Cytomegalovirus increases collagen synthesis in chronic rejection in the rat

Kaija Inkinen1, Anu Soots1, Leena Krogerus2, Cathrien Bruggeman4, Juhani Ahonen1 and Irmeli Lautenschlager1,3,

Departments of 1 Surgery, 2 Pathology and 3 Virology, Helsinki University Central Hospital and Helsinki University, Helsinki, Finland and 4 Department of Medical Microbiology, University of Maastricht, Maastricht, The Netherlands



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. We have demonstrated previously that cytomegalovirus (CMV) infection enhances chronic renal allograft rejection in a rat model. Interstitial fibrosis, a characteristic finding for chronic rejection, was also more prominent in CMV-infected grafts. The effect of CMV on the development of fibrosis in this model was investigated here at the molecular level. The collagen/DNA ratio, gene expression of type I and III collagen mRNAs and the presence of myofibroblasts were examined.

Methods. Transplantations were performed under triple drug immunosuppression in a rat strain combination of DA(RT1a) and BN(RT1n). One group of animals was infected with rat CMV and the other was left uninfected. The grafts were harvested at different time points post-transplantation. Graft histology was evaluated according to the Banff criteria and quantified by the chronic allograft damage index (CADI). Total collagen was measured and DNA and RNA were extracted from the grafts. Type I and III collagen mRNAs were determined by slot blot and in situ hybridizations. Myofibroblasts were demonstrated by immunohistochemistry.

Results. The time-related increase of the collagen/DNA ratio in the CMV-infected grafts was higher than in the uninfected animals, correlating with the development of fibrosis at the histology. The expression of type I and III collagen mRNAs peaked shortly after transplantation, together with the presence of myofibroblasts, with significantly higher peaks in the CMV-infected grafts compared with the non-infected ones.

Conclusions. CMV increases the expression of both type I and III collagens and the accumulation of myofibroblasts, and enhances total collagen synthesis in the development of interstitial fibrosis in chronic renal allograft rejection.

Keywords: chronic rejection; collagens; cytomegalovirus; fibrosis; kidney



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Cytomegalovirus (CMV) infection is thought to be one of the risk factors for chronic allograft rejection [1]. A great number of clinical studies on chronic rejection in heart [2], lung [3] and liver [4], as well as experimental models [5], support the link between CMV and allograft rejection. In renal transplantation, an association between CMV and acute rejection has been reported [6]. However, little is known about CMV and chronic rejection in renal transplantation. Previously we have developed an experimental model in which rat renal allografts, after an early inflammatory episode at 5–10 days post-operation, develop chronic rejection under triple-drug immunosuppression within 40–60 days [7]. In this experimental model, we have demonstrated that CMV prolongs and increases graft inflammation, accelerates and enhances the development of chronic rejection, and results in prominent interstitial fibrosis within 20 days [5].

The histological findings of chronic renal allograft rejection are well defined and include interstitial inflammation of various degrees, fibrosis, glomerulosclerosis, vascular intimal thickening and tubular atrophy [8]. Interstitial fibrosis is one of the most prominent findings in cases of chronic rejection of the kidney. The involvement of interstitial myofibroblasts in chronic rejection has been described [9]. These highly fibrogenic cells play a major role in the deposition of collagen and other matrix proteins in renal fibrosis [9].

In general, renal fibrosis and glomerulosclerosis are classic disturbances of extracellular matrix (ECM) regulation, and manifest themselves as an accumulation of collagens in the kidney. The collagens are of great physiological importance as a support for the renal parenchyma and as a component of the basement membrane [10]. The normal renal interstitium is composed of a loose ECM containing type I and type III collagen. These collagens are present in reticular fibres throughout the interstitium, surrounding the collecting ducts, in the media and the adventitia of the blood vessels, and in the renal capsule [11]. Previous studies on renal pathology indicate that in interstitial fibrosis of any aetiology, the newly deposited collagen is almost exclusively type III [10].

In our previous study, we demonstrated that CMV infection enhanced chronic renal allograft rejection in a rat model [5]. Interstitial fibrosis, which is one of the characteristic findings for chronic rejection, was also more prominent in CMV-infected grafts. In the present study, the effect of CMV infection on the development of interstitial fibrosis was investigated at the molecular level. The collagen/DNA ratio, gene expression of type I and III collagen mRNAs and accumulation of myofibroblasts at various time-points were examined in the presence of rat CMV.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Rats
Inbred DA (RT1a) and BN (RT1n) male rats of 200–300 g were used. The animals were fed with regular rat food and tap water ad libitum. The animals were treated according to the international principles of laboratory animal care. The study was approved by the committee for experimental research of Helsinki University Central Hospital and regional authorities.

Renal transplantations
Transplantations were performed in a rat strain combination of DA and BN as described previously [7]. The animals were anaesthetized with midazolam (Dormicum®, Roche, Basel, Switzerland) and fentanyl-fluanisone (Hypnorm®, Janssen, Buckinghamshire, UK). The microsurgical technique of Lee [12] was used: the renal artery and vein were anastomosed, end to side and ureter to ureter. The grafts were flushed and stored in heparinized Euro-Collins' solution on ice. The total ischaemic time was 30±10 min. Differences between the ischaemic times of the CMV-infected and non-CMV-infected groups were not statistically significant. The immunosuppression consisted of triple drug therapy of methylprednisolone (2 mg/kg), azathioprine (2 mg/kg) and cyclosporine (5 mg/kg) daily, subcutaneously. One group of animals was infected with rat CMV (RCMV) and the other was left uninfected (see below).

One group of allograft recipients that also received triple treatment were infected with the RCMV Maastricht strain by inoculation with 105 plaque-forming units of RCMV intraperitoneally, 1 day after renal transplantation. The characteristics of the rat virus and the RCMV infection, as well as the inoculation of the virus, have been described in detail previously [13]. The presence of RCMV infection in the graft was confirmed 6–7 days after inoculation by viral culture [5]. The CMV-infected and non-infected rats were killed and the grafts were harvested at different times after transplantation: 3–5, 6–7, 10–14, 20, 30 and 40 days post-operation. The total number of grafts was 59 (30 and 29 rats in the CMV- and non-infected groups, respectively). There were five rats in both groups harvested 3–4, 6–7 and 10–14 days after transplantation. On day 20 there were three rats in the CMV group and four rats in the non-infected group, and on day 30 there were six and five rats, and on day 40 there were five and six rats, respectively.

Autotransplantations with the same cold and warm ischaemia times were performed on 20 animals, with three to four animals per each time point, to which triple drug immunosuppression was also given and the autografts were used as control material for the time-related follow-up harvested at the same time points. Differences between the ischaemic times of the auto-, non-infected and CMV-infected transplantations were not statistically significant. Non-transplanted normal rat kidneys served as the day 0 control samples.

Histology
Histological examination of the graft was performed on the explants in parallel. The autografts were used as negative controls. The specimens were fixed in normal buffered formalin and stained with haematoxylin–eosin and Masson's trichrome. Graft histology was evaluated according to the Banff criteria [14]. The numerical chronic allograft damage index (CADI) was used to quantify the chronic alterations in the graft [15]. The CADI was formed based on the six histopathological changes characteristic of chronic rejection as described previously [15]: interstitial inflammation, fibrosis, glomerular sclerosis, mesangial matrix increase, vascular intimal thickening and tubular atrophy.

Determination of total tissue collagen and DNA
The total collagen and DNA concentrations of the grafts were analysed to calculate the net gain of matrix in the grafts. The CMV-infected and non-infected grafts and the own native kidneys were homogenized in distilled water. The amount of total collagen and DNA in the tissue was determined from the homogenate as described earlier [16]. The collagen/DNA ratio was calculated, assuming that DNA directly reflects the number of nucleated cells and the amount of tissue in the graft.

Determination of various mRNAs
For the determination of specific mRNAs, total RNA was extracted from the grafts [17]. Aliquots of total RNA were fractionated by electrophoresis on agarose gels and transferred by blotting (PosiBlot, Stratagene, CA, USA) onto MagnaGraph Nylon Transfer Membranes (Micron Separations Inc., MA, USA). The filters were pre-hybridized and hybridized with cDNA clones labelled by random priming (Random Primed DNA Labeling Kit, Boehringer Mannheim, Mannheim, Germany) using [32P]dCTP. The following cDNA clones were used as hybridization probes: clone p{alpha}1R2 containing sequences complementary to rat pro{alpha}1(I) collagen mRNA [18], clone pRGR5 for rat pro{alpha}1(III) collagen mRNA [19] and rat cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [20]. The bound probe was then detected by autoradiography using Kodak X-omat X-ray films.

For the accurate quantitation of pro{alpha}1(I) and pro{alpha}1(III) collagen mRNAs, slot blot hybridizations were employed. Serial dilutions of total RNAs were denatured and dotted onto nylon filters (MagnaGraph Nylon Transfer Membrane) using a vacuum manifold (Hoefer). Dilution series of purified DNA inserts of plasmids p{alpha}1R2 and pRGR5 were applied onto the filters for standards. The filters were hybridized with the same probes as described above. The amounts of pro{alpha}1(I) and pro{alpha}1(III) collagen mRNAs were estimated by densitometric scanning of the exposed films using a densitometer (HP ScanJet IIc Scanner, Hewlett-Packard, CA, USA) connected to a computer to quantify the bands (Bio Image, UK). The results were corrected for minor variations in the amount of GAPDH expression in the samples. The mRNA levels were calculated using standard curves obtained from serial dilutions of the insert DNAs. To allow comparison of the relative expression of genes coding for type I and type III collagen, the densitometric units were corrected for sizes of the cDNAs and expressed as pmol/g of total RNA dotted.

In situ hybridization for type I and III collagen mRNAs
In situ hybridization analysis was performed as described previously [21]. In brief, the plasmids (clone p{alpha}1R2 for pro{alpha}1(I) collagen mRNA and clone pRGR5 for pro{alpha}1(III) collagen RNA) containing the inserted probe were linearized with restriction enzymes to allow transcription of digoxigenin-labelled sense and antisense RNA probes according to the Riboprobe Synthesis Kit method (Boehringer Mannheim). Paraffin sections (5 µm) of the kidney transplants were deparaffinized and hydrated through descending ethanol concentrations. Pre-treatment included incubation with proteinase K and paraformaldehyde post-fixation. Sections were hybridized with labelled RNA-probe. Digoxigenin-labelled probes were detected using the DIG-detection kit (Boehringer Mannheim) following the manufacturer's instructions. After a colour substrate incubation the slides were counterstained with haematoxylin. Staining was considered positive when seen with the antisense probe only. The number of positive interstitial cells was counted per high power visual field (magnification x400).

Demonstration of interstitial myofibroblasts
Interstitial myofibroblasts were demonstrated in frozen sections (3–5 µm) of the explanted grafts by immunohistochemistry. Indirect immunoperoxidase staining using a monoclonal antibody against {alpha}-smooth muscle actin (Dako, Copenhagen, Denmark) was used. Before staining, the sections were treated with chloroform to eliminate non-specific reactions due to endogenous peroxidase. A peroxidase-conjugated rabbit anti-mouse antibody (Dako) and a peroxidase-conjugated goat anti-rabbit antibody (Zymed, San Francisco, CA, USA) were used as secondary antibodies. The reaction was revealed using 3-amino-9-ethyl carbazole solution containing hydrogen peroxide. Mayer's hemalum was used as a counterstain. The number of positive interstitial cells was counted per high power visual field.

Statistics
The data were expressed as mean±SD and the Mann–Whitney U-test was used to compare the results between the groups. P values of <0.05 were considered significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Histological findings
Within 20 days of transplantation with CMV-infected allografts chronic rejection occurred, with characteristic vascular changes and prominent interstitial fibrosis (Figure 1Go). With the non-infected grafts, chronic rejection was seen only after 40 days. The histological findings have been described in detail in our previous publication [5]. The histological changes were examined blindly and the results were quantified and expressed as CADI values (Table 1Go). The CADI value in CMV-infected allografts was already significantly higher on days 6–7 (5.8±0.6 vs 2.9±1.5, P<0.01) when compared with the non-infected allografts due to the significantly more intense inflammation. In CMV-infected grafts, the CADI value (9.0±0.5 vs 5.9±1.7, P<0.05) reached its maximum value, together with maximal histological signs of chronic rejection and prominent interstitial fibrosis on day 20. In the non-infected allografts, the maximal CADI value was reached on day 40, together with the histological criteria of chronic rejection (Table 1Go). In the autotransplanted control animals, no significant inflammatory response nor any changes in chronic rejection were recorded. Reactive changes due to the surgical procedure, which amounted to low CADI values (<2.0), were seen.



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Fig. 1.  Histological findings in the grafts harvested on day 20 after transplantation: (a and b) CMV-infected grafts; (c and d) non-infected grafts; and (e and f) autografts. Note the arterial intimal proliferation (between arrows) (a), and the interstitial fibrosis (red colour) and tubular atrophy (b) in the CMV-infected grafts. Note the intima with one layer of endothelial cells (c and e) in the non-infected grafts and in autografts. Weigert-van Gieson staining was used throughout. Original magnification: x400 (a, c and e) and x200 (b, d and f).

 

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Table 1.  CADI index in rat renal autografts, chronic rejection (ChRx), and ChRx with CMV

 

Generation of collagen
The total collagen and DNA concentrations of the grafts were analysed. The collagen/DNA ratio was counted, assuming that DNA directly reflects the number of nucleated cells and the amount of tissue in the graft. Figure 2Go demonstrates the time-related collagen/DNA ratio of the grafts over a period of 40 days after transplantation. A higher collagen/DNA ratio in the CMV-infected grafts compared with the uninfected animals was clearly evident, indicating the net gain of matrix in the kidneys. However, the difference between the two groups was statistically significant (P<0.05) only on day 40, due to the large biological variations. On day 30 in particular, there was one animal in the non-infected group showing much a higher collagen/DNA ratio than the other animals in the same group. With the autografts, the collagen content did not change significantly during the follow-up time of 40 days (Figure 2Go).



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Fig. 2.  The collagen/DNA ratio in the CMV-infected allografts, non-CMV-infected allografts and autografts without CMV over a period of 40 days after transplantation. Each point represents the mean+SD. Asterisk represents P<0.05.

 

Expression of collagen genes demonstrated by slot blot
The expression levels (pmol/g of total RNA) of types I and III pro-collagen mRNAs in the CMV- and non-CMV-infected allografts are shown in Figure 3Go. Both of the collagen genes were expressed at all time points measured. The expression of type I collagen mRNA was at its maximum on days 3–5 in the CMV-infected and non-infected grafts, but in the CMV-infected grafts the expression levels remained at a higher level than in the non-infected grafts between days 6 and 20. The difference was clear, but was statistically significant only on days 10–14 (P<0.05) due to the large variance between individual animals. The expression of type III collagen mRNA was seen on days 3–5 in both groups, but in CMV-infected grafts the level was increased up to 20 days after transplantation. The difference between the groups was clear and significant. In the CMV-infected animals, the expression of type III collagen mRNA increased to a significantly higher level than in the uninfected grafts, at the time points of days 10–14 (P<0.05). In the autografts, the expression of type I collagen mRNA was seen only on days 3–7 and type III collagen mRNA at all time points measured, but the expressions were lower than in the allografts (Figure 3Go).



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Fig. 3.  Pro-collagen mRNA levels (pmol/g of total RNA) in rat renal allografts in CMV-infected and non-infected animals and autografts (non-infected animals), analysed by slot blot hybridization using cDNA probes for pro{alpha}1(I) collagen (a) and pro{alpha}1(III) collagen (b) over a period of 40 days post-transplantation. Each point represents the mean+SD. Asterisk represents P<0.05.

 

Type I and III collagen mRNA expression in the interstitial cells demonstrated by in situ hybridization
The location of gene expression was equal for both collagen types I and III. The mRNA signal was mainly recorded in the interstitial, fibroblast-like cells of the cortex but also in the medullocortical junction. In addition, positive fibroblast-like cells were constantly seen around the large arteries throughout the follow-up.

The number of collagen type I and III mRNA expressing interstitial cells peaked shortly after transplantation, at days 3–7, in both CMV-infected and non-infected grafts (Figure 4Go). Thereafter the number of mRNA expressing cells decreased, but were constantly seen in lesser amounts throughout the follow-up until day 40. In the CMV-infected kidneys, significantly increased numbers of the type I collagen mRNA positive cells were seen 6–7 days after transplantation, compared with the non-infected grafts (83+15 vs 42+18, P<0.05). Thereafter the number of positive cells was also higher throughout the follow-up, but the difference was not statistically significant. The number of type III collagen mRNA expressing interstitial cells was also higher at days 6–7 in the CMV-infected kidneys (86±3 vs 64±30), but thereafter no difference was recorded between the groups. Throughout the study, the differences in expression of type I and III collagen mRNA between the CMV-infected and non-infected kidneys was quantitative but not qualitative. Type I and III collagen mRNA expressing fibroblast-like interstitial cells, demonstrated by in situ hybridization, are shown in Figure 6Go. In the autografts, the expression of type I and III collagen mRNAs were seen only on days 3–7, and the numbers of expressing cells were much lower than in the allografts (Figure 4Go).



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Fig. 4.  The number of pro-collagen mRNA positive cells in the interstitium of the CMV-infected and non-infected rat renal allografts and autografts (non-infected animals), analysed by in situ hybridization using cDNA probes for pro{alpha}1(I) collagen (a) and pro{alpha}1(III) collagen (b). Each point represents the mean+SD. Asterisk represents P<0.05.

 


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Fig. 6.  The expression of pro{alpha}1(I) collagen mRNA (a) and pro{alpha}1(III) collagen mRNA (b) in fibroblast-like cells (arrows) of the cortical interstitium in the CMV-infected renal allograft at day 7 post-transplantation (original magnification: x200). (c) A closer view of pro{alpha}1 (III) collagen positive cells (arrows) in the interstitum. Original magnification: x1000. (d) Myofibroblasts (arrows) in the interstitium of a CMV-infected renal allograft on day 5 post-transplantation demonstrated by immunohistochemistry. Original magnification x1000.

 

Accumulation of myofibroblasts
Accumulation of myofibroblasts in the interstitium of the kidney allografts, visualized by anti-{alpha}-actin antibody in immunohistochemistry (Figure 5Go), was also recorded shortly after transplantation. The number of positive cells peaked at days 3–5 in both groups, but significantly (P<0.05) higher numbers of {alpha}-actin expressing myofibroblasts were recorded in the CMV-infected kidneys on days 3–5 (50±13 vs 33±8), days 6–7 (33±8 vs 18±7) and days 10–14 (25±5 vs 12±7) after transplantation (Figure 6Go). During the follow-up, the number of myofibroblasts decreased in both groups concomitantly with the increase of interstitial fibrosis at histology. The expression of {alpha}-actin was also constantly recorded in the smooth muscle cells of the arteries in both CMV-infected and non-infected grafts. In the autografts, the number of myofibroblasts did not change significantly during the 40-day follow-up (Figure 5Go).



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Fig. 5.  The number of myofibroblasts in the interstitium of CMV-infected and non-infected renal allografts and autografts (non-infected animals), analysed by immunohistochemistry using anti-{alpha}-actin antibody. Each point represents the mean+SD. Asterisk represents P<0.05.

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We have previously used RCMV Maastricht strain to study the effect of viral infection on the development of chronic renal allograft rejection in the rat [5]. The RCMV model is suitable since the pathogenesis of infection in HCMV-infected humans is quite similar to that in RCMV-infected rats [22]. However, there are different RCMV strains, which may have an impact in the animal models, as has been shown in rat heart transplantation [23].

In this experimental model of chronic renal allograft rejection, CMV clearly accelerated the development of interstitial fibrosis and collagen synthesis in the grafts. At the molecular level, the expression of both type I and type III collagen mRNAs was more prominent in the CMV-infected kidneys than the expression of these collagen types in the non-infected grafts. CMV also increased interstitial accumulation of myofibroblasts, which have been shown to be involved in the deposition of collagen and other matrix proteins in renal fibrosis [9]. The previous observation that myofibroblasts together with type III collagen accumulate in the fibrosis of chronic renal allograft rejection is in accordance with our findings [9].

Various mechanisms by which CMV infection enhances fibrosis in kidney allograft rejection could be suggested. We have previously shown in the same model of chronic rejection that CMV prolongs and increases inflammation and number of macrophages [5], as well as increasing T-cell activation and the expression of adhesion molecules in the graft [24]. Pro-inflammatory cytokines and inflammatory growth factors, such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF-ß) are highly fibrogenic and stimulate collagen synthesis in fibroblasts [25]. Cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor-{alpha} (TNF-{alpha}), have an important role in the stimulation of the synthesis and release of the growth factors PDGF and TGF-ß [26,27]. One of the growth factors associated with renal fibrosis is connective tissue growth factor (CTGF) [28], which is secreted by fibroblasts after activation by TGF-ß [29]. Induction of the generation of interstitial fibrosis in our model can thus be explained by the CMV-induced increase of the inflammatory reaction in the graft. CMV is known to induce the inflammatory cytokines IL-1 and TNF-{alpha} [30,31], which then stimulate the growth factors. On the other hand, CMV infection has been shown to induce the transcription and secretion of TGF-ß1 directly [32]. The increased fibrogenesis in the CMV-infected grafts can also be mediated further by the TGF-ß–CTGF interactions. Our preliminary data also demonstrated that CMV increases the expression of TGF-ß, PDGF and CTGF in this experimental kidney transplant model [33].

The increased type I and III collagen gene expression and early development of allograft fibrosis in the CMV-infected grafts is also in accordance with other experimental and clinical studies. Interstitial up-regulation of both type I and type III collagens has been reported in human and mouse renal allografts with chronic rejection [34,35]. In particular, up-regulation of type III collagen expression within the cortical interstitium seems to be involved in the development of fibrosis [34,35]. In our study, both type I and type III collagens were expressed, and CMV infection increased this expression in rat kidney allografts. The number of mRNA expressing cells peaked in both groups during the first week after transplantation; however, the amount of mRNA of collagen types I and III, as demonstrated by slot blot, remained significantly higher in the CMV-infected grafts somewhat later. Thus, the difference in expression was quantitative but not qualitative. The increased collagen synthesis correlated with the enhanced and more prominent interstitial fibrosis at histology.

In conclusion, CMV infection increased the expression of both type I and type III collagens, promoted accumulation of myofibroblasts and enhanced total collagen synthesis in the development of interstitial fibrosis in chronic renal allograft rejection. Further studies are necessary to investigate the relationship between the viral infection and the mechanism of the fibrogenesis, including a detailed analysis of the involvement of cytokines and growth factors in this process.



   Acknowledgments
 
The authors wish to thank Saara Merasto, Tarja Markov, Raisa Loginov and Stephen Venn for technical assistance, and Kari Savelius for the animal care. This work was supported by grants from the Sigrid Juselius Foundation and Helsinki University Central Hospital Research Funds (EVO).



   Notes
 
Correspondence and offprint requests to: Dr I. Lautenschlager, Transplant Unit Research Laboratory, Fourth Department of Surgery, Helsinki University Central Hospital, Kasarmikatu 11–13, FIN-00130 Helsinki, Finland. Email: Irmeli.Lautenschlager{at}hus.fi Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
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Received for publication: 14. 4.01
Accepted in revised form: 5.12.01