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

274 Abstracts 21176-21180

12th World AIDS Conference

a different effect on CD4+ cell proliferation and chemokine production.
Methods: NSI and SI viruses were grown in peripheral blood mononuclear cells
and titered for TCID50. A low amount of virus was inoculated onto PHA-stimulated
CD4+ cells and the effects on cell proliferation and production of beta-chemokines
(RANTES, MIP-la, MIP-1b) were measured at the peak of virus replication (7-10
days). Cell proliferation was monitored by thymidine incorporation and cell surface markers as well as changes in viable cell number. Replication of virus was
measured by reverse transcriptase activity. Chemokine production was assessed
by ELISA for protein and Northern blot analyses for mRNA; intracellular cytokine
levels were measured using monoclonal antibodies. Virus-infected cells were
enumerated by FACS analysis using antibody to HIV p24 antigen.
Results: Infection by NSI isolates of HIV-1 enhanced cell proliferation at
least 2 fold and increased production of all 3 beta-chemokines 3-6 fold when
compared to control uninfected cultures. The chemokine increase was mirrored
by an enhanced expression of chemokine mRNA and intracellular protein. Both
virus-infected (p24) and uninfected cells had these findings on chemokine expression. During high level replication of SI viruses, cell proliferation and chemokine
production per viable cell were reduced.
Conclusions: NSI and SI viruses affect cell proliferation and beta-chemokine
production differently. Since beta-chemokines block NSI infection, the association
of enhanced beta-chemokine production with NSI virus replication does not support a major role of these chemokines in controlling HIV replication. The increased
production could be involved in enhancing an inflammatory response following
infection by NSI HIV strains.
221*/21176 Patterns of cytokine production analysed at single
cell level during highly active antiretroviral therapy
(HAART)
Rui Victorino1, Ana Sousa2, A.F. Chaves2, A.C. GraCa2, M. Doroana3,
F. Antunes3, R.M.M. Victorino4. 1 Medicinaz Piso2 Faluldade De Medicina AV
Prof Egas Moniz 1600 Lisbon; 2Cel Immunology Lab Faculty of Medicine Lisbon;
3Dep Infectious Diseases FML-HSM Lisbon; 4Medicinez-Cel Immunology Lab
FML-HSM Lisbon, Portugal
Background: We have previously documented alterations in the potential for cytokine production at single cell level by flow cytometry in HIV1 infected subjects.
Here we investigated the effect of triple antiretroviral therapy in the patterns of
cytokine production at single cell level by flow cytometry.
Methods: Peripheral blood mononuclear cells from 14 HIV1 infected patients
were intracytoplasmicaly stained for IL2, IFNy, IL4 and IL10 after a 4 hour culture
in the presence of PMA+I and brefeldin A, and the frequency of cytokine producing cells was assessed by flow cytometry in total T cells, CD4, CD8, CD28 and
CD45RO subsets at day 0 and 2, 4, 8, 16, 24 and 32 weeks after therapy with
nelfinavir, lamivudine and stavudine.
Results: HAART induced drops in viral load over one log in all patients which
reached levels below 400 copies/ml in 12 patients and significant increases in
CD4 T cell counts in all patients. The proportion of IL2 producing T cells increased at week 4 and this increase was more marked within the CD8 T cell
subset. Curiously, there was an increase in the percentage of T cells producing
IL4 mainly due to cells with a ThO phenotype (IL4+IFNy+) at week 4 in all but
two patients who exhibited very high levels of IL4 production at time zero and a
marked eosinophilia. The increase in IL4+ cells was much more obvious within
the CD45RO subset and persists at week 24. Interestingly, the percentage of
IFNy producing CD8 T cells, which we have previously shown to be markedly
increased in HIV1+ patients, was unaltered during therapy and this is mainly due
to the persistency of the expansion of CD28 negative CD8 T cells producing
IFNy. In contrast, IFNy producing CD4+ cells increased slightly at week 4 and
this was maintained in the following weeks of the study.
Conclusion: The effects of HAART on the potential for cytokine production
analysed at single cell level by flow cytometry are not consistent with a simple Thl
to Th2 shift model and, furthermore, no significant alteration in the CD8 cytokine
production disturbances was observed.
1 21177 Semiquantitation of human chemokine nmRNAs by
reverse transcription/competitive PCR using a newly
constructed multispecific competitor
Franz Ludwig Dumoulin, Markus Alfredo, Jurgen K. Rocustroh,
Tilman Sauerbruch, Ulrich Spengler. Dept. of Medicine, University of Bonn,
Sigmund Freud Str. 25, 53127 Bonn, Germany
Background/Objectives: Chemokines are importantly involved in the pathogenesis of HIV infection. To facilitate further research, we have constructed a
multispecific competitor fragment for use in reverse transcription/semiquantitative
polymerase chain reaction (PCR).
Methods: With three successive rounds (25 cycles each) of PCR using composite primers and a high fidelity enzyme we added primer binding sites for
human MCP-1, MCP-2, MCP-3, MIPla, MIP2a, RANTES and /3-actin to linker
sequences from v-erbB. The primer positions on the construct were designed to

yield amplicons which were approximately 100 bp longer than amplicons from the
wild type sequence.
Results: Sequence analysis confirmed the correct positioning of the different
primer binding sites on the competitor fragment. Further validation showed that
co-amplification of reverse transcribed RNA from stimulated peripheral blood
mononucleated cells along with the multispecific competitor fragment resulted in
PCR products readily separated by agarose electrophoresis. The relative band

intensities from competitor and cDNA amplicon can be quantitated directly from
agarose gels using video-camera imaging and a densitometry software system; in addition, semiquantitive comparison of the levels of reserse transcribed
chemokine mRNA is possible by normalization relative to the housekeeping gene
p-actin.
Conclusion: The constructed multispecific competitor fragment should be helpful in further studies on the role of chemokines in the pathogenesis of HIV infection
in small tissue specimens
21178 1 Correlation between HIV RNA and levels of cytokines,
chemokines and soluble factors in the plasma
Gillese Hittinger1, A. Lafeuillade2, A. Djediouane2, C. Poggi3, N. Profizi3,
0. Costes2. Unite Infectiologie, Hopital Chalucet, Toulon; 2Hopital Chalucet,
Toulon; 3 Hopital Toulon-La Seyne, Toulon, France
Objectives: to evaluate the correlation between the levels of HIV-1 replication in
plasma and those of circulating cytokines, chemokines and soluble factors.
Design: 80 patients naive of antiretroviral therapy have been analysed once.
Methods: Patients did not have recent concurrent infections. HIV-1 RNA was
quantitated using the Roche PCR kit and levels of cytokines, chemokines and
soluble factors by ELISA. We measured tumor necrosis factor a (TNF-u) and
its soluble receptor type 2 (sRII-TNF-u) levels, soluble receptor of Interleukin 4
(sR-IL4), Interleukin 6 (IL6) and its soluble receptor (sR-IL6) levels, RANTES,
macrophage inhibitory protein 1 alpha and beta (MIP-la, MIP-1p).
Results: mean CD4 count was 437 ~ 34 cells/p/L, mean plasma RNA was
4.52 ~ 0.09 copies/ml. Log plasma RNA was correlated with TNF-a (r = 0.31, p
= 0.005) and sRII-TNF-a levels (r = 0.40, p < 0.001). No correlation was found
between plasma RNA and sR-IL4, IL-6 or sR-IL6. High plasma RANTES levels
were found in most cases and mean values were higher in patients with <500
CD4 cells//jL (p = 0.02). However, plasma RANTES did not correlate with plasma
RNA. RANTES levels were correlated with CD8 cells in patients with <200 CD4
cells and with platelet counts in patients with >200 CD4 cells. MIP-1a did not
correlate with plasma RNA. MIP-la was correlated with CD4 cells. MIP-1i was
correlated with plasma RNA in patients with <200 CD4 cells. Neither MIP-1a nor
MIP-1p were correlated with CD8 cells or platelet counts.
Conclusion: this study shows a correlation between the TNF-a system and
levels of HIV-1 replication in plasma. However, this statistically significant correlation is not sufficient to establish a cause-effect relationship and further studies
are needed. On the contrary, no correlation was found for other markers, like
levels of chemokines which have been shown capable to inhibit viral growth in
vitro.
21179 | TNFa production by alveolar macrophages from
HIV-seropositive patients is modulated through
NF-KB
PaulR. Skolnik, Jean-Marie Mathys, J.M. Mathys, P.R. Skolnik. TUFTS-New
England Medical Center, 750 Washington St., NEMC 67 Boston, MA, USA
Objective: To assess the effects of chemical protease inhibitors on the production of the proinflammatory cytokine TNFa by alveolar macrophages (AM) and
peripheral blood mononuclear cells (PBMC) from HIV-infected persons.
Methods: PBMC, obtained by venipuncture, and AM obtained by bronchoalveolar lavage (BAL) from HIV-infected persons (N = 5), were cultured for 16 hours
before addition of LPS (10ng/ml), and the chemical protease inhibitors TPCK
(N-tosyl-L-phenylalaninechloromethylketone) (1-1001 g/ml), BTEE (N-benzoylL-tyrosine ethylester)(1-100 tpg/ml), or D609 (50p/M). TNFa protein in supernatant
fluids was measured by ELISA. Total TNFa and NF-KB mRNA were determined
by PCR ELISA with actin as an internal standard. NF-KB activity was determined
by gel shift assay. To assess whether NF-KB activity in AM is dependent on the
phospholipase C (PLC) pathway, D609, a specific PLC inhibitor, was added to
the cultures.
Results: Addition of the inhibitors to LPS-stimulated AM and PBMC resulted
in complete suppression of TNFa production in AM and PBMC at the concentrations tested. LPS stimulation induced an increase in NF-KB activity which was
inhibited by either TPCK or BTEE. The level of TNFa mRNA remained constant
in the presence of the inhibitors. KB mRNA was unchanged after incubation with
the inhibitors, but NF-KB activity as measured by gel shift assay was reduced.
Addition of D609 resulted in a decrease in TNFu and NF-K-B activity expression
in AM and PBMC.
Conclusions: These data suggest that, as previously shown in peripheral blood
mononuclear cells, TNFo mRNA expression in LPS-stimulated AM is mediated
via NF-KB and that NF-KB activity is modulated, at least in part, by the PLC
signaling pathway.
S21180 Early up-regulation of TNF-a in naive patients treated
with potent anti-HIV therapies
Vicenzo Vullo, C.M. Mastoianni, F. Mengoni, P. Santopadre, M. Lichtner,

C. D'Agostino, A. Corpolongo. Dept of Infect Trop Diseases, La Sapienza
University, Policlinico Umberto I 00161, Rome, Italy
Background: Short term administration of highly active antiretroviral treatment
(HAART) increased circulating memory CD4+ cells and naive CD8+ cells, ameliorated immune activation and enhanced CD4+ lymphocyte function. The early
cytokine response by the innate immune system has not yet evaluated.