Effect of Heating and Glycation on the Allergenicity of 2S Albumins (Ara h 2/6) from Peanut

Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins

Allergenic and immunogenic potential of cow's milk beta-lactoglobulin and caseins evidenced without adjuvant in germ-free mice

Scope: In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ-free (GF) mice exhibit Th2-polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant.[br/] Methods and results: beta-lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward kappa- and alpha S1-caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG-reactivation, IL-4, IL-5, IL-13 and IFN-gamma productions by splenocytes were higher in GF mice than in CV mice. Heat-treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat-treatment increased protein immunogenicity and led to the production of anti-BLG and anti-kappa-casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL-4, IL-5 and IL-13 in BLG-reactivated splenocytes from CV mice.[br/] Conclusion: Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins

Impact of delayed bacterial colonisation of the gut on susceptibility to allergic sensitisation to cow's milk in mice

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Delayed bacterial colonization of the gut alters the host immune response to oral sensitization against cow's milk proteins

Scope Cow's milk allergy is the most prevalent food allergy in infants whose immune system development is critically stimulated during postnatal gut colonization by commensal bacteria. Allergenic potential of cow's milk beta-lactoglobulin (BLG) and caseins (CAS) was investigated in germ-free (GF) BALB/c mice and in GF mice conventionalized (CVd) at 6 weeks of age. Methods and results Oral sensitization to cow's milk in the presence of cholera toxin led to higher BLG-specific IgE, IgG1, and IgG2a responses in GF mice than in conventional (CV) mice. No significant difference was observed for CAS-specific IgE responses although IgG1 responses to alpha S1- and kappa-caseins were higher in GF mice than in CV mice. CVd mice, orally inoculated with fecal preparations from CV mice, also displayed biased antibody responses compared to CV mice. Secretion of Th2 cytokines by BLG- and CAS-reactivated splenocytes of CVd mice was similar to that of GF mice whereas cytokine production by reactivated cells from mesenteric lymph nodes of CVd mice was equivalent to that of CV mice. Conclusion Oral sensitization to BLG and CAS was differentially affected by the absence of gut microbiota and delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens

Influence of the route of administration on immunomodulatory properties of bovine beta-lactoglobulin-producing Lactobacillus casei

International audienceBecause of their intrinsic immunomodulatory properties, some lactic acid bacteria were reported to modulate allergic immune responses in mice and humans. We recently developed recombinant strains of Lactobacillus casei that produce beta-lactoglobulin (BLG), a major cow's milk allergen. Here, we investigated immunomodulatory potency of intranasal and oral administrations of recombinant lactobacilli on a subsequent sensitization of mice to BLG. Intranasal administration of the BLG-producing Lb. casei stimulated serum BLG-specific IgG2a and IgG1 responses, and fecal IgA response as well, but did not inhibit BLG-specific IgE production. In contrast, oral administration led to a significant inhibition of BLG-specific IgE production while IgG1 and IgG2a responses were not stimulated. After both oral and intranasal administrations, production of IL-17 cytokine by BLG-reactivated splenocytes was similarly enhanced, thus confirming the adjuvant effect of the Lb. casei strain. However, a mixed Th1/Th2 cell response was evidenced in BLG-reactivated splenocytes from mice intranasally pretreated, with enhanced secretions of Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-4 and IL-5) whereas only production of Th1 cytokines, but not Th2 cytokines, was enhanced in BLG-reactivated splenocytes from mice orally pretreated. Our results show that the mode of administration of live bacteria may be critical for their immunomodulatory effects

Allergic sensitization to bovine beta-lactoglobulin: comparison between germ-free and conventional BALB/c mice

International audienceBackground: The 'hygiene hypothesis' suggests that high hygienic standards met in western countries lead to a lack of microbial exposure, thus promoting the development of atopy by preventing the proper maturation of the immune system. Germ-free animals are deprived of the immune stimulation that occurs during postnatal gut colonization by commensal bacteria. Germ-free mice could thereby provide an attractive model for studying the impact of gut microbiota on the development of Th2-mediated disorders such as allergy. Methods: Germ-free and conventional BALB/c mice were sensitized to beta-lactoglobulin (BLG), a major cow's milk allergen, by means of intraperitoneal injections in the presence of incomplete Freund's adjuvant. Time courses of serum and fecal BLG-specific antibody responses were monitored and cytokine production was assayed in BLG-reactivated splenocytes. Results: Serum BLG-specific IgG1 and IgE concentrations were significantly higher in germ-free mice during the primary immune response and IgE production persisted longer in germ-free mice. Furthermore, secretion of BLG-specific IgA was evidenced only in feces from germfree mice while, in contrast, fecal IgG1 concentrations were at least 3-fold higher in conventional mice than in germ-free mice. Production of IL-5, IL-10 and IFN-gamma was 3-fold enhanced in BLG-reactivated splenocytes from germ-free mice. Conclusion: The absence of gut microbiota significantly affects the BLG-specific immune response in BALB/c mice, thus suggesting that this model might be of interest for further studies exploring the influence of gut colonization by different bacterial strains on the development of an allergic-type sensitization

Serological and clinical information for the 34 peanut allergic patients recruited in different European centres, within the Europrevall European project, used in EAST studies, basophil histamine release tests and/or PBMC proliferation tests.

<p>HEP: histamine equivalent prick test; nd: not determined. Where known, other allergies of the subjects are indicated.</p><p>The origin of the sera is as follows: 2A: Zurich, 52A: Amsterdam, 54A-82: Arnhem, 1682-2305: Vienna; 03-0043 till 12-0048 EuroPrevall Serum Bank.</p><p>Grading is based on Brockow and Ring and on Sampson <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023998#pone.0023998-Brockow1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023998#pone.0023998-Sampson1" target="_blank">[42]</a>. Grade 1 = dermal symptoms; grade 2 = gastro-intestinal problems like nausea and or cramping; grade 3 = any of the former grades plus vomiting/diarrhoea and respiratory tract problems like throat pruritis or tightness, grade 4 = any of the former grades and respiratory arrest plus cardiovascular problems like hypotension.</p

Effect of heating on secondary structure and oligomeric state of Ara h 2/6.

<p>Far-UV CD spectra of Ara h 2/6 heated alone (<b>A</b>, H-Ara h 2/6) or in the presence of 100 mM glucose (<b>B</b>, G-Ara h 2/6) before heating (N-Ara h 2/6, —) and for heated-cooled protein after heating to 110°C for 15 min (−−−) and 60 min (---). Inset graphs show change in molar residue ellipticity at 228 nm with heating time. Size exclusion chromatography profiles (<b>C</b>) are shown of the Ara h 2/6 before and after heating for 15 min at 110°C in the presence or absence of glucose. Retention volumes of molecular weight standards (size indicated in kDa) are shown by arrow heads. <b>D</b>. Far-UV CD spectra of Ara h 6 purified from roasted peanut.</p

Effect of thermal treatment on cytokine induction capacity.

<p>Fresh PBMC were left unstimulated (Med) or stimulated with N-, H- or G-Ara h 2/6 and production of IL-5 (<b>A</b>), IL-13 (<b>B</b>), IL-10 (<b>C</b>) and IFN-γ (<b>D</b>) was measured after 7 days of culture. Symbols represent peanut-allergic patients #65 (▪), #66 (♦), #70 (▴), #67 (•) and #73 (×). Horizontal bars represent the mean of the five PA responder subjects (solid) and the mean of 7 NA controls (dotted). Asterisks indicate statistically significant differences between the PA and the NA group and # indicate statistically significant differences between the medium and allergen-stimulated cultures for the PA group (*, #: <i>P</i><0.05). No differences between the medium and allergen-stimulated cultures were observed for the NA group.</p

Effect of thermal treatment on the activation of effector cells induced by Ara h 2/6.

<p>(<b>A</b>) Human stripped basophils were passively sensitized with individual sera (#2305) and then incubated with increasing concentrations of N-Ara h 2/6 (•), H-Ara h 2/6 (⧫), and G-Ara h 2/6 (▴), or whole peanut extract from raw (★) or roasted (○) peanut. Histamine release was assayed in corresponding supernatants. Twenty-three sera out of the 35 available corresponding to all sera from Zurich, Amsterdam, Vienna and EPSB, and sera 54A to 58A from Arnhem were used, giving similar results (not shown). No histamine release was induced by the different allergens when using a serum from patients not sensitized to peanut (data not shown). (<b>B</b>) Humanized RBL-2H3 cells were passively sensitized with sera from peanut-allergic patients (#70) and stimulated with increasing concentrations of native (N; ―•―), heated (H; – –⧫– –) or glycated (G; - - -▴- - -) Ara h 2/6. Error bars represent the SD of triplicate values. No β-hexosaminidase release was induced by the different allergens when using sera from non-allergic patients (data not shown). The table presented within the figure represents the average protein concentrations (ng/ml, n = 6) to obtain 50% of the maximum allergen release of the native allergen (EC50). Means without a common letter differ (P<0.05).</p