Rat Cytomegalovirus Infection Research Articles

Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation.

BackgroundCongenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells.Objectives and MethodsIn order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments.ResultsRat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b– lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1.ConclusionIn line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental, those putative double-edge swords events actually play.

The Role of Alloresponsive Ly49+NK Cells in Rat Islet Allograft Failure in the Presence and Absence of Cytomegalovirus

There are still many factors to discover to explain the low success rates of islet allografts. In this study, we demonstrate that specific subpopulations of alloreactive NK cells may be involved in the failure of islet allografts. By performing allotransplantation in rats (n = 13), we observed peripheral expansion and infiltration of alloreactive Ly49i2(+) NK cells in the grafts. An effective strategy in rats to enhance the expansion of Ly49i2(+) NK cells is performing a rat cytomegalovirus infection (n = 6). Cytomegalovirus infection was associated with an early expansion of the Ly49i2(+) NK cells and accelerated islet graft failure. The Ly49i2(+) NK cells are both alloreactive and involved in virus clearance. The expansion of this subpopulation could not be blocked by cyclosporin A immunosuppression. Also alloreactive KLRH1(+) NK cells infiltrated the grafts, but nonalloreactive NKR-P1B(+) cells were not observed in the islet allografts. Perforin staining of the infiltrating NK cells demonstrated the cytotoxic capacity of these cells. Our data suggest a role for this NK subpopulation in rat islet allograft destruction.

Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4(+) T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.

Effects of rat cytomegalovirus on the nervous system of the early rat embryo

The purpose of the study was to investigate the impact of rat cytomegalovirus (RCMV) infection on the development of the nervous system in rat embryos, and to evaluate the involvement of Wnt signaling pathway key molecules and the downstream gene neurogenin 1 (Ngn1) in RCMV infected neural stem cells (NSCs). Infection and control groups were established, each containing 20 pregnant Wistar rats. Rats in the infection group were inoculated with RCMV by intraperitoneal injection on the first day of pregnancy. Rat E20 embryos were taken to evaluate the teratogenic rate. NSCs were isolated from E13 embryos, and maintained in vitro. We found: 1) Poor fetal development was found in the infection group with low survival and high malformation rates. 2) The proliferation and differentiation of NSCs were affected. In the infection group, NSCs proliferated more slowly and had a lower neurosphere formation rate than the control. The differentiation ratio from NSCs to neurons and glial cells was significantly different from that of the control, showed by immunofluorescence staining. 3) Ngn1 mRNA expression and the nuclear β-catenin protein level were significantly lower than the control on day 2 when NSCs differentiated. 4) The Morris water maze test was performed on 4-week pups, and the infected rats were found worse in learning and memory ability. In a summary, RCMV infection caused abnormalities in the rat embryonic nervous system, significantly inhibited NSC proliferation and differentiation, and inhibited the expression of key molecules in the Wnt/β-catenin signaling pathway so as to affect NSCs differentiation. This may be an important mechanism by which RCMV causes embryonic nervous system abnormalities.

Relevance of maintenance triple‐drug immunosuppression to bridle the amplification of rat cytomegalovirus infection after experimental lung transplantation

Immunosuppressive therapy required to treat rejection after lung transplantation (LTx) contributes significantly to the pathogenesis of cytomegalovirus (CMV) infection and disease. In a weak allogeneic left LTx model in the rat (Fisher 344 [F344] to Wistar Kyoto [WKY] rats) we analyzed the influence of acute CMV infection on postoperative day (POD) 3, with application of standard triple-drug immunosuppression (TD-IS) (cyclosporin A, azathioprine, prednisolone) on late outcome after LTx. Native right lungs and syngeneic grafts (WKY to WKY) served as controls. Rats were sacrificed on POD 15, 30, 60, and 100. TD-IS completely prevented acute and chronic rejection in non-infected rats. Allografts of CMV-infected rats treated with TD-IS showed only mild perivascular infiltrations in 6/10 rats (POD 15 and 30), which persisted up to POD 100 in 4/10 rats. In the long-term course, mild isolated interstitial and alveolar changes were found in 40% of these animals. In conclusion, rat CMV infection partially neutralized the immunosuppressive effect of TD-IS. However, an amplification of CMV infection under TD-IS can be controlled and does not result in fatal outcome.

Rat Pancreatic β Cells and Cytomegalovirus Infection

Cytomegalovirus (CMV) infection has been suggested to accelerate beta-cell destruction and thereby to contribute to new-onset diabetes and failure of islet allografts in both humans and rodents. Surprisingly, direct CMV infection of beta cells has received only minor attention. Therefore, we investigated the susceptibility of rat beta cells for rat CMV (RCMV) infection and the direct effects on the regulation of immune cell-activating ligands. Primary rat beta cells, the rat beta-cell line Rin-m5F, and fibroblasts were RCMV-infected in vitro. The viral gene and protein expression levels were determined as a measure for RCMV susceptibility. Gene expression levels of intracellular adhesion molecule 1, lymphocyte function associated antigen 3, rat major histocompatibility complex region A, rat major histocompatibility complex region E, toll like receptor 2, and clustered domain 14 were determined as a measure for cellular immunogenicity. We demonstrate that beta cells are susceptible for RCMV infection but allow only low levels of viral gene expression. In contrast, infected fibroblasts demonstrated productive viral infection and formation of viral progeny. After RCMV infection, beta-cell immunogenicity was markedly increased, as demonstrated by the increased cellular expression of immune cell-activating ligands. Direct beta-cell infection by RCMV and subsequent low-grade viral gene expression may lead to increased immunogenicity of native or transplanted beta cells in vivo. An infection-induced enhanced beta-cell recognizability may have important consequences for beta-cell survival and the development of diabetes or rejection of islet grafts.

Rat cytomegalovirus infection depletes MHC II in bone marrow derived dendritic cells

While cytomegalovirus (CMV) infects and replicates in a multitude of cell types, the ability of the virus to replicate in antigen presenting cells (APCs) is believed to play a critical role in the viral dissemination and latency . CMV infection of APCs and manipulation of their function are important areas of investigation. CMV down regulation of MHC II is reportedly mediated by the HCMV proteins US2, US3, UL83, UL111a (vIL10) or through the induction of cellular IL10. In this study, we demonstrate that rat CMV (RCMV) significantly reduces MHC II expression neither by mechanisms that do not involve orthologues of the known HCMV genes nor by an increase in cellular IL10. Rat bone marrow derived dendritic cells (BMDC) were highly susceptible to infection with RCMV and a recombinant RCMV expressing eGFP. RCMV infection of BMDCs depleted both surface and intracellular MHC II to nearly undetectable levels as well as reduced surface expression of MHC I. The effect on MHC II only occurred in the infected GFP positive cells and is mediated by an immediate early or early viral gene product. Furthermore, treatment of uninfected immature DCs with virus-free conditioned supernatants from infected cells failed to down regulate MHC II. RCMV depletion of MHC II was sensitive to treatment with lysosomal inhibitors but not proteasomal inhibitors suggesting that the mechanism of RCMV-mediated down regulation of MHC II occurs through endocytic degradation. Since RCMV does not encode homologues of US2, US3, UL83 or UL111a, these data indicate a novel mechanism for RCMV depletion of MHC II.

Cytomegalovirus Evasion of Innate Immunity by Subversion of the NKR-P1B:Clr-b Missing-Self Axis

Cytomegaloviruses are known to encode several gene products that function to subvert MHC-dependent immune recognition. Here we characterize a rat cytomegalovirus (RCMV) C-type lectin-like (RCTL) gene product with homology to the Clr ligands for the NKR-P1 receptors. RCMV infection rapidly extinguished host Clr-b expression, thereby sensitizing infected cells to killing by natural killer (NK) cells. However, the RCTL protein functioned as a decoy ligand to protect infected cells from NK killing via direct interaction with the NKR-P1B inhibitory receptor. In vivo, an RCTL mutant virus displayed diminished virulence in an NK-dependent and strain-specific manner, suggesting that host NKR-P1 polymorphisms have evolved to avert the viral decoy mechanism while maintaining Clr-b recognition to preserve self tolerance. These findings reveal a unique strategy adopted by cytomegaloviruses to evade MHC-independent self-nonself discrimination. The existence of lectin-like genes in several poxviruses suggests that this may represent a common theme for viral evasion of innate immunity.

Cytomegalovirus Accelerates Chronic Allograft Nephropathy in a Rat Renal Transplant Model With Associated Provocative Chemokine Profiles

Background Studies have shown that rat cytomegalovirus (RCMV) infection accelerates transplant vascular sclerosis (TVS) in rat heart and small bowel allotransplants. In these models, RCMV-accelerated TVS results from increased graft infiltration of inflammatory cells through up-regulation of chemokine expression. The aim of this study was to determine if RCMV infection accelerates renal transplant chronic allograft nephropathy (CAN), and the role of chemokines in this process. Methods F344 kidneys were transplanted into Lewis recipients with and without RCMV infection. To monitor CAN, serum creatinine (Cr) levels were measured starting at 4 weeks posttransplantation. At 7 and 21 days, and at terminal rejection, grafts were examined for histologic changes, inflammatory cell infiltrates, viral load, and chemokine expression profiles. Results By week 8, serum Cr showed significant elevation ( P < .01) in the RCMV-infected group vs uninfected group, and remained significantly elevated through the end of the study. RCMV+ renal allografts had significant inflammatory cell infiltration and increased CAN at postoperative day (POD) 28. The CC chemokines RANTES, MCP-1, and MIP-1α, and the CXC chemokine IP-10 were up-regulated in RCMV-infected vs uninfected allografts. IP-10 was significantly up-regulated early in the process, whereas RANTES and MCP-1 were induced at a later time. Conclusions RCMV infection accelerates CAN, with associated graft inflammatory infiltrates, which is paralleled by an increase in expression of CC and CXC chemokines. Our findings suggest that the early induction of IP-10 in the infected allografts promotes alterations in T-cell and monocyte migration to the graft, which initiates accelerated inflammatory and fibrotic changes associated with CAN.

Rat Cytomegalovirus Infection Interferes with Anti-CD4 mAb-(RIB 5/2) Mediated Tolerance and Induces Chronic Allograft Damage

In order to assess the role of heterologous immunity on tolerance induction (TI) by signal 1 modification, the influence of rat cytomegalovirus infection (RCMVI) on TI by a non-depleting monoclonal anti-CD4 mAb (monoclonal antibody) (RIB 5/2) in a rat kidney transplant (KTx) model was investigated. Orthotopic rat KTx (Dark Agouty (DA)-->Lewis (LEW)) was performed after TI with RIB 5/2 [10 mg/kg body weight (BW); day -1, 0, 1, 2, 3; i.p. (intraperitoneal route)]. RCMVI (5x10E5 Plaque forming units [PFU] i.p.) was simultaneously conducted to KTx, 50 days after KTx, and 14 days before and after KTx. RIB 5/2 induced robust allograft tolerance even across the high-responder strain barrier. RCMVI broke RIB 5/2-induced tolerance regardless of the time of RCMVI but did not induce acute graft failure during the 120 days follow-up. RCMVI induced a significant chronic deterioration of allograft function (p<0.01) and enhanced morphological signs of chronic allograft damage (p<0.05). Cellular infiltrates and major histo-compatibility complex (MHC)-expression were more pronounced (p<0.05) in the infected groups. RCMVI induced not only RCMV-specific T-cell response but also enhanced the frequency of alloreactive T cells. RCMV interferes with anti-CD4 mAb-induced tolerance and leads to chronic allograft damage. The data we presented suggest a potentially important role of viral infections and their prophylaxis in clinical TI protocols.

Cytomegalovirus-enhanced development of transplant arteriosclerosis in the rat; effect of timing of infection and recipient responsiveness

Cytomegalovirus (CMV) is put forward as a risk factor for transplant arteriosclerosis (TA). In this article, we studied CMV-enhanced development of TA in rats in different donor/recipient combinations in relation to the timing of infection. Recipient rats transplanted with an aortic allograft (BN to Lew) were infected with rat CMV (RCMV) at different time-points relative to transplantation. The virus-induced effects on TA development were also determined in other strain combinations (PVG to AO and DA to WF). Finally, transmission of RCMV from aortic grafts and its effect on TA was studied. RCMV infection enhanced TA development only in Lew recipients and only after infection early post-transplantation (days 1-5). Virus transmission to the recipient only occurred from 5 and 10 days infected aortic donor-grafts, however without affecting TA development. These data indicate that the acute alloresponse and acute CMV infection need to occur simultaneously to enhance TA. This effect, however, appears to be strain combination dependent and therefore cannot be generalized.

Cytomegalovirus aggravates intimal hyperplasia in rats by stimulating smooth muscle cell proliferation

Epidemiological and animal studies suggest a role for cytomegalovirus (CMV) in restenosis. Previously, we demonstrated that proliferating smooth muscle cells (SMCs) in the injured arterial wall are particularly susceptible to CMV-induced effects. Therefore, we hypothesised that, depending on the time point of infection after vascular injury, CMV infection may affect cell proliferation either in the media or in the neointima, thereby aggravating the process of restenosis. In the present study, we focused on the individual layers of the arterial wall by evaluating, besides the neointima-to-media ratio, the medial and neointimal area and cellularity in the rat femoral artery. Vascular injury was photochemically induced in rat femoral arteries. Immediately or 14 days thereafter, rats were infected with rat CMV (RCMV) or mock infected. The presence of RCMV in the vascular wall was determined at 3, 5, 14 and 35 days after infection by quantitative real-time PCR. When rats were infected immediately after injury, a significant increase was seen only in the medial but not in the neointimal cross-sectional area. On the other hand, when rats were infected 14 days after the initial injury, a significant increase was only seen in the neointimal area, thereby confirming our hypothesis that RCMV infection primary affects proliferating SMCs. As the mean number of SMCs per μm 2 in both cell layers was unchanged despite an increase in cross-sectional area, this implies that RCMV stimulated SMC proliferation. Furthermore, these vascular effects were observed without the virus being abundantly present in the vascular wall, suggesting that inflammatory and immune-mediated responses to RCMV infection are more important in aggravating the response to vascular injury than the virus itself.

The r131 gene of rat cytomegalovirus encodes a proinflammatory CC chemokine homolog which is essential for the production of infectious virus in the salivary glands.

Rat cytomegalovirus (RCMV) possesses two adjacent genes, r131 and r129, which have the potential to encode CC chemokine homologs. Interestingly, the amino acid sequences encoded by both genes show similarity to the sequence of the murine CMV (MCMV) MCK-2 protein, which is encoded by the m131/129 gene. In order to study the significance of the r131 gene in the pathogenesis of RCMV infection, we generated two different virus strains in which the r131 open reading frame is disrupted. Replication of these null mutant strains, designated RCMVdeltar131a and RCMVdeltar131b, was evaluated in vitro and in vivo. Both strains were found to replicate with a similar efficiency as wild-type (WT) RCMV in vitro. However, in contrast to WT virus, neither RCMVdeltar131a nor RCMVdeltar131b established a high-titer infection in the salivary glands of immunocompromised rats. Furthermore, in a local, rat footpad infection model, both recombinant viruses induced a significantly lower amount of paw swelling than did WT RCMV. Also, a higher number of infiltrating macrophages was observed in paws infected with WT RCMV than in those infected with the recombinants. Taken together, these results suggest that r131 (i) promotes inflammation at initial sites of inoculation and, subsequently, efficient virus dissemination to or infection of the salivary glands and (ii) might be involved in the persistence of virus infection, at least in the spleen. In addition, our data indicate that r131 represents the functional homolog of the MCMV m131/129 gene.

Platelet-derived growth factor regulates cytomegalovirus infection-enhanced obliterative bronchiolitis in rat tracheal allografts.

Cytomegalovirus (CMV) infection is a risk factor for the development of obliterative bronchiolitis (OB) after lung transplantation. In the rat tracheal allograft model, rat CMV (RCMV) infection is associated with accelerated OB through enhanced alloimmune activation and increased smooth muscle cell (SMC) proliferation. Using this model, we investigated the role of platelet-derived growth factor (PDGF) in RCMV infection-enhanced OB. Immunohistochemistry and in situ hybridization revealed that RCMV infection significantly up-regulates PDGF ligand and receptor expression in inflammatory and SMC-like cells in tracheal allografts. Selective inhibition of PDGF receptor tyrosine kinase activity by CGP 53716 prevents the development of OB in RCMV-infected allograft recipients. The results of this study emphasize the key regulatory role of PDGF in the pathogenesis of RCMV infection-enhanced OB, suggesting a novel strategy for the prevention of this fibroproliferative disorder.

Infection with rat cytomegalovirus (CMV) in the immunocompromised host is associated with the appearance of a T cell population with reduced CD8 and T cell receptor (TCR) expression.

Infection with human cytomegalovirus (HCMV) mostly results in a chronic subclinical infection; the immune system is unable to eliminate the virus and is apparently in equilibrium with the persistent virus. In the immunosuppressed host this equilibrium is disturbed, resulting in clinical infection. Rat cytomegalovirus (RCMV) infection in its host can be used as a model for HCMV infection. Using flow cytometry we examined the effect of acute RCMV infection on the composition of leucocyte subsets in the peripheral blood of both immunocompetent and immunosuppressed (5 Gy total body irradiation) Lewis rats. Special attention was paid to the natural killer (NK) cells and the CD8+ T cells known to be involved in the control of viral infections. Furthermore, we determined the presence of leucocyte subsets in the internal organs by immunohistochemistry. In immunocompetent rats, infection caused a small increase in NK cells and a large increase in CD8+ T cells. In contrast, infection of immunosuppressed rats caused a marked increase in NK cells and a small increase in CD8+ T cells, consisting of T cells with reduced expression of both CD8 and TCR. This phenomenon is characteristic of anergic CD8+ T cells, possibly explaining the ability of the virus to escape elimination by the immune system. The increase of NK cells in the peripheral blood of immunosuppressed, RCMV-infected rats could also be detected in kidney, liver, lung and pancreas, but not in salivary gland. This could explain the long persistence of infectious virus in the salivary gland.

Induction of cyclo-oxygenase-2 by acute liver allograft rejection and cytomegalovirus infection in the rat.

Cytomegalovirus (CMV) infection has been shown to increase inflammation in rat liver allografts. In-vitro CMV has been shown to transactivate cyclo-oxygenase-2 (COX-2), while COX-2 plays a role in the CMV replication cycle. Our aim was to investigate the expression of COX-2 in liver allograft rejection and concomitant CMV infection. Expression of COX-2 was studied immunohistologically in rat liver allografts with or without rat CMV infection, in isografts, and in normal rat liver. There were small amounts of COX-2-positive mononuclear inflammatory cells in the normal liver and isografts. Acute rejection increased the amount of COX-2-expressing cells in the portal areas only, whereas concomitant CMV infection did this also in the sinusoid area. COX-2 may play a role in CMV infection in vivo as well. The possible role of COX-2 in the association between CMV infection and allograft rejection warrants further study.

Elimination of donor-specific alloreactivity prevents cytomegalovirus-accelerated chronic rejection in rat small bowel and heart transplants.

The primary cause for late failure of vascularized allografts is chronic rejection (CR) characterized by transplant vascular sclerosis (TVS). Cytomegalovirus (CMV) infection accelerates TVS and CR by unclear mechanisms involving direct effects of CMV, indirect effects of the recipient's immune response to CMV, or interactions between CMV and the recipient's alloreactivity. This study examined the role of CMV and the alloreactive response in the development of TVS using bone marrow chimerism (BMC) in rat small bowel (SB) and heart transplantation models. Fisher 344 (F344) rat heart or SB grafts were transplanted into F344/Lewis bone marrow chimera. F344 heart or SB grafts transplanted into Lewis recipients (low-dose cyclosporine) were positive controls for the development of TVS. Lewis heart or SB grafts transplanted into Lewis recipients (+/-cyclosporine) were transplantation controls. The effect of rat CMV (RCMV) (5x105 plaque-forming units) on TVS (neointimal index, NI) and graft survival was studied in these groups. RCMV infection was assessed by serologic analysis and quantitative polymerase chain reaction techniques (TaqMan). RCMV infection accelerated the time to graft CR (SB 70-38 days; hearts 90-45 days) and increased the severity of TVS in both the SB allografts (day 38, NI=27 vs. 52) and the heart allografts (day 45, NI=43 vs. 83). Grafts from CMV-infected syngeneic recipients failed to develop TVS and CR. Donor-specific tolerance induced by BMC prevented allograft TVS and CR in both transplant models. In contrast to naïve Lewis recipients, RMCV infection failed to cause allograft TVS and CR in bone marrow (BM) chimeras. The events in CMV-induced acceleration of TVS involve a crucial interplay between CMV infection and the recipient's alloreactive immune response.

Rat cytomegalovirus open reading frame R44 is an early-late gene that encodes a nuclear protein.

Rat cytomegalovirus (RCMV) open reading frame R44 is the homolog of human cytomegalovirus gene UL44, which encodes the DNA polymerase accessory protein. Here, we show that R44 is transcribed as a 3.6-kb mRNA within the early and late phases of infection in vitro. In order to find potential monoclonal antibodies (MoAbs) directed against the R44-encoded protein (pR44), a panel of anti-RCMV MoAbs was screened for binding to pR44 recombinant proteins. Thus, an anti-pR44 MoAb, termed RCMV8, was identified. By using this MoAb, pR44 could be detected as early as 8 h after RCMV infection in vitro. The pR44 protein was determined to have a molecular mass of approximately 55 kDa and was found to be localized to the nucleus of RCMV-infected cells.

Prevention of cytomegalovirus infection-enhanced experimental obliterative bronchiolitis by antiviral prophylaxis or immunosuppression in rat tracheal allografts.

In this study, the prevention of rat cytomegalovirus (RCMV) infection-enhanced experimental obliterative bronchiolitis in rat tracheal allografts was investigated. RCMV infection markedly enhanced cell proliferation and histological changes of obliterative bronchiolitis, a form of chronic rejection after lung transplantation. These alterations were linked to increased interleukin (IL)-2 and tumor necrosis factor-alpha (TNF-alpha) immunoreactivity, and reduction of IL-10 expression. In recipient rats with acute RCMV infection, prophylaxis with either ganciclovir (DHPG) or hyperimmune serum (HIS) totally prevented RCMV infection-enhanced tracheal occlusion. DHPG treatment initiated during acute RCMV infection also reduced lesion development but markedly less than DHPG prophylaxis. Treatment of acute RCMV infection with HIS alone or in combination with DHPG had no significant effect on tracheal occlusion. Inhibition of the transcription of cytokines by high doses of cyclosporine A significantly reduced RCMV infection-enhanced tracheal obliteration. In rats with chronic RCMV infection, obliterative alterations were prevented by DHPG prophylaxis initiated at the time of transplantation. Prophylaxis either with DHPG or HIS did not affect the amount of infectious RCMV recovered from host salivary glands, nor were there differences seen in RCMV major immediate early DNA expression in tracheal allografts between different antiviral drug regimens. Immunohistochemical analysis of allografts revealed that inhibition of tracheal occlusion by antiviral prophylaxis was associated with a reduction in the number of ED1(+) macrophages and cells staining for Th1 cytokines and TNF-alpha, while immune modulation by cyclosporine A up-regulated IL-10 production. In conclusion, the results of the present study suggest that the CMV infection-enhanced chronic rejection develops independently of viral load but requires both immune activation and simultaneous CMV gene expression beyond immediate early genes.

Induction of Vascular Adhesion Protein-1 during Liver Allograft Rejection and Concomitant Cytomegalovirus Infection in Rats

Vascular adhesion protein-1 (VAP-1) is an adhesion molecule controlling lymphocyte recirculation through high endothelial venules of the lymph nodes. It has also been shown to be induced and to mediate lymphocyte adhesion at sites of inflammation. We studied the expression of VAP-1 and two other inducible adhesion molecules ICAM-1 and VCAM-1 in our experimental model of rat liver allograft rejection and, in addition, the effect of concomitant rat cytomegalovirus (RCMV) infection on this expression. Expression of VAP-1, ICAM-1, and VCAM-1 was studied in rat liver allografts with or without RCMV infection, isografts, and normal rat liver. Immunoperoxidase technique and monoclonal antibodies including a novel anti-VAP-1 reagent were used. VAP-1 expression was induced by acute rejection in sinusoids, hepatocytes, and also in bile ducts, when compared to the isografts or normal liver, where only blood vessels were consistently positive. Sinusoidal and hepatocyte expression of VAP-1 was prolonged by the presence of RCMV. ICAM-1 and VCAM-1 expression was also induced by acute rejection. However, RCMV increased sinusoidal VCAM-1 expression compared to uninfected grafts. The present experimental study shows that VAP-1 is up-regulated in acute rejection of liver allografts, and that this up-regulation is prolonged by RCMV infection.