Complementary feeding practices are related to the risk of food allergy in the ELFE cohort.

BACKGROUND: Conflicting results have been obtained when analyzing the relationship between complementary feeding (CF) practices and allergic diseases in childhood. This study aims to further explore the association between allergic diseases in early childhood (10.1016/j.jaci.2012.02.036) and the age at CF introduction (10.1016/S0140-6736(15)00149-X), food diversity in the first year of life (10.1016/j.ijporl.2019.109759) and the delayed introduction of major allergenic foods. METHODS: This analysis focused on 6662 children from the French nationwide ELFE cohort. Data on feeding practices were collected monthly from 3 to 10 months old. Their age at CF introduction was calculated alongside a diversity score, and the number of major allergenic foods (out of eggs, fish, wheat, and dairy products) not introduced at 8 and 10 months. Their associations with parent-reported allergy-related health events between 1 and 5.5 years were assessed using logistic regressions adjusted for confounding factors. A sensitivity analysis excluding early allergic cases (occurring between 2 months and 1 or 2 years) was conducted. RESULTS: Late CF (>6 months) was related to a higher risk of food allergy (OR [95% CI] = 1.35 [1.02; 1.78]), a low diversity score at 8 months to a higher risk of asthma (OR [95% CI] = 1.22 [1.01; 1.48]), and two allergenic foods or more not being introduced at 10 months to a higher risk of rhinoconjunctivitis (OR [95% CI] = 1.20 [1.00; 1.44]) and food allergy (OR [95% CI] = 2.46 [1.77; 3.42]). Only this last association remained significant after the exclusion of early cases. CONCLUSION: The delayed introduction of major allergenic foods is related to a higher risk of food allergy, which supports the updated guidelines for allergy prevention.

Basal Ti level in the human placenta and meconium and evidence of a materno-foetal transfer of food-grade TiO2 nanoparticles in an ex vivo placental perfusion model.

BACKGROUND: Titanium dioxide (TiO2) is broadly used in common consumer goods, including as a food additive (E171 in Europe) for colouring and opacifying properties. The E171 additive contains TiO2 nanoparticles (NPs), part of them being absorbed in the intestine and accumulated in several systemic organs. Exposure to TiO2-NPs in rodents during pregnancy resulted in alteration of placental functions and a materno-foetal transfer of NPs, both with toxic effects on the foetus. However, no human data are available for pregnant women exposed to food-grade TiO2-NPs and their potential transfer to the foetus. In this study, human placentae collected at term from normal pregnancies and meconium (the first stool of newborns) from unpaired mothers/children were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopy (STEM) coupled to energy-dispersive X-ray (EDX) spectroscopy for their titanium (Ti) contents and for analysis of TiO2 particle deposition, respectively. Using an ex vivo placenta perfusion model, we also assessed the transplacental passage of food-grade TiO2 particles. RESULTS: By ICP-MS analysis, we evidenced the presence of Ti in all placentae (basal level ranging from 0.01 to 0.48 mg/kg of tissue) and in 50% of the meconium samples (0.02-1.50 mg/kg), suggesting a materno-foetal passage of Ti. STEM-EDX observation of the placental tissues confirmed the presence of TiO2-NPs in addition to iron (Fe), tin (Sn), aluminium (Al) and silicon (Si) as mixed or isolated particle deposits. TiO2 particles, as well as Si, Al, Fe and zinc (Zn) particles were also recovered in the meconium. In placenta perfusion experiments, confocal imaging and SEM-EDX analysis of foetal exudate confirmed a low transfer of food-grade TiO2 particles to the foetal side, which was barely quantifiable by ICP-MS. Diameter measurements showed that 70 to 100% of the TiO2 particles recovered in the foetal exudate were nanosized. CONCLUSIONS: Altogether, these results show a materno-foetal transfer of TiO2 particles during pregnancy, with food-grade TiO2 as a potential source for foetal exposure to NPs. These data emphasize the need for risk assessment of chronic exposure to TiO2-NPs during pregnancy.

Intragastric administration of Lactobacillus casei BL23 induces regulatory FoxP3+RORγt+ T cells subset in mice.

Many studies have highlighted the immunomodulatory properties of the probiotic strain Lactobacillus casei BL23. Recently, we demonstrated the ability of this strain to modulate the Th2-oriented immune response in a mouse model of cow's milk allergy based on the induction of a Th17-biased immune response. The probiotic function of L. casei has been also linked to gut-microbiota modifications which could been potentially involved in the immune regulation; however, its precise mechanism of action remains poorly understood. In this regard, recent studies suggest that gut microbiota induces a specific subset of CD4+FoxP3+ Treg cells that also express RORγt+, the specific transcription factor of Th17 cells. This new type of regulatory T cells, called type 3 Treg, displays suppressive function during intestinal inflammation, participating in inflammation control. We thus explored the ability of L. casei BL23 to specifically induce type 3 Treg cells, both in vitro and in vivo. Our results showed that intragastric administration of L. casei BL23 to mice induces local and systemic FoxP3+ RORγt+ type 3 Treg cells that could then participate in the beneficial effects of L. casei BL23 in different intestinal-related disorders.

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.

Respiratory allergen from house dust mite is present in human milk and primes for allergic sensitization in a mouse model of asthma.

There is an urgent need to identify environmental risk and protective factors in early life for the prevention of allergy. Our study demonstrates the presence of respiratory allergen from house dust mite, Der p 1, in human breast milk. Der p 1 in milk is immunoreactive, present in similar amounts as dietary egg antigen, and can be found in breast milk from diverse regions of the world. In a mouse model of asthma, oral exposure to Der p through breast milk strongly promotes sensitization rather than protect the progeny as we reported with egg antigen. These data highlight that antigen administration to the neonate through the oral route may contribute to child allergic sensitization and have important implications for the design of studies assessing early oral antigen exposure for allergic disease prevention. The up-to-now unknown worldwide presence of respiratory allergen in maternal milk allows new interpretation and design of environmental control epidemiological studies for allergic disease prevention.

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.

Production of human antibodies by in vitro immunization using a fusion protein containing the transcriptional transactivator of HIV-1.

Antigen-specific activation of human B cells represents a key step for the production of monoclonal antibodies. Several approaches have been developed over the last thirty years in order to improve the process of lymphocyte activation in vitro. In the present study, we investigated whether the transcriptional transactivator (Tat) of human immunodeficiency virus, which possesses numerous biological activities, is able to trigger antibody secretion when incubated with human peripheral blood mononuclear cells. No such effect was observed when using Tat as a free protein. However, we found a significant IgM antibody production when Tat was previously fused to a double domain, called ZZ, derived from protein A of Staphylococcus aureus. The effect was also observed when the fusion protein, called ZZTat101, was incubated with purified B cells, indicating that the phenomenon does not require T-cell help. Antibody secretion was observed in the absence of cytokines that are usually used during in vitro immunization experiments, indicating that ZZTat101 provides the signals required for the initiation of the immune response. Antibody secretion was observed using a ZZTat mutant, containing only the Tat residues 22 to 57, called ZZTat22-57, indicating that this region is sufficient to initiate the immune response. In contrast, the effect was not found with a ZZTat22-57 mutant devoid of the seven Tat cysteines located between residues 22 and 37, demonstrating that these residues play a crucial role in the phenomenon. Our results pave the way to the development of a new in vitro immunization method based on antigens associated with ZZTat.

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.

Immunoglobulin-E-binding epitopes of wheat allergens in patients with food allergy to wheat and in mice experimentally sensitized to wheat proteins.

BACKGROUND: At present, B cell epitopes involved in food allergy to wheat are known only for a few allergens and a few categories of patients. OBJECTIVE: To characterize the epitopes of different wheat kernel allergens: α-, γ, ω2, and ω5-gliadin, a low-molecular-weight (LMW) glutenin subunit, and a lipid transfer protein (LTP1) recognized by allergic patients and by sensitized mice and provide further understanding of the role of structure in determining allergic response. METHODS: Sera were obtained from 39 patients suffering from food allergy to wheat. BALB/c mice were sensitized to gliadins or LTP1 by intraperitoneal immunizations. Continuous epitopes bound by IgE were delineated by the Pepscan technique. The response to reduced, alkylated LTP1 was compared with that of the native form to evaluate the importance of protein folding on IgE reactivity. RESULTS: Few continuous epitopes of LTP1 reacted with IgE from allergic patients and mice, but one of them was common to several patients and sensitized mice. The unfolded protein was not recognized by either patient or mouse IgE, emphasizing the major role of LTP1 folding and discontinuous epitopes in IgE-binding. In contrast, many continuous epitopes were detected by patient and mouse IgE especially for an ω5-gliadin, which is an unstructured protein, and to a lesser extent, for the other gliadins and a LMW-glutenin subunit. CONCLUSION AND CLINICAL RELEVANCE: The conformation of LTP1 appeared to have a strong impact on the type of IgE-binding epitopes elicited by this protein in both man and mouse. The responses in mice sensitized to gliadins or LTP1 were sufficiently comparable with the human response in terms of IgE-binding epitopes to provide support for the use of the mouse model in further investigations.

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.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel 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.

Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors.

BACKGROUND: Peanut is a most common and potent food allergen. Many peanut allergens have been characterized using, in particular, IgE-binding studies. OBJECTIVES: We optimized an in vitro functional assay to assess the capacity of peanut allergens to degranulate humanized rat basophilic leukaemia cells, RBL SX-38 cells, after sensitization by serum IgE from peanut-allergic patients. We thus compared the activity of the main peanut allergens, i.e. Ara h 1, Ara h 2, Ara h 3 and Ara h 6, purified from roasted peanut. METHODS: Sera of 12 peanut-allergic patients were collected and total and peanut-specific IgE were measured. They were used to sensitize RBL SX-38 cells and the degranulation was induced by incubation with ranging concentrations of a whole peanut protein extract or of purified peanut allergens. The mediator release was quantified by the determination of beta-hexosaminidase activity in the supernatant. The intensity of the degranulation was expressed as maximum release and as EC50, corresponding to the dose of allergen that induced 50% of the maximum release. RESULTS: For each serum, only 10 IU/mL of human IgE was necessary to sensitize the cells and obtain an optimal degranulation. With all the allergens, the release was positively correlated with the concentration of allergen-specific IgE in the serum used to sensitize the cells. The medians of EC50 obtained for Ara h 2 and Ara h 6 were 2.1 and 2.8 pm, respectively, while they were much higher for Ara h 3 and Ara h 1 (65 and 150 pm, respectively). CONCLUSION: The RBL SX-38 release assay proved to be sensitive, specific and reproducible. It allowed the comparison of the degranulation potential of different peanut allergens. For all the sera tested, Ara h 2 and Ara h 6 were more potent than Ara h 1 or Ara h 3.

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.

Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei.

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.

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.