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Scope: During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β-lactoglobulin (BLG), in their interactions with cells crucially involved in allergy., Methods and Results: BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T-cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco-2 monolayer. Uptake of glycated BLG by bone marrow-derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor-mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4 + T-cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG-specific IgE sensitized basophil cells., Conclusions: This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy., (© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The current methodology to identify allergenic food proteins is effective in identifying those that are likely to cross-react with known allergens. However, most assays show false positive results for low/non-allergens. Therefore, an ex vivo/in vitro DC-T cell assay and an in vivo mouse model were used to distinguish known allergenic food proteins (Ara h 1, β-Lactoglobulin, Pan b 1, bovine serum albumin, whey protein isolate) from low/non allergenic food proteins (soy lipoxygenase, gelatin, beef tropomyosin, rubisco, Sola t 1). CD4+ T cells from protein/alum-immunized mice were incubated with corresponding protein-pulsed bone marrow-derived DC and analyzed for cytokine release. All known allergens induced Th2 responses in vitro, whereas soy lipoxygenase, gelatin or beef tropomyosin did not. Sola t 1 and rubisco induced a more generalized T cell response due to endotoxin contamination, indicating the endotoxin-sensitivity of the DC-T assay. To analyze responses in vivo, mice were orally sensitized on days 0 and 7. Known allergens induced IgE and mMCP-1 release upon oral challenge at day 16, whereas the low/non-allergens did not. Both the DC-T cell assay and the mouse model were able to distinguish 5 known allergens from 5 low/non-allergens and may be useful to identify novel allergenic food proteins., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Intestinal epithelial stress or damage may contribute to allergic sensitization against certain food antigens. Hence, the present study investigated whether impairment of intestinal barrier integrity by the mycotoxin deoxynivalenol (DON) contributes to the development of whey-induced food allergy in a murine model. C3H/HeOuJ mice, orally exposed to DON plus whey once a week for 5 consecutive weeks, showed whey-specific IgG1 and IgE in serum and an acute allergic skin response upon intradermal whey challenge, although early initiating mechanisms of sensitization in the intestine appeared to be different compared with the widely used mucosal adjuvant cholera toxin (CT). Notably, DON exposure modulated tight-junction mRNA and protein levels, and caused an early increase in IL-33, whereas CT exposure affected intestinal γδ T cells. On the other hand, both DON- and CT-sensitized mice induced a time-dependent increase in the soluble IL-33 receptor ST2 (IL-1R1) in serum, and enhanced local innate lymphoid cells type 2 cell numbers. Together, these results demonstrate that DON facilitates allergic sensitization to food proteins and that development of sensitization can be induced by different molecular mechanisms and local immune responses. Our data illustrate the possible contribution of food contaminants in allergic sensitization in humans.

Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models., (Copyright © 2016 Elsevier B.V. All rights reserved.)