Biomedical subjects

B Barbeau

Publications and source records attributed to B Barbeau.

At least 19 recordsLinked to original sources

PubMed2005

The impact of selected water quality parameters on the inactivation of Bacillus subtilis spores by monochloramine and ozone.

Selected water quality parameters-pH, dissolved organic carbon, turbidity (NTU), and temperature-were tested for their potential effects on ozone and monochloramine inactivation of Bacillus subtilis spores. In oxidant demand-free phosphate-buffer, temperature had the strongest influence on inactivation kinetics when using ozone, pH had a smaller but significant impact on B. subtilis spore inactivation with both monochloramine and ozone. Where monochloramine was applied, modeling and experimental measurements confirmed that dichloramine levels were too low to produce significant inactivation effects under these experimental conditions. It was demonstrated that oxidant demand-free phosphate buffer may not be an adequate environmental analogue for inactivation responses in natural waters.

Bacillus subtilis↗

PubMed2003

Regulation of procollagen I (alpha1) by interleukin-4 in human bronchial fibroblasts: a possible role in airway remodelling in asthma.

BACKGROUND: In bronchial mucosa, T cells are in close association with fibroblasts. This cell contact raises the possibility of cross-talk between the two cell types through cytokines, such as interleukin-4 (IL-4). OBJECTIVE: We postulated that IL-4 may modulate collagen synthesis and degradation in the fibroblasts of asthmatics. METHODS: Bronchial fibroblasts from asthmatics (BAF) and normal controls (BNF) were stimulated with IL-4. Procollagen I gene expression and protein production were measured by real-time PCR, RT-PCR, and radioimmunoassay. The effect of IL-4 on the regulation of procollagen I (alpha1) promoter was studied through transient cell transfections. The implication of Sp1 and AP-1 in regulating IL-4-induced procollagen I (alpha1) production was determined. The effect of IL-4 on metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) production and gene expression was evaluated. RESULTS: Following IL-4 stimulation, there was a significant increase in the expression of mRNA of procollagen I (alpha1) by human bronchial fibroblasts of asthmatics and controls. IL-4 has a dose-response effect on mRNA, with a maximal effect at 5 ng/mL, as determined by real-time PCR. The maximal increase in procollagen I (alpha1) was observed at 6 h after IL-4 stimulation in both BNF and BAF. BAFs have a greater increase in the procollagen I (alpha1)/beta2 microglobulin ratio after 6 h of IL-4 stimulation (4.1 x 10-2+/-0.03 to 20.8 x 10-2+/-0.1) compared with BNF (2.9 x 10-2+/-0.006 to 9.2 x 10-2+/-0.08) (P=0.001). In transient transfection experiments, IL-4 increased promoter activity by threefold in BAF and BNF. Sp1 was up-regulated after IL-4 stimulation and AP-1 was down-regulated as shown by electrophoretic mobility shift assay. IL-4 decreased MMP-2 protein and mRNA levels, and did not alter TIMP-2 production. CONCLUSIONS: IL-4 positively regulates procollagen I (alpha1) transcription by direct promoter activation and increases the TIMP-2/MMP-2 ratio, thereby supporting the profibrotic effect of this cytokine. Thus, this study emphasizes that IL-4 may be considered as a link between inflammation and collagen deposition observed in asthmatic airways.

Asthma↗

PubMed2003

Evaluation of Bacillus subtilis and coliphage MS2 as indicators of advanced water treatment efficiency.

The assessment of water treatment facilities for their efficiency using alternate indicators is of paramount importance. Current methods for assessing efficiency are limited by the specific characteristics of the microorganisms, such as their different sensitivities to disinfectants. A pilot study was carried out to compare different treatment scenarios for the future upgrade of the Sergio Cuevas Water Treatment plant (the largest in the Caribbean) in San Juan, Puerto Rico. The treatment units under investigation included a coagulation-flocculation-sedimentation unit, dual-media filters, micro-filtration units, intermediate ozone injection and contact columns as well as a biological filtration unit. The plant was challenged at different stages of treatment with Bacillus subtilis spores and MS2 coliphages in an attempt to test them as possible alternate indicators of treatment plant performance. These organisms were chosen because of their resistance to disinfection and desiccation, their low analysis costs and ease of detection. The removal of spores and coliphages by each treatment unit tested was calculated by seeding a known concentration (5-7 log10) of spores and coliphages and following the removal or disinfection rates. The seeded indicators were detected using traditional culture techniques. Ballasted clarification was shown to be highly efficient at removing 99.1% (approximately 3 log10) of the spores and 85.1% (approximately 0.86 log10) of MS2. Ozone treatment inactivated 80.37% (approximately 1.4 log10) spores and 99.95% (approximately 3.07 log10) coliphages. The coliphage inactivation rate obtained confirmed data obtained by previous studies indicating that MS2 was less resistant to ozonation than B subtilis spores. The membrane technology had the best efficiency in terms of physical removal of spores achieving over 99.9% (> 3 log10) removal. Coliphage removal mechanisms remain to be determined and will be a future focus of the study. Preliminary results indicate that aerobic spores and coliphages may be useful as indicators to determine the efficiency of different drinking water treatment technologies.

Bacillus subtilis↗

PubMed2001

Negative regulation of the NFAT1 factor by CD45: implication in HIV-1 long terminal repeat activation.

HIV-1 gene regulation is greatly dependent on the presence of the -104/-81 enhancer region which is regulated by both NF-kappaB and NFAT transcription factors. We have found that a greater induction in HIV-1 long terminal repeat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulation of a CD45-deficient cell line (J45.01) in comparison to the parental Jurkat cells. Unlike NF-kappaB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transcriptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-induced NFAT activation, NFAT translocation and calcium influx peaked at similar time points for both Jurkat and J45.01 cell lines. The NFAT-dependent promoters from the IL-2 and TNF-alpha genes were also more potently activated by PMA/Iono in J45.01 cells. Interestingly, higher levels of intracellular calcium were consistently demonstrated in PMA/Iono-induced CD45-deficient cell lines (J45.01 and HPB45.0). Furthermore, PMA/Iono induction of calcium mobilization in both Jurkat and J45.01 cell lines was observed to be EGTA-sensitive. Mechanistic studies revealed that CD3zeta and ZAP-70 were more heavily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was present at higher levels in J45.01 nuclear extracts and that the NFAT1 member was predominant. In conclusion, our results indicate that NFAT activation by stimuli acting in a more distal fashion from the TCR-mediated signaling pathway can be down-regulated by CD45 and that this CD45-dependent regulation in turn affects HIV-1 long terminal repeat activation.

Calcium Signaling↗

PubMed2001

Neuraminidase from a bacterial source enhances both HIV-1-mediated syncytium formation and the virus binding/entry process.

Neuraminidases, also termed sialidases, which catalyze the removal of sialic acid residues from various glycoconjugates, have been previously reported to modulate HIV-1 replication. Given that some of the known opportunistic microbes found in patients infected with HIV-1 harbor neuraminidase (NA) activity, we speculated that pathogen-derived NA might be envisaged as an important factor in the pathogenesis of this retroviral infection. In the present study, we have monitored the putative modulation of HIV-1-mediated syncytium formation and virus replication by highly purified bacterial-derived NA from Arthrobacter ureafaciens. Taking advantage of a luciferase-based syncytium quantitative assay, we demonstrate here that the level of HIV-1-mediated syncytium formation is enhanced in the presence of NA and that it necessitates interaction between gp120 and CD4/chemokine coreceptor. By using pseudotyped recombinant luciferase-encoding HIV-1 particles, we found that NA treatment of human CD4-positive target cells (i.e., T lymphoid, monocytoid, and peripheral blood mononuclear cells) significantly augmented single-round infection by T- and macrophage-tropic isolates of HIV-1. The observed increase in HIV-1 infection was linked with an enhancement in the initial steps of the virus replicative cycle as monitored by viral binding and entry assays. Interestingly, NA treatment also enhances infectivity of HIV-1 pseudotypes with envelope glycoprotein from the amphotropic murine leukemia virus or the vesicular stomatitis virus. Taken together, our results provide useful information regarding the possible contribution of microbial agents carrying NA activity to HIV-1 pathogenesis.

Arthrobacter↗

PubMed2001

Regulation of nuclear factor of activated T cells by phosphotyrosyl-specific phosphatase activity: a positive effect on HIV-1 long terminal repeat-driven transcription and a possible implication of SHP-1.

Although protein tyrosine phosphatase (PTP) inhibitors used in combination with other stimuli can induce interleukin 2 (IL-2) production in T cells, a direct implication of nuclear factor of activated T cells (NFAT) has not yet been demonstrated. This study reports that exposure of leukemic T cells and human peripheral blood mononuclear cells to bis-peroxovanadium (bpV) PTP inhibitors markedly induce activation and nuclear translocation of NFAT. NFAT activation by bpV was inhibited by the immunosuppressive drugs FK506 and cyclosporin A, as well as by a specific peptide inhibitor of NFAT activation. Mobility shift assays showed specific induction of the NFAT1 member by bpV molecules. The bpV-mediated NFAT activation was observed to be important for the up-regulation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) and the IL-2 promoter; NFAT1 was demonstrated to be particularly important in bpV-dependent positive action on HIV-1 LTR transcription. The active participation of p56(lck), ZAP-70, p21(ras), and calcium in the bpV-mediated signaling cascade leading to NFAT activation was confirmed, using deficient cell lines and dominant-negative mutants. Finally, overexpression of wild-type SHP-1 resulted in a greatly diminished activation of NFAT by bpV, suggesting an involvement of SHP-1 in the regulation of NFAT activation. These data were confirmed by constitutive NFAT translocation observed in Jurkat cells stably expressing a dominant-negative version of SHP-1. The study proposes that PTP activity attenuates constitutive kinase activities that otherwise would lead to constant NFAT activation and that this activation is participating in HIV-1 LTR stimulation by PTP inhibition.

Active Transport, Cell Nucleus↗

PubMed2001

Inhibition of HIV-1-mediated syncytium formation and virus replication by the lipophosphoglycan from Leishmania donovani is due to an effect on early events in the virus life cycle.

Previous findings have indicated that the major surface molecule of Leishmania, lipophosphoglycan (LPG), could abrogate HIV-1-induced syncytium formation and virus replication. In the present work, we were interested in characterizing this inhibitory process. Data from a new luciferase-based semiquantitative assay for syncytium formation, relying on the coincubation of a T-cell line containing an HIV-1 LTR-driven luciferase construct with a cell line chronically infected with HIV-1, confirmed that LPG was indeed a strong inhibitor of HIV-1-dependent syncytium formation and that this inhibition was dose-dependent. As determined by flow cytometric analyses, this inhibition was not apparently due to downregulation of CD4, CXCR4 or LFA-1, three distinct surface glycoproteins known to be important in HIV-1 mediated syncytium formation. Furthermore, LPG did not seem to affect signal transduction pathways in T cells as judged by measurement of HIV-1 LTR-driven reporter gene activity upon treatment with different stimuli. However, pretreatment of either of the cell lines used in the assay with LPG led to a significant decrease of virus-mediated syncytium formation, which was further accentuated when both cell lines were pretreated. LPG inhibition of HIV-1 replication was next assessed. When measuring either infection with luciferase-encoding recombinant HIV-1 particles or multinucleated giant cell formation following an acute virus infection, we again observed that LPG was efficient at blocking HIV-1 replication. Specific assays probing different steps of viral entry demonstrated that attachment was not hindered by LPG but that viral entry was modulated, suggesting that LPG targets a postbinding step. Hence, incorporation of LPG into a target cell membrane could influence its fluidity and diminish both the virus-cell and cell-to-cell fusion processes initiated by HIV-1.

Animals↗

PubMed2000

Presence of new alternative exons in human and mouse Fli-1 genes.

The mouse Fli-1 proto-oncogene is activated by proviral integration of four murine leukemia retroviruses and its human counterpart is translocated (11,22) in Ewing tumors. We have identified two alternative exons 1 by RACE analysis from a human neuroectodermal tumor. Exons 1a and 1b are located respectively 1.3 and 2.5 kb upstream from the published exon 1. Translation of these alternative messengers is predicted to generate very similar proteins. The sequence upstream from exon 1b showed functional promoter activity. Exon 1b was not conserved in the mouse but was detected in every analyzed human cell, whereas exon 1a was present only in a subset of them and also in various mouse cell lines. These results suggest that both mouse and human Fli-1 gene expression might be under the control of several independent promoter regions.

Alternative Splicing↗

PubMed2000

Occurrence of pathogenic microorganisms in the Saint Lawrence River (Canada) and comparison of health risks for populations using it as their source of drinking water.

A 300-km portion of the Saint Lawrence hydrological basin in the province of Québec (Canada) and 45 water treatment plants were studied. River water used by drinking water treatment plants was analyzed (6-L sample volumes) to determine the level of occurrence of bacterial indicators (total coliforms, fecal coliforms, and Clostridium perfringens) and pathogens (Giardia lamblia, Cryptosporidium, human enteric viruses). Pathogens and bacterial indicators were found at all sites at a wide range of values. Logistic regression analysis revealed significant correlations between the bacterial indicators and the pathogens. Physicochemical and treatment practices data were collected from most water treatment plants and used to estimate the level of removal of pathogens achieved under cold (0 degree C-4 degrees C) and warm (20 degrees C-25 degrees C) water temperature conditions. The calculated removal values were then used to estimate the annual risk of Giardia infection using mathematical models and to compare the sites. The estimated range of probability of infection ranged from 0.75 to less than 0.0001 for the populations exposed. Given the numerous assumptions made, the model probably overestimated the annual risk, but it provided comparative data of the efficacy of the water treatment plants and thereby contributes to the protection of public health.

Animals↗

PubMed1999

p56(lck), ZAP-70, SLP-76, and calcium-regulated effectors are involved in NF-kappaB activation by bisperoxovanadium phosphotyrosyl phosphatase inhibitors in human T cells.

This study investigates the second messengers involved in NF-kappaB activation by the bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors. We first initiated a time course analysis of bpV-mediated activation of the human immunodeficiency virus type-1 long terminal repeat- and NF-kappaB-driven reporter gene. Our results showed a slower and more transient activation of both kappaB-regulated luciferase-encoding vectors by bpV compounds when compared with the action of tumor necrosis factor-alpha (TNF). Time course analyses of NF-kappaB translocation by shift assay experiments further confirmed these results, hence implying distinct pathways of NF-kappaB activation for bpV compounds and TNF. Attempts to characterize the bpV-dependent signaling cascade revealed that the src family protein tyrosine kinase p56(lck) was critical for NF-kappaB induction by bpV. Furthermore, p56(lck) interaction with the intracytoplasmic tail of CD4 markedly enhanced such induction. Optimal activation of NF-kappaB following bpV treatment necessitated downstream effectors of p56(lck) such as the syk family protein tyrosine kinase ZAP-70 and the molecular adaptor SLP-76. Importantly, reduced NF-kappaB activation was observed when capacitative calcium entry was deficient but also upon pharmacological inhibition of calmodulin and calcineurin. Altogether, these results suggest that induction of NF-kappaB by phosphotyrosyl phosphatase bpV inhibitors necessitates both proximal and distal effectors of T cell activation.

Adaptor Proteins, Signal Transducing↗

PubMed1999

The GATA-1 and Spi-1 transcriptional factors bind to a GATA/EBS dual element in the Fli-1 exon 1.

Fli-1 is a proto-oncogene which is rearranged in tumors induced by three different retroviruses, Cas-Br-E, F-MuLV, and 10A1. This gene is a member of the Ets gene family, a class of transcription factors that recognize and bind to a DNA motif known as the Ets binding site (EBS). Our laboratory has previously cloned and characterized the promoter region of both human and mouse Fli-1 genes. We had then identified several regulatory elements conserved between the two species. Two of them, an exon 1 GATA/EBS dual element and an EBS element located in the 5' end of intron 1, were analysed in the present study. EMSA analysis performed with nuclear extracts from different cell lines showed that the EBS element in intron 1 (EBSi) was bound by one potential Ets-related ubiquitous factor. The GATA/EBS element was bound by several factors that seemed Ets-related, one of which was found to be specifically expressed in hematopoietic cells. the GATA/EBS dual element was thus chosen for further analysis. A human Fli-1-derived genomic fragment containing the GATA/EBS led to enhanced transcription when positioned upstream of the SV40 promoter in the erythroleukemic HEL cell line. In addition, an increasing number of GATA/EBS oligonucleotides upstream of this same promoter resulted in a copy number-dependent increase in luciferase activity which was greatly reduced when the EBS consensus sequence was mutated. One of the factors binding to the GATA/EBS region was identified to be Spi-1 by supershift analysis and was also shown to bind to the EBS element of the human Ets-2 gene. Supershift analysis also demonstrated the binding of the GATA-1 factor to the GATA/EBS dual element. Our results suggest that Spi-1 and GATA-1 might play a key role in the regulation of Fli-1.

Animals↗

PubMed1999

Role of the leukocyte function antigen-1 conformational state in the process of human immunodeficiency virus type 1-mediated syncytium formation and virus infection.

Human immunodeficiency virus type 1 (HIV-1)-mediated syncytium formation is recognized as being highly dependent on intercellular adhesion molecule (ICAM)-1-leukocyte function-associated molecule 1 (LFA)-1 interaction, whereas the process of infection with cell-free virions is independent of such complementary interaction. Our group has recently demonstrated that an antibody-mediated induction of the high affinity state of LFA-1 for ICAM-1 renders target T cells more prone to HIV-1-dependent syncytium formation and infection by ICAM-1-bearing virions. To further substantiate these results, we made use of mutant cell lines expressing LFA-1 in either a low (parental HPB-ALL and HAmut) or a high affinity state for ICAM-1 (HAP4) and compared syncytium formation and virus infection. Cells expressing the activated form of LFA-1 were found to be more susceptible to HIV-1-induced syncytium formation and to infection by ICAM-1-bearing HIV-1 particles. The observed increase was solely due to the LFA-1 activation state because it was abrogated by anti-LFA-1 or anti-ICAM-1 antibodies and not due to variations in surface expression of LFA-1, CD4, or the chemokine coreceptor CXCR4. However, a linear relation was seen between the level of CXCR4 surface expression and susceptibility to syncytium formation/virus infection when ICAM-1-LFA-1 interaction was either absent (i.e., infection with ICAM-1-negative virions) or abrogated (treatment with anti-LFA-1 or anti-ICAM-1 antibodies). These results emphasize the important role of the LFA-1 activation state with respect to virus-induced syncytium formation and HIV-1 infection.

Cell Line↗

PubMed1999

LIVE/DEAD BacLight : application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water.

A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit (BacLight) was applied to estimate both viable and total counts of bacteria in drinking water. BacLight is composed of two nucleic acid-binding stains: SYTO 9 and propidium iodide. SYTO 9 penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal incubation conditions were found to be 15 to 20 min, at room temperature in the dark. Total (red + green) and viable (green) cells can hence be counted simultaneously. Factors affecting the staining procedure were tested (addition of glutaraldehyde, staining time, chlorine impact). In the absence of stress, BacLight viable counts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. BacLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, growth rate or temperature) induced a decrease in viability that was more pronounced for CTC and plate counts than for BacLight viable counts.

Chlorine↗

PubMed1998

Prostaglandin E2 Up-regulates HIV-1 long terminal repeat-driven gene activity in T cells via NF-kappaB-dependent and -independent signaling pathways.

Replication of human immunodeficiency virus type-1 (HIV-1) is highly dependent on the state of activation of the infected cells and is modulated by interactions between viral and host cellular factors. Prostaglandin E2 (PGE2), a pleiotropic immunomodulatory molecule, is observed at elevated levels during HIV-1 infection as well as during the course of other pathogenic infections. In 1G5, a Jurkat-derived T cell line stably transfected with a luciferase gene driven by HIV-1 long terminal repeat (LTR), we found that PGE2 markedly enhanced HIV-1 LTR-mediated reporter gene activity. Experiments have been conducted to identify second messengers involved in this PGE2-dependent up-regulating effect on the regulatory element of HIV-1. In this study, we present evidence indicating that signal transduction pathways induced by PGE2 necessitate the participation of cyclic AMP, protein kinase A, and Ca2+. Experiments conducted with different HIV-1 LTR-based vectors suggested that PGE2-mediated activation effect on HIV-1 transcription was transduced via both NF-kappaB-dependent and -independent signaling pathways. The involvement of NF-kappaB in the PGE2-dependent activating effect on HIV-1 transcription was further confirmed using a kappaB-regulated luciferase encoding vector and by electrophoretic mobility shift assays. Results from Northern blot and flow cytometric analyses, as well as the use of a selective antagonist indicated that PGE2 modulation of HIV-1 LTR-driven reporter gene activity in studied T lymphoid cells is transduced via the EP4 receptor subtype. These results suggest that secretion of PGE2 by macrophages in response to infection or inflammatory activators could induce signaling events resulting in activation of proviral DNA present into T cells latently infected with HIV-1.

Calcium↗

PubMed1998

The lipophosphoglycan of Leishmania donovani up-regulates HIV-1 transcription in T cells through the nuclear factor-kappaB elements.

We have recently demonstrated that the parasite Leishmania donovani and its surface molecule, lipophosphoglycan (LPG), can activate HIV-1 replication in monocytoid cells. Our present interest was to determine whether LPG could also up-regulate HIV-1 transcription in T cells. Using a CD4-positive human lymphoid T cell line (1G5) containing a stably integrated HIV-1 long terminal repeat (LTR)-luciferase construct, we found that LPG is a potent inducer of HIV-1 LTR activity. Treatment of 1G5 cells with signaling antagonists revealed that protein tyrosine kinase- and protein kinase A-dependent pathways were actively participating in the LPG-induced enhancement of HIV-1 LTR-driven activity. Transfection of Jurkat E6.1 cells with plasmids containing wild-type and nuclear factor-kappaB (NF-kappaB)-mutated HIV-1 LTR-luciferase constructs has suggested a role for NF-kappaB binding sites in the LPG-mediated induction of HIV-1 LTR activity. An LPG-induced binding factor specific to the NF-kappaB consensus sequences could be observed using electrophoretic mobility shift assay. Finally, transfection experiments performed with a vector containing HIV-1 kappaB binding sites only showed similar LPG-mediated induction, which was abrogated by sodium salicylate, a known NF-kappaB inhibitor. We thus demonstrate that the LPG-mediated induction of HIV-1 LTR activity in T cells involves several second messengers culminating in activation of HIV-1 LTR-driven transcription via NF-kappaB-binding consensus sequences. In conclusion, these results reinforce the idea that L. donovani is a putative cofactor in HIV-1 pathogenesis.

Animals↗

PubMed1998

Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kappaB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells.

Tuberculosis has emerged as an epidemic, extended by the large number of individuals infected with human immunodeficiency virus type 1 (HIV-1). The major goal of this study was to determine whether the mycobacterial cell wall component mannose-capped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis (M. tuberculosis) could activate transcription of HIV-1 in T cells with the use of an in vitro cell culture system. These experiments are of prime importance considering that CD4-expressing T lymphocytes represent the major virus reservoir in the peripheral blood of infected individuals. Using the 1G5 cell line harbouring the luciferase reporter gene under the control of the HIV-1 LTR, it was first found that culture protein filtrates (CFP) from M. tuberculosis or purified ManLAM could activate HIV-1 LTR-dependent gene expression unlike similarly prepared CFP extracts devoid of ManLAM. The implication of protein tyrosine kinase(s), protein kinase A and/or protein kinase C was highlighted by the abrogation of the ManLAM-mediated activation of HIV-1 LTR-driven gene expression using herbimycin A and H7. It was also determined, using electrophoresis mobility shift assays, that M. tuberculosis ManLAM led to the nuclear translocation of the transcription factor NF-kappaB. M. tuberculosis ManLAM resulted in clear induction of the luciferase gene placed under the control of the wild-type, but not the kappaB-mutated, HIV-1 LTR region. Finally, the ManLAM-mediated activation of HIV-1 LTR transcription was found to be independent of the autocrine or paracrine action of endogenous TNF-alpha. The results suggest that M. tuberculosis can upregulate HIV-1 expression in T cells and could thus have the potential to influence the pathogenesis of HIV-1 infection.

Binding Sites↗

PubMed1998

Modulation of human immunodeficiency virus type 1-induced syncytium formation by the conformational state of LFA-1 determined by a new luciferase-based syncytium quantitative assay.

The ICAM-1/LFA-1 interaction has been clearly demonstrated to play an active role in syncytium formation induced by human immunodeficiency virus type 1 (HIV-1). Since it is known that a high-affinity state of LFA-1 for ICAM-1 can be induced through conformational change, such a high-affinity state may also contribute to the process of syncytium formation. In this study, we have investigated the involvement of the conformational status of LFA-1 in HIV-1-dependent syncytium formation by using the anti-LFA-1 antibody NKI-L16, which is known to activate the high-affinity state. Initial visual observations by light microscopy indeed suggested that the addition of the NKI-L16 antibody led to bigger and more numerous syncytia when different cell lines were tested. To further analyze this NKI-L16-dependent increment of syncytium formation in a quantitative assay, a new luciferase-based assay was developed by using a T-cell line containing an HIV-1 long terminal repeat (LTR)-driven luciferase construct (1G5) in coincubation with an HIV-1-positive cell line (J1.1). Upon fusion, the viral Tat protein could diffuse to the 1G5 cells, leading to a transcriptional increase of the HIV-1 LTR-driven luciferase gene. Initial evaluation of this assay showed a good correlation between the level of syncytium formation determined by microscopic observation and the level of measured luciferase activity. In addition, this assay showed a greater induction of enzymatic activity correlating with syncytium formation in comparison to a similar incubation with the HeLa-CD4-LTR-beta-gal indicator cell line. By using this test, NKI-L16 treatment of 1G5/J1.1 cells led to a three- to sevenfold increase in HIV-1 LTR-driven luciferase activity. The syncytium-dependent luciferase activity in NKI-L16-treated cells could be blocked by classical syncytium inhibitors such as soluble CD4, anti-CD4, and anti-gp120 antibodies. Inhibition could also be observed with specific blocking agents for the chemokine receptor CXCR4, as well as with soluble ICAM-1, anti-LFA-1, anti-ICAM-1, and anti-ICAM-2 blocking antibodies, indicating the requirement for the LFA-1/ICAM interaction. Treatment of peripheral blood mononuclear cells with NKI-L16 resulted in a higher level of syncytium formation in the presence of the cell line J1.1. Conversely, when PBMCs were infected with two different syncytium-inducing HIV-1 primary isolates, coincubation with NKI-L16-pretreated 1G5 cells led to higher levels of luciferase activity for both virus isolates. Our results therefore show for the first time a direct role for the LFA-1 high-affinity state in virus-mediated syncytium formation. Based on the demonstration that an increase in ICAM-1 binding is induced by T-cell activation, these data suggest an in vivo involvement of the high-affinity state of LFA-1 in HIV-1-induced syncytium formation. Moreover, syncytia might preferentially occur in lymph nodes, since this microenvironment harbors a high proportion of activated T cells.

Antibodies↗

PubMed1997

Activation of HIV-1 long terminal repeat transcription and virus replication via NF-kappaB-dependent and -independent pathways by potent phosphotyrosine phosphatase inhibitors, the peroxovanadium compounds.

Replication of human immunodeficiency virus type 1 (HIV-1) is increased by different cytokines and T cell activators, also known to modulate tyrosine phosphorylation levels. A novel class of protein tyrosine phosphatase (PTP) inhibitors, peroxovanadium (pV) compounds, were tested for a putative effect on HIV-1 long terminal repeat (LTR) activity. We found that these PTP inhibitors markedly enhanced HIV-1 LTR activity in 1G5 cells, a stably transfected cell line that harbors an HIV-1 LTR-driven luciferase construct. A direct correlation between the extent of tyrosine phosphorylation and the level of HIV-1 LTR inducibility was seen after treatment with three different pV compounds. Transient transfection experiments were carried out in several T cell lines, and after addition of pV, a marked increase in HIV-1 LTR activity was measured. Monocytoid cells were tested using U937-derived cell lines and were also found to be sensitive to the pV-mediated potentiating effect on HIV-1 LTR activity. A significant reduction of the pV-mediated increase in HIV-1 LTR activity was seen in cells transiently transfected with an HIV-1 LTR-driven luciferase construct bearing a mutation in both NF-kappaB binding sites although detectable levels of induction remained. Electrophoretic mobility shift assays allowed the identification of the nuclear translocation of the NF-kappaB p50.p65 heterodimer complex induced by pV compounds. A dominant negative version of the repressor IkappaBalpha mutated on serines 32 and 36 impeded pV-induced NF-kappaB-dependent luciferase activity. Western blot analysis showed a clear diminution in the protein level of IkappaBalpha starting 30 min after pV treatment of Jurkat E6.1 cells which is indicative of its degradation. On the other hand, no increase in tyrosine phosphorylation was observed on IkappaBalpha itself. Finally, we tested the PTP inhibitors on four cell lines latently infected with HIV-1 and showed a consistent pV-mediated increase in virion production. Thus, our studies suggest that pV-mediated activation of HIV-1 LTR activity is controlled by the nuclear translocation of the NF-kappaB transcription factor, which is mediated by IkappaBalpha serine phosphorylation and degradation, but also by a still undefined NF-kappaB-independent pathway.

Cell Line↗