Cognitive and behavioral abnormalities are a consequence of AIDS- associated neurological disease. HIV has been implicated in the etiology of this neurological dysfunction. Several groups have reported blood-brain barrier (BBB) abnormalities in AIDS patients. AIDS patient autopsies reveal that brain capillary endothelium is a cellular target for HIV in the CNS in vivo. Human brain capillary endothelial (HBCE) cells possess specialized structural characteristics that regulate the passage of cells and molecules across the capillary walls and facilitate formation of the blood-brain barrier (BBB). These properties distinguish HBCE cells from endothelial cells of other organs but may make them susceptible to unique manifestations of HIV infection. For example, HIV infection of HBCE cells may disrupt the barrier function or selective permeability of the BBB and contribute to neurologic dysfunction. Infected cells may also constitute a direct route of entry of HIV into the CNS. We have established pure primary cultures of HBCE cells as an in vitro model to examine the interaction between HIV and HBCE cells. Using this system we have shown significant HIV infection of HBCE cells but not large vessel endothelial cells from other organs. The infection persists in culture and is productive yet non-cytopathic. Recently we have shown that T cell but not macrophage tropic HIV strains infect HBCE cells. HIV infection of HBCE cells is not blocked with MAB to the two known HIV receptors: CD4 and galactocerebroside. Infection is however neutralized with MAB against defined epitopes on gp120 distinct from the CD4-binding domain. This implicates the presence of a novel HIV receptor which may be important for entry into the CNS. We will extend these observations to more clearly define the mechanisms and consequences of HIV infection on HBCE cells. Chimeric viruses with hybrid envelope sequences will be used to map regions of gp120 involved in HBCE tropism. We will also assess the ability of HIV to alter HBCE surface antigenic properties which affect inflammatory reactions and leukocyte trafficking. Since cytokines produced by HBCE cells may influence other CNS cells, we will examine constitutive, induced,and HIV infected cellular production of these molecules.We will also use dual chamber culture methods utilizing HBCE cells and astrocytes to establish in vitro BBB conditions maintaining apical and basal polarity of cells. Using this culture system, we will examine efficiency of HIV passage from luminal and abluminal surfaces as well as the ability of virus to perturb permeability of the BBB. The latter studies will assess the ability of HIV to interfere with transendothelial cells transport of radiolabeled metabolites and amino acids. Final we will utilize scatchard analyses to determine if HIV entry into HBCE cells is due to a specific receptor. We will identify the moiety of the HBCE surface receptor and clone the receptor from a cDNA library. For the latter aim we will either microinject expression libraries into HeLa cells and screen with HIV gpt or utilize an extensive HBCE monoclonal library to identify an antibody which specifically inhibits viral infection. The monoclonal which inhibits HIV infection will be used to screen HBCE expression libraries. In summary experiments in this project will elucidate potential mechanisms involved in HIV induced dementia as well as identify viral and cellular components which mediate this process leading to a rational design for site directed therapy.
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