Biomedical subjects

C Leclerc

Publications and source records attributed to C Leclerc.

At least 19 recordsLinked to original sources

PubMed2000

Immune responses induced by recombinant BCG strains according to level of production of a foreign antigen: malE.

A variety of viral, bacterial and parasitic antigens have been expressed in BCG and the capacity of these recombinant bacteria to induce immune responses has been well documented. However, little is known about the parameters influencing the induction of immune responses by recombinant BCG (rBCG), such as level of production and localization of the recombinant antigen. In the present study, we have constructed several rBCG strains expressing the malE gene from Escherichia coli which is either secreted or targeted to the cytoplasm or plasma membrane. Expression of malE was quantified by ELISA and localization was analyzed by flow cytometry. Even when using the same promoter, levels of cytoplasmic or membrane MalE production were far less than those from secreting strains using either mycobacterial or E. coli secretion signals. Stronger and more rapid immune responses were induced by rBCG strains with the highest levels of secreted MalE compared to cytoplasmic or membrane constructs, including both good humoral and proliferative responses in BALB/c, C57BL6 and even C3H mice, previously shown to be poor MalE responders. These results suggest that the levels of foreign antigen production play an important role in the induction of immune responses by rBCG strains.

ATP-Binding Cassette Transporters↗

PubMed2000

Brain functional reorganization in early blind humans revealed by auditory event-related potentials.

Visually challenged individuals often compensate for their handicap by developing supra-normal abilities in their remaining sensory systems. Here, we examined the scalp distribution of components N1 and P3 of auditory evoked potentials during a sound localization task in four totally blind subjects who had previously shown better performance than sighted subjects. Both N1 and P3 waves peaked at their usual positions while blind and sighted individuals performed the task. However, in blind subjects these two components were also found to be robust over occipital regions while in sighted individuals this pattern was not seen. We conclude that deafferented posterior visual areas in blind individuals are recruited to carry out auditory functions, enabling these individuals to compensate for their lack of vision.

Acoustic Stimulation↗

PubMed2000

[Mesenteric vein and inferior vena cava thrombosis: disseminated intravascular coagulation and/or G 20210 A mutation of the prothrombin gene?].

The authors report the case of a 30-year-old man who died from pulmonary embolism and multiorgan failure caused by mesenteric and inferior vena cava thrombosis. The patient was found heterozygous for the prothrombin gene variant (G 20210 A). The family study showed the same asymptomatic anomaly in his brother. This recently described mutation is associated with an increased risk for venous thrombosis. The investigations and treatment of mesenteric venous thrombosis are discussed.

Adult↗

PubMed2000

Short synthetic glycopeptides successfully induce antibody responses to carcinoma-associated Tn antigen.

Glycopeptides containing a tumor-associated carbohydrate antigen (mono-, tri- or hexa-Tn antigen) as a B-cell epitope and a CD4+ T-cell epitope (PV: poliovirus or TT: tetanus toxin) were prepared for immunological studies. Several Tn antigen residues [FmocSer/Thr (alpha-GalNAc)-OH] were successively incorporated into the peptide sequence with unprotected carbohydrate groups. The tri- and hexa-Tn glycopeptides were recognized by MLS128, a Tn-specific monoclonal antibody. The position of the tri-Tn motif in the peptide sequence and the peptide backbone itself do not alter its antigenicity. As demonstrated by both ELISA and FACS analysis, the glycopeptides induced high titers of anti-Tn antibodies in mice, in the absence of a carrier molecule. In addition, the generated antibodies recognized the native Tn antigen on cancer cells. The antibody response obtained with a D-(Tn3)-PV glycopeptide containing three alpha-GalNAc-D-serine residues is similar that obtained with the Tn6-PV glycopeptide. These results demonstrate that short synthetic glycopeptides are able to induce anticancer antibody responses.

Animals↗

PubMed2000

Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites.

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8(+) T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.

Adenylate Cyclase Toxin↗

PubMed2000

Extending the CD4(+) T-cell epitope specificity of the Th1 immune response to an antigen using a Salmonella enterica serovar typhimurium delivery vehicle.

We analyzed the CD4 T-cell immunodominance of the response to a model antigen (Ag), MalE, when delivered by an attenuated strain of Salmonella enterica serovar Typhimurium (SL3261*pMalE). Compared to purified MalE Ag administered with adjuvant, the mapping of the peptide-specific proliferative responses showed qualitative differences when we used the Salmonella vehicle. We observed the disappearance of one out of eight MalE peptides' T-cell reactivity upon SL3261*pMalE immunization, but this phenomenon was probably due to a low level of T-cell priming, since it could be overcome by further immunization. The most striking effect of SL3261*pMalE administration was the activation and stimulation of new MalE peptide-specific T-cell responses that were silent after administration of purified Ag with adjuvant. Ag presentation assays performed with MalE-specific T-cell hybridomas showed that infection of Ag-presenting cells by this intracellular attenuated bacterium did not affect the processing and presentation of the different MalE peptides by major histocompatibility complex (MHC) class II molecules and therefore did not account for immunodominance modulation. Thus, immunodominance of the T-cell response to microorganisms is governed not only by the frequency of the available T-cell repertoire or the processing steps in Ag-presenting cells that lead to MHC presentation but also by other parameters probably related to the infectious process and to the bacterial products. Our results indicate that, upon infection by a microorganism, the specificity of the T-cell response induced against its Ags can be much more effective than with purified Ags and that it cannot completely be mimicked by purified Ags administered with adjuvant.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2000

Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis.

The adenylate cyclase (CyaA) of Bordetella pertussis delivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8(+) T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8(+) T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8(+) CD4(+) T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.

Adenylate Cyclase Toxin↗

PubMed2000

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity.

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.

Amino Acid Sequence↗

PubMed1999

Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses.

Amino Acid Sequence↗

PubMed1999

A fully synthetic immunogen carrying a carcinoma-associated carbohydrate for active specific immunotherapy.

Aberrant glycosylation of mucins leads to the exposure of cryptic carbohydrate antigens at the surface of carcinoma cells, which, therefore, represent potent targets for anticancer therapeutic vaccines. To date, the development of immunogens to stimulate immune response to such saccharidic antigens is based on carbohydrate conjugation to carrier proteins. However, these traditional protein conjugates are poorly defined in chemical composition and structure. As an alternative, we synthesized a multiple antigenic O-linked glycopeptide (MAG) carrying the carbohydrate Tn antigen associated with a CD4+ T-cell epitope (MAG:Tn-PV). This fully synthetic immunogen is highly defined in composition and carries a high saccharidic epitope ratio over the entire molecule. The MAG:Tn-PV was able to induce anti-Tn IgG antibodies that recognize human tumor cell lines. A therapeutic immunization protocol performed with this fully synthetic immunogen increased the survival of tumor-bearing mice. Thus, the accurately defined and versatile MAG system represents an efficient strategy to induce carbohydrate-specific antitumor immune responses but may also be applicable to the prevention of infectious diseases, if it is based on bacterial oligosaccharides.

Animals↗

PubMed1999

Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope.

Bordetella pertussis secretes an invasive adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain into the cytosol of eukaryotic target cells directly through the cytoplasmic membrane. We have shown previously that recombinant CyaA can be used to deliver viral CD8+ T cell epitopes to the MHC-class I presentation pathway to trigger specific CTL responses in vivo. In the present study, we show that mice immunized with a detoxified but still invasive CyaA carrying a CD8+ T cell epitope of OVA developed strong epitope-specific CTL responses, which kill tumor cells expressing this Ag. Treating mice with this recombinant molecule after the graft of melanoma cells expressing OVA induced a strong survival advantage compared with control animals. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic antitumor immunity.

Adenylate Cyclase Toxin↗

PubMed1999

Direct delivery of the Bordetella pertussis adenylate cyclase toxin to the MHC class I antigen presentation pathway.

Among bacterial toxins, the adenylate cyclase toxin of Bordetella pertussis (CyaA) has a unique mechanism of entry that consists in the direct translocation of its catalytic domain across the plasma membrane of target cell, a mechanism supposed to be independent of any endocytic pathway. Here, we report that the CyaA toxin is delivered to the cytosolic pathway for MHC class I-restricted Ag presentation. Using peritoneal macrophages as APC, we show that the OVA 257-264 CD8+ epitope genetically inserted into a detoxified CyaA (CyaA-OVA E5) is presented to CD8+ T cells by a mechanism requiring 1) proteasome processing, 2) TAP, and 3) neosynthesis of MHC class I. We demonstrate that the presentation of CyaA-OVA E5, like the translocation of CyaA into eukaryotic cells, is dependent on extracellular Ca2+ and independent of vacuolar acidification. Moreover, inhibitors of the phagocytic and macropinocytic endocytic pathways do not affect the CyaA-OVA E5 presentation. The absence of specific cellular receptors for CyaA correlates with the ability of various APC to present the recombinant CyaA toxin, including dendritic cells, macrophages, splenocytes, and lymphoid tumoral lines. Taken together, our results show that the CyaA presentation pathway is not cell type specific and is unrelated to a defined type of endocytic mechanism. Thus, it represents a new and unconventional delivery of an exogenous Ag into the conventional cytosolic pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Immunogenicity of viral B-cell epitopes inserted into two surface loops of the Escherichia coli K12 LamB protein and expressed in an attenuated aroA strain of Salmonella typhimurium.

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different 'permissive' sites of a carrier protein. By using the outer membrane protein LamB of Escherichia coli K12 as a carrier, we were able to express a number of different foreign epitopes at the bacterial surface. In the present work, taking advantage of the recent determination of the crystal structure of LamB, we inserted two model B-cell epitopes i.e.--the C3 epitope from poliovirus (residues 93 to 103 of VP1) and the preS2 epitope from hepatitis B virus, (residues 132 to 145)--at the tip of the most distal and largest surface exposed region of LamB (after residues 386, into loop L9). We also used two previously constructed LamB hybrids, corresponding to the insertion of the C3B or preSB epitope into permissive site 153 (lying in the middle of the fourth surface loop of LamB), to construct two LamB proteins corresponding to the simultaneous insertion of the two different epitopes (with one epitope per site). The LamB hybrids were placed under the control of the anaerobically inducible pnirB promoter and expressed in a LamB-negative derivative of the aroA attenuated strain of S. typhimurium, SL3261. In vitro, the recombinant proteins were expressed at a high level (up to 10% of whole cell proteins) and in vivo the recombinant plasmids were stably maintained. For both epitopes, genetic coupling at site 386 appeared to be more favorable for the induction of anti-epitope antibodies than coupling at site 153. Moreover, the LamB hybrid corresponding to the simultaneous insertion of the preSB epitope at site 153 and of the C3B epitope at site 386 allowed the induction of both anti-poliovirus and anti-hepatitis B antibodies.

Animals↗

PubMed1999

Engineering parvovirus-like particles for the induction of B-cell, CD4(+) and CTL responses.

An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine (PPV) or canine parvovirus (CPV) expressed in insect cells with the baculovirus system has been developed. PPV:VLPs containing a CD8(+) epitope from the LCMV nucleoprotein evoked a potent CTL response and were able to protect mice against a lethal infection with the virus. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a CD4(+)3 log(10) units) against poliovirus. The possibility of combining different types of epitopes in different positions of a single particle to stimulate different branches of the immune system paves the way to the production of more potent vaccines in a simple and cheap way.

Animals↗

PubMed1999

Noggin upregulates Fos expression by a calcium-mediated pathway in amphibian embryos.

In amphibia, noggin, one of the neural inducers expressed in the Spemann organizer, acts by neutralizing the effects of bone morphogenetic protein-4 (BMP-4). It is shown that noggin is able to activate L-type calcium channels. The fos proto-oncogene is known to be induced within minutes by calcium signaling. Here it is reported that in animal cap explants of the amphibian Pleurodeles waltl, noggin can induce upregulation of a FOS-related protein in a calcium-dependent manner. Specific inhibition of the dihydropyridine sensitive L-type calcium channels blocked both calcium influx and the induction of FOS-related protein. When animal cap explants were treated with caffeine in order to release calcium from an internal store or with a specific agonist of the L-type calcium channels, FOS-related protein could be detected in cell nuclei by 5 or 15 min, respectively. Additionally, the calcium calmodulin kinase inhibitor. KN62, could block the upregulation of FOS-related protein induced by agents that increased intracellular calcium ([Ca2+]i). The present results suggest that transcription factors from the FOS family are downstream targets of neural inducer noggin.

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

PubMed1999

mAb against hen egg-white lysozyme regulate its presentation to CD4(+) T cells.

Specific antibodies increase antigen uptake and presentation by antigen-presenting cells via the B cell receptor in B cells or FcgammaR in dendritic cells. To determine whether the interaction between antibody and antigen could influence the set of peptides presented by MHC II molecules, we analyzed the presentation of different CD4(+) T cell epitopes of hen egg-white lysozyme (HEL) after the capture of immune complexes formed between HEL and seven different specific mAb. The 103-117 T cell epitope (I-E(d)) was specifically and selectively up-regulated by the D1.3 and F9.13.7 mAb that binds to proximal loops in the native structure of HEL. Furthermore, Ii-independent T cell epitopes exposed on the HEL surface (116-129 and 34-45, I-A(k) restricted) which require a mild processing involving the recycling of MHC II molecules were selectively up-regulated by mAb that overlap those T cell epitopes (D1.3 and D44.1). However, F10.6.6, somatically derived from the same germ line genes as D44.1 and exhibiting an higher affinity for HEL, was without effect on the presentation of the 34-45 epitope. An Ii-dependent T cell epitope buried into the tertiary structure of HEL (45-61, I-A(k) restricted) and requiring the neosynthesis of MHC II was up-regulated by high-affinity mAb recognizing epitopes located at the N- or C-terminus of the T cell epitope. These results strongly suggest that (i) the spatial relationship linking the T cell epitope and the B cell epitope recognized by the mAb, (ii) the intrinsic processing requirements of the T cell epitope, and (iii) the antibody affinity influences the presentation of a given T cell epitope.

Animals↗

PubMed1999

Efficacy of a self-esteem module in the empowerment of individuals with schizophrenia.

To enhance empowerment and improve self-esteem among individuals with severe and persistent mental illness, a 12-week "module" (a self-contained program of activities) was created and tested in a randomized clinical trial. Fifty-one individuals with schizophrenia were assigned to the experimental group in addition to regular treatment, and 44 individuals participated in a control group that continued with regular treatment only. Psychosocial, diagnostic, neurocognitive, and symptomatology measures were taken for all 95 subjects before treatment (T0), after treatment (T1), and at a 6-month follow-up (T2). Results indicated module effects on coping skills (active coping skills significantly increased) and psychotic symptoms (positive symptoms significantly decreased), demonstrating the efficacy of this particular type of intervention. Interpretation of the results highlighted the significance of the environment and the role it could potentially play in supporting the empowerment of severely mentally ill individuals.

Adaptation, Psychological↗

PubMed1999

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying multiple copies of a viral CD8(+) T-cell epitope.

Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo.

Adenylate Cyclase Toxin↗