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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 &lt, 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.

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.

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.

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.

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.

From Pubmed: " BACKGROUND: Antigen-specific immunotherapy (AIT) is a promising therapeutic approach for both cow's milk allergy (CMA) and peanut allergy (PNA), but needs optimization in terms of efficacy and safety. AIM: Compare oral immunotherapy (OIT) and subcutaneous immunotherapy (SCIT) in murine models for CMA and PNA and determine the dose of allergen needed to effectively modify parameters of allergy. METHODS: Female C3H/HeOuJ mice were sensitized intragastrically (i.g.) to whey or peanut extract with cholera toxin. Mice were treated orally (5 times/week) or subcutaneously (3 times/week) for three consecutive weeks. Hereafter, the acute allergic skin response, anaphylactic shock symptoms and body temperature were measured upon intradermal (i.d.) and intraperitoneal (i.p.) challenge, and mast cell degranulation was measured upon i.g. challenge. Allergen-specific IgE, IgG1 and IgG2a were measured in serum at different time points. Single cell suspensions derived from lymph organs were stimulated with allergen to induce cytokine production and T cell phenotypes were assessed using flow cytometry. RESULTS: Both OIT and SCIT decreased clinically related signs upon challenge in the CMA and PNA model. Interestingly, a rise in allergen-specific IgE was observed during immunotherapy, hereafter, treated mice were protected against the increase in IgE caused by allergen challenge. Allergen-specific IgG1 and IgG2a increased due to both types of AIT. In the CMA model, SCIT and OIT reduced the percentage of activated Th2 cells and increased the percentage of activated Th1 cells in the spleen. OIT increased the percentage of regulatory T cells (Tregs) and activated Th2 cells in the MLN. Th2 cytokines IL-5, IL-13 and IL-10 were reduced after OIT, but not after SCIT. In the PNA model, no differences were observed in percentages of T cell subsets. SCIT induced Th2 cytokines IL-5 and IL-10, whereas OIT had no effect. CONCLUSION: We have shown clinical protection against allergic manifestations after OIT and SCIT in a CMA and PNA model. Although similar allergen-specific antibody patterns were observed, differences in T cell and cytokine responses were shown. Whether these findings are related to a different mechanism of AIT in CMA and PNA needs to be elucidated."

The prevalence of allergic diseases in western countries is increasing. One of the first allergies to develop early in life, is cow’s milk allergy (CMA). Oral tolerance is generated against harmless food proteins such as cow’s milk proteins, leading to systemic immunological non-responsiveness. However, one to three percent of children generate a Th2 driven immune response against these proteins and develop CMA. Upon oral ingestion of the culprit food symptoms range from gastro-intestinal and behavioral changes, to cutaneous and respiratory reaction and even anaphylaxis may occur. Most children outgrow CMA, however they still are more susceptible to develop other allergies later in life. Currently there is no cure for cow’s milk allergy. Therefore, it is important to study ways aiming to prevent or treat CMA. Non-digestible oligosaccharides (NDO) present in human milk, have a beneficiary influence on the intestinal microbiome and the immune system. Cow’s milk-based formula is nowadays supplemented with NDO mimicking some structural and functional aspects of human-milk NDO. In the current studies, the CMA preventive and treatment effects of three different enzymatically produced or isolated NDO, namely short-chain Galacto oligosaccharides (G), long-chain Fructo oligosaccharides (F) and pectin derived Acidic oligosaccharides (A) were examined. These three NDO were used singularly or in combination (GF or GFA) to investigate whether CMA could be diminished in a murine model of CMA. The supplemented diets were fed two weeks prior and during the whole sensitization and challenge phase (prevention) or only during the challenge phase (treatment). Both GF or GFA were effective in reducing the CMA symptoms only in a preventive setting. GF modulated immune markers in the intestinal mucosa, downregulating Th2 related GATA3 expression, while GFA enhanced Th1 related Tbet expression, the expression of regulatory cytokines IL10 and TGFβ and the frequency of intestinal FoxP3 positive regulatory T-cells. The contribution of IL10 and TGFβ in the CMA protective effect of GFA were further investigated by using antibodies that block the IL10 receptor or TGFβ in vivo. This inhibition abrogated the preventive effect of GFA on CMA symptoms completely. Beyond the effects of NDO also dietary supplementation with vitamin D or vitamin A was studied since these are known for their immunoregulatory capacities. Vitamin D did not add to the preventive effect of GFA, whereas vitamin A supported GFA in its capacity to reduce CMA symptoms. Since GFA supports the immune system, it was also studied if GFA affects the vaccination response in mice. Indeed, GFA was found to enhance the influenza vaccination response via inhibition of a specific regulatory T cell subtype, which normally downregulates the Th1 cell population. These studies help to understand the mechanisms by which dietary supplementation with GFA (1 w/w%) influences the immune system leading to reduced CMA development and boosting the influenza vaccination response. These results pave the way to further optimize NDOs for immune support in early life, which could contribute to a lower risk to develop CMA.

Food allergies impact the quality of life of both patients and their families and cause limitations in dietary and social habits. Moreover, food allergies can lead to life-threatening situations in case of food-induced systemic anaphylaxis. The world-wide prevalence of IgE-mediated food allergies has been estimated to be 4% in children and 1-3% in the adult population. To date, no curative treatments exist and food allergy management relies primarily on avoidance of the offending food(s). Novel therapeutic strategies aiming for induction of specific oral tolerance to food proteins are under study. In particular, oral immunotherapy (OIT) has shown promising results in relation to effective desensitization and sustained unresponsiveness after discontinuation of the treatment. Nevertheless, safety concerns and a lack of (long-term) efficacy have hampered translation to the clinic thus far. Non-digestible oligosaccharides (prebiotics) are fermented into bacterial metabolites, e.g. short-chain fatty acids (SCFA), by bacteria residing in the gut. SCFA can exert anti-inflammatory functions, both locally and at distinct sites in the body after uptake in the systemic circulation. SCFA can directly communicate with immune cells and intestinal epithelial cells via specific surface-bound receptors. In addition to stimulation of the growth of beneficial microbes, non-digestible oligosaccharides were shown to induce tolerance-associated immune cells and support homeostasis in the gut. In this PhD thesis, it was investigated whether a diet supplemented with non-digestible oligosaccharides can support the efficacy of OIT to treat cow’s milk allergy in a murine model. The data indicated that the combination of OIT and a non-digestible oligosaccharide supplemented diet effectively reduced acute allergic symptoms upon allergen challenge in cow’s milk allergic mice. The Th2-dominated allergic immune response was suppressed and Th1 and regulatory T cell responses were increased. Lower levels of allergen-specific IgE were detected in mice after the combination therapy and mast cell and basophil responses toward the allergen were decreased. Moreover, bone marrow-derived mast cell development was altered after in vivo exposure to non-digestible oligosaccharides. In addition to immunological parameters, higher levels of the SCFA butyrate were detected in mice treated with the combination therapy. Oral supplementation with butyrate during OIT was as effective as supplementation with non-digestible oligosaccharides in terms of desensitization. Specific alterations in bacterial species were observed in cow’s milk allergic mice treated with the combination of OIT and the prebiotic diet. Interestingly, higher levels of the dysbiosis-associated bacterial phylum Proteobacteria were observed in mice only treated with OIT. It was therefore hypothesized that providing the diet supplemented with non-digestible oligosaccharides during OIT improves bacterial composition and metabolism, leading to improved immune function and control of allergies. In conclusion, the research conducted in this PhD thesis supports the use of food components with immunomodulatory capacities to improve the efficacy of immunotherapy strategies. Future (pre-clinical) studies are needed to investigate the potential of non-digestible oligosaccharide supplementation to enhance safety of immunotherapy and to retain long-term allergy protection.

Cow’s milk allergy (CMA) is one of the earliest and most common food allergies. It affects approximately 3.5% of children. β-Lactoglobulin (BLG) is one of the main allergenic proteins in cow’s milk. Although many infants spontaneously outgrow CMA throughout childhood, an increasing number of cow’s milk allergic children remain permanently allergic. On the other hand, suffering from CMA in early life is associated with an increased risk of developing asthma later in life. Currently, no treatment is available for CMA and allergen avoidance is the gold standard in the clinic. New preventive and therapeutic approaches are needed to improve the quality of life of patients. Early oral exposure to allergenic foods is important for the development of oral tolerance and is therefore of interest for the primary prevention of food allergies. Protein fragments and peptides are suggested as a safer alternative for the use of the whole protein. Additionally, the use of dietary interventions with specific immunomodulatory components is suggested to beneficially affect the course of allergy development. In this thesis, results are presented on the possibility to induce oral tolerance to the whole whey protein by prior oral exposure to protein fragments or peptides. Furthermore, the possible beneficial effects of a dietary intervention with non-digestible oligosaccharides alone or in combination with health-promoting bacteria on the development of oral tolerance were studied in a mouse model of orally-induced whey allergy. Furthermore, a novel delivery system based on poly(lactic-co-glycolic acid) (PLGA) was used for enhancing the bioavailability of the peptides. The findings of this thesis show that a hydrolyzed whey protein partially prevented the whey-induced allergic response in mice and this was further enhanced when combined with a dietary intervention with non-digestible galacto-, long-chain fructo- and pectin-derived acidic (GFA) oligosaccharides. Similarly, the allergic response was reduced when specific T cell epitope-containing peptides from BLG (PepMix) were administered to mice in parallel with a diet containing short- and long-chain fructo-oligosaccharides and Bifidobacterium breve M-16V (FF/Bb) prior to allergic sensitization. The preventive effect of the PepMix+FF/Bb intervention was associated with enhanced propionate and butyrate concentrations in the cecum and increased markers for regulatory T cells (Tregs) in the Peyer’s patches. Oral exposure to PepMix and FF/Bb before sensitization contributed to an increased percentage of Tregs in the small intestine, enhanced Th1/Th2 ratio in the Peyer’s patches and partial non-responsiveness of splenocytes upon whey sensitization and challenge. Results from in vitro studies with human intestinal epithelial cells (IEC) suggested that IEC-derived galectin-9 is involved in the mechanisms of the non-digestible galacto- and long-chain fructo- (GF) oligosaccharides and a synthetic toll-like receptor 9 ligand by stimulating the activity of the aldehyde dehydrogenase activity in dendritic cells. The use of PLGA nanoparticles for peptide delivery demonstrated partial prevention the whey-induced allergic skin response, suggesting the potential beneficial role of a delivery vehicle in facilitating the tolerogenic capacity of the T cell epitopes. Altogether, this thesis highlights several dietary interventions which might support the development of oral tolerance and contribute to CMA prevention strategies.