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A Determination of Tumor Necrosis Factor Expression in TMJ Inflammation with the Use of Microarray Analysis
R. Spears*,
R. Oakes,
C. Moore,
L.L. Bellinger and
B. Hutchins
Department of Biomedical Sciences, Baylor College of Dentistry-The Texas A&M University System Health Science Center, 3302 Gaston Avenue, Dallas, TX 75246, USA;
Correspondence: *corresponding author, rspears{at}tambcd.edu
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ABSTRACT
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Many different factors can lead to inflammatory changes within temporomandibular joint tissues. This investigation examined if the expression of TNF- and its receptors was altered in TMJ tissues during inflammation. Adult male rats were injected bilaterally with complete Freund’s adjuvant (CFA) into the TMJ or served as uninjected controls and were killed two days after CFA treatment. TMJ tissues were removed, and expression of TNF- and its receptors was examined via gene microarray analysis, RT-PCR, Western blot, and ELISA. Gene microarray analysis provided evidence for changes in gene expression, notably that TNF- and TNF-R1, but not TNF-R2, were significantly elevated in CFA-treated TMJ tissues. However, protein levels of TNF-, TNF-R1, and TNF-R2 were all significantly increased in CFA-treated TMJ tissues. These results indicate that the pro-inflammatory cytokine TNF- may play a significant role in the onset of inflammatory conditions associated with adjuvant-induced arthritis of the TMJ.
Key Words: tumor necrosis factor • inflam-mation • adjuvant • TMJ • microarray
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INTRODUCTION
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Adjuvant-induced arthritis has gained widespread use as an animal model for the study of rheumatoid arthritis and inflammatory responses in joint tissues (Harper et al., 2000, 2001). Studies show that rats with adjuvant-induced arthritis have elevated levels of tumor necrosis factor- (TNF-) and that TNF- levels are elevated in synovial tissue and fluid of patients with rheumatoid arthritis (Husby and Williams, 1988; Saxne et al., 1988; Neidel et al., 1995; Feldmann et al., 1996; Takahashi et al., 1998; Mannel and Echtenacher, 2000; Ulfgren et al., 2000). The extent of TNF- increase appears to precede the progression of inflammation and bone erosion within affected joints (van den Berg et al., 1999). While studies have examined changes that occur in CFA-induced arthritis (Smith-Oliver et al., 1993; McComb et al., 1999; Feige et al., 2000), there are no reports in the literature analyzing the mechanistic changes that occur in the TNF- signaling pathway during the onset of TMJ inflammation.
A recently developed method for analyzing cellular changes in response to various stimuli is with DNA microarrays. This technique measures changes in the expression of thousands of genes and may provide information about regulatory mechanisms and functional pathways. Used in combination with protein-based methods that measure the final translated product, gene arrays provide a valuable tool for determining possible changes during initiation of the inflammatory process.
In this study, cDNA array analysis was used to examine differences in gene expression after the application of CFA in TMJ tissues, followed by identification of changes in mRNA and protein levels indicated by the array data. The data presented here focused upon changes in the expression of TNF- and its receptors, TNF-R1 and TNF-R2, in response to CFA-induced inflammation, because of their role in the development of the inflammatory response.
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MATERIALS & METHODS
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Surgical Procedures and Adjuvant Injection
Twelve adult male Sprague-Dawley rats (200-250 g, Harlan Industries, Houston, TX, USA) were anesthetized and given bilateral, intra-articular injection (into the TMJ) with a mixture of 50 µg CFA (Mycobacterium tuberculosis, Sigma, St. Louis, MO, USA) in 50 µL of paraffin oil. An additional 12 uninjected rats served as controls, to prevent any contralateral cross-over effect from the CFA. To mimic conditions of acute inflammation, all animals were killed two days after CFA treatment via carbon dioxide inhalation and decapitation, and TMJ tissues (synovium, retrodiscal tissues, and articular disc) were removed. Both TMJ tissues from one animal were pooled to form a sample, then 6 samples from both the CFA-treated and the control animals were used for either RNA or protein extraction. All surgical procedures and death protocols were approved by the Baylor College of Dentistry Institutional Animal Care and Use Committee.
cDNA Expression Microarray
Total RNA was extracted from TMJ tissues via a modified method of Chomczynski and Sacchi (1987) with use of TRIZOL Reagent (Gibco BRL, Rockville, MD, USA) and treated with RNAse-free DNAse I (Roche Molecular Biochemicals, Indianapolis, IN, USA). First-strand cDNA synthesis was run with 2 µg of RNA along with [-32P] dATP and the cDNA synthesis primer mix at 50°C. The radioactively labeled probes were then hybridized, along with 3 µg of Cot-1 DNA, at a final concentration of 2-4 x 106 counts/min, onto individual AtlasTM Array membranes that contain 1148 cDNAs spotted onto positively charged nylon membranes. The membranes were washed, exposed to Kodak BioMax x-ray film (Kodak, Rochester, NY, USA) for 6 days at -80°C, then analyzed by means of the AtlasImageTM Software (Clontech Laboratories, Inc.). Plasmid and bacteriophage DNAs were used as negative controls to confirm hybridization specificity, while numerous housekeeping cDNAs acted as positive control sites for normalizing RNA abundance.
The signal from a blank portion of the cDNA array was taken as background, and each individual gene expression on the arrays was adjusted for background. Normalization of relative gene intensities between the uninjected and CFA-injected array membranes was established via globalization with the housekeeping gene ubiquitin. To minimize false-positives, we set the threshold level of significant changes in mRNA levels at ± 3.0-fold difference compared with controls. Three separate samples for both the CFA-treated and control animals were run on separate membranes, and the process was repeated 3 times, with results presented as means and standard deviations from all of the membranes. The data were statistically analyzed via the unpaired Student’s t test, with the significance defined as P  0.05.
RT-PCR
A 2-µg quantity of total RNA from each of 3 separate CFA-treated and control samples was run in triplicate, subjected to DNAse treatment, and reverse-transcribed by means of an M-MuLV reverse transcriptase system with random hexamer primers (Perkin Elmer) at 42°C for 1 hr. PCR was performed with 2 µL of the cDNA, 0.5 µL of Taq polymerase (Promega), 2 µL of each dNTP, and 50 pmol of the appropriate oligonucleotide primer pair, with the use of relative RT-PCR kits (Ambion, Inc., Austin, TX, USA). Amplification of the cDNA was performed by a hot-start method under conditions of 94°C for 5 min, then melting for 30 sec at 94°C, followed by extension for 1 min at 57°C for TNF- and β-actin, 59°C for TNF-R1, and 61°C for TNF-R2. Annealing was then carried out at 72°C for 1 min, with 28 cycles for TNF-, 26 cycles for TNF-R1 and β-actin, and 30 cycles for TNF-R2. A 20-µL aliquot of each PCR product was separated onto a 2% agarose gel, stained with ethidium bromide, photographed under UV fluorescence, and analyzed by densitometry. The RT-PCR band densities (239 bp for TNF-, 343 bp for TNF-R1, 261 bp for TNF-R2, and 294 bp for β-actin) were quantified via computer-assisted two-dimensional densitometric scanning with the public domain NIH Image Analysis Program 1.59b4f. Results were expressed as means and standard deviations and were statistically analyzed via the unpaired Student’s t test, with the significance defined as P 0.05.
Western Blot and ELISA Analysis for TNF-, TNF-R1, and TNF-R2
Three separate samples for both the CFA-treated and control TMJ tissues were homogenized in ice-cold protein-extraction buffer, then centrifuged at 7500 g for 15 min at 4°C, and the resulting supernatant was collected. Total protein concentration of the lysates was determined via a standard Folin Lowry protein assay. Protein (25 µg) was electrophoresed onto gels then transferred to polyvinylidine difluoride membranes (ImmunobilonTM NC Transfer Membranes, Millipore Co., Bedford, MA, USA) according to the standard protocol. Polyclonal primary antibodies recognizing the amino terminus epitope—either rabbit anti-TNF-, -TNF-R1, or -TNF-R2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA)—were then added at a dilution of 1:1000 overnight at 4°C, followed by incubation for 2 hrs with a secondary antibody of goat anti-rabbit-horseradish peroxidase at a dilution of 1:2000. The products were detected by means of an enhanced chemiluminescence light detection kit (Bio-Rad Labs) on Kodak BioMax film. We stripped and reprobed the membranes using a primary antibody for β-actin to verify equal protein loading. Western blot densities were quantified with the use of the NIH Image Analysis Program 1.59b4f.
Western analyses were run in triplicate and repeated 3 times, and representative data are shown. Quantitative measurement of TNF-, TNF-R1, and TNF-R2 was performed with the use of commercially available sandwich ELISA assays (R&D Systems, Minneapolis, MN, USA) and measured by means of a Molecular Devices SpectraMax 250 ELISA Plate Reader (Sunnyvale, CA, USA). Protein content among all samples was normalized. Each sample was run in triplicate, and the results were expressed as means and standard deviations and were statistically analyzed via the unpaired Student’s t-test, with the significance defined as P 0.05.
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RESULTS
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Changes in Gene Expression after CFA Treatment as Measured by cDNA Microarrays
To identify changes in gene expression during CFA-induced inflammation of the TMJ, we used cDNA microarrays to examine expression levels of hundreds of full-length genes. The majority of gene-expression signals did not exceed background hybridization level or were not at least three-fold different between control and CFA-treated. However, results from the gene microarrays demonstrated that CFA treatment produced at least a ± 3.0-fold change in expression levels in 111 genes ( 10% of all genes monitored), with 81 genes (73%) up-regulated and 30 genes (27%) down-regulated relative to the uninjected controls (Table). Interestingly, CFA produced an up-regulation in TNF- (6.8-fold) and TNF-R1 (3.1-fold) in comparison with the uninjected control gene expression. TNF-R2 gene expression remained unchanged 48 hrs after CFA treatment.
Expression of TNF-, TNF-R1, and TNF-R2 mRNA in TMJ Tissues after CFA Treatment
To verify results obtained from the microarrays, we analyzed the expression of TNF-, TNF-R1, and TNF-R2 mRNA in TMJ tissues by RT-PCR (Fig. 1A ). The results of densitometric analysis (Fig. 1B) demonstrated that mRNA levels for TNF- and TNF-R1 were increased in the CFA-injected animals in comparison with controls. TNF-R2 mRNA levels in CFA-treated tissues were not significantly different from control levels. mRNA for both TNF receptors was detectable in the untreated and the CFA-treated TMJ tissues; however, the level of TNF-R2 was less than that of TNF-R1, suggesting that TNF-R1 was the major receptor subtype for TNF- in TMJ tissues.
Changes Produced in Protein Expression of TNF-, TNF-R1, and TNF-R2 after CFA Treatment
We used Western blot analysis to determine protein expression following a CFA-induced acute TMJ inflammation for TNF-, TNF-R1, and TNF-R2 48 hrs after CFA treatment. The TNF-, TNF-R1, and TNF-R2 protein levels were increased in CFA-treated animals in comparison with uninjected controls (Figs. 2A, 2B). These changes were quantified by ELISA (Fig. 3). The ELISA results demonstrated a significant increase in the protein expression of TNF-, TNF-R1, and TNF-R2, suggesting that CFA administration produced an acute inflammatory response characterized by increased expression of TNF- and its receptors.
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DISCUSSION
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The goal of this study was to determine changes in gene, mRNA, and protein expression for TNF- and its receptors, TNF-R1 and TNF-R2, in TMJ tissues during the acute stage of CFA-induced inflammation. While previous studies have reported changes associated with increased activity of cytokines during conditions such as rheumatoid arthritis (Husby and Williams, 1988; Saxne et al., 1988; Neidel et al., 1995; Feldmann et al., 1996; Mannel and Echtenacher, 2000; Ulfgren et al., 2000), there are few reports in the literature investigating the role that TNF- plays during the onset of inflammation at the TMJ. Additionally, with the advent of gene microarray technology, a valuable tool is available that can be utilized to analyze expression differences in hundreds of genes. This is the first report of gene microarray analysis used for the examination of changes that occur in TMJ tissues after the initiation of an inflammatory response.
The findings of this study demonstrate that protein expression of TNF- and its two receptors is increased in TMJ tissues after CFA injection. A few studies have shown that TNF- is present in moderate to high concentrations in synovial fluid and tissues during inflammation (Kubota et al., 1998; Takahashi et al., 1998; Gravallese and Goldring, 2000; Arend, 2001; Choy and Panayi, 2001). However, the results from this study are the first to report that protein levels of not only TNF- but also its receptors are elevated during inflammatory proliferation at the TMJ. Since elevated levels of TNF- can lead to increased damage at joints affected by inflammation, the present results support the possibility that TNF- may play a significant role in TMJ damage.
Interestingly, results from the determination of mRNA and gene expression for TNF- and its receptors in TMJ tissues after CFA treatment differed somewhat from the observed changes in protein levels. RT-PCR results confirmed the results of the gene array, since mRNA of TNF- and the TNF-R1 receptor were increased in response to CFA. However, TNF-R2 mRNA, unlike the protein, was not altered in CFA-treated TMJ tissues, according to the criteria set forth for microarray analysis. This may be an example of translational gene regulation rather than transcriptional regulation and, therefore, does not produce detectable changes in mRNA expression.
TNF- delivers its signal through binding to both TNF-R1 and TNF-R2. Most of the biological activities associated with TNF- are mediated through TNF-R1, while TNF-R2 often will bind then subsequently pass TNF- to TNF-R1 (Bazzoni and Beutler, 1996). In some respects, the finding in the present study that TNF-R1 is increased relative to TNF-R2 is not surprising, since TNF-R1 plays a more active role during inflammation and is more ubiquitous than TNF-R2 (Kwon et al., 1993; Wajant et al., 2001). Also, this difference in the protein and mRNA expression for TNF-R2 may represent differences in the need for expression of TNF-R2 in TMJ tissues relative to other areas of the body where TNF-R2 may play a more important role (Lung et al., 2001). The TMJ tissues of the CFA-treated animals contain numerous inflammatory cells that have migrated into the inflamed tissues. These cells are likely to possess many more TNF-R1 receptors than TNF-R2, further increasing the likelihood that TNF-R1 will be the predominant receptor located within the inflamed tissues.
Since development of TMJ inflammation involves a complex cascade of interrelated events, analysis of microarray data allows for a comparison in the expression of hundreds of different genes (Burke, 2000; Cox, 2001; Meltzer, 2001; Quackenbush, 2001). Gene microarrays have been used to determine changes in gene expression during inflammatory conditions (Heller et al., 1997; Glynne and Watson, 2001). The findings from this study indicate that a large number of genes are differentially expressed following treatment with CFA and can be categorized into functional groups (Table). While general areas such as metabolism, cell-surface antigens, and ion channels/transporters had the greatest number of genes affected, changes in several particular genes, such as caspases-1 and -3, are noteworthy in relationship to the role they may play in CFA-induced inflammation. When TNF- binds to TNF-R1, it can lead to activation of a caspase cascade and eventual apoptosis. The effects of this process have yet to be elucidated and may be a potential mediator of changes observed during inflammation. In conjunction with this, the observation in this study that numerous genes involved in energy metabolism have been down-regulated may suggest that mitochondrial changes, such as those observed during apoptosis, may also be taking place.
However, it is important to keep in mind that many genes that do not display changes in differential expression between conditions may also be of functional importance and should not be disregarded on the basis of unobserved changes. The expression profile presented in this study represents data from several different cell types present in the TMJ tissues. Among these are cells of the immune system that have migrated to the area and are responsible for propagation of the inflammatory response, such as macrophages, monocytes, and leukocytes. Inflammation within these tissues is also linked to increased proliferation of fibroblasts within the inflamed areas (Joosten et al., 1999). Many of the observed changes in the microarray data are likely due to the influx and presence of these different cell types within the inflamed tissues that are not present in the uninflamed control tissues. However, this most likely represents what happens during the typical inflammatory process during joint inflammation of the TMJ. Therefore, while this study provides valuable information about overall changes within the TMJ tissues, further research is necessary to determine which cell types are responsible for the effects reported in this study. Taken together, the data from this study indicate that a complex cascade of events takes place during the onset of TMJ inflammation and that TNF- plays a significant role during this process.
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ACKNOWLEDGMENTS
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This study was supported in part by NIH Traineeship grant T35 DE07188-06 and the Center for Craniofacial Research and Diagnosis at Baylor College of Dentistry-Texas A&M University System Health Science Center.
Received for publication March 7, 2003.
Revision received June 27, 2003.
Accepted for publication July 10, 2003.
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REFERENCES
|
|---|
- Arend WP (2001). Physiology of cytokine pathways in rheumatoid arthritis. Arthritis Rheum 45:101–106.[CrossRef][Medline]
[Order article via Infotrieve]
- Bazzoni F, Beutler B (1996). The tumor necrosis factor ligand and receptor families. N Engl J Med 334:1717–1725.[Free Full Text]
- Burke HB (2000). Discovering patterns in microarray data. Mol Diagn 5:349–357.[CrossRef][Medline]
[Order article via Infotrieve]
- Chomczynski P, Sacchi N (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.[Medline]
[Order article via Infotrieve]
- Choy EH, Panayi GS (2001). Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 344:907–916.[Free Full Text]
- Cox JM (2001). Applications of nylon membrane arrays to gene expression analysis. J Immunol Methods 250:3–13.[CrossRef][Medline]
[Order article via Infotrieve]
- Feige U, Hu YL, Gasser J, Campagnuolo G, Munyakazi L, Bolon B (2000). Anti-interleukin-1 and anti-tumor necrosis factor-alpha synergistically inhibit adjuvant arthritis in Lewis rats. Cell Mol Life Sci 57:1457–1470.[CrossRef][Medline]
[Order article via Infotrieve]
- Feldmann M, Brennan FM, Maini RN (1996). Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14:397–440.[CrossRef][Medline]
[Order article via Infotrieve]
- Glynne RJ, Watson SR (2001). The immune system and gene expression microarrays—new answers to old questions. J Pathol 195:20–30.[CrossRef][Medline]
[Order article via Infotrieve]
- Gravallese EM, Goldring SR (2000). Cellular mechanisms and the role of cytokines in bone erosions in rheumatoid arthritis. Arthritis Rheum 43:2143–2151.[CrossRef][Medline]
[Order article via Infotrieve]
- Harper RP, Kerins CA, Talwar R, Spears R, Hutchins B, Carlson DS, et al. (2000). Meal pattern analysis in response to temporomandibular joint inflammation in the rat. J Dent Res 79:1704–1711.
- Harper RP, Kerins CA, McIntosh JE, Spears R, Bellinger LL (2001). Modulation of the inflammatory response in the rat TMJ with increasing doses of complete Freund’s adjuvant. Osteoarthritis Cartilage 9:619–624.[Medline]
[Order article via Infotrieve]
- Heller RA, Schena M, Chai A, Shalon D, Bedilion T, Gilmore J, et al. (1997). Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proc Natl Acad Sci USA 94:2150–2155.[Abstract/Free Full Text]
- Husby G, Williams RC Jr (1988). Synovial localization of tumor necrosis factor in patients with rheumatoid arthritis. J Autoimmun 1:363–371.[CrossRef][Medline]
[Order article via Infotrieve]
- Joosten LA, Helsen MM, Saxne T, van De Loo FA, Heinegard D, van Den Berg WB (1999). IL-1 alpha beta blockade prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF-alpha blockade only ameliorates joint inflammation. J Immunol 163:5049–5055.[Abstract/Free Full Text]
- Kubota E, Kubota T, Matsumoto J, Shibata T, Murakami KI (1998). Synovial fluid cytokines and proteinases as markers of temporomandibular joint disease. J Oral Maxillofac Surg 56:192–198.[CrossRef][Medline]
[Order article via Infotrieve]
- Kwon J, Chung IY, Benveniste EN (1993). Cloning and sequence analysis of the rat tumor necrosis factor-encoding genes. Gene 132:227–236.[CrossRef][Medline]
[Order article via Infotrieve]
- Lung HL, Leung KN, Stadlin A, Ma CM, Tsang D (2001). Induction of tumor necrosis factor receptor type 2 gene expression by tumor necrosis factor-alpha in rat primary astrocytes. Life Sci 68:2081–2091.[CrossRef][Medline]
[Order article via Infotrieve]
- Mannel DN, Echtenacher B (2000). TNF in the inflammatory response. Chem Immunol 74:141–161.[Medline]
[Order article via Infotrieve]
- McComb J, Gould T, Chlipala E, Sennelo G, Frazier J, Kieft G, et al. (1999). Antiarthritic activity of soluble tumor necrosis factor receptor type I forms in adjuvant arthritis: correlation of plasma levels with efficacy. J Rheumatol 26:1347–1351.[Medline]
[Order article via Infotrieve]
- Meltzer PS (2001). Spotting the target: microarrays for disease gene discovery. Curr Opin Genet Dev 11:258–263.[CrossRef][Medline]
[Order article via Infotrieve]
- Neidel J, Schulze M, Lindschau J (1995). Association between degree of bone-erosion and synovial fluid levels of tumor necrosis factor alpha in the knee-joints of patients with rheumatoid arthritis. Inflamm Res 44:217–221.[CrossRef][Medline]
[Order article via Infotrieve]
- Quackenbush J (2001). Computational analysis of microarray data. Nat Rev Genet 2:418–427.[CrossRef][Medline]
[Order article via Infotrieve]
- Saxne T, Palladino MA Jr, Heinegard D, Talal N, Wollheim FA (1988). Detection of tumor necrosis factor alpha but not tumor necrosis factor beta in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 31:1041–1045.[Medline]
[Order article via Infotrieve]
- Smith-Oliver T, Noel LS, Stimpson SS, Yarnall DP, Connolly KM (1993). Elevated levels of TNF in the joints of adjuvant arthritic rats. Cytokine 5:298–304.[CrossRef][Medline]
[Order article via Infotrieve]
- Takahashi T, Kondoh T, Fukuda M, Yamazaki Y, Toyosaki T, Suzuki R (1998). Proinflammatory cytokines detectable in synovial fluids from patients with temporomandibular disorders. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:135–141.[CrossRef][Medline]
[Order article via Infotrieve]
- Ulfgren AK, Grondal L, Lindblad S, Khademi M, Johnell O, Klareskog L, et al. (2000). Interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment. Ann Rheum Dis 59:439–447.[Abstract/Free Full Text]
- van den Berg WB, Joosten LA, van de Loo FA (1999). TNF alpha and IL-1 beta are separate targets in chronic arthritis. Clin Exp Rheumatol 17(6 Suppl 18):S105–S114.[Medline]
[Order article via Infotrieve]
- Wajant H, Henkler F, Scheurich P (2001). The TNF-receptor-associated factor family: scaffold molecules for cytokine receptors, kinases and their regulators. Cell Signal 13:389–400.[CrossRef][Medline]
[Order article via Infotrieve]
Journal of Dental Research, Vol. 82, No. 10,
807-813 (2003)
DOI: 10.1177/154405910308201009
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ABSTRACT
INTRODUCTION
