A prime target of HIV is the CNS. To enter the CNS, HIV must negotiate the blood-brain barrier (BBB). Our goal is to determine how HIV crosses the BBB and ultimately to develop therapeutic strategies for blocking passage. Previously, we have used gp l2O as a model of HIV and shown that it crosses the BBB by a process resembling adsorptive endocytosis (AE), a vesicular response of the brain endothelial cell (BEC) to pathological insults such as toxic glycoproteins, which provides routes into and across the BEC. Our unpublished work shows that HIV interacts with the BBB essentially as predicted by gp l2O. We have shown that the uptake and transport across the BBB of gp 120 and HIV has five indentifiable stages (surface binding, internalization, post-internalization fusion, luminal exocytosis, and transcytosis [exocytosis to the abluminal membrane]), is dependent on cAMP, cytoskeletal binding, glucose, and potassium and is stimulated by wheatgerm agglutinin (WGA) and lipopolysaccharide (LPS). Fusion involves a protamine sulfate-sensitive co-receptor. Based on the literature, these findings suggest that nitric oxide (NO), phospholdnases (PK), cGMP, the NMDA glutatmate receptor, and prostaglandin E2 (PGE2) mediate transport. Here, we propose to elucidate the mechanisms used by gp 120 and FHV to cross the BBB. We hypothesize that the uptake and transport of gp l2O and HIV by brain endothelial cells is dependent on binding to sialic acid and N-acetyl-B-D-glucosaminyl acid and is mediated through specific cell pathways (NO, NMDA, PK, PGE2). We also hypothesize that the potentiation of gp 120 and HIV transport by cAMP is dependent on NO, NMDA, PK, and PGE2 pathways whereas LPS acts primarily through the release of TNF. We will test these two hypotheses in vivo and in vitro for gp I2O and HIV in four specific aims SAl: To detemiine whether sialic acid or N-acetyl-B-D-glucosaminyl acid are the sugars to which gp l2O and HIV bind at the BEC; SA 2: To deiemiine whether in vivo transport or in vitro surface binding, internalizatior, post-internalization fusion, exocytosis, or transcytosis is dependent on NO, NMDA, PK, or PGE2 pathways; SA 3: To detemiine whether cAMP potentiated gp l2O and HIV transport is opposed by cGMP and mediated through NO, NMDA, PK, or PGE2 pathways; SA 4: To detemiine whether LPS stimulates transport of gp I2O and HIV by acting directly on the endothelial cell or is dependent on release of TNF and other proinflammatory cytokines and whether LPS stimulation is mediated through NO or NMDA.
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