154 research outputs found

    Influence de la présence et de la composition du microbiote intestinal sur le développement et la prévention des allergies alimentaires

    Get PDF
    Le dĂ©veloppement de l allergie peut ĂȘtre influencĂ© par le microbiote intestinal qui est impliquĂ© dans la maturation du systĂšme immunitaire de l hĂŽte lors de la colonisation du tractus digestif dĂšs la naissance. L objectif de mon travail a Ă©tĂ© d Ă©tudier l impact du microbiote intestinal sur le dĂ©veloppement d une sensibilisation allergique Ă  des protĂ©ines de lait de vache Ă  l aide d un modĂšle de souris BALB/c gnotoxĂ©niques. Dans une premiĂšre Ă©tude, nous avons montrĂ© que les souris axĂ©niques (Ax, sans germe) sont plus rĂ©actives que les souris conventionnelles (CV) au potentiel immunogĂ©nique et allergĂ©nique de la b-lactoglobuline (BLG) et de la casĂ©ine (CAS), lorsque ces deux protĂ©ines sont injectĂ©es intrapĂ©ritonĂ©alement sans adjuvant. A l aide d un autre modĂšle de sensibilisation par voie orale au lait, nous avons confirmĂ© que les souris Ax dĂ©veloppent des rĂ©ponses IgE contre la BLG plus fortes que celles des souris CV. Les mĂ©canismes de sensibilisation contre la BLG et la CAS sont alors diffĂ©remment affectĂ©s par la prĂ©sence ou non d un microbiote intestinal. Par ailleurs, une colonisation tardive du tractus digestif de souris Ax Ă  l Ăąge de 6 semaines par le microbiote de souris CV induit chez les souris conventionnalisĂ©es (CVd) le dĂ©veloppement, aprĂšs sensibilisation, de rĂ©ponses humorales toujours plus fortes que celles observĂ©es chez les souris CV. A l inverse, une conventionnalisation des souris Ax au moment du sevrage Ă  l Ăąge de 3 semaines, induit un niveau de sensibilisation plus faible que celui des souris CV. Dans ce cas, des diffĂ©rences de composition du microbiote intestinal entre souris CV et CVd pourraient jouer un rĂŽle dans le faible niveau de sensibilisation des souris CVd. Nous avons enfin Ă©valuĂ© l impact de l implantation dĂšs la naissance d une souche de Lactobacillus casei en monoxĂ©nie (souris Mx). La rĂ©ponse humorale contre la CAS, mais pas contre la BLG, est alors significativement plus Ă©levĂ©e chez les souris Mx que chez les souris Ax. Ces diffĂ©rentes Ă©tudes suggĂšrent que l influence du microbiote sur le dĂ©veloppement d une sensibilisation aux protĂ©ines du lait de vache diffĂšre selon les allergĂšnes et selon le mode d exposition aux allergĂšnes. Ces rĂ©sultats soulignent Ă©galement qu un retard de colonisation du tractus digestif peut perturber durablement la rĂ©activitĂ© du systĂšme immunitaire Ă  une sensibilisation contre des antigĂšnes alimentaires.The development of allergic responses can be influenced by the gut microbiota, which critically stimulates the maturation of the host immune system during colonization of the digestive tract at birth. We thus aimed to study the impact of the gut microbiota on the development of an allergic sensitization to cow's milk proteins by using a gnotobiotic BALB/c mouse model. First, we showed that germ-free (GF) mice are more responsive than conventional mice (CV) to the immunogenic and allergenic potential of b-lactoglobulin (BLG) and casein (CAS) when these proteins are injected intraperitoneally without adjuvant. With another model of oral sensitization to cow s milk, the development of higher BLG-specific IgE responses in GF mice compared to CV mice was confirmed. We also observed that the mechanisms leading to oral sensitization to BLG and CAS are differentially affected by the absence of gut microbiota. Furthermore, a delayed colonization of the digestive tract of 6-week-old GF mice by a conventional microbiota was studied. The conventionalized mice (CVd) still developed, after sensitization, higher antibody responses than those measured in CV mice. In contrast, GF mice conventionnalized just after weaning, at 3 week of age, displayed a level of sensitization lower than that of CV mice. Differences in the gut microbiota composition evidenced between CVd and CV mice could also play a role in the lower level of sensitization of CVd mice. Finally, we evaluated the impact of the neonatal mono-colonization of mice by a strain of Lactobacillus casei. The antibody responses against CAS, but not against BLG, were then significantly higher in mono-associated mice than in GF mice. These studies suggest that the influence of microbiota on the development of sensitization to cow's milk proteins depends on the nature of the allergens and the mode of exposure. These results also underline that delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. Ă©lectronique (751069903) / SudocSudocFranceF

    Efficient production and secretion of bovine ÎČ-lactoglobulin by Lactobacillus casei

    Get PDF
    BACKGROUND: Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. The model LAB Lactococcus lactis has been intensively used to produce and deliver such heterologous proteins. However, compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. Here, we compared different strategies to optimize the production of bovine ÎČ-lactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23. RESULTS: Using a nisin-inducible plasmid system, we first showed that L. casei BL23 strain could efficiently secrete a reporter protein, the staphylococcal nuclease (Nuc), with the lactococcal signal peptide SP(Usp45 )fused to its N-terminus. The fusion of SP(Usp45 )failed to drive BLG secretion but led to a 10-fold increase of intracellular BLG production. Secretion was significantly improved when the synthetic propeptide LEISSTCDA (hereafter called LEISS) was added to the N-terminus of the mature moiety of BLG. Secretion rate of LEISS-BLG was 6-fold higher than that of BLG alone while intracellular production reached then about 1 mg/L of culture. The highest yield of secretion was obtained by using Nuc as carrier protein. Insertion of Nuc between LEISS and BLG resulted in a 20-fold increase in BLG secretion, up to 27 ÎŒg/L of culture. Furthermore, the lactococcal nisRK regulatory genes were integrated into the BL23 chromosome. The nisRK insertion allowed a decrease of BLG synthesis in uninduced cultures while BLG production increased by 50% after nisin induction. Moreover, modification of the induction protocol led to increase the proportion of soluble BLG to around 74% of the total BLG production. CONCLUSION: BLG production and secretion in L. casei were significantly improved by fusions to a propeptide enhancer and a carrier protein. The resulting recombinant strains will be further tested for their ability to modulate the immune response against BLG via mucosal delivery in a cow's milk allergy model in mice

    Effect of simulated gastro-duodenal digestion on the allergenic reactivity of beta-lactoglobulin

    Get PDF
    <p>Abstract</p> <p>Background</p> <p>Cow's milk (CM) allergy affects about 2% of infants. The allergenicity of dietary proteins, including those from CM, has been related to their digestibility although the generality of the link and its causality remains to be demonstrated. In this study we use an in vitro digestion system, to investigate the digestibility of ÎČ-lactoglobulin (blg) during gastrointestinal transit and to assess the impact of this process on blg allergenic reactivity in CM allergic children.</p> <p>Methods</p> <p>Blg digesta were prepared using an <it>in vitro </it>digestion protocol simulating either gastric digestion alone or followed by duodenal digestion with or without phosphatidylcholine (PC). Biochemical analysis of blg digesta was performed by SDS-PAGE and their concentration was measured by a sandwich ELISA. Assessment of their allergenic reactivity was done <it>in vitro </it>by EAST inhibition, specific basophil activation (basotest) and lymphocyte proliferation (PCNA-flow cytometry) assays using sera and cells from patients allergic to blg and <it>in vivo </it>by skin prick testing (SPT) of these patients.</p> <p>Results</p> <p>Blg was only broken down to smaller peptides after gastro-duodenal digestion although a sizeable amount of intact protein still remained. Digestion did not modify the IgE binding capacity of blg except for gastro-duodenal digestion performed in the absence of PC. These results are consistent with the quantity of intact blg remaining in the digesta. Overall both gastric and gastroduodenal digestion enhanced activation of sensitized basophils and proliferation of sensitized lymphocytes by blg. However, there was a tendency towards reduction in mean diameter of SPT following digestion, the PC alone during phase 1 digestion causing a significant increase in mean diameter.</p> <p>Conclusions</p> <p>Digestion did not reduce the allergenic reactivity of blg to a clinically insignificant extent, PC inhibiting digestion and thereby protecting blg allergenic reactivity. SPT reactivity was reduced compared to blg immunoreactivity in <it>in vitro </it>tests.</p

    Risk assessment of new sequencing data on GM maize event MIR604

    Full text link
    In 2009 and 2010, the EFSA GMO Panel concluded the assessment of genetically modified (GM) maizes MIR604, MIR604 × GA21, MIR604 × Bt11 and MIR604 × GA21 × Bt11. These maizes were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 23 July 2015, the European Commission (EC) received from Syngenta new nucleic acid sequencing data on maize event MIR604 and updated bioinformatic analyses using the new sequencing data. EC tasked EFSA to analyse these data and to indicate whether the previous conclusions of the EFSA GMO Panel on the above-listed GM maizes remain valid. The EFSA GMO Panel used the appropriate principles described in its guidelines for the risk assessment of GM plants to analyse the received data. The new sequencing data indicated a single base pair difference compared to the sequencing data originally provided, located in a non-coding region of the insert. which had already been present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence did not give rise to safety issues. Therefore, the GMO Panel concludes that the original risk assessment of event MIR604 as a single and as a part of stacked events remains valid

    Genetic basis and detection of unintended effects in genetically modified crop plants

    Get PDF
    In January 2014, an international meeting sponsored by the International Life Sciences Institute/Health and Environmental Sciences Institute and the Canadian Food Inspection Agency titled “Genetic Basis of Unintended Effects in Modified Plants” was held in Ottawa, Canada, bringing together over 75 scientists from academia, government, and the agro-biotech industry. The objectives of the meeting were to explore current knowledge and identify areas requiring further study on unintended effects in plants and to discuss how this information can inform and improve genetically modified (GM) crop risk assessments. The meeting featured presentations on the molecular basis of plant genome variability in general, unintended changes at the molecular and phenotypic levels, and the development and use of hypothesis-driven evaluations of unintended effects in assessing conventional and GM crops. The development and role of emerging “omics” technologies in the assessment of unintended effects was also discussed. Several themes recurred in a number of talks; for example, a common observation was that no system for genetic modification, including conventional methods of plant breeding, is without unintended effects. Another common observation was that “unintended” does not necessarily mean “harmful”. This paper summarizes key points from the information presented at the meeting to provide readers with current viewpoints on these topics
    • 

    corecore