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Keywords:

  • Bronchiolitis obliterans syndrome;
  • epithelial mesenchymal transition;
  • flow cytometry;
  • lung transplantation;
  • obilterative bronchiolitis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Bronchiolitis obliterans syndrome (BOS) compromises lung transplant outcomes and is characterised by airway epithelial damage and fibrosis. The process whereby the normal epithelial configuration is replaced by fibroblastic scar tissue is poorly understood, but recent studies have implicated epithelial mesenchymal transition (EMT). The primary aim of this study was to assess the utility of flow cytometry in detecting and quantifying EMT in bronchial epithelial cells.

Large airway brushings were obtained at 33 bronchoscopies in 16 BOS-free and 6 BOS grade 1–3 patients at 2–120 months posttransplant. Flow cytometry was used to assess expression of the mesenchymal markers αSMA, S100A4 and ED-A FN and HLA-DR. TGF β1 and HGF were measured in Bronchoalveolar lavage (BAL). Expression of all three mesenchymal markers was increased in BOS, as was HLA-DR. BAL HGF, but not TGF β1 was increased in BOS. Longitudinal investigation of one patient revealed a 100% increase in EMT markers concurrent with a 6-fold increase in BAL TGF β1 and the diagnosis of BOS at 17 months posttransplant.

Flow cytometric evaluation of bronchial epithelium may provide a novel and rapid means to assess lung allografts at risk of BOS.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Although lung transplantation is a well-accepted therapeutic option for selected patients with advanced lung disease, long-term survival is limited largely by progressive and treatment refractory airway remodeling manifest clinically as bronchiolitis obliterans syndrome (BOS) (1). The predominant histopathologic finding in patients with BOS is of fibroproliferative small airway obliteration (obliterative bronchiolitis (OB)), although large airways are also affected with the eventual development of bronchiectasis. Unfortunately, there has been no substantial improvement in the reported incidence of BOS over the last 10 years, despite improvements in immunosuppression, surgical techniques and patient management (2,3). There is therefore an urgent need to understand the pathogenesis of airway remodeling in order to identify new targets for therapeutic development.

Although the precise pathogenesis of BOS is unclear, there are recognized epidemiologic risk factors all of which lead to airway injury. The strongest risk factor is acute rejection, with the development of antibodies to HLA-DR being particularly important (4,5). These associations suggest that immune mechanisms underlie BOS and have led to the hypothesis that the disorder is a form of chronic rejection. However, many patients with acute rejection do not develop BOS, and some patients with BOS have never experienced acute rejection. Several nonimmune associations also exist, particularly cytomegalovirus and other viral infections and gastroesophageal reflux disease (2). BOS thus appears to represent a final common pathway of response to a variety of noxious insults. This heterogeneity of associations has made it difficult to determine a single treatment strategy that will benefit all patients (2). Identifying the origin of activated airway fibroblasts may be a key step in achieving this aim.

Airway fibroblasts potentially originate through a number of pathways including proliferation and activation of resident fibroblasts, recruitment of circulating progenitors and transition of airway epithelial cells. Epithelial-mesenchymal transition (EMT) has been implicated as a source of fibroblasts in renal (6) and pulmonary fibrosis (7). EMT is a biologic option available to epithelial cells suffering chronic or repeated injury and involves the loss of epithelial proteins such as E-cadherin and the gain of mesenchymal proteins such as S100A4 and vimentin and eventually myofibroblastic markers such as alpha-smooth muscle actin (α-sma) and the ED-A splice variant of fibronectin (ED-A FN) (8). These changes in protein expression are associated with increased cellular motility and the production of collagen and are driven by key profibrotic cytokines such as transforming growth factor β (TGF β1) (9) and antifibrotic cytokines such as hepatocyte growth factor (HGF) (10). Since TGF β1 is a key cytokine driving BOS (11) and given the known risk factors for BOS, all of which lead to epithelial injury, we hypothesized that EMT may play a central role in BOS pathogenesis. We further hypothesized that TGF β1 levels may be increased and HGF decreased in bronchoalveolar lavage (BAL) and epithelial HLA-DR expression increased in patients with BOS.

In order to test this hypothesis, our primary aim was to look for evidence of EMT in patients with and without BOS following lung transplantation using flow cytometric evaluation of bronchial brushing samples. Flow cytometry is a particularly attractive technique to investigate the expression of mesenchymal proteins by epithelial cells, as the coexpression of proteins of interest can be rapidly, accurately and quantitatively assessed cell by cell. Secondary aims were to look for changes in EMT-related cytokines TGF β1 and HGF in BAL and HLA-DR expression on bronchial epithelial cells from BOS patients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients

This was a collaborative study between the Western and South Australian lung transplant programs. The programs have similar surveillance and directed bronchoscopy schedules and immunosuppressive protocols. The study protocol was reviewed and approved by Human Research and Ethics Committees at both centers. BOS was defined and graded according to international guidelines (12). BAL and bronchial brushings were obtained from 16 stable transplant subjects and 6 subjects with BOS. Patients with evidence of acute rejection or infection requiring specific therapy were excluded, except for one patient with acute rejection followed by cytomegalovirus infection, who was followed longitudinally.

Bronchoscopy

Bronchoscopy, BAL and transbronchial biopsies were performed as previously described (13). Histological assessment was performed by pathologists who were blinded to the results of flow cytometric testing. Bronchial brushings were obtained using a cytology brush (Olympus Australia Pty Ltd, Mt Waverley VIC; outer diameter 2 mm, length 10 mm) from proximal (divisions 2–4) bronchi. Cells were agitated into 2.5 mL RPMI 1640 supplemented with 20% fetal calf serum (Gibco, Berlin, Germany). BAL was performed using 3 × 50 mL aliquots of warmed saline. The first aliquot was aspirated and used for clinical testing. The second and third aliquots were pooled, centrifuged (600 g (4°C) 10 min) and the supernatant stored at −80°C. Specimens were transported at 4°C and processed within 20 h of collection. We have previously shown that markers of EMT are unchanged for over 24 h when cells are stored in supplemented media (unpublished observations). Cell counts were performed using a modified Neubauer hemocytometer.

Immunological reagents

PE-CY5-conjugated Mabs to the leukocyte marker CD45 (Immunotech/Coulter, Marseille, France) was used to identify leukocyte contamination in bronchial brushings. The following monoclonal antibodies (Mabs) and immunological reagents were also employed: Epithelial cell antigen [FITC] (Dako-Cytomation, Glostrup, Denmark), anti-α-sma [FITC] (Sigma, St Louis, MO), polyclonal ED-A FN (Sapphire Bioscience, Redfern, Australia), Rat anti-mouse IgG1 [PE] (BD Biosciences, San Jose), polyclonal rabbit anti-human S100A4 (Dako), goat ant-rabbit IgG [PE] (Southern Technology Associates Inc., Birmingham, AL) IgG1/IgG1[FITC/PE] (BD) was employed as a negative control. Cell membrane lysing agent [FACSlyse] and permeabilizing agent [FACSperm] were obtained from BD.

Flow cytometric assessment of EMT and MHC expression

Five hundred microliter aliquots of prepared bronchial brushing were added to FACS tubes and red blood cells lysed by addition of 1 mL FACSlyse (BD) for 1 min followed by centrifugation at 1500 × g for 90 s. Cell membranes were then permeabilized by addition of 500 μL FACSperm (BD) for 10 min. Cells were washed with a buffer containing 10 mmol/L HEPES (Sigma)/NaOH, pH 7.4, 150 mmol/L NaCl, 1 mmol/L MgCl 2, 1.8 mmol/L CaCl (wash buffer), centrifuged, and the supernatant discarded. To block Fc receptors and reduce nonspecific binding, 20 μL of normal human immunoglobulin (60 g/L, Intragam, Commonwealth Serum Laboratories, Australia) was added to each tube for 20 min at room temperature. Cells were then incubated for 20 min with unconjugated Mabs to S100A4 or ED-A FN. Secondary staining for 15 min with goat anti-rabbit IgG [PE] (diluted 1/20 with wash buffer) or rat anti-mouse IgG1 [PE] was performed for S100A4 and ED-A FN, respectively. Cells were again washed and the cell surface stained with directly conjugated Mabs (α-sma [FITC], epithelial cell antigen [FITC], CD45 [PC5], HLA DR [PE]). Cells were rewashed then wash buffer (20 μL) was added, and events (50 000 for BAL and 10 000 for bronchial brushing) acquired immediately using a FACScalibur flow cytometer (BD) and analyzed using Cell Quest software (BD). As a negative control, cells were stained with primary antibodies (no secondary) or IgG (FITC) IgG (PE) control Mab and Mabs to identify cell type (Figure 1).

image

Figure 1. Flow cytometric evaluation of EMT after lung transplantation. (A) For analysis of EMT markers by flow cytometry, debris, lymphocytes and cell fragments were firstly excluded in region 1. (B) A further region (R2) defined cells that exhibited negative staining for CD45 (to further exclude leucocyte contamination). Using boolean logic, all subsequent analysis was carried out in cells from R1 and R2. (C) A control tube of epithelial cells stained with IgG control was included and the staining patterns used to set quadrant markers for flow cytometric analysis of (D) α-SMA (FITC) and HLA-DR (PE) (E) epithelial cell antigen (FITC) and S100A4 (PE) or (F) FN EDA+ (PE).

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Measurement of soluble TGF β1 and HGF

BAL supernatant was thawed and concentrated using Amicon Ultra-4 10 kDa centrifugal filter devices (Millipore, Billerica, MA) (4000 g, 10 to 25 min). Cytokine levels (corrected for concentration factor) were determined using a DuoSet ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions and were not corrected for urea or albumin (14). Latent TGF β1 was first activated through the addition at 1.5 of 0.1 mL 1N HCl to supernatant and neutralized with 1.5 1.2N NaOH/0.5M HEPES. A quality control sample of human serum was run on each ELISA plate with the interassay coefficient of variation for the plates remaining within ± 15%.

Statistical analysis

In patients studied at more than one time point, analysis was performed on all data, except for the one patient who developed BOS during the study. In that case only data obtained after the diagnosis of BOS were included. Data were found to be nonparametric so are presented as median (range). Statistical analysis was performed using the Mann–Whitney U-test and Pearson correlation tests using SPPS software. Differences between groups where p < 0·05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Six patients with BOS (three grade 3, two grade 2) and 16 BOS-free patients were studied. Patient demographics are presented in Table 1. There were no significant differences in baseline characteristics between the two groups except that patients with BOS were studied later posttransplant (33.3 ± 11.25 vs. 9.7 ± 1.0 month, p < 0.01, Table 1). Representative flow cytometric dot plots of expression of EMT markers by bronchial epithelial cells are presented in Figure 1.

Table 1.  Demographic characteristics of the study population
IndicationAge at transplantGenderTransplant typeTime post transplant (month)BOS grade
  1. UIP = usual interstitial pneumonia; α-1AT = alpha -1 antitrypsin.

  2. *p < 0.01 versus BOS-free group.

  3. BOS diagnosed at 17 months.

BOS-free
UIP39MBilateral120
Bronchiectasis56MBilateral4, 100
Emphysema61MBilateral30
UIP50ML single60
UIP58MR single20
UIP54FL single90
α-1ATdeficiency55MBilateral180
UIP60MBilateral6, 9, 120
Emphysema54MBilateral100
Emphysema45FBilateral6, 9, 180
Emphysema63MBilateral120
Bronchiectasis52FBilateral60
Emphysema65FBilateral9, 120
Emphysema65FBilateral60
Eisenmenger's26FHeart lung12,180
Emphysema58FR single90
Median (range)55.5 (26–65)  9 (3–18)* 
BOS
UIP58ML single252
UIP57FR single8, 11, 12, 14,173
Congenital heart disease41MHeart lung1202
Congenital heart disease40MHeart lung723
Emphysema58MBilateral281
Emphysema45MBilateral263
Median (range)51 (40–58)  27 (17–120) 

Bronchial epithelial cell mesenchymal protein expression is increased in BOS

There was low-level expression of mesenchymal proteins by epithelial cells in stable patients; however, BOS patients had markedly increased expression of all mesenchymal proteins studied, in particular ED-A Fn (Figure 2A–C). No cells expressed only mesenchymal proteins without coexpression of epithelial antigen (we have previously validated epithelial cell antigen as a reliable marker of epithelial cells using both primary brushing-derived epithelial cells and a 16 HBE epithelial cell line (14)).

image

Figure 2. Bronchial epithelial cell mesenchymal protein expression is increased in BOS. Mesenchymal markers (α-SMA –α-smooth muscle actin; S100A4 and EDA+ FN – EDA splice variant of fibronectin) were assessed by flow cytometry in 6 patients with BOS and 16 BOS-free patients. Box plots present median (% of epithelial cells expressing the mesenchymal marker) ±25th and 75th percentiles (solid box) with the 10th and 90th percentiles shown by whiskers outside the box. *Significant increase (p < 0.05) in mesenchymal protein expression by epithelial cells from patients with BOS compared to BOS-free patients.

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BAL HGF, but not TGF β1, is increased in BOS

There was no difference in BAL TGF β1 between the two groups (BOS 17.4 (4.4–41.6) pg/mL vs. BOS-free 14 (3.2–34.3) pg/mL) (Figure 3), however, BAL HGF was increased in BOS patients compared with patients who were BOS-free (67.9 (35.4–1601.7) vs. 14 (6.1–108.4) pg/mL, respectively, p < 0.05) (Figure 3A).

image

Figure 3. Bronchoalveolar lavage (BAL) hepatocyte growth factor (HGF), but not TGF β1 is increased in BOS. HGF and TGF β1 levels in BAL were assessed in six patients with BOS and 16 BOS-free patients. Data summarized in box plots as described in Figure 2. Patients with BOS had significantly increased BAL HGF (p = 0.02). *Significant increase (p < 0.05) in HGF in BAL from patients with BOS compared to BOS-free subjects.

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Epithelial HLA-DR expression is increased in BOS

Epithelial cells from patients with BOS had significantly increased expression of HLA-DR when compared to epithelial cells from stable patients (BOS 57% (50–85) vs. BOS-free 32% (14–62), p = 0.002, (Figure 4)).

image

Figure 4. Bronchial epithelial cell HLA-DR expression is increased in BOS. HLA-DR expression by bronchial epithelial cells was assessed by flow cytometry in six patients with BOS and 16 BOS-free patients. Data summarized in box plots as described in Figure 2. Patients with BOS had significantly increased epithelial HLA-DR expression (p = 0.02). *Significant increase (p < 0.05) in HLA-DR expression by epithelial cells from patients with BOS compared to BOS-free subjects.

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Longitudinal assessment of EMT

In 7 patients, all of whom were initially BOS-free, evaluation of lung function, S100A4, α-sma, ED-A FN, BAL [TGF-β1], BAL [HGF] as well as clinical evaluation including transbronchial biopsy was performed on a minimum of two occasions. For 6 of these patients low-level expression of mesenchymal markers by epithelial cells and BAL HGF and TGF β1 did not change with time (data not shown) consistent with their stable clinical status. For 1 patient, an increase in S100A4, α-sma, ED-A FN, and BAL TGF-β1 and HGF (Figure 5) were noted concurrent with the diagnosis of BOS. This patient was a highly sensitized female with a history of recurrent acute rejection and CMV pneumonitis prior to the development of treatment refractory BOS.

image

Figure 5. Longitudinal analysis of EMT markers in bronchial brushing-derived epithelial cells. Epithelial cell expression of the EMT markers (α-sma and S100A4) and BAL cytokines (TGF-β1 and HGF) were measured in a highly sensitized (pretransplant panel reactive antibody 90%) 57-year-female recipient of a single lung transplant for usual interstitial pneumonia (UIP). The patient developed treatment refractory BOS following an episode of acute rejection (A2B0) at 6-month posttransplant and an episode of CMV pneumonitis at 10-month posttransplant. Note increased expression of EMT markers, TGF β1 and HGF concurrent with the diagnosis of BOS at 17 months after transplantation. No bronchial brushing samples were obtained at the last bronchoscopy at 20 months. αSMA =α-smooth muscle actin; S100A4; TGF β1 = transforming growth factorβ; HGF = hepatocyte growth factor; FEV1 = forced expiratory volume in 1 second; BAL = bronchoalveolar lavage; CMV = cytomegalovirus.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We have demonstrated increased expression of mesenchymal proteins by large airway bronchial epithelial cells in patients with BOS after lung transplantation. This patient group also had increased epithelial expression of HLA-DR and increased BAL HGF levels. Longitudinal data in one patient who developed BOS during the study revealed marked increases in mesenchymal protein expression by epithelial cells and increased BAL TGF β1 and HGF at the time of BOS diagnosis. While we acknowledge that there are many difficulties in establishing the existence and importance of EMT in vivo, our data provide support for a role for EMT in the pathogenesis of BOS.

Observational studies have identified multiple risk factors for BOS including recurrent acute allograft rejection, cytomegalovirus infection, respiratory viral infections, infection with P. aeruginosa and gastro-esophageal reflux disease (reviewed by Boehler et al. (2)). However, these studies provide little insight into how apparently disparate insults can lead to a relatively homogeneous phenotype of chronic allograft dysfunction characterized predominantly by fibroproliferative obliteration of small airways. Of central importance in elucidating the pathogenic mechanisms underlying BOS is identification of the source of the activated fibroblasts leading to airway obliteration. Studies examining fibrosis in other organs, particularly kidney (6), have challenged the assumption that fibrosis occurs through activation and migration of local fibroblasts and have instead suggested that these profibrotic cells can arise from circulating progenitors and from dedifferentiation of epithelial cells to a mesenchymal phenotype in response to recurrent or persistent injury (EMT). The latter pathogenic process is particularly attractive since it ties together the well recognized but diverse risk factors for BOS. In this study, we provide evidence that the differentiation state of bronchial epithelial cells in lung transplant patients and particularly patients with BOS, is indeed unstable with expression of both epithelial and mesenchymal proteins. These cells appear to have been caught in the act of transition.

Despite a large body of in vitro data, there is only very limited information regarding the importance of EMT in human lung disease and only one study has examined the potential role of EMT in lung transplantation (16). Ward et al. studied endobronchial biopsy specimens from 16 stable, BOS-free patients using immunohistochemistry. A median 15% (0–48%) of epithelial cells from stable lung transplant patients expressed S100A4 (16)—similar to the level expression in our study (19% (11–40%)). Our study goes further to demonstrate associated increases in the expression of the myofibroblastic markers αSMA and ED-A FN in lung transplant patients, further increments in expression of these key mesenchymal proteins in patients with BOS, and longitudinal data demonstrating upregulation of mesenchymal protein and cytokine expression in one patient who developed BOS during the study.

TGF β1 is produced by a wide range of inflammatory, epithelial and mesenchymal cells, and is a potent activator of fibroblasts, inducing extracellular matrix production. It is also heavily immunosuppressive, with the ability to deactivate immune effector cells. TGF β1 is therefore a key cytokine with the ability to halt the inflammatory process and to orchestrate tissue repair. Since TGF β1 has been implicated in BOS pathogenesis (11,17) it is surprising that we found no increase in TGF β1 in BAL from patients with BOS. Since there did appear to be a trend to an increase, this may simply reflect the relatively low number of patients with BOS in our study. Alternatively, one group found BAL TGF β1 to be increased during OB evolution but not in stable or ‘burnt-out’ OB (17). In favor of this explanation in our study, all but one of our patients fell into the stable or ‘burnt-out’ category, and longitudinal data in the remaining patient demonstrated a progressive increase in BAL TGF β1 during BOS evolution (Figure 5).

HGF is predominantly expressed in mesenchymal cells and acts on epithelial and other cells through its tyrosine kinase receptor c-met. It is a potent inducer of cellular turnover and plays a central role in epithelial repair after injury (18). In lung transplantation, serum HGF levels increase with episodes of pulmonary infection and particularly rejection (19). HGF generally acts as an inhibitor of EMT (20), although in some contexts HGF can induce at least partial EMT (a phenomenon known as ‘reversible scatter’ (6)). We hypothesized that levels of HGF in BAL from patients with BOS may be reduced, but instead found a significant increase. This could represent a reparative response to the recurrent and/or persistent epithelial injury that characterizes BOS, or alternatively may simply indicate an increased number of HGF producing mesenchymal cells in allografts with BOS.

Our finding of increased epithelial HLA-DR expression in patients with BOS is in line with the increasing body of literature implicating this component of the HLA system in BOS pathogenesis (4,5). HLA-DR expression is usually restricted to professional antigen presenting cells such as dendritic cells, macrophages and activated T cells, but can be induced in bronchial epithelial cells by cytokines such as interferon gamma (IFNγ) to assist in the attraction, localization and adhesion of leukocytes. It remains unclear whether HLA-DR expression is a primary or secondary event in BOS pathogenesis. Specifically, it is unclear whether HLA-DR expression is an early and central event associated with antigen presentation by epithelial cells with subsequent amplification of the immune response to the graft or is simply a marker of on-going epithelial inflammation.

In vitro, EMT is characterized by gain of mesenchymal proteins, loss of key epithelial proteins and increased cellular motility (9). These processes are much more difficult to study in vivo. We are therefore unable to fully confirm EMT as it is defined in in vitro studies. It is possible, for instance, that bronchial epithelium only transiently expresses mesenchymal proteins at the time of injury (‘reversible scatter’ (6)), but that full-blown EMT, including increased cellular motility with invasion of the basement membrane and production of collagen, does not occur. Reversible scatter could explain the very few cells in the large airway epithelium that expressed mesenchymal proteins in the absence of epithelial markers. If this is the case, the circumstances that lead to epithelium undergoing full EMT may be key to understanding BOS pathogenesis. An alternative explanation is that once epithelial cells have undergone EMT and have become motile, they migrate through the basement membrane and are beyond the reach of the bronchial brushing technique. Since the patients with BOS in our study were all studied later posttransplant than the stable patients, we can not exclude the possibility that increased mesenchymal protein expression is a function of time posttransplant rather than a feature of BOS. Although there was no effect of time posttransplant on mesenchymal protein expression in the patients without BOS (data not shown), larger studies including BOS-free patients further posttransplant will be required to exclude this possibility.

In this study, we provide evidence for EMT as the final common pathway to airway obliteration following epithelial injury. The commonality of this response to injury may provide the opportunity for broadly effective therapeutic strategies. Another contributor to the poor prognosis is the insensitivity of current diagnostic techniques. The novel techniques described in this study, particularly the identification of epithelial cells coexpressing the EDA splice variant of fibronectin, may provide a powerful, less invasive (compared with transbronchial biopsy) and more sensitive method for evaluating the pulmonary allograft.

In conclusion, flow cytometric analysis of bronchial brushing samples provides a simple, safe, rapid, quantitative and potentially important additional method for assessment of the health of the lung allograft. Using this technique, we describe increased expression of mesenchymal proteins in epithelial cells from patients with BOS, suggesting that EMT may be important in BOS pathogenesis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Sarah Wong and Suzanna Temple for assistance in performing the TGF β1 and HGF assays and Jessica Ahern for her expert technical assistance with the flow cytometric analyses.

Funding sources:

This work was supported by the National Health and Medical Research Council of Australia, grant #5018121 and the University of Western Australia.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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