Persistent cytomegalovirus infection is associated with increased expression of TGF-ß1, PDGF-AA and ICAM-1 and arterial intimal thickening in kidney allografts

Ilkka Helanterä1,2, Raisa Loginov1, Petri Koskinen2, Tom Törnroth2, Carola Grönhagen-Riska2 and Irmeli Lautenschlager1

1 Department of Virology, Helsinki University Hospital and University of Helsinki, Helsinki, Finland and 2 Department of Medicine, Division of Nephrology, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

Correspondence and offprint requests to: Dr I. Lautenschlager, MD, PhD, Transplant Unit Research Laboratory, Helsinki University Hospital, Meilahti, P-Floor, P.O. Box 340, FIN-00029 HUS, Helsinki, Finland. Email: irmeli.lautenschlager{at}hus.fi



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Cytomegalovirus (CMV) is a suggested risk factor for the development of chronic allograft nephropathy. Transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) are important molecules in this process. We analysed the impact of persistent CMV infection in kidney allografts on the expression of growth factors, adhesion molecules and inflammation markers.

Methods. In a population of 172 renal transplant recipients, CMV was diagnosed in 82 patients by pp65 antigenaemia test and viral cultures. Biopsies taken after CMV infection were available from 48 of the 82 patients for the demonstration of CMV antigens by immunohistochemistry and in situ DNA hybridization. Biopsy material for further analyses was available from 16 CMV patients. Five patients with no previous CMV infection were used as controls. Biopsy histology was scored according to Banff 97 classification.

Results. In 11 out of 16 patients, persistent CMV antigens and/or DNA were demonstrated in the biopsy >2 months after the last positive finding in blood or urine. Increased expression of TGF-ß1 was recorded in tubuli and in arterial endothelium in biopsies with a positive CMV finding compared with controls. Also, the expression of PDGF-AA was increased in tubuli and somewhat in arterial endothelium in CMV-positive biopsies. The expression of intercellular adhesion molecule-1 (ICAM-1) was increased significantly in peritubular capillary endothelium. Vascular intimal thickening was increased in the biopsies with persistent CMV infection.

Conclusions. Persistent CMV infection in kidney allografts was associated with increased vascular changes and increased expression of TGF-ß1, PDGF-AA and ICAM-1.

Keywords: chronic allograft nephropathy; cytomegalovirus; intercellular adhesion molecule-1; platelet-derived growth factor; renal transplantation; transforming growth factor-ß1



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Chronic allograft nephropathy (CAN) is characterized histologically by mononuclear interstitial inflammation, tubular atrophy, glomerular lesions, arterial intimal thickening and interstitial fibrosis [1]. The aetiology of CAN is multifactorial [1]: risk factors include acute rejection episodes, ischaemia–reperfusion injury and viral infections, especially cytomegalovirus (CMV) infections [2].

CMV is a major cause of morbidity in renal transplant recipients. CMV has been associated with the development of acute rejections and CAN [2], but controversial data have been reported also [3]. Recently we showed that the presence of CMV proteins or genome in the kidney allograft together with a previous history of acute rejection episodes was associated with increased vascular changes in 6-month protocol biopsies [4].

Transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) are thought to be key stimulating growth factors in the pathogenesis of CAN [5,6]. In vitro, CMV has been shown to directly induce the expression and secretion of TGF-ß [7]. In our recent study, we showed that urinary excretion of TGF-ß was increased in kidney transplant recipients during CMV infection and that the increased excretion of TGF-ß was associated with increased fibrosis in 6-month protocol biopsies (unpublished data). PDGF, an important mesenchymal cell mitogen, consists of two polypeptide chains (A and B) and can form three different isoforms (PDGF-AA, -BB and -AB). In vitro, CMV has also been shown to increase the production of PDGF [8].

Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and VCAM-1, are involved in cell–cell interactions, T-cell activation and extravasation of inflammatory cells from the blood and are important mediators in the early phase of the alloresponse in renal transplants. Furthermore, studies in experimental models of renal transplantation have shown that CMV is associated with increased expression of ICAM-1 and VCAM-1 [9], together with accelerated development of chronic changes.

The association of persistent CMV with vascular changes in the graft was investigated in our previous study [4]. The present research was undertaken to study the mechanisms behind this recent observation. The purpose of this study was to examine the impact of persistent CMV in the kidney allograft on the expression of growth factors (TGF-ß1, PDGF-AA and PDGF-BB), adhesion molecules (ICAM-1, VCAM-1 and ELAM-1) and inflammation markers [CD4, CD8, MHC class-II molecule and interleukin (IL)-2 receptor] and to correlate findings with biopsy histology.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
Approximately 150–200 renal transplantations are performed annually in the Helsinki University Hospital and, annually, approximately 40 patients remain in follow-up at the hospital. The patients included in our retrospective study were selected from a population of renal transplant recipients, transplanted between 1992 and 2000, who were being followed up at Helsinki University Hospital Division of Nephrology and in who there had been a suspicion of CMV infection (n = 172). The patients were not monitored regularly for CMV, but samples for the detection of CMV were always obtained when infection was suspected. No antiviral prophylaxis for CMV or other herpes viruses was given post-operatively. This retrospective material is from a period prior to the introduction of CMV prophylaxis. Nowadays, high-risk patients (CMV seronegative recipients receiving allograft from a seropositive donor, D+/R–) receive valganciclovir prophylaxis for the first 6 months in our clinic. Clinically significant CMV infections were treated with intravenous (i.v.) ganciclovir. Trimethoprim–sulfamethoxazole prophylaxis against Pneumocystis carinii was given routinely during the first 6 months after transplantation or if the level of serum creatinine was >200 µmol/l, pentamidine inhalations were usually used instead.

Primary immunosuppression therapy by the time of transplantation consisted of cyclosporin A, azathioprine and methylprednisolone in every recipient. Diagnosis of acute rejection was based on biopsy histology [10] and on clinical criteria. Biopsy-proven acute rejections of grades I–III were treated with high doses of i.v. methylprednisolone, OKT3 or plasmapheresis. None of the patients used angiotensin-converting enzyme inhibitors or angiotensin II-receptor blockers during the first 6 months after transplantation.

Demonstration of CMV infection
The diagnosis of CMV infection was based on the standard CMV pp65 antigenaemia test and rapid shell vial cultures from blood and urine. Specimens for detection of CMV antigenaemia and viral cultures from blood and urine were obtained only when clinical signs of CMV infection were suspected (fever, unexplained increase of serum creatinine, leukopenia, thrombocytopenia, hepatopathy, gastroenteritis and pneumonia). In a population of 172 renal transplant recipients, CMV was diagnosed in 82 patients by pp65 antigenaemia test and viral cultures from blood and urine.

Demonstration of CMV in the biopsies
Frozen biopsy material from biopsies taken after CMV infection was available from 48 out of the 82 patients for the demonstration of CMV antigens by immunohistochemistry and in situ DNA hybridization. The demonstration of CMV antigens (pp65) is only successful on frozen material and the demonstration of CMV DNA is also more sensitive in frozen sections than in standard formalin-fixed material. For the detection of CMV antigens in kidney allograft biopsies, monoclonal antibody against CMV structural protein (pp65) and indirect immunoperoxidase staining was applied. The biopsy material was snap-frozen and 3–4-µm thick sections were cut, acetone fixed and stored at –20°C until used. Before staining, the sections were treated with chloroform to avoid unspecific reactions with endogenous peroxidase. The presence of CMV in kidney allograft biopsies was also demonstrated by in situ DNA hybridization using a biotinylated probe (Enzo Biochem, Inc., New York, NY, USA) prepared from a mixture of two clones of CMV sequences in the BamHI site of pBR22, as described previously [4].

Biopsy histology
A protocol biopsy at 6 months post-transplantation was performed on every kidney allograft recipient according to the policy of our clinic. For grading of histological parameters associated with CAN, criteria of the Banff 97 classification were used [10]. All biopsies were scored by two independent observers in a blinded fashion.

Demonstration of growth factors and cytokines in the biopsies
Frozen biopsy material for further immunohistochemical stainings was available from 16 CMV patients and from five control patients, in whom CMV antigenaemia tests and viral cultures from blood and urine were all negative in several samples at different time-points. Of these biopsies, only three out of 16 were protocol biopsies in the CMV group and two out of five in the control group. Altogether, 13 out of 16 biopsies in CMV patients and three out of five in control patients, used for immunohistochemical staining, were taken due to graft dysfunction or suspicion of acute rejection >6 months after transplantation. All of these patients with available frozen biopsy material were from the same patient material as in our recent publication [4]. The expression of TGF-ß1, PDGF-AA, PDGF-BB, ICAM-1, ELAM, VCAM-1, IL-2 receptor, CD4, CD8 and MHC class-II antigen was demonstrated in frozen sections (3–5 µm thick) of the kidney allograft biopsies by immunoperoxidase stainings. Monoclonal antibodies were used for ICAM-1, VCAM-1, ELAM-1 (R&D Systems, Abingdon, UK), IL-2R (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA), CD4, CD8 (Southern Biotechnology Associates, Inc., Birmingham, USA) and MHC class-II (DAKO A/S, Clostrup, Denmark) and polyclonal antibodies for TGF-ß1, PDGF-AA and PDGF-BB (R&D Systems, Abingdon, UK). Expression was scored semi-quantitatively with a scale from 0 to 3 or by counting positive leukocytes cells per high-power visual field (in the case of IL-2R, CD4 and CD8). All biopsies were scored by two observers. Expression was scored separately in tubuli, in glomeruli, in arterial and peritubular capillary endothelial cells and in vascular smooth muscle cells.

Statistical analyses
All data are expressed as means±SD. Statistical significance between the two groups was measured by the non-parametric Mann–Whitney U-test. A non-parametric test was chosen because of the small sample sizes and inability to determine whether the samples were distributed normally.



   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
CMV infections
CMV infection was diagnosed in 15 out of 16 patients with a positive antigenaemia test and viral cultures from blood and urine and in one out of 16 patients with a positive culture from urine only (which was also considered as indicative of the presence of CMV in this patient with serostatus D+/R+). The onset of the first CMV infection episode was at a mean 49±24 days after transplantation. Altogether, four primary infections and 12 reinfections/reactivations were recorded. In the CMV group, nine patients developed several CMV infection episodes (range: 1–3). All except three patients in the CMV group received ganciclovir treatment. These three patients demonstrated occasional low-level CMV antigenaemia (<10/50 000) only, which subsided during a follow-up period of a few days. None of these patients developed acute rejection after CMV infection. One of these three patients who did not receive ganciclovir treatment had persistent CMV infection during follow-up. The five patients in whom CMV infection was not diagnosed at any time-point were used as controls. In 11 of 16 patients, CMV persisted in the kidney allograft, as CMV antigens and/or DNA were demonstrated in the biopsy 2–12 months after the last positive finding in blood or urine. In eight patients, both CMV antigens and DNA were found in the biopsy and in three patients only CMV DNA was found in the biopsy. The demographic data of the 11 patients with persistent CMV infection in the graft and of the five control patients, which were included in the further analysis, are presented in Table 1.


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Table 1. Demographic data of the patientsa

 
Acute rejections
Acute rejection was diagnosed in six out of 11 patients in the persistent CMV group (at a mean 41±37 days after transplantation) and in three out of five patients in the control group (at 102±104 days after transplantation; P = NS). All except one of the acute rejection episodes were mild and fully reversible with high-dose i.v. corticosteroid treatment. One patient from the persistent CMV group suffered from grade II vascular rejection and was treated successfully with plasmapheresis.

Impact of CMV on graft histology
In the 6-month protocol biopsies, the degree of arterial intimal thickening was increased significantly in the biopsies of patients with persistent CMV found in the graft (1.3±0.4 vs 1.0±0.3, P = 0.02; Figure 1 and Table 2). Vascular changes were seen in all biopsies from recipients with persistent CMV. The degree of interstitial fibrosis was also slightly increased, although this did not reach statistical significance.



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Fig. 1. Photomicrographs from the biopsies. (A) Normal small arteries (arrow) and normal renal architecture from a biopsy of a patient with no history of CMV infection. (B) Totally occluded small arteriole (arrow) in a biopsy from a patient with persistent CMV infection in the kidney allograft. (May–Grünwald–Giemsa stain; original magnification: x400.)

 

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Table 2. Impact of persistent CMV on the histopathological changes

 
No differences were found in the other histopathological parameters evaluated.

Impact of persistent CMV on the expression of growth factors and inflammation markers
Biopsies for the immunohistochemical analyses were taken 290±226 days after transplantation in the persistent CMV group and 202±110 days after transplantation in the control group (P = NS). Biopsies were taken at 233±231 days after CMV infection in the persistent CMV group, 280±217 days after the acute rejection episode in the persistent CMV group and 103±69 days after the rejection episode in the control group (P = NS). No signs of acute rejection were present in any of the biopsies used for the immunohistochemical stainings and none of the patients had had acute rejection episodes during the last 3 months before the biopsy. The expression of ICAM-1, TGF-ß1 and PDGF-AA is shown in Table 3. The expression of TGF-ß1 was significantly increased in arterial endothelial cells in the group of patients with persistent CMV compared with controls (P = 0.036; Figure 2). Also, the expression of PDGF-AA, but not that of PDGF-BB, was increased significantly in tubuli (P = 0.040) and the expression of ICAM-1 was increased significantly in peritubular capillary endothelium (P = 0.003) in patients with persistent CMV in the graft, compared with controls (Figure 2). In biopsies of patients with a previous history of CMV infection but no CMV found in the graft after the infection (non-persistent CMV), expression of TGF-ß1 and PDGF-AA were increased only slightly and did not reach statistical significance (Table 3) and this was not associated with any histological changes in the biopsies. Expression of ICAM-1 was increased significantly also in the non-persistent CMV group, compared with controls, in peritubular capillary endothelium and arterial endothelium (Table 3). The biopsies used for the immunohistochemical stainings in the non-persistent CMV group were obtained a mean 712±669 days after transplantation. All biopsies from recipients with persistent CMV showed significant expression of ICAM-1, TGF-ß1 and PDGF-AA, whereas borderline expression of TGF-ß1 in tubuli was seen in only one of the control patients and marginal expression of PDGF-AA was seen in two biopsies from control patients. Marginal expression of ICAM-1 was seen in all of the biopsies from the control patients. No differences were recorded in the expression of the other molecules analysed (i.e. IL-2R, CD4, CD8, MHC class-II antigens, VCAM-1, ELAM-1 and PDGF-BB) between patients with persistent CMV and control patients (data not shown). Previous acute rejection did not have any impact on the expression of ICAM-1, TGF-ß 1 or PDGF-AA. The expression of ICAM-1 in peritubular capillary endothelium in patients with persistent CMV and previous acute rejection episodes vs patients with persistent CMV but no previous acute rejection was 2.8±0.4 vs 2.8±0.4, the expression of TGF-ß1 in arterial endothelium was 0.8±1.1 vs 1.2±1.3 and the expression of PDGF-AA in tubuli was 2.0±0.7 vs 1.3±1.0, respectively (P = NS).


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Table 3. The impact of persistent CMV infection on the expression of TGF-ß1, PDGF-AA and ICAM-1

 


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Fig. 2. The expression of TGF-ß, PDGF-AA and ICAM-1 in frozen sections of the biopsies. The expression, as demonstrated by immunoperoxidase staining, is seen as red staining. The anatomic structures are visualized by using Mayer's haemalum as counterstain. (A) Expression of TGF-ß in a biopsy from a control patient and (B) positive staining in medial smooth muscle cells and endothelial cells in an artery (arrow) and in tubular epithelial cells in a biopsy from a patient with persistent CMV infection in the graft. (C) Expression of PDGF-AA in a biopsy from a control patient and (D) strong positive staining in vascular endothelial and medial smooth muscle cells, tubular epithelial cells and glomerular cells in a biopsy from a patient with persistent CMV infection in the graft. (E) Expression of ICAM-1 in a biopsy from a control patient and (F) strong positive staining in peritubular capillary structures and also in glomerular structures in a biopsy from a patient with persistent CMV infection in the kidney allograft. (Original magnification: x200.)

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study we analysed the impact of persistent CMV infection in kidney allografts on the expression of various growth factors and cytokines, which are known to play a role in the development of CAN, within the graft. We found that persistent CMV infection was associated with increased expression of TGF-ß1, PDGF-AA and ICAM-1 in the allograft biopsies compared with biopsies from control patients with no evidence of CMV infection. These molecules are all thought to be key mediators of inflammatory, fibrogenic and vasculopathic responses in the development of CAN [5,6,11]. Furthermore, we found that persistent CMV infection in the graft was associated with increased vascular intimal thickening in 6-month protocol biopsies compared with controls. Previous, mostly mild acute rejections did not have any correlation with the expression of these molecules or with the histopathological findings in the biopsies and none of the patients had had acute rejection episodes during the previous 3 months before the biopsy. No differences were found in previous pre- or peri-operative factors, medications used or in the blood trough levels of cyclosporin A between the recipients with persistent CMV infection in the graft and the control group. The ideal control group for the purpose of this study would be CMV seronegative patients who received an allograft from a CMV seronegative donor (D–/R–). However, the seroprevalence of CMV in the Finnish population is between 70% and 80% and such a combination is rare (2%) [4]. Only one patient in this material was D–/R–.

CMV is a suggested risk factor for the development of CAN [2], although some studies have failed to show this correlation in clinical transplantation [3]. In our previous study we showed that the presence of CMV proteins or genome in the kidney allograft together with a previous history of acute rejection episodes was associated with increased vasculopathic changes in 6-month protocol biopsies [4]. In an experimental rat model of chronic renal allograft rejection, rat CMV (RCMV) infection increased inflammation and enhanced the development of transplant vasculopathy and also increased the development of interstitial fibrosis in renal allografts [2]. Several studies support the role of CMV in the development of chronic rejection in other solid organ transplantations, i.e. cardiac allograft arteriosclerosis [12], obliterative bronchiolitis in lung allografts [13] and vanishing bile-duct syndrome in liver allografts [14].

Holma et al. [15] have demonstrated that CMV genome may persist in the kidney allograft for several months (≤1 year) and that this persistence is usually associated with the development of chronic changes in the graft. Persistent expression could be found in various tubular, glomerular and vascular structures of the graft during a latent period (without a positive CMV finding in blood or urine) several weeks or even months after an active infection. We found CMV antigen expression and, especially, CMV DNA in tubular epithelial cells, in endothelial cells as well as in interstitial inflammatory cells in the kidney allograft. In our study, the persistence of CMV was associated with histopathological features of CAN, recorded as vascular intimal thickening.

Several growth factors are thought to be important in the development of CAN. TGF-ß is secreted by monocytes/macrophages and mesangial cells and it directly stimulates the synthesis of extracellular matrix proteins and also prevents matrix degradation, resulting in accumulation of extracellular matrix [16]. It also triggers smooth muscle cells to proliferate and is suggested to play a role in the development of atherosclerosis [16]. TGF-ß1 is thought to be a key molecule in the development of CAN [5]. In vitro, CMV immediate early gene product IE2 has been shown to directly induce the transcription and secretion of TGF-ß1 [7]. Furthermore, CMV has been shown to induce the production of tumour necrosis factor-{alpha} (TNF-{alpha}) and IL-1 in monocytes and macrophages [17,18] and these proinflammatory cytokines are known to stimulate the synthesis and release of growth factors TGF-ß and PDGF. In an experimental rat model of chronic kidney allograft rejection, RCMV was shown to increase the expression of TGF-ß, PDGF and connective tissue growth factor in the graft. RCMV was also shown to enhance collagen synthesis and generation of fibrosis in rat renal allografts [2]. In our recent study, we found that the urinary excretion of TGF-ß1 was increased during CMV infection and that this was associated with increased fibrosis in 6-month protocol biopsies (unpublished data). In the present study, increased expression of TGF-ß1 was seen in vascular endothelial cells and less markedly in tubular epithelial cells in patients with persistent CMV infection in the kidney allograft.

PDGF exists as a disulphide-linked dimer of two polypeptide chains (A and B), which can form three isoforms (PDGF-AA, -BB and -AB). PDGF is an important stimulating growth factor for smooth muscle cells and fibroblasts and has also been suggested to play a role in the development of CAN [6]. PDGF-AA is expressed by intimal and medial smooth muscle cells in arteries in chronic renal vascular rejection, whereas PDGF-BB is expressed mostly in infiltrating monocytes [19]. PDGF-AA is also thought to play a role in intimal proliferation in atherosclerosis [20]. At a molecular level, CMV has been shown to increase the production of PDGF in vitro [8]. In our study, increased expression of PDGF-AA was recorded in tubular epithelial cells and also somewhat in the arterial endothelium, in biopsies of patients with a persistent CMV infection in the allograft. Expression of PDGF-BB was scarce and seen mostly in the infiltrating inflammatory cells.

Adhesion molecules, such as ICAM-1, play a key role in the extravasation of inflammatory cells from the bloodstream through capillary walls at the site of inflammation. They are also involved in cell–cell interactions and T-cell activation and are shown to be important in the early phase of the alloresponse in the transplanted organ [11]. Cytokines, such as IL-1 and TNF-{alpha}, directly induced by CMV [17,18], are known to induce the production of adhesion molecules, such as ICAM-1. In experimental rat models of chronic renal allograft rejection, RCMV increased the expression of ICAM-1 in the process of accelerated development of chronic rejection [9]. In our study, increased expression of ICAM-1 was seen in peritubular capillaries in patients with persistent CMV infection in the graft.

Acute rejection is considered as the most important risk factor for the development of CAN [1]. Increased expression of ICAM-1 and PDGF-AA is also seen during acute rejection [11,19]. None of the biopsies analysed in this study showed any signs of acute rejection and none of the patients had had acute rejection episodes during the last 3 months before the biopsy. In our study, previous acute rejection episodes did not have any impact on the histological changes seen or on the expression of TGF-ß1, PDGF-AA or ICAM-1 in recipients with persistent CMV infection in the kidney allograft. Thus, the changes seen in our study were not due to previous acute rejection episodes.

The focus of this study was to analyse the impact of persistent CMV infection in the kidney allograft on the expression of molecules thought to be important in the development of CAN and on the histological changes, compared with controls with no evidence of CMV infection at any time-point. Recipients with previous CMV infection, but no CMV found in the graft after the systemic infection (non-persistent CMV), showed slightly increased expression of TGF-ß1 and PDGF-AA, but to a lesser extent than in patients with persistent CMV infection. Expression of ICAM-1 also was increased significantly in recipients with non-persistent CMV infection. However, on average, these biopsies in the non-persistent CMV group were obtained later than those in the persistent CMV and control groups. No histological changes were recorded in those patients with previous transient CMV infection. Transient CMV infection might also have a direct impact on the graft due to the inflammatory response to infection, in addition to all other injuries to the graft. However, we suggest that persistent CMV infection causes long-term increased expression of cytokines and growth factors, which could be associated with increased vasculopathic changes, as seen in this study and also in our recent study [4].

The results of our study support the hypothesis that the increased expression of ICAM-1 in peritubular capillaries, associated with persistent CMV infection in the kidney allograft, might result in an increased number of inflammatory cells capable of producing various cytokines, such as TNF-{alpha} and IL-1, also induced directly by CMV. We hypothesize that indirectly via these cytokines, but also directly, CMV infection may stimulate the production of TGF-ß and PDGF-AA, which are known to stimulate the proliferation and migration of smooth muscle cells, resulting in the vasculopathic and fibrotic changes seen in CAN.

In conclusion, the results of the present study show that persistent CMV infection in the kidney allograft is associated with increased expression of TGF-ß, PDGF-AA and ICAM-1, molecules thought to be important in the development of CAN. We suggest that the proinflammatory and profibrotic effect of chronic CMV infection in the kidney allograft might be one mechanism by which CMV accelerates the development of CAN, recorded in this study as increased vascular wall thickening. However, this small retrospective study is not sufficient to answer all the questions about the possible association between persistent CMV infection and CAN or the mechanisms involved. Further studies with larger numbers of patients are needed to show the association of CMV and CAN.



   Acknowledgments
 
This study was supported by grants from Biomedicum Helsinki Foundation, Kidney Foundation Finland, the Finnish Medical Society Duodecim, the Finnish Society of Transplant Surgeons and from Helsinki University Hospital Research Funds (EVO).

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 2. 7.04
Accepted in revised form: 1.12.04





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