HIV- I can lead to a series of devastating clinical conditions in the central nervous system (CNS) of certain infectedindividuals characterized by cognitive and motor dysfunction. The molecular mechanisms underlying this HIV-1-induced dementia complex remain enigniatic. In addition, in the era of highly active anti-retroviral therapy (HAART), the CNS may also act as a reservoir or sanctuary site for HIV- I during combination therapy. Our laboratories have developed recently an in vitro blood:brain barrier (BBB) system comprised totally of human CNS cell-tvpes, which successfully models the cellular interactions of the BBB in vivo. In addition, we have demonstrated recently that the HIV- I regulatory protein, Vpr, has significant effects on inducing apoptosis in human neuronal cells, as well as activating bone marrow-derived macrophages. Utilizing complementary techniques based on these preliminary studies, the effects of selected lentiviral proteins on the BBB will be analyzed. It is hypothesized that Vpr, and possibly other specific viral proteins including Tat, Nef and the envelope protein gp I 2O which have been demonstrated to induce apoptosis in other human cells, may have effects on inducing programmed cell-death in BBB-constitudng cells, including microvascular endothelial cells (MVEC) and astrocytes.
In specific aim one, this hypothesis will be evaluated using the in vitro BBB system with read-outs for apoptosis and BBB permeability changes including the TUNEL, Annexin V and Caspase-8 assays, in addition to trans-endothelial electrical resistance (TEER) and ZO] ti,-,ht-junction protein alterations. Both free recombinant ]antiviral proteins, in addition to mutant viruses lacking the genes of interest, will be analyzed in this system. In addition, utilizing a series of HIV- I -based lentiviral vector systems, these selected viral proteins will be expressed in the relevant BBB cell-types. In the complementary specific aim two, HIV-1-infected cells will also be analyzed for their inductions of permeability changes and apoptosis generation in the in vitro BBB system. In particular, mutant viruses lacking each specific viral regulatory and structural gene will also be used in a complementary approach, after infection of a CD4+ T-cells and primary macrophages for effects on the BBB. Finally, the effects of combination anti-retroviral chemotherapy on HIV- 1-infected cell interactions with the BBB will be analyzed. This will be a model svstem for different regimens of chemotherapeutics, in altering infected-cells' induced alterations on permeability and programmed cell-death within the human BBB. In the era of HAART, these will be critical studies in understanding how changes in combinations of HAART regimens affect HIV-I:BBB interactions, which will also be modeled in the system with resistant viral strains in infected peripheral blood cells. Within these inter-related studies, it is proposed that a series of new technologies, including HIV- I vector systems and an in vitro BBB system, be brought to bear towards determining the precise effects of HIV- I proteins, and thus the molecular mechanisms involved, in inducing alterations in the BBB.
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