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Biomedical subjects

A Doglio

Publications and source records attributed to A Doglio.

At least 19 recordsLinked to original sources
PubMed2005

Tumor necrosis factor-alpha stimulates HIV-1 production in primary culture of human adipocytes.

Adipose tissue of HIV-1-infected patients shows severe abnormalities such as profound changes in adipose tissue morphology and metabolism. Does HIV-1 infect the adipose cell remains an unsolved question since previous attempts showed that HIV-1 poorly infects human adipocytes in vitro. In the present study, preadipose cells from human subcutaneous fat pads were differentiated in vitro, checked for HIV receptor expression, then infected with R5 and X4 HIV1 strains. Using a sensitive RT-PCR assay, we showed that HIV-1 tat and rev early viral transcripts were expressed in infected adipocytes giving a clear evidence of HIV-1 transcriptional activity in these cells. However, at the same time, no sign of productive infection was demonstrated since infected adipocytes did not efficiently produce Gag p24 antigen. We hypothesized that such a limitation could result from the lack of activation of adipocyte-signaling pathways able to stimulate HIV-1 gene expression in quiescent adipocytes. Indeed, a significant increase in Gag p24 production was observed after stimulation of infected adipocytes with pro-inflammatory cytokines, such as tumor necrosis factor alpha or interleukin-1-beta. Taken together, these results demonstrate that HIV-1 does infect human adipose cells in vitro and suggest that the initial limited infection can be overcome upon pro-inflammatory cytokine treatment.

Adipocytes↗
PubMed2001

Terminal minihelix, a novel RNA motif that directs polymerase III transcripts to the cell cytoplasm. Terminal minihelix and RNA export.

Determining the cis-acting elements controlling nuclear export of RNA is critical, because they specify which RNA will be selected for transport. We have characterized the nuclear export motif of the adenoviral VA1 RNA, a small cytoplasmic RNA transcribed by RNA polymerase III. Using a large panel of VA1 mutants in both transfected COS cells and injected Xenopus oocytes, we showed that the terminal stem of VA1 is necessary and sufficient for its export. Surprisingly, we found that the nucleotide sequence within the terminal stem is not important. Rather, the salient features of this motif are its length and its relative position within the RNA. Such stems thus define a novel and degenerate cytoplasmic localization motif that we termed the minihelix. This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that export of one such RNA, like hY1 RNA, is specifically competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and that this motif could be exported via a novel and specific nuclear export pathway.

Animals↗
PubMed2001

A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor.

We have previously shown that secreted phospholipases A2 (sPLA2) from bee and snake venoms have potent anti-human immunodeficiency virus (HIV) activity. These sPLA2s block HIV-1 entry into host cells through a mechanism linked to sPLA2 binding to cells. In this study, 12 synthetic peptides derived from bee venom sPLA2 (bvPLA2) have been tested for inhibition of HIV-1 infection. The p3bv peptide (amino acids 21 to 35 of bvPLA2) was found to inhibit the replication of T-lymphotropic (T-tropic) HIV-1 isolates (ID(50) = 2 microM) but was without effect on monocytotropic (M-tropic) HIV-1 isolates. p3bv was also found capable of preventing the cell-cell fusion process mediated by T-tropic HIV-1 envelope. Finally, p3bv can inhibit the binding of radiolabeled stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and the binding of 12G5, an anti-CXCR4 monoclonal antibody. Taken together, these results indicate that p3bv blocks the replication of T-tropic HIV-1 strains by interacting with CXCR4. Its mechanism of action however appears distinct from that of bvPLA2 because the latter inhibits replication of both T-tropic and M-tropic isolates and does not compete with SDF-1alpha and 12G5 binding to CXCR4.

Amino Acid Sequence↗
PubMed1999

Second generation of the automated Cobas Amplicor HCV assay improves sensitivity of hepatitis C virus RNA detection and yields results that are more clinically relevant.

The first and second generations of the Cobas Amplicor HCV assay were compared among patients at risk of hepatitis C virus (HCV) infection. The second-generation test was found to be of greater sensitivity and of good specificity among clinical specimens containing HCV RNA of different genotypes. Finally, this new test is shown to predict the outcome of interferon therapy better.

Hepacivirus↗
PubMed1999

Secreted phospholipases A(2), a new class of HIV inhibitors that block virus entry into host cells.

Mammalian and venom secreted phospholipases A(2) (sPLA(2)s) have been associated with a variety of biological effects. Here we show that several sPLA(2)s protect human primary blood leukocytes from the replication of various macrophage and T cell-tropic HIV-1 strains. Inhibition by sPLA(2)s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA(2)s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA(2)s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA(2) have no effect on HIV-1 infection, suggesting that sPLA(2) catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA(2)s to host cells. Indeed, of 11 sPLA(2)s from venom and mammalian tissues assayed, 4 venom sPLA(2)s were found to be very potent HIV-1 inhibitors (ID(50) < 1 nM) and also to bind specifically to host cells with high affinities (K(0.5) < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA(2)s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA(2)s are being characterized in humans.

Animals↗
PubMed1998

T cells chronically infected with HIV do not contain sufficient Nef to promote CD4 downmodulation in the absence of envelope-mediated effects.

Among HIV viral proteins, envelope glycoproteins and Nef have been both suggested to participate in CD4 downregulation during the course of HIV infection. In a previous study, we provided evidence that a mutant form of CD4 that does not bind gp120 was never downregulated in chronically HIV-1- and HIV-2-infected CEM cells. To further investigate the relative effects of Nef or glycoproteins in CD4 downregulation, recombinant vaccinia virus (VV) vectors were used to express high levels of HIV-1 viral proteins in cells expressing both wild-type and mutant CD4. It was demonstrated that during HIV infection, overexpression of Nef, achieved through the VV expression system, was necessary to induce CD4 downregulation in the mutant CD4-expressing cell model. These results are consistent with the hypothesis that Nef-mediated CD4 downregulation depends on the cellular levels of Nef expression. We concluded that during the late stage of viral replication, CD4 downregulation is mostly due to gp120 and not to Nef because of a low level of Nef expression.

CD4 Antigens↗
PubMed1998

Hepatitis G and C viruses respond to interferon-alpha with different virologic kinetics.

The aim of this work was to specify the time course of response to interferon (IFN) of hepatitis G virus (HGV) and hepatitis C virus (HCV) in coinfected individuals. A group of 33 patients, undergoing 12 months of IFN therapy for chronic hepatitis C, was screened for the presence of both HGV and HCV RNAs to select seven coinfected patients. Spontaneous recovery from HGV infection was excluded through the detection of antibodies to the envelope-2 protein of HGV and HCV isolates were genotyped. Within three months of treatment, we found that HGV RNA was transiently cleared in 6/7 patients, but the rate of long-term favorable response was very low (1/7). In addition, considering the same individuals separately, it was shown that HGV and HCV responded to IFN with different kinetics in 5/7 patients. Taken together, these results underscore the importance of the virological basis of the resistance to IFN treatment.

Antiviral Agents↗
PubMed1998

Lack of influence of hepatitis G virus infection on alcohol-related hepatic lesions.

BACKGROUND: The purposes of this study were to analyse the prevalence and histologic impact of hepatitis G virus (HGV), a newly discovered virus, in alcoholic patients, a population known to be at risk for viral hepatitis. METHODS: One hundred and thirty-nine consecutive alcoholics admitted to our liver unit (106 men and 33 women; mean age, 47.1 +/- 10.9 years) were included in the study. All patients had consumed more than 60 g of ethanol per day for at least 1 year. One hundred healthy blood donors constituted a control group. Antibodies to HGV E2 protein and HGV-RNA testing by reverse transcription-polymerase chain reaction (RT-PCR) with primers derived from the NS5 coding region were performed in all serum samples. RESULTS: A significantly higher seroprevalence of anti-E2 antibodies was observed in alcoholic patients than in healthy blood donors (41 (29.5%) versus 8 (8%); P < 0.0001). Moreover, the prevalence of HGV-RNA was significantly higher in alcoholic patients (13 (9.3%) versus 1 (1%); P = 0.01). HGV-RNA and anti-HGV antibodies were never detected simultaneously. HGV viraemia was not associated with an increased risk of cirrhosis or hepatocarcinoma in alcoholic subjects. CONCLUSIONS: Our study reports a high prevalence of HGV in alcoholic patients. HGV infection does not modify or aggravate the course of alcoholic liver disease.

Case-Control Studies↗
PubMed1998

3'-End modification of the adenoviral VA1 gene affects its expression in human cells: consequences for the design of chimeric VA1 RNA ribozymes.

Polymerase III (pol III)-dependent genes, like the adenoviral VA1 gene, are of particular interest for expressing small therapeutic RNAs into cells. A new VA1 RNA carrier molecule was generated through the deletion of the VA1 RNA central domain to give rise to the VAdeltaIV RNA vector that was devoid of undesirable physiologic activity (i.e., inhibition of the interferon-induced protein kinase, PKR). This vector was used to express in human cells hammerhead ribozymes targeted against the human immunodeficiency virus (HIV). Eight anti-HIV ribozymes were inserted at the 3'-end of this vector immediately before the four T-residues that serve as a transcription termination signal. Although the constructs were active in vitro, they failed to inhibit HIV replication in transient assays. Analysis of the intracellular ribozyme expression in cells revealed several anomalies. First, using mutant derivatives, we showed that the presence of two or three consecutive T-residues in the ribozyme portion was sufficient to promote the release of anomalous short transcripts. Second, when the ribozyme did not contain T-rich sequence, full-length transcripts were produced, but these transcripts were very unstable and were retained in the cell nucleus. In contrast, insertion of the ribozyme in place of the central domain of VA1 RNA led to production of full-length transcripts that were stable and located in the cytoplasm but that were not found to be active in vitro. Taken together, these results have important consequences for the future design of active intracellular ribozymes based on the use of pol III-transcribed genes.

3' Untranslated Regions↗
PubMed1997

Phenotyping of intrahepatic and peripheral blood lymphocytes in patients with chronic hepatitis C.

The host immune responses have been suggested to play a role in liver injury occurring in patients with chronic hepatitis C. In order to explore the relationship between the relative proportions of intrahepatic and peripheral blood lymphocytes (IHL, PBL), the levels of viremia, and the histological hepatitis activity score, three-color fluorescence-activated cytometric analysis was performed for 36 patients with chronic hepatitis C and six control subjects without chronic hepatitis. The liver biopsy was performed before any antiviral therapy. Each liver specimen was divided into two parts: one for histological examination and one for immunological analysis. Tricolor CD45 was used to improve "lymphogating." Fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies with specificity for CD3, CD4, CD8, and CD20 (lymphocyte subpopulations), for CD69 (activated lymphocytes), and for CD16/56 (natural killer cells) were used. The livers of patients with chronic hepatitis C contained a greater proportion of CD4+ lymphocytes that exhibited marked expression of CD69 than in control subjects (20.7 +/- 7.3% vs 10.2 +/- 4.6%, P = 0.027). Moreover, in patients with chronic hepatitis C, the proportion of CD4+ IHL correlated with the histological hepatitis activity evaluated by the Knodell score (r = 0.48, P = 0.004). No correlation was found between the percentage of CD4+ IHL and the level of viremia or transaminase activities. Our findings clearly indicate that a cellular immune response does take place in HCV-infected livers and could thus contribute to the outcome of hepatitis C virus infection.

Adult↗
PubMed1996

Alteration of CD44 expression in HIV type 1-infected T cell lines.

CD44 is known to interfere in HIV replication and to participate in many physiological processes such as lymphocyte binding to high endothelial venules of lymphoid tissue, lymph nodes, and mucosal endothelium. The T cell lines MOLT-4 and CEM, and CEM subclones were infected with the HIV-1 LAI strain and monitored for the expression of CD44 during the course of chronic virus production until the infected cells were at the stage of latent infection. The levels of CD44 protein expression were quantified using cell surface immunostaining and biotinylation. The maturation of CD44 molecules was evaluated by metabolic sulforadiolabeling and CD44 mRNA was visualized by Northern blot analysis. We show a downmodulation of CD44 expression in infected T cell lines and subclones. This phenomenon was most evident at the stage of latent infection. Then, CD44 molecules were undetectable at both the protein and mRNA levels in latently infected CEM cells and CEM subclones. In addition, the 97-kDa standard CD44 isoform showed a shift upward, while detectable during the stage of chronic virus production. In latently infected MOLT-4 cells, the CD44 protein levels were dramatically decreased; CD44 mRNA was detected, but the sizes differed from the mRNA in uninfected cells. Since CD44 is known to regulate in part lymphocyte homing and HIV replication, the alterations that were observed in the expression of this molecule could interfere with the particular homing of HIV-infected cells and/or viral latency.

Alternative Splicing↗
PubMed1995

Autoantibodies directed against CD43 molecules with an altered glycosylation status on human immunodeficiency virus type 1 (HIV-1)-infected CEM cells are found in all HIV-1+ individuals.

Autoantibodies to lymphocytes have been detected in sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals, and several autoantigens have been described. Among them, hyposialylated CD43 has been shown to be a target for autoantibodies in up to 47% of HIV+ individuals. However, the corresponding autoantigen (ie, the incompletely sialylated CD43) has not been isolated from blood cells of HIV-1-infected individuals. Recently, we have observed in vitro that HIV-1 productively or latently infected CEM cells (CEMLAI/NP) express CD43 molecules with modified glycosylation (mogly CD43). Using CEMLAI/NP cells, which do not express any structural viral antigen, we show now that all of the tested HIV+ sera from asymptomatic individuals, and up to 86% of those from subjects at the acquired immunodeficiency syndrome stage contain antibodies (mainly IgM and, to a lesser degree, IgG) that recognize the surface of CEMLAI/NP cells, and precipitate mogly CD43 molecules from the cells lysates. Taken together with our previous demonstration of altered glycosylation of CD43 from HIV-1-infected CEM cells in vitro, the constant antimogly CD43 autoimmune response observed from asymptomatic HIV-1+ subjects is likely to illustrate the occurrence of an altered glycosylation in vivo of the major lymphocyte surface CD43 glycoprotein, associated with HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Protection of a T-cell line from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule.

Adenovirus VA1 gene is efficiently transcribed by RNA polymerase III and gives rise to a small highly ordered RNA. To inhibit replication of human immunodeficiency virus (HIV), a chimeric VA1 RNA molecule was designed that contained a short antisense RNA sequence complementary to a conserved region of the HIV-1 rev encoding mRNA (28 nucleotides). This sequence, which was inserted into a projecting loop of the VA1 RNA central domain, was mainly single stranded and available for binding with its complementary sequence. The chimeric VA1 antisense was abundantly expressed in human cells constituting 3% of mRNA and promoted strong and specific inhibition of HIV-1 gene replication. The stable expression of antisense RNA in human T cells (CEM) protected these cells from HIV-1 multiplication for at least 3 months. No side effects were detected because of the lack of antisense effect upon replication of the closely related HIV-2. The VA1 gene may provide a suitably compact gene cassette for the intracellular expression of short antisense RNA directed against HIV.

Adenoviridae↗
PubMed1995

Induction by human immunodeficiency viruses types 1 and 2 of degradation of CD4 but not of a CD4 mutant unable to bind viral envelope glycoproteins.

HIV-1 appears to use a multiple gene strategy to regulate CD4 receptor expression, which emphasizes the importance of this regulation in the viral life cycle. The cytoplasmic interaction between gp160 and CD4 is probably the major event governing CD4 down-regulation, although other viral proteins, such as Nef (CD4 cell surface localization) and Vpu (CD4 degradation), are thought to participate as well. Because of the lack of vpu in HIV-2, we investigated the effects of two HIV-2 isolates (ROD 10 and EHO) on CD4 expression in the CEM T-cell line. We found that these HIV-2 strains induce CD4 degradation to a similar extent as that induced by an HIV-1 isolate (BRU). To assess the role of each viral protein involved in CD4 regulation (gp, Nef and Vpu), we developed cell lines expressing a mutated form of CD4 unable to efficiently bind gp160, in addition to their endogenous CD4. Using this system, we provide evidence that the mutated CD4 is always expressed in HIV-1-, and HIV-2-infected cells, independent of the presence of Nef, while the endogenous CD4 is completely lost. These results highlight the key role of intracytoplasmic gp-CD4 interaction, explaining in vitro the CD4 down-regulation in T-cell lines.

Base Sequence↗
PubMed1994

Altered glycosylation of leukosialin, CD43, in HIV-1-infected cells of the CEM line.

CD43 (leukosialin, gpL115, sialophorin) is a major sialoglycoprotein widely expressed on hematopoietic cells that is defective in the congenital immunodeficiency Wiskott-Aldrich syndrome. It is thought to play an important role in cell-cell interactions and to be a costimulatory molecule for T lymphocyte activation. Using a metabolic 35SO4(2-) radiolabeling assay or biotinylation of cell surface proteins, we describe here that CD43 are sulfated molecules the glycosylation of which is altered in human immunodeficiency virus type 1 (HIV-1)-infected leukemic T cells of the CEM line. Hyposialylation of O-glycans and changed substitution on N-acetylgalactosamine residues are observed. The glycosylation defect is associated with an impairment of CD43-mediated homotypic aggregation which can be restored by resialylation. The hyposialylation of CD43 on HIV-1+ cells may explain the high prevalence of autoantibodies directed against nonsialylated CD43 that have been detected in HIV-1-infected individuals. A defect in glycosylation of important molecules such as CD43 or, as we recently described, CD45 may explain alterations of T cell functions and viability in HIV-1-infected individuals. In addition, a possible implication of hyposialylation in the HIV-1-infected cells entrapment in lymph nodes could be envisioned.

Antibodies, Monoclonal↗