 1
in Small Airway Epithelium from Tobacco Smokers
and Patients with Chronic Obstructive Pulmonary
Disease (COPD)
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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Tobacco smoke is believed to cause small airway disease and then
chronic obstructive pulmonary disease (COPD), but the molecular mechanisms by which small airway obstruction occurs remain unknown. To study the gene expression levels of transforming
growth factor (TGF)-
1, a potent fibrogenic factor, in small airway
epithelium from smokers and patients with COPD, we harvested
highly pure samples of epithelial cells from small airways under direct vision by using an ultrathin bronchofiberscope BF-2.7T (outer
diameter 2.7 mm with a biopsy channel of 0.8 mm in diameter).
The expression levels of TGF-1 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of
TGF-1 corrected by -actin transcripts were significantly higher
in the smoking group and patients with COPD than those in nonsmokers (p < 0.01). Furthermore, among smokers and patients
with COPD, TGF-1 mRNA levels correlated positively with the extent of smoking history (pack-years) and the degree of small airway obstruction as assessed by measurements of flow-volume
curves. Immunocytochemistry of the cells demonstrated more intense stainings for TGF-1 in samples from smokers and patients
with COPD than from nonsmokers. Spontaneously released immunoreactive TGF-1 levels from cultured epithelial cells were more
elevated in subjects with a history of smoking and patients with
COPD than in nonsmokers. Our study showed a close link between smoking and expression of TGF-1 in small airways. Our results also suggested that small airway epithelial cells might be involved in obstructive changes found in smokers and patients with COPD.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Tobacco smoke has been implicated as one of the most important factors that can cause small airway disease followed by
chronic obstructive pulmonary disease (COPD) (1, 2). Animal
models of tobacco exposure showed inflammatory responses
along the airways as well as lung parenchymal structures (3-
5), but the molecular mechanisms by which small airway obstruction occurs remain unknown. Recent studies suggest
some fibrogenic growth factors may be involved in the remodeling processes of the small airways (6, 7). One of the most potent and extensively studied growth factors is transforming
growth factor (TGF)-1, which induces fibroblast proliferation, increased production of collagen and other extracellular matrix proteins, and decreased collagen degradation (8). This growth factor is also chemotactic for macrophages (9) and
mast cells (10). TGF-1 has an inhibitory effect on growth
of epithelial cells including bronchial epithelial cells (11, 12).
TGF-1 is usually released as inactive forms, which are activated by cleavage of SH-bonds with proteolytic enzymes. Airway epithelial cells constitutively express these polypeptides
in both active and inactive forms, and thereby can function as
autocrine growth inhibiting factors for themselves (13, 14).
TGF-1 mRNA and proteins are localized within peripheral
bronchial epithelial cells in normal lungs (15). de Boer and coworkers (16) recently demonstrated that TGF-1 mRNA and
proteins were increased in peripheral lung tissues from smokers with and without COPD by immunostaining and in situ hybridization techniques. Peripheral airway epithelial cells are,
therefore, important possible sources of this growth factor and
may play a role in airway mucosal repair and increased collagen deposition along the airway walls.
We harvested living epithelial cells from small airway mucosa under direct vision by using an ultrathin bronchofiberscope BF-2.7T (outer diameter 2.7 mm with a biopsy channel
0.8 mm in diameter) (17, 18). Although we found that expression of interleukin-8 (IL-8) and ICAM-1 was significantly
higher in small airway epithelium from tobacco smokers than
from nonsmokers (19), there was no correlation between these
inflammatory markers and airway obstruction in small airways
as assessed by 
25.
In the present study, we evaluated the mRNA levels of
TGF-1 in small airway epithelium from nonsmokers, smokers, and patients with COPD by the reverse transcription and
polymerase chain reaction (RT-PCR) technique, and correlated the magnitude of the expression of this fibrogenic factor
to the degree of small airway obstruction.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Subjects
Thirty-three Japanese healthy volunteers and 17 patients with COPD
were included in the study. Among healthy volunteers, 18 people (14 males and 4 females, mean age 58.0 yr) were current smokers and 15 people were never smokers (12 males and 3 females, mean age 53.3 yr). All the subjects were free from respiratory diseases, with normal
chest X-ray films and with no respiratory symptoms for at least 3 mo
before this study. Spirometry was performed in all subjects with no
abnormal results in percentage FVC and FEV1 among healthy people.
Maximal flow rates at 50% and 25% lung volumes (50 and 25, respectively) were also evaluated and expressed as percent predicted
values (20). The diagnosis of COPD basically depended on the reported criteria (16) including decreased percentage FEV1 (< 75%)
and no response to inhalation of bronchodilators. The data for 25
were significantly greater in never smokers than in smokers and patients with COPD (p < 0.01, ANOVA). The clinical data of these subjects are summarized in Table 1. The study was planned according to
the ethical guidelines following the declaration of Helsinki and given
institutional approval and an informed consent was obtained from
each subject.
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Bronchoscopy with an Ultrathin Scope
The subjects underwent a bronchofiberscopic examination with a BF-XT20 fiberscope (Olympus, Tokyo, Japan) in a standard fashion (17, 18). Under fluorographic guidance an ultrathin fiberscope (BF-2.7T) was inserted through a 2.8-mm-diameter biopsy channel. A newly modified BC-0.7T brush was then inserted to collect cells by brushing the airway mucosal surfaces several times. Brushing of the mucosa was routinely performed at three or four different ninth or tenth lower lobe bronchioles. The cells were immediately collected by vortexing the brush in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS, heat inactivated; GIBCO, Grand Island, NY). The cells were centrifuged for 5 min at 1000 rpm. The recovered cells were washed twice in Hanks' balanced salt solution without calcium and magnesium (HBSS; GIBCO). The number of the cells was counted by a standard hemocytometer and the cell viability was assessed by the trypan blue dye exclusion technique (18).
Differential Counting of the Cells
The cytospin preparations from harvested cells were obtained by a cytocentrifuge, and were routinely stained by Diff-Quick stain (modified Wright-Giemsa stain; Midorijuji, Kobe, Japan). The cytospin preparations were also stained by periodic acid-Schiff (PAS) stain for the detection of secretory granules. For detection of keratin in the cells, the specimens were stained with antikeratin (KL-1; Immunotech, Marseille, Cedex, France), or with control IgG1 monoclonal antibodies using the avidin-biotin complex method (21, 22). The differential counts of the harvested cells basically depended on the Diff-Quik and PAS stainings and were divided into four categories: ciliated, secretory, and nonciliated epithelial cells as well as other inflammatory cells (23).
RT-PCR for TGF-1 mRNA in Small Airway Epithelial Cells
To assess the TGF-1 mRNA levels in human small airway epithelial
cells, a semiquantitative assay utilizing RT-PCR was performed as
previously reported (18, 24). We used the epithelial cell samples for
RT-PCR only when the samples contained less than 5% nonepithelial
cells as evaluated by Diff-Quik and keratin staining. Total RNA was
isolated by the guanidinium thiocyanate-phenol-chloroform extraction method as described by Chomczynski and Sacchi (25). Briefly, after cell counting and assessment of cell viability, the cells (5.0 × 105
viable cells) were lysed in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7; 0.5% sarcosyl, 0.1 M 2-mercaptoethanol) and RNA was extracted from the solution by chloroform extraction. After that, the isopropanol precipitate RNA was washed twice with 70% ethanol, dried, and resuspended in diethylpyrocarbonate-treated water. Extracted RNA was reverse transcribed to cDNA by using a
Takara RNA-PCR kit according to the manufacturer's recommendations. Briefly, total RNA, random hexadeoxynucleotides as primer,
and avian myeloblastosis virus reverse transcriptase were used for cDNA synthesis. The following specific primer pairs were used for
PCR amplification:
TGF-1 (5'primer) 5'-GCCCTGGACACCAACTATTGCT-3'
(3'primer) 5'-AGGCTCCAAATGTAGGGGCAGG-3'
-actin (5'primer) 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3'
(3'primer) 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'
(Clontech, Palo Alto, CA).
The reaction mixture contained 10 mM Tris-HCl (pH 8.3 at 25° C),
50 mM KCl, 1.5 mM MgCl2, 1 mg/ml gelatin, 0.4 µM of each primer,
0.25 M diethyl-p-nitrophenyl monothiophosphate (dNTP), 1.0 µg
cDNA, and 1 U of Taq polymerase (Perkin-Elmer-Cetus, Norwalk, CT) in 25 µl. Amplification was performed for allotted cycles of denaturing (94° C, 2 min), annealing (60° C, 30 s), and extension (72° C,
1.5 min) using a thermal cycler (Progene; Techne, Cambridge). The
PCR cycle was determined by preliminary experiments showing a linear relationship between PCR cycles and intensity of signals on ethidium bromide-stained agarose gels. For semiquantitative evaluation of
TGF-1 and -actin mRNAs, 30 and 25 cycles were chosen, respectively. PCR product was run on a 1.0% agarose gel, and the intensity
of ethidium bromide fluorescence was evaluated by NIH Image version 1.61.
It was possible that the results of RT-PCR described above might
be influenced by an artifact during the amplification processes. To improve confidence in the results, we reevaluated the mRNA levels of
-actin and TGF-1 by doing the amplification processes in the same
tube. In these experiments, PCR amplification processes were set to
25 cycles.
Cell Culture
In some experiments, where enough epithelial cells were obtained, the cells were plated onto collagen-coated 48-well flat-bottom tissue culture plates (Koken, Tokyo, Japan) at a density of 5 × 104 cells/well with hormonally defined SAGB medium (Clonetics; SankoJunyaku Co., Ltd., Tokyo, Japan). Morphological changes during culture were studied by phase-contrast microscopy showing polygonal, nonciliated cells with a tight connection to each other. Confluent monolayers of epithelial cells were stained with antikeratin (KL-1; Immunotech), antivimentin (DAKO-Vimentin; DAKOPatts, Glostrup, Denmark), or with control IgG1 monoclonal antibodies using the avidin-biotin complex method (21, 22) to show that the cells were of epithelial cell origin.
Evaluation of TGF-1 Release by Cultured Epithelial Cells
On confluency, the epithelial cell-conditioned media were harvested
after different time periods. Immunoreactive TGF-1 was measured
by specific ELISA (R&D Systems, Inc., Minneapolis, MN) and was
expressed in pg/106 cells/24 h. For measurement of total amounts of
TGF-1, the samples were acidified by the addition of 1 N HCl followed by neutralization with NaOH as recommended by the manufacturer.
Immunocytochemistry for TGF-1
The cell samples were cytocentrifuged onto glass slides by Cytospin 2 (Shandon Southern Products, Cheshire, England) and fixed with 4%
paraformaldehyde. Immunocytochemistry was performed using avidin-biotin complex peroxidase (ABO-PO) as described previously in detail (26, 27). Briefly, the fixed cells were preincubated with 10% normal goat serum (Nichirei Corp., Tokyo, Japan) for 15 min to prevent
nonspecific binding. The cells were then incubated with an anti-TGF-1-specific antibody (1:100 diluted, 10 µg/ml; R&D Systems) for 30 min at room temperature. After rinsing in phosphate-buffered saline
(PBS), the slides were incubated with biotin-conjugated goat anti-rabbit immunoglobulin G antibody (Nichirei) and ABC-PO solution for
30 min. After rinsing in PBS, the reaction products were visualized using diaminobenzidine (Sigma). The staining intensity was graded and
expressed as 0 = absence of staining, 1 = moderate staining, 2 = intense staining, and 3 = very intense staining, as reported by de Boer
and coworkers (16).
Statistics
The results were analyzed by nonparametric equivalents of analysis of variance (ANOVA) for multiple comparison as reported (18). Spearman's rank correlation test was used for correlation analysis between the two data.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell Harvest, Viability, and Morphology
The total cell number and cell differentials evaluated by Diff-Quick, PAS and keratin stainings were shown in Table 2. The total cell number, viability and cell differential counts were not statistically different among the non-smokers, smokers and patients with COPD. The number of the recovered cells ranged from 1.20 × 106 to 2.30 × 106 with the mean of 1.78 × 106. The cell viability ranged from 62.5% to 79.5% with the mean of 68.0%. Approximately 70% of the viable cells were non-ciliated round cells which were positive to keratin staining as reported previously (18). As shown in Table 2, the major contaminating cells were neutrophils, but the percent of the non-epithelial cells were less than 5% in all cases.
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Small Airway Epithelial Cells in Smokers and Patients with
COPD Showed Increased TGF-1 mRNA as Compared with
Those in Nonsmokers by RT-PCR
The signals for TGF-1 and -actin were detected in all cases,
and the relative intensity of TGF-1 mRNA/-actin was statistically higher in smokers and patients with COPD than in nonsmokers as shown in Figure 1a and 1b. The TGF-1 mRNA
levels in patients with COPD were significantly higher than
those of healthy smokers.
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We reevaluated the mRNA levels of TGF-1 and -actin in
the same tube when a large enough amount of cDNA was obtained. Among 7 nonsmokers, 11 smokers, and 8 patients with
COPD, the levels of TGF-1 corrected by -actin transcripts
were again statistically higher in smokers and in patients with
COPD than in nonsmokers (Figure 1c).
RT-PCR Analysis Using Attached Cells
To exclude the possibility that the above observations were
caused by the contamination of a few nonepithelial cells such as neutrophils, we incubated the cells on collagen-coated tissue culture plates for 90 min to allow the epithelial cells to attach, and then the plates were rinsed twice and RNA was extracted (n = 5 in each group among never smokers, smokers,
and patients with COPD). By this technique, the keratin-positive cells were always more than 98.5% in all the samples as
assessed by immunostain on collagen-coated LabTec chamber
slides. The results again elucidated that the signals for TGF-1
mRNA were significantly increased in smokers and in patients
with COPD than in nonsmokers (Figure 1d).
Correlation between the Levels of TGF-1 mRNA and
Smoking History
Among current smokers without airway obstruction, TGF-1
mRNA levels correlated positively with the extent of smoking
history when the signals were normalized by -actin transcripts (r = 0.620, p < 0.001) (Figure 2a). The magnitude of
TGF-1 expression in patients with COPD also showed a significant correlation with smoking history as shown in Figure
2b (r = 0.653, p < 0.001). Such was also the case when the data
from PCR in the same tubes were evaluated (data not shown).
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Correlation between TGF-1 mRNA Levels and
Lung Function Data
The mRNA levels of TGF-1 significantly correlated with
percentage 25 and 50/25 in smokers (Figure 3a and 3c), but
there was no relationship to percentage 50 (Figure 3b), FVC,
or FEV1 (data not shown). Levels in patients with COPD
showed significant correlation with percentage 50 as well as
25, but not with 50/25 (Figure 4), FVC, or FEV1 (data not
shown). Such was also the case when the data from PCR in the
same tubes were evaluated (data not shown).
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Immunocytochemistry
The recovered epithelial cells were subject to TGF- staining.
As shown in Figure 5, airway epithelial cells from smokers and
patients with COPD showed more intense staining than from nonsmokers.
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TGF-1 Release versus Smoking
The cells from nonsmokers (n = 5), smokers (n = 5), and patients with COPD (n = 6) were cultured until confluence in
SAGB medium. Immunocytochemical studies demonstrated
that the cells were keratin positive, but vimentin negative,
showing the cells were epithelial cells. Inactive and active
forms of TGF-1 proteins spontaneously released by the epithelial cells were evaluated after different time periods by specific enzyme-linked immunosorbent assay (ELISA). As shown
in Figure 6, there was a time-dependent accumulation of immunoreactive TGF-1 in culture supernatants and total (inactive and active) amounts of TGF-1 were greater in smokers
and patients with COPD than in nonsmokers after 24 h. However, the levels of active form of TGF-1 were not different among the three groups. We also studied the effects of stimulation with IL-1 (10 ng/ml) and tumor necrosis factor (TNF)-
(10 ng/ml) in some samples. Neither stimulus affected the release of TGF-1 (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present studies, we found that the levels of TGF-1
mRNA in small airway epithelium were significantly higher in
smokers and patients with COPD compared with nonsmokers.
Importantly, the magnitude of the TGF-1 signals showed a
positive correlation with burden of cigarette smoking. Our results further showed that TGF-1 gene expression levels correlated with the degrees of peripheral airway obstruction as
evaluated by measurements on flow-volume curves. Immunocytochemistry also showed that the TGF-1 protein was more
intensely detected in epithelial cells from smokers and patients with COPD than from nonsmokers. Spontaneous release of total TGF-1 protein from cultured epithelial cells was
also increased in tobacco smokers and patients with COPD
compared with nonsmokers.
It is well known that cigarette smoking causes inflammatory responses in small airways (1, 2, 28). These changes included infiltration of inflammatory cells such as neutrophils, macrophages, and mast cells, and thickening of the airway walls with increased collagen deposition (1, 2). Such inflammatory and remodeling processes are believed to be related to the obstruction in small airways. The tobacco-related changes in the peripheral airways impose a major risk for development of COPD, especially pulmonary emphysema. Local migration of neutrophils might be induced by the direct effect of tobacco contents (29), however, additional data suggested that cigarette smoke stimulated airway epithelial cells to release chemotactic activities for neutrophils (30, 31) such as IL-8. In fact, the mRNA levels of IL-8 and one of the important adhesion molecules ICAM-1 were increased in small airway epithelial cells from smokers as compared with nonsmokers (19). However, it remained unclear whether these cells were also involved in the steps of tissue repair and remodeling, and eventual obstructive changes in the small airways. Recently, Stefano and coworkers (30) demonstrated that severity of airflow limitation is associated with severity of airway inflammation in smokers.
TGF- has been hypothesized to be involved in airway remodeling found in chronic airway inflammatory disorders such
as COPD and asthma (31, 32). de Boer and coworkers (16)
studied the expression of TGF-1 mRNA and proteins in resected lungs from smokers and patients with COPD by in situ
hybridization and immunostaining techniques. They showed
that semiquantitative histological scores of TGF-1 mRNA
and protein levels assessed by visual analogue scoring system
were significantly increased in bronchiolar and alveolar epithelium as well as endothelium in these subjects. There were
significant correlations between the scores of TGF-1 mRNA
or proteins in bronchiolar epithelium, and the number of macrophages or mast cells, suggesting a role of this growth factor in the accumulation of these inflammatory cells. Aubert and
coworkers (33) studied the expression levels of TGF-1 in lungs
from patients with asthma, nonobstructive tobacco smokers,
and patients with COPD by Northern blot analysis. They
found no difference among the three study groups. These apparent discrepancies of these results might be due to the different techniques utilized for evaluation. Aubert's group studied the magnitude of mRNA by Northern blot analysis using
lung tissues, and de Boer and coworkers (16) used in situ hybridization and immunostaining techniques. In the present
studies, we obtained highly pure populations of small airway
epithelial cells by an ultrathin scope from volunteers and patients with COPD who were free of lung cancer or other lung diseases necessitating surgery. Then, we compared the levels
of TGF-1 mRNA among nonsmokers, smokers without COPD,
and patients with COPD. The levels of TGF-1 mRNA were
statistically increased in people who smoked compared with
nonsmokers. We also studied the TGF-1 protein expression
by immunocytochemical analysis, and observed results comparable to the findings of de Boer and coworkers (16). Total
amounts of spontaneously released TGF-1 were also increased in smokers and patients with COPD, but the active
forms of this peptide did not change among the three groups, possibly because its activation is mainly dependent on proteolytic enzymes in the local microenvironments.
Our present findings needed careful considerations, as
there were several methodological limitations. First, RT-PCR
itself was not a quantitative technique for the evaluation of
certain gene expression levels to reconfirm the findings. To improve the technique, we repeatedly evaluated expression levels
by amplifying -actin and TGF-1 genes in the same tubes.
We also studied the expression of TGF-1 protein by immunocytochemical analysis. Although it was difficult to evaluate
the intensity of staining, these studies again showed increased
TGF-1 expression in peripheral bronchial epithelial cells
when a visual analogue scale reported previously (16) was applied. Second, a small number of nonepithelial cells in the cell
samples might have led to the misleading results. To exclude
this possibility, attached cells were used for RT-PCR analysis.
The cells were more than 98.5% epithelial cells and the results
again showed that the levels of TGF-1 gene expression were
higher in the epithelial cells from smokers and patients with
COPD than from nonsmokers. Immunocytochemistry also
demonstrated that the positive cells were virtually all epithelial cells. In accordance with the findings by RT-PCR, increased release of total TGF-1 from cultured epithelial cells
was found in smokers and patients with COPD. However, the
cultured epithelial cells might have been stimulated by attachment to the culture plates, or might have been changed as a result of being retrieved from microenvironments in the airways.
Results of immunostaining showed that the freshly recovered
epithelial cells from smokers and patients with COPD showed
increased staining as compared with nonsmokers, which was in accordance with the findings from cultured epithelial cells. Other approaches such as an in situ hybridization technique
will better elucidate gene expression in the specific cell types
in the airways.
In conclusion, we demonstrated an increased expression of
TGF-1 mRNA in small airway epithelium from healthy smokers and patients with COPD. It was suggested that small airway epithelial cells might be involved in the processes of airway remodeling and the resultant obstructive changes in the
small airways.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. H. Takizawa, Department of Laboratory Medicine, University of Tokyo, School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail: TAKIZAWA-PHY{at}h.u-tokyo.ac.jp
(Received in original form August 30, 1999 and in revised form August 17, 2000).
Acknowledgments: Supported in part by the Adult Diseases Memorial Foundation and the Manabe Medical Foundation.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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