Role of CXCR4 in HIV-1-induced apoptosis of cells with a CD4+, CXCR4+ phenotype.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
C Devaux
Publications and source records attributed to C Devaux.
Monoclonal antibodies against the Androctonus australis hector scorpion neurotoxin I: characterisation and use for venom neutralisation.
A series of monoclonal antibodies (mAbs) specific for the alpha-neurotoxin I (Aah I) from the venom of the dangerous Androctonus australis hector scorpion were obtained using carrier protein-coupled toxin. Competitive RIA, receptor assays and mouse toxicity tests were performed to characterise mAbs in terms of affinity and neutralisation. Cross-reactivity studies and two-site ELISA results allowed some classification of mAbs into three groups. One mAb, 9C2, was particularly interesting since it recognised the parent toxin I with a K(D) of 0.15 nM and was also reactive with toxins of the same immunological group. Its ability to neutralise the toxic effect of the parent toxin and the venom fraction has been investigated. This anti-Aah I mAb 9C2, associated with anti-Aah II mAb 4C1, provides a valuable tool to neutralise the toxicity of the venom.
Amino Acid Sequence↗
A new potent HIV-1 reverse transcriptase inhibitor. A synthetic peptide derived from the interface subunit domains.
The biologically relevant and active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase found in infectious virions are heterodimers produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. We have shown that the dimeric nature of reverse transcriptase represents a important target for the design of a new class of antiviral agents. In this work, we propose a new strategy for its inhibition by targeting protein/protein interactions during viral formation in infected cells. From the screening of peptides derived from the tryptophan cluster at the interface of the connection subdomain, we have designed a short peptide (10 residues) corresponding to residues 395-404, which can block dimerization of reverse transcriptase in vitro and in infected cells. This peptide is highly efficient in abolishing the production of viral particles, without any adverse toxic side effects, when transduced into human immunodeficiency virus type 1-infected cells together with a new peptide carrier.
Amino Acid Sequence↗
Oyster IKK-like protein shares structural and functional properties with its mammalian homologues.
In our search for genes involved in oyster immunity we isolated a cDNA encoding a polypeptide closely related to the mammalian IkappaB kinase (IKK) family. IKK proteins play a central role in cell signaling by regulating nuclear factor-kappaB (NF-kappaB) activation. We report here the cloning of an oyster IKK-like protein (oIKK) which possesses the characteristic organization of the mammalian IKK proteins, namely an amino-terminal kinase domain followed by a leucine zipper region and a carboxyl-terminal helix-loop-helix motif. When transfected into human cell lines, oIKK activated the expression of NF-kappaB-controlled reporter gene, whereas transfections with mutants of oIKK deleted within the kinase domain or within the helix-loop-helix motif respectively abolished and greatly reduced reporter gene activation. These results indicate that oIKK can replace the hIKK-alpha in catalyzing NF-kappaB nuclear translocation, and in triggering gene expression. Our results sustain the concept of an evolutionarily conserved signaling machinery in which IKK plays a major role.
Amino Acid Sequence↗
Exercise improves flow-mediated vasodilatation of skeletal muscle arteries in rats with chronic heart failure. Role of nitric oxide, prostanoids, and oxidant stress.
BACKGROUND: Flow-mediated dilatation (FMD) of the peripheral arteries may be impaired in chronic heart failure (CHF), and this could contribute to the increased peripheral resistance and exercise intolerance that occur with this disease. Physical exercise improves the FMD of large conduit arteries in CHF, but whether a similar impairment also occurs in smaller arteries is unknown. The mechanisms of the changes in FMD after CHF or exercise are also unknown. METHODS AND RESULTS: FMD was assessed in isolated, perfused, and preconstricted gracilis muscle arteries from sham-operated rats or CHF rats (coronary artery ligation) who were either sedentary or exercised (30-minute swimming period twice a day for 10 weeks, starting 7 days after ligation). In animals with hemodynamic and echographic signs of CHF, FMD was abolished and converted into vasoconstriction (percent change in diameter after 370 microL/min flow: sham, 42+/-5%; CHF, -4+/-3%; P<0.05). Exercise partially restored FMD (18+/-3%; P<0.05 versus CHF). In sham rats, FMD was abolished by the nitric oxide-synthase inhibitor Nomega-nitro-L-arginine (L-NA) but unaffected by the cyclooxygenase inhibitor diclofenac or the free radical scavenger N-(2-mercaptopropionyl)-glycine (MPG). In arteries from sedentary CHF rats, FMD was not modified by L-NA, but it was partially restored by diclofenac or MPG. In exercised CHF rats, FMD was abolished by L-NA and only moderately improved by diclofenac or MPG. Likewise, endothelial nitric oxide synthase mRNA expression (determined by reverse transcription polymerase chain reaction at the level of the gracilis muscle) was reduced by CHF, and this was prevented by exercise. CONCLUSIONS: CHF abolishes the FMD of small arteries by impairing the nitric oxide pathway, increasing oxidant stress, and releasing a prostanoid-contracting factor. Exercise partially restores FMD by increasing expression of endothelial nitric oxide synthase and preventing the production of vasoconstrictor prostanoids and free radicals. Such restoration of FMD might contribute to the increase in exercise capacity after physical exercise in CHF.
Animals↗
Multiple control levels of cell proliferation by human T-cell leukemia virus type 1 Tax protein.
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Carrier Proteins↗
HIV-1 reactivation in resting peripheral blood mononuclear cells of infected adults upon in vitro CD4 cross-linking by ligands of the CDR2-loop in extracellular domain 1.
HIV-1 infects resting peripheral blood mononuclear cells (PBMCs) but remains inactive state until subsequent cell activation. We have demonstrated that the cross-linking of cell surface CD4 by gp120-anti-gp120 immune complexes or heat-inactivated HIV-1 (iHIV-1) is sufficient to trigger activation signals leading to virus reactivation (9). In this study, we demonstrate that NF-kappaB nuclear translocation and stimulation of virus production by iHIV-1 were strictly linked to the concentrations of viral proteins used as exogenous stimuli. Moreover, we further investigated the physiologic relevance of these observations. When submitted to an in vitro CD4 cross-linking by iHIV-1, PBMCs from HIV-1-infected patients were found to produce virus. This viral reactivation was associated with increased NF-kappaB nuclear translocation in patients' PBMCs. Additionally, virus reactivation in resting PBMCs infected in vitro with HIV-1 was found to be specifically induced by ligands of the CDR2-loop in domain 1 (D1) of CD4 (virus envelope and anti-CD4 monoclonal antibodies). In contrast, virus reactivation was not observed following CD4 oligomerization by antibodies that bind other epitopes in D1, including the D1/CDR3-loop. Finally, soluble CD4 (sCD4) prevented virus reactivation by D1/CDR2-loop ligands. Our results indicate that the signaling events initiated in PBMCs by oligomerization of CD4 at the D1/CDR2-loop can trigger HIV-1 upregulation in infected individuals.
Adult↗
Synthetic peptides derived from the variable regions of an anti-CD4 monoclonal antibody bind to CD4 and inhibit HIV-1 promoter activation in virus-infected cells.
The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven beta-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat beta-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.
Amino Acid Sequence↗
A recombinant single-chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector.
Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis. The cDNAs encoding the variable regions of this antibody were isolated by PCR-mediated cloning. A single-chain Fv gene was engineered and expressed in Escherichia coli. The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo. We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single-chain Fv, which opens new perspectives for the treatment of envenomizations.
Amino Acid Sequence↗
Cyclosporine A and purinergic receptors in rat kidney.
Previous reports have demonstrated that Cyclosporine A (CyA) chronically administered induces an increase in adenosine plasma concentration by inhibiting adenosine uptake by red blood cells (RBC). We hypothesized that this effect may modulate, by a down regulation, the mRNA expression of adenosine receptors in rat kidney. Since high blood pressure (HBP) is a classical side effect of CyA treatment, nicardipine, a dihydropyridine calcium channel blocker, is often associated with CyA in treatment. To distinguish between the effects of CyA-induced HBP and the effects of CyA by itself, we have evaluated the effects of CyA and/or nicardipine on the mRNA expression of A1 and A2a adenosine receptors. The study was performed on five groups of rats (n= 8) receiving during 21 days either serum saline (0.5 ml i.p), CyA (12 mg/kg/day, i.p), nicardipine (1.2 mg/kg i.p) or nicardipine + CyA. The last (or fifth) group was injected with vehicle (0.5 ml i.p). Blood samples for adenosine assay were collected in the renal artery at day 21, just before the rat kidneys were removed for quantitation of adenosine A1 and A2a mRNA concentration by RT-PCR. We make two conclusions :i) Nicardipine induces a decrease in mRNA expression of A1 but not of A2a adenosine receptors. However, because nicardipine lowered both blood pressure and A1 mRNA expression, it is not possible to conclude if A1 mRNA decrease is implicated in the nicardipine effects on blood pressure.ii) CyA induces an increase in renal artery adenosine concentration and a decrease in mRNA expression of A1 and A2a adenosine receptors.
Adenosine↗
Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat.
The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data.
Antigens, Plant↗
The synthetic CD4 exocyclic CDR3.AME(82-89) inhibits NF-kappaB nuclear translocation, HIV-1 promoter activation, and viral gene expression.
We have previously shown that the synthetic aromatically modified exocyclic (AME) analog (CDR3.AME(82-89), derived from the CDR3 (residues 82-89) region of CD4 domain 1, inhibits replication of human immunodeficiency virus type 1 (HIV-1) in infected cells. In this work, we investigated the mechanism by which this inhibition is achieved. Although cells exposed to HIV-1 and treated with the CDR3.AME(82-89) peptide did not release viral particles for more than a week and kept surface expression of CD4, viral DNA was found in those cells 24 h after virus exposure, indicating that the CDR3.AME(82-89) analog does not prevent virus entry. However, virus transcription remained extremely low in infected cells, as demonstrated by the study of spliced HIV-1 mRNA in cultures treated with CDR3.AME(82-89) 72 h postinfection. Finally, the CDR3.AME(82-89) peptide was found to be a potent inhibitor of HIV-1 promoter activity and nuclear factor-kappaB translocation, indicating that the antiviral property of this peptide is, at least in part, linked with the ability of the molecule to prevent HIV-1 transcription.
Anti-HIV Agents↗
Three cases of canine leptospirosis in Quebec.
Three dogs from different locations with acute renal failure were hospitalized in autumn 1996 and 1997. Leptospira interrogans serovar pomona was detected by the microscopic agglutination test. All dogs recovered after antibiotic treatment. The importance of the development of vaccines adapted to emerging serovars in dogs should be addressed.
Agglutination Tests↗
Activation of E2F-mediated transcription by human T-cell leukemia virus type I Tax protein in a p16(INK4A)-negative T-cell line.
The human T-cell leukemia virus type I (HTLV-I) is a causative agent of adult T-cell leukemia. Although the exact mechanism by which HTLV-I contributes to leukemogenesis is still unclear, the Tax protein is thought to play a major role in this process. This 40-kDa polypeptide is able to interact with the tumor suppressor p16(INK4A). Consequently, Tax can activate the signaling pathway that lead to the release of E2F that in turn induces expression of factors required for cell cycle progression. In this paper, we demonstrate that Tax can also activate E2F-mediated transcription independently of p16(INK4A). Indeed, when Tax is coexpressed with the E2F-1 transcription factor in CEM T-cells, which lack expression of p16(INK4A), it strongly potentiates the E2F-dependent activation of a reporter construct driven by a promoter containing E2F binding sites. This stimulation is abrogated by mutations affecting the E2F-binding sites. In addition, Tax also stimulates the transcription of the E2F-1 gene itself. Using Tax mutants that fail to activate either ATF- or NF-kappaB-dependent promoters and different 5' truncation mutants of the E2F-1 promoter, we show that the Tax-dependent transcriptional control of the E2F1 gene involves, at least in part, the ATF binding site located in the E2F-1 promoter.
Activating Transcription Factors↗
An anti-CD4 (CDR3-loop) monoclonal antibody inhibits human immunodeficiency virus type 1 envelope glycoprotein-induced apoptosis.
Inhibition of human immunodeficiency virus type 1 (HIV-1)-inducing programmed cell death (PCD) by anti-CD4 monoclonal antibodies (mAbs) was investigated using DNA intercalant YOPRO-1 assay. We found that 13B8.2, an mAb that binds the CDR3-like loop in domain 1 (D1) of CD4, protected infected CEM cell cultures against HIV-1-induced PCD. Protection was not observed using another anti-CD4 mAb (BL4) that binds D1-D2, suggesting that the mechanism involved in cell protection against HIV-1-induced PCD requires engagement of precise CD4 epitopes. Because 13B8.2 is known to inhibit syncytia formation and virus transcription, this mAb could inhibit HIV-1-induced PCD by (1) inhibiting virus gene expression, (2) preventing viral envelope-CD4 interaction, and/or (3) interfering with apoptotic signals. Our data indicated that the absence of enhanced PCD in infected cell cultures treated with 13B8.2 mAb probably was the result of inhibition of HIV-1 replication and virus spread. Moreover, 13B8.2 mAb was found to inhibit PCD mediated by membrane-expressed HIV-1 envelope glycoproteins. Finally, we found that 13B8.2 mAb displayed no protective interference with apoptotic signal induced by Fas, dexamethasone, and serum withdrawal.
Antibodies, Monoclonal↗
Inhibition of HIV-1 replication by a monoclonal antibody directed toward the complementarity determining region 3-like domain of CD4 in CD45 expressing and CD45-deficient cells.
Monoclonal antibodies directed toward the complementarity determining region (CDR)3-like loop of the aminoterminal domain of CD4 have been shown to inhibit the replication of human immunodeficiency virus (HIV) in CD4 positive T cells. The mechanism of action of these antibodies is not yet elucidated, although several observations suggest that they inhibit viral transcription by signal transduction through the CD4 molecule, potentially implicating the activation of a protein tyrosine kinase (PTK) cascade. Since CD45 is the major protein tyrosine phosphatase associated to the plasma membrane in T cells, and has been shown to regulate the activity of several PTK, we postulated that CD45 may be necessary for the inhibitory action of the CDR3-like specific anti-CD4 antibodies. Therefore we tested the effect of one such anti-CD4 monoclonal antibody, 13B8.2, in repressing HIV replication in CD45 positive cell lines and CD45 deficient variants. Our data show that cells respond to 13B8.2 postinfection treatment regardless of CD45 expression, indicating that neither CD45 nor PTK regulated by CD45 are implicated in the mechanism of action of this antibody.
Antibodies, Monoclonal↗
Involvement of extracellular signal-regulated kinase module in HIV-mediated CD4 signals controlling activation of nuclear factor-kappa B and AP-1 transcription factors.
Although the molecular mechanisms by which the HIV-1 triggers either T cell activation, anergy, or apoptosis remain poorly understood, it is well established that the interaction of HIV-1 envelope glycoproteins with cell surface CD4 delivers signals to the target cell, resulting in activation of transcription factors such as NF-kappa B and AP-1. In this study, we report the first evidence indicating that kinases MEK-1 (MAP kinase/Erk kinase) and ERK-1 (extracellular signal-regulated kinase) act as intermediates in the cascade of events that regulate NF-kappa B and AP-1 activation upon HIV-1 binding to cell surface CD4. We found that CEM cells transfected with dominant negative forms of MEK-1 or ERK-1 do not display NF-kappa B activation after HIV-1 binding to CD4. In contrast, NF-kappa B activation was observed in these cells after PMA stimulation. Although the different cell lines studied expressed similar amounts of CD4 and p56(lck), HIV-1 replication and HIV-1-induced apoptosis were slightly delayed in cells expressing dominant negative forms of MEK-1 or ERK-1 compared with parental CEM cells and cells expressing a constitutively active mutant form of MEK-1 or wild-type ERK-1. In light of recently published data, we propose that a positive signal initiated following oligomerization of CD4 by the virus is likely to involve a recruitment of active forms of p56(lck), Raf-1, MEK-1, and ERK-1, before AP-1 and NF-kappa B activation.
Apoptosis↗
In vitro selection of HIV-1 resistant to an anti-CD4 monoclonal antibody that inhibits virus transcription.
Phase I studies using monoclonal antibodies (mAbs) that bind to the Ig-CDR3-like loop in domain 1 of CD4 (e.g., 13B8-2 mAb) have already been documented for HIV-1-infected patients. In vitro, such mAbs do not inhibit virus to cell fusion but are able to inhibit virus envelope-mediated syncytia formation. Moreover, these mAbs inhibit Tat-induced activation of HIV-1 promoter and HIV-1 transcription in infected CD4+ cells. Here, we report the selection of escaped mutant virus or viruses derived from HIV-1Lai capable of replicating in vitro in the presence of concentrations of 13B8-2 mAb, that usually inhibit HIV-1Lai particle production. The escaped mutant virus or viruses, termed HIV-1Lai13EM, kept the major enzymatic restriction sites found in HIV-1Lai and remained sensitive to anti-CD4 mAb-, soluble CD4-, and recombinant gp120-mediated inhibition of syncytia formation. Possible genetic changes affecting the tat gene or the 5' long terminal repeat (LTR) were investigated. Partial sequence analysis of HIV-1Lai13EM and a control HIV-1Lai grown for 85 days in CEM cells, demonstrated that the first tat exon of these two viruses encoded identical proteins. Although a point mutation G>A was frequently encountered (6 of 13 sequences) in the LTRs of HIV-1Lai13EM at position -188 within the negative regulatory element (NRE), this mutation did not confer the escape mutant phenotype. Our study indicates that the mutant phenotype probably requires genetic changes in a region or regions outside the LTRs.
Anti-HIV Agents↗