Search PubMed⌕ Search

Biomedical subjects

C Desgranges

Publications and source records attributed to C Desgranges.

At least 37 records · Page 2Linked to original sources
PubMed1999

Semiquantitative analysis of residual disease in patients treated for adult T-cell leukaemia/lymphoma (ATLL).

Many adult T-cell leukaemia/lymphoma (ATLL) patients who respond to induction treatment, then relapse. Knowing the clonality pattern of residual tumourous clones during treatment could help understand disease evolution and aid therapeutic decisions. We developed a sensitive and semi-quantitative molecular analysis of these clones in ATLL patients. DNA samples from PBMCs derived from eight ATLL patients were studied over time by quadruplicate linker mediated PCR (LMPCR) amplification of HTLV-1 integration sites. Patients were treated with combination chemotherapy, zidovudine-interferon-alpha and/or by peripheral stem cell transplantation or allogeneic bone marrow transplantation. Persistence of tumourous clones at a high frequency (>1/300 PBMCs) was frequently observed, even in complete responders, and was invariably correlated with relapse and/or poor outcome. Fluctuation in the frequency of some tumourous clones was observed with evidence for clonal change under treatment in one patient, indicating that treatment of ATLL can result in the selection of resistant clones. Finally, allogeneic bone marrow transplantation (BMT) using an HTLV-1 infected sibling as donor was found to be associated with long-lasting disappearance of tumourous clones and a possible cure of the disease. Long-term persistent clonal expansion of circulating HTLV-1 bearing T cells which derived from the donor bone marrow was evidenced in this patient. In conclusion, variable success in treatment of ATLL is probably due to the clonal heterogeneity which results in the selection of resistant clones. Semi-quantitative assessment of residual disease (RD) through LMPCR may predict treatment failure. Accordingly, additional therapy may be tailored to the clonality pattern observed after first-line therapy.

Adult↗
PubMed1998

Characterization of human monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in vitro binding neutralization properties.

Both linear and conformational determinants of hepatitis C virus (HCV) are believed to be involved in viral neutralization. After immortalization of B cells from HCV chronically infected patients with Epstein-Barr virus, we obtained two polyclonal lymphoblastoid cell lines (LCL) secreting human monoclonal antibodies (HMabs). One clone was derived from a patient infected with a genotype 4 isolate while the second was isolated from a genotype 1b-infected patient. Immunoprecipitation studies, Western blot, and immunofluorescence analysis, peptide scanning, and ELISA studies indicated that the HMabs (1) recognized conformation-dependent determinant(s), (2) were capable of recognizing genotype 1a and 1b derived antigens, and (3) were able to precipitate noncovalently associated E1E2 complexes believed to exist on the surface of virion particles. The HMab derived from the genotype 4-infected patient was in addition shown to neutralize the in vitro binding of recombinant E2 protein onto susceptible cells suggesting a potential for in vivo neutralization. These data indicate that anti-E2 antibodies directed at conserved conformational-dependent determinant(s) exist in chronic HCV infection.

Antibodies, Monoclonal↗
PubMed1998

Identification of exposed epitopes on the envelope glycoproteins of human T-cell lymphotropic virus type I (HTLV-I)

Several lines of evidence underscore the important role of the humoral response specific for HTLV-I envelope protein in the protection against viral infection. One approach to producing efficient immunogens is to synthesize peptides corresponding to the primary amino-acid sequence of neutralizing epitopes found in the external sub-unit gp46. In this study, we have selected synthetic peptides overlapping the major linear neutralizing determinants described earlier and used them as immunogens in rabbits and mice. All rabbit polyclonal anti-sera raised against peptides recognized epitopes in a denaturated context as well as MAbs raised against the HB peptide (aa287-311). By contrast, synthetic peptides O (aa89-110), HH (aa190-209), T (aa190-212) and HB (aa287-311) have generated antibodies efficiently binding their epitopes in a native context, suggesting that these domains are well exposed both at the heterodimer and at the oligomer surface. None of the antibodies induced by synthetic peptides show in vitro neutralizing properties, even those with a good capacity to bind the native form of HTLV-I envelope proteins.

Amino Acid Sequence↗
PubMed1998

Intracellular neutralization of HIV transcytosis across tight epithelial barriers by anti-HIV envelope protein dIgA or IgM.

Human immunodeficiency virus, generated during contact between HIV-infected cells and the apical surface of an epithelial cell, can cross a tight epithelial barrier by transcytosis. We show that transcytosis of primary HIV isolates is blocked by dimeric IgA or IgM against HIV envelope proteins. Neutralization occurs intracellularly within the apical recycling endosome, and immune complexes are specifically recycled to the mucosal surface. One epitope involved in neutralization is a conserved sequence of the gp41 HIV envelope protein subunit. Finally, transcytosis also occurs across functional human mucosal tissue in a process inhibited by a serosal internalization of IgM against the HIV envelope protein. These results suggest that induction of mucosal immunity to HIV envelope proteins may impair the transcytotic route of HIV mucosal transmission.

Antigen-Antibody Complex↗
PubMed1998

Investigations into the cross-reactivity of rabbit antibodies raised against nonhomologous pairs of synthetic peptides derived from HIV-1 gp120 proteins.

The immunological cross-reactivity of several peptides with specific pattern-property characteristics related to the epitopes of human immunodeficiency virus type 1 (HIV-1) gp160/ 120 envelope proteins has been investigated. Proteins with similar primary structures can be expected to show functional or topographic similarities, such as specific epitopes which may cross-react with antibodies derived from the immunisation of animals with other members of the same protein family. These structure-function characteristics may be revealed as periodicities derived from presentations based on the discrete Fourier transformation of the distributions of various physico-chemical amino acid descriptors, constituting the polypeptide backbone and amino acid side-chains of the protein molecule. Such approaches, for example, have permitted prediction of periodicities corresponding to secondary structural motifs, including amphipathic alpha-helices and beta-sheets, within protein sequences, and have helped to clarify potential binding sites for ligands, substrates or cofactors with interacting macromolecules. Based on this approach, characteristic periodicities have been identified which represent common Fourier transform spectral properties of the envelope (ENV) gp160/120 glycoproteins from a range of HIV-1 isolates. In addition, similar periodicities have been detected as components of the discrete Fourier transform representation of the corresponding amino acid descriptors of the CD4 binding domain of gp120. Accordingly, we have synthesised several peptides having periodic characteristics in their discrete Fourier transform representations similar to these HIV-1 proteins. These nonhomologous synthetic peptides induced cross-reactive antibodies in New Zealand White rabbits. Polyclonal antibodies raised to one of these peptides reacted with HIV-1 ENV gp120-related proteins, as determined by enzyme-linked immunosorbent assay and Western blotting techniques. These findings provide further evidence for a role of immunological cross-reactivity and molecular biomimicry in the development of peptide-based vaccines directed against viral or bacterial pathogens.

Animals↗
PubMed1997

Mouse monoclonal antibodies directed against the HTLV-I protease recognize epitopes internal to the dimer.

The proteases (PR) of retroviruses are expressed as gag-PR fused polyprotein. The active PR is a dimer obtained after the aggregation of the gag and gag-pro precursors, which leads to the formation and the release of the viral particle. Subsequently, in the cell, the PR is present essentially as a monomeric polyprotein. To mimic the antigenic properties of such an intracellular form of the PR, we produced a monomeric form of the HTLV-I (human T-cell leukemia virus, type-I) PR fused to the maltose binding protein (MBP-PR). Monoclonal antibodies (mabs) directed against MBP-PR were developed. Three mabs were obtained that recognized different epitopes. Two were directed against the NH2-terminus, a region that contributes to the dimerization interface. The other was specific to a peptide that lines the substrate binding pocket. This latter epitope is located just downstream of the D-T-G peptide of the catalytic site. The two identified regions contained the amino acids Asp6, Arg10 and Asp36, which were previously shown to be important in the stabilization of the dimer. In view of the localization of the recognized epitopes, these mabs will be useful for inhibition studies of the HTLV-I PR by intracellular immunization.

Animals↗
PubMed1997

Transfusion transmission of human T-lymphotropic virus type I (HTLV-I) from an asymptomatic blood donor: conservation of LTR U3, env, and tax nucleotide sequences in a recipient with HTLV-I-associated myelopathy.

BACKGROUND: Transfusion of human T-lymphotropic virus type I (HTLV-I) contaminated blood is sometimes linked with the rapid onset of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in immunocompromised recipients. STUDY DESIGN AND METHODS: This study addressed the question of whether HTLV-I variants could emerge in an immunocompromised patient who developed HAM/ TSP after the transfusion of blood from an asymptomatic HTLV-I carrier. Base pairs (n = 2327) of the HTLV-I genome located in the LTR U3 region and parts of the env and tax genes were sequenced and compared to the isolated identified in the asymptomatic blood donor and to well-known ATK-1 and H5 strains. The same analysis was performed on another set of samples from an asymptomatic HTLV-I-positive blood donor and a similar blood recipient. RESULTS: No critical changes in nucleotide sequences were identified in the immunosuppressed HAM/TSP patient when compared with the nucleotide sequences of the corresponding blood donor, the other asymptomatic blood donor and recipient or the ATK-1 and H5 strains. CONCLUSION: Immunosuppression does not seem to favor the emergence of particular nucleotide sequences in the genome located in the LTR U3 region or in parts of the env and tax genes in transfused patients who develop HAM/TSP.

Base Sequence↗
PubMed1997

Induction of neutralizing antibodies against HTLV-I envelope proteins after combined genetic and protein immunizations in mice.

Direct DNA inoculation can induce both protective humoral and cellular responses against several viruses. The HTLV-I envelope glycoproteins are the major antigens recognized by sera of HTLV-I infected patients that generate neutralizing immune responses in vitro and in vivo. We compared immune responses elicited after a single inoculation of two plasmids encoding the complete HTLV-I envelope proteins followed or not by gp62 Baculovirus recombinant protein boosts in BALB/c mice. First, we observe that the coexpression of env and rex genes is not sufficient to raise a detectable specific humoral response after a single DNA inoculation. Protein boosts generated a high antibody response in mice primed with DNA expressing HTLV-I envelope proteins as compared to naive and negative control vector primed groups. This humoral response presented high neutralizing antibody titers. These results suggest that a single inoculation of DNA expressing HTLV-I env gene can stimulate memory B-cell clones that are able to respond effectively to subsequent encounters with HTLV-I envelope proteins and a specific cellular T helper cell response in mice.

Amino Acid Sequence↗
PubMed1997

Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles.

Here we report the presence of a protein kinase activity associated with human immunodeficiency virus type 1 (HIV-1) particles. We observed phosphorylation of five major proteins by the endogenous protein kinase activity. Phosphoamino acid analysis revealed phosphorylated serine and threonine residues. In addition, we observed autophosphorylation of two proteins in the presence of gamma-ATP in an in-gel phosphorylation assay. These two proteins are not linked by a disulfide bond, suggesting that two different protein kinases are associated with HIV-1 virions. Our results indicate the presence of ERK2 mitogen-activated protein kinase and of a 53,000-molecular-weight protein kinase associated with virions. Moreover, the use of different HIV strains derived from T cells and promonocytic cells, as well as the use of human T-cell leukemia virus type 1 particles, demonstrates that ERK2 is strongly associated with retrovirus particles in a cell-independent manner. Exogenous substrates, such as histone proteins, and a viral substrate, such as Gag protein, are phosphorylated by virus-associated protein kinases.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Overexpression of P2Y2 purinoceptor in intimal lesions of the rat aorta.

Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y2 (P2u) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y2 cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y2 expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y2 mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y2 mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y2 mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y2 expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y2 purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.

Amino Acid Sequence↗
PubMed1997

Zidovudine treatment is not associated with HTLV-1 reverse transcriptase gene mutations in HTLV-I/HIV-1 co-infected patients.

Zidovudine treatment of human immunodeficiency virus (HIV) infection induces drug-resistant viral strains harbouring specific amino acid substitutions in the reverse transcriptase (RT). To investigate whether this phenomenon could be observed in the case of human T lymphotropic virus type I (HTLV-I) infection, we analysed the HTLV-I RT proviral gene sequence in five HTLV-I/HIV-1 co-infected patients treated with zidovudine for HIV-1 infection and in one untreated co-infected subject. In the 816 bp of HTLV-I pol gene sequence determined, no particular nucleotide mutation associated with zidovudine therapy could be identified in the treated subjects. Moreover, the dominant HTLV-1 deduced amino acid sequences determined in treated subjects were identical to that from the untreated subject. Our data show that in the co-infected patients already presenting well-defined mutations associated with zidovudine resistance in HIV-1, no mutations were observed in a part of the pol gene coding for the RT activity of HTLV-I.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Nucleotide receptor P2u partially mediates ATP-induced cell cycle progression of aortic smooth muscle cells.

mRNA of the P2u purinoceptor (or nucleotide receptor) is detected both by polymerase chain reaction or Northern blot analyses in cultured aortic smooth muscle cells. When added to the culture medium of these cells, UTP, a specific ligand of the P2u receptor, induces an increased expression of both immediate-early and delayed-early cell cycle-dependent genes. This induction demonstrates similar features (kinetics, concentration dependence) to those obtained after stimulation of aortic smooth cells by exogenous ATP, a common ligand for most P2 purinoceptors. In contrast, 2-methylthioATP, a preferential ligand for P2y purinoceptors, induces only a significant increase of immediate-early genes but not of delayed-early genes. Moreover, the 2-methylthioATP-induced responses (c-fos mRNA increase, free intracellular calcium transient) are lower than those induced by ATP or UTP and are complementary to those of UTP. These results demonstrate that functional P2u receptors are present on cultured aortic smooth muscle cells and suggest that the bulk of responses induced by extracellular ATP on cell cycle progression are mediated via P2u purinoceptors, a hypothesis confirmed by cytofluorometric studies. Since some ATP- or UTP-induced genes code for chemotactic proteins (monocyte chemoattractant protein-1 and osteopontin), this study suggests that these nucleotides may contribute to vascular or blood cell migration and proliferation and consequently to the genesis of arterial diseases.

Adenosine Triphosphate↗
PubMed1996

Identification of differentially expressed mRNA species during HIV infection by RNA arbitrarily primed PCR.

BACKGROUND: A number of strategies, such as subtractive cDNA libraries and high through-put sequencing, have been devised to assess differential gene expression. Most of these approaches, however, are cumbersome and/or require tremendous technological power. In this paper, we describe a method, RNA fingerprinting using arbitrarily primed polymerase chain reaction (RAP-PCR), that is rapid, less cumbersome and can differentiate low levels of mRNA expression. OBJECTIVES: To identify genes that are differentially expressed following human immunodeficiency virus type 1 (HIV-1) infection in different cell types by RAP-PCR. STUDY DESIGN: RNA was extracted from both HIV-1-infected and uninfected HUT78 cells and peripheral blood mononuclear cells (PBMCs), reverse transcribed, and RAP-PCR amplified using numerous primer sets. RESULTS: Three genes, gamma-actin, the HIV-1 nef and an unknown sequence, were identified as being differentially expressed in HUT78 cells. The level of gamma-actin mRNA expression is increased after HIV infection and, as expected, the nef gene was solely expressed in HIV-infected cells. In contrast, the unknown mRNA is down-regulated by HIV. Northern blot analysis and/or specific PCR confirmed the differential expression of these three genes. RNA fingerprinting using phytohemagglutinin (PHA)-activated PBMCs infected by HIV in vitro, revealed that gamma-actin is still up-regulated by HIV, whereas the unknown product no longer shows down-regulation. CONCLUSIONS: These results illustrate the usefulness of the RAP-PCR method for isolating and identifying differentially expressed genes during HIV-1 infection of primary lymphocytes.

Actins↗
PubMed1996

Complement-dependent cytotoxic antibodies to human T lymphotropic virus type I (HTLV-I)-infected cells in the sera of HTLV-I-infected individuals.

To investigate whether HTLV-I induces the development of complement-dependent cytotoxic antibodies in humans, sera of asymptomatic HTLV-I carriers and of patients suffering from tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) or adult T cell leukaemia (ATL) were used in a cytotoxicity assay against a panel of target cells. This panel included uninfected cell lines (CEM, Jurkat, Molt and H9), cell lines chronically infected with HTLV-I (MT2, MT4, C9IPL and HUT102), as well as lines H36 (H9 infected with HTLV-I), H9-IIIB (H9 infected with HIVms) and H9-MN (H9 infected with HIMVMN). HTLV-I+ sera induced lysis of H36 and of lines expressing HTLV-I antigens in the presence of rabbit complement, but did not lyse cells in presence of human complement. The HTLV-I+ sera also failed to lyse the HTLV-I- lines and H9 cells, suggesting that lysis was specific for HTLV-I. H36 cell lysis was prevented by IgG depletion of the sera and by dialysis of rabbit complement against EGTA or EDTA. Rabbit complement-dependent cytotoxic antibodies were present in the sera of 14/14 HTLV-I-infected individuals; the highest titres were predominantly found in the sera of the TSP/HAM patients. Such antibodies were also detected in 5/5 individuals coinfected with HIV-1 and HTLV-I, although no cytotoxic antibody could be found against HIV-infected cells. Vice versa, sera of HIV-1-infected individuals did not exert a lytic effect in the presence of complement (of human or rabbit origin) against HIV-1- or HTLV-I-infected cells. Incubation of the sera of four HTLV-I-infected patients with HTLV-I env-specific synthetic peptides demonstrated that some of the complement-dependent cytotoxic antibodies recognized epitopes located on gp46 between amino acids 190 and 209. There is no correlation of rabbit complement-dependent cytotoxic HTLV-I antibodies with the development of disease.

Antibodies, Viral↗