Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

266 Abstracts 21136-21140

12th World AIDS Conference

confocal microscopy were used to study proteins that form an activation complex
in response to HIV envelope treatment.
Results: Tyrosine phosphorylation of pyk2, focal adhesion kinase (FAK),
ZAP70, paxillin, Ick, and CC chemokine receptor 5 (CCR5) was observed following HIV envelope treatment; all of these phosphorylation events were inhibited
by pre-incubation with soluble (s) CD4. Inhibition by anti-V3 loop antibodies of
envelope-induced phosphorylation of ZAP70 and CCR5 suggested partial dependence of these phosphorylation events on signaling through CCR5. Intracellular
redistribution of FAK to focal adhesion complexes as well as physical association
of FAK and CCR5 were induced by HIV envelope treatment.
Conclusions: Novel intracellular signaling events have been identified that
are initiated by ligation of CD4 and CCR5 by HIV envelope. The HIV envelope-induced phosphorylation and association of proteins involved in cellular
adhesion and chemotaxis (i.e. FAK and CCR5) suggest a molecular mechanism
whereby HIV may recruit uninfected target cells to areas with high concentration
of virus such as lymphoid tissue.
21136 In vitro evidence of HIV-2 protection from subsequent
HIV-1 infection
Phyllis Kanki1, E. Kokkotov1, A. Gueye-Noiaye2, D. Schwartz3, S. Mboup2,
M.E. Essex1. 1 Harvard AIDS Institute 651 Huntington Ave Boston MA 02115;
3Johns Hopkins School of Public Health Baltimore MD, USA; 2Universite Cheikh
Anta Diop Dakar, Senegal
Objectives: A 12 year prospective study (1985-97) of HIV-2 infected individuals
in Senegal, West Africa has demonstrated a reduced risk of subsequent HIV-1
infection ranging from 52-74% (Science 268:1612; Science 272:1959). We used
an HIV-1 in vitro challenge system to determine if PBMCs from HIV-2 infected
individuals showed altered susceptibility to HIV-1 infection. Upon demonstration
of resistant HIV-2 infected PBMCs, we further sought to determine the mechanism
for this protection.
Methods: Cryopreserved PBMCs from 15 HIV-2 infected and 13 HIV negative
individuals were PHA stimulated and infected with 600 TCID-50 of HIV-1 (JRCSF)
or HIV-1 (IIIB) and monitored for HIV-1 p24 antigen on days 4, 7, 10 and 14.
Endogenous HIV-2 virus production was measured by p27 SIV ELISA. Levels of
RANTES, MIPla, MIP1/ were measured in culture supernatant by ELISA.
Results: 9 of 15 (60%) HIV-2 PBMCs demonstrated over 90% inhibition of
HIV-1 JRCSF compared to 0 of 13 HIV negative controls (Fisher exact test, p
value =.007). Similarly, resistance was observed with E50 another CCR5 virus.
In contrast, all HIV-2 positive and HIV negative PBMCs were equally susceptible
to HIV-1 (Illb) infection. Supernatant levels of MIP-la (r = 0.56, p =.03) and
MIP-1/ (r = -0.69, p =.004) were inversely correlated with HIV-1 replication;
defined by log peak p24. Using polyclonal antibodies to RANTES, MIPla and
MIP1/1, resistance was neutralized in 7 of 7 resistant PBMCs evaluated with no
change in the infectivity of similarly treated HIV negative PBMC controls.
Conclusions: HIV-2 infected PBMCs demonstrated resistance to HIV-1 virus
challenge. This effect was restricted to CCR5 HIV-1 viruses and mediated via
Beta-chemokines. This may be the first evidence that Beta -chemokine inhibition
of HIV, correlates with protection in people.
21137 1HIV-lnduced apoptosis of renal tubular epithelial cells
involving Fas sensitization and ice-protease activation
Pier Giulio Conaldi1, L. Biancone', A. Bottelli1, A. Wade-Evans2, L.C. Racusen3,
G. Camussi1, A. Toniolo'. 'Dept. of Clinical and Biological Sciences, Viale Borri
57-21100, Varese, Italy; 2Div Retrovirology-NIBSC, Potters Bar-Herts, UK;
3Dept. Pathology, John Hopkins University, Baltimore, MD, USA
Objectives: HIV-infected patients may suffer several kidney diseases, which are
characterized by rapid progression to renal insufficiency and end-stage renal disease. The main feature of HIV-induced nephropathy is the prominent tubulopathy
with apoptotic changes of tubular cells. This paper describes the effects of HIV-1
infection on human proximal tubular epithelial cells (PTEC) observed in vitro with
a view to explaining the occurrence of tubular damage in vivo.
Methods: The study has been done with primary and immortalized cultures
of PTEC and mesangial cells (MC). The infection has been carried out with
syncytium-inducing HIV-1 strain adapted to growth on T cell lines.
Results: PTEC and MC express surface CD4, CXCR-4, CCR5, CCR3 and
other chemokine receptors. HIV-1 does actively replicate in PTEC and MC, but the
effect of HIV infection was different in the two cell types. Whereas viral replication
in MC caused only minor cytopathic effects, HIV-infected PTEC underwent death
by apoptosis in 10-15 days. These findings are consistent with the histopathological features of tubular injury, which represents the hallmark of HIV-associated
renal damages.
The exposure to gp120, gp160 and Tat did not cause the death of PTEC.
HIV-infected PTEC showed enhanced expression of Fas and were sensitized to
Fas stimulation. In fact, the addition of anti-Fas agonistic antibody to the cultures,
in the early phase of infection, relevantly increased and precipitated cell death.
Apoptosis of HIV-infected PTEC was prevented by the inhibition of ICE protease

activity: cell death resulted to be arrested although virus replication persisted in
the tubular cells.
Conclusions: We found that PTEC are permissive to HIV-1 entry and are
able to sustain active replication of the virus. HIV-1 kills the infected PTEC by
triggering an apoptotic pathway involving Fas upregulation, sensitization to Fas
signaling, and activation of ICE protease. The inhibition of ICE protease prevents
PTEC death in spite of persistent viral replication. These results may explain the

development of tubulopathy, a common pathology in the infected patients, and
may suggest novel therapeutic hints.
21138 1 Microglia secrete chemoattractants affecting monocyte
migration through the blood-brain barrier during HIV-1
encephalitis
Yuri Persidsky', A. Ghorpade', J. Limoges', M. Stins2, K.S. Kim2, M. Fiala2,
H.E. Gendelman'. 'Center for Neurovirology 600 S. 42nd St. Omaha NE
68918-5215; 2Univ. of Southern California Los Angeles CA, USA
Objectives: Clinical neurological impairments in HIV-1 dementia (HD) parallel
numbers of infiltrating blood monocytes to brain. How monocytes access the brain
during HD is, therefore, critical to unraveling viral neuropathogenic mechanisms.
Design: To investigate this cellular process, we constructed an artificial BBB.
In this system, brain microvascular endothelial cells and autologous astrocytes
are placed on opposite sides of the matrix-coated porous membrane (Persidsky
et al. J. Immunol. 1997).
Methods: The transendothelial passage of monocytes was assessed after application of monocyte-derived macrophages or microglia with/out HIV-1 infection
to the astrocyte "brain compartment" of the model. Observations with the artificial
BBB were substantiated in a SCID mouse model of HIV-1 encephalitis (Persidsky
et al. Am. J. Pathol. 1996) in which we stereotactically inoculated microglia or
monocytes into SCID mouse brains.
Results: Blood monocytes penetrated 2-3.5 times more efficiently (p < 0.004)
through the BBB constructs containing microglia as compared to membranes
containing monocytes. HIV-1 infected microglia cells further enhanced migration of monocytes. In SCID mouse brains at 7 days following inoculation equal
numbers of HIV-infected microglia or monocytes were distributed in the basal
ganglia. Eighty percent of the total cells identified expressed HIV-1 p24 antigen.
A pronounced accumulation of mouse blood-derived macrophages was seen in
areas surrounding virus-infected human microglia as compared to brains containing HIV-1 infected monocytes. A prominent astrogliosis was seen 7 days
after microglia/monocyte inoculation and was greater in mouse brains with HIV-1
infected microglia.
Conclusions: We conclude that microglia are a richer source of monocyte
chemoattractants than monocytes. This may explain, in nature, the predilection
of monocytes to enter the brain during progressive HIV dementia.
|21139 Upregulation of HIV-1 replication by /-chemokines
Mark Kelly', H.M. Naif2, A.L. Cunningham2, A.R. Lloyd'. 'Inflammation
Research Unit University, Sydney 17/69 Birriga Road Bellevue Hill NSW 2023;
2Centre for Virus Research Westmead Hospital, Sydney, Australia
Background: Clinical trials examining potential antiviral effects of p-chemokines
in patients with HIV disease have commenced. These are based on the in vitro
inhibition of M-tropic HIV-1 entry into susceptible cells by /1-chemokines when
added simultaneously with HIV. However, cells recruited to lymph nodes during
HIV disease are likely to be exposed to / -chemokines prior to being infected.
Aim: To contrast the effects of /-chemokines on HIV replication when added
prior to or simultaneously with HIV infection.
Methods: Monocytes and CD4 T lymphocytes were isolated from whole blood
of seronegative donors. Cells were exposed to p -chemokines for 48 hours prior
to or simultaneously with infection with M-tropic HIV isolates. Subsequent HIV
replication was assessed by measurement of culture supernatant p24 antigen by
ELISA.
Results: Upregulation of HIV replication occurred in cells exposed to
/-chemokines prior to HIV infection. These effects occurred at physiological
concentrations, were CCR5 independent and involved cell-signalling. In contrast,
exposure of cells to /-chemokines simultaneously with HIV infection resulted in
down-regulation of HIV replication. These effects occurred at supraphysiological
concentrations, were CCR5 dependent and did not involve cell signalling.
Conclusions: Exposure of monocytes and CD4 T cells to /-chemokines prior
to HIV infection results in upregulation of viral replication. These effects occur at
physiological concentrations, are CCR5 independent and involve cell-signalling.
Trials of f/-chemokines in patients with HIV disease should proceed with caution
given the capacity of these proteins to stimulate HIV replication.
|21140 Signal transduction due to HIV-1 envelope interactions
with the CD4/CXCR4 receptor complex
Christian De Vaux, M. Biard-Piechaczyk, N. Coudronniere, C. Guillerm,
J. Roland, V. Robert-Hebmann, L. Briant. CRBM/CNRS, Lab. Inf. Retrovir. Sigal.
Cell., Inst. de Biologie, 4 Boulevard HENRI IV 34060 Montpellier, France
Objectives: to study the nature of signaling pathways that are activated following
HIV-1 envelope binding to the CD4/CXCR4 receptor complex, and to discriminate
between signals triggered through CD4 and signals that require CXCR4.
Design: Human cell lines stably transfected with different forms of CD4,

CXCR4, and/or cellular kinases and human T cells were used to investigate
signal transduction following engagement of HIV-1 receptor molecules. Signal
transduction was monitored by specific kinase activation assays, activation of
transcription factor (AP-1 and NF-k B), and promoter activation assays. Cell activation statut was also monitored by cell-surface phenotyping (i.e. CD25) or
analysis of cell cycle stage, and cell death was studied with YOPRO-1, TUNEL
assay and propidium iodide staining.