The discovery of neurologic disease associated with the human retro viruses HIV and HTLV- I has presented an urgent need to understand the mechanisms involved in retro virus invasion and damage of the CNS. A favored hypothesis for retro virus-induced neuropathogenesis is that neuronal damage occurs by an indirect mechanism. Nitric oxide (NO), a potent cell signaling molecule and mediator of cytotoxicity, has been implicated as an effector of neurotoxicity. To investigate the role of NO in retro virus-induced neurologic disease, the investigators will employ a polytropic murine retro virus model that includes three coisogenic viruses: Fr98, EC and EC-1. Each virus infects the CNS, by only Fr98 and EC are neurovirulent and cause a disease characterized by imbalance and ataxia in infected mice. Preliminary studies have demonstrated that Fr98 infection in mice results in up regulation of the proinflammatory cytokines IL-I and TNF-in the CNS. These cytokines are known to up-regulate inducible nitric oxide synthase (iNOS) expression with concomitant production of NO, suggesting a potential role for iNOS and NO in murine retro virus- induced neuropathogenesis. Thus, this murine model provides a unique opportunity to examine iNOS expression and NO production during retro virus infection, and to determine whether this response correlates with neurovirulence. The overall goal of this project is to investigate the role of NO in the pathogenesis of retro virus-induced CNS disease.
Specific Aim 1 will characterize cytokine and iNOS expression in the CNS of mice infected with the highly neurovirulent retro virus Fr98. A comparative analysis of iNOS expression in response to infection by neurovirulent (Fr98 and EQ and non-neurovirulent (EC-1) viruses will be conducted in Specific Aim 2 to determine whether iNOS expression correlates with neurovirulence in vivo.
In Specific Aim 3 an in vitro system using primary mixed glial cell cultures will be developed to further examine the NO response to retro virus infection. In addition to conventional NO detection methods, this study will employ novel NO probes developed as part of Project 1 of this NIH IDeA COBRE proposal to quantify NO and examine the kinetics of NO production in glial cell cultures infected with neurovirulent or non-neurovirulent viruses. Finally, Specific Aim 4 will measure the ability of conditioned medium from the infected glial cell cultures to induce neurotoxicity in an in vitro neuronal cell death assay, and determine whether inhibition of iNOS expression can ameliorate this activity.
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