Final Program and Oral Abstracts [International Conference on AIDS (8th: 1992: Amsterdam, Netherlands)]

TUESDAY, 21 JULY 1992

TuD 0548-TuD 0553

TuD 0548 HIV-1 SUPPRESSES ERYTHROPOIETIN PRODUCTION IN VITRO
Scadden, David T.*; Wang, Z*, Goldberg, MA**
* Div. of Hematology/Oncology, New England Deaconess Hospital,
Harvard Medical School **Brigham & Women's Hospital, Harvard Med.
Serum erythropoietin (Epo) levels are depressed in anemic AIDS patients
relative to controls. The basis for abnormal modulation of Epo has not been
defined. The hepatoma cell line Hep3B produces Epo in response to hypoxia
and serves as a model for the study of Epo regulation. Hep3B cells are infectible
with HIV-1 and were used as a model for evaluating potential direct effects of
Epo regulation. HIV-1 infected or transfected Hep3B cells produced Epo at
significantly lower levels than uninfected Hep3B cells under low 02 conditions
or following exposure to cobalt chloride. Epo production by hypoxia induced
HIV-1 infected Hep3B cells was depressed compared with non-HIV containing
Hep3B cells when normalized for cell number, total cellular protein or albumin,
but not depressed when normalized for alpha-fetoprotein production. The
cellular levels of Epo mRNA were not diminished in the HIV-1+ Hep3B cells
indicating a probable post-transcriptional effect of HIV-1 on Epo expression.
HIV-1 appeared to depress the production of Epo and some, but not all, other
cellular proteins. These results suggest that impaired production of Epo is a
direct effect of HIV-1 infection and may contribute to anemia in AIDS.

TuD 0549 MUCOSAL CYTOKINE EXPRESSION DURING PROGRESSION OF HIV INFECTION.
Reka S. Garro M, Kotler DP. St. Luke's-Roosevelt Hosp, Columbia U, NY, NY, USA.
Oblectives To determine the pattern of cytokidne expression In rectal mucosa of HIV-Infected Individuals and
to correlate cytoklne expression with disease stage and HIV expression. Previous studles have correlated
HIV antigen detection in the Intestine with clinical symptoms and with hlastologlc and bIochemical evidence
of Inflammation In the absence of other enteric pathogens. Cytokine modulation of HIV production has been
demonstrated in experimental systems but not in clinical specimens.
Methods Rectal biopales from 24 subjects were studied; 5 with early disease (CD4>400/mm), 6 with
moderately advanced disease (CD4 < 400), 9 AIDS (3 with no colonic pathogens, 3 with cryptosporldlosls,
3 with CMV colitis), and 4 HIV-seronegative controls. All HIV-infected subjects complained of altered bowel
habits. Mucosal lymphold hyperplasla or Inflammatory changes were seen In all cases. RNA In situ
hybridization was performed using 35S rlboprobes of HIV, TNF, IL-1, IL-2, IL-5, and 11-6.
Control Early Middle AIDS-no 01 CMV CRYPTO
HIV 0% 20% 83% 67% 0% 33%
TNF 0% 0% 67% 67% 67% 33%
11-1 0% 40% 50% 67% 100% 67%
IL-2 75% 80% 50% 67% 0% 33%
IL-5 0% 60% 80% 33% 33% 0%
IL-6 0% 80% 100% 67% 33% 67%
Results HIV RNA was detected most frequently In non-AIDS subjects with moderately advanced disease. The
changes In IL-5 expression mirrored HIV. IL-2 and IL-6 mRNA expression were most common early in the
disease, while TNF and IL-1 expression were most common In advanced disease.
Conclusion Cytokine mRNA expression Is detectable In rectal mucosa of HIV-lnfected Individuals and is not
related to enteric pathogens. The expression of cytokines varied during disease stage. Cytokine modulation
of HIV expression In early disease might be related to IL-2 or IL-6, rather than TNF or IL-1. Intestinal mucosa
might be an Important reservoir of HIV during the preclinical phase of HIV infection.
Safak Reka - St. Luke's-Roosevelt Hospital Center
421 W. 113th St. - New York, New York 10025
[phone] FAX # [phone]
TuD 0551 Tumor Necrosis Factor Causes Apoptotic Cell Death in HIV-Infected
CD4 Positive Cells. Nobuvuki. Kobavashi. Department of Virology and
Parasitology, Yamaguchi University, School of Medicine, Ube,
Yamaguchi, Japan.
Objective: We have proposed the hypothesis (Virus Genes,4,183,1990, AIDS 5,1405,1991)
that Tumor Necrosis Factor ( TNF ) possibly involved in the progression of AIDS. We
found that TNF augment HIV replication (Jpn.J.Cancer Res. 79,156,1988,
Med.Microbiol.Immunol,177,181,1988, J.Virol.63,2504,1989 ) and at the same time
selectively kill HIV-infected cells. We ( AIDS Res.and Human Retroviruses 5,131,1989)
and others cleared that TNF augment HIV replication via activating cellular
transcriptional factor termed NF-icB which bind to the enhancer region of HIV-LTR.
However, the mechanism of cell death by TNF is not cleared yet. Here, we investigated the
mechanism of HIV- infected cell's death by TNF.
Methods: HIV-infected human T-cell lines treated with TNF were examined by electron
microscopy and the specific degradation of chromosomal DNA was analyzed by agarose
gel-electrophoresis.
Conclusion: Typical features of apoptotic cell death,including condensation of chromatin,
were observed within 3 hr after TNF treatment in HIV -infected cells. And specific
degradation of chromosomal DNA was visualized by 24 hr after TNF treatment.
Essentially identical results were obtained by anti-Fas Ab. However, TNF induced cell
death was not inhibited by actinomycin D, a RNA synthesis inhibitor. Thus, our data
indicate that HIV-infected cells were depleted by apoptosis but not by necrosis such as
over production of viruses.
Nobuyuki Kobayashi. Department of Virology and Parasitology,Yamaguchi
University, School of Medicine. 1144 Kogushi,Ube, Yamaguchi 755,Japan.
TEL: [phone] FAX: [phone]

TuD 0550 RP 55778, A PLATELET ACTIVATING FACTOR (PAF) ANTAGONIST, INHIBITS
EXPRESSION OF HIV IN CHRONICALLY INFECTED PROMONOCYTIC CELLS.
Weissman, Drew, PoliG., Bousseau, A., and Fauci, A.S. LIR, NIAID, NIH, Bethesda,
Maryland 20892 USA. Rhone-Poulenc Rorer, Centre de Recherches de Vitry/Alfortville,
13, qual Jules Guesde, 94403, Vitry sur Seine - France
Objectives: To investigate the potential role of a pharmacological antagonist of PAF, RP 55778, on HIV
replication in acutely as well as chronically infected cells.
Methods: CD4+ T and promonocytic cell lines (Jurkat, MT4, U937), mitogen-stimulated peripheral blood
mononuclear cells were infected with HIV-1 in the presence or absence of RP 55778. RP 55778 was also
tested as a modulator of cytokine- or phorbol myristate acetate (PMA)-dependent virus expression in
chronically infected cell lines of T cell (ACH-2, J1) and monocytic (Ul) origin. Virus expression was monitored
by reverse transcriptase activity, immunofluorescence, Northem blot analysis of HIV RNA, long terminal repeatchloramphenicol-acetyl-transferase transient transfection experiments.
Results: No significant effects of RP 55778 on HIV replication/expression were observed in either acutely
infected cells or in chronically infected T cell lines. However, RP 55778 showed a strong suppressive effect
on HIV expression in monocytic U1 cells stimulated either with PMA, TNF-n, or other cytokines such as
interleukin-6 or granulocyte macrophage-colony stimulating factor. PMA stimulation of U1 cells stimulated the
secretion of endogenous TNF-a leading to an autocrine upregulatory loop of HIV expression which was
suppressed by RP 55778. At the molecular level, RP 55778 blocked HIV expression in U1 cells at both
transcriptional and post-transcriptional levels. RP55778 is likely to act independently from the surface PAF
receptor in that PAF did not either directly stimulated HIV expression in U1 or block the RP 55778 suppressive
effect.
Conclusions: RP 55778 inhibited HIV expression in a persistently infected monocytic cell line by multiple
mechanisms, including the inhibition of TNF-a production and TNF-a-mediated induction of HIV expression.
RP 55778, an orally bioavailable compound already shown to be well tolerated in phase 1, may result a
beneficial tool in the treatment of HIV infection.
Dr. Drew Weissman
LIR, NIAID, National Institutes of Health, 9000 Rockville Pike,
Bethesda, MD 20892 USA - [phone] FAX: [phone]

TuD 0552 MULTIPLE TRANSCRIPTIONAL PATHWAYS REGULATE HIV EXPRESSION TuD 0553 INTERFERON-y(IFN-y) EXERTS DICHOTOMOUS EFFECTS ON DIFFERENT STAGES
IN PERSISTENTLY INFECTED CELLS. Poll. Guido; Kinter A.L.; Bressler, P.; OF HIV EXPRESSION IN PERSISTENTLY INFECTED CELLS. Biswas, Priscilla*; Poli,
Biswas, P.; Stanley, S.K.; Fauci, A.S.. LIR, NIAID, NIH, Bethesda, MD G.*; Kinter, A.L.*; Bressler, P.*; Orenstein, J.M.**; Fauci, A.S**LIR, NIAID, NIH, Bethesda,
20892 USA. MD 20892 USA; "George Washington Univ., Depart. Pathol, Washington, DC USA.

Obiective: To investigate the molecular mechanisms governing HIV expression in persistently infected cells of both
T lymphocytic and monocytic origin.
Methods: Chronically infected cell lines were generated by limiting dilution cloning of the cell population surviving
the acute phase of in vitro infection with either virus stocks or molecular infectious clones of HIV-1. Virus
expression was monitored by reverse transcriptase activity assay, Northem blot, nuclear run-on and long terminal
repeat (LTR)-chloramphenicol-acetyl-transferase (CAT) assays. Mobility shift assays (MSA) followed by UV
crosslinking were performed to characterize the presence of cellular transcription factors.
Results: Certain T lymphocytic or monocytic cell lines exhibited detectable constitutive levels of HIV expression.
In these cells, baseline transcription was not sustained by NF-icB, but was found highly dependent on the Spl
transcription factor. In cell lines characterized by low to undetectable constitutive expression of HIV, viral
transcription could be induced by either phorbol myristate acetate (PMA) or tumor necrosis factor-a (TNF-a) which
have been reported to trigger HIV transcription via activation of NF-xB. Both PMA and TNF-a induced virus
expression in the Jurkat-derived T cell line J1, which was infected with a wild type HIV-1. However, PMA, but not
TNF-a, retained the ability to upregulate virus expression in a second Jurkat-derived cell line (JaK-1) which was
originally infected with an infectious molecular clone of HIV-1 lacking the NF-xB consensus sequences in its LTR.
Similarly, PMA but not TNF-a could transactivate an HIV-LTR-CAT construct which was mutagenized at the NF-KB
consensus sites and was transfected in promonocytic U1 cells. No detectable levels of NF-xB binding were seen
by MSA up to several hours after PMA stimulation whereas high levels of binding were present as early as 5 min
post-TNF-o treatment in Ul cells. Finally, transforming growth factor-P and retinoic acid significantly blocked PMAmediated, but not TNF-n-mediated induction of HIV production in Ul cells Indicating again that these two inducers
may activate virus expression by functionally distinct transcriptional pathways.
Conclusions: Expression of integrated HIV proviruses is under the control of several transcriptional pathways.
Baseline transcription of HIV in both T lymphocytic and monocytic cells was correlated with Spl binding to the HIV
LTR. TNF-a induction of HIV expression was strictly dependent upon the activation of NF-KB, whereas PMA also
activated a yet unidentified NF-rKB-independent pathway in both T lymphocytic and monocytic cells.
Guido Poli, LIR, NIAID, National Institutes of Health
9000 Rockville Pike, Bldg 10, Room 11B-13 Bethesda, MD 20892 USA
[phone]

Obiectives: To investigate the effects of IFN-y on persistently HIV infected cell lines of T lymphocytic (J1) and
promonocytic origin (U1).
Methods: U1 and Ji cells were cultured with IFN-y either alone or in the presence of phorbol myristate acetate
(PMA) or tumor necrosis factor-an/- (TNF-oa-P). Virus expression was detected by measuring reverse
transcriptase activity, ultrastructural studies, Western blot analysis of viral proteins, Northern blot analysis of HIV
RNA, long terminal repeat (LTR)-chloramphenicol-acetyl-transferase (CAT) transfection and nuclear run-on
analyses. Mobility shift assays for the presence of cellular transcription factors were also performed.
Results: IFN-y inhibited RT activity produced by unstimulated J1 and PMA-stimulated J1 and U1 cells in the
absence of inhibition of their cell-associated viral proteins. IFN-y, as well as IFN-a, redirected virion budding and
accumulation from the plasma membrane to intracytoplasmic compartments in Ul cells. IFN-y, but not IFN-a
or -3, induced virus expression in U1 cells in the absence of PMA and synergized with both TNF-o/-p and
glucocorticoid hormones in this effect. IFN-y strongly induced the accumulation of steady state HIV mRNA and
strongly synergized with TNF-a in terms of de novo viral transcription. In addition, co-stimulation of Ul cells with
IFN-y and TNF-a, but not with IFN-y and glucocorticolds, resulted in the terminal differentiation into macrophagelike cells with features of intracytoplasmic virion budding in vacuoles, and, ultimately in cell death.
Conclusions: IFN-y is a complex regulator of HIV expression capable of modulating different stages of the
retrovirus life cycle. Both suppressive (in the presence of PMA) as well as inductive effects were seen in
monocytic cells stimulated with IFN-y either alone or in combination with TNF-n or glucocorticoids.
Priscilla Biswas, LIR, NIAID, National Institutes of Health
9000 Rockville PIke, Bldg 10, Room 11B-13, Bethesda, MD 20892 USA
[phone]

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