Behavioral and cognitive dysfunctions are commonly observed in patients with AIDS known as dementia complex (ADC). Increased replication of HIV in brain cells is correlated with development of ADC. Thus, factors that can enhance HIV synthesis in brain are likely to contribute to ADC. Cocaine abuse is considered to be a contributing risk factor for AIDS, and is shown to increase infectious HIV production in monocytes; possibly by inducing transforming growth factor beta. Effects of neurotransmitters, brain hormones, and cocaine on the replication of HIV and synthesis of cytokines in resident microglia and macrophage are not well understood. Effects of some of these factors were studied on trans- activation of HIV promoter (LTR) as well as on the synthesis of cytokines in human fetal microglia derived from legally terminated pregnancies. When microglia were transfected with HIV-LTR and exposed to either substance-P (SP), beta endorphin or GP-120, they significantly enhanced the trans-activation of HIV-LTR (p>0.05). More over, SP and GP-120 induced the synthesis of interleukin (IL)-1, IL-6 and tumor necrosis factor alpha (TNFalph); heat-inactivated GP-120 failed to do so. When simulataneously added with GP-120, SP synergistically enhanced the induction of cytokines. Cytokines, thus, released in the brain may influence the replication of HIV since exposure of HIV-LTR transfected microglia to IL-1 or TNF-alpha resulted in an increased trans-activation. In vitro exposure of HIV-LTR transfected fetal microglia to cocaine, also, enhanced trans-activation. In utero exposure to cocaine is likely to render fetus more susceptible for HIV infection since microglia derived from one fetus of cocaine abuser when transfected with HIV-LTR, showed enhanced HIV-promoter activity. This proposal aims to investigate in microglia the (1) effects of SP, beta-endorphin, cocaine and GP-120 on trans-activation of HIV promoter; (2) mechanisms of enhanced HIVtrans-activation by these agents; (3) effects of these agents on the synthesis of p-24 antigen and reverse transcriptase (RT) enzyme of HIV; and (4) induction of cytokines by these agents. Cultures of microglia will be prepared from brain tissue collected from legally aborted fetuses and rapid autopsy by a method established in PI's laboratory. HIV expression will be determined by the trans-activation of HIV-LTR, synthesis of p-24 and RT. Trans-activation and mechanisms will be evaluated by the expression of reporter gene chloramphenicol acetyl transferase which is linked to either HIV-LTR or to two transcriptional elements (Kappa B or Activated Protein-1). p-24 antigen and RT synthesis will be determined by Enzyme Linked Immunosorbent Assay (ELISA) kits and by in vitro radiometric analysis, respectively. Cytokines will be identified by ELISA, MRNA synthesis and appropriate bioassays.
