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C Leclerc

Publications and source records attributed to C Leclerc.

At least 127 records · Page 7Linked to original sources
PubMed1995

Expression of L-type Ca2+ channel during early embryogenesis in Xenopus laevis.

The mechanisms involved in the first step of neurogenesis, i.e. neural induction, are poorly understood, particularly in terms of the signalling pathway. In a recent work it has been shown that in urodeles the activation of L-type calcium channels is sufficient to trigger neural induction. In order to substantiate a possible role of this channel in early development in anurans, we have detailed the kinetics of the expression and the localization of the alpha 1 subunit of L-type calcium channel in the early stages of Xenopus laevis embryogenesis using immunological techniques. We observed that the expression of the alpha 1 subunit started during blastulation, where a cytoplasmic labeling was observed. At the onset of gastrulation alpha 1 was targeted to the plasma membrane of the dorsal and the ventral ectoderm. Some labeling was found in the mesoderm but never in the endoderm. This expression seems to be general, since similar results have been obtained in anurans (Xenopus) and in urodeles (Pleurodeles). In addition, we found that the alpha 0 subunit of the G(o) protein is expressed simultaneously and strictly colocalized with the alpha 1 subunit of the L-type calcium channel. The role of this channel and its regulation by G(o) protein during early neurogenesis is discussed.

Animals↗
PubMed1994

Increased internal Ca2+ mediates neural induction in the amphibian embryo.

The molecular mechanism of neural induction is still unknown and the identity of the natural inducer remains elusive. It has been suggested that both the protein kinase C and cAMP signal transduction pathways may be involved in mediating its action. Here we provide evidence that Ca2+ is implicated in the process of transduction of the neuralizing signal. We find that an increase in intracellular Ca2+ concentration [Ca2+]i occurs during neural induction provoked in vitro by the lectin Con A in Pleurodeles waltl embryo. We demonstrate that specific L-type Ca2+ channel agonists also trigger neural induction. Conversely, noninducing lectins do not raise [Ca2+]i. Ryanodine and caffeine trigger neural induction. An increase in [Ca2+]i was also observed after treatment with the phorbol 12-myristate 13-acetate, which has been reported to be inductive. The [Ca2+]i increase triggered by phorbol ester and Con A was abolished by staurosporine and by L-type Ca2+ channel antagonists. Our findings demonstrate that the [Ca2+]i increase occurs via L-type Ca2+ channels. We suggest an amplification of this increase by a Ca(2+)-induced Ca2+ release mechanism which involves intracellular ryanodine-sensitive stores. We propose that Ca(2+)-dependent processes controlled by protein kinase C are implicated in the regulation of gene expression in response to neural induction.

Alkaloids↗
PubMed1994

Molecular context of a viral T cell determinant within a chimeric bacterial protein alters the diversity of its T cell recognition.

We genetically introduced two different viral CD4+ T cell epitopes within two internal sites of the Escherichia coli maltose-binding (MalE) protein. Affinity-purified hybrid MalE proteins were used to analyze the influence of the molecular environment on the presentation of inserted epitope to T cells. In the first model, the 120 to 132 PreS T cell epitope was inserted alone or with its C-terminal B cell epitope (132-145) at site 133 or 303 of MalE. The maltose-binding protein with PreS peptide inserts expressing the 120 to 132 sequence were able to induce in vivo and in vitro peptide-specific T cell response, whatever the length and the position of the insert. In the second model, the 103 to 115 T cell epitope from the C3 region of poliovirus type 1 (PV1) was inserted, with various flanking sequences, either at site 133 or 303 of MalE protein. The longer C3:86 to 115 insert induced poliovirus-specific T cell responses at both sites of MalE, whereas the C3:93 to 115 insert did it only at site 303 but not at site 133. Moreover, C3:103 to 115 specific T cell hybridomas discriminated between the processed peptides generated from the different chimeric proteins, as a result of differences in the length and the position of the inserted sequence. Therefore, in this experimental model the loss of in vivo immunogenicity of an antigenic determinant within a chimeric protein is related to the activation of a reduced T cell repertoire. These observations involve important consequences for the engineering of recombinant vaccines.

ATP-Binding Cassette Transporters↗
PubMed1994

Low intracellular pH is involved in the early embryonic death of DDK mouse eggs fertilized by alien sperm.

Intracellular pH was measured in normal 8-cell stage mouse embryos and in embryos from a cross between DDK females and C3H males. DDK/C3H embryos display the DDK syndrome and spontaneously begin to decompact toward the late 16-cell stage. Ultimately, 90% fail to form blastocysts. Normal embryos have a resting intracellular pH close to neutrality. In DDK/C3H embryos a substantial proportion (46%) has an intracellular pH below 6.7. An equivalent proportion of DDK/C3H embryos was found previously to show slow communication through gap junctions at the 8-cell stage. This is probably a consequence of low intracellular pH. In normal embryos the weak acid, butyric acid, decreased intracellular pH and slowed the transfer of Lucifer Yellow through gap junctions. Normal embryos treated with butyrate for between 1 and 6 hr beginning at the 8-cell stage and cultured for 24 hr, reproduced the DDK/C3H phenotype. After 48 hr some butyrate treated embryos recovered, while others remained as decompacted morulae. Treatment of control and DDK/C3H 8-cell stage embryos with dibutyryl cyclic AMP or forskolin, which will increase intracellular cyclic AMP, speeded gap junctional communication. Forskolin treatment prevented expression of the DDK syndrome in DDK/C3H embryos, although the rescue was transient and the syndrome returned when forskolin was removed. The finding that the DDK syndrome is manifested as low intracellular pH may provide clues to the molecular basis of the defect.

Animals↗
PubMed1994

Reception and transduction of the serotonin signal responsible for meiosis reinitiation in oocytes of the Japanese clam Ruditapes philippinarum.

Prophase-arrested oocytes of Ruditapes philippinarum are triggered to undergo germinal vesicle breakdown under the influence of the neurohormone serotonin (5HT) and then arrest in metaphase 1. Our data show that these oocytes possess a single class of original 5HT receptors. Their binding parameters have been determined on semipurified membrane preparations incubated with [3H]5HT. No significant differences were observed when comparing 5HT-competent and -incompetent batches as well as prophase- or metaphase-arrested oocytes. Specific experiments including incubation with mastoparan or mas 7, GTP iontophoresis, and IP3 quantification strongly suggest that these receptors must be coupled with G-proteins to be functional. Peak change in IP3 mass occurs at 3 min and is likely to trigger the 5HT-dependent Ca2+ transient that begins at this time. In metaphase-arrested oocytes, binding of 5HT to its receptors no longer produces a Ca2+ surger. This is likely to result from a negative retrocontrol loop which would involve kinase C and exert its effect upstream of the Ca2+ surge. Indeed, the phorbol ester PMA proved able to reduce the Ca2+ response and to block 5HT action when applied during the first 3 min corresponding to the hormone-dependent period. Such an inhibition was reversed in the presence of 5 microM of the C kinase inhibitor GF109203X and could be bypassed by ionophore, ammonia, and thapsigargin, which trigger a receptor-independent Ca2+ surge.

Animals↗
PubMed1994

Stabilization and enhancement of interleukin-2 in vitro bioactivity by new carriers: supramolecular biovectors.

Human recombinant interleukin-2 can be associated and released from supramolecular biovectors (SMBVs), consisting of particles made of polymerized polysaccharides. The particles are substituted with phosphate residues and contain bound lipid molecules (palmitic acid) buried near their surfaces. The association of IL-2 with SMBVs modifies its in vitro bioactivity. SMBVs prolong the growth of IL-2-dependent cells, enhance IL-2 proliferative activity and restore the activity of impaired IL-2. These properties mainly depend on the presence of lipids linked to the SMBV and on both the degree of acylation and the SMBV: IL-2 ratio. SMBVs are therefore good candidates for the stabilization and enhancement of the biological activity of IL-2.

Acylation↗
PubMed1994

Genetic control of antibody responses induced against an antigen delivered by recombinant attenuated Salmonella typhimurium.

Recombinant derivatives of nonpathogenic bacteria such as attenuated Salmonella typhi have the potential to be used for delivery of heterologous antigens to the immune system. Genetic factors may modulate the immune responses to these live attenuated organisms and could therefore modify the immunogenicity of future human vaccines. In the present study, we compared the antibody responses of Ity or H-2 congenic strains of mice to a foreign antigen expressed by the murine attenuated aroA S. typhimurium strain. Our results demonstrate that the Ity gene may modulate the antibody responses to the foreign antigen but that the major genetic influence is exerted by H-2 genes, which control the capacity of mice to respond to the antigen expressed by recombinant attenuated Salmonella cells. This genetic control is related to differences in responsiveness of different strains of mice to low doses of antigen. Increasing the amount of foreign antigen expressed by recombinant Salmonella cells overcame the genetic restriction of these responses. These findings are potentially of great importance for the design of live vaccines for humans and show that care must be taken to optimize the amount of foreign antigen delivered to the immune system.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1994

Immunogenicity of viral B- and T-cell epitopes expressed in recombinant bacterial proteins.

Foreign polypeptides can be expressed as genetic inserts in several permissive sites of MalE and LamB, two Escherichia coli envelope proteins. Several viral B and T-cell epitopes have been inserted in these proteins and we analyzed the role of the molecular environment on the immunogenicity of the foreign epitopes. These studies demonstrated that the antigenicity and immunogenicity of B-cell epitopes depend on their site of insertion in the carrier protein. Using bacteria expressing B-cell epitopes either at the cell surface or in the periplasm, it was also shown that the cellular location of a foreign B-cell epitope expressed by recombinant bacteria determines its T-cell dependent or independent characteristics. Analysis of in vivo immunogenicity of purified LamB or MalE hybrid proteins expressing two different T-cell epitopes established that the immunogenicity of recombinant T-cell epitopes may be strongly affected by both the insertion site and inserted adjacent residues. The in vitro analysis of specific T-cell hybridoma response to hybrid MalE proteins also showed that the molecular context of a T-cell determinant alters the diversity of its T-cell recognition.

ATP-Binding Cassette Transporters↗
PubMed1994

Molecular and cellular parameters controlling the immunogenicity of foreign B- or T-cell epitopes expressed by recombinant vectors.

Extensive work is being performed to develop live recombinant bacterial vaccines. The use of non-pathogenic bacteria or attenuated strains derived from pathogens may allow protection against the pathogen and at the same time induce immunity against one or several foreign antigens expressed by the recombinant micro-organism. Several bacteria such as attenuated Salmonella or BCG have been used successfully in several experimental models to induce protective immune responses against several pathogens. However, the presentation to the immune system of a foreign antigen in a context different from the natural one may greatly modify the characteristics of elicited immune responses. It is therefore of the utmost importance to establish rules concerning the influence of the vector on the immunogenicity of recombinant antigens. Using a bacterial system that allows the expression of genetically engineered hybrid proteins, we have analysed: 1) the role of the molecular environment on the immunogenicity of foreign B- or T-cell epitopes; 2) the influence of the cellular location of a foreign B-cell epitope on the induction of specific immune responses; 3) the role of the bacterial vector (E. coli or Salmonella typhimurium) on the isotypic characteristic of antibody responses induced against the recombinant antigen. These studies revealed the complexity of the mechanisms which control the immunogenicity of foreign B- or T-cell epitopes expressed by recombinant vectors.

ATP-Binding Cassette Transporters↗
PubMed1993

Induction of T cell responses by chimeric bacterial proteins expressing several copies of a viral T cell epitope.

A viral T cell epitope was genetically inserted within the periplasmic MalE protein of Escherichia coli in two different permissive insertion sites and resulting hybrid proteins were used to study the in vitro and in vivo immunogenicity of the foreign T cell epitope. Purified hybrid MalE proteins containing the T cell epitope 120-132 (PreS:T) from PreS2 region of hepatitis B virus HBsAg inserted alone or with its adjacent B cell epitope (132-145) were able to induce strong peptide-specific T cell responses in mice. In vitro stimulation of primed lymph node cells or specific T cell hybridomas by the hybrid proteins required processing of the inserted T cell epitope and was inhibited by antigen-presenting cells fixation. The inserted T cell epitope was presented in vitro, in association with appropriate major histocompatibility complex molecules, as efficiently as free synthetic peptide. The in vitro immunogenicity of MalE hybrid proteins was increased by inserting four tandemly repeated copies of PreS:T, either at site 133 or 303. These results were confirmed in vivo by comparing the proliferative responses of lymph node cells from DBA/1 mice primed with MalE hybrid proteins containing one or four copies of PreS:T. Thus, the use of MalE hybrid proteins expressing multiple copies of a given foreign T cell epitope allows the induction of peptide-specific T cell response with a lower dose of priming antigen.

ATP-Binding Cassette Transporters↗
PubMed1993

Identification of helper T cell epitopes of dengue virus E-protein.

The T cell proliferative response to dengue 2 (Jamaica) E-glycoprotein (495 amino acids) was analyzed in vitro using either killed virus or E-protein fragments or synthetic peptides. Inactivated dengue virus stimulated dengue-specific lymph node (LN) CD4+T cell proliferation in BALB/c (H-2d), C3H (H-2k) and DBA/1 (H-2q) but not in C57BL/6 (H-2b) mice. Moreover, LN cells from dengue-virus primed BALB/c mice proliferated in vitro in response to three purified non-overlapping E-protein fragments expressed in E. coli as polypeptides fused to trpE (f22-205, f267-354, f366-424). To further determine T cell epitopes in the E-protein, synthetic peptides were selected using prediction algorithms for T cell epitopes. Highest proliferative responses were obtained after in vitro exposure of virus-primed LN cells to peptides p135-157, p270-298, p295-307 and p337-359. Peptide p59-78 was able to induce specific B and T cell responses in peptide-primed mice of H-2d, H-2q and H-2k haplotypes. Two peptides p59-78 corresponding to two dengue (Jamaica and Sri Lanka) isolates and differing only at position 71 cross-reacted at the B but not at the T cell level in H-2b mice. This analysis of murine T helper cell response to dengue E-protein may be of use in dengue subunit vaccine design.

Amino Acid Sequence↗
PubMed1993

Immunodominance of a recombinant T-cell epitope depends on its molecular environment.

In the present study, we have investigated the influence of the molecular environment of a T-cell epitope on its immunogenicity. We genetically inserted into different sites of two bacterial recipient proteins, LamB or MalE, an immunodominant T-cell epitope: the 120-132 T-cell epitope from the PreS2 region of HBV. The T-cell epitope was introduced, either alone (PreS:T) or with an adjacent B-cell epitope (PreS:TB). After purification, the hybrid proteins were injected into mice and we studied the immunogenicity of recombinant T-cell epitopes by analyzing the in vitro proliferative responses of LN cells from these mice to the inserted peptides. The immunization of mice with recombinant MalE protein containing the PreS:T or PreS:TB peptides at two different sites induced strong peptide-specific proliferative responses, indicating that the insertion sites did not affect the immunodominance of the inserted T-cell epitope. A strong T-cell proliferative response was also obtained after immunization of mice with hybrid LamB protein containing the PreS:TB epitope at position 153. In contrast, the recombinant proteins which contained only the PreS:T epitope at positions 153 or 374 failed to stimulate T-cell responses. Therefore, this study demonstrates that the immunogenicity of recombinant T-cell epitopes may be strongly affected both by the insertion site and by inserted adjacent residues.

Amino Acid Sequence↗
PubMed1993

Immune responses to hybrid maltose-binding proteins.

The Escherichia coli maltose-binding protein is a highly versatile carrier protein allowing the construction of genetically engineered hybrid proteins. It accepts large fusions to both C- and N-termini as well as the insertion of shorter peptides at 'permissive sites' within the continuity of the protein. We have genetically inserted immunogenic peptides corresponding to defined viral B- and T-cell epitopes into two permissive sites: one at amino acid site 133, the other at site 303. The hybrid proteins are easily purifiable and immunogenic, inducing peptide-specific B- and T-cell responses. When delivered by live bacteria (E. coli K12 and aroA Salmonella typhimurium) antibody responses can be induced against both the MalE carrier and the inserted B-cell epitope. We discuss the induction of T-cell responses by bacterial delivery systems.

ATP-Binding Cassette Transporters↗
PubMed1993

Expression and immunogenicity of the V3 loop from the envelope of human immunodeficiency virus type 1 in an attenuated aroA strain of Salmonella typhimurium upon genetic coupling to two Escherichia coli carrier proteins.

A peptide comprising residues glu293 to ser334 from the principal neutralization determinant (V3 loop) of the envelope of human immunodeficiency virus type 1 (HIV1 LAVBRU isolate) has been inserted within internal permissive sites of either LamB or MalE, two envelope proteins from Escherichia coli K12. The MalE hybrid protein (MalE133-V3 loop) was stably expressed in the periplasm of Escherichia coli K12, and the V3 loop peptide was detectable on the surface of the native protein by an anti-gp160 monoclonal antibody (mAb 110-A). The disulfide bridge between the two cysteines of the loop was formed. In contrast, genetic coupling to the outer membrane protein LamB did not allow the expression of a stable hybrid protein, and major proteolytic cleavage products of the LamB153-V3 loop were detected by mAb 110-A. The two plasmid-encoded hybrid genes were transferred to an aroA mutant of Salmonella typhimurium. Constitutive expression of the MalE133-V3 loop had no detectable effect on cell growth and on the survival in vivo of the recipient strain. The LamB153-V3 loop was not stably expressed in Salmonella, either in vitro or in vivo. Live recombinant salmonellas expressing MalE-V3 and LamB-V3 loop hybrids were used to immunize mice. The MalE-V3 loop hybrid induced anti-HIV1 envelope antibodies detectable by Western blot and ELISA, while the anti-HIV1 envelope antibodies induced by the LamB-V3 loop hybrid were only detectable by Western blot. In addition, purified MalE-V3 loop hybrid protein was able to stimulate in vitro and induce in vivo a V3 loop-specific T-cell proliferative response.

AIDS Vaccines↗
PubMed1993

Oral immunization with recombinant BCG induces cellular and humoral immune responses against the foreign antigen.

It has been shown recently that BCG can be used as a live recombinant vaccine to stimulate immune responses. Proliferative or cytotoxic T-cell responses against several viral proteins such as HIV Gag, Env or Nef were obtained after parenteral immunization with BCG expressing these proteins. Antibody responses were also obtained after immunization of mice with recombinant BCG strain which expressed lac Z under the control of a promoter sequence isolated from Mycobacterium paratuberculosis. We have used this recombinant vaccine in guinea-pigs to investigate the influence of various routes of immunization on the immunogenicity of a foreign antigen expressed by recombinant BCG. Guinea-pigs were immunized by oral, respiratory or intradermal routes and proliferative responses, delayed-type hypersensitivity and antibody responses specific for beta-galactosidase were followed for 16 weeks. Results demonstrated that humoral and cellular immune responses specific for beta-galactosidase can be produced in all groups of guinea-pigs. However, the respiratory and especially the oral route of administration induced higher local and systemic immune responses than the intradermal route of immunization. Moreover, the oral immunization of mice with this recombinant BCG induced IgA responses which could be detected in both sera and intestinal secretions. Therefore, this study demonstrates for the first time that oral immunization with recombinant BCG can induce strong cellular and humoral immune responses.

Administration, Inhalation↗
PubMed1993

B cells do not present antigen covalently linked to microspheres.

B cells have been shown to present antigen to T cells very efficiently through their capacity to capture antigens by their membrane immunoglobulin. This direct cognate interaction of T and B cells results in the proliferation and differentiation of B cells. This concept has been established using soluble proteins. However, most of the antigens to which the immune system is exposed are included in complex particulate structures such as bacteria or parasites. The capacity of B cells to present these large and complex antigens is still unclear. To address this question we have studied the presentation by trinitrophenyl (TNP)-specific B cells of the same antigen TNP-KLH (keyhole limpet haemocyanin), either in a soluble form or covalently linked to poly(acrolein) microspheres, from 0.25 to 1.5 microns in diameter. In the presence of irradiated splenocytes or purified macrophages as a source of antigen-presenting cells (APC), KLH-specific T cells proliferated in response to soluble TNP-KLH or to TNP-KLH coupled to beads. In contrast, TNP-specific memory B cells were totally ineffective in presenting the TNP-KLH beads to KLH-specific T cells whereas they presented very efficiently soluble TNP-KLH. Similar results were obtained with the A20 B lymphoma or with lipopolysaccharide (LPS)-activated TNP-specific B cells. These results therefore indicate that B cells are unable to present large size particulate antigens such as bacteria or parasites.

Animals↗
PubMed1992

Expression of heterologous peptides at two permissive sites of the MalE protein: antigenicity and immunogenicity of foreign B-cell and T-cell epitopes.

We previously determined a number of 'permissive' sites in the periplasmic maltose-binding protein (MalE) from Escherichia coli. These sites accept the insertion of heterologous peptides without major deleterious consequences for the activities, structure and cellular location of the protein. This study explores the versatility of two such permissive sites for the synthesis of foreign peptides, and examines the antigenicity and the immunogenicity of the inserts. One site is located after amino acid 133 (aa133) of MalE, and the other after aa303. Both sites tolerate inserts of up to at least 70 aa and accept sequences of different natures. Hydrophobic aa sequences are accepted, although strongly hydrophobic sequences, such as the Sendai virus F protein membrane anchor, affected export. We compared the antigenic and the immunogenic properties of peptides derived from the coat proteins of HBV and poliovirus which contain well defined B-cell epitopes. Specific monoclonal antibodies show that the antigenic properties of the inserted B-cell epitopes were different at the two sites. Despite these differences, the inserted peptides elicited strong and comparable antibody responses in mice against the corresponding synthetic peptides. In this case, and with these criteria, the molecular context of the peptides did not affect the immunogenicity of B-cell epitopes. We show for the first time that when a foreign peptide carrying a T-cell epitope was inserted in MalE, the hybrid proteins can elicit a T-cell response against the foreign peptide both in vivo and in vitro. Furthermore, the MalE hybrid was as efficient as free peptide in stimulating T-cell hybridomas in vitro. The MalE vectors provide a powerful genetic system to study how the position and the conformation of a peptide within a protein affect the B-cell and T-cell responses.

ATP-Binding Cassette Transporters↗