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International audience; Despite a high level of sequence identity between cow's, goat's, and sheep's milk (CM, GM, and SM, respectively) proteins, some patients tolerant to CM are allergic to GM and SM. In most cases, this specificity is due to the presence of IgE antibodies that bind only to caprine and ovine caseins. The patients may then develop severe allergic reactions after ingestion of CM products contaminated with low amounts of GM or SM. We thus aimed to develop an assay able to detect traces of caprine/ovine β-caseins in different food matrices, irrespective of the presence of the bovine homolog. We produced monoclonal antibodies (mAbs) specific to caprine caseins in mice tolerized to the bovine whole casein then sensitized to the caprine whole casein. In order to develop a two-site immunometric assay, we selected mAbs that could discriminate the caprine β-casein from its bovine homolog. Characteristics and performances of two tests were determined with various dairy products. Results were analyzed in relation with the IgE-immunoreactivity of the food matrices, thanks to sera from CM, GM/SM allergic patients. Our two-site immunometric assays demonstrated a high sensitivity with a detection limit of 1.6–3.2 ng/mL of caprine and ovine β-caseins. The tests were able to detect contaminations of GM in CM at the ppm level. Heat-treatment, ripening and coagulation processes, usually applied to dairy products that exhibit a very high IgE-immunoreactivity, did not impair the test sensitivity. These quantitative assays could then be useful for the risk assessment of food products potentially contaminated with GM and SM in order to prevent adverse reactions in patients specifically allergic to these milks.

International audience; Background: Early nutrition may influence the development of food allergies later in life. In the absence of breastfeeding, hydrolysates from cow’s milk proteins (CMP) were indicated as a prevention strategy in at risk infants, but their proof of effectiveness in clinical and pre-clinical studies is still insufficient. Thanks to a validated mouse model, we then assessed specific and nonspecific preventive effects of administration of extensive hydrolysates from caseins (eHC) on the development of food allergy to CMP. The additional nonspecific effect of the probiotic Lactobacillus GG (LGG), commonly used in infant formula, was also assessed.Methods: Groups of young BALB/cByJ female mice were pretreated by repeated gavage either with PBS (control mice), or with PBS solution containing non-hydrolyzed milk protein isolate (MPI), eHC or eHC+LGG (eq. of 10 mg of protein/gavage). All mice were then experimentally sensitized to CMP by gavage with whole CM mixed with the Th2 mucosal adjuvant Cholera toxin. All mice were further chronically exposed to cow’s milk. A group of mice was kept naïve. Sensitization to both caseins and to the non-related whey protein β-lactoglobulin (BLG) was evaluated by measuring specific antibodies in plasma and specific ex vivo Th2/Th1/Th17 cytokine secretion. Elicitation of the allergic reaction was assessed by measuring mMCP1 in plasma obtained after oral food challenge (OFC) with CMP. Th/Treg cell frequencies in gut-associated lymphoid tissue and spleen were analyzed by flow cytometry at the end of the protocol. Robust statistical procedure combining non-supervised and supervised multivariate analyses and univariate analyses, was conducted to reveal any effect of the pretreatments.Results: PBS pretreated mice were efficiently sensitized and demonstrated elicitation of allergic reaction after OFC, whereas mice pretreated with MPI were durably protected from allergy to CMP. eHC+/-LGG pretreatments had no protective effect on sensitization to casein (specific) or BLG (non-specific), nor on CMP-induced allergic reactions. Surprisingly, eHC+LGG mice demonstrated significantly enhanced humoral and cellular immune responses after sensitization with CMP. Only some subtle changes were evidenced by flow cytometry.Conclusion: Neither specific nor nonspecific preventive effects of administration of casein-derived peptides on the development of CMP food allergy were evidenced in our experimental setup. Further studies should be conducted to delineate the mechanisms involved in the immunostimulatory potential of LGG and to clarify its significance in clinical use.

International audience; Background: The high incidence of food allergy in childhood points to the need of elucidating early life factors dictating allergy susceptibility. Here, we aim to address in a mouse model how the exposure to a major cow's milk allergen through breastmilk of mothers with different immune status influences food allergy outcome in offspring. Methods: BALB/cJ future dams were either kept naive, or sensitized through the oral route using cholera toxin ("orally sensitized") or through the i.p. route using alum ("i.p. sensitized"), or rendered fully tolerant (oral gavage without any adjuvant) to bovine beta-lactoglobulin (BLG). After mating with naive males and delivery, mothers were orally exposed or not to BLG during the whole lactation. Then, eight groups of lactating mothers were considered: naive, i.p. sensitized, orally sensitized, or tolerant, each exposed or not during lactation. In order to specifically address breastmilk effects on their allergy susceptibility, pups from naive-synchronized mothers were cross-fostered by the different groups of treated dams and lactating mothers at delivery. In some experiments, mothers kept their own pups to address a possiblein uteroeffect. BLG antigen, BLG-specific antibodies, and BLG-immune complexes were measured in breastmilk from the different lactating mother groups. Allergic sensitization was monitored in 5-weeks old female offspring (n= 7-8/group of lactating mothers) by determining BLG-specific antibodies in plasma and splenocytes cytokine secretion after i.p. injections of BLG/alum. Allergic reaction to oral BLG challenge was evaluated by measuring mMCP1 in plasma.Results: Offspring was protected from one allergic i.p. sensitization when nursed by i.p. sensitized mothers, independently of BLG exposure during lactation. Orally sensitized dams conferred protection in offspring solely when exposed to BLG during lactation, while naive mothers did not provide any protection upon BLG exposure. The levels of protection correlated with the levels of BLG-specific antibodies and BLG-immune complex in breastmilk. There was a trend for decreased sensitization in offspring breastfed by tolerant and exposed mothers, which was not associated with transfer of specific antibodies through breastmilk. Protection provided by nursing by treated/exposed mothers was not persistent after a boost i.p. injection of the progeny and then did not protect them from an allergic reaction induced at this time point. No additionalin uteroeffects were evidenced. Conclusion: Our study demonstrates the strong potential of breastmilk to modulate immune response to a major cow's milk allergen in the progeny. It highlights the importance of maternal immune status and of her consumption of the allergen during lactation in dictating the outcomes in offspring. This opens perspectives where modulating maternal immune status might increase the chance of cow's milk allergy prevention in breastfed children.

Additional file 1. Cytokine secretion after mitogen reactivation of PBMC

Resumen del trabajo presentado a la 3rd International ImpARAS Conference, celebrada en Helsingør (Dinamarca) del 10 al 12 de octubre de 2017., [Background] Protein-induced enterocolitis syndrome (FPIES) is a non-IgE mediated food allergy of increasing prevalence for which immune mechanisms are not elucidated and no biomarkers are available., [Objectives] We aimed 1) to further characterize immune response and 2) to carry complementary and innovative approaches using metabolomics to go deeper in pathogenesis and to identify specific biomarkers of cow’s milk induced-FPIES (CM-FPIES), [Methods] Children with active CM-FPIES (n=9) or active IgE-mediated CM allergy (IgE-CMA, n=6; also avoiding CM consumption) were recruited in Hospital Necker day care Unit when visiting to perform an oral food challenge (OFC). Blood samples were collected before the OFC. Plasma from age-matched patients tolerant to CM but presenting an IgE-mediated peanut allergy (n=6) were used as controls. The total and CM-allergens specific IgE, IgG1-4, IgA, IgM and IgD were assessed in all plasma, as the same as IgE and IgG4 specific to digestats obtained after in vitro gastric and gastro-duodenal digestion of CM. Specific ex-vivo stimulation of PBMC from CM-FPIES and IgE-CMA children were performed and cytokine secretion and cellular proliferation induced analyzed. Additionally, T cells and innate lymphoid cells (ILC) were analyzed in intestinal biopsies obtained from children with active (n=2) vs non active (n=2) FPIES. Finally, individual metabolic profiles were obtained using two complementary LC-MS methods analysis on plasma from CM-FPIES patients and compared to that obtained from patients suffering from active or resolved IgE-CMA (negative OFC, n=3)., [Results] Systemic antigen-specific T-cells and humoral responses were evidenced in IgE-CMA patients whereas they were weak or even absent in CM-FPIES patients, an absence of response that cannot then be restricted to a lower exposure to cow’s milk. Preliminary data evidenced activated Th1, Th2 and Th17 cells and activated innate lymphoid cells showing a mixed ILC1/2 phenotype within the intestinal mucosa of active FPIES. Moreover, discriminant statistical analysis of metabolic profiles clearly separated CM-FPIES versus IgE-CMA patients. Further univariate analysis allowed the identification of discriminant metabolites, demonstrating notably dysregulation of fatty acids metabolism in CM-FPIES children., [Conclusions] Children with CM-FPIES have a weak or absent systemic specific immune responses. Our preliminary data suggest that new studies analysing innate and adaptive cells in the mucosa may help to delineate the pathophysiology of FPIES. Metabolomics could further help to identify biomarkers for FPIES, by highlighting altered metabolic pathways.