Proteomics Analysis Reveals Altered Nutrients in the Whey Proteins of Dairy Cow Milk with Different Thermal Treatments.

Thermal treatments of milk induce changes in the properties of milk whey proteins. The aim of this study was to investigate the specific changes related to nutrients in the whey proteins of dairy cow milk after pasteurization at 85 °C for 15 s or ultra-high temperature (UHT) at 135 °C for 15 s. A total of 223 whey proteins were confidently identified and quantified by TMT-based global discovery proteomics in this study. We found that UHT thermal treatment resulted in an increased abundance of 17 proteins, which appeared to show heat insensitivity. In contrast, 15 heat-sensitive proteins were decreased in abundance after UHT thermal treatment. Some of the heat-sensitive proteins were connected with the biological immune functionality, suggesting that UHT thermal treatment results in a partial loss of immune function in the whey proteins of dairy cow milk. The information reported here will considerably expand our knowledge about the degree of heat sensitivity in the whey proteins of dairy cow milk in response to different thermal treatments and offer a knowledge-based reference to aid in choosing dairy products. It is worth noting that the whey proteins (lactoperoxidase and lactoperoxidase) in milk that were significantly decreased by high heat treatment in a previous study (142 °C) showed no significant difference in the present study (135 °C). These results may imply that an appropriately reduced heating intensity of UHT retains the immunoactive proteins to the maximum extent possible.

Comparative effect of bovine buttermilk, whey, and lactoferrin on the innate immunity receptors and oxidative status of intestinal epithelial cells.

Milk contains bioactive molecules with important functions as defensive proteins; among them are the whey protein lactoferrin and proteins of the milk fat globule membrane (MFGM) present in buttermilk. The aim of this study has been to investigate the effects of lactoferrin, whey, and buttermilk as modulators of intestinal innate immunity and oxidative stress on intestinal epithelial cells, to evaluate its potential use for the development of functional foods. The mRNA expression levels of innate immune system Toll-like receptors (TLR2, TLR4, and TLR9), lipid peroxidation (malondialdehyde + 4-hydroxyalkenals) and protein expression levels of carbonyl were analyzed in enterocyte-like Caco-2/TC7 cells treated for 24 h with different concentrations of lactoferrin, whey, or buttermilk. None of the substances analyzed caused oxidative damage; however, whey significantly decreased the levels of lipid peroxidation. Furthermore, both lactoferrin and whey reduced the oxidative stress induced by lipopolysaccharide. With respect to TLR receptors, lactoferrin, whey, and buttermilk specifically altered the expression of TLR2, TLR4, and TLR9 receptors, with a strong decrease in the expression levels of TLR4. These results suggest that lactoferrin, whey, and buttermilk are potentially interesting ingredients for functional foods because they seem to modulate oxidative stress and the inflammatory response induced by the activation of TLRs.

Decreased Histone Acetylation Levels at Th1 and Regulatory Loci after Induction of Food Allergy.

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.

Liposomal and CpG-ODN formulation elicits strong humoral immune responses to recombinant Staphylococcus aureus antigens in heifer calves.

Bovine mastitis caused by Staphylococcus aureus is a serious problem in dairy production and effective immunoprophylaxis is an unmet goal so far. The objective of this work was to assess the humoral immune response of heifer calves against two recombinant S. aureus antigens: Clumping factor A (ClfA) and Fibronectin Binding Protein A (FnBPA), formulated with a novel adjuvant based on cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODN). Six groups of 6-8 months old heifer calves received three doses biweekly of antigens, formulated with Al(OH)3, liposomes, CpG-ODN or Lip + CpG-ODN. Animals also received a fourth dose after a year (day 410) and a booster before calving. The administration of Al(OH)3+FnBPA/ClfA and Lip + FnBPA/ClfA + CpG-ODN induced the highest specific IgG levels, after the first 3 doses and induced a fast increase of antibodies after the fourth dose. All the formulations stimulated the production of specific IgG1, after the third and fourth dose. Specific IgG2 for both proteins was only stimulated after the fourth dose by Lip + FnBPA/ClfA + CpG-ODN. Pre-calving immunisation with Lip + FnBPA/ClfA + CpG-ODN led to the highest IgG levels during the calving period and to the production of the IgG2 subclass. The formulation was also able to stimulate the highest antibody levels in milk, 30 and 45 days after pre-calving booster. The combination of liposomes and CpG-ODN as adjuvant for a subunit vaccine, together with the immunisation schedule described, induced a strong humoral immune response with production of specific IgG2. The formulation demonstrated to induce immune memory allowing the application of a single pre-calving booster to maintain high antibody levels throughout the period of increased susceptibility to intramammary infections.

Evaluation of the efficiency of hydrolyzed whey formula to prevent cow's milk allergy in the BALB/c mouse model.

BACKGROUND: Partially hydrolyzed milk formulas have been proposed for primary prevention in at-risk infants, but evidence of their efficiency and elucidation of the underlying mechanisms are still lacking. Thanks to a Th2-biased mouse model mimicking at-risk patients, we aimed to assess the potency of a partially hydrolyzed whey formula (pHWF) to induce oral tolerance thus preventing further cow's milk (CM) allergy. METHODS: BALB/c mice were gavaged with pHWF, standard milk formula (SF), or vehicle only (PBS+). All mice were then orally sensitized to CM using cholera toxin and further chronically exposed to CM. Humoral (IgE, IgG1, IgG2a) and cellular (Th2/Th1/Th17 cytokine secretion; frequency of CD4+GATA3+ and CD4+CD25+Foxp3+ T cells in the spleen) responses against ß-lactoglobulin (BLG) and whole caseins (CAS) were assessed, as well as a marker of elicitation of allergic reaction (mMCP-1) released after an oral challenge with CM. RESULTS: All markers of sensitization and of allergic reaction were evidenced in the PBS+ mice and were significantly enhanced upon chronic exposure. Gavage with SF totally and durably prevented sensitization and elicitation of the allergic reaction. Conversely, pre-treatment with pHWF only reduced BLG-specific sensitization (IgE, Th2 cytokines), with no significant effect on sensitization to caseins. However, pHWF pre-treatment significantly reduced mMCP-1 concentration in plasma after CM challenges. CD4+CD25+Foxp3+ Treg cell frequency could not be correlated with tolerance efficiency. CONCLUSION: Partially hydrolyzed whey formula only partially prevents the further development of CM allergy in this Th2-biased model. A hydrolysate from both whey and casein fractions may be more efficient.

IL-10 Receptor or TGF-ß Neutralization Abrogates the Protective Effect of a Specific Nondigestible Oligosaccharide Mixture in Cow-Milk-Allergic Mice.

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 ß (TGF-ß) 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-ß, 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-ß 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-ß 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-ß in CMA mice.

Bodybuilding protein supplements and cow's milk allergy in adult.

Summary: We report a case of a previously healthy 24-year-old man with a 3-month history of gastrointestinal symptoms during exercise and also few minutes after the ingestion of cow's milk (CM) without exercise. He reported the ingestion of a blend of hydrolyzed whey and casein proteins for bodybuilding for the last 2 years. The in vivo tests showed positivity to CM, α-lactalbumin, ß-lactoglobulin and casein extracts, and also to the protein supplement. The serum specific IgE was positive for CM, ß-lactoglobulin and α-lactalbumin. The in vivo and in vitro tests results suggested an IgE-mediated CMA. Adult-onset CMA has been rarely reported, and to our knowledge this is the first case possibly related to bodybuilding supplements. The authors theorize that the presentation of large amounts of proteins in the gastrointestinal tract may favor sensitization.

The mycotoxin deoxynivalenol facilitates allergic sensitization to whey in mice.

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.

Hydrolyzed whey protein prevents the development of food allergy to ß-lactoglobulin in sensitized mice.

Food allergy is an adverse immune response to dietary proteins. Hydrolysates are frequently used for children with milk allergy. However, hydrolysates effects afterwards are poorly studied. The aim of this study was to investigate the immunological consequences of hydrolyzed whey protein in allergic mice. For that, we developed a novel model of food allergy in BALB/c mice sensitized with alum-adsorbed ß-lactoglobulin. These mice were orally challenged with either whey protein or whey hydrolysate. Whey-challenged mice had elevated levels of specific IgE and lost weight. They also presented gut inflammation, enhanced levels of SIgA and IL-5 as well as decreased production of IL-4 and IL-10 in the intestinal mucosa. Conversely, mice challenged with hydrolyzate maintained normal levels of IgE, IL-4 and IL-5 and showed no sign of gut inflammation probably due to increased IL-12 production in the gut. Thus, consumption of hydrolysate prevented the development of clinical signs of food allergy in mice.

Development of ß-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

BACKGROUND: Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. OBJECTIVE: An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. METHODS: Mice were immunized with bovine ß-lactoglobulin, and subsequently the variable domains of dissimilar anti-ß-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. RESULTS: After sensitization with this pool of anti-ß-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa ß-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-ß-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on ß-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. CONCLUSION: Usage of our 'unlimited' source and well-defined pool of ß-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.