Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Track A: Basic Science

Tu.A.2039 - Tu.A.2043

(TMX), for periods of I to 5 days. Using a flow cytometric techiqiue with 3 colors (CD4 -FITC/CD8-PE/Apoptosis-7AAD and CD4-FITC/Fas-PE/Apoptosi --/AA D)), the percentages
of surviving and apoptotic cells expressing CD4, CD8 and Fas were dietermined.
Results:There was a time-dependent increase in apoptosis in PBMCs ftor AIDS patients
that is correlated with disease severity At day 5 of incubation the percent of apoptotic
PBMCs was 25 ~ 4%, 39 ~+ 7% and 57 ~+ 4% for controls, CDC -B 6and CDC-C patients,
respectively. 2) Accelerated apoptosis occurred predominantly ir CD-4 cells in patients
with AIDS. At day 5 of incubation, spontaneous apoptosis of CDt4 cells (expressed as a
percent of total T cells) was 17 ~+ 4%, 22 ~+ 3% and 43 + 5% in controls CDC-B and CDCC patients, respectively 3) The calmodulin antagonists,TFP and TtX, inhibited the accelerated apoptosis in CD4+ cells from AIDS patients, resulting in an inc ease in the ratio of viable
CD4 +/CD8+ cells. 4) The inhibitory effect of TFP and TMX on apoptosis was enhanced in
advanced disease (CDC-C) compared to less severe disease (CDC--B). At dlay 5 of incubation,TFP increased the ratio of viable CD4+/CD8+ cells by 2.2 and 3.4 fold in CDC-B and
CDC-C patients, respectively 5) Abnormal CD4 cell apoptosis was associated with
increased Fas expression in HIV+ patients.
Conclusions: These investigations imply that calmodulin and Fas n;a play an important role
in the depletion of CD4+ cells in AIDS patients and that calmodulin antagonists may provide a novel therapy for AIDS.
J.M. McDonald,The University of Alabama at Birmingham, Department of Pathology, 509
LHRB, 701 South 19th Street, Birmingham, AL 35294-0007 (USA) Telephone: [phone]
Fax: [phone] Email: [email protected]
Tu.A.2039
Bcl-2 AND RELATED PROTEINS ARE SELECTIVELY MODIFIED BY HIV INFECTION
PRIOR TO VIRUS INDUCED APOPTOTIC CELL DEATH
Virk Abinash*, Badley AD*, Paya CV*. *Mayo Clinic, Rochester, MN
Objective: Apoptosis of CD4 T cells plays an important role in AIDS pathogenesis.
Accumulating data indicates that CD4 T cells from HIV-infected individuals are inherently
susceptible to apoptosis. However, the mechanisms regulating susceptibility to apoptosis
remain unknown. Bcl-2 and bcl-XL have been demonstrated to be anti-apoptotic whereas
their counterparts bax and bcl-XS are pro-apoptotic molecules.
Methods: We have recently characterized a model of HIV mediated death wherein HIV
mediates apoptotic death of infected U937 cells specifically through Fas ligand six days
postinfection. Using this model, we propose to study that bcl-2 and its homologues regulate
susceptibility to cell death mediated by HIV.
Results: I) The anti-apoptotic molecules, bcl-2 and bcI-XL, are downregulated by day 4
postinfection, and II) the pro-apoptotic molecules, bax and bcl-XS. are upregulated at the
same time.The relative role of these molecules in the HIV-mediated apoptosis is being tested by overexpression of the decreased anti-apoptotic molecules (bcl-2 and bcI-XL) driven
by a heterologous promoter or downregulation of the increased proapoptotic molecules.
Stable expression of bcl-2 under the expression of a murine retrovirus promoter (SFFV) in
U937 cells does not inhibit HIV-induced cell death but renders the cell refr-actory to antiFas crosslinking antibody, suggesting that the modification by HIV of pro- and anti-apoptotic
molecules other than both may be responsible for the virus-induced susceptibility to apoptosis. Current work focuses on double overexpression of bcl-2 and bcl-XL and downregulation of bax and bcl XS by antisense oligonucleotides.
Conclusions: The above results suggest that HIV infection selectively modifies bcl-2 and
related proteins to possibly result in a net state of susceptibility to apoptosis. Reversal of
these modifications by genetic manipulation will allow to determine their role in HIV mediated apoptosis.
A.Virk, Mayo Clinic, 200 First St. SW, Guggenheim 501, Rochestei, MN 55905 Telephone:
507-284-9646; Fax: [phone]
Tu.A.2040
REDUCTION OF HIV PRODUCTION AND CYTOPATHIC EFFECTS BY INHIBITORS
OF THE NA+/K+/2CI" COTRANSPORTER
Garry, Robert F,Voss T Gatti R Fermin C. Tulane University Schoo! of Medicine, New
Orleans, Louisiana, USA.
Objective: Infection of CD4+ T-lymphoblastoid cells by cytopathic strains of HIV- I results in
an increase in cell volume that leads to lysis and cell death.The increase in cell volume is
attributable in part to an HIV-induced increase in activity of the plasma membrane-associated Na+/K+/2CI- cotransporterThe objective of this study was to elucidate the role of the
Na+/K+/2CI- cotransporter in HIV replication and cytopathogenesis.
Methods: CD4+ T-lymiphoblastoid cells were infected with HIV- I, then incubated in medium
containing furosemide or bumetanide, loop diuretics that are specific inhibitors of the
Na+/K+/2CI' cotransporter. Intracellular ion concentrations were quantitated using ion-sensitive fluorescent probes and video imaging techniques. HIV specific cytopathic effects were
quantitated by photomicroscopy and HIV production was measured by antigen capture EIA.
Results: HIV infection increases the intracellular concentrations of both sodium (Na+) and
potassium (K+). Cells treated with micromolar concentrations of furosernide or bumetanide
had reduced intracellular Na~ arid K~, and showed mairkedly rducecd smecy~tium formation
and single cell killing.The majority of ti-eased cells were able to suvivc a ate HIV infection
from two to four weeks longer than untreated cels.The loop diuretic aiso reduced HIV
production by grea{ter than 90% at these physiologically attainable.or en itration.
Conclusions: Increased expression of the Na~/K~/2CI- cotranspoirter iniajor regulator of
cell volume, plays a role in the development of DIV-specific cytepathic elfects.The HIVinduced increases in intracellular cation content mediated by this tiranspc:mt are also
important for effective progeny virus production. Loop diuretics may hive potential in AIDS
treatment.
Robert F. Garry, Dept. Micro/In-aunolTulane Medical School, 14301uane Avenue, New
Orleans, Lociisiana, UflATelephone: 504-587-2027 Fax: 504-588- laI-i.t emai:
rgarr ytmcpop.tmc.tulane.edu
Tu.A.204 I
THE RAPID INDUCTION OF APOPTOSIS IN UNINFECTED CD4 LYMPHOCYTES BY
HIV-INFECTED CELLS OCCURS WITHOUT FORMATION OF SYNCYTIA
Nardelli, B., Gonzalez, C.J., Valentine FredT. NewYork University Medical Centec New
York, NY U.S.A.

Objective:To examine the possible role of syncytia formation in the rapid killing of uninfected CD4+ lymphocytes.
Methods and Background:We have demonstrated (PNAS 92:7312,'95) that cells productively infected with HIV are able to trigger the rapid death by apoptosis of a larger number
of resting as well as activated CD4+ cells.This infected cell-mediated killing (ICMK) occurs
between infected and uninfected autologous PBMC T cells or T cell lines. ICMK is inhibited
by soluble gp120 or sCD4 but not by AZT Infected and uninfected cells are cocultured for
I to 72 hrs. Death of uninfected cells is measured by prelabeling with C 51, by the release
of H3-labeled fragmented DNA, by terminal deoxynucleotidyl transferase (TUNEL) labeling
of DNA fragments, or by enumeration of cells prelabeled with PKH26 dye.
Results: ICMK not only occurs in cells before syncytia are formed, but can be shown to
occur in experimental conditions when syncytia are not formed. a) Calcium is required for
the formation of syncytia, but not ICMK; b) anti-CD7 MoAb partially inhibits syncytia, but
does not affect killing; c) HeLa cells chronically infected with a mutant L.AV that does not
form syncytia, still cause the death of uninfected target cells; d) a peptide firom gp4 I (provided by Dr Matthews) blocks syncytia formation, but does not inhibit ICMK: e) prototype,
non-syncytia-forming isolates of HIV are able to manifest ICMK. In the presence of AZT and
the peptide, the co-culture of I infected cell per 20 uninfected, results in the death of 65%
of the uninfected cells within 72 hours.
Conclusions: Syncytia are not required for ICMK, and this process may contribute to the
large destruction of CD4+ lymphocytes occurring in patients.
FT Valentine, Dept. of Medicine, 550 First Ave, New York, NY 100 I 6, U.S.A.
Tu.A.2042
INCREASED IN-VITRO TETANUS SPECIFIC ISOLATION AFTER IN-VIVO TETANUS
IMMUNIZATION OF HIV INFECTED INDIVIDUALS
Ostrowski, Mario, Stanley S, Justement J, Gantt K, Goletti D. Fauci, AS. National Institute of
Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland 20892, USA
Objective:To delineate the role of in-vivo immunization with a common recall antigen on
in-vitro antigen specific isolation of HIV firom seropositive individuals.
Methods: HIV infected individuals were immunrized with tetanus toxoid. Blood was drawn
at multiple time points after immunization and unfriactionated or CD8 depleted PBMCs
were placed in culture for virus isolation.Tetanus specific isolation of virus was defined as an
increased ability to isolate HIV in-vitro following in-vivo immunization compared to preimmunization in the presence of exogenous tetanus as opposed to no added stimulus or
control antigen alone.
Results:We have previously demonstrated an increased ability to isolate HIV in-vitro after
in-vivo immunization when IL2+1L4 were added to the cultures in the absence of antigen.
This study examines the role of antigen recognition in the in-vitro isolation of HIV 7/13
(54%) patients demonstrated tetanus specific isolation of HIV In four patients, virus was isolated in the presence of tetanus without addition of any exogenous cytokine. In two
patients virus could be isolated from unfractionated PBMCs when tetanus antigen was
added to the culture; isolation from unfractionated PBMCs is generally quite difficult in the
absence of added PHA or PHA blasts. HIV was demonstrated to be produced mainly from
CD4+ cells with a CD45RO,CD25+ phenotype.Tetanus specific isolation of virus was associated with elevated levels of IFN gamma, IL-6,TNFa and IL4 in culture supernatants when
compared to control cultures.
Conclusions:We demonstrate increased isolation of HIV in-vitro after in -vivo immunization
when the immunizing antigen is placed in the culture.These findings have important implications with regards to the role of ongoing antigen specific immune responses in the upregulation of HIV expression.
M. Ostrowski, Bldg. 10, Rm. 6A I 1, 10 Center Drive, MSC- 1576 Bethesda, MD 20892 USA,
Tel: [phone]; Fax: [phone]
Tu.A.2043
PRODUCTIVE HUMAN IMMUNODEFICIENCY VIRUS INFECTION OF HIGHLY
PURIFIED MEGAKARYOCYTIC PRECURSORS AND MATURE MEGAKARYOCYTES
C. Cheucci, M. Federico, R. Guerriero, E. Pelosi, U.Testa, H.J. Hassan, C.Peschle. Dept. of
Hematology-Oncology and Virology Istituto Superiore di Sanita, Rome, Italy
Objective: To study the effect of HIV- I on human megakaryocytopoiesis.
Methods: CD34+ hematopoietic progenitor cells (HPCs), 90% prified fr-om human peripheral blood (PB) were grown in serum-free liquid suspension culture in the presence of
thrombopoietin (TPO) to induce terminal MK differentiation/maturation. PB HPCs and MK
precursors were challenged with the lympho-monocytotropic NL4-3 HIV- I strain at different days from purification (0,5 or 10).The presence of HIV tat mRNA was analyzed by RT
PCR; production of functional viral particles was tested by antigen capture of p24 protein
and by titration of supernatants from challenged MKs e on C8 166 T lynplrocytic cell line.
Inhibition of HIV infection was performed pretred pretreating the cells with anti-CD4
MoAb.
Results: MKs generated by PB HPCs are productively irnfected by HIV at various stages of
differentiation. Particularly, the presence and release of IV was observed in MKs infected at
days 5-10 after purification whereas the cells challenged at day 0 were negative.
Morphological analysis demonstrated cytopathnogenic effects only in day 0 DIV treated cells
that showed a highly reduced size and nuclei number Conversely cells challenged at day 5,
although pioduetively infected, fully differentiated to platelet producing cells. Infection of MK
precursors pretreated with anti-CD4 MoAb and challenged with the virus resalted only
partially inhibited.
Conclusions: Duman MKs represent 0.03% of total fbone nrairowe cells arid 0.0196 oh PB
cells. Studies on megakaryocytopoiesis have been harnpered so Gin by lackc of a aelatively
pure and abundant MK population. A recently established PB HPC differentiation culture
system allows to generate a relatively large number of highly purified (97-99%) MK precursors and then mature MKs (Guerriero R. et al. Blood, 1995), thus providing an in vitro
experimental tool to analyze the effect of HIV- I on megakaryocytopoiesis. Our results indicate that human MKs could be productively infected by HIVThe effect of the virus on MK
proliferation and differentiation depends on the stage at which the cells are challenged. At
day 0 HPCs are small undifferentiated blasts: in these cells the virus may induce a delay in
the differentiative process. At day 5, most cells are large and mononuclear: this population
represents putative MK precursors (as indicated also by the presence of >60%

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