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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 651-656, (2002)
© 2002 American Thoracic Society

Original Article

Tumor Necrosis Factor-{alpha} and Angiostatin Are Mediators of Endothelial Cytotoxicity in Bronchoalveolar Lavages of Patients with Acute Respiratory Distress Syndrome

Jürg Hamacher, Rudolf Lucas, H. Roger Lijnen, Susanne Buschke, Yves Dunant, Albrecht Wendel, Georges E. Grau, Peter M. Suter and Bara Ricou

Department of Anaesthesiology, Pharmacology, and Surgical Intensive Care, University Medical Center, Geneva, Switzerland; Division of Biochemical Pharmacology, University of Konstanz, Konstanz, Germany; Cardiovascular Research, VIB, University of Leuven, Leuven, Belgium; and Department of Physiology, Université de la Méditerranée, Marseille, France

Correspondence and requests for reprints should be addressed to Rudolf Lucas, Ph.D., Biochemical Pharmacology, Department of Biology, Universitätsstrasse 10, Box M668, D-78457 Konstanz, Germany. E-mail: rudolf.lucas{at}uni-konstanz.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute respiratory distress syndrome (ARDS) is characterized by an extensive alveolar capillary leak, permitting contact between intra-alveolar factors and the endothelium. To investigate whether factors contained in the alveolar milieu induce cell death in human lung microvascular endothelial cells, we exposed these cells in vitro to bronchoalveolar lavage fluid (BALF) supernatants from control patients, patients at risk of developing ARDS, and patients with early- and late-phase ARDS. In contrast to BALF from control patients, a significant cytotoxicity was found in BALF from patients at risk of developing ARDS, with late-phase ARDS, and especially from patients with early-phase ARDS. Subsequently, we determined the levels of factors known to exert cytotoxicity in endothelial cells, i.e., tumor necrosis factor (TNF)-{alpha}, transforming growth factor (TGF)-ß1, and angiostatin. BALF from patients at risk of developing ARDS, with early-phase ARDS, and with late-phase ARDS, contained increased levels of TNF-{alpha} and angiostatin, but not of TGF-ß1, as compared with BALF from control patients. Whereas inhibition of TGF-ß1 had no effect in this setting, neutralization of TNF-{alpha} or angiostatin inhibited the cytotoxic activity on endothelial cells of part of the early-phase ARDS BALF. These results indicate that TNF-{alpha} and angiostatin may contribute to ARDS-related endothelial injury.

Key Words: respiratory distress syndrome • cytotoxicity • endothelium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute respiratory distress syndrome (ARDS) develops in 20–50% of patients suffering from severe conditions such as major trauma, sepsis, or shock (1) and is associated with a mortality rate of about 40% (2, 3). This syndrome may develop either after direct lung injury, such as pulmonary infection or aspiration, or secondary to an indirect insult, such as sepsis, shock, or multiple trauma. The lung injury is characterized by an activation of both intrapulmonary and circulating cells. Activation and persistence of neutrophils and their products play a central pathogenic role in its initiation and amplification (4–6), but monocytes and macrophages are also implicated (7, 8).

ARDS is clinically characterized by a severe pulmonary gas exchange disturbance that usually requires mechanical ventilation. It is functionally based on an alveolar cell injury with disruption of the integrity of both the endothelial–interstitial barrier and interstitial–alveolar barrier (4), which results in flooding of the alveolar spaces with an inflammatory exudate that may become as protein-rich as blood plasma. The critical role of cell death in early phases of ARDS is supported by the protection against such injury by caspase inhibitors in an animal model (9). Late ARDS (> 21 days after onset) is generally associated with marked remodeling of the interstitial and alveolar spaces, with marked fibrosis in certain cases.

Human lung microvascular endothelial cells (HL-MVEC) are both morphologically and functionally different from large-vessel endothelial cells (10). They form a huge internal pulmonary surface similar in magnitude to the alveolar surface, represent a crucial part of the air–blood barrier, and may experience changes by proinflammatory stimuli that include thrombogenic features or even cell death (11, 12).

Because the capillary leak, i.e., the loss of capillary tightness for fluid and substances, is a characteristic feature of the early phase of ARDS, a direct contact between factors contained in the bronchoalveolar lavage fluid (BALF) and the endothelium may occur. Such diffusion of molecules from the alveoli into the capillaries and vice versa has clearly been shown in an animal model of acute oleic acid lung injury (13).

Inflammatory cytokines may lead to major changes in endothelial cells (11). Therefore, in the present study, we investigated the potential direct cytotoxicity of the inflammatory cytokines tumor necrosis factor (TNF)-{alpha} (14–16) and transforming growth factor (TGF)-ß1 (17), as well as of angiostatin (18) on the endothelium during ARDS. Indeed, it was recently shown that angiostatin, a cleavage product from plasminogen with strong antimetastatic and antiangiogenic activity in vivo (18), could specifically induce apoptosis in endothelial cells (19, 20).

Because an increased presence of neutrophil granulocytes–derived elastase, as well as of matrix metalloproteinases, i.e., enzymes that can cleave angiostatin from plasminogen, has been demonstrated in the alveolar space of patients with ARDS (21–25), we also investigated the possible implication of the apoptosis-inducing factor angiostatin in ARDS-related endothelial damage. We used primary HL-MVEC as an indicator system for cytotoxic activity of ex vivo BALF from patients with ARDS and determined the causal role of TNF-{alpha}, TGF-ß1, and angiostatin in blocking experiments.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Primary HL-MVEC (Clonetics, San Diego, CA) were cultured according to the manufacturer's protocol. Human lung fibroblasts (ATCC 210; American Type Culture Collection, Rockville, MD) were grown in Dulbecco modified Eagle medium (GIBCO, Grand Island, NY) with L-glutamine and antibiotics-supplemented 10% heat-inactivated fetal calf serum.

Antibodies
Neutralizing mouse anti-human TNF-{alpha} (clone B-C7) was a kind gift from Dr. J. Wijdenes, Besançon, France. Mouse anti-human TGF-ß1 monoclonal antibody (clone TB21) and mouse anti-human intercellular adhesion molecule–1 (ICAM-1) (clone RR1/1) monoclonal antibody were from Biosource (Fleurus, Belgium), and an agonistic monoclonal anti-CD95 antibody (clone CH-11) was obtained from Immunotech (Marseille, France). A neutralizing monoclonal mouse anti-human angiostatin antibody was used (26).

Patient Population and BAL Protocol
BALF of patients admitted to the Surgical Intensive Care Division of the University Hospital of Geneva and presenting risk factors for developing ARDS were obtained. The study was approved by the local ethics committee. Patients who presented the same risk factors but did not develop ARDS constituted the "at risk" group (23, 27). ARDS was defined according to standard criteria (2). "Early ARDS" was defined as the first 5 days since the onset of ARDS, and "late phase" refers to patients meeting ARDS criteria after 21 days or more of evolution. BAL were performed and processed using a standard technique (28). BAL of 10 control patients was used: 7 patients investigated for chronic cough, 1 for hilar masses, and 2 for pulmonary nodules. Anthropometric data of patient groups are described in Table 1 .


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  		TABLE 1. Anthropometric data of patient groups (mean ± sd)

 
Cytotoxicity Assay
HL-MVEC were incubated at 2 x 105 cells/ml in 96-well plates (Falcon, BD, Bedford, MA) for 18 hours with 50% (vol/vol) of BALF supernatants in the presence of 1 µg/ml of Actinomycin D (Sigma, Buchs, Switzerland). Negative controls were wells where BALF was replaced by the same volume of 0.9% NaCl. BALF were preincubated for 30 minutes with 10 µg/ml of neutralizing monoclonal antibodies for TNF-{alpha}, TGF-ß1, and angiostatin or with isotype controls. In the experiments comparing the transluminal versus the luminal cytotoxicity, HL-MVEC were grown to confluence in either transwells (transluminal cytotoxicity; Costar V, Corning Inc., Corning, NY, Transwell-COL, 6.5 mm diameter, 0.4 mm pore size), which were inserted into wells of a 24-well culture plate (Costar), or directly in 24-well plates (Costar). When confluence was reached (checked by the diffusion of fluorescein isothiocyanate–labeled bovine serum albumin [100 µg/ml] in the transwell set-up), the cells were treated with TNF, angiostatin, or early ARDS BALF, all incubated from the abluminal side of the cells, in the presence of 1 µg/ml of Actinomycin D. Positive controls in both settings consisted of cells treated with 33% (vol/vol) ethanol for 1 hour. Finally, 10% (vol/vol) Alamar blue (AccuMed International, East Grinstead, UK) was added and fluorescence read 2 hours later (Cytofluor II, PerSeptive Biosystems, Framingham, MA).

Detection of TNF-{alpha}, Soluble TNF Receptor 2, and TGF-ß1
Human TNF-{alpha}, soluble TNF receptor 2 (sTNFR2), and TGF-ß1 were detected using the hTNF-{alpha}, shTNFR2, and the hTGF-ß1 ELISA kits, respectively (Biosource Europe, Fleurus, Belgium).

Cell-based Enzyme-linked Immunosorbent Assay
To assess ICAM-1 induction of BALF, HL-MVEC (4 x 105/ml) were incubated for 18 hours with 50 vol% of the BALF. A cell-based enzyme-linked immunosorbent assay using a mouse anti-human ICAM-1 monoclonal antibody (3 µg/ml; Biosource International, Fleurus, Belgium) and the fluorescent substrate Attophos (Europa Products, Cambridge, UK) was performed (29).

Statistical Analysis
Analyses were performed with Systat version 9, (SPSS, Inc., Chicago, IL), giving mean (SD) or, if not normally distributed, median and range. Analysis of non-normally distributed continuous variables between groups was performed by Kruskal–Wallis test, followed by Mann–Whitney U test for differences between two specific groups if overall difference was significant. A p value less than or equal to 0.05 was considered significant. Boxplots were used, indicating median and 10th, 25th, 75th, and 90th percentiles of distribution.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BALF from Patients at Risk of Developing ARDS, Patients with Early and Late ARDS Display Cytotoxicity in HL-MVEC
As shown in Figure 1 , BALF from patients with early-phase ARDS and, to a lesser extent, from patients at risk of developing ARDS and patients with late-phase ARDS (> 21 days of ARDS) (Table 1) caused a significantly higher cytotoxicity in HL-MVEC than did BALF from control patients (% cell death, control: median [range] 0 [-1, 5]; at risk: 7 [0, 26]; early ARDS: 16 [0, 100]; late-phase ARDS: 8 [3, 20]), when incubated for 18 hours in the presence of 1 µg/ml of Actinomycin D. Using the same incubation protocol in the presence of Actinomycin D, the same BALF did not exert any measurable cytotoxicity in the primary human lung fibroblast cell line ATCC 210. Interestingly, under transcriptional inhibition, BALF from patients with early ARDS exerted a significant cytotoxicity in confluent monolayers of HL-MVEC, when incubated from both the luminal ([mean ± SEM of percentage of cell death] 16.6 ± 9.9%, n = 9) and the transluminal side (15.1 ± 7.9%, n = 9).



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  		Figure 1. BALF-induced cytotoxicity on (HL-MVEC).

 
Early-Phase ARDS BALF Contain Increased TNF-{alpha}, sTNFR2, and Angiostatin Levels, as Compared with Controls
As shown in Figure 2A , BALF from patients with early-stage ARDS have significantly higher TNF-{alpha} levels compared with control patients, confirming earlier findings by others (22, 30) (control: median [range] 6 [0, 26] pg/ml; at risk: 8 [3, 66] pg/ml; early ARDS: 48 [0, 318] pg/ml; late-phase ARDS: 6 [0, 92] pg/ml). The bioactivity of TNF-{alpha} in vivo can be blocked by its soluble receptors sTNFR1 and sTNFR2. Moreover, previous work has indicated that lung MVEC from patients with ARDS express a significantly higher level of TNFR2, but not of TNFR1, than did their counterparts in control patients (31). Because this TNF receptor is cleaved by the same enzyme that generates soluble TNF-{alpha} from its membrane-bound form (TNF-{alpha} converting enzyme [32]), we have also measured levels of sTNFR2 in BALF. As shown in Figure 2B, we have found significantly increased levels of sTNFR2 in BALF of patients at risk of developing ARDS (1.1 [0.28, 2.8] ng/ml) or with early-phase ARDS (1.9 [0, 23.3] ng/ml), but not with late-phase ARDS (0.44 [0, 5.8] ng/ml), as compared with control patients (0.10 [0, 2.0] ng/ml).





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  		Figure 2. (A) TNF-{alpha} levels in BALF of the four different patient groups. (B) Levels of soluble TNF receptor 2 in BALF of the different patient groups. (C) TGF-ß1 levels in BALF of the different patient groups.

 
In contrast to TNF-{alpha} and sTNFR2, TGF-ß1 levels were not significantly different between the control and the at risk or ARDS groups (Figure 2C; control: 0 [0, 50] pg/ml; at risk: 30 [0, 300] pg/ml; early-phase ARDS: 10 [0, 300] pg/ml; late-phase ARDS: 31 [0, 203] pg/ml).

The increased alveolar concentration of elastases (21, 22) and matrix metalloproteinases (23–25), together with an increased presence of plasminogen in patients with ARDS could generate the endothelial cell–specific apoptotic factor angiostatin (18, 19). In a parallel study, we determined the levels of this protein and found that angiostatin concentrations are indeed significantly increased in BALF from patients at risk of developing ARDS and patients with late- and especially early-phase ARDS (33).

Because the highest cytotoxicity was present in the early-phase ARDS BAL group, we selected this group to test the inhibitory capacity of neutralizing anti–TNF-{alpha}, anti–TGF-ß1, and antiangiostatin monoclonal antibodies.

TNF-{alpha} and Angiostatin Are Mediators of Cytotoxicity in Endothelial Cells
Figure 3 shows that a neutralizing TNF-{alpha} monoclonal antibody inhibited > 90% of the cytotoxic activity in three out of eight early-stage ARDS BALF. Moreover, a monoclonal antibody neutralizing the apoptotic activity of angiostatin (36E6 monoclonal antibody) (19, 34) partially inhibited the cytotoxicity of five out of eight samples. In contrast, a monoclonal antibody neutralizing the cytotoxicity of TGF-ß1 did not have any significant inhibitory activity on the tested BALF. Note that in the three BALF in which anti–TNF-{alpha} neutralized a significant part of the cytotoxicity, only moderate inhibition by a neutralizing antiangiostatin monoclonal antibody could be observed (19, 35, and 0%); on the other hand, the two BALF in which anti-angiostatin monoclonal antibody significantly inhibited cytotoxicity, anti–TNF-{alpha} treatment had only a minor effect (17, 0%). We could demonstrate the presence of CD95 on our assayed cells by fluorescence-activated cell sorter but could not detect any CD95-mediated cytotoxicity in the HL-MVEC using 100 ng/ml of the agonistic anti-CD95 monoclonal antibody CH-11, either alone or on costimulation with interleukin-1ß and/or TNF (not shown).



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  		Figure 3. Inhibition of cytotoxicity in eight patients with early ARDS of the group shown in Figure 1 by neutralizing antibodies to TNF-{alpha}, angiostatin, or TGF-ß1. Cytotoxicity was 40% (SD 25%) in this group.

 
ICAM-1 is Upregulated by BALF from Patients at Risk of Developing ARDS and Patients with Early- and Late-Phase ARDS, but Does Not Correlate with Cytotoxicity
Figure 4 shows the upregulation of ICAM-1 (CD 54) by BALF on HL-MVEC. Since ICAM-1 is constitutively expressed in HL-MVEC, we assessed the times increase of basal expression upon treatment of the cells with the BALF. Compared with controls (median [range] x basal ICAM-1 expression: 1.49 [1.0, 1.87], BAL from patients at risk of developing ARDS (2.9 [1.5, 17.6] x basal), with early ARDS (2.5 [0.7, 9.3] x basal), or late ARDS (2.5 [2.0, 3.6] x basal) showed a significantly higher level of ICAM-1 expression. However, no relationship between cytotoxicity and ICAM-1 upregulation could be found (rho = 0.19).



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  		Figure 4. Upregulation of ICAM-1 on HL-MVEC by BAL from the different patient groups.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During ARDS, a diffuse transmural alveolar wall damage occurs, characterized by a severe epithelial injury, that was proposed to be mainly mediated by Fas ligand (35–37) and by endothelial cell death and dysfunction, particularly in pulmonary microvessels (2, 5, 38, 39). Besides vascular lesions in pulmonary arteries during ARDS (40), up to 50% of lung capillaries may be destroyed (41). The importance of cell death is further supported by the demonstration of endothelial cell death after lipopolysaccharide injection (9, 42) and a protection against lung injury by caspase inhibitors in animal models (9). The finding that TNF-binding protein protected against lipopolysaccharide-induced death and endothelial cell demise in mice further suggests that systemic TNF-{alpha} is involved in both effects (43).

Many causes of ARDS are indirect, i.e., "extrapulmonary," like sepsis or multiple trauma, and not of direct pulmonary origin as in pneumonia or pulmonary contusion. This implies that factors stemming most probably from the blood are linked to lung failure in those indirect injuries. Therefore, endothelial cells, representing blood–tissue contact, may be crucial in the corresponding lung damage. Whereas mechanisms of adhesion and migration of neutrophil granulocytes on and through the endothelial cell layer have been postulated to be important for endothelial injury (44), direct cytotoxicity of intra-alveolar factors in endothelial cells, as observed in a study using bovine macrovascular cells in vitro (45), has not been investigated in human microvascular endothelial cells. Therefore, in this study, we used these cells incubated with BALF from control patients versus patients with ARDS to test the cytotoxic activity of the ex vivo BALF and to determine the contribution of TNF-{alpha}, TGF-ß1, and angiostatin in blocking experiments. The roles of both CD95 and endostatin were not investigated further because despite the observed presence of CD95 on our HL-MVEC, CD95 stimulation did not induce cytotoxicity in our HL-MVEC assay, and the endostatin content in ARDS BAL did not differ from that in controls (33).

Our in vitro indicator system of HL-MVEC showed a significant cytotoxic activity of BAL samples isolated from patients at risk of developing ARDS, patients with late ARDS, and especially patients with early ARDS but not from control patients. Cytotoxicity was lower in patients at risk of developing ARDS but subsequently not developing ARDS, as compared with patients who had developed ARDS. Out of the three factors described as inducing apoptosis in endothelial cells, increased levels of TNF-{alpha} and angiostatin, but not of TGF-ß1, were observed in BALF from patients at risk of developing ARDS and patients with early and late ARDS. Although the level of sTNFR2, which binds and inactivates TNF-{alpha}, was significantly higher in early ARDS BALF than in controls, TNF-{alpha} still displayed a major cytotoxicity in HL-MVEC in some of the samples. This effect could be efficiently blocked by a neutralizing anti–TNF-{alpha} monoclonal antibody. These data thus indicate that in the alveolar milieu in some patients, bioactive TNF-{alpha} is present despite high sTNFR2 levels and that it may be an important factor in the early ARDS-associated endothelial damage. In contrast, in early-phase ARDS BALF, we could not identify a substantial cytotoxic role for TGF-ß1 in endothelial cells using our assay. Neither did we detect any significant increase in the levels of this cytokine during ARDS nor did we find any important inhibitory effect of antibodies neutralizing the activity of TGF-ß1 in early-phase ARDS BAL cytotoxicity. However, as TGF-ß1 action requires the dissociation from its latency-associated peptide, by means of an {alpha}vß6 integrin-binding and possibly thrombospondin-1 interaction (46–48), a cytotoxic effect in endothelial cells of this pleiotropic factor during ARDS cannot be ruled out by our assay.

Angiostatin has been shown to be a negative regulator of angiogenesis. In vitro, it induces apoptosis in endothelial cells but not in fibroblasts or epithelial cells (19). This cleavage product of plasminogen can be generated by several enzymes, one of which is the neutrophil-derived elastase, the concentration of which was found to be high in BAL during ARDS (21, 22). Furthermore, matrix metalloproteinases, which have been found in increased levels in ARDS (23), may cleave plasminogen to angiostatin (49–51). In patients with ARDS, due to sepsis, it was observed that besides fibrin and microthrombi, endothelial lesions were mainly in close contact with accumulated neutrophil granulocytes in capillaries (52). Consequently, we speculate that polymorphonuclear leucocyte–derived enzymes, including angiostatin-generating proteases, are implicated in the development of these lesions. Both the increased level of angiostatin during ARDS (33) and the partial inhibition of endothelial in vitro cytotoxicity by a neutralizing monoclonal antibody to angiostatin in some patients with early ARDS suggest harmful effects of angiostatin in ARDS. Whether angiostatin's activating proteases also directly contribute to the endothelial damage (53) has not been investigated in our study. Furthermore, we did not define the source of the implicated factors. The question whether factors from specific cell types act as regulators of demise of other cell types not only in vitro (54) but also in vivo (55) remains open.

For ex vivo BAL samples of patients with early-phase ARDS, no correlation was found between the time since the onset of ARDS and the extent of in vitro cytotoxicity. Neither was there a difference between in vitro cytotoxicity of material from surviving versus subsequently dying patients nor was there one between patients with early ARDS with a direct pulmonary cause of ARDS, e.g., pneumonia or contusion, versus patients with an indirect cause, such as sepsis or multiple trauma (data not shown).

It was previously shown that the constitutive expression of ICAM-1 and, to a lesser extent, vascular adhesion molecule–1 was significantly increased in MVEC isolated from patients with ARDS, as compared with MVEC from control patients (31). The in vitro BALF-induced ICAM-1 upregulation observed in our study is in line with this previous study. ICAM-1 upregulation may increase neutrophil sequestration and migration through the endothelium and may thus contribute to an alternative mechanism for endothelial injury (10, 44). However, neither the in vitro capacity of the ARDS BALF to up-regulate ICAM-1 (CD54) nor their concentrations of interleukin-1ß correlated with the endothelial cytotoxicity. Furthermore, the findings of specific mediators believed to be responsible for endothelial cell death and the lack of a relationship between total protein concentration contained in the early ARDS BALF (data not shown) and corresponding cytotoxic activity let us exclude the theory that the observed cytotoxicity was nonspecific.

As shown in Figure 3, an important part of the observed cytotoxicity could not be blocked by a single specific blocking substance. This indicates that mediators other than the ones tested in this study may account for part of the cytotoxicity or that the setup used does not allow detecting the bioactive form of some of them, as could be the case with TGF-ß1. In addition, an important further explanation would be that additive or supra-additive concerted effects might account for endothelial cell death, which has also been suggested by recent work (56). Moreover, it has recently been shown that a dramatic counterbalance of inflammatory and apoptotic cytokines in the form of soluble receptors or antagonists is present in BALF from patients with early ARDS (27). Antiapoptotic substances might thus also have counterbalanced part of the cytotoxic activity in our setting.

In conclusion, our study indicates that the increased levels of active TNF-{alpha} and angiostatin in BAL during ARDS cause death of primary HL-MVEC but not primary human lung fibroblasts. Such endothelial cell death may contribute in vivo to endothelial–interstitial barrier dysfunction, expose interstitial and alveolar epithelial cells to cytotoxic–apoptotic substances, and thereby amplify acute lung injury or on the other hand disseminate (57), as also suggested ex vivo (58), and thus contribute to multiorgan failure (59). Together with the findings suggesting that the soluble Fas ligand, but not TNF (60), causes lung epithelial cell death, our findings support the hypothesis of an endothelial cell–selective injury by TNF and angiostatin during ARDS. Strategies to prevent, decrease, or heal endothelial cell injury may therefore modify or even avoid acute lung injury or ARDS.


   Acknowledgments
 
The authors thank Dr. Laurent P. Nicod, P.D., M.D., Pulmonary Division, University of Geneva, Geneva, Switzerland, for his help and critical comments.


   FOOTNOTES
 
Supported by grant WE686/18 from the Deutsche Forschungsgemeinschaft (research group "Endogenous tissue injury: Mechanisms of autodestruction"; J.H. and A.W.), by grant 32.22.902.92 from the Swiss National Science Foundation (B.R. and P.M.S.), and by grant G.0138.00 from FWO-Vlaanderen (H.R.L.).

Received in original form September 4, 2001; accepted in final form May 30, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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