Partha S. Biswas

Inhibition of Ocular Angiogenesis by siRNA Targeting Vascular Endothelial Growth Factor Pathway Genes : Therapeutic Strategy for Herpetic Stromal Keratitis

Ocular neovascularization often results in vision impairment. Frequently vascular endothelial cell growth factors (VEGFs) are mainly responsible for the pathological neovascularization as in the case in neovascularization induced by CpG oligodeoxynucleotides and herpes simplex virus infection in this report. siRNAs targeting either VEGFA, VEGFR1, VEGFR2, or a mix of the three were shown to significantly inhibit neovascularization induced by CpG when given locally or systemically. The efficacy of systemic administration was facilitated by the use of a polymer delivery vehicle. Additional experiments showed a significant inhibitory effect of the siRNAs mix when given either locally or systemically in vehicle against herpes simplex virus-induced angiogenesis as well as against lesions of stromal keratitis. These results indicate that the use of VEGF pathway-specific siRNAs represents a useful therapy against neovascularization-related eye diseases.

CXCR2−/− Mice Show Enhanced Susceptibility to Herpetic Stromal Keratitis: A Role for IL-6-Induced Neovascularization

Ocular infection with HSV results in a blinding immunoinflammatory lesion known as herpetic stromal keratitis (HSK). Early preclinical events include inflammatory cell, mainly neutrophils, infiltration of the stroma, and neovascularization. To further evaluate the role of neutrophils in pathogenesis, HSV infection was compared in BALB/c and mice of the same background, but lacking CXCR2, the receptor for chemokines involved in neutrophil recruitment. Our results show clear differences in the outcome of ocular HSV infection in CXCR2−/− compared with control BALB/c mice. Thus, CXCR2−/− animals had minimal PMN influx during the first 7 days postinfection, and this correlated with a longer duration of virus infection in the eye compared with BALB/c mice. The CXCR2−/− mice were also more susceptible to HSV-induced lesions and developed HSK upon exposure to a dose of HSV that was minimally pathogenic to BALB/c mice. The basis for the greater HSK lesion susceptibility of CXCR2−/− mice was associated with an elevated IL-6 response, which appeared in turn to induce the angiogenic factor, vascular endothelial growth factor. Our results serve to further demonstrate the critical role of angiogenesis in the pathogenesis of ocular lesions.

Mice Transgenic for IL-1 Receptor Antagonist Protein Are Resistant to Herpetic Stromal Keratitis: Possible Role for IL-1 in Herpetic Stromal Keratitis Pathogenesis

Ocular infection with HSV may result in the blinding immunoinflammatory lesion stromal keratitis (SK). This represents a CD4+ T cell-mediated immunopathologic lesion in both humans and a mouse model. Early events in the pathogenesis that set the stage for SK are poorly understood. The present study evaluates the role of IL-1 using a transgenic mouse that overexpresses the IL-1 receptor antagonist (IL-1ra) protein. Such transgenic mice were markedly resistant to SK compared with IL-1ra−/− and C57BL/6 control animals. The resistance was shown to be the consequence of reduced expression of molecules such as IL-6, macrophage-inflammatory protein-2, and vascular endothelial growth factor, normally up-regulated directly or indirectly by IL-1. A critical event impaired in IL-1ra transgenic mice was vascular endothelial growth factor production with a consequent marked reduction in angiogenesis, an essential step in SK pathogenesis. Targeting IL-1 could prove to be a worthwhile therapeutic approach to control SK, an important cause of human blindness.

Herpetic eye disease: immunopathogenesis and therapeutic measures.

Infection of the cornea with herpes simplex virus (HSV) can result in a chronic disease called herpetic stromal keratitis (HSK). The disease represents one of the leading causes of infectious blindness in the Western world. Immune-mediated cellular damage is suspected in the pathogenesis of human HSK. The murine model has been pivotal in further establishing HSK as an immunopathological disease. This article reviews understanding of HSK, both in humans and in the mouse model, with an emphasis on possible future therapeutic strategies to counteract this blinding immunoinflammatory disease.

Elucidating the protective and pathologic T cell species in the virus‐induced corneal immunoinflammatory condition herpetic stromal keratitis

Herpetic stromal keratitis (HSK) results in postinfection with Herpes simplex virus type 1 (HSV‐1). The pathogenesis involves tissue damage by the host immune system, classifying HSK as an immunopathological disease. The crucial disease orchestrating cells is thought to be the T lymphocytes. The present study elucidates pathogenic and protective T cell subsets involved in the development of HSK using the gBT mice, which possess a monoclonal population of CD8+ T cells reactive to a HSV immunodominant epitope. Results show that HSV‐reactive CD8+ T cells enter infected corneas during the acute but not the chronic phase of the disease during which the predominant population is CD4+ T cells. Adoptive transfer experiments in T and B cell‐deficient recombination‐activating gene knockout mice revealed that HSV‐reactive CD8+ T cells are capable of ocular virs clearance, possibly through a combination of corneal and peripheral nervous system antiviral effects, but are not involved in lesion development. CD4+ T cells of the virus‐specific or nonspecific species emerged as the pathogenic T cells capable of precipitating disease. These observations have the potential to yield important treatment strategies by targeting specific cell types in HSK.

Counteracting corneal immunoinflammatory lesion with interleukin‐1 receptor antagonist protein

Herpetic stromal keratitis (HSK) is a T cell‐orchestrated, immunoinflammatory lesion that results from corneal Herpes simplex virus infection. Previous reports indicate an essential role for proinflammatory cytokine interleukin (IL)‐1 in HSK pathogenesis. The present study evaluates the efficacy of IL‐1 receptor antagonist (IL‐1 ra) protein in the management of HSK. Mice receiving IL‐1 ra had diminished disease severity. The administration of IL‐1 ra was shown to reduce the influx into the cornea of cells of the innate and adaptive immune response. In addition, the treatment diminished corneal vascular endothelial growth factor levels, resulting in reduced angiogenic response. Our results show the importance of targeting early proinflammatory molecules such as IL‐1 to counteract HSK and advocate IL‐1 ra as an effective agent to achieve this.

Concomitant Helper Response Rescues Otherwise Low Avidity CD8+ Memory CTLs to Become Efficient Effectors In Vivo

This report seeks a means of maximizing memory CD8 T cell responses to peptide immunization. Delivery of the CD8 peptide epitope by stress protein, heat shock protein (hsp)70, results in excellent immunogenicity at the acute phase but memory responses were poor both in terms of the number of responding cells as well as their functional avidity. We demonstrate for the first time that hsp70 can also be used as a vehicle to achieve CD4 T cell responses to loaded peptide epitopes and that coimmunization with hsp70 loaded with both CD8 and CD4 peptide epitopes may increase memory up to 3-fold. Furthermore, CD8+ T cell memory responses were of higher avidity measured both by in vitro cytotoxicity assays and a new methodology that measures the avidity of CTL activity in vivo in mice. Our results emphasize that peptide immunization remains a viable approach to induce long-term CD8+ T cell function, providing steps are taken to assure appropriate stimulation of Th cell responses.

Role of Inflammatory Cytokine-Induced Cycloxygenase 2 in the Ocular Immunopathologic Disease Herpetic Stromal Keratitis

ABSTRACT Ocular infection with herpes simplex virus (HSV) results in a blinding immunoinflammatory stromal keratitis (SK) lesion. Early preclinical events include polymorphonuclear neutrophil (PMN) infiltration and neovascularization in the corneal stroma. We demonstrate here that HSV infection of the cornea results in the upregulation of the cycloxygenase 2 (COX-2) enzyme. Early after infection, COX-2 was produced from uninfected stromal fibroblasts as an indirect effect of virus infection. Subsequently, COX-2 may also be produced from other inflammatory cells that infiltrate the cornea. The induction of COX-2 is a critical event, since inhibition of COX-2 with a selective inhibitor was shown to reduce corneal angiogenesis and SK severity. The administration of a COX-2 inhibitor resulted in compromised PMN infiltration into the cornea, as well as diminished corneal vascular endothelial growth factor levels, likely accounting for the reduced angiogenic response. COX-2 stimulation by HSV infection represents a critical early event accessible for therapy and the control of SK severity.

Protective and pathological roles of virus-specific and bystander CD8+ T cells in herpetic stromal keratitis.

Herpetic stromal keratitis (HSK), resulting from corneal HSV-1 infection, represents a T cell-mediated immunopathologic lesion. In T cell transgenic mice on a SCID or RAG knockout background, the T cells mediating lesions are unreactive to viral Ags. In these bystander models, animals develop ocular lesions but are unable to control infection. Transfer of HSV-immune cells into a CD8+ T cell bystander model resulted in clearance of virus from eyes, animals survived, and lesions developed to greater severity. However, the adoptively transferred CD8+ T cells were not evident in lesions, although they were readily detectable in the lymphoid tissues as well as in the peripheral and CNS. Our results indicate that viral-induced tissue damage can be caused by bystander cells, but these fail to control infection. Immune CD8+ T cells trigger clearance of virus from the eye, but this appears to result by the T cells acting at sites distal to the cornea. A case is made that CD8+ T cell control is expressed in the trigeminal ganglion, serving to curtail a source of virus to the cornea.