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Introduction: Cow’s milk allergy (CMA) is one of the most common food allergies especially early in life. A mixture of nondigestible short-chain galacto-oligosaccharides, long-chain fructo-oligosaccharides, and pectin-derived acidic-oligosaccharides (GFA) may reduce allergy development and allergic symptoms in murine CMA. Recently, vitamin D (VitD) has been suggested to have beneficial effects in reducing allergy as well. Objective: In this study, the immune modulatory effect on allergy prevention using the combination of GFA and VitD was investigated. Methods: Female C3H/HeOuJ mice were fed a control or GFA-containing diet with depleted, standard (1,000 IU/kg), or supplemented (5,000 IU/kg) VitD content for 2 weeks before and during whey sensitization (n = 10–15). Mice were sensitized 5 times intragastrically with PBS as a control, whey as cow’s milk allergen, and/or cholera toxin as adjuvant on a weekly interval. One week after the last sensitization, mice were intradermally challenged in both ear pinnae and orally with whey, subsequently the acute allergic skin response and shock symptoms were measured. After 18 h, terminal blood samples, mesenteric lymph nodes, and spleens were collected. Whey-specific immunoglobulin (Ig) E and IgG1 levels were measured by means of ELISA. T cell subsets and dendritic cells (DCs) were studied using flow cytometry. Results: Additional VitD supplementation did not lower the allergic symptoms compared to the standard VitD diet. CMA mice fed the GFA diet supplemented with VitD (GFA VitD+) significantly decreased the acute allergic skin response of whey sensitized mice when compared to the CMA mice fed VitD (VitD+) group (p < 0.05). The effect of GFA was not improved by extra VitD supplementation even though the CMA mice fed the GFA VitD+ diet had a significantly increased percentage of CD103+ DCs compared to the VitD+ group (p < 0.05). The VitD-deprived mice showed a high percentage of severe shock and many reached the humane endpoint; therefore, these groups were not further analyzed. Conclusions: High-dose VitD supplementation in mice does not protect against CMA development in the presence or absence of GFA.

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective. Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice. Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined. Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3+CD4+ Th2 and CD4+RORγt+ Th17 cells in the lungs of the allergic mice. Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation.

BACKGROUND: Failure to induce oral tolerance may result in food allergy. Hydrolysed cow's milk-based infant formulas are recommended in subjects with a high risk of developing allergic disease. Presentation of T cell epitopes is a prerequisite to generate regulatory T cells that could contribute to oral tolerance. OBJECTIVE: To investigate whether a specific hydrolysed whey-based infant formula contains peptides that function as T cell epitopes to support the development of oral tolerance to whey. METHODS: First, a novel liquid chromatography-mass spectrometry (LC-MS) method was developed to characterize β-lactoglobulin-derived peptides present in a specific infant formula with a focus on region AA#13-48 of β-lactoglobulin, which has previously been described to contain T cell epitopes with tolerogenic potential. Second, the formula was subjected to the ProImmune ProPresent® antigen presentation assay and MHC class II binding algorithm to identify relevant HLA-DRB1-restricted peptides. Third, identified peptides were tested on human cow's milk protein-specific T cell lines to determine T cell recognition. RESULTS: Thirteen peptides of minimal 9AAs long that overlap with AA#13-48 of β-lactoglobulin were identified. Six of them were found across all batches analysed. It was further confirmed that these peptides were processed and presented by human dendritic cells. The identified HLA-DRB1-restricted peptides were correlated to AA#11-30 and AA#23-39 of β-lactoglobulin. Importantly, the proliferation assay showed that the synthetic peptides were recognized by cow's milk protein-specific T cell lines and induced T cell proliferation. CONCLUSION AND CLINICAL RELEVANCE: This study demonstrates that the tested hydrolysed infant formula contains functional HLA-DRB1-restricted T cell epitopes, which can potentially support the development of oral tolerance to whey.

Background Dietary nondigestible, short-chain galacto-, long-chain fructo-, and pectin-derived acidic oligosaccharides (GFAs) lower the effector response in cow-milk-allergic (CMA) mice; and forkhead box P3 (Foxp3)–positive regulatory T cells (Tregs) were shown to contribute to this. Objective The aim of this study was to assess the contribution of interleukin 10 (IL-10) and transforming growth factor β (TGF-β) to the protective effect of the GFA diet in CMA mice. Methods Female C3H/HeOuJ mice, 3–4 wk old, were orally sensitized with cholera toxin (Sham) or whey and cholera toxin (Whey) 1 time/wk for 5 consecutive weeks and challenged with whey 1 wk later. The mice were fed a control or 1% GFA (9:2:1) (Whey+GFA) diet starting 2 wk before the first sensitization. In a second experiment, the mice were also injected with αIL-10 receptor (αIL-10r), αTGF-β, or isotype control antibodies 24 h before each sensitization. The acute allergic skin response, anaphylaxis score, whey-specific IgE, mucosal mast cell protease 1 (mMCP-1), and Treg frequency in the mesenteric lymph nodes (MLNs) and intestinal Foxp3, Il10, and Tgfb mRNA expression were determined. Results In Whey+GFA mice, intestinal Il10, Tgfb, or Foxp3 mRNA expression was 2–10 times higher (P

Human milk oligosaccharides (HMOs) are absorbed into the blood (about 1% of the HMO intake) and subsequently excreted in urine, where they may protect the infant from pathogen infection. As dietary galacto-oligosaccharides (GOS) have partial structural similarities with HMOs, this study investigated the presence of GOS and oligosaccharides originating from milk replacer in blood serum, urine, and cecal and fecal samples of piglets, as a model for human infants. Using liquid chromatography-mass spectrometry and capillary electrophoresis with fluorescence detection, oligosaccharides originating from piglet diet including 3'-sialyllactose and specific GOS ranging from degree of polymerization 3 to 6 were detected in blood serum and in urine of piglets. In blood serum, GOS levels ranged from 16 to 23 μg/mL, representing about 0.1% of the GOS daily intake. In urine, approximately 0.85 g of GOS/g of creatinine was found. Cecum digesta and feces contained low amounts of oligosaccharides, suggesting an extensive GOS intestinal fermentation in piglets.