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C Leclerc

Publications and source records attributed to C Leclerc.

At least 109 records · Page 6Linked to original sources
PubMed1996

Overcoming the crypticity of a viral T cell determinant by insertion into a chimeric bacterial protein.

Among the potential T cell determinants contained in a protein antigen, the T cell response only focuses on a few immunodominant T cell determinants, whereas cryptic epitopes remain hidden to the immune system. In the present work, we have studied the antigen processing and presentation of the C3:93-115 sequences of Mahoney poliovirus VP1 protein, which is immunodominant in H-2d but cryptic in H-2s and H-2q mouse MHC haplotypes. For this purpose, we genetically inserted the C3 determinant into five internal sites of a bacterial protein, the maltose binding protein of Escherichia coli (MalE). In four out of five insertion sites of MalE, the C3 determinant retained its immunodominance when the purified hybrid proteins were injected to BALB/c (H-2d) mice. Moreover, in SJL/J (H-2s) mice, in three out of five MalE-C3 constructs, the new structural environment of the cryptic C3 epitope rescued its processing and its in vivo presentation to T cells. In contrast, in DBA/1 (H-2q) mice, although MalE-C3 chimeric proteins were correctly processed in vitro, the C3 epitope remained cryptic in vivo. In this case, the impairment to stimulate a T cell response in vivo was correlated with a short time persistence of C3 peptides bound to Aq molecules at the surface of live antigen-presenting cells. These results emphasize the role of flanking residues on the lack of processing of cryptic determinants and the importance of the life span of peptide-MHC complexes to stimulate T cell responses.

ATP-Binding Cassette Transporters↗
PubMed1996

Characterization of T-cell immune responses of Echinococcus multilocularis-infected C57BL/6J mice.

Specific and non-specific parasite-induced changes in lymphocyte responses were analysed in C57/BL/6J mice after intrahepatic infection with Echinococcus multilocularis. Spleen cells harvested at selected times after infection were in vitro stimulated with mitogens or a crude soluble parasite extract (EmAg) at an optimized dose. Cell proliferative responses to Con-A were not modified by the infection over the first 22 weeks. In contrast, LPS-induced responses were decreased from the 13th week. A strong CD4+ proliferative T-cell response to the parasitic extract of infected mouse spleen cells was observed at the early stage of infection. This response then progressively decreased but remained significantly higher than that of control mice until the 19th week of infection. Cytokine production was investigated after in vitro EmAg stimulation of spleen cells. IFN-gamma, IL-2, IL-5 were produced within the first weeks after infection whereas the detection of IL-10 was slightly delayed. Thus, the promotion of the disease does not appear associated with the expansion of one rather than another T-cell subset in C57BL/6J mice. A general immunosuppression affecting both mitogenic and parasite-specific T-cell responses was observed at the end of the infection.

Animals↗
PubMed1996

[Adaptation to the community by young adults after one or more psychiatric hospitalizations].

This longitudinal study aimed at elaborating a model explaining the adaptation to the community of young adults suffering from mental illness. During their sick leave, an interview evaluated their social support, the stress experienced, coping strategies and emotional distress. Six months later, therapists were interrogated as to the recourse of these young adults to psychiatric services, their loyalty to therapeutic and medical treatment as well as how they kept busy during that period. Analyses by structural equations (LISREL) have allowed to develop a model which adequately reproduces relations observed between variables and explains 43% of the total variance. This model also indicates that the level of psychological distress has a direct impact on a psychiatric rehospitalization.

Adolescent↗
PubMed1995

DNA-mediated immunization to the hepatitis B surface antigen. Activation and entrainment of the immune response.

The use of plasmid vectors expressing the HBsAg, along with improved protocols for transfection of muscle fibers (Refs. 3-6 and Davis et al., this volume), have provided the reagents and methods with which to investigate the characteristics of the strong immune response given by this antigen after DNA-mediated immunization. Analysis of the fine specificity of the humoral response provides support for the idea that the HBsAg-bearing particles are formed such that the B and T epitopes are presented to the immune system in a way resembling that of the natural viral or subviral particles. As shown here and elsewhere, DNA-mediated immunization with the HBsAg-expressing plasmid vectors induces strong CTL responses as well as a dominant Th1 phenotype among the splenic lymphocytes of immunized mice. The Th1 cytokine profile can be obtained in two different strains of mice and with two types of proteins, HBsAg and beta-galactosidase. One important line of investigation in the future will be to determine the mechanism of this generic Th1 response to DNA-based immunization. Circumstantial evidence, discussed by Pisetsky et al. (this volume), suggests that the chemical nature of DNA may play a role as an adjuvant (see also Ref. 31), and this hypothesis to explain the cytokine profiles observed after DNA-mediated immunization must now be taken seriously. All the questions raised by this novel method of immunization are of interest for the design of future vaccines, even if DNA itself is ultimately not the vaccinating moiety. The question of antigen presentation is particularly intriguing, since the small amounts of protein produced by DNA-mediated immunization (on the order of nanograms) are capable of inducing strong immune responses at the level of B and T cells. Although initially it seemed obvious that endogenous protein synthesis in cells transfected with plasmid DNA would account for the observed induction of CTL activity, this idea must be examined in light of two well established sets of experimental results. First, the primary events in activation of CD8+ (as well as CD4+) T lymphocytes normally require professional APC capable of furnishing co-stimulatory signals to supplement the consequences of interaction of the T-cell receptor with MHC surface molecules. Second, endogenous synthesis and processing is not the only mechanism of class I epitope presentation, and numerous examples are now known whereby particulate exogenous proteins, such as HBsAg, can be taken up and processed in such a way as to allow class I presentation of peptides. Consideration of these two points suggests that a major contribution to the observed CTL induction afforded by DNA-mediated immunization could come from the sustained presence of the antigenic protein in interstitial spaces or in the circulation, coupled with the ability of the exogenous protein to be processed for class I presentation. This could be true for many other proteins in addition to the HBsAg. This hypothesis eliminates the inconvenient notion that muscle fibers (or other nonleukocyte cells) present antigen in a way compatible with primary activation of T cells. However, muscle tissue can be an important reservoir of the antigen because of the potential for prolonged synthesis of the protein; this could therefore explain the immune entrainment observed after DNA-mediated immunization. Muscle fibers or other cells could also serve to present class I epitopes for the purpose of restimulating and thus expanding the pool of activated CD8+ T lymphocytes. These explanations, though certainly plausible, will require experimental investigation. The small numbers of the transfected cells in vivo, as well as the potential mobility of transfected cells other than muscle fibers, may well render such experimentation difficult. DNA-mediated immunization clearly offers opportunities for obtaining novel insights into immunological mechanisms and immunization processes. It is also likely to promote vacc

Animals↗
PubMed1995

Stimulation of a memory B cell response does not require primed helper T cells.

The use of universally immunogenic T cell epitopes, such as those identified in tetanus toxin or malaria circumsporozoite protein, could represent a major improvement in the development of synthetic vaccines. However, one limitation of this approach is the lack of T cell cross-reactivity between the vaccine and the pathogen. To determine whether the memory B cell response elicited by immunization with a synthetic peptide containing a B cell epitope linked to a T cell epitope can be restimulated by the same B cell epitope linked to different T cell epitope(s), we used a synthetic peptide which contains non-overlapping B and T cell determinants from hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV). The results of this study clearly show that primed T cells can increase the antibody response against a B cell epitope linked to the priming T cell determinant. However, the antibody response obtained was weaker than that obtained after two injections of the peptide containing both B and T cell epitopes, showing the important role played by memory B cells in secondary antibody responses. Moreover, a strong antibody response against the B cell epitope was elicited by boosting mice with the B cell epitope linked to a heterologous carrier, thus demonstrating that a strong B cell memory response can be revealed in the absence of primed T cells. These results therefore provide new important information for the design of synthetic or recombinant vaccines.

Animals↗
PubMed1995

Introduction.

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Journal Article↗
PubMed1995

In vivo labelling of L-type Ca2+ channels by fluorescent dihydropyridine: correlation between ontogenesis of the channels and the acquisition of neural competence in ecotderm cells from Pleurodeles waltl embryos.

The ability of the ectodermal cells to be induced and to differentiate toward neural tissue, called neural competence, is acquired shortly before gastrulation and lost during late gastrula stages in Pleurodeles waltl embryos. We have examined ectodermal cells' neural competence in relation to the evolution of the density of L-type calcium channels using the fluorescent labelled dihydropyridine probe (STBodipy-DHP). We find that the appearance of dihydropyridine sensitive calcium channels (L-type Ca2+ channels) is correlated with the acquisition of neural competence by the ectoderm cells. The highest density of these channels is reached when competence of the ectoderm is optimal. Conversely, the decrease of L-type Ca2+ channel density occurs simultaneously with the normal loss of competence. In addition, we show that these channels are functional since stimulation by S(-)-Bay K 8644 triggered an increase in [Ca2+]i revealed by fluorescence measurements using Fluo-3. This increase in [Ca2+]i is a function of the L-type Ca2+ channels' density. We propose that the molecular basis of the gain and loss of neural competence is linked to the presence of L-type Ca2+ channels in ectodermal cell membranes of Pleurodeles waltl embryos.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Progressive development of gap junctions during growth of human pancreatic duct cells (Capan-1) in vitro and in vivo.

Among their numerous functions, gap junctions play a crucial role in proliferation, differentiation and secretion processes, although their existence and potential role in ion secretion in human pancreatic ducts have yet to be established. To investigate the morphogenesis and the role of gap junctions in human pancreatic duct cells, the Capan-1 cell line maintained in culture or heterotransplanted into nude mice was employed as model system. Capan-1 cells polarize during their growth in vivo and in vitro forming duct-like structures. Furthermore in culture, after confluence, these cells form domes, which is indicative of ion exchange processes. After treatment with tannic acid and freeze-fracture, gap junctions were observed along the basolateral membranes of Capan-1 cells on electron microscopic examination. The presence of alkaline phosphatases on gap junctions was demonstrated cytoenzymatically. In addition, cell-to-cell communication was visualized by microinjection of Lucifer yellow. During differentiation of Capan-1 cells in culture, the frequency of intercellular communications increased markedly over the period (days 11-13) when the cells form duct-like structures. The increase in gap junctions was demonstrated by analysis of the polarized cells organized in duct-like structures that are commonly observed in the tumors formed by heterotransplantation of Capan-1 cells into nude mice. Furthermore, gap junctions associated with tight junctions were also observed in the cells forming such structures. The role of gap junctions in ion exchange was evaluated by counting the number of domes in cultures treated with heptanol. Heptanol (an uncoupling agent of gap junction communication) completely inhibited dome formation in a reversible way, and reduced the frequency of intracellular communications by 44%. These results suggest that the gap junctions expressed by Capan-1 cells are involved in ion secretion by the human cancerous pancreatic duct cell line, Capan-1. In the present study, we show that: i) the expression of gap junctions is linked to development of the spatial conformation of the cells; and ii) gap junctions may be involved in ion secretion.

Adenocarcinoma↗
PubMed1995

Recombinant BCG strains expressing the SIVmac251nef gene induce proliferative and CTL responses against nef synthetic peptides in mice.

CTL responses are known to be important for the control of HIV and SIV infections. Such responses are targeted against various components of these viruses including regulatory proteins like Nef. The SIVmac251nef gene was cloned in Mycobacterium bovis BCG under the control of P(AN), a promoter from Mycobacterium paratuberculosis. Nef was expressed as a fused polypeptide with ORF2, an open reading frame adjacent to P(AN). Mice inoculated with rBCG(SIVmac251nef) exhibited proliferative and CD8+ cytotoxic T-cell (CTL) responses against several Nef synthetic peptides. A mapping of the epitopes recognized by CTLs revealed that the central region of Nef is mainly involved in responses. This region had already been demonstrated to induce CTLs in experimentally SIV-infected macaques as well as in HIV-infected individuals. These results demonstrate the feasibility of constructing BCG vaccine strains expressing nef for eliciting cytotoxic responses.

Animals↗
PubMed1995

Intranasal immunization of mice against influenza with synthetic peptides anchored to proteosomes.

Synthetic vaccines that are based on peptides representing immunogenic epitopes require a carrier molecule as well as an adjuvant in order to be effective. The choice of carriers or adjuvants approved for use in humans is very limited, and a considerable effort is devoted to develop new and efficient delivery systems. One of these vehicles utilizes preparations of outer membranes of meningococci, that form hydrophobic interactions, denoted proteosomes. Immunogenic proteins and peptides can be anchored to these proteosomes vesicles, which may serve as both carrier and adjuvant functions. In the present study we examined the ability of proteosomes to present epitopes of influenza, to elicit specific anti-influenza responses and to protect mice against viral challenge after intranasal immunization. Three influenza peptides were used--one corresponding to amino acid residues 91-108 of the haemagglutinin surface glycoprotein of H3 subtype, which comprises a B-cell epitope, and two from the internal nucleoprotein--a T-helper cell (Th) epitope (residues 55-69) and a cytotoxic T-lymphocyte (CTL) epitope (147-158). Mice were immunized intranasally (i.n.) with preparations containing each of the above epitopes, or various combinations thereof. The results obtained with this system demonstrate that influenza epitopes represented by synthetic peptides anchored to a proteosome carrier elicit both humoral and cellular specific immune responses, that can lead to partial protection of the mice from viral challenge. The importance of immunizing with vaccines containing both B- and T-cell peptide epitopes was emphasized by the demonstration that such vaccines elicited longer lasting immunity and led to more effective protection against influenza viral challenge.

Administration, Intranasal↗
PubMed1995

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant.

Macrophages, dendritic cells or B lymphocytes have been shown to play a major role in the presentation of soluble antigens to CD4+ T cells. In contrast, the capacity of these cells to present particulate antigens such as bacterial or parasitic antigens to T cells remains controversial. To investigate this question, well defined particulate antigens were prepared by covalent linkage of proteins or peptides to 1 micron in diameter synthetic microspheres. The T cell immunogenicity of such particulate antigens was analyzed in vitro and in vivo. In vitro, a soluble protein such as hen egg lysozyme (HEL) coupled to beads stimulated a strong proliferative T cell response of lymph node cells from HEL-primed mice or of specific T cell hybridomas. HEL coupled to beads was presented to the specific T cell hybridomas by splenocytes or by peritoneal macrophages, but not by lymphoma B cells. Immunization of mice with several different protein antigens or with a synthetic peptide covalently linked to beads induced strong CD4+ T cell responses in the absence of adjuvant. The strong in vivo immunogenicity of proteins coupled to beads did not result from a non-specific adjuvant effect of beads since covalent linkage of the antigen to beads was strictly required to induce T cell responses in the absence of adjuvant. In vivo treatment by carrageenan showed that macrophages are required for the in vivo stimulation of T cell responses by these particulate antigens. Thus, these results demonstrated the role of phagocytic cells, especially macrophages, for in vivo presentation of particulate antigens. These particulate antigens represent an interesting approach for the development of new vaccines, and for the in vivo analysis of the role of various antigen presenting cells in T cell activation and differentiation.

Adjuvants, Immunologic↗
PubMed1995

Cytotoxic T cell response to Mengo virus in mice: effector cell phenotype and target proteins.

The Mengo virus specific cytotoxic T lymphocyte (CTL) response was investigated after intraperitoneal infection of mice with the attenuated Mengo virus strain vMC24. A high level of CTL activity was detected in spleen cell cultures obtained from infected C3H/HeJ (H-2k) or C57BL/6 (H-2b) mice after a secondary in vitro stimulation with Mengo virus-infected cells. The CTL activity, which was MHC class I-restricted, was shown to be mediated by CD8+ T cells. Recombinant vaccinia viruses that expressed capsid proteins VP0, VP1 or VP3 were produced and used to identify the protein(s) recognized by the Mengo virus-specific CTLs. In both C3H/HeJ and C57BL/6 mice, analysis of CTL activity against target cells expressing each capsid protein showed that VP0 was the only capsid protein recognized by the CD8+ CTLs. The CTL epitope(s) could be further located in the C-terminal half of VP0, i.e. in capsid protein VP2. Moreover, using unlabelled target cells expressing VP0 as cold competitors, we were able to almost completely inhibit recognition and lysis of Mengo virus-infected cells by specific CD8+ CTLs. Thus, the CTL response directed against VP2 was immunodominant in both C3H/HeJ- and C57BL/6-infected mice.

Animals↗
PubMed1995

Immunogenicity of poliovirus B and T cell epitopes presented by hybrid porcine parvovirus particles.

We have analysed the potential capacity of hybrid porcine parvovirus (PPV) capsids to present foreign epitopes to the immune system. Foreign sequences were introduced into the N and C termini of PPV VP2, which was previously shown to assemble spontaneously into parvovirus-like particles. The integrity of the C terminus was shown to be essential for preserving the structure of the capsid and therefore could not be used for epitope fusion. In contrast, insertion of sequences corresponding to T and B cell poliovirus epitopes in the N terminus did not alter the formation of particles. Moreover, the chimeric capsids containing the C3:T epitope were able to induce a T cell response in vivo. However, hybrid particles containing the C3:B epitope fused to the N terminus did not induce any peptide-specific antibody response, suggesting that the inserted B cell epitope was not exposed at the surface of the particles. These results show that the N terminus in PPV empty capsids is not an adequate site for insertion of B cell epitopes, but may be useful for T cell epitope presentation and suggest that the N terminus is located in an internal position.

Animals↗
PubMed1995

Cell-invasive activity of epitope-tagged adenylate cyclase of Bordetella pertussis allows in vitro presentation of a foreign epitope to CD8+ cytotoxic T cells.

The adenylate cyclase (AC) toxin (CyaA) of Bordetella pertussis has an invasive catalytic domain (AC domain) which penetrates the cytoplasmic membrane of a variety of eukaryotic cells and intoxicates them by unregulated synthesis of cyclic AMP. Previous work led to identification of five permissive sites in the AC domain at which heterologous peptides are accommodated without affecting its enzymatic properties. We have constructed a set of CyaA toxins tagged at these permissive sites by insertion of a CD8+ T-cell epitope, RPQASGVYMGNLTAQ, from the nucleoprotein of lymphocytic choriomeningitis virus. Introduction of the epitope at any of the five sites did not affect the capacity of the toxin to deliver its AC domain into target cells. Moreover, the toxin with the inserted epitope was shown to sensitize target cells for lysis by epitope-specific CD8+ cytotoxic T lymphocytes in vitro, showing that the tagged AC was processed for presentation of the lymphocytic choriomeningitis virus epitope in association with the major histocompatibility complex class I molecules. This finding indicates that by virtue of delivery of foreign epitopes into the antigen-presenting cells, purpose-designed recombinant CyaAs may be useful for induction of specific major histocompatibility complex class I-restricted cell-mediated immunity also in vivo.

Adenylyl Cyclases↗
PubMed1995

4-aminopyridine acts as a weak base and a Ca2+ mobilizing agent in triggering oocyte meiosis reinitiation and activation in the Japanese clam Ruditapes philippinarum.

Ovarian oocytes of the prosobranch mollusc Patella vulgata and the pelecypod Ruditapes philippinarum are arrested during prophase of the first maturation division. Release from this blockade, which is revealed by germinal vesicle breakdown, drives these oocytes to a second arrest in metaphase I, at which time the oocytes become fertilizable. The respective roles of Ca2+ and H+ ion movements during this early step in meiosis reinitiation has not been fully established yet. In this work we reveal the presence of acidic vesicles and report that bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, two inhibitors of the vacuolar-type H(+)-ATPase, applied to Ruditapes oocytes, produce a significant inhibition of their response to the natural neurohormone serotonin. Since sodium deprivation did not affect this response, this suggests that a v-type ATPase pump, possibly located in the membrane of these acidic vesicles, may play a subtle role in the cascade of events that releases oocytes from their prophase block. We then describe how 4-aminopyridine, a drug reputed to be a K+ channel antagonist, triggers both meiosis reinitiation and activation of Patella and Ruditapes oocytes. This agent acts as a weak base, its effect depending on external pH. Moreover, using the fluorescent probes BCECF and Fluo-3/AM, we observe that this drug both alkalinizes the endoplasm and promotes an intracellular Ca2+ surge. This dual effect may explain why Ruditapes oocytes no longer stop in metaphase under these conditions and behave like other bivalve species which are directly fertilizable at the germinal vesicle stage.

4-Aminopyridine↗
PubMed1995

Thimerosal triggers meiosis reinitiation in oocytes of the Japanese clam Ruditapes philippinarum by eliciting an intracellular Ca2+ surge.

Ovarian oocytes of the bivalve mollusc Ruditapes philippinarum are arrested during first meiotic prophase. Release from this blockade is triggered by the neurohormone serotonin (5HT or 5-hydroxytryptamine), which promotes germinal vesicle breakdown and drives these oocytes to a second arrest in metaphase I. 5HT action involves binding to a specific G protein-coupled receptor which results in a transient rise in IP3 and in the intracellular free Ca2+ concentration. Here we analyze the cytological effects and mode of action of the sulphydryl reagent thimerosal which could also trigger meiosis reinitiation in Ruditapes. No metaphase I spindle formed under these conditions since thimerosal was found to be able to preclude or reverse tubulin polymerization when applied to prophase- or to metaphase-arrested oocytes, respectively. Our results strongly suggest that the common final target for 5HT and thimerosal actions consists in a transient rise in internal free Ca2+ level that we could follow using Fluo3/AM as a probe. The effect of thimerosal in promoting oocyte maturation and increasing intracellular free Ca2+ concentration was improved by excess KCI. In addition, thimerosal, but not KCI, was found to facilitate 5HT-induced maturation at subthreshold hormone concentrations which, by themselves, did not produce an intracellular Ca2+ surge. These data suggest that thimerosal may inhibit Ca2+ pumps of the endoplasmic reticulum and unmask the plasma membrane voltage-sensitive Ca2+ channels which also appear after 5HT-induced GVBD.

Aniline Compounds↗
PubMed1995

Expression of membrane targeted aequorin in Xenopus laevis oocytes.

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

Aequorin↗