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.

Peanut allergens are rapidly transferred in human breast milk and can prevent sensitization in mice.

BACKGROUND: Food allergens have been evidenced in breast milk under physiological conditions, but the kinetic and the role of this passage in food allergies are still unclear. We then aimed to analyze the passage of peanut allergens in human breast milk and their allergenicity/immunomodulatory properties. METHODS: Human breast milk was collected from two non-atopic peanut-tolerant mothers before and at different time points after ingestion of 30 g of commercial roasted peanut. Ara h 6, Ara h 6 immune complexes, and the IgE binding capacity of breast milk samples were measured using specific immunoassays. Their allergenic functionality was then assessed using cell-based assay. Finally, human breast milk obtained before or after peanut ingestion was administered intragastrically to BALB/c mice at different ages, and mice were further experimentally sensitized to peanut using cholera toxin. RESULTS: Ara h 6 is detected as soon as 10 min after peanut ingestion, with peak values observed within the first hour after ingestion. The transfer is long-lasting, small quantities of peanut allergens being detected over a 24-h period. IgG-Ara h 6 and IgA-Ara h 6 immune complexes are evidenced, following a different kinetic of excretion than free allergens. Peanut allergens transferred in milk are IgE reactive and can induce an allergic reaction in vitro. However, administration of human breast milk to young mice, notably before weaning, does not lead to sensitization, but instead to partial oral tolerance. CONCLUSION: The low quantities of immunologically active allergens transferred through breast milk may prevent instead of priming allergic sensitization to peanut.

Goat's milk allergy without cow's milk allergy: suppression of non-cross-reactive epitopes on caprine ß-casein.

BACKGROUND AND OBJECTIVE: Goat's milk (GM) allergy associated with tolerance to cow's milk (CM) has been reported in patients without history of CM allergy and in CM-allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM-allergic/CM-tolerant patients recognize caprine ß-casein (ßcap) without cross-reacting with bovine ß-casein (ßbov) despite a sequence identity of 91%. In this study, we investigated the non-cross-reactive IgE-binding epitopes of ßcap. METHODS: Recombinant ßcap was genetically modified by substituting caprine domains with the bovine counterparts and by performing site-directed mutagenesis. We then evaluated the recognition of modified ßcap by IgE antibodies from 11 GM-allergic/CM-tolerant patients and 11 CM-allergic patients or by monoclonal antibodies (mAb) raised against caprine caseins. The allergenic potency of modified ßcap was finally assessed by degranulation tests of humanized rat basophil leukaemia (RBL)-SX38 cells. RESULTS: Non-cross-reactive epitopes of ßcap were found in domains 44-88 and 130-178. The substitutions A55T/T63P/L75P and P148H/S152P induced the greatest decrease in IgE reactivity of GM-allergic/CM-tolerant patients towards ßcap. The pivotal role of threonine 63 was particularly revealed as its substitution also impaired the recognition of ßcap by specific mAb, which could discriminate between ßcap and ßbov. The modified ßcap containing the five substitutions was then unable to trigger the degranulation of RBL-SX38 cells passively sensitized with IgE antibodies from GM-allergic/CM-tolerant patients. CONCLUSIONS: Although IgE-binding epitopes are spread all over ßcap, a non-cross-linking version of ßcap was generated with only five amino acid substitutions and could thus provide new insight for the design of hypoallergenic variants.

Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine ß-lactoglobulin.

BACKGROUND: Food allergy is considered as resulting from an impaired development or a breakdown of oral tolerance. We aimed to induce oral tolerance to the major cow's milk allergen bovine ß-lactoglobulin (BLG) or corresponding trypsin hydrolysates (BLG-Try) and to investigate the mechanisms involved. METHODS: Wild-type BALB/cJ mice were gavaged on days 1-3 and 8-10 with different doses of native BLG (nBLG) or with nBLG-Try and were then sensitized on day 14 by i.p. administration of BLG in alum. Sensitization was assessed by measurement of BLG-specific antibodies in sera and of cytokines secreted by BLG-reactivated splenocytes. Elicitation of the allergic reaction was assessed by measurement of cytokines and mMCP-1 in sera collected 35 min after an oral challenge. Cellular and biochemical markers of the allergic reaction were also analysed in bronchoalveolar lavage fluids (BAL) collected 24 h after intra-nasal challenge. Analysis of the CD4(+) CD25(+) Foxp3(+) cells in different organs obtained 3 days after gavage and in vivo depletion of CD25(+) cells before oral tolerance induction were then performed. RESULTS: Systemic sensitization and elicitation of the allergic reaction were totally inhibited in mice gavaged with 2 mg of nBLG whereas nBLG-Try was far less efficient. A high percentage of CD4(+) Foxp3(+) cells were observed in BAL from tolerant mice, and a negative correlation between the number of eosinophils and the percentage of Foxp3(+) cells was evidenced. Efficient induction of CD4(+) CD25(+) Foxp3(+) cells after nBLG gavage and impaired oral tolerance induction after in vivo depletion of CD25 cells were then demonstrated. CONCLUSION: For the first time, allergen-induced Treg cells that inhibited both the sensitization and the elicitation of the allergic reaction were evidenced in gavaged wild-type mice.

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.

Sensitization and elicitation of an allergic reaction to wheat gliadins in mice.

We developed a mouse model of allergy to wheat flour gliadins, a protein fraction containing major wheat allergens. We compared the antibody responses (i.e., specific IgE and IgG1) and the profiles of cytokines secreted by reactivated splenocytes induced after intraperitoneal injections of gliadins in three strains of mice, namely, Balb/cJ, B10.A, and C3H/HeJ. The intensities of the allergic reactions elicited by intranasal challenge were also compared. Both the sensitization and elicitation were the highest in Balb/cJ mice, whereas weak or no reaction was observed in the others strains. Interestingly, the specificity of the mouse IgE against the different gliadins (i.e., alpha-, beta-, gamma-, omega 1,2-, and omega 5-gliadin) was similar to that observed in children allergic to wheat flour. Balb/cJ mice may thus provide a relevant model for the study of sensitization and elicitation by wheat gliadins and for improving our understanding of the specific role and mechanisms of action of the different classes of gliadins.

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.

In vivo transfer of plasmid from food-grade transiting lactococci to murine epithelial cells.

We recently demonstrated that noninvasive food-grade Lactococcus lactis (L. lactis) can deliver eukaryotic expression plasmid in mammalian cells in vitro. Here, we evaluated, in vivo, whether a eukaryotic expression plasmid carried by lactococci can translocate to the epithelial cells of the intestinal membrane. The strain LL(pLIG:BLG1) carrying one plasmid containing a eukaryotic expression cassette encoding beta-lactoglobulin (BLG), a major allergen of cow's milk, was orally administered by gavage to mice. BLG cDNA was detected in the epithelial membrane of the small intestine of 40% of the mice and BLG was produced in 53% of the mice. Oral administration of LL(pLIG:BLG1) induced a low and transitory Th1-type immune response counteracting a Th2 response in case of further sensitization. We demonstrated for the first time the transfer of a functional plasmid to the epithelial membrane of the small intestine in mice by noninvasive food-grade lactococci.

Allergy to goat and sheep milk without allergy to cow's milk.

BACKGROUND: Cow's milk (CM) allergy is the most frequent cause of food allergy in infants. Most children who are allergic to CM are also sensitized to whey proteins and/or to the casein fraction and many of them cannot tolerate goat's or sheep's milk (GSM) either. Conversely, the GSM allergies that are not associated with allergic cross-reactivity to CM are rare. METHODS: Twenty-eight children who had severe allergic reactions, including anaphylaxis, after consumption of GSM products but tolerated CM products were recruited in a retrospective study. Whole casein and whey proteins were fractionated from CM and GSM. beta-Lactoglobulin and the different caseins were isolated, purified and used to perform enzyme allergosorbent tests (EAST) and EAST inhibition studies with the sera of the allergic children. RESULTS: Clinical observations, skin prick testing and immunoglobulin (Ig)E-binding studies confirmed the diagnosis of GSM allergy without associated CM allergy. EAST determinations demonstrated that GSM allergy involves the casein fraction and not whey proteins. Cow's milk caseins were not at all or poorly recognized by the patient's IgE, while alphaS(1)-, alphaS(2)- and beta-caseins from GSM were recognized with a high specificity and affinity. In all cases, increasing concentrations of CM caseins failed to inhibit the binding of patient's IgE to sheep or goat milk caseins, whereas this binding was completely inhibited by GSM caseins. CONCLUSIONS: The characteristics of GSM allergy differ from those of the CM allergy because it affects older children and appears later. CM products do not elicit any clinical manifestation in GSM allergic patients, whereas CM allergic patients, usually cross-react to GSM. In all the GSM allergic children, the IgE antibodies recognized the caseins but not the whey proteins. Moreover, IgE specificity and affinity was high to GSM and lower to CM caseins despite their marked sequence homology. Doctors and allergic individuals should be aware that GSM allergy requires a strict avoidance of GSM and milk-derived products because reactions could be severe after ingestion of minimal doses of the offending food.

Skin localization of cow's milk proteins delivered by a new ready-to-use atopy patch test.

PURPOSE: Atopy patch tests (APTs) allow the detection of delayed allergies at the skin level. The localization of beta-lactoglobulin delivered into the skin by an innovative ready-to-use APT (E-patch was investigated and the efficacy and safety of this device were assessed. METHODS: The E-patch containing beta-lactoglobulin was placed for 24 h in contact with hairless rat skin mounted in a Franz diffusion cell. Transdermal passage was monitored by measurement of beta-lactoglobulin A-[methyl-(14)C] or by two-site enzyme immunoassay. An iterative skin stripping allowed measurement of the beta-lactoglobulin penetrating the first external skin layers. RESULTS: After 24 h, 92% of beta-lactoglobulin remained on the skin. The iterative skin strippings showed a 135-fold higher concentration of beta-lactoglobulin in the stratum corneum than that found in the epidermis-dermis. Analysis of the solution in the receiver compartment by radioactivity assays or immunoassays indicates that intact protein did not cross the skin. CONCLUSIONS: The E-patch system allows native beta-lactoglobulin to concentrate in the stratum corneum, in the vicinity of immunological cells, but does not lead to its systemic delivery. Therefore, it is suggested that this delivery system creates ideal conditions for promoting a positive topical response with reduced risk of systemic anaphylactic reactions caused by the native form of the beta-lactoglobulin A.

Influence of thermal processing on the allergenicity of peanut proteins.

Peanuts are one of the most common and severe food allergens. Nevertheless, the occurrence of peanut allergy varies between countries and depends on both the exposure and the way peanuts are consumed. Processing is known to influence the allergenicity of peanut proteins. The aim of this study was to assess the effect of thermal processing on the IgE-binding capacity of whole peanut protein extracts and of the major peanut allergens Ara h 1 and Ara h 2. Whole proteins, Ara h 1, and Ara h 2 were extracted and purified from raw, roasted and boiled peanuts using selective precipitation and multiple chromatographic steps, and were then characterized by electrophoresis and mass spectrometry. The immunoreactivity of whole peanut extracts and purified proteins was analyzed by the enzyme allergosorbent test (EAST) and EAST inhibition using the sera of 37 peanut-allergic patients. The composition of the whole protein extracts was modified after heat processing, especially after boiling. The electrophoretic pattern showed protein bands of low molecular weight that were less marked in boiled than in raw and roasted peanuts. The same low-molecular-weight proteins were found in the cooking water of peanuts. Whole peanut protein extracts obtained after the different processes were all recognized by the IgE of the 37 patients. The IgE-binding capacity of the whole peanut protein extracts prepared from boiled peanuts was 2-fold lower than that of the extracts prepared from raw and roasted peanuts. No significant difference was observed between protein extracts from raw and roasted peanuts. It is noteworthy that the proteins present in the cooking water were also recognized by the IgE of peanut-allergic patients. IgE immunoreactivity of purified Ara h 1 and Ara h 2 prepared from roasted peanuts was higher than that of their counterparts prepared from raw and boiled peanuts. The IgE-binding capacity of purified Ara h 1 and Ara h 2 was altered by heat treatment and in particular was increased by roasting. However, no significant difference in IgE immunoreactivity was observed between whole protein extracts from raw and roasted peanuts. The decrease in allergenicity of boiled peanuts results mainly from a transfer of low-molecular-weight allergens into the water during cooking.

Peanut- and cow's milk-specific IgE, Th2 cells and local anaphylactic reaction are induced in Balb/c mice orally sensitized with cholera toxin.

BACKGROUND: The development of animal models developing specific immunoglobulin (Ig)E presenting the same specificity as human IgE and similar clinical symptoms as those observed in allergic patients are of great interest for the understanding of mechanisms involved in the induction and regulation of food allergy. METHODS: Balb/c female mice were sensitized with whole peanut protein extract (WPPE) by means of intraperitoneal (i.p.) injections with alum or gavages with cholera toxin (CT). The WPPE specific IgE, IgG1 and IgG2a were monitored. Th2 cells activation was analysed assaying interleukin (IL)-4 and IL-5 vs IFNgamma on reactivated splenocytes. Local anaphylactic reaction was evaluated by assaying histamine in faecal samples. The oral sensitization protocol was further extended to cow's milk proteins (CMP). RESULTS: Balb/c mice developed high peanut-specific IgE and IgG1 responses either after i.p. or oral sensitizations. In both cases, antibodies were specific to polymer of glycinin fragments, containing polypeptides from Ara h3/4, and to a lesser extent to Ara h1 and Ara h2. Interleukin-4 and IL-5 production were evidenced. Balb/c mice could also be sensitized to CMP, as demonstrated by CMP-specific IL-4 and IL-5 secretions and induction of IgE specific for whole caseins, beta-lactoglobulin, serum bovine albumin and lactoferrin. Of interest was the occurrence of a local anaphylactic reaction in the peanut and CM models. CONCLUSIONS: In contrast with previous authors, Balb/c mice were sensitized and evidenced an allergic reaction after oral administrations of peanut or CMP plus CT, providing an interesting model for further studies on immunopathogenic mechanisms.

Oral administration of recombinant Lactococcus lactis expressing bovine beta-lactoglobulin partially prevents mice from sensitization.

BACKGROUND: The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response. OBJECTIVE: We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy. METHODS: Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations. RESULTS: Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein. CONCLUSION: Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.