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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Asthma presents a variable clinical response to corticosteroids (CS). Because CS more likely act on inflammation than on tissue remodeling, the presence of bronchial structural changes in certain asthmatics may explain their limited clinical response to CS. Matrix metalloproteinase-9 (MMP-9) and its
inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), are, respectively, involved in tissue inflammatory processes and fibrogenic processes. Previous reports have suggested that MMP-9:TIMP-1 ratio may reflect the balance between these two processes in various diseases. This study evaluated the
relation of this ratio and the response to CS in severe asthma. Twenty asthmatics with low baseline
FEV1 (59 ± 4% predicted) and 
30 % increase with 
2-agonist were recruited. Serum MMP-9 and
TIMP-1 levels were measured and correlated with response to an oral CS trial (methylprenisolone 40 mg/d for 14 d). With oral CS, FEV1 changes (
FEV1) ranged from 
15 to +43%. The FEV1 closely
correlated with the MMP-9:TIMP-1 ratios (rho = 0.79, p = 0.0006). In conclusion, serum MMP-9:
TIMP-1 ratio could predict the response of oral CS therapy in asthma. The low MMP-9:TIMP-1 ratio
observed in subjects with little or no FEV1 improvement with CS supports the hypothesis that, in
these asthmatic subjects, bronchial fibrogenesis predominates over inflammation.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The extracellular matrix (ECM) is involved in tissue homeostasis and in pathologic conditions such as tumor invasion, wound healing, and inflammatory and fibrogenic diseases (1). Matrix metalloproteinases (MMPs) digest different ECM components. Among them MMP-2 and MMP-9, named gelatinase A and B, respectively, degrade type IV collagen of basal membrane (2, 3). Eosinophils express MMP-9, and their migration through basal membrane in vitro is reduced by metalloproteinase inhibition (4). T-cell transmigration through basal membrane is also mediated by MMP-2 and MMP-9 (5). Consequently, these enzymes could be released in tissue by inflammatory cells and may represent a degenerative burden for the ECM.
In counterbalance, the tissue inhibitors of matrix metalloproteinase (TIMPs) bind noncovalently in 1:1 proportion MMPs and inhibit their enzymatic activity (6, 7). TIMPs have other biologic activities: recombinant TIMP-1 induces proliferation of skin fibroblasts isolated from patients with systemic sclerosis and these fibroblasts release TIMP-1 that could be responsible for the autocrinous promotion of fibrogenesis found in this disease (8). Appropriate regulation of MMPs and their inhibitors during the tissue repair processes is important for tissue healing. Bullen and colleagues (9) reported that TIMP-1 level in wound fluids increased significantly 48 h after a surgical intervention. In healing wounds, the gelatinase activity:TIMP-1 ratio diminishes over time, whereas in chronic wounds it remains high.
Imbalance between MMP and TIMP activities has also
been observed in different pathologic conditions. In rheumatoid arthritis, serum level MMP-9 is increased, likely resulting
from a sustained and intense inflammatory process that causes
joint tissue degeneration. In contrast, in systemic sclerosis, an
increased level of serum TIMP-1 is related to the severity of
the disease, to a higher incidence of lung fibrosis, and to the
extent of skin sclerosis and fibroblast activity (10, 11). An excess TIMP-1 over MMP-1 is also observed in a rat model of
liver fibrosis (12). In chronic hepatitis C, serum TIMP-1 levels correlated positively with the degree of liver fibrosis, serum MMP-2 levels with periportal necrosis, and the MMP-2:
TIMP-1 ratio with the response to the interferon- treatment
(13). In cervical carcinoma, high MMP-9:TIMP-1 and MMP-2:
TIMP-2 ratios are associated with a poor prognosis, which may
be related to the tumor invasiveness (14). These data suggest that the balance between tissue MMP and TIMP biologic activities may represent an important determinant of the nature
of the remodeling process and the MMP:TIMP ratio has been
proposed as a prognostic indicator in these diseases. Consequently, an excess of MMPs may be responsible for structural
degradation of tissues, whereas an excess of TIMPs may promote excessive tissular repair process and fibrosis.
Asthma is a chronic bronchial inflammatory disease characterized by lymphocyte and eosinophil submucosa infiltrate (15). Importantly, this inflammatory process likely activates bronchial fibroblasts and induces airway wall remodeling (16). An inadequate bronchial mucosa repair process initiated by chronic inflammation-induced tissue damages and resulting mucosal structural changes likely play an important role in the pathogenesis of asthma (16, 17). Consequently as in other diseases, the MMP and TIMP levels, and more specifically the balance between these two biologic compounds, may reflect asthma pathophysiology and its clinical features.
Corticosteroids (CS) are the basic treatment of asthma. They influence both inflammatory and remodeling processes by reducing inflammatory cell infiltrates and changes in extracellular matrix (18). Laitinen and Laitinen (19) have suggested that CS have a normalizing effect on different cell types and extracellular matrix components, thereby preventing asthma. However, the impact of long-term inhaled CS therapy on subepithelial fibrosis might be minor (20). In moderate to severe asthma, high doses of inhaled CS or even oral CS are required to achieve sufficient control of symptoms. Some of these subjects respond poorly to CS and present marked fluctuations of their airflow obstruction despite high doses of CS. Recent studies on CS-resistant asthma have suggested that the molecular mechanism of poor sensitivity to CS therapy could be due to a glucocorticoid signal transduction pathway defect caused by an intense airway inflammatory process (21).
To our knowledge, no study reported the influence of airway remodeling on the asthma response to CS. We hypothesize that not only the inflammation but also the fibrogenic process modulate the response of asthma to treatment with CS. An ongoing airway tissue remodeling could induce an excessive fibrogenic process, reducing CS-induced improvement in pulmonary functions (22). In this study, we evaluated the possibility that in severe asthma, serum MMP-9 and TIMP-1, used as interdependent indicators of inflammatory and fibrogenic processes, respectively, correlate with the clinical response to CS.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Clinical Evaluation
Twenty subjects were recruited for this study, which was approved by
the Laval Hospital Ethics Committee. They were asthmatics according to the ATS guidelines for the diagnosis of asthma (25). Patients
with symptoms or signs of bronchial or lung infection, other respiratory diseases such as cystic fibrosis, interstitial lung fibrosis, bronchiectasis, and history of lung infections, or diseases such as diabetes,
hypertension, and history of psychiatric disorder were excluded. The
selection of asthmatics for a CS trial to identify CS-sensitive and CS-resistant subjects was based on the criteria of Corrigan and colleagues
(26): significant airflow obstruction with an important reversibility
( 30% FEV1 improvement over baseline) to inhaled 2-adrenoreceptor agonists (BD) (salbutamol, 100 µg, two puffs). After signing an
informed consent, the patients were submitted to a CS trial of 40 mg
methylprednisolone daily for 14 d.
Venous blood was sampled early in the morning prior to the first
oral dose of CS and 24 h after the last dose of CS. Compliance of the
subjects during the CS trial was evaluated by the decrease of serum
cortisol at the end of the CS therapy compared with their baseline values. Clinical characteristics of the selected subjects are given in Table
1. Serum samples collected at the entry of the study were kept for
measurement of gelatinase activity and MMP-9 and TIMP-1 proteins.
Serum samples from a healthy subject, a subject with mild asthma
(baseline FEV1 85% of predicted, using only a 2-agonist on demand), and two subjects with severe asthma (one with baseline FEV1
58% of predicted using 100 mg of prednisone daily, one with baseline
FEV1 74% of predicted using 25 mg of prednisone daily) were used
for zymographic gelatinase assay.
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Measurement of the Serum Gelatinase Activity by Cytometry
In order to evaluate the total serum gelatinase activity in solution, the serum was activated with an organomercuric compound (APMA, p-aminophenyl mercuric acetate; Sigma, St. Louis, MO) and buffered with calcium and zinc (27). Each serum sample was thawed on ice and diluted 1:1 (vol/vol) in digestion buffer containing 10 mM TRIS-HCl, 2 mM CaCl2, 1 mM ZnCl2, and 10 mM APMA. A method based on fluorochrome-labeled substrate coated on polystyrene microspheres was used to determine the gelatinase activity in sera by flow cytometry (28). The microspheres coated with fluorescein-labeled gelatin were incubated 24 h at 37° C with buffered sera. After incubation, the reaction was stopped by adding a phosphate-buffered solution (PBS) with 0.5% of gelatin and 10 mM ethylene diamine tetraacetic acid. The microsphere fluorescence analyses were performed on an ELITE ESP (Coulter, Burlington, ON, Canada) using a 15 mW argon laser as the source of excitation, and the fluorescein emission was measured by a photomultiplier with a 520 nm band-pass filter. The loss of microsphere fluorescence was proportional to the digestion of fluorescein-labeled gelatin coated on microspheres and reflected the gelatinase activity. The relative gelatinase activity was calculated using the peak position value of the sample (n = 5,000 events) on a log-scaled fluorescence histogram. The peak position value of gelatin-fluorescein-coated microspheres (n = 5,000 events) incubated in the digestion buffer without serum was used as reference to evaluate the relative gelatinase activity.
The porcine skin gelatin (Sigma) (2 mg/ml) was labeled with FITC (fluorescein-5 isocyanate) (Molecular Probes, Eugene, OR) at 0.2 mg/ ml in high pH buffer (50 mM boric acid, 200 mM NaCl at pH 9.2) for 24 h at 4° C. Gelatin-FITC conjugates were purified on a Sephadex G-75 column (Pharmacia Biotech AB, Baie d'Urfé, PQ, Canada) equilibrated with PBS (pH, 7.4) and optical density (OD) was measured at 280 and 492 nm on each collected fraction (1 ml). Fractions of purified FITC-gelatin conjugates with a FITC/protein molar ratio above 5, considered as suitable for cytometry analysis, were then pooled for microsphere coating. Polystyrene microspheres of 15 µm (Polysciences, Markham, ON, Canada) at a density of 108 beads/ml were incubated for 2 h at room temperature with the purified gelatin-FITC conjugates (adjusted to 1 mg/ml).
Detection of Serum MMP-9 and TIMP-1 Proteins by Immunoassays
The serum detection of total MMP-9 and TIMP-1 was done using the Biotrak ELISA-based detection system (Amersham, Oakville, ON, Canada). Assays were performed following the manufacturer's recommendations; aliquots of serum were diluted 1:20 or 1:40 in the assay buffer. MMP-9 and TIMP-1 concentrations were evaluated using Softmax software (Molecular Devices, Eugene, OR). The detection limit was, respectively, 1.25 ng/ml for TIMP-1 and 1.3 ng/ml for MMP-9. TIMP-1 detection kit does not cross-react with TIMP-2; the assay detected the total TIMP-1, free TIMP-1, and MMPs-TIMP-1 complexes. MMP-9 detection kit had < 3% cross-reactivity with pro-MMP-1, pro-MMP-2, and pro-MMP-3. The MMP-9 assay detected the pro-MMP-9 and pro-MMP-9-TIMP-1 complexes.
Zymography
Gelatin zymographs were performed on patients with severe asthma, on a normal subject, and on a subject with mild asthma in order to confirm that the gelatinase activity evaluated by flow cytometry is mostly due to the level of MMP-9 detected by ELISA. Each serum was diluted (1:20) in nonreducing sample buffer containing sodium dodecyl sulphate (SDS), glycerol, and bromophenol blue and subjected to electrophoresis on 10% polyacrylamide SDS gels containing 1 mg/ml of porcine skin gelatin (Sigma). After electrophoresis, the polyacrylamide gels were washed in 50 mM TRIS-HCl (pH, 7.4) and then in 2.5% Triton X-100, 50 mM TRIS-HCl (pH, 7.4) to remove all traces of SDS. They were rinsed again in 50 mM TRIS-HCl (pH, 7.4) for 10 min and finally incubated overnight in digestion buffer containing 10 mM CaCl2 and 100 mM NaCl. The gels were fixed in 10% methanol solution containing 10% acetic acid and then stained with 0.05% Coomassie brilliant blue. Gelatin digestion was identified as clear zones of lysis against a blue background. Molecular weights of the gelatinolytic bands were estimated using SDS-polyacrylamide gel electrophoresis (PAGE) protein standards, and they ranged from 45 to 200 kD (BioRad, Mississauga, ON, Canada). Gelatinolytic activity was measured on zymography-digitalized images using the NIH image shareware v.1.6.1 (http://rsb.info.nih.gov/nih-image/). The density (number of pixels) and the surface area digested were taken in account to measure the gelatinase activity (gelatinase activity = number of pixels × surface area).
Statistical Analysis
Serum parameters measured before the CS trial were correlated with
the pre-BD FEV1 improvement with the CS trial (FEV1) expressed in percentages: the percentage of change in FEV1 = FEV1 after CS
therapy FEV1 before CS therapy/FEV1 before CS therapy × 100. Correlation analysis was performed using linear regression calculation and Spearman's nonparametric correlation test. Bartlett's sphericity test was used to confirm correlation. All graphic representations show
individual values. Data of subgroups of subjects were analyzed using
nonparametric tests (Mann-Whitney U test) (29). All data were analyzed using the StatView software version 4.5 (Abacus Concepts, Berkeley, CA).
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RESULTS |
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Clinical Characteristics of Enrolled Subjects and Their Physiologic Responses to Oral CS
The selected asthmatics required high daily doses of inhaled
CS (1,288 ± 161 µg, all 500 µg of beclomethasone or equivalent). Despite this inhaled CS treatment, they still had low
baseline FEV1 (59 ± 4%), which increased by 30% after inhalation of 2-agonist. The FEV1 with the CS trial ranged
from 15 to +43%. As suggested by Corrigan and colleagues
(26), we divided subjects based on their CS response into CS
responders (CSR) (FEV1 improved by 25% after CS) and
CS nonresponders (CSNR) (FEV1 improved 
15% after CS).
Two subjects showed an improvement in FEV1 of 23% and we deliberately included them in the CSR group. Their individual values appear as triangles in the graphic representations,
except for the gelatinase assay, which was not tested in these
two subjects. No significant difference in sex, age, duration of
asthma, inhaled CS usage, and baseline FEV1 before oral CS
therapy was observed between CSR (n = 9) and CSNR (n = 11) (Table 1). Oral CS treatment improved baseline FEV1 by
a mean of 30.2 ± 2.5% in CSR and did not change it in CSNR,
0.8 ± 3.2% (Table 2). After the oral CS trial, CSR almost
reached their predicted FEV1 value after BD (94.3 ± 6.9%),
whereas CSNR did not improve their post-BD FEV1 (77.8 ± 5.1%). After the CS trial, the serum cortisol was decreased in
all subjects (CSR: 83 ± 13% of pre-CS values, p = 0.001;
CSNR: 70 ± 12%, p = 0.003).
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Serum Gelatinase Activity and MMP-9 Level, and Their Correlation
Serum gelatinase activity measured by cytometry varied from 68 to 88 arbitrary units. Sera of CSR (83 ± 1.4 arbitrary units, n = 7) showed higher gelatinase activity than did those of CSNR (76.7 ± 2.5, n = 8, p = 0.03). Serum MMP-9 protein levels measured by ELISA varied from 1.4 to 413 ng/ml. The sera of CSR had significantly higher MMP-9 levels (231 ± 17 ng/ml, n = 9) than did the sera of CSNR (158 ± 33 ng/ml, n = 11, p = 0.03). The level of MMP-9 significantly correlated with the gelatinase activity (r = 0.91, p = 0.0001) (Figure 1, top panel).
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Association of Serum Gelatinase Activity and MMP-9 Protein by Zymography
On the zymograms, the gelatinase activity observed was near the 97 kD reference protein that corresponds with the molecular weight of MMP-9 or gelatinase B (pro-MMP-9 = 92 kD and MMP-9 = 83 kD) (Figure 1, bottom panel). MMP-2 (72 kD), or gelatinase A, also has a high gelatinolytic activity, but no such band was detected even with the individual having the highest level of gelatinolytic activity. Furthermore, a linear correlation was found between the zymographic gelatinase activity measured by image analysis of the 92 kD band in the sera of the selected subjects and the respective level of MMP-9 measured by immunoassay (r = 0.83, p = 0.04; chi-square = 7.2, p = 0.03, n = 6).
Serum TIMP-1 Level and Its Correlation to MMP-9
Serum TIMP-1 level varied from 553 to 1,797 ng/ml. The mean TIMP-1 values for CSR and CSNR were 904 ± 80 ng/ml and 1,137 ± 118 ng/ml, respectively, and were not statistically different. The linear regression of TIMP-1 versus MMP-9 demonstrates that there is a direct relationship between MMP-9 and its inhibitor (CSR: r = 0.90, p = 0.0009 and CSNR: r = 0.74, p = 0.009) (Figure 2). It also shows that CSR are different from CSNR in that CSNR have more TIMP-1 relative to their level of MMP-9.
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MMP-9 and TIMP-1 Level and Ratio Correlation with the Change in FEV1 after Oral CS Therapy
A correlation between the level of MMP-9 and the clinical response to CS therapy (FEV1 changes) was found using Spearman's test (rs = 0.56, p = 0.01) (Figure 3, top panel). However,
this correlation could not be confirmed by Bartlett's test (chi-square = 4.6, p = 0.10). No correlation was observed between
TIMP-1 level and the FEV1. When the MMP-9: TIMP-1 ratios were plotted against the FEV1, a highly significant positive correlation was found between the clinical response to CS
therapy (FEV1 changes) and the serum MMP-9: TIMP-1 ratios measured before oral CS therapy (rs = 0.79, p = 0.0006;
chi-square = 14.2, p = 0.0008) (Figure 3, bottom panel). When
data were divided for their CS responsiveness, the mean
MMP-9:TIMP-1 ratio of CSNR (0.14 ± 0.02) was significantly lower than that of CSR (0.26 ± 0.01, p = 0.0003).
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The main goal of this study was to evaluate factors that are representative of the tissue inflammatory and fibrogenic activities and to correlate these factors with the response of subjects with incompletely controlled asthma to oral CS therapy. We report that baseline serum MMP-9:TIMP-1 ratio strongly correlates with the airflow obstruction improvement after oral CS therapy in severe asthma. To our knowledge, this is the first observation correlating markers of inflammatory and fibrogenic tissue-remodeling processes with the response to CS in asthma and suggesting a modulatory effect of structural repair mechanism in asthma CS response.
The efficacy of CS on FEV1 improvement likely depends
on the presence of a significant airway inflammatory process.
Data obtained in other chronic diseases such as rheumatoid
arthritis, systemic sclerosis, and hepatitis C suggest that the
dosage of serum MMP-9 could be used as a noninvasive marker
to monitor the intensity of tissue inflammation and/or degenerative burden caused by asthma and that serum MMP-9:
TIMP-1 ratio could be used as a noninvasive test to evaluate
the inflammatory and tissue remodeling patterns. In the subjects with incompletely controlled asthma studied herein, the
CS-induced improvement in FEV1 was closely correlated with the serum MMP-9:TIMP-1 ratio, but weakly correlated with
the MMP-9 level, and not with the level of TIMP-1 individually. Indeed, these two markers are interdependent, MMP-9
being found associated with its inhibitor (TIMP-1) that tightly
regulates its enzymatic activity (6, 7). As observed here the resulting inflammatory degenerative burden likely depends more
on the combined effect of both factors than on MMP-9 level
alone. Moreover, TIMPs also promote fibroblast proliferation
(8, 30). In some asthmatics, as in subjects with chronic wound
and rheumatoid arthritis, the predominant inflammatory component can be controlled by CS. In others, as in those with
systemic sclerosis and chronic hepatitis, an abnormal repair
process may lead to an excessive fibrogenesis and modify the
bronchial structure. As observed in systemic sclerosis, this fibrosis may be resistant to CS. The fact that MMP-9:TIMP-1 ratio predicts the CS-induced FEV1 suggests that the balance between inflammation and fibrogenesis is an important factor modulating the response to CS. In systemic sclerosis, the
TIMP-1 serum level correlates with the level of TIMP-1 secreted from their fibroblasts (8). Gelatinase activity has been
detected in sputum of children with uncontrolled asthma (31).
Recently, MMP-9 mRNA and protein were detected in bronchial biopsies of asthmatics; the majority of the cells expressing
MMP-9 mRNA were eosinophils (32). Eosinophils and CD4+
lymphocytes infiltrating bronchial mucosa could be a source
for MMPs, whereas fibroblasts could be the source of TIMPs. It would be interesting to identify the cell types expressing MMP-9 and TIMP-1 in the bronchial mucosa and correlate
bronchial MMP-9 and TIMP-1 expressions with serum levels.
The serum MMP-9:TIMP-1 ratio of CSNR is low compared
with that of CSR, suggesting that in CSNR the inflammatory
process is not predominant and that increasing the dose of inhaled CS might not be the best treatment alternative. Based
on the MMP-9:TIMP-1 ratio and the CS-nonresponse to high
doses of oral CS, these CSNR would not need higher doses of
CS but, rather, could benefit from a long-acting bronchodilator. The recent study of Pauwels and colleagues (33) suggests
that long-acting 2-agonist could represent an acceptable alternative treatment to an increase of inhaled CS in subjects with
persistent symptoms of asthma despite moderate to high doses
of inhaled CS. To date there are no data confirming that this is
an adequate therapy in all asthmatics. One has to worry that
such treatment would not control inflammation and thus allow
some patients to progressively develop irreversible airway scarring and fibrosis. Assuming a role for MMP-9 and TIMP-1 in regulating the asthma bronchial-inflammation-remodeling process, the MMP-9:TIMP-1 could represent a valuable noninvasive index suggesting who would better benefit of an increased
dose of CS or of the introduction of long-acting bronchodilator therapy.
Recently, a remarkable effort has been made to better understand the molecular basis of resistance to CS in asthma. In CS-resistant asthma, the lower glucocorticoid receptor (GR) binding affinity to CS may be associated with an ongoing inflammatory process that may be due to a persistent inflammatory cell infiltration (34). Increased nuclear factor interference with CS-receptor complexes via protein-protein interactions has been proposed by Barnes and Adcock (21) as an important molecular mechanism of CS resistance. It is therefore conceivable to hypothesize that in some asthmatics the molecular mechanism of CS resistance is due to a defect in glucocorticoid signal transduction pathway induced by an ongoing intense inflammatory process. We believe that this mechanism is likely involved mostly in subjects with severe asthma and CS dependency who require high doses of CS to acceptably control asthma symptoms (35). It could also be involved at least in part in some of our subjects with intense airway inflammation.
According to the above observations, we propose a hypothetical model of clinical responsiveness to CS therapy based on the relationship between inflammatory and remodeling features of asthma and MMP-9:TIMP-1 ratio. In most asthmatic subjects, the bronchial lung inflammatory process is predominant and is effectively controlled by CS therapy; we thus find a range of patients with severe asthma who respond significantly to high doses of oral CS (the CS responders). At their entry into the study, they may represent subjects who had a dose of inhaled CS insufficient to overcome their inflammatory component and subsequently improved their FEV1 with oral CS. Patients whose airflow obstruction is not responsive to CS therapy (the CS nonresponders) may have preponderant structural changes. As in systemic sclerosis, CS have little effect on fibrotic structural changes. As feedback inhibition, the fibrogenic events initiated by the inflammatory process and leading to structural changes may have an antagonistic effect on the inflammatory process. The preponderant level of TIMP-1 may reflect this fibrogenic activity.
In conclusion, this study has identified serum markers that correlate with the response to CS in asthmatic subjects. Assuming a role for these factors in asthma inflammation and fibrogenic remodeling, an intense inflammation responds to high doses of CS and a fibrogenic remodeling leads to the reduced response to CS. This report stresses the importance of the balance between intimately related inflammatory and remodeling processes that likely modulate the physiologic response to CS and provides new hypotheses for research development in the understanding of severe asthma and CS resistance in asthma. Consequently, we have described herein a novel potential non-invasive tool to evaluate the pathologic profile of asthma and help to predict its response to CS.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Michel Laviolette, Hôpital Laval, 2725, Chemin Ste-Foy, Ste-Foy, PQ, G1V 4G5 Canada .
(Received in original form February 11, 1998 and in revised form August 18, 1998).
M. Bossé has a FRSQ (Fonds de la Recherche en Santé du Québec) studentship.M. Audette holds a FRSQ senior scholarship.
Acknowledgments: The writers thank Drs. Yves Lacasse and Rémi Laliberté for revising the manuscript and for their constructive criticism, and Geneviève Laflamme and Geneviève Ross for their excellent technical assistance.
Supported by Astra Canada.
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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