Abstract
Asthma and chronic bronchitis are inflammatory diseases with extracellular matrix (ECM) remodeling and collagen deposition. Collagen homeostasis is controlled by metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). We evaluated MMP and TIMP balance in induced sputum of 10 control, 31 untreated asthmatic, and 16 chronic bronchitic subjects. We first performed zymographic analysis to identify the profile of MMPs. Zymography revealed a similar MMPs profile in all populations studied and that MMP-9 was the major enzyme released. We then measured, using enzyme immunoassay, the concentrations of MMP-9 and of its inhibitor TIMP-1 and evaluated whether airflow limitation may be associated with an imbalance between these enzymes. MMP-9 and TIMP-1 concentrations were greater in sputum of patients with asthma and chronic bronchitis than in control subjects. The molar ratio between MMP-9 and TIMP-1 was lower in asthmatics and chronic bronchitics than in control subjects, and positively correlated with FEV1 values. In asthma, MMP-9 levels were significantly correlated with the number of macrophages and neutrophils. This study shows that airway inflammation in asthma and chronic bronchitis is associated with an imbalance between MMP-9 and TIMP-1 which may have a role in the pathogenesis of ECM remodeling and airflow obstruction.
Connective tissue cells produce and secrete an array of macromolecules forming a complex network filling the extracellular space of the submucosa called the extracellular matrix (ECM) (1). ECM macromolecules are secreted locally and include fibrous proteins (collagen, elastin) and structural or adhesive proteins (fibronectin and laminin) embedded in a hydrated polysaccharide gel containing glycosaminoglycans. The ECM is a dynamic structure, and an equilibrium between synthesis (2) and degradation of ECM components is required for the maintenance of its homeostasis. Although many proteases can cleave ECM molecules, Zn2+-matrix metalloproteinases (MMPs) and their inhibitors are likely to be the normal physiologically relevant mediators of ECM degradation (3, 4). At least three subclasses of MMPs have been identified: collagenases, gelatinases, and stromelysins. They are mainly synthesized by connective tissue cells, granulocytes, and monocyte macrophages (5). The major physiologic inhibitors of MMPs are the broad-spectrum serum inhibitor α2-macroglobulin and a special class of tissue inhibitors of metalloproteinases (TIMPs) (3). The balance between MMPs and their inhibitors is critical in tissue repair and remodeling, and its homeostasis plays an important role in the breakdown and deposition of ECM in the airways wall.
Asthma and chronic bronchitis are inflammatory diseases of the airways characterized by airways remodeling, due, at least in part, to an excess of ECM deposition in the airway wall (6, 7), which leads to subepithelial collagen deposition in asthma (8), and to fibrosis of the small airways in chronic bronchitis (7, 9). These structural changes of the airways may represent an important cause of airflow obstruction in asthma (10) and chronic bronchitis.
In the present study we evaluated whether in asthma and chronic bronchitis the degree of airflow obstruction, as assessed by FEV1 values, was associated with an imbalance between the levels of MMP-9 and its tissue inhibitor, TIMP-1. MMP-9 and TIMP-1 were measured in sputum samples induced by inhalation of hypertonic saline by 10 control subjects, 31 asthmatic subjects, and 16 chronic bronchitic patients.
Thirty-one untreated asthmatic subjects ranging in age from 18 to 69 yr (median and 25 to 75% percentiles: 41, 25 to 61.5 yr) were selected for the study. Asthma was diagnosed on the basis of criteria previously described in detail (11). None of the subjects was a current or previous smoker. Patients were excluded from the study if they had undergone a severe exacerbation of asthma requiring hospitalization during the month preceding the study. Inhaled corticosteroids or oral corticosteroids had been withdrawn for at least 2 mo before the commencement of the study; the use of nedocromil sodium or cromoglycate had been stopped for at least 2 wk and theophylline in the previous 48 h.
Sixteen patients with chronic bronchitis and/or chronic obstructive pulmonary disease (COPD) ranging from 48 to 76 yr of age (median and 25 to 75% percentiles: 68.5; 60 to 72.5 yr) were selected for the study. Chronic bronchitis and COPD were defined according to criteria previously described in detail (12). Patients diagnosed as having COPD had a FEV1 below 70% predicted, and displayed a 10% or smaller increase in their FEV1 at the time of the procedure after an inhaled dose of 200 mg of albuterol. They were all smokers (29 to 75 annual packs, mean ± SD: 38.8 ± 11.9 packs). Patients were excluded if they had undergone a bronchial infection during the month preceding the study; no subject had received corticosteroids of any form during the 2 mo prior to the study. All chronic bronchitis patients underwent routine chest X-ray and computed tomography (CT) scan. Patients with obvious emphysema, as assessed by routine chest X-ray and CT scan, were excluded.
Ten normal subjects ranging from 25 to 39 yr of age (median and 25 to 75% percentiles: 29.5, 27 to 33 yr) were selected for the study. None of these subjects had ever suffered from asthma or chronic bronchitis. They had not undergone any bronchial or respiratory tract infection during the month preceding the trial. All the subjects were lifelong nonsmokers and their pulmonary function was within the normal range.
The study was approved by the ethics committee and the patients gave their informed consent for participation.
Sputum induction and processing were performed according to the methods of Fahy and colleagues (13) with slight modifications (14). Each subject was submitted to spirometry before sputum induction. Patients were exposed to an aerosol of 3% hypertonic saline solution, early in the morning, in a fasting condition for 20 min. The subjects were encouraged to cough throughout the procedure, and regularly interrupted their inhalation of hypertonic saline in order to expectorate sputum into 50-ml sterile ampoules. Subjects were asked to accurately wash their oral cavity with saline solution before expectorating sputum, as well as to blow the nose in order to minimize the salivary contamination. The aerosol was administered by an ultrasonic nebulizer (Fisoneb; Fisons Italchimici Spa, Rome, Italy), which generates particles with a median diameter of 2.5 μm and has an output of 1 ml/ min. In all subjects a sample of saliva was collected before the sputum induction procedure.
The volume of the induced sputum and saliva sample was determined and an equal volume of dithiothreitol (Sigma Chemical Co., St. Louis, MO) diluted with saline solution to obtain 0.1% concentration, was added. The samples were then mixed gently by vortex mixer and placed in a water bath at 37° C for 15 min to ensure a complete homogenization. The samples were removed from the water bath periodically for further brief gentle vortex mixing. The homogenized sputum and saliva were centrifuged at 800 × g for 10 min to separate the supernatants from the cell pellet. The supernatants were then aspirated and frozen at −20° C for subsequent biochemical analysis.
The cell pellet was resuspended in saline solution. The total cell count and cell viability were assessed in 10-μl aliquots, using a standard hemocytometer and by trypan blue exclusion, respectively. Then the cells were cytocentrifuged (Cytospin 2; Shandon Instruments, Runcorn, UK) and stained by Diff-Quik staining (Merz-Dade, Dudingen, Switzerland) to perform the differential cell counts. The slides were read by two independent investigators (P.D., A.M.) blindly who counted at least 400 cells per slide. The number of the squamous cells was subtracted from the total cell counts and the differential cell counts were expressed as corrected percentage.
MMPs present in sputum samples were detected by their capacity to degrade gelatin (15) according to a method previously described (16). Briefly, supernatants obtained from sputum were subjected to electrophoresis on 11% polyacrylamide gels containing 1 mg/ml of gelatin, in the presence of sodium dodecyl sulfate (SDS-PAGE) under nonreducing conditions. After electrophoresis, gels were washed in Triton X100 for 1 h, rinsed briefly, and incubated at 37° C for 24 h in buffer containing 100 mM TRIS HCl pH 7.40 and 10 mM CaCl2. After incubation the gels were stained with Coomassie Brilliant Blue R250 and then destained in a solution of 7.5% acetic acid with 5% methanol. Zones of enzymatic activity were indicated by negative staining: areas of proteolysis appeared as clear bands against a blue background. Molecular weight of the gelatinolytic bands was estimated relative to the U937 MMP-9 reference and to prestained molecular-weight markers from Novex (San Diego, CA) as previously described (16). To identify the gelatinolytic activities, samples were subjected to immunoprecipitation using mouse monoclonal antibodies anti-human MMP-9 and MMP-2 (Fuji Chemical Industries, Toyama, Japan) as described previously before being subjected to electrophoresis (16).
Determinations of the absolute value of MMP-9 and TIMP-1 in induced sputum and saliva were performed by enzyme-linked-immunosorbent assay (ELISA) (MMP9-BIOTRAK ELISA and TIMP1-BIOTRAK ELISA; Amersham International plc, Little Chalfont, Buckinghamshire, UK). These assays measure the total amount of each respective protein (MMP-9 and TIMP-1), whether free or complexed to one another or to matrix. The limits of detection are 4 to 128 ng/ml for MMP-9 and 3.13 to 50 ng/ml for TIMP-1.
Results are expressed as medians and percentiles. Statistical analysis was performed using Mann-Whitney U test to assess differences between groups. Spearman's rank correlation was calculated to assess the correlation between data.
The median ages of patients with asthma and of control subjects were similar, but chronic bronchitis patients were significantly older than the subjects of the two other groups (p < 0.001). FEV1 values of untreated asthmatic patients ranged from 60 to 126% of predicted values (median and percentiles: 86; 75.2 to 96%), and in chronic bronchitis or COPD from 41 to 98% of predicted values (median and percentiles: 70.5, 56.5 to 88%). Among untreated asthmatics, 10 had a FEV1 value below 80%. In addition, among chronic bronchitis eight patients had COPD since their FEV1 values were below 70%. All patients with chronic bronchitis or COPD had normal total lung volume and diffusion capacity, thus excluding superimposed emphysema.
The percentage of squamous cells was not significantly different in sputum samples obtained from control subjects, asthmatic patients, or chronic bronchitis patients (Table 1). The corrected median total cell count was similar for control subjects, and asthmatic patients; it was higher in chronic bronchitis patients, however, the difference with other groups was not statistically significant. The median viability of sputum cells was 69.3% (percentiles: 61 to 76.6) in control subjects, 71.4% (percentiles: 61.2 to 80) in asthmatic, and 73.5% (percentiles: 60.1 to 84.4) in chronic bronchitis. There was no significant correlation between the cell viability and concentrations of MMP-9 and TIMP-1 measured in the three study groups. The percentage of neutrophils was significantly greater in chronic bronchitis than in asthmatic patients and in control subjects (p < 0.002 and p < 0.0002, Mann-Whitney U test, respectively). The percentage of eosinophils was significantly higher in asthma than in chronic bronchitis and in control subjects (p < 0.0004 and p < 0.0001, Mann-Whitney U test, respectively).
| Control Subjects | Untreated Asthma | Chronic Bronchitis | p Value† | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| p C/UA | p C/CB | p UA/CB | ||||||||||
| Squamous cells, % | 25.3 (16–35) | 16.1 (11.6–43.4) | 15.1 (7.6–32) | NS | NS | NS | ||||||
| Total cell counts, million cells/ml | 1.5 (0.7–2.7) | 1.5 (1–2.7) | 2.6 (1.3–6.3) | NS | NS | NS | ||||||
| Corrected cell counts, million cells/ml | 1.1 (0.4–2.3) | 1.1 (0.7–2.1) | 2.2 (1.1–3.7) | NS | NS | NS | ||||||
| Differential cell counts, % | ||||||||||||
| Macrophages | 83.5 (79–89.4) | 63.5 (44.4–81.4) | 38.6 (17–51.3) | < 0.010 | < 0.001 | < 0.020 | ||||||
| Neutrophils | 10.3 (8–15) | 31.2 (10.4–54.9) | 60 (42–82) | < 0.045 | < 0.0002 | < 0.002 | ||||||
| Lymphocytes | 0 (0–2) | 1.2 (0–2.9) | 0 | NS | NS | < 0.001 | ||||||
| Eosinophils | 0 | 5 (1.9–12.8) | 0.2 (0–1.6) | < 0.0001 | < 0.010 | < 0.0004 | ||||||
| Epithelial cells | 4.2 (0–10) | 1.2 (0–3.4) | 1 (0–1.7) | NS | NS | NS | ||||||
The total cell count in saliva was not significantly different in control subjects, asthmatic patients, and chronic bronchitis patients (median and percentiles: control subjects: 0.8; 0.4 to 1 106 cells/ml; asthma: 0.7; 0.5 to 0.9 106 cell/ml; chronic bronchitis: 0.9; 0.7 to 1 106 cells/ml); similarly the median percentage of squamous cells in saliva was not significantly different in the three groups (median and percentiles: control subjects: 99; 97 to 100%; asthma: 98; 97 to 100%; chronic bronchitis: 99; 96 to 100%).
Zymographic analysis of the sputum samples showed the presence of a major gelatinase species having a molecular weight similar to that of MMP-9. Representative examples of sputum samples from one patient in each group are shown in Figure 1. Sputum samples were also examined for the presence of activated MMP-9, but no active form of the metalloproteinase was observed. In addition, no gelatinolytic activity was immunoprecipitated with anti-MMP-2 antibody in any of the sputum samples analyzed.
Fig. 1. Analysis of MMPs in induced sputum by zymography. The figure shows a representative zymographic analysis of sputum supernatant samples obtained from one control (lane 2), one asthmatic (lane 3), and one chronic bronchitis (lane 4) subject. Arrow indicates the position of MMP-9. Gelatin zymography revealed only a major band at 92 kD in all groups. Lane 1 shows the U937 MMP-9 reference used as a standard.
[More] [Minimize]The concentrations of MMP-9 were significantly greater in sputum of asthmatics (median and percentiles: 60; 38 to 108 ng/ml) and chronic bronchitis patients (median and percentiles: 80.5; 37 to 102 ng/ml) than in sputum of control subjects (median and percentiles: 23; 8 to 37 ng/ml) (p < 0.006 and p < 0.008, Mann-Whitney U test, respectively) (Figure 2). In asthma and chronic bronchitis the levels of sputum MMP-9 were significantly correlated with the absolute number of macrophages and neutrophils (asthma: p < 0.0005 and p < 0.03 respectively; chronic bronchitis: p < 0.03 and p < 0.05, respectively; Spearman's rank correlation test) (Figure 3). On the other hand, the concentrations of MMP-9 did not correlate with the number of eosinophils in both asthma and chronic bronchitis.
Fig. 2. Concentrations of MMP-9 and of TIMP-1 (expressed as ng/ml) in sputum obtained from control, asthmatic, and chronic bronchitis subjects, as well as the MMP-9/TIMP-1 molar ratio in sputum samples. Statistical analysis by Mann-Whitney U test.
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Fig. 3. Correlation between the number of neutrophils and of macrophages and the concentrations of MMP-9 in induced sputum of asthmatic and chronic bronchitis patients. Statistical analysis by Spearman's rank test.
[More] [Minimize]TIMP-1 levels were significantly higher in asthmatics (median and percentiles: 140; 87 to 706 ng/ml) and chronic bronchitis patients (median and percentiles: 475; 348 to 1,295 ng/ ml) than in control subjects (median and percentiles: 60; 6.3 to 96 ng/ml) (p < 0.0003, Mann-Whitney U test) (Figure 2). In asthma, the concentrations of TIMP-1 were significantly correlated with the absolute number of macrophages (p < 0.02; Spearman's rank correlation test). The concentrations of TIMP-1 did not correlate with the number of eosinophils in both asthma and chronic bronchitis. In saliva, both TIMP-1 and MMP-9 levels were very low or undetectable and no significant differences were found between the groups studied.
The molar ratio MMP-9/TIMP-1 was significantly lower in asthmatics (median and percentiles: 0.1; 0.04 to 0.2) and chronic bronchitis patients (median and percentiles: 0.1; 0.03 to 0.1) than in control subjects (median and percentiles: 0.3; 0.1 to 0.41) (p < 0.008 and 0.001, respectively, Mann-Whitney U test) (Figure 2). The molar ratio was not statistically different between asthmatic and chronic bronchitis patients.
In asthmatic and chronic bronchitis patients, there was no significant correlation between sputum MMP-9 concentrations and FEV1 values but there was a significant positive correlation between the sputum TIMP-1 levels and the FEV1 values (p < 0.002 and p < 0.03, Spearman's rank correlation test) and between the sputum MMP-9/TIMP-1 molar ratios and the FEV1 values (p < 0.0005 and p < 0.003, respectively, Spearman's rank correlation test) (Figure 4). In addition, in asthmatic patients having airway obstruction (FEV1 < 80% predicted), the concentrations of TIMP-1 were significantly greater than in asthmatic patients without airway obstruction (FEV1 > 80% predicted) (p < 0.01, Mann-Whitney U test).
Fig. 4. Correlation between the concentrations of MMP-9 and FEV1 values in asthmatic and chronic bronchitis patients. Statistical analysis by Spearman's rank test.
[More] [Minimize]This study shows that in induced sputum obtained from untreated asthmatic and chronic bronchitis patients the concentrations of MMP-9 and TIMP-1 are significantly increased as compared with those from control subjects. However, by comparison with control subjects, in asthma and chronic bronchitis the concentrations of TIMP-1 exceed those of MMP-9 and are inversely correlated with FEV1 values.
It is increasingly accepted that airway inflammation and remodeling which occur in asthma and chronic bronchitis can cause functional alteration because of quantitative changes in airway wall compartments and/or by changes in the biochemical composition of the various constituents of the airway wall. The structural changes of the airways can represent an important cause of airflow obstruction in asthma (10) and chronic bronchitis. Among structural changes, the remodeling of the ECM components can have important functional consequences because it can exaggerate airway narrowing for a given degree of smooth muscle shortening (17-19). Both asthma and chronic bronchitis are characterized by ECM remodeling caused by increased collagen deposition which can lead to airway wall thickening. In asthma, collagen deposition is mainly localized under a normal subepithelial basal lamina forming a dense layer rich in fibrillar collagens (8). In chronic bronchitis, collagen deposition is more diffused in the airway wall, and can lead to the fibrosis of the small airways (7, 9). These histologic alterations suggest an altered ECM homeostasis leading to an increased collagen deposition within the airway wall. Although the mechanisms underlying these changes are not completely known, it is likely that in both asthma and chronic bronchitis the remodeling of ECM is characterized by an imbalance between metalloproteases and their inhibitors which determines an excessive collagen deposition in the airways.
The results of the present study support this hypothesis and show that in sputum of asthmatic and chronic bronchitis subjects the concentrations of TIMP-1 are significantly increased as compared with those of control subjects. In addition, in both asthmatic and chronic bronchitis subjects the molar ratio between MMP-9 and TIMP-1 is significantly lower than in control subjects, suggesting the existence of a protease–antiprotease imbalance in these diseases. High concentrations of TIMP-1 can lead to an increased deposition of ECM components, and may increase myofibroblast proliferation acting through its cell growth promoting activity (20). These data, together with the absence of activated MMP-9 due to the excess of TIMP-1, suggest a trend toward fibrosis in both asthma and chronic bronchitis. Hence, the high levels of TIMP-1 may potentially contribute to the pathogenesis of the increased thickness of the basement membrane in asthma (8) and of the ECM deposition in the airway wall in chronic bronchitis (9). In addition, in chronic bronchitis and asthma the inverse correlation between MMP-9/TIMP-1 molar ratio and airway obstruction as well as the higher TIMP-1 concentrations in obstructed than nonobstructed asthmatic patients, support the concept that MMP-9/TIMP-1 imbalance may have important consequences for airway obstruction, probably because of collagen deposition and the increased airway wall thickness (17– 19). It should, however, be highlighted that the intensity of airway wall remodeling and “scar formation” is quite unpredictable and seems to vary greatly from one patient to another. Such a variability is also suggested by our results that show TIMP-1 values widely dispersed in both asthmatic and chronic bronchitis patients, probably because of the heterogeneity of the clinical severity of the diseases. Thus, the present study may represent a potentially useful model to measure and monitor in induced sputum the levels of “biological factors” that may identify those patients with an increased risk of rapid deterioration of lung function.
The increased concentrations of TIMP-1 found in asthma and chronic bronchitis can be the result of the effects of several mediators released during the development of airway inflammation in these diseases. Among these mediators, an important role may be played by transforming growth factor-β (TGF-β) which is capable of increasing the production of TIMPs (21), and the expression of which is increased in the airways of asthmatic and chronic bronchitis patients (22). This study also shows that macrophages are an important cellular source of TIMP-1, and points out the involvement of these cells in airway remodeling in asthma and chronic bronchitis. The results of the present study extend previous evidence obtained by our group, showing that airways macrophages isolated from bronchoalveolar lavage of asthmatic and chronic bronchitis patients release high concentrations of the profibrotic growth factor TGF-β and of fibronectin (23) and, therefore, lend support to the concept that macrophages actively participate in the remodeling of the airways (24).
Airway wall remodeling in asthma and chronic bronchitis must be accompanied by degradation of ECM in addition to synthesis and deposition of new matrix. The results of this study support this concept and demonstrate that MMP-9 is the major metalloproteinase detectable in sputum, and that its levels are higher in asthmatic and chronic bronchitis patients than in control subjects. However, using zymography we observed that there was almost no 83 kD MMP-9 (activated form). This result is not surprising and might be due to a very large excess of TIMP-1 over MMP-9. It is likely that MMP-9 activation occurs in the inflamed airways because of the presence of several inflammatory mediators, such as oxygen free radicals which can activate endogenous metalloproteinase (25). MMP-9 activation may also occur on the surface of inflammatory cells without releasing into the extracellular milieu. However, it is important to note that the mechanisms of MMP activation in vivo still remain unknown, and it is unlikely that MMP activation in vitro mimics processes that occur in vivo (26). The detection of the active MMP-9 form is also difficult owing to the lack of commercially available antibodies recognizing such form of the enzyme, and Ohno and coworkers (27) have shown that MMP-9 is rapidly secreted extracellularly after its synthesis, precluding sufficient accumulation of the protein to be detected by immunohistochemistry. Because it is conceivable that MMP-9 activation depends upon the extent of airway inflammation and, possibly, the clinical severity of asthma, it may be suggested that while in asthmatic subjects without exacerbations MMP-9 is in an inactive form, during acute episodes of asthma, MMP-9 may undergo activation.
The major source of MMP-9 in human lung are macrophages (5) but this enzyme may also be released by eosinophils (27). Our results suggest that in addition to macrophages and eosinophils, neutrophils are the major cell sources of MMP-9 in the airways. The ability of neutrophils to release a variety of extracellular matrix-degrading proteases has been widely demonstrated. In the induced sputum of asthmatic and chronic bronchitis patients, we have previously shown a significant correlation between the percentage of neutrophils and the concentrations of free elastase (14). The percentage of neutrophils has also been found to be correlated with the content of MMP-9 in several lung diseases (28, 29). Thus, this study confirms previous evidence and suggests that neutrophils have the potential to destroy ECM and determine chronic injury of the lung in asthma and chronic bronchitis.
We acknowledge that a potential weakness of our study is that induced sputum technique is an indirect approach to study remodeling processes that are likely to occur within the airway tissues, and may not reflect what is going on in the body. However, it should be considered that airway remodeling is the end result of a chronic inflammatory process involving cells located in the airway tissues as well as in the lumen. In pulmonary emphysema which is characterized by progressive and extensive destruction and remodeling of airway tissues, alveolar macrophages release greater quantities of MMP and gelatinase B than in control subjects (30), and can degrade all the components of the alveolar matrix. In addition, the ability of alveolar macrophages to release greater amounts of TGF-β, fibronectin, and MMP-9 in patients with asthma and chronic bronchitis than in control subjects (16, 23) supports the hypothesis that cells located in the airway lumen can contribute to the “turn over” of the extracellular matrix, and have the potential to regulate inflammatory and remodeling processes occurring within the bronchial tissues.
As for bronchoalveolar lavage fluid, induced sputum has been recently proposed as a relatively standardized method useful to assess airway inflammation (13, 31). Some biological parameters, such as the number of eosinophils, have been found to be correlated in bronchial biopsies and in induced sputum (32), indicating that, at least in some circumstances, induced sputum may reflect processes occurring within the bronchial wall. Recently, we have shown that the induced sputum of asthmatic patients contains higher levels of active elastase than induced sputum of normal subjects (14), suggesting a possible mechanism responsible for the increased degradation of elastin observed in bronchial biopsies obtained from large airways (33). Finally, the evaluation of matrix metalloproteases and of their inhibitors has also been proposed in other recent studies, suggesting that this approach, which is certainly indirect, may be useful for the measurement of markers of airway inflammation and remodeling (34, 35).
In conclusion, this study shows that asthma and chronic bronchitis are characterized by an imbalance between MMP-9 and TIMP-1 which may play an important role in the pathogenesis of tissue remodeling and of airway obstruction.
The authors gratefully acknowledge the expert assistance of Dr. F. Capony in data analysis and manuscript preparation.
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Sponsored by a joint grant from C.N.R. (Italy) and INSERM (France).
