B lymphopenia in uraemia is related to an accelerated in vitro apoptosis and dysregulation of Bcl-2
Gema Fernández-Fresnedo1,
María Angeles Ramos1,
Maria Consuelo González-Pardo1,
Angel Luis Martín de Francisco1,
Marcos López-Hoyos2,* and
Manuel Arias1,*
1 Nephrology and
2 Immunology Units, Hospital Universitario Marqués de Valdecilla, INSALUD, Santander, Spain
Correspondence and offprint requests to:
Dr Manuel Arias, Servicio de Nefrologia, Hospital Universitario Marqués de Valdecilla, ES-39008 Santander, Spain.
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Abstract
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Background. Lymphopenia has been described in patients with chronic renal failure (CRF). It is postulated that the decline in lymphocytes is due to accelerated apoptosis. We investigated whether dysregulation of programmed cell death plays a role in the immunodeficiency described in CRF.
Methods. Peripheral blood lymphocytes (PBL) from pre-dialysis uraemic patients (nHD) and haemodialysed patients (HD) were cultured with no stimulus for 96 h. Apoptosis of lymphocytes was measured by propidium iodide staining and flow cytometry. Expression of Fas and Bcl-2 was also analysed by flow cytometry.
Results. Peripheral blood B cells were significantly lower in pre-dialysis and haemodialysis uraemic patients compared to control. Lymphocytes from both groups of patients had a higher rate of apoptosis in vitro than those from healthy controls. This effect was more pronounced in B lymphocytes and a significant correlation between the B lymphopenia and the percentage of apoptotic B cells after 48 h of culture without stimulus was observed. The increased lymphocyte apoptosis in CRF was accompanied by a significantly lower in vitro Bcl-2 expression. However, Fas did not seem to play a role in spontaneous lymphocyte apoptosis in end-stage renal disease.
Conclusions. Our data indicate that B lymphopenia in CRF may be partially attributed to an increased susceptibility to cell death by apoptosis that is associated with a decreased expression of Bcl-2.
Keywords: apoptosis; Bcl-2; chronic renal failure; Fas; lymphopenia
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Introduction
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Lymphoid homeostasis is based on a tight equilibrium between cell growth and cell death. Control mechanisms exist which serve to select fully functional lymphocytes and to discharge either incompetent or potentially autoreactive lymphocytes. This elimination is mostly accomplished by a particular type of cell death known as apoptosis [1] that plays a central role in the development and shape of a functional peripheral lymphoid repertoire [2,3]. Thus, the dysregulation of apoptotic lymphoid death can lead to an ever-expanding number of immune disorders, such as autoimmunity, immunodeficiencies or lymphomas [4,5].
Chronic renal failure (CRF) variably alters many organs and systems, the immune system being one of the most affected [6]. Although there is some controversy, these changes have been mostly related to a defect in the cellular arm of the immune response, as demonstrated by a prolonged survival of skin grafts, a higher incidence of neoplasias and lymphopenia, and a low mitogenic response to different stimuli in vitro [711]. Although the pathogenic mechanism for this CRF-associated lymphopenia is unknown, disturbances in the regulatory mechanisms of apoptosis may be involved. Indeed, increased susceptibility to apoptosis has been observed in other cells in the uraemic state such as monocytes [12,13] and neutrophils [14].
Apoptosis is finely tuned by a great number of signals that either activate or repress the genetic programme leading to cell death [15]. Moreover, all these processes are controlled by a complex network of proteins grouped in different and expanding families located in the different cell membranes or in the cytosol: TNFR, caspases and Bcl-2 families [16]. Among them, Fas and Bcl-2 are the most broadly investigated molecules in the pathogenesis of different diseases. Fas, a abiquitous protein belonging to the TNFR family, is located in the cytoplasmic membrane [17]. Fas binds to a TNF-like protein called Fas ligand (FasL) in such a way that both cell surface proteins are upregulated in activated lymphocytes. The interaction of both molecules triggers apoptosis and contributes to the regulation of immune responses [18]. In this way, alterations of the FasFasL interaction may allow a prolonged survival of pathological lymphocytes leading to the development of lymphadenopathy and immune disturbances [19]. On the other hand, Bcl-2, the first member of the Bcl-2 family described, functions as a repressor of cell death [20]. It is expressed in a highly regulated pattern during T and B cell maturation, being involved in the control and regulation of the correct development of lymphocytes and its dysregulation may explain many immunodeficiencies, lymphomas or autoimmune diseases [2,4,5].
The aim of the present work was to investigate whether apoptotic death plays a role in the pathogenesis of the lymphopenia described in uraemic patients. We investigated whether lymphocytes from patients with CRF had a higher susceptibility to undergo spontaneous apoptosis in vitro compared with normal donor lymphocytes. In addition, we measured the expression of apoptosis-related proteins, such as Bcl-2 and Fas, in different lymphocyte subsets in these patients. Moreover, we studied the effect of haemodialysis therapy on the induction of apoptotic death in lymphocytes from uraemic patients.
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Subjects and methods
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Patients
After informed consent was obtained, clinical information was recorded and blood was drawn into heparinized tubes. Immunohaematological and biochemical studies were carried out on the following population groups (numbers in parenthesis): non-dialysed uraemic patients (nHD, 32); haemodialysis patients (HD, 36) and healthy control donors (C, 41). All of them were matched for gender, age, and nutritional state (plasma proteins >6 g/dl, plasma albumin >3 g/dl, plasma cholesterol >150 mg/dl). Likewise, different parameters were determined in order to characterize disease activity. Subjects with a history of either atopia, recent or concurrent acute disease, autoimmune disease, immunosuppressive treatment, hyperparathyroidism or iron overload were excluded from the study. nHD patients with a creatinine clearance >20 ml/min were selected and HD patients entered the study if they were stabilized in haemodialysis replacement for at least 6 months. HD patients who had lost renal graft transplants were included if this had occurred at least 6 months previously.
In vitro studies were undertaken in samples from 10 uraemic patients of the nHD group and 14 patients of the HD group. Fourteen healthy donors were employed as a control group. To ensure that inter-experimental variability did not introduce systematic error in the data, patient and control peripheral blood lymphocytes (PBL) were paired and analysed on the same day under the same experimental conditions.
Clinical and biochemical features
Table 1
shows data on age, gender, aetiology of renal disease, duration of disease, time into haemodialysis therapy, serum creatinine and urea levels, and serum immunoglobulin levels. It should be noted that patients with renal failure of autoimmune aetiology were removed from the study. Likewise, different biochemical parameters were determined in order to discard any factor that could interfere in the study. No significant differences for any of the serological parameters between the groups of patients were found.
Cell cultures
Peripheral blood mononuclear cells (PBMC) were isolated from 40 ml heparinized blood with Ficoll-Paque Plus density gradient (Pharmacia Biotech, Uppsala, Sweden). Subsequently, samples were depleted of monocytes by adherence to the plastic during a 1-h incubation at 37°C in 5% CO2 in complete medium comprising RPMI 1640 (Flow, Irvine, Scotland) supplemented with penicillin (200 U/ml), streptomycin (200 mg/ml), glutamine (2 mM) and 10% heat-inactivated fetal calf serum (Biowhittaker, Verviers, Belgium). Then, PBL purified from PBMC were placed in 24-well, flat-bottomed plates (Falcon®, Becton-Dickinson, Franklin Lakes, NJ) and cultured at a final concentration of 106 cells/ml in complete medium with no stimulus for 96 h. Cells were harvested every 24 h until the end of the study and quantitation of apoptosis was assessed by flow cytometry as described below.
Measurement of apoptotic cells
The percentages of apoptotic cells were calculated immediately after isolation of PBL, and every 24 h of incubation up to the fourth day according to a method previously described [21,22]. Cells were harvested from cultures and incubated with 5 µl of either anti-human CD4 (clone SK3), CD8 (clone SK1) or CD19 (clone HD37) mAbs coupled to FITC (Becton Dickinson, Mountain View, CA), followed by fixing in 2 ml cold 70% ethanol at 4°C for 45 min. Cells were then pelleted, washed in phosphate-buffered saline (PBS) and resuspended in 400 µl, PBS containing RNAase-A at 1 mg/ml (Sigma, St Louis, MO) and propidium iodide (PI) (100 µg/ml, Sigma). Thereafter, cells were incubated for 15 min at room temperature in the dark and then were kept at 4°C in the dark until analysis. Finally, at least 20 000 cells were analysed in a FACScalibur flow cytometer using the CellQuest 3.0.1 software (Becton Dickinson), and percentages of cells undergoing apoptosis were determined by dual-colour analysis. In contrast with non-apoptotic cells that show two small peaks with high fluorescence intensity, nuclei of apoptotic cells were stained with lower intensity by PI and can be detected in a broad peak with lower fluorescence intensity [21].
Fas and Bcl-2 expression
In order to investigate the expression of surface Fas and intracellular Bcl-2 in different subpopulations of lymphocytes, staining for surface molecules (either CD4 or CD8 or CD19, and Fas), followed by Bcl-2 staining was performed on whole blood lymphocytes, as previously described [23]. In brief, cells were stained with FITC-mAbs against CD4, CD8 or CD19, and with anti-human Fas phycoeritrin (PE)-coupled mAb (clone UB2, Immunotech, Marseille, France). For Bcl-2 staining, after incubation with mAbs for surface markers, cells were fixed with 1% paraformaldehyde to avoid loss of the surface markers. Following fixation, staining with a hamster anti-human-Bcl-2 mAb (clone 6C8, Pharmingen, San Diego, CA) was performed after permeabilizing cells with 0.03% saponin. Subsequently, cells were incubated with a biotinylated F(ab')2 goat anti-hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), followed by incubation with StrepTavidin-RED 670TM (GIBCO BRL, Gaithersburg, MD). Viable cells were analysed as described above and dead cells and debris were excluded based on forward and sideways light scatter.
Activation phenotype analysis
Activation markers on freshly isolated B and T cells from the patients under study were examined by staining with mAbs, conjugated to FITC, for either CD4, CD8 or CD19 molecules, together with mAbs against either CD25 (clone 1HT44H3) or HLA-DR (clone NS1/1-AG4) coupled to PE. Subsequently, cells were acquired and analysed by cytometry as described above.
Statistical analysis
Statistical analyses included correlation by the Pearson test and one-way ANOVA for comparisons among groups. P values <0.05 were considered significant.
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Results
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Haematologic and immunologic studies
A greater number of blood leukocytes was observed in both groups of uraemic patients compared to the healthy group (P<0.05) which was mostly due to granulocytes (P<0.01) (Table 2
). Lymphopenia could not be detected in absolute numbers, except very weakly in the HD group (not significant). However, percentages of lymphocytes were clearly decreased in the nHD and HD groups compared to the C group (P<0.001). Analysis of peripheral lymphoid subpopulations revealed no difference between the C group and nHD and HD groups with regard to the T cell subsets, CD3+, CD4+ or CD8+. However, a significant reduction of the absolute B cell counts (CD19+ cells) was found for the uraemic patients (Table 3
). This decrease was more marked in the HD group (87±83/mm3) than in the nHD group (134±94/mm3) compared to the C group (188±97/mm3) (P<0.001 C vs HD; P<0.05 C vs nHD). Furthermore, differences were significant between both groups of patients (P<0.05). Despite this B lymphopenia, there were no significant differences in serum immunoglobulin levels between groups (Table 1
).
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Table 2. Total white blood cell counts and numbers and percentages of mononuclear cells and granulocytes in control subjects, non-haemodialysis patients and haemodialysis patientsa
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Table 3. Absolute and relative values of the main lymphocyte subsets in peripheral blood of control subjects, and non-haemodialysis or haemodialysis patientsa
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In vitro apoptosis
The lower number of peripheral blood B lymphocytes in patients with CRF led us to suspect their higher susceptibility to die by apoptosis. To investigate this possibility, PBL isolated from patients and control subjects were placed in culture with no stimulus and harvested every 24 h up to 96 h to analyse the percentages of apoptotic cells by PI staining (Figure 1
).

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Fig. 1. Quantification of apoptotic lymphocytes in culture by flow cytometry after staining with propidium iodide (PI). Total lymphocytes were collected from cell cultures with only medium every 24 h and stained with PI for DNA analysis. The flow cytometry histograms represent six different experiments. Percentages of hypodiploid cells are shown in each histogram.
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A higher percentage of apoptotic lymphocytes was found in the uraemic patients throughout the culture period (Figure 2
). This phenomenon was more pronounced for B lymphocytes since we detected >55% of apoptotic B cells at 48 h in culture in both groups of patients while not reaching 40% in the C group (Figure 2
). Interestingly, the increased rate of apoptosis in B cells from nHD and HD groups was negatively correlated with the absolute B cell numbers in whole blood (Figure 3
). In the case of CD4+ or CD8+ lymphocytes, the rates of spontaneous apoptosis were also higher in uraemic patients, but differences were not so important and only noticeable after 96 h of culture (Figure 2
). Furthermore, we did not find any correlation between apoptosis of T cells and peripheral blood T cell absolute numbers.
Expression of Fas and Bcl-2 proteins
We next analysed the expression of apoptosis-related gene products, Fas and Bcl-2, in peripheral blood lymphocytes by cytofluorometry. Figure 4
shows a representative staining of Bcl-2 and Fas proteins within peripheral blood CD19+ cells from subjects from the three groups of the study. As previously reported [24,25], three populations can be defined according to the intensity of Bcl-2 expression: low, normal and high expression of Fas or Bcl-2 molecules. In healthy subjects the higher percentage of lymphocytes express normal Bcl-2 levels (Figure 4
) [24,25]. We measured the proportions of Fas+ and Bcl-2low cells that are more susceptible to undergo apoptosis.

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Fig. 4. Expression of Fas and Bcl-2 by peripheral blood CD19+ B lymphocytes. Freshly isolated PBL obtained from a C patient (left), nHD patient (centre) and one HD patient (right) were analysed for coexpression of Bcl-2 (upper) or Fas (lower) and CD19 molecules by cytofluorometry. Expression of Fas was measured as percentage of positive cells. For Bcl-2, three subsets were defined according to the level of expression: Bcl-2low: Bcl-2normal and Bcl-2high. Levels of expression are indicated by markers and proportions in each histogram. Isotype control histograms are overlaid in each histogram. The figure shows a representative experiment with PBL from one subject from each group studied.
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In concordance with the frequency of apoptotic cells, the proportion of total lymphocytes from patients with CRF expressing the pro-apoptotic molecule Fas, was higher than from healthy subjects (Figure 5
). Nevertheless, we could not attribute this increased Fas expression to any subset of lymphocytes since there was a trend toward increased frequencies of Fas+ cells in both T and B lymphocytes in nHD and HD groups, which did not reach statistical significance (Figure 5
).
To determine whether this upregulated Fas expression could be induced by lymphocyte activation, expression of CD25 on CD4+ and CD8+ cells, and HLA-DR on CD19+ cells was studied. Although percentages of CD4+CD25+ and CD19+HLA-DR+ cells from HD patients (17.42±10 and 9.6±4.9 respectively) were higher than from nHD (10±9.6 and 5.05±3 respectively) and C (6.5±6.2 and 4±1.29 respectively) subjects, differences were not statistically significant as observed for Fas expression.
Unlike Fas expression, we detected an increase in the percentages of T and B cells expressing Bcl-2low in both groups of patients with CRF before placing cells in culture (Figure 6
). The difference was statistically significant for the CD19+ subpopulation in both groups of patients with CRF compared to the C group (P=0.03 C vs HD; P=0.04 C vs nHD), but it was only significant between the HD and C group for the CD4+ and CD8+ subsets (P=0.01).
Remarkably, the percentages of Bcl-2low B lymphocytes in uraemia were correlated with percentages of apoptotic B lymphocytes at 48 h of culture (r=0.5) although it was not statistically significant. Nevertheless, there was no correlation between proportions of Bcl-2low T cells and T cells apoptosis in vitro. There was neither correlation between Fas expression and apoptosis for any lymphocyte subset.
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Discussion
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Immunodeficiency secondary to CRF is a well-established finding, paradoxically associated with signs of cell activation. Such an immunodeficient state has been related to lymphopenia and, especially, to low numbers of some lymphocyte subsets [2630]. On the other hand, control of programmed cell death is crucial in maintaining homeostasis in the immune system [13], as demonstrated by the occurrence of immune disturbances when an apoptotic programme is altered in AIDS and some autoimmune diseases [4,5]. Here we attempted to investigate the possible mechanism involved in the lymphopenia observed in uraemic patients, particularly its relation with the susceptibility of lymphocytes to die spontaneously by apoptosis in vitro.
There are no clear data about the presence of B lymphopenia in uraemia [26,29,30] and when it exists it is usually accompanied by a more significant T lymphocyte reduction [30]. In contrast, we observed that the B cells were the most decreased lymphocyte population without an alteration in T cell numbers. However, this B lymphopenia was not reflected by decreased plasma levels of IgG, IgM and IgA which is in agreement with previous studies [30].
Particularly, B cells from HD patients were more affected than B lymphocytes from the nHD group. This effect could be explained by mechanical damage of B cells in the dialysis membrane. However, we have found no differences in B lymphopenia related to the biocompatibility of the dialysis membrane (unpublished observations). Moreover, previous works have not shown mechanical destruction of B lymphocytes in the dialysis membrane [31].
The observations reported here strongly suggest that accelerated cell death through apoptosis plays an important role in the B lymphopenia detected. At the same time, we found an increased in vitro apoptosis rate for both CD4+ and CD8+ T lymphocytes in both groups of patients with CRF, although this was not so marked. In this context, our results differ from those reported by Matsumoto et al. [32]. These authors observed a higher susceptibility of T lymphocytes from nHD uraemic patients to die through apoptosis than those from HD uraemic patients. This discrepancy may be related to the differences in the methods used to isolate the T lymphocytes since Matsumoto et al. obtained T lymphocytes after passage through an anti-CD3 mAb coated column. Thus, it is possible that T lymphocytes obtained in this way could be activated and some findings could be due to the phenomenon known as activation-induced cell death (AICD) [33,34]. Our time-course study used lymphocytes isolated with minimal manipulation. Moreover, Matsumoto and colleagues did not study the apoptotic phenomenon in B cells from uraemic patients [32].
One possible explanation for the susceptibility of B and T cells to apoptosis is that in uraemic patients there are enough cytokines essential for T cell survival, but growth factors for B cells are lacking, making them more susceptible to apoptosis by neglect. In fact, a very recent study demonstrates that the production of Th1 cytokines (i.e. IL-2, IFN-
) is preserved in patients with end-stage renal disease and on low-flux HD, whereas Th2 cytokine (i.e. IL-4, IL-10) production, so necessary for B cell survival, is impaired [35].
Finally, apoptosis has been shown to be one of the mechanisms responsible for lymphopenia associated with aging [22]. However, all the patients and controls employed in the present study were age and gender matched.
Fas receptor cross-linking results in the death of Fas+ T cells upon FasL ligation [17,18]. This interaction helps terminate an ongoing immune response by inducing apoptosis in activated lymphocytes. Our data showed slight increased frequencies of Fas+/CD4+ T cells at basal time from patients with CRF, although this was also observed for CD8+ T cells and CD19+ B cells. The increased percentage of Fas+ cells was more pronounced in HD than in nHD patients which is in disagreement with the findings of Matsumoto et al. [32], who reported a higher expression of Fas in nHD than in HD patients. Again, the discrepancy with their work may be due to differences in the methods of isolation and detection of Fas, as commented above. Nevertheless, their findings and ours may be explained by the phenomenon of AICD which may occur in patients with CRF, in which there are a number of stimuli that can induce the lymphocyte activation. In our case, the slight increase of ex vivo expression of Fas in lymphocytes, due to a possible in vivo stimulation, could explain the increased susceptibility to spontaneously die through apoptosis at 48 h of culture. However, there was no correlation between Fas expression and spontaneous lymphocyte apoptosis. An increased expression of FasL on lymphocytes from freshly isolated PBL would be consistent with such a hypothesis [33,34], but preliminary studies in our laboratory indicate no increase in the expression of FasL on freshly isolated cells (data not shown). Furthermore, no significant increase in the expression of activation markers, which could indicate the presence of AICD, was found on B or T cells in the present work. In addition, AICD seems to participate in the elimination of self-reactive cells but its role in the homeostasis of the immune response against foreign antigens is unclear [16]. Further investigation to define the role of Fas/FasL interaction in the induction of apoptosis of the lymphocytes from patients with CRF and to clearly define the relationship that it has with the AICD phenomenon is needed.
Bcl-2 plays an important role as an anti-apoptotic molecule and is regulated developmentally in the B cell lineage [2,20]. Moreover, it is fundamental in the control of spontaneous apoptosis or death by neglect [16,20], i.e. the phenomenon which we examined in this work. We therefore studied this molecule with a similar approach to that followed for Fas. As significantly decreased ex vivo expression of Bcl-2 was observed in CD4+, CD8+ and CD19+ lymphocytes. The differences in B lymphocyte apoptosis were predominantly observed after 48 h of culture, when these lymphocytes need those growth factors that are lacking in the culture medium, and the diminished expression of Bcl-2 might fail to rescue uraemic B lymphocytes from apoptosis by neglect. Experiments with the addition of necessary survival factors to the culture medium, such as IL-4, IL-13 or anti-CD40 mAbs [36], to suppress the spontaneous apoptosis of B lymphocytes in vitro need to be performed. On the other hand, the higher percentage of apoptotic T cells was observed after 96 h of culture. In this case, it might be that any other Bcl-2 family member [20] could be protecting T cells from apoptosis by neglect. Thus, we did not address the possibility of dysregulation in the Bcl-2/bax balance [16,20] causing the immune dysregulation described. In the same way, the balance between other anti-apoptotic molecules expressed on B cells, such as BclxL or A1, and pro-apoptotic molecules such as bak, bid, bad or Apaf-1 [37,38], could be interfering in the apoptotic programme of T lymphocytes in end-stage renal disease.
In summary, our data demonstrate that the increased susceptibility of B lymphocytes to die by apoptosis in vitro may be responsible, at least in part, for the B lymphopenia detected in CRF. We also provide evidence that the specific dysregulation of Bcl-2 predispose CD19+ B cells from uraemic patients to undergo in vitro apoptosis. Nevertheless, a direct effect of apoptosis on the B lymphopenia in vivo remains to be demonstrated. However, apoptosis in vivo is very complex to study since apoptotic cells are rapidly removed by the neighbouring phagocytic cells. Furthermore, given current and expanding knowledge about the molecular mechanisms of programmed cell death, the roles of other molecules controlling apoptosis remain for further investigation.
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Acknowledgments
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We are very grateful to Drs Jesús Merino and Ramón Merino for helpful comments on the manuscript. This work was supported by the `Fundación Marqués de Valdecilla', Santander, Spain. Dr Gema Fernández-Fresnedo is a fellow of the `Fundación Marqués de Valdecilla'.
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Notes
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* The last two authors share senior authorship. 
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Received for publication: 6. 4.99
Accepted in revised form: 18.11.99