Yvan Boutin

Inactivation of Adhesion and Invasion of Food-Borne Listeria monocytogenes by Bacteriocin-Producing Bifidobacterium Strains of Human Origin

ABSTRACT Three bacteriocin-producing bifidobacterial isolates from newborns were identified as Bifidobacterium thermacidophilum (two strains) and B. thermophilum (one strain). This study was undertaken to evaluate the ability of these strains to compete with food-borne Listeria monocytogenes for adhesion and invasion sites on Caco-2 and HT-29 cells. The bifidobacteria adhered at levels ranging from 4% to 10% of the CFU added, but none of the bifidobacteria were able to invade cells. The abilities of Listeria to adhere to and to invade cells varied widely depending on the strain tested. Three groups of Listeria were identified based on invasiveness: weakly invasive, moderately invasive, and highly invasive strains. One strain from each group was tested in competition with bifidobacteria. B. thermacidophilum RBL70 was the most effective in blocking invasion of Listeria, and the decreases in invasion ranged from 38% to 90%. For all three bifidobacterial strains, contact between the cell monolayer and the bifidobacteria for 1 h before exposure to Listeria increased the degree of inhibition. Finally, visualization of competition for adhesion sites on cells by fluorescent in situ hybridization suggested that the two bacteria tended to adhere in close proximity.

A bioactive (1→3)-, (1→4)-β-d-glucan from Collybia dryophila and other mushrooms

Polysaccharides from higher Basidiomycete mushrooms, mainly β-d-glucans, are considered to be potent bioactive fungal compounds. In this study a β-glucan (1.237 × 106 Da) consisting of (1→ 3) and (1→ 4) glucosidic linkages, named Collybia dryophila polysaccharide (CDP), was extracted from the wild mushroom C. dryophila. CDP was shown to strongly inhibit nitric oxide production in activated macrophages suggesting that this polysaccharide displays a potential anti-inflammatory activity. In addition it was shown that polysaccharides similar to CDP (CDP-like) are present in Lentinus edodes and different wild mushrooms collected in northeastern North America.

Lol p I–specific IgE and IgG synthesis by peripheral blood mononuclear cells from atopic subjects in SCID mice

BACKGROUND The development of an animal model representative of the in vivo situation of human atopic diseases is always of interest for a better understanding of IgE production and regulation. Along these lines, mice with severe combined immunodeficiency (SCID mice) engrafted with lymphocytes from atopic subjects might be a suitable model for such studies. OBJECTIVE This study aims to analyze the production of Lol p I-specific IgE and IgG antibodies in SCID mice after transplantation of human peripheral blood mononuclear cells from atopic patients sensitive to grass pollens and from nonatopic donors. METHODS Peripheral blood mononuclear cells were transplanted into SCID mice, which were then challenged with Lol p I, and antibody responses (IgG and IgE) were analyzed over a 6-week period. RESULTS Total IgG antibody was measured in each mouse serum after transplantation. Also, most mice (regardless of whether donors were atopic) that were challenged with Lol p I produced specific IgG antibody. Total IgE antibody production was observed only in mice grafted with cells from atopic patients. Lol p I-specific IgE antibodies were also produced after immunization with Lol p I. Although IgG antibody/response tended to plateau, the IgE antibody response increased until it peaked and declined thereafter. Interferon-gamma was detected in sera from mice producing IgE antibody, which supports a possible role of interferon-gamma in the decrease of IgE response. CONCLUSION This study suggests that the SCID mouse model could represent an interesting approach to studying specific, total IgG and IgE antibody production, and ultimately their regulation.

Effects of bifidobacterial cytoplasm peptide and protein fractions on mouse lymphocyte proliferation and cytokine production

Abstract The ability of peptides and proteins from Bifidobacterium lactis Bb12 cytoplasm to elicit immune responses was measured in terms of murine splenocyte proliferation and cytokine secretion. Peptides and proteins from B. lactis Bb12 cytoplasm were separated into acidic and basic fractions by liquid-phase electrofocalization based on isoelectric pH. Both fractions were further fractionated by RP-HPLC based on hydrophobicity. Major peaks (of low hydrophobicity) selected in each fraction were found by mass spectrometry to contain an abundant peptide or protein with contaminants. The acidic fraction gave a stimulation index of 1.61±0.08 compared to 1.33±0.02 for the basic fraction and 2.96±0.35 for crude extract. The basic fraction stimulated IFN-γ secretion more than the acidic fraction (579±195 vs. 191±7 pg ml−1). The results demonstrate that peptides and proteins from B. lactis Bb12 exhibit immunostimulating activity, suggesting the use of cytoplasm as a nutraceutical product to enhance immune responses in children or elderly persons with reduced immune function.

Immunological and Biological Properties of Recombinant Lol p 1

BACKGROUND Current forms of allergy diagnosis and therapies are based on the use of natural allergenic extracts. Despite strong evidence that higher therapeutic efficacy may be achieved with purified allergens, the purification of multiple allergic components from extracts is a fastidious and sometimes an impossible task. However, the use of recombinant allergens may be an alternative to overcome this problem. OBJECTIVE In this study, we compared the immunological properties of recombinant (r) Lol p 1 with those of the natural protein. METHOD We cloned directly the gene encoding Lol p 1 from genomic DNA of ryegrass pollen. This gene was subcloned into the expression vector pMAL-c and expressed as fusion protein. Subsequently, rLol p 1 was cleaved from maltose-binding protein using factor Xa. Using binding inhibition and proliferative assays, we assessed the immunological properties of the recombinant allergens. The capacity of rLol p 1 to trigger basophil histamine release and to elicit a skin reaction was also assessed and compared to those of its natural counterpart. RESULTS We found that the Lol p 1 gene has no introns since we amplified this gene directly from genomic DNA. We demonstrated that the binding sites of anti-Lol p 1 monoclonal antibody, specific human IgG and IgE antibody are well conserved on rLol p 1 as no difference in the binding inhibition profile was observed when using either natural or recombinant protein. At the T-cell level, rLol p 1 elicited a T-cell response in mice comparable to that observed with the natural protein. In addition, we demonstrated that the biological characteristics of rLol p 1 were comparable to those of the natural counterpart, in that rLol p 1 elicited a skin wheal reaction and induced basophil histamine release in grass-allergic patients only. CONCLUSION The data indicate that natural Lol p 1 and rLol p 1 shared identical immunological and biological properties.

Characterization of Allergenic Determinants on the C-Terminal Region of the r-Lol p 1

Current forms of allergy diagnosis and therapies are based on the use of natural allergenic extracts. Such extracts represent mixtures of more than 50 different molecules, mostly proteins. Despite strong evidence that higher therapeutic efficacy may be achieved with purified extracts, the purification of multiple allergic components from a given extract is a fastidious and sometimes an impossible task. However, the use of re-combinant allergens may be an alternative to overcome this problem. To date, cDNA from several allergic proteins have been cloned and the corresponding recombinant allergens have been synthesized. The characterization of these allergens by cDNA technology provides important information about the primary structure of allergens and about the similarities with already known proteins. In the case of ryegrass pollen (Lolium perenne), which is responsible for a large portion of grass pollen allergies worldwide, at least three major classes of allergenic proteins (Lol p 1, Lol p 2 and Lol p 3)(1— 5) have been isolated and characterized. The major allergen, Lol p 1, is a glycoprotein of about 32 kDa and represents the major IgE-binding protein, as 85–90% of ryegrass sensitive patients react against this protein. Moreover, elevated levels of Lol p 1-specific IgE have been detected in the sera of up to 95% of grass pollen sensitive patients. Recently, the cDNA of two isoforms of Lol p 1 have been sequenced and found to encode a protein of the same size (240 residues). However a lack of information still persists about the IgE-binding epitopes on the Lol p 1. The present study has therefore further characterized the IgE binding epitopes on the rLol p 1 using recombinant technology, mAbs, and synthetic peptides.

Biological activity of monoclonal anti-idiotypic antibody representing the internal image of the major allergenic component of Lolium perenne pollen

Upon immunization with an anti-Lol p I (major allergenic component of Lolium perenne pollen) monoclonal antibody, we have previously produced anti-idiotypic monoclonal antibody (A7H2) displaying some internal image properties. The present study was designated to evaluate the capacity of this anti-idiotypic monoclonal antibody to mimic functionally the antigen by triggering histamine release from basophils of patients allergic to Lol p I. Anti-idiotypic monoclonal antibody, as the antigen, could induce histamine release in a dose-response fashion in all of the atopic patients (6/6). The inhibition of this histamine release by the addition of the idiotype (290A-167) confirmed the specificity of the reaction. Binding inhibition of human IgE to Lol p I demonstrated that the anti-idiotypic antibody recognized an idiotope expressed in the antigen-combining site of IgE molecules. Altogether, these data confirmed the internal properties of our anti-idiotypic antibody and it can mimic the original antigen in its capacity to trigger histamine release.

Suppression of immune response to Lol p I by administration of idiotype

BACKGROUND Allergic diseases are characterized by an increased production of specific IgE antibodies. Suppression of IgE antibody production may be accomplished through idiotypic manipulation. OBJECTIVE Using an animal model, we explored the effects of anti-Lol pI monoclonal antibody administration on the subsequent IgE and IgG antibody response against Lol pI. METHODS Mice were treated with an anti-Lol pI monoclonal antibody (290A-167), which resulted in the production of anti-idiotypic antibodies as evidenced by their ability to bind to the Fab fraction of 290A-167 and to inhibit the binding of rabbit polyclonal anti-idiotypic antibodies to 290A-167. The animals were then immunized with Lol pI adsorbed onto alum, and the immune response to the protein was analyzed. RESULTS Antigen-specific IgG1 and IgE responses were strongly suppressed as determined by immunoassay. Suppression of anti-Lol pI IgE antibodies was confirmed by a reduction of end-point titers measured by passive cutaneous anaphylaxis. The suppression of antigen-specific antibody was accompanied by a reduction of anti-Lol pI antibody-producing spleen cells. CONCLUSION These data indicate that pretreatment with 290A-167 can strongly downregulate the IgE response to the main allergen of ryegrass pollen, which is associated with an increase in anti-idiotypic antibodies. This approach could provide rapid, long-term hyposensitization in patients with grass pollen allergy.

ANTI-IDIOTYPIC ANTIBODIES IN THE TREATMENT OF ALLERGIES

An increased IgE synthesis and local inflammatory response are the hallmarks of the atopic immune response. Therefore a better understanding of the mechanisms regulating the IgE production and multiple events or factors leading to the inflammation is needed to define strategies of treatment targeting the abnormal response.