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Background: Cytomegalovirus (CMV) viremia that is resistant or refractory to the standard antiviral therapy still constitutes a major threat to high-risk transplant recipients. In addition, multiple CMV recurrences may lead to neutropenia because of repeated courses of therapy with ganciclovir derivatives. Leflunomide, a drug for rheumatoid arthritis, has been reported to have anti-CMV activity. This study reports on its use in 17 transplant recipients with complex CMV syndromes who had failed or were intolerant to other therapies., Methods: Single-center, retrospective study. Clinical data were extracted from the electronic medical record. CMV DNA viral loads were performed by quantitative hybrid capture assay., Results: Leflunomide was initiated after a median of three episodes of CMV viremia, with a mean peak viral load of 245,826 copies/mL. Initial clearance of CMV viremia was observed in 14 of 17 patients (82%), and 9 of 17 (53%) patients achieved a long-term suppression of CMV recurrences. Higher peak viral load and higher viral load at the start of leflunomide therapy were associated with failure to suppress viremia. The duration of leflunomide therapy ranged from 1 to 24 months (median 3.5 months, interquartile range 2.6-7 months), and the mean time to an undetectable CMV-DNA was 1.9 months. Adverse effects included diarrhea (35%), anemia (18%), and increased liver function tests (12%)., Conclusions: Leflunomide, alone or in combination, has potential utility in treatment of complex CMV syndromes and in long-term suppression of viremia. The optimal duration of therapy and the balance of risks and benefits are not yet known.

Background: FK778 is a malononitrilamide, a class of immune suppressive compounds with antiviral features and experimental activity in chronic rejection, a potentially interesting combination for organ transplantation. The goal of this project was to study the tolerability, immune suppressive efficacy, and anti-cytomegalovirus (CMV) activity of FK778 and to assess the in vivo relevance of its previously described inhibition of de novo pyrimidine synthesis., Methods: Heart transplants were performed in rats (Brown Norway [BN] to Lewis) and treated with varying doses of FK778 or leflunomide for 28 days. At 28 days, at the time of rejection or at the death of the animal, the allograft and other vital organs were obtained for study by light microscopy and immunohistochemistry. In separate experiments, Lewis rats were given sublethal irradiation, inoculated with rat CMV (Maastricht strain), and treated with varying doses of FK778 and leflunomide. In both the transplant and CMV studies, IP uridine was given at 250 mg/kg to cohorts or animals receiving FK778 and leflunomide., Results: FK778 controls acute rejection and inhibits CMV replication at 20 mg/kg but is toxic at 25 mg/kg. Toxicity is manifested as anemia, changes in hepatic and intestinal histology, and mortality. The toxicity but not the immune suppressive or antiviral efficacy, is reduced significantly by exogenous uridine administration., Conclusion: FK778 has both immune suppressive and antiviral activities, neither of which is entirely dependent on inhibition of pyrimidine synthesis. These, and other published observations, suggest that the antiviral activity and a considerable part of the efficacy of the malononitrilamide family of drugs is attributable to activities other than drug induced pyrimidine deficiency.

Background: Cellular immunity is the reputed mechanism of lung allograft failure. Humoral immunity is not a commonly recognized pathway., Material and Methods: We describe 22 patients who developed a posttransplantation septal capillary injury syndrome in the absence of panel-reactive antibodies. Factor VIII levels served as an index of microvascular injury. Routine light microscopic studies were performed in a total of 73 biopsies; 54 biopsy specimens were analyzed for deposition of C1q, C4d, C5b-9, and immunoglobulin (IgG, IgM, and IgA). Indirect immunofluorescent testing to assess for antiendothelial cell antibodies was performed using patient serum and human pulmonary microvascular endothelial cell cultures as substrate. Control samples were based on patients who were clinically well at the time of the biopsy., Results: All presented with a deterioration in respiratory function. All patients had elevated factor VIII levels; the levels were significantly greater compared with pretransplantation baseline values (P =<0.03). The biopsy specimens were remarkable for septal capillary necrosis with significant septal capillary deposition of C1q, C3, C4d, and/or C5b-9 along with immunoglobulin, including IgG, with variable endothelial cell localization. The degree of septal capillary necrosis was significantly less in posttransplantation patients who were clinically doing well ( P<0.0001) as was the degree of C1q, C3, C4d, and C5b-9 ( P<0.05). Indirect antiendothelial cell antibody studies were positive in most patients. Treatment interventions included plasmapheresis, resulting in functional improvement: the postpheresis biopsy specimens showed a reduction in both the degree of septal capillary injury (P <0.0003) and the amount of C1q, C3, C4d, and C5b-9 deposition (P <0.05)., Conclusions: Septal capillary injury accompanied by direct and indirect immunofluorescent evidence of humoral immunity is a frequent finding on transbronchial biopsies. The findings suggest that humoral immunity to endothelial-based alloantigen is a common occurrence in lung grafts and may be a critical factor in chronic graft dysfunction.

Background: Cytomegalovirus (CMV), a common infectious complication in transplant recipients, has been implicated as an exacerbating agent in the development of transplant vascular sclerosis (TVS); however, mechanisms defining this role remain to be fully resolved. Our previous studies suggest that CMV-infected graft endothelial cells (EC) can initiate a host T-cell activation cascade, and that cytokines produced as a consequence enhance graft endothelial alloimmunogenicity. Because antiviral therapy has not uniformly attenuated CMV-associated risk of TVS development, we have tested the hypothesis that antiviral agents do not affect T-cell activation by allogeneic CMV-infected EC., Methods: Human umbilical vein EC, inoculated with CMV VHL/E and incubated in the presence or absence of ganciclovir (GCV) or foscarnet (PFA), were cocultured with CMV-seropositive or -seronegative donor-derived T cells in the presence of these agents, then labeled with [(3)H]thymidine. Cocultures were harvested, and radiolabel incorporation was assayed by scintillation counting., Results: Limiting dilution analysis demonstrated that proliferation frequencies of CMV-seropositive donor-derived T cells in response to CMV-infected EC (approximately 80 cells/106, compared with approximately 4 cells/10(6) in response to uninfected EC) were not significantly modified by GCV (approximately 86 cells/10(6)) or PFA (approximately 82 cells/10(6)). Likewise, T-cell proliferation curves generated in response to stimulator cell titrations were essentially identical regardless of drug treatment., Conclusions: Results of these experiments suggest that although these drugs limit CMV replication and dissemination, they do not attenuate the inflammatory potential of infected EC, a force that may be a major factor in CMV-mediated exacerbation of the development of TVS.

Background: Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective in the prevention/control of acute and chronic rejection in animal models and currently in phase I clinical trials in human transplant recipients. This agent is also effective in the control of graft-versus-host disease, autoimmune reactions, and the growth of certain tumors. The importance of the endothelium in these disease processes led us to hypothesize that leflunomide might act directly upon the endothelial cell (EC)., Methods and Results: Assay of human EC colony formation demonstrated dose-dependent, leflunomide-mediated inhibition of EC proliferation. In addition, the organization of EC into capillary-like networks, which occurs during 18 hr of incubation on Matrigel, was progressively disrupted with increasing concentrations of leflunomide. Finally, fibrin-embedded transverse sections of murine aorta, which sprout numerous microvessels during an 11-day incubation, were inhibited from doing so in the presence of this agent. All drug concentrations used in these experiments were nontoxic and pharmacologically relevant, and none of these effects were reversible by exogenous uridine, implying that inhibition of these processes was not due to intracellular pyrimidine depletion. Furthermore, neither cyclosporine nor tacrolimus exerted inhibitory activity in any of the experiments described above., Conclusions: Data generated by these studies distinguish leflunomide among immunosuppressants as uniquely capable of inhibiting angiogenesis-related endothelial functions and suggest additional mechanisms by which this agent might intervene in the diverse array of disease processes against which it has shown therapeutic potential.

Background: Despite advances in antiviral chemotherapy, herpes simplex virus type 1 (HSV-1), continues to complicate the clinical course of many allograft recipients. We have previously demonstrated that the experimental immunosuppressive agent leflunomide inhibits production of cytomegalovirus by interference with virion assembly. We test the hypothesis that this agent exerts similar antiviral activity against HSV-1., Methods and Results: Plaque assay of virus yield from endothelial or Vero cells after inoculation with each of four clinical HSV-1 isolates demonstrated a dose-dependent reduction of virus production in the presence of pharmacologic concentrations of A77 1726, the active metabolite of leflunomide. DNA dot blot and biochemical assay of viral DNA polymerase activity indicated that A77 does not inhibit viral DNA synthesis. Rather, as visualized by transmission electron microscopic method, this agent seems to disrupt virion assembly by preventing nucleocapsid tegumentation., Conclusions: These findings, in demonstrating that leflunomide exerts antiviral activity against HSV-1 by mechanisms similar to those we have previously shown with cytomegalovirus, imply that this agent may possess broad spectrum activity against other herpesviruses.

Background: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression., Results: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination., Conclusions: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.

Background: Cytomegalovirus (CMV), a betaherpesvirus associated with allograft rejection, infects the endothelium, the cellular interface between allograft tissue and the host immune system. Because of recent appreciation of the phenotypic diversity of endothelial cells (EC) from different vascular compartments, controversy now exists on the universality of CMV-mediated adhesion molecule induction previously described on umbilical vein EC. Therefore, we herein extend these previous studies to arterial and microvascular EC, which represent sites of vascular rejection., Methods: Human coronary artery, aortic, umbilical artery, and microvascular EC were mock or CMV infected and/or treated with tumor necrosis factor-a before flow cytometric and immunohistochemical analysis., Results: CMV directly enhanced intercellular adhesion molecule-1 on all EC isolates but did not induce E-selectin or vascular cell adhesion molecule-1. Furthermore, CMV-infected EC were refractory to tumor necrosis factor-alpha-mediated induction of these molecules., Conclusion: CMV-induced modulations of adhesion molecule expression, which may affect allograft immunogenicity, seem common to all EC regardless of vascular origin.

Background: Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed organ transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic allograft rejection in animal models. Because a number of CMV proteins are known to be phosphorylated, we tested the hypothesis that this agent might exert inhibitory activity against CMV., Methods and Results: Plaque assays demonstrated dramatic dose-dependent attenuation of production of multiple clinical CMV isolates in leflunomide-treated human fibroblasts and endothelial cells, common targets for CMV infection in vivo. As shown by Northern blot analysis and immunohistochemical staining, leflunomide neither interferes with transcription of immediate early or late viral genes, nor with expression of corresponding proteins. CMV-specific DNA dot blots and biochemical enzyme assays indicated that, in contrast to currently approved anti-CMV drugs, leflunomide exerts no inhibitory effect on the accumulation of viral DNA in infected cells, or on viral DNA polymerase activity. Rather, as visualized by transmission electron microscopy, this agent appears to act at a late stage in virion assembly by preventing tegument acquisition by viral nucleocapsids. Finally we have demonstrated equivalent inhibitory activity of leflunomide against multi-drug-resistant CMV isolates., Conclusions: These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

Background: Cytomegalovirus (CMV) has been implicated as an exacerbating agent in the development of transplant vascular sclerosis; however, specific etiologic mechanisms remain unresolved. Based upon our previous observations that CMV-infected endothelial cells (ECs) stimulate proliferation and cytokine production by allogeneic T cells, we now test the hypothesis that CMV-driven cytolytic activity may contribute to graft endothelial injury., Methods: Limiting dilutions of CMV-seropositive or -seronegative donor-derived T cells were stimulated with CMV-infected or uninfected allogeneic ECs in the presence of interleukin-2. T-cell proliferation was monitored by assay of [3H]thymidine incorporation and stimulated T cells were tested for lytic activity against CMV-infected or uninfected radiolabeled EC targets by 51Cr release assay. Natural killer (NK) cell activity was examined by incubating freshly isolated peripheral blood mononuclear cells with 51Cr-labeled targets, followed by assay of radiolabel release., Results: CMV-infected ECs were resistant to T cell- and NK-mediated cytolysis regardless of donor serostatus, nature of stimulation, or level of T-cell proliferation. In contrast, although uninfected ECs were unharmed by NK cells, these targets experienced significant lysis by T cells stimulated with either uninfected or CMV-infected ECs., Conclusions: These results implicate CMV-infected graft endothelium as a persistent source of infectious virus, a chronic stimulus for potentially destructive host inflammatory activity, and a potential trigger for the generation of lytic injury to uninfected bystander endothelia, suggesting multiple mechanisms by which this virus might perturb equilibrium at the graft/host interface.

Human cytomegalovirus (CMV) has been associated with allograft rejection and, in particular, with transplant-associated arteriosclerosis. However, the role CMV plays in the development of transplant-associated arteriosclerosis remains unclear. CMV can infect the endothelium, the interface between allograft tissue and the host immune cells, but the direct induction of endothelial human leukocyte antigen (HLA) class II by CMV remains controversial. Our previous studies with venous endothelial cells (EC) have shown that CMV does not directly induce this antigen on infected EC and, furthermore, renders these cells refractory to interferon (IFN)-gamma induction. However, questions have arisen regarding the relevance of these findings to arterial endothelia. Thus, we have extended these studies to determine whether similar interactions occur in arterial EC. EC derived from human coronary artery, aorta, and umbilical artery were assayed by immunofluorescence flow cytometry and dual immunohistochemical staining following IFN-gamma treatment and/or inoculation with CMV. Data generated by these experiments demonstrate that regardless of vascular origin: (1) CMV does not directly induce endothelial surface or cytoplasmic HLA class II, and (2) although uninfected arterial EC are HLA class II inducible by IFN-gamma, infected cells are completely refractory to this effect. These results suggest that CMV-mediated inhibition of HLA class II expression is a phenomenon shared by human arterial and venous endothelia of both fetal and adult origin.

We have investigated the contribution of monocytes (Mo) to the activation of purified human T cells by allogeneic human vascular endothelial cells (HUVEC). We have previously demonstrated that allogeneic HUVEC stimulate IL-2 production by CD8+ helper T lymphocytes (HTL), but not CD4+ HTL, in the absence of accessory Mo. We now show that addition of responder-autologous Mo to such cultures stimulates a high frequency of CD4+ HTL (1/6500), but no additional CD8+ HTL (1/35,000), as detected by limiting dilution analysis (LDA). The CD4+ HTL production of IL-2 increased with increasing numbers of Mo. Monoclonal antibodies to MHC class II interfered with HTL responses to allogeneic HUVEC in the presence, but not in the absence of autologous Mo. In contrast, IFN-gamma-treated HUVEC stimulated a high frequency of CD4+ HTL in the absence of autologous Mo. However, deletion experiments revealed that the population of HTL responsive to IFN-gamma-treated HUVEC is distinct from the population that responds to HUVEC in the presence of autologous Mo. These data suggest that Mo promote IL-2 production by presenting HUVEC-derived alloantigens via MHC class II molecules to CD4+ HTL, rather than by providing cytokines that promote more efficient IL-2 production by CD8+ HTL, or by inducing MHC class II expression on the HUVEC. In general, these data demonstrate that autologous Mo can play a significant role in the response of T cells to allogeneic HUVEC. Further, they demonstrate that the activation of human HTL by allogeneic HUVEC is complex, and can occur by at least three pathways: (1) direct stimulation of a small number of CD8+ HTL, but no CD4+ HTL, by quiescent HUVEC, (2) direct stimulation of a large number of CD4+ HTL by IFN-gamma-treated HUVEC, and (3) indirect stimulation of a different subset of CD4+ HTL by HUVEC in the presence of autologous monocytes. These three pathways of alloactivation are not unique to allogeneic HUVEC, but this experimental system provides a convenient and relevant model with which each pathway can be easily and independently investigated.

CMV has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system. Although endothelial HLA class II expression is a hallmark of vascular rejection, CMV does not directly induce these antigens on infected endothelial cells (EC) and, indeed, renders them refractory to HLA DR induction by IFN-gamma. Our earlier studies have demonstrated, however, that CMV-infected EC are capable of eliciting vigorous activation responses by allogeneic, CMV-seropositive donor-derived CD4+ T cells. We now show that T cells thus activated can induce HLA DR expression on noninfected EC. Human umbilical vein endothelial cells (HUVEC) were inoculated at low titer with CMV strain VHL/E, cocultured with allogeneic, CMV-seropositive or CMV-seronegative donor-derived CD4+ T cells, then dual immunohistochemically stained for CMV antigen and HLA DR, or assayed for HLA DR expression by fluorescence flow cytometry. Results demonstrated that HLA DR was induced in 20-70% of HUVEC in monolayers containing less than 0.5% CMV-infected EC after coculture with CMV-seropositive donor-derived T cells. No such induction was observed in experiments employing T cells isolated from CMV-seronegative individuals. Expression of this antigen was strictly limited to noninfected cells. Rare HLA DR induction was observed in virus-free cocultures. To determine whether endothelial HLA DR was induced by a soluble factor(s) elaborated by activated T cells, noninfected HUVEC monolayers were treated for 72 hr with cell-free supernatant from CMV-infected or noninfected HUVEC/T cell cocultures and assayed as above. Again, HLA DR expression was induced by supernatant from CMV-positive cocultures (only when cocultured T cells were isolated from CMV-seropositive donors), but not by supernatant from CMV-negative cocultures or from T cells cultured alone. The soluble factor was identified as IFN-gamma by the complete attenuation of HLA DR induction by anti-IFN-gamma mAb. These findings suggest that allograft endothelium harboring low level CMV may be capable of eliciting host CD4+ T cell activation, and that consequent release of IFN-gamma is capable of inducing endothelial HLA class II expression, as observed in vascular rejection and transplantation-associated arteriosclerosis. Results of these studies thus provide insight into mechanisms that may help elucidate the association between CMV and rejection-related immune events in the allograft.

Cytomegalovirus, a source of serious complications among immunosuppressed individuals, infects endothelial cells in vivo, and has been epidemiologically associated with allograft rejection and transplantation-associated accelerated arteriosclerosis (TxAA). As the interface between the host immune system and allograft tissues, the endothelium may be of primary importance in mediating pathogenic immune responses to CMV. We have therefore investigated T lymphocyte responses to CMV-infected allogeneic human umbilical vein endothelial cells (HUVE) in vitro. Proliferation assays demonstrated dramatically enhanced responses by CMV-seropositive donor-derived PBMC or purified T cells cocultured with CMV-infected HUVE, as compared with those elicited by noninfected stimulator cells. As determined by limiting dilution analysis of IL-2-producing cells, the frequency of T cells responding to infected HUVE was generally found to exceed by approximately one order of magnitude those responding to uninfected cells. Similar analyses of isolated T cell subsets revealed that these responses (proliferation and IL-2 production) were nearly entirely accountable to the CD4+ fraction. Responses of CD8+ populations, however, varied among donors. The marked activation of CD4+ cells is particularly intriguing since we have shown that CMV-infected HUVE do not express HLA class II antigens. Responses of lymphocytes derived from CMV-seronegative donors were minimal in all assays. These studies show that purified T cells can respond to CMV in the exclusive context of allogeneic endothelial cells. Furthermore, we demonstrate the heretofore undescribed phenomenon of CD4+ T cell activation in the absence of serologically detectable HLA class II antigen. As vascular rejection and TxAA are characterized by subendothelial T cell infiltration, these findings support a role for CMV/endothelial interaction in their pathogenesis.

Cytomegalovirus infection in the renal allograft recipient has been associated with the initiation of acute rejection. The mechanism of this induction is unknown. It may be related to renal tubular epithelial and endothelial expression of HLA class II antigens or a CMV immediate-early antigen that exhibits immunologic crossreactivity with HLA DR. In this study the ability of CMV to both infect and subsequently induce class II antigen expression on cultured human umbilical-vein endothelial cells (HUVEs), in the absence of cytokines, particularly gamma interferon, was tested. Individual HUVE cell lines were first proven to express HLA class II antigens in the presence of 10, 100, and 200 units of recombinant IFN-gamma as early as 24 hr postincubation by an immunohistochemical technique and by flow cytometry. These cell lines were successfully infected with CMV strains AD169 and CMV3 as determined by the presence of early and late viral antigens and CMV DNA. The degree of infection was dose and incubation-time dependent. Infection of HUVEs with these CMV strains and a nonattenuated clinical isolate failed to induce HLA DR, DP, or DQw1 expression in the absence of IFN-gamma. These findings support the hypothesis that endothelial cells in vivo may serve as reservoirs of CMV infection. They do not support the hypothesis that CMV produces an immediate-early antigen that has immunologic cross-reactivity with HLA DR. Furthermore, there is no support for the hypothesis that CMV independently induces HLA class II antigens in the absence of IFN-gamma.