Protection against TNFα-dependent liver toxicity by intraperitoneal liposome delivered DsiRNA targeting TNFα in vivo.

Tumor necrosis factor-alpha (TNFα) is a classic proinflammatory cytokine implicated in the pathogenesis of several autoimmune and inflammatory diseases including viral encephalitis. Macrophages being major producers of TNFα are thus attractive targets for in vivo RNA interference (RNAi) mediated down regulation of TNFα. The application of RNAi technology to in vivo models however presents obstacles, including rapid degradation of RNA duplexes in plasma, insufficient delivery to the target cell population and toxicity associated with intravenous administration of synthetic RNAs and carrier compounds. We exploited the phagocytic ability of macrophages for delivery of Dicer-substrate small interfering RNAs (DsiRNAs) targeting TNFα (DsiTNFα) by intraperitoneal administration of lipid-DsiRNA complexes that were efficiently taken up by peritoneal macrophages and other phagocytic cells. We report that DsiTNFα-lipid complexes delivered intraperitoneally altered the disease outcome in an acute sepsis model. Down-regulation of TNFα in peritoneal CD11b+ monocytes reduced liver damage in C57BL/6 mice and significantly delayed acute mortality in mice treated with low dose LPS plus d-galactosamine (D-GalN).

Association of simian virus 40 vp1 with 70-kilodalton heat shock proteins and viral tumor antigens.

Proper folding of newly synthesized viral proteins in the cytoplasm is a prerequisite for the formation of infectious virions. The major capsid protein Vp1 of simian virus 40 forms a series of disulfide-linked intermediates during folding and capsid formation. In addition, we report here that Vp1 is associated with cellular chaperones (HSP70) and a cochaperone (Hsp40) which can be coimmunoprecipitated with Vp1. Studies in vitro demonstrated the ATP-dependent interaction of Vp1 and cellular chaperones. Interestingly, viral cochaperones LT and ST were essential for stable interaction of HSP70 with the core Vp1 pentamer Vp1 (22-303). LT and ST also coimmunoprecipitated with Vp1 in vivo. In addition to these identified (co)chaperones, stable, covalently modified forms of Vp1 were identified for a folding-defective double mutant, C49A-C87A, and may represent a "trapped" assembly intermediate. By a truncation of the carboxyl arm of Vp1 to prevent the Vp1 folding from proceeding beyond pentamers, we detected several apparently modified Vp1 species, some of which were absent in cells transfected with the folding-defective mutant DNA. These results suggest that transient covalent interactions with known or unknown cellular and viral proteins are important in the assembly process.

Effects of CXCR3 signaling on development of fatal encephalitis and corneal and periocular skin disease in HSV-infected mice are mouse-strain dependent.

PURPOSE: The host inflammatory response to ocular infection with herpes simplex virus (HSV) can be either protective, with disease-free survival, or it can promote diseases such as HSV corneal disease (or herpes stromal keratitis [HSK] in humans) and encephalitis (HSE), depending on mouse strain. The role of CXCR3 chemokine signaling in HSV-induced central nervous system (CNS) inflammation and corneal disease was evaluated, and responses in genetically susceptible and resistant strains of mice were contrasted. METHODS: Resistant C57BL/6J (B6) and susceptible 129S6 (129) mice were given monoclonal antibodies (mAbs) to neutralize the CXCR3 ligands monokine induced by interferon-gamma (MIG, CXCL9) and interferon inducible protein-10 (IP-10, CXCL10) during HSV infection. In addition, the development of HSV disease was monitored in CXCR3-null mutant mice derived from resistant (B6) and susceptible (BALB/c) strains. Inflammatory cells infiltrating the cornea and brain stem were isolated and stained for flow cytometric analysis. RESULTS: MIG and IP-10 were induced in nervous system tissue after HSV inoculation by the corneal route. HSV-infected 129 mice treated with MIG- or IP-10-neutralizing mAbs showed significantly enhanced survival compared with mice treated with control isotype antibody, whereas survival of the B6 mice was unaltered. Similarly, greater survival was observed for BALB.CXCR3(-/-) mice compared with control BALB/c mice. Reduced CNS inflammation was documented that extended to the cornea, such that HSV corneal disease severity was reduced in susceptible BALB.CXCR3(-/-). In contrast, although survival of B6 and B6.CXCR3(-/-) mice was indistinguishable, B6.CXCR3(-/-) mice developed more severe corneal and periocular skin disease. CONCLUSIONS: The effects of CXCR3 signaling in HSV infection are strongly dependent on mouse strain.