Region-specific regulation of central histaminergic H3 receptor expression in a mouse model of cow's milk allergy.

Central histaminergic H3 receptor (H3R) has been extensively investigated as a potential therapeutic target for various neurological and neurodegenerative disorders. Despite promising results in preclinical rodent models, clinical trials have not provided conclusive evidence for the benefit of H3R antagonists to alleviate cognitive and behavioral symptoms of these disorders. Inconsistent pharmacological efficacies may arise from aberrant changes in H3R over time during disease development. Because H3R is involved in feedback inhibition of histamine synthesis and secretion, the expression of the autoreceptor may also be reciprocally regulated by altered histamine levels in a pathological condition. Thus, we investigated H3R expression in a mouse model of cow's milk allergy, a condition associated with increased histamine levels. Mice were sensitized to bovine whey proteins (WP) over 5 weeks and H3R protein and transcript levels were examined in the brain. Substantially increased H3R immunoreactivity was observed in various brain regions of WP-sensitized mice compared to sham mice. Elevated H3R expression was also found in the thalamic/hypothalamic region. The expression of histaminergic H1, but not H2, receptor subtype was also increased in this and the midbrain regions. Unlike the brain, all three histaminergic receptors were increased in the small intestine. These results indicated that the central histaminergic receptors were altered in WP-sensitized mice in a subtype- and region-specific manner, which likely contributed to behavioral changes we observed in these mice. Our study also suggests that altered levels of H3R could be considered during a pharmacological intervention of a neurological disease.

Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

Tolerogenic Effect Elicited by Protein Fraction Derived From Different Formulas for Dietary Treatment of Cow's Milk Allergy in Human Cells.

Several formulas are available for the dietary treatment of cow's milk allergy (CMA). Clinical data suggest potentially different effect on immune tolerance elicited by these formulas. We aimed to comparatively evaluate the tolerogenic effect elicited by the protein fraction of different formulas available for the dietary treatment of CMA. Five formulas were compared: extensively hydrolyzed whey formula (EHWF), extensively hydrolyzed casein formula (EHCF), hydrolyzed rice formula (HRF), soy formula (SF), and amino acid-based formula (AAF). The formulas were reconstituted in water according to the manufacturer's instructions and subjected to an in vitro infant gut simulated digestion using a sequential gastric and duodenal static model. Protein fraction was then purified and used for the experiments on non-immune and immune components of tolerance network in human enterocytes and in peripheral mononuclear blood cells (PBMCs). We assessed epithelial layer permeability and tight junction proteins (occludin and zonula occludens-1, ZO-1), mucin 5AC, IL-33, and thymic stromal lymphopoietin (TSLP) in human enterocytes. In addition, Th1/Th2 cytokine response and Tregs activation were investigated in PBMCs from IgE-mediated CMA infants. EHCF-derived protein fraction positively modulated the expression of gut barrier components (mucin 5AC, occludin and ZO-1) in human enterocytes, while SF was able to stimulate the expression of occludin only. EHWF and HRF protein fractions elicited a significant increase in TSLP production, while IL-33 release was significantly increased by HRF and SF protein fractions in human enterocytes. Only EHCF-derived protein fraction elicited an increase of the tolerogenic cytokines production (IL-10, IFN-γ) and of activated CD4+FoxP3+ Treg number, through NFAT, AP1, and Nf-Kb1 pathway. The effect paralleled with an up-regulation of FoxP3 demethylation rate. Protein fraction from all the study formulas was unable to induce Th2 cytokines production. The results suggest a different regulatory action on tolerogenic mechanisms elicited by protein fraction from different formulas commonly used for CMA management. EHCF-derived protein fraction was able to elicit tolerogenic effect through at least in part an epigenetic modulation of FoxP3 gene. These results could explain the different clinical effects observed on immune tolerance acquisition in CMA patients and on allergy prevention in children at risk for atopy observed using EHCF.

Differential Effects of Dry vs. Wet Heating of ß-Lactoglobulin on Formation of sRAGE Binding Ligands and sIgE Epitope Recognition.

The effect of glycation and aggregation of thermally processed ß-lactoglobulin (BLG) on binding to sRAGE and specific immunoglobulin E (sIgE) from cow milk allergic (CMA) patients were investigated. BLG was heated under dry conditions (water activity < 0.7) and wet conditions (in phosphate buffer at pH 7.4) at low temperature (<73 °C) and high temperatures (>90 °C) in the presence or absence of the milk sugar lactose. Nε-(carboxymethyl)-l-lysine (CML) western blot and glycation staining were used to directly identify glycation structures on the protein fractions on SDS-PAGE. Western blot was used to specify sRAGE and sIgE binding fractions. sRAGE binding was highest under wet-heated BLG independent of the presence of the milk sugar lactose. Under wet heating, high-molecular-weight aggregates were most potent and did not require the presence of CML to generate sRAGE binding ligands. In the dry system, sRAGE binding was observed only in the presence of lactose. sIgE binding affinity showed large individual differences and revealed four binding profiles. Dependent on the individual, sIgE binding decreased or increased by wet heating independent of the presence of lactose. Dry heating required the presence of lactose to show increased binding to aggregates in most individuals. This study highlights an important role of heating condition-dependent protein aggregation and glycation in changing the immunogenicity and antigenicity of cow's milk BLG.

Many Patients With Irritable Bowel Syndrome Have Atypical Food Allergies Not Associated With Immunoglobulin E.

BACKGROUND & AIMS: Confocal laser endomicroscopy (CLE) is a technique that permits real-time detection and quantification of changes in intestinal tissues and cells, including increases in intraepithelial lymphocytes and fluid extravasation through epithelial leaks. Using CLE analysis of patients with irritable bowel syndrome (IBS), we found that more than half have responses to specific food components. Exclusion of the defined food led to long-term symptom relief. We used the results of CLE to detect reactions to food in a larger patient population and analyzed duodenal biopsy samples and fluid from patients to investigate mechanisms of these reactions. METHODS: In a prospective study, 155 patients with IBS received 4 challenges with each of 4 common food components via the endoscope, followed by CLE, at a tertiary medical center. Classical food allergies were excluded by negative results from immunoglobulin E serology analysis and skin tests for common food antigens. Duodenal biopsy samples and fluid were collected 2 weeks before and immediately after CLE and were analyzed by histology, immunohistochemistry, reverse transcription polymerase chain reaction, and immunoblots. Results from patients who had a response to food during CLE (CLE+) were compared with results from patients who did not have a reaction during CLE (CLE-) or healthy individuals (controls). RESULTS: Of the 108 patients who completed the study, 76 were CLE+ (70%), and 46 of these (61%) reacted to wheat. CLE+ patients had a 4-fold increase in prevalence of atopic disorders compared with controls (P = .001). Numbers of intraepithelial lymphocytes were significantly higher in duodenal biopsy samples from CLE+ vs CLE- patients or controls (P = .001). Expression of claudin-2 increased from crypt to villus tip (P < .001) and was up-regulated in CLE+ patients compared with CLE- patients or controls (P = .023). Levels of occludin were lower in duodenal biopsy samples from CLE+ patients vs controls (P = .022) and were lowest in villus tips (P < .001). Levels of messenger RNAs encoding inflammatory cytokines were unchanged in duodenal tissues after CLE challenge, but eosinophil degranulation increased, and levels of eosinophilic cationic protein were higher in duodenal fluid from CLE+ patients than controls (P = .03). CONCLUSIONS: In a CLE analysis of patients with IBS, we found that more than 50% of patients could have nonclassical food allergy, with immediate disruption of the intestinal barrier upon exposure to food antigens. Duodenal tissues from patients with responses to food components during CLE had immediate increases in expression of claudin-2 and decreases in occludin. CLE+ patients also had increased eosinophil degranulation, indicating an atypical food allergy characterized by eosinophil activation.

The potential anti-inflammatory role of adiponectin in food allergy: a case-control study on children.

We aimed to assess the possible relationship between food allergy and two key adipokines - leptin and adiponectin - in children with food allergy. A total of forty patients with definite diagnosis of food allergy according to clinical history and specific IgE (sIgE) for food allergens (group I) were enrolled in this pilot study. The control group (group II) included thirty children with no evidence of allergic symptoms. Serum levels of leptin and adiponectin were measured by ELISA. Meanwhile, sIgE was measured for the eight most common food allergens by the immunoblot method in all participants. The median ages in groups I and II were 18·5 and 23·5 months, respectively. The respective Caesarean section rate was 64·9 and 16·7 % in groups I and II (P<0·001). Serum levels of adiponectin were significantly higher in the patient group compared with controls (24·11 (sd 12·14) v. 10·67 (sd 12·23) µg/ml, P<0·001), whereas no statistically meaningful difference was detected in serum leptin concentrations (P=0·92). There was a significant inverse relationship between age and adiponectin levels in group I (P=0·002, r -0·479) and group II (P=0·04, r -0·365), and it was more significant in group I. The most common allergens in the patient group were wheat (52·5 %), hazelnut (52·5 %), cow's milk (50 %) and egg white (30 %). The results of this study suggest an essential link between adiponectin and food allergy that is probably unlikely to be affected by obesity as a confounding factor.

Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA.

The whey protein ß-lactoglobulin (BLG) is a major milk allergen which is absent in human milk. Here, we for the first time generated DNA-free BLG bi-allelic knockout cow by zinc-finger nuclease (ZFNs) mRNA and produced BLG-free milk. According to the allergenicity evaluation of BLG-free milk, we found it can trigger lower allergic reaction of Balb/c mice including the rectal temperature drop and the allergen-specific immunoglobulin IgE production; BLG free-milk was easily digested by pepsin at 2 min, while BLG in control milk was still not completely digested after 60 min, and the binding of IgE from cow's milk allergy (CMA) patients to BLG free-milk was significantly lower than that to the control milk. Meanwhile, the genome sequencing revealed that our animal is free of off-target events. Importantly, editing animal genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to foods produced by genome edited animals. Finally, the ZFNs-mediated targeting in cow could be transmitted through the germline by breeding. These findings will open up unlimited possibilities of modifying milk composition to make it more suitable for human health and also improve the functional properties of milk.

Oral exposure to the free amino acid glycine inhibits the acute allergic response in a model of cow's milk allergy in mice.

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.

Gut microbiota composition and butyrate production in children affected by non-IgE-mediated cow's milk allergy.

Cow's milk allergy (CMA) is one of the earliest and most common food allergy and can be elicited by both IgE- or non-IgE-mediated mechanism. We previously described dysbiosis in children with IgE-mediated CMA and the effect of dietary treatment with extensively hydrolyzed casein formula (EHCF) alone or in combination with the probiotic Lactobacillus rhamnosus GG (LGG). On the contrary, the gut microbiota in non-IgE-mediated CMA remains uncharacterized. In this study we evaluated gut microbiota composition and fecal butyrate levels in children affected by non-IgE-mediated CMA. We found a gut microbiota dysbiosis in non-IgE-mediated CMA, driven by an enrichment of Bacteroides and Alistipes. Comparing these results with those previously obtained in children with IgE-mediated CMA, we demonstrated overlapping signatures in the gut microbiota dysbiosis of non-IgE-mediated and IgE-mediated CMA children, characterized by a progressive increase in Bacteroides from healthy to IgE-mediated CMA patients. EHCF containg LGG was more strongly associated with an effect on dysbiosis and on butyrate production if compared to what observed in children treated with EHCF alone. If longitudinal cohort studies in children with CMA will confirm these results, gut microbiota dysbiosis could be a relevant target for innovative therapeutic strategies in children with non-IgE-mediated CMA.

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.

PLGA nanoparticles loaded with beta-lactoglobulin-derived peptides modulate mucosal immunity and may facilitate cow's milk allergy prevention.

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-ß 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.

Partially hydrolyzed whey proteins prevent clinical symptoms in a cow's milk allergy mouse model and enhance regulatory T and B cell frequencies.

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.

Eliciting Th1 Immune Response Using Casein- (Alpha S1) loaded Dendritic Cells.

Allergen-specific immunotherapy (AIT) has been recently considered as an alternative approach to ameliorate the symptoms of allergen exposure and improvement the patients' quality of life. Dendritic cells (DC) in the forms of tolerogenic or Th1-induced cells have been investigated in several studies as one of the promising approaches of AIT in allergic diseases. The aim of this study was to evaluate the potency of casein-loaded DCs in eliciting the Th1 immune responses in Balb/c mice as a potential therapeutic approach in allergic condition. Immature bone marrow-derived DCs were loaded with casein (protein or mRNA) or green fluorescent protein (GFP) mRNA. DCs were evaluated based on the expression of specific markers and production of proinflammatory cytokines.  Expression of DC markers in all groups was significantly higher than immature DCs, but lower than LPS-activated DCs. Despite an increase in TNF-α and IL-12, IL-6 was decreased in casein-DC treatments. Caseinloaded DCs could induce proliferation in lymphocytes and stimulate them to produce higher amounts of IFN-γ and in some extent IL-10 and TGF-ß, while they could not stimulate IL-4 secretion. Casein-loaded DCs could partially elicit the Th1 responses; this would be a promising approach to use them as an allergic protective way for applying immune cell therapy in cow's milk allergy.

Assessment of IgE Reactivity of ß-Casein by Western Blotting After Digestion with Simulated Gastric Fluid.

Cow's milk allergy is defined as an immunologically mediated adverse reaction to cow's milk proteins and it is usually, along with hen's egg allergy, the first food allergy identified in childhood.One of the main aspects to consider when evaluating the allergenic potential of food proteins is the effect of gastric digestion. It is known that allergens are usually able to survive the harsh acidic environment of the stomach, tolerate the presence of surfactants, and resist digestion by pepsin. They might also be digested into high molecular weight peptide fragments, which retain the same, or sometimes increased, IgE-binding. In this respect, western blotting is a highly sensitive and efficient technique that we have used to detect IgE-binding to the digests of milk and egg proteins. Given the importance of the resistance of food proteins to gastric digestion in their capacity to modulate the immune response, we describe in this chapter the assessment of IgE reactivity of a relevant cow's milk allergen, ß-casein, by western blotting after simulated digestion under relevant physiological conditions.

Interleukin-13 suppresses interleukin-10 via inhibiting A20 in peripheral B cells of patients with food allergy.

The regulatory B cells (Breg) are important in the body immunity. The differentiation process of Breg is not fully understood yet. Ubiquitin A20 has immune regulatory functions. This study aims to investigate the role of A20 in the regulation of interleukin (IL)-10 in B cells. In this study, B cells were isolated from the peripheral blood samples of healthy subjects and patients with food allergy (FA). The B cells were analyzed by flow cytometry, real time RT-PCR, Western blotting and chromatin immunoprecipitation. We observed that the frequency of Breg and the levels of A20 in B cells were markedly lower in FA patients than in healthy controls. In vitro deletion of A20 compromised the expression of IL-10. B cells in FA patients showed higher levels of histone deacetylase (HDAC)-11 than in healthy subjects. Exposure to IL-13 in the culture induced high levels of HDAC11 in B cells. IL-13 also repressed the expression of A20 in B cells, in which HDAC11 played a critical role via inducing the chromatin remoldeling at the IL-10 promoter locus. Mice with A20-deficient B cells are prone to FA. In summary, ubiquitin A20 can increase the IL-10 expression in B cells, which can be affected by the IL-13-induced HDAC11. To inhibit HDAC11 may have therapeutic potential for FA.

Mesenteric IL-10-producing CD5+ regulatory B cells suppress cow's milk casein-induced allergic responses in mice.

Food allergy is a hypersensitive immune reaction to food proteins. We have previously demonstrated the presence of IL-10-producing CD5(+) B cells and suggested their potential role in regulating cow's milk casein allergy in humans and IgE-mediated anaphylaxis in mice. In this study, we determined whether IL-10-producing CD5(+) regulatory B cells control casein-induced food allergic responses in mice and, if so, the underlying mechanisms. The induction of oral tolerance (OT) by casein suppressed casein-induced allergic responses including the decrease of body temperature, symptom score, diarrhea, recruitment of mast cells and eosinophils into jejunum, and other biological parameters in mice. Notably, the population of IL-10-producing CD5(+) B cells was increased in mesenteric lymph node (MLN), but not in spleen or peritoneal cavity (PeC) in OT mice. The adoptive transfer of CD5(+) B cells from MLN, but not those from spleen and PeC, suppressed the casein-induced allergic responses in an allergen-specific and IL-10-dependent manner. The inhibitory effect of IL-10-producing CD5(+) B cells on casein-induced allergic response was dependent on Foxp3(+) regulatory T cells. Taken together, mesenteric IL-10-producing regulatory B cells control food allergy via Foxp3(+) regulatory T cells and could potentially act as a therapeutic regulator for food allergy.

Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut.

Evaluation of the antigenicity of hydrolyzed cow's milk protein formulas using the mouse basophil activation test.

Hypoallergenic infant formulas are widely used for infants with cow's milk allergy. The aim of this study was to assess the utility of the mouse basophil activation test (BAT) in the evaluation of residual antigenicity in these formulas. Whole blood samples derived from ß-lactoglobulin- or casein-immunized mice were incubated with one of the following formulas: conventional, partially hydrolyzed, or extensively hydrolyzed. Basophilic activation was analyzed by flow cytometry using an IgE-dependent activation marker CD200R1 and an IgG-dependent activation marker CD200R3. Systemic anaphylaxis was induced by i.v. injection of milk formula and results were compared. Conventional formula induced pronounced changes in CD200R1 and CD200R3 expression on basophils, whereas extensively hydrolyzed formulas did not elicit any changes in these markers. Similarly, challenge with conventional formula induced anaphylaxis, whereas extensively hydrolyzed formulas did not induce anaphylaxis. Although the partially hydrolyzed formula also induced basophilic activation and systemic anaphylaxis, the magnitude of these effects was smaller than that observed with the conventional formula. Compared to CD200R1, the observed trend in CD200R3 expression resembled the results obtained from systemic anaphylaxis test more closely. These findings show that mouse BAT, in particular using CD200R3, is highly useful for the evaluation of antigenicity of milk formulas.

Histopathologic findings in children diagnosed with cow's milk protein allergy.

BACKGROUND: Cow's milk protein allergy is the most common cause of food allergy. The challenge test, either open or doubled-blind with a placebo control, is regarded as the criterion standard. Endoscopy and histologic findings are considered a method that can aid in the diagnosis of this entity. AIMS: The aim of this study was to describe the histopathologic findings in children suspected of cow's milk protein allergy that were seen at our hospital. MATERIAL AND METHODS: A descriptive, observational study was conducted on 116 children clinically suspected of presenting with cow's milk protein allergy that were seen at the Department of Gastroenterology and Nutrition of the Instituto Nacional de Pediatría. Upper endoscopy and rectosigmoidoscopy with biopsies were performed and the findings were described. RESULTS: Of the 116 patients, 64 (55.17%) were girls and 52 (44.83%) were boys. The rectum was the site with the greatest presence of eosinophils per field in both groups, followed by the duodenum. In general, more than 15 eosinophils were found in 46% of the patients. CONCLUSIONS: Between 40 and 45% of the cases had the histologic criterion of more than 15 to 20 eosinophils per field and the rectosigmoid colon was the most affected site. Therefore, panendoscopy and rectosigmoidoscopy with biopsy and eosinophil count are suggested.

Successful oral tolerance induction to cow’s milk in a child with allergy to extensively hydrolysed formula

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