Daniel M. Miller

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.

Viral Inhibition of Interferon Signal Transduction

The type I and II interferons (IFNs) are potent stimulators of antigen processing and presentation and are essential in antiviral immunity. IFNs upregulate the transcription of major histocompatibility complex (MHC) class I and II molecules, associated antigen-processing proteins, and induce the production of direct antiviral effector molecules such as 2′,5′-oligoadenylate synthetase, double-stranded-RNA-dependent protein kinase and Mx proteins. It is increasingly evident that viruses have evolved mechanisms to globally inhibit the actions of IFNs through disruption of their signal transduction pathways. Herein, we review the ability and novel mechanisms of several diverse viruses to inhibit IFN-induced JAK/STAT signal transduction.

Outcomes Of Transconjunctival Sutureless 25-gauge Vitrectomy With Silicone Oil Infusion

Purpose: To evaluate the outcomes and complications of surgical management with 25-gauge pars plana vitrectomy (PPV) and silicone oil (SO) tamponade in complex vitreoretinal diseases. Methods: Retrospective review of a consecutive, interventional case series at a single center. Results: Thirty-five eyes of 35 patients were included in the study. The indications for vitrectomy included tractional retinal detachment (11 eyes), macular hole (6 eyes), proliferative vitreoretinopathy or recurrent retinal detachment (9 eyes), neovascular glaucoma (3 eyes), giant retinal tear (3 eyes), and pathologic myopia with epiretinal membrane or macular hole (3 eyes). All patients underwent 25-gauge PPV with either 1,000-centistoke (n = 31) or 5,000-centistoke (n = 4) SO tamponade infused through a 24-gauge angiocatheter. No intraoperative complications were noted. The median preoperative visual acuity was counting fingers (range, 20/50 to light perception). The median postoperative visual acuity after a median follow-up of 6 months (range, 1–19 months) was 20/200 (range, 20/30 to light perception). A small subconjunctival SO bleb was identified in two patients. Recurrent retinal detachment occurred in three patients. No significant complications relating to the use of SO in the setting of 25-gauge PPV occurred. Conclusions: Advances in 25-gauge PPV instrumentation have enabled expanding indications for 25-gauge PPV. 25-Gauge PPV with SO tamponade is safe and efficient and can be considered in the surgical management of complex vitreoretinal disease.

PRIMARY REPAIR OF RETINAL DETACHMENT WITH 25-GAUGE PARS PLANA VITRECTOMY

Purpose: To evaluate 25-gauge pars plana vitrectomy (PPV) for primary repair of rhegmatogenous retinal detachment (RRD). Study Design and Participants: This retrospective, consecutive case series included 42 eyes of 41 patients who underwent primary repair of RRD utilizing transconjunctival 25-gauge PPV without scleral buckling at the Cincinnati Eye Institute from July 2004 through January 2007. Methods: The medical records were retrospectively reviewed, and the corresponding demographic data, preoperative ophthalmic diagnoses, surgical management, and postoperative course and treatment were recorded. Main outcome measures included single surgery anatomical success, preoperative and postoperative visual acuity, and complications. Results: Most patients had pseudophakic RRD (36 [85.7%] of 42 eyes). The crystalline lens was present in the remaining 6 eyes (14.3%). Of 42 eyes, 28 (66.7%) had macula-on RRD, while 14 (33.3%) had macula-off RRD. Four surgeons contributed to this study, and 25-gauge PPV instrumentation, a wide-angle viewing system, endolaser photocoagulation, and gas tamponade were used in each case. The single surgery anatomical success rate was 92.9% (39 of 42 eyes). For eyes with macula-on RRD, best-corrected visual acuity was 20/50 (0.43 logMAR [logarithm of the minimum angle of resolution]) preoperatively and 20/30 (0.23 logMAR) postoperatively (P = 0.24). For eyes with macula-off RRD, best-corrected visual acuity was 5/200 (1.56 logMAR) preoperatively and 20/30 (0.23 logMAR) postoperatively (P = 0.001). Three eyes required additional surgery for final reattachment. Final reattachment was achieved in 100% of patients (mean follow-up, 8 months). Conclusions: Twenty-five–gauge PPV with laser retinopexy and gas tamponade is effective for primary repair of RRD. The single operation anatomical success rate is comparable with rates reported for primary vitrectomy with 20-gauge instrumentation, scleral buckling, and combined vitrectomy/scleral buckling.

Human Cytomegalovirus Disrupts Constitutive MHC Class II Expression

CD8+ and CD4+ T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.

Viral effects on antigen processing

Viruses have evolved numerous mechanisms that modulate MHC-mediated antigen presentation, which in turn protect infected cells from T-lymphocyte-mediated immunosurveillance. Recent studies of previously identified viral immunomodulatory proteins reveal the allelic specificity of these proteins, their ability to function in xenogeneic systems and the difficulty in translating in vitro data to in vivo models; moreover, new mechanisms of viral modulation of MHC expression have emerged.

Treatment of diabetic macular edema with an inhibitor of vascular endothelial-protein tyrosine phosphatase that activates Tie2

PURPOSE AKB-9778 is a small-molecule competitive inhibitor of vascular endothelial-protein tyrosine phosphatase (VE-PTP) that promotes Tie2 activation and reduces vascular leakage and neovascularization in mouse models. The purpose of this study was to test the safety, tolerability, pharmacokinetics, and biological activity of AKB-9778 in patients with diabetic macular edema (DME). DESIGN Open-label, dose-escalation clinical trial. PARTICIPANTS Four dose cohorts of 6 patients with DME self-administered subcutaneous injections of 5 mg, 15 mg, 22.5 mg, or 30 mg AKB-9778 twice daily for 4 weeks. METHODS Patients were seen weekly during a 4-week treatment period for safety assessments, best-corrected visual acuity (BCVA) assessment by Early Treatment Diabetic Retinopathy Study protocol, and measurement of central subfield thickness (CST) by spectral-domain optical coherence tomography. Additional safety assessments were performed at 6, 8, and 12 weeks. MAIN OUTCOME MEASURES Safety assessments, change from baseline BCVA, and change from baseline CST. RESULTS All doses were well tolerated. A modest, transient reduction in blood pressure and adverse events consistent with vasodilatory activity of AKB-9778 emerged at doses of 22.5 mg or more twice daily. At the week 4 primary end point, BCVA improved 5 letters or more from baseline in 13 of the 18 patients receiving 15 mg or more twice daily; 1 patient improved by 10 to 15 letters, and 2 patients improved by more than 15 letters. Among 18 patients receiving 15 mg or more twice daily, CST decreased by more than 100 μm in 5 patients and by 50 to 100 μm in 2 patients. There was a significant correlation between reduction in CST and improvement in BCVA. CONCLUSIONS No safety concerns were identified after systemic administration of AKB-9778 for 4 weeks in patients with DME, and doses of 15 mg or more twice daily reduced macular edema and improved vision in some patients. This is a preliminary demonstration of clinical safety and efficacy of a VE-PTP inhibitor and Tie2 activator.

Cytomegalovirus induces sialyl Lewis(x) and Lewis(x) on human endothelial cells.

BACKGROUND Cytomegalovirus (CMV) is the primary viral cause of complications in transplant recipients. We sought to understand the mechanisms of its dissemination and induction of vascular disease, which may lead to transplant complications. Sialyl Lewis(x) (sLe(x)) and Lewis(x) (Le(x)) are known for their roles in mediating cell adhesion and as tumor-associated carbohydrate antigens. Herein we explore whether CMV induces surface expression of these important molecules in endothelial cells (EC). METHODS Flow cytometry was used to detect surface expression of sLe(x) and Le(x) on CMV-infected human umbilical vein endothelial cells (HUVEC), with or without ultraviolet inactivation of the virus. To elucidate mechanisms of CMV-mediated induction, mRNA coding for predominant HUVEC sialyltransferases (ST) and fucosyltransferases (FT), key enzymes in sLe(x) and Le(x) synthesis, was analyzed by Northern blot. Dual immunohistochemical staining for sLe(x) and Le(x) expression of human colon and placental tissue was performed to investigate in vivo relevance. RESULTS sLe(x) expression on CMV-infected HUVEC was strongly up-regulated by 8 days after inoculation. Le(x) expression was detectable earlier and increased steadily over time. In contrast, ultraviolet-inactivated CMV did not induce expression of these molecules. Northern blot assays demonstrated higher levels of important EC glycosyltransferases ST-IV, FT-III, and FT-IV in CMV-infected EC. Finally, high levels of sLe(x) and Le(x) were expressed in CMV-infected EC in vivo. CONCLUSIONS Given the known biologic functions of sLe(x) and Le(x), we suggest that CMV induction of these molecules may have widespread consequences ranging from CMV dissemination to induction of CMV-associated vascular disease, including thrombosis.

Human Cytomegalovirus Inhibition of Major Histocompatibility Complex Transcription and Interferon Signal Transduction

Pathogens have evolved diverse mechanisms for escaping host innate and adaptive immunity. Viruses that maintain a persistent infection are particularly effective at disabling key arms of the host immune response. For example, the herpesviruses establish a persistent infection in human and animal hosts, in part through critical immunoevasive strategies. Cytomegalovirus, a beta-herpesvirus, impairs major histocompatibility complex (MHC) class I and class II antigen presentation by decreasing MHC expression on the surface of the infected cell, thus enabling infected cells to escape CD8+ and CD4+ T lymphocyte immunosurveillance. Moreover, cytomegalovirus blocks the interferon signal transduction pathway, thereby limiting the direct and indirect antiviral effects of the interferons. In this review, we focus on an emerging paradigm in which the effectiveness of viruses, particularly human cytomegalovirus, to escape antiviral immune responses is significantly enhanced by their ability to inhibit MHC transcription and interferon (IFN)-stimulated (JAK/STAT) signal transduction.