Further studies on the biological activity of hazelnut allergens.

BACKGROUND: Sensitization to hazelnut allergens vary depending on the geographic origin and age of the patients. The objective of this study was to further investigate the allergenic activity of hazelnut allergens using sera from patients recruited in various European regions and presenting different sensitization patterns to hazelnut proteins. METHODS: Natural Cor a 11 and Cor a 9 were purified from hazelnut whereas Cor a 1 and Cor a 8 were produced as recombinant proteins (rCor a 1.04 and rCor a 8). Sera from hazelnut allergic patients were collected in France (n = 5), Switzerland (n = 2), Greece (n = 11) and Spain (n = 3), within the Europrevall project. Total and allergen-specific IgE were quantified by enzyme allergosorbent test and IgE immunoblot were performed using pooled sera from birch-pollen endemic region or from Greece. Histamine Release (HR) assays were performed with stripped basophils passively sensitized with individual sera and challenged by a hazelnut extract or the different hazelnut allergens. RESULTS: As previously described, hazelnut allergic patients from Mediterranean countries are mainly sensitized to the nsLTP Cor a 8 whereas patients from France and Switzerland are sensitized to pollen-related allergens. Interestingly, an intermediate profile was evidenced in patients from Madrid. Hazelnut 7S globulin (Cor a 11) and 11S globulin (Cor a 9) were found to be minor allergens, recognized only by patients from Mediterranean countries. The biologic activity of the 4 tested allergens, analysed by HR assay, further confirmed the sensitization patterns, but also demonstrated the very high elicitation potency of Cor a 8. CONCLUSIONS: This work, extending previously published researches, represents a step towards the better understanding of the complexity of hazelnut allergy and provides new data on the biological activity of hazelnut allergens and extracts.

Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays.

BACKGROUND: Peanuts are often consumed after roasting, a process that alters the three-dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity. OBJECTIVE: We aimed to establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut (Ara h 2/6). METHODS: Ara h 1 and Ara h 2/6 were purified from raw peanuts and heated in a dry form for 20 min at 145°C in the presence (R+g) or absence (R-g) of glucose, and soluble proteins were then extracted. Sera obtained from 12 well-characterized peanut-allergic patients were used to assess the IgE binding and degranulation capacities of the allergens. RESULTS: Extensive heating at low moisture resulted in the hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble R+g Ara h 1 formed large aggregates. Although the IgE-binding capacity of R+g and R-g Ara h 1 was decreased 9000- and 3.6-fold, respectively, compared with native Ara h 1, their capacity to elicit mediator release was increased. Conversely, both the IgE-binding capacity and the degranulation capacity of R-g Ara h 2/6 were 600-700-fold lower compared with the native form, although the presence of glucose during heating significantly moderated these losses. CONCLUSIONS AND CLINICAL RELEVANCE: Extensive heating reduced the degranulation capacity of Ara h 2/6 but significantly increased the degranulation capacity of Ara h 1. This observation can have important ramifications for component-resolved approaches for diagnosis and demonstrates the importance of investigating the degranulation capacity in addition to IgE reactivity when assessing the effects of food processing on the allergenicity of proteins.

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

Because 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.

BACKGROUND: 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 germ-free 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.