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N Yahi

Publications and source records attributed to N Yahi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Direct effect of type 1 human immunodeficiency virus (HIV-1) on intestinal epithelial cell differentiation: relationship to HIV-1 enteropathy.

Human immunodeficiency virus (HIV)-infected patients display severe impairments of gastrointestinal functions, including diarrhea and malabsorption, even in the absence of opportunistic infections. Since HIV-1 proteins and nucleic acids have been detected in several cell types of the intestinal mucosa, it has been postulated that HIV-1 itself could alter enterocytic functions. In the present study, we analyzed the effect of HIV-1 on the differentiation process of the epithelial intestinal cell clone HT-29-D4, which mimics the maturation of enterocytes along the crypt-villus axis of the small intestine. We found that HIV-1 infection impairs cellular differentiation (i) by affecting the barrier function of the epithelium, as evidenced by a decrease in the transepithelial electrical resistance, and (ii) by inhibiting the activity of one major glucose absorption function, i.e., sodium/glucose cotransport. At the morphological level, HIV-1 infection of HT-29-D4 cells was associated with the formation of lumina, which are representative of a defect in cellular organization. These morphofunctional perturbations induced by HIV-1 could be mimicked by nocodazole, a microtubule-disrupting agent. Correspondingly, HIV-1 exposure of HT-29-D4 cells evoked a massive disruption of microtubules, as revealed by alpha-tubulin indirect immunofluorescence staining. A similar effect was observed after incubation of the cells with either recombinant gp120 or a monoclonal antibody against galactosylceramide (GalCer), the intestinal receptor for HIV-1 gp120, suggesting that the effect of HIV-1 was mediated by the binding of gp120 to GalCer. Based on these data, we propose that HIV-1 may selectively alter enterocytic functions through a direct effect on the intracellular architecture of the cells. In contrast with previous theories for HIV-1 enteropathy, our data support the concept that HIV-1 may perturb intestinal functions without necessarily infecting intestinal epithelial cells.

Cell Differentiation↗
PubMed1997

Synthetic soluble analogs of galactosylceramide (GalCer) bind to the V3 domain of HIV-1 gp120 and inhibit HIV-1-induced fusion and entry.

Galactosylceramide (GalCer) is an alternative receptor allowing human immunodeficiency virus (HIV)-1 entry into CD4-negative cells of neural and colonic origin. Several lines of evidence suggest that this glycosphingolipid recognizes the V3 region of HIV-1 surface envelope glycoprotein gp120. Since the V3 loop plays a key role in the fusion process driven by HIV-1, we decided to synthesize soluble analogs of GalCer with the aim to develop a new class of anti-HIV-1 agents that could neutralize HIV-1 infection through masking of the V3 loop. We describe a short route, in three steps, for the synthesis of soluble analogs of GalCer, using unprotected lactose as the starting sugar. The analogs were prescreened in an assay based on the interaction between a V3 loop-derived synthetic peptide and [3H]suramin, a polysulfonyl compound displaying high affinity for the V3 loop. One of the soluble analogs, i.e. CA52(n15), strongly inhibited the binding of [3H]suramin to the V3 peptide, with an IC50 of 1.2 microM. This molecule was also able to inhibit [3H]suramin binding to recombinant gp120 with similar activity. Using a competition enzyme-linked immunosorbent assay with highly specific anti-gp120 monoclonal antibodies, the region recognized by CA52(n15) could be mapped to amino acids 318-323, which corresponds to the highly conserved consensus motif GPGRAF. Interestingly, the region recognized by suramin, i.e. IQRGP-R-F, was partially overlapping this motif. CA52(n15) was able to inhibit HIV-1-induced cell fusion as well as HIV-1 entry into both CD4(+) and CD4(-)/GalCer+ cells. A structure-activity relationship study showed that: (i) the antiviral activity of soluble analogs of GalCer correlates with V3 loop binding, and (ii) the hydrophobic moiety of the molecule plays an important role in this activity. Taken together, these data show that synthetic analogs of GalCer can inhibit HIV-1 entry into both CD4(-) and CD4(+) cells through masking of the V3 loop.

Amino Acid Sequence↗
PubMed1997

Perturbations of glucose metabolism associated with HIV infection in human intestinal epithelial cells: a multinuclear magnetic resonance spectroscopy study.

OBJECTIVE: To analyse the effect of HIV-1 infection on the glucose metabolism of human intestinal epithelial cells. METHODS: HT-29 cells were infected with HIV-1NDK and studied 3 weeks (acutely infected cells) or 9 months (chronically infected cells) post-infection. Perchloric acid extracts were analysed by high-resolution 1H, 31P and 13C nuclear magnetic resonance spectroscopy. Metabolite concentrations and specific 13C enrichments were quantified for chronically infected, acutely infected and control cells grown in Dulbecco's modified Eagle's medium containing natural-abundance or 1-13C-enriched glucose to determine significant differences between infected and non-infected cells. RESULTS: Chronically HIV-infected cells showed alterations in glycerol-3-phosphate (+40%), fructose-1,6-diphosphate (-66%), uridine diphosphate glucuronic acid (-33%), lactate (+75%) and [1-13C]glucose (+181%) levels, and in specific lactate 3-13C enrichment (+19%) when compared with controls. Acutely infected cells exhibited decreased fructose-1,6-diphosphate (-58%) and increased nicotinamide adenine dinucleotide (+33%) levels relative to controls. CONCLUSION: HIV-1 infection results in a disturbance of glycolytic and oxidative activities in human intestinal epithelial cells. This finding supports the concept that HIV-1 may directly impair some metabolic functions of the intestinal epithelium, and that it can be considered a potential aetiological agent for HIV-associated enteropathy.

Glucose↗
PubMed1997

Quantification of HIV-1 viral load in lymphoid and blood cells: assessment during four-drug combination therapy.

OBJECTIVE: To assess the antiretroviral effect of a combination of zidovudine, didanosine, lamivudine and saquinavir in plasma, peripheral blood mononuclear cells (PBMC) and lymph-node mononuclear cells (LNMC) after 8 weeks. METHODS: Ten HIV-1 antiretroviral therapy-naive patients were given a combination of oral zidovudine (200 mg three times daily), oral didanosine (200 twice a day), oral lamivudine (150 mg twice a day) and oral saquinavir (600 mg three times daily). HIV-1 plasma RNA was measured by quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR). Infectious HIV-1 in PBMC and LNMC was measured by a coculture technique. HIV-1 RNA in PBMC and LNMC was quantified by RT-PCR. Proviral DNA titres in PBMC and LNMC were measured by endpoint dilution PCR. CD4 T-cells were analysed by flow cytometry. RESULTS: CD4 cell counts rose in all patients (mean increase of 125 +/- 71 CD4 cells x 10(6)/l) and the benefit was greater for patients with fewer than 350 CD4 cells x 10(6)/l (mean increase of 159 +/- 74 CD4 cells x 10(6)/l). Plasma HIV-1 RNA decreased exponentially in all patients (mean decrease of 3.1 log10 after 8 weeks with a mean half-life of 2.2 +/- 0.6 days). HIV-1 RNA showed a decrease of 3.07 log10 in PBMC and of 2.1 log10 in LNMC. The decrease in plasma HIV-1 RNA was consistently associated with the decrease in LNMC. These data were supported by a concomitant drop of HIV-1 infectious titres in PBMC (mean decrease of 1.41 log10) and in LNMC (mean decrease of 2.54 log). CONCLUSIONS: These data show a significant antiretroviral effect of this four-drug combination in blood and lymphoid tissues. However, a greater decrease in HIV-1 RNA was observed in PBMC and in plasma than in lymph node cells.

Adult↗
PubMed1997

Co-expression of CXCR4/fusin and galactosylceramide in the human intestinal epithelial cell line HT-29.

OBJECTIVE: To detect the expression CXCR4/fusin in human intestinal epithelial cells and to assess its potential role in the pathway of HIV-1 infection mediated by the alternative gp120 receptor galactosylceramide (GalCer). METHODS: GalCer+ (HT-29, HT-29/CD4+) and GalCer- (Caco-2/Cl2, Cl14 and Cl14/CD4+) human intestinal cell lines were analysed for CXCR4/fusin expression using the monoclonal antibody (MAb) 12G5. This MAb was then evaluated for its ability to inhibit HIV-1 infection in permissive cells. HIV-1 infection was measured by detection of p24 antigen, polymerase chain reaction amplification, and cocultivation with CD4+ cells. RESULTS: CXCR4/fusin was detected on the surface of HT-29 and HT-29/CD4+, but not on Caco-2/Cl2, Cl14 and Cl14/CD4+ cells. Ninety per cent of CXCR4/fusin+ HT-29 and HT-29/CD4+ cells co-expressed GalCer. Infection of HT-29 cells by laboratory isolates of HIV-1 was inhibited by both anti-GalCer and anti-CXCR4/fusin MAbs. Expression of CD4 rendered HT-29 cells sensitive to HIV-1(89.6), a macrophage-tropic isolate that does not recognize GalCer. The 12G5 MAb blocked HIV-1 infection of HT-29/CD4+ cells. In contrast, the expression of HIV-1 receptors, i.e., CD4 GalCer or both, into CXCR4/fusin-negative intestinal cells did not confer sensitivity to HIV-1 infection. The resulting receptor-positive cell lines could, however, bind HIV-1, whereas the original cell lines could not. CONCLUSION: HIV-1 entry into human intestinal cells involves both GalCer and CXCR4/fusin. HIV-1 isolates such as 89.6 that are able to use CXCR4/fusin as coreceptor, but do not bind to GalCer, do not infect these cells. These data raise the possibility that CXCR4/fusin may function as a coreceptor for HIV-1 entry into CD4-/GalCer+ intestinal epithelial cells.

Antibodies, Blocking↗
PubMed1996

SPC3, a V3 loop-derived synthetic peptide inhibitor of HIV-1 infection, binds to cell surface glycosphingolipids.

Synthetic multibranched peptides derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120 inhibit HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms: competitive inhibition of HIV-1 binding to CD4-/GalCer+ colon cells and postbinding inhibition of HIV-1 fusion with CD4+ lymphocytes. In the present study, we have characterized the cellular binding sites for the V3 peptide SPC3, which possesses eight V3 consensus motifs GPGRAF radially branched on a neutral polyLys core matrix. These binding sites are glycosphingolipids that share a common structural determinant, i.e., a terminal galactose residue with a free hydroxyl group in position 4: GalCer/sulfatide on CD4-/GalCer+ colon cells; LacCer and its sialosyl derivatives GM3 and GD3 on CD4+ human lymphocytes. These data suggest that the V3 peptide binds to the GalCer/sulfatide receptor for HIV-1 gp120 on HT-29 cells and thus acts as a competitive inhibitor of virus binding to these CD4- cells, in full agreement with previously published virological data. In contrast, SPC3 does not bind to the CD4 receptor, in agreement with the data showing that the peptide inhibits HIV-1 infection of CD4+ cells by acting at a postattachment step. The binding of SPC3 to LacCer, GM3, and GD3, expressed by CD4+ lymphocytes, suggests a role for these glycosphingolipids in the fusion process between the viral envelope and the plasma membrane of CD4+ cells. Since the multivalent peptide can theoretically bind to several of these glycosphingolipids, we hypothesize that the resulting cross-linking of membrane components may affect the fluidity of the plasma membrane and/or membrane curvature, altering the virus-cell fusion mechanism.

Antibodies, Monoclonal↗
PubMed1996

A new method for the determination of specific 13C enrichment in phosphorylated [1-13C]glucose metabolites. 13C-coupled, 1H-decoupled 31P-NMR spectroscopy of tissue perchloric acid extracts.

A 31P-NMR method for the determination of 13C enrichment in phosphorylated [1-(13)C]glucose metabolites was developed by taking advantage of the 13C satellites detectable for 31P-NMR signals of metabolites such as UDP-hexoses, UDP-N-acetylhexosamines and other phosphorylated compounds generated during glycolysis and subsequent anabolism. HT-29 cells were incubated in culture medium containing 4.5 g/l [1-(13)C]glucose for 24 h prior to cell extraction, and high-resolution 31P-NMR spectra were acquired from perchloric acid extracts. Since glucose and its phosphorylated products are key metabolites for many different metabolic processes, this method may be very helpful for studying specific metabolic pathways involving phosphorylated glucose metabolites.

Carbon Isotopes↗
PubMed1996

Analysis of individual purine and pyrimidine nucleoside di- and triphosphates and other cellular metabolites in PCA extracts by using multinuclear high resolution NMR spectroscopy.

This work demonstrates that individual purine and pyrimidine NDP and NTP can be assigned in high resolution 31P NMR spectra from tissue extracts. To the best of our knowledge, it is shown for the first time that ATP, GTP, UTP, CTP, and the corresponding diphosphates can be quantitated in cell extracts without using HPLC or other biochemical methods. This work provides the basis for further optimization of nucleotide quantitation by 31P NMR spectroscopy, and for a full assessment of this method. Furthermore, a new technique was developed for 1H, 31P, and 13C NMR signal assignment and quantitation in cell extracts by using the same external reference capillary for all three nuclei. This allows for efficient, quantitative, multinuclear NMR spectroscopy without extract contamination by standard material.

Adenosine Diphosphate↗
PubMed1995

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Antigens, CD↗
PubMed1995

Production of a highly cytopathic HIV-1 isolate from a human mucosal epithelial cell line cultured on microcarrier beads in serum-free medium.

The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins.

Adenocarcinoma↗
PubMed1995

Intracellular calcium release induced by human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein in human intestinal epithelial cells: a putative mechanism for HIV-1 enteropathy.

Intracellular Ca2+ ([Ca2+]i) was measured in single human epithelial intestinal HT-29-D4 cells with the Ca2+ probe Fura-2 and digital imaging microscopy. Treatment of these cells with HIV-1 surface envelope glycoprotein gp120 (or a soluble form of its precursor gp160) induced an important increase of [Ca2+]i. This effect was abolished by preincubation of the viral glycoprotein with neutralizing antibodies specific for the V3 domain of gp120. These antibodies inhibited the binding of both gp120 and gp160 to galactosylceramide (GalCer), the alternative HIV-1 receptor in HT-29-D4 cells. Moreover, treatment of HT-29-D4 cells with an anti-GalCer mAb induced an increase in [Ca2+]i and rendered the cells insensitive to HIV-1 glycoprotein stimulation. The calcium response resulted from release of Ca2+ from caffeine-sensitive intracellular stores. Finally, the viral glycoprotein specifically abrogated the calcium response to the neuropeptide agonist neurotensin, a stimulator of chloride secretion via inositol trisphosphate-mediated calcium mobilization. Reciprocally, after neurotensin stimulation, the cells did not respond to gp120, showing that neurotensin and gp120 stimulate a common pathway of [Ca2+]i mobilization. These results suggest that HIV-1 may directly alter ion secretion in the intestine and thus be the causative agent of the watery diarrhea associated with HIV-1 infection.

Calcium↗
PubMed1995

Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4-negative mucosal epithelial cell line.

The glycosphingolipid galactosylceramide (GalCer), which binds gp120 with high affinity and specificity, is a potential alternative receptor for human immunodeficiency virus type 1 (HIV-1) in some CD4-negative neural and epithelial human cells, including the human colonic epithelial cell line HT-29. In the present study, we demonstrate that synthetic multibranched peptides derived from the consensus sequence of the HIV-1 V3 loop block HIV-1 infection in HT-29 cells. The most active peptide was an eight-branched multimer of the motif Gly-Pro-Gly-Arg-Ala-Phe which at a concentration of 1.8 microM induced a 50% inhibition of HIV-1 infection in competition experiments. This peptide was not toxic to HT-29 cells, and preincubation with HIV-1 did not affect viral infectivity, indicating that the antiviral activity was not due to a nonspecific virucidal effect. Using a high-performance thin-layer chromatography binding assay, we found that multibranched V3 peptides recognized GalCer and inhibited binding of recombinant gp120 to the glycosphingolipid. In addition, these peptides abolished the binding of an anti-GalCer monoclonal antibody to GalCer on the surface of live HT-29 cells. These data provide additional evidence that the V3 loop is involved in the binding of gp120 to the GalCer receptor and show that multibranched V3 peptides are potent inhibitors of the GalCer-dependent pathway of HIV-1 infection in CD4-negative mucosal epithelial cells.

Amino Acid Sequence↗
PubMed1994

Interferon-gamma decreases cell surface expression of galactosyl ceramide, the receptor for HIV-1 GP120 on human colonic epithelial cells.

HT-29-A7, a CD4-negative clonal derivative of the human colonic adenocarcinoma cell line HT-29, is particularly sensitive to infection by several isolates of HIV-1 and, correspondingly, expresses high amounts of galactosylceramide (galactocerebroside, GalCer). GalCer is a neutral glycolipid which binds to the HIV-1 envelope glycoprotein gp120 and is present at abundant levels in normal human epithelial cells of the small and large intestine. Treatment of the HT-29-A7 cells with recombinant gamma-interferon (rlFN gamma) induced a dose-dependent inhibition of GalCer expression on the cell surface, as demonstrated by indirect immunofluorescence and by enzymatic labeling of cell surface glycoconjugates with oxidase-tritiated sodium borohydride. The rIFN gamma effect was not associated with any toxicity and was specific for GalCer, since expression of carcinoembryonic antigen did not decrease following treatment. The decrease in GalCer expression was associated with resistance of the cells to HIV-1 infection. In contrast, rIFN gamma did not alter cell surface expression of CD4, the classical HIV receptor, in HT-29-A7 cells that had been transduced with a retroviral vector expressing full-length CD4, and there was no effect on their infection. These results strongly suggest that rIFN gamma blocks HIV-1 infection of HT-29-A7 cells by decreasing GalCer synthesis and expression. This effect on expression of a viral receptor is a novel antiviral property of rIFN gamma which should be exploited for antiviral therapeutics.

Colon↗
PubMed1994

Suramin inhibits binding of the V3 region of HIV-1 envelope glycoprotein gp120 to galactosylceramide, the receptor for HIV-1 gp120 on human colon epithelial cells.

The infection of human colonic epithelial cells HT-29 by human immunodeficiency virus type 1 (HIV-1) occurs independently of CD4, the main HIV-1 receptor expressed on lymphocytes and macrophages. Recent studies from our group have shown that HT-29 cells express the glycosphingolipid galactosylceramide (GalCer), a potential alternative receptor for the HIV-1 envelope glycoprotein gp120. The binding of recombinant gp120 to GalCer was blocked by monoclonal antibodies directed against the third variable region (V3) of gp120, suggesting that the V3 domain was implicated in GalCer recognition. In the present report, we show that suramin, a polysulfonyl naphtylurea known to inhibit retroviral reverse transcriptases in vitro, blocks HIV-1 infection in HT-29 cells. The effect is dose dependent, with a half-maximal inhibition (IC50) achieved for a suramin concentration of 54 micrograms/ml. Since [3H]suramin was not significantly internalized into HT-29 cells during our infection assay (i.e. 2 h), we have considered the possibility that the drug could act at an extracellular step of the HIV-1 cycle. Using a high performance thin layer chromatography binding assay, we show that suramin inhibits binding of HIV-1 gp120 to purified GalCer with an IC50 of 25 micrograms/ml. Suramin does not bind to GalCer, since preincubation of GalCer with suramin did not prevent the subsequent attachment of gp120. Using a solid-phase assay, we show that [3H]suramin specifically binds to recombinant gp120 and that this binding could be blocked by a monoclonal antibody specific for the conserved GPGRAF motif of the V3 domain of gp120. We also demonstrate that [3H]suramin binds to multibranched synthetic GPGRAF peptides that block HIV-1 infection in HT-29 cells. Binding of [3H]suramin to V3 peptides is specific and inhibited by unlabeled suramin (IC50 of 28 micrograms/ml). In contrast, the suramin derivative NF036, that is unable to block HIV-1 infection in HT-29 cells, does not inhibit the binding of [3H]suramin to V3 peptides. Taken together, these results suggest that suramin blocks HIV-1 infection in HT-29 cells because it binds to the V3 domain of gp120 and hence prevents the interaction between gp120 and the GalCer receptor.

Amino Acid Sequence↗
PubMed1994

Physical contact with lymphocytes is required for reactivation of dormant HIV-1 in colonic epithelial cells: involvement of the HIV-1 LTR.

HIV-1 transmission from mucosal epithelial cells to lymphocytes is a potential mechanism of HIV-1 contamination during sexual intercourse. The human colon epithelial cell line HT-29, that is infectable by various HIV-1 strains, is a useful model for studying the molecular mechanisms involved in this process. In the present study, we show that HT-29 cells, when exposed to either HIV-1(LAI) or HIV-1(NDK) at a low multiplicity of infection, became infected but did not produce infectious virions. Using two-compartment cell culture chambers separated by a porous membrane, we showed that PBL were able to rescue infectious HIV-1 from latently infected HT-29 cells following a physical interaction between the two cell populations. In contrast, HT-29 cells, infected with the same viruses at a high multiplicity of infection, were able to produce mature viral particles that were infectious to PBL in absence of cellular contacts. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that cell-to-cell contact induced an activation of the HIV-1 promoter. These observations provide a putative molecular mechanism for transmission of HIV-1 from mucosal epithelial cells to lymphocytes.

Cell Communication↗