Deborah A. Knight

Novel mechanism of inhibition of cytomegalovirus by the experimental immunosuppressive agent leflunomide.

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.

Inhibition of Cytomegalovirus in vitro and in vivo by the Experimental Immunosuppressive Agent Leflunomide

Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed 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 rejection in animal models. Herein we summarize our recent studies demonstrating that leflunomide inhibits the production of multiple clinical CMV isolates (including multi-drug-resistant virus) in both human fibroblasts and endothelial cells. In contrast to all other anti-CMV drugs currently in use, leflunomide does not inhibit viral DNA synthesis, but rather appears to interfere with virion assembly. Finally, preliminary studies in a rat model suggest that this agent reduces viral load in vivo. 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.

Use of C4d as a Diagnostic Adjunct in Lung Allograft Biopsies

Purpose: Humoral allograft rejection is a defined mechanism for cardiac and renal graft dysfunction; C4d deposition, a stable component of complement activation, inversely correlates with graft survival. With the recent recognition of humoral rejection in lung grafts, we examined C4ds role as a prognostic adjunct in lung allografts. Material and Methods: Twenty‐three lung recipients underwent biopsies for deterioration in clinical status or routine surveillance. Clinically unwell patients possessed acute rejection or bronchiolitis obliterans syndrome (BOS). Biopsies attributable to infection were excluded from the study. In addition to routine light microscopy, an attempt was made to correlate the clinical status and morphologic findings with the pattern of C4d deposition and also to compare these clinical and morphologic parameters with the other assessed immunoreactants. Panel reactive antibody testing was also carried out at various points in their post transplantation course whereby in 6 of the cases the samples were procured at exactly the same time as the tissue samples. Results: The patients were segregated into two groups: those patients with recurrent acute rejection and those with BOS. In those patients with symptomatic acute rejection, all biopsies showed light microscopic and immunofluorescent evidence of humoral allograft rejection. The level of C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal capillary necrosis. In addition, high and intermediate levels of C4d correlated with a clinical diagnosis of acute rejection. C4d was the strongest predictor of parenchymal injury and of the clinical status (p < .0001) compared to other the immunoreactants C1q, C5b‐9 and immunoglobulin. There was no specific correlation between C4d deposition and the presence of acute cellular rejection. In those patients fulfilling clinical criteria of BOS, deposits of C4d as well as other immunoreactants were found in the bronchial wall as opposed to the rarity of this finding in bon‐BOS patients. However the only statistically significant predictor of BOS was bronchial wall deposition of C1q. In no case were panel reactive antibodies at significant levels discovered post transplantation. Conclusions: In the context of acute rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection assessed pathologically; there is no association with the presence of histocompatibility related antibodies. It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition within the bronchial wall is a feature of BOS and hence may be used as a marker of chronic graft dysfunction. The antigenic target resulting in C4d deposition may not be histocompatibility related.

Inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide

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.

Concurrent Antiviral and Immunosuppressive Activities of Leflunomide In Vivo

We previously reported that the immunosuppressive malononitrileamides leflunomide and FK778 exert antiviral activity against cytomegalovirus (CMV). In the current investigation, we tested the hypothesis that leflunomide exerts concurrent antiviral activity and immune suppression in CMV‐infected cardiac allograft recipients. Lewis rats were transplanted with Brown Norway hearts and then inoculated with rat CMV. Plaque assay demonstrated that leflunomide (30 mg/kg/day) reduced viral loads by 4–6 logs, and that the reduction in viral load was unaffected by administration of uridine. Leflunomide was as effective as cyclosporine A (CsA) or tacrolimus in preservation of allograft integrity through day 28. These studies directly demonstrate the bifunctionality of leflunomide as concurrently immunosuppressive and antiviral, enhancing the promise of this agent as a clinical option for treatment of transplant recipients.

Evidence That Humoral Allograft Rejection in Lung Transplant Patients Is Not Histocompatibility Antigen‐Related

We have recently recognized humoral rejection (HR) in lung allograft recipients and its association with acute and chronic graft dysfunction. We have shown that C4d, a stable marker of classic complement activation, is deposited in lung allografts, correlating with clinical rejection and parenchymal injury. The antigenic target may be endothelium in the setting of recurrent acute rejection while varying components of the bronchial wall may be important in chronic graft dysfunction. We sought to establish whether there is a role for antibodies with histocompatibility antigen specificity in the lung humoral allograft phenomenon. Flow cytometric and ELISA assays to assess donor‐specific antigens were conducted on sera from 25 lung transplant recipients who had experienced one or more episodes of clinical rejection; in addition, the serum samples were tested for evidence of antiendothelial cell antibody activity. Morphologically, each case had biopsies showing septal capillary injury with significant deposits of immunoreactants with microvascular localization and positive indirect immunofluorescent antiendothelial cell antibody assay. Panel‐reactive antibody testing showed absence of MHC Class I/II alloantibodies; ELISA based crossmatch detecting donor‐specific MHC Class I/II specific antibodies was negative. HR can occur in the absence of antibodies with HLA specificity; antigenic targets may be of endothelial cell origin.

Cytomegalovirus-mediated modulation of adhesion molecule expression by human arterial and microvascular endothelial cells

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.

In vitro induction of endothelial HLA class II antigen expression by cytomegalovirus-activated CD4+ T cells.

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-γ. 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 do

Minimal intestinal epithelial cell toxicity in response to short- and long-term food-relevant inorganic nanoparticle exposure.

Toxicity of commercial nanoparticles of titania, silica, and zinc oxides is being investigated in this in vitro study. Particles of these compositions are found in many food items, and thus this study is directed toward particle behavior in simulated digestion media and their interaction with intestinal epithelial cell line C2BBe1, a clone of Caco-2 cells, originally isolated from a human colon cancer. Even though the primary particle size of all three particles was below 50 nm, the particles appeared as aggregates in culture media with a negatively charged surface. In the presence of pepsin (pH 2), the charge on the titania became positive, and silica was almost neutral and aggregated extensively, whereas ZnO dissolved. For silica and titania, treatment with simulated intestinal digestive solution led to a strongly negatively charged surface and particle sizes approaching values similar to those in media. On the basis of infrared spectroscopy, we concluded that the surface of silica and titania was covered with bile salts/proteins after this treatment. Transmission electron microscopy indicated that the C2BBe1 cells internalized all three particles. Toxicity assays included investigation of necrosis, apoptosis, membrane damage, and mitochondrial activity. Titania and SiO₂ particles suspended in media at loading levels of 10 μg/cm² exhibited no toxicity. With ZnO at the same loading level, mild toxicity was observed based only on the LDH assay and decrease of mitochondrial activity and not necrosis or apoptosis. Titania particles exposed to the simulated digestion media exhibited mild toxicity based on decrease of mitochondrial activity, likely due to transport of toxic bile salts via adsorption on the particle surface.

The Role of Anti–Endothelial Cell Antibody–Mediated Microvascular Injury in the Evolution of Pulmonary Fibrosis in the Setting of Collagen Vascular Disease

We encountered 16 patients with connective tissue disease in whom pulmonary fibrosis developed. Routine light microscopic, ultrastructural, and direct immunofluorescent analyses were conducted, and circulating antibodies, including those of endothelial cell derivation, were assessed using indirect immuno-fluorescence and Western blot assays. Underlying diseases were dermatomyositis, scleroderma, mixed connective tissue disease, sclerodermatomyositis, Sjögren syndrome, rheumatoid arthritis, and anti-Ro-associated systemic lupus erythematosus. Antibodies to one or more Ro, RNP, Jo 1, OJ, and/or nucleolar antigens were seen in all cases and antiphospholipid antibodies in half. All biopsies revealed microvascular injury in concert with intraparenchymal fibrosis; in some cases, there were corroborative ultrastructural findings of microvascular injury. Patterns of fibroplasia represented nonspecific interstitial pneumonitis and usual interstitial pneumonitis. We noted IgG, IgA, and/or complement in the septal microvasculature. In 6 cases with available serum samples, indirect immunofluorescent endothelial cell antibody studies were positive and Western Blot studies showed reactivity of serum samples to numerous endothelial cell lysate-derived proteins. Pulmonary fibrosis, a recognized complication of systemic connective tissue disease, develops in connective tissue disease syndromes with pathogenetically established immune-based microvascular injury at other sites. A similar mechanism of antibody-mediated endothelial cell injury may be the basis of the tissue injury and fibrosing reparative response.