BLOOD BRAIN BARRIER DYSFUNCTION--AIDS NEUROLOGIC DISEASE

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.