Development of a low allergenic product for patients with milk allergy and assessment of its specific IgE reactivity.

BACKGROUND: Milk oral immunotherapy is the riskiest and most unpredictable form of oral immunotherapy. We aimed to produce a low allergenic product than conventional once baked-cake/muffin, to develop indirect in-house ELISA to check the tolerance status with milk products and evaluate IgE reactivity of patients' sera via western blotting (WB) and indirect in-house ELISA. METHOD: A low allergenic product named biscotti-twice baked-cake was developed, and the total protein concentration was determined. The protein content was studied by SDS-PAGE and proteomics. Milk-specific IgE (sIgE) binding assays were performed by WB and indirect in-house ELISA by using patients' sera. RESULTS: Casein band intensity was observed to be lower in the biscotti-twice baked-cake than in the once baked-cake (p = .014). Proteomics analysis and αS1-casein measurement showed that the lowest intensity of casein was found in biscotti. The low binding capacity of milk sIgE to biscotti compared with once baked-cake was shown by WB (p = .0012) and by indirect in-house ELISA (p = .0001). In the ROC analysis, the area under the curve (AUC) of the in-house ELISA IgE was comparable with Uni-CAP milk and casein sIgE. The AUC of the in-house ELISA IgE for cake (0.96) and biscotti (1) was slightly better than Uni-CAP milk sIgE (0.94; 0.97) and casein sIgE (0.96; 0.97), respectively. CONCLUSION: The low allergenicity of the newly developed low allergenic product "biscotti-twice baked-cake" has been demonstrated by in vitro experiments. Biscotti could be a safe treatment option than once baked-cake/muffin in patients who are reactive to once baked-milk products.

[Influence of lactoferrin and enzymatiс hydrolysates of cow's and mare's milk proteins on anaphylactic sensitivity and cytokine profile of rats].

The development of innovative ingredients of specialized formula for children with intolerance to cow's milk proteins requires accounting the influence of the protein component on the allergic sensitivity. The aim of the research was to study the effect of lactoferrin (LF) from cow colostrum, cow's milk protein hydrolysate (CMPH) and mare's milk protein hydrolysate (MMPH) on the severity of the systemic anaphylaxis reaction, the levels of specific IgG antibodies and cytokines in rats parenterally sensitized with ovalbumin (OVA). Material and methods. The experiment was carried out on 4 groups of 26 male Wistar rats, which were sensitized intraperitoneally with chicken egg OVA and a systemic anaphylaxis reaction was induced on the day 29 by intravenous administration of a challenge dose of the antigen (6 mg per kg body weight). LF, CMPH and MMPH were introduced into the diet in doses of 1.4-2 g/kg body weight per day (on an average 1.59±0.04, 1.53±0.05 and 1.48±0.05 g per kg body weight respectively). The content of IgG antibodies in the blood serum was determined by an indirect ELISA; the levels of cytokines IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, GM-CSF, IFN-γ and TNF-α were detected by multiplex immunoassay. Results. Dietary LF did not have a significant effect on the severity of active anaphylactic shock (AAS), concentrations of antibodies and cytokines in the blood of animals. As a result of CMPH consumption, there were no significant changes in AAS severity and IgG antibodies to OVA but significant increase in TNF-α level was observed as well as a significant decrease in IL-1α (p<0.05). In animals receiving MMPH, there were also no significant changes in the severity of AAS, but a 1.9-fold decrease in the level of IgG antibodies to OVA (p<0.001) was noticed along with a significant increase in IL-12(p70) (p<0.05) and IL-10 (p<0.10) level. Conclusion. Cosumption of LF by sensitized rats didn't significantly affect their anaphylactic sensitivity and cytokine profile, while CMPH intake induced some signs of pro-inflammatory processes. Consumption of MMPH was accompanied by the formation of an anti-inflammatory cytokine profile, which corresponds to a decrease in the intensity of the humoral immune response to the model allergen. Differences in the effects of two hydrolisates, which have similar degrees of hydrolysis, may be associated with the specific composition of glycopeptides formed during the enzymatic cleavage of milk protein produced by these two species of dairy animals.

Basophil allergen threshold sensitivity to casein (casein-specific CD-sens) predicts allergic reactions at a milk challenge in most but not all patients.

BACKGROUND: The basophil activation test is an emerging clinical tool in the diagnosis of cow's milk allergy (CMA). The aim was to assess the association between the basophil allergen threshold sensitivity to the major milk protein casein (casein-specific CD-sens), the levels of milk- and casein-specific Immunoglobulin E antibodies (IgE-ab), and the severity of allergic reactions at milk challenges. METHODS: We enrolled 34 patients aged 5-15 (median 9) years who underwent a double-blind placebo-controlled milk-challenge (DBPCMC) as screening before inclusion in an oral immunotherapy study for CMA. The severity of the allergic reaction at the DBPCMC was graded using Sampson's severity score. Venous blood was drawn before the DBPCMC. Milk- and casein-specific IgE-ab were analyzed. Following in vitro stimulation of basophils with casein, casein-specific CD-sens, was determined. RESULTS: Thirty-three patients completed the DBPCMC. There were strong correlations between casein-specific CD-sens and IgE-ab to milk (rs = 0.682, p < .001), and between casein-specific CD-sens and IgE-ab to casein (rs = 0.823, p < .001). There was a correlation between the severity of the allergic reaction and casein-specific CD-sens level (rs = 0.395, p = .041) and an inverse correlation between casein-specific CD-sens level and the cumulative dose of milk protein to which the patient reacted at the DBPCMC (rs = -0.418, p = .027). Among the 30 patients with an allergic reaction at the DBPCMC, 67% had positive casein-specific CD-sens, 23% had negative casein-specific CD-sens, and 10% were declared non-responders. CONCLUSION: Two thirds of those reacting at the DBPMC had positive casein-specific CD-sens, but reactions also occurred despite negative casein-specific CD-sens. The association between casein-specific CD-sens and the severity of the allergic reaction and cumulative dose of milk protein, respectively, was moderate.

Clinical characteristics, laboratory findings, and tolerance acquisition in infants with cow's milk protein allergy in a private center in Lima, Peru for the period 2021-2022.

BACKGROUND: Cow's milk protein allergy (CMPA) remains relatively understudied in Latin America. METHODS: In this observational study, we enrolled 64 patients with a median age of 3 months, of whom 60% were male. Patients included had a history of IgE-mediated reactions with IgE sensitization or non-IgE-mediated reactions or symptoms following exposure to cow's milk. They underwent skin prick test, ImmunoCAP, fecal calprotectin (FC), and fecal eosinophil-derived neurotoxin (EDN), in addition to double-blinded placebo-controlled oral food challenges (DBPCFC), with clinical evolution and tolerance acquisition observed over 1 year. RESULTS: Malnutrition was present in 78.1% of patients, and 87.5% had a family history of atopy, with 51.6% receiving exclusive breastfeeding. Gastrointestinal manifestations were prevalent in 90.6% of patients, followed by dermatological manifestations (10.9%), with only 2 experiencing anaphylaxis. IgE-mediated CMPA was observed in only six patients. In those with non-IgE-mediated CMPA, FC had a median of 284 mg/dL (IQR: 138.5-415.5), while EDN had a median of 508.5 mg/dL (IQR: 160.25-868). One year after diagnosis, median FC significantly decreased (p < 0.0001), and malnutrition prevalence reduced to 17.1%. Moreover, 81% of patients acquired tolerance following DBPCFC, with 52% utilizing nutritional replacement formulas at diagnosis. Notably, 94% of those extensively hydrolyzed casein-based formulas achieved tolerance (p = 0.08). CONCLUSION: Our findings provide a foundational framework for future investigations into CMPA diagnosis, tolerance acquisition, and the utilization of hypoallergenic formulas tailored to the unique characteristics of our region.

The association of milk and multiple food avoidance with growth parameters in infants and children.

BACKGROUND: Recent studies reported that strict avoidance of milk products in cow's milk allergy (CMA) affects growth and bone turnover, causing negative calcium balance and changes in bone metabolism. OBJECTIVE: To investigate biochemical parameters to predict bone turnover and its relations with height and weight measurements and nutritional intake. METHODS: Height, weight, and body mass index z scores were plotted for age according to the World Health Organization. A 3-consecutive day food record was analyzed for nutritional values of foods. The blood levels of calcium, phosphorus, alkaline phosphatase, vitamin D, and parathyroid hormone (PTH) were determined. RESULTS: The study included 69 controls, 66 children with isolated CMA, and 59 children with multiple food allergy (FA). The z scores for weight, height, and body mass index were lower in isolated CMA and multiple FA groups than controls (P < .001, P = .004, and P = .002, respectively). The nutritional intakes of protein, fat, carbohydrates, vitamins B2 and B12, niacin, calcium, and phosphorus were significantly lower in isolated CMA and multiple FA than controls. In infants (≤2 years of age), although blood calcium level was in normal range, it was significantly lower in isolated CMA and multiple FA than in controls (P < .001). In children older than 2 years, PTH level was significantly higher in isolated CMA and multiple FA groups than in controls (P = .003). CONCLUSION: Our study revealed that children with isolated CMA and multiple FA had a high nutrition gap, growth deceleration, and unbalanced bone metabolism, as illustrated by low blood calcium and elevated PTH levels.

Metabolomics identifies phenotypic biomarkers of amino acid metabolism in milk allergy and sensitized tolerance.

BACKGROUND: A substantial proportion of sensitized individuals tolerate suspected foods without developing allergic symptoms; this phenomenon is known as sensitized tolerance. The immunogenic and metabolic features underlying the sensitized-tolerant phenotype remain largely unknown. OBJECTIVE: We aimed to uncover the metabolic signatures associated with clinical milk allergy (MA) and sensitized tolerance using metabolomics. METHODS: We characterized the serum metabolic and immunologic profiles of children with clinical IgE-mediated MA (n = 30) or milk-sensitized tolerance (n = 20) and healthy controls (n = 21). A comparative analysis was performed to identify dysregulated pathways associated with the clinical manifestations of food allergy. We also analyzed specific biomarkers indicative of different sensitization phenotypes in children with MA. The candidate metabolites were validated in an independent quantification cohort (n = 41). RESULTS: Metabolomic profiling confirmed the presence of a distinct metabolic signature that discriminated children with MA from those with milk-sensitized tolerance. Amino acid metabolites generated via arginine, proline, and glutathione metabolism were uniquely altered in children with sensitized tolerance. Arginine depletion and metabolism through the polyamine pathway to fuel glutamate synthesis were closely associated with suppression of clinical symptoms in the presence of allergen-specific IgE. In children with MA, the polysensitized state was characterized by disturbances in tryptophan metabolism. CONCLUSIONS: By combining untargeted metabolomics with targeted validation in an independent quantification cohort, we identified candidate metabolites as phenotypic and diagnostic biomarkers of food allergy. Our results provide insights into the pathologic mechanisms underlying childhood allergy and suggest potential therapeutic targets.

Development of a light-initiated chemiluminescence assay for the quantitative detection of cow's milk allergen-specific IgE.

BACKGROUND: Cow's milk allergy is a common food allergy in children. Clinically, cow's milk-specific IgE (CM-sIgE) antibody test is often used to diagnose milk allergy. An inexpensive light-initiated chemiluminescence assay (LICA), with fast detection speed and small sample volume demand, has application prospects in the field of quantitative detection of CM-sIgE. METHODS: Chemibeads coated with five major milk allergens, serum samples, biotinylated anti-human IgE antibodies, and streptavidin-coated sensibeads constitute a system to establish a LICA method for the quantitative detection of CM-sIgE. A series of experiments were performed to optimize its reaction conditions and evaluated its performance. RESULTS: The optimal conditions for LICA were 10:4 mass ratio of chemibeads to milk allergen, 20 µg/mL chemibeads, 1.0 µg/mL biotinylated anti-human IgE antibodies and a 1/10 dilution of serum for 30-min incubation. The limit of Quantitation (LoQ) was 0.22 kUA/L. For repeatability, the CV ranged from 3.71% to 8.11%. For intermediate precision, the CV ranged from 4.08% to 14.71%. It was linear within 0.20-18.20 kUA/L. This method did not interfere with common interfering substances and total IgE in serum, and there was no obvious cross-reaction with milk-specific IgG and non-milk-specific IgE. CONCLUSION: We have established a method to quantitatively detect CM-sIgE based on light-initiated chemiluminescence assay, which has good analytical performance and could meet the needs of clinical laboratories.

Successful Milk Oral Immunotherapy Promotes Generation of Casein-Specific CD137+ FOXP3+ Regulatory T Cells Detectable in Peripheral Blood.

Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (TREG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific TREG in a cohort of subjects undergoing OIT. Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4+ T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4+ T cell phenotypes were quantified by flow cytometry. Results: Our culture system induced activated casein-specific FOXP3+Helios+ TREG cells and FOXP3- TEFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific TREG cells increased significantly with escalating doses of milk during OIT while casein-specific TEFF cell frequencies remained constant. Moreover, expanded casein-specific TREG cells expressed higher levels of FOXP3 compared to polyclonal TREG cells, suggesting a more robust TREG phenotype. The induction of casein-specific TREG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific TREG cells negatively correlated with the time required to reach the maintenance phase of desensitization. Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3+ TREG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro TREG response to casein may correlate with the time to reach maintenance in CMP-OIT.

Chronic Milk-Dependent Food Protein-Induced Enterocolitis Syndrome in Children from West Pomerania Region.

Characteristics of chronic milk-dependent food protein-induced enterocolitis syndrome (FPIES) in children from the region of Western Pomerania were studied. Prospectively, 55 children were diagnosed at a median of 2.2 months. The open food challenges (OFC), morphologies, milk-specific IgE (sIgE) (FEIA method, CAP system), and skin prick tests (SPTs) were examined. Vomiting and diarrhea escalated gradually but quickly led to growth retardation. Of the infants, 49% had BMI < 10 c, 20% BMI < 3 c; 25% had anemia, and 15% had hypoalbuminemia. During the OFCs we observed acute symptoms that appeared after 2-3 h: vomiting diarrhea and pallor. A total of 42% children required intravenous hydration. Casein hydrolysates or amino acids formulae (20%) were used in treatment. In 25% of children, SPT and milk sIgE were found, in 18%-other food SPTs, and in 14% allergy to other foods. A transition to IgE-dependent milk allergy was seen in 3 children. In the twelfth month of life, 62% of children had tolerance to milk, and in the twenty-fifth month-87%. Conclusions. Chronic milk-dependent FPIES resolves in most children. By the age of 2 children are at risk of multiple food sensitization, and those who have milk sIgE are at risk to transition to IgE-mediated milk allergy. Every OFC needs to be supervised due to possible severe reactions.

A light-initiated chemiluminescent assay for the detection of children's milk protein-specific IgE with excellent ability to avoid interference of specific IgG.

The detection of allergen-specific IgE is of value for the diagnosis of children''s milk allergy. However, its accuracy will interfere with the presence of high levels of specific IgG in the serum of children with milk allergy. To solve this problem, we established a light-initiated chemiluminescent assay (LICA) based on nanomicrospheres, which neutralized the interference of specific IgG by increasing the amount of antigen coated on the microspheres. The ability of this method to resist IgG interference was confirmed by adding extra specific IgG to the serum of allergic patients. Finally, the positive rate of allergen-specific IgE was increased to 85%, which was better than the indirect ELISA (70%), indicating that this method has certain advantages for the detection of specific IgE in children with milk allergy.

Selenium Modulates the Allergic Response to Whey Protein in a Mouse Model for Cow's Milk Allergy.

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.

Assessment of Inflammatory Markers in Children with Cow's Milk Allergy Treated with a Milk-Free Diet.

BACKGROUND: The aim of the study was to establish whether the use of a strict milk-free diet in children with cow's milk allergy, resulting in the resolution of clinical symptoms of the disease, also extinguishes the inflammatory reaction induced by the allergy. METHODS: We examined 64 children (aged 3-6 years) with a diagnosed cow's milk allergy who had been treated with an elimination diet for at least six months and showed remission of the disease's clinical symptoms as a result of the treatment. The control group consisted of 30 healthy children of the same age following an unrestricted age-appropriate diet. Concentrations of cytokines, calprotectin, and adipokines (leptin, resistin, chemerin, neutrophilic lipocalin associated with gelatinase-NGAL) were determined in the serum samples obtained from the studied children by immunoenzymatic assays. RESULTS: Patients with CMA had significantly higher median values of serum IL-6, TNF-α, resistin, chemerin and NGAL in comparison to the healthy children (p < 0.05, p < 0.001, p < 0.05, p < 0.01, p < 0.001, respectively). Serum concentrations of IL-10, leptin, calprotectin and CRP as well as in WBC count were in the same range in both studied groups. We observed direct statistically significant correlations between levels of IL-10 and CRP (p = 0.005), IL-10 and WBC (p = 0.045), TNF-α and WBC (p = 0.038), calprotectin and WBC (p < 0.001), chemerin and CRP (p < 0.001) as well as between NGAL and WBC (p = 0.002) in children with CMA. CONCLUSION: The use of a strict milk-free diet by children with CMA, resulting in the resolution of clinical symptoms of the disease, does not seem to extinguish the inflammation induced by the allergy. The findings of this study-elevated IL-6, TNF-α, resistin, chemerin and NGAL levels in patients with CMA-suggest that these parameters seem to be involved in the generation of a low-grade proinflammatory environment observed in cow's milk allergy and could be used to monitor the effectiveness of treatment.

Evaluation of some predictive parameters for baked-milk tolerance in children with cow's milk allergy.

BACKGROUND: Inclusion of baked-milk products to the diet appears to markedly accelerate tolerance to unheated milk compared to a strict avoidance diet. OBJECTIVE: : The present study aims to investigate the predictors of baked-milk tolerance in children with Immunoglobulin E (IgE)-mediated cow's milk (CM) allergy. METHODS: The study included 80 patients diagnosed with IgE-mediated CM allergy upon oral food challenge (OFC) testing at our clinic. Patients who developed and did not develop reactions during OFC with baked milk were compared considering clinical and laboratory parameters. RESULTS: Eighty patients with CM allergy comprised 48 male and 32 female infants with an average age of 7.25 ± 2.45 (3-13) months. We found that 62.5% of them showed tolerance to baked milk in the OFC test performed with cakes containing 2.6-g milk protein. When the patients who tolerated and could not tolerate baked-milk products were compared for test results, we detected a statistically significant intergroup difference regarding diameter of wheal in skin prick test (SPT) performed with muffin slurry, levels of specific Immunoglobulin E (sIgE) in CM, sheep's milk (SM), goat's milk (GM), casein, and the amount of unheated milk consumed until a reaction developed in the OFC test performed with unheated milk (P < 0.05). CONCLUSION: We defined novel decision points based on CM, SM, GM, casein sIgE levels, wheal diameter in SPT with muffin slurry, and the amount of milk ingested during OFC performed with unheated milk that may be useful in predicting outcomes of baked-milk ingestion.

Effect of oral immunotherapy in children with milk allergy: The ORIMA study.

BACKGROUND: This study was aimed at evaluating the efficacy and safety of oral immunotherapy (OIT) in children with severe cow's milk allergy. METHODS: The subjects comprised 28 children (aged 3-12 years) with allergic symptoms that were induced by ≤ 10 mL of cow's milk in an oral food challenge test (OFC). The subjects were randomly allocated to the treatment group (n = 14) and control group (n = 14); the former received rush immunotherapy for 2 weeks, followed by a gradual increase of cow's milk volume to 100 mL for 1 year, and the latter completely eliminated cow's milk for 1 year. Both groups underwent an OFC with 100 mL of cow's milk after 1 year. RESULTS: The treatment group had significantly higher rates of a negative OFC [7/14 (50%) vs. 0/14 (0%), p < 0.01] compared with the control group. The cow's milk-specific IgE level significantly decreased in the treatment group (p < 0.01) but not in the control group (p = 0.63). During the study period, adrenaline was required in 6/14 patients (43%) of the treatment group and in 0/14 patients (0%) of the control group. Long follow-up data were available at the 2-year point after the study for 8 in the treatment group and 7 (87.5%) of these continued to ingest milk (>100 mL). CONCLUSIONS: The effect of immunotherapy was 50%, but the incidence of adverse events was not low. Further studies focusing on safety is necessary to standardize OIT for cow's milk allergy.

Induction of Hypersensitivity with Purified Beta-Lactoglobulin as a Mouse Model of Cow's Milk Allergy.

Cow's milk allergy is one of the most prevalent food allergies in both children and adults. As dairy products are common dietary ingredients and the prevalence of chronic conditions is on the rise, milk allergy is a growing public health concern. To elucidate underlying mechanisms and develop therapeutic strategies, reliable animal models are essential research tools. Sensitization to a milk protein is the principal procedure for establishing animal models of cow's milk allergy. However, the methods of sensitization vary from laboratory to laboratory, using different milk proteins with different amounts, routes, and durations of allergen exposure during sensitization of varying sex and strains of mice, likely resulting in diverse immunological and physical responses. Furthermore, the sources and potential impurities of milk protein may also produce variable responses. Thus, standardization of sensitization protocol is important, particularly when results are compared across studies. Here, we describe a method to generate a mouse model of cow's milk allergy using purified ß-lactoglobulin as the milk allergen with cholera toxin as an adjuvant in a 5-week oral sensitization protocol.

Isotype-Specific Detection of Serum Immunoglobulins Against Allergens.

Type-I hypersensitivity is commonly characterized by increased levels of antigen-specific immunoglobulin (Ig) E. Therefore, it is important for clinical and research investigators to reliably measure serum levels of IgE in allergic patients and animal models. While current ELISA-based methods are simple and commonly performed for the detection of allergen-specific IgE using serum or plasma, they may produce misleading results. This is in part due to decreased sensitivity for IgE in the presence of other Ig isotypes in the same sample, such as IgG, that are typically more abundant than IgE. When assessment of multiple Ig isotypes is necessary, performing optimized assays for individual isotypes requires high sample volumes. Here, we describe an approach to increase the sensitivity for IgE detection while conserving the sample volume needed. This method not only improves the accuracy of serum IgE measurements but also allows simultaneous analysis of other allergen-specific immunoglobulins.

Administration of Extensive Hydrolysates From Caseins and Lactobacillus rhamnosus GG Probiotic Does Not Prevent Cow's Milk Proteins Allergy in a Mouse Model.

Background: Early nutrition may influence the development of food allergies later in life. In the absence of breastfeeding, hydrolysates from cow's milk proteins (CMP) were indicated as a prevention strategy in at risk infants, but their proof of effectiveness in clinical and pre-clinical studies is still insufficient. Thanks to a validated mouse model, we then assessed specific and nonspecific preventive effects of administration of extensive hydrolysates from caseins (eHC) on the development of food allergy to CMP. The additional nonspecific effect of the probiotic Lactobacillus GG (LGG), commonly used in infant formula, was also assessed. Methods: Groups of young BALB/cByJ female mice were pretreated by repeated gavage either with PBS (control mice), or with PBS solution containing non-hydrolyzed milk protein isolate (MPI), eHC or eHC+LGG (eq. of 10 mg of protein/gavage). All mice were then experimentally sensitized to CMP by gavage with whole CM mixed with the Th2 mucosal adjuvant Cholera toxin. All mice were further chronically exposed to cow's milk. A group of mice was kept naïve. Sensitization to both caseins and to the non-related whey protein ß-lactoglobulin (BLG) was evaluated by measuring specific antibodies in plasma and specific ex vivo Th2/Th1/Th17 cytokine secretion. Elicitation of the allergic reaction was assessed by measuring mMCP1 in plasma obtained after oral food challenge (OFC) with CMP. Th/Treg cell frequencies in gut-associated lymphoid tissue and spleen were analyzed by flow cytometry at the end of the protocol. Robust statistical procedure combining non-supervised and supervised multivariate analyses and univariate analyses, was conducted to reveal any effect of the pretreatments. Results: PBS pretreated mice were efficiently sensitized and demonstrated elicitation of allergic reaction after OFC, whereas mice pretreated with MPI were durably protected from allergy to CMP. eHC+/-LGG pretreatments had no protective effect on sensitization to casein (specific) or BLG (non-specific), nor on CMP-induced allergic reactions. Surprisingly, eHC+LGG mice demonstrated significantly enhanced humoral and cellular immune responses after sensitization with CMP. Only some subtle changes were evidenced by flow cytometry. Conclusion: Neither specific nor nonspecific preventive effects of administration of casein-derived peptides on the development of CMP food allergy were evidenced in our experimental setup. Further studies should be conducted to delineate the mechanisms involved in the immunostimulatory potential of LGG and to clarify its significance in clinical use.

Effectiveness of allergy testing in milk induced eosinophilic esophagitis. Description and follow-up of patients.

INTRODUCTION: Eosinophilic esophagitis (EoE) is a chronic, local immune-mediated esophageal disease that has been on the increase lately. There is currently enough evidence to conclude that EoE is an allergic disorder triggered by food allergens, with cow's milk (CM) being the most frequent. Dietary intervention is the first-line approach. This study aimed to assess the clinical characteristics, the diagnostic method, and the prognosis of patients whose culprit food was CM, as opposed to other triggers. METHODS: Children with EoE evaluated in our pediatric Allergy Department were retrospectively studied from 2004 to 2017. We collected clinical variables, diagnostic protocol, treatment, and follow-up data. We compared patients whose culprit food was CM and patients with EoE due to other causative agents. RESULTS: We analyzed 31 children with EoE and found the causative food to be cow's milk in 14 (45%). Clinical characteristics were similar in patients with EoE due to milk or any other cause. Eight of 14 patients with milk-induced EoE (57.14%) presented positive skin prick test results against cow's milk. All patients had positive IgE against cow's milk. None of the patients had any other food as the trigger. The median follow-up was 2.68 years (6 months to 9 years) with initial remission of 100%. CONCLUSION: Testing-based elimination diets effectively treated all of the patients with milk-induced EoE. The advantage of this diagnostic protocol is that it required a mean of only two foods to be tested, significantly smaller number than in empiric diets.

Predictors of a positive oral food challenge to cow's milk in children sensitized to cow's milk.

INTRODUCTION AND OBJECTIVES: The diagnosis of IgE-mediated cow's milk allergy (CMA) is often based on clinical history and on specific IgE levels and/or skin-prick tests (SPT), both of which are sensitive but not specific. The gold standard, oral food challenge (OFC), is expensive and time-consuming and involves a risk of severe allergic reactions. This study aimed to determine the value of specific IgEs, ratios of specific IgEs for cow's milk and its components to total IgE, and wheal size on SPT for predicting a positive OFC for CMA. MATERIAL AND METHODS: We retrospectively studied 72 patients [median age, four years; age range 0.75-15 years] sensitized to cow's milk who underwent OFCs to milk. predictive variables between patients with positive and negative OFCs were compared. Receiver operator characteristic (ROC) curves were uses to assess variables' discriminatory capacity and Youden's index to determine the best cut-offs for predicting CMA. RESULTS: The OFC was positive in 39 (54%) patients. Wheal size on SPT and all specific IgEs and specific-to-total IgE ratios were significantly different between patients with positive OFCs and those with negative OFCs (p<0.001). The variable with the greatest area under the ROC curve was casein-specific IgE (0.98), followed by ß-lactoglobulin-specific IgE (0.923), casein-specific-to-total-IgE ratio (0.919), and α-lactalbumin-specific IgE (0.908). Casein-specific IgE ≥0.95kU/L yielded 88.9% sensitivity and 90.9% specificity. CONCLUSIONS: In our center, casein-specific IgE >0.95kU/L can obviate an OFC to cow's milk for the diagnosis of CMA in patients sensitized to cow's milk with a compatible history.