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In many parts of the world, goat milk has been part of the human diet for millennia. Allergy to goat's milk, not associated with allergy to cow's milk, is a rare disorder, although some cases have been described. Goat milk proteins have substantial homology with cow's milk proteins and even show cross-reactivity; therefore, they are not advised as an alternative to cow's milk for infants with IgE-mediated cow's milk allergies. However, there are indications that, due to the composition of the goat milk proteins, goat milk proteins show lower allergenicity than cow's milk due to a lower αS1-casein content. For this reason, goat milk might be a better choice over cow's milk as a first source of protein when breastfeeding is not possible or after the breastfeeding period. Additionally, some studies show that goat milk could play a role in specific types of non-IgE-mediated cow milk allergy or even in the prevention of sensitization to cow's milk proteins. This review discusses a possible role of goat milk in non-IgE mediated allergy and the prevention or oral tolerance induction of milk allergy.

Cow's milk allergy is a common food allergy among infants. Improved hygiene conditions and loss of microbial diversity are associated with increased risk of allergy development. The intestinal immune system is essential for oral tolerance induction. In this respect, bacterial CpG DNA is known to drive Th1 and regulatory T-cell (Treg) development via Toll-Like-Receptor 9 (TLR-9) signaling, skewing away from the allergic Th2 phenotype. We aimed to induce allergen specific tolerance via oral delivery of poly (lactic-co-glycolic acid) nanoparticles (NP) co-encapsulated with a selected β-lactoglobulin derived peptide (BLG-Pep) and TLR-9 ligand CpG oligodeoxynucleotide (CpG). In vivo , 3-4-week-old female C3H/HeOuJ mice housed in individually ventilated cages received 6-consecutive-daily gavages of either PBS, whey, BLG-Pep/NP, CpG/NP, a mixture of BLG-Pep/NP plus CpG/NP or co-encapsulated BLG-Pep+CpG/NP, before 5-weekly oral sensitizations with whey plus cholera toxin (CT) or only CT (sham) and were challenged with whey 5 days after the last sensitization. The co-encapsulated BLG-Pep+CpG/NP pretreatment, but not BLG-Pep/NP, CpG/NP or the mixture of BLG-Pep/NP plus CpG/NP, prevented the whey-induced allergic skin reactivity and prevented rise in serum BLG-specific IgE compared to whey-sensitized mice. Importantly, co-encapsulated BLG-Pep+CpG/NP pretreatment reduced dendritic cell (DC) activation and lowered the frequencies of PD-L1+ DC in the mesenteric lymph nodes compared to whey-sensitized mice. By contrast, co-encapsulated BLG-Pep+CpG/NP pretreatment increased the frequency of splenic PD-L1+ DC compared to the BLG-Pep/NP plus CpG/NP recipients, in association with lower Th2 development and increased Treg/Th2 and Th1/Th2 ratios in the spleen. Oral administration of PLGA NP co-encapsulated with BLG-Pep and CpG prevented rise in serum BLG-specific IgE and symptom development while lowering splenic Th2 cell frequency in these mice which were kept under strict hygienic conditions., Competing Interests: JG is employed by Nutricia Research B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Thijssen, Hennink, Garssen, van Nostrum and Willemsen.)

Background: Extensively hydrolyzed formulas are recommended for the dietary management of infants with cow's milk allergy (CMA)., Objectives: Hypoallergenicity, growth, and gastrointestinal (GI) tolerability of a new extensively hydrolyzed whey-protein formula (eHWF) in CMA children were assessed., Methods: In this prospective, randomized, international, multi-center study (Trial NL3889), 34 children with confirmed CMA (74% IgE-mediated) underwent a double-blind, placebo-controlled food challenge (DBPCFC) with an eHWF developed with non-porcine enzymes, supplemented with prebiotic short-chain galacto- and long-chain fructo-oligosaccharides (0.8 g/L, ratio 9:1), arachidonic acid (0.35/100 g), and docosahexaenoic acid (0.35/100 g). If tolerant to the eHWF, children participated in a 7-day open food challenge with this eHWF. Anthropometrics and GI tolerability were assessed in an optional 16-weeks follow-up., Results: Of the 34 children who started the DBPCFC with the eHWF, 25 subjects (19 boys, mean age: 61 weeks, 18 with IgE-mediated CMA) completed the DBPCFC and 7-day open challenge without major protocol deviations and tested negative at both challenges. One child experienced a late moderate eczematous allergic reaction in the optional follow-up period, indicating the need for close monitoring of subjects starting new formula. Weight and length gain followed the World Health Organization growth curves. Changes in frequency and consistency of stools upon test formula intake were transient., Conclusions: The newly developed eHWF is a suitable option in CMA treatment as all subjects tolerated the product. This result is in line with the international criteria for hypoallergenicity (American Academy of Pediatrics) that state that more than 90% of CMA children must tolerate the formula. Use of the formula is also associated with normal growth curves and GI tolerability., Trial Registration: Trial NL3889, https://www.trialregister.nl/trial/3889., (© 2022 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Tolerance development is an important clinical outcome for infants with cow's milk allergy., Objective: This multicenter, prospective, randomized, double-blind, controlled clinical study (NTR3725) evaluated tolerance development to cow's milk (CM) and safety of an amino acid-based formula (AAF) including synbiotics (AAF-S) comprising prebiotic oligosaccharides (oligofructose, inulin) and probiotic Bifidobacterium breve M-16V in infants with confirmed IgE-mediated CM allergy., Methods: Subjects aged ≤13 months with IgE-mediated CM allergy were randomized to receive AAF-S (n = 80) or AAF (n = 89) for 12 months. Stratification was based on CM skin prick test wheal size and study site. After 12 and 24 months, CM tolerance was evaluated by double-blind, placebo-controlled food challenge. A logistic regression model used the all-subjects randomized data set., Results: At baseline, mean ± SD age was 9.36 ± 2.53 months. At 12 and 24 months, respectively, 49% and 62% of subjects were CM tolerant (AAF-S 45% and 64%; AAF 52% and 59%), and not differ significantly between groups. During the 12-month intervention, the number of subjects reporting at least 1 adverse event did not significantly differ between groups; however, fewer subjects required hospitalization due to serious adverse events categorized as infections in the AAF-S versus AAF group (9% vs 20%; P = .036)., Conclusions: After 12 and 24 months, CM tolerance was not different between groups and was in line with natural outgrowth. Results suggest that during the intervention, fewer subjects receiving AAF-S required hospitalization due to infections., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In the western world the prevalence of atopic diseases such as food allergies is increasing highly significantly. One of the earliest and most prevalent food allergies occurring in the first year of life is cow's milk allergy. No treatment is available and only avoidance of the cow's milk allergens prevents the occurrence of an allergic reaction. Since cow's milk allergic children have an increased risk of developing other allergies later in life, investigating nutritional strategies to prevent the development of cow's milk allergy by developing oral tolerance is of high interest. Nutritional components such as prebiotics, probiotics, synbiotics and long-chain polyunsaturated fatty acids possess potential to support the maturation of the immune system early in life that might prevent the development of cow's milk allergy. The available research, so far, shows promising results particularly on the development of eczema. However, the preventive effects of the nutritional interventions on the development of food allergy are inconclusive. Future research may benefit from the combination of various dietary components. To clarify the preventive effects of the nutritional components in food allergy more randomized clinical trials are needed., (© 2021. Springer Nature Switzerland AG.)

Background: Two to four percentage of infants are affected by cow's milk allergy (CMA), which persists in 20% of cases. Intervention approaches using early oral exposure to cow's milk protein or hydrolysed cow's milk formula are being studied for CMA prevention. Yet, concerns regarding safety and/or efficacy remain to be tackled in particular for high-risk non-exclusively breastfed infants. Therefore, safe and effective strategies to improve early life oral tolerance induction may be considered., Objective: We aim to investigate the efficacy of CMA prevention using oral pre-exposure of two selected 18-AA β-lactoglobulin-derived peptides loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in a whey-protein induced CMA murine model., Methods: The peptides were loaded in PLGA NPs via a double emulsion solvent evaporation technique. In vivo, 3-week-old female C3H/HeOuJ mice received 6 daily gavages with PBS, whey, Peptide-mix, a high- or low-dose Peptide-NPs or empty-NP plus Peptide-mix, prior to 5 weekly oral sensitizations with cholera toxin plus whey or PBS (sham). One week after the last sensitization, the challenge induced acute allergic skin response, anaphylactic shock score, allergen-specific serum immunoglobulins and ex vivo whey-stimulated cytokine release by splenocytes was measured., Results: Mice pre-treated with high-dose Peptide-NPs but not low-dose or empty-NP plus Peptide-mix, were protected from anaphylaxis and showed a significantly lower acute allergic skin response upon intradermal whey challenge compared to whey-sensitized mice. Compared with the Peptide-mix or empty-NP plus Peptide-mix pre-treatment, the high-dose Peptide-NPs-pre-treatment inhibited ex vivo whey-stimulated pro-inflammatory cytokine TNF-α release by splenocytes., Conclusion & Clinical Relevance: Oral pre-exposure of mice to two β-lactoglobulin-derived peptides loaded PLGA NPs induced a dose-related partial prevention of CMA symptoms upon challenge to whole whey protein and silenced whey-specific systemic immune response. These findings encourage further development of the concept of peptide-loaded PLGA NPs for CMA prevention towards clinical application., (© 2021 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Cow's milk allergy is a common food allergy in infants, and is associated with an increased risk of developing other allergic diseases. Dietary selenium (Se), one of the essential micronutrients for humans and animals, is an important bioelement which can influence both innate and adaptive immune responses. However, the effects of Se on food allergy are still largely unknown. In the current study it was investigated whether dietary Se supplementation can inhibit whey-induced food allergy in an animal research model. Three-week-old female C3H/HeOuJ mice were intragastrically sensitized with whey protein and cholera toxin and randomly assigned to receive a control, low, medium or high Se diet. Acute allergic symptoms, allergen specific immunoglobulin (Ig) E levels and mast cell degranulation were determined upon whey challenge. Body temperature was significantly higher in mice that received the medium Se diet 60 min after the oral challenge with whey compared to the positive control group, which is indicative of impaired anaphylaxis. This was accompanied by reductions in antigen-specific immunoglobulins and reduced levels of mouse mast cell protease-1 (mMCP-1). This study demonstrates that oral Se supplementation may modulate allergic responses to whey by decreasing specific antibody responses and mMCP-1 release.

Background: beta-lactoglobulin (BLG) is one of the major cow's milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity., Primary Objective: to analyze and identify lactosylation sites by capillary electrophoresis mass spectrometry (CE-MS)., Secondary Objective: to assess the effect of lactosylated BLG on antigenicity and degranulation of mast cells., Methods: BLG was lactosylated at pH 7, a water activity (aw) of 0.43, and a temperature of 65 °C using a molar ratio BLG:lactose of 1:1 by incubating for 0, 3, 8, 16 or 24 h. For the determination of the effect on antibody-binding capacity of lactosylated BLG, an ELISA was performed. For the assessment of degranulation of the cell-line RBL-hεIa-2B12 transfected with the human α-chain, Fcε receptor type 1 (FcεRI) was used., Results: BLG showed saturated lactosylation between 8 and 16 incubation hours in our experimental setup. Initial stage lactosylation sites L1 (N-terminus)-K47, K60, K75, K77, K91, K138 and K141-have been identified using CE-MS. Lactosylated BLG showed a significant reduction of both the IgG binding ( p = 0.0001) as well as degranulation of anti-BLG IgE-sensitized RBL-hεIa-2B12 cells ( p < 0.0001)., Conclusions and Clinical Relevance: this study shows that lactosylation of BLG decreases both the antigenicity and degranulation of mast cells and can therefore be a promising approach for reducing allergenicity of cow's milk allergens provided that the process is well-controlled.

Immunoglobulin E (IgE)-mediated allergy against cow's milk protein fractions such as whey is one of the most common food-related allergic disorders of early childhood. Histone acetylation is an important epigenetic mechanism, shown to be involved in the pathogenesis of allergies. However, its role in food allergy remains unknown. IgE-mediated cow's milk allergy was successfully induced in a mouse model, as demonstrated by acute allergic symptoms, whey-specific IgE in serum, and the activation of mast cells upon a challenge with whey protein. The elicited allergic response coincided with reduced percentages of regulatory T (Treg) and T helper 17 (Th17) cells, matching decreased levels of H3 and/or H4 histone acetylation at pivotal Treg and Th17 loci, an epigenetic status favoring lower gene expression. In addition, histone acetylation levels at the crucial T helper 1 (Th1) loci were decreased, most probably preceding the expected reduction in Th1 cells after inducing an allergic response. No changes were observed for T helper 2 cells. However, increased histone acetylation levels, promoting gene expression, were observed at the signal transducer and activator of transcription 6 ( Stat6 ) gene, a proallergic B cell locus, which was in line with the presence of whey-specific IgE. In conclusion, the observed histone acetylation changes are pathobiologically in line with the successful induction of cow's milk allergy, to which they might have also contributed mechanistically., Competing Interests: L.M., D.H.V.-G., T.M.D.W., L.M.J.K., J.G., B.C.A.M.v.E., and M.M.T. are/were (partly) employed at Danone Nutricia Research. D.H.V.-G reports personal fees from Merck Sharp & Dohme outside of the submitted work. All other authors report no potential conflict of interest.

Introduction: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas., Aim: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers., Methods: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study., Results: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers., Conclusions: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The allergy-protective capacity of raw cow's milk was demonstrated to be abolished after heat treatment. The heat-sensitive whey protein fraction of raw milk is often implied to be the source of this allergy-protective effect, but a direct link between these proteins and the protection against allergic diseases is missing. This study therefore aimed at investigating the mechanistic relation between heat damage to whey proteins and allergy development. Raw cow's milk was heated for 30 min at 50, 60, 65, 70, 75, or 80 °C and the native whey protein profile of these differentially heated milk samples was determined using LC-MS/MS-based proteomics. Changes in the native protein profile were subsequently related to the capacity of these milk samples to prevent the development of ovalbumin-induced food allergy in a murine animal model. A substantial loss of native whey proteins, as well as extensive protein aggregation, was observed from 75 °C. However, whey proteins with immune-related functionalities already started to denature from 65 °C, which coincided with the temperature at which a loss of allergy protection was observed in the murine model. Complement C7, monocyte differentiation antigen CD14, and polymeric immunoglobulin receptor concentrations decreased significantly at this temperature, although several other immunologically active whey proteins also showed a decrease around 65 °C. The current study demonstrates that immunologically active whey proteins that denature around 65 °C are of importance for the allergy-protective capacity of raw cow's milk and thereby provides key knowledge for the development of microbiologically safe alternatives to raw cow's milk.

Background: Amino acid-based formulas (AAFs) are used for the dietary management of cow's milk allergy (CMA). Whether AAFs have the potential to prevent the development and/or symptoms of CMA is not known., Objective: The present study evaluated the preventive effects of an amino acid (AA)-based diet on allergic sensitization and symptoms of CMA in mice and aimed to provide insight into the underlying mechanism., Methods: C3H/HeOuJ mice were sensitized with whey protein or with phosphate-buffered saline as sham-sensitized control. Starting 2 weeks before sensitization, mice were fed with either a protein-based diet or an AA-based diet with an AA composition based on that of the AAF Neocate, a commercially available AAF prescribed for the dietary management of CMA. Upon challenge, allergic symptoms, mast cell degranulation, whey-specific immunoglobulin levels, and FoxP3 + cell counts in jejunum sections were assessed., Results: Compared to mice fed with the protein-based diet, AA-fed mice had significantly lower acute allergic skin responses. Moreover, the AA-based diet prevented the whey-induced symptoms of anaphylaxis and drop in body temperature. Whereas the AA-based diet had no effect on the levels of serum IgE and mucosal mast cell protease-1 (mMCP-1), AA-fed mice had significantly lower serum IgG2a levels and tended to have lower IgG1 levels (P = .076). In addition, the AA-based diet prevented the whey-induced decrease in FoxP3 + cells. In sham-sensitized mice, no differences between the two diets were observed in any of the tested parameters., Conclusion: This study demonstrates that an AA-based diet can at least partially prevent allergic symptoms of CMA in mice. Differences in FoxP3 + cell counts and serum levels of IgG2a and IgG1 may suggest enhanced anti-inflammatory and tolerizing capacities in AA-fed mice. This, combined with the absence of effects in sham-sensitized mice indicates that AAFs for the prevention of food allergies may be an interesting concept that warrants further research., (© 2020 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

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., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Background: Several studies demonstrated the adverse effect of milk processing on the allergy-protective capacity of raw cow's milk. Whether milk processing also affects the allergenicity of raw milk is hardly investigated., Objective: To assess the allergenicity of raw (unprocessed) and processed cow's milk in a murine model for food allergy as well as in cow's milk allergic children., Methods: C3H/HeOuJ mice were either sensitized to whole milk (raw cow's milk, heated raw cow's milk or shop milk [store-bought milk]) and challenged with cow's milk protein or they were sensitized and challenged to whey proteins (native or heated). Acute allergic symptoms, mast cell degranulation, allergen-specific IgE levels and cytokine concentrations were determined upon challenge. Cow's milk allergic children were tested in an oral provocation pilot with organic raw and conventional shop milk., Results: Mice sensitized to raw milk showed fewer acute allergic symptoms upon intradermal challenge than mice sensitized to processed milk. The acute allergic skin response was low (103 ± 8.5 µm vs 195 ± 17.7 µm for heated raw milk, P < 0.0001 and vs 149 ± 13.6 µm for shop milk, P = 0.0316), and there were no anaphylactic shock symptoms and no anaphylactic shock-induced drop in body temperature. Moreover, allergen-specific IgE levels and Th2 cytokines were significantly lower in raw milk sensitized mice. Interestingly, the reduced sensitizing capacity was preserved in the isolated native whey protein fraction of raw milk. Besides, native whey protein challenge diminished allergic symptoms in mice sensitized to heated whey proteins. In an oral provocation pilot, cow's milk allergic children tolerated raw milk up to 50 mL, whereas they only tolerated 8.6 ± 5.3 mL shop milk (P = 0.0078)., Conclusion and Clinical Relevance: This study demonstrates that raw (unprocessed) cow's milk and native whey proteins have a lower allergenicity than their processed counterparts. The preclinical evidence in combination with the human proof-of-concept provocation pilot provides evidence that milk processing negatively influences the allergenicity of milk., (© 2019 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

The conditionally essential amino acid glycine functions as inhibitory neurotransmitter in the mammalian central nervous system. Moreover, it has been shown to act as an anti-inflammatory compound in animal models of ischemic perfusion, post-operative inflammation, periodontal disease, arthritis and obesity. Glycine acts by binding to a glycine-gated chloride channel, which has been demonstrated on neurons and immune cells, including macrophages, polymorphonuclear neutrophils and lymphocytes. The present study aims to evaluate the effect of glycine on allergy development in a cow's milk allergy model. To this end, C3H/HeOuJ female mice were supplemented with glycine by oral gavage (50 or 100 mg/mouse) 4 hours prior to sensitization with cow's milk whey protein, using cholera toxin as adjuvant. Acute allergic skin responses and anaphylaxis were assessed after intradermal allergen challenge in the ears. Mouse mast cell protease-1 (mMCP-1) and whey specific IgE levels were detected in blood collected 30 minutes after an oral allergen challenge. Jejunum was dissected and evaluated for the presence of mMCP-1-positive cells by immunohistochemistry. Intake of glycine significantly inhibited allergy development in a concentration dependent manner as indicated by a reduction in; acute allergic skin response, anaphylaxis, serum mMCP-1 and serum levels of whey specific IgE. In addition, in-vitro experiments using rat basophilic leukemia cells (RBL), showed that free glycine inhibited cytokine release but not cellular degranulation. These findings support the hypothesis that the onset of cow's milk allergy is prevented by the oral intake of the amino acid glycine. An adequate intake of glycine might be important in the improvement of tolerance against whey allergy or protection against (whey-induced) allergy development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 < 0.05) and the MLN Treg frequency was 25% higher compared with Whey mice (P < 0.05). The acute allergic skin response was 50% lower in Whey+GFA mice compared with Whey mice (P < 0.01), and IL-10 receptor (IL-10r) or TGF-β neutralizing antibodies prevented this protective effect (P < 0.001). The Whey mice had higher serum mMCP-1 concentrations and whey-immunoglobulin E (-IgE) levels than Sham mice (P < 0.01), whereas these were not higher in Whey+GFA mice, and neutralizing antibodies partially interfered with these responses., Conclusions: Dietary GFAs enhance the Treg frequency in the MLNs and mucosal IL-10 and TGF-β transcription while suppressing the allergic effector response. Neutralizing antibodies showed that the allergy-protective effect of the GFA diet was mediated by IL-10 and TGF-β in CMA mice.

BackgroundPrebiotics and probiotics (synbiotics) can modify gut microbiota and have potential in allergy management when combined with amino-acid-based formula (AAF) for infants with cow's milk allergy (CMA).MethodsThis multicenter, double-blind, randomized controlled trial investigated the effects of an AAF-including synbiotic blend on percentages of bifidobacteria and Eubacterium rectale/Clostridium coccoides group (ER/CC) in feces from infants with suspected non-IgE-mediated CMA. Feces from age-matched healthy breastfed infants were used as reference (healthy breastfed reference (HBR)) for primary outcomes. The CMA subjects were randomized and received test or control formula for 8 weeks. Test formula was a hypoallergenic, nutritionally complete AAF including a prebiotic blend of fructo-oligosaccharides and the probiotic strain Bifidobacterium breve M-16V. Control formula was AAF without synbiotics.ResultsA total of 35 (test) and 36 (control) subjects were randomized; HBR included 51 infants. At week 8, the median percentage of bifidobacteria was higher in the test group than in the control group (35.4% vs. 9.7%, respectively; P<0.001), whereas ER/CC was lower (9.5% vs. 24.2%, respectively; P<0.001). HBR levels of bifidobacteria and ER/CC were 55% and 6.5%, respectively.ConclusionAAF including specific synbiotics, which results in levels of bifidobacteria and ER/CC approximating levels in the HBR group, improves the fecal microbiota of infants with suspected non-IgE-mediated CMA.

Beta-lactoglobulin (BLG)-derived peptides may facilitate oral tolerance to whey and prevent cow's milk allergy (CMA). Loading of BLG-peptides in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Pep-NP) may improve this. Here we studied the uptake of NP and the capacity of NP and Pep-NP to activate bone marrow dendritic cells (BMDC). Furthermore, CMA prevention was evaluated by orally exposing three-week-old female C3H/HeOuJ mice to Pep-NP, NP or free peptides (PepMix) for 6 days before oral sensitization with whole whey protein and effects on the spleen and small intestine lamina propria (SI-LP) were studied. In BMDC, NP and Pep-NP enhanced CD40 expression and IL-6 and TNF-α secretion, while tended to decrease CD80 expression and prevented PepMix-induced IL-12 secretion. In vivo, oral exposure to Pep-NP, but not NP or PepMix, prior to whey sensitization tended to partially prevent the acute allergic skin response to whole whey protein. Splenocytes of NP-pre-exposed mice secreted increased levels of whey-specific IL-6, but this was silenced in Pep-NP-pre-exposed mice which also showed reduced TNF-α and IFN-γ secretion. In the SI-LP, Pep-NP pre-exposure reduced the CD4 + T cell frequency in CMA mice compared to PBS pre-exposure. In addition, while NP increased whey-specific IL-6 secretion in the SI-LP, Pep-NP did not and maintained regulatory TGF-β secretion. This study presents a proof-of-concept that PLGA nanoparticles facilitate the capacity of BLG peptides to suppress the allergic response to whole whey protein. Hence, PLGA nanoparticles may be further developed as an adjunct strategy for BLG-peptide-based oral tolerance induction and CMA prevention., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Scope: Partially hydrolyzed cow's milk proteins are used to prevent cow's milk allergy in children. Here we studied the immunomodulatory mechanisms of partial cow's milk hydrolysates in vivo., Methods and Results: Mice were sensitized with whey or partially hydrolyzed whey using cholera toxin. Whey-specific IgE levels were measured to determine sensitization and immune cell populations from spleen, mesenteric lymph nodes and Peyer's patches after oral whey administration were measured by flowcytometry. Whey-specific IgE and IgG1 levels in partial whey hydrolysate sensitized animals were enhanced, but challenge did not induce clinical symptoms. This immunomodulatory effect of partial whey hydrolysate was associated with increased regulatory B and T cells in the spleen, together with a prevention of IgM-IgA class switching in the mesenteric lymph nodes and an increased Th1 and activated Th17 in the Peyer's patches., Conclusion: Partial hydrolysate sensitization did not induce whey-induced clinical symptoms, even though sensitization was established. Increased regulatory cell populations in the systemic immune system and a prevention of increased total Th1 and activated Th17 in the intestinal immune organs could contribute to the suppression of allergic symptoms. This knowledge is important for a better understanding of the beneficial effects of hydrolysates., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Autism spectrum disorder (ASD) is multifactorial, with both genetic as well as environmental factors working in concert to develop the autistic phenotype. Immunological disturbances in autistic individuals have been reported and a role for food allergy has been suggested in ASD. Single gene mutations in mammalian target of rapamycin (mTOR) signaling pathway are associated with the development of ASD and enhanced mTOR signaling plays a central role in directing immune responses towards allergy as well. Therefore, the mTOR pathway may be a pivotal link between the immune disturbances and behavioral deficits observed in ASD. In this study it was investigated whether the mTOR pathway plays a role in food allergy-induced behavioral and immunological deficits. Mice were orally sensitized and challenged with whey protein. Meanwhile, cow's milk allergic (CMA) mice received daily treatment of rapamycin. The validity of the CMA model was confirmed by showing increased allergic immune responses. CMA mice showed reduced social interaction and increased repetitive self-grooming behavior. Enhanced mTORC1 activity was found in the brain and ileum of CMA mice. Inhibition of mTORC1 activity by rapamycin improved the behavioral and immunological deficits of CMA mice. This effect was associated with increase of Treg associated transcription factors in the ileum of CMA mice. These findings indicate that mTOR activation may be central to both the intestinal, immunological, and psychiatric ASD-like symptoms seen in CMA mice. It remains to be investigated whether mTOR can be seen as a therapeutic target in cow's milk allergic children suffering from ASD-like symptoms., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Increased intake of vegetable oils rich in n-6 PUFA, including soyabean oil, has been associated with an increase in allergic disease. The present study aimed to determine the effect of an increasing dose of dietary vegetable oil on allergic outcomes in mice. To study this, mice received a 7 v. 10 % soyabean oil diet before and during oral sensitisation with whey or whey hyperimmune serum transfer. Another group of mice received partial whey hydrolysate (pWH) while being fed the diets before oral sensitisation. The acute allergic skin response, serum Ig level, mouse mast cell protease-1 (mMCP-1) concentration and/or splenic T-cell percentages were determined upon whey challenge. When the diets were provided before and during oral sensitisation, the acute allergic skin response was increased in mice fed the 10 % soyabean oil diet compared with the 7 % soyabean oil diet. Whey IgE and IgG1 levels remained unaltered, whereas mMCP-1 levels increased in mice fed the 10 % soyabean oil diet. Furthermore, allergic symptoms were increased in naive mice fed the 10 % soyabean oil diet and sensitised with whey hyperimmune serum. In addition to enhancing the mast cell response, the 10 % soyabean oil diet increased the percentage of activated Th1 and Th2 cells as well as increased the ratios of Th2:regulatory T cells and Th2:Th1 when compared with the 7 % soyabean oil diet. Oral tolerance induction by pWH was abrogated in mice fed the 10 % soyabean oil diet compared with those fed the 7 % soyabean oil diet during pretreatment with pWH. In conclusion, increased intake of soyabean oil rich in n-6 PUFA suppresses tolerance induction by pWH and enhances the severity of the allergic effector response in whey-allergic mice. Dietary vegetable oils rich in n-6 PUFA may enhance the susceptibility to develop or sustain food allergy.

Background: Cow's milk allergy is a common food allergy in childhood and no effective preventive or curative treatment is available. This study aimed at comparing single short-chain galacto- (scGOS), long-chain fructo- (lcFOS) or pectin-derived acidic oligosaccharides (pAOS) and/or mixtures of scGOS/lcFOS (GF) or scGOS/lcFOS/pAOS (GFA) to prevent or treat food allergy., Methods: In the preventive protocol, C3H/HeOuJ mice were fed diets containing single oligosaccharides or mixtures GF or GFA throughout the study protocol. In the treatment protocol, GF or GFA was provided for 4 wk starting after the last sensitization. The allergic skin response and anaphylaxis scores were determined, after oral challenge whey-specific immunoglobulins were measured, and qPCR for T-cell markers and Foxp3 counts using immunohistochemistry were performed on the small intestine and colon., Results: Only in the preventive setting, the GF or GFA mixture, but not the single oligosaccharides, reduced the allergic skin response and whey-IgG(1) levels in whey-sensitized mice, compared to the control diet. Both GF and GFA increased the number of Foxp3+ cells in the proximal small intestine of whey - compared to sham-sensitized mice. Expression of Th2 and Th17 mRNA markers increased in the middle part of the small intestine of whey-sensitized mice, which was prevented by GF. By contrast, GFA enhanced Tbet (Th1), IL-10 and TGF-β mRNA expression compared to GF which was maintained in the distal small intestine and/or colon., Conclusions: Dietary supplementation with scGOS/lcFOS or scGOS/lcFOS/pAOS during sensitization, both effectively reduce allergic symptoms but differentially affect mucosal immune activation in whey-sensitized mice., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Supplementation with long-chain n-3 polyunsaturated fatty acids (LCPUFAs) has been found to reduce the development of allergic disease., Objective: The aim of this study was to compare the effectiveness of fish oil diets rich in eicosapentaenoic acid (20:5n-3; EPA) or docosahexaenoic acid (22:6n-3; DHA) in suppressing food allergic symptoms., Methods: Mice were fed a control diet (10% soybean oil) or fish oil diet rich in EPA (4% soybean oil + 6% EPA oil containing 28.8% EPA and 13.7% DHA) or DHA (4% soybean oil + 6% DHA oil containing 7% EPA and 27.8% DHA), starting 14 d before and for 5 wk during oral sensitization with peanut extract (PE) or whey. Acute allergic skin responses, serum immunoglobulins (Igs), and mucosal mast cell protease-1 (mmcp-1) were assessed. Hyperimmune serum was transferred to naive recipient mice fed the different diets., Results: The DHA diet effectively reduced the acute allergic skin response compared with the control or EPA diet in PE-allergic mice (control, 159 ± 15, or EPA, 129 ± 8, vs. DHA, 78 ± 7 μm; P < 0.0001 or P < 0.05, respectively). In contrast, both the DHA and EPA diets reduced the allergic skin response in whey allergic mice (control, 169 ± 9, vs. DHA, 91 ± 13, or EPA, 106 ± 14 μm; P < 0.001 or P < 0.01, respectively); however, only the DHA diet reduced mmcp-1 and whey-specific IgE and IgG1. The DHA and EPA diets also reduced the acute skin response in passively immunized mice., Conclusions: The DHA-rich fish oil diet reduced allergic sensitization to whey and allergic symptoms in both PE- and whey-allergic mice. These data suggest that DHA-rich fish oil is useful as an intervention to prevent or treat food allergy symptoms., (© 2014 American Society for Nutrition.)

Background: Several studies investigated whether hydrolysed proteins can induce tolerance to cow's milk (CM) in children at risk of developing CM allergy. Due to methodological problems and inconsistent findings, the evidence for a tolerogenic effect is limited. A major problem is that different hydrolysates may give different outcomes due to variations in their production and composition., Objective: The aim of the study was to investigate the effect of the degree of hydrolysis on the allergenicity and immunogenicity of whey hydrolysates., Methods: The hydrolysis of whey was stopped at different time-points between 1 and 60 min. In 18 CM allergic patients, the allergenicity of the hydrolysates was determined by immunoblot and the basophil activation test. To test immunogenicity, CM-specific T cell lines were generated., Results: In most patients, increasing time of hydrolysis decreased IgE recognition and basophil activation. However, in five patients, hydrolysed proteins induced more basophil activation than non-hydrolysed proteins. The immunoblot data indicated that these patients recognized either a 25- to 30-kDa degradation product of casein or a 10-kDa degradation product of whey. Although T cell activation was decreased in all patients over time, half of them still showed a positive response to the proteins after 60 min of hydrolysis., Conclusion: Increasing the time of hydrolysis reduces both allergenicity and immunogenicity of whey hydrolysates in most but not all patients. This indicates that not the degree of hydrolysis is decisive but the presence and stability of IgE and T cell epitopes in the hydrolysate recognized by individual patients., (© 2013 John Wiley & Sons Ltd.)

Food allergy is an aberrant immune-mediated reaction against harmless food substances, such as cow's milk proteins. Due to its very early introduction, cow's milk allergy is one of the earliest and most common food allergies. For this reason cow's milk allergy can be recognized as one of the first indications of an aberrant inflammatory response in early life. Classically, cow's milk allergy, as is true for most other allergies as well, is primarily associated with abnormal humoral immune responses, that is, elevation of specific immunoglobulin E levels. There is growing evidence indicating that cellular components of both innate and adaptive immunity play significant roles during the pathogenesis of cow's milk allergy. This is true for the initiation of the allergic phenotype (stimulation and skewing towards sensitization), development and outgrowth of the allergic disease. This review discusses findings pertaining to roles of cellular immunity in allergic inflammation, and tolerance induction against cow's milk proteins. In addition, a possible interaction between immune mechanisms underlying cow's milk allergy and other types of inflammation (infections and noncommunicable diseases) is discussed.

Background: Recent studies have indicated that peptides containing T cell epitopes may be used for immunotherapy. While for several cow's milk allergens the T cell epitopes have been described, the T cell epitopes in the major allergen α-lactalbumin (α-LAC) are unknown. Therefore, the aim of this study was to determine the T cell epitopes in α-LAC., Methods: Nineteen synthetic peptides spanning α-LAC were obtained. Cow's milk-specific T cell lines (TCLs) of 46 subjects were generated and tested for their specificity for α-LAC. The lines responding to α-LAC were subsequently tested to determine their activation in response to the peptides., Results: More than half of the TCLs generated did not respond to α-LAC or lost their responsiveness during subsequent experiments, which indicates that α-LAC has low immunogenicity. Only 8 TCLs recognized 1 or more peptides. The recognition of the peptides was diverse and no major epitopes could be defined., Conclusion: The immunogenicity of α-LAC is very low compared to other major allergens in cow's milk. Moreover, there seems to be no dominant epitope present in the protein. Therefore, it seems unlikely that peptides of this protein can be used for immunotherapy., (© 2014 S. Karger AG, Basel.)

Background: Recently, we have shown that dietary long-chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) largely prevent allergic sensitization in a murine model for cow's milk allergy. The aim of this study was to assess the contribution of regulatory T cells (Treg) in the prevention of food allergy by n-3 LCPUFA., Methods: C3H/HeOuJ female donor mice were fed a control or fish oil diet before and during oral sensitization with cow's milk protein whey. Acute allergic skin response (ASR), anaphylaxis, body temperature, serum immunoglobulins, and mouse mast cell protease-1 (mmcp-1) were assessed. Splenocytes of sham- or whey-sensitized donor mice fed either control or fish oil diet were adoptively transferred to naïve recipient mice. Recipient mice received a whole splenocyte suspension, splenocytes ex vivo depleted of CD25+ cells, or MACS-isolated CD4+ CD25+ Treg. Recipient mice were sham- or whey-sensitized and fed control diet., Results: The ASR as well as whey-specific IgE and whey-specific IgG1 levels were reduced in whey-sensitized donor mice fed the fish oil diet as compared to the control diet. Splenocytes of control-diet-fed whey-sensitized donors transferred immunologic memory. By contrast, splenocytes of fish-oil-fed whey-sensitized - but not sham-sensitized - donors transferred tolerance to recipients as shown by a reduction in ASR and serum mmcp-1, and depletion of CD25+ Treg abrogated this. Transfer of CD25+ Treg confirmed the involvement of Treg in the suppression of allergic sensitization., Conclusions: CD25+ Treg are crucial in whey allergy prevention by n-3 LCPUFA., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Prior exposure to partial whey hydrolysates has been shown to reduce the allergic response to whey in mice. This effect was more pronounced in combination with a diet containing non-digestible oligosaccharides (scGOS/lcFOS/pAOS). It is unknown which fractions/epitopes are responsible for this effect. Therefore, the prophylactic ability of synthetic peptides of β-lactoglobulin with/without a scGOS/lcFOS/pAOS-containing diet to reduce the allergic response in a mouse model for cow's milk allergy was investigated., Methods: Of 31 peptides, nine peptides were selected based on human T cell data. Mice were pre-treated orally with three peptide mixtures or single peptides for six consecutive days. During this period, they received a control or scGOS/lcFOS/pAOS-containing diet. Subsequently, mice were orally sensitized to whey and received an intradermal and oral challenge. After sacrifice, serum and mesenteric lymph nodes (MLN) were collected for further analysis., Results: Prior exposure to peptide mixtures 1 and 3 significantly reduced the acute allergic skin response to whey. Mixture 2 showed no effect. An additive effect of the scGOS/lcFOS/pAOS-containing diet was only observed for mixture 1. Of the peptides in mixture 1, one peptide (LLDAQSAPLRVYVEELKP) showed the strongest effect on the acute allergic skin response. This peptide also tended to decrease whey-specific antibody levels and to increase the percentages of CD11b+CD103+ dendritic cells and CD25+Foxp3+ T cells in the MLN., Conclusions: Prior exposure to specific peptides of β-lactoglobulin reduces the allergic response to whey, which may involve regulatory dendritic and T cells. Combining peptides with a sGOS/lcFOS/pAOS-containing diet enhances this effect., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Cow's milk allergy is one of the most common food allergies in children and no treatment is available. Dietary lipid composition may affect the susceptibility to develop allergic disease., Objective: Assess whether dietary supplementation with long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) prevents the establishment of food allergy., Methods: Mice were fed a control or fish oil diet before and during oral sensitization with whey. Acute allergic skin response, serum immunoglobulins as well as dendritic cell (DC) and T cell subsets in mesenteric lymph nodes (MLN), spleen and/or small intestine were assessed., Results: The acute allergic skin response was reduced by more than 50% in sensitized mice fed the fish oil diet compared to the control diet. In addition, anti-whey-IgE and anti-whey-IgG1 levels were decreased in the fish oil group. Serum transfer confirmed that the Th2-type humoral response was suppressed since sera of fish oil fed sensitized mice had a diminished capacity to induce an allergic effector response in naïve recipient mice compared to control sera. Furthermore, the acute skin response was diminished upon passive sensitization in fish oil fed naïve recipient mice. In addition, the percentage of activated Th1 cells was reduced by fish oil in spleen and MLN of sham mice. The percentage of activated Th2 cells was reduced in both sham- and whey-sensitized mice. In contrast, whey-sensitized mice showed an increased percentage of CD11b+CD103+CD8α- DC in MLN in association with enhanced FoxP3+ regulatory T cells (Treg) in spleen and intestine of fish oil fed whey-sensitized mice compared to sham mice., Conclusions and Clinical Relevance: Dietary n-3 LCPUFA largely prevented allergic sensitization in a murine model for cow's milk allergy by suppressing the humoral response, enhancing local intestinal and systemic Treg and reducing acute allergic symptoms, suggesting future applications for the primary prevention of food allergy., (© 2013 John Wiley & Sons Ltd.)

This study describes two phases of a multi-phase project aiming to validate a mouse model for cow's milk allergy to assess the potential allergenicity of hydrolysed cow's milk based infant formulas (claim support EC-directive 2006/141/E). The transferability and the discriminatory power of this model was evaluated in 4 research centers. Mice were sensitized by oral gavage with whey or extensively hydrolysed whey (eWH) using cholera toxin as an adjuvant. Whey-specific antibodies, mMCP-1 levels, anaphylactic shock symptoms, body temperature and the acute allergic skin response were determined upon whey challenge. In phases I and II, all 4 centers detected elevated levels of whey-specific IgE/IgG1 in whey sensitized animals. Elevated levels of mMCP-1, anaphylactic symptoms, body temperature drop and acute allergic skin response were scored upon whey challenge in 3 out of 4 research centers. In contrast, none of the evaluated parameters were elevated in eWH orally exposed groups. The cow's milk allergy mouse model is capable to distinguish the sensitizing capacity of complete or hydrolysed cow's milk protein. The model uses straightforward parameters relevant to food allergic responses and can be effectively transferred between different laboratories. We propose this mouse model as a new strategy for the screening of new hypoallergenic cow's milk formulas., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

A transglutaminase cross-linked caseinate was designed for use in dairy products to increase the viscosity of food matrices. The difference in structure of cross-linked caseinate might have implications for the risk of developing cow's milk allergy. The sensitizing capacity and the allergenicity (the potency to induce an allergic effector response) of cross-linked sodium caseinate was investigated using a mouse model for cow's milk allergy. Mice were orally sensitized with cross-linked caseinate or caseinate using cholera toxin as adjuvant. Anaphylactic shock reactions, change in body temperature, acute allergic skin response, caseinate-, cross-linked caseinate-IgE and mMCP-1 concentrations were determined after challenge with cross-linked caseinate or caseinate. Sensitization with cross-linked caseinate did not result in anaphylactic shock symptoms, drop in body temperature or release of serum mMCP-1. A tendency toward decreased casein-specific IgE levels was observed. The allergenicity did not differ between both products. These results indicate that in already caseinate-sensitized mice, cross-linked caseinate did not provoke more pronounced allergenic reactions compared to sodium caseinate. On top of that, reduced sensitization to cross-linked caseinate was observed. Cross-linked caseinate might therefore be an interesting new dietary concept for humans at risk for food allergy although more mechanistic studies and clinical trials are needed for validation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Cow's milk (CM) hydrolysates are frequently used as milk substitutes for children with CM allergy. In hydrolysates, allergenic epitopes within CM proteins are diminished by enzymatic treatment. The aim of this study was to examine the allergenic and immunogenic properties of whey proteins during hydrolysis., Methods: During hydrolysis, samples were obtained at 0, 10, 15, 30, 45, 60, 75 and 90 min. Degradation was checked by HPLC and SDS-PAGE. Allergenic potential was analyzed by IgE crosslinking capacity of human Fcε receptor type 1-transduced rat basophilic leukemia cells sensitized with serum of CM-allergic patients. Whey-sensitized C3H/HeOuJ mice were ear challenged intracutaneously with the hydrolysates. Immunogenicity was tested using whey-specific human T-cell clones and T-cell lines at the level of proliferation and release of IL-4, IL-10, IL-13 and IFN-γ., Results: After 15 min of hydrolysis, the majority of the proteins were degraded. Hydrolysis for 15 min resulted in 92% inhibition of mast cell degranulation and in 82% reduction of ear swelling in the mouse model. In contrast, T-cell-stimulatory capacity was less affected by hydrolysis: reduction of human T-cell proliferation was only 9%. This was further reduced to 57 and 74% after 30 and 45 min of hydrolysis, respectively. Cytokine production followed the pattern of T-cell proliferation., Conclusion: Via differential analysis of allergenic versus immunogenic properties of the time kinetics of hydrolysis of whey proteins, we have demonstrated specific hydrolysis conditions with reduced IgE-crosslinking responses but retained T-cell activating properties. This approach might be useful in better defining CM hydrolysates., (Copyright © 2012 S. Karger AG, Basel.)

Background: Little is known about the contribution of the invariant natural killer T (iNKT) cells in the onset of food allergy. Using a mouse model for cow's milk allergy the function of iNKT cells was investigated., Methods: Mice were sensitized orally with casein or whey proteins. One hour before the sensitizations the mice were injected intraperitoneally with α-galactosylceramide (αGalCer) or control. One week after the last sensitization acute allergic skin reactions were measured. Furthermore, in the liver, spleen and mesenteric lymph nodes (MLN) percentages of iNKT cells were analyzed and liver lymphocyte restimulation assays were performed., Results: Whey- or casein-sensitized mice treated with αGalCer showed enhanced acute allergic skin reactions. The percentage of iNKT cells in the liver of sensitized mice was reduced compared to sham-sensitized mice. αGalCer treatment was found to deplete iNKT cells in the liver of sensitized as well as sham-sensitized mice, and these hepatocytes did not respond to ex vivo restimulation with αGalCer. αGalCer treatment did not reduce iNKT cell percentages in the spleen and MLN of sham-sensitized mice but abrogated the increase in iNKT cell percentage in the spleen upon whey sensitization, whereas it enhanced the iNKT cell percentage in the MLN of casein-sensitized mice. Due to the repeated application of αGalCer, livers were functionally depleted of iNKT cells. This resulted in an increased allergic effector response which was most pronounced in whey-sensitized mice and associated with enhanced whey-specific immunoglobulin levels., Conclusion: iNKT cells may suppress cow's milk allergic symptoms in mice and may differentially regulate oral sensitization for casein and whey., (Copyright © 2012 S. Karger AG, Basel.)

Dietary non-digestible carbohydrates reduce the development of cows' milk allergy in mice. In the present study, the contribution of CD25+ regulatory T-cells (Treg) was investigated using in vivo Treg depletion and adoptive transfer studies. Mice were orally sensitised with casein and fed a diet containing 2 % short-chain galacto-, long-chain fructo- and acidic oligosaccharides (GFA) or a control diet. Donor splenocytes of mice sensitised with casein and fed the GFA or control diet were adoptively transferred to naive recipient mice, which were casein- or sham-sensitised and fed the control diet. In addition, in vivo or ex vivo CD25+ Treg depletion was performed using anti-CD25 (PC61). The acute allergic skin response upon intradermal casein challenge and casein-specific Ig were determined. Furthermore, T-helper (TH) 1 and TH2 cell numbers were analysed in the mesenteric lymph nodes. The oligosaccharide diet strongly reduced the development of the acute allergic skin response, which was abrogated by the in vivo anti-CD25 treatment. The diet enhanced the percentage of TH1 cells and tended to reduce the percentage of TH2 cells in casein-sensitised mice. Recipient mice were protected against the development of an acute allergic skin response when transferred with splenocytes from casein-sensitised GFA-fed donor mice before sensitisation. Ex vivo depletion of CD25+ Treg abrogated this transfer of tolerance. Splenocytes from sham-sensitised GFA-fed donor mice did not suppress the allergic response in recipient mice. In conclusion, CD25+ Treg contribute to the suppression of the allergic effector response in casein-sensitised mice induced by dietary intervention with non-digestible carbohydrates.

Background: Hypoallergenic formulas are considered a good option for infants at risk for cow's milk allergy. The aim of this animal study was to investigate whether whey hydrolyzates (WH) have the capacity to induce oral tolerance to whey., Methods: Whey, partial or extensive WH was given via gavages to naïve mice prior to oral whey sensitization using cholera toxin as an adjuvant. The acute allergic skin response, mouse mast cell protease-1 (mMCP-1), whey-specific IgE, IgG(1) and effector Th2-cells, Th1-cells, and Foxp3(+) regulatory T-cells were determined in the mesenteric lymph nodes (MLN). MLN cells from tolerized mice were adoptively transferred to naïve recipient mice prior to whey sensitization., Results: In contrast to the extensive WH, pre-treatment of naïve mice with whey or partial WH reduced the acute allergic skin response and mast cell degranulation after whey challenge. However, only treatment with whey prevented the generation of serum-specific IgE/IgG(1) . In partial WH tolerized mice, Foxp3(+) regulatory T-cell numbers in the MLN were increased compared to whey-sensitized mice. Both whey and partial WH treatment showed a tendency toward a decreased number of effector Th2-cells. Transfer of MLN cells from tolerized mice protected recipient mice from developing an acute allergic skin response., Conclusion: These results show that partial WH with limited sensitizing properties reduced the effector response upon whey challenge. This effect is transferable using MLN cells and was associated with enhanced Foxp3(+) regulatory T-cell numbers in the MLN. Partial WH retained the capacity to induce active immune suppression in mice which may be relevant for allergy prevention., (© 2011 John Wiley & Sons A/S.)

Background: Symptoms of allergy are largely attributed to an IgE-mediated hypersensitivity response. However, a considerable number of patients also exhibit clinical features of allergy without detectable systemic IgE. Previous work showed that Ig-free light chains (IgLC) may act as an alternate mechanism to induce allergic responses. CD4+CD25+ T cells are crucial in the initiation and regulation of allergic responses and compromised function might affect the response to allergens., Objective: To examine the contribution of CD4+CD25+ T cells and IgLC towards the whey-allergic response., Methods: Mice were sensitized orally with whey using cholera toxin as an adjuvant. CD25+ T cells were depleted in vivo using a CD25 mAb. The acute allergic skin response to whey and ex vivo colon reactivity was measured in the presence or absence of F991, a specific inhibitor of IgLC. Serum whey-specific antibodies and IgLC in serum and mesenteric lymph node (MLN) supernatants were measured. Depletion of CD4+CD25+ T cells was confirmed in the spleen., Results: Anti-CD25 treatment strongly reduced whey-specific antibody levels and resulted in a partial depletion of effector T cells and a major depletion of Foxp3(+) regulatory T cells. Surprisingly, despite the abolished specific IgE response, the acute allergic skin response to whey was not affected. IgLC levels were enhanced in the serum and MLN supernatants of CD25-depleted sensitized mice. F991 inhibited the acute skin response and colon hyperreactivity in anti-CD25-treated mice, indicating that these responses were mainly IgLC dependent., Conclusions: Depletion of CD4+CD25+ T cells resulted in a switch from an IgE- to an IgLC-dependent acute skin response and functional hyperresponsiveness of the colon. Our data suggest that CD25+ T cells play a crucial role in balancing cow's milk allergy between IgE and IgE-independent responses and both mechanisms might play a role in allergic responses to the same allergen.

Background: Cow's milk allergy (CMA) affects 2.5% of young infants. In previous murine studies it was observed that allergic sensitization to the major cow's milk allergens casein and whey led, respectively, to IgE-independent and IgE-dependent clinical responses., Objectives: In this study the involvement of immunoglobulin free light chains (Ig-fLCs) in the hypersensitivity response to cow's milk proteins was explored in mice, and Ig-fLC serum levels were determined in children affected by CMA or atopic dermatitis (AD)., Methods: Mice were orally sham, casein, or whey sensitized. Acute allergen-specific skin responses were determined, and serum immunoglobulin and Ig-fLC concentrations were measured. Ig-fLC dependency was validated by using the Ig-fLC blocker F991 in actively and passively sensitized mice. Ig-fLC serum concentrations were measured in a cohort of infants with CMA and infants with AD., Results: After sensitization, no specific IgE was detectable in sera of casein-sensitized mice, whereas specific IgE levels were enhanced in whey-sensitized mice. Instead, Ig-fLC levels were increased in sera from casein-sensitized mice. Furthermore, blocking Ig-fLCs strongly diminished the allergic skin responses not only in casein-sensitized mice but also in mice transferred with splenocyte supernatants of casein-sensitized mice. In both patients with CMA and patients with AD, serum Ig-fLC concentrations were significantly enhanced., Conclusions: This study indicates that sensitization with cow's milk proteins can lead to both IgE-dependent and Ig-fLC-dependent allergic hypersensitivity responses. Also, in children affected with CMA or AD, serum Ig-fLC concentrations were increased, implying the relevance of Ig-fLC measurements in the diagnoses of human allergic disease., (Copyright (c) 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Hypoallergenic milk formulae are used for cow's milk allergic infants and may be a good option for infants at risk. Clinical studies have shown that the protein source or the hydrolysis methodology used may influence the effectiveness in infants stressing the importance of adequate pre-clinical testing of hypoallergenic formulae in an in vivo model of orally induced cow's milk allergy. This study was undertaken to introduce a new read-out system to measure the residual allergenicity of whey hydrolysates on both the sensitization and challenge phase of orally induced cow's milk allergy in mice. Mice were sensitized orally to whey or a partial whey hydrolysate (pWH) to measure the residual sensitizing capacity. To predict the residual allergenicity of hydrolysates, whey allergic mice were challenged in the ear with pWH, extensive whey hydrolysate or an amino acid-based formula. An acute allergic skin response (ear swelling at 1 h), whey-specific serum antibodies, and local MCP-1 concentrations were measured. In contrast to whey, oral sensitization with pWH did not result in the induction of whey-specific antibodies, although a minor residual skin response to whey was observed after challenge. Skin exposure to whey hydrolysates showed a hydrolysation dependent reduction of the acute allergic skin response in whey allergic mice. In contrast to whey, skin exposure to pWH did not enhance tissue MCP-1 levels. The acute allergic skin response in mice orally sensitized to cow's milk proteins reveals a new pre-clinical tool which might provide information about the residual sensitizing capacity of hydrolysates supporting the discussion on the use of hypoallergenic formulae in high risk children. This mouse model might be a relevant model for the screening of new hypoallergenic formulae aimed to prevent or treat cow's milk allergy.

Dietary intervention with a unique prebiotic nondigestible carbohydrate mixture has been shown to reduce the development of allergic disease in infants at risk. In this study, the involvement of CD25(+) regulatory T-cells (Treg) in the carbohydrate-induced effects was investigated in mice orally sensitized with whey using adoptive transfer experiments. Donor mice were sensitized with whey and fed a diet containing short-chain galacto-, long-chain fructo- and acidic-oligosaccharides, or a control diet starting 2 wk before sensitization. The acute allergic skin reaction upon intradermal whey challenge was determined and whey-specific Ig were measured. Splenocytes of the donor mice were transferred to naïve recipient mice after partial ex vivo depletion of CD25(+) Treg. The prebiotic diet clearly diminished the acute allergic skin reaction (P < 0.001). Whey-sensitized recipient mice transferred with splenocytes from whey-sensitized, prebiotic-fed donor mice displayed almost complete prevention of the acute allergic skin reaction compared with mice receiving cells from sham-sensitized, prebiotic-fed donor mice (P < 0.001). Partial depletion of CD25(+) T-cells inhibited these effects (P < 0.001), although IgE sensitization was not prevented. This study indicates the involvement of whey-specific CD25(+) Treg in the suppression of the allergic effector response induced by dietary intervention with prebiotics.

Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. The purpose of this study was to compare effects of dietary supplementation with a prebiotic mixture (Immunofortis), a probiotic strain [Bifidobacterium breve M-16V], or a synbiotic diet combining both on the outcome of the allergic response when provided during oral sensitization with whey in mice. Mice were fed diets containing 2% (wt:wt) Immunofortis and/or the B. breve M-16V (n = 6/group). The acute allergic skin response was determined by measuring ear swelling. Antigen-induced anaphylaxis was scored. Furthermore, whey-specific serum immunoglobulins and mouse mast cell protease-1 (mMCP-1) were determined. In mice fed the synbiotic mixture, the allergic skin response and the anaphylactic reaction were strongly reduced compared with whey-sensitized mice fed the control diet (P < 0.01). Immunofortis or B. breve M-16V alone were significantly less effective in reducing the allergic skin response than the synbiotic diet and did not reduce the anaphylactic reaction. The whey-specific IgE and IgG(1) responses were not affected; however, IgG(2a) was greater in all treated groups than in the control group (P < 0.05). Serum mMCP-1 concentrations, reflecting mucosal mast cell degranulation, were lower in mice fed synbiotics compared with those fed the control diet (P < 0.01). Dietary supplementation with Immunofortis, B. breve M-16V, and particularly the synbiotic mixture, provided during sensitization, reduces the allergic effector response in a murine model of IgE-mediated hypersensitivity that mimics the human route of sensitization. This model shows the potential for dietary intervention with synbiotics in reducing the allergic response to food allergens.

Background: Cow's milk allergy (CMA) is characterized by hypersensitivity against casein or whey, affecting 2.5% of young infants. The pathogenesis of CMA involves IgE as well as non-IgE-mediated reactions and clinical symptoms are found in the skin, lungs and gastrointestinal tract. In this study, local and systemic immunopathology was determined in whey- or casein-allergic mice., Methods: Mice were orally sensitized with casein or whey using cholera toxin as an adjuvant. Serum immunoglobulins and the acute allergic skin reaction (ear swelling 1 h after intradermal allergen challenge) were determined to reveal systemic hypersensitivity. Furthermore, pathophysiological changes were assessed within the intestine., Results: An acute allergic skin reaction was induced in both whey- and casein-sensitized mice. In these mice, whey-specific IgE, IgG(1), IgG(2a) and casein-specific IgG(1) levels were found to be increased. In addition, the serum mouse mast cell protease-1 (mMCP-1) concentration was enhanced, reflecting mast cell degranulation. Indeed, the number of mMCP-1-positive mast cells within the colon was diminished in both whey- and casein-sensitized mice. Only in casein-sensitized mice isometric contraction of the colon was reduced, reflecting motility alterations., Conclusion: Mice, orally sensitized against casein or whey, revealed an allergen-specific acute allergic skin reaction. In casein-sensitized mice, hypocontractility of the colon reflected pathophysiological changes within the intestine. Allergen-induced ear swelling and intestinal contractility changes are novel parameters in animal models of CMA which may add to the search for new therapeutic strategies to relieve symptoms of CMA., ((c) 2008 S. Karger AG, Basel.)

Background: The central role of specific IgE in cow's milk allergy (CMA) is well documented. However, less is known about the function of other immunoglobulin isotypes in allergy and tolerance to cow's milk proteins (CMPs)., Objective: To determine differences in the antibody responses that are associated with allergy and tolerance to cow's milk in allergic, atopic and non-atopic individuals of different age groups., Methods: Nineteen infants (<1 year), 18 children (6-14 years) and 41 adults (21-68 years) were included. Each age group was comprised of subjects with CMA, atopic individuals without a history of CMA and non-atopic subjects. Levels of specific IgE, IgG4, IgG1 and IgA to whole cow's milk and the six most abundant individual CMPs were determined in plasma by ELISA. For comparison, specific IgE and IgG4 were measured to ovomucoid and house dust mite (HDM) in individuals allergic for the respective allergens, and in atopic and non-atopic subjects without allergy., Results: In infants and children with CMA, alphas1-casein and beta-lactoglobulin induced the highest specific IgE response, whereas alphas1-casein was the most allergenic CMP in adult patients. Specific IgG4 and IgG1 responses were the highest to alphas1-casein and beta-lactoglobulin in all age groups, while kappa-casein and alpha-lactalbumin induced the highest levels of IgA. CMP-specific IgG4 was higher in atopic children and adults without CMA, as compared with non-atopic individuals. A similar difference between tolerant atopic and non-atopic subjects was observed for IgG4 specific to ovomucoid, whereas HDM-specific IgG4 was not detectable in these subjects., Conclusion: Maintenance of tolerance to cow's milk in atopic children and adults without CMA is associated with elevated levels of specific IgG4, in combination with low specific IgE. The up-regulation of specific IgG4 in tolerant atopic individuals may be related to the type of allergen and its regular dose of exposure.

Background: Little is known about the association between human leucocyte antigen (HLA) and cow's milk allergy (CMA). The aim of the present study was to determine the HLA restriction of T cell clones (TCCs) specific to alphas1-casein, the most abundant milk protein, and to study possible HLA class II allele associations with CMA., Methods: alphas1-Casein-specific TCCs were derived from 6 children with CMA, 9 atopic children without CMA and 5 non-atopic children. T cell epitope specificity was defined by stimulation with overlapping peptides, spanning the alphas1-casein molecule. HLA restriction was determined in proliferation assays using antibodies blocking either HLA-DP, HLA-DQ or HLA-DR. HLA genotyping was performed in 32 subjects with CMA, 23 atopic and 22 non-atopic individuals., Results: Ten TCCs were restricted to HLA-DQ, 6 TCCs to HLA-DR and 4 TCCs to HLA-DP. The sequence in alphas1-casein that was most immunogenic to T cells from children with CMA contained T cell epitopes restricted to DQB1*0201, DPB1*0401 and DRB1*1501. The DQB1*0501 allele frequency was lower in children with CMA than in non-atopic children, but this difference could not be confirmed in an additional group of subjects with and without CMA., Conclusions: HLA-DQ plays a substantial role in the presentation of T cell epitopes in alphas1-casein. However, HLA class II allele frequencies do not show major differences between cow's milk allergic, atopic and non-atopic subjects. T cell epitopes in the most immunogenic region are presented by various abundantly present HLA genotypes. Therefore, this sequence may be a suitable target for peptide immunotherapy.

Background: One to two percent of infants suffer from IgE-mediated allergic reactions against cow's milk proteins. Most children develop clinical tolerance, but approximately 15% are still allergic by the age of 10 years. Little is known about the T cell epitopes in individual cow's milk protein in relation to allergy and tolerance., Objective: To identify T cell epitopes in alphas1-casein, the most abundant milk protein, and to investigate T cell responses toward these epitopes in allergic, atopic and non-atopic children., Methods: Allergen-specific T cell lines (TCLs) were derived from peripheral blood mononuclear cells of 11 cow's milk allergic, nine atopic and nine non-atopic children. T cell responses were measured to alphas1-casein and to overlapping peptides (18-mers), spanning the alphas1-casein molecule. Proliferation was determined by incorporation of (3)H-thymidine, and cytokine production (IL-10, IL-13 and IFN-gamma) was measured by ELISA., Results: Four main regions (amino acid (AA) residues 43-66, 73-96, 91-114 and 127-180) in the alphas1-casein molecule were immunogenic to T cells, among which the AA residues 133-156 spanned the immunodominant part. Only subtle differences were found in peptide recognition between the subject groups. Some of the peptides induced slightly Th1- or Th2-skewed cytokine responses. The increased levels of IL-10 in response to alphas1-casein observed in TCLs from atopic children appeared not to be linked to recognition of specific IL-10-inducing epitopes., Conclusions: The immunodominant sequence in alphas1-casein is spanned by AA residues 133-156. Tolerance towards alphas1-casein in atopic children may be mediated by an overall induction of IL-10 and not by recognition of certain T cell epitopes. The identified T cell epitopes in children with cow's milk allergy may be useful targets in developing peptide immunotherapy.

Background: The role of antigen-specific T cells in the mechanism of food allergy or maintenance of tolerance toward an innocuous antigen, such as cow's milk, is not yet fully understood., Objective: The cow's milk-specific T-cell response of donors with various allergic backgrounds was investigated., Methods: Cow's milk-specific T-cell clones (TCCs) were generated from the blood of children with persistent cow's milk allergy (CMA) and the blood of cow's milk-tolerant allergic and nonallergic control subjects. The TCCs were characterized by their antigen-specific proliferation, cytokine production, and activation status., Results: Cow's milk-specific TCCs of children with persistent CMA were T(H)2 skewed, and the production of IL-4 and IL-13 was significantly correlated with the expression of the activation marker CD25. TCCs of the allergic control subjects were characterized by a high production of IL-10, which was positively correlated with the production of IL-4 and IFN-gamma and with the expression of CD25. TCCs derived from nonallergic control subjects had an attenuated response toward cow's milk in that they did not produce high levels of cytokines nor did they express high levels of surface markers. As in the allergic control subjects, in the nonallergic control subjects IL-10 production was positively correlated with the expression of CD25., Conclusion: The activation status of T cells derived from persistent donors with CMA was associated with the production of IL-4 and IL-13, whereas activated TCCs of cow's milk-tolerant control subjects were characterized by the production of IL-10 and, to a lesser extent, IFN-gamma. These findings suggest that activated CD4(+) T cells (characterized by a high CD25 expression) might contribute to the tolerogenic immune response toward an antigen, such as cow's milk, through the production of IL-10.