Abstract Book Vol. 2 [International Conference on AIDS (14th: 2002: Barcelona, Spain)]

XIV International AIDS Conference

Abstracts WePeA5803-WePeA5807 27

tion to RANTES, MIP-lo, MIP-1 1 as well to IFN-y at the stage A of the disease. In
addition PBMCs from the same patients gave the same cytokine and chemokine
profile. It was recorded a progressive loss of the IFN-y, RANTES, MIP-lt, MIP1 as the infection moved to stages B and C of the disease. In addition it was
recorded a progressive increase in the production of IL-4 and IL-10 at the late
stages of the disease. We recorded a positive correlation of P-chemokines and
IFN-y. The significant finding in this study is that we found the same chemokine
and cytokine profile in PBMCs and semen in all clinical stages of the disease.
Seronegative HIV-1 individuals generated strong Thl- type cytokine response
and no chemokine responses.
Conclusion: The findings suggest that 1-chemokines have the potential to limit
HIV replication in vivo and that they do not offer any protection against the progression of the HIV-1 disease.
Presenting author: Christina Panagiotidi, National School of Public Health, 196
Alexandras Avenue, 115 21, Athens, Greece, Tel.: [phone], Fax: +301
6444870, E-mail: epidim @ otenet.gr
WePeA58O3 Cloning and functional analysis of the promoter of
hiv-1 inhibitory chemokine stromal-cell derived
factor1
C. Garcia-Moruja 1, J.M. Alonso2, M.C. Torres1, N. Gonzalez2, F.J. Luque1,
F. Arenzana-Seisdedos3, J. Alcami2, A. Caruz1. 1Area de Genetica, Dpto.
Biologia Experimental Universidad de Jaen, Jaen, Spain; 2Centro Nacional de
Microbiologia, Instituto de Salud Carlos ///, Madrid, Spain; 3lnstitute Pasteur,
Paris, France
Background: Stromal-cell derived factor (SDF1) is a CXC-type chemokine that
binds specifically to CXCR4. SDF1 is essential for the development and it has
chemotactic activities on lymphocytes and monocytes. Also SDF1 has antiviral
activity against X4 dependent HIV strains by inducing HIV co-receptor endocytosis. SDF1 is constitutively expressed in many organs but the details of its regulation are poorly understood. To elucidate the regulation of SDF1 expression we
cloned and analyzed its promoter region.
Methods: We performed a screening of human genomic DNA library and the
5'proximal region of SDF1 gene were isolated and cloned. The identification of
5'ends of mRNA were done by primer extension. A series of 10 truncated portions of the promoter were created extended from 1129 bp to 135 bp from the
translation start site of SDF1 gene. These constructs were inserted upstream
of a luciferase reporter gene and transfected by electroporation in stromal cells
(MS5), astrocytes (U373) and fibroblasts (LC5).
Cells were stimulated by a series of different stimuli and luciferase activity was
measured 24 h post-treatment.
Results: and conclusions: We have isolated a 1129 bp of the 5'-flanking region
of SDF1 gene. Two transcription start points were located at 27 and 42 bp downstream of a non-consensus TATA box. Several potential binding sites for transcription factors occurs within the sequence like IK2, E12/E47, AP1, SP1, NFAT,
Oct-1, Myo D and CACCC-BF The full length promoter exhibited strong activity in
a variety of cell lines incluiding MS5, LC5, U373 and it was partially inhibited by
y-interferon and FGF in LC5 and U373. This inhibitory effect was eliminated by
the deletion of the -1129/-711 region. PMA+ionomycin activated SDF1 promoter
in LC5 and U373 but in contrast it has an inhibitory effect in MS5 cells. ILlP induced a strong activation of the promoter in U373 cells and y irradiation induced
promoter upregulation in MS5 stromal cells.
Presenting author: Antonio Caruz, Dpto. Biologia Experimental, Edificio B-3,
Universidad de Jaen, Spain, Tel.: [phone], Fax: [phone], E-mail:
[email protected]
WePeA584 IL-8 production in patients infected with HIV-1
and/or M.tuberculosis
R. Markoval, V. Terzieva1, S. Nikolaeva1, Y. Todorova1, D. Stefanova2,
K. Kostov3. 1Natona/ Center of Infectious and Parasitic Diseases, National
Center of Infectious and Parasitic Diseases, Central Immunology Lab., 26 Yanko
Sakazov Blvd, Sofia 1504, Bulgaria; 2University Hospital for Lung Diseases,
Sofia 1606, Bulgaria; 3Hospital for Infectious Diseases, Sofia 1606, Bulgaria
Background: HIV and MTB act synergistically both clinically and pathogenically.
IL-8 plays an important role in the pathogenesis of HIV-1 disease and TB, particularly in the context of secondary microbial infections. Our aim was to study
IL-8 production as an antimicrobial chemokine in the presence of HIV-1 infection,
pulmonary TB and co-infection evaluating the spontaneous and stimulated IL-8
production.
Methods: Four groups (each of 8 pts, aged 18-52 years) that included healthy
donors, pts with pulmonary TB, pts seropositive for HIV-1 and HIV-1 pts with pulmonary TB, were enrolled. Isolated PBMCs and PMNs were stimulated with PHA
(Sigma), PPD or LPS (Sigma). The level of IL-8 in each supernatant was determined in ELISA Cytoscreen (Biosource). Neutrophil function was measured by

the NBT assay (Sigma) too. Differences in IL-8 production between groups were
compared by non-parametric Mann-Whitney U-test.
Results: No significant differences were found in spontaneously produced IL-8
in PMN cultures between different groups. Upon stimulation of PMN with PPD
IL-8 production was significantly reduced only in HIV/TB pts. The levels of spontaneously released IL-8 by PBMC cultures of infected persons were higher than
those found in cultures of healthy controls. PHA and PPD showed similar patterns

of stimulation of PBMC from different groups, but the PHA response was found
more reduced. There was a high degree of correlation (r=0.8) between PMN production of IL-8 and the Neu function measured by the NBT test in all pts.
Conclusions: Our results, even preliminary and from a small number of pts indicate that PBMC and PMN IL-8 production in HIV-1, TB and HIV/TB pts was
strongly suppressed with lowest values found in co-infected persons. Evaluation
of IL-8 production may have important implications for estimation of antimicrobial
defenses in HIV/TB pts. Further studies concerning specific anti-TB and anti-HIV
therapy are in progress.
Presenting author: Roumiana Markova, National Center of Infectious and Parasitic Diseases, Central Immunology Lab., 26 Yanko Sakazov Blvd, Sofia 1504,
Bulgaria, Tel.: [phone], Fax: [phone], E-mail: markova@ncipd.
netbg.com
WePe0A5806 1Characterization of a CD4+ T-cell Clone
Expressing both CXCR4 and CCR5 Coreceptors
S. Sanchez Palomino1, M. Bermejo 1, J. Garcia Perez1, M. Beltran1,
A. Valenzuela2, F Arenzana2, J. Alcami1. 1 Centro Nacional/de Microbiologia.
Instituto de Salud Carlos I///, Centro Nacional de Microbiologia, Instituto de Salud
Carlos I///, Ctra Majadahonda-Pozuelo km 2, Majadahonda, 28220 Madrid,
Spain; 22Unite d7mmunologie Virale. Institut Pasteur, Paris, France
Background HIV-1 entry is mediated through the interaction with CD4 and with
either CCR5 or CXCR4 coreceptors which enable to define strains according to
their tropism. HIV tropism is a clear determinant of virulence, as it has been widely
documented that a switch from R5 to X4 tropism results in CD4 decline and poor
clinical prognosis. However, testing HIV tropism remains difficult, as cell lines
lack the expression of CCR5. We have produced and characterized lymphocytic
clones derived from cell lines expressing both HIV-1 coreceptors in order to develop a target cellular model susceptible of been infected by both R5 and X4 HIV
strains.
Methods MT-2 cells, a cell line expressing CD4 and CXCR4 with high permisivity
to HIV-1 infection, were transfected by electroporation with the expression vector pcDNA3 (Neo) carrying the cDNA for the CCR5 correceptor. Transfected cells
were subcloned by limiting dilution and selected in the presence of geneticine.
Cell clones were characterized based on the expression of CCR5 level as quantified by flow cytometry using the monoclonal antibody 2D7.
Results Several clones expressing high levels of CCR5 were generated. The
SSPA-B7 clone was selected among the transfected cells because the high expression of CCR5 in 86% of the cellular population. The newly expressed CCR5
was fully functional with respect to its biochemical properties as well as an HIV
correceptor. This cellular clone exhibited a high susceptibility to infection with X4
and R5 HIV strains which induced typical cytopathic effect. HIV infection with BaL
and NL 4.3 strains was blocked by treatment with chemokines specific for CCR5
and CXCR4 receptors respectively. Taking advantage of these properties we have
been able to characterize the viral tropism of primary HIV-1 isolates from patients.
Conclusions The SSPA-B7clone provides a sensitive and stable system to isolate and characterize HIV strains with both R5 and/or X4 tropism.
Presenting author: Sonsoles Sanchez Palomino, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo km 2, Majadahonda, 28220 Madrid, Spain, Tel.: [phone]ext3948, Fax: [phone],
E-mail: [email protected]
WePeA587 Up-regulation of the LD780 isoform of MIP-la in
PBMC cultures obtained from HTLV-2 infected/
HIV-1 exposed seronegative individuals
E. Pilotti1, E. Elviri2, B. Campanini3, M. Sassi 1 E. Vicenzi4, M. Careri 2,
P. Dall'Aglio1, U. Bertazzoni5, G. Poli4, C. Casoli1. Institute of Medical
Pathology University of Parma, Parma, Italy; 2Dep. of Analytical Chemistry,
University of Parma, Parma, Italy; 3Dep. of Biochemistry and Molecular Biology
University of Parma, Parma, Italy; 4AIDS Immunopathogenesis Unit, San
Raffaele Scientific Institute, Milano, Italy; 5Dep. of Mother and Child, Biology and
Genetic section, University of Verona, Verona, Italy
Background: Through a co-cultivation system based on IL-2 stimulated PBMCs
from individuals both mono- and co-infected with HIV-1 and HTLV-2, we have
demonstrated a negative interference between these two retroviruses via upregulation of inhibitory CC-chemokines and particularly MIP-Ia, which alone
seems to account for most the anti-HIV activity (Casoli et al., Blood 95:2760,
2000). In the present study we have investigated whether HTLV-2 infection affects
the expression of the two different isoforms of MIP-1ci in spontaneous PBMC cultures obtained from three HTLV-2 infected individuals who remain HIV-1 seronegative despite multiple high risk exposures (ESN).
Methods: The MIP-1ct isoforms LD78t and LD78t were purified to homogeneity from PBMC cultures of the three HTLV-2/HIV-1 ESN subjects by a multistep procedure isolation and identified by RP-HPLC-Electrospray or MALDI-TOF

Mass Spectrometry. gpl20Env-C5 peptide-driven MIP-lot isoforms production by
PBMC short-term cultures were measured by immunoassay.
Results: From PBMC-derived supernatants, Electrospray and MALDI-TOF-MS
analysis provided evidence of co-existence of LD78 a and LD78P. Quantitative
analysis from MALDI-TOF mass spectra of isoforms purified from HTLV-2/HIV-1
ESN individuals revealed that the f form was preferentially expressed (1.6 fold).
Stimulation of PBMCs with gpl20-C5 peptide induced the release of both iso