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Biomedical subjects

M Hervé

Publications and source records attributed to M Hervé.

At least 19 recordsLinked to original sources
PubMed2001

Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization.

DNA vaccination induces antigen-specific immune responses with characteristics distinct from other vaccination modes. In the present study, the contribution of the plasmid backbone adjuvant effect to the quality of the DNA-raised antibody response was investigated. For this purpose, three intradermal primings were compared in mice using: (1) the recombinant Schistosoma haematobium glutathione S-transferase antigen (rSh28GST): (2) rSh28GST supplemented with a non-coding plasmid; and (3) a Sh28GST-encoding plasmid. In contrast to immunization with the protein, DNA immunization elicited a very stable antibody (Ab) response over a prolonged period of time. This feature was attributed to the plasmid backbone, because co-administration of the non-coding plasmid with rSh28GST allowed the maintenance of the specific Ab response. A strong anamnestic Ab response was induced after intradermal boost with rSh28GST only in the mice primed with pMSh. This indicated that the selective ability of DNA vaccination to induce memory humoral response was independent of the plasmid backbone. In contrast the plasmid backbone was found to strongly participate in the preferential IgG2a Ab production observed. These results suggest that, following DNA immunization, the Th1-biased profile and the maintenance of the long-lived Ab response could be attributed to an adjuvant effect of the plasmid backbone during priming, whereas the strength of B-cell memory was independent of this effect.

Adjuvants, Immunologic↗
PubMed2000

HLA-DR restricted peptide candidates for bee venom immunotherapy.

T cell epitopes containing peptides have been recently proposed as an alternative to conventional immunotherapy of allergic diseases because they are expected to be better tolerated than allergen extracts. A principal limitation to their clinical use is that they present an important diversity, which primarily results from the polymorphism of HLA class II molecules. In Caucasian populations, however, seven alleles of the most expressed molecules (namely DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominate. Peptides from allergens that would efficiently bind to them should be potential candidates for specific immunotherapy. In this paper, we have determined the peptides present in the major bee venom allergen by investigating the capacity of synthetic peptides that encompass its whole sequence to bind to each allele. Several efficient binders have been identified and are either allele-specific or common to several HLA-DR molecules. Interestingly enough, the 81-97 sequence is universal in the sense that it binds to all studied molecules. This sequence is surrounded by several active regions, which make the 76-106 sequence particularly rich of binding determinants and a good candidate for specific immunotherapy. Statistical analyses of the binding data also provide an overview of the preponderant HLA-DR alleles specificity.

Alleles↗
PubMed1999

On the diversity and heterogeneity of H-2(d)-restricted determinants and T cell epitopes from the major bee venom allergen.

One of the main limitations of using synthetic peptides for immunotherapy in allergic patients is the difficulty to delineate the immunodominant T cell epitopes which are necessarily dependent on HLA molecules. We have thus addressed the question of the role of MHC II molecules in immunodominant epitopes selection in the particular case of the major bee venom allergen (API m1). To exhaustively and easily explore it, we used BALB/c mice whose H-2 haplotype is associated with high IgE and IgG responses to API m1. By means of extensive sets of synthetic peptides, we investigated the specificity of polyclonal T cells and monoclonal hybridomas from mice immunized with API m1 and delineated four immunodominant regions, restricted to either the I-E(d) or the I-A(d) molecule. All the peptides were also tested for their capacity to bind to immunopurified MHC II molecules. Eight determinants of high affinity were identified. They clustered into three distinct regions and were largely overlapping. They included all the immunodominant epitopes, but half of them were not capable of stimulating T cells. Strikingly, interacting surfaces with either the TCR or MHC II molecule greatly differed from one determinant to another. In one case, we observed that flanking regions exerted a particular action on T cell stimulation which prevented the fine epitope localization. Our results underline the diversity and complexity of MHC II-restricted determinants and T cell epitopes from the major bee venom allergen, even in a single haplotype. These data also participate in the development of alternative approaches to conventional immunotherapy.

Allergens↗
PubMed1998

[Exploration of 112 children suspected of amoxicillin allergy. Indications and efficacy of oral provocation test].

BACKGROUND: One to 10% of treatments using betalactams, particularly synthetic penicillin, are complicated by allergic reactions, usually cutaneous, and not easily imputable to immunologic sensitization in children. PATIENTS AND METHODS: The aim of this study was to identify, using cutaneous and biological tests, those from a group of 112 children suspected of amoxicillin allergy (evidenced by rash) who were actually sensitized, and to confirm the absence of allergy in others by an oral provocation test (OPT) associated to a long-term survey. The cutaneous tests were made by prick test and intra-dermo reaction (IDR) with Allergopen and with amoxicillin or amoxicillin + clavulanic acid. The biological tests included examination for penicillin and amoxicillin antibodies by using various techniques including enzyme-linked immunosorbent assay (ELISA) immunoglobulin G (IgG) and IgE, FARR, radioallergo sorbent test (RAST) and a histaminoliberation. When these tests were negative, an OPT with the suspected antibiotic was subsequently performed. RESULTS: Thirty-nine children (36.4%) confidently presented at least one positive cutaneous test (38 Allergopen, ten amoxicillin); 25 biological tests were positive (16 ELISA IgE, one ELISA IgG and eight histaminolibarations), seven times with negative cutaneous test. Forty-five children were judged to be sensitized to amoxicillin, with only one who subsequently took amoxicillin again. Among the 67 others, 52 received an OPT, six of them with moderate cutaneous reactions. Fifty-one (45.5%) children were allergic and 46 (41%) were allowed to take amoxicillin again; 17 did, one of them with a benign cutaneous reaction. CONCLUSION: Efficacy and safety of this type of investigation seems clear; it will have to be confirmed by other studies.

Adolescent↗
PubMed1998

Pseudopeptide ligands for MHC II-restricted T cells.

The potential therapeutic use of peptides to activate or anergize specific T cells is seriously limited by their susceptibility to proteolytic degradation. Classically, peptides are stabilized by incorporation of non-natural modifications including main chain modifications. In the case of MHC II-restricted peptides, the peptide backbone actively participates to the interaction with the MHC molecule and hence may preclude the peptidomimetic approach. We thus investigated whether a single amide bond modification influenced the peptide capacity to bind to a MHC II molecule and to stimulate specific T cells. Twenty pseudopeptide analogs of the I-Ed binder 24-36 peptide, whose sequence was derived from a snake neurotoxin, were obtained by replacing each amide bond of the peptide central part, by either a reduced psi[CH2-NH] or N-methylated psi[CO-NMe] peptide bond. In agreement with the major interacting role played by the peptide backbone, several peptides displayed a low, if any, capacity to bind to the MHC II molecule and did not lead to T cell stimulation. However, one-third of the peptides were almost as active as the 24-36 peptide in I-Ed binding assays and one-fifth in T cell stimulation assays. Among them, two pseudopeptides displayed native-like activity. Good binders were not necessarily good at stimulating T cells, demonstrating that main chain modification also affected T cell recognition. We thus showed that a peptidomimetic approach could create a new type of MHC II ligand to control T cell responses.

Animals↗
PubMed1997

On the immunogenic properties of retro-inverso peptides. Total retro-inversion of T-cell epitopes causes a loss of binding to MHC II molecules.

Retro-inversion is considered an attractive approach for drug and vaccine design since it provides the modified peptides with higher resistance to proteolytic degradation. We therefore investigated in detail the effect of retro-inversion on the immunological properties of synthetic peptides. We have synthesized retro-inverso analogues of MHC II restricted peptides that thus contained the correct orientation of the side chains but an inverse main chain. Retro-inversion made the peptides unable to compete in I E(d) or I A(d) binding tests, demonstrating a very low, if any, capacity to bind to MHC II molecules. These results confirm previous structural data that hydrogen bonds between residues of MHC II molecules and the main chain of antigenic peptides play a major interacting role. In vito experiments further showed that retro-inversion of a T-cell epitope causes its inability to either sustain in vitro T-cell stimulation or to prime specific T cells. Moreover, the retro-inverso peptide was not recognized by antibodies raised against the native peptide and did not elicit antibodies when injected into BALB/c mice. Retro-inverso peptides appear to be poor immunogens as a result of their weak capacity to bind to MHC II molecules. As an advantage, they are not expected to trigger undesirable humoral responses such as hypersensitivity or allergic disease. These results also provide a molecular explanation regarding the weak immunogenicity of D-amino acids containing polypeptides.

Amino Acid Sequence↗
PubMed1997

The specificity of antibodies raised against a T cell peptide is influenced by peptide amidation.

Peptides used for immunization are designed on the basis of combination of B and T cell epitopes. They are sometimes acetylated and amidated in order to mimic the protein insertion of the B cell epitope, but to our knowledge the effect of modifying the N- and C-termini is not clearly identified. In this paper, we have investigated in detail the influence of amidation and acetylation on the immunogenic properties of the T cell epitope 24-36 which is derived from a snake neurotoxin. Acetylation enhanced the capacity of the peptides to bind to I-Ed and to stimulate specific T cells in vitro but both modifications did not influence in vivo the T cell priming ability of the peptides. However, amidation of the peptides 24-36 provoked a dramatic effect on the antibody specificity they elicited, whereas acetylation did not. Antibodies recruited by amidated peptides weakly recognized the non amidated ones, while the latter elicited antibodies which hardly bind to the former. These results show how a subtle chemical change of a peptide immunogen modifies the reactivity of the elicited antibodies in an unrelated manner from the peptide MHC II binding ability and T cell stimulating capacity. We thus amplify the previously described polarity of chimeric TB peptides that raise antibodies mainly against their C-terminal part. Finally, these results may also facilitate the choice of the status of N and C termini of the peptides designed for immunization which at present have their extremities indifferently free or modified by acetylation and/or amidation.

Acetylation↗
PubMed1995

Immunogenicity of a disulphide-containing neurotoxin: presentation to T-cells requires a reduction step.

It is known that production in a host of antibodies against a protein is associated with various molecular events. These include the stimulation of specific T-lymphocytes, a step that implies the processing of the protein into peptides by various endosomal/lysosomal enzymes, such as cathepsins. Strikingly, however, we observed in vitro that cathepsins B and D have no degrading effect on toxin alpha from Naja nigricollis, a curaremimetic toxin of 61 amino acids and four disulphides. In sharp contrast, the enzymes exert an efficient cleavage of the toxin polypeptide chain once the toxin disulphides are reduced. We also found that the fully reduced toxin and the native toxin were presented with comparable efficiency to two different T-hybridomas by antigen-presenting cells (APC). Together, the data suggest that presentation of toxin fragments to T-cells requires a reduction step of toxin disulphides and, in agreement with previous findings, that this step may be achieved by APC. We wish to suggest that this phenomenon may commonly occur for any toxic proteins that contain disulphides.

Amino Acid Sequence↗
PubMed1995

Probing immunogenicity of a T cell epitope by L-alanine and D-amino acid scanning.

All residues of the I-Ed restricted fragment 24-36 of a snake toxin were individually changed into L-alanine and the corresponding D-enantiomer. Four analogs substituted with L-Ala at positions 25;30, 31 and 33, and nine analogs substituted with a D-residue along the stretch 25-33 lost most (position 28) or all their capacity to stimulate a toxin-specific T hybridoma. None of these analogs stimulated splenocytes from mice immunized with the peptide 24-36. Only the L-A31 and D-W29 modified analogs could prime a T cell response which, however, showed no cross-reactivity with the native peptide, demonstrating that T cell response selectivity can be deeply modified by mutation or configuration inversion of a single residue. Our data suggest that (i) the region 25-33 is the core of the T epitope that binds to I-Ed, and (ii) Y25 R30 and R33 contribute to the peptide binding by anchoring into pockets of I-Ed. In agreement with T cell priming observations, only the L-A31 and D-W29 modified analogs elicited strong antibody responses, just like the peptide 24-36, whereas nearly all other analogs were less immunogenic. All but the L-Ala30 and L-Ala33 modified analogs were recognized by a 24-36 specific antiserum as well as the native peptide. Altogether, our results show that substitution by D-amino acid in a peptide could be particularly well-suited for either minimizing the risk of hypersensitivity or designing peptidic vaccines.

Alanine↗
PubMed1995

Fine chemical modifications at N- and C-termini enhance peptide presentation to T cells by increasing the lifespan of both free and MHC-complexed peptides.

We investigated the effect of modifying the N- and/or C-termini of the snake toxin peptide 24-36 on its presentation to T cells. Acetylation at the N-terminus as well as amidation at the C-terminus enhanced the capacity of the peptide to activate T cells. Simultaneous modifications further increased the stimulating activity, the peptide becoming approximately 100-fold more potent than the unmodified peptide. Clearly, the introduced modifications increased the lifetime of the peptide free in solution, by decreasing its proteolytic degradation, during the T cell stimulation assays. Paradoxically, however, at similar concentrations of free peptides, the modified ones, especially those having an acetylated N-terminus, were much more active than the unmodified peptide, irrespective of the experimental conditions. These observations suggested that components other than protection from proteolytic degradation should be associated with the higher stimulating activities of the modified peptides. Accordingly, chasing experiments with APC revealed that acetylation at N-terminus caused a higher persistence of the peptides at APC surface. Together, our data indicate that (i) the T cell stimulating capacity of a peptide is associated with its lifespans in the free and MHC II bound states; and (ii) these lifespans can be greatly enhanced by introducing fine chemical modifications at N- and C-termini. These data may have some implications in designing more potent peptidic immunomodulators.

Amino Acid Sequence↗
PubMed1994

Boc-Cys(Npys)-OH (BCNP): an appropriate reagent for the identification of T cell epitopes in cystine and/or cysteine-containing proteins.

Some T cell epitopes become inactive when their thiols are blocked with various irreversible reagents (Régnier-Vigouroux, 1988; Maillère, 1992; Maillère et al., 1993). Blocking protein and peptide thiols with BCNP (Boc-Cys(Npys)-OH) constitutes a most appropriate strategy when searching for thiol-containing T cell epitopes. Free cysteines can thus be readily transformed into disulphide-like moieties which not only resist undesirable oxidative reactions but which also remain susceptible to reduction by antigen presenting cells, a prerequisite for the activity of thiol-dependent T cell epitopes. We describe the use of this reagent in a study of the intact disulphide-rich protein, toxin alpha from Naja nigricollis, and also two disulphide-containing toxin fragments.

Amino Acid Sequence↗
PubMed1994

A 13C solid-state NMR study of the structure and auto-oxidation process of natural and synthetic melanins.

This paper presents a 13C CP/MAS NMR study of the melanin pigments obtained through natural and synthetic origins: sepia-melanin from squid ink and three synthetic 5,6-dihydroxyindole-melanins prepared using different non-enzymatic oxidation pathways. The synthetic pigments can be distinguished from natural melanin by the absence of aliphatic carbons, thereby confirming the unreacted 3,4-dihydroxyphenylalanine and the proteinaceous origins of the aliphatic resonances in natural eumelanin. The spectra of selected non-protonated carbon resonances and those with only protonated carbon signals led to a quantitative analysis. An auto-oxidative experiment using a synthetic melanin, over a period of 130 h, has shown an unusually slow disappearance of hydrogen peroxide formed in situ. The 13C-NMR spectrum of the insoluble oxidized synthetic melanin compared to that before auto-oxidation clearly demonstrates that the oxidation process is associated with chemical changes within the pigment; i.e., carbonyl functional group formation and an increase of the non-protonated carbons fraction.

Animals↗
PubMed1994

Cross-polarization dynamics and spin diffusion in some aromatic compounds.

The inversion-recovery cross-polarization (IRCP) magic-angle spinning experiment has been applied to study the 13C-1H cross-polarization dynamics of protonated aromatic carbons in ferrocene, 5,6-dimethoxyindole (DMI) and some indole derivatives. Using the 13C-detected proton spin diffusion (SD) experiment recently developed by Zhang et al. [Solid State Nucl. Magn. Reson., 1 (1992) 313], the slow decaying or incoherent stage of the IRCP experiment is shown to be controlled by the spin diffusion process at the directly bound proton. Moreover, a simple phenomenological model treating spin diffusion as a relaxation process provides an excellent agreement with both the IRCP and SD experimental data for all the different C-H pairs of DMI and its derivatives. The resulting time constants of the non-exponential spin diffusion decays are related to the local intra- and intermolecular network of dipolar interactions. This model is nevertheless found to be inadequate for ferrocene because intramolecular spin diffusion then has an inhomogeneous character.

Diffusion↗
PubMed1993

Influence of glucose on the deoxyglucose metabolism in S cerevisiae: detection and identification of deoxyglucose and trehalose derivatives by 1H- and 13C-NMR spectroscopy.

The metabolism of 2-deoxy-D-glucose (DG) in glucose grown repressed Saccharomyces cerevisiae cells was studied in the absence and presence of glucose (Glc) at 30 degrees C in a standard pyrophosphate medium containing 4.5 x 10(7) cells/ml. 1H- and 13C-NMR spectroscopy were successfully used to distinguish and identify several derivatives of DG and trehalose. Using [1-13C]DG, alpha- and beta-DG, alpha- and beta-DG6P, dideoxy-trehalose (DG-DG) and deoxy-trehalose (DG-Glc) can be simultaneously observed in the intracellular medium. The [DG6P]/[DG] ratio is about 5-6. The results seem to indicate the existence of an equilibrium between DG and DG6P, which limits the production of DG6P in cells. Glucose was found to exert a great influence on the metabolism of DG. It favours the formation of DG-DG and DG-Glc.

Carbon Isotopes↗
PubMed1992

Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site-directed mutagenesis and photolabelling experiments.

The positive charge of Lys27 was suppressed by chemical means in two short-chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin alpha from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6-15-fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site-directed mutagenesis. This change provokes a 120-fold decrease in the affinity, which reflects a major alteration of toxin-receptor cognate events. Using toxin-alpha derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor-bound state by photocoupling experiments. The delta chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the alpha chain. The toxin derivative weakly labelled the alpha and gamma chains but not the beta chain. The toxin may therefore interact with subunits other than the alpha chain, at least in the vicinity of Lys27.

Amino Acid Sequence↗