistical comparisons between ethnic groups, however, revealed no significant differences on study variables. Daughters ranged in age from 28 to 77 (M = 50.19, SD = 10.77). Many were married (41%), a large majority had children (75%), and of them 63% had children currently living at home. As a group daughters were well educated, with 95% having completed at least high school and 69% having at least a college degree. Two thirds of the daughters were working, with most in sales, technical, or administrative support positions. Fifty-one percent of the daughters were residing with their mother. Daughters had been providing care for an average of 4.65 years (SD = 4.09). Mothers ranged in age from 60 to 99 (M = 78.60, SD = 9.30) and represented a broad range of functional dependence (activities of daily living [ADL] score M = 17.16, SD = 8.43, range = 0–28). Measures Care provided to mother. The assessment of caregiving focused on two content areas, instrumental and emotional care. An inventory of 31 instrumental activities and 22 emotional activities was presented to daughters. Examples of instrumental activities included preparing meals, performing housework, assisting with grooming, and obtaining medical equipment and supplies. Examples of emotional activities included providing gifts, hugging and kissing, voicing one's love, and offering comfort and sympathy. Daughters rated how often they had engaged in each activity with their mother in the past month on a 5-point scale: 1 ("not at all"), 2 ("once or twice"), 3 ("about once a week"), 4 ("several times a week"), and 5 ("about every day"). I summed ratings to provide two caregiving scores for each daughter: total frequency of instrumental care (α = .87) and total frequency of emotional care (α = .86), with higher scores representing more frequent caregiving. Adult attachment. I used two instruments to assess adult attachment. The Adult Attachment Scale (AAS; Cicirelli 1995) contains 16 items about the relationship between daughter and mother. Items reflect the theoretical underpinnings of attachment behavior, such as distress upon separation, joy upon reunion, and felt security provided by the relationship. Daughters rate items on a 7-point Likert-type scale ranging from 1 ("not at all true, disagree completely") to 7 ("definitely true, agree completely"). Items are summed to yield a total score that can range from 16 to 112, with higher scores indicating a stronger attachment to mother according to Cicirelli. Internal consistency reliability of the AAS has been reported as .95, with a 1-year test-retest reliability of .73 (Cicirelli 1995). In the current sample, the coefficient alpha was .95. The Relationship Questionnaire (RQ; Bartholomew and Horowitz 1991) is based on Bartholomew's theory of adult attachment (Bartholomew 1990; Griffin and Bartholomew 1994). Drawing on Bowlby 1988 conception of internal working models, Bartholomew has suggested there are two dimensions, or models, at work in an individual's attachment style. The other model represents the degree to which other people are expected to be available when needed and the disposition to seek or avoid close relationships. The self model embodies the degree to which individuals possess an internalized sense of self-worth and experience anxiety in relationships. The other and self models interact to yield four attachment styles: Secure, Preoccupied, Fearful, and Dismissive. To complete the RQ, participants rate the extent to which descriptions of the four styles correspond to the relationship they have with a particular individual. In this study the text was modified so that descriptions pertained to mothers. Ratings are made on a 7-point Likert-type scale, ranging from 1 ("not at all like me") to 7 ("very much like me"; see Appendix, Note 1). Scharfe and Bartholomew 1994 reported moderate stability of self-reported attachment patterns. In the current study internal consistency reliability among the four attachment style ratings was .81. Mother's level of functional dependency. Functional dependence was measured with a 14-item scale from the Duke Older Americans Resources and Services Multidimensional Functional Assessment Questionnaire (OARS; Fillenbaum 1988). Each daughter was asked to rate her mother's abilities on physical and instrumental ADLs using a 3-point scale: 2 ("able to perform the activity without help"), 1 ("able to perform the activity with some help"), or 0 ("completely unable to perform the activity"). Total ADL score was the sum of the 14 items, with higher scores representing greater functional independence. Internal consistency reliability for the scale was .95. Caregiver burden. The seven-item scale used by Cicirelli 1993 to measure caregiver burden was used in this study. Daughters rate the extent to which caring for mother interferes with their social life, relationship with partner, physical health, and mental health. Ratings are made on a 5-point Likert-type scale ranging from 1 ("not at all") to 5 ("very much"). Ratings are summed, and higher scores indicate greater burden. Cicirelli reported an internal consistency for the scale of .83; in the current study coefficient alpha was .80. Daughters also were asked how many months they had been providing the level of care they were currently providing. Duration of caregiving was included because some studies have documented a relationship between length of caregiving and negative caregiver outcomes (Hannappel, Calsyn, and Allen 1993). Demographic and family structure. Additional information that was collected included daughter's age, ethnicity, marital status, number of years of education, current or most recent occupation, number of children currently living with daughter, mother's age, and whether mother was currently living with daughter. A rating of socioeconomic status (SES) was based on Duncan 1961 socioeconomic index (SEI), which was updated by G. Stevens and Cho 1985 to reflect the 1980 census. I calculated an overall SES score for each daughter by summing standard scores for SEI and education, with higher scores representing higher SES. Data Analysis I calculated descriptive statistics to examine the characteristics of the sample. Preliminary exploration of the relationships among demographic variables, functional dependence, attachment variables, caregiving, and burden were performed with bivariate Pearson product-moment correlations and analyses of variance (ANOVAs). I used post-hoc comparisons to examine mean differences in caregiving across attachment styles, with the Scheffe method used to control for Type I errors. Multivariate analyses included a series of hierarchical least squares linear regressions to examine the variance in caregiving accounted for by attachment. I performed two hierarchical regressions using the same demographic, contextual, and attachment independent variables. Instrumental caregiving was the dependent variable in the first regression; emotional caregiving was the dependent variable in the second. Independent variables were entered in four blocks. Demographic and contextual variables were entered in the first step, on the rationale that they are the least modifiable. I entered mother's ADL score in the next step to account for caregiving driven by actual functional need. In the next step the amount of caregiving of the type complementary to the dependent variable was entered (emotional care when instrumental care was the dependent variable, and vice versa) in recognition of the probable overlap between types of care. In the last step I entered two attachment dimensions (discussed later) to examine their impact on caregiving independent of the preceding variables. I performed a final hierarchical regression using a similar logic to predict caregiver burden based on demographic and contextual variables, mother's level of functional dependence, amount of instrumental and emotional caregiving, and attachment dimensions. Results Care Provided to Mothers The possible range of scores for the instrumental caregiving scale was 31–115, and the mean in the current sample was 62.29 (SD = 16.81, range = 33–106). The possible range of scores for the emotional caregiving scale was 22–110, and the mean in the current sample was 64.51 (SD = 14.60, range = 27–98). Overall, daughters in this sample were providing a range of care in both domains. The bivariate correlation between the two types of caregiving was not significant (r = .22, p > .05). Adult Attachment AAS scores ranged from 29 to 112 and had a distribution (M = 81.13; SD = 24.01) similar to that reported by Cicirelli 1993(M = 81.23, SD = 20.86). As a group, daughters in this sample appeared to experience moderate to strong feelings of attachment toward their mother, although there was wide variability in the attachment scores. On the RQ, using the highest dimensional rating as an indicator of dominant attachment style, I found that the distribution of attachment styles was as follows: Secure, 66%, n = 53; Fearful, 9%, n = 7; Preoccupied, 6%, n = 5; Dismissing, 19%, n = 15. These figures are consistent with the distribution of attachment styles found in other studies with adults (e.g., Collins and Read 1990; Feeney and Noller 1990). Correlations among the AAS and RQ ratings appear in Table 1 . Scores on the AAS were significantly positively correlated with self-ratings on the Secure style and significantly negatively correlated with self-ratings on the Fearful and Dismissive styles. Correlations within the RQ were also significant and in expected directions. To maximize variance accounted for by the two highly correlated attachment measures and to maintain a concise number of variables, I combined the AAS and RQ ratings into factor scores from a principal components analysis. Factor scores were calculated with unit weighting. The first factor was composed of the following scores (rotated factor loadings are in parentheses): AAS score (.90), RQ Secure rating (.83), and RQ Dismissive rating (−.82). The eigenvalue for the first factor was 2.91, which accounted for 58% of the variance. On the basis of its components, this factor seemed to represent the security of attachment to mother and a daughter's tendency to seek or avoid a close relationship with her. This factor was named the Security dimension, and it corresponds to Bartholomew's other model of internal representations (Bartholomew and Horowitz 1991). Although the eigenvalue for the second factor (.97, 20% of the variance) was below the standard 1.0 cutoff, the factor accounted for a significant portion of variance and was therefore included. In addition, it mapped coherently onto Bartholomew 1990 two-dimension theory of internal attachment representations. The second factor was composed of RQ Fearful (factor loading = .67) and RQ Preoccupied (factor loading = .92) ratings and appeared to symbolize anxiety and self-doubt experienced by daughter in her relationship with her mother. This factor was named the Anxiety dimension, and it corresponds to Bartholomew's self model of internal representations. Univariate Analyses of Attachment and Caregiving Estimated marginal mean caregiving levels and standard errors for each attachment style appear in Table 2 . A one-way ANOVA revealed no significant differences between groups on instrumental care, F(3,76) = 1.84, p = .15, although the low sample size in the Fearful group limited the power to detect differences. Significant group differences were found among the groups in terms of emotional care, F(3,76) = 13.64, p < .001. Post-hoc comparisons revealed that daughters with a secure attachment style provided more emotional care than daughters with any of the insecure attachment styles. No significant differences were found among the insecure attachment styles. A similar pattern of relationships among attachment dimensions and caregiving is apparent in the bivariate correlations in Table 1 . The only attachment variable significantly associated with instrumental care was the RQ Fearful index (r = −.26, p < .05), with higher scores associated with less instrumental care. Meanwhile, every attachment variable was significantly correlated with emotional care. Multivariate Analyses of Attachment and Caregiving In the next set of analyses I used hierarchical linear regression to explore the relationship between caregiving and demographic, family structure, contextual, and attachment variables. Prior to the regressions two cases with univariate outliers were identified: one daughter with a high instrumental caregiving score and one daughter with a high score on the Anxiety dimension. These cases were retained with their original data because they were not statistically influential cases, as indicated by a Cook's distance less than 1.0 (Cook and Weisberg 1982), and they did not produce a substantial change in the regression coefficients when they were dropped (J. Stevens 1996). As a set, the independent variables were found to be neither singular nor adversely multicollinear. Residual scatterplots suggested adequate fulfillment of the assumptions of linearity, homoscedasticity, and independence of residuals. Bivariate correlations for the variables in these analyses appear in Table 3 . The large number of correlations precluded direct interpretation because of the possibility of a Type I error, but relationships throughout the correlation matrix were in the directions expected. Table 4 contains results from the three hierarchical regressions, the first with instrumental care as the dependent variable, the second with emotional care as the dependent variable, and the third with caregiver burden as the dependent variable. For each independent variable the table contains the unstandardized regression coefficient (b); the standard error of the regression coefficient (SE b); the standardized regression coefficient (β); and the squared semipartial correlation (sr2), which represents the unique proportion of variance contributed by the variable. In the prediction of instrumental care, number of coresident children emerged as a significant predictor; daughters living with more children provided more instrumental care. In the second step, mother's level of functional dependence was a significant predictor; mothers with more impairment received more instrumental care. In the third step, amount of emotional care was a significant predictor, indicating the overlap between both types of care. In the final step, when the Security and Anxiety attachment dimensions were added, neither added significantly to the explanation of instrumental caregiving, ΔR2 = .00, F(10,69) = .08, p = .94. The variance contributed by the independent variables that were significant at this final step was as follows: number of children in the household (3% of the variance), mother's functional dependence (32%), and amount of emotional care (6%). To summarize the interpretation of this first regression, demographic and family structure variables appeared unrelated to the amount of instrumental care daughters were providing to their mothers with the exception that daughters with more children in their household were providing more instrumental care. Mothers who were more functionally dependent also received more instrumental care. And mothers who were receiving more emotional care from their daughters tended to be receiving more instrumental care as well. Attachment dimensions were unrelated to the amount of instrumental care daughters were providing. In the first step in the prediction of emotional care, work status was significant; working daughters provided less care. In subsequent steps mother's functional dependence emerged as a significant predictor (more impaired mothers received more emotional care), as did instrumental care, again indicating overlap between types of care. In the final step, when the Security and Anxiety attachment dimensions were added to the regression equation, there was a significant change in the multiple correlation, ΔR2 = .21, F(10,69) = 14.69, p < .001. Both attachment dimensions contributed unique variance: Higher scores on the Security dimension were associated with more emotional care (15% of the variance), and higher scores on the Anxiety dimension were associated with less emotional care (6% of the variance). Three other independent variables made significant contributions: work status (3% of the variance), mother's functional dependence (6%), and instrumental care (11%). To summarize the results of this second regression, the only demographic variable that added significantly to the explanation of emotional care was work status, where working daughters tended to provide less emotional care. Mothers who were more functionally dependent were receiving more emotional care, which also accompanied larger amounts of instrumental care. Finally, daughters high on the Security dimension and low on the Anxiety dimension provided more emotional care (see Appendix, Note 2). In a final regression to predict caregiver burden, only two independent variables emerged as significant at the end of the sequence. The number of children currently living with daughters accounted for 6% of the variance in caregiver burden; daughters with more children at home reported more burden. The Security attachment dimension accounted for 6% of the variance in caregiver burden; daughters with a higher degree of security reported less burden. Discussion In the current study I used the life-span developmental framework of attachment theory to examine associations between socioemotional bonds and the caregiving that daughters were providing to their older mothers. As predicted, attachment patterns are unrelated to the amount of instrumental care daughters provide. In contrast, attachment patterns are significantly associated with the amount of emotional care daughters provide, with securely attached daughters providing more emotional care than insecurely attached daughters. Daughters with a more secure attachment bond to their mothers also report less caregiver burden. Attachment patterns appear to play a role in the amount and nature of care provided to parents and in the psychological outcomes for caregiving children, although there are important theoretical and methodological issues that remain to be addressed in future research. Attachment and Caregiving In the current study I extend work on attachment and parent care by exploring the relationship between specific attachment dimensions, Security and Anxiety, and two types of care, instrumental support and emotional support. First, adult daughters' attachment patterns are unrelated to the provision of instrumental care to their older mothers. Assistance with shopping, housework, medication management, and social service arrangement are offered to mothers regardless of the quality of the attachment bond between daughter and mother. This finding is consistent with research that has shown a continued provision of instrumental care despite its sometimes overwhelming physical and psychological burden (Cantor 1983). One explanation for this finding may be that genuine functional needs supercede attachment dynamics. A daughter in the current study admitted, "I know what I have to do, and I don't mind doing it, but sometimes it stresses me out … She and I were never really close, but you have to do what you have to do." Social and familial norms may propel instrumental caregiving regardless of the quality of the child–parent relationship (Ikkink, Van Tilburg, and Knipscheer 1999). In fact, practical support may be just the kind of care that enables some daughters to be involved yet emotionally safe. Concrete, task-focused activities such as picking up prescriptions and doing laundry require relatively little direct contact with a parent and can be managed so that they involve minimal emotional exchange and, consequently, minimal risk of friction or disappointment. In contrast, in the current study the provision of emotional care is associated with two attachment dimensions, Security and Anxiety. Higher scores on the Security dimension correspond to a more secure attachment to mother, an internalized sense of her supportiveness, and a willingness to seek intimacy with her. Daughters who score high on this dimension provide more emotional care. As Cicirelli 1991 has suggested, secure daughters may provide emotional support as a way to protect their mother as old age compromises health and hints at the attachment relationship's inevitable end. Insecure daughters, on the other hand, may be less motivated to invest emotional energy in a relationship that has been unsupportive or painful and whose end may be unremarkable or even a relief. A converse relationship exists between attachment Anxiety and emotional care. Higher scores on this dimension represent feelings of poor self-worth and apprehension in relationship with mother, and daughters who score high on this dimension provide less emotional care. They may be fearful of providing emotional care because they doubt whether they can do so effectively or whether they deserve to be in a supportive relationship. One daughter commented, "It never seems like I can do enough. And if I don't go over there for a couple days I end up feeling guilty … It's like I'm still trying to please her." With their precarious self-esteem, insecurely attached daughters may withdraw from their mothers rather than risk conflict or rejection. Overall these results are consistent with Cicirelli 1991, Cicirelli 1993 contention that adult children may provide care to preserve the attachment relationship. Yet the current study also provides evidence that attachment patterns may have more complex associations with caregiving than previously considered. Namely, the provision of care appears to depend on the nature of the attachment bond with parents (i.e., whether it is characterized by security or insecurity). Moreover, the current study clarifies that different types of care (instrumental vs emotional) have different associations with attachment patterns. Instrumental care is provided regardless of attachment patterns, yet the amount of emotional care depends on attachment, with more care provided by securely attached children who want to prolong the nurturance they receive back from their parent (Kramer 1997). Indeed, this study demonstrates that daughters with more secure attachment bonds report lower levels of caregiver burden, as have other studies (Crispi et al. 1997). The correlational nature of these data do not allow causal conclusions, but the data do suggest that secure attachment bonds may be associated with positive psychological outcomes for adult children, adding to a broader literature of similar findings (Main 1996). Altogether, adult attachment patterns appear to have a dual impact on child–parent caregiving interactions: an impact on the nature of care that older adults receive and an influence on how successfully children cope with parent care responsibilities. To conclude the findings from this study, two other factors also are related to parent care. First, there is a significant association between caregiving and daughter's work status, although that relationship differs depending on the domain of care: Working daughters provide less emotional care, but work status is unrelated to instrumental care. This result suggests that when daughters experience multiple demands on their time emotional care for a parent may seem secondary to pragmatic needs. Sitting with mother, reminiscing with her, listening to her talk about things that are important to her—these activities may seem less essential than more conspicuous needs such as dinner that must be provided or laundry that must be done. One indirect effect of the multiple burdens on caregiving children may be, then, that parents experience a qualitative difference in how their children interact with them. Second, the only family structure characteristic that is significantly associated with caregiving is the number of children living with daughters: Daughters with more children at home provide more instrumental care (no systematic relationship emerged for emotional care). With children at home daughters may have the liberty to devote more time to caring for their mother as they delegate responsibilities in their own household to their children. More children at home, however, was also associated with greater caregiver burden, suggesting that the stress of caring for a parent may exist regardless of the supports a woman has in her own home. It is important to return for a moment to review the effect sizes of factors that are associated with caregiving (Rosenthal and Rosnow 1991). Demographic factors have small effect sizes (number of children at home, sr2 = .03 for caregiving, sr2 = .06 for burden; work status, sr2 = .03 for caregiving). Mother's functional dependence has an effect size that is large in regard to instrumental care (sr2 = .32) but small to medium in regard to emotional care (sr2 = .06). Finally, the Security and Anxiety attachment dimensions have small to medium effect sizes for emotional caregiving (sr2 = .15 and .06, respectively) and a small effect size in relation to burden (sr2 = .06). In terms of clinical significance, then, changes in functional dependence can be associated with quite substantial shifts in the amount of instrumental care that is provided to mothers. When an older parent develops incontinence, for instance, a substantial increase in caregiving time and energy may occur. In addition, even small variations in attachment patterns are associated with significant differences in emotional care and burden. The effect sizes for attachment dimensions are particularly noteworthy because those factors represent possible points of intervention. Implications and Limitations Results from this study suggest that attachment patterns may provide some indication of how likely daughters are to provide emotional care to their mothers, thereby making it possible to identify older adults who may be at risk for receiving less emotional support from their families. What to do with that information is, of course, another question. The extent to which adult children's attachment patterns can be changed is unclear (Slade 1999). Yet Bowlby's term, internal working model, was carefully chosen to reflect its nature as a "dynamic representation" (Bretherton 1993, p. 239) that could be modified by direct intervention or in response to reparative attachment relationships. Other authors have described earned attachments and the positive modification of attachment representations suggesting the possibility of creating adaptive relationships between adult children and their parents even when before they were contentious (Byng-Hall 1999; Krause and Haverkamp 1996). Even if attachment modification is not a primary goal, family education programs that examine interpersonal dynamics before caregiving becomes a necessity might offer families an opportunity to discuss caregiving expectations and address discrepancies across generations before they evolve into disappointment or conflict. Results of the current study also have implications for policy efforts that address the division of responsibility for caregiving between families and other sources. Cicirelli 1991 has suggested that interaction between adult children and older parents is an important, ongoing developmental process with benefits for both sides, and policy "should be formulated to assist adult children to maintain attachment and protective behavior toward the parent" (p. 38). How might this proposition be met by children who do not get along with their parents, children with less secure and more anxious attachments? To encourage extensive caregiving in those circumstances, and to expect that care to have a positive emotional tone, may be unrealistic. Strategies that address family involvement in caregiving will need to recognize that not all families want or can tolerate close, emotionally focused interactions (Magai and Cohen 1998). In their recent work on long-term care, Kane, Kane, and Ladd 1998 stated that some family members should not be caregivers and that public policies should recognize the contribution of informal caregivers but not rely on them exclusively. The current study also brings into relief questions that require further empirical attention. First, the theoretical and measurement questions in adult attachment remain numerous and complex. The psychometric properties of the AAS and the RQ are not firmly established, and what they measure remains open to a certain degree of speculation. For instance, the AAS and RQ were worded to measure daughters' attachments to their mothers, but it is possible that an overarching attachment representation rather than a relationship-specific one might be more directly responsible for daughters' behavior (Cicirelli 1998). Future applications of attachment theory to the study of adult child–parent relationships also will require consideration of how those relationships differ in adulthood from their earlier incarnation: changes in the power dynamic, differences in resources and the direction in which they are shared, the newly real potential end of the relationship, and the host of intervening events and relationships both inside and outside the family that can influence contemporary interactions. Another limitation of the current study is that it did not include the perspective of mothers receiving care. Attachment has always been conceived of as a behavioral system, with one party influencing the other in mutual feedback. The reciprocal nature of the caregiving dynamic demands a transactional approach in future research (Magai and Cohen 1998). In fact, it is probably important for investigators to examine the entire system of attachments within a family to understand how attachments influence relationships across and within generations. Some additional caveats deserve mention. The conceptual overlap between the questions I used to measure mother's level of functional dependence and those that assessed instrumental caregiving might explain their relationship in the regressions. In addition, researchers have pointed out the potential error inherent in self-reports of caregiving (Brody, Litvin, Albert, and Hoffman 1994). Regarding the sample itself, this was a group of homogeneous daughters who volunteered to discuss their caregiving and family relationships and therefore may represent a particular kind of family, one with relatively positive relationships. Missing, of course, were daughters who refused to participate and might have more troubled relationships and different patterns of attachment and caregiving. Future research will need to include more diverse caregiving dyads and will need to use creative strategies to recruit families in which caregiving takes place in the context of more overt discord. Final considerations are the cross-sectional nature of these data and the reality that hierarchical regression is not a substitute for experimental control. Variables in the hierarchical regression are entered in a logical sequence, but the technique does not preclude the possibility of reciprocal causality. Attachment may influence caregiving, but caregiving also may influence attachment. At the beginning of caregiving a daughter may feel relatively comfortable with the situation, but her attitude may change if the demands of caregiving become unmanageable (Walker, Acock, Bowman, and Li 1996). Conversely, a daughter who spends more and more time helping her mother may experience burgeoning affection for her, a stronger sense of security, and a more positive attachment bond (Davila, Burge, and Hammen 1997). The possible bidirectional influence of attachment patterns points to the importance of longitudinal designs in clarifying the role of attachment across the caregiving history. Despite these limitations, the current study does suggest that attachment theory is a useful framework for understanding caregiving in adult family relationships. In approaching decades families are likely to experience expanded responsibility caring for older adults, and research that explores connections between emotional and behavioral family patterns will continue to be vital. Notes In a complementary method of administration, respondents can indicate which one description best represents the relationship they have with their mother. In this study the categorical ratings were uniformly consistent with the highest dimensional rating. Because the dimensional ratings provide more detail about the interplay among attachment styles, they were used in analyses. In follow-up analyses potential interaction effects between the attachment dimensions and mother's functional dependence were explored, on the basis of Cicirelli 1993 findings. No significant interactions were found. Table 1. Zero-Order Correlations Among Attachment Variables and Caregiving 1 2 3 4 5 6 7 8 9 1. AAS — .80** −.49** −.19 −.60** .76** .40** .16 .51** 2. RQ–Secure — −.60** −.35** −.59** .83** .64** .21 .49** 3. RQ–Fearful — .43** .34** −.65** −.77** −.26* −.40** 4. RQ–Preoccupied — .19 −.02 −.73** −.04 −.34** 5. RQ–Dismissive — −.80** −.01 .02 −.51** 6. Security dimension — .31** .15 .51** 7. Anxiety dimension — −.13 −.30** 8. Instrumental care — .22 9. Emotional care — Note: AAS = Adult Attachment Scale; RQ = Relationship Questionnaire. * p < .05; **p < .01. Table 2. Estimated Marginal Means and Standard Errors for Instrumental and Emotional Caregiving Instrumental Care Emotional Care Attachment Style M SE M SE Secure .10 .14 .45 .12 Fearful −.77 .35 −.68 .29 Dismissive .12 .40 −.89 .34 Preoccupied .01 .25 −.75 .21 Note: Scores are standardized because of different scales of measurement for instrumental and emotional caregiving. Table 3. Correlations Among Demographics, Attachment Dimensions, Caregiving, and Burden 2 3 4 5 6 7 8 9 10 11 12 13 1. Ethnicity .24* .16 .28* .14 −.13 .17 .03 −.19 .12 −.07 .04 .00 2. SES — .13 .07 −.02 −.08 .21 −.08 −.16 −.06 −.20 −.08 −.03 3. Work status — .20 .05 −.25* .32** .08 −.18 .13 −.21 −.21 −.03 4. Marital status — .34** −.10 .06 −.07 −.19 −.06 .00 .00 .04 5. No. of coresident children — −.06 .06 −.04 −.06 −.10 .12 −.02 .26* 6. Mother's coresidence — −.66** −.05 .10 −.07 .48** −.09 .34* 7. ADL score — .19 .02 .06 −.76** .11 −.52** 8. Duration of caregiving — −.10 .04 −.21 .06 −.11 9. Security dimension — −.01 .15 .51** .26* 10. Anxiety dimension — −.13 −.30** .00 11. Instrumental care — .22* .49** 12. Emotional care — −.15 13. Caregiver burden — Notes: Ethnicity, 1 = African American, 2 = European American; work status, 0 = not working, 1 = working; marital status, 0 = unmarried, 1 = married; coresidence, 0 = no, 1 = yes. ADL = activities of daily living; SES = socieconomic status. * p < .05; **p < .01. Table 4. Summary of Hierarchical Regression Analyses of Demographic, Caregiving, and Attachment Dimensions on Instrumental Care, Emotional Care, and Caregiver Burden Instrumental Carea Emotional Careb Caregiver Burdenc Predictor Variables b SE b β sr 2 b SE b β sr 2 b SE b β sr 2 Step 1 Ethnicity 0.73 2.42 0.02 .00 3.92 2.61 0.14 .01 0.42 1.16 −0.04 .00 SES −0.25 0.66 −0.03 .00 −0.18 0.72 −0.02 .00 0.20 0.32 0.06 .00 Work status 4.82 2.57 0.14 .01 −6.48 2.78 −0.21 .03* 1.20 1.26 0.10 .01 Marital status −1.69 2.47 −0.05 .00 2.96 2.70 0.10 .01 −1.12 1.19 −0.10 .01 No. of coresident children 3.25 1.21 0.19 .03* −2.54 1.36 0.17 .02 1.71 0.61 0.29 .06** Mother's coresidence 0.48 2.95 0.01 .00 −4.05 3.20 −.014 .01 0.92 1.41 0.08 .00 Duration of caregiving −0.02 0.01 −0.09 .01 0.03 0.02 0.15 .02 0.00 0.01 −0.03 .00 Step 2 ADL score −1.64 0.19 −0.82 .32*** 0.82 0.28 0.48 .06** −0.24 0.13 −0.35 .03 Step 3 Instrumental care 0.49 0.12 0.57 .11*** 0.07 0.06 0.22 .01 Emotional care 0.41 0.10 0.35 .07*** 0.03 0.05 0.07 .00 Step 4 Security dimension −0.06 1.40 −0.00 .00 6.37 1.32 0.44 .15*** −1.82 0.67 −0.32 .06** Anxiety dimension 0.43 1.24 0.03 .00 −4.02 1.27 −0.27 .06** 0.58 0.59 0.10 .01 Notes: Coefficients are from the final Step 4 model. Only two significant differences in coefficients were noted from the final results: In the regression on instrumental care, coresidence was significant in Step 2 but in no subsequent steps, and in the regression on caregiver burden, coresidence was significant in Step 1 and ADL score was significant in Step 2, but neither was significant in subsequent steps. ADL = activities of daily living; SES = socioeconomic status; sr2 = squared semi-partial correlation. a For the regression on instrumental care, R2 = .33 for Step 1 (p < .001); ΔR2 = .29 for Step 2 (p < .001); ΔR2 = .11 for Step 3 (p < .001); ΔR2 = .00 for Step 4 (p = .94). b For the regression on emotional care, R2 = .08 for Step 1 (p = .48); ΔR2 = .01 for Step 2 (p = .30); ΔR2 = .25 for Step 3 (p < .001); ΔR2 = .21 for Step 4 (p < .001). c For the regression on caregiver burden, R2 = .21 for Step 1 (p < .05); ΔR2 = .17 for Step 2 (p < .001); ΔR2 = .02 for Step 3 (p = .41); ΔR2 = .06 for Step 4 (p < .05). * p < .05; **p < .01; ***p < .001. Brian D. Carpenter is now at the Department of Psychology, Washington University in St. Louis. Support for this research was provided by a grant from the Retirement Research Foundation and Division 20 of the American Psychological Association. Generous cooperation was provided by Linda Noelker and MaryAnn Caston at the Benjamin Rose Institute, Cleveland, OH. This study was the dissertation research of the author, conducted at Case Western Reserve University under the guidance of Milton E. Strauss. Portions of these data were presented at the 1997 Annual Meeting of the Gerontological Society of America. Address correspondence to Brian D. Carpenter, Department of Psychology, Washington University, Box 1125, St. Louis, MO 63130. Ainsworth M. D. S., 1989. Attachments beyond infancy. American Psychologist 44:709-716. Ainsworth M. D. S., Blehar M. C., Waters E., Wall S., 1978. Patterns of attachment: A psychological study of the strange situation Erlbaum, Hillsdale, NJ. Bartholomew K., 1990. Avoidance of intimacy: An attachment perspective. Journal of Social and Personal Relationships 7:147-178. Bartholomew K., Horowitz L. M., 1991. Attachment styles among young adults: A test of a four-category model. 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W., Bartholomew K., 1994. Models of the self and other: Fundamental dimensions underlying measures of adult attachment. Journal of Personality and Social Psychology 67:430-445. Hannappel M., Calsyn R. J., Allen G., 1993. Does social support alleviate the depression of caregivers of dementia patients?. Journal of Gerontological Social Work 20:35-51. Hazan C., Shaver P. R., 1990. Love and work: An attachment-theoretical perspective. Journal of Personality and Social Psychology 59:270-280. Ikkink K. K., Van Tilburg T., Knipscheer K. C. P. M., 1999. Perceived instrumental support exchanges in relationships between elderly parents and their adult children: Normative and structural explanations. Journal of Marriage and the Family 61:831-844. Kane R. A., Kane R. L., Ladd R. C., 1998. The heart of long-term care Oxford University Press, New York. Kramer B. J., 1997. Gain in the caregiving experience: Where are we? What next?. The Gerontologist 37:218-232. Krause A. M., Haverkamp B. E., 1996. 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Journal of Gerontology 52B: (Special Issue) 102-109. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0B9BFCC313049B368C3A2E4BDE041AEFF8CA4CD9.txt b/test/dataset/in/resources/corpus/Clean_0B9BFCC313049B368C3A2E4BDE041AEFF8CA4CD9.txt new file mode 100644 index 0000000..89127c0 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0B9BFCC313049B368C3A2E4BDE041AEFF8CA4CD9.txt @@ -0,0 +1 @@ +]>Nature45071692007112249750260028-08361476-46872007Nature Publishing GroupSupplementary InformationThe file contains Supplementary Figures 1-9 with Legends, Supplementary Tables 1-5 and the MIAME Checklist. Supplementary VideoThe file contains Supplementary Video 1 showing contracting cardiomyocytes derived from differentiated CRES cell lines. 10.1038/nature06357Producing primate embryonic stem cells by somatic cell nuclear transferJ. A.ByrneJ AD. A.PedersenD AL. L.ClepperL LM.NelsonMW. G.SangerW GS.GokhaleSD. P.WolfD PS. M.MitalipovS MOregon National Primate Research Center and,Oregon Stem Cell Center, Oregon Health & Science University, 505 N.W. 185th Avenue, Beaverton, Oregon 97006, USAMunroe-Meyer Institute, 985450 Nebraska Medical Center, Omaha, Nebraska 68198, USAWhitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USAPresent address: Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Palo Alto, California 94304, USA.Correspondence and requests for materials should be addressed to S.M.M. (mitalipo@ohsu.edu).Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.&e071122-1; &nature06357-s1;ES cells can differentiate into multiple cell types, representatives of which could be used in replacement therapy for ageing or diseased cells and tissues. However, ES cells derived from in vitro fertilized (IVF) embryos are genetically divergent from the patient (allogenic) and thus any resultant transplanted cell would be rejected without the continual application of immunosuppressive drugs. One way to resolve completely the transplant rejection issue would be to generate ES cells that are genetically identical to the patient. Recent reports suggest that somatic cells can be directly reprogrammed into ES-cell-like cells in mice, after transfection with key stem-cell-specific (stemness) genes. However, the ability to extrapolate this approach to other mammals, including primates, remains in question, and the propensity of such cells to develop into tumours owing to c-myc transgene reactivation remains a concern. Alternatively, ES cells can be derived by epigenetic reprogramming of somatic cells via SCNT in spindle-free oocytes, a process commonly referred to as therapeutic cloning (Supplementary Fig. 1). However, despite encouraging results in the murine model, the feasibility of therapeutic cloning in primates remains in question. Although many mammals have been successfully produced by means of SCNT no successful primate reproductive cloning has been achieved. Moreover, the efficiency of blastocyst formation after human and non-human primate SCNT has typically been very low, suggesting lack or incomplete nuclear reprogramming with existing SCNT protocols. We recently reported incomplete nuclear remodelling, including nuclear envelope breakdown and premature chromosome condensation, after standard SCNT in rhesus macaques (Macaca mulatta) and correlated this observation with a decline in maturation promoting factor activity. SCNT protocols designed to prevent premature cytoplast activation and maturation promoting factor decline resulted in robust nuclear envelope breakdown and premature chromosome condensation and in significantly increased blastocyst development, suggesting that maturation promoting factor activity is instrumental for efficient nuclear reprogramming. Here we used these SCNT protocols to demonstrate therapeutic cloning in the rhesus macaque and provide a translational model for testing the feasibility, therapeutic effectiveness and long-term safety of therapeutic cloning as a concept.Embryo development and ES cell derivationThe primary culture of skin fibroblasts, used as the source of nuclear donor cells for SCNT, was established from a nine-year-old adult rhesus macaque male (male 1) housed at the Oregon National Primate Research Center (Supplementary Fig. 2). Mature metaphase II rhesus macaque oocytes were rendered spindle-free with the Oosight spindle imaging system that uses polarized light to visualize the oocyte meiotic spindle (Supplementary Fig. 3). Analysis of the removed karyoplasts, for the presence of the meiotic spindles, consistently confirmed a 100% efficiency of spindle removal using this approach. The donor fibroblast nuclei were introduced into cytoplasts by electrofusion, incubated for 2 h to allow nuclear remodelling to occur, and subsequently activated and cultured to the blastocyst stage as described previously (see also Methods). We observed a 16% (35 out of 213) blastocyst formation rate with this nuclear donor cell line (Supplementary Table 1). SCNT blastocysts demonstrated a similar morphology to low-grade IVF blastocysts (Supplementary Fig. 4). Twenty expanded or hatching SCNT blastocysts were used for ES cell derivation via mechanical inner cell mass (ICM) isolation (n = 2), immunosurgery (n = 15) or direct culture of intact blastocysts (n = 3) on mouse embryonic fibroblast (MEF) feeder layers. Two ES cell lines (cloned rhesus embryonic stem: CRES-1 and CRES-2) were derived, both after immunosurgery (10% derivation efficiency from blastocysts). Overall, 304 oocytes collected from 14 rhesus macaque females were used to generate two ES cell lines, a 0.7% derivation efficiency from oocytes.Genetic analysis and pluripotencyAs the number of mitochondria, each with 16.6 kilobases (kb) of mitochondrial DNA (mtDNA), in the cytoplast dwarfs any mitochondrial contribution from the donor somatic cells, embryos derived by SCNT should predominantly, if not exclusively, possess mitochondria inherited from the oocyte. Therefore, ES cells derived from SCNT embryos should contain mtDNA identical to the female providing the recipient cytoplasts and nuclear DNA genetically identical to the male providing the nuclear donor cells. To investigate whether the CRES-1 and CRES-2 cell lines contained the same nuclear DNA as the donor (male 1) fibroblasts, we performed microsatellite typing using 39 short tandem repeat (STR) loci and analysis of 56 single nucleotide polymorphisms (SNPs), 30 of which were informative for inheritance. Both the STR analysis—which included 25 common STR loci (Table 1) and 14 major histocompatibility complex (MHC)-linked STRs (Supplementary Table 2)—and the SNP analysis (Supplementary Table 3) demonstrated a complete match of both CRES lines to each other and to the nuclear DNA isolated from skin fibroblasts and peripheral blood leucocytes of male 1. In contrast, DNA obtained from the oocyte donor females for CRES-1 (female 1) and CRES-2 (female 2) demonstrated no significant similarity to CRES-1 or CRES-2 (Table 1 and Supplementary Tables 2 and 3). The genomic constitution of an IVF-derived rhesus macaque ES cell line (ORMES-22; ref. 17), and the ORMES-22 oocyte donor female (female 3) and sperm donor male (male 2), were also included to demonstrate STR allele inheritance (Table 1 and Supplementary Table 2).To investigate whether the CRES-1 and CRES-2 cell lines contained the same mtDNA as their respective oocyte donor females, we performed mtDNA sequence analysis investigating an informative domain 1 (ID1) in the rhesus macaque mitochondrial D-loop hypervariable region 2 (RhDHV2). This RhDHV2 sequence contained multiple informative SNPs including at ID1 nucleotide positions 4, 22 and 28 (Fig. 1). Analysis of SNP22 (an A-to-G polymorphism) demonstrated that CRES-1 mtDNA was derived from the oocyte donor female 1 and not from the nuclear donor for CRES-1. Similarly, analysis of SNP4 (a C-to-T polymorphism) and SNP28 (an A-to-G polymorphism) confirmed that the CRES-2 mtDNA was derived from the CRES-2 oocyte donor female 2 and not from the nuclear donor. Thus, microsatellite, SNP and mtDNA analyses verified that CRES-1 and CRES-2 contained nuclear DNA genetically identical to the nuclear donor fibroblasts and mtDNA inherited from oocytes, a hallmark of SCNT-produced ES cells and offspring.Both CRES lines demonstrated typical ES cell morphology (Fig. 2a–d), maintained an undifferentiated morphology after repeated manual passaging (>20 passages per line so far) and expressed key primate stemness markers including OCT4 (also called POU5F1), SSEA-4, TRA1-60 and TRA1-81 (Fig. 2). Moreover, transcripts of other stemness genes including NANOG, SOX2, LEFTYA (also called LEFTY2), TDGF and TERT were detected by polymerase chain reaction with reverse transcription (RT–PCR) analysis in both IVF-derived ES cell controls (ORMES-10 and ORMES-22) and CRES cell lines (Supplementary Fig. 5). Conventional cytogenetic G-banding analysis of the nuclear donor fibroblasts used for SCNT (Supplementary Fig. 6) and the CRES-2 cell line (Fig. 3a) demonstrated a normal male rhesus macaque chromosome (42, XY) complement in all cells analysed. However, analysis of the CRES-1 cell line indicated the presence of three metaphase cells representing a hypodiploid clone characterized by loss of the Y chromosome (Supplementary Fig. 7), and seventeen cells representing a diploid clone characterized by an isochromosome comprised of two copies of the long arm of the Y chromosome (41,X[3]/42,X,i(Y)q10)[17]) (Fig. 3b). Subsequent fluorescent in situ hybridization (FISH) analysis confirmed the G-banding findings; metaphase cells revealed the presence of a signal for bacterial artificial chromosome (BAC) CH250-283K14 on both arms of the Y chromosome (Fig. 3d), indicating the presence of the i(Y)(q10) observed in the G-banding study. Additional studies were positive for loss of the Y chromosome in 12% of the CRES-1 cells analysed.Transcriptional profilingGlobal transcription profiles of three biological replicates each of male 1 skin fibroblasts (nuclear donor for both CRES lines), both CRES-1 and CRES-2 cell lines and two control ES cell lines derived from fertilized embryos (ORMES-10 and ORMES-22) were examined by Affymetrix microarray analysis. For the primary microarray comparison (Supplementary Data 3), three types of analyses were performed: (1) replicates of each cell line were compared against each other, (2) each cell line was compared against the somatic donor cell line; and (3) each cell line was compared to a control IVF-derived ES cell line (see Methods). For each comparison, the detected signal for each present ‘P’ probe set (P < 0.05) was plotted in a scatter graph, the number of present probe sets (PP) used was recorded and the correlation value was calculated. All comparisons of control ORMES biological replicates with each other demonstrated a correlation value of greater than 60% and all unrelated sample comparisons (that is, between ES cell and somatic cell biological replicates) demonstrated a correlation value of significantly less than 60%; therefore a correlation value of 60% or greater was considered indicative of a significant transcriptional correlation. When the replicates of the somatic donor cells were compared, 99% transcriptional correlation was observed (Supplementary Fig. 8a), suggesting that minimal artificial variation was introduced via the protocols used. Although it was not possible to determine with certainty the degree of technical versus biological variation between replicates, it should be noted that all samples were processed identically and at the same time, and the level of technical variation between the donor somatic cell samples was 1% or less, suggesting that most of the 20–30% transcriptional variation observed between ES cell replicates was biological in origin. If so, ES cells show significant transcriptional plasticity not observed in somatic cells (Supplementary Fig. 8a). However, further transcriptional profiling will be required to confirm this observation. Comparisons of the CRES cell lines to the somatic donor cells and control IVF-derived ES cells demonstrated that both CRES lines had fully reprogrammed into an ES cell transcriptional state, with no significant transcriptional correlation between the CRES lines and the donor somatic cells (Supplementary Fig. 8b) but a significant correlation between CRES cells and the control ES cells (Supplementary Fig. 8c).To identify ES-cell-specific genes, comparison analysis was performed between each of the three control ORMES-10 replicates and each of the three somatic donor cell replicates, to give a total of nine ES cell–somatic comparisons (Supplementary Data 4). This set of ES cell comparisons identified 4,998 somatic-cell-specific probe sets/genes (Supplementary Data 5) and 6,178 ES-cell-specific probe sets/genes (Supplementary Data 6 and Methods). Over 90% of the somatic-cell-specific genes were significantly downregulated in the CRES cell line replicates (Supplementary Table 4), and over 85% of the ES-cell-specific genes demonstrated significantly greater expression in the CRES cell line replicates (Table 2) when compared with the somatic donor cells. Transcriptional analysis of the control ORMES-22 replicates also demonstrated that over 90% of the somatic-specific genes had significantly less expression (Supplementary Table 4) and over 85% of the ES-cell-specific genes had significantly greater expression (Table 2) when compared with the somatic donor cells.The final microarray analysis involved examining the level of expression of 12 putative rhesus macaque stemness genes identified in previous transcriptional profiling. These putative stemness genes had the highest average fold change in gene expression when undifferentiated ES cell biological replicates were compared to their in vitro differentiated counterparts, and all 12 were significantly upregulated in the five different ES cell lines examined. All 12 stemness genes were significantly upregulated in all of the ORMES-10, ORMES-22, CRES-1 and CRES-2 replicates (Supplementary Data 7), and the average fold change in gene expression for both CRES-1 and CRES-2 was comparable to that for ORMES-10 and ORMES-22 when compared to somatic donor cell replicates (Table 3). As a control, the relative fold change in expression for these putative stemness genes between the donor somatic cell replicates was insignificant (Table 3).Our overall conclusion after analysis of global transcriptional profiles is that both CRES-1 and CRES-2 cells are transcriptionally similar to control ES cell lines derived from IVF-produced blastocysts.Differentiation potentialTo define pluripotency further, both CRES lines were exposed to conditions for cardiomyocyte differentiation in vitro. CRES-1 and CRES-2 efficiently produced contracting aggregates (Supplementary Video) expressing markers of cardiac muscle tissue (Supplementary Fig. 9). Directed neural differentiation resulted in efficient formation of various neural phenotypes, demonstrating elongated cellular morphology and expression of neural markers, including microtubule-associated protein 2 (MAP2; Fig. 2e), &bgr;-III-tubulin (Fig. 2f) and tyrosine hydroxylase (Fig. 2g). When injected into severe combined immune deficiency (SCID) mice, both CRES lines formed teratomas and subsequent histological analysis identified representatives of all three germ layers (Fig. 2h–k), confirming their pluripotent status.DiscussionOur results demonstrate successful reprogramming of primate somatic cells into pluripotent ES cells, provided that an efficient SCNT method is available. We achieved a significant increase in the blastocyst formation rate (from 1% to 16%) when the use of Hoechst 33342 was avoided during the oocyte spindle removal step. The detrimental effect of fluorochrome bisbenzimide (Hoechst 33342) and ultraviolet light on cytoplast quality has been previously documented, and we speculate that the impaired blastocyst formation rate after conventional SCNT in primates may result from one or more of the following factors: Hoechst 33342 and/or ultraviolet damage to the relatively transparent primate oocyte; Hoechst 33342/UV-induced oocyte activation and/or maturation promoting factor degradation; reaction of the residual Hoechst 33342 in cytoplasts with the introduced donor cell DNA, thereby impairing reprogramming; and/or Hoechst 33342 contact with mitochondrial DNA, thus reducing cytoplast mitochondrial function.Recognizing the importance of high-quality cytoplasts for successful reprogramming, we have sought non-invasive approaches for spindle detection and removal. ‘Blind’ enucleation techniques involving ‘squish’ or ‘one-step manipulation’ were inefficient, at least in our hands, because they failed to enucleate all oocytes. In fact, our initial effort to derive ES cells from SCNT blastocysts produced with these protocols resulted in the isolation of parthenogenetic ES cells owing to failed spindle removal. Fortunately, recent developments in high-performance imaging resulted in an Oosight spindle imaging system supporting rapid and highly efficient real-time enucleation of primate oocytes. Introduction of the donor nucleus can be accomplished by either direct injection or electrofusion—the latter was dictated here by the relatively large donor cells used. The SCNT blastocyst development rate was significantly lower than fertilized controls but within the 7–29% range described by us when fetal fibroblasts, adult ear fibroblasts, cumulus and oviductal epithelial cells were used as nuclear donor cells in the monkey. Blastocysts produced by SCNT usually demonstrated poor morphology and have, so far, failed to support a term pregnancy after embryo transfer despite considerable efforts (D.P.W., unpublished data). This result may not be predictive for ES cell isolation, however, as the requirements for reproductive and therapeutic cloning are different in that a normal trophectoderm is not required for the latter.With an adequate supply of SCNT blastocysts, the final challenge in therapeutic cloning is ES cell isolation, and although several methods were examined, the conventional method involving immunosurgical dispersal of the trophectoderm seemed to be the best. The derivation efficiency in the present study is within the range reported in the mouse, where the ES cell derivation efficiency from SCNT embryos was 0.2–3.4% per oocyte and 4–10% per blastocyst.Regarding the origin of the CRES lines, in addition to the 100% spindle removal efficiency, karyotype, microsatellite and SNP analyses confirmed that both CRES lines originated from SCNT embryos and not from parthenotes. CRES lines demonstrated typical ES cell morphology, self-renewal capacity and expression of stemness markers. These cell lines were also transcriptionally similar to ES cells derived from fertilized blastocysts, and pluripotent, as demonstrated by the generation of representatives of all three germ layers after in vivo teratoma formation. Our results parallel findings in the mouse and confirm the possibility of complete nuclear reprogramming of somatic cells into pluripotent ES cells via SCNT. The CRES-1 cell line revealed a translocation of unknown origin characterized by an isochromosome comprised of two copies of the long arm of chromosome Y, whereas CRES-2 exhibited a normal euploid male karyotype. In our previous study, five out of seven analysed SCNT blastocysts were karyotypically normal with two embryos demonstrating aneuploidy. Karyotyping individual SCNT embryos based on either biopsied blastomere or trophectodermal cell analysis followed by ES cell isolation could address whether chromosomal abnormalities in SCNT-derived ES cells originate from aneuploid embryos or occur during ES cell isolation and culture.The primary rationale for therapeutic cloning is transplantation of histocompatible ES-cell-derived phenotypes back into the patient. The MHC profile of both CRES lines perfectly matched the donor male in all MHC loci examined via microsatellite analysis, suggesting that transplantation of differentiated derivatives back into the donor animal would not lead to rejection. It is possible that immune response will occur in the donor animal resulting from the epitopes derived from the allogenic recipient oocyte mitochondria contribution; although preliminary research with cloned bovine and porcine cells and tissues with allogenic mtDNA suggests that grafting back into the nuclear donor organism can occur without destruction by the immune system. The use of biological materials from other species, such as fetal bovine serum, mouse embryonic fibroblasts and guinea-pig complement, may also contaminate ES cells with xeno-epitopes and pathogens that would make such cells unsuitable for clinical applications. Other possibly minor factors that should nevertheless also be investigated include the problems that may arise from the lack of sperm-derived centrosomes, the possible effects of non-random X-inactivation and the possibility that cloned ES cells may possess the shortened telomeres of their donor somatic cell source. The possibility has also been suggested that SCNT embryos are epigenetically abnormal based on DNA methylation patterns. Therefore, the epigenetic status of CRES cell lines including imprinted gene expression would also be an interesting area for future research.Assuming that our modified SCNT protocols are applicable to humans, the low efficiency of ES cell derivation (0.7%) along with restricted availability and high cost of human oocytes suggest that a significant increase in the overall SCNT embryo generation and ES cell derivation rates would be required before a clinical implementation of human therapeutic cloning could occur. Recently, mouse ES cells were relatively efficiently cloned using aged, failed-to-fertilize oocytes, and substantial numbers of such human oocytes augmented by immature oocytes are routinely discarded, representing an untested source for human SCNT. In addition to therapeutic cloning, human SCNT-derived ES cells would also be extremely valuable in studies devoted to understanding disease mechanisms and developing possible treatments through in vitro experimentation.Methods SummaryA primary culture of fibroblasts was established from a skin biopsy of an adult rhesus macaque male (male 1) and prepared for SCNT as previously described. Mature metaphase II oocytes were rendered spindle-free using the Oosight imaging system (CRI, Inc.) and a donor somatic cell nucleus was introduced into a cytoplast through electrofusion. Reconstructed embryos were activated 2 h after fusion by exposure to 5 &mgr;M ionomycin (CalBiochem) for 5 min followed by a 5-h incubation in 2 mM 6-dimethylaminopurine (DMAP), placed in HECM-9 medium and cultured at 37 °C in 6% CO2, 5% O2 and 89% N2 until the expanded blastocyst stage. The ICMs of selected SCNT blastocysts were plated onto MEF feeder layers and cultured in ES cell culture medium for 5–7 days. ICMs that attached to the feeder layer and initiated outgrowth were manually dissociated into small clumps with a microscalpel and replated onto fresh MEFs. After the first passage, colonies with ES-cell-like morphology were selected for further propagation, characterization, low-temperature storage and in vitro and in vivo differentiation, as previously described.AnimalsAdult rhesus macaques housed in individual cages were used in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee at the ONPRC/OHSUOvarian stimulation of rhesus macaquesControlled ovarian stimulation and oocyte recovery was performed as has previously been described. Briefly, cycling females were subjected to follicular stimulation using twice-daily intramuscular injections of recombinant human FSH as well as concurrent treatment with Antide, a GnRH antagonist, for days 8–9. Unless indicated otherwise, all reagents were from Sigma-Aldrich and all hormones were from Ares Advanced Technologies Inc. Females received recombinant human luteinizing hormone on days 7-9 and recombinant human chorionic gonadotrophin (hCG) on day 10.Somatic cell nuclear transferA primary culture of fibroblasts was established from an adult rhesus macaque male (male 1) as previously described. Briefly, a small skin biopsy was surgically derived, washed in Ca2+- and Mg2+-free Dulbecco PBS (Invitrogen) and minced into pieces before incubation in Dulbecco Modified Eagle’s Medium (DMEM, Invitrogen) containing 1 mg ml-1 collagenase IV (Invitrogen) at 37 °C in 5% CO2 for 40 min. Tissue pieces were then vortexed, washed, seeded into 75 cm3 cell culture flasks (Corning) containing DMEM supplemented with 100 IU ml-1 penicillin, 100 &mgr;g ml-1 streptomycin (Invitrogen), 10% FBS (DMEM/FBS culture media) and cultured at 37 °C in 5% CO2 until reaching confluency. Fibroblasts were then disaggregated with trypsin treatment and frozen down in aliquots of 1 × 106 cells in medium containing 10% dimethyl sulphoxide (DMSO).Fibroblasts were subsequently thawed, plated onto 4-well dishes (Nunc) and cultured under standard conditions until reaching 50–90% confluency. Cells were then synchronized in the G0/G1 phase of the cell cycle by culturing in DMEM medium with 0.5% FBS for 4 days before SCNT. Cumulus–oocyte complexes were collected from anaesthetized animals by laparoscopic follicular aspiration (28–29 h after hCG) and placed in TALP/HEPES medium (modified Tyrode solution with albumin, lactate and pyruvate) containing 0.3% BSA (TH3) at 37 °C. Oocytes were stripped of cumulus cells by mechanical pipetting after brief exposure (<1 min) to hyaluronidase (0.5 mg ml-1) and placed in chemically defined, protein-free HECM-9 medium (hamster embryo culture medium) at 37 °C in 6% CO2, 5% O2 and 89% N2 until further use.Recipient MII oocytes were transferred to 30 &mgr;l manipulation droplets of TH3 with 5 &mgr;g ml-1 cytochalasin B on a glass bottom manipulation dish (http://www.willcowells.com) covered with paraffin oil (Zander IVF) and incubated at 37 °C for 10–15 min before spindle removal. The chamber was then mounted on an inverted microscope (Olympus) equipped with the Oosight imaging system (CRI, Inc.), glass stage warmer (Tokai Hit, http://www.tokaihit.com) and Narishige micromanipulators. The spindle was located and extracted by aspiration into an enucleation pipette (20–25 &mgr;m outer diameter). Metaphase spindle removal was confirmed by its presence in the enucleation pipette. The Oosight imaging system allows non-invasive, polarized light imaging and detection of the spindle based on birefringence. Using this innovative approach, we were able to locate and quickly remove the oocyte spindle real-time with 100% efficiency. After oocyte spindle removal a disaggregated donor somatic cell was aspirated into a micropipette and placed into the perivitelline space of the cytoplast on the side opposite the first polar body. Cell fusion was induced by two 50 &mgr;s DC pulses of 2.7 kV cm-1 (Electro Square Porator T-820, BTX, Inc.) in 0.25 M d-sorbitol buffer containing 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.5 mM HEPES and 1 mg ml-1 fatty-acid-free BSA. Successful fusion was confirmed visually 30 min after electroporation by the disappearance of the donor cell in the perivitelline space. All nuclear transfer micromanipulation and fusion procedures were conducted on microscope stage warmers (Tokai Hit) maintaining 37 °C. Reconstructed embryos were activated by exposure to 5 &mgr;M ionomycin for 5 min in TALP/HEPES medium supplemented with 1 mg ml-1 fatty-acid-free BSA and then transferred for 5 min in TALP/HEPES medium supplemented with 30 mg ml-1 fatty-acid-free BSA and 2 mM 6-dimethylaminopurine (DMAP) followed by a 5 h incubation in HECM-9 medium containing 2 mM DMAP at 37 °C in 6% CO2. Activated oocytes were placed in 4-well dishes containing HECM-9 medium supplemented with 10% FBS and 12 &mgr;M &bgr;-mercaptoethanol (BME) and cultured at 37 °C in 6% CO2, 5% O2 and 89% N2 for a maximum of 10 days with medium change every other day.ES cell derivation and cultureZonae pellucidae of selected expanded SCNT blastocysts were removed by brief exposure (45–60 s) to 0.5% pronase in TH3 medium. A small proportion of embryos were either transferred directly to MEFs as whole blastocysts or after mechanical dissection of the ICM. Remaining blastocysts were subjected to immunosurgical isolation of the ICMs as previously described. Briefly, zona-free blastocysts were exposed to rabbit anti-rhesus spleen serum (a gift from J. A. Thomson) for 30 min at 37 °C. After extensive washing in TH3, embryos were incubated in guinea-pig complement reconstituted with HECM-9 (1:2, v/v) for an additional 30 min at 37 °C. Partially lysed trophectodermal cells were mechanically dispersed by gentle pipetting with a small bore pipette (125 &mgr;m inner diameter; Stripper pipette, Midatlantic Diagnostics Inc.) followed by the rinsing of ICMs three times with TH3 medium. Isolated ICMs were plated onto Nunc 4-well dishes containing mitotically inactivated feeder layers consisting of MEFs and cultured in DMEM/F12 medium with glucose and without sodium pyruvate supplemented with 1% nonessential amino acids, 2 mM l-glutamine, 0.1 mM &bgr;-mercaptoethanol and 15% FBS at 37 °C, 3% CO2, 5% O2 and 92% N2. ICMs that attached to the feeder layer and initiated outgrowth were manually dissociated into small cell clumps with a microscalpel and replated onto new MEFs. After the first passage, colonies with ES-cell-like morphology were selected for further propagation, characterization and low-temperature storage as previously described. Medium was changed daily and ES cell colonies were split every 5–7 days by manual dissociation and replating collected clumps onto dishes with fresh MEFs.In vitro and in vivo differentiation of ES cellsFor embryoid body formation, entire ES cell colonies were loosely detached from feeder cells and transferred into feeder-free, 6-well, ultra-low adhesion plates (Corning Costar) and cultured in suspension in ES cell medium for 5–7 days. To induce further differentiation, embryoid bodies were transferred into collagen--well culture dishes (Becton Dickinson) to allow attachment. For neuronal differentiation, medium was replaced with serum-free DMEM/F12 containing ITS supplement (insulin, transferrin and sodium selenite, Invitrogen) and fibronectin (5 &mgr;g ml-1; Invitrogen). Cultures were maintained for 7 days, with medium replenishment every 2 days. The resulting cultures were disaggregated with collagenase or trypsin treatment and replated onto polyornithine- and laminin-coated plates or glass coverslips in N2 medium consisting of DMEM/F12 supplemented with laminin (1 &mgr;g ml-1; Invitrogen), bFGF (10 ng ml-1; R&D Systems), and N2 supplement (Invitrogen). Cultures were maintained for an additional 7 days with a daily medium change. After 7 days, bFGF was omitted from the medium and cultures were maintained for an additional 7–12 days to induce differentiation into mature neuronal phenotype. Differentiation into cardiac cells was initiated by embryoid body formation in suspension as described above. Embryoid bodies were then plated into collagen-coated dishes and cultures were maintained in ES cell medium for 2–4 weeks. In order to obtain teratomas, 3–5 million undifferentiated ES cells from each cell line were harvested and injected into the hind-leg muscle or subcutaneously into 4-week-old, SCID, beige male mice using an 18 gauge needle. Six to seven weeks after injection, mice were killed and teratomas were dissected, sectioned and histologically characterized for the presence of representative tissues of all three germ layers.Immunocytochemical proceduresUndifferentiated and differentiated ES cells were fixed in 4% paraformaldehyde for 20 min. After permeabilization with 0.2% Triton X-100 and 0.1% Tween-20, nonspecific reactions were blocked with 10% normal serum (Jackson ImmunoResearch Laboratories, Inc.). Cells were then incubated for 40 min in primary antibodies, washed three times and exposed to secondary antibodies conjugated with fluorochromes (Jackson ImmunoResearch) before co-staining with 2 &mgr;g ml-1 4',6-diamidino-2-phenylindole (DAPI) for 10 min, whole-mounting onto slides and examination under epifluorescence microscopy. Primary antibodies for OCT4, SSEA-4, TRA1-60 and TRA1-81 were from Santa Cruz Biotechnology Inc. Neural-specific antibodies including microtubule-associated protein (MAP2C), &bgr;-III-tubulin and tyrosine hydroxylase were from Chemicon International Inc.Cytogenetic analysisMitotically active ES cells in log phase were incubated with 120 ng ml-1 ethidium bromide for 40 min at 37 °C, 5% CO2, followed by 120 ng ml-1 colcemid (Invitrogen) treatment for 20–40 min. Cells were then dislodged with 0.25% trypsin, and centrifuged at 200g for 8 min. The cell pellet was gently re-suspended in 0.075 M KCl solution and incubated for 20 min at 37 °C followed by fixation with methanol:glacial acetic acid (3:1) solution. Cytogenetic analysis was performed on 20 metaphase cells from each ES cell line following standard GTW-banding procedures. Images were acquired using the Cytovision Image Analysis System (Applied Imaging) and karyotypes were arranged as previously described. FISH analysis was performed on metaphase cells from the donor somatic cell line and CRES-1 using BAC CH250-283K14, specific for the rhesus macaque Yq11.21 region. The clone was obtained from the CHORI-250 rhesus macaque BAC library (Children’s Hospital Oakland Research Institute, BACPAC Resources), grown in LB media supplemented with 25 µg ml-1 chloramphenicol and isolated using a mini-prep protocol adapted from the standard Qiagen plasmid purification method (Qiagen Inc.). Probes were nick-translated with rhodamine-5-dUTP (Enzo Life Sciences) using a modification of the manufacturer’s protocol (Vysis). Approximately 200 ng of the probe was precipitated along with 2 &mgr;g human Cot-I DNA (Invitrogen). Before hybridization, slides were pre-treated at 73 °C in 2× SSC for 2 min, followed by digestion at 37 °C in a protease solution for 5 min, post-fixation in 10% buffered formaldehyde with 2 M MgCl, and dehydration. The slide and probe mixture were co-denaturated at 74 °C for 2 min and incubated overnight at 37 °C using the HYBrite system (Vysis). Images were acquired using the Cytovision image analysis system.RT–PCRTotal RNA was extracted from cells using an RNA purification kit (Invitrogen) according to the manufacturer’s instructions. Total RNA was treated with DNase I before cDNA preparation using the SuperScript III first-strand synthesis system for RT–PCR (Invitrogen) according to the manufacturer’s instructions. The first strand cDNA was further amplified by PCR using individual primer pairs for specific genes. The primer sequence, annealing temperature and amplicon size for each pair of primers are listed in Supplementary Table 5. All PCR samples were analysed by electrophoresis on 2% agarose gel containing 0.5 &mgr;g ml-1 ethidium bromide and visualized on a transilluminator.Microsatellite analysisFor STR genotyping, DNA was extracted from blood or cultured cells using commercial kits (Gentra). Six multiplexed PCR reactions were set up for the amplification of 39 markers representing 25 autosomal loci and 14 autosomal, MHC-linked loci. On the basis of the published rhesus macaque linkage map, these markers are distributed in 19 chromosomes. Two of the markers included in the panel, MFGT21 and MFGT22, were developed from Macaca fuscata and do not have a chromosome assignment. PCRs were set up in 25 &mgr;l reactions containing 30–60 ng DNA, 2.5 mM MgCl2, 200 &mgr;M dNTPs, 1× PCR buffer II, 0.5 U Amplitaq (Applied Biosystems) and fluorescence-labelled primers in concentrations ranging from 0.06 to 0.9 &mgr;M, as required for each multiplex PCR. Cycling conditions consisted of four cycles of 1 min at 94 °C, 30 s at 58 °C, 30 s at 72 °C, followed by 25 cycles of 45 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C and a final extension at 72 °C for 30 min. PCR products were separated by capillary electrophoresis on the ABI 3730 DNA analyser (Applied Biosystems) according to the manufacturer’s instructions. Fragment size analysis and genotyping was done with the computer software STRand (http://www.vgl.ucdavis.edu/informatics/STRand/). Primer sequences for MHC-linked STRs 9P06, 246K06, 162B17(A and B), 151L13, 268P23 and 222I18 were designed from the corresponding rhesus macaque BAC clone sequences deposited in GenBank (accession numbers AC148662, AC148696, AC148683, AC148682, AC148698 and AC148689, respectively). Loci identified by the letter ‘D’ prefix were amplified using heterologous human primers.SNP analysisSNP analysis was performed as previously described. Briefly, SNP genotyping was performed using iPLEX reagents and protocols for multiplex PCR, single base primer extension and generation of mass spectra, as per the manufacturer’s instructions (for complete details see iPLEX Application Note, Sequenom). Four multiplexed assays containing 56 SNPs were included, 30 which were informative for CRES inheritance (see Supplementary Table 3). All 56 SNPs demonstrated a 100% match between male 1 donor cells and the CRES-1 and CRES-2 cells lines. Initial multiplexed PCRs were performed in 5 &mgr;l reactions on 384-well plates containing 5 ng of genomic DNA. Reactions contained 0.5 U HotStar Taq polymerase (QIAGEN), 100 nM primers, 1.25× HotStar Taq buffer, 1.625 mM MgCl2 and 500 &mgr;M dNTPs. After enzyme activation at 94 °C for 15 min, DNA was amplified with 45 cycles of 94 °C × 20 s, 56 °C × 30 s, 72 °C × 1 min, followed by a 3-min extension at 72 °C. Unincorporated dNTPs were removed using shrimp alkaline phosphatase (0.3 U, Sequenom). Single-base extension was carried out by addition of single base primer extension primers at concentrations from 0.625 &mgr;M to 1.25 &mgr;M using iPLEX enzyme and buffers (Sequenom). Single base primer extension products were measured using the MassARRAY Compact system, and mass spectra were analysed using TYPER software (Sequenom).Mitochondrial DNA analysisDNA was extracted from cell lines using commercial kits (Gentra). The rhesus macaque mitochondrial D-loop hypervariable region 2 (RhDHV2) sequence was amplified for each sample using primers RhDF2 (5′-TAACATATCCGATCAGAGCC-3′) and RhDR (5′-TTAAACACCCTCTACGCCG-3′). PCR for each sample was performed using Platinum PCR SuperMix (Invitrogen) containing 0.5 &mgr;M of each primer (final volume 50 &mgr;l). Reaction conditions were initial denaturation at 94 °C for 2 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s and a final extension at 72 °C for 3 min, generating 544 bp of sequence covering the RhDHV2 region. Products from these reactions were then sequenced to determine polymorphic variation in the RhDHV2 region by direct sequencing. The informative domain 1 (ID1) sequence encompassing Macaca mulatta mtDNA nucleotide positions 451–480 (GenBank NC_005943) was identified as containing SNPs informative for the mitochondrial inheritance of both CRES-1 and CRES-2. Each ID1 sequence was confirmed by three other sequencing reactions and all of the RhDHV2 chromatograms used in this project were obtained with Sequencher v. 4.7 (GeneCodes).Microarray analysisThe MIAME (Minimum Information About a Microarray Experiment) guidelines for microarray research were incorporated into the design and implementation of these studies. All the microarray comparison analysis data files cited in the text are available online at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE7748). All the microarray comparison analysis data cited in the text are available at ftp://ftp.ncbi.nih.gov/pub/geo/DATA/supplementary/series/GSE7748. Total RNA was isolated from cell colonies selected for the appropriate ES cell morphology (flat monolayer colony with distinctive cobbled stem cell morphology and a high nucleo-cytoplasmic ratio) using the Invitrogen TRIZOL reagent, followed by further purification with the Qiagen RNeasy MinElute Cleanup Kit. For each cell line examined three biological replicates were used and each replicate represented either a different passage or different sub-line (which had been cultured independently and without mixing for several passages). The RNA samples were quantified using the NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies) and the quality of the RNA was assessed using Lab-on-a-Chip RNA Pico Chips and the 2100 Bioanalyser (Agilent Technologies). Samples with electropherograms that showed a size distribution pattern predictive of acceptable microarray assay performance were considered to be of good quality. Two micrograms of total RNA from each sample were amplified and labelled using a single-cycle cDNA synthesis and an in vitro transcription cRNA-RNA labelling system (GeneChip one-cycle target labelling and control reagents; Affymetrix). Following successful cRNA amplification, 10 &mgr;g of labelled target cRNA was hybridized to rhesus macaque Genome Arrays (Affymetrix) using standard protocols, as described in the GeneChip Expression Analysis manual (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). The rhesus macaque array contains 52,865 probe sets, representing over 20,000 genes. The arrays were scanned using the GeneChip laser scanner (Affymetrix) and image processing, normalization and expression analysis were performed with the Affymetrix GCOS version 1.4 software. MAS-5 statistical analysis was performed to calculate the signal log ratio (SLR) for each probe set comparison, and the gene expression fold changes between two samples were calculated from the SLR using the following formula: fold change = (2SLR). The GCOS 1.4 MAS 5.0 software was used to calculate statistically significant differences in gene expression (P < 0.002) between samples.For the primary microarray comparison analysis (Supplementary Data 3) the log10 of the absolute detected signal for each present ‘P’ probe set (P < 0.05) was plotted in a scatter graph (Supplementary Fig. 8) using Affymetrix GeneChip Operating Software (GCOS) version 1.4. For the correlation value calculations, the microarray data for each individual cell line comparison was filtered to only include probe sets present (P < 0.05) in both cell lines. The present probe sets (PP) value details the number of probe sets, post filtering, with a present (P < 0.05) signal in both compared cell lines. The correlation value for each cell line comparison was calculated using MAS-5 (Affymetrix microarray suite 5) analysis to calculate the proportion of compared probe sets which demonstrated no significant change ‘NC’ in gene expression (Supplementary Data 3). Each cell line had three biological replicates and the letter after the cell line name details which replicate was used in the primary microarray comparison analysis.For the secondary gene-specific analysis, comparison analysis was performed between each of the three control ORMES-10 biological replicates and each of the three somatic donor cell replicates, to give a total of nine somatic–ES cell comparisons (Supplementary Data 4). The following selection criteria were used to identify rhesus somatic-specific genes: (1) genes that were considered to be present (P < 0.05) in all three somatic donor cell replicates; and (2) genes that demonstrated statistically significant decrease ‘D’ in gene expression in the ORMES-10 replicates in all nine comparisons with the somatic donor cell replicates after GCOS comparisons with MAS-5 statistical analysis. A total of 4,998 somatic-specific probe sets were identified in this way (Supplementary Data 5). The following selection criteria were used to identify rhesus ES-cell-specific genes: (1) genes that were considered to be present (P < 0.05) in all three ORMES-10 replicates; and (2) genes that demonstrated a statistically significant increase ‘I’ in gene expression in the ORMES-10 ES cell replicates in all nine comparisons with the somatic donor cell replicates after GCOS comparisons with MAS-5 statistical analysis. A total of 6,178 ES-cell-specific probe sets were identified in this way (Supplementary Data 6). It should be noted that the general approximation when working with large numbers of probe sets is to assume that each probe set represents hybridization to a single gene. However, multiple probe sets can exist for certain genes, so the actual number of genes included in the analysis is significantly lower than the number of probe sets analysed. The somatic-specific and ES-cell-specific genes identified from this comparison analysis were then used to investigate whether the CRES cell lines had successfully downregulated somatic-specific genes (Supplementary Table 4) and successfully upregulated ES-cell-specific genes (Table 2) after comparison analysis with the three somatic donor cell replicates.For the tertiary stemness gene analysis, stemness genes were derived from our previous transcriptional profiling work with rhesus macaque embryonic stem cell lines. These 12 putative stemness genes had the highest average fold change in gene expression when three undifferentiated biological replicates of ORMES-6 were compared to their in vitro differentiated counterparts, and all 12 were significantly upregulated in five different rhesus macaque embryonic stem cell lines examined. Comparison analysis was performed between the ORMES and CRES cell line replicates and the donor somatic cell replicates (Supplementary Data 7), and the average fold change increase in gene expression of the 12 stemness genes in the ORMES and CRES cell lines was calculated (Table 3). All of the normalized microarray data sets generated from these studies can be found on the GEO website, as noted above.Statistical analysisNon-microarray results were analysed by chi-squared and unpaired t-test using Statview Software (SAS Institute, Inc.) with statistical significance set at 0.05. Microarray results were analysed by MAS-5 statistical analysis in Affymetrix GeneChip Operating Software (GCOS) version 1.4.Chromatograms of the rhesus macaque mitochondrial D-loop hypervariable region 2 informative domain 1 (RhDHV2 ID1).The ID1 sequence encompassed Macaca mulatta mtDNA nucleotide positions 451–480 (GenBank accession number NC_005943). Sequence analysis revealed that SNP22 (A-to-G) was informative for the mitochondrial inheritance for CRES-1 whereas SNP4 (C-to-T) and SNP28 (A-to-G) were informative for the mitochondrial inheritance for CRES-2. Results were confirmed by three independent sequences.Morphological and immunocytochemical analysis of CRES cells and their differentiated derivatives.a–d, Morphology of CRES colonies and expression of primate ES-cell-specific markers: OCT4, SSEA-4, TRA1-60 and TRA1-81. For each stemness marker analysed, a positive signal was only observed in CRES colonies, not in mouse embryonic fibroblast feeder cells, demonstrating antibody specificity. e–g, Cellular morphology and expression of neural markers in differentiated CRES cells, including microtubule-associated protein 2 (MAP2), &bgr;-III-tubulin and tyrosine hydroxylase. The left hand column of a–g represents phase contrast images and the right hand column demonstrates marker expression as detected via immunocytochemistry. The scale bars in a–d, g represent 100 &mgr;m and the scale bars in e, f represent 50 &mgr;m. h, j, Presence of ectoderm (Ec)-derived neuroepithelium (×400, haematoxylin and eosin (H&E)) in teratomas produced by injection of CRES-1 and CRES-2 into SCID mice. i, k, Presence of mesoderm (Me)-derived cartilage and endoderm (En)-derived glandular epithelium (×100, H&E) in CRES-1 and CRES-2 teratomas.Cytogenetic analysis of CRES cells.a, G-banding of CRES-2 cells demonstrating a normal euploid rhesus karyotype (42, XY). b, CRES-1 cell line with aneuploid karyotype, characterized by an isochromosome comprised of two copies of the long arm of the Y chromosome (41,X[3]/42,X,i(Y)q10)[17]). c, FISH analysis of the nuclear donor fibroblasts demonstrating a normal karyotype with two red fluorescent signals on the long (q) arm of the Y chromosome. d, FISH analysis of CRES-1 cells indicating the presence of four signals for the Y chromosome long (q) arm, confirming the presence of the i(Y)(q10) aneuploidy.Short tandem repeat (STR) analysis of CRES cell linesSTR lociFemale 1oocyte donor for CRES-1Female 2oocyte donor for CRES-2Male 1 somatic nuclear donor for CRES-1 and CRES-2CRES-1CRES-2Female 3oocyte donor for ORMES-22Male 2 sperm donor for ORMES-22ORMES-22 CRES-1 is an ES cell line isolated from a SCNT blastocyst produced by fusion of a cytoplast donated by female 1 and a skin fibroblast originated from male 1. Similarly, CRES-2 was derived from a cytoplast donated by female 2 and a donor somatic nucleus from male 1. ORMES-22 is an ES cell line derived from an embryo produced by IVF of a female 3 oocyte with a male 2 sperm. AME, amelogenin. Sex (AME)XXXXXYXYXYXXXYXXD1S548190/206190/198190/190190/190190/190190/190190/190190/190D2S1333301/301293/301289/301289/301289/301273/293285/289273/285D3S1768221/221205/213193/217193/217193/217205/213205/205205/205D4S2365283/283275/287283/283283/283283/283283/283283/283283/283D4S413131/131133/145131/139131/139131/139131/145125/141131/141D5S1457136/136132/136132/136132/136132/136132/136132/140136/140D6S501176/180176/180176/180176/180176/180188/192180/180180/188D7S513191/205205/209189/191189/191189/191189/217193/199199/217D7S794108/124124/128128/128128/128128/128108/108108/128108/128D8S1106144/144148/160144/148144/148144/148148/168160/168168/168D9S921183/195183/191179/179179/179179/179183/195175/195183/195D10S1412157/166160/160157/157157/157157/157157/157160/160157/160D11S2002256/256256/256260/264260/264260/264252/252256/260252/256D11S925308/338310/316308/310308/310308/310308/308338/338308/338D12S364282/290282/288281/290281/290281/290282/290268/296268/290D12S67121/129192/204117/125117/125117/125117/133109/117109/133D13S765228/240212/220228/256228/256228/256216/236228/228228/236D15S823333/349329/353329/361329/361329/361357/385345/353345/385D16S403164/168156/158158/164158/164158/164152/164152/152152/164D17S1300232/280244/280272/276272/276272/276248/252228/284252/284D18S537178/178178/178174/178174/178174/178174/178162/174162/178D18S72306/308306/322306/308306/308306/308308/308306/308308/308D22S685315/319291/303315/327315/327315/327311/311327/327311/327MFGT21113/115117/119115/115115/115115/115111/113115/125113/125MFGT22104/104104/104100/104100/104100/104100/104104/110104/104Analysis of ES-cell-specific gene expression in rhesus macaque stem cell linesCell lineBiological replicateNumber of ES cell genes* upregulated compared to donor line replicate A†Number of ES cell genes* upregulated compared to donor line replicate B†Number of ES cell genes* upregulated compared to donor line replicate C† *The general approximation when working with large numbers of probe sets is to assume that each probe set represents hybridization to a single gene. However, multiple probe sets can exist for certain genes, so the actual number of genes included in the analysis is significantly lower than the number of probe sets analysed. †The percentage value represents the proportion of probe sets, out of the 6,178 ES-cell-specific probe sets, significantly upregulated after comparison analysis. Nuclear donor cellsAN/A21 (0.3%)47 (0.8%)Nuclear donor cellsB30 (0.5%)N/A77 (1.2%)Nuclear donor cellsC18 (0.3%)13 (0.2%)N/AORMES-22A5,482 (89%)5,388 (87%)5,389 (87%)ORMES-22B5,558 (90%)5,607 (91%)5,644 (91%)ORMES-22C5,766 (93%)5,672 (92%)5,723 (93%)CRES-1A5,974 (97%)6,001 (97%)5,984 (97%)CRES-1B5,896 (95%)5,919 (96%)5,926 (96%)CRES-1C5,748 (93%)5,845 (95%)5,784 (94%)CRES-2A5,931 (96%)5,843 (95%)5,850 (95%)CRES-2B5,658 (92%)5,552 (90%)5,483 (89%)CRES-2C5,863 (95%)5,933 (96%)5,889 (95%)Expression analysis of putative rhesus macaque stemness genesAffymetrix probe set IDStemness gene*Nuclear donor cellsfold change†ORMES-10fold change†ORMES-22fold change†CRES-1fold change†CRES-2fold change† *The stemness genes were identified in previous transcriptional profiling of rhesus macaque ES cell lines. These 12 genes had the highest average fold change in gene expression of five different rhesus macaque ES cell lines. †Microarray suite-5 (MAS-5) statistical analysis was performed to calculate the signal log ratio (SLR) for each cell line in comparison to the somatic donor cell line biological replicates, and the averaged gene expression fold change between compared samples was calculated from the SLR using the formula: fold change = (2SLR). All 12 stemness genes were significantly upregulated (P < 0.002) in CRES cell lines. MmugDNA.26523.1.S1_s_atNFE2L31429389532378MmuSTS.2285.1.S1_atPOU5F1 (OCT4)0315288320281MmugDNA.9427.1.S1_atNR5A21282310278325MmugDNA.32128.1.S1_atNANOG1246180256190MmuSTS.1436.1.S1_atLCK117920621894MmugDNA.11728.1.S1_atVTCN11245139153125MmugDNA.42677.1.S1_atDPPA4415411717878MmugDNA.28461.1.S1_atSLC12A1171128185169MmugDNA.6836.1.S1_atC14orf115 (LOS699513)181878564MmuSTS.3122.1.S1_atMYRIP071517553MmugDNA.15193.1.S1_atADH4068135258MmuSTS.2310.1.S1_atPRDM14128444640The authors acknowledge the Division of Animal Resources and the Endocrine Services Cores at the Oregon National Primate Research Center for assistance and technical services. We thank M. Sparman, C. Ramsey and V. Dighe of the Assisted Reproductive Technology Core for their embryological and logistical assistance; J. Fanton and D. Jacobs for laparoscopic oocyte retrievals; B. Ferguson for performing the SNP analysis; C. Penedo for microsatellite analysis; and R. Stouffer, M. Grompe and R. Reijo Pera for reviewing this manuscript. Microarray assays were performed in the Affymetrix Microarray Core of the OHSU Gene Microarray Shared Resource. This study was supported by funds from ONPRC and NIH grants to S. Mitalipov, R. Stouffer and D. Dorsa.Author Contributions S.M.M. and J.A.B. designed experiments, conducted SCNT and ES cell derivation. L.L.C. performed DNA/RNA isolations and stemness gene expression. J.A.B. analysed the microarray data and performed the mitochondrial DNA analysis. D.A.P. assisted with ES cell derivation and performed ES cell culture, characterization and differentiation. W.G.S. and M.N. performed the cytogenetic analysis. S.G. analysed teratomas. 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Nature Genet. 29, 365–371 (2001) \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BAE243B509F5F3E735D4774B38A8EF81EABBEC1.txt b/test/dataset/in/resources/corpus/Clean_0BAE243B509F5F3E735D4774B38A8EF81EABBEC1.txt new file mode 100644 index 0000000..f1e41e6 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BAE243B509F5F3E735D4774B38A8EF81EABBEC1.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 9902310.1093/gerona/55.5.B220 Journal of Gerontology: Biological Sciences Increase of Oxidatively Modified Protein Is Associated With a Decrease of Proteasome Activity and Content in Aging Epidermal Cells Petropoulos Isabelle a Conconi Mariangela a Wang Xin b Hoenel Batya b Brégégère François a b Milner Yoram b Friguet Bertrand a aLaboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Université Denis Diderot, Paris, France bDepartment of Biological Chemistry, The Hebrew University, Jerusalem, Israel Bertrand Friguet, Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universit\|[eacute]\| Denis Diderot-Paris 7, CC 7128, 2 place Jussieu, 75251 Paris Cedex 05, France E-mail: bfriguet@paris7.jussieu.fr. 1 5 2000 55 5 B220 B227 20 9 1999 1 3 1999 The Gerontological Society of America 2000 For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker β-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins. hwp-legacy-fpage B220 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: Jay Roberts, PhD KERATINOCYTES, which are the main cell type in the epidermis, not only constitute the physicochemical barrier that protects the body from environmental assaults (e.g., by pathogens and toxins) but also have more complex immune functions, such as the expression of class II antigens and interaction with T lymphocytes during inflammation (1). The epidermis is also very sensitive to age-related alterations leading to various major pathologies, including hyperproliferative and autoimmune diseases (2)(3)(4). Numerous theories have been proposed to explain the mechanisms of cellular aging. Among them, the free-radical hypothesis suggests that oxygen free radicals are causal factors in aging, inflicting molecular damages, some of which are not repaired and accumulate with age (5). Indeed, it has been shown that aging cells do accumulate damaged DNA, lipids, and proteins, leading to cellular functional alterations that are strongly believed to contribute to the aging process (6)(7). Among biological macromolecules, proteins constitute a particular target for the reactive oxygen species attacks that generate protein carbonyl derivatives. There is an age-related increase in the carbonyl content of protein in the human brain (8), gerbil brain (9), rat hepatocyte (10), human red blood cells (11), and in the whole body of flies (12). Moreover, the carbonyl content of protein in cultured human fibroblasts increases exponentially as a function of donor age (11). There are other protein modifications, such as glycation, that cause alterations to cellular and organ function (13). Aging is also characterized by a decline of the specific activity and thermostability of enzymes, which is due to oxidative damage as well as to other modifications (11)(13)(14)(15). The accumulation of modified proteins reflects a dysregulation of the steady-state level of damaged proteins in aged tissues, which can be explained by a higher modification rate, or a lower degradation rate of these proteins, or both. The half-lives of proteins in senescent animals have been shown to be significantly extended as compared with those in younger ones (16). It is now well established that the main proteolytic system, responsible for the intracellular degradation of oxidized or misfolded proteins, is the multicatalytic proteinase (MCP), also called 20 S proteasome (17)(18)(19). The 20 S proteasome is a high-molecular-mass (700 kDa) proteolytic complex, made of 28 subunits arranged as four stacked rings (see ref. (20) for a review). The two outer rings are formed by α subunits and the two inner rings are formed by the β subunits that carry the proteolytic activities (21)(22). Recently, we and other investigators have shown that the peptidylglutamyl-peptide hydrolase (PGPH) activity of rat liver proteasome exhibited the most remarkable reduction with age (23)(24)(25)(26). These results strongly suggest a potential participation of the MCP in the slower turnover of proteins, thus contributing to the age-related accumulation of altered proteins. In this study, we present evidence for an impaired proteasome function in human keratinocytes upon aging. We have found a decrease of proteasome peptidase activities in epidermis extracts from old donors as compared with young ones and, as expected, an age-related increase of damaged proteins. We also found a similar decrease in senescent keratinocyte cultures. This can be explained, at least in part, by the decrease of proteasome content found in old cells. Materials and Methods Reagents and Chemicals Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-AMC), N-Cbz-Leu-Leu-Glu-β-naphthylamine (LLE-NA), 7-aminomethylcoumarin, and β-naphthylamine were purchased from Sigma-Aldrich (L'Isle d'Abeau, France). N-Cbz-Leu-Leu-Leucinal (MG 132) was kindly supplied by Dr. F. Arenzana-Seisdedos. The rabbit anti-glycation products polyclonal antibodies (anti-GP) (27), a gift from Dr. H. Bakala, were raised against advanced glycated endproducts conjugated RNase. Rabbit immune sera raised against pure rat MCP and against HNE (4-hydroxynonenal)-modified keyhole limpet hemocyanin (anti-HNE) were obtained from Assay Research (Silver Spring, MD) as previously described (23)(28). Epidermis and Keratinocyte Extracts Preparations Epidermal cells were prepared from skin biopsies or plastic surgery procedures (mammary gland reductions) from healthy donors from 17 to 67 years of age. Skin samples were incubated 2 hours at 4°C in 2 M of NaCl and 20 mM of ethylenediamine tetra-acetic acid (EDTA). Then, the epidermis was separated from the dermis and homogenized with a Dounce homogenizer (Prolabo, Fontenay, France). Primary cultures of keratinocytes were derived from newborn foreskins and cultivated in Dulbecco's modified eagle medium nutrient mixture F-12 ham (1:1) at 37°C and 5% CO2 by using a 3T3 fibroblast feeder layer, as previously described (29). Cells were usually passaged up to six times. Cells were collected with a rubber policeman and homogenized in an extraction buffer (20 mM of Tris, pH 7.4, 250 mM of sucrose, 1 mM of EDTA, and 5 mM of 2-mercaptoethanol) with a Dounce homogenizer. Cell debris and organelles were removed from the crude extracts by centrifugation for 1 hour at 10,000 × g at 4°C. Protein and Peptidase Assays Protein concentrations were determined by a bicinchoninic acid assay (Pierce). Chymotrypsin-like and PGPH activities of the MCP in crude extracts were assayed with fluorogenic peptides LLVY-AMC and LLE-NA, respectively, as previously described (30). Typically, assay mixtures contained 75-μg proteins of epidermal extract, or 50-μg proteins of keratinocyte extract, in extraction buffer with the appropriate peptide substrate (10 μM of LLVY-AMC or 200 μM of LLE-NA) in a final volume of 200 μl. The mixtures were incubated 30 minutes at 37°C and analyzed by spectrofluorometry for release of aminomethylcoumarin (AMC) with excitation and emission wavelengths at 350 nm and 440 nm, respectively, or for release of β-naphthylamine (NA) with excitation and emission wavelengths at 333 nm and 410 nm, respectively. MCP proteolytic activity was determined as the difference between the total activity of crude extracts and the remaining activity in the presence of 20-μM proteasome inhibitor MG 132 (31). Gel Electrophoresis and Western Blots Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) was done by using the Laemmli method (32) on a 12% acrylamide (wt/vol) separating gel. Immunoblot experiments using anti-MCP, anti-GP, or anti-HNE antibodies were performed after SDS/PAGE separation of 20 μg of total cellular proteins from keratinocyte cultures or epidermis followed by electrotransfer onto Hybond nitrocellulose membrane (Amersham-Pharmacia-Biotech, Les Ulis, France). Immunoblot detection of carbonyl groups was performed with the OxyBlot oxidized protein detection kit (Appligene-Oncor, Strasbourg, France), according to the manufacturer. Briefly, 10 μg of proteins were incubated for 15 minutes at room temperature with 2,4-dinitrophenylhydrazine (DNPH) to form the carbonyl derivative dinitrophenylhydrazone before SDS/PAGE separation. After transfer onto nitrocellulose, modified proteins were revealed by anti-DNP antibodies. The detection of bands in immunoblots was carried out with the ECL (Enhanced ChemiLuminescence) Western blotting analysis system of Amersham (Les Ulis, France), using peroxidase conjugated anti-rabbit, secondary antibodies. The densitometric analyses of the autoradiographies were performed by using Lecphor software on a Biocom system (Biocom, Les Ulis, France). Enzyme-linked Immunosorbent Assays and β-Galactosidase Assay A competitive enzyme-linked immunosorbent assay (ELISA) was performed to measure the amount of modified glycated products in skin epidermis upon aging. Microplates were coated with 1 μg/ml of modified glycated bovine serum albumin (BSA) diluted in a buffer carbonate (20 mM, pH 9.6) for 14 hours at 4°C. After they were washed with 0.05% Tween in phosphate-buffered saline (PBS) and blocked with 6% milk (wt/vol) in PBS, epidermis extracts from various donors were added and incubated with polyclonal anti-GP antibodies (27) for 2 hours at room temperature. After washing, peroxidase-conjugated secondary antibodies were added and incubated for 2 hours at room temperature. After washing, the substrate 2, 2′Azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid was added and the absorbance of the reaction product was measured at 405 nm in a SLT Rainbow microplate reader (SLT Labinstruments, Salzburg, Austria). A calibration curve was obtained by using glycated BSA. For the proteasome amount to be determined, cell suspensions were obtained by trypsinization of skin epidermis from donors of different ages, or cultured keratinocytes after different passages. For ELISA tests, 5 × 105 suspended cells were fixed in 3% paraformaldehyde in Eppendorf tubes for 15 minutes at room temperature, then incubated for 15 minutes with 0.1 M of glycine (pH 7.0), and permeabilized by 1% Triton X-100 for 30 minutes at room temperature. Suspended cells were pretreated for 10 minutes with 3% H2O2 and blocked with 5% BSA in PBS for 2 hours at 37°C. They were incubated with anti-MCP antibodies, properly diluted in PBS containing 3% BSA and 0.05% NaN3, overnight at 4°C, and then washed four times with 0.05% Tween in PBS for 10 minutes each. Peroxidase-conjugated secondary antibodies were diluted in PBS, 3% BSA, 0.05% NaN3, and added to the cells for 2 hours at room temperature. Finally, the cells were washed and incubated with 0.4 mg/ml of tetramethyl benzidine and 1.3 mM of H2O2 in 0.1 M of citric acid Na2HPO4 buffer (pH 5.5). The reaction was stopped in 0.33 M of H3PO4 and the absorbance was measured at 450 nm in a BIO-TEK (BIO-TEK Instruments, Winooski, VT) microplate reader (33)(34). For β-galactosidase activity to be measured, 5 × 105 suspended cells were fixed with 3% paraformaldehyde in PBS for 5 minutes, washed, and incubated in 0.5 ml of the following substrate solution: 0.4 mg/ml of p-nitrophenyl-β-galactopyranoside, 2 mM of MgCl2, 150 mM of NaCl, and 0.1% NaN3 in 50 mM of Tris buffer (pH 8.0) supplemented with a mixture of protease inhibitors at 37°C. The reaction product (p-nitrophenol) was measured spectrophotometrically at 405 nm. Statistical Analysis Differences for proteasome and β-galactosidase activities and proteasome antigen data were evaluated by using analysis of variance (ANOVA) procedures with the StatView software (Abacus Concepts, Berkeley, CA). A p value of <.05 was considered significant. Results Increase of Modified Proteins in Aging Epidermis Protein oxidation has been implicated in different biological processes, both physiological, such as aging or apoptosis, and pathological, such as cancer and neurodegenerative diseases (see ref. 6 for a review). In order to investigate the status of oxidatively modified proteins in keratinocytes during aging, protein carbonyl content was monitored by using the OxyBlot detection kit as described in the Materials and Methods section. Proteins of epidermis extracts from young donors (17, 20, and 26 years old), middle-aged donors (39 and 42 years old), and old donors (50, 60, and 67 years old) were treated with DNPH to derivatize their carbonyl groups into 2,4-dinitrophenylhydrazone. Western blots revealed oxidized polypeptides by using anti-DNP antibodies. As shown in Fig. 1, an age-related increase of carbonyl content was observed in some proteins. In samples from old cells in particular, high-molecular-weight polypeptides appeared on the top of the gel (molecular weights above 100 kDa), possibly resulting from cross-linking reactions. As a way to assess whether other modifications occur and to determine the origin of carbonyl groups in these proteins, Western blots were performed by using anti-HNE and anti-GP polyclonal antibodies. A general increase in modified protein adducts was visible with age, and anti-HNE antibodies recognized modified groups in high-molecular-weight proteins (Fig. 1 and Fig. 1). Because the patterns of glycated proteins did not show qualitative changes, a competitive ELISA was performed to measure their amounts and confirmed their increase with age (Fig. 1). Taken together, these results show that glycation and modification by the lipid peroxidation product HNE are implicated in the age-related accumulation of damaged proteins. Age-Related Decrease of Proteasome Activity and Content in Epidermis There is considerable evidence that the 20 S proteasome is responsible for the degradation of oxidatively modified intracellular proteins (35)(36) and that an age-related accumulation of these proteins may be due to a decline of proteasome peptidase activities (23)(24)(25)(26). For this hypothesis to be examined in epidermal cells, the chymotrypsin-like and PGPH activities of proteasome were monitored in epidermis extracts of various donors of from 17 to 67 years of age. As shown in Fig. 2, both activities decreased gradually with the age of donors. As a way to determine whether the age-related decline in the proteasome peptidase activities was due to decreased proteasome content, the amounts of proteasome were estimated by Western blot analysis of epidermis extracts from donors of different ages, using anti-rat MCP. Several proteasome subunits from human keratinocytes were recognized by these antibodies. A representative experiment is shown in Fig. 2, where the proteasome content was indeed reduced by ∼10% and 60% in the epidermis from 50- and 67-year-old donors, respectively, when compared with the sample from the 17-year-old donor. ELISA measurements of MCP antigens performed on epidermal cell suspensions from various donors (see Fig. 5 below) showed a similar age-related decrease. Increase of Protein Damage and Decrease of Proteasome in Keratinocytes Upon Serial Passaging Normal diploid cells undergo numerous physiological and biochemical changes during serial passaging in vitro. These changes may reflect aging events that occur in organisms (37)(38). We used keratinocytes in culture to examine the possible modification of proteasome activity and content in relation to protein damage upon aging. To investigate the status of oxidatively modified proteins in keratinocytes during serial passages, we monitored protein carbonyl content by using the OxyBlot detection kit as described in the Materials and Methods section. Cellular extracts were prepared at different passages and treated with DNPH; the derived products were revealed by an anti-DNP antiserum after SDS PAGE separation and Western blotting. As shown in Fig. 3, an increase of high-molecular-weight modified proteins was observed in passages 3 and 4 as compared with passages 1 and 2. Western blots were also performed by using an anti-HNE polyclonal antibody. This antibody also reacted with more proteins of high molecular weight in later passages than in earlier ones (Fig. 3). The accumulation of such modified high-molecular-weight polypeptides presumably results from protein cross-linking events. Proteasome activity was measured by monitoring the chymotrypsin-like and the PGPH activities in cellular extracts of keratinocytes. The results, summarized in Fig. 4, show a drop in chymotrypsin-trypsin-like activity in passages 4 and 5, compared with passages 1 and 2. A similar profile is observed for the PGPH activity. As a way to determine if the decline of activity can be correlated to a decreased proteasome content in different keratinocytes extracts, a Western blot analysis was performed. Fig. 4 clearly shows a decrease of proteasome level during keratinocyte serial passages. In the passages 3 and 4, the residual level was approximately 30% and 20% respectively, of that observed in the first passage. Decrease of Proteasome and Increase of β-Galactosidase in Aging Epidermis and Keratinocyte Cultures Dimri and colleagues described β-galactosidase as a senescence marker in human fibroblasts and keratinocytes (39). The intracellular β-galactosidase activity was measured in suspensions of keratinocytes prepared from skin epidermis or from cell cultures of different ages, as described in the Materials and Methods section. The amount of proteasome was measured in similar suspensions obtained from the same samples, by ELISA tests using anti-rat MCP antibodies (see the Materials and Methods section). As shown in Fig. 5, β-galactosidase activity increased with serial passages of keratinocytes, while proteasome antigen, detected by ELISA, decreased. An age-related increase of β-galactosidase activity and a decrease of proteasome antigen were also observed in skin epidermal cells of old donors (Fig. 5). Discussion The accumulation of damaged or oxidized protein reflects not only the rate of protein modification but also the rate of damaged or oxidized protein degradation that is dependent, at least in part, on the activity of intracellular proteases that preferentially degrade damaged proteins. The proteasome is the major protease that degrades oxidatively modified cytosolic proteins (35)(36), and it has been reported that an age-related accumulation of damaged or oxidized proteins may be explained by an impaired activity of the proteasome (23)(24)(25)(26). In this work, we have studied the fate of the proteasome in the cellular aging of human epidermis, a tissue very sensitive to aging alterations. We first showed that there is an age-related increase in the protein carbonyl content in the epidermis and in cultured keratinocytes, the main cellular component of the epidermis. This result corroborates previous reports that have established such a correlation in the mammalian brain (8)(9), in rat hepatocytes (10), and in human fibroblasts (11). As demonstrated in this study, in epidermis, as well as in serial passages of keratinocytes, protein carbonyl groups that can be generated by direct oxidation of amino acids are also produced by protein glycation and reactions with peroxidation products of polyunsaturated fatty acids such as HNE (see Fig. 1 and Fig. 3). Interestingly, these modifications seem to be highly selective, because only a few proteins are the target of oxidative damage. This finding is in agreement with previous observations in aging flies, which showed that two mitochondrial proteins, aconitase and adenine nucleotide translocase, are selectively oxidized during aging (40)(41). Furthermore, among these modified proteins that accumulate with age, some are high-molecular-weight proteins. Such high-molecular-weight species might result from cross-linking reactions that occur upon production of bityrosine by the hydroxyl radical (42), of intermolecular lysyl-HNE-lysyl linkages (43)(44), or of Maillard reaction adducts (45). We then addressed the question of whether the accumulation of damaged proteins reflects an alteration in the activity of the proteolytic machinery in charge of damaged or oxidized protein degradation, that is, the 20 S proteasome. Modifications of proteasome activities and content have been extensively studied in rat liver during aging by us and by other groups (23)(24)(25)(26). Shibatani and colleagues (24) reported a significant decrease of the SDS-treated proteasome PGPH activity in cytosolic extracts upon aging. Using purified proteasome preparations, we demonstrated that the PGPH activity declined in old animals to ∼50% compared with young animals (23)(25). More recently, Hayashi and Goto (26) also reported an age-related decline of the PGPH activity and of the chymotrypsin-like activity, although to a lesser extent. In addition, Wagner and Margolis showed an age-related decrease in proteasome activity in the bovine lens (46). We assayed both the chymotrypsin-like and the PGPH activities of proteasome in extracts of epidermal cells, and we found that they were notably reduced in aged cells. Using the specific proteasome inhibitor MG 132, we determined that proteasome accounts for ∼90% of both activities (data not shown). Therefore, this decrease represents a net reduction of proteasome activity. To determine whether the decline of proteasome activity may be due to a decrease in proteasome content upon aging, we performed both ELISA and Western blots by using rabbit polyclonal antibodies raised against rat proteasome. These antibodies recognize the human proteasome and reveal two distinct major bands. The intensities of the two major bands decreased with age (Fig. 2). Similarly, Fig. 4 shows that, in keratinocyte cultures, more than one proteasome subunit decreased with age. However, we cannot yet conclude whether there is a general decline of proteasome content, or a differential reduction in some subunits. It would be of interest to identify among proteasome subunits those that show an age-related decrease. A depletion of proteasome subunits with age has not been detected in rat liver (25)(26), leading us to propose that the activity decline resulted from posttranslational modifications of proteasome subunits. To our knowledge, this is the first time that an age-related decline of proteasome subunit content has been observed. However, it has been shown that the β-subunits X (also called MB 1 or epsilon) and Y (also called delta) are downregulated under pathologic conditions such as infection (47) and after γ-interferon treatment (48). It is interesting to note that these subunits have been involved in chymotrypsin-like and PGPH activities, respectively (49). Furthermore, decreased proteasome content is likely to explain the observed decline in activity but does not rule out the possibility that some subunits are also posttranslationally modified. Moreover, the proteasome activity was tested in crude extracts, and it is possible that the age-related alteration of proteasome activity could also be due to a loss of specific activators, the presence of endogenous inhibitors, or both. In fact, it has been pointed out that HNE cross-linked proteins may act as proteasome inhibitors (43)(50). Thus, further work is needed to ascertain why the proteasome is declining in activity with age. A decrease of proteasome activity and content was also observed in serial passages of keratinocytes. Cells were tested at various passages for β-galactosidase activity, which was used as a marker of replicative senescence in human fibroblasts, keratinocytes (39), and more recently in endothelial and smooth muscle cells from humans and rabbits (51). The inverse relationship between β-galactosidase activity and proteasome content strongly suggests that proteasome is downregulated in replicative senescence. Interestingly, Bosman and colleagues, in pioneering studies in replicative senescence of WI-38 fibroblasts, have shown a correlated increase of the β-galactosidase activity and a loss of a “critical neutral protease” that might be the proteasome (52). The accumulation of oxidized and modified proteins and the correlative decrease in proteasome activity and content during epidermial aging take place in the context of complex cellular events leading to skin alterations. The fact that a similar status of protein modification and of proteasome decline was found in senescent keratinocytes in culture and in aged epidermal cells in vivo establishes cell culture as a valuable tool for research on proteasomes in keratinocyte aging. Further investigations will be necessary to elucidate the actual role of proteasomes in the aging process and may lead to an original approach of aging control in skin. Figure 1. Modification of cytosolic proteins upon epidermal cell aging. A: The OxyBlot was performed with 10 μg of cytosolic protein extracts from epidermal cells from young, middle-aged, and old donors. Extracts were first treated with 2,4-DNPH, then subjected to SDS/PAGE on a 12% polyacrylamide gel, and electrotransferred onto a nitrocellulose membrane. The blots were processed with anti-DNP antibodies as described by the manufacturer. For a densitometric analysis, the measurements corresponding to the 17-year-old donor were taken as 100%. The intensity of the major band was increased to ∼300% in the oldest sample, and the high-molecular-weight bands appeared only in epidermal extracts from middle-aged donors and to a larger extent in those from old-aged donors. B and C: 20 μg of cytosolic protein extracts were subjected to SDS/PAGE on a 12% polyacrylamide gel and electrotransferred onto a nitrocellulose membrane. The blots were processed with anti-HNE (B) or anti-GP (C) polyclonal antibodies at a dilution of 1:2000 or 1:1000, respectively. For a densitometric analysis, the measurements corresponding to the 17-year-old donor were taken as 100%. The intensities of the major bands, reacting with anti-HNE antibodies and ranging from 50 kDa to 80 kDa in molecular weights, were increased to 800% for the 67-year-old donor, and a band of higher molecular weight was only present in the three old samples. The band with a molecular weight ∼80 kDa reacting with anti-GP was increased to 130% for the 50-year-old donor and 160% for the 67-year-old donor, respectively. D: A competitive ELISA was performed as described in the Materials and Methods section to determine the amount of glycated adducts in epidermal extracts. The assay was calibrated by using modified glycated BSA. The increase of glycated adduct is significant (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(F\ =\ 12.073\) \end{document}, p < .05) between the young (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}) and the old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}) donors. Figure 2. Effect of age on proteasome peptidase activities and content in epidermal extracts. A: The chymotrypsin-like (▴) and PGPH (○) activities of the proteasome were assayed in epidermal extracts by using LLVY-AMC and LLE-NA as fluorogenic peptide substrates, respectively. The enzymatic assays were performed as described in the Materials and Methods section. For the chymotrypsin-like activity, a significant difference (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(F\ =\ 9.534\) \end{document}, p < .05) was found between the young donors, from 17 to 23 years old (n = 4) and the old donors, from 50 to 67 years old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 5\) \end{document}). A similar trend to an age-related decline was observed for the PGPH activity. B: 20 μg of soluble proteins from epidermal extracts from 17, 50, and 67-year-old donors were subjected to SDS/PAGE on a 12% polyacrylamide gel, electrotransferred onto a nitrocellulose membrane, and immunoblotted with rabbit anti-MCP polyclonal antibodies at a dilution of 1:5000, as described in the Materials and Methods section. Figure 3. Modification of cytosolic proteins in keratinocytes upon serial passages. A: The OxyBlot was achieved with 10 μg of cytosolic protein extracts from passage 1 to passage 4 keratinocyte cultures. Protein extracts were first incubated with 2,4-DNPH followed by SDS/PAGE, electrotransferred, and Western blotted with anti-DNP antibodies as described by the manufacturer. For a densitometric analysis, the measurement corresponding to the first passage was taken as 100%. The intensity of the high-molecular-weight band reacting with anti-DNP antibodies was increased to 180% for passage 4. B: 20 μg of protein cytosolic extracts from passage 1 to passage 4 keratinocyte cultures were subjected to SDS/PAGE, electrotransferred, and Western blotted as described in the Materials and Methods section. The blots were processed with anti-HNE polyclonal antibodies at a dilution of 1:2000. For a densitometric analysis, the measurement corresponding to the first passage was taken as 100%. The intensity of the high-molecular-weight band reacting with anti-HNE antibodies was increased to 620% for passage 4. The experiments, reported in each panel, were done twice with cells obtained from two different cultures. Figure 4. Effect of serial passaging on the proteasome chymotrypsin-like activity and content in keratinocytes in culture. A: The chymotrypsin-like (♦) and PGPH (○) activities of the proteasome were assayed in cytosolic extracts by using 10 μM of LLVY-AMC and 200 μM of LLE-NA as the peptide substrate with 75 μg of total protein as described in the Materials and Methods section. A significant difference in the chymotrypsin-like activity (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(F\ =\ 18.622\) \end{document}, p < .05) was found between early passages 1 and 2 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}) and late ones 4 and 5 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}). A similar trend to a decline of activity in serial passages is observed for the PGPH activity. B: 20 μg of cytosolic protein extracts from keratinocytes at passages 1 to 4 were subjected to SDS/PAGE on a 12% polyacrylamide gel, electrotransferred onto a nitrocellulose membrane, and immunoblotted with rabbit anti-MCP polyclonal antibodies at a dilution of 1:5000, as described in the Materials and Methods section. The experiments, reported in each panel, were done twice with cells obtained from two different cultures. Figure 5. Proteasome antigen vs β-galactosidase activity in serial keratinocyte cultures and epidermis donors. Cell suspensions were obtained from the epidermis of various age donors or from keratinocytes in serial culture passages. For each sample from skin A or cell culture B, ELISA tests were performed for MCP, and β-galactosidase was assayed as described in the Materials and Methods section. The two series of result values were plotted on a single graph. In A, the symbols used are ♦, young donors; ○, middle-aged donors; and ▴, old donors. In B, the symbols used are ♦, early passages 1 and 2; ○, intermediate passage 3; and ▴, late passages 4 and 5. The decrease of proteasome antigen as well as the increase of β-galactosidase activity are significant between the young donors from 17 to 23 years old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 4\) \end{document}) and the old donors from 50 to 67 years old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 6\) \end{document}), p < .001 A, and in passages 4 and 5 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 5\) \end{document}) as compared with passages 1 and 2 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 6\) \end{document}), p < .001 B. 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Nature.261:499-501. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BAFDDE8709B9A764528AAD5B1AEF1DEA250AE6F.txt b/test/dataset/in/resources/corpus/Clean_0BAFDDE8709B9A764528AAD5B1AEF1DEA250AE6F.txt new file mode 100644 index 0000000..0128912 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BAFDDE8709B9A764528AAD5B1AEF1DEA250AE6F.txt @@ -0,0 +1 @@ +jpartjopartJournal of Public Administration Research and Theory1477-98031053-1858Oxford University Press10.1093/jopart/mun016ArticlesLeadership, Service Reform, and Public-Service Networks: The Case of Cancer-Genetics Pilots in the English NHSMartinGraham P.CurrieGraemeFinnRachaelUniversity of NottinghamAddress correspondence to the author at graham.martin@nottingham.ac.uk.1020091382008194769794© The Author 2008. Published by Oxford University Press on behalf of the Journal of Public Administration Research and Theory, Inc. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org2009In attempting to reform public services, governments worldwide have sought to effect change through policies aimed at both transforming structures of public-service provision and facilitating the agency of public servants working within these. Various obstacles have been found, however, to impede the effectiveness of such efforts. In this article, the authors examine the role of organizational networks and distributed leadership—two prominent policies aimed at structure and agency, respectively—in the establishment and consolidation of service reform in the English National Health Service. Using a comparative case-study approach, they contrast the trajectories of two attempts to introduce and gain acceptance for service reform, noting important differences of context, process, and outcome between the sites. The findings indicate the importance of dispersed, as well as distributed, leadership in achieving change in a networked public-service setting. Effective leaders may indeed achieve change through the structures and processes of the network. However, the coexistence alongside the network of other organizational forms constrains the ability of leaders to achieve change without complementary action beyond the boundaries of the network.INTRODUCTIONAcross the economically developed world over the last 10–15 years, governments have pursued increasingly interlaced strategies aimed at reforming public-service provision. Transformative efforts to “Reinvent Government” (Osborne and Gaebler 1992) in the United States and “modernize public services” (Newman 2001) in the United Kingdom are exemplary of policies seeking to improve both the effectiveness and efficiency of public-service delivery. In pursuit of this aim, policies addressing the “structures” of public-service delivery—the way public services are organized—have accompanied policies addressing the “agency” of public servants—the skills and attributes of the individuals employed in public services. Often, these two approaches to public-service reform are seen as mutually dependent. Organizational reforms may give rise to new roles necessitating new skills sets; the broadening of public-service staff's skills may in turn allow them to act more dynamically and autonomously in identifying opportunities for service improvement and acting upon them.This apparent synergy between structural reconfigurations and upskilling individuals as agents of reform, however, is not always so straightforward in practice. Many commentators have noted the contradictions inherent in certain organizational changes within the public services and the inhibitive effect these have on the possibilities for collaboration and joined-up service provision in reformed public services. In particular, there is the well-documented tension between hierarchies, markets, and networks. The collaborative network, as an organizing principle for public-service provision which might bridge gaps between state agencies and assist in addressing social problems that defy the efforts of a single agency (Huxham and Vangen 2000; Jennings and Ewalt 1998; Provan and Sebastian 1998), clashes with traditional top-down hierarchical organizational forms, and with the increasingly prominent role of markets and quasi-markets in national contexts subject to the Anglo-American influence (Currie and Suhomlinova 2006; Ferlie et al. 2003; Hood et al. 2000). In the United Kingdom, a policy focus on networks has coexisted with the reinforcement of a hierarchical performance-management regime and the increasing marketization of public-service provision, mirroring developments elsewhere (Kickert and Koppenjan 1997; Tenbensel 2005).In this article, we empirically examine the degree of convergence (or otherwise) between a particular “structural” reform—the centrally mandated network—and a particular facet of individual “agency”—leadership of change—which have both been central to government reform policies, in the United Kingdom and elsewhere. Networks and leadership have been advocated as means of reinvigorating state-run services, especially in the United States, the United Kingdom, and developed-world countries subject to Anglo-American influences such as Canada, Australia, and New Zealand (Agranoff and McGuire 2001; Hennessey 1998; Kakabadse et al. 2003; Kickert et al. 1997; O'Toole 1997; Shortell et al. 2000). In theory, leadership and networks should complement each other, with the looser organization and less hierarchical ordering logic of the network allowing leadership of change, distributed among network members rather than led from a single organizational apex, to flourish. This would affirm the findings of Denis et al. (1996) on the potential of distributed leadership in complex organizations with plural, ambiguous aims (cf. Bryson and Kelley 1978). In practice, as we set out in the sections that follow, this may not be the case. Moreover, as we explore in the empirical section of this article, in the ever more complex and unstable context of networks in the National Health Service (NHS) in England,1 both the possibilities of distributed leadership and the barriers it faces are increasing. Drawing on a wider comparative study of the implementation of pilot cancer-genetics services in the NHS, we focus on the divergent courses of two case-study sites in which similar service reforms were introduced, highlighting the importance of differences in network organization and leadership style, and both the synergies and incongruities between these. Our analysis suggests that in itself, the structural reform of the network does little to achieve public-service reconfiguration. It does, however, create a “space” within which a certain enactment of leadership might be effective in linking diverse actors across organizational boundaries in pursuit of joint agenda. To the extent that the network competes with other organizational forms and modes of governance, however, this space is a constricted one; consequently, there are limits to the effectiveness of the leaders and their agency, at the points where the network overlaps with other organizational forms (hierarchies and markets), which have their own norms of influence and authority which differ from those which are effective within the network.We continue first by reviewing the policy and academic literature on networks and leadership, before describing the specific empirical field in which our study was located. We then present our methods and findings before discussing the theoretical and policy implications of these.NETWORKS AND LEADERSHIP: THEORY AND POLICY IN THE PUBLIC SERVICESThe increasing attention paid to the role of networks and leadership in public administration has its roots in the waves of reforms to public services over recent decades often grouped under the heading “New Public Management” (Ferlie et al. 1996; Hood and Peters 2004; Peters and Pierre 1998). Though all characterized by efforts to improve the effectiveness and efficiency of public services, these reforms were heterogeneous in nature. The evolution of the approach, exemplified in approaches such as “Reinventing Government” and “modernization” that aim for a more totalizing, transformative effect on public services, has precipitated an increasing need for new organizational forms and new forms of leadership. As Ferlie et al. (1996, 13) have it, this model of New Public Management that emerged from the 1990s onward moves away from hierarchical forms and leadership approaches towards “new management styles, such as management by influence; an increased role for network forms of organization; stress on strategic alliances between organizations as a new organizational form.” Collaborative organizational forms and associated new forms of leadership have gained currency among governments of many developed-world countries (Agranoff and McGuire 2001; Hall and O'Toole 2004; Kickert et al. 1997; Meier 2004; Provan and Milward 1995; Rhodes 1997; Scharpf 1993). In the United Kingdom, the public-service network has been seen as a means of “joining up” service provision even as hierarchy and market have remained, and indeed been strengthened, as modes of governance. Though the notions of the intra- and interorganizational networks originate in the private sector (see Nohria and Eccles 1992; Reed 1992), there is a growing literature on their role in public-service provision (Ferlie and Pettigrew 1996; O'Toole 1997; Peters and Pierre 1998; Provan and Milward 1995; Rashman and Hartley 2002). Private-sector networks are largely explained in terms of reduced transaction costs and interdependence between firms, but for public-service organizations, network-level outcomes for clients are additionally seen as a motivation for integration (Provan and Sebastian 1998). In the literature, moreover, networks are seen as more flexible and adaptable in the face of organizational turbulence, as opposed to hierarchies that orient toward stability (Thompson et al. 1991), though empirical evidence for this is not strong (Bate 2000; Rashman and Hartley 2002).Networks have had a particular appeal for policymakers under “new Labour” governments in the United Kingdom since 1997 as an adaptable organizational form which combined the dynamism of the market with the co-ordination of the hierarchy in delivering the “modernization” of public services. By facilitating joined-up working across professional and organizational boundaries and by assisting the sharing of knowledge, good practice might be translated into rapid service improvement, spread across the public services. As we will see in the next section, though, the reality of the implementation and practice of organizational networks is not quite so straightforward.Alongside this focus on the reconfiguration of organizations and the relationships between them, efforts at public-service reform have also been based on complementary attempts to transform the workforce and its capacities: empowering public-service employees to act intelligently and autonomously within the enabling structures of networked organizations to effect change. The “Reinventing Government” initiative of the Clinton-led administration of the United States is exemplary of the expectations placed on this synergy, as a means of “[changing] the culture of our national bureaucracy away from complacency and entitlement toward initiative and empowerment.”2 In the United Kingdom, new Labour's ambitions for “modernization” similarly envisaged a public-service workforce empowered to act intelligently and autonomously to effect change within the new organizational forms (Currie et al. 2005).Within such policies, “leadership” has been an ongoing emphasis. Research on leadership in the private sector has tended to give precedence to transformational leadership, embodied in a single, heroic figure at the apex of an organization, as both a subject of study and a prescription for effectiveness (Bryman 1992; 1999). Such norms and ideals of leadership have to some extent permeated public-service leadership policy (Currie et al. 2005), but there is recognition also (among academics and policymakers) of the limits to this model in the more pluralistic and political context of the public services. For Heifetz (1994), leadership theory premised on the idea of a single inspirational leader is inapplicable to the public services, and attempts to conceptualize public-service leadership in this way serve only to perpetuate this myth. Rather, leadership is about “adaptive work” which requires buy-in and enactment by individuals at all levels of an organization. In the policy and practice of leadership in the public services, then, there is an onus on “distributed” leadership, “from apex to front line” (Hartley and Allison 2000, 39), even as power and responsibility are formally concentrated in the roles of the heads of public-service organizations. Arguably, the need for distributed leadership is all the more pronounced in public-service networks since “the notion of a leader with a hierarchical relationship to followers does not apply in collaborations, so the potential for exercising ‘decisive leverage’ by virtue of a formal position is reduced” (Huxham and Vangen 2000, 1167; cf. O'Toole 1997).Although the need for an understanding of leadership as distributed within public-service organizations, and especially networks, is accepted, exactly what constitutes distributed leadership is less obvious, and the subject of ongoing clarification in the literature. Van Wart (2005) offers a typology of distributed leadership, variously emphasizing the deliberate delegation of leadership from the top-down through “substitutes for leadership” and “superleadership,” and the more “organic” growth of “self-leadership” and “self-managed teams.” In all cases, though, shared leadership ultimately requires both a willingness to cede leadership to others on the part of organizational heads and the capacity of other actors to take it on (van Wart 2005, 372–3). As Crosby and Bryson (2005, 29) suggest, “potential for effective leadership lies alike with those who do and do not have formal positions of power and authority. Indeed, this view of leadership may be most useful in reminding those with little authority how powerful they can be through collaboration […] and in reminding those in a supposedly powerful position just how much they rely on numerous stakeholders for any real power they have.”Thus, even if we accept Bennett et al.’s (2003, 7) notion of distributed leadership as “the product of concertive or conjoint activity, […] an emergent property of a group or network,” it is important to recognize that such emergence occurs within the boundaries set by other parties, including those in whom leadership is traditionally concentrated. Furthermore, distribution of leadership emphatically does not imply equal spread of leadership among all parties. Power and influence may remain concentrated in certain “nodes” of leadership, who “lead up and out rather than down” (Crosby and Bryson 2005, 32). This is, indeed, the finding of Vangen and Huxham (2003), who find that the leadership qualities required by network managers are as much about “thuggery”—manipulation and politicking—as about facilitation and mobilization. These contours of distributed leadership—in particular, its relation to traditional top-down authority and its potential concentration in new nodes of power—are important to acknowledge in an understanding of the role of leadership in networks. As with transformational leadership, distributed leadership is seen in the literature as having the potential to improve organizational performance (Denis et al. 1996; 2000; Pearce and Conger 2003; Rainey and Steinbauer 1999).Government policy in England has emphasized the centrality of leadership in the modernization of all fields of public services (Cabinet Office 1999). Calls for individualistic leadership from the top and distribution of leadership coexist in government policy, so that in health, for example, programs supporting transformational leadership from organizational heads are accompanied by efforts to empower staff and “lead change through people.”3Together, structural reforms such as the introduction of networks and reforms aimed at harnessing the agency of staff, such as leadership development, are seen by policymakers as having the potential to contribute positively to reforms in the United States, the United Kingdom, and elsewhere. Furthermore, they are seen not just as complementary but to some extent as mutually dependent. Without equipping and empowering staff with the leadership and other skills to inseminate, manage and adapt to change, organizational reforms to public services are bound to fail. To this extent, then, the logic of these approaches to public service reform is evident. As we see in the next section, however, the practice is not so clear-cut.NETWORKS AND LEADERSHIP: OBSTACLES AND IMPEDIMENTSIn implementing networks in the public services, certain practical difficulties come to light. First, and most obviously, the resilience of the hierarchy and the increasing role of the market in many national contexts mean that networks have coexisted alongside two other organizational forms, with certain consequent tensions. Top-down performance management and quasi-market–based competition have the potential to drive a wedge between individuals and organizations in a way that is inimical to the collaborative ideals of the network (Currie and Suhomlinova 2006; Ferlie and Pettigrew 1996). To the extent that their performance is managed according to their core duties and judged in competition with their colleagues, professionals will tend to orient toward their own silos rather than reach out to foster collaborative relationships which may not benefit them. The same is true of organizations (Ferlie et al. 2003; Hood et al. 2000; McNulty and Ferlie 2002) and is reflected in various national contexts where network-based reforms coexist with other organizational forms, such as Australia (Ryan and Walsh 2004), the Scandinavian countries (Christensen et al. 2007), the Netherlands (Brandsen and van Hout 2006; Kickert et al. 1997), and the United States (Provan et al. 2004). Competition between organizations in contexts governed by market or quasi-market relations, as exhibited, for example, in managed care networks in the United States, will have a similarly divisive effect (Shortell et al. 2000), with the risk that agencies relapse into relationships based on contracting rather than collaboration (Brandsen and van Hout 2006; Keast and Brown 2006).Furthermore, many public-service fields, such as health and education, are professional bureaucracies in which these kinds of difficulties are compounded (Riccucci 2005). Each profession has its own pervasive socialization, which might work against a network orientation in a number of ways: a professional knowledge domain that is esoteric, to some extent tacit, and difficult to share (Currie and Suhomlinova 2006); a career pathway that tends to be based on specialization rather than broadening of knowledge (Nancarrow and Borthwick 2005); and a place in a well established and institutionalized hierarchy of professions and subprofessions (Bate 2000). Consequently, professions have long been acknowledged as averse to managerial interventions (e.g., Lipsky 1980; Riccucci 2005). Notably for our empirical field, physicians are often characterized as the quintessential autonomous profession (e.g., Riccucci 2005) and tend to be individualistic in their practice and attitude toward integration (Shortell et al. 2000). Furthermore, the status, power, and expertise of the medical profession mean that any set of organizational reforms requires the compliance of physicians for its effectiveness (Greco and Eisenberg 1993; Shortell et al. 1998). This is arguably all the more so in the NHS, where in contrast to the relative erosion of medical power and autonomy in the United States (Hafferty and Light 1995), physicians have retained an authority that gives them power of veto over NHS administrators in regard of any organizational reconfigurations that they do not welcome (Bate 2000; Ferlie and Pettigrew 1996; Harrison et al. 1992).Moreover, there is the risk that, in insisting that organizations adopt a more networked form, policymakers undermine the very essence that makes the network a functional organizational form in the first place. If they rely for their effectiveness and dynamism upon their voluntaristic, informal, and organic nature, then efforts to impose networks may have perverse consequences. In particular, as Kickert and Koppenjan (1997, 39) point out, in networks, “government is not the single dominant actor that can unilaterally impose its will. Hierarchicial, central top-down steering does not work in networks.” Rather, governance mechanisms must fit within the network structure (Bruijn and Heuvelhof 1997). Yet the ongoing influence of top-down performance management regimes, so strong in countries such as the United Kingdom where vertical accountability remains characteristic of public services, may militate against this need, to the extent that the network may become little more than an instrument of surveillance and performance management, to the detriment of collaborative efforts by network managers and members (Addicott et al. 2007).The necessity of more lateral means of governing and guiding networks toward common aims creates particular challenges for the leader within the public-service network. The widely noted ambiguity of objectives, power and accountability that characterizes the public sector as compared to the private sector (Denis et al. 1996; 2000; Heifetz 1994) are compounded, as we have seen, by the network. This suggests a need for the distribution of leadership across individuals in collaborating organizations. However, organizational objectives are frequently set by stakeholders “external” to networks, such as policymakers. This complicates any such effort to pluralize change agency and distribute leadership since actors across networks will be constrained in their actions by the parameters set by these external stakeholders (Currie and Lockett 2007), which may not encourage collaborative activity. Furthermore, the hierarchical order of professions—in health, for example, the ongoing subservience of nursing to medicine, at least in the view of many physicians, and the endurance of intraprofessional hierarchies and power relationships (e.g., between surgery and medicine) (Currie and Suhomlinova 2006)—is also likely to, at best, complicate any effort to distribute leadership beyond traditional leaders.NETWORKS AND LEADERSHIP: TRANSFORMING STRUCTURE AND AGENCY?The preceding section indicates the difficulties of establishing both networks and corresponding modes of leadership in public services characterized by the coexistence of other organizational forms and various tensions between professions and policy aims. Ostensibly, distributed leadership and networked organizational forms seem well suited to each other, but the complications of the public-services context mean that their effectiveness and synergy in effecting service reform are compromised. Equally, however, each policy might have the potential to surmount the difficulties presented by the contingencies of the public services by reconfiguring power relationships to foster a more dynamic organization than one governed by hierarchy or competition.What is suggested in particular is the need for an enactment of leadership that seeks to engage a multiplicity of stakeholders, facilitating and consolidating change by concentrating on processes and outcomes together (Huxham and Vangen 2000; Vangen and Huxham 2003). Even as this suggests a distribution of leadership, it also implies personal investment by those charged with network leadership tasks: as Huxham and Vangen (2000, 1171) have it, “it is paradoxical that the single-mindedness of leaders appears to be central to collaborative success.” At the same time, though, in a professional bureaucracy characterized by looseness of accountability and practitioner autonomy, there is a need to engage professionals for organizational change to be achieved and consolidated (Riccucci 2005; Shortell et al. 2000). Any change needs to ensure that “organizational players are empowered enough to be able to avoid the frustration and chaos that leads to the reassertion of pervious forms and processes” (Ferlie et al. 1996, 14).In other words, the “structural change” of the network is reliant on the “agency” of leaders—distributed or otherwise—in making it functional since “formal bureaucratic structure, as defined by rules and regulations, has little impact on the behaviors of front-line workers” (Riccucci 2005, 64). For all the collaborative rhetoric of the network form, then, it will have little tangible impact on practice in the absence of effective leadership. In making this rhetoric reality, leaders can, however, draw on the resources of the network: in the language of Huxham and Vangen (2000, 1171), the “leadership media” of “structures, processes and participants.” It is through the properties of the network that leadership might be enacted, and although these may well be outside the control of any one leader, they are also to some extent malleable. Structures, for example, “normally emerge out of the practical reality of the tasks that they tackle” (Huxham and Vangen 2000, 1167), so that even within an overarching organizational structure, certain “substructures” might be assembled in pursuit of the leader's task. Similarly, O'Toole (1997, 48) suggests to network managers the possibilities of a number of routes to influence: “find ways to shift network membership toward more supportive coalitions; locate key allies at crucial nodes; try to alter agreements between the parties to heighten program salience; and buffer well-functioning arrays to limit uncertainty and complexity.” Crosby and Bryson (2005) indicate the potential for influence in networks through “forums,” “arenas,” and “courts,” each subject to their own rules of conduct and therefore amenable to particular enactments of leadership. These are the concrete opportunities within interorganizational collaborations for leaders to be effective, for example, through visionary leadership, “creating and communicating shared meaning” in formal and informal network forums that inseminate ideas, and political leadership, “making and implementing decisions” in the legislative and executive arenas that determine formal policy (Crosby and Bryson 2005, 35).Although the nature of the structures, processes, and participants of the network will vary, and with them the opportunities for influence, what these ideas do encompass is an emergent image of what the effective leader within a network might look like, in terms of skills, style, and personality. The remainder of this article considers this question, as well as the more general question of the interaction between the structural reform of the network and the agency of leaders in achieving change. In the next section, we introduce the specific field in which we conducted our fieldwork and describe the service reform that the pilots studied attempted to introduce into this context.CANCER NETWORKS IN THE NHS, MODERNIZATION, AND SERVICE REFORMAlthough in the United Kingdom networked public services are often characterized by commentators as being a particular feature of policies of new Labour governments since 1997, the form has earlier roots. Reflecting wider trends for networked organization in the policies of various developed-world countries, UK public services were already moving towards networked governance in the early 1990s under the previous Conservative administration (Newman 2001). To this extent, networks can be seen as a logical development of the New Public Management policies prevalent across developed-world governments, especially those subject to Anglo-American influences (Ferlie et al. 1996; Kickert et al. 1997; O'Toole 1997). In the United Kingdom, health policy was an early adopter of the network principle, with “managed clinical networks” introduced in a variety of specialties, including cancer, in the NHS from the mid-1990s, mirroring trends toward networked health-service delivery in Europe (Wijngaarden et al. 2006) and the United States (Shortell et al. 1994).Though funded from general taxation and offering universal access to British citizens, the NHS is far from a monolithic public-service organization. In common with other fields of public-service provision, the introduction of the internal market to the NHS in the early 1990s created an increasingly fragmented organizational scene. From a system based on geographically determined organizational divisions, the NHS was subdivided into purchaser and provider “trusts,” each effectively an organization in its own right, with autonomy over its human resources policies, contracting arrangements, and finances, and the potential (in theory) for bankruptcy (though the political unacceptability of hospital closures ensured that in practice this was rarely threatened, with mergers and takeovers a more palatable alternative). As with public-service networks elsewhere in Europe and the world (Kickert et al. 1997), then, the network represented one means of mediating such organizational fragmentation: effectively, managed clinical networks in the NHS were to be interorganizational in character (between autonomous NHS trusts), albeit technically within a single overarching organization, the NHS.In relation to cancer, the 1995 Calman-Hine report envisaged new service structures in England and Wales “based on a network of expertise in cancer care reaching from primary care through Cancer Units in district hospitals [acute secondary-care hospitals] to Cancer Centres [specialist tertiary-care facilities]” (Department of Health 1995, 7). The vision here was of a network that would coexist with the emerging internal market in healthcare by encouraging competing providers to work together with purchasers to reduce duplication of services and promote clearer patient pathways. Good practice was to spread across the network through leadership from the medical lead in collaboration with colleagues across the network. In the interests of better clinical outcomes, though, concentration of expertise was also on the agenda, with cancer units needing a critical mass of patient throughput to maintain surgical subspecialization, and expertise in rarer cancers and specialist diagnostic and treatment processes to be concentrated in cancer centers. Consequently, although they were intended to be collaborative ventures between the different partners in the networks, “big players” in the networks—hospitals with much to gain or lose from the concentration of resources—have in many places come to dominate their agenda over smaller hospitals and primary care organizations. From the start, cancer networks were intended to promote both service rationalization and collaboration—with all the potential tensions this could create. However, as Addicott et al. (2007, 95) point out, in recent years the role of cancer networks has been significantly managerialized, becoming “largely focused on the structural configuration of services, performance targets and workforce planning.” In four of the five cancer networks they study, a preoccupation with meeting top-down performance-management agenda meant that collaboration was limited.Despite their government-driven mandate, cancer networks mirrored the function of two rather different network forms in the United States. First, they reflected the role of integrated networks of health-care providers that emerged in response to the market pressures created by the advent of managed care, as a means of rationalizing services and offering cost-effectiveness through economies of scale (Shortell et al. 1994). Second—and as Addicott et al. (2007) point out, in tension with the first role—they also serve a function similar to that of the community clinical oncology programs and special populations networks funded by the National Cancer Institute, with their aim of fostering knowledge-sharing relationships between research institutions and community-based intervention, education, and research projects (Baquet et al. 2005; McKinney et al. 1993).This was the context faced by the pilot program of cancer-genetics services cosponsored by the Department of Health and Macmillan Cancer Support,4 which sought to improve provision in this field by introducing a structured care pathway for those with a suspected family history of cancer. The Harper report (Department of Health 1998) identified inequalities, inefficiencies, and poor practice in cancer-genetics provision and suggested a division of labor and flow of knowledge between primary care, cancer units, and cancer centers which mirrored the set-up of cancer networks. This would result in smoother patient experiences and the elimination of poor practice, such as inaccurate risk assessments resulting in unnecessary screening (if genetic risk is overestimated) or inappropriate reassurance (if underestimated). An expert group convened by Macmillan and the Department of Health operationalized this in a framework known as the “Kenilworth model,” and in turn, this was adapted to local conditions and piloted in seven locations across England. With endorsement from an expert committee comprising policymakers, hospital clinicians, family physicians (GPs), nurses, service users, and others, it had credibility and a degree of “top-down push,” though it was not mandated; rather, the expectation was that each pilot would shape the care pathway in accordance with the particularities of its own locality.In principle, then, the Kenilworth model represented just the kind of improvement to and rationalization of service organization that cancer networks were designed to help to achieve and reproduce. In terms of innovation diffusion, the overall setting augured well. With the change agency of Macmillan and the Department of Health behind it, the innovation of the Kenilworth model seemed to have some leverage behind it and addressed an identifiable problem (Rogers 2003). Given the preceding discussion, however, there are clear obstacles to the operation of networked collaboration and to leadership that might confound uptake and establishment of the new care pathway. In the remainder of this article, we compare the trajectories of two of the Kenilworth pilots, considering the roles played by leadership and by the cancer networks in which they were located in their course. The degree of success of the two pilots was divergent, as assessed in terms of two key outcomes: throughput of patients, and whether the model was sustained beyond the pilot period. “Derton” had a throughput that was several times that of “Nottley”; the project (and therefore the Kenilworth-based care pathway) in Derton was sustained with local money following the pilot period, whereas Nottley's project and pathway were not.5METHODSThese two cases were among the 11 that were selected as case studies for a wider evaluation of a program of pilot genetics services in the NHS. This program encompassed 28 government-funded pilots working in various clinical areas, including the seven cancer-genetics pilots working to implement the Kenilworth model in different localities. The pilots followed a genetics white paper (Secretary of State for Health 2003), which set out the aim of “mainstreaming” genetics knowledge and practice in everyday NHS provision. The pilots were to be one means of achieving this ambition.The brief of the evaluation was to identify the barriers and facilitators faced by the pilots in realizing this aim. In pursuit of this, a comparative case-study approach was adopted, using qualitative methods to facilitate intra- and intercase analysis to illuminate key differences of context and mechanism that give rise to differences of outcome. This enables generalization through process analysis and the development of theory (Eisenhardt 1989; Yin 2003). More generally, where quantitative approaches permit the statistically based identification of apparent correlations between cause and effect, qualitative methods such as these are particularly useful in elucidating the mechanisms which give rise to such correlations (Lee 1999; Silverman 2004). As such, they are well suited to the study of “processes” within organizations (Langley 1999; Pettigrew 1997): in this case, the establishment of service reform within a network and the enactment of leadership in facilitating this. Our study thus addresses the gap identified by Bryman (2004) in relation to research on leadership, by adopting a qualitative approach which does not simply attempt to replicate quantitative research in identifying the key variables on which effectiveness rests, but rather focuses on the mechanisms in given contexts (such as the network) that give rise to certain outcomes.Our selection of 11 case studies from 28 funded pilots was theoretically guided (Eisenhardt 1989). The intention was to select a sample that was representative of the variation present among the 28 pilots in terms of a number of variables. These included clinical field, host organization (primary care organization [PCT], “mainstream” clinical department in a hospital, specialist genetics center), profession of pilot lead (medical geneticist, mainstream physician, GP, nurse, manager), characteristics of the area served (urban/rural, socioeconomic profile, ethnic profile), and so on. Potential cases were assessed by members of the research team on the basis of documentary materials provided by the pilots and preliminary interviews with pilot leads in every site. Following this, the research team selected its sample of 11, which included four (of a total of seven) cancer-genetics pilots aiming to implement the Kenilworth model. The focus of this article is on the pilots in Derton and Nottley, which were both based in hospitals and served areas covered by their cancer networks. The other two cancer-genetics cases were considerably smaller in scale, each serving the population of the single PCTs which hosted them and therefore engaging less with the wider cancer networks. Whereas the issues relating to cancer networks in this article are specific to Derton and Nottley—since no other pilot among the 11 cases was embedded within a network in this way—other findings presented (e.g., the range of leadership behaviors exhibited) have much in common with the wider findings in the other case-study sites.In each case-study site, our research included a number of qualitative methods: in-depth interviews with pilot stakeholders, participant, and non-participant observation of meetings at pilot and program level, and documentary analysis. The interviews covered a number of issues, grouped under the following headings: collaborative working, information technology, human resources management, leadership, user involvement, knowledge management, organizational culture, and policy. Although the interview schedule included a number of possible questions under each heading, in line with in-depth interview methodology, the interviewers tended to use these as prompts only, deviating from the list of questions to discuss issues as they were relevant to respondents and their organizational contexts, but seeking to frame questions as openly as possible so as not to “lead” interviewees (Silverman 2004). In the course of observational research, we paid particular attention to exchanges of knowledge between involved parties and barriers identified in the course of meetings to collaboration and service reform in their “mainstreaming genetics” ambitions. Documentary analysis encompassed bid documents, reports to sponsors and evaluation reports, and focused on the same issues as those explored through interviews and observation, with particular attention paid to change through time.Some 88 interviews have been conducted across the 11 sites, including 12 each in Derton and Nottley. Interviews were tape recorded and transcribed in their entirety and lasted between 40 min and 3 hours. In Derton and Nottley, similar respondents were interviewed, reflecting the similar aims and organizational contexts of the two pilots. They included pilot leads (medical geneticists in both cases), nurses and genetic counselors involved in delivering the pilots, primary-care–based practitioners such as GPs, managers, and clinicians within the cancer networks and the host hospitals, and service users involved as members of the projects’ steering groups. Data drawn from observation and documentary analysis activities both informed these interviews and complemented them by illuminating the approaches taken to establishing service reform within wider networks and the barriers encountered in attempting this.We undertook an iterative analysis process, rereading and coding transcripts, notes and documents, generating themes, and cross-checking these through discussions between authors. Thematically related parts of the embedded analysis in each data source were grouped together. Each of the three authors had engaged in fieldwork, and all three engaged in analysis. In Derton, GPM developed an initial analysis that was checked by GC; in Nottley, RF developed an initial analysis that was checked by GPM. We discussed the coding of transcripts, document, and meeting notes with each other, ensuring inter-researcher reliability of interpretation and enhancing analysis. Subsequently, the analysis agreed across the authorial team for each case was considered against the over-arching research questions. As a means of triangulating this analysis, findings were presented to both the commissioners of and participants in the research, providing a check on the authenticity of our analysis (Yin 2003).In presenting qualitative analysis, a number of approaches are possible, each seeking in its way to produce a summary that is both comprehensible and explicitly derived from primary data and thus amenable to critical appraisal by the reader (Eisenhardt 1989). Our approach in this article involves presenting the narratives from the two cases examined in turn: in-depth descriptions that deploy the primary data to illustrate the dimensions of leadership in the establishment of service reform they emerged in the cancer networks of Derton and Nottley, which reflect many of the key findings from the other nine case-study sites.RESULTS: DERTONThis pilot was led by a specialist geneticist, but he was keen to ensure that the service was delivered through and “owned” by the cancer network, which covered three acute hospitals (comprising two cancer units and one cancer center) and 10 PCTs. Genetics provision here had previously been delivered from another hospital, some 40 miles from the cancer center: consequently, patients and clinicians in the network had seen genetics as a remote specialty, its perceived esoteric knowledge compounded by its geographical distance. Recently, however, a new genetics “satellite unit” had been set up in the hospital that hosted the cancer center, and the geneticist leading the project had been appointed to a post here with a view to improving genetics coverage in this underserved area. The prior inaccessibility of cancer-genetics expertise had led to the setting up of a plethora of local protocols within the network for dealing with suspected inherited cases of cancer.This variation, together with the arrival of the genetics satellite unit 2 years earlier, had created the political will within the cancer network for a more coherent way of dealing with suspected family histories of cancer and an opportunity to do something about it. A colorectal surgeon (and, notably, the current medical director of the cancer network) described the dissatisfaction with the incumbent system:The ones with the really high risk we would have sent to [the genetics service], obviously. But there's a lot of them who didn't fit the high-risk criteria, for example for bowel cancer, and we give them advice which—you're not quite, 100 percent sure whether it is scientifically based.Colorectal surgeonClinicians in cancer-related specialties outside genetics had taken on responsibility for genetic risk assessment. But they encountered criteria for assessing genetic risk in breast, bowel, and ovarian cancers which were far from universally agreed upon and limited access to specialist clinical knowledge which might assist adjudication. Here, then, was a potentially receptive structure in terms of the attitudes of the network's members, with political will exemplified by the attitude of the colorectal surgeon and medical director of the network quoted above.Having taken up responsibility for genetic risk assessment reluctantly, existing services had nevertheless grown into an important part of cancer-service provision locally. Growing patient numbers and dissatisfaction over the variation in protocols saw the issue rise in importance within the cancer network, for practitioners and managers alike. Increasing numbers of patients were being admitted to screening services, without a “proper” (i.e., universally agreed) system of weighing the benefits and risks that might accrue from this radiation exposure. Moreover, this was without the explicit agreement of the primary-care purchasers who paid for the service, and this was a concern for the hospitals providing the service, who feared that in the absence of an agreed contract, funding might be withdrawn. As with the attitudes of clinicians, the organizational context too, then, was potentially receptive to the kind of change in care pathways augured by the Kenilworth model.Consequently—and in contrast to the experience of Nottley below, and indeed of other case-study sites not detailed here—the cancer network was proactive in seeking to establish the pilot as an integral part of local cancer care pathways, as a means of solving these issues. The pilot's lead clinician, meanwhile, was acutely aware of the need to ensure ownership by the network, given his status as an “outsider” associated with the distant genetics service. He sought to ensure “ownership” of the service by the network and adopted a highly dialogical and diplomatic stance in putting his ideas to colleagues within the network:Lead: [Previously] there was no continuity, and no discussion between genetics and the surgeons. I think if any lesson's come out of this project, it's that this sort of very detailed day-to-day discussion underpins a successful service. If you don't have that networking, then you can't sustain a service.GPM: The discussion between geneticists and their referrers?Lead: Yes, the stakeholders, the ability to sit down with somebody and explain exactly what you want to do, and for them to be able to pick up the phone and say, ‘I've got this patient: what do you think?’, any different niggles that happen. If you've got someone who's not happy, it's easy to go and see them or pick up the phone: it's just simple human communication.Lead (clinical geneticist)Through these kinds of “forums”, in the lexicon of Crosby and Bryson (2005), the clinical lead provided a vision in a politically sensitive way. These dialogical, quiet leadership skills had some success in paving the way for the service reform, as confirmed by the geneticist's new colleagues:It sounds a bit like I'm blowing [his] trumpet, but seriously, he was a knight in shining armor, and was really swamped with demands from people like me, who wanted to have this pathway set up, like, yesterday. It was very efficient very quickly. And [he] is a particularly good public-relations individual: his ability to make complicated genetics understandable is almost unique.Gynecological oncologistIn persuading physicians in these forums, then, a “quiet” approach to leadership was important, alongside firm, quantitative evidence of the potential benefits of the service reform in terms of patient outcomes and conforming to national guidance on risk assessment: “speaking their language,” as the pilot lead put it. In the terminology by Crosby and Bryson (2005), this was about visionary leadership using forums adroitly in order to create a compelling, shared vision of service reform and utilizing existing “norms of relevance and pragmatic communication, media and modes of argument” (Crosby and Bryson 2005, 120) to good effect.Credibility with physicians, through quiet leadership and the language of clinical audit, was however only one aspect of consolidating the place of the service within the network. Equally important was winning over the nurses who had taken on responsibility for risk assessment under the extant system, on whom the pilot relied for much of its throughput. This rather different professional group necessitated a different approach, requiring that the entire pilot team be versed in the arts of quiet leadership and negotiation. So one of the pilot's nurses took on the task of delicately persuading other nurses about the improvements that the new system might bring, deploying her understanding of “nursing culture,” as she put it.Thus, the pilot team members adapted their leadership to the different constituencies to be persuaded. “Objective evidence” was a key means of convincing medical specialists. Nurses, as the delivers of the service, were generally more concerned about the effects of the new model on the patient experience. Persuading each group, though, was crucial to the pilot's success, and each group could be engaged through its own informal and formal forums, with their own unwritten rules of engagement. And once key physicians and nurses elsewhere in the network had been convinced of the worth of the new system, they themselves were likely to spread acceptance among their colleagues. A breast-care nurse specialist in one of the cancer units, who had previously taken on much of the local risk-assessment work, explained how her colleagues had had concerns that the new care pathway was “just a way of getting people out of the system.” Following her own discussions with the lead nurse on the pilot, she was now convinced of its worth and had been instrumental in persuading other nurses in the unit, acting as a “link” between the pilot and her colleagues. In this way, the initial visionary work of the pilot team members as leaders gave rise to a more diffuse leadership, in the terms of Huxham and Vangen (2000) enacted through the structures and processes of the network as well as embodied in the wider participants beyond the core leaders from the pilot team.The success of this process also rested on the effective structure of the cancer network, recognized by respondents as being functional, democratic, and consensual, at least compared with others.6 Prior to the start of the pilot, the network had convened a “visioning event” attended by stakeholders (physicians, nurses, managers, patients, others) from every hospital and PCT in the network, and this was seen to have promoted the sense of “network ownership” so desired by the pilot lead. This, then, had provided an important forum for the development of a collective vision. More generally, the pilot lead highlighted the role of the network in establishing the service across the cancer units, citing the leadership of one network manager in particular:When I met her, I thought, ‘Goodness, this woman is so networked!’ She's been a senior nurse for years and she knows everyone. She knows the system: she's got an amazing knowledge of how to exert mild pressures here and there to achieve different aims.Lead (clinical geneticist)Alongside the receptive structural context provided by the network and its forums, and the delicate, visionary leadership work of the pilot team, the pilot's establishment also rested on the slightly more directive leadership enacted by this manager. Occasionally, individual clinicians were reluctant to accept the implications of the pilot for their own practice, despite the efforts of pilot staff through quiet leadership and audit evidence. On such occasions, the consensus of the wider network, once achieved, could be deployed as a means of persuasion in its own right, as the manager explained:The objectivity of the network is useful to say, ‘You're not supposed to be doing that kind of work on patients’: we bring intelligence from elsewhere in the network. […] Often just by bringing that objectivity, it can be made to work, by bringing in other clinical views.Cancer-network managerIn these ways, the collaborative rhetoric of the notional “network” was made real. Where collegiate, professional leadership through the network's structures and processes did not succeed, a certain amount of more directive, manipulative, even “thuggish” (Vangen and Huxham 2003) leadership could fill in, using this “objectivity” of the network as a tool of persuasion of more reluctant actors.The network itself, then, and the shrewd and varied leadership tactics of various agents within it, was effective at gaining sign-up willingly or reluctantly from a range of hospital practitioners. Engaging with primary-care practitioners, though, was less straightforward. Crucially, primary-care stakeholders were not such central actors in the network, with its mandated focus on service rationalization. The pilot had held various educational and publicity events for GPs and community nurses, but ensuring longer-term engagement was a challenge. Notably, there were fewer forums at which primary-care actors might be engaged; furthermore, there were fewer obvious common interests between the pilots and GPs, on which visionary leadership might draw. To this extent, primary-care practitioners were on the fringes of the network, both in terms of its “infrastructure” and in terms of the commonalities of interest on which it was built.Similar difficulties were present in the pilot's efforts to obtain ongoing funding, which involved dealing with purchasers who were also based in primary care. Though it had sign-up “in principle” from purchasers from the start, making post-pilot financial arrangements was not straightforward. Notably, although formally included in the network, those in charge of purchasing decisions in primary care were also responsible for funding a range of services beyond cancer. Consequently, negotiations about ongoing funding were subject to a range of considerations emanating from outside the cancer network. In the terms of Crosby and Bryson (2005), this “arena” was one that was subject to a range of different authorities and influences, including not only competing services but also centrally determined guidance on funding priorities. Furthermore, reorganization and management changes within primary care meant that not all purchasers were willing to follow through on their original commitment to the pilot, and indeed, only about half the PCTs in the network eventually funded the service and care pathway on an ongoing basis. This partial success highlights the fact that the structure of the network, and the styles of leadership this facilitates, is limited where it meets other principles of organization and governance, a theme which we will take up in the discussion. And as we discover in the next section, this space of operation can be even more constricted by the overlapping imperatives of other organizational forms.RESULTS: NOTTLEYThis was a joint pilot led by two geneticists at neighboring hospitals, part of a large cancer network containing six hospital trusts and covering a total population of around two million (twice that of the network in Derton). The pilot involved two, largely separate, projects aimed at increasing the accessibility of cancer genetics in primary care. Here we focus on one of these—which used advertising in GPs’ surgeries to attract self-referrals from concerned individuals, especially those from “hard-to-reach” ethnic-minority backgrounds—though many of the issues we raise apply to both. In common with Derton, both projects followed the core principles of the Kenilworth model to rationalize care pathways. Given the primary-care focus, however, the principal constituencies to whom they needed to appeal were quite different. The catchment of each project was also smaller than in Derton, partly because of concerns about the risk of overwhelming the genetics service with new referrals by “opening the door” in primary care.As in Derton, the pilot had obtained sign-up from the cancer network, but here the network was somewhat less proactive in encouraging and spreading the work of the two projects. There was a sense that the cancer network was preoccupied with conforming to central-government targets and with the need to find funding for existing services from cash-strapped purchasers:Because it hasn't got a definitive work stream or target attached to it, no-one has said to me in the three-and-a-half years I've been here, ‘Account for what you're doing on cancer genetics’—and they ask me that about lots of other things. I've got to have action plans on a million things, but cancer genetics isn't one of them. […] If I go to the [purchasers] with any service development, unless it is linked to a target or is completely unavoidable, they just say, ‘You must be mad.’ What they say—which is true—is: ‘We haven't got the money to run the services we've got: why would we fund new services?’Cancer network managerCompared to Derton, the network seemed much more governed by hierarchical principles—especially conformity to centrally determined priorities—and this was reflected in the nature of the leadership enacted by this network manager and others.The pilot thus sought to forge its own relationships with primary-care practitioners and the target communities, but with mixed success. The project successfully negotiated space in the premises of a number of GP practices to carry out its surgeries and to display advertising materials. But contact with GPs went little further than this, due to difficulties similar to those experienced by the pilot staff in Derton, around the lack of buy-in from primary-care professionals. So pilot staff decided to concentrate on self-referrals, exempting GPs from the care pathway and avoiding the need to bring them onboard. However, engaging the target communities in the pilot also proved problematic, and so efforts to increase uptake from minority-ethnic groups had only marginal success:Administrator: It's always been the aim but it hasn't necessarily come to the outcome we would like.RF: In terms of?Administrator: Because gaining access to some ethnic minorities is very difficult. I still think there's a lot of understanding yet to be achieved as to why we don't get these people coming forward.With little experience of such community-engagement strategies, inseminating interest among the public was problematic.Efforts, then, to engage the key stakeholders—potential patients and GPs—were marginal, by accident and design, respectively. This was in contrast to Derton, where the cancer network enabled the pilot to gain buy-in from clinicians “within hospitals” whose direct importance to the service (as referrers) matched their indirect influence (in creating a critical mass of consensus and support among the powerful constituents of the network).7 An absence of common interest was exacerbated by the absence of the opportunities for effective leadership that the structures and processes of the network might provide. Over time in Nottley, the realization dawned that given a relatively low throughput of self-referrals, buy-in from primary-care practitioners (especially GPs, with their influence on purchasing decisions) was something that the project needed after all:GPM: Is it an issue if GPs don't know about it? Do they need to know?Genetic counselor: Yeah, they do. For any possibility of extending the service, they do. Every person we've seen has had a letter, […] copied to their GP, so the GP knows they've been seen as part of the project. But it's just along with the other thousand [things] they look at per day! That's the side that has been a little disappointing, I thought we might get more involvement and a bit of ownership of the project amongst the practice nurses, and the other staff in the GP surgeries.Pilot genetic counselorThe pilot staff had little experience of engagement with primary care, with its rather different set of pressures, policies, and relationships. The same culture shock had affected Derton to some extent, but there the project could rely on the proactive work of the cancer network or amenable groups of hospital-based stakeholders. Within Nottley, the structure of the network did not support the pilot team's forlorn efforts at influencing practitioners. The managerial priorities of the network were compounded by the fact that primary-care practitioners, though formally part of the network, were also subject to many other pressures and priorities.Furthermore, the pilot staff lacked experience of the competitive funding mechanisms for this kind of project, being based in a specialist genetics department which received its funding through separate arrangements. Bidding for money from mainstream purchasers was thus a foreign experience for the project lead, and the departmental business manager. So, as a cancer-network manager explained, the pilot was “outside the mainstream cancer community” of the wider hospital and therefore marginal to cancer clinicians, the cancer network, and purchasers. The manager continued that although she wanted the project to work, “in terms of my overall job [in the cancer network]—the things I am meant to lose sleep about—it is quite a small part.” The pilot, then, found itself falling between stools. To use the lexicon of Crosby and Bryson, it was estranged from the core concerns of the cancer network, and the opportunities for effecting visionary leadership through the network's “forums.” Like Derton, it was also estranged from the primary-care–based purchasers. Unlike Derton, though, it lacked the critical mass of support from stakeholders within the cancer network, which had provided Derton's project with sufficient political leadership and leverage to succeed in the “arenas” outside the cancer network which determined decisions on ongoing funding.Consequently, despite various applications, neither this part of the pilot nor the other obtained ongoing funding. The absence of money or political will across the cancer network led the project lead to surmise, rather gloomily, that proactive efforts to lead this kind of reform were doomed to failure. It would only be when genetics centers began to place directive limits on the numbers of referrals that purchasers would be forced, reactively, to engage with the need for reformed care pathways and effective demand management.DISCUSSIONThe divergent trajectories of these ostensibly similar service reforms, based on a common care-pathway model, can be traced to a number of interacting factors. Both sought to introduce service-delivery reforms to improve client focus, and differences in the adaptation of this ambition to existing provision were significant in this divergence in themselves. However, the divergent trajectories also related to differences in the contexts of the two pilots—particularly the networks—and the agency of those seeking to implement them—notably, the presence or absence of particular forms of leadership among pilot staff and wider stakeholders.In both cases, the pilot staff attempted to draw on the resources of networks that should, in principle, be supportive of organizational reforms to rationalize care pathways, improve patient outcomes, and reduce inconsistency of provision. In practice, though, and reflecting the findings of Addicott et al. (2007), the network in Nottley was dysfunctional in this regard, with a focus on performance management and efficiencies that squeezed out any nonmandated initiatives. In Derton, the cancer network was more supportive of the pilot. In part, this seemed due to the more productive relationships already present, but it was notable also that the issue addressed by the pilot was already high on the political agenda—not least, due to financial concerns. This confirms the important point made by Van de Ven and Rogers (1988) that we should not overemphasize the role of organizational resistance in impeding innovation diffusion. Rather, it may be more instructive to view organizational resistance as the aggregate resistance of members or the inappropriateness of the innovation to the core concerns of the organization, as seemed to be the case in Nottley (Van de Ven and Rogers 1988). In Derton, in contrast, the pilot was aligned with the managerial agenda of the wider network, indicating that the susceptibility of networks to top-down pressures (Addicott et al. 2007; Keast and Brown 2006) is not necessarily an obstacle to collaboration within them (cf. Moynihan and Ingraham 2003; Olsen 2006; Provan and Milward 1995)—provided those concerned know which “mild pressures” to exert, as the lead here put it. Rather, there was complementarity between the collegiate, “quiet” leadership embodied by the pilot team and the slightly more directive leadership deployed by the network manager, who recognized the synergy between the aims of the network and those of the pilot. Here we echo the findings of various authors about the diversity of skills needed by leaders in networked settings (Huxham and Vangen 2000; Riccucci 1995; Vangen and Huxham 2003), the importance of influential champions to innovation diffusion (Rogers 2003), and indeed the need for leaders with different skills and organizational positions in relation to different leadership tasks (Crosby and Bryson 2005; Shortell et al. 2000). Furthermore, we highlight that public-service networks in particular, with diffuse objectives and loci of power, may require this combination of leadership styles and all the more so if they are professional bureaucracies with the extant complexities of power and accountability that these entail. Especially important in this context was the ability of the pilot team to make their service relevant to the concerns of others, increasing the likelihood of acceptance (Ferlie et al. 2005; Fitzgerald and Dopson 2005; Harrison 1998) by creating, as Crosby and Bryson (2005, 115) have it, “communal stories that help diverse stakeholder groups develop a sense of what they have in common with each other and what they might do to tackle common problems and create a better future.” This was a crucial way in which heterophilous links, in Rogers’ (2003) terms, were created to ensure that the Kenilworth model was adopted and accepted by diverse stakeholders.Evident in Derton was the way in which the structure of the network facilitated effective leadership through formal and informal forums which provided pilot members with the opportunity to foster a collective vision with other actors, mirroring closely the role for visionary leadership articulated by Crosby and Bryson (2005). Through the work of the pilot team in engaging with others through these forums and the directive leadership of the network manager, they were able to make effective use of the three “leadership media” identified by Huxham and Vangen (2000): the structure of the network provided a normative tool that could be used to bring “deviant” practitioners into line; the processes of the network offered the opportunity for the development of a shared vision; and, crucially, through other participants in the network, the vision was spread further and became established across diverse stakeholders. Central to this, then, was not just the quiet, “distributed” leadership within the pilot, but also the quiet, “dispersed” leadership (Buchanan, Caldwell, et al. 2007; cf. Hartley 2005) of stakeholders in the wider network. The former was to some extent under the control of the project, whereas the latter was more diffuse, but nevertheless important to widespread uptake. To this extent, then, the agency of the leaders relied on the structures (and processes and participants) of the network. This finding also reflects the intimations of Alexander et al. (2001) on the importance of what they call “collateral leadership” among both staff and community groups in the community-care networks they study, as a complement to—not a substitute for—more vision-based leadership. Our findings here converge with and develop existing literature in Europe and North America on distributed leadership in health-care settings (Buchanan, Addicott, et al. 2007; Denis et al. 2000; Huxham and Vangen 2000), local government (Hartley and Allison 2000), and education (Leithwood et al. 1999; Spillane et al. 2001). These studies illustrate the importance of subtle, distributed forms of leadership in settings characterized by the ambiguity of objectives and power relationships that typifies public-service organizations, particularly professional bureaucracies. Our study shows how, with the added complication of the interorganizational network, multiple forms of distributed and dispersed leadership are required to engage and inculcate diverse, powerful stakeholders, given the powerlessness to achieve change of any single actor or group of actors. This combination of leadership styles—and, perhaps more importantly, “leaders”—seems from our study crucial to achieve transformative change in such a context.Thus, the structures, processes, and participants of the network were essential resources for the effectiveness of leadership. It is in this sense, perhaps, that, as O'Toole (1997, 49) has it, there is a need for “a consideration of networks as causal forces in the administrative setting.” Certainly, the efficacy of leadership in Derton relied in part on the opportunities and normative framework provided by the network. However, what is also clear, from the comparison with Nottley, in particular, is the impotence of the network form in itself. Without the will and the agency of its actors, as expressed through leadership at various organizational levels, the network has no causal force. Formal structures, as scholars such as Lipsky (1980), Riccucci (2005), and Sandfort (2000) have found, have little determining effect on the behavior of front-line staff, especially where those staff enjoy professional autonomy. As Sandfort (2000) suggests, it is the “informal” structures of professionals’ collective schemas of understanding that are determinant of behavior, not the reconfiguration of formal, organizational structures. As such, transformative agency is crucial, embodied in leadership of some form, even as leaders employ the structures, processes, and participants of the network in achieving their ends. To this extent, the policymakers seem correct in their assessment of the need for complementarity.However, what our findings also illustrate are the limits to the space in which this synergistic relationship between structure and agency can operate. In Nottley especially, but to some extent also in Derton, the influence of coexisting structural forms was always present. This was most obvious in relation to physicians and purchasers in primary care. The division between primary-care and hospital physicians has been noted as a significant barrier to integration elsewhere (Grumbach et al. 1999; Shortell et al. 2000). The purchaser-provider split is a ubiquitous one in networks in any field which is at least partly characterized by a contractual, market or quasi-market relationship (Kickert et al. 1997). Though formally part of the network, it was clear in both sites that physicians and purchasers were susceptible to neither the principles of collaboration nor the performance of leadership in the way that the network's more central stakeholders were. It is evident, then, that the space created for effective leadership by the structural intervention of the network is a partial, constricted one, that is less open in the margins of the network where there are competing pressures on behavior from other organizational forms, such as hierarchical performance management and competition between providers. Crosby and Bryson (2005) recognize this when they identify the rather different leadership skills required in arenas and courts as compared to forums. Notable in relation to their example of the African-American Men Project in Minnesota was the administrative, decision-making power borne by a key leader as an elected official, enabling him to enact political leadership in the executive arenas concerned (Crosby and Bryson 2005). Nottley's lack of such political leadership saw it fail in its bid for resources in the arena of the purchasing decision-makers; Derton's project almost met a similar fate, saved only by the collective political clout of the network's powerful stakeholders. The more general, theoretical point in relation to the categorization of Crosby and Bryson (2005) of effective leadership strategies is that whereas forums may be susceptible to the shaping influence of leaders within a network, arenas and courts frequently are not. As such they require not just a different style of leadership but potentially also a different set of leaders altogether, with legitimacy and leverage within their domains.Our contribution to theory may thus be summarized as follows. The need for distributed leadership, highlighted in the literature in relation to various public-service professional bureaucracies, is compounded by the interorganizational network form. Leadership needs to be dispersed as well as distributed to engage diverse, powerful stakeholder groups, and this implies a variety of leaders as well as leadership styles. The interorganizational network itself is ineffective as a means of achieving change, but it does provide the space and the media for leaders to effect change. However, this space is a constricted one, and at its boundaries are other organizational forms, with their pressures on actors and corresponding norms of behavior. Consequently distributed and dispersed leadership are often not enough to achieve change, if change rests also on the decisions and behaviors of those governed by the pressures of these other organizational forms. The arenas and courts outside the network, in terms of Crosby and Bryson (2005), may require not only a different form of leadership but also a different tranche of leaders. This last point may relate especially to countries such as the United Kingdom and much of continental Europe, where the remnants of traditional, centralized systems coexist alongside the network form, though as we have seen, it is also relevant in relation to the coexistence of networks and markets.On a practical level, certain specific lessons for the health sector also have broader implications for the operation of public-service networks more generally. In the health services of the United Kingdom, United States, and other countries, “the relative power of specialists has diminished as primary care physicians have assumed a more central role as the initial contact point with patients and as major decision makers regarding referrals to specialists and sites of care throughout the system” (Shortell et al. 2000, 79). In the United Kingdom, policymakers are currently seeking to consolidate this new-found power of primary-care physicians and purchasers, so that they might become the drivers of health-service change. However, evident from both these studies was the degree to which primary-care actors were sidelined within the networks and in service reform. The marginality of this locus of power was instrumental in Nottley's failure and almost derailed Derton's project too, and in more general terms, the need for policymakers to align incentives and levers, through integration or centralization within the network, is strong (Keast and Brown 2006; Milward and Provan 1998; Provan and Milward 1995; Provan et al. 2004). Client-level networking between practitioners (Provan and Sebastian 1998) needs to be supported by organizational integration that supports practitioners’ efforts and enables the establishment of change in the face of prevailing institutional pressures. “Inter alia,” this means that notwithstanding their contractually based relationship, organizations on both sides of purchaser-provider relationships need to be incorporated in the structures, and inculcated in the ethos, of the network.CONCLUSIONOur study is based on relatively thin empirical data, and so should be interpreted cautiously, especially in terms of the additions to existing theory suggested. Many of the findings, though, confirm existing evidence; moreover the findings on the importance of distributed leadership from these two case studies are mirrored in data from our wider study of 11 sites. Our findings indicate that despite the well-documented obstacles to networked governance and distributed leadership in the public services, a careful alignment of objectives with managerial agenda can bring success to reforms in service provision. A combination of clear benefits to wider stakeholders within the network, and distributed and dispersed leadership, can give rise to effective collaboration and establishment of reforms, through structural integration and the harnessing of agency. To this extent, policies invoking structure and agency were indeed synergistic. However, the relationship was a contingent one, illustrating how network-based reforms to organizational structure are both potentially powerful and simultaneously impotent: powerful in creating a space within which certain enactments of leadership might flourish and achieve service reform; impotent as instruments of that reform in themselves, as a means of changing practice in the face of the much more powerful, informal structures that are determinant of behavior (Riccucci 2005; Sandfort 2000). Furthermore, the space for effective leadership created was a limited one, which became more and more constricted in the face of coexistent organizational forms and modes of governance. Networks compound the need for distributed leadership in public-service contexts since they introduce new loci of power that might be influenced by a more dispersed form of leadership. However, the coexistence of networks alongside other organizational forms constricts the effectiveness of this mode of leadership, and it is likely that in many settings, complementary modes of leadership that conform with the requirements of these other organizational forms may be required.Our study adds to a growing literature on the practice of leadership and the role of networks in public-service organizations and brings these literatures together to highlight the potentials for and impediments to leadership in public-service networks. As we noted in our opening section, as policies focused on transforming the structures of public-service provision and the agency of public servants respectively, networks and leadership seem to some extent complementary. The tangible success of distributed, quiet leadership, and dispersed change agency, in one case, indicates how these two policies can indeed work synergistically. The less successful efforts of the second case, illustrate the fragility of this complementarity.In terms of policy, our study reaffirms the importance of carefully aligned policy levers that ensure that organizations within networks have drivers which incline them toward networked collaboration rather than operation within their silos. In health, the gap between hospital and primary care would seem to be a particularly important one in terms of divergent drivers and ill-aligned incentives, reflecting the uneven influence of network-based organizational forms in other sectors, especially those characterized by market or quasi-market relationships. For organizations and practitioners, then, the key message is around ensuring the functionality of networks and the importance of locating those crucial change agents whose influence might be harnessed toward service reform. Finally, we hope our study illustrates the distinctive contribution of qualitative study (cf. Bryman 2004), even as it highlights the need for further investigation of the processes by which individual agency interacts with structural reconfiguration to succeed or fail in consolidating modernizing service reforms of this kind.FUNDINGUK Department of Health.AddicottRachaelMcGivernGerryFerlieEwanThe distortion of a managerial technique? 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H. vande BontAntoinette AHuijsmanRobbertLearning to cross boundaries: The integration of a health network to deliver seamless careHealth Policy20067920313YinRobert KCase study research: Design and methods2003LondonSage1We refer throughout this article to the public services in England only since policies in the devolved administrations of the other three nations of the United Kingdom have diverged significantly from English public-service reforms, particularly in relation to health-service reorganization.2Bill Clinton, Announcement of the initiative to streamline government, March 1993. http://govinfo.library.unt.edu/npr/library/speeches/030393.html, accessed January 18, 2008.3See, for example, http://www.nhsleadershipqualities.nhs.uk/3, accessed September 18, 2007.4Macmillan is a British cancer charity with a particular interest in contributing to improving the management of cancer and ensuring an improved patient experience by working toward more joined-up cancer provision within the NHS and between NHS and other providers of care.5We use pseudonyms and do not give exact figures regarding patient throughput here, in order to protect the identities of our case-study sites, in accordance with the terms of ethical approval for the wider evaluation from which this article derives.6As one cancer network manager here confided: “I've been to other networks where they haven't got relationships with hospitals: they're not allowed in through the doors.”7It was also in contrast to another case-study site not detailed here, where mobilization of involved service users had seen a proactive publicity campaign resulting in large numbers of self-referrals and sustained media coverage for the service. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BB74813D12C151A76D786E766714354809B160E.txt b/test/dataset/in/resources/corpus/Clean_0BB74813D12C151A76D786E766714354809B160E.txt new file mode 100644 index 0000000..e4e4076 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BB74813D12C151A76D786E766714354809B160E.txt @@ -0,0 +1 @@ +]>EXG5327S0531-5565(00)00139-X10.1016/S0531-5565(00)00139-XElsevier Science Inc.Fig. 1Possible causes of aging in the mechanisms of life, and the target of “Gerontology Proteomics”. Cascade of cellular aging may proceed in an intracellular environment that is made with cell-type-specific “proteome”. Many primary causes of cellular aging are commonly speculated for all cell types including mitotic and post-mitotic cells. Genetic background is one of the most important factor that determine the quality of the defense system against environmental attacks: Cosmic rays, UV light, reactive oxygen species (ROS), and reducing sugars are significant elements of environmental attacks. Results of the battle against these environmental attacks may be revealed in their proteome profiles.Fig. 2Detection of age-related protein alteration by 2D gel electrophoresis and quantitation of relative intensity by image processing in proteome analysis. The relative intensity of ssp7001 shows a transitional increase around 65PDL, which is the border between phases 2 and 3 of cellular aging of normal human diploid fibroblasts TIG-3.Fig. 3Identification of spot protein by MS. The spot protein was transferred to a PVDF membrane, and digested with lysylendoproteinase Lys-C. The mass spectrum of peptide fragments was obtained using a Micromass Q-Tof MS system. The mass fingerprint database search was executed on the internet MS-FIT site. The ssp7001 was identified as phosphoprotein stathmin.Fig. 4Determination of methionine oxidation in human amyloid beta peptide (1–40) by MALDI-TOF-MS. The sample solution was prepared as described in Methods. One-μl aliquot was applied to the MALDI target plate. The mass spectrum was recorded with a Voyager-DE STR MALDI-TOF-MS system (PE Biosystems, Foster City, CA) in a reflection mode. The spectrum indicated that the authentic preparation of human amyloid beta peptide (1–40) contained both native and Met35-oxidized forms. Detected ions were all monovalent and the 15.96-Da mass shift was derived from the methionine oxidation.Current status and perspectives of proteomics in aging researchTToda*ttoda@tmig.or.jpDepartment of Gene Regulation and Protein Function, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-Ku, Tokyo 173-0015, Japan*Tel. +81-3-39643241; fax: +81-3-35794776AbstractThe accumulation of non-enzymatic modifications on both DNA and protein molecules under the attack of reactive oxygen species (ROS), is one of the most possible factors responsible for the functional deterioration in aged cells. Direct protein modifications as well as DNA damages may be detectable, in part, by proteome analysis if the gene expression is affected by the damages on DNA. The novel term “proteome”, which is a compound of “protein” and “genome”, means a whole set of proteins expressed in a tissue or a cell strain to be investigated. Proteomics is a methodology for analyzing proteomes. In proteomics, two-dimensional gel electrophoresis is performed primarily to separate constitutive proteins, followed by mass spectrometry to identify each protein of interest and to determine a possible post-translational modification. Proteomics has offered us an innovative tool for investigating the molecular mechanisms of cellular aging.KeywordsProteomeTwo-dimensional gel electrophoresisMass spectrometryProtein oxidation1IntroductionThe age-dependent deterioration of cell function appears in both replicative and post-mitotic cells, in living organisms. The major function of stem cells is cell division in the regeneration of cell population and tissues. Most replicative blast cells play another role: in the excretion of cytokines for inter-cellular signal transduction. Post-mitotic cells in the brain, heart, kidney and many other organs have tissue-specific functions. Although a downstream of the cascade in cellular aging may be cell-type specific, and may proceed in a given intracellular environment that is made with all constituents of the proteome, there may be common factors in the upstream of the cascade. The accumulation of molecular damages on DNAs and proteins under environmental attacks including oxidative stress, may be one of the major events that trigger the cascade of cellular aging as described in Fig. 1.Oxidative protein modification may alter methionine residue to sulfoxide, phenylalanine to diphenyl, and lysyl residue to carbonyl (Smith et al., 1997; Wells Knecht et al., 1997; Stadtman and Berlett, 1998). In proteomics, such protein altered by post-translational modification, may be separated from the original form by high resolution two-dimensional gel electrophoresis (2-DE), and the modification may be determined by mass spectrometry (MS).2Materials and methodsImmobiline DryStrip and Pharmalyte were purchased from Amersham Pharmacia Biotech KK (Tokyo, Japan). Sequi-Blot PVDF membrane was obtained from the Nippon Bio-Rad Laboratories (Tokyo, Japan). Acrylamide, N,N′-methylenebisacrylamide and TEMED were from Daiichi Pure Chemicals (Tokyo, Japan). Sequencing grade endoproteinase Lys-C was from Roche Molecular Biochemicals (Indianapolis, IN, USA). Trizma base, Tricine, SDS, Triton X-100, iodoacetamide, acetonitrile, trifluoroacetic acid and alpha-cyano 4-hydroxy-cinnamic acid were from Sigma (St. Louis, MO, USA). Quick CBB staining reagent, silver staining reagent kit “Wako”, urea, and other chemicals of reagent grade were obtained from Wako Pure chemicals (Osaka, Japan).2.1Two-dimensional gel electrophoresisConstitutive proteins in a cell extract were separated into isolated spots by 2-DE in an “immobilized pH gradient isoelectric focusing (IPG-IEF)/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system” according to the standard protocol (Toda and Kimura, 1997), with minor modifications. The details of the modified protocol are shown in our web homepage: http://proteome.tmig.or.jp/2D/2DE_method.html. In brief, the first-dimensional IPG-IEF was carried out on a re-swollen Immobiline DryStrip, pH 4–8, 18-cm long (Code No. 18-1004-34) in the CoolPhoreStar IPG-IEF Type P horizontal IEF apparatus (Anatech, Tokyo, Japan). A 20-μl aliquot of the sample solution, which was absorbed in a small piece of filter paper, was applied near the cathode wick on the IPG gel. Spot proteins on a gel plate for “differential protein display” were subsequently visualized by silver staining. For the preparative use of 2-DE gel, the IPG gel was swollen in a solution containing 0.2ml of cell extract. After 46,700-Vh electrofocusing, the IPG gel was equilibrated with the SDS-treatment solution (50mM Tris–HCl, pH 6.8, 6M urea, 0.5% (w/v) dithiothreitol, 2% (w/v) SDS, 0.005% (w/v) BPB, 25% (v/v) glycerol), initially for 30min, and then followed by carbamoylmethylation in an iodoacetamide-containing buffer (50mM Tris–HCl, pH 6.8, 6M urea, 4.5% (w/v) iodoacetamide, 2% (w/v) SDS, 0.005% (w/v) bromophenol blue (BPB), 25% (v/v) glycerol) for 10min. An equilibrated gel strip was placed on top of a gel slab (7.5%T, 3%C, 20×18cm), and firmly contacted to the top of a gel slab by pressing the gel strip with a shark-teeth comb. SDS-PAGE was run vertically in the CoolPhoreStar SDS-PAGE Tetra-200 vertical slab gel electrophoresis apparatus (Anatech, Tokyo, Japan) using a tricine buffer system.2.2Protein staining and image processingProtein spots were visualized on the gel slab for a differential protein display analysis by silver staining, using the original version of the reagent kit “Wako” as it showed the widest dynamic range in optical density among all the commercially available reagents tested. The 2-DE gel images were acquired using a Sharp JX-330 scanner. Noise reduction, background subtraction, spot detection, spot quantification, gel-to-gel matching and differential protein analysis were carried out using a PDQuest software (Bio-Rad Laboratories, Hercules, CA, USA).2.3Endoproteinase digestion and peptide mass finger printingAfter scanning, Coomassie-stained proteins in the gel slab were re-solubilized and transferred onto a PVDF membrane as follows. The stained gel was rinsed in pure water thrice, each time for 30min, incubated in a re-solubilization buffer (0.2% (w/v) SDS, 0.3% (w/v) Tris, 0.7% (w/v) glycine) for 10min and mounted on an electrotransfer blotting chamber. The electrotransfer was carried out overnight at 4V/cm in 0.1% (w/v) SDS, 0.3% (w/v) Tris and 1.5% (w/v) glycine.Protein spots were excised from the PVDF membrane and the Coomassie dye was removed by rinsing in 60% (v/v) methanol. For the MS analysis, the piece of membrane was incubated with 4.5% (w/v) polyvinyl pyrolidone 25 in 2.5mM Tris–HCl, pH 7.7, for 30min, followed by a brief rinsing in 5% (v/v) acetonitrile. The digestion was carried out overnight with 0.1μg of sequencing grade endoproteinase Lys-C in 30μl of 8% (v/v) acetonitrile, 2.5mM Tris–HCl, pH 7.7, at 37°C. After digestion, the reaction mixture was supplemented with 10μl of acetonitrile and sonicated for 5min.After removing the acetonitrile by blowing with nitrogen gas, peptides in the digestion mixture were trapped on C18 resin packed in a ZipTipC18 (Millipore Corporation, MA, USA), and eluted in 5μl of 75% (v/v) methanol, 1% (v/v) formic acid. Nano-ESI-MS for peptide-mass fingerprinting was performed using a Micromass Q-Tof system, equipped with a NanoFlow Probe Tip Type F (Micromass UK Ltd., Manchester, UK). The peptide solution was put into a bolosilicate capillary tip and subjected to electrospray ionization (ESI) at a flow rate of 10nl/min. The MS spectrum was analyzed with the ProteinLynx software. Protein identification through a database search was carried out using the MS-FIT proteomic tool at the UCSF web server, through the internet.3Results3.1Screening of age-related protein alteration by proteome profiling and differential protein displayAlterations in the relative intensity of protein spots appearing on proteome profiles, may be the results of various molecular alterations that occur during aging. Quantitation of the integrated optical density of each protein spot is required for the 2D gel electrophoresis of protein, to screen out the molecular events that may be responsible for the functional deterioration in senescent cells.Coomassie Blue staining is the most reliable method for the quantitative demonstration of proteins on a gel slab. However, the sensitivity of Coomassie staining is not high enough to detect minor components of cellular proteins. Autoradiography of [35S]methionine-labeled proteins shows the highest sensitivity. However, it is not applicable to the detection of post-translational modification of proteins. This is due to the fact that, only the fresh proteins that are de novo synthesized during the period of labeling incubation are detected by autoradiography. Cypro ruby fluorescent staining shows almost the same sensitivity as silver staining, and quantitativity as Coomassie staining. Therefore, fluorescent staining will be included in the standard protocol of proteomics in the future. Silver staining is a practical method for spot protein visualization in proteome analysis at present, because it shows the highest sensitivity and practical reliability in relative quantitation for differential protein display, as shown in Fig. 2.In this 2D gel area, the transitional increase of spot protein ssp7001 was observed around 65PDL; at that level, the normal human diploid fibroblasts (TIG-3) transfer into phase 3 of the replicative cell aging and the doubling time was delayed.3.2Identification of spot protein by mass spectrometry in proteomicsIn general, western blotting has been performed for the identification of proteins in spots for the last several years. However, the immunochemical technique was not applicable to the unknown protein for which a specific antibody was not available. Co-electrophoresis is another way to assign a spot to a candidate protein when its authentic protein standard protein is available. In modern proteomics, a sequence database search queried with peptide mass fingerprint data and/or partial sequence tag data obtained by MS, is generally performed to identify proteins on 2D gel patterns. Fig. 3 shows an example of identification of ssp7001 spot protein by peptide mass fingerprinting. The mass spectrum of Lys-C digests of ssp7001 was recorded using a Micromass ESI-Q-Tof-MS system (MICROMASS UK, Manchester, UK). The database search was queried to MS-FIT in the Protein Prospector Server at California University, and subsequently it was assigned to a microtubule associating phosphoprotein stathmin. When no candidate protein is hit by the database search, because it is really novel, molecular cloning should be done to clarify the meaning of the protein alteration in the aging process. In that case, Edman-degradation microsequencing has an advantage over mass spectrometry in sequencing longer peptide fragments of the protein in order to design proves and/or primers in molecular cloning of its corresponding cDNA.3.3Determination of protein modification by mass spectrometry in proteomicsAge-related protein alterations in heat stability and specific activity have been reported by many groups (Holliday and Tarrant, 1972; Gershon and Gershon, 1973; Chetsanga and Liskiwskyi, 1977; Pigeolet and Remacle 1991). Further, the accumulation of detergent-insoluble protein was also observed in aged cells and tissues (Yang and Wang, 1994). It was suggested that these age-related alterations reflect post-translational modifications such as oxidation by attacking of reactive oxygen species (Gordillo et al., 1989). Oxidative modification may produce carbonyl groups, ortho-tyrosine structures and methionine sulfoxide residues that increase the hydrophobicity of the protein surface (Chao et al., 1997). In the proteome analysis, structures of oxidative modification on spot proteins, isolated by 2D gel electrophoresis, can be determined by MS. Human amyloid beta (Aβ)1–40 peptide contains a methionine, and its oxidation induces the alteration of the 3D structure. The Met35-oxidized Aβ shows a higher molecular mass of 16Da than native Aβ, as shown in Fig. 4. Carbonylation does not result in a sufficient shift in molecular mass (−1Da); however, theoretically its dinitrophenylhydrazine derivatives gives an increase of 180Da.4DiscussionWe have obtained an excellent methodology, proteomics, which is most suitable for investigating protein factors in molecular mechanisms of cellular aging. Alterations in gene expression that are the results of DNA damages, and accumulation of altered proteins that are made by oxidative modification can be detected by methods in proteomics. The advanced method of two-dimensional gel electrophoresis, in which isoelectric focusing is carried out on an immobilized pH gradient for the first dimensional separation, offers the highest resolution of proteins. More than thousands of spots are detected on a gel slab in an optimized condition. The proteome database of normal human diploid fibroblasts TIG-3 has been established, and is displayed on our Gerontology Informatics database: the PDL-dependent alterations of protein spots are demonstrated here. Although the resolution of 2D gel electrophoresis is still not high enough to separate all the proteins in a cell extract, it will be improved by partitioning the pH and molecular mass range using narrow pH range IPG-IFE gel strips and various concentrations of acrylamide gel slabs for SDS-PAGE. The multiplication of 2D gel electrophoresis for partitioning the range of separation may yield better results than the simple enlargement of a gel size to cover the overall range of separation. Identification of the protein by making inquiries on the proteomic database with a peptide mass fingerprint data, will be more successful after completion of the genome-sequencing projects. Most post-translational modifications that are accumulated in aged cells under attacks of reactive oxygen species (ROS), are easily detected by proteomics. The extensive application of proteomics in the investigation of altered proteins in aged cells has led to the next stage of research: the molecular mechanisms of aging.ReferencesChao et al., 1997C.CChaoY.SMaE.RStadtmanModification of protein surface hydrophobicity and methionine oxidation by oxidative systemProc. Natl. Acad. Sci. 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Invest.1001997839846Yang and Wang, 1994GYangEWangTerminin (Tp 63/60), a novel cell senescence-related protein, is present in the aging human hippocampusBrain Res.6441994188196 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BCBE54EBC6305C3FAC6AD0B9E7B93DBB6F31E5B.txt b/test/dataset/in/resources/corpus/Clean_0BCBE54EBC6305C3FAC6AD0B9E7B93DBB6F31E5B.txt new file mode 100644 index 0000000..49b7e9a --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BCBE54EBC6305C3FAC6AD0B9E7B93DBB6F31E5B.txt @@ -0,0 +1 @@ +AMGP61210S1064-7481(12)61210-X10.1097/00019442-199905000-00009American Association for Geriatric PsychiatryTABLE 1Comparison of demographics between the early-onset-of-depression (EOD) and the late-onset-of-depression (LOD) samples (Student's t-test and chi-square)Onset of DepressionTotal Group (TG) EOD and LOD (N = 245)EOD (n = 114)LOD (n = 131)PAge, years, mean ± SD75.28±7.1372.19±6.9477.96±6.170.001Length of stay, days, mean ± SD118.69±63.42125.75 ±60.52112.53±65.440.100Ham-D, baseline, mean ± SD19.89 ±6.8420.03 ±7.0919.76 ±6.630.759Ham-D, discharge, mean ± SD10.30±6.599.35 ±6.4911.28±6.580.048Sex, n (%)0.604 Male64 (26.1%)28 (24.6%)36 (27.5%) Female181 (73-9%)86 (75.4%)95 (72.5%)Level of education, n (%)0.062 No formal education7 (2.9%)1 (0.9%)6 (4.6%) Elementary, partial or complete90 (36.7%)36 (31.6%)54 (41.2%) High school or technical school, partial45 (18.4%)24 (21.1%)21 (16%.0) High school, complete68 (27.8%)34 (29.8%)34 (26.0%) College/University, partial10 (4.1%)6 (5.3%)4 (3.1%) College/University, complete20 (8.2%)10 (8.8%)10 (7.6%) Graduate studies, partial or complete5 (2.0%)3 (2.6%)2 (1.5%)DSM-IV diagnoses, n (%) Primary Axis I0.187  Major depression240 (98.0%)110 (96.5%)130 (99.2%)  Othera5 (2.0%)4 (3.5%)1 (0.8%) Axis II0.224  No diagnosis217 (88.6%)104 (91.2%)113 (86.3%)  Personality disorderb28 (11.4%)10 (8.8%)18 (13.7%)Note: EOD: onset 0.05).TABLE 3Comparison of cognitive functioning between early-onset-of-depression (EOD) and late-onset-of-depression (LOD) groups with age and level of education as covariates (ANCOVA)Onset of DepressionEODLODPMini-Mental State Exam (MMSE), baseline n110119 Adjusted mean26.7927.370.271Mini-Mental State Exam (MMSE), discharge n7154 Adjusted mean27.5127.520.980Mattis Dementia Rating Scale (MDRS), baseline n9299 Adjusted mean127.72127.810.969Note:EOD: onset < age 60; LOD: onset ≥ age 60. Multivariate analysis of covariance revealed no significant difference between the two groups (EOD vs. LOD) with respect to performance on the MDRS and the MMSE (Wilks F[1,98] =0.98; P1>0.05).TABLE 4Comparison of cognitive functioning (per subscales of the MMSE and MDRS at baseline) between early-onset-of-depression (EOD) and late-onset-of-depression (LOD) groups, mean±SD (ANOVA)Total GroupOnset of DepressionEODLODPMini-Mental State Exam (MMSE), baseline Orientation956±0.909.76±0.54938± 1.100.002 Registration2.99±0.112.98±0.132.99±0.900.148 Calculation3.82± 1.553-90 ± 1.383-74 ± 1.700.381 Language7.24 ±0.847.40 ±0.787.10±0.960.006 Recall2.10±0.932.22 ±0.901.99±0.940.418 Construction0.70 ±0.480.75 ±0.440.66±0.510.131Mattis Dementia Rating Scale (MDRS), baseline Attention34.73 ±2.5835.03 ± 1.8134.44±3-ll0.232 Initiation and Perseveration30.88 ±5.7231.40±5.2630.38±6.110.318 Construction5.39± 1-325.57±0.985.21 ± 1.560.090 Conceptualization3305 ±5.5134.48 ±4.8731.70 ±5.740.001 Memory20.03 ±4.3320.92 ±3-6119-18±4.780.009Note:EOD: onset 0.05).TABLE 6Chi-square comparison of the proportions of early-onset-of-depression (EOD) vs. late-onset-of-depression (LOD) patients with dementia as defined by a Mattis Dementia Rating Scale (MDRS) score <123Onset of DepressionTotal Group (N=191)EOD (n = 92)LOD (n = 99)PMDRS≥123115 (60.2%)63 (68.5%)52 (52.5%)0.025MDRS<12376 (39-8%)29 (31.5%)47 (47.5%)Note:EOD: onset 50% of individuals with a PTSD at some point will have a co-morbid major depressive disorder and as outlined above depression is not an infrequent consequence of trauma exposure.ConclusionThe predictable risk from traumatic exposure and the many barriers to care pose a particular challenge in the management of traumatic events in the workplace. Optimally, an occupational health service should identify and manage the risks at an organizational level as well as providing readily accessible evidence-based treatment in a timely manner to those individuals identified to be symptomatic. There is remarkably little literature examining screening, monitoring and the effectiveness of evidence-based treatments in different occupational groups.The absence of direct studies in occupational settings means that there is a critical need for research in these populations. Nonetheless, there is considerable indirect evidence to shape the practice of employers so that evidence accrued from civilian and military settings may form the basis of risk assessments, monitoring practices and interventions.FundingNHMRC programme grant number 300304 held by A.C.M. and R.A.B.Conflicts of interestNone declared.1.SorensonSBPreventing traumatic stress: public health approachesJ Trauma Stress200215372.McCleeryJMHarveyAGIntegration of psychological and biological approaches to trauma memory: implications for pharmacological prevention of PTSDJ Trauma Stress2004174854963.National Institute for Clinical ExcellencePost-Traumatic Stress Disorder: The Management of PTSD in Adults and Children in Primary and Secondary Care, National Clinical Practice Guideline Number 262005LondonCromwell Press Limited4.Australian Centre for Posttraumatic Mental HealthAustralian Guidelines for the Treatment of Adults with Acute Stress Disorder and Posttraumatic Stress Disorder2007Melbourne, VictoriaACPMH5.U.S. Preventive Services Task ForceScreening and behavioral counseling interventions in primary care to reduce alcohol misuseAnn Intern Med20041405555576.McFarlaneACKledberRJFigleyCRGersonsBPRThe severity of the trauma: issues about its role in post traumatic stress disorderBeyond Trauma: Cultural and Societal Dynamics1995New YorkPlenum Press31547.MarmarCRMcCaslinSEMetzlerTJPredictors of posttraumatic stress in police and other first respondersAnn N Y Acad Sci200610711188.BryantRAGuthrieRMMaladaptive appraisals as a risk for posttraumatic stress: a study of trainee firefightersPsychol Sci2005167497529.HuizinkACSlottjePWitteveenABLong term health complaints following the Amsterdam air disaster in police officers and fire-fightersOccup Environ Med20066365766210.RodgersLMA five-year study comparing early retirements on medical grounds in ambulance personnel with those in other groups of service staff. 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Part II: causes of retirementsOccup Med (Lond)19984811913212.SterudTEkebergØHemEHealth status in the ambulance services: a systematic reviewBMC Health Serv Res200668213.van der PloegEKleberRJAcute and chronic job stressors among ambulance personnel: predictors of health symptomsOccup Environ Med200360Suppl 1i40i4614.McCaslinSERogersCEMetzlerTJThe impact of personal threat on police officers' responses to critical incident stressorsJ Nerv Ment Dis200619459159715.CarlierIVLambertsRDGersonsBPThe dimensionality of trauma: a multidimensional scaling comparison of police officers with and without posttraumatic stress disorderPsychiatry Res200097293916.BenedekDMHollowayHCBeckerSMEmergency mental health management in bioterrorism eventsEmerg Med Clin North Am20022039340717.ThamKYTanYHLohOHTanWLOngMKTangHKPsychiatric morbidity among emergency department doctors and nurses after the SARS outbreakAnn Acad Med Singapore200433Suppl 5S78S7918.McCaslinSEMetzlerTJBestSRAlexithymia and PTSD symptoms in urban police officers: cross-sectional and prospective findingsJ Trauma Stress20061936137319.MarshallRDGarakaniAA psychobiology of the acute stress response and its relationship to the psychobiology of post-traumatic stress disorderPsychiatr Clin North Am20022538539520.ElizingaBMBremnerJDAre the neural substrates of memory the final common pathway in posttraumatic stress disorder (PTSD)?J Affect Disord20027011721.BrewinCRAndrewsBValentineJDMeta-analysis of risk factors for posttraumatic stress disorder in trauma-exposed adultsJ Consult Clin Psychol20006874876622.BryantRALongitudinal psychophysiological studies of heart rate: mediating effects and implications for treatmentAnn N Y Acad Sci20061071192623.AdlerABHuffmanAHBliesePDCastroCAThe impact of deployment length and experience on the well-being of male and female soldiersJ Occup Health Psychol20051012113724.GrayMJBoltonEELitzBTA longitudinal analysis of PTSD symptom course: delayed-onset PTSD in Somalia peacekeepersJ Consult Clin Psychol20047290991325.GershonRRLinSLiXWork stress in aging police officersJ Occup Environ Med20024416016726.WeisaethLAcute posttraumatic stress: nonacceptance of early interventionJ Clin Psychiatry200162Suppl 17354027.ShalevABiological responses to disastersPsychiatr Q20007127727828.GriegerTACozzaSJUrsanoRJPosttraumatic stress disorder and depression in battle-injured soldiersAm J Psychiatry20061631777178329.RonaRJHooperRJonesMMental health screening in armed forces before the Iraq was and prevention of subsequent psychological morbidity: a follow-up studyBr Med J200633399130.DuckworthDHManaging psychological trauma in the police service: from the Bradford fire to the Hillsborough crush disasterJ Soc of Occup Med19914117117331.WesselySRisk, psychiatry and the militaryBr J Psychiatry200518645946632.DemariaTBarrettMRyanDMedical screenings as a trigger for PTSD in public safety workersAnn N Y Acad Sci2006107147848033.TurpinGDownsMMasonSEffectiveness of providing self-help information following acute traumatic injury: randomised controlled trialBr J Psychiatry2005187768234.GilbodyDSReview: disease management programmes improve detection and care of people with depressionEvid Based Ment Health200478035.WrightKMHuffmanAHAdlerABCastroCAPsychological screening program overviewMil Med200216785386136.DowlingFGMoynihanGGenetBLewisJA peer-based assistance program for officers with the New York City Police Department: report of the effects of Sept. 11, 2001Am J Psychiatry200616315115337.FullertonCSUrsanoRJWangLAcute stress disorder, posttraumatic stress disorder, and depression in disaster or rescue workersAm J Psychiatry20041611370137638.Christopher Burton v. The State Of New South Wales 9889/01.39.DavidsonJRLong-term treatment and prevention of posttraumatic stress disorderJ Clin Psychiatry200465Suppl 1444840.BoscarinoJAAdamsREFoaEBLandriganPJA propensity score analysis of brief worksite crisis interventions after the World Trade Center disaster: implications for intervention and researchMed Care20064445446241.BissonJIMcFarlaneACRoseSFoaEBKeaneTMFriendmanMJPsychological debriefingEffective Treatments for PTSD2000New YorkGuilford Publications395942.van EmmerikAAKamphuisJHHulsboschAMEmmelkampPMSingle session debriefing after psychological trauma: a meta-analysisLancet200236076677143.Daubert v. Merrell Dow Pharmaceuticals Inc. 1993, Supreme Court of the United States. 579.44.MarkMMSocial science evidence in the courtroom: daubert and beyond?Psychol Public Policy Law1999517519345.ShumanDWSalesBDThe impact of daubert and its progeny on the admissibility of behavioural and social science evidencePsychol Public Policy Law1999531546.BryantRAPsychosocial approaches of acute stress reactionsCNS Spectr20051011612247.GillespieKDuffyMHackmannAClarkDMRelated articles, links community based cognitive therapy in the treatment of posttraumatic stress disorder following the Omagh bombBehav Res Ther20024034535748.ResickPANishithPWeaverTLAstinMCFeuerCAA comparison of cognitive-processing therapy with prolonged exposure and a waiting condition for the treatment of chronic posttraumatic stress disorder in female rape victimsJ Consult Clin Psychol20027086787949.DuvalFLebowitzBDMacherJPTreatments in depressionDialogues Clin Neurosci2006819120650.KessingLAndersonPPredictive effects of previous episodes on the risk of recurrence in depressive and bipolar disordersCurr Psychiatry Rep20057413420 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BE8560AC98800DB170664ADA09913085B56B1D1.txt b/test/dataset/in/resources/corpus/Clean_0BE8560AC98800DB170664ADA09913085B56B1D1.txt new file mode 100644 index 0000000..688fa2d --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BE8560AC98800DB170664ADA09913085B56B1D1.txt @@ -0,0 +1 @@ +]>NBA5195S0197-4580(98)00050-510.1016/S0197-4580(98)00050-5Elsevier Science Inc.Fig. 1Variation (mean ± SD) in NK cell cytotoxic activity (expressed as lytic units = L.U.) under basal conditions (spontaneous activity) and after cortisol incubation (10−6 M) in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).Fig. 2Variation (mean ± SD) in NK cell cytotoxic activity (expressed as lytic units = L.U.) under basal conditions (spontaneous activity) and after IL-2 incubation (100 IU/mL) in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).Fig. 3Variation (mean ± SD) in NK cell cytotoxic activity (expressed as lytic units = L.U.) under basal conditions (spontaneous activity) and after IFN-β incubation (650 IU/mL) in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).Fig. 4Correlations between NK cytotoxic cell activity and Mini Mental State Examination (MMSE) score in patients with SDAT. rs = Spearman’s correlation coefficient.Fig. 5Calcium-dependent PKC activity (mean ± SD) after cortisol, IL-2, and IFN-β incubation in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).Fig. 6Representative Western blot analyses of PKC α and βII immunoreactivities in soluble NK cell fractions from healthy elderly subjects after 20 h exposure to solvent, cortisol, IFN-β, and IL-2. Five micrograms of protein were loaded and diffused by electrophoresis on the gels. The arrowheads on the left denote the size of the specific immunoreactive band for each PKC isoform, whereas those on the right show the migration positions of the molecular size standards.Table 1Clinical and biochemical characteristics of healthy subjects and patients with SDAT (standard Deviation between brackets)Healthy subjectsPatients with SDATn1211men:women7:56:5age (years)78 (7)77 (6)BMI (kg/m2)22.5 (1.5)21.2 (1.7)Ht (%)44 (5)42 (3)Hb (g/dL)13.9 (1.4)13.7 (1.3)serum albumin (g/L)43 (3)41.7 (2.6)serum pre-albumin (g/L)0.33 (0.02)0.31 (0.02)transferrin (g/L)3.1 (0.2)3.0 (0.3)total lymphocytes (cells/mm3)1973 (51)1925 (51)Table 2Variations in NK cytotoxic activity evaluated in PBMC before immunomagnetic separationHealthy elderly subjectsPatients with SDATSpontaneous lysisL.U./107 cells19.1 ± 3.818.6 ± 3.7NK cytotoxic activity after cortisol (10−6 M)L.U./107 cells11.2 ± 2.714.1 ± 2.9percent inhibition (%)41.3 ± 6.024.1 ± 2.3aNK cytotoxic activity after IL-2 (100 IU)L.U./107 cells32.8 ± 5.637.6 ± 8percent stimulation (%)71.7 ± 3.7102.1 ± 7.3bNK cytotoxic activity after IFN-β (650 IU)L.U./107 cells34.2 ± 4.843.7 ± 9.6percent stimulation (%)79.1 ± 9.6134.9 ± 11.3bResults are mean ± SD (ap < 0.01,bp < 0.001 vs. healthy subjects).Table 3Effect of cortisol, IL-2, and INF-β on NK cell cytosolic PKC α and βII immunoreactivities in healthy elderly subjectsPKC αPKC βIIBasal0.328 ± 0.0410.470 ± 0.051Cortisol0.313 ± 0.048 (−5%)0.482 ± 0.068 (+2%)IL-20.099 ± 0.013 (−70%)a0.282 ± 0.034 (−40%)aIFN-β0.100 ± 0.027 (−70%)a0.157 ± 0.028 (−67%)aThe densitometry of each immunoreactive band was normalized to the immunoreactivity of a rat brain cerebellum cytosolic fraction, utilized as internal positive control in each blot (ratio of NK/cerebellum PKC immunoreactivity). Values are mean ± SEM of three different assays.ap < 0.05 ANOVA and post hoc Dunnet’s test.Original ArticlesIncreased Natural Killer Cell Cytotoxicity in Alzheimer’s Disease May Involve Protein Kinase C DysregulationSolerteS.BSolerteA*FioravantiMFioravantiAPascaleAPascaleBFerrariEFerrariAGovoniSGovoniCBattainiFBattainiDADepartment of Internal Medicine, Geriatrics and Gerontology Clinic, University of Pavia, Pavia, ItalyBInstitute of Pharmacological Sciences, University of Milano, ItalyCInstitute of Pharmacology, University of Pavia and IRCCS S. Giovanni di Dio, Alzheimer Unit, Sacred Heart Hospital-FBF, Brescia, ItalyDDepartment of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Italy*Bruno Solerte, Department of Internal Medicine, University of Pavia, Ospedale S. Margherita, Piazza Borromeo, 2, 27100 Pavia, Italy.AbstractIncreased cytokine-mediated cytotoxic natural killer (NK) cell activity has recently been demonstrated in patients with senile dementia of the Alzheimer’s type (SDAT). In the present study, we evaluated whether protein-kinase C (PKC), a main regulatory enzyme involved in the mechanism of exocytosis by NK cells, has a role in the cytotoxic response of NK cells (during IL-2 and IFN-β exposure) from SDAT patients. Our data demonstrate the presence of an increased cytotoxic response by NK cells to IL-2 (mean increase +102%) and IFN-β (mean increase +132%) in SDAT patients in comparison with healthy elderly subjects (+75% and +88% for IL-2 and IFN-β, respectively). A smaller suppression of NK cytotoxicity after cortisol was also observed in SDAT (mean decrease −24%) than in the control group (−44%). The NK cell activity of SDAT patients was inversely correlated with the cognitive status as evaluated by the analysis of MMSE (Mini Mental State Examination) score. A comparison of young and elderly healthy subjects revealed no variations in NK cell activity. A physiological decrease in cytosolic PKC activity was demonstrated in healthy old subjects after IL-2 and IFN-β incubation, but not in SDAT patients, while no variations in kinase activity were observed after cortisol incubation. The decreased activity with cytokines was associated with reduced levels of PKC α and βII isoforms. An alteration in cytokine-mediated NK cell activity associated with PKC dysregulation is therefore suggested to occur in patients with SDAT. These changes may indicate the existence of an immunological component to the pathogenesis and progression of the disease.KeywordsAlzheimer’s diseaseDementiaAgingProtein kinase CNatural killerCytotoxicityCytokinesCortisolMagnetic cell sorterA growing body of evidence indicates that neuroimmune mechanisms, involving both immunological and neurobiological factors, may be associated with the pathophysiology and progression of Alzheimer’s disease (AD) [27, 39, 54]. Among the cellular components of the immune system, natural killer (NK) cells are considered to be important effectors in the host defenses which express non major histocompatibility complex (MHC)-restricted cytotoxic activity against tumoral and viral targets [25, 36, 46, 48, 62, 63]. NK cytotoxic activity is mainly dependent on biochemical events leading to the exocytosis of preformed proteolytic granules [13, 53, 67], and therefore can be modulated by cytokines and hormonal factors [5, 18, 20, 24, 26, 63, 64].A protein kinase C (PKC)-dependent signaling pathway has been implicated in the Ca2+-dependent mobilization of cytotoxic granules from NK cells [19, 32, 59, 61]. In fact, the PKC activator phorbol myristate acetate (PMA) induces a prompt cytotoxic response that is sensitive to the highly specific PKC inhibitor, PKC pseudosubstrate antagonist peptide [61]. Recent research has also demonstrated that NK cell cytotoxicity toward tumor cell line K562 is specifically regulated by a PKC-dependent pathway, independently of other signal transduction systems [9].There are two separate lines of evidence to support the existence of altered immune response and PKC activity in the brain and peripheral tissues of demented patients, suggesting that there may be an interaction between these systems. Thus, changes in NK number and function have been reported in normal aging [1, 8, 30, 31, 35, 43, 57]and in patients with senile dementia of the Alzheimer’s type (SDAT) [3, 55, 56]. At the same time, PKC alterations have been found in neuronal and non-neuronal peripheral tissues [14, 17, 21, 44, 51], while decreased PKC activity has been demonstrated in cultured fibroblasts [22]and in platelets [10]of patients with AD.In light of these considerations, the aim of the present study was to investigate whether the altered NK cell cytotoxic function observed in SDAT was associated with disorders of the PKC signal transduction pathway. To this end, NK cells derived from SDAT patients and healthy elders were isolated by means of negative magnetic separation and assayed for cytotoxicity against K562 targets after incubation with IL-2, IFN-β, and cortisol. Calcium-dependent PKC activity in soluble and particulate fractions of NK cells, and NK cytolytic function were also evaluated.1Methods1.1SubjectsThe study was approved by the Ethical Committee of the Department of Internal Medicine of the School of Medicine of the University of Pavia. Written informed consent was obtained from all subjects and patients or, where appropriate, from their caregivers.Eleven patients with senile dementia of the Alzheimer type (SDAT) were carefully selected from the Alzheimer’s Unit of our department and were recruited for the study. The diagnosis of SDAT was initially performed in agreement with the criteria described in the Diagnostic and Statistical Manual of Mental Disorders, 3rd edition-revised (DSM III-R) [2], and then each patient was confirmed for probable or possible AD using the diagnostic standards of the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRA) criteria [40]. The evaluation of the Hachinski ischemic score [23]was used to exclude multi-infarct dementia, and all SDAT patients achieved a score of 4 or less (mean ± SD, 2.2 ± 0.9). Diagnosis of SDAT was further supported by physical and neurological examination, by laboratory evaluation (including a complete biochemical assessment of nutritional status) and by the determination of brain imaging (MRI or CT scan). Cognitive status and the severity of dementia were also quantified by using the Mini Mental State Examination (MMSE) test [16]. The score of patients with SDAT ranged from 6 to 15 (mean ± SD = 10.3 ± 2.4). All patients with SDAT were free of any medication (for at least 2 weeks) and no diseases (e.g., respiratory and urinary tract infections) known to affect immune function and PKC were observed 2 months before the inclusion in the study.Patients with SDAT were compared to 12 age and sex-matched healthy elderly subjects. These subjects were recruited in agreement with the criteria of the SENIEUR protocol [34]in order to exclude clinical and immunological alterations. The clinical and biochemical characteristics of healthy elderly subjects and patients with SDAT are reported in Table 1. Ten normal-weight healthy young subjects (6 women and 4 men, age = 32.2 ± 4 years) were also chosen as a further control group in order to compare the data on NK cytotoxic activity obtained in SDAT and in healthy elders. No differences concerning sex distribution, age, body weight (expressed as body mass index = BMI) and biochemical parameters were demonstrated between healthy elderly subjects and patients with SDAT (Table 1).1.2ProceduresAfter an overnight fast, human peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood samples (Vacutainer, Hemogard, lithium heparin, Becton Dickinson, Meyland, France).PBMC were isolated by Ficoll-Hypaque density gradient (Lympholyte-H, Cedarlane Laboratories Ltd., Ornby, Ontario, Canada) [11]. Plastic-adherent cells were removed by incubation at 37°C in Petri-culture dishes for 1 h.The remaining non-adherent cell population was passed through nylon wool columns preincubated for 1 h with RPMI 1640 supplemented with 10% heat-inactivated autologous serum (RPMI/AS) at 37°C (5% CO2 in air). T/NK cells were obtained by rinsing the columns with tissue culture medium, which leaves B cells and remaining monocytes attached to the nylon wool [28]. The enriched fraction of PBMC containing T/NK cells was used for the separation in the magnetic field.The NK cells were separated under sterile conditions. The magnetic cell separation system (MACS) and the NK cell isolation kit (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) were used for immunomagnetic separation of NK. Washed PBMC were resuspended in 80 μL buffer (per 107 total cells), containing PBS supplemented with 0.5% BSA, and incubated for 15 min. at 6°C with 20 μL of reagent composed of modified CD3, CD4, CD19, and CD33 antibodies of mouse IgG1 isotype in order to label non-NK cells. Afterwards, PBMC were washed once in PBS and incubated for 15 min. at 6°C with 20 μL of colloidal superparamagnetic MACS microbeads recognizing non-NK cells. Labeled and unlabeled cells were separated in a high-gradient magnetic field, generated in a steel wool matrix inserted into the field of a permanent magnet [42, 47]. The columns were sterilized in an autoclave at 120°C shortly before use.The negative unlabeled cells, representing the enriched non-magnetic NK cell fraction, were eluted from the separation column outside the magnetic field in a laminar flow hood to ensure appropriate asepsis. The efficiency of separation was evaluated by flow cytometry, using a FACScan (Becton Dickinson, Mountain View, CA, USA). The sample obtained from the negative fraction was stained with FITC-conjugated NK cell antibodies (CD56, CD16+) and counted for total NK cell number. Anti-Leu 11b (anti-CD16) and anti-Leu 19 (anti-CD56) were purchased from Becton Dickinson. The MACS procedure allowed us to separate the negative NK cell population in approximately 2 h with yields of over 95% and purity of 97 ± 1% for CD16, CD56+ NK cells. Viability of the NK subpopulation was determined by Trypan blue uptake prior to the cytotoxicity assay against K562 tumor cells and PKC activity assay.The human myelogenous leukemia cell line K562 was used as the target in the cytotoxicity assays [50, 59]and was grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% glutamine, and 100 μg/mL gentamycin. The K562 cell line was maintained in suspension culture flasks at 37°C in a 5% CO2 incubator (Heraeus BB 6220, Hanau, Germany).All K562 target cells used were > 90% viable as measured by Trypan blue dye exclusion (Trypan blue solution 0.4% SIGMA Chimica, Milano, Italy).Immunological procedures were performed in a completely sterile manner in a class II biological safety cabinet (Microflow 51426, MDH Ltd., Andover, UK). Complete medium containing RPMI 1640 medium (HyClone Laboratories Inc, Logan, UT, USA) enriched with 10% heat-inactivated FCS (HyClone Laboratories Inc, Logan, UT, USA), 1% glutamine (HyClone Laboratories Inc, Logan, UT, USA) and 100 μg/mL gentamycin (Irvine Scientific, Santa Ana, CA, USA) was used for cytotoxicity assays.After magnetic separation, NK cells were washed three times (with 0.9% saline and complete RPMI medium) and finally resuspended to a measured (Sysmex Toa F800 Microcell Counter, Dasit, Bareggio, Italy) density of 7.75 × 106 cells/mL of complete medium. NK cells were incubated for 20 h [18, 26]at 37°C in a humidified atmosphere of 95% air and 5% CO2, with cortisol, IL-2, and IFN-β and without the use of modulators (to measure the spontaneous basal cytotoxicity). Cortisol (Hydrocortisone, SIGMA Chimica, Milano, Italy), IL-2 (recombinant human IL-2, Proleukin, Chiron Corporation, Emeryville, USA), and human IFN-β (Betantrone, Italfarmaco S.p.A., Milano, Italy) were diluted in a complete fresh medium (in a 0.1 mL final volume) and used at a final concentration of 10−6 M, 100 IU/mL, and 650 IU/mL, respectively. After incubation, the cells were washed twice with 0.9% saline and then once with complete medium containing Medium 199 modified and 5% fraction V bovine albumin (SIGMA Chemical Co., St. Louis, MO, USA) counted, assessed for viability by Trypan blue dye exclusion, and then assayed for cytotoxicity against K562 target cells.After washing three times with 0.9% saline and complete medium (Medium 199+5% Albumin fraction V), 3 × 104 target cells in 0.1 mL complete medium were mixed in triplicate with various concentrations of NK effector cells in the wells of a round-bottomed 96-hole standard microtiter plate (TPP®, Celbio, Pero-Milano, Italy), at a final total volume of 0.2 mL. These mixtures gave final effector:target ratios (E:T) of 25:1, 12.5:1, 6.25:1, 3.125:1. After a second incubation for 4 h at 37°C in a 5% CO2 atmosphere, the microtiter plate was centrifuged and a fixed aliquot (0.1 mL) of supernatant was extracted from each well and transferred to the corresponding wells of a flat-bottomed microtiter plate. The cytotoxicity assay of NK cells was based on the kinetic measurement by a computer-assisted (Milenia Kinetic Analyzer DPC, Los Angeles, CA, USA) microtiter plate reader of the amount of lactate dehydrogenase (LDH) released into the supernatant of target cells, according to the calculation of Korzeniewski and Callewaert [29]. Subsequently, 0.1 mL of lactic acid dehydrogenase substrate mixture was added to each well at intervals of 3 s. Data on NK cytotoxic activity were expressed as lytic units (LU)/107 cells [49]and as percentage of increase or decrease of specific lysis.After a 20-h incubation at 37°C (with 5% CO2) with cortisol, IL-2, and IFN-β and without the use of modulators, four different aliquots of NK cells were frozen on dry ice and stored at −80°C until the kinase assay (no more than 1 week). Cells were homogenized using a teflon glass homogenizer in 500 μL 0.32 M sucrose buffered with 20 mM Tris-HCl pH 7.4 containing 2 mM EDTA, 0.5 mM EGTA, 50 mM mercaptoethanol, 0.3 mM phenylmethylsulfonylfluoride, and 20 μg/mL leupeptin. The homogenate was centrifuged at 100,000 × g for 1 h: the supernatant constituted the soluble fraction. The resulting pellet was treated with the same homogenization buffer containing 10 mg/mL CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate} for 20 min., sonicated, and centrifuged at 100,000 × g for 1 h: the supernatant represented the solubilized particulate fraction.Calcium-dependent PKC activity was assessed as previously described and the antibodies against PKC isoforms were provided by W.C. Wetsel [7]. Briefly, 4 μg of soluble and particulate fractions were incubated at 37°C in 100 μL final volume of a buffer containing 3 μmol Tris-HCl pH 7.5, 0.8 μmol magnesium acetate, 0.1 μmol CaCl2, and 50 μg histone type III S as kinase substrate (basal activity). Stimulated activity was evaluated by introducing 40 μg phosphatidylserine and 4 μg diolein into the same incubation buffer. The reaction was started by adding 20 μM [γ−32P]-ATP (0.3 μCi/sample, S.A. 3000 Ci/mmole—Amersham, Italy) and stopped after 5 min. with 100 μL of 25% trichloroacetic acid and filtration through Schleicher and Schull ME 25 0.45 μm filters under vacuum. Samples were washed 3 times with a solution containing 10% trichloroacetic acid, 10 mM NaH2PO4, and 5 mM EGTA. Radioactivity retained by filters was determined by liquid scintillation using Formula 989 (NEN, Cologno Monzese, Italy). Protein content was measured by the Bradford’s micromethod [12]. Specific PKC activity was evaluated as the difference between stimulated and basal activities and expressed as nmol/min./mg protein.Cytosolic fractions were prepared from NK cells as previously described.Sample aliquots were diluted with sample buffer, boiled for 5 min., and processed as described [7]. Antibody to PKC α was diluted at 1:10000; PKC βI, βII, and γ at 1:5000. Western blot was developed using enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Milano, Italy). With regard to the densitometric analysis, the image of the Western blot was acquired with a Nikon CCD video camera module. The optical density of the immunoreactive bands was calculated and the peak area of a given band was analyzed by means of the Image 1.47 program for digital image processing (Wayne Rasband, NIH, Research Services Branch, NIMH, Bethesda, MD, USA).One-way analysis of variance (ANOVA F-test) was employed to measure differences in clinical and biochemical parameters among healthy subjects and patients with SDAT. ANOVA was also used to evaluate differences in the cytotoxicity and PKC activity of NK cells recorded under basal conditions (spontaneous lysis) and after incubation with cortisol, IL-2, or IFN-β. Tukey’s and Dunnet’s post hoc tests were used when ANOVA proved significant. Non-parametric Spearman’s correlation coefficient was employed to evaluate the correlation between MMSE score and NK cytotoxic activity (spontaneous and after cortisol, IL-2, and IFN-β). Multiple regression analysis was performed to assess the relationship between clinical and biochemical parameters (as independent variables) with the spontaneous NK cytotoxicity and the percent of variation of NK cell activity after cortisol, IL-2, and IFN-β (as dependent variables). p < 0.05 was accepted as the threshold of statistical significance (two-tailed). All analyses were run with the SPSS/PC+ V3.0 statistical package.2ResultsSpontaneous NK cytotoxic activity, evaluated in the entire PBMC cell population before the immunomagnetic separation (MACS) and in NK cells after MACS, were compared in healthy young and elderly subjects and in patients with SDAT. No differences in spontaneous cytotoxicity were found between healthy elderly subjects (17.5 ± 3.3 L.U./107 cells) and patients with SDAT (17.9 ± 3.6 L.U./107 cells) (Fig. 1 Fig. 2 Fig. 3 ). The data obtained in PBMC before and after MACS were superimposable (19.1 ± 3.8 vs. 18.6 ± 3.7 L.U./107 cells) (Table 2 ). The spontaneous NK cytotoxicity of both groups was also similar to that found in healthy young subjects (17.7 ± 3.4 L.U. 107 cells).A significantly (p < 0.01) reduced suppressive effect on cytotoxic activity (mean percent inhibition of −24%) was demonstrated in patients with SDAT as compared to healthy elderly subjects (mean percent inhibition of −44%) when NK cells and PBMC cells were cultured in the presence of hydrocortisone, 10−6 M (Fig. 1, Table 2). The results obtained in NK and PBMC cells were comparable (Table 2). Moreover, a similar pattern of NK cytotoxic activity (during cortisol incubation), was demonstrated when healthy elders were compared to healthy young subjects (percent inhibition of NK cell activity after cortisol = 42.8 ± 5.1%).NK cytotoxic activity, evaluated in NK cells (Fig. 2) and in the whole PBMC cell population (Table 2), was analyzed in healthy elderly subjects and in patients with SDAT during IL-2 incubation (100 IU/mL/cells). Higher NK and PBMC cell cytotoxic response to IL-2 was found in patients with SDAT (mean percent stimulation of +102%) than in healthy elderly subjects (mean percent stimulation of +75%) (p < 0.001). No difference in cytolytic response during IL-2 incubation was demonstrated when NK cells (Fig. 2) were compared to PBMC cells (Table 2). The modulatory effect of IL-2 was also similar when healthy elderly subjects and healthy young subjects were compared (percent stimulation on NK cell activity after IL-2 = 77.1 ± 3.7%).NK cytotoxic activity measured in NK cells (Fig. 3) and in the entire PBMC cell population (Table 2) was also evaluated in healthy elderly subjects and in patients with SDAT during IFN-β incubation (650 IU). Significantly increased NK and PBMC cell cytotoxic response to IFN-β was demonstrated in patients with SDAT (percent stimulation of +132%) in comparison with healthy elderly subjects (percent stimulation of +88%; p < 0.001). No variations in cytotoxic responses (during IFN-β incubation) were demonstrated when NK (Fig. 3) and PBMC cells (Table 2) were compared. There were also no differences in the modulatory effects of IFN-β on NK cytotoxic activity when healthy elderly subjects were compared to healthy young subjects (percent stimulation of NK cell activity after IFN-β = 86.4 ± 9.3%).Non-parametric Spearman’s correlation coefficient (rs) analysis demonstrated several inverse correlations between NK cell cytotoxic activity and the impairment of cognitive function (evaluated by MMSE) in patients with SDAT. In these patients, there were significant correlations between spontaneous NK cytotoxicity (p = 0.0208) and the percent stimulation of NK cytotoxicity after IL-2 (p = 0.0056) and IFN-β (p = 0.0044) and MMSE (Fig. 4 ); whereas cortisol did not correlate with MMSE (Fig. 4). On the contrary, no correlations were demonstrated between NK activity and the other clinical and biochemical parameters incorporated as independent variables into the multiple regression analysis.Lower basal values of PKC activity of NK cells were found in SDAT patients than in healthy elderly subjects (−33%), even if the differences were not statistically significant (Fig. 5 ).Fig. 5 represents the effects of cortisol (10−6 M), IL-2 (100 IU/mL), and IFN-β (650 IU/mL) on cytosolic Ca2+-dependent PKC activity in healthy elderly subjects and in patients with SDAT. The PKC data are presented as specific activity in nmoL/min./mg protein. No significant variations in PKC activity were measured after cortisol incubation in healthy elderly subjects and in patients with SDAT. On the contrary, IL-2 and IFN-β significantly decreased cytosolic PKC activity in healthy elderly subjects (percent variation of −45% and −47%, respectively). This response was completely lacking in patients with SDAT, where PKC activity remained unchanged after exposure to IL-2 or IFN-β. The enzyme activity in the membrane compartment was unmodified in basal conditions and throughout all treatments (data not shown) when healthy elders and SDAT patients were compared (0.35 ± 0.21 and 0.34 ± 0.21 nmoL/min./mg protein, respectively).The decreased PKC activity after IL-2 and IFN-β in healthy elderly subjects was associated with decreased levels of PKC α and βII isoforms (Fig. 6 ); whereas these isoforms were not influenced by cortisol. PKC βI immunoreactivity also migrated at 80 kDa and was of lower intensity than that for PKC βII, while PKC γ was not expressed in NK cells (data not shown). The quantification of the PKC bands in healthy elderly subjects is reported in Table 3. While cortisol did not modify PKCα and βII levels, IL-2 decreased cytosolic PKCα by 70% and PKCβII by 40% and IFN-β decreased PKCα by 70% and PKCβII by 67%.The basal levels of soluble PKCα and βII in NK cells of SDAT patients were not different from healthy subjects and cortisol, IL-2, and IFN-β treatment did not change isoform levels (data not shown).3DiscussionThe results of the present study indicate that NK cells of patients with SDAT were more sensitive to stimulation of cytotoxic activity during IL-2 and IFN-β exposure than NK cells of healthy elderly subjects. In the latter group, the functional activity of NK cells was similar to that of healthy young subjects. The increased cytokine-mediated NK cytotoxicity in SDAT was also associated with an impaired suppression of NK cell activity by cortisol. These effects on functional activity were evident in both NK and PBMC cells. In fact, with our experimental design we were able clearly to demonstrate that cytotoxic activity, as expressed by immunoseparated NK cells (under basal conditions and after the use of modulators), was similar to that observed in the whole population of PBMC cells. In agreement with previous data [55, 56], the increase in NK cytotoxic response to IL-2 and IFN-β would seem to be specific to SDAT patients, as NK cells exhibited a normal response to cytokines in healthy elders, as well as in patients with dementia of multi-infarct origin. The reduction in the suppressive effect of cortisol on NK cytotoxicity may contribute further to activating the NK cellular compartment in SDAT, and in addition compromise any capacity for physiological suppression of excessive activation. These results reveal some interesting new insights into the involvement of the NK cellular compartment in the neuroimmune hypothesis of SDAT [27, 39, 54–56]. Moreover, the specificity of NK cell dysregulation in SDAT might provide a new biological marker of the disease.In healthy subjects and SDAT patients, the increase in NK cell cytotoxicity with IFN-β exposure is more pronounced by comparison to that found during IL-2 and IFN-γ [55, 56], suggesting that IFN-β should be used in clinical models investigating NK cytotoxic activity [33, 37].In addition to supporting the hypothesis of a linkage between immunity and neuropathology in SDAT [27, 39, 54–56], the evidence of specific NK functional disorders in our patients with SDAT may be of clinical relevance. These observations may provide a useful aid in the early diagnosis and in the understanding of pathogenetic mechanisms, and also introduce new perspectives for therapeutic strategies [38].We investigated the activity and levels of PKC in the NK cells, as this is a specific enzyme involved in the regulation of NK cytotoxic activity toward tumor cells [9]. The basal cytosolic PKC activity was reduced in NK cells derived from patients with SDAT, although the effect was not statistically significant. This observation agrees qualitatively with the reported data of decreased PKC activity in fibroblasts and platelets of SDAT patients [6, 10, 22, 65]. In spite of the reduction in PKC activity, the spontaneous cytotoxic activity of NK cells was similar to that found in healthy elders, suggesting no effect of PKC on the regulation of spontaneous cytotoxic function. On the contrary, PKC seems to be associated mainly with NK activation by cytokines. Following 20-h treatment with IL-2 and IFN-β, the cytosolic PKC activity of NK cells was decreased in healthy elderly subjects but not in SDAT patients, and this decrease was associated with diminished levels of both PKCα and βII isoforms.As mentioned, the activation of PKC leads to exocytosis of preformed cytotoxic granules. It is tempting to speculate that after 20 h of incubation PKC is physiologically down-regulated in healthy old subjects, thus contributing to the extinction of the NK function. On the other hand, this mechanism is suggested to be lacking in SDAT. The down-regulatory process might be altered in the NK cells derived from these patients (and therefore not yet detectable at the end of incubation period selected in our experiments). Another control mechanism might also be envisaged. Experimental data have shown that PKC activation can inhibit IL-2 responses in NK cells and IFN α/β responses in monocytes [52]. This control mechanism may be operant in our system and we might speculate that, in order to activate feed-back inhibition, PKC must be activated and subsequently, metabolized. This could result in decreased PKC activity in healthy elders after long-term exposure to IL-2 and IFN-β. In SDAT patients, where the mechanism of PKC activation of NK might be deficient, as shown in other tissues [6, 10, 22, 65, 66], PKC cannot feed-back cytokine activity efficiently, thereby leading to permanent NK activation. In support of these suggestions, the cytokine-mediated decrease in PKC activity that was observed in healthy elderly subjects may be related to decreased levels of the major calcium-dependent isoforms present (i.e., PKC α and βII), as shown by Western blot analysis. It remains to be established which steps are involved in coupling cytokine activation to PKC signal transduction in NK cells. Obviously it should be recalled that the PKC signal transduction system is not the only one that regulates NK cell activity toward different targets. Other kinases [9, 45, 60]and G protein (PKC-independent) signals [19, 61]may mediate the regulatory action of cytokines.The biochemical events leading to cytokine-dependent mechanisms of NK overactivation in SDAT may be related to the clinical course of the disease and in particular to the development of cognitive derangement. In this context, the spontaneous and IL-2 and IFN-β induced NK cytotoxic responses were inversely correlated with MMSE, suggesting a linkage between cytokine-mediated NK activity and the deterioration of cognitive status in these patients. Moreover, it is well known that IL-2 negatively influences cognitive processes in a dose-dependent manner by reducing cholinergic control of learning and memory [4, 15].Finally, with regard to the neuroimmune hypothesis of SDAT [27, 38, 39, 54], it is tempting to speculate that the NK overactivation [55, 56]might play a role in the development of immune-mediated brain damage in this disease, by means of the interaction of these cells with astrocytes and microglia, as these cells also directly express cytokine receptors [41]. In this context, the neurobiological basis for immune effects on neuropathology and cognitive derangement in SDAT may provide important information for creating strategies to delay the clinical progression of the disease.[58]AcknowledgementsWe wish to thank Dr. Silvia Severgnini for technical support with the immunological procedures; Dr. Teresa Ricciardi for clinical assistance in the Alzheimer’s Centre of our Department and Dr. W.C. 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Immunol.411987269332 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BE9E89E3B9DC04A046D733FF17E3CE41CEACFF4.txt b/test/dataset/in/resources/corpus/Clean_0BE9E89E3B9DC04A046D733FF17E3CE41CEACFF4.txt new file mode 100644 index 0000000..42e4877 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BE9E89E3B9DC04A046D733FF17E3CE41CEACFF4.txt @@ -0,0 +1 @@ +]>MAT1601S0378-5122(01)00241-910.1016/S0378-5122(01)00241-9Elsevier Science Ireland LtdFig. 1Steep mode evaluation of skin tensile strength. Recorded mechanical parameters during variations in the elevation of skin (E, mm) in time (T, s). Ue=elastic (immediate) distension of the skin during the first traction (0.15 s); Uv=visco-elastic deformation until the maximum distension (MD) of the skin is reached at completion of the first traction (5 s); RD=residual deformation of the skin at the end of the first cycle (10 s).Fig. 2Progressive mode evaluation of skin strength. Typical recording of the elevation (E, mm) of skin during a progressive increase (25 mbar/s) in a 20 s suction-on (S, mbar) until the maximum distension (MD) of the skin obtained under the 500 mbar suction, followed by a 20 s suction-off decrease at the same rate. Hysteresis (HY) is defined by the area delimited by the suction–relaxation curves.Table 1Demographic dataMenopausal womenNumberAgeMenopause ageDuration of HRTUntreated6059.3±6.850.6±2.90HRT6057.9±7.149.9±2.37.7±4.6Time is expressed in years (mean±S.D.).Table 2Values of BMD and tensile strength of the skin in the untreated menopausal groupMethod/parameterMean±S.D.CV (%)MedianRangeBone mass densityHip0.799±0.136170.7850.546–1.113Lumbar spine0.872±0.144170.8480.598–1.233Femoral neck0.681±0.113170.6620.504–0.964Skin, steep modeMD (μm)357.5±131.637385100–500BE (%)67.4±15.82364.014–100VER (%)63.2±59.69436.76–240Skin, progressive modeMD (μm)363.0±127.335395100–610BE (%)40.5±17.94433.70–81.8HY (AU)2023±549.72720221075–3292Abbreviations: MD, maximum distension; BE, biologic elasticity; VER, visco-elastic ratio; HY, hysteresis; AU, arbitrary units.Table 3Values of BMD and tensile strength of the skin in the HRT-treated menopausal groupMethod/parameterMean±S.D.CV (%)MedianRangeBone mass densityHip0.838±0.136160.8520.412–1.136Lumbar spine0.932±0.161170.9480.620–1.365Femoral neck0.734±0.117160.7040.567–0.959Skin, steep modeMD (μm)272.4±153.25620570–630BE (%)77.8±14.51981.745.9–100VER (%)105.0±68.465110.113.9–270Skin, progressive modeMD (μm)277.6±147.953220100–630BE (%)52.9±18.43551.920.0–86.2HY (AU)1560±600.2381378989–3714Abbreviations: MD, maximum distension; BE, biologic elasticity; VER, visco-elastic ratio; HY, hysteresis; AU, arbitrary units.Table 4Mann–Whitney U-test assessing the difference between the untreated and HRT-treated menopausal groupsMethod/parameterPBone mass densityHipNSLumbar spineNSFemoral neckNSSkin, steep modeMD0.0037BE0.0011VER0.0032Skin, progressive modeMD0.0018BE0.0013HY<0.0001Abbreviations: MD, maximum distension; BE, biologic elasticity; VER, visco-elastic ratio; HY, hysteresis; NS, not significant.Table 5Untreated menopausal group. Coefficient of correlation (r) assessing linear correlations between BMD values and parameters of skin tensile strengthSkin tensile strengthBMD valuesHipFemoral neckLumbar spineSteep modeMD−0.0430.140−0.084BE0.429**0.351*0.200VER−0.276−0.395*0.057Progressive modeMD0.0810.343*−0.0003BE0.2020.2500.183HY0.0400.1410.036(*P<0.05, **P<0.01). Abbreviations: MD, maximum distension; BE, biologic elasticity; VER, visco-elastic ratio; HY, hysteresis.Table 6HRT menopausal group. Coefficient of correlation (r) assessing linear correlations between BMD values and parameters of skin tensile strengthSkin tensile strengthBMD valuesHipFemoral neckLumbar spineSteep modeMD0.1030.394−0.030BE0.2050.4460.168VER−0.118−0.327−0.151Progressive modeMD0.0310.204−0.040BE0.338*0.620*0.385*HY−0.1060.003−0.123(*P<0.05). Abbreviations: MD, maximum distension; BE, biologic elasticity; VER, visco-elastic ratio; HY, hysteresis.Comparative effect of hormone replacement therapy on bone mass density and skin tensile propertiesG.EPiérard*SVanderplaetsenCPiérard-FranchimontDepartment of Dermatopathology, Belgian SSTC Research Center 5596, University Medical Centre Sart Tilman, B-4000 Liege, Belgium*Corresponding author. +32-4-3662408; fax: +32-4-3662976AbstractObjectives: connective tissues constitutive of skin and bones are affected during the climacteric. Hormone replacement therapy (HRT) can help mitigate their atrophy. The aim of this study was to compare the HRT effect on the skin tensile properties and bone mass density. Methods: a total of 120 postmenopausal women (60 untreated, 60 receiving HRT) were enrolled in the study. Skin tensile properties were assessed on the volar forearm using a computerized suction device. A 500 mbar suction was applied through a 4-mm diameter hollow probe. Two operating modes were applied using a steep and a progressive suction, respectively. BMD was measured on the hip, femoral neck and lumbar spine using dual X-ray absorptiometry. Results: in both groups of women skin elasticity was correlated with BMD. HRT significantly reduced the climacteric-associated decline in skin elasticity. A trend in better preserved BMD was also found in these women without, however, reaching significance. Conclusions: it is concluded that measures of the skin tensile properties can be sensitive enough to disclose HRT efficacy upon connective tissue atrophy. Any decrease in skin elasticity during the climacteric should prompt to perform a BMD assessment.KeywordsHormone replacement therapyBone mass densitySkin tensile property1IntroductionBoth skin and bones are collagen-rich structures particularly affected by the climacteric. Indeed, dermal atrophy and osteoporosis are not unfrequently found in the postmenopausal years. Some studies have addressed these biological aspects and their relationships [1–10]. The positive effect of hormone replacement therapy (HRT) to prevent such changes has been shown repeatedly [11–19].It is acknowledged that a relationship exists between the biomechanical properties of connective tissues and their molecular composition and structural organizations [20,21]. In these respects, non-invasive assessments of bone and skin atrophy are useful in clinical practice [22–26]. The measurement of bone mass density (BMD) and skin tensile characteristics are convenient because they are sensitive and reproducible.The aim of the present study was to compare the effect of HRT on BMD and skin tensile strength in postmenopausal women.2Patients and methodsA total of 120 healthy postmenopausal Causasian women were enrolled in this study (Table 1). They were allocated to two groups, namely 60 untreated women and 60 women receiving oral or transdermal HRT. The medical files documented low estradiol level and high serum FSH in untreated women and before HRT initiation in the others. Presence of osteoporotic crusched vertebrae was the exclusion criterion.A computerized suction device (Cutometer SM 474, C+K Electronic, Cologne, Germany) was used to measure the skin tensile properties. Briefly, the device consists of a hand-held hollow probe fitted to a spring, which ensures that the head is applied to the skin at constant pressure. The main unit consists of a vacuum pump that generates a negative pressure up to 500 mbar. Measurements of the skin deformation inside the suction head are monitored by an optical measuring unit with an accuracy of 0.01 mm [27]. In the present study, the probe had a 4-mm diameter aperture. Measurements were taken from the mid volar aspect of the volar forearm in order to minimize the influence of photoageing on the results. Two modalities of measurements bound to distinct graphic representations were used, namely the steep and the progressive suction modes.The steep suction mode was used with one cycle 500 mbar suction for 5 s followed by a 5-s relaxation period. The skin deformation was plotted as a function of time (Fig. 1). The immediate elastic deformation of the skin (Ue, μm) corresponding to the steep linear part of the curve was computed after 0.15 s of suction. The delayed visco-elastic part of the skin deformation under suction (Uv, μm) corresponded to the mechanical creep between 0.15 and 5 s. The maximum distension of skin (MD, μm) was obtained after 5 s of 500 mbar suction and the residual deformation (RD, μm) at completion of the test cycle. The biologic elasticity (BE, %) was derived following BE=102(MD−RD)/MD. The visco-elastic ratio during the suction phase (VER, %) was calculated after VER=Uv/Ue.The progressive suction mode applied an increasing negative pressure at a linear rate of 25 mbar/s for 20 s, followed by a release of the depression at the same rate (Fig. 2). It was recorded as a stress/strain relationship. MD and BE were measured as in the steep suction mode. In addition, hysteresis (HY) corresponding to the area delimited by the traction and relaxation curves was measured in arbitrary units (AU) using image analysis of the stress/strain graphs (MOP Videoplan Kontron, Eching, Germany).BMD was measured by dual X-ray absorptiometry (DXA) of the trabecular bone of the hip, femoral neck and L1–L4 lumbar spine.Magnitude, spread and symmetry of the data were assessed using the Shapiro–Wilk test. The distribution of the values of each parameter was characterized by the range, mean and standard deviation (S.D.). The Mann–Whitney U-test was used to compare the medians from all parameters between untreated and HRT-recipient menopausal women. The coefficient of variation (CV, %) was calculated following CV=(102×S.D.)/M. The coefficient of correlation (r) was applied to evaluate the relationships between BMD and each biomechanical parameter. A P value lower than 0.05 was considered statistically significant.3ResultsAn overview of the data is presented in Tables 2 and 3. The range in BMD values was quite large at each of the three test sites. The interindividual variability in the skin tensile strength was large as well. Significant differences were yielded between the untreated and the HRT-recipient menopausal women with regard to all the skin tensile strength parameters (Table 4). A decrease in MD and HY, and an increase in BE and VER were present in the HRT group compared with the untreated women. By contrast, no difference was disclosed between the BMD values of the untreated and HRT groups.Significant linear correlations were found in untreated menopausal women between both the hip and femoral neck BMD and some of the skin tensile parameters (Table 5). Indeed, positive relationships were found between these BMD and BE given by the steep suction mode. A negative correlation was yielded between BMD values of femoral neck and VER in the steep mode. Another positive correlation was present between femoral neck BMD and MD given by the progressive suction mode. No correlations were disclosed between on the one hand any of the BMD and on the other hand BE and HY in the progressive mode and MD in the steep mode.Significant positive linear correlations were found in the HRT-recipient menopausal women between each set of BMD evaluations and BE given by the progressive suction mode (Table 6). No significant correlations were found between any of the other skin biomechanical parameters and the BMD values.4DiscussionIn an earlier study, we reported for the first time the relationship between BMD and the skin tensile properties as assessed by a computer-assisted suction method [28]. In that overall evaluation conducted in a rather heterogeneous population at risk of developing osteoporosis, the viscoelastic properties of skin were altered when BMD values were abnormal. Particularly BE decreased in parallel with bone atrophy. At variance with the earlier study, the present was focused on menopausal women receiving or not HRT.It is acknowledged that the overall elastic properties of skin decrease with age [21,27,29]. In addition, age-related bone and skin atrophies are well documented and the negative impact of the climactric upon such an evolution is beyond doubt [1,7,8,10,12,16]. As a counterpart, the beneficial effect of HRT to alleviate the trend in osteoporosis and cutaneous climacteric ageing is generally acknowledged [9,17–19,23,24,26]. In the present study, higher BMD values were found in HRT-treated patients, although the differences with unteated women did not reach significance. This contrasts with other longitudinal studies demonstrating the beneficial effect of HRT in the prevention of the climacteric bone atrophy.Skin slackness developing after menopause is likely related to several factors, including the declines in skin collagen content and dermal thickness [23,24]. Some studies have shown that HRT could limit the extent of skin collagen loss in the initial postmenopausal years [12,17,23]. Another factor influencing skin slackness deals with the elastic fibre alteration with estrogen deprivation. However, the possibility of their repair during HRT is controversial [6,13]. The present data confirm the trend towards a better preserved skin elasticity in women receiving HRT. It is noteworthy that the interindividual variations in BE and VER were higher in the untreated group than in the HRT group. Hence, HRT appears to limit, at least to some extent, the range of variations in BE and VER, thus seemingly limiting one facet of intrinsic ageing. A negative correlation was found between the femoral neck BMD and VER. This relationship was earlier reported [28]. A positive correlation between BMD at all locations and BE in the steep and progressive suction modes was earlier shown [28]. Such a result is confirmed by the present data, except for the spine BMD and BE in the steep mode.In conclusion, earlier observations about the relationship between bone and skin are supported by the present findings. We now point out that the beneficial effect of HRT on skin elasticity may prevail on the effects on bone or at least can be detected earlier using the current non-invasive methods. In addition, there is some evidence that the assessment of the skin tensile properties may predict the effect of HRT on osteoporosis prevention. The present study does not promote measurements of the skin tensile properties as a surrogate of BMD determination. However, it appears that a decrease in skin elasticity may represent a clue inciting BMD assessment. Further studies are needed to assess the value of measuring skin tensile properties in predicting osteoporosis.AcknowledgementsWe thank Professor U. Gaspard from the Gynecology Department for his helpful comments.References[1]M.M.BlackS.ShusterE.BottomsOsteoporosis, skin collagen, and androgensBr. Med. J.41970773774[2]M.BrincatC.F.MonizJ.W.W.StuddA.J.DarbyA.MagosD.CooperSex hormones and skin collagen content in postmenopausal womenBr. Med. J.287198313371338[3]M.BrincatC.F.MonizJ.W.W.StuddA.J.DarbyA.MagosG.EmburyE.VersiLong-term effects of the menopause and sex hormones on skin thicknessBr. J. Obstet. Gynaecol.921985256259[4]M.BrincatC.F.MonizS.KabalanE.VersiT.O'DowdA.L.MagosJ.MontgomeryJ.W.W.StuddDecline in skin collagen content and metacarpal index after the menopause and its prevention with sex hormone replacementBr. J. Obstet. Gynaecol.941987126129[5]L.NilasC.ChristiansenRates of bone loss in normal women: evidence of accelerated trabecular bone loss after the menopauseEur. J. Clin. Invest.181988529534[6]J.L.BologniaI.M.BravermanM.E.RousseauP.M.SarrelSkin changes in menopauseMaturitas111989295304[7]D.ChappardC.AlexandreJ.M.RobertG.RiffatRelationships between bone and skin atrophies during agingActa. Anat.1411991239244[8]C.Castelo-BrancoF.PonsE.GratacosA.FortunyJ.A.VanrellJ.Gonzalez-MerloRelationship between skin collagen and bone changes during ageingMaturitas181994199206[9]G.E.PiérardThe quandary of climacteric skin ageingDermatology1931996273274[10]S.E.WhitmoreM.A.LevineRisk factors for reduced skin thickness and bone density. Possible clues regarding pathophysiology, prevention, and treatmentJ. Am. Acad. Dermatol.381998248255[11]B.EttingerH.K.GenantC.E.CannLong-term estrogen replacement therapy prevents bone loss and fracturesAnn. Intern. Med.1021985319324[12]M.BrincatE.VersiC.F.MonizA.MagosJ.TraffordJ.W.W.StuddSkin collagen changes in postmenopausal women receiving different regimens of estrogen therapyObstet. Gynecol.701987123127[13]R.PunnonenP.VaajalahtiK.TeisalaLocal oestriol treatment improves the structure of elastic fibres in the skin of postmenopausal womenAnn. Chir. Gynaecol.7619873941[14]T.StormG.H.ThamsborgT.SteinickeH.K.GenantO.H.SorensenEffect of intermittent cyclical etidronate therapy on bone mass and fracture rate in women with postmenopausal osteoporosisNew Engl. J. Med.322199012651271[15]C.Castelo-BrancoM.DuranJ.Gonzalez-MerloSkin collagen changes related to age and hormone replacement therapyMaturitas151992113119[16]E.F.HollandJ.W.StuddJ.P.ManseelA.LeatherA.J.BaileyChanges in collagen composition and cross-links in bone and skin of osteoporotic postmenopausal women treated with percutaneous estradiol implantsObstet. Gynecol.831994180183[17]R.MaheuxF.NaudM.RiouxR.GrenierA.LemayJ.GuyM.LangevinA randomized double-blind, placebo-controlled study on the effect of conjugated estrogens on skin thicknessAm. J. Obstet. Gynecol.1701994642649[18]M.GibaldiHormone replacement therapy: estrogen after menopausePharmacotherapy161996366375[19]C.B.HammondMenopause and hormone replacement therapy: an overviewObstet. Gynecol.8719962S15S[20]H.OxlungRelationship between the biomechanical properties, composition and molecular structure of connective tissuesConnect. Tissue Res.1519866572[21]G.E.PiérardEEMCO guidance of the in vivo assessment of tensile functional properties of the skin. Part 1: relevance to the structures and ageing of the skin and subcutaneous tissuesSkin Pharmacol. Appl. Physiol.121999352362[22]S.HagiwaraS.O.YangC.GluerE.BendavidH.K.GenantNon-invasibe bone mineral density measurement in the evaluation of osteoporosisRheum. Dis. Clin. North Am.201994651669[23]G.E.PiérardC.LetaweA.DowlatiC.Piérard-FranchimontEffect of hormone replacement therapy for menopause on the mechanical properties of skinJ. Am. Geriatr. Soc.431995662665[24]A.CallensL.VaillantP.LecomteM.BersonY.GallG.LoretteDoes hormonal aging exists? A study of the influence of different hormone therapy regimens on the skin of postmenopausal women using non-invasive measurement techniquesDermatology1931996289294[25]F.HenryC.Piérard-FranchimontG.CauwenberghG.E.PiérardAge-related changes in facial skin contours and rheologyJ. Am. Geriatr. Soc.451997220222[26]C.Piérard-FranchimontF.CornilJ.DehavayF.Deleixhe-MauhinB.LetotG.E.PiérardClimacteric skin ageing of the face. A prospective longitudinal comparative trial on the effect of oral hormone replacement therapyMaturitas3219998793[27]A.B.CuaK.P.WilhelmH.MaibachElastic properties of human skin: relation to age, sex and anatomical regionArch. Dermatol. Res.2821990283288[28]Piérard GE, Piérard-Franchimont C, Vanderplaetsen S, Franchimont N, Gaspard U, Malaise M. Relationships between bone mass density and tensile properties of skin in women. Eur J Clin Invest, in press.[29]C.EscoffierJ.de RigalA.RochefortR.VasseletJ.L.LévêqueP.G.AgacheAge-related mechanical properties of human skin: an in vivo studyJ. Invest. Dermatol.931989353357 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BF0C19A0F8A042FDA537D63BC3FC62AAD27F9AD.txt b/test/dataset/in/resources/corpus/Clean_0BF0C19A0F8A042FDA537D63BC3FC62AAD27F9AD.txt new file mode 100644 index 0000000..457814c --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BF0C19A0F8A042FDA537D63BC3FC62AAD27F9AD.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 01002010.1093/gerona/57.1.M45 Journal of Gerontology: Medical Sciences Prevalence and Incidence of Cardiovascular Disease in 1160 Older Men and 2464 Older Women in a Long-term Health Care Facility Aronow Wilbert S. a b Ahn Chul c Gutstein Hal a aHebrew Hospital Home, Bronx, New York bDepartment of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, New York cDivision of Clinical Epidemiology, University of Texas Medical School at Houston, Houston, Texas Wilbert S. Aronow, CMD, Department of Medicine, New York Medical College, 23 Pebble Way, New Rochelle, NY 10804 E-mail: WSAronow@aol.com. 1 1 2002 57 1 M45 M46 27 2 2001 12 2 2001 The Gerontological Society of America 2002 Background. We report the prevalence and incidence of cardiovascular disease in older men and women in a long-term health care facility. Methods. The prevalence of hypertension, chronic atrial fibrillation, pacemaker rhythm, coronary artery disease (CAD), thromboembolic stroke, and symptomatic peripheral arterial disease (PAD) and the incidence of new coronary events, thromboembolic stroke, and congestive heart failure (CHF) were investigated in 1160 men, mean age 80 ± 8 years, and in 2464 women, mean age 81 ± 8 years, in a long-term health care facility. Mean follow-up was 46 ± 30 months. Results. The prevalences of hypertension, pacemaker rhythm, CAD, and thromboembolic stroke were similar in men and women. The prevalence of atrial fibrillation was higher in men (16%) than in women (13%; p = .019). The prevalence of PAD was higher in men (32%) than in women (26%; p = .0001). At the 46-month follow-up, the incidences of new coronary events, thromboembolic stroke, and CHF were similar in men and women. Conclusions. Older men and women in a long-term health care facility have a high prevalence and incidence of cardiovascular disease. The prevalences of hypertension, pacemaker rhythm, CAD, and thromboembolic stroke and the incidences of new coronary events, thromboembolic stroke, and CHF were similar in men and women. However, the prevalences of atrial fibrillation and of PAD were higher in men than in women. hwp-legacy-fpage M45 hwp-legacy-dochead RESEARCH ARTICLE Decision Editor: John E. Morley, MB, BCh CARDIOVASCULAR disease is the most common cause of death in older persons in a long-term health care facility (1). We report data from a prospective study investigating the prevalence of cardiovascular disease and the incidence of cardiovascular disease at 46-month mean follow-up in 1160 older men and 2464 older women in a long-term health care facility. Methods In a prospective study, we investigated the prevalence and incidence of cardiovascular disease in 1160 men, mean age 80 ± 8 years (range 60 to 100 years), and in 2464 women, mean age 81 ± 8 years (range 60 to 103 years), in a long-term health care facility. The 3624 persons were unselected persons 60 years of age and older who were not terminally ill at admission. Of the 3624 persons, 2162 (60%) were white, 950 (26%) were African American, 503 (14%) were Hispanic, and 9 (<1%) were Asian. Follow-up was 46 ± 30 months (range 1 to 196 months). All 3624 persons had 12-lead electrocardiograms with 1-minute rhythm strips taken at entry into the study, routinely every year, and whenever clinically indicated. Atrial fibrillation was diagnosed from the baseline 1-minute rhythm strips of the electrocardiograms (2). The prevalence of a pacemaker rhythm was also investigated in all 3624 persons (3). Systemic hypertension was diagnosed according to the criteria of the Sixth Joint National Committee (JNC VI) Report on the Detection, Evaluation, and Treatment of Hypertension (4). Coronary artery disease (CAD) was diagnosed if the person had a documented clinical history of myocardial infarction or electrocardiographic evidence of Q-wave myocardial infarction (n = 1483) or typical angina pectoris without previous myocardial infarction (n = 38). Sixteen of the 38 persons with typical angina pectoris without prior myocardial infarction also had coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty. New coronary events were diagnosed if the person developed nonfatal or fatal myocardial infarction or sudden cardiac death. Myocardial infarction was diagnosed as previously described (5). Sudden cardiac death was defined as an unexpected cardiac death in a person with heart disease found dead within 1 hour of being clinically stable (6). Persons with fatal primary ventricular fibrillation documented by electrocardiogram were classified as having sudden cardiac death. All coronary events were reviewed by the senior investigator with the physician taking care of the person. Previous thromboembolic stroke was diagnosed by a neurologist (2). New thromboembolic stroke was diagnosed by a neurologist if a focal neurological event occurred suddenly but without prolonged unconsciousness, nuchal rigidity, pronounced leukocytosis, or bloody spinal fluid (2). The focal neurological signs of ischemic stroke were explained by loss of function in a restricted area of the brain corresponding to a particular vascular territory (2). Ischemic stroke was also confirmed by computerized axial tomography in 766 of 787 persons (97%) with thromboembolic stroke. Symptomatic peripheral arterial disease (PAD) was diagnosed if the person had a documented history of surgery for PAD or if the person had ischemic pain at rest, ulceration or gangrene in an extremity, intermittent claudication, numbness, coldness, cyanosis, or pallor in an extremity, or trophic changes with dry, scaly, and shiny atrophic skin, diminished hair growth, thickened, brittle toenails, or subcutaneous atrophy in an extremity associated with absent or weak arterial pulses (7). Congestive heart failure (CHF) was diagnosed if two criteria were satisfied: (1) pulmonary basilar rales were heard by at least two physicians, including the senior investigator, and (2) pulmonary vascular congestion was present on the chest x-rays interpreted by both an experienced radiologist and the senior investigator (8). For analyses comparing the two groups of men versus women, chi-square tests were used. Results Table 1 shows the prevalences of hypertension, atrial fibrillation, pacemaker rhythm, CAD, prior thromboembolic stroke, and PAD in older men and in older women and, at the 46-month mean follow-up, the incidences of new coronary events, thromboembolic stroke, and CHF. Table 1 also lists levels of statistical significance. Discussion The data from the present prospective study showed that the prevalence of hypertension was 60% in women (39% with isolated systolic hypertension and 21% with systolic and diastolic hypertension), mean age 81 years, and 57% in men (37% with isolated systolic hypertension and 20% with systolic and diastolic hypertension), mean age 80 years, in a long-term health care facility. This difference is not significant. The prevalence of a pacemaker rhythm was 5% in the men and 5% in the women in this study. However, the prevalence of chronic atrial fibrillation was significantly higher in the older men (16%) than in the older women (13%) (p = .019). The higher prevalence of chronic atrial fibrillation in older men than in older women was present at all ages ≥60 years of age. CAD is the leading cause of death of older persons in a long-term health care facility (1). The data from the present study showed that the prevalence of CAD was 43% in men and 41% in women and the incidence of new coronary events at the 46-month follow-up was 46% in men and 44% in women. These differences are not significant. There was no significant difference in prevalence or incidence of angina pectoris or myocardial infarction between older men and older women. Our study also showed that the prevalence of prior thromboembolic stroke was 33% in men and 31% in women and the incidence of new thromboembolic stroke was 23% in men and 21% in women. These differences are not significant. The incidence of CHF was also not significant between older men (29%) and older women (26%). However, older men had a significantly higher prevalence of symptomatic PAD (32%) than older women (26%; p = .0001). The significantly higher prevalence of current cigarette smoking in older men (21%) than in older women (9%; p < .0001) contributed to the significantly higher prevalence of symptomatic PAD in older men than in older women. Table 1. Prevalence and Incidence of Cardiovascular Disease in Older Men and Women in a Long-term Health Care Facility Men (n = 1160) Women (n = 2464) Variable No. % No. % p Value Prevalence of hypertension 665 57 1471 60 NS Prevalence of chronic atrial fibrillation 181 16 314 13 .019 Prevalence of pacemaker rhythm 61 5 125 5 NS Prevalence of coronary artery disease 502 43 1019 41 NS Prevalence of thromboembolic stroke 370 32 761 31 NS Prevalence of peripheral arterial disease 374 32 637 26 .0001 Incidence of coronary events 529 46 1086 44 NS Incidence of thromboembolic stroke 268 23 519 21 NS Incidence of congestive heart failure 335 29 643 26 NS ≥1 of the above 991 85 2078 84 NS Note: NS = not significant. 1 Aronow WS, 2000. Clinical causes of death of 2372 older persons in a nursing home during 15-year follow-up. J Am Med Dir Assoc.1:95-96. 2 Aronow WS, Ahn C, Gutstein H, 1996. Prevalence of atrial fibrillation and association of atrial fibrillation with prior and new thromboembolic stroke in older patients. J Am Geriatr Soc.44:521-523. 3 Aronow WS, 1991. Correlation of arrhythmias and conduction defects on the resting electrocardiogram with new cardiac events in 1,153 elderly patients. Am J Noninvas Cardiol.5:88-90. 4 Joint National Committee1997. The Sixth Report of the Joint National Committee on the Detection, Evaluation, and Treatment of High Blood Pressure (JNC VI). Arch Intern Med.157:2413-2444. 5 Aronow WS, 1987. Prevalence of presenting symptoms of recognized acute myocardial infarction and of unrecognized healed myocardial infarction in elderly patients. Am J Cardiol.60:1182 6 Roberts WC, 1986. Sudden cardiac death: definitions and causes. Am J Cardiol.57:1410-1413. 7 Aronow WS, Sales FF, Etienne F, Lee NH, 1988. Prevalence of peripheral arterial disease and its correlation with risk factors for peripheral arterial disease in elderly patients in a long-term health care facility. Am J Cardiol.62:644-646. 8 Aronow WS, Ahn C, Kronzon I, 1999. Comparison of incidences of congestive heart failure in older African-Americans, Hispanics, and whites. Am J Cardiol.84:611-612. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BF103D7E74AF2EBAB8CFC4582AA9D106F5232FC.txt b/test/dataset/in/resources/corpus/Clean_0BF103D7E74AF2EBAB8CFC4582AA9D106F5232FC.txt new file mode 100644 index 0000000..7250d69 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BF103D7E74AF2EBAB8CFC4582AA9D106F5232FC.txt @@ -0,0 +1 @@ + geronb J Gerontol B Psychol Sci Soc Scigeronb The Journals of Gerontology Series B: Psychological Sciences and Social Sciences J Gerontol B Psychol Sci Soc Sci 1079-5014 1758-5368 Oxford University Press S15410.1093/geronb/63.3.S154 Journal of Gerontology: Social Sciences Routine Assistance to Parents: Effects on Daily Mood and Other Stressors Savla Jyoti Almeida David M. Davey Adam Zarit Steven H. 1Department of Human Development and Center for Gerontology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia. 2Department of Human Development and Family Studies, Pennsylvania State University, University Park. 3College of Health Professions, Temple University, Philadelphia, Pennsylvania. Address correspondence to Jyoti Savla, Center for Gerontology, Virginia Polytechnic and State University, 237 Wallace Hall (0426), Blacksburg, VA 24061. E-mail: JSavla@vt.edu 5 2008 63 3 S154 S161 22 1 2008 15 8 2007 Copyright 2008 by The Gerontological Society of America 2008 Objectives. The present study examined the association of providing assistance to older parents amid everyday circumstances and short-term psychological consequences for adult children providing assistance. Methods. We explored this association using 824 daily diary interviews of 119 adult children providing assistance in the National Study of Daily Experiences by using a left-censored random effects tobit regression model that accounted for the clustered data and floor effects in reported psychological distress. Results. Psychological distress was higher on days adult children provided assistance to their parent (b = 0.88, p <.05) even after we controlled for situational variables such as time spent on daily paid work, time spent on leisure activities, and assistance provided to individuals other than parents. Demographic and psychosocial variables such as having resident children (b = 2.14, p <.01), less education (b = −0.54, p <.05), and neuroticism (b = 2.08, p <.05), also predicted daily psychological distress. Discussion. Even after we controlled for within-person (daily situational variables) and between-person factors (background characteristics), the act of providing assistance itself had immediate associations with daily mood for helpers, particularly for those with fewer resources and greater demands on time. Feasibility and success of programs that provide respite and relief services to older adults and their children should be assessed in light of daily living. Routine assistance Daily diary Stress proliferation Caregiving hwp-legacy-fpage S154 hwp-legacy-dochead RESEARCH ARTICLE VARIATION in assistance between adult children and their older parents has emerged as an important topic in social gerontology (e.g., Davey, Janke, & Savla, 2005; Pillemer & Suitor, 2006). Whereas studies of continuous care to chronically ill family members can contribute to researchers' general understanding of care-related stress and its psychological consequences on the younger generations providing assistance, most of these studies are specific to certain types of care experiences (e.g., for Alzheimer's patients, stroke patients) and focus on intense caregiving. This focus has provided an important, but limited, perspective on the range of possible outcomes (Ory, Hoffman, Yee, Tennstedt, & Schulz, 1999). There is growing recognition that family members are involved in a range of activities: from infrequent to daily provision and from casual routine assistance to very intensive care for family members that could still affect the daily lives of people providing assistance (e.g., Szinovacz & Davey, 2007). Furthermore, a preponderance of the literature has relied on retrospective accounts of assistance that are often collected over a relatively long period of time, covering weeks, months, and years. As a result, these accounts are likely to be confounded by other events that happened during the time period. This study examined the more immediate impact of routine assistance against the context of daily living and its cumulative effects over time for adult children providing assistance. Understanding of daily stressors and their immediate and cumulative influence over time has benefited from developments in daily diary designs in which repeated measures are collected from individuals during their daily lives (Almeida, 2005; Bolger, Davis, & Rafaeli, 2003). In this design, individuals reported regarding a number of everyday events, behaviors, symptoms, and emotional states to capture the ongoing experiences in the natural context of daily living (Larson & Almeida, 1999). Unlike traditional designs that require respondents to recall experiences over long time frames, this design alleviates memory distortions, especially those related to current emotional state, and improves accuracy of recall. But the most valuable feature of diary methods is that they allow assessment of within-person processes and permit researchers to shift their focus from considering mean levels of stressors and well-being in a group of individuals to charting the day-to-day fluctuations in stress and well-being within an individual himself or herself (Reis & Gable, 2000). Because individuals serve as their own controls, investigators can now examine how daily provision of assistance to a parent is associated with changes in individuals' own well-being from one day to the next. For example, research has suggested that competing roles may increase psychological distress among caregivers (e.g., Murphy et al., 1997). Most of these studies have used a between-person design, comparing one caregiver to another. These studies could be confounded by within-person differences, such as amount of time spent on competing roles or experience of poor health or physical ailment on a day when assistance is provided. In contrast, in the present study, we were able to test whether psychological distress is higher on days when adult children provide assistance than on days they do not. Similarly, between-person designs have found that higher education may buffer helpers from psychological distress. Our within-person design, however, allowed us to determine the buffering effect of education on a day when assistance is provided versus one when it is not, as well as to compare helpers on other potentially important background characteristics. Stress Proliferation on Everyday Living Providing routine assistance to an older parent has salient psychological consequences such as increased distress and burden for the adult child providing help (e.g., Aneshensel, Pearlin, Mullan, Zarit, & Whitlatch, 1995; Antonucci, Akiyama, & Lansford, 1998; Marks, 1998; Walen & Lachman, 2000). Many researchers believe that this distress and burden is partly due to the impingement of the helping role on other everyday roles and experiences, which makes up the structural underpinning of stress spillover or proliferation (Pearlin, 1989; Pearlin, Aneshensel, & LeBlanc, 1997). According to the stress process model (Aneshensel et al., 1995), individuals bear multiple social roles; for instance, they may simultaneously be breadwinners, parents, employees or employers, members of voluntary organization, and so forth. Each of these roles imposes time commitments, responsibilities, and obligations. Many of these roles have been a part of the daily plan of an individual for a long time and have been accommodated into the flow of daily living. The helping role emerges after these primary roles have already been acclimatized (Pearlin, Mullan, Semple, & Skaff, 1990). Initially, the demands of providing routine assistance to a parent may be minimal and sporadic; however, they can continue to grow over time, making reordering of priorities and reallocating energies increasingly difficult. It is this feature of the helping role that contributes to stress proliferation, wherein the new role imposes demands on one's time and energy and requires restructuring and juggling of the primary roles in daily life, making it particularly challenging and eventually undermining the health and well-being of the individual providing routine assistance to parents (Pearlin, 1989). Extant research supporting this theory has found that whether providing help is situation specific and occasional or repetitive and chronic, it is still powerful enough to disrupt an array of roles, activities, and relationships that are only proximally related to the helping role. Using longitudinal data, McKinlay, Crawford, and Tennstedt (1995) found that providing assistance exerted the greatest toll on an individual's personal life, followed by family life and employment. They found that providing assistance was particularly stressful for those who had multiple responsibilities and for those who spent less time on themselves. Murphy and her colleagues (1997) found that role overload was highest for women helpers with multiple roles of parent or worker. At the same time, resentment in the helping role was highest for those women who had fewer roles apart from providing help, those who had to quit work to fulfill their role, and those without a partner. More recently, Stephens, Townsend, Martire, and Drule (2001) examined role conflict experienced by 278 helping women who simultaneously played the role of mother, wife, and employee. Their results suggested that part of the stress that these women experienced was due to conflicts between the helping role and the other roles they were playing. Situational and Background Characteristics Although we expect to find evidence of stress proliferation in the everyday lives of all helpers, we believe that the nature and extent of exposure and reactivity to stress will vary with the background and situational characteristics of the person involved. For instance, the social and economic characteristics of helpers as well as the possession of resources from which they can draw are important covariates to consider. We consider several important ones in this article. Adult children's age is an important predictor of the provision of routine assistance. Older adult children give more support than younger adult children (Ikkink, van Tilburg, & Knipscheer, 1999; Wong, Capoferro, & Soldo, 1999) and might have fewer conflicting demands. Although the effect of adult children's gender on routine exchanges is not as well understood (Pinquart & Sörensen, 2006), it is quite clear that women, in particular daughters, spend more time providing assistance to their older parents than do adult sons (Campbell & Martin-Matthews, 2003). Household structure also plays an important role in the determination of exchanges between the generations and vulnerability to stressors. An adult child's marital status is an important aspect of intergenerational exchanges, wherein unmarried daughters are more likely to engage in an exchange of household assistance than married siblings for whom the opportunity cost of providing assistance is higher (Couch, Daly, & Wolf, 1999). Moreover, the presence of minor children in married-couple households leads to increased time spent in domestic work, reduced time in the labor market, decreased monetary transfers to parents, and increased role overload (Ikkink et al., 1999). Economic theories also suggest that the educational or financial status of adult children facilitates the provision of assistance to older parents. For instance, empirical studies (Couch et al., 1999; Johnson, 2008) have found that siblings with little education or earning lower wages provide hands-on assistance to older parents, whereas their higher earning counterparts provide financial resources or use paid services. We therefore expect that adult children with lower education may be more distressed on days when assistance is provided. Finally, several researchers have asserted that personal dispositions interact with stressful situations in determining individuals' own appraisals of a stressor (Ben-Porath & Tellegen, 1990; Costa, Somerfield, & McCrae, 1996). Studies of personality traits have shown that neuroticism predicts increased exposure and lowered adjustment to interpersonal daily stressors (Bolger & Zuckerman, 1995; Mroczek & Almeida, 2004). Perceived mastery and control, in contrast, are known to buffer the emotional effects of chronic daily stressors (Lachman & Weaver, 1998; Pearlin & Schooler, 1978; Yates, Tennstedt, & Chang, 1999). We therefore expect that adult children with higher mastery and lower scores on the neuroticism trait will experience less significant psychological distress on days when assistance is provided to parents. Whereas some of the factors listed above increase one's vulnerability to stressors, others may serve as protective factors. Study Purpose, Hypotheses, and Research Questions Daily diary designs provide a unique opportunity to simultaneously study within-person and between-person differences by examining psychological distress in helpers on days when assistance is provided versus days when it is not (within-person), as well as by comparing helpers as a function of their background and contextual factors (between-person). Given that the proliferation of stressors on primary role-related activities is a key determinant of stress and well-being among individuals providing assistance, in the present study we hypothesized that psychological distress would be higher on days helpers encountered more daily situational factors (spent more time on activities, encountered stressors, provided help to other family members) in addition to providing assistance to a parent. We also hypothesized that women; younger individuals; African Americans; unmarried individuals; persons with young children; as well as individuals with lower education, higher neuroticism, and lower mastery would be more susceptible to psychological distress. Using a representative sample of the population, we identified adult children that engage in a full range of assistance in order to address two main questions. First, how are the daily role-related experiences affected by the type of day (i.e., helping vs non-helping day)? Second, after we control for daily role-related experiences and responsibilities and person-level variables, is psychological distress higher on days when assistance is provided than on days when it is not? Methods Data and Sample We used data from participants in the National Study of Daily Experiences (NSDE). The NSDE is a randomly selected subsample of the National Survey of Midlife Development in the United States (MIDUS) carried out under the John D. and Catherine T. MacArthur Foundation Network for Successful Midlife Development (for detailed descriptions of the MIDUS project, see Brim, Ryff, & Kessler, 2004). Out of the 1,242 MIDUS participants contacted, 1,031 (562 women, 469 men) participated in the NSDE daily diary study, yielding a response rate of 83%. Over the course of eight consecutive evenings, participants completed short telephone interviews about their daily experiences. The initiation of interviews was staggered across days of the week to control for the possible confounding between day of study and day of week. Participants completed an average of seven of the eight interviews, resulting in a total of 7,221 daily interviews. Participants received $20 for their participation (for more details on the study, see Almeida, Wethington, & Kessler, 2002). Out of the 1,031 NSDE participants, we identified 119 individuals who reported providing either instrumental or emotional assistance to their parent on at least 1 of the 8 days of the diary interview. The 119 individuals completed an average of 6.9 days of interviews out of the 8 days, resulting in 824 daily interviews. Measures Outcome variable Daily psychological distress was operationalized using an inventory of 10 emotions from the Non-Specific Psychological Distress Scale (Kessler et al., 2002; Mroczek & Kolarz, 1998), which was conducted on each of the 8 days of telephone interviews. Participants rated these mood-related questions on a 5-point scale (0 = none of the time; 4 = all of the time). The inventory included emotions such as sadness, hopelessness, anxiety, and restlessness. The scale was developed using item response models and factor analysis, yielding a single-factor structure representing current psychological distress (for complete information on psychometric properties of the scale and validation, refer to Kessler et al., 2002; Mroczek & Kolarz, 1998). Cronbach's alphas ranged from.70 to.89 across the 8 days of administration. Psychological distress in this sample had a positively skewed distribution (M = 1.97, SD = 3.51, skewness = 3.55) because the scores clustered toward the lower end of the scale. Other studies of more serious caregivers have seen this low rating of psychological distress as well (Schulz et al., 1997). To adjust for the skewness, we attempted to transform this variable to a more symmetric distribution by adding a constant of unity to the score before taking the natural logarithm of psychological distress (Mlogged psychological distress = 0.67, SD = 0.83). We used the logged psychological distress score for descriptive and univariate analyses and relied on random effects tobit regression with left-side censoring for the multivariate model. Predictor variables Using a broad definition of helping, we assessed providing routine assistance to a parent as help provided with emotional or instrumental tasks on each of the 8 days of the study. Two questions were asked regarding assistance to people living outside the house, in particular a parent. The first question was “Did you provide any unpaid assistance or instrumental assistance to someone outside the house?” (e.g., help with shopping, etc.). If participants agreed, then they were asked to name each of those to whom they provided assistance on that day. The list included parents. Likewise, the second question was about providing emotional support (e.g., giving advice, comforting them). Providing either emotional or instrumental support or both on a given day to a parent was coded as 1 if yes and 0 if no. Providing emotional or instrumental support to someone else other than a parent was also used as a control variable and was coded similar to the previous variable, namely 1 if provided support and 0 if provided no support on a given day. In this way, we could distinguish assistance provided to parents from assistance provided to others. Three time-use variables measured competing everyday situational demands. These variables were related to time spent each day on tasks and activities other than providing assistance to a parent. The first variable, routine chores, assessed the amount of time the participant spent on routine chores in the house, such as yard work. The second variable reflected the amount of time spent on activities related to business, paid work, or school, which included time traveling and thinking about the work. The third variable considered the use of time on activities related to leisure, such as relaxing, taking a nap, or engaging in physical exercise or leisure activities. Time spent on these activities was coded in hours and minutes. Background variables From the MIDUS survey, which was collected approximately a year before the NSDE diary interviews, variables that acted as reasonably stable background characteristics were included as between-person covariates in these analyses. This study included demographic variables such as gender (0 = male, 1 = female), African American race (0 = no, 1 = yes), and education (1 = less than high school, 2 = high school, 3 = college, 4 = college and higher). Apart from these individual characteristics, we utilized family-related variables such as marital status (1 = married, 0 = not married) and whether the participant had any children younger than 18 years (0 implying no children). We coded age reported during the NSDE diary interview into categories: 1 = 25 to 35 years (24.37%), 2 = 36 to 45 years (30.25%), 3 = 46 to 55 years (26.89%), 4 = 56 to 64 years (15.97%), 5 = 65 to 74 years (2.52%). Finally, we included trait neuroticism and personal mastery as two personal characteristics variables. The neuroticism items included the following four adjectives: moody, worrying, nervous, and calm (Lachman & Weaver, 1997). Participants indicated how well each of the four items described them on a 4-point scale from 1 (a lot) to 4 (not at all). All but the last item were reverse-coded, and the mean across the items was taken such that a higher value indicated higher levels of neuroticism. Scores ranged from 1 to 4. Cronbach's alpha for this sample was.76. The personal mastery scale consisted of two items from Pearlin and Schooler (1978) and two items from Lachman and Weaver (1998). See Lachman and Weaver (1998) for more information on this scale. Respondents rated on a 7-point scale (1 = strongly agree; 7 = strongly disagree) how strongly they agreed with the following questions: (a) I can do just about anything I really set my mind to; (b) When I really want to do something, I usually find a way to succeed at it; (c) Whether or not I am able to get what I want is in my own hands; and (d) What happens to me in the future depends mostly on me. We recoded responses so that higher scores indicated greater personal mastery. The scale was constructed by calculating the mean across each set of items. Scores ranged from 1 to 7. Cronbach's alpha for this sample was.63. Table 1 provides descriptive statistics for the total sample. Results Descriptive Statistics Table 1 presents the descriptive statistics for the participants in the study. The average age of participants was 45 years (SD = 11.41, range = 25–74), 67% were female, 65% were married, 38% had resident children younger than 18, and the majority of the sample was Caucasian (94%), with high school education or higher (M = 2.90, SD = 0.93). These participants were on average moderately high on neuroticism (M = 2.24, SD = 0.74, range = 1–4) and on mastery (M = 5.91, SD = 0.92, range = 1–7). On average, participants provided assistance on 28% of the study days (approximately 2 out of 8 days). A total of 66 participants (55%) provided routine assistance to their parents on only 1 of the days, compared to 53 (45%) who provided assistance on more than 1 day of the study (not shown in Table 1). On average, each day participants spent approximately 2 hr on routine chores (M = 2.19, SD = 2.25); 4.5 hr on work-related activities (M = 4.26, SD = 4.60); and approximately 11 hr on leisure activities, sleep, and exercise (M = 10.68, SD = 3.92). These participants also provided assistance to other family members, friends, and work colleagues on 4% of the study days. Out of the 119 participants, only 27 (22.69%) provided assistance to other family members on parent care days. A total of 39 participants (32.77%) provided instrumental support to a parent, 60 participants (50.42%) provided emotional support, and 20 participants (16.81%) provided both instrumental and emotional support to a parent. Univariate Analysis Examining Psychological Distress and Role-Related Experiences In order to examine if engaging in the helping role has an immediate association with psychological distress and other role-related experiences, we next considered time-use data and role-related experiences to test using paired t tests whether days when assistance was provided to parents were different from days when it was not, as shown in Table 2. Participants reported significantly higher psychological distress (t = −2.01, p <.05) on days when assistance was provided (Mlog = 0.76) than on days when it was not (Mlog = 0.66). With regard to everyday living activities, on average participants spent the same amount of time on routine chores and leisure activities, however they spent significantly less time (t = 2.54, p <.01) on work-related activities on days when assistance was provided (M = 3.67) than on days when it was not (M = 4.61). Participants also reported a significantly greater number of stressors on days when assistance was provided (t = −3.00, p <.01). On average, they experienced stressors on 57% of days when assistance was provided, compared with 43% of the days when it was not (t = −2.90, p <.01). Out of all stressors, network stressors (i.e., events that occurred in the lives of close family members and friends) occurred more on days when assistance was provided (experienced on 17% of helping days) than on days when it was not (experienced on 7% of non-helping days). Multivariate Analysis Examining Psychological Distress and Role-Related Experiences To take into account the clustering of participants on 8 days of the study and in order to adjust for floor effects in reported negative affect, in the next set of analysis we used a left-censored random effects tobit regression model to predict daily psychological distress. One of the main reasons for using daily diary designs is that we expected there would be as much within-person variation (i.e., helping days are different from non-helping days) as between-person variation (i.e., participants differ from one another) in psychological distress among participants. To examine this, we began our analysis with the standard unconditional model that estimated the average psychological distress and tested whether there was significant variation in daily psychological distress. As we expected, we found as much intraindividual variability as interindividual variability. The intraclass correlation coefficient (rho) of 0.49 suggested that approximately half of the total variation in daily psychological distress was within-person and the other half of the variation was between-person. Model 1 in Table 3 indicated that on average psychological distress was higher on days when assistance was provided than days when it was not (p <.05). We next estimated a series of models, beginning with a model with everyday situational factors (within-person predictors) followed by background characteristics (between-person predictors) predicting daily psychological distress. Model 2 in Table 3 presents the results including the everyday situational factors measured on 8 days of the study. Results indicated that on days that participants devoted more time to paid work (b = 0.12, p <.05) and leisure activities and sleep (b = 0.18, p <.01), they reported higher psychological distress as compared to days when they spent less time on these activities. Spending time on routine household chores was not related to psychological distress. Additionally, psychological distress was higher on days when the participant provided assistance to another family member other than the parent (b = 2.70, p <.01). Even after we controlled for the amount of time spent on paid work, on leisure activities and sleep, and engaging in other helping tasks, providing assistance to a parent was associated with the experience of greater psychological distress (b = 0.87, p <.05). Model 3 in Table 3 presents the results including situational (within-person) as well as background factors (between-person), such as age of adult child, gender, marital status, race, parental status, education, and personality factors such as neuroticism and mastery. Examination of the between-person variables showed that having less education (b = −0.54, p <.01), being single (b = −0.96, p =.07), and having young children (b = 2.14, p <.01) increased the chances of experiencing psychological distress across all days. Conversely, having lower neuroticism (b = 2.08, p <.001) and higher mastery (b = −0.53, p <.06) acted as protective factors against psychological distress. Age and gender of the adult child as well as race did not predict daily psychological distress among participants. Finally, even after we controlled for the situational factors as well as the background characteristics, providing assistance to a parent continued to be associated with greater psychological distress on the day help was given compared to days when it was not (b = 0.88, p <.05). We explored several interactions between everyday situational factors and background characteristics of the adult children; however, perhaps due to the limited sample size, we did not find any in the present study. Discussion Previous research has examined assistance between generations primarily using retrospective accounts of assistance provided over long time spans. These methods do not capture the everyday hassles and disturbances that are associated with the act of providing daily assistance to a parent. In the present study, we took a microlevel approach to examining the association of providing routine assistance amid everyday circumstances and the psychological consequences for the adult child over shorter time spans. In order to address our first research question, we began by examining how daily role-related experiences were affected on days when assistance was provided compared with when it was not. Past studies that have examined the relationship between work roles and helping roles have found that helpers often have to give up or cut down on personal roles (employee or parental role) to provide assistance (Murphy et al., 1997; Stephens et al., 2001). Consistent with this idea, we found that our sample of helpers was reasonably involved in routine daily chores and work-related activities, but on days when assistance was provided they spent less time on work-related activities. Notably, we also found that helpers reported more stressors on days when assistance was provided than on days when it was not. Many of the stressors revolved around participants' social networks. This is an interesting finding, because network stressors are events that occur in the lives of others. Perhaps helpers' expression of care and compassion further exposes them to the stressors of friends and family. To address our second research question, we used a multivariate analysis approach to examine psychological distress on days when assistance was provided versus days when it was not, taking into account daily role-related experiences and responsibilities and background characteristics. Our results clearly show that even at the microlevel, the enactment of the role of providing routine assistance to a parent is in itself stressful. Consistent with our first hypothesis and in line with previous research that suggests that conflicting demands of helping and fulfilling other personal roles (e.g., employee, parent) are important factors that account for negative effects on the well-being of the helper, we found that any increase in the amount of time spent on work on a given day increased psychological distress on days when assistance was provided. A surprising finding was that time spent on leisure and sleep was also related to higher psychological distress on days when assistance was provided. Because leisure and sleep are also planned activities that may cause role conflict and overload on days when assistance is provided, it is not surprising to find that distress was higher on those days. Further research is required to examine the implications of these findings. Some researchers have suggested that if these factors are held constant, providing assistance might lead to positive appraisals of the helping role (Marks, 1998). Our findings do not support this. Even after we controlled for the amount of time spent on activities such as household chores, work, as well as providing help to others, providing assistance to a parent was still significantly related to higher daily psychological distress. Consistent with our second hypothesis, we found clear evidence that being single, being non-White, and having lower education was associated with higher daily psychological distress (Couch et al., 1999; Pinquart & Sörensen, 2006). Moreover, we also found evidence that personal characteristics such as low neuroticism and high mastery buffered the effects of care enactment on psychological distress. However, we did not find any effects for age, gender, and race in our study. This could be because of the relative homogeneity of this sample, as the study was not specifically designed to study assistance provided by adult children. The age range within a sample of adult children would also be more restricted than in a sample that also included assistance provided by spouses. It is also possible that differences between sons and daughters are less pronounced than those found between husbands and wives (e.g., Aneshensel et al., 1995; Miller, 1990; Zarit, Todd, & Zarit, 1986; Zarit & Whitlatch, 1992) or across a wider continuum of assistance (Davey & Szinovacz, 2007). Likewise, the small proportion of non-Whites in the sample made it difficult to find any race effects and also restricts the generalizability of the results. The present study is among the first systematic studies that have examined the daily impact of providing routine assistance to a parent living outside the house; however, there are several limitations that we need to acknowledge. First of all, the present data were not collected with the intention of understanding routine assistance to parents. We therefore had to rely on global measures of assistance instead of specific dimensions of care, such as number of hours of assistance to a parent. Because measures of intensity of providing care were not available, we are not able to clarify whether it is the act of providing care or the intensity of providing care that is more important for predicting psychological distress. Future research should examine the daily intensity of help provision and its consequences on indicators of distress. Additionally, variables that are important predictors of intergenerational exchanges, such as parent's health status and proximity to the parent, were not known and so could not be included. Providing assistance to a parent who lives in closer proximity might be physically draining and exhausting; however, being far away from a parent might not give adult children immunity from feeling overwhelmed on days the parent requires care. The lack of information about parent's health status and proximity to parents did not allow us to assess variability in everyday distress due to these factors. Finally, we tested several interactions between providing daily assistance, daily exposure to stressors, and resilience variables, but due to the low power of the study, these could not be estimated and remain to be explored in future diary studies. Despite these limitations, the current study clearly suggests the possible link between assisting a parent and the downward trajectory of health and well-being in caregiving. The accumulation of small and large daily stressors may build up and spill over into other areas of life, eventually undermining psychological resources and well-being. Our results also imply that individuals who experience greater role conflict and demand on their time as well as those with the fewest resources experience the most distress on days when assistance is provided to parents. These results also suggest new strategies for supporting people assisting parents and other older relatives. Rather than designing respite and support programs in a nonspecific way, experts could design programs that specifically target the everyday care events that are stressful. By building on an understanding of the daily events that people find stressful, this approach could make daily life easier for older adults and the individuals who support them and prevent the depletion of care resources. By focusing on stressors in this way, support programs may be more effective in relieving caregiver burden while also giving financial and other support for those with the fewest resources. Decision Editor: Kenneth F. Ferraro, PhD Table 1. Descriptive Statistics for the Analytic Sample (N = 119). Variable M SD Day assistance provided to parent (proportion) 0.28 0.45 Everyday situational factors     Time spent on routine chores (hr) 2.19 2.25     Time spent on work (hr) 4.26 4.60     Time spent on leisure and sleep (hr) 10.68 3.92     Engagement in additional helping tasks (proportion) 0.04 0.20 Background characteristics     Age of adult child (years) 44.51 11.41     Daughter (proportion) 0.67 0.47     Married (proportion) 0.65 0.48     Black (proportion) 0.06 0.23     Any children (proportion) 0.38 0.48     Education category 2.90 0.93     Neuroticism 2.24 0.74     Mastery 5.91 0.92 Table 2. Paired t Tests Comparing Psychological Distress, Daily Activities, and Stressor Variables on Days When Assistance Was Provided Versus Not (N = 119). Days Assistance Provided Days Assistance Not Provided Variable M SD M SD t Psychological distress (log) 0.66 0.63 0.76 0.72 −2.01* Everyday tasks     Time spent on routine chores 2.07 1.61 2.30 2.11 −1.25     Time spent on work 4.61 3.79 3.67 4.06 2.54**     Time spent on leisure 10.81 3.29 10.73 3.73 0.26 Routine assistance to others     Provision of assistance to others 0.04 0.10 0.06 0.21 −1.06     Provision of emotional assistance to others 0.43 0.39 0.45 0.68 −0.33     Provision of instrumental assistance to others 0.11 0.22 0.15 0.33 −1.24 Stressor variables     Number of stressors 0.57 0.49 0.79 0.80 −3.00**     Any stressors 0.43 0.31 0.57 0.43 −2.90**     Arguments 0.24 0.23 0.29 0.39 −1.27     Work stressors 0.13 0.21 0.12 0.29 0.41     Home stressors 0.09 0.16 0.13 0.29 −1.50     Network stressors 0.07 0.15 0.17 0.32 −3.12** Note: *p <.05; **p <.01. Table 3. Random Effects Tobit Regression Predicting Daily Psychological Distress. Model 1 (Baseline) Model 2 (Time-Varying Covariates) Model 3 (Full Model) Fixed Effect b (SE) b (SE) b (SE) Day assistance provided 0.84 (0.40)* 0.87 (0.39)* 0.88 (0.39)* Everyday situational factors     Time spent on routine chores −0.01 (0.09) −0.03 (0.09)     Time spent on work 0.12 (0.05)* 0.13 (0.05)*     Time spent on leisure and sleep 0.18 (0.05)** 0.19 (0.05)**     Engagement in additional helping tasks 2.70 (0.85)** 2.68 (0.86)** Background characteristics     Age of adult child 0.40 (0.25)     Daughter 0.13 (0.57)     Married −0.96 (0.56)     Black −0.01 (1.04)     Any children 2.14 (0.60)**     Education −0.54 (0.27)*     Neuroticism 2.08 (0.38)**     Mastery −0.53 (0.29) Intercept 1.01 (0.31)** −1.59 (0.81)* −3.24 (2.76) Variance components Between-person (Level 2)     Variance (Intercept) 16.83 (0.34)** 15.73 (0.33)** 9.32 (0.29)** Within-person across days (Level 1)     Variance (Intercept) 17.33 (0.16)** 16.69 (0.16)** 16.83 (0.16)** Notes: N = 119. Days of interviews = 824. *p <.05; **p <.01. This research was supported in part by National Institute of Mental Health Training Grant T32 MH18904 to Jyoti Savla and MacArthur Foundation Research Network on Successful Midlife Development and National Institute on Aging Grant AG19239 awarded to Adam Davey. J. Savla conceptualized the paper, analyzed the data, and wrote the paper. D. M. Almeida designed the research, performed the data collection, and assisted in writing the paper. A. Davey and S. H. Zarit assisted with the conceptualization, analysis, and interpretation of the paper and contributed to writing the paper. References Almeida, D. M. (2005). Resilience and vulnerability to daily stressors assessed via diary methods. Current Directions in Psychological Science, 14,64-68. Almeida, D. M., Wethington, E., Kessler, R. C. (2002). The Daily Inventory of Stressful Events (DISE): An interview-based approach for measuring daily stressors. Assessment, 9,41-55. 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Contributors to and mediators of psychological well-being for informal caregivers. Journal of Gerontology: Psychological Sciences, 54B,P12-P22. Zarit, S. H., Todd, P. A., Zarit, J. M. (1986). Subjective burden of husbands and wives as caregivers: A longitudinal study. The Gerontologist, 26,260-270. Zarit, S. H., Whitlatch, C. (1992). Institutional placement: Phases of the transition. The Gerontologist, 32,665-672. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0BF4D215BE8EC646F25CF2D801A253009A2EE6E4.txt b/test/dataset/in/resources/corpus/Clean_0BF4D215BE8EC646F25CF2D801A253009A2EE6E4.txt new file mode 100644 index 0000000..2fc8a36 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0BF4D215BE8EC646F25CF2D801A253009A2EE6E4.txt @@ -0,0 +1 @@ +]>MAT1245S0378-5122(98)00056-510.1016/S0378-5122(98)00056-5Elsevier Science Ireland LtdFig. 1Reductions in lipoprotein(a) during hormone replacement therapy.Table 1Reduction in homocysteine during hormone replacement therapyHRT regimen*Reduction (%)AuthorReferenceOestradiol–dydrogesterone (sc)10.9Van der MoorenEur J Clin Invest 1994;24:733–736Conj. oestrogen–medrogestone (sc)6.8Van der MoorenFertil Steril 1997;67:67–73Oestradiol–dydrogesterone (cc)13.5MijatovicFertil Steril 1998;69:876–882Oestradiol–dydrogesterone (sc)12.6MijatovicObstet Gynecol 1998;91:432–436*Oral HRT regimen; sc, sequentially combined; cc, continuously combined.Hormone replacement therapy in postmenopausal women with specific risk factors for coronary artery diseaseMarius Jvan der Mooren*VeljaMijatovicWMarchien van BaalCoen D.AStehouwerProject ‘Ageing Women’, Departments of Obstetrics and Gynaecology and Internal Medicine, University Hospital-Vrije Universiteit, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands*Corresponding author. Tel: +31 20 4443244; fax: +31 20 4444422; e-mail: mj.vandermooren@azvu.nlAbstractHormone replacement therapy (HRT) in postmenopausal women is associated with a reduction in the risk of developing coronary artery disease (CAD) of about 50%. Women with an elevated risk for CAD appear to benefit most by HRT. The HRT-associated cardiovascular protection may be related to favourable changes in several important cardiovascular risk estimators, such as circulating blood concentrations of cholesterol, lipoprotein(a) (Lp(a)) and homocysteine. This paper reviews the literature presently available on the effects of HRT on cholesterol, Lp(a) and homocysteine concentrations, and special attention will be given to the effects on their elevated concentrations. The effect of HRT in women with hypertension is reviewed as well. From this overview it can be concluded that risk factors such as cholesterol, Lp(a), and homocysteine can be favourably modulated by HRT, and especially, that the strongest reductions can be achieved in those women with the highest concentrations. Although clinical trials still need to demonstrate the impact of lowering concentrations of Lp(a) and homocysteine, HRT appears to be a promising risk reduction strategy in this respect.KeywordsCoronary artery diseaseOestrogensHormone replacement therapyRisk factorsCholesterolLipoprotein(a)HomocysteineHypertension1IntroductionIn industrialised countries coronary artery disease (CAD) is the major cause of death in women. The mortality rate from CAD is about 3–4 fold that related to cancer. The risk of developing atherothrombotic CAD is associated with various factors. Modulation of these factors is a potential tool for the primary and secondary prevention of CAD, and may therefore be of great importance for the cardiovascular health in the individual, but it may also have a substantial socio-economic impact.In women as compared to men, the incidence of CAD is low before the age of 50. In contrast, in women older than 50, CAD risk strongly increases, indicating that the postmenopausal oestrogen deficiency state is a major risk factor in the development of CAD. Moreover, young ovariectomised women also suffer from an increased risk of dying from CAD [1], which supports the hypothesis that loss of ovarian function increases CAD risk.Accumulating evidence supports the suggested cardiovascular protective role of postmenopausal hormone replacement therapy (HRT) [2, 3]. Many epidemiological studies and several meta-analyses have reported a 50% lower incidence of CAD in oestrogen users compared with non-users [4], and comparable data have recently been published for combined oestrogen–progestogen users [5]. In addition, experimental studies in monkeys [6], in rabbits and rats have demonstrated that hormone administration reduces the arterial cholesterol accumulation.The cardiovascular protective influence of HRT appears to be of special benefit in women at high risk for CAD [7–12]. In the Lipid Research Clinic study, Bush et al. [7]reported a stronger reduction in CAD mortality in oestrogen-using hyperlipidaemic women compared with oestrogen-using normo-lipidaemic women. Ross et al. [8]demonstrated, in oestrogen-using women, that CAD mortality was relatively more reduced in heavy smokers when compared with non-smoking women. Ten-year mortality in oestrogen-using versus non-using women with angiographically proven CAD was more reduced in women with severe coronary stenosis than in women with normal angiography [9].2Risk factorsAn increasing number of factors have been identified which influence the risk of developing CAD. For many years, most attention was given to the impact on CAD risk due to changes in serum lipids and lipoproteins, and it was demonstrated that postmenopausal women have a more atherogenic lipid profile when compared with premenopausal women. Furthermore, HRT was found to have favourable effects on the lipid profile reflected by a reduction in the atherogenic LDL-cholesterol and an increase in the anti-atherogenic HDL-cholesterol.However, the alterations in lipids and lipoproteins appear to explain only 30–50% of the cardiovascular benefit of HRT [7]. Many other cardiovascular risk factors (haemostatic and fibrinolytic factors, parameters of endothelial function and parameters of vascular and cardiac function) have recently been reported to be modulated by sex steroids, and therefore may contribute to the cardiovascular protection mediated by postmenopausal HRT [3, 13, 14]. Recent reports have demonstrated that plasma concentrations of lipoprotein(a) (Lp(a)) and homocysteine are independent cardiovascular risk factors that play a role in the modulating influence of sex steroids on CAD risk.The next paragraphs will review the literature presently available on the effects of HRT in women with increased cardiovascular risk based on specific risk factors, namely hypercholesterolaemia, high Lp(a), hyperhomocysteinaemia, and hypertension. Although HRT has been reported to beneficially influence the fibrinolytic potential without disturbing coagulation, and to reduce postmenopausal insulin resistance, most of these studies were done in healthy women [14, 15]. However, in postmenopausal women with non-insulin-dependent diabetes mellitus short-term treatment with 17β-oestradiol results in an improvement of insulin sensitivity in the liver, glycaemic control, the lipoprotein profile and fibrinolysis [16, 17]. Until now no data were available on the effects of HRT in women with risk factors such as a high fibrinogen level or a disturbed coagulation-fibrinolysis balance, heavy smoking, and obesity, and therefore these risk factors will not be reviewed here.2.1CholesterolThe effects of HRT on conventional lipids and lipoproteins, such as total cholesterol, LDL- and HDL-cholesterol, triglycerides and apolipoproteins, have been thoroughly investigated and published in numerous reports [15, 18–20]. In general it can be concluded that during oral HRT, plasma concentrations of total and LDL-cholesterol decrease by about 10–15% and HDL-cholesterol increases by about 10–15%, all changes that favour cardiovascular health. Triglyceride concentrations however increase during oral HRT, the clinical relevance of which is unknown. The effects of transdermal HRT on lipids and lipoproteins are generally less marked, but do not show an increase in triglycerides.2.2HRT and hypercholesterolaemiaIn hypercholesterolaemic women, HRT, both oral and transdermal, is effective in improving the lipid and lipoprotein profile [21–27].Slowinska-Srzednicka et al. [23]observed an increase of 62% in HDL2-cholesterol and a decrease of 10% in total cholesterol during treatment of 11 postmenopausal non-obese hypercholesterolaemic women with transdermal 17β-oestradiol (100 μg/day), sequentially combined with oral chlormadinone acetate (2 mg/day).Tonstad et al. [24]demonstrated reductions of 14 and 19% in total cholesterol and in LDL-cholesterol, respectively, during treatment of hypercholesterolaemic women with oral sequentially combined 17β-oestradiol plus norethisterone acetate, in addition to a lipid-lowering diet. The reduction in LDL-cholesterol correlated positively with its initial level, indicating stronger reductions in women with high LDL-cholesterol.Denke [25]reported continuous combined conjugated oestrogens (0.625 mg/day) plus medroxyprogesterone acetate (2.5 mg/day) to be very effective in addition to a lipid-lowering diet in 32 hypercholesterolaemic women. Total cholesterol was reduced by 17%, and in 62% of women the high LDL-cholesterol concentrations were normalised.Recent studies [26, 27]have demonstrated that treatment with statins, when compared with HRT, are more potent in reducing total cholesterol and LDL-cholesterol in hypercholesterolaemic postmenopausal women, and do also reduce triglyceride levels, whereas oestrogen replacement increases HDL-cholesterol and triglycerides. Treatment with conjugated oestrogens plus pravastatin resulted in the combined favourable effects of both individual treatments [27].It can be concluded that, from the data available so far, HRT, oral as well as transdermal, appears to provide an effective approach in the management of hypercholesterolaemia in postmenopausal women.2.3Lipoprotein(a)Lp(a) is an independent risk factor for atherothrombotic cardiovascular disease [28–30]. The Lp(a) particle structurally greatly resembles low density lipoprotein. However, in Lp(a), the protein moiety consisting of apoprotein B-100 is covalently linked to the glycoprotein Apo(a). Due to the similarity of Apo(a) to plasminogen, Lp(a) competes for the binding of plasminogen in the fibrinolytic cascade. Furthermore, Lp(a) may accumulate in the arterial wall and contribute to the formation of atherosclerotic plaques. Therefore, Lp(a) has both thrombogenic and atherogenic properties.Genetic constitution strongly determines circulating levels of Lp(a). Unlike other lipoproteins, Lp(a) concentrations have been demonstrated to be moderately reduced by only a few drugs [31].After natural and surgical menopause, Lp(a) concentrations increase [32–34], indicating that the postmenopausal elevation in CAD risk may be mediated, at least in part, by changes in Lp(a) concentrations. During the last decade, several studies have reported that Lp(a) can be reduced by postmenopausal HRT [34–49].2.4HRT and high lipoprotein(a)The Lp(a)-lowering effects of HRT are reported to vary widely from 10 to 50% [34–49](Fig. 1), and some of these are already demonstrable after 2 months of intervention [38]. This probably is related to the diversity of HRT regimens studied, and it has been suggested that androgenic progestogens induce the largest fall in Lp(a). Crook et al. [50]reported a mean reduction of 79% in premenopausal women treated for endometriosis with the anabolic steroid danazol. Some of the studies have demonstrated a correlation between the reduction in Lp(a) and its level before intervention.Soma et al. [35]were the first to report a 51% fall in mean Lp(a) after 12 months of treatment with sequentially combined oral conjugated oestrogen (1.25 mg/day)-medroxyprogesterone acetate (10 mg/day). They later reported a significant correlation between absolute decrease in Lp(a) and baseline concentrations (r=0.59; P<0.001) in the same trial [36, 37].Farish et al. [38]observed a 47% reduction in Lp(a) after 2 months of treatment with oral norethisterone (10 mg/d). Their data indicated a larger fall in the women (n=2) with the highest pretreatment value.Lobo et al. [39]found marginally significant reductions of 31.7 and 26.9%, respectively, during 6 months of treatment with oral conjugated oestrogen (0.625 mg/d) only whether or not combined with excercise. They also observed a non-significant reduction of 10.6% during 6 months treatment with transdermal oestradiol (50 μg/day). In contrast to others [36–38, 41, 45, 48, 49], the authors found the largest fall (38%) in women with low Lp(a) values compared with a fall of 8% in women with ‘high’ values. However, they chose a threshold value of only 100 mg/l, which is rather low.Van der Mooren et al. [40]demonstrated a significant reduction of 17.5% after 6 months of treatment with sequentially combined oral oestradiol (2 mg/day)-dydrogesterone (10 mg/day), and in their 2-years’ report they showed this reduction to persist for 24 months, and also to be present in women with Lp(a) concentrations higher than 300 mg/l, a threshold level that appears to confer a significant higher risk of atherothrombotic disease [51].Marsh et al. [41]reported a decrease of 17.6% after 12 months treatment with continuously combined oral oestradiol (1 mg/d)-desogestrel (0.15 mg/d), and found a significant correlation between absolute change in Lp(a) and the pretreatment value (r=0.66; P<0.001).Taskinen et al. [45]observed mean decreases in Lp(a) of 31 and 16% after 12 months of treatment with, respectively, continuous combined oral oestradiol (2 mg/day)-norethisterone (1 mg/day) therapy and sequentially combined transdermal oestradiol (50 μg/day)-oral medroxyprogesterone acetate (10 mg/day) regimen. The decrement in Lp(a) was significantly correlated with the baseline Lp(a) levels (r=0.96 and 0.88, respectively; P<0.001). The authors observed a larger reduction in subjects with high baseline Lp(a) values during both HRT regimens.Mijatovic et al. [48]reported a mean decrease of 13.0% after 6 months’ treatment with a continuous combined oral oestradiol (2 mg/d)-dydrogesterone (2.5–15 mg/day) regimen. The reduction in Lp(a) correlated significantly with baseline Lp(a) (r=0.65; P<0.001), and women with high pretreatment values (>250 mg/l) performed better (P=0.055) than women with with normal baseline values.Tuck et al. [49]found a mean decrease of 23% (P=0.0002) after 4 weeks treatment with oral conjugated oestrogen (0.625 mg/d), and in their study the absolute amount of Lp(a) lowering correlated with the initial Lp(a) level (r=0.6; P=0.018) although they could not detect a difference between women with Lp(a) levels>40 mg/dl compared with subject with Lp(a) levels<30 mg/dl.How HRT interacts with Lp(a) metabolism still needs to be elucidated. Possibly LDL-receptor-mediated degradation of Lp(a) increases as the LDL-receptor is upregulated by oestrogens [52–54]. Alternatively, increased oestrogen-induced VLDL-synthesis in the liver may hamper the production of Lp(a) and be responsible for lower Lp(a) levels during oestrogen administration [55, 56].2.5HomocysteineHomocysteine is the demethylated derivative of the essential amino acid methionine. Mildly elevated plasma concentrations of homocysteine (hyperhomocysteinaemia) are an independent risk factor for vascular occlusive disease [57–65]. In addition, there is increasing evidence that homocysteine may affect the coagulation system and the resistance of the endothelium to thrombosis [66], and that it may interfere with the vasodilator and anti-thrombotic functions of nitric oxide [67].Plasma levels of homocysteine are, in part, genetically determined [65, 68], but acquired states such as folic acid and vitamin B deficiences as well as renal and liver failure may increase homocysteine levels too [59]. Homocysteine concentrations have also been reported to be higher in postmenopausal women compared to premenopausal women [69, 70], indicating that the low CAD risk before menopause may be related, in part, to favourable effects of oestrogens and/or progestogens on homocysteine metabolism. On the other hand, HRT reduces homocysteine concentrations in postmenopausal women [71–75].2.6HRT and hyperhomocysteinaemiaOnly few studies have been published on the relation between HRT and homocysteine metabolism [71–75](Table 1). The first report by Van der Mooren et al. [71]demonstrated a significant reduction of 10.9% in fasting serum homocysteine concentrations after 6 months of treatment with sequentially combined oestradiol (2 mg/day)-dydrogesterone (10 mg/day) therapy in 21 healthy postmenopausal women. Most of the demonstrated reduction could be attributed to the decrease of 17% observed in the women with elevated baseline homocysteine concentrations (higher than 13.8 μmol/l). The observed decrease was maintained during 24 months of treatment.Another observation from the same group was done in women treated with transdermal oestradiol in two dosages, 50 and 80 μg/day [72]. Although the decreases in homocysteine levels were modest, again reductions (about 10%) were observed especially in women with elevated homocysteine values at baseline.Furthermore, a significant decrease of 6.8% in fasting plasma homocysteine concentrations was demonstrated after 6 months of treatment with 3-monthly sequentially combined conjugated oestrogen (0.625 mg/day)-medrogestone (10 mg/day) therapy in 23 healthy postmenopausal women [73]. In this study, the decrease was also found especially in the hyperhomocysteinaemic women in whom a significant 12% reduction was observed. However, in this study the reduction in homocysteine was temporary and after 12 months of treatment no significant changes versus baseline were found. A large uncontrolled study by Mijatovic et al. [74]in 135 healthy postmenopausal women treated with continuously combined oestradiol (2 mg/day)-dydrogesterone (2.5–15 mg/day) therapy demonstrated a significant reduction in homocysteine levels of 13.5% after six months of treatment. They also observed the largest reduction in the women with the highest baseline homocysteine concentrations. The abolute decrease in homocysteine significantly correlated with the pretreatment concentration (r=0.42; P<0.001) indicating a larger reduction in women with high homocysteine levels. However, all these studies were uncontrolled, and three of them consisted of relatively small numbers. They remained therefore inconclusive.Very recently, our group demonstrated, for the first time in a controlled study, that HRT, given as sequentially combined oestradiol (1 mg/day)-dydrogesterone (5–10 mg/day) therapy during 15 months, reduced homocysteine concentrations with 12.6% versus baseline, whereas no changes were observed in a control group [75].The mechanisms underlying the observed decrease in plasma homocysteine by HRT are still unknown. It may be related to an increase in kidney methionine synthase activity [76]and to changes in the transamination of methionine [77], but it may also result from anabolic/catabolic effects [78].From the data currently available we can conclude that homocysteine concentrations can be reduced by postmenopausal HRT and that women with elevated levels demonstrate the largest reduction. Although the clinical relevance of lowering homocysteine concentrations still has to be elucidated, indirect data based on B-vitamin (folate, vitamin B6 and B12) administration (which are potent homocysteine-lowering agents) induced reduction in atherosclerotic plaque formation [79]and reduction in non-fatal myocardial infarction and fatal coronary heart disease [80]are very promising. Future prospective randomised clinical trials on cardiovascular outcome in hyperhomocysteinaemic women are needed before HRT can indeed be recommended as risk reduction strategy in hyperhomocysteinaemic women.3Blood pressureThe effects of HRT on blood pressure have scarcely been investigated. Most studies have reported no change or a small decrease in systolic and diastolic blood pressures [13, 81–91]. Proposed mechanisms to explain these observations are vasodilation [92], possibly related to calcium antagonistic effects of oestrogens [93], and inhibition of the angiotensin-converting-enzyme [94].3.1HRT and hypertensionIn an open prospective study Lip et al. [85]reported no changes in systolic and diastolic blood pressures in 75 hypertensive women on HRT for 8–32 months.Although Pines et al. [86]observed no changes in blood pressure after 6–9 months of oral conjugated oestrogens (0.625 mg/day) or oestradiol (2 mg/day) in 14 postmenopausal women with borderline to mild hypertension, they demonstrated a significant improvement in several parameters of cardiac function as measured by echocardiography at rest and during exercise.Van der Mooren [90]reported significant reductions of respectively 6.8 and 8.6 mmHg in systolic and diastolic blood pressures of 99 postmenopausal women with baseline diastolic blood pressure of 90 mmHg or more, and treated for 6 months with sequentially combined oral 17β-oestradiol (2 mg/day) plus dydrogesterone (5–20 mg/day).In a double-blind, randomised, placebo-controlled trial Kornhauser et al. [91]observed no changes in blood pressure after 90 days treatment with two HRT regimen in hypertensive postmenopausal women who had interrupted their antihypertensive medication.It can be concluded from the data available so far that HRT has no unfavourable effects in (mildly) hypertensive postmenopausal women. 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Acta Obstet Gynecol Scand 1997;76 Suppl 167:5;60 (abstract).91CKornhauserJMMalacaraMEGarayELPerex-LuqueThe effect of hormone replacement therapy on blood pressure and cardiovascular risk factors in postmenopausal women with moderate hypertensionJ Hum Hypertens11199740541192PCollinsCMCRosanoPMSarrel17β-Estradiol attenuates acetylcholine-induced coronary arterial consriction in women but not in men with coronary heart diseaseCirculation921995243093PCollinsGMRosanoCJiangCardiovascular protection by oestrogen—a calcium antagonist effect?Lancet34119931264126594AJProudlerAIHasib AhmedDCrookHormone replacement therapy and serum angiotensin-converting-enzyme activity in postmenopausal womenLancet34619958990 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0C0FB6C999438348D3A0896D4538904E65A7E680.txt b/test/dataset/in/resources/corpus/Clean_0C0FB6C999438348D3A0896D4538904E65A7E680.txt new file mode 100644 index 0000000..234971f --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0C0FB6C999438348D3A0896D4538904E65A7E680.txt @@ -0,0 +1 @@ + gerona J Gerontol A Biol Sci Med Scigerona The Journals of Gerontology Series A: Biological Sciences and Medical Sciences J Gerontol A Biol Sci Med Sci 1079-5006 1758-535X Oxford University Press 154610.1093/gerona/60.12.1546 Journal of Gerontology: Medical Sciences Foot and Ankle Characteristics Associated With Impaired Balance and Functional Ability in Older People Menz Hylton B. 1 Morris Meg E. 2 Lord Stephen R. 3 1Musculoskeletal Research Centre, Faculty of Health Sciences, La Trobe University, Bundoora, Victoria, Australia. 2School of Physiotherapy, University of Melbourne, Parkville, Victoria, Australia. 3Prince of Wales Medical Research Institute, University of New South Wales, Randwick, New South Wales, Australia. Address all correspondence to Hylton B. Menz, PhD, Musculoskeletal Research Centre, School of Physiotherapy, Faculty of Health Sciences, La Trobe University, Bundoora, Victoria 3086, Australia. E-mail: h.menz@latrobe.edu.au 12 2005 60 12 1546 1552 15 3 2005 18 10 2004 The Gerontological Society of America 2005 Background. Ageing is associated with changes to the structure and function of the foot and ankle, and there is preliminary evidence that foot problems impair balance and increase the risk of falls. To explore this in more detail, we conducted a study to determine the relative contribution of several foot and ankle characteristics to performance on a range of balance and functional tests. Methods. One hundred seventy-six people (56 men and 120 women, mean age 80.1 years, standard deviation 6.4 years) residing in a retirement village underwent tests of foot and ankle characteristics (including foot posture, range of motion, strength, and deformity), sensorimotor function (including vision, sensation, strength, and reaction time), and balance and functional ability (including tests of standing balance, leaning balance, stepping, sit-to-stand, and walking speed). Results. Many foot and ankle characteristics and sensorimotor measures were associated with performance on the balance and functional tests in univariate analyses. Multiple regression analysis consistently revealed that ankle flexibility, plantar tactile sensitivity, and toe plantarflexor strength were significant and independent predictors of balance and functional test performance, explaining up to 59% of the variance in these test scores. Conclusions. Foot and ankle characteristics, particularly ankle flexibility, plantar tactile sensation, and strength of toe plantarflexor muscles, are significant independent predictors of balance and functional ability in older people. Programs to improve the strength and flexibility of the foot and interventions to augment plantar sensation may be beneficial in improving mobility and reducing the risk of falls. hwp-legacy-fpage 1546 hwp-legacy-dochead RESEARCH ARTICLE MAINTAINING balance and performing functional tasks depend on the interaction of multiple sensory, motor, and integrative systems. These systems include vision, vestibular function, peripheral sensation, strength, and reaction time (1). Functioning of each of these factors declines with age (2). By directly assessing an individual's physiological abilities, impairments in one or more physiological domains can be identified and their contribution to physical ability can be determined. In previous studies, this approach has been used to examine the relative contribution of sensorimotor factors to balance (3), functional ability (4,5), and risk of falling (1). However, there are a number of factors not included in this conceptual model. In particular, it is likely that foot problems have a detrimental effect on mobility that is independent of the influence of these factors. Foot problems are reported by approximately 30% of community-dwelling older people (6–8), and are associated with reduced walking speed (7), difficulty in performing activities of daily living (7,9), and increased risk of falls (10–12). Despite the recognition of the detrimental effect of foot problems on mobility, the mechanism by which foot problems increase risk of falling has not been explored in detail. As the only source of direct contact with the ground during weight-bearing tasks, the foot contributes to the maintenance of stability in two main ways: (i) by providing mechanical support for the body via the osteoligamentous architecture of the arch and the coordinated function of lower limb muscles, and (ii) by the provision of sensory information regarding body position from plantar tactile mechanoreceptors. It is therefore likely that deficits in foot posture, flexibility, strength, or sensation impair this support function and predispose to loss of balance. We have previously demonstrated that an overall measure of foot impairment based on the observation of foot lesions and structural deformities was significantly and independently associated with performance on clinical tests of balance and functional ability (13). However, very little is known about whether foot posture, range of motion (ROM), sensation, and strength may influence mobility. There is some evidence that excessively flat feet (14) or highly arched feet (15) may impair standing balance in healthy young persons, and a significant association between ankle ROM and balance has been reported in older women (16). The aim of this study was to build on previous work to determine whether a broader range of tests of foot characteristics are significant determinants of balance and functional ability in a sample of older people. In particular, we were interested to ascertain whether these tests are capable of explaining additional variance in balance and functional performance after well established sensorimotor factors are considered. Methods Participants The study sample comprised 176 people (56 men and 120 women) aged 62–96 years (mean 80.1 years, standard deviation = 6.4) who were residing in a retirement village. One hundred fifty-five resided in independent living units, and 21 resided in serviced apartments. Residents in serviced apartments attended a communal dining room for all meals and had their rooms maintained by staff; however, all were independent in dressing, bathing, and toileting (Table 1). Residents were deemed ineligible for the study if they were unable to ambulate household distances without an assistive device or scored <7 on the Short Portable Mental Status Questionnaire (17). The participation rate was 54%. Age and sex data were available for nonresponders. Nonresponders were of similar age (80.1 years, standard deviation = 7.3) and gender (38% men) to participants. The Human Studies Ethics Committee at La Trobe University approved the study, and informed consent was obtained from all persons prior to their participation. Foot and Ankle Characteristics Foot and ankle characteristics were tested across five domains: foot posture, foot ROM, foot deformity and lesions, foot strength, and foot sensation. Foot posture was assessed using the foot posture index (18), arch index (19), and navicular height (20). Navicular height was corrected for differences in foot size by dividing it by the length of the foot (20). Ankle flexibility was measured in degrees using a modified version of the weightbearing lunge test (20). Participants rested their hand on a bench placed alongside them at waist height to reduce the demands for balance control when undertaking the test. First metatarsophalangeal joint (1st MPJ) ROM was measured in a nonweightbearing position with a goniometer while the examiner maximally extended the hallux (21). Foot deformity was evaluated by documenting the presence of hallux valgus, lesser toe deformities, corns, and calluses. The presence and severity of hallux valgus was determined using the Manchester scale (22). An overall measure of hallux valgus severity was determined by summing the scores for right and left feet. Presence of lesser digital deformity, corns, and calluses was determined according to previously published criteria (23), and a sum of the total number of each of these abnormalities for both feet was documented. The strength of the plantarflexor muscles of the hallux and lesser toes was determined using the paper grip test (24). Tactile sensitivity of the 1st MPJ was evaluated using an aesthesiometer using a two-alternative forced choice protocol (1). Reliability coefficients for each of these tests when performed by older people are reported elsewhere (20). Sensorimotor Function Assessment Sensorimotor function was assessed across four domains: vision (visual acuity and contrast sensitivity), sensation (tactile sensitivity of the lateral malleolus and proprioception), strength (knee extension and ankle dorsiflexion), and finger-press reaction time. Descriptions of the apparatus and procedures for these tests and their test–retest reliability are reported elsewhere (1). Balance and Functional Assessment The balance and functional tests are shown in Figure 1. Postural sway was measured using a sway meter that measured displacements of the body at the level of the waist (1). Testing was performed with participants, with eyes open, standing on the floor and on a medium-density foam rubber mat. Leaning balance was measured using the maximum balance range test and coordinated stability tests (25). Functional ability was evaluated using the alternate stepping test (the time taken to alternately place each foot on a 19 cm-high step eight times), sit-to-stand (time taken to rise from a 43-cm-high chair five times without using the arms), and walking speed over 6 meters. Reliability coefficients for each of these tests when performed by older people are reported elsewhere (1,5,25). All tests were performed barefoot to exclude the influence of footwear (26). Maximum balance range, coordinated stability, and walking speed were corrected for height prior to analysis. Statistical Analysis Variables with right-skewed distributions were log transformed. Pearson correlation coefficients were computed to examine the relationships between foot and ankle characteristics, sensorimotor measures, and balance and functional test performance scores. Independent samples t tests were performed to assess for differences in balance and functional test scores according to the paper grip test performance. All variables found to be significantly associated with the balance and functional tests were then entered into a series of stepwise multiple regression analyses to determine their relative importance in explaining variance in each of the tests. Age was then forced into the model to determine whether it could explain any more variance in the balance and functional test scores. To avoid the inclusion of misleading or unhelpful variables due to covariance among some independent variables, only the most highly correlated variable from each domain was included as a possible predictor at the entry of each block. Beta weights and signs for all variables entered into the regression model were also examined to ensure they made meaningful contributions to functional test performance. Change in the amount of variance (R2) was assessed following the addition of age into the model. The standardized beta weights provided give an indication of the relative importance of the various measures entered into the model in explaining variance in balance and functional test scores. The data were analyzed using SPSS for Windows (SPSS, Inc., Chicago, IL). Results Descriptive Statistics Descriptive statistics for each of the foot and ankle, sensorimotor, and balance and functional test measurements are shown in Table 2. Associations Between Foot and Ankle Characteristics and Balance and Functional Tests Table 3 shows the associations between the foot and ankle tests and balance and functional test scores. The foot and ankle characteristics with the most consistent associations with balance and functional test scores were plantar tactile sensitivity, ankle flexibility, the presence of lesser toe deformities, and the severity of hallux valgus. Separating the sway values into anteroposterior and mediolateral components made little difference to these associations, with the exception of hallux valgus being associated with mediolateral sway on the floor (r = 0.158, p =.037). Table 4 shows the differences in functional test scores according to performances on the two paper grip tests. Independent sample t tests revealed that participants who failed the two paper grip tests performed worse on each of the balance and functional tests. Associations Between Sensorimotor Testsand Balance and Functional Tests Table 5 shows the associations between the sensorimotor tests and balance and functional tests. Most sensorimotor measures were associated with balance ability functional performance, with strength and reaction time tests exhibiting the strongest correlations. Multiple Regression Analyses Table 6 shows the results of the stepwise multiple regression analysis for each of the balance and functional tests. One or more of the foot and ankle test scores were found to be significant independent predictors of each of the tests. In particular, ankle flexibility was found to be an independent predictor of each test, exhibiting the highest ß weight for sway on the floor, coordinated stability, alternate step test, and sit-to-stand. One of the two paper grip tests and tactile sensitivity of the 1st MPJ were also found to be independent predictors of the balance and functional tests. With the exception of coordinated stability, the inclusion of age explained further variance in all tests. Discussion The findings of this study indicate that foot and ankle characteristics significantly contribute to balance and functional ability in older people. In particular, tactile sensitivity of the plantar surface of the foot and ankle flexibility were strongly correlated with postural sway, whereas ankle flexibility and the strength of toe plantarflexor muscles were consistently associated with the leaning tests and functional measures. The association between plantar sensation and standing balance is consistent with previous reports of increased postural sway in persons with peripheral neuropathy (27,28) and impaired postural responses when sensory input is blocked in healthy persons (29,30), and indicates that plantar mechanoreceptors provide functionally important information regarding body position (31). Similarly, the association between toe plantarflexor strength and maximal balance range is consistent with Endo and colleagues (32), who reported a strong correlation between force plate measures of toe plantarflexor strength and the anterior limit of the functional base of support. Toe muscle function may play a particularly important role in the maintenance of balance in older people. Tanaka and colleagues (33) have shown that when standing, older people exert greater pressure with their toes than do younger people, possibly in an attempt to intensify sensory information to maintain balance. Contrary to previous findings in young persons (14,15), foot posture was not found to be a predictor of balance or functional ability. The presence of corns and calluses was also poorly correlated with the functional measures; however, this is most likely because these foot problems are almost ubiquitous in this age group (7). Consistent with our previous study (23), toe deformities (including hallux valgus) were significantly associated with balance and functional ability in univariate analyses; however, the inclusion of ankle flexibility and toe strength in this study precluded them from being included in the multivariate models due to their relatively weaker associations. Despite the broad range of possible predictors included in the regression models, it is acknowledged that much of the variance in the balance and functional measures remains unaccounted for. The sway tests were relatively poorly predicted, explaining between 11% and 24% of the variance in test performance. The amount of explained variance in the leaning balance and functional tests was considerably greater (between 47% and 59%); however, other factors such as vestibular function and pain may have provided additional information. Nevertheless, the multiple R2 values reported here compare favorably to other investigations of these functional measures (3–5), indicating that the foot and ankle measurements are useful additions to the sensorimotor test battery used previously. The major foot and ankle predictors revealed by the study are potentially modifiable. Although peripheral sensory loss is generally irreversible, there is emerging evidence that augmenting tactile sensory information from the sole of the foot using insoles with raised projections (34) or vibrating pads (35) may improve balance in older people. Furthermore, interventions directed at increasing ankle and 1st MPJ ROM and increasing the strength of toe plantarflexor muscles may have some value in decreasing the risk of falls in older people. Previous studies indicate that ankle motion can be increased with stretching (36–38), Tai Chi (39), and water exercise (40) programs in older people; however, the effect of increasing ankle flexibility on balance and falls risk has yet to be explored. Similarly, preliminary evidence suggests that “grasping” exercises to strengthen toe muscles results in improved standing balance in older people (41); however, it is not known whether this translates to a decreased risk of falls. Conclusion Foot and ankle characteristics, particularly tactile sensitivity, ankle flexibility, and toe strength, are important determinants of balance and functional ability in older people. Intervention studies to reduce risk of falling may possibly benefit from augmenting sensory information from the foot and the inclusion of stretching and strengthening exercises for the foot and ankle. Decision Editor: John E. Morley, MB, BCh Figure 1. Balance and functional tests. A, postural sway; B, maximal balance range; C, coordinated stability; D, sit-to-stand; E, alternate step test; F, walking speed Table 1. Prevalence of Major Medical Conditions, Medication Use, Participation in Physical Activity, and Mobility and ADL Limitations in Study Population. Condition No. (%) Medical conditions     Heart condition 61 (34.7)     Poor vision 44 (25)     Stroke 9 (5.1)     Osteoarthritis 133 (75.6)     Diabetes 19 (10.8)     Incontinence 29 (16.5) Medication use     Cardiovascular medications 124 (70.5)     Psychoactive medications 45 (25.6)     Musculoskeletal medications 28 (15.9)     More than four medications 115 (65.3) Physical activity     Planned walks <1 day/wk 72 (40.9)     Incidental physical activity <1 h/day 34 (19) Mobility and ADL limitations     Occasionally use a walking aid 44 (25)     Difficulty with housework 89 (50.6)     Difficulty shopping 36 (20.5)     Difficulty cooking 30 (17) Note: ADL = activities of daily living. Table 2. Descriptive Statistics for Each of the Variables Measured. Test Mean (SD) Range Foot and ankle tests     Foot posture index 5.01 (4.74) −7 to 16     Arch index 0.24 (0.06) 0.02–0.36     Navicular height/foot length 0.105 (0.032) 0.003–0.176     Ankle flexibility, ° 33.43 (10.77) 0–56     1st MPJ ROM, ° 55.52 (16.85) 15–100     Hallux valgus severity 1.92 (1.89) 0–6     Corns 0.16 (0.57) 0–4     Calluses 1.57 (2.34) 0–13     Lesser toe deformities 2.74 (2.53) 0–8     1st MPJ tactile sensitivity, gm 4.49 (0.61) 3.27–5.86 Sensorimotor tests     Visual acuity–high contrast (log MAR) 1.97 (2.16) 0.74–19.26     Visual acuity–low contrast (log MAR) 4.41 (3.71) 1.56–19.95     Contrast sensitivity, dB 20.31 (3.40) 5–24     Proprioception, ° error 1.65 (1.2) 0.1–5.8     Tactile sensitivity–ankle, gm 4.61 (0.59) 3.72–5.86     Knee extension strength, kg 21.68 (7.39) 6–46     Ankle dorsiflexion strength, kg 9.77 (5.19) 2–23     Reaction time, ms 273.90 (82.82) 169.6–703.5 Balance and functional tests     Sway on floor, mm2 61.47 (42.74) 32–329     Sway on foam, mm2 165.75 (111.79) 54–1013     Maximum balance range, mm 138.03 (46.84) 35–246     Coordinated stability, errors 11.45 (10.38) 0–39     Alternate step test, s 16.58 (9.95) 5.75–35.27     Sit-to-stand, s 19.32 (10.72) 6.09–46.02     Walking speed, m/s 0.88 (0.24) 0.20–1.41 Note: SD = standard deviation; MPJ ROM = metatarsophalangeal joint range of motion; log MAR = log minimum angle resolvable in minutes of arc. Table 3. Associations Between Balance and Functional Tests and Foot and Ankle Characteristics (Pearson's r). Test FPI AI NH AF 1st MPJ ROM Corns Calluses Toe Deformities Hallux Valgus 1st MPJ Tactile Sway on floor‡ 0.049 −0.050 −0.107 −0.226† −0.129 −0.001 0.049 0.063 0.131 0.207† Sway on foam‡ 0.103 −0.013 −0.067 −0.301† −0.160* −0.029 0.041 0.154* 0.259† 0.292† Maximum balance range −0.151* −0.106 0.154* 0.513† 0.219† −0.125 −0.189* −0.239† −0.363† −0.229† Coordinated stability 0.024 0.030 −0.094 −0.540† −0.152* 0.081 0.050 0.159* 0.274† 0.324† Alternate step test‡ 0.090 0.105 −0.194† −0.545† −0.116 0.019 0.075 0.179* 0.267† 0.228† Sit-to-stand‡ 0.054 0.019 −0.156* −0.511† −0.046 0.100 0.070 0.194† 0.276† 0.176* Walking speed −0.176* −0.068 0.183* 0.550† 0.176* −0.105 −0.153* −0.237† −0.339† −0.176* Note: *p <.05. †p <.01. ‡Log transformed. FPI = foot posture index; AI = arch index; NH = navicular height; AF = ankle flexibility; 1st MPJ ROM = first metatarsophalangeal joint range of motion; 1st MPJ tactile = first metatarsophalangeal joint tactile sensitivity. Table 4. Balance and Functional Test Scores According to Performance on Paper Grip Tests. Paper Grip Test–Hallux Paper Grip Test–Lesser Toes Test Passed (N = 112) Failed (N = 64) Passed (N = 95) Failed (N = 81) Sway on floor, mm2 57.38 (40.63) 68.63 (45.66)* 57.09 (42.91) 66.60 (42.22)* Sway on foam, mm2 156.28 (120.27) 182.33 (93.75)* 149.33 (115.31) 185.01 (104.97)† Maximum balance range, mm 150.96 (46.05) 115.39 (39.24)† 158.56 (43.01) 113.95 (39.22)† Coordinated stability, errors 8.87 (9.43) 15.98 (10.48)† 6.33 (6.92) 17.45 (10.58)† Alternate step test, s 14.05 (7.28) 21.00 (9.88)† 13.57 (6.91) 20.11 (9.77)† Sit-to-stand, s 16.56 (8.88) 24.17 (11.97)† 16.27 (8.55) 22.90 (11.89)† Walking speed, m/s 0.92 (0.22) 0.79 (0.25)† 0.96 (0.20) 0.78 (0.24)† Notes: *Significant difference at p <.05. †Significant difference at p <.01. Table 5. Associations Between Balance and Functional Tests and Sensorimotor Assessments. Test Visual Acuity (High) Visual Acuity (Low)‡ Contrast Sensitivity Reaction Time‡ Proprioception‡ Tactile Sensitivity (Ankle) Ankle Dorsiflexion Strength Knee Extension Strength Sway on floor‡ 0.180* 0.189* −0.127 0.082 −0.048 0.148* −0.095 −0.040 Sway on foam‡ 0.216† 0.212† −0.176* 0.351† 0.227† 0.295† −0.266† −0.275† Maximum balance range −0.329† −0.323† 0.328† −0.454† −0.259† −0.263† 0.608† 0.600† Coordinated stability 0.407† 0.408† −0.421† 0.433† 0.310† 0.403† −0.425† −0.409† Alternate step test‡ 0.319† 0.306† −0.349† 0.402† 0.279† 0.209† −0.477† −0.542† Sit-to-stand‡ 0.246† 0.233† −0.318† 0.363† 0.274† 0.144 −0.491† −0.505† Walking speed −0.289† −0.298† 0.370† −0.464† −0.272† −0.164* 0.535† 0.599† Notes: *p <.05. †p <.01. ‡Log transformed. Table 6. Results of Multiple Regression Analyses. Test Predictor Variable β Weight Multiple R2 Sway on floor Ankle flexibility −0.226† Tactile sensitivity 1st MPJ 0.165* 0.077 Age 0.197* 0.110‡ Sway on foam Reaction time 0.351† Tactile sensitivity 1st MPJ 0.261† Ankle flexibility −0.172* 0.216 Age 0.159* 0.238‡ Maximum balance range Ankle dorsiflexion strength 0.608† Ankle flexibility 0.337† Paper grip test–lesser toes∥ 0.252† Knee extension strength 0.262† Reaction time −0.127* 1st MPJ ROM 0.114* 0.572 Age −0.162† 0.594§ Coordinated stability Ankle flexibility −0.540† Paper grip test–lesser toes∥ −0.396† Reaction time 0.253† 0.492 Tactile sensitivity 1st MPJ 0.160† 0.515§ Alternate step test Ankle flexibility −0.545† Knee extension strength −0.417† Visual acuity–high contrast 0.209† Paper grip test–hallux∥ −0.157† 0.518 Age 0.161† 0.537§ Sit-to-stand Ankle flexibility −0.511† Knee extension strength −0.387† Paper grip test–hallux∥ −0.191† Contrast sensitivity −0.131* 0.445 Age 0.193† 0.474§ Walking speed Knee extension strength 0.599† Ankle flexibility 0.407† Contrast sensitivity 0.192† Reaction time −0.121* 0.554 Age −0.200† 0.585§ Note: *Significance of β weight p <.05. †Significance of β weight p <.01. ‡Indicates change in R2 when age entered into model (p <.05). §Indicates change in R2 when age entered into model (p <.01). ∥Indicates participant passed the test. MPJ = metatarsophalangeal joint; ROM = range of motion. Dr. Menz is currently National Health and Medical Research Council (NHMRC) Australian Clinical Research Fellow (id: 234424). This study was supported by an R.M. Gibson Fellowship from the Australian Association of Gerontology and the NHMRC Health Research Partnerships Grant: Prevention of Injuries in Older People. We thank Michael Smythe (Manager) and Margaret Smythe (Director of Nursing) of the La Trobe Retirement Village for their assistance. References 1 Lord SR, Menz HB, Tiedemann A. A physiological profile approach to falls risk assessment and prevention. Phys Ther.2003;83:237-252. 2 Lord SR, Ward JA. Age-associated differences in sensori-motor function and balance in community-dwelling women. Age Ageing.1994;23:452-460. 3 Lord SR, Clark RD, Webster IW. Postural stability and associated physiological factors in a population of aged persons. J Gerontol Med Sci.1991;46:M69-M76. 4 Lord SR, Lloyd DG, Li SK. 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Risk factors for falls among elderly persons living in the community. New Eng J Med.1988;319:1701-1707. 11 Koski K, Luukinen H, Laippala P, Kivela SL. Physiological factors and medications as predictors of injurious falls by elderly people: a prospective population-based study. Age Ageing.1996;25:29-38. 12 Leveille S, Bean J, Bandeen-Roche K, Jones R, Hochberg M, Guralnik J. Musculoskeletal pain and risk of falls in older disabled women living in the community. J Am Geriatr Soc.2002;50:671-678. 13 Menz HB, Lord SR. The contribution of foot problems to mobility impairment and falls in older people. J Am Geriatr Soc.2001;49:1651-1656. 14 Cobb SC, Tis LL, Johnson BF, Higbie EJ. The effect of forefoot varus on postural stability. J Orthop Sports Phys Ther.2004;34:79-85. 15 Hertel J, Gay MR, Denegar CR. Differences in postural control during single-leg stance among healthy individuals with different foot types. J Athlet Train.2002;37:129-132. 16 Mecagni C, Smith JP, Roberts KE, O'Sullivan SB. Balance and ankle range of motion in community-dwelling women aged 64 to 87 years: a correlational study. Phys Ther.2000;80:1001-1011. 17 Pfieffer E. A short portable mental status questionnaire for the assessment of organic brain deficit in elderly patients. J Am Geriatr Soc.1975;23:433-441. 18 Redmond AC, Burns J, Crosbie J, Ouvrier R. An initial appraisal of the validity of a criterion based, observational clinical rating system for foot posture (abstract). J Orthop Sports Phys Ther.2001;31:160. 19 Cavanagh PR, Rodgers MM. The arch index: a useful measure from footprints. J Biomech.1987;20:547-551. 20 Menz HB, Tiedemann A, Kwan MMS, Latt MD, Lord SR. Reliability of clinical tests of foot and ankle characteristics in older people. J Am Podiatr Med Assoc.2003;93:380-387. 21 Hopson MM, McPoil TG, Cornwall MW. Motion of the first metatarsophalangeal joint: reliability and validity of four measurement techniques. J Am Podiatr Med Assoc.1995;85:198-204. 22 Garrow AP, Papageorgiou A, Silman AJ, Thomas E, Jayson MI, Macfarlane GJ. The grading of hallux valgus. The Manchester Scale. J Am Podiatr Med Assoc.2001;91:74-78. 23 Menz HB, Lord SR. Foot pain impairs balance and functional ability in community-dwelling older people. J Am Podiatr Med Assoc.2001;91:222-229. 24 de Win M, Theuvenet W, Roche P, de Bie R, van Mameren H. The paper grip test for screening on intrinsic muscle paralysis in the foot of leprosy patients. Int J Lepr Other Mycobact Dis.2002;70:16-24. 25 Lord SR, Ward JA, Williams P. Exercise effect on dynamic stability in older women: a randomised controlled trial. Arch Phys Med Rehabil.1996;77:232-236. 26 Arnadottir SA, Mercer VS. Effects of footwear on measurements of balance and gait in women between the ages of 65 and 93 years. Phys Ther.2000;80:17-27. 27 Lord SR, Caplan GA, Colagiuri R, Ward JA. Sensori-motor function in older persons with diabetes. Diabet Med.1993;10:614-618. 28 Richardson JK, Ashton-Miller JA, Lee SG, Jacobs K. Moderate peripheral neuropathy impairs weight transfer and unipedal balance in the elderly. Arch Phys Med Rehabil.1996;77:1152-1156. 29 Magnusson M, Enbom H, Johansson R, Pyykko I. Significance of pressor input from the human feet in anterior-posterior postural control. The effect of hypothermia on vibration-induced body-sway. Acta Otolaryngol.1990;110:182-188. 30 Perry SD, McIlroy WE, Maki BE. The role of plantar cutaneous mechanoreceptors in the control of compensatory stepping reactions evoked by unpredictable, multi-directional perturbation. Brain Res.2000;877:401-406. 31 Kavounoudias A, Roll R, Roll JP. The plantar sole is a ‘dynamometric map’ for human balance control. NeuroReport.1998;9:3247-3252. 32 Endo M, Ashton-Miller J, Alexander N. Effects of age and gender on toe flexor muscle strength. J Gerontol Med Sci.2002;57A:M392-M397. 33 Tanaka T, Noriyasu S, Ino S, Ifukube T, Nakata M. Objective method to determine the contribution of the great toe to standing balance and preliminary observations of age-related effects. IEEE Trans Rehabil Eng.1996;4:84-90. 34 Maki BE, Perry SD, Norrie RG, McIlroy WE. Effect of facilitation of sensation from plantar foot-surface boundaries on postural stabilization in young and older adults. J Gerontol Med Sci.1999;54:M281-M287. 35 Priplata AA, Niemi JB, Harry JD, Lipsitz LA, Collins JJ. Vibrating insoles and balance control in elderly people. Lancet.2003;362:1123-1124. 36 Mills EM. The effect of low-intensity aerobic exercise on muscle strength, flexibility, and balance among sedentary elderly persons. Nurs Res.1994;43:207-211. 37 Swank AM, Durham MP. Adding weights to stretching exercise increases passive range of motion for healthy elderly. J Strength Cond Res.2003;17:374-378. 38 Sohng KY, Moon JS, Song HH, Lee KS, Kim YS. Fall prevention exercise program for fall risk factor reduction of the community-dwelling elderly in Korea. Yonsei Med J.2003;44:883-891. 39 Wolf SL, Barnhardt HX, Kutner NG, McNeely E, Coogler C, Xu T. Reducing frailty and falls in older persons: an investigation of Tai Chi and computerized balance training. Atlanta FICSIT Group. Frailty and Injuries: Cooperative Studies of Intervention Techniques. J Am Geriatr Soc.1996;44:489-497. 40 Alexander MJL, Butcher JE, MacDonald PB. Effect of a water exercise program on walking gait, flexibility, strength, self-reported disability and other psycho-social measures of older individuals with arthritis. Physiother Can.2001;53:203-211. 41 Kobayashi R, Hosoda M, Minematsu A, et al. Effects of toe grasp training for the aged on spontaneous postural sway. J Phys Ther Sci.1999;11:31-34. \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0C1B762A904A33974ED09F1DCB01DE08D525F735.txt b/test/dataset/in/resources/corpus/Clean_0C1B762A904A33974ED09F1DCB01DE08D525F735.txt new file mode 100644 index 0000000..0079c0d --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0C1B762A904A33974ED09F1DCB01DE08D525F735.txt @@ -0,0 +1 @@ +EXG5534S0531-5565(01)00163-210.1016/S0531-5565(01)00163-2Elsevier Science Inc.Aging Research WorldwideUK research on the biology of agingAubrey D.N.J.de Grey*ag24@gen.cam.ac.ukDepartment of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK*Tel.: +44-1223-333963; fax: +44-1223-333992AbstractOnly a few years ago, it could fairly be said that biogerontology research in the UK was in a sorry state. With the exception of the evolutionary biology of aging, which was revolutionized by Britons in the 1950s and in which the UK has remained paramount ever since, the number of research groups whose main focus was biogerontology had waned to single digits, and even those groups were generally very small. This situation has been transformed during the past decade, with the result that the UK arguably leads Europe in this field, in terms of both the quality and the quantity of its output. Moreover, the health of UK biogerontology research seems secure for the foreseeable future. Its one potential Achilles heel is the overemphasis on compression of morbidity as a goal, since further compression is highly unlikely to occur and is anyway inconsistent with the public's demonstrated desires.KeywordsAgingBiogerontologyUnited KingdomFunding1IntroductionIs there such a thing as biogerontology? This ostensibly rhetorical question has become hard to answer in the UK in recent years, as work on age-related physiological decline has become interdigitated with fields once viewed — by biogerontologists, but especially by their own workers — as quite distinct. The refrain “I'm not really a gerontologist, but…” is heard almost comically often at British aging meetings. This is a natural occurrence, given that the boundary between aging and age-related diseases is extremely fuzzy (if it exists at all), but in this gerontologist's experience, it has happened more abruptly in the UK than elsewhere.Not only is it a natural occurrence, it is an exceptionally good thing for biogerontology in respect of its standing in biology at large. The vast complexity of the aging process, in some ways outstripping even that of development, has traditionally discouraged young and/or successful biologists from becoming involved in its study, for fear that they (and their careers) will become mired in uninterpretable data. This has, in decades past, given rise to the complementary prejudice that those who do study the biology of aging do so because they could not compete in more tractable subfields of biology. In short, biogerontology has not been respected by biologists in general. This was as true in other countries as in the UK, and is being progressively dispelled there as here; again, however, the abruptness of that process in the UK in the past few years is striking. The bulk of this article is a survey of the many examples that comprise this phenomenon. Several such researchers focus more on specific age-related diseases than on ‘aging itself’; this again is a step forward, since research into either area can (and frequently does) inform the other.A major driving force that has brought about this change is the one that might be guessed, money, and especially an improvement in the structure of how it is disbursed. Two initiatives — one governmental, one by a charity — have dominated the transformation of UK biogerontology; they will be described below.The one area of UK biogerontology that has remained healthy throughout the post-war period is the evolutionary biology of aging. Here, too, however, a transformation has occurred, with specialists in the ‘why’ and the ‘how’ of aging interacting more intensively and productively than ever before. Examples of this will also be noted below.The number of groups now working on aging or age-related disease in mammals is now so great that an article of this length cannot be comprehensive; accordingly, I have preferentially cited below those groups with whose work I am most familiar, and I apologize to those whose work is omitted. However, I hope to have fulfilled the major purpose of this survey (and of this series on Aging Research Worldwide) by describing the major themes in which research on biogerontology in the UK is particularly strong.2Research on the evolutionary biology of agingThe post-war revolution in our understanding of why aging has evolved, or not been evolved away, was due principally to the insights of the British researchers Medawar and Williams (Medawar, 1952; Williams, 1957) and is an area of which the UK has remained consistently at the forefront (Hamilton, 1966; Kirkwood and Holliday, 1979; Charlesworth, 1993; Partridge and Barton, 1993). A particularly welcome development in recent years is that research groups originally dedicated mainly to the theoretical aspects of this topic have become increasingly active on the experimental side, thereby producing work that combines the best of both (e.g. Martin et al., 1998; Sgro and Partridge, 1999). The evolution of aging, like that of any other aspect of life subject to Darwinian selection, occurs ultimately at the molecular and cellular level; hence, evolutionary biogerontologists who are prepared to broaden their expertise to include molecular and cellular gerontology will be better equipped to test their (and others') ideas. As a result they will progressively eclipse any who remain wedded to an approach based exclusively on mortality data; there is evidently no danger of the UK evolutionary biogerontology community falling into the latter category.3Invertebrate model organismsCompared to many other countries, a relatively small proportion of UK biogerontology is focused on the standard invertebrate models (flies, worms and yeast). Exceptions are the group of Partridge (see above), working with Drosophila, and Gems (e.g. Gems and Riddle, 2000), working with C. elegans. Both groups are based at University College London, and they have recently collaborated to explore the remarkably similar influence of the insulin/IGF signaling pathway on aging in the two organisms (Clancy et al., 2001). S. cerevisiae aging is researched by the groups of Smart (e.g. Powell et al., 2000) in Oxford, Piper (e.g. Harris et al., 2001) in London, and Morgan (e.g. Mankouri and Morgan, 2001) in Liverpool.4Research on mechanisms of aging in mammalsThough evolutionary biogerontology has remained pre-eminent in the UK, the number of researchers involved in it has not greatly increased in recent years. This certainly cannot be said of molecular and cellular gerontology.4.1Oxidative damageSeveral groups, both long and newly involved in biogerontology, work on the etiology of oxidative stress and damage in aging. Among the longest standing are those of Merry (e.g. Lambert and Merry, 2000), focused on caloric restriction in rodents, and Jackson (e.g. McArdle et al., 2001), with specific interests in age-related muscle dysfunction; both groups are based in Liverpool. A valuable complement to Merry's work is that of the group of Speakman, based in Aberdeen, who study the relationship between metabolic rate and lifespan in mice and other small mammals (e.g. Selman et al., 2001).The main origin of oxidative damage, including that leading to aging, is generally agreed to be the mitochondrion. It is therefore essential for biogerontology to involve researchers with a detailed understanding of how this most complex of subcellular structures functions and malfunctions. The Cambridge group of Brand, long prominent in bioenergetics in general and mitochondrial proton leak in particular, has recently broadened its interest in the role of mitochondria in aging (e.g. Brand, 2000).Additionally, Turnbull's group in Newcastle studies the deleterious effects of mitochondrial DNA mutations in aging and age-related disease (e.g. Cottrell et al., 2001), as well as in early-onset mtDNA-linked diseases.A target of oxidative damage that may be just as important as DNA is proteins; this damage typically results in the production of carbonyl moieties, which are highly reactive and can cause protein–protein cross-linking. One of our most powerful natural defences against this process may be the dipeptide carnosine, which is present at millimolar levels in many tissues. The UK is fortunate to be home to Alan Hipkiss, whose expertise in carnosine biochemistry is second to none and whose work is increasingly revealing how carnosine exerts its effects (e.g. Hipkiss et al., 2001).4.2Cellular senescence and nuclear DNA damageCellular senescence, the subfield of biogerontology that has perhaps enjoyed (or suffered?) the highest public profile in recent years, is a prime example of the recent resurgence of British aging research, with the sole exception that its growth somewhat predated that of most other disciplines. Before about 1990 only one group, that of Shall, was highly active in the cellular senescence field in the UK (e.g. Karatza et al., 1984); now, by contrast, we can boast highly productive home-grown research groups in numerous locations around the country (e.g. Bridger et al., 2000; Wyllie et al., 2000; James et al., 2000), as well the group of von Zglinicki, previously based in Berlin (e.g. von Zglinicki et al., 2000) but recently transplanted to Newcastle. These are in addition to work on telomere structure and function not directly connected with aging, such as that pursued by Jackson's group in Cambridge (e.g. Teo and Jackson, 2001).It is now widely appreciated that other forms of chromosomal damage, particularly double-strand breaks, can trigger cellular responses very similar to those associated with telomere shortening. This has accentuated the interest of biogerontologists in DNA damage over and above its role in cancer, and conversely the interest of DNA damage specialists in aging. The work of Shall's group must again be noted as having led the way in this regard, with their interest in the role of poly(ADP-ribos)ylation in DNA strand break repair (e.g. Durkacz et al., 1980). This is the main focus of the group of Burkle (e.g. Beneke et al., 2000), which, like that of von Zglinicki (see above), has recently transferred from Germany to Newcastle. Prominent among groups whose focus on DNA damage and repair has only recently become integrated into biogerontology is that of Cox (e.g. Ongkeko et al., 1999), based in Oxford and now researching the molecular basis of Werner's syndrome (e.g. Rodriguez-Lopez et al., 2001).4.3Aging of specific organ systemsThe age-related decline of the immune system has become a particularly strong branch of UK biogerontology in recent years. Groups involved include those of Aspinall (e.g. Aspinall and Andrew, 2000), Akbar (e.g. Plunkett et al., 2001) and Dunn-Walters (e.g. Banerjee et al., 2000) in London, Lord (e.g. Butcher et al., 2000) in Birmingham, Goyns (e.g. Lavery and Goyns, 2001) in Sunderland and Barnett (e.g. Hyland et al., 2001) in Coleraine, Northern Ireland. Aspinall's group stands out among all UK biogerontology workers — not only those involved in immunosenescence — as maintaining an interest in the actual reversal, rather than the mere retardation, of age-related decline, something that is also a major focus of the present author (de Grey et al., 2001).The heart is an organ whose aging is distinctive in several important ways: for example, it loses cellularity without losing mass. Boyett's group in Leeds is prominent in research on the mode of action of the heart's pacemaker, the sinoatrial node (e.g. Boyett et al., 2000).Skeletal muscle, on the other hand, loses considerable mass during aging, with multifarious downstream consequences. Aging of muscle is the focus of the groups of Jackson in Liverpool (e.g. McArdle et al., 2001) and Smith in Birmingham (e.g. Smith et al., 2000).The eye suffers from several major types of aging, of which the most universal is macular degeneration. The group of Boulton in Manchester studies aspects of this process (e.g. Beatty et al., 2001). Glaucoma, another extremely widespread disease of the aging eye, is studied by the group of Webster in Liverpool (e.g. Grierson et al., 2000).An area often neglected due to its non-life-threatening nature, but no less deserving of basic research for that, is urinary incontinence. This is studied by the group of Ferguson in Cambridge (e.g. Burton et al., 2000).Groups involved in studying the aging of the nervous system include those of Cowen (e.g. Gavazzi et al., 2001) in London, Edwardson (e.g. Singleton et al., 2001) in Newcastle, Davies (e.g. Fotheringham et al., 2000) in Manchester and Franklin (e.g. Hinks and Franklin, 2000) in Cambridge. Of all aspects of aging, this is perhaps the one where the overlap with diseases often regarded as distinct from ‘aging itself’ is least clear, so highly relevant work on neurodegeneration by UK groups too numerous to list here must also be noted.Last but not least, the various components of the extracellular matrix are the subject of research by several groups. A prominent example is that of Clark in Norwich, which studies cartilage aging leading to osteoarthrosis (e.g. Dean et al., 2000).5Funding of biogerontology research in the UKAs noted in the Introduction, the ‘growth spurt’ of UK biogerontology has occurred at least in part because of the improved environment for obtaining funding for such research. Much of the credit for this must go to the major charity funding biogerontology research, Research into Ageing (RiA). Founded a quarter of a century ago, it has enjoyed a surge of financial success in recent years under the inspirational leadership of Elizabeth Mills, with its income rising nearly threefold between financial years 1994/1995 and 1999/2000. This money has been used to fund research into all the aspects of aging mentioned in Section 4.3 and more. It has recently merged with the much larger, but hitherto not biology-oriented, charity Help the Aged, a move which is set to provide further sharp increases in its budget for the next two years at least. Mrs Mills has now stepped down as its head and has been succeeded by Dr Susanne Sorensen, under whose direction we can be confident that its influence will continue to be considerable.Partly as a result of RiA's success, an effort was begun in 1996–1997 to address the problem, so familiar to biogerontologists, that our field falls between the two stools of medicine and basic biology. (The UK situation in this regard is exacerbated by our lack of a governmental organization specifically focused on aging, equivalent to the United States' National Institute on Aging.) This resulted in the SAGE (Science of Ageing) initiative, whereby the BBSRC (Biotechnology and Biological Sciences Research Council), the government agency with responsibility for funding basic biology research, funded a total of 29 grants totalling £5 million starting in 1998. Many of the groups mentioned in Section 4.3 were funded by this initiative despite never having been funded to work on aging previously, so by that measure it is the main cause of the surge in biogerontology research in the UK.6Future prospectsThe conspicuous success of the SAGE initiative has led at once to the BBSRC organizing a follow-up, Experimental Research on Ageing (ERA). Applications for this new initiative, which like SAGE will disburse up to £5 million over three years, closed in May 2001; it is rumored to have been fivefold over-subscribed. Thus, we can look forward with confidence to a continuation of the remarkable volume of high-quality research into aging that is presently undertaken in the UK.A further measure of the UK's success in this area is the relatively small number of UK researchers who have recently moved abroad: only two major recent emigrants are known to the present author. This virtual absence of a ‘brain drain’ is a vital requirement for the long-term health of any area of research, so we must hope — and can expect — that it will continue.The only note of caution that must be raised is with regard to the confusion presently widespread in the UK concerning what research on aging is intended, or likely, to achieve in the medium and long term. Descriptions or ‘mission statements’ published by the funding bodies described above include the following:“The aim of ERA is to understand the basic biology of healthy aging. It is hoped that such information could eventually lead to new treatments that could reduce age related decline and thus increase ‘healthspan’ and improve quality of life for the elderly. ERA is not aimed at lengthening life span or addressing specific age related diseases such as Alzheimer's.” (BBSRC, 2001).“Both life expectancy and healthy life expectancy increased between 1981 and 1995; but healthy life expectancy — the number of expected years of life in good or fairly good general health — did not increase by as much as life expectancy. This means that both men and women are living more years in poor health or with a limiting long-standing illness. Research into Ageing is committed to funding research that will help to close this gap: to achieve a healthspan to fit our lifespans.” (Research into Ageing, 2000).By these words, the UK's major funders of biogerontology research are committing themselves to contribute to the continuation of the trend termed ‘compression of morbidity’ (Fries, 1980) — the increase of healthspan but not (or, at least, to a lesser degree) lifespan. Noble though this aspiration may be, it is increasingly clear that nothing of the kind will result from our research, simply because medicine that works on robust people (postponing their frailty) tends also to work on frail people (postponing their death). Healthspan is much harder to measure than lifespan, whose ‘rectangularization’ is now firmly established to have ceased in the western world about 50 years ago (Wilmoth and Horiuchi, 1999), but the evidence that there is also no ongoing compression of morbidity is likewise now solid (Research into Ageing, 2000; Crimmins, 2001). Thus, the mission statements quoted above are almost certain to be unfulfilled; since it is the public's money that such bodies are spending, this seems decidedly unsatisfactory (at least to the present author). Moreover, it is wholly unnecessary: the failure of morbidity to be further compressed is due not only to the availability of medicine that prolongs frailty but to its take-up by frail individuals, who thereby demonstrate that they predominantly prefer frailty to death. 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Med.2820006474 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0C1BD68C40FC2CE322889091B7C53C34AE0CA2E5.txt b/test/dataset/in/resources/corpus/Clean_0C1BD68C40FC2CE322889091B7C53C34AE0CA2E5.txt new file mode 100644 index 0000000..691246c --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0C1BD68C40FC2CE322889091B7C53C34AE0CA2E5.txt @@ -0,0 +1 @@ +epirevepirevEpidemiologic Reviews1478-67290193-936XOxford University Press10.1093/epirev/mxn003ARTICLESComorbid Forms of Psychopathology: Key Patterns and Future Research DirectionsCerdáMagdalena123SagdeoAditi2GaleaSandro2341Robert Wood Johnson Foundation Health and Society Scholars Program, Department of Epidemiology, University of Michigan, Ann Arbor, MI2Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI3Center for Urban Epidemiologic Studies, New York Academy of Medicine, New York, NY4Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NYCorrespondence to Dr. Magdalena Cerdá, Department of Epidemiology, School of Public Health, University of Michigan, 109 South Observatory, 3639 SPH Tower, Ann Arbor, MI 48109-2029 (e-mail: mcerda@umich.edu or mcerda@nyam.org).111200810720083011551772942008Epidemiologic Reviews © The Author 2008. Published by the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.2008The purpose of this review is to systematically appraise the peer-reviewed literature about clustered forms of psychopathology and to present a framework that can be useful for studying comorbid psychiatric disorders. The review focuses on four of the most prevalent types of mental health problems: anxiety, depression, conduct disorder, and substance abuse. The authors summarize existing empirical research on the distribution of concurrent and sequential comorbidity in children and adolescents and in adults, and they review existing knowledge about exogenous risk factors that influence comorbidity. The authors include articles that used a longitudinal study design and used psychiatric definitions of the disorders. A total of 58 articles met the inclusion criteria and were assessed. Current evidence demonstrates a reciprocal, sequential relation between most comorbid pairs, although the mechanisms that mediate such links remain to be explained. Methodological concerns include the inconsistency of measurement of the disorders across studies, small sample sizes, and restricted follow-up times. Given the significant mental health burden placed by comorbid disorders, and their high prevalence across populations, research on the key risk factors for clustering of psychopathology is needed.anxietycomorbidityconduct disorderdepressionsocial conditionssubstance-related disordersINTRODUCTIONPsychiatric comorbidity is the presence, simultaneously or in sequence, of two or more disorders in an individual within a certain time period (1, 2). Psychiatric comorbidity is a prevalent phenomenon across age groups and types of populations (3–5). The Epidemiologic Catchment Area (ECA) Survey found that 35 percent of respondents with at least one lifetime disorder had one or more additional disorders during their lifetime (6); the National Comorbidity Survey Replication found that 27.7 percent of the respondents had two or more disorders during their lifetime (7).Understanding psychiatric comorbidity has important research, clinical, and nosologic implications. From a research perspective, epidemiologic research conducted on isolated disorders may underestimate the burden imposed by mental health problems because this group may represent a select portion of the population that suffers from psychiatric disorders (8). Documenting the relation between key psychiatric disorders over the life course can provide a more accurate assessment of the psychiatric burden experienced by different population groups.With respect to clinical concerns, comorbidity is associated with more severe psychiatric symptoms, more functional disability, longer illness duration, less social competence, and higher service utilization (9–11). Given the high prevalence of comorbidity and its clinical consequences, research efforts that aim to better understand the patterns of comorbidity over time are clearly essential to help guide its prevention.Investigation into the distribution of comorbidity across key psychiatric disorders can also help us elucidate the structure of phenotypic psychopathology. The cooccurrence of disorders has called into question the validity of current categorical classifications of mental disorders, with critics suggesting that a dimensional approach to classifying comorbid disorders is more appropriate (12). Studying comorbid patterns can thus help us discern commonalities and distinctions in the developmental pathways of comorbid disorders and in the etiologic profiles of each comorbid disorder—elements that will provide the basis to understand whether the disorders are different expressions of the same underlying disturbance or are distinct disorders in their own right (13).This review aims to examine current knowledge on the epidemiology of clustered forms of psychopathology over the life course, focusing on four of the most common mental disorders: anxiety, depression, conduct disorder, and substance abuse. According to the National Comorbidity Survey Replication, 28.8 percent of the US population suffered from an anxiety disorder sometime in their life, whereas 20.8 percent suffered from a mood disorder, 14.6 percent had a substance use disorder, and 9.5 percent presented symptoms of conduct disorder (7). The cooccurrence of these conditions has been consistently documented in clinical and population samples (5).This paper systematically reviews comorbidity studies within a life course framework, focusing exclusively on prospective population-based studies. This presents a novel approach to address comorbidity and enables us to study similarities and differences in comorbidity patterns at key life stages as well as to understand how the normative developmental trajectories of disorders may influence the magnitude of comorbidity from childhood to adulthood. Focusing on population-based studies overcomes one of the key limitations of clinical studies: selection bias that may determine the patterns of comorbidity observed in the sample.REVIEW OF THE LITERATUREThis review encompasses the peer-reviewed literature published between 1970 and 2007. We limited our review to these years to reflect current thinking about psychiatric comorbidity and to include studies that use methods considered standard today. The literature reviewed was identified through the Social Science Citation Index and Science Citation Index, and it covered studies about concurrent and sequential comorbidity between anxiety, depression, substance use/abuse, and conduct disorder. We restricted our review to longitudinal, population-based studies that provided information about the temporal associations between disorders. As stated by Kraemer et al. (14), cross-sectional studies, particularly those that use lifetime prevalence with mixed-age samples to estimate comorbidity, run the risk of identifying instances of “pseudocomorbidity” when no real comorbidity exists.The literature on comorbidity is vast and encompasses many different fields, from medicine to public health to psychology. We restricted our review to psychiatric definitions of the disorders because a wider review would have been beyond the scope of this project. For example, for conduct disorder, we considered measurement of symptoms or diagnoses of conduct disorder, oppositional defiant disorder, and antisocial personality disorder but not aggression or delinquency. The search was limited to English-language studies in biomedical research. Keywords and terms used for the search included primarily the following: 1) for substance abuse: substance abuse, alcohol abuse, drug abuse, cannabis, cocaine, heroin, street drugs, smoking, injection drug use; 2) for depression: depression, depression symptomatology, depression symptoms, mood disorders; 3) for conduct disorder: conduct disorder, antisocial personality disorder, antisocial, opposition defiant disorder, externalizing behavior/disorder; 4) for anxiety disorder: anxiety, anxiety symptomatology, anxiety symptoms, internalizing disorder; and 5) for comorbidity: comorbidity, joint trajectories, discordant trajectories, concurrent, cooccurrence, risk pathways, chain of risk, launch factors, transitions, risk pathways.FINDINGSIn this paper, we review concurrent and sequential comorbidity between the four disorders, and we present available data on concurrent comorbidity, sequential comorbidity, and contributions of each disorder to the deflection of developmental trajectories of the other three disorders. This review covers 58 studies, including those using child and adolescent samples, and then adult samples, to present available knowledge on comorbidity at key life stages. The original search provided 103 articles, of which 45 were removed because of the absence of a prospective study design. Although studies such as Kessler et al.'s (15) on the National Comorbidity Survey Replication or the cross-national comparison of comorbidity conducted by Merikangas et al. (16) offer groundbreaking data on the distribution of comorbid disorders in population-based samples, they were not included because they used retrospective measures of disorder onset in cross-sectional study designs.The studies reviewed here (summarized in tables 1 and 2) approach the issue of causality between comorbid disorders by investigating the relative timing of the onset of each disorder and the impact that the prevalence of one disorder has on the course of the other. The strength of the causal link depends on whether the cooccurrence of the disorders persists after accounting for common risk factors (3, 17).TABLE 1.Population studies on comorbid patterns between substance abuse, anxiety, depression and conduct disorder among children and adolescentsComorbidityCitationSampleFollow-up timeMeasurement of disordersConclusionsAnxiety and substance use/abuseGoodwin et al. (20)1,265 children (635 males, 630 females) from Christchurch, New Zealand21 yearsCIDI* and custom-written survey items used; alcohol dependence: DSM-IV* criteria; illicit drug dependence: DSM-IV criteria; nicotine dependence: DSM-IV; anxiety disorders (generalized anxiety disorders, panic disorder, agoraphobia, social phobia, and specific phobia): CIDI diagnostic to assess DSM-IV criteriaYoung people with anxiety disorders are at increased risk of substance dependence; association is largely noncausal and is explained by 1) presence of fixed childhood and family factors associated with both anxiety and substance dependence; and 2) time-dynamic associations including lagged effects of substance dependence and development of comorbid depression. OR* of substance dependence associated with anxiety disorder = 1.8 (95% CI*: 1.2, 2.6).Anxiety and substance use/abuseZimmerman et al. (22)2,548 adolescents aged 14–24 years in Munich, Germany4 yearsDiagnostic assessment based on the computer-assisted version of the DIA-X/M-CIDI*; DSM-IV anxiety disorders assessed; DSM-IV alcohol use disorders assessed include alcohol abuse without dependence and alcohol dependence without alcohol abuseAnxiety disorders are significant predictors of the subsequent onset and persistence of regular and hazardous alcohol use and alcohol use disorders. Panic disorder and social phobia are the only anxiety disorders involved in these predictive associations.Depression and substance use/abuseSilberg et al. (71)307 monozygotic and 185 dizygotic same-sex male twin pairs from Virginia; 392 monozygotic and 187 dizygotic like-sex female pairs4 yearsSubstance use based on ratings given by multiple informants during a home interview using the DSM-based CAPA,* conduct disorder measured with a composite rating based on child's response to CAPA symptoms, a diagnosis of conduct disorder and oppositional defiant disorder, and ratings on the Olweus aggression scale; depression based on CAPA symptoms associated with a diagnosis of major depressive episodeAmong boys, the cross-lagged correlations between conduct disturbance and substance use and between substance use and depression were approximately equal, indicating that a common factor contributed to these behaviors; among girls, there were differences in the pattern of the cross-lagged correlations for conduct disorder and depression, substance use and conduct disorder, and substance use and depression, indicating that conduct disorder preceded substance use and that depression followed substance use. Among girls, the cross-lagged associations were also consistent with conduct disorder leading to depression.Depression and substance use/abuseFergusson et al. (33)Birth cohort of 1,265 children (635 males, 630 females) born in Christchurch, New Zealand in mid-1977Assessed at birth, 4 months, 1 year, annually to age 16 years, and at ages 18 and 21 yearsSmoking based on written survey, nicotine dependence based on DSM-III-R* criteria at age 16 years and DSM-IV criteria at ages 18 and 21 years; major depression measured with DISC* with DSM-III-R criteria at age 16 years and CIDI with DSM-IV criteria at ages 18 and 21 yearsMajor depression was associated with increased rates of daily smoking (IRR* = 1.19 (95% CI: 1.03, 1.39)). and elevated rates of nicotine dependence (OR = 1.75 (95% CI: 1.13, 2.70)).Depression and substance use/abuseRao et al. (34)155 women aged 17–19 years recruited from three high schools in Los Angeles, CaliforniaFollow-ups conducted every 6 months up to 5 yearsAxis I disorders assessed with the Structured Clinical Interview for DSM-III-R; self-reported depressive symptoms assessed with the Beck Depression Inventory; self-reported problems of substance use assessed at the end of follow-up with the Rutgers collegiate Substance Abuse Screening TestDepression and substance use disorder were related concurrently; major depressive disorder and substance use disorder were significantly associated in follow-up; substance use disorder problem score and Beck Depression Inventory (self-reported depressive symptoms) during follow-up were also correlated. Major depressive disorder before the study did not predict substance use disorder during the follow-up period, whereas prior substance use disorder did predict major depressive disorder at follow-up. Prior substance use disorder also predicted the worst Beck Depression Inventory score during follow-up.Depression and substance use/abuseWindle and Davies (35)975 high school students from western New York State in the first wave, 220 added in the second wave; average age, 15.54 years (data collected from primary caregivers: 90% mothers)Time 1: 1 year since study; time 2: 2 years since studyDepression: CES-D* Scale criterion score of >23, “heavy drinking”: Quality Frequency Index—adolescents who consumed >45 drinks in the last 30 days24–27% of adolescents identified as depressed met the criteria for heavy drinking (33–37% of boys, 16–18.5% of girls); 23–27% of adolescent heavy drinkers were depressed (18–20% of boys, 27–33% of girls). Approximately 5% of participants had depression and were heavy drinkers at either wave. The mixed drinking and depression group showed the lowest stability between time 1 and time 2: only 20% of those in the mixed group at time 1 persisted in this group at time 2.Depression and substance use/abuseAseltine et al. (13)900 adolescents (9th, 10th, and 11th graders) from Boston, Massachusetts3 yearsDepressive symptoms measured with CES-D Scale; measures of alcohol and drug use adapted from Monitoring the Future studiesRate of cooccurring depression and substance use problems was 4% (n = 39); those with prior substance problems were more than twice as likely to develop cooccurring problems (9%) at time 2 than were the depressed (4%).Anxiety, depression, and substance use/abuseKaplow et al. (28)1,420 children in the total study from western North Carolina; for this analysis, only 936 included who completed child or parent report measures of generalized anxiety, separation anxiety, and depressive symptomatology as well as reported alcohol use at all four time points4 yearsCAPA used to assess psychiatric symptomsChildren with early symptoms of generalized anxiety were found to be at increased risk for initiation of alcohol use (OR = 1.14 (95% CI: 1.03, 1.25)), whereas children with early symptoms of separation anxiety were at decreased risk (OR = 0.71 (95% CI: 0.51, 0.94)); depressive symptomatology was associated with increased risk for initiation of alcohol use in adolescence (OR = 1.60 (95% CI: 1.29, 1.99)).Anxiety, depression, and substance use/abuseHayatbakhsh et al. (21)3,239 Australian young adultsBirth to age 21 yearsAnxiety and depression symptoms in the last 6 months measured in the 21-year follow-up by using the Young Adult Self-Report; cannabis use retrospectively assessed, occasional use = use of cannabis once a month or not in the past month, frequent use = use of cannabis every day or every few daysFrequent use of cannabis, either without (OR = 2.1 (95% CI: 1.1, 4.0)) or with (OR = 2.7 (95% CI: 1.8, 4.1)) use of other illicit drugs, predicts more than a twofold increase in anxiety and depression in young adults. No association was found between symptoms of anxiety and depression at age 14 years and either occasional or frequent use of cannabis by young adults. Frequent use of cannabis and early onset of use are associated with symptoms of anxiety and depression in young adults.Anxiety and depressionRepetto et al. (51)579 African-American adolescents in Ann Arbor, Michigan, who were at risk of dropping out of school4 yearsDepressive symptoms assessed with items from the Brief Symptom Inventory; anxiety symptoms assessed with six items from the Brief Symptom InventoryAdolescents who presented consistently high levels of depressive symptoms were more likely to report higher anxiety symptoms compared with adolescent members of other trajectories. Adolescents whose depressive symptoms followed the decreasing trajectory had higher levels of anxiety than those in the consistently low or increasing groups.Anxiety and depressionStein et al. (2)2,548 adolescents and young adults aged 14–24 years from Munich, GermanyOne follow-up 34–50 months laterMunich-CIDI used to diagnose by using DSM-IV criteria; social anxiety disorder defined as meeting DSM-IV criteria per the Munich-CIDI diagnostic criteria; depressive disorder defined as meeting DSM-IV criteria for one or more episodes of major depression or dysthymiaPersons with a combination of social anxiety disorder and depression are at the greatest risk of depression in early adulthood. Persons with depression and social anxiety disorder (current or previous) were significantly more likely (OR = 8.7 (95% CI: 4.5, 16.8)) than persons with no mental disorder to have experienced a depressive disorder during the follow-up period.Anxiety and depressionWittchen et al. (26)3,021 adolescents and young adults aged 14–24 years at first interview in Munich, GermanyThree waves over 4–5 yearsDiagnostic assessment based on the computer-assisted version of the Munich-CIDI; diagnoses made by using DSM-IV criteriaThe proportion of major depressive episodes among those with an anxiety disorder was low for those aged 14–17 years but increased at ages 18–24 years. Almost two thirds of cases with anxiety disorder were comorbid with major depression at follow-up (OR = 3.9 for generalized anxiety disorder, OR = 3.4 for panic disorder, and OR = 1.7 for specific phobia). The number of anxiety disorders, baseline impairment associated with anxiety, the frequency of avoidance, and the presence of panic-like attacks all increased the risk of secondary depression among those with primary anxiety disorders.Anxiety and depressionPine et al. (50)776 young people (aged 9–18 years) in upstate New YorkAssessed in 1983, 1985, and 1992: 9 yearsUsed DISC to assess DSM-III criteria from parents and children at times 1 and 2; at time 3, DISC used with the children onlyMajor depression at time 2 predicted generalized anxiety disorder at time 3. Overanxious disorder predicted major depression at time 3. Conduct disorder at time 1 was related to major depression at time 3 (OR = 2.54 (95% CI: 1.42, 7.68)). Conduct disorder at time 2 was related to major depression at time 3 (OR = 2.38 (95% CI: 1.10, 5.16)).Conduct disorder and substance use/abuseCohen et al. (60)749 adolescents aged 13.7 years at the first interview from two upstate New York countiesInterviewed in 1983, 1985–1986, and 1991–1994Substance use disorders and conduct disorder assessed with DISC-I; instrument administered to both the child and a parent; DSM-III-R diagnoses madeAdolescents with conduct disorder had 2.59 times the hazard of initiating marijuana use over the study period in comparison to adolescents without conduct disorder. Conduct disorder in early adolescence was independently associated with significantly elevated mean symptoms of alcohol abuse/dependency and marijuana use disorder across the age range.Conduct disorder and substance use/abuseJuon et al. (75)952 African-American children who began 1st grade in Woodlawn, Chicago, in 1966–1967 and remained in the Woodlawn school during their 1st year1966–1967; 1975–1976; 1992–1994Self-report measures of smoking frequency and quantity (instrument not specified); substance use disorder measured by using the modified version of CIDI based on DSM-III-R criteria; major depressive disorder measured with CIDI; antisocial behavior operationalized as ratings from 1st-grade teachers as aggressive or shy and aggressiveThose who had been rated as aggressive (OR = 2.10) or as both shy and aggressive (OR = 1.93) in the 1st grade were more likely to be current smokers/early adopters than nonsmokers. Those who had comorbidity of major depressive disorder and drug problems (OR = 4.70) were more likely to be current smokers/early adopters than nonsmokers compared with those who had no problems.Conduct disorder and substance use/abuseHussong et al. (66)461 men from the Dunedin, New Zealand, Multidisciplinary Health and Development Study8 years, at ages 18, 21, and 26 yearsAlcohol abuse and marijuana abuse assessed by symptoms from the DIS*; antisocial behaviors assessed via the self-report offending interview, which ascertains illegal behaviors and conduct problems; DSM-IV criteria used for these measuresSubstance abuse exerts both proximal and distal effects on desistance of antisocial behavior over young adulthood. Men with greater substance abuse at the end of adolescence showed greater antisocial behavior across young adulthood. Periods in which men reported greater symptoms of substance abuse corresponded to higher-than-normal levels of antisocial behavior.Conduct disorder and substance use/abuseMarmorstein and Iacono (67)289 families of male twins and 337 families of female twins, first assessed when the twins were approximately 17 years of age in Minnesota3 yearsConduct disorder measured from an adapted version of the Structured Clinical Interview for DSM-III-R Personality Disorders; substance dependence symptoms assessed by using adolescents' reports on a modified version of the Substance Abuse Module of CIDI; DSM-III-R criteria usedYouths with late-onset antisocial behavior had higher rates of substance dependence than controls for all three classes of substances. The late-onset group also had more nicotine and cannabis dependence than those with desisting behavior. Youths with persisting antisocial behavior had more nicotine, alcohol, and cannabis dependence than youths with no antisocial behavior and youths with desisting antisocial behavior.Conduct disorder and substance use/abuseWhite et al. (64)506 boys in the Pittsburgh Youth Study6 yearsDrug use: assessed with the 16-item substance-use scale adapted from the National Youth Survey; psychopathologic characteristics: assessed with the Self-Report Delinquency Scale, the Youth Self-Report, and the caretaker-completed DISC-Parent Version; depressed mood = number of symptoms from the Recent Moods and Feelings QuestionnaireMarijuana use was associated with higher overall level of conduct disorder but did not affect the rate of growth of conduct disorder. No association was found between oppositional defiant disorder scores and marijuana use; oppositional defiant disorder was a significant predictor of level of alcohol use. Number of symptoms of depression was positively related to level of alcohol use but not marijuana use.Conduct disorder and depressionIngoldsby et al. (68)431 school-age children from Durham, North Carolina; Nashville, Tennessee; Seattle, Washington; central Pennsylvania5th−7th gradeConduct problems: youth completed the 32-item Things You Have Done survey at the end of 5th grade, youth completed the Self-Report Delinquency Scale at the end of 7th grade; parents rated Child Behavior Checklist, teachers completed the externalizing scale on the Teacher's Report Form; depressive symptomatology: youth completed the Reynolds Child Depression Scale at the end of 5th and 7th grades and parents completed a subscale of the Child Behavior ChecklistThis study did not find support for the hypothesis that conduct problems predict increases in depression in early adolescence. Cases of comorbid conduct problems and depressive symptoms were significantly persistent over time. A substantial portion of youth in the conduct problems group in 5th grade moved to the cooccurring group by 7th grade. Serious depression or conduct problems by 7th grade occurred more frequently in comorbid cases than in cases with either disorder alone.Conduct disorder and depressionKim et al. (70)206 boys recruited through 4th-grade classes (depression studied from ages 14–15 to 23–24 years) from Oregon10 yearsDepressive symptoms: assessed with the CES-D Scale; antisocial behavior in late childhood: antisocial construct from items from the Teacher Child Behavior Checklist, telephone interview scores, peer nominations, home observations, and interviewer impressionEarly antisocial behavior was associated with depressive symptoms in adulthood.Conduct disorder, substance use/abuse, and depressionMeaselle et al. (36)Community sample of 496 female adolescents who varied in age from 13 to 15 years at first assessment from the metropolitan area of the southwestern United States5 yearsDepressive symptoms: adapted version of the K-SADS*; antisocial behavior symptoms: Externalizing Syndrome of the Child Behavior Checklist; substance abuse symptoms: items adapted from Stice et al. (76); DSM-IV criteria used for allInitial depression level predicted increases in substance abuse symptoms and slower decreases in antisocial behavior; initial levels of antisocial symptoms predicted increases in depression and substance abuse; initial substance abuse level predicted slower decelerations in antisocial behavior.Conduct disorder, anxiety, and depressionGregory et al. (49)1,037 members of a birth cohort in Dunedin, New Zealand (baseline beginning at age 11 years)21 years: follow-ups at ages 11, 13, 15, 18, 21, 26, 32 years (N = 972 at this follow-up)At ages 11–15 years, DISC used to diagnose mental health according to DSM-II; at ages 18–32 years, DIS used to assess mental health by using DSM-IV criteriaAdult anxiety cases were also more likely to have experienced juvenile depression relative to those without adult anxiety. Adults with social phobia, agoraphobia, and posttraumatic stress disorder were more likely to have experienced externalizing spectrum disorders than those without these disorders: adults with posttraumatic stress disorder were likely to have met diagnostic criteria for conduct or oppositional defiant disorder.Conduct disorder, anxiety, and depressionLavigne et al. (61)510 children aged 2–5 years at baseline from Chicago, Illinois5 yearsExternalizing and internalizing behavior problems assessed by the Child Behavior Checklist; DSM-III-R diagnoses conducted with the Diagnostic Interview for Children and AdolescentsSingle-diagnosis oppositional defiant disorder at wave 1 was associated with later comorbidity of oppositional defiant disorder and mood disorders and of comorbidity of oppositional defiant disorder and an anxiety disorder. There was moderate-to-high stability for oppositional defiant disorder comorbid with anxiety and low stability for oppositional defiant disorder comorbid with mood disorder.Conduct disorder, anxiety, depression, and substance use/abuseLoeber et al. (74)Pittsburgh Youth Study of three samples of boys in grades 1 (age 6.9 years), 4 (age 10.2 years), and 7 (age 13.4 years) (each sample including 250 of the most antisocial boys in each grade plus 250 randomly selected others)Youngest group assessed at screening and at eight assessment waves spaced at 6-month intervals; middle group assessed at screening and six assessment waves; oldest group assessed at screening and seven assessment wavesChild Behavior Checklist and the DISC-Parent Version administered to caretaker; teachers given the Teacher Report Form; boys assessed with the National Youth Survey 40-item Self-Reported Delinquency Scale and the 16-item Substance Use Scale based on the National Youth Survey; oppositional defiant disorder: DSM-III-R based on DISC-Parent VersionPersistent substance use in preadolescence was predicted by persistent delinquency and internalizing problems, while persistent substance use in adolescence was predicted by persistent delinquency only. Comorbid persistent substance use and delinquency were predicted by oppositional defiant disorder in middle childhood and by persistent internalizing problems in middle-to-late childhood.Conduct disorder, anxiety, depression, and substance use/abuseRohde et al. (77)1,507 participants selected from high schools in western Oregon aged 14–18 yearsMean = 13.8 monthsAdolescents interviewed with a version of K-SADS that combined features of the epidemiologic version (K-SADS-E) to derive diagnoses based on DSM-II-R criteria; alcohol use: four symptoms for alcohol use and seven symptoms for dependence assessed; psychopathology: diagnoses clustered among DSM subdivisionsOf adolescents with alcohol abuse/dependence, 20% had an internalizing disorder, 35% had an externalizing disorder, and 45% had both internalizing and externalizing disorders. 87.5% of the time, anxiety disorders preceded alcohol disorders, and, 80.0% of the time, disruptive behavior disorders preceded alcohol disorders. Comorbidity was associated with an earlier age at onset of alcohol disorder; 16% of adolescents with a current psychiatric disorder moved into a more problematic alcohol use group from time 1 to time 2 vs. 5.8% of adolescents with no diagnosis between the assessment points.Conduct disorder, anxiety, depression, and substance use/abusePardini et al. (25)506 boys in the Pittsburgh Youth StudyAverage of 12.5 years; boys screened, assessed at ages 13.9, 20.4, and 25.4 yearsDepression: assessed with the self-report Recent Moods and Feelings Questionnaire (contains items with DSM-III criteria); anxiety/withdrawal: assessed with a scale that combined the child (youth self-report), teacher (Teacher Report form), and parent-report Child Behavior Checklist; alcohol use disorders (assessed with the DIS on two occasions in early adulthood (DSM-III-R diagnoses)Early conduct disorder symptoms predicted increased alcohol use disorder symptoms and alcohol dependence diagnosis by young adulthood. Adolescent boys with high levels of anxiety/withdrawal had lower levels of alcohol use disorder symptoms and were less likely to develop alcohol dependence by young adulthood. Increased depression in early adolescence was associated with higher alcohol use disorder symptoms and alcohol abuse and dependence diagnoses by young adulthood, but only for boys with high levels of conduct disorder symptoms.Conduct disorder, anxiety, depression, and substance use/abuseCostello et al. (18)1,420 children from western North Carolina3–5 years (children aged 9–13 years assessed annually until age 16 years)CAPA used to assess psychiatric symptomsSignificant comorbidity was found among the behavioral disorders and anxiety and depression; depression was comorbid with conduct disorder in girls but not boys; depression was comorbid with substance use disorder in boys but not girls; anxiety predicted later substance use disorder, but depression did not; in girls, but not boys, anxiety predicted depression even when controlling for current comorbidity.Conduct disorder, anxiety, depression, and substance use/abuseKrueger et al. (8)961 members of an unselected birth cohort from Dunedin, New ZealandAged 18–21 yearsMental disorders (in the prior 12 months) assessed by using the DIS to generate diagnoses according to DSM-III-R criteriaA two-factor model provided the best fit to the data: major depressive disorder, dysthymia, generalized anxiety disorder, agoraphobia, social and simple phobia, and obsessive compulsive disorder were presumed to reflect internalizing problems; conduct disorder and marijuana and alcohol dependence were presumed to reflect externalizing problems. Subjects retained their positions regarding the latent factors over the 3-year period.Conduct disorder, anxiety, depression, and substance use/abuseWittchen et al. (27)1,395 community subjects from Munich, Germany, aged 14–17 years at baselineThree waves over 10 yearsSubstance use, abuse, and dependence, as well as mental disorders, assessed with the DIA-X/M-CIDI, according to DSM-IVMajor depression was associated with a higher risk of cannabis use and dependence: (OR for cannabis use = 2.2 (95% CI: 1.3, 3.7); OR for cannabis dependence = 2.4 (95% CI: 1.3, 4.5)). Hypomania and mania were associated with incident cannabis use and cannabis dependence (OR for cannabis use = 2.4 (95% CI: 1.0, 5.4); OR for cannabis dependence = 2.7 (95% CI: 1.1, 6.3)). Among anxiety disorders, only panic-anxiety was associated with cannabis use (OR = 3.1 (95% CI: 1.4, 6.8)). Conduct problems predicted cannabis use (OR = 2.3 (95% CI: 1.2, 4.2)).Conduct disorder, anxiety, depression, and substance use/abuseDierker et al. (19)173 high- and low-risk youths in New Haven, Connecticut (identified on the basis of parental history of substance abuse) aged 7–17 years1988–1998Child diagnoses based on K-SADS and independent ratings from DSM-III-R obtained from a child and adolescent psychiatrist; tobacco assessed using K-SADSPsychiatric disorders were significantly associated with nicotine dependence: the association between having a psychiatric disorder and dependence vs. lower levels of use were as follows: OR for anxiety = 4.5 (95% CI: 1.71, 11.94), OR for affective disorder = 10.7 (95% CI: 3.79, 30.25), OR for conduct disorder = 5.0 (95% CI: 1.6, 15.73), OR for oppositional defiant disorder = 5.8 (95% CI: 1.80, 18.43); in the low-risk population, oppositional defiant disorder was the only risk factor that predicted transition to nicotine dependence.Conduct disorder, anxiety, depression, and substance use/abuseCostello et al. (30)1,420 children from western North CarolinaFollowed from ages 9, 11, and 13 years to age 16 yearsCAPA used to assess psychiatric symptomsDisruptive behavior disorders and depression were associated with a higher rate and earlier onset of substance use and abuse in both sexes, but anxiety predicted later onset of smoking. Onset of conduct disorder and anxiety was well before the first use of any substance. Substance use and abuse of every type were more common among girls with behavior disorders; depressed boys, compared with nondepressed boys, had significantly higher rates of every type of substance use.Conduct disorder, anxiety, depression, and substance use/abuseSung et al. (62)1,420 children aged 9, 11, and 13 years from North Carolina3–7 years (until participant aged 16 years, reviewed annually)CAPA used in interviews; substance use, abuse, and dependence items used; psychiatric disorders classified by using DSM-IV criteria for symptoms experienced in the past 3 monthsConduct disorder, particularly at earlier ages (14 and 15 years) was the strongest psychiatric predictor of transition from substance use to a disorder (OR = 2.0 (95% CI: 1.2, 3.6)). Boys, but not girls, with a history of depression were at increased risk of substance use disorder; anxiety increased the risk of substance use disorder in girls at age 16 years.*CIDI, Composite International Diagnostic Interview; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; OR, odds ratio; CI, confidence interval; DIA-X/M-CIDI; Munich-Composite International Diagnostic Interview; CAPA, Child and Adolescent Psychiatric Assessment; DSM-III-R, Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised; DISC, Diagnostic Interview Schedule for Children; IRR, incidence rate ratio; CES-D, Center for Epidemiological Studies Depression; DIS, Diagnostic Interview Schedule; K-SADS, Schedule for Affective Disorders and Schizophrenia and School-Age Children.TABLE 2.Population studies on comorbid patterns between substance abuse, anxiety, depression and conduct disorder among adultsComorbidityCitationSampleFollow-up timeMeasurement of disordersConclusionsAnxiety and substance use/abusede Graaf et al. (78)7,076 adults aged 18–64 years from the NetherlandsRetrospective; used age at onset dataDiagnoses based on DSM-III-R* from the CIDI*The association between major depression and alcohol or drug dependence was stronger than that for alcohol or drug abuse. Men were more likely than women to report major depression temporally secondary to any other disorder; of those who had ever experienced a mood disorder, 46% of males and 57% of females had a history of anxiety disorders, and 43% of males and 15% of females had a substance use disorder; in most anxiety-comorbid cases, mood disorder arose after anxiety disorder.Anxiety and substance use/abuseKushner et al. (24)454 incoming freshmen at a large universityAt years 1, 4, and 7Anxiety disorders (generalized anxiety disorder, social phobia, agoraphobia with panic attacks, panic attacks in the absence of agoraphobia): assessed with DIS* version III-R to use criteria from DSM-III; alcohol abuse/dependence: used DIS and DSM-IIIStrong evidence was found for reciprocal causal influences between anxiety disorders and alcohol dependence; having an anxiety disorder at years 1 or 4 quadrupled the risk for new onset of alcohol dependence at year 7.Anxiety and substance use/abuseNewcomb et al. (31)470 adults from Los Angeles, California4 yearsPolydrug problems assessed with items based on DSM-IV* criteria for drug abuse and dependence; anxiety (included in the agitation measure) measured by three items on a 5-point Likert rating scale, taken from the Hopkins Symptom ChecklistEarly dysphoria directly increased agitation as an adult. Polydrug problems in young adulthood increased anxiety in adulthood. Social conformity accounted fully for the relation between polydrug problems and agitation. Marijuana problems in early adulthood increased later suicidal ideation. Early cocaine problems increased later hostility. Dysphoria in young adulthood increased alcohol problems but decreased cocaine abuse in later adulthood.Anxiety and substance use/abuseSartor et al. (79)1,269 offspring (average age, 20.1 years) of male twins from the Vietnam Era Twin Registry in the United StatesRetrospectiveDrinking outcomes measured with Semi-Structured Assessment for the Genetics of Alcoholism (onset defined as age at which full DSM-IV criteria were met); lifetime diagnoses for DSM-IV mood, anxiety, and conduct-related disorders also obtainedNicotine dependence (HR* = 3.91 (95% CI*: 2.48, 6.17)) and generalized anxiety disorder (HR = 3.45 (95% CI: 2.08, 5.72)) were robust predictors of progression from first drink to alcohol dependence; conduct disorder (HR = 1.75 (95% CI: 1.10, 2.77)) and cannabis abuse (HR = 1.88 (95% CI: 1.22, 2.90)) were also associated with rapid transition to alcohol dependence.Anxiety and substance use/abuseSonntag et al. (29)2,548 individuals aged 14–15 years at baseline from Munich, GermanyAverage, 19.7 monthsPsychopathologic and diagnostic assessments based on the computer-assisted personal interview version of the DIA-X/M-CIDI*When age/gender/comorbid depressive disorder were controlled for, baseline nonusers with at least one social fear but no social phobia diagnosis showed elevated odds for an occurrence of nicotine dependence in the follow-up period (OR* = 3.85 (95% CI: 1.34, 11.00)). All baseline, nondependent, regular smokers with social fears but not social phobia also showed higher odds for nicotine dependence (OR = 1.5 (95% CI: 1.01, 2.23)).Anxiety and substance use/abuseBovasso (80)1,920 individuals in the Baltimore (Maryland) Epidemiologic Catchment Area Survey15 yearsDIS assessed symptoms of DSM-III disorders at baseline and DSM-III-R symptoms at baseline; outcomes measured by using the 20-item version of Goldberg's General Health Questionnaire to assess general distress, DIS for psychiatric disorders, and DIS for symptoms of substance abuse/dependenceAnxiety/depression did not predispose individuals to substance use, and substance use did not predispose individuals to anxiety/depression.Depression and substance use/abuseCrum et al. (40)1,920 adults from Baltimore, Maryland (average age, 41.7 years at baseline; range, 18–86)Median 12.6 years of follow-upDepression measured with DIS by using DSM-III criteria; alcohol dependence measured as 1) lifetime and 2) new onset; DSM-III criteria used at baseline and 1 year follow-up, DSM-III-R criteria used at 12.6-year follow-upDepressive syndrome was associated with lifetime prevalence of alcohol dependence: for women, OR = 1.89 (95% CI: 1.16, 3.06), while, for men, OR = 1.83 (95% CI: 1.14, 2.93). Depressive syndrome was not associated with onset of alcohol dependence. Having a current or prior anxiety disorder increased the odds of alcohol dependence among women: OR = 2.35 (95% CI: 1.28, 4.32).Depression and substance use/abuseDixit and Crum (41)1,383 women from Baltimore, Maryland, ranging in age from 18 to ≥65 years, who did not report heavy alcohol use at baseline1 yearCenter for Epidemiologic Studies Depression Scale heavy alcohol use measured as five or more drinks at least once in the past month; depression measured with DIS by using DSM-III criteriaThe estimated risk of heavy alcohol use for women with major depression, depressive syndrome, and/or dysthymia was 2.22 greater than the risk for women without such a history (95% CI: 1.00, 4.92). The adjusted relative risk of heavy alcohol use associated with an incremental number of lifetime-experienced depressive symptoms was estimated to be 1.09 (95% CI: 1.01, 1.18).Depression and substance use/abuseGilman and Abraham (42)14,480 respondents not meeting DSM-III criteria for alcohol dependence or lifetime major depression, mean age, 43 years (range, 18–96), in five US community sites located near Duke University; Johns Hopkins University; University of California, Los Angeles; Washington University; and Yale University1 yearBoth alcohol dependence and major depression measured with the DIS by using DSM-III criteria; administered by nonclinician interviewersThe odds of alcohol dependence associated with number of baseline depressive symptoms were, for females, 1–3 symptoms, OR = 2.75 (95% CI: 1.52, 5.00), 4–6 symptoms, OR = 3.52 (95% CI: 1.86, 6.66), ≥7 symptoms, OR = 7.88 (95% CI: 2.86, 21.70); for males, 1–3 symptoms, OR = 1.50 (95% CI: 1.08, 2.08). The odds of depression associated with 1–3, 4–6, and ≥7 alcoholic symptoms, respectively, were, for females, OR = 1.66 (95% CI: 1.05, 2.64), OR = 3.98 (95% CI: 2.37, 6.69), and OR = 4.32 (95% CI: 1.92, 9.75); for males, OR = 1.19 (95% CI: 0.76, 1.88) OR = 2.49 (95% CI: 1.26, 4.92), and OR = 2.12 (95% CI: 0.90, 5.00). The odds of major depression corresponding to a DSM-III alcohol dependence diagnosis were 3.52, 95% CI: 2.16, 5.72 for females and 1.77 (95% CI: 1.08, 2.91) for males.Depression and substance use/abuseHettema et al. (43)2,926 male and 1,929 female adult twin subjects aged 21–62 years in Richmond, VirginiaRetrospective (based on age-at-onset data)Lifetime diagnoses for DSM-III-R major depressive disorder, generalized anxiety disorder, panic disorder, phobias, alcohol dependence, and psychoactive substance use disorder assessed via telephone and face-to-face structured psychiatric interviews based on the modified SCID*Generalized anxiety disorder and major depressive disorder had the highest same-year onsets (about 41%); panic disorder tends to occur after major depressive disorder in females but before major depressive disorder in males; the majority of phobias precede major depressive disorder in both genders. Onset of alcohol dependence precedes that of major depressive disorder in about one third of cases; for psychoactive substance use disorder, onset for females was about evenly divided between those occurring before (44%) and after (49%) major depressive disorder, significantly more males (54%) reported onset before that of major depressive disorder. Generalized anxiety disorder and panic disorder cases had the highest risk of developing concurrent major depressive disorder, with same-year-onset HRs of 38.07 and 12.28, respectively.Depression and substance use/abuseKuo et al. (37)7,477 twins, including 3,302 females with a mean age when last assessed of 36.5 years and 4,175 males with a mean age of 37.0 years in VirginiaTwo measurements: waves 1 and 4 for female-to-female twin pairs and waves 1 and 2 for male-to-male and male-to-female twin pairsDSM-III-R diagnoses of major depression and alcohol dependence assessed by using adapted versions of standard structured interviews (not specified)Risk of developing alcohol dependence associated with lifetime or concurrent major depression was higher for males than for females. Risk of alcohol dependence in year 1 associated with major depression onset in the prior year was higher than that associated with lifetime major depression: twofold for males and fourfold for females. Among females, the risk of alcohol dependence associated with prior major depression was higher than that associated with concurrent major depression (HR = 5.1 vs. HR = 3.1). Lifetime alcohol dependence predicted increasing HR of major depression for both genders, and the effect was stronger for females. Concurrent alcohol dependence had the highest effects on major depression risk. However, compared with lifetime alcohol dependence, preceding alcohol dependence did not increase major depression risk.Anxiety, depression, and substance use/abusede Graaf et al. (81)4,796 adults from the NetherlandsBaseline, 1 year, 3 yearsCIDI usedOf respondents who developed an incident disorder, 16.1% developed comorbid disorders; anxiety-comorbid mood disorder was the most frequent incident comorbid disorder among females, and both anxiety-comorbid and substance use-comorbid mood disorders were the most frequent among males.Anxiety, depression, and substance use/abuseJohn et al. (38)Random adult population sample aged 18–64 years (n = 4,075) from northern Germany (Lubeck and surrounding communities)Baseline, 30 months, and 36 monthsNicotine dependence, affective and anxiety disorders according to DSM-IV assessed with the computer-assisted DIA-X/M-CIDIMale former or current daily smokers had higher odds ratios of affective and anxiety disorders: compared with male never smokers, male current daily smokers had 4.5 higher odds of having an anxiety disorder. Having 1–2 nicotine dependence symptoms was associated with higher odds of affective (OR = 1.7 (95% CI: 1.2, 2.4)) and anxiety (OR = 1.6 (95% CI: 1.2, 2.2)) disorders; having ≥3 symptoms was associated with 3.6 times higher odds of affective disorders (95% CI: 2.6, 5.0) and 3.7 higher odds of anxiety disorders (95% CI: 2.8, 5.0).Anxiety and depressionBeesdo et al. (53)3,021 individuals from Munich, Germany, aged 14–24 years at baseline and 21–34 years at follow-up10 yearsIndividuals assessed with the DIA-X/M-CIDISocial anxiety disorder was consistently associated with subsequent depression, independent of age at onset of social anxiety disorder (relative risk range: 1.49–1.85).Anxiety and depressionBreslau et al. (48)1,007 individuals from a list of all members of a large health maintenance organization aged 21–30 years in Michigan3.5 yearsDIS used, following DSM-III-R criteriaHigher lifetime prevalence of major depressive disorder in females vs. males was primarily for major depressive disorder comorbid with anxiety; prior history of an anxiety disorder was associated with increased risk of major depressive disorder in males and females. Prior anxiety disorder accounted for the higher prevalence of major depressive disorder in females.Anxiety and depressionBreslau et al. (47)1,007 individuals from a list of all members of a large health maintenance organization aged 21–30 years in Michigan3.5 yearsDIS used, following DSM-III-R criteriaPrior anxiety was associated with a higher risk of major depressive disorder in both sexes. Women were not more vulnerable than men to depression because of an anxiety disorder. Substance use disorder does not play the comparable role in men that anxiety plays in women in contributing to depression onset.Anxiety and depressionde Graaf et al. (9)7,065 adults aged 18–65 years in the NetherlandsBaseline, 1 year, 3 yearsCIDI used15.2% of pure mood, 10.5% of anxiety, and 6.8% of substance use disorder cases became comorbid; half the transitions from pure substance use disorders were into mood-comorbid and half into anxiety-comorbid.Anxiety and depressionGrant et al. (57)102 undergraduates with an average age of 18.64 years from an undergraduate college in the United States1 yearSocial anxiety measured by using the Social Phobia and Anxiety Inventory; depressive symptoms measured by using the Beck Depression Inventory22% of the total effect of time 1 social anxiety on time 2 depressive symptoms was accounted for by avoidance of expressing emotions. Individuals with social anxiety disorder may develop depressive symptoms to the extent that they withhold strong emotions, which may lead to a feeling of loss of self.Anxiety and depressionMerikangas et al. (55)4,547 subjects (2,201 men and 2,346 women) aged 19 and 20 years representative of the canton of Zurich, Switzerland15 yearsThe Structured Diagnostic Interview for Psychopathologic and Somatic Syndromes used to make diagnoses according to DSM-III and DSM-III-R criteria for most major categoriesConcurrent comorbidity between anxiety and depression was highly stable across the 15 years of the study (OR range: 3.0–6.0); subjects with anxiety only had a moderate probability of developing depression only (OR range: 1.5–7.8), whereas those with depression only did not develop anxiety only (OR range: 0.3–1.8); substantial transitions from pure anxiety or pure depression to comorbid depression/anxiety (OR range: 1.5–10.0); women were more likely than men to transition from pure anxiety or depression to comorbid conditions at follow-up.Anxiety and depressionMoffitt et al. (54)1,037 members of a birth cohort (972 were assessed by the last measurement) in New Zealand21 years (studied at ages 11, 13, 15, 18, 21, 26, and 32 years)At ages 11, 13, and 15 years, diagnoses with DSM-III criteria made with the DISC*-Child Version and, at older ages, with the DIS (at ages 18 and 21 years, DSM-III-R; at ages 26 and 32 years, DSM-IV); both generalized anxiety disorder and major depressive disorder diagnosedCumulative prevalence of adult comorbid major depressive disorder + generalized anxiety disorder was 12% in the cohort; sequential and cumulative comorbidity was balanced: ≤37% of cases of depression were preceded by anxiety disorders, but 63% were not; the two disorders had strong cumulative lifetime comorbidity from ages 11 to 32 years (OR = 5.3 (95% CI: 3.9, 7.2)); of lifetime major depressive disorder cases, 48% had lifetime anxiety disorder; of lifetime anxiety cases, 72% had lifetime major depressive disorder.Anxiety and depressionWetherell et al. (46)1,391 Swedish twins with an average age of 60.9 years at first measurementInterviewed in 1987, 1990, and 1993Depressive symptoms assessed with the Center for Epidemiologic Studies Depression Scale; anxiety assessed with the State Anxiety subscale of the State-Trait Personality InventoryA two-factor model with latent depression and anxiety factors provided the best fit to the data; the two factors were highly correlated (0.84). Between 1987–1990 and 1990–1993, the parameters for the paths linking anxiety to subsequent depression were significant, whereas the paths linking depression to subsequent anxiety were not significant and could be dropped from the model.Anxiety and depressionWilhelm et al. (56)156 young adults enrolled in a postgraduate teachers' training program in the United States15 yearsDIS and subsequently its newer form, CIDI, used to find cases of depression and anxiety; DIS and CIDI used to generate DSM-III and then DSM-III-R diagnosesSubjects with two or more episodes of major depression had higher rates of meeting “major anxiety” and “all anxiety” status than those with one episode. Those with one episode had higher rates than subjects in the “no depression” group. Mean age at onset of simple phobia was significantly younger among those who had multiple episodes of depression. One of the best predictors to distinguish between “no depression” and “one or more episodes” was major anxiety.Anxiety and depressionSchoevers et al. (12)2,173 subjects aged 65–84 years from the Amsterdam Study of the Elderly3 yearsDutch translation of the Mini-Mental State Examination, the Geriatric Mental State Examination, the Activities of Daily Living scale, the Instrumental Activities of Daily Living scale, and the Cambridge Mental Disorders of the Elderly Examination interview used; diagnoses of depression, organic “caseness,” and generalized anxiety reached by use of the Geriatric Mental State–AGECAT systemDepression and generalized anxiety disorder together had a significantly worse prognosis (27% remission) than the “pure” categories: 4 of 199 subjects with depression developed generalized anxiety disorder (pure generalized anxiety disorder), but 27 (13.6%) developed pure depression/generalized anxiety disorder; subjects with pure depression/generalized anxiety disorder at baseline were more likely to keep the combined disorder than have it change into pure forms.Conduct disorder and substance use/abuseNock et al. (82)3,199 respondents aged 18–44 years in the National Comorbidity Survey Replication in the United StatesRetrospective study (based on age-at-onset data)Mental disorders assessed by using CIDI to generate diagnoses according to DSM-IV criteria and the ICD-10* diagnostic systemOppositional defiant disorder is associated with substantial risk of secondary mood, anxiety, impulse-control, and substance use disorders; 92.4% of respondents with lifetime oppositional defiant disorder met criteria for at least one other lifetime disorder; oppositional defiant disorder was significantly comorbid with agoraphobia without panic disorder (OR = 2.1) and conduct disorder (OR = 12.6). The ORs for impulse control disorders were 4.0–12.6, and the ORs for anxiety disorders were 2.1–4.5.Conduct disorder and substance use/abuseAngst et al. (39)591 adults (aged 19–20 years at baseline) selected from a representative sample of Zurich, Switzerland: two thirds were high scorers in the Symptom Checklist 90-R Global Severity Index (GSI), and one third were a random sample of those whose GSI score was below the 85th percentile20 yearsAlcohol abuse and dependence classified according to DSM-IV criteria; major depressive episodes defined by DSM-III criteria in the first two interviews (1979 and 1981) and DSM-III-R criteria in the last four interviews (1986, 1988, 1993, and 1999)Major depressive episodes were associated with alcohol dependence but not with alcohol abuse: for men, OR = 3.8 (95% CI: 2.0, 7.1); for women, OR = 5.2 (95% CI: 1.8, 14.5).Conduct disorder and substance use/abuseKratzer and Hodgins (63)6,449 males and 6,268 females from Sweden30 yearsConduct problems in school: teachers rating students on a 3-point scale at grades 6 and 9; conduct problems in the community: defined as children referred to the Child Welfare Committee; mental disorders: Stockholm county register screened to document admissions to psychiatric wardsMales identified with conduct problems in childhood were more likely than the no-conduct-problems group to develop substance abuse in adulthood; females identified with conduct problems in childhood were at greater risk than no-conduct-problems females for crime and for mental disorders in adulthood; a diagnosis of substance abuse accounted for most of the mental disorders among conduct-problem-school but not conduct-problem-community females.Conduct disorder and depressionJohnson et al. (69)658 individuals from New York18–21 years total: assessed in 1983, 1985–1986, 1991–1993, and finally in 2001–2004Axis I disorders assessed with DISC-I, the nonpatient version of the structured clinical interview for DSM-IV (SCID-IV-NP) at age 33 years; assessment of personality disorders with items from the Personality Diagnostic Questionnaire, the Structured Clinical Interview for Personality Disorders (SCID-II) and DISC-IIndividuals with a DSM-IV cluster A (paranoid, schizoid, or schizotypal) or cluster C (avoidant, dependent, obsessive-compulsive) panic disorder by mean age 22 years were at elevated risk of recurrent or chronic unipolar depression; antisocial, borderline, dependent, depressive, histrionic, and schizotypal panic disorder traits, identified between the ages of 14 and 22 years, were significantly associated with risk of dysthymic disorder or major depressive disorder.Conduct disorder and anxietyJohnson et al. (72)658 individuals from Albany and Saratoga counties, New York18–21 years total: assessed in 1983, 1985–1986, 1991–1993, and finally in 2001–2004Axis I disorders assessed with DISC-I at ages 14–21 years, the nonpatient version of the structured clinical interview for DSM-IV (SCID-IV-NP) at age 33 years; personality disorders: Personality Diagnostic Questionnaire, the Structured Clinical Interview for Personality Disorders (SCID-II), and DISC-I usedAntisocial disorder between 14 and 22 years of age was associated with elevated risk of anxiety disorders by mean age 33 years. Antisocial personality traits were associated with risk of agoraphobia at age 33 years (OR = 1.68 (95% CI: 1.14, 2.47)).Conduct disorder and anxietyNock et al. (82)3,199 adults aged ≥18 years in the coterminous United StatesRetrospectiveCIDI to diagnose based on DSM-IV and ICD-10Conduct disorder typically precedes mood and substance use disorders but occurs most often after impulse and anxiety disorders; presence of conduct disorder is associated with an elevated risk of substance use disorders (OR = 5.9); conduct disorder predicted the presence of comorbid anxiety disorders (OR = 1.5, p = 0.038).*DSM-III-R, Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised; CIDI, Composite International Diagnostic Interview; DIS, Diagnostic Interview Schedule; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; HR, hazard ratio; CI, confidence interval; DIA-X/M-CIDI; Munich-Composite International Diagnostic Interview; OR, odds ratio; SCID, Structured Clinical Interview for DSM-III-R; DISC, Diagnostic Interview Schedule for Children; ICD-10, International Statistical Classification of Disease and Related Health Problems, Tenth Revision.Anxiety and substance useThe evidence on the link between anxiety and substance use is mixed: most studies have been cross-sectional or retrospective in design and used dimensional rather than diagnostic measures of anxiety. The few existing prospective, diagnostic studies (18–22) propose several alternative causal associations between anxiety and substance abuse: 1) individuals with an anxiety disorder may be more likely to develop or sustain a substance use disorder, in part as a way to manage the symptoms of anxiety (21, 23, 24); 2) having an anxiety disorder may reduce or delay the onset of behavioral problems such as substance abuse either because of the high level of anxious social cognitions that serve as a reminder of the negative consequences of the behavior or because of behavioral inhibition that results from anxiety disorders (25); 3) substance use may exert physiologic effects that increase vulnerabilities to anxiety disorders; or 4) a third variable may promote the development or maintenance of both (20).Children and adolescents.Childhood and adolescence is a critical time to investigate the timing of onset of the comorbid disorders since anxiety has a relatively early age at onset (26). Few studies have looked prospectively at the link between anxiety and substance abuse among the younger age groups. Several studies have found support for the “self-medication” thesis: that persons with anxiety use alcohol and drugs to suppress their anxiety symptoms (18, 22, 27–29). Costello et al. (18) found that anxiety predicted later substance use disorders among a representative population sample of adolescent girls, but not among boys. However, several studies have also found support for the reverse association, in which substance dependence promotes the development of anxiety (20, 21). A particularly compelling study that followed children over 21 years and obtained prospective reports of anxiety and substance dependence found that the association between anxiety and substance dependence was largely or entirely noncausal, while substance dependence predicted anxiety disorder at follow-up. Finally, some evidence also exists for a negative relation between anxiety and substance use: Pardini et al. (25) followed the Pittsburgh Youth Study sample of boys from early adolescence to young adulthood, and they found that higher levels of anxiety/withdrawal were inversely related to symptoms and diagnoses of alcohol use disorder; a cohort study of a representative population sample aged 9–13 years at baseline found that anxiety predicted a later onset of smoking (30).Lack of conclusive evidence is due to the heterogeneity of substance use and anxiety subtypes examined in the few available population-based studies: associations may differ between subtypes. Kaplow et al. (28) proposed that, in studies that consider anxiety as a general syndrome and fail to differentiate within its dimensions, the opposing functions of different types of anxiety may offset each other, resulting in null findings. In fact, Kaplow et al. found opposing effects for generalized anxiety disorder and separation anxiety disorder: whereas generalized anxiety disorder was positively associated with alcohol use initiation, separation anxiety disorder resulted in a lower risk of onset of alcohol use. A study of 2,548 adolescents in Germany also found that the association depended on the type of anxiety disorder examined; although the seven lifetime anxiety disorders taken as a set predicted subsequent onset of hazardous use, abuse, and persistence, only panic and social phobia were independent predictors of hazardous use and abuse and the persistence of an alcohol use disorder (22).Adults.Studies conducted among adults provide support for a potential reciprocal relation between anxiety and substance abuse. A study of 454 college students followed over 7 years, for example, found that having an anxiety disorder was associated with 3.5–5 times higher odds of developing a new alcohol dependence, while the odds of developing a new anxiety disorder were about four times higher for those diagnosed with alcohol dependence in previous years (24). The direction of the relation between anxiety and substances may depend on the type of substance. The Kushner et al. study (24) was limited by use of a select population that may have a more similar mental health status than the general population. In a study of a random sample of the adult population, however, Newcomb et al. (31) also found a bidirectional relation between anxiety and substance use: polydrug problems in young adulthood increased anxiety in adulthood, but anxiety was actually associated with fewer cocaine problems later in life—possibly because individuals who experience high levels of arousal would be less likely to use a drug that exacerbated that arousal.Depression and substance abuseDepressive symptoms are associated with substance use disorders throughout adolescence and adulthood (25). As with anxiety, the directionality and causal nature of the association remains in question: depression and substance use disorders may be linked through common confounding factors, or the association may be causal—either that alcohol and illegal substances are used as a form of self-medication to manage symptoms of depression or that substance use may increase vulnerability to depression through behavioral changes or neurophysiologic alterations (32).Children and adolescents.Several studies have reported that depression and substance abuse tend to cooccur in adolescence (18, 19, 21, 33–35). In a study of high school students in western New York, Windle and Davies (35) found that 24–27 percent of adolescents identified as depressed also met the criteria for heavy drinking, whereas Rao et al. (34) found that 21.1 percent of adolescent women with depression also had a substance use disorder. The nature of the association between depression and substance abuse among adolescents remains in question. Several studies found that depressed adolescents were more vulnerable to substance use; this association has been found for marijuana (27), smoking (33), alcohol abuse (25), and substance abuse problems (36). Fergusson et al. (33) and Pardini et al. (25), authors of two of the most promising studies on issues of depression–substance abuse comorbidity among adolescents, highlight the lack of conclusive evidence on this issue. Fergusson et al. followed a birth cohort of 1,265 children from birth to age 21 years and found that major depression was associated with increased rates of daily smoking and nicotine dependence, although the association could be due to confounders that affected the development of both outcomes. Pardini et al. also followed up 506 boys until young adulthood and found that increased depression in early adolescence was associated with more alcohol use disorder symptoms and alcohol abuse and dependence diagnoses by young adulthood, but only for boys with high levels of conduct problems.Evidence also exists to support the hypothesis that substance use may increase vulnerability to depression. Hayatbakhsh et al. (21) followed a comparable cohort of 3,239 children up to age 21 years and found that symptoms of depression in childhood did not predict use of marijuana, but use of marijuana was associated with depression. A small study of 155 women aged 17–19 years also found that substance use disorders predicted worse depression symptoms at follow-up, but depression did not predict substance use disorders at follow-up (34).Adults.Comorbidity between depression and substance use persists until adulthood: there is a two- to fourfold higher risk of developing one disorder if the other has occurred (37, 38). As has been detected in adolescence and young adulthood, support also exists for the use of alcohol and illegal substances as a way to manage depressive symptoms in adulthood (37, 39–41). Several studies also document a reciprocal association between the two, as was the case with anxiety (42, 43). Gilman and Abraham (42) followed up participants from the Epidemiologic Catchment Area Survey and found that, although the odds of alcohol dependence increased in relation to baseline depressive symptoms, the odds of major depression also increased in response to the number of alcohol symptoms and a diagnosis of alcohol dependence.Anxiety and depressionHigh rates of comorbidity have been found between anxiety and depression. An important fraction of depression cases also present symptoms of anxiety, to the point that Krueger et al. (8) proposed that “pure” cases of depression or anxiety would be unrepresentative of the entire spectrum of these disorders. In a 40-year follow-up of levels of anxiety and depression, the probability of having anxiety for those who already suffered from depression ranged from 0.54 to 0.98, while, for those who did not suffer from depression, the probability ranged from 0.03 to 0.10 (44). Anxiety and depression function in a cohesive manner and have been classified as internalizing disorders, which are stable over time (8, 45). The comorbidity of these disorders has raised some controversy. While some argue that anxiety and depression are distinct disorders with different etiologic profiles and high levels of cooccurrence, others interpret the overlap as indicative of similar psychopathologic processes and shared risks or as different manifestations of the same disease (26, 46).Children and adolescents.Strong heterotypic continuity exists between depression and anxiety from childhood to adulthood (2, 18, 26, 47–51). In the Great Smoky Mountains study that followed a cohort of children aged 9–13 years for 5 years, anxiety predicted depression and vice versa, even when controlling for current comorbidity (18). A gender-specific analysis indicated, however, that the associations independent of current comorbidity persisted among girls only. Another study that followed those aged 9–18 years over 9 years found that overanxious disorder in adolescence was associated with adult major depression, while major depression in adolescence predicted generalized anxiety in adulthood (50). These associations were independent of gender.The degree of comorbidity between depression and anxiety may vary as a function of age. Wittchen et al. (26) followed 3,021 respondents aged 14–24 years at baseline over 4 years and found that, although only 9 percent of those with an anxiety disorder at ages 14–17 years also had major depressive episodes, the proportion was considerably higher among those aged 18–24 years. Moreover, depression increased considerably among those with anxiety disorder at final follow-up: almost two thirds of cases were comorbid with major depression. The variation in comorbidity by age might be a function of age at onset of the two disorders; whereas anxiety disorders tend to start in childhood and early adolescence, depressive disorders increase in late adolescence and continue to rise in those aged 20 years or older (2, 26, 52).The frequency and type of comorbidity differ by disorder. Wittchen et al. (26) found that the odds ratio for presenting major depression at follow-up ranged from 1.7 for specific phobia to 3.4 for panic disorder and 3.9 for generalized anxiety disorder. Pine et al. (50) also found that major depression predicted generalized anxiety disorder but not simple or social phobia.Comorbidity is related to symptom severity, degree of impairment, course, and outcome. Wittchen et al. (26) found, in a study of 3,021 respondents aged 14–24 years at first interview followed over 4–5 years, that the number of anxiety disorders present, persistence of anxious avoidance behavior, and degree of psychosocial impairment were the characteristics most strongly associated with development of secondary depression. Repetto et al. (51) confirmed, in a more restricted study of African-American adolescents at risk of school dropout, that adolescents who presented consistently high levels of depressive symptoms were more likely to present a higher number of anxiety symptoms.Adults.Most studies have identified anxiety as a primary disorder that precedes secondary depression, perhaps related to the difference in ages at onset as well as to the higher degree of stability of anxiety. A critical review of longitudinal studies of representative samples, however, indicates that this question remains unsettled and may depend on life stage examined, length of study follow-up, and type of anxiety disorder studied. A follow-up of a representative population sample over 10 years from early adolescence to young adulthood found that social anxiety disorder was temporally primary relative to depression in most of the cases and that the risk of subsequent depression was twofold for individuals with social anxiety disorder in comparison to those without social anxiety disorder (53). However, a study that followed up a birth cohort until age 32 years (54) found that major depression preceded generalized anxiety disorder almost as often as generalized anxiety disorder preceded major depression. Two studies in older adulthood found that anxiety preceded depression within short time intervals. A study of older Swedish twins found that over two 3-year intervals, anxiety symptoms led to depressive symptoms, while the opposite did not occur (46). The same phenomenon was reported among the elderly for depression and generalized anxiety disorder: a follow-up of a sample of community-living elderly over 3 years found that generalized anxiety disorder either remitted or progressed into depression or mixed anxiety/depression, whereas subjects with depression or depression/generalized anxiety disorder were unlikely to develop noncomorbid generalized anxiety disorder at follow-up (12).Comorbid disorders may be more stable than “pure” cases of disease. A study that followed adults aged 19–20 years at baseline over 15 years found that substantial transitions occurred from anxiety and depression alone to comorbidity; an average of 21 percent of those with depression alone and 24 percent of those with anxiety alone developed comorbid anxiety and depression at baseline, and, once comorbidity developed, the recurrence of either disorder alone was much lower (55).Specificity also exists in the degree of comorbidity between different types of anxiety disorders and depression. Generalized anxiety disorder has been identified as the type of anxiety disorder most closely connected with major depression (54). Wilhelm et al. (56) followed a cohort of subjects involved in teacher training in 1978–1993 and found a stronger association between number of depressive episodes and panic disorder, generalized anxiety disorder, and agoraphobia than between number of depressive episodes and either simple or social phobia. Gregory et al. (49) followed a representative birth cohort from ages 11 to 32 years and found that adult anxiety cases were more likely to have experienced juvenile depression than those without adult anxiety, except for specific phobia and panic disorder cases.The specific nature of the associations between depression and anxiety may depend on certain interpersonal features of anxiety disorders. Two studies have found, for example, that behavioral inhibition (53) and avoidance of expressing emotions (57), rather than lack of assertion or interpersonal dependency, explained part of the transition from social anxiety disorder to depression. Grant et al. (57) hypothesized that inhibition of strong emotions to significant others as a strategy to maintain associations may lead to a loss of self, which may increase depressive symptoms.Conduct disorder and substance useConduct disorder has been defined in the Diagnostic and Statistical Manual of Mental Disorders: DSM-IV as a pervasive pattern of aggressive and deceptive behavior that begins in adolescence (58). Conduct disorder and substance use have been classified under the same latent construct of externalizing disorders (8). Measelle et al. (36) found, for example, that an externalizing factor accounted for 79 percent and 95 percent of the baseline levels of antisocial behavior and substance abuse, respectively. Both disorders tend to increase through late adolescence and peak in young adulthood, and they are more prevalent among males (59). Several studies have proposed that a major fraction of the association can be attributed to common genetic factors (45).Children and adolescents.Most studies on conduct disorder and substance use concern adolescence and young adulthood. Similar to anxiety and depression, the directionality of the link between these conditions and conduct seems to vary across studies (18, 30, 49, 60–62). Cohen et al. (60) followed up 749 adolescents from ages 13 to 24 years and found that participants with conduct disorder in early adolescence experienced significantly elevated rates of alcohol abuse/dependency and marijuana use disorder between early adolescence and young adulthood. Kratzer and Hodgins (63) found a similar phenomenon when they followed an unselected birth cohort from pregnancy to 30 years of age: childhood conduct problems were associated with a higher risk of severe substance abuse. White et al. (64) moved beyond examining the influence of conduct disorder on levels of substance abuse and investigated its influence on both mean levels of use and change in use over time. Although conduct disorder was associated with higher overall levels of alcohol and marijuana use throughout the study period, it predicted change in levels of alcohol use only. Participants with higher levels of conduct disorder began adolescence with higher levels of alcohol use, but their level of alcohol use grew slower than that among participants who had lower initial levels of conduct disorder—possibly because of regression to the mean.Some of the most promising studies on comorbidity have investigated the impact that conduct disorder and substance abuse have on the developmental patterns of the comorbid disorder. Conduct disorder, for example, has been found to play an important role in the shift from substance use to substance use disorders (30, 65). The Great Smoky Mountains study found that conduct disorder, particularly at earlier ages (14 and 15 years), was the strongest psychiatric predictor of the transition from substance use to a disorder (62). Substance abuse can play a role in crime desistance in young adulthood (45, 66). Hussong et al. (66) found that substance abuse may exert both proximal and distal effects on antisocial desistance over young adulthood: 1) substance abuse early in young adulthood can slow an individual's pattern of criminal desistance relative to the lifetime pattern of criminality; and 2) substance abuse can result in “time-specific” elevations in antisocial behavior relative to an individual's own antisocial trajectory. A community sample of female adolescents aged 13–15 years found that, although initial levels of antisocial behavior predicted increases in substance abuse, substance abuse also predicted slower deceleration in antisocial behavior (36); a study of female and male twin pairs reported that youths with late-onset and persisting antisocial behavior had higher rates of substance use than youths in groups without a history of antisocial behavior or a pattern of antisocial desistance (67).AdultsWe found one study that examined the influence of conduct disorder on substance abuse but used retrospective reports of age at onset for comorbid disorders. The National Comorbidity Survey Replication indicated that lifetime conduct disorder was associated with 5.9 times higher odds for substance use disorders (58). Conduct disorder was more likely to occur before substance use disorders 88.5 percent of the time. Although both active and remitted conduct disorder were significant predictors of substance use disorders, the risk of substance use disorders was significantly higher for those with active conduct disorder. The persistent effect of remitted conduct disorder may indicate that conduct disorder is a risk marker for unmeasured common causes of substance use disorders or that active conduct disorder has long-lasting consequences that persist even after the disorder has remitted. The higher risk associated with active conduct disorder may also, however, point to a causal role that conduct disorder plays in related disorders.Conduct disorder and anxiety and depressive disordersThe limited number of studies identified that addressed the connections between conduct disorder and either depression or anxiety led us to group the review of these disorders into one section. Factor analyses of internalizing disorders, notably anxiety and depression, and externalizing disorders, of which conduct disorder constitutes an important fraction, have identified distinct developmental patterns for these two groups of disorders but have also detected a correlation across time between the two types of disorders (8, 45). Little is understood about the mechanisms that may connect conduct disorder with mood disorders. Conduct disorder may disrupt interpersonal functioning, contributing to greater conflict with parents and peers and greater social rejection and academic failure, which may generate a sense of repeated failure and feelings of anxiety or depression (68). At the same time, depression and anxiety may impede social development and generate interpersonal conflict, which may contribute to the onset of conduct problems. Common etiologic factors may also underlie both types of disorders (69).The developmental course of conduct disorder tends to be intertwined with internalizing problems, particularly depression (36). For example, a study involving a community sample of female adolescents followed from early to late adolescence found that initial depressive and antisocial behavior symptoms predicted future increases in the other: while initial levels of antisocial symptoms predicted increases in depressive symptoms, initial levels of depression predicted a slower deceleration in antisocial problems (36). Early antisocial problems have also been found to predict depressive symptoms in adulthood (69–71). Kim et al. (70) found that, among young men followed from early adolescence to young adulthood, early antisocial behavior was associated with depressive symptoms in adulthood; Silberg et al. (71) detected a correlation between early conduct problems and later depression among adolescent female twin pairs. The link may be explained by a failure model, whereby early developmental failures associated with antisocial behavior increase vulnerability to depressive symptoms (36).Some evidence also exists of a link between conduct disorder and specific types of anxiety (49, 58, 61, 72). A birth cohort of a representative sample followed from ages 11 to 32 years showed an association between a juvenile history of conduct disorder and posttraumatic stress disorder at age 32 years (49). A longitudinal study of a population sample assessed from ages 14 to 33 years also found that the presence of antisocial personality disorder traits at ages 14–22 years was associated with a higher risk of anxiety disorders by age 33 years, most particularly for agoraphobia (72). The National Comorbidity Survey Replication found mixed evidence about the temporal order between conduct disorder and anxiety: conduct disorder occurred after specific and social phobia but prior to all other types of anxiety disorders (58). The findings from the National Comorbidity Survey Replication require replication within a longitudinal study because they are based on retrospective reports of age at onset.Putting it all together: internalizing and externalizing disorders as predictors of substance abuseMost studies on comorbidity assess the interaction between pairs of disorders concurrently and over time, but they fail to determine how clusters of disorders may promote onset of psychopathology. One exception has been the area of youth risk behaviors; selected studies have investigated the relative influence of externalizing and internalizing disorders on the prevalence of substance abuse or conduct disorder. Adolescence has long been considered the stage at which risk behaviors such as delinquency and substance use are initiated and when disorders such as substance abuse become consolidated (36, 73). It is a time when multiproblem youth emerge, characterized by persistent substance use, persistent delinquency, and/or persistent internalizing problems (74). This peak in comorbidity has motivated studies of the intersections between multiple forms of psychopathology in adolescence and young adulthood.Loeber et al. (74), for example, used longitudinal data from three samples of the Pittsburgh Youth Study to examine the cooccurrence of persistent substance use with delinquent and persistent internalizing problems in boys aged 7–18 years. The joint occurrence of mood dysregulation, in the form of internalizing problems, and behavioral dysregulation, in the form of delinquency, emerged in preadolescence and played a significant role in the persistence of substance use. During adolescence, however, only persistent delinquency predicted persistent substance use. Another publication using the Pittsburgh Youth Study expanded this investigation by examining how the cooccurrence of conduct disorder and depressive symptoms identified high-risk youth (25). The authors found that higher levels of depression led to the development of alcohol use disorder symptoms but among only those subjects who had high levels of early conduct disorder symptoms. The interaction between depression and conduct disorder may indicate a need to manage negative affective states without a high regard for social norms, or it may indicate a subgroup of individuals at particularly high risk of developing a severe form of substance use.SummaryProspective population studies that investigate the sequential links between comorbid disorders remain scarce. The bulk of prospective studies assess the influence of the prevalence of one disorder on the future prevalence of the comorbid disorder. Few studies actually move beyond this topic to investigate the influence that comorbidity may have on changes in the state of psychopathology, that is, in shifts from persistence to desistance, or from substance use to abuse, or from disorder-free status to onset. Such information would provide initial evidence of the specific mechanisms by which disorders influence each other. Moreover, differences in the duration of study follow-up, choice of age groups, and selections of measurement instruments and diagnostic criteria make comparability across studies more difficult.Despite these limitations, several points emerge from our review of population-based studies of comorbid disorders. The concurrent and sequential links among conduct disorder, substance use/abuse, anxiety, and depression are neither random nor a result of bias from help-seeking clinical samples. The nature of comorbidity is specific to the type of disorder under study: evidence exists for a reciprocal relation between substance use and depression, anxiety, and conduct disorder, while the bulk of studies on depression and anxiety indicate that anxiety may precede onset of depression. The studies that address comorbidity of conduct disorder with anxiety and depression indicate that conduct disorder may precede these mood disorders, but the paucity of studies that test a reciprocal association impede us from making conclusive statements.The mechanisms that connect disorders remain in question. Three hypotheses link comorbid disorders: 1) the association may be indirectly causal, so that a primary disorder may exert neurophysiologic, individual, or social changes that increase vulnerability to the secondary disorder; 2) disorders may be directly linked, so a primary disorder may, for example, exert neurophysiologic changes that contribute to onset of the secondary disorder; or 3) a set of common risk factors, such as common genes or the experience of childhood trauma, may explain the connection between two disorders. However, these hypotheses are difficult to distinguish from one another. For example, a social factor such as affiliation with deviant peers could predispose adolescents to both substance use disorder and conduct disorder (hypothesis 3), but conduct disorder could also lead to association with deviant peers, which would contribute to substance use disorder (hypothesis 1). A review of the risk factors associated with the initiation and maintenance of comorbid conditions can help illuminate the mechanisms that underlie comorbidity in psychopathology.CONCLUSIONSUnderstanding patterns of comorbidity is essential in order to evaluate psychiatric nosology and fully understand the developmental trajectories of key forms of psychopathology. To date, a number of studies have shown that anxiety, depression, substance abuse, and conduct disorder cluster in individuals across the life course. This critical review is one of the first focused on population-based, longitudinal studies of comorbidity between these four key disorders.The four psychiatric disorders we reviewed have been classified under two latent constructs: externalizing and internalizing disorders. Although factor analyses indicate a higher correlation between disorders within each construct, clustering also exists between externalizing and internalizing disorders. Approaching first the issue of internalizing disorders, anxiety and depression function jointly from childhood to adulthood. Comorbidity seems to be a function of age; it increases from late adolescence onward, possibly because of the later age at onset of depression relative to anxiety. Comorbidity also depends on the type of anxiety disorder concerned: several studies found a higher clustering between major depressive disorder and generalized anxiety disorder, for example, than with other anxiety disorders. In terms of externalizing disorders, conduct disorder and substance abuse show a strong reciprocal link, particularly in adolescence and young adulthood. Conduct disorder and substance abuse not only influence the onset and prevalence of the other but also influence the shift between developmental stages of the other disorder, that is, in shifts from substance use to a substance use disorder, or in the likelihood of moving from conduct disorder to desistance.Studies have documented a link between disorders across the internalizing/externalizing divide. Studies indicate that the relation between substance abuse and anxiety or depression is reciprocal: substance abuse alone or depression/anxiety alone predicts onset of the other disorder. The impact of anxiety on substance abuse remains in question: anxiety disorders have been associated with both a higher and a lower likelihood of substance use disorders. The direction of the association may depend on the type of anxiety disorder and substance in question. Research has also addressed the question of clustering between conduct disorder and anxiety/depression, although research is less developed in this area. Depression and conduct disorder play a role in shaping the developmental trajectory of the other. Initial levels of depression have been associated with a slower rate of desistance from conduct disorder, while conduct disorder predicted increases in depressive symptoms from adolescence to adulthood. The links between anxiety and conduct disorder are less understood. The few existing studies focus on adolescence and young adulthood and have indicated that initial levels of conduct disorder were associated with higher levels of anxiety.Existing research on comorbidity has a number of strengths. Prospective studies linking the presence of one disorder to onset of the other, as well as examining the influence that a disorder may have on developmental shifts in comorbids, are growing, and they offer promise in enabling us to understand the specific ways in which comorbid disorders are linked over the life course. Moreover, research is shifting from a focus on a pair of disorders to a more holistic approach that analyzes how clusters of disorders interact with each other. We think this line of investigation will be key in understanding the complexity of comorbid relations and in estimating the health burden that results from multiple comorbidities.At the same time, the epidemiologic literature falls short in its consideration of key issues within comorbidity research. First, although early research in this area is promising, a need exists for study designs that make it possible to test the timing and specific nature of comorbid relations—that is, shifting from merely investigating whether disorders are “associated” with each other to understanding the ways that disorders deflect a comorbid pair from specific stages in its normative trajectory. This process involves studying the influence of disorders in promoting qualitative shifts in the comorbid disorder, from alcohol use initiation to abuse to dependence, for example. It also implies accounting for the spectrum of relevant comorbid disorders within the same study to control for spurious associations and understand how the presence of a third disorder may influence the causal link between comorbid pairs.Second, our understanding is limited in terms of the specific comorbid patterns between subtypes of disorders. Although we recognize that specific types of anxiety disorders, for example, may show different comorbidity patterns with depression, or that alcohol abuse may have different levels of comorbidity with anxiety than illicit drug abuse does, the scarcity of available, population-based, longitudinal studies on each specific subtype combination prevented us from making critical distinctions between comorbid patterns. Studies that compare comorbid patterns between disorder subtypes across key developmental stages, from childhood to adulthood, offer a promising way to address this gap.Third, one of the key questions that remains relates to the causes of comorbidity: the direction and mechanisms underlying causal links, as well as the potential spurious nature of such links. Investigating factors at multiple levels of influence as potential confounders, mediators, triggers, or modifiers of comorbid associations would enable us to identify groups at highest risk of the presentation of multiple disorders and distinguish features of the individual and his or her environment that are most malleable to preventive intervention. Moreover, it would enable us to establish whether associations between comorbid disorders are truly causal or are spurious phenomena resulting from common risk factors. Little effort has been invested in systematically documenting the associations between these disorders across different age groups or in reviewing the factors associated with the cooccurrence of such comorbid forms of psychopathology. 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results from the National Comorbidity Survey ReplicationJ Child Psychol Psychiatry20074870313 \ No newline at end of file diff --git a/test/dataset/in/resources/corpus/Clean_0C1C12E1156F7025DBB91BEDD4B8D1E7BB81C36A.txt b/test/dataset/in/resources/corpus/Clean_0C1C12E1156F7025DBB91BEDD4B8D1E7BB81C36A.txt new file mode 100644 index 0000000..99050a8 --- /dev/null +++ b/test/dataset/in/resources/corpus/Clean_0C1C12E1156F7025DBB91BEDD4B8D1E7BB81C36A.txt @@ -0,0 +1 @@ +annoncannoncAnnals of Oncology1569-80410923-7534Oxford University Press10.1093/annonc/mdl050reviewsMerkel cell cancer of the skinPectasidesD*PectasidesMEconomopoulosTSecond Department of Internal Medicine, Oncology Section, University of Athens, ‘Attikon’ University Hospital, Haidari, 1 Rimini, Athens, Greece*Correspondence to: Dr D. Pectasides, Second Department of Internal Medicine, Propaedeutic, Oncology Section, Attikon University Hospital, Rimini 1, Haidari, Athens, Greece. Tel: +3210-5831691; Fax: +3210-5831690; E-mail: pectasid@otenet.gr102006243200617101489149525920051220061022006© 2006 European Society for Medical Oncology2006Merkel cell carcinoma (MCC) is a rare malignant cutaneous tumor of the elderly with rapidly growing skin nodules found predominantly on sun-exposed areas of the body. The vast majority of patients present with localized disease, while up to 30% have regional lymph node metastases. Despite local excision and the incidence of local recurrence, regional lymph node metastases and distant metastases is high and usually occurs within 2 years of primary diagnosis. The optimal treatment for patients with MCC remains unclear. The best outcome is achieved with multidisciplinary management including surgical excision of primary tumor with adequate margins and post-operative radiotherapy (RT) to control local and regional disease. Patients with regional nodal metastases should be treated with lymph node dissection plus RT. Adjuvant chemotherapy (CT) should be considered as part of the initial management. In case of metastatic disease CT based on regimens used for small-cell lung cancer is the standard treatment of care.chemotherapyMerkel cell carcinomaprognosisradiotherapysurgeryintroductionMerkel cell carcinoma (MCC) is a rare malignant cutaneous tumor of the elderly that is characterized by an aggressive course with regional nodal involvement, distant metastases and a high rate of recurrence. MCC was first described by Toker in 1972 [1] as trabecular carcinoma of the skin and, since then, has also been considered as cutaneous APUDoma, neuroendocrine tumor of the skin, Merkel cell tumor, primary small cell carcinoma of the skin, primary undifferentiated carcinoma of the skin, anaplastic carcinoma of the skin and murky cell carcinoma [2]. MCC and neuroendocrine carcinoma of the skin are the most widely used terms and may best reflect postulated origin and immunocytologic characteristics of this neoplasm.epidemiologyEvery year in the United States, there are approximately 470 new recorded cases. The estimated annual incidence according to US Surveillance data, Epidemiology, and the End-Results Program is 0.23 per 100 000 people for whites [3]. From 1987 to 2001, the Finnish Cancer Registry recorded 141 cases of MCC in a population of 5 000 000, which gives an annual prevalence of 0.2 cases per 100 000 [4]. The incidence in blacks (0.01 cases per 100 000) as well as for Polynesians seems to be lower. Both sexes are affected, but there seems to be a male predominance (2.3:1) [3, 5–7]. In some series the incidence in women was reported higher than that in men [8, 9]. MCC occurs in elderly patients, with an average age of diagnosis at 69 years (range 7–104 years). Only less than 5% of cases occur before the age of 50 years [7, 9–13].clinical manifestation and diagnosisMost patients present with rapidly growing, painless, firm, non-tender, dome-shaped red, occasionally ulcerated skin nodules, which have a red or bluish color, measuring up to several centimeters, on predominantly sun-exposed areas of the body [11]. The overlying skin is smooth and shiny, sometimes exhibiting ulcerative, acneiform or telangiectatic features [14]. However, the clinical feature of Merkel cell tumor is typically not acneiform. MCC can spread through the dermal lymphatic system, resulting in the development of multiple satellite lesions. MCC involves predominantly the head and neck (>50% of cases) [9, 15]; it involves the extremities in 40% of cases and the trunk in less than 10% of cases [2, 16–20]. On the face, the eyelids are involved more frequently [21]. MCC involving other sites not exposed to sunlight has been reported in a small percentage of cases [22–24]. At presentation most patients with MCC present with localized disease (70%–80%), 9%–26% have regional lymph node metastases and 1%–4% distant metastases [11, 18]. Despite local excision, the incidence of local recurrence (27%–60%), regional lymph node metastases (45%–91%) and distant metastases (18%–52%) is high and usually occurs within 2 years of primary diagnosis [11, 25, 26]. The majority of patients die from distant metastases involving liver, bone, lung, brain or distant lymph nodes [11, 26, 27]. Because the early course of MCC is asymptomatic, diagnosis may be delayed until the detection of regional lymphadenopathy and distant metastases. Symptoms are related to local spread or to involvement of regional lymph nodes. Superior vena cava syndrome due to obstruction by tumor has been reported [28]. Spontaneous regression of the primary lesion has been reported [29] and 20%–30% of patients present without obvious primary lesion. Patients who present with no obvious primary tumor seem to have a better prognosis [30, 31]. The differential diagnosis of MCC includes basal cell carcinoma, lymphoma, melanoma and squamous cell carcinoma, with MCC usually being overlooked [14]. The correct diagnosis of MCC is not made until tissue sampling and pathologic evaluation are performed.staging and prognosisThere is no universal accepted staging system for MCC, but tentative classification relies on clinical manifestation and includes: stage I, localized cutaneous involvement (IA: ≤2 cm, IB: >2 cm); stage II, regional nodal involvement; stage III, systemic metastases [7, 32]. Even though the diagnosis of MCC is exclusively pathologic, imaging is useful for staging, surgical guidance, therapeutic management and follow-up. This staging system allows therapeutic approaches according to the stage: stage I and II should be treated with curative intent and stage III as a palliative treatment.The most important prognostic factor for survival and development of distant metastases is the presence of lymph node involvement [6, 33]. In the series of Poulsen et al. [30], most important predictors for local control and survival were the tumor site and the presence of regional lymph node metastases. Morrison et al. [34] reported that the median survival of patients with lymph node metastases was 13 months and for those without lymph node involvement it was 40 months. Other reported poor prognostic factors include location on the lower extremities [31] because of difficulties in local control and the high incidence of local failure due to the poor blood supply in the legs, which limits big surgical procedures, and the subsequent poor tolerability of radiotherapy (RT) by the elderly. Furthermore, they include tumor size ≥2cm [4, 35], male sex [6, 9, 11, 18], age ≥60 years [7], no treatment with radiotherapy [36] and positive surgical margins [37].etiologyRisk factors include exposure to sunlight and ultraviolet (UV) light. MCC seems to have a relationship with sun exposure, both by its anatomic predilection and geographic distribution. Miller and Rabkin reported a correlation of the UV B index and the incidence of MCC [3]. An association has been reported between MCC and other UV light-related skin cancers (squamous cell carcinoma, basal cell carcinoma) previous irradiation and exposure to radiant infrared heat [10, 38–43]. The development of MCC in non-sun-exposed regions of the body suggests that factors other than sunlight have been implicated in the pathogenesis of MCC. Lunder and Stern [44] reported a 100-fold increased incidence of MCC in patients treated with methoxsalen and UVA for psoriasis. Exposure to arsenic has also been implicated [45]. Immunocompromise and immunosuppression may represent additional risk factors for MCC, whose prevalence is unusually high in organ recipients compared with the general population [46, 47]. The risk of MCC in renal recipients has been estimated at 0.13/1000 person-years [48]. A high prevalence of second neoplasm occurring before, concurrent with or after the diagnosis of MCC (25%–28%) has recently been reported. These neoplasms include squamous cell carcinoma, hematological malignancies, breast cancer and ovarian cancer [43]. MCC has also been reported in patients with HIV infection and in those with chronic lymphocytic leukemia, suggesting that these diseases have an association with MCC. Engels et al. [49] reported that the relative risk of developing MCC in patients with HIV infection is approximately 13.4.pathologyMCC involves the dermis and frequently extends into the subcutaneous fat. Sometimes, MCC involves the overlying epidermis [50]. The tumor consists of small blue, ovoid cells with hyper-chromatic nuclei, a very fine chromatine and minimal cytoplasm. Mitoses are frequent and the apoptotic index high. The triad of vesicular nuclei with small nucleoli, abundant mitosis and apoptosis is suggestive of MCC.The most widely accepted origin of MCC is the Merkel cell [51], whose function is not clear. This clear, oval cell is located within or close to the basal layer of the epidermis and functions as a cutaneous mechanoreceptor [52]. The ultrastructural and immunohistochemical similarities between MCC and Merkel cells support the Merkel cell origin of MCC [53–55]. However, there is evidence that runs counter to this postulate: MCC and Merkel cells have different location (dermis and epidermis, respectively) and MCC does not express vasoactive intestinal peptides and metenkephalin specific to Merkel cells [56]. An intermediate consideration suggests that the origin of MCC is associated with an immature, totipotential stem cell that acquires neuroendocrine characteristics during malignant transformation [56, 57]. The same concept of precursor stem cells may explain the coexistence of MCC and squamous cell carcinoma of the skin [38].Although MCC can usually be diagnosed histologically rather easily, it sometimes may be difficult using light microscopic findings due to its similarities with other small cell tumors. These include metastatic oat cell carcinoma, metastatic carcinoid tumor, neuroblastoma, and some types of melanoma, lymphoma and squamous cell carcinoma [11, 57]. MCC can be definitively diagnosed with hematoxylin–eosin and immunohistochemical staining, electron microscopy or both [15, 25]. Immunohistochemical staining can distinguish MCC from these tumors. MCC express both neuroendocrine (neuron-specific enolase, synaptophysin, chromogranin) and cytokeratin markers (cytokeratin 20, as a paranuclear dot, CAM 5.2) and is negative for S100 and common leukocyte antigen. Epithelial membrane antigen and BEP-EP4 may also be expressed in MCC. Cytokeratin 7, which identifies bronchial small-cell carcinoma, is negative in MCC. In addition, a homeodomain-containing nuclear transcription factor expresses in bronchial small-cell carcinoma and is negative in MCC [58, 59]. On the contrary, neurofilament protein is commonly expressed in MCC and often not in bronchial small-cell carcinoma [60, 61]. CD 117 (KIT receptor) is also present in 95% of MCC, but its expression does not correlate with more aggressive tumor [62].Three histological subtypes of MCC have been reported: intermediate, small cell and trabecular, but these variants have no clinical relevance [63]. Intermediate variant is the most common seen subtype; the small-cell variant is histologically similar to other small-cell carcinomas and should be distinguished from the bronchial small-cell carcinomas.molecular biology of MCCNumerous chromosomal abnormalities have been reported in MCC, but the exact pathways of neuroendocrine differentiation are yet unclear. The most interesting chromosomal abnormality is the deletion of the short arm of chromosome 1 (1p36) [64, 65], which is also common in melanoma and neuroblastoma [66, 67]. These findings lead to arguments about the neurocrest origin of MCC [68]. Another abnormality is the loss of heterozygosity in chromosome 3p21, the same region that is also affected in small-cell lung cancer [69].Several other abnormalities have been reported, including trisomy 1, trisomy 6, trisomy 11, trisomy 18 and deletion of chromosome 7 [70–73]. Loss of heterozygosity has also been reported in chromosomes 10q and 13 [74, 75]. The tumor suppressor gene p53 is also occasionally present in MCC, and the expression of mutant p53 may be associated with poor outcome [76]. P73 has been localized in 1p36.33 and, as in melanoma, is expressed infrequently [77, 78].Recently, Leonard et al. [79] reported that MNF (Merkel nuclear factor) contains the POU-IV family member Brn-3c and that Brn-3c is expressed in normal Merkel cells. In addition, Brn-3c protein reactivity is restricted to a subset of MCC biopsies and is not seen in biopsies revealing adherent, variant cell lines lacking neuroendocrine markers. In mice POU-IV Brn-3 and the transcription factor ATOH1 are very important for the function of normal Merkel cells and for neuroendocrine differentiation [31]. The precise role of these abnormalities in the prognosis of MCC has to be determined.imagingAfter diagnosis, patients should be assessed by imaging techniques to define the exact extent of disease. Because MCC behaves like malignant melanoma, lymphoscintigraphy may be used to localize sentinel nodes, which may harbor micrometastases. Lymphoscintigraphy in combination with intra-operative lymphatic mapping allows accurate and selective sampling of sentinel nodes [80]. Distant metastases are present in some cases at presentation. The pattern of metastases is not specific for MCC and seems to mimic multifocal invasion by other small-cell carcinomas. Ultrasonography of soft tissue may show hypoechoic nodules arising from the dermis. CT scan may demonstrate regional lymph node involvement or systematic metastases in the lungs, liver, bones and other sites [81]. Invasion of CNS is rare but should not be overlooked. In such cases, it is best to perform MRI.Distant metastases and locoregional recurrence may be evaluated with somatostatin receptor scintigraphy (SRS) based on the neuroendocrine characteristics of MCC [80]. SRS is helpful in pre- and post-therapeutic evaluation of MCC. Compared with CT scan and MRI, SRS is less affected by inflammation, edema and tissues at the surgical and irradiated site [80, 82]. However, SRS may be limited in evaluating metastases in liver, kidneys and spleen. The presence of somatostatin receptors has also been used therapeutically in MCC [83]. Guitera-Rovel et al. [84] reported a sensitivity of 78% and a specificity of 96% in 20 MCC patients assessed by SRS.Positron emission tomography with the glucose analog 2-[fluorine-18] fluoro-2-deoxy-D-glucose (FDG) also plays an important role in assessing and monitoring MCC [85].treatmentTreatment options will be discussed according to the disease stages. Because of the rarity of MCC, there are no prospective trials investigating surgery, RT or chemotherapy (CT).stage IFor stage I disease the main treatment strategies include surgery and radiation therapy. In these cases the reported 5-year survival reached 64%. Controversies exist regarding the best excision margins in MCC, as there are no prospective, controlled studies dealing with this aspect. Margins 2–3 cm wide and 2 cm deep have been generally accepted [2, 11, 18, 32, 86–88]. In the older series [14, 32], the recommended margins were ≥3 cm. In the studies by O'Connor et al. [14] and by Yiengpruksawan et al. [32], margins ≥3 cm were associated with reduction in local recurrence. Updating results were reported based on 102 patients with MCC treated at Memorial Sloan Kettering Cancer Center [35]. After a median follow-up of 35 months, the overall 5-year disease-specific survival rate was 74%. Recurrence of disease occurred in 55 patients (55%), and the most common site of first recurrence was within the draining lymph nodes (n = 35). Elective lymph node dissection was the only independent predictor of improved relapse-free survival. However, in the study by Ott et al. [9] and in the study by Gillenwater et al. [89], 66 patients with MCC of the head and neck region, no difference in outcome (locoregional control and survival) was noted when the resection margins were >2 cm or <2 cm or the patients had an adequate RT or <1 cm, or 1–2 cm, or >2 cm, respectively. In contrast, in the latest study, a comparison of the patients who did (n = 26) and did not (n = 34) receive post-operative radiation therapy revealed a significant difference in local [3 (12%) versus 15 (44%), respectively; P <0.01] and regional [7 (27%) versus 29 (85%), respectively; P <0.01] recurrence rates. There was, however, no significant difference in the disease-specific survival between these groups (P = 0.30). Distant disease developed in 36% of all patients regardless of therapy [89].Mohs micrographic surgery has been considered as the best method of wide clearance. However, no controlled clinical trials comparing this method with the traditional wide excision have shown its benefit. O'Connor et al. [90] evaluated the use of Mohs micrographic surgery for this aggressive neoplasm. Standard surgical excision for local disease was associated with high rates of local persistence [13 of 41 (31.7%)] and regional metastasis [20 of 41 (48.8%)]. Only one of 12 (8.3%) Mohs-treated patients, with histologically confirmed clearance, had local persistence of disease. Regional metastasis developed in four of 12 cases (33.3%). Regional metastasis developed in none of the four patients treated with RT after Mohs surgery and in four of eight patients treated with Mohs surgery without post-operative RT. Boyer et al. [91] retrospectively conducted a study; the study group consisted of 45 patients with stage I MCC who were histologically and clinically free of disease after Mohs excision. Twenty patients subsequently received elective post-operative RT to the primary site and 25 patients had no adjuvant radiation therapy. One marginal recurrence (4%) and three in-transit metastases were observed in the Mohs surgery alone group, whereas none were observed in the Mohs surgery and RT group. The proportion of patients with these events was not significantly different between treatment groups. Overall survival, relapse-free survival and disease-free survival were not significantly different between treatment groups [91]. They concluded that adjuvant RT appears unessential to secure local control of primary MCC lesions completely excised with Mohs micrographic surgery.MCC is a radiosensitive malignancy and adjuvant RT has been advocated for local and regional disease control [6]. Because of the high rate of local or regional relapse, RT has been used post-operatively, in an adjuvant basis in patients with MCC. However, most of the studies argued for the benefits of post-surgery RT [6, 33, 92–97]. Allen et al. [35] found that local recurrence occurred in approximately 12% of patients and was not more common in those who did not receive adjuvant RT after resection of the primary lesion. Adjuvant RT should be considered in patients with primary disease in whom wide surgical margins are unattainable or in those found to have pathologically close or involved margins after excision. In contrast, Wilder et al. [97] reported in a review that the administration of RT post-operatively to both the surgical bed and the draining lymph nodes improves locoregional control and may result in long-term disease-free survival when administered after the initial surgical resection. Similarly, Shaw et al. [18] in a review of 30 reports of MCC reported that after excision alone of the primary lesion, local recurrence occurred in 39% of patients and regional failure occurred in 46%. In contrast, patients treated by excision plus prophylactic treatment (adjuvant node dissection and/or adjuvant RT), experienced local recurrence in 26% and regional failure in 22%. Locoregional recurrence carried an ominous significance with 67% of patients subsequently dying of the disease. For patients who either presented with regional disease or later developed regional disease, the best outcome (44% survival with mean follow-up of 40 months) was obtained following treatment by therapeutic node dissection with or without RT. On the contrary, treatment of regional disease with RT alone was associated with only a 20% survival rate. In addition, Ott et al. [9] treated 31 patients with MCC of the head and neck region. Therapy included local excision with or without RT in 19 patients, local resection and lymphadenectomy with or without RT in eight patients, and RT alone in four patients with head and neck tumors. They concluded that locoregional recurrence correlates with inadequate margins and lack of RT, but remission is possible with multimodality therapy. Similarly, Meeuwissen et al. [6] demonstrated the importance of surgery plus RT over surgery alone in preventing local recurrence of this highly malignant skin cancer. Medina-Franco et al. [98] reviewed 1024 cases with MCC and reported that adjuvant RT was associated with a reduced risk of local recurrence (P < 0.00001). RT has also been used alone and has been reported to achieve local control [92, 94, 99, 100]. The administered dose of RT ranges from 30 to 70 Gy, but the majority of patients received 45–50 Gy [31, 70].Patients with MCC have at presentation 10%–30% regional lymph node involvement [11] and micrometastatic disease in prophylactic lymph node dissection has been found in 100% of patients [31]. Because the incidence of regional lymph node metastases is extremely high, ranging from 45% to 91% at diagnosis [11, 25, 26], elective nodal surgery with RT should be considered in patients with stage I disease. Controversy regarding the management of clinically negative lymph nodes has been eliminated with the use of sentinel lymph node mapping (SLN) for patients with MCC [15, 101, 102]. Approximately 25% of patients with MCC have metastatic disease in the SLN [15, 35]. Prophylactic lymph node dissection is not recommended as standard management [63, 87]. However, some investigators have recommended the prophylactic use of lymph node dissection for tumors at high risk for recurrence [87]. If metastatic disease is proved in SLN, management includes complete lymphadenectomy plus post-operative RT. Early removal of microscopic disease in regional lymph nodes detected by SLN biopsy may afford the patient a greater opportunity for cure than expectant management of the regional nodal basin, although this remains unproven [103]. Unfortunately, over half of the patients who undergo regional lymphadenectomy for clinically positive nodes will die of distant metastases [18, 32]. Lymphoscintigraphy and sentinel node biopsy have been proposed to overcome the problem of unpredictable lymphatic drainage and to minimize the need for node dissection [104, 105]. A meta-analysis of case series of 60 patients with Merkel cell carcinoma managed with sentinel lymph node biopsy was performed by Mehrany et al. [101]. Forty of 60 patients (67%) had a biopsy-negative sentinel lymph node; 97% of this group had no recurrence at 7.3 months median follow-up. Twenty patients (33%) had a biopsy-positive sentinel lymph node; 33% of this group experienced local, regional or systemic recurrence at 12 months median follow-up. Risk of recurrence or metastasis was 19-fold greater in biopsy-positive patients (odds ratio, 18.9; P = 0.005). None of 15 biopsy-positive patients who underwent therapeutic lymph node dissection experienced a regional recurrence. They concluded that sentinel lymph node positivity is strongly predictive of a high short-term risk of recurrence or metastasis in patients with MCC. Therapeutic lymph node dissection appears effective in preventing short-term regional nodal recurrence. The role of adjuvant CT in stage I MCC remains experimental. However, aggressive adjuvant treatment should be considered for patients with positive sentinel lymph nodes [101].stage IIIf stage II disease has been documented, management should include simultaneous excision of the primary site, regional lymph node dissection and RT to the primary tumor and regional nodal site. Lymph node involvement is frequently bulky and the decision for operation should be based on the operability of the nodal mass, the patient's performance status and the site of lymph node involvement [31]. RT at a dose of 50–60 Gy should be used for the regional nodal mass.CT can be given in high-risk patients [106–109]. In a small series of 35 patients [86] no benefit from adjuvant CT was obtained. In contrast, Boyle et al. [7] reported a 40% (eight of 20) RR to CT, particularly CBDCA plus VP-16 given to patients with regional lymph node disease. They proposed that for such poor-prognosis tumors, further investigation of adjuvant RT and CT is warranted, as responsiveness of recurrent disease has been confirmed. In patients with recurrent or locally advanced disease and adequate performance status, Tai et al. [110] feel that chemoradiation (radiotherapy plus CAV-cyclophosphamide, doxorubicin or epirubicin, and vincristine or cisplatin and etoposide) may be considered as a treatment option.stage IIIDistant metastases are frequent at initial presentation [11, 32]. These lesions may be found incidentally at routine radiographic work-up or imagine evaluation for unrelated causes. The pattern of metastasis is not specific for MCC and mimics multifocal invasion by other small-cell carcinomas. However, organs most frequently affected are the liver, bone, lung, brain and skin. The prognosis of patients with stage III MCC is poor, with an average interval of 8 months between diagnosis and death [10]. Treatment consists of CT, palliative RT and possibly surgery. CT for MCC includes several regimens consisting of cyclophosphamide (CTX), doxorubicin (ADR), vincristine (VCR), 5-fluoruracil (5-FU), cisplatin (CDDP), carboplatin (CBDCA) and etoposide (VP-16) [10, 86, 106, 110, 111]. CT has only short-lived success in patients with MCC [10, 14].RT can be given for palliation of bone and brain metastases or for palliation of cutaneous deposits that are bleeding or fungating. CT is usually reserved for those patients with generalized disease or advanced local or regional disease. Several investigators have reported favorable responses using a variety of regimens [110, 111]. Complete response rates of 20%–35% (median duration 6 months) and partial response rates of 23%–50% have been reported in patients with stage III disease [86, 110–112]. Voog et al. [111] reviewed 37 reports on the use of CT for 107 patients with MCC. The reported response rate was 60% for patients with locally advanced disease and 57% for those with metastatic disease. Cisplatin, ADR and 5-FU were the agents most commonly associated with a significant response. Cyclophosphamide was utilized in 56% of cases, ADR in 49% of cases and platinum derivatives in 25% of cases. Tai et al. [110] confirmed these results, reporting RR in 68% of patients with locally advanced disease and 59% in those with metastatic disease. Cyclophosphamide, ADR or epirubicin (EPI) and VCR appeared to be the most active agents. The reported RR was 75.7% (CR 35.1%). The combination of CDDP or CBDCA plus VP-16 had a RR of 60% (CR 36%). These responses did not differ significantly (P = 0.19). In our study [112] the RR was 83% (CR 33%, PR 50%) for patients both with locally advanced and metastatic disease.conclusionsMCC is a rare and aggressive neuroendocrine tumor of the skin. The optimal treatment for patients with MCC remains unclear. The best outcome is achieved with multidisciplinary management. Optimal management of patients with MCC consists of optimal surgery of primary tumor with adequate margins and post-operative RT to control local and regional disease. SLN mapping is essential in patients with clinically negative lymph nodes because it provides important prognostic information and allows selection of patients for additional nodal therapy. Patients with localized disease should be treated with conservative surgery followed by post-operative RT. The dose of post-operative RT ranges from 45 Gy to 60 Gy in 25–30 fractions. Patients with resectable nodal disease should be treated with regional node dissection followed by post-operative RT. Patients with fixed, unresectable nodal metastases should be treated with pre-operative CT plus RT followed by a nodal dissection. 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This research assessed how the attitudes of Americans toward government programs that serve older people changed between the mid-1980s and late 1990s and how much of the shift was due to intracohort change and how much was due to cohort replacement. Methods. Data come from three nationally representative cross-sectional samples, surveyed by telephone in 1986 (N = 1,209), 1990 (N = 1,500), and 1997 (N = 1,559). Results. Attitudes of Americans have become less supportive of expanding entitlement programs for older people and more supportive of cutting their costs and benefits. Between 1986 and 1997, most cohorts, particularly older adults, grew more in favor of maintaining Social Security benefit levels but less in favor of expanding them. Young adults tended to be driving the societal shift in attitudes toward decreasing benefits. Intercohort change was more important than cohort replacement in this process. Analyses of change in 2 attitude domains between 1990 and 1997 revealed that the general population felt less strongly that older people are entitled to benefits and expressed greater opposition to the associated costs. However, young adults moderated their concerns about costs as they got older, although the young adults in the cohort replacing them had become more critical of the principle of entitlement. Discussion. These findings enhance the understanding of the roles that historical conditions and aging play in shaping the attitudes of adult cohorts toward public programs for older citizens. Discrepant findings based on the intercohort change in younger age groups are reconciled by differentiating maturation effects from period effects on impressionable youth. hwp-legacy-fpage S36 hwp-legacy-dochead RESEARCH ARTICLE IN the United States, as in almost all developed nations, support of the older population has largely been collectivized through government-sponsored income maintenance and health care programs. However, over the last several decades, rising life expectancies and the impending aging of the baby-boom cohort have created anxiety concerning the economic viability of entitlement programs serving a swelling older population. Messages in the mass media through much of the early 1990s (e.g., Becker 1994; D'Antonio 1993; Duff 1995; Jackson 1994; Romero 1994; Samuelson 1994; Wolfe 1995) have reflected—as well as stirred—public fears concerning the anticipated demands placed on the retirement system by aging baby boomers (Cornman and Kingson 1996; Duff 1995; Wolfe 1995). It is not unusual for today's elderly to be blamed for these funding dilemmas, for instance, when they are portrayed as a leisure class of affluent retirees unfairly benefitting from the taxes paid by a younger and poorer working-age population (D'Antonio 1993; Jackson 1994; Marshall, Cook, and Marshall 1993; Romero 1994; Samuelson 1994). Public anxiety over the consequences of a swelling older population, combined with greater wariness of "big government" on the part of the public, has revived the debate concerning the desirability of large-scale, universal, and increasingly expensive public programs that provide services and benefits as an entitlement of old age. Scholars have expressed concern that insecurity over the future of entitlement programs for the elderly may erode public support for these programs, particularly in younger generations (Achenbaum 1986; Bengtson 1993; Bengtson and Achenbaum 1993; Kingson and Williamson 1993; Myles 1995). If the late 20th century was truly a watershed period in age-group relations, then one would expect a change on the part of the public in their support for programs that benefit older people. We asked two basic questions in this investigation: (a) Have Americans grown less supportive of entitlement programs serving older people? (b) If so, how much of this weakening is the result of shifts in the attitudes of individuals through time (intracohort change), and how much is due to the emergence of a young adult cohort with a distinctly more negative attitude toward entitlements of older people than the cohort it replaced (cohort replacement)? Two general explanations for why age strata may vary in their attitudes toward public benefits for older people have to do with their stage in the life cycle and the cohort into which they were born. Life-cycle effects on attitudes are those that correspond to the imperatives imposed by the needs and incentives associated with chronological age. Cohort effects are those that correspond to the disproportionate influence that historical periods have on the attitudes of certain age groups, particularly on the attitude formation of impressionable young adults. Life-cycle factors are addressed by Ponza, Duncan, Corcoran, and Groskind 1988, who advance three mechanisms for explaining variations in attitudes by age: self-interest, insurance, and altruism. In terms of self-interested motivations, one would expect the younger individuals to be less strongly in favor of protecting old-age benefits than would the older population because they derive little direct benefit from these programs (Rhodebeck 1993; Shapiro, Patterson, Russell, and Young 1987). An insurance model proposes that individuals who anticipate their own old-age needs would be just as protective of benefits to older people as are older persons themselves. Under the assumptions of this model, one would expect that elevated support for protecting old-age benefits would begin in middle age. Altruism would be evident if age groups indirectly benefitting from the policy, such as young adults, support liberal benefits just as strongly—or more strongly—than the older population. Models that mix altruism and self-interest propose that the interests of the middle aged are served by old-age programs that accommodate the needs of older generations and that allow them to shift the potential costs of their own elder-care responsibilities to publically funded programs. We are not in a position to directly test these models, although they inform our discussion concerning the effects of age on public attitudes toward entitlement programs for older people. The influence of cohort membership is rarely articulated as an explanation for age-strata differences in attitude formation around issues of significance to older people. We maintain that without differentiating chronological age from cohort membership it is not entirely possible to understand whether support for public programs and privileges for older people is influenced by life-stage processes or by ideologies permeating the sociohistorical period in which birth cohorts adopt their attitudes. This may be why there is little consensus in the literature concerning the attitudes of younger adults toward programs targeting the older population, with some research finding higher support in this age group (Day 1993; Logan and Spitze 1995), some finding lower support (Kronebusch and Schlesinger 1994; Silverstein and Parrott 1997), and others finding few differences from other age groups (Cook and Barrett 1992). In this context, our principal interest focused on the ideological crystallization of a young cohort that is rooted in its exposure to particular economic, cultural, and historical experiences. If young adults are particularly impressionable to the values and ideologies of the contemporary culture, then the upsurge in "elder-bashing" during the 1980s and 1990s would have created social change through the emergence of a new cohort of young adults into the population, one with values distinctly more negative on this issue than the one it replaced (Logan and Spitze 1995; Ponza et al. 1988; Schlesinger and Kronebusch 1994). For example, it has been suggested that young adults who had trouble entering the labor market during the recession of the early 1990s may have blamed economic stagnation on older people by relying on stereotypes of older people as affluent and selfish (Binstock 1983). Young adults may have been particularly susceptible to contemporary media messages that reinforce negative stereotypes of older people and engender a culture of ageism (Bishop and Krause 1984; Vasil and Wass 1993). Our argument for the importance of cohort replacement derives from the ideas of Ryder 1965, who outlined how population turnover—the continuous succession of new cohorts into the adult population and the exit of older cohorts—operates as an engine of social change. As young cohorts reach adulthood, their "fresh contact" with the world serves to refresh social institutions and promote innovation (Mannheim, 1922/Mannheim 1952). This approach provides an important point of departure for understanding how cohort membership shapes attitudes. In short, the cohort perspective suggests that historical conditions leave an indelible imprint on the attitudes of young adults at a time when they are most susceptible to absorbing the social values of the period, a phenomenon known as the impressionable youth hypothesis (Alwin, Cohen, and Newcomb 1991; Clausen 1993; Elder 1994; Marwell, Aiken, and Demerath 1987). Crucial to this argument is the way personal biography aligns with historical contingencies to produce sharp deviations in the attitudes of cohorts emerging into adulthood. The life-course perspective provides a useful point of departure for speculating about sources of aggregate social change (if it does exist) in the values of adults toward programs serving older people, leading to the following questions: Has the public become increasingly skeptical about the value and utility of entitlement programs serving older people? Have the attitudes of all cohorts on this issue shifted consistently? Have younger cohorts with uniquely different attitudes replaced older cohorts to, at least partially, produce this societal change? In this investigation we examined sources of aggregate change in the attitudes of Americans toward public programs providing support for the older population. We heeded Glenn 1976 admonition against trying to disentangle the well-known confound among age, period, and cohort effects. Instead, we used an approach outlined by Firebaugh 1997, in which social or aggregate change is decomposed into two components: intracohort change and cohort replacement. Intracohort (or individual) change refers to that component of societal change resulting from the maturation of cohorts over historical time, and cohort replacement refers to that component resulting from the process of population turnover—the successive replacement of younger cohorts into the population and the successive removal of older cohorts. We first tracked whether preferences of the population for public spending on the Social Security program changed between 1986 and 1997 and identified the components of change due to intracohort change and cohort replacement during a period of heightened generational tension. Second, we investigated whether the attitudes of members of the post-baby-boom cohort (those born between 1966 and 1972) changed as they grew older over the 7 years between 1990 and 1997 and whether the next cohort of young adults that replaced them in 1997 (those born between 1973 and 1979) had distinctly different attitudes than their predecessors. In summary, we charted the degree of social change in the orientations of the public toward support of older people over the late 20th century and identified the components of that change related to the aging of cohorts and the dynamics of cohort replacement. Methods Samples We used data from three nationally representative telephone surveys of adults aged 18 years and older in the United States conducted in 1986, 1990, and 1997. Each sample was generated by random digit dialing of households within the contiguous United States. One adult aged 18 years or older was randomly selected from each eligible household as the designated respondent. The first survey (N = 1,209) was conducted in 1986 by Cook and colleagues (for details, see Cook and Barrett 1992) and lasted approximately 45 min on average. The second and third surveys were conducted in 1990 (N = 1,500) and in 1997 (N = 1,559), and each lasted 35 min on average (for details, see Bengtson and Harootyan 1994; Silverstein and Parrott 1997). Participation rates were 71% for the 1986 survey, 79% for the 1990 survey, and 64% for the 1997 survey. Because our analysis compared responses to a question regarding Social Security spending that was asked in 1986 and replicated in 1997 only, the first part of our investigation contrasted the full samples from these two surveys. We present the unweighted frequency distributions of sample characteristics for the 1986 and 1997 surveys in Table 1 . The sample in 1997 had significantly more education, were less likely to be married, and had a different age distribution with proportionately fewer young adults. The second part of the investigation focused on young adults aged 18–24 years, an age group with which we were able to test the hypothesis that youthful persons are particularly impressionable when exposed to historical change. Because the 1990 survey recorded respondents' ages for those older than 24 in 10-year categories, we were not able to investigate cohort change in older groups given the 7 years between surveys. Thus, we here describe the process of cohort change and replacement in these cohorts between 1990 and 1997 when they comprised, respectively, 210 and 168 individuals. The later cohort was significantly more likely to be male and showed trends toward having more education and more minority representation than its predecessor. In addition, we used the group that was aged 25–31 in 1997 (n = 241) to demonstrate the degree of intracohort change. Dependent Variables In the first stage of the analysis, public attitudes toward the Social Security program were assessed with the following question, asked in both 1986 and 1997: "Do you feel Social Security benefits should be increased, decreased, or maintained at their current levels?" Distributions of the three responses are shown for 1986 and 1997 surveys in Table 1 . There was a statistically significant shift away from advocating increases in benefits and toward maintaining or reducing benefits. Indeed, the percentage of respondents who supported reducing benefits almost doubled, from 3.3% to 6.3%, whereas those advocating an increase in benefits went from a majority to less than half of all respondents. In the second part of this investigation, we focused solely on the cohort change and replacement with respect to the youngest cohort between 1990 and 1997. In this section we took a more nuanced approach to assessing attitudes by taking into account the possibility that judgments about the worthiness of the target group of a social program are made independently of judgments about its financing—what has been termed ideological schizophrenia in public policy evaluation (Day 1993; Silverstein and Parrott 1997). Thus, we considered attitudes along two basic dimensions: the perceived legitimacy and utility of programs targeted at older people, and the perceived costliness and fiscal inequity of programs serving older people. Level of agreement with the following three statements reflected attitudes toward the legitimacy and utility of programs for older people: (a) "Social Security and Medicare are an earned right for persons aged 65 and older." (b) "If the government cut back on spending on programs for older people, we would all be hurt." (c) "The government should add coverage of nursing home care and home health care to Medicare, even if it means higher federal taxes for everyone." We used the level of agreement with the next three statements to illustrate attitudes toward the perceived costliness and fiscal inequity of programs for older people: (a) "Federal programs that provide benefits to older persons are too costly." (b) "Persons aged 65 and older get more than their fair share of government programs and tax benefits." (c) "All people over age 65 should pay a larger share of their medical costs than they do today." All statements were rated on a scale from 1 to 5, where 1 = "strongly disagree" and 5 = "strongly agree." To determine whether these six items corresponded to the two underlying dimensions of interest, we performed factor analysis with oblique rotation on these items. The solution and factor loadings from this analysis (Table 2 ) revealed that for each survey year the items scaled in a way that was consistent with our expectations: The first three items loaded on the first factor (Entitlement to Programs), and the second three items loaded on a second factor (Costliness of Programs), with an interfactor correlation of −.242. Using structural equation modeling with latent variables, we replicated this factor structure and were able to show that constraining factor loadings to be equivalent between 1990 and 1997 did not produce a significant decline in model fit, χ2(4, n = 2907) = 5.9, p = .21. This suggested that the measurement model was invariant over the two waves of measurement and permitted a meaningful analysis of change in these constructs over time. Therefore, for each of the two dimensions in each time period, we computed an average score from its three corresponding scale items. Independent Variables Our goal of decomposing the components of aggregate change in the population required only that age and historical time be used as variables. To match the age ranges with the 11-year interval between 1986 and 1997, we constructed the following age groups at both periods: 18–28, 29–39, 40–50, 51–61, 62–72, and 73 and older. We recognized that each of the samples may have had different compositions over time owing to shifts in population characteristics over time (e.g., increases in education) as well as sampling variability. Firebaugh and Davis 1988 pointed out that these compositional changes may or may not be of interest depending on whether the goal of the investigation is simply to estimate the amount of change due to cohort replacement or to discover why cohort replacement promotes social change. However, differences between samples due to sampling variability may prove problematic to the degree that these differences are also related to the attitudes that are being studied. Therefore, we present our findings both unadjusted and adjusted for the following variables: gender, marital status (married vs. unmarried), race (African American vs. other), and education (having attended college vs. high school graduate or less). As an adjunct to our analyses with respect to the entire U. S. population, we also examined intracohort and cohort replacement in attitudes toward aging policy among members of the youngest cohort of American adults, those aged 18–24 years, between 1990 and 1997. We present these results without control variables applied. Procedure We used the decomposition method outlined by Firebaugh 1997 to disaggregate net (or social) change into components of intracohort change and cohort replacement. Because net change is the sum of intracohort change and cohort replacement, we calculated the size of cohort replacement as CR = NC − IC, where CR = cohort replacement, NC = net change, and IC = intracohort change. The orthogonal intracohort change and cohort replacement components can be contrasted to determine how much of the net change is due to individual change and how much is due to replacement of older cohorts with younger cohorts. It is important to note that intracohort change is not the same as an aging effect and that cohort replacement is not the same as a cohort effect because period changes are confounded with maturation and cohort replenishment. Thus, it was important for us to rely on theory and "side" information to make educated guesses as to the mechanism underlying these two components of social change (see Firebaugh 1992; Glenn 1976). Results Our first step in setting up the cohort table that describes the components of change was to form a series of six adjacent cohorts. In Table 3 , we assembled separate tables showing the proportion of each age group in each year who responded with each of the following three responses concerning desired change in Social Security benefits: "increase," "maintain," and "decrease." By taking row and diagonal differences between years, we calculated, respectively, the size of net social change between adjacent cohorts and the size of intracohort change. To more formally assess the change within cohorts, we also tested whether intracohort change was statistically significant for each cohort. In estimating the components of social change for the sample, we took unweighted averages of the columns. Firebaugh 1997 noted that cohort replacement effects can derive both from changes in the values expressed by succeeding cohorts and from the restructuring of cohort representation in the population. Because we were concerned with understanding the extent to which social change derived from the unique imprint of historical context on young cohorts and not from fluctuations in the relative size of cohorts, we followed the lead of Alwin and Scott 1996 and equated the size of cohorts by calculating averages unweighted by group size to estimate the magnitude of social change and intracohort change (and by extension, cohort replacement). Change between 1986 and 1997 in the percentage of respondents advocating for increase in Social Security benefits is shown in the first panel of Table 3 . These results show that unadjusted net change in the population was −11.6%. This drop was further separated into intracohort and cohort replacement components. The component of net change due to intracohort change was −13.5%. Thus, individuals within cohorts were changing at a rate of change that exceeded the aggregate amount of social change because of a slightly positive cohort replacement effect of 1.9%. Essentially all the change in the population between 1986 and 1997 could be attributed to historical and developmental influences that occurred within cohorts, and virtually none of the almost 12% reduction could be attributed to cohort replacement. Adjusting for demographic variables reduced the amount of change observed but did not alter this conclusion. When we examined patterns of change across different cohorts, we found a large amount of variation in the size of the intracohort effect. Most notable was the substantial reduction—by more than 27%—among those aging from 62–72 to 73 and older in their support for increasing benefits. By contrast, young adults aged 18–28 declined only 7% in their support by the time they were aged 29–39, a reduction that was not significant at the .05 level. We next turned to those in the population who believed that Social Security benefits should be maintained. The second panel of Table 3 reveals a net increase of 9.1% in those advocating this position, and a somewhat smaller increase of 7.2% with demographic factors controlled. The intracohort component was slightly greater than the net change at an 11.8% increase, with the cohort replacement component at −2.7%. This implies that cohorts were changing toward greater protection of benefits at a rate that outpaced aggregate social change. This occurred because, after intracohort change was taken into account, more recent cohorts were, on average, slightly less in favor of maintaining benefits. With regard to change within specific cohorts, only the youngest group, those aged 18–28, did not significantly strengthen their preference for maintaining benefits. Especially striking was the large intracohort change among those 62–72 years old in 1986 toward greater protection of benefits. Because this change (24.6%) was similar in magnitude but opposite in sign to the change observed in this cohort toward increasing benefits (−27.1%), it appears that there was a substantial shift away from advocating for the expansion of benefits toward their preservation following the transition to late old age. Finally, the last panel in Table 3 presents results for those who were in favor of reducing Social Security benefits. Although advocacy for benefit reduction clearly represented a minority opinion, there was a net 2.6% increase in this response choice, of which the intracohort component was 1.8%. The only significant increase occurred in the 18–28 cohort (in 1986), which registered a 4.2% rise in the proportion advocating the reduction of benefits—more than double the average percentage increase across all cohorts. The cohort replacement component was 0.8%, representing one third of the aggregate social change. Thus, population turnover accounted for a relatively greater amount of social change in the populations' increased preference for cutting benefits. As before, introducing control variables served to reduce the amount of change across the two surveys but left the components of change largely proportional to each other. Young Adults: 1990–97 Given our interest in the extent to which impressionable young adults were susceptible to growing public concern over how support to older people will be financed in coming generations, we used a more nuanced approach to analyze several dimensions of these attitudes within two cohorts of young adults across 1990 and 1997: the strength of attitudes supporting the legitimacy of entitlement programs for older people and concerns over the costs of such programs. Because only the youngest cohorts were considered, intracohort and cohort replacement effects were more straightforward than in the previous section of this investigation. An assumption of the cohort replacement model is that the attitudes of the youngest adults are not influenced by intracohort (maturation or historical) change because they are just entering the system of adult cohorts. Thus, the aggregate difference between these two similarly aged young-adult cohorts separated by 7 years was attributed solely to cohort replacement. Intracohort change was calculated as before, by comparing members of the same cohort across the two periods as they aged to the next age category. We present in Table 4 the mean agreement scores among young adult cohorts with statements reflecting entitlement and cost dimensions of aging policy. As a basis for comparison, means are also shown for the whole population (as net aggregate change). Results are presented without adjusting for gender, education, race, and martial status because negligible differences were found in the means when these variables were controlled. With respect to the strength of attitudes toward entitlement of older people, we noted a significant decline of one fifth of a point in support for entitlement due to cohort replacement of young adults. That is, compared with their predecessors, the more recent cohort of those aged 18–24 years weakened in their support of programs targeting older people. This change was consistent with the trend observed in the general population of more than one quarter of a point decline in support for entitlement. The intracohort change in the 18–24-year-old group was not statistically significant, implying that the passage of members of this cohort to a later stage of life did not produce a change in their attitudes toward entitlement of older people to programs and benefits. Attitudes on the part of young adults toward the cost of programs for older people are shown in the second part of Table 4 . Neither cohort replacement nor intracohort change of young adults was statistically significant, although both effects showed a slight trend of declining concern with the cost of programs serving older people. These declines were in contrast to a significant increase of more than one tenth of a point in the population's level of anxiety over cost issues. To test whether the effects of intracohort change and cohort replacement among young adults were significantly different from the aggregate change for the full population, we conducted regression analyses in which intracohort change and cohort replacement factors (effect coded) were interacted with a dummy variable representing the two periods of measurement. In these models interaction terms reveal whether intracohort change and cohort replacement effects were different from the average social change observed in the population as a whole. These four regressions (not shown) produced significant interaction terms for intracohort change by year, for both attitudes toward entitlement and attitudes toward cost. These results implied that the trend in attitudes of members of the 18–24 cohort (as they aged to 25–31) ran counter to the average trend in the population of increasing wariness over the legitimacy of entitlement and the associated costs. Remarkably, these patterns revealed that attitudes in the general population were converging with those of this young adult cohort along both dimensions over the 7-year period. Despite an overall increase in the wariness expressed by the public toward the legitimacy and the cost of programs for older people, it is still noteworthy that, on average, Americans tended to be sympathetic toward programs serving older people. On the basis of the scale means, the support for entitlement was consistently stronger than the level of concern over cost. Further, when we examined individual scale items, we found that even in 1997 nearly three quarters of the population agreed that entitlement programs for older people were an "earned right" of older people and less than a third agreed that they were "too costly." Thus, any discussion of the social change in the public's attitude on aging policy should be placed in the context of generally widespread support for the role of the state in taking care of older citizens (Cook and Barrett 1992; Day 1990, Day 1993; Marmor, Mashaw, and Harvey 1990). Discussion In this investigation we used three nationally representative samples surveyed over an 11-year period to examine the degree to which cohort turnover and cohort change are related to changes in public attitudes toward programs that serve older people. Contrary to expectations, we found that cohort replacement played a small role in explaining the trend through the 1980s and 1990s of increasing opposition to the expansion of Social Security benefits. Instead, being embedded within a society that, as a whole, is moving toward being less generous to older people is primarily responsible for this social change in the population. The most dramatic shift in this regard occurred among those making the transition from early to late old-age, who most sharply moved away from a preference for increasing benefits levels. Whether or not this change is the result of growing concern about retrenchment in these benefits, increasing satisfaction with current benefit levels, or strengthening altruistic motivations cannot be known from the available data. The wholesale shift of this "young old" age group toward a preference for maintaining benefits may mean that this benefit-eligible group has become more protective of their benefits at a time when these programs are considered to be at risk but may also imply increasing satisfaction with current payment levels. However, that this elderly cohort also exhibited a relatively large (though not significant) increase in the percentage advocating for the reduction of benefits may imply that benefits are viewed as overly generous by their very recipients, especially if seen as imposing a burden on younger generations. A modest impact of cohort replacement was found with regard to the increase of those advocating reductions in benefits, much of that due to the cohort turnover among young adults aged 18–28 years. More notable is the relatively large increase in this cohort calling for a reduction in benefits—more than twice the average intracohort increase. Earlier we noted that intracohort change potentially reflects the effects of maturation (life-cycle transitions) and the effects of changing historical period. Young adults may have been particularly apt to absorb political messages of the 1980s and 1990s that framed the debate over entitlement programs as a crisis that would put future generations at risk—an example of the impressionable youth hypothesis posed by Alwin and Krosnick 1991. Even though members of this young age group started out in 1986 with greater skepticism toward public programs benefitting older people, they further increased their opposition to expanding benefits. This cohort of young adults was disproportionately swayed by the ideological shift toward questioning the legitimacy of entitlement programs for older people. Evidence of the importance of cohort turnover emerged in our examination focusing on the attitudes of the young adult cohort toward the entitlement programs for the aged. Our results provide strong evidence that the cohort more recently emerging into adulthood is substantially more skeptical than its predecessor concerning the deservingness of older people. We found no corresponding effect with regard to attitudes toward cost. However, there were intracohort differences observed with the passage of members of this young adult cohort into the next older age category, because their attitudes toward entitlement and costs did not become more conservative as did those of the overall population. Thus, although members of this cohort started out being more skeptical about entitlement and more critical about the costs of programs for older people, their attitudes stabilized so that over time they came to more closely resemble those of the population in general. It is likely that members of this cohort have moved into work and family roles that have caused them to "age out" of their substantially more negative opinions about entitlements and costs associated with the older population and adopt values more consistent with the mainstream opinion. Putting the results of this analysis together presents an apparent contradiction concerning the importance of cohort membership in the changing attitudes of the public toward programs targeting older people. Why does the transition of members of the youngest cohort to the next age category result in the strengthening in their preference for reducing Social Security benefits and a relative weakening in their concerns over the costs of programs for older people? The confound mentioned earlier between aging and historical change in the intracohort effect may provide a clue for solving this apparent contradiction. In addressing the issue, we first draw attention to the fact that in each analytical section the young adult cohorts reflect different age intervals and are assessed over different, if overlapping, historical periods. In the first analysis the interval began in 1986, a time when public support for Social Security was the highest level it had been in more than a decade (see Cook and Barrett 1992). If young adults are, in fact, especially sensitive to changes in sociopolitical milieu (Alwin and Krosnick 1991), then the values of this age group would be most affected by the media messages, proliferating in the decade following 1986, that questioned the viability and equity of public programs for the aged. Thus, the increased support for cuts in Social Security benefits in this cohort can more safely be attributed to historical forces than to maturation. Liberalization in the attitudes of the youngest age group toward entitlements and costs is out of step with the trend in the overall population and therefore may be the result of maturation. This interpretation is strengthened by the fact that it is in the age range subsequent to 18–24, following the transition to full adulthood, that some of the most fundamental life-style changes occur. This research is clearly not the last word on the subject of cohort change and attitudes toward aging policy. The availability of data from multiple birth cohorts collected over more than two time periods would go far toward determining the enduring effects of population turnover and maturation on attitudes toward public programs serving older people. Further, it is possible that several political events over the period studied (including the failure of Clinton's attempt at health care reform), as well as the changing tone of messages in the media and popular press, are responsible for observed changes in attitudes. However, the relative contribution of any one event is difficult to infer without data collected over many time points during the interval. Our inferences must, therefore, be speculative concerning the ultimate causes of social change in the population on this issue. In general our findings help resolve the question of whether attitudes of young adults toward programs for the aged are a product of youth itself (and thus will change with age) or the imprint of a unique historical period (and thus will be different among future young adults; Silverstein and Parrott 1997). The answer seems to be that both are true. The attitudes of young adults are changing as they age, suggesting that the imprint of historical conditions on impressionable youth is not indelible. On the other hand, historical forces appear to be selectively shaping the attitudes of new cohorts of young adults. The more contemporary young-adult cohort was especially likely to absorb societal messages of anxiety over public programs for the aged compared with the cohort it was replacing. Will members of the current cohort of young adults also age out of their skeptical attitudes toward benefit programs for older people, and will a new skeptical younger generation replace them? Addressing this question with multipanel data will be necessary to ultimately determine the consistency with which historical contingencies act on young adults to mold their attitudes toward public programs serving the older population. Table 1. Characteristics of U.S. Samples for 1986 and 1997 Surveys Year of Survey 1986 1997 Characteristic n % n % p Gender .295 Male 518 42.8 699 44.8 Female 691 57.2 860 55.2 Education <.001 High school graduate or less 592 49.0 611 39.2 Some college or more 617 51.0 948 60.8 Race/Ethnicity .751 African American 111 9.2 138 8.9 Hispanic 52 4.3 76 4.9 White non-Hispanic and other 1,046 86.5 1,345 86.3 Marital Status <.05 Not currently married 515 42.6 726 46.6 Currently married 694 57.4 833 53.4 Age <.001 18–28 269 22.3 290 18.6 29–39 343 28.4 426 27.3 40–50 200 16.6 373 23.9 51–61 166 13.8 214 13.7 62–72 144 11.9 157 10.1 73+ 85 7.0 99 6.4 Attitude Toward Social Security Spending <.001 Increase 680 56.9 717 46.9 Maintain 476 39.8 717 46.9 Decrease 40 3.3 96 6.3 Total 1,207 100.0 1,559 100.0 Table 2. Factor Analysis With Oblique Rotation Performed on Six Items Measuring Attitudes Toward Programs for Elderly Persons: 1990 and 1997 Pooled Data Rotated Factor Loadings Variable Entitlement Factor Cost Factor Social Security and Medicare are earned right 0.644 −0.045 Cuts in spending for older people would hurt all 0.635 −0.055 The government should add coverage of long-term care 0.701 −0.031 Federal programs benefitting older persons are too costly 0.262 0.802 Persons aged 65 and older get more than their fair share −0.171 0.644 All people aged 65 and older should pay a larger share of medical costs −0.230 0.545 Eigenvalue 1.797 1.039 % variance explained 29.9 17.3 Notes: Factor loadings above .5 are shown in bold. Interfactor correlation = −.242. Table 3. Percentage of U.S. Adults Responding That Social Security Benefits Should Be Increased, Maintained, or Decreased: By Age and Year of Measurement (1986 and 1997) Increase Benefits Maintain Benefits Decrease Benefits Age 1986 1997 NC IC 1986 1997 NC IC 1986 1997 NC IC 18–28 54.7 50.9 −3.8 — 40.4 41.5 1.1 — 4.9 7.7 2.8 — 29–39 60.2 47.7 −12.5 −7.0* 35.7 43.2 7.5 2.8 4.1 9.1 5.0 4.2** 40–50 55.8 48.8 −7.0 −11.4** 40.2 45.8 5.6 10.1** 4.0 5.5 1.5 1.4 51–61 57.2 43.5 −13.7 −12.3** 41.0 51.2 10.2 11.0** 1.8 5.3 3.5 1.3 62–72 56.9 47.4 −9.5 −9.8* 42.4 51.3 8.9 10.3* 0.7 1.3 .6 −0.5 73+ 53.1 29.8 −23.3 −27.1** 45.7 67.0 21.3 24.6** 1.2 3.2 2.0 2.5 Mean changea — — −11.6 −13.5 — — 9.1 11.8 — — 2.6 1.8 Mean change adjusteda,b — — −9.5 −12.0 — — 7.2 10.3 — — 2.3 1.6 Cohort replacementc 1.9/2.5 −2.7/−3.1 .79/.70 Notes: NC = net change; IC = intracohort change. Significance tests performed only for IC. See Table 1 for sample sizes. a Means unweighted by age-group size; tests of statistical significance not conducted. b Means when adjusted (within age group) by gender, education, marital status, and race. c Cohort replacement evaluated with unadjusted means/adjusted means. * p < .10; **p < .05. Table 4. Attitudes Held by Young Adults Toward Entitlement and Costs of Programs Serving Older People Between 1990 and 1997 Age 1990 1997 IC CR NC Mean support for entitlement 18–24 3.74 3.53 — −0.21* — 25–31 — 3.78 0.04 — — Total sample 4.02 3.75 — — −0.27* Mean concern over cost 18–24 2.44 2.40 — −0.04 — 25–31 — 2.36 −0.08 — — Total sample 2.27 2.38 — — 0.11* Notes: IC = intracohort change; CR = cohort replacement; NC = net change as measured by total mean difference. Statements were rated between 1 and 5, with 1 = "strongly agree" and 5 = "strongly disagree." 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How to think and: (t to think) \ No newline at end of file diff --git a/test/run.js b/test/run.js new file mode 100644 index 0000000..2377a37 --- /dev/null +++ b/test/run.js @@ -0,0 +1,56 @@ +/* global __dirname, require, process, it */ +'use strict'; + +var pkg = require('../package.json'), + myObject = require('../index.js'), + TU = require('rd-tu'), + fs = require('fs'), + path = require('path'); + +// Données de test +var dataset = require('./dataset/in/data/dataset.json'); + +// Mapping indiquant quelle fonction de test et quelles données utiliser pour chaque fonction +var wrapper = { + "indexAll": testOf_indexAll, + "graphs": { + "docToDoc": testOf_graphsDocToDoc, + } +}; + +/** + * Test de chaques fonctions de : + * - indexAll + * - graphs. + * - docToDoc() + */ +TU.start({ + description: pkg.name + '/index.js', + root: 'myObject', + object: myObject, + dataset: dataset, + wrapper: wrapper +}); + +/** + * Fonction de test à appliquée pour : + * - indexAll() + */ +function testOf_indexAll(fn, item, cb) { + return fn(item.arguments.directory, item.arguments.output, function(err, res) { + return cb(err); + }); +} + +/** + * Fonction de test à appliquée pour : + * - graphs.docToDoc() + */ +function testOf_graphsDocToDoc(fn, item, cb) { + return fs.readFile(item.arguments.filePath, 'utf-8', function(err, res) { + if (err) return cb(err); + return fn(JSON.parse(res), item.arguments.options, function(err, res) { + return cb(err); + }); + }); +} \ No newline at end of file