Project #3: In the central nervous system, HIV-1 induced disorders are coupled with dysregulation of cytokines as a result of their abnormal pattern of expression. Among these cytokines, Tumor Necrosis Factor a (TNFa), has captured much attention due to the positive feedback interplay between the HIV-1 regulatory protein, Tat, and the downstream executors of the TNFa signaling pathway, including NFKB transcription factors. Expression of TNFa can also be dysregulated by opiates suggesting that cooperativity between HIV-1 and opiates at the molecular level may influence the neuropathogenesis of AIDS in drug-addicted patients. However, the molecular mechanism involved in the activation of TNFa upon HIV-1 infection in the context of drugs of abuse such as morphine remains unknown. Our preliminary results support the possible involvement of the p65 subunit of NFKB and p38 MARK signaling in this event. Activation of TNFa can promote oxidative stress in cells which, in turn, initiates a positive feedback cascade of events that further enhances TNFa production. Morphine has been shown to induce oxidative stress via an unknown pathway leading us to envision a role for TNFa in morphine-induced oxidative stress during the course of viral infection. Together with activated TNFa, the HIV-1 regulatory protein (Tat) and the envelope protein (gp120) can exert a toxic effect on neurons causing neuronal injury and death and morphine can accelerate this by affecting several key regulatory events that control neuronal cell survival. We plan to identify the mechanism by which morphine and HIV-1 infection upregulate TNFa gene expression and to determine the signaling events that are involved in Tat, gp120, and TNFa induced neuronal cell dysfunction. In the context of this program project, we will be able to develop and utilize, in parallel, cell culture models from HIV-1 and SIV-1 infected macrophages from human and monkey to unravel the various regulatory pathways that are affected upon drug treatment and viral infection, and cross examine the biological relevance of the in vitro cell culture findings to the results from the experimental SIV/macaque model that is the central focus of this program. Through such an integrated synergism with the other participants of this program, the outcome of these molecular studies will provide critical information that can be translated toward the development and use of therapeutic approaches in addicted AIDS patients.