Cow's Milk Allergy Patients Research Articles

Fecal Calprotectin Determination in a Cohort of Children with Cow's Milk Allergy.

Cow's milk allergy (CMA) is the most common food allergy among children. An oral food challenge (OFC) remains a mainstay of the diagnosis of CMA, especially for the non-IgE-mediated type; however, this test can be risky and time-consuming. Hence, there is a need to identify biomarkers. Fecal calprotectin (FC) showed variable results, with good reliability and reproducibility in CMA patients. In this prospective study, we enrolled 76 children (aged 5-18 months) with CMA-related gastrointestinal and cutaneous symptoms following guidelines from the European Society of Pediatric Gastroenterology, Hepatology and Nutrition. Clinical assessments included history, physical examination, skin prick tests, and IgE assays. FC levels and the Cow's Milk Related Symptom Score (CoMiSS) were measured in 51 patients pre (T1) and post-diet (T2), with a subgroup analysis of 15 patients with elevated baseline FC (>50 mg/kg). The results showed that FC levels significantly decreased after the elimination diet (median: 30 mg/kg at T1, 16 mg/kg at T2; p < 0.01). In the subgroup with higher FC levels, median values dropped from 90 mg/kg to 33 mg/kg (p < 0.01). CoMiSS also improved (median: 8.50 at T1, 3.00 at T2; p < 0.01). Linear regression analysis showed no correlation between FC values and the CoMiSS at T1 and T2. In conclusion, the reduction in FC value after an elimination diet suggests that it could be considered a possible biomarker of bowel inflammation in CMA patients. Further studies are necessary to confirm these data and to evaluate and standardize the use of FC for diagnosis and follow-up of CMA.

Serum levels of specific IgE to cow's milk and its components as predictors of anaphylaxis in Chinese children with cow's milk allergy.

Cow's milk allergy (CMA) is one of the most common food allergies in young children. As improved diagnostic tools, allergic tests are inconsistent and limited in predicting anaphylaxis. To explore risk factors for anaphylaxis and to determine practical cut-offs for allergic tests in predicting anaphylaxis. This is a prospective cohort study. Children with IgE-mediated CMA were enrolled and divided into three groups (Group 1: non-anaphylaxis; Group 2: GRADE I anaphylaxis; Group 3: GRADE II-IV anaphylaxis that warranted epinephrine). Prick-to-prick tests (PTPs) using fresh cow's milk (CM) were performed. Serum specific IgE (sIgE) against CM and its components, including casein, alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin were measured. The 90% and 95% positive predictive value (PPV) decision points for predicting anaphylaxis were determined. Potential predictors of anaphylaxis were evaluated in logistic regression models. This study included 134 CMA patients with a median age of 14.4 months. The sensitization rate to any CM component was 89%. Group 3 was more likely to be sensitized to multiple CM components and have higher sIgE levels. The 95% PPV diagnostic decision points of casein-sIgE in predicting anaphylaxis was 13.0 kUA/L. For GRADE II-IV anaphylaxis, casein-sIgE ≥ 54.9 kUA/L could provide a PPV of 88.9%. The elevated casein-sIgE level (OR 14.0, P=0.025) and complicating respiratory allergic diseases (OR 4.8, P=0.022) were independent risk factors for GRADE II-IV anaphylaxis. High casein-sIgE levels are strongly associated with CM anaphylaxis. Detection of casein-sIgE may offer an additional value for the prediction of CM anaphylaxis.

Measurement of oxidative stress-related markers in gastro-intestinal damages in Bulgarian pediatric patients

Cow's milk allergy (CMA), affects ~2 - 7.5% of the infants, and results in an immunological response to casein and α-/β-lactalbumins such as: skin rashes, respiratory and gastrointestinal disturbances. Inflammatory bowel disease (IBD), affecting adolescents, is sustained by an impaired immune/ inflammatory response against intestinal microorganisms. Reactive oxygen/nitrogen (ROS/RNS) overproduction in the gastrointestinal tract damages the mucosa in CMA and IBD. The aim of the present study was to determine ROS/RNS and oxidative disturbances in the intestinal mucosa influence in CMA and IBD. We investigated the levels of advanced protein (OPC), protein carbonyls (PCC), nitric oxide (•NO) and total antioxidant capacity (TAC) in blood serum in CMA and IBD patients. The results showed a significant increase in PCC (p &lt; 0.0001), OPC (p &lt; 0.0001), and increased •NO deposition (p &lt; 0.001) and TAC (p &lt; 0.0001) that likely induce oxidative damage in CMA versus IBD patients. In conclusion, the ROS/RNS accumulation, and oxidative damage to the protein skeleton in the colonic mucosa play a key role in the CMA and IBD pathophysiology, especially in the initiation, duration and maintenance of mucosal inflammation.

A personalized approach to the management of breastfed infants with functional gastrointestinal disorders and cow’s milk allergy. The role of nutritional recommendations for a nursing mother

Introduction. Functional gastrointestinal disorders (FGID) in infants are a complex of symptoms from the gastrointestinal tract that cannot be explained by structural or biochemical disorders. To alleviate the symptoms of functional disorders, a set of measures is required, first of all, psychological support for the mother and, of course, rational feeding, which includes maintaining breastfeeding, optimizing the nutrition of the nursing mother. Despite the abundance of professional information on this issue, previous studies demonstrate a low level of women’s awareness of nutrition during breastfeeding. The aim of the work is to determine the effectiveness of recommendations on nursing mother’s nutrition in the complex treatment of breastfed children with FGID and allergy to cow’s milk proteins.&#x0D; Materials and methods. Seventy five mother–child pairs were included in the study. 1–4 months infants exclusively breastfed during the study were divided into two groups: 1 — FGID infants (n = 51) and 2 — infants with suspected cow’s milk allergy (CMA) n = 24. In addition to the comprehensive treatment of the child, mothers of both groups were given recommendations on the nursing woman’s nutrition. Mothers of children from group 2 were assigned a diagnostic dairy-free diet for a period of 1 month. &#x0D; Results. On the 2nd visit, when analyzing the symptoms of the FGID in group 1, the number of children with symptoms of regurgitation of more than 2 episodes per day decreased by 28% (p &lt; 0.001), crying for more than 3 hours per day — by 52% (p &lt; 0.001). Children of group 2 with a clear positive effect from the diagnostic dairy-free diet therapy of the mother (n = 20) were diagnosed with Allergic and alimentary gastroenteritis and colitis (K52.2 — 10th revision of ICD-10), the continuation of the dairy-free diet for the mother and the supervision of an allergist were prescribed. The survey of women showed that all nursing women were interested in nutrition issues during lactation, 92% of the surveyed mothers searched for this information on the Internet. At the same time, only 12% of women considered this information to be sufficient. Evaluating the presented materials on nutrition, the overwhelming majority of respondents replied that they helped them to adjust their diet and expand nutrition, as well as to maintain breastfeeding.&#x0D; Conclusion. Thus, an integrated approach in the management of FGID infants and gastrointestinal manifestations of food allergies, including the use of nutrition recommendations developed in the National Medical Research Center of Children’s Health for nursing women, allows achieving a positive effect on the side of symptoms both in FGID and CMA patients, and also contributes to the improvement of the psycho-emotional state of a woman and prolonged breastfeeding.

One-Step Purification of IgE Epitope-Specific Antibody Using Immunomagnetic Beads and Highly Sensitive Detection of Bovine β-Lactoglobulin for the Prediction of Milk Allergenicity in Foods.

Bovine β-lactoglobulin (BLG) is a common allergen found in milk, and the immunoglobulin E (IgE) epitope plays a crucial role in cow milk allergy. Therefore, targeting the IgE epitope could be useful in accurately detecting BLG and assessing its allergenicity. However, producing an IgE epitope-specific antibody (IgE-EsAb) through traditional methods requires complex and time-consuming procedures. Here, IgE-EsAb was purified from rabbit anti-BLG sera by immunomagnetic beads in one step. Then, a sandwich ELISA (sELISA) based on the IgE-EsAb was developed to detect BLG and predict the potential milk allergenicity in foods. The obtained IgE-EsAb could specifically recognize the target IgE epitope of BLG and exhibited high affinity and specificity. The developed IgE-EsAb-based sELISA demonstrated an ultra-wide linear range of 3.9-1.28 × 105 ng/mL, with a limit of detection of 0.49 ng/mL for BLG. Additionally, the proposed immunoassay showed high specificity and recoveries (91.24-109.61%). The ability of the IgE-EsAb-based sELISA to evaluate the potential milk allergenicity in foods was validated using sera from cow milk allergy patients. These results suggest that immunomagnetic beads are an effective tool for rapidly obtaining the IgE-EsAb, and our proposed sELISA could be a reliable and user-friendly method for monitoring trace amounts of BLG and predicting the potential milk allergenicity of food samples.

What is the tolerance age of patients with cow's milk protein allergy? What are the parameters that can predict its persistence?

Objective: Cow’s milk allergy (CMA) is one of the most common food allergies in infants. The natural history of CMA indicates that resolution is common. Some studies suggest that food allergies are now being resolved at an older age. The aim of this study was to investigate the tolerance and persistence characteristics, as well as the timing of tolerance, in our own CMA patients. Material and Methods: Patients who were diagnosed with CMA and followed up in our pediatric immunology and allergy outpatient clinic between 2016-2021 years were evaluated retrospectively. Patients’ demographic characteristics, laboratory results, and tolerance time were recorded. Results: The 632 patients’ files were reviewed. 192 (30%) of the patients were followed for more than two years. The tolerance to baked products was observed with a median of 14 months, while the tolerance to fermented products was observed with a median of 17 months. Full tolerance was observed with a median of 25 months. The number of patients older than 2 years of age who could not consume any milk protein-containing product was 72 (11.4%) and 56 (77.7%) of them were IgE mediated CMA. The rate of girls with tolerance (43.5%) was found to be significantly higher than nontolerant girls (25%) (p=0.001). The mean skin test diameter of the tolerant group was significantly higher than the mean of the non-tolerant group (p=0.022). The percentage of eosinophils (p=0.003) and total IgE level (p=0.004) in the tolerant group were significantly lower compared to the intolerant group. There was no significant difference between the presence of tolerance in terms of allergy number, eosinophil count and specific IgE count (p&gt;0.05). Conclusion: Identifying the factors influencing tolerance can lead to changes in the management of the disease. We observed that 88.6% of patients older than 2 years were tolerant to milk proteins, and all patients with non-IgE mediated CMA achieved tolerance by 2 years of age. The ratio of total IgE to specific IgE, age, total IgE and specific IgE levels, presence of non-IgE or IgE mediated allergy, and gender were factors that influenced the development of tolerance in our study. Age at diagnosis and having a single or multiple food allergies did not impact the tolerance status. The prognosis for CMA remains favorable. The use of the milk ladder in managing CMA is an important approach for promoting early tolerance.

Development of a Two-Step Hydrolysis Hypoallergenic Cow's Milk Formula and Evaluation of Residue Allergenicity by Peptidomics and Immunoreactivity Analysis.

Cow's milk allergy (CMA) is an abnormal immune response that severely affects the nutritional supplementation of allergic infants. Currently, only a limited number of hypoallergenic formulas are available on the market, and these are only categorized according to their degree of hydrolysis, which still poses an allergy risk and cannot be consumed by CMA patients, especially infants. To address this issue, we developed a two-step hydrolysis hypoallergenic formula targeting destruction of allergen epitope from whey protein. Then, a comprehensive evaluation system was constructed, including peptidomics analysis, in vivo and in vitro allergenicity assessments, revealing allergic changes in the product from the epitope structure level to the immunological level. The results showed that 97.14% of hydrolyzed peptides from α-lactalbumin and β-lactoglobulin did not contain allergenic epitopes after treatment with trypsin and flavourzyme. In vitro and in vivo allergenicity assessment results confirmed that the two-step hydrolysis method effectively reduced the allergenicity of whey protein. Compared with the common milk powder, the hypoallergenic formula induced lower levels of basophil degranulation and relieved the body's anaphylactic symptoms caused by cow milk. This study provides a promising solution to the limited hypoallergenic formula problem and may benefit allergic infants who require nutritional supplements.

Development of a novel polyclonal antibody against bovine αS1-casein IgE epitopes for prediction of potential allergenicity of milk in foods

ABSTRACT Bovine αS1-casein (αS1-CN) is the main allergen in cow's milk, and immunoglobulin E (IgE) epitopes are the material basis to trigger allergic reactions. Thus, IgE epitopes could be as key targets for precise αS1-CN detection and allergenicity assessment. Here, we designed a tandem containing nine IgE linear epitopes of αS1-CN (tαS1-CN), expressed the recombinant tαS1-CN in Escherichia coli, and prepared the rabbit polyclonal antibody against recombinant tαS1-CN (pAb-tαS1-CN). The obtained recombinant tαS1-CN had a purity of 94.43% and retained the antigenicity and potential allergenicity of IgE epitopes. The pAb-tαS1-CN could recognize the nine IgE epitopes of αS1-CN, and also showed high specificity. The ability of pAb-tαS1-CN to evaluate the potential allergenicity of milk in foods was validated by sera from cow's milk allergy patients. These findings indicate that our prepared pAb-tαS1-CN may represents a valuable tool for the evaluation of milk allergenicity in processed foods.

Bifidobacteria modulate immune response in pediatric patients with cow's milk protein allergy.

In children with an allergy to cow's milk proteins (CMA), the altered composition of intestinal microbiota influences the immune tolerance to milk proteins (CMP). This study aims to investigate the effect of probiotics on the phenotype and activation status of peripheral basophils and lymphocytes in a pediatric CMA cohort. CMA children underwent 45 days of treatment with Bifidobacteria. The basophil degranulation and the immune phenotype of B cells, T helper cells, and regulatory T cells were analyzed in peripheral blood at diagnosis (T0), after a 45-day probiotic treatment (T1), and 45 days after the probiotic wash-out (T2). We observed in probiotic-treated CMA patients a decrease in naive T lymphocytes. Among the CD3+ cell subsets, both naive and activated CD4+ cells resulted markedly reduced after taking probiotics, with the lowest percentages at T2. A decreased basophil degranulation was observed in response to all analyzed CMP at T1 compared to T0. The probiotic treatment resulted in a decrease of circulating naive and activated CD4+ T cells, as well as degranulating basophils. These data suggest that the Bifidobacteria could have a beneficial effect in the modulation of oral tolerance to CMP. ISRCTN69069358. URL of registration: https://www.isrctn.com/ISRCTN69069358 . Probiotic treatment with Bifidobacteria induces a reduction of both naive and activated circulating CD4+ T cells in pediatric patients with cow's milk allergy (CMA). The probiotic supplementation induces a decreased basophil degranulation. The immunological tolerance persists even after 45 days of the probiotic wash-out. Bifidobacteria in vivo supplementation down-modulates the activation of innate and adaptive immunity in pediatric patients with cow's milk allergy. Bifidobacteria contribute to the development of immune tolerance in CMA patients.

The use of amino acid formulas in pediatric patients with allergy to cow's milk proteins: Recommendations from a group of experts.

One of the most common food allergies in children is cow's milk allergy (CMA). In breast-fed infants with CMA, the mother is encouraged to avoid dairy products. If this is not possible, or in formula fed infants, use of hypoallergenic replacement formulas such as extensively hydrolyzed formulas (EHF) is recommended. However, in ∼5% of patients EHFs are not tolerated and/or allergy symptoms can persist. When EHFs are ineffective and in severe forms of CMA, amino acid-based formulas (AAF) should be considered. Six pediatric gastroenterologists with extensive experience in food allergy management reviewed scientific publications and international clinical practice guidelines to provide practical recommendations on AAF. The guidelines reviewed had discrepancies and ambiguities around the specific indications for using formulas as a milk substitute. The panel recommends AAFs as the first therapeutic option in anaphylaxis due to CMA, in acute and chronic severe food protein-induced enterocolitis syndrome, in CMA associated with multiple food allergy, and in cases of eosinophilic esophagitis not responding to an extended exclusion diet or not eating solids. The main benefit of AAF is its absence of residual allergenicity, making it a safe treatment option in severe CMA patients who do not tolerate or respond to an EHF.

Characteristics of cow's milk allergy and sensitization in Chinese patients.

Characteristics of cow's milk allergy and sensitization in Chinese patients.

Cut-Off Values of Specific IgE and Skin Prick Test to Predict Oral Food Challenge Positivity in Children with Cow's Milk Allergy.

The cut-off values for the skin prick test diameters and cow's milk-specific IgE measurements are used to predict the result of the oral food challenge test for the diagnosis of cow's milk allergy. This study aimed to determine the diagnostic values of skin prick test and cow's milk-specific IgE according to age groups and compare the diagnostic powers of these 2 methods. In total, 153 children who had a preliminary diagnosis of cow's milk allergy were evaluated. Group A (n = 90) consisted of cow's milk allergy patients whose diagnosis was confirmed by a positive oral food challenge or a history of anaphylaxis. Group B (n = 63) was composed of patients with a negative oral food challenge. The demographic, clinical, and laboratory findings of 2 groups were compared. The cut-off points for cow's milk-specific IgE and cow's milk-skin prick test were determined as >2.12 kUA/L and >5 mm, respectively. The area under the curve was 0.844 for cow's milk-skin prick test (sensitivity 73%, specificity 84%) and 0.745 for cow's milk-specific IgE (sensitivity 67%, specificity 86%). The diagnostic power of skin prick test was determined to be higher when compared to cow's milk-specific IgE (P = .02). According to the predicted probability curves, decision points for cow's milk-specific IgE and cow's milk-skin prick test with 95% probability were determined as follows, respectively: for ≤24 months: 22 kUA/L, 11.3 mm; for >24 months: 44.1 kUA/, 15.1 mm. The lowest cut-off value with a positive predictive value of 95% and a specificity of 96% was found in patients 3.3 kUA/L) Conclusion: The use of high probability diagnostic values of communities for specific IgE and skin prick test along with a significant clinical history may provide accurate and rapid diagnosis of cow's milk allergy and facilitate patient follow-up.

Is oral food challenge test useful for avoiding complete elimination of cow's milk in Japanese patients with or suspected of having IgE-dependent cow's milk allergy?

BackgroundCow's milk, along with hen's egg, are common causes of food allergies in children worldwide. Accidental ingestion of milk is common and often induces severe allergic reactions. Oral food challenge test (OFC) is usually performed in patients with or suspected of having a food allergy. However, the evidence of whether cow's milk OFC is useful in IgE-dependent cow's milk allergy patients to avoid total elimination is not known. MethodsAfter setting the clinical question and outcomes, we performed a systematic review for relevant articles published from January 1, 2000 to August 31, 2019 using PubMed® and Ichushi-Web databases. Each article was then evaluated for the level of evidence. All positive results of the OFC were defined as adverse events. ResultsForty articles were selected in this study. Our review revealed that cow's milk OFC was able to avoid the complete elimination of cow's milk in 66% of the patients with cow's milk allergy. We also found that adverse events occurred frequently (50.5%). ConclusionsThis analysis supports the recommendation of conducting cow's milk OFC to avoid complete elimination of cow's milk, however the test should be conducted with careful consideration of the patient's safety. As the methods of OFC and subjects varied among the articles selected in this study, further studies are needed to obtain higher quality evidence.

DL-propargylglycine administration inhibits TET2 and FOXP3 expression and alleviates symptoms of neonatal Cows’ milk allergy in mouse model

Background Cows’ milk allergy (CMA) is a hypersensitivity immune reaction brought on by specific immunologic mechanisms to cow’s milk proteins. As one of the most common food allergies in infants, the incidence of CMA during the first year of life is estimated to be nearly 7.5%. Due to the limitation in the knowledge of the pathological mechanism underlying CMA, however, the clinical interventions and therapies remain very unsatisfactory. Aim of the study The transcriptional factor FOXP3 possesses crucial roles in CMA, and increased FOXP3 mRNA expression has a predictive function in faster acquisition of tolerance in infants with CMA. But the exact mechanism remains not fully elucidated. Methods For PAG treatment, PAG (dissolved in saline 30 mg/mL, 0, 5, 10, 20 mg/kg BW) was administered daily intraperitoneally (ip) for one week at the time that 6 weeks after the CMP sensitisation. Results In the present study, we revealed that the expression of FOXP3 is significantly up-regulated in PBMCs from CMA patients and CMA mice on mRNA and protein level. Furthermore, a dramatic reduction in the FOXP3 TSDR methylation and a significant increase in the expression of TET2 are observed in CMA patients and CMA mice. More importantly, we found that propargylglycine (PAG) significantly alleviates symptoms of CMA in mice by suppressing the expression of FOXP3 through restoring TET2 expression. Conclusions Our work revealed a novel function of PAG on CMA, which may provide a deeper insight into the pathomechanism of CMA and a novel therapy target for CMA clinical interventions.

Individual Sensitization Pattern Recognition to Cow's Milk and Human Milk Differs for Various Clinical Manifestations of Milk Allergy.

Cow’s milk allergy (CMA) belongs to one of the most common food allergies in early childhood affecting 2–3% of children under 3 years of age. However, approximately 1% of adults remain allergic to cow’s milk, often showing severe reactions even to traces of milk. In our study, we recruited patients with different clinical manifestations of CMA, including patients with anaphylaxis and less severe symptoms. We assessed the sensitization patterns and allergic responses of these subgroups through different immunological and cell-based methods. Sera of patients were investigated for IgE against whole cow’s milk and its single allergens by CAP- FEIA. In a newly developed in-house multiplex dot assay and a basophil activation test (BAT), cow’s milk allergens, in addition to human breast milk and single allergens from cow’s and human milk were analyzed for IgE recognition and severity of CMA in the included patients. Both the CAP-FEIA routine diagnostic and the multiplex dot test could differentiate CMA with severe from milder allergic reactions by means of the patients’ casein sensitization. The BAT, which mirrors the clinical response in vitro, confirmed that basophils from patients with severe reactions were more reactive to caseins in contrast to the basophils from more moderate CMA patients. By means of this improved component-resolved diagnosis of CMA, individual sensitization patterns could be assessed, also taking sensitization against human milk into consideration.

Higher Polygenetic Predisposition for Asthma in Cow's Milk Allergic Children.

Cow’s milk allergy (CMA) is an early-onset allergy of which the underlying genetic factors remain largely undiscovered. CMA has been found to co-occur with other allergies and immunological hypersensitivity disorders, suggesting a shared genetic etiology. We aimed to (1) investigate and (2) validate whether CMA children carry a higher genetic susceptibility for other immunological hypersensitivity disorders using polygenic risk score analysis (PRS) and prospective phenotypic data. Twenty-two CMA patients of the Dutch EuroPrevall birth cohort study and 307 reference subjects were genotyped using single nucleotide polymorphism (SNP) array. Differentially genetic susceptibility was estimated using PRS, based on multiple P-value thresholds for SNP inclusion of previously reported genome-wide association studies (GWAS) on asthma, autism spectrum disorder, atopic dermatitis, inflammatory bowel disease and rheumatoid arthritis. These associations were validated with prospective data outcomes during a six-year follow-up in 19 patients. We observed robust and significantly higher PRSs of asthma in CMA children compared to the reference set. Association analyses using the prospective data indicated significant higher PRSs in former CMA patients suffering from asthma and related traits. Our results suggest a shared genetic etiology between CMA and asthma and a considerable predictive sensitivity potential for subsequent onset of asthma which indicates a potential use for early clinical asthma intervention programs.

Gut microbiota composition and butyrate production in children affected by non-IgE-mediated cow\u2019s 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.

Altered miR-193a-5p expression in children with cow's milk allergy.

Cow's milk allergy (CMA) is one of the most common food allergies in children. Epigenetic mechanisms have been suggested to play a role in CMA pathogenesis. We have shown that DNA methylation of Th1/Th2 cytokine genes and FoxP3 affects CMA disease course. Preliminary evidence suggests that also the miRNome could be implicated in the pathogenesis of allergy. Main study outcome was to comparatively evaluate miRNome in children with CMA and in healthy controls. Peripheral blood mononuclear cells were obtained from children aged 4-18 months: 10 CMA patients, 9 CMA patients who outgrew CMA, and 11 healthy controls. Small RNA libraries were sequenced using a next-generation sequencing-based approach. Functional assessment of IL-4 expression was also performed. Among the miRNAs differently expressed, 2 were upregulated and 14 were downregulated in children with active CMA compared to healthy controls. miR-193a-5p resulted the most downregulated miRNA in children with active CMA compared to healthy controls. The predicted targets of miR-193a-5p resulted upregulated in CMA patients compared to healthy controls. Peripheral blood CD4+ T cells transfected with a miR193a-5 inhibitor showed a significant upregulation of IL-4 mRNA and its protein expression. Children who outgrew CMA showed miRNA-193a-5p level, and its related targets expression, similar to that observed in healthy controls. Our results suggest that miR-193a-5p is a post-transcriptional regulator of IL-4 expression and could have a role in IgE-mediated CMA. This miRNA could be a novel diagnostic and therapeutic target for this common form of food allergy in childhood.

Oral Food Desensitization in Children With IgE-Mediated Cow's Milk Allergy: Immunological Changes Underlying Desensitization.

PurposeThis study aimed to evaluate the safety and efficacy to induce clinical desensitization to cow's milk (CM) of an oral immunotherapy (OIT) protocol in a pediatric population with cow's milk allergy (CMA). In addition, the immune responses against β-casein, of peripheral blood mononuclear cells (PBMCs) from CMA patients, before and after the protocol were evaluated and compared to a nonallergic population.MethodsA group of 20 children with IgE-mediated CMA and 15 nonallergic children were recruited. Allergic subjects underwent an OIT protocol based on weekly doses of commercial semi-skimmed ultra-high temperature treated (UHT) CM, followed by a maintenance phase. Immune profiles and changes in all subjects were investigated by measuring Th1, Th2, and Treg cytokines, transcription factors, and specific IgE and IgG4 levels.ResultsThe CM-OIT protocol enabled to desensitize 70% of the allergic patients. Successful OIT was accompanied by significant increases in casein-specific IgG4 levels, together with a reduction in the concentration of antigen-specific IgE and in IL-5, IL-13, and IL-10 production by β-casein-stimulated PBMCs. Baseline significant differences observed between allergic and nonallergic children in IL-13 and IL-5 levels were no longer found once the protocol had finished.ConclusionsThe OIT protocol was safe and effective in inducing milk desensitization in 70% of the children with CMA, leading to alterations in their immune profiles toward a nonallergic phenotype.

Assessment of IgE and IgG4 Binding Capacities of Cow's Milk Proteins Selectively Altered by Proteases.

Specific IgE and IgG4 have been reported to play key roles in the context of IgE-mediated cow's milk allergy (CMA), but the persistence of their epitopes in milk hydrolysates has not been evaluated. Using sera from 32 CMA patients, 6 CMA patients treated by epicutaneous therapy (CM-treated), and 4 CM-tolerant peanut allergic patients, we analyzed the IgE and IgG4 binding capacities related to major milk allergens in processed milk. Different proteases (plasmin, chymosin, α-chymotrypsin, or pepsin) were used progressively and selectively to hydrolyze β-lactoglobulin (β-LG) and casein (CN) in milk. We then showed that proteases differentially affect IgE or IgG4 immunoreactivities of CN and β-LG and also that we could not relate IgE and/or IgG4 levels or specificities to milk hydrolysates to the clinical status of the patients.