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.

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.

Immune Effects of the Nitrated Food Allergen Beta-Lactoglobulin in an Experimental Food Allergy Model.

Food proteins may get nitrated by various exogenous or endogenous mechanisms. As individuals might get recurrently exposed to nitrated proteins via daily diet, we aimed to investigate the effect of repeatedly ingested nitrated food proteins on the subsequent immune response in non-allergic and allergic mice using the milk allergen beta-lactoglobulin (BLG) as model food protein in a mouse model. Evaluating the presence of nitrated proteins in food, we could detect 3-nitrotyrosine (3-NT) in extracts of different foods and in stomach content extracts of non-allergic mice under physiological conditions. Chemically nitrated BLG (BLGn) exhibited enhanced susceptibility to degradation in simulated gastric fluid experiments compared to untreated BLG (BLGu). Gavage of BLGn to non-allergic animals increased interferon-γ and interleukin-10 release of stimulated spleen cells and led to the formation of BLG-specific serum IgA. Allergic mice receiving three oral gavages of BLGn had higher levels of mouse mast cell protease-1 (mMCP-1) compared to allergic mice receiving BLGu. Regardless of the preceding immune status, non-allergic or allergic, repeatedly ingested nitrated food proteins seem to considerably influence the subsequent immune response.

Assessment of the Sensitizing Potential of Proteins in BALB/c Mice: Comparison of Three Protocols of Intraperitoneal Sensitization.

Most food allergy cases are associated with a limited group of allergens. This could be attributed to an increased ability of some foods to sensitize and trigger allergic reactions. However, there are no validated animal models to evaluate the sensitizing or allergenic potentials of proteins. Our aim was to evaluate three protocols of adjuvant-free intraperitoneal sensitization that differ in the time points for sample collection (days 14, 28 and 35 from beginning of the sensitization) and also in the number of immunizations (2, 5 and 3, respectively). Ovalbumin (OVA; 0.05 mg), cow milk proteins (CMP; 0.025, 0.05 and 0.25 mg), and potato acid phosphatase (PAP; low allergenic protein; 250.0 mg) were administered intraperitoneally (ip) to BALB/c mice (n = 4⁻6) and the protein-specific IgE and IgG antibody responses were evaluated using ELISA. Additional serum protein-specific IgE antibodies evaluations were carried out after IgG depletion. Anti-OVA IgE antibodies were detected in mice from all three protocols. The responses were higher in the group of mice that underwent the 28-day protocol than in those that underwent the 14- or 35-day protocols (p < 0.01 and p < 0.05, respectively). Anti-CMP IgE antibodies were detected in both the 14- and 28-day protocols, but the response was higher in the group that underwent the 28-day protocol (p < 0.001). The anti-CMP IgE antibody response detection was improved after serum IgG depletion (p < 0.001). Anti-PAP IgE antibodies were not detected. Mice with undetectable serum levels of protein-specific IgE triggered anti-OVA, -CMP, and -PAP IgG responses. An adjuvant-free 28-day protocol with five ip immunizations seems appropriate for evaluation of the inherent sensitizing or allergenic capacity of the studied proteins. Reproducible results were obtained utilizing the BALB/c mouse strain. Inter-laboratory studies including a larger number of proteins should be carried out to validate this model.

Accuracy of serum IgE concentrations and papule diameter in the diagnosis of cow's milk allergy / Acurácia de concentrações de IgE séricas e de diâmetros de pápulas no diagnóstico de alergia ao leite de vaca

Abstract Objective To compare serum concentrations of specific IgE and mean papule diameters induced in the immediate skin reactivity test with cow's milk and its fractions with results of the oral challenge test, and to establish cutoff points capable of predicting clinical reactivity to cow's milk in patients treated at a referral service. Methods One hundred and twenty-two children (median of 17 months) with a history of immediate reactions to cow's milk and presence of specific IgE for cow's milk and/or its fractions (positive skin and/or IgE serum tests) were submitted to open oral challenge test with cow's milk. Results The oral challenge test was positive in 59.8% of the children, 49% of whom were males. Serum levels of specific IgE, as well as mean cow's milk papule diameters, were significantly higher in allergic patients (medians: 3.39 kUA/L vs. 1.16 kUA/L, 2.5 mm vs. 0 mm). The optimal cutoff points (Youden's index) of serum IgE specific for cow's milk and its fractions capable of predicting cow's milk reactivity (positive oral challenge test) were: 5.17 kUA/L for cow's milk, 0.95 kUA/L for α-lactalbumin, 0.82 kUA/L for β-lactoglobulin, and 0.72 kUA/L for casein, whereas for papule diameters the cutoff points were 3.5 mm for cow's milk and 6.5 mm, 9.0 mm, and 3.0 mm for the α-lactalbumin, β-lactoglobulin, and casein fractions, respectively. Conclusions The cutoff points capable of predicting clinical reactivity to cow's milk were: 5.17 kUA/L for serum-specific IgE and 3.5 mm for papule diameter measurement, values considered discriminatory for the diagnosis of cow's milk allergy.

Resumo Objetivo Comparar concentrações séricas de IgE específica e diâmetros médios das pápulas induzidas no teste cutâneo de leitura imediata com leite de vaca e suas frações com resultados do teste de provocação oral e estabelecer pontos de corte, capazes de predizer reatividade clínica ao leite de vaca em pacientes atendidos em um serviço de referência. Métodos Cento e vinte e duas crianças (mediana 17 meses), com história de reações imediatas ao leite de vaca e presença de IgE específicas para leite de vaca e/ou frações (testes cutâneos e/ou IgE sérica positivos) foram submetidas ao teste de provocação oral aberto com leite de vaca. Resultados O teste de provocação oral foi positivo em 59,8% das crianças, 49% eram do sexo masculino. Os níveis séricos de IgE específica, assim como os diâmetros médios das pápulas para leite de vaca, foram significantemente maiores nos alérgicos (medianas: 3,39kUA/L vs. 1,16 kUA/L; 2,5 mm vs. 0 mm). Os "pontos de corte ótimos" (Índice de Youden) das IgE séricas específicas para o leite de vaca e suas frações capazes de predizer a reatividade ao leite de vaca (teste de provocação oral positivo) foram: 5,17kUA/L para o leite de vaca, 0,95 kUA/L para α-lactoalbumina, 0,82kUA/L para β-lactoglobulina e 0,72kUA/L para caseína e para os diâmetros de pápulas foram 3,5 mm para leite de vaca e 6,5 mm, 9,0 mm e 3,0 mm para as frações α-lactoalbumina, β-lactoglobulina e caseína, respectivamente. Conclusões Os níveis de corte capazes de predizer reatividade clínica ao leite de vaca foram: 5,17kUA/L para IgE sérica específica e 3,5 mm para a medida do diâmetro da pápula, valores considerados discriminatórios para o diagnóstico da alergia ao leite de vaca.

Do infants with cow's milk protein allergy have inadequate levels of vitamin D? / Lactentes com alergia à proteína do leite de vaca apresentam níveis inadequados de vitamina D?

Abstract Objective: To verify whether infants with cow's milk protein allergy have inadequate vitamin D levels. Methods: This cross-sectional study included 120 children aged 2 years or younger, one group with cow's milk protein allergy and a control group. The children were recruited at the pediatric gastroenterology, allergology, and pediatric outpatient clinics of a university hospital in the Northeast of Brazil. A questionnaire was administered to the caregiver and blood samples were collected for vitamin D quantification. Vitamin D levels <30 ng/mL were considered inadequate. Vitamin D level was expressed as mean and standard deviation, and the frequency of the degrees of sufficiency and other variables, as proportions. Results: Infants with cow's milk protein allergy had lower mean vitamin D levels (30.93 vs.35.29 ng/mL; p = 0.041) and higher deficiency frequency (20.3% vs.8.2; p = 0.049) than the healthy controls. Exclusively or predominantly breastfed infants with cow's milk protein allergy had higher frequency of inadequate vitamin D levels (p = 0.002). Regardless of sun exposure time, the groups had similar frequencies of inadequate vitamin D levels (p = 0.972). Conclusions: Lower vitamin D levels were found in infants with CMPA, especially those who were exclusively or predominantly breastfed, making these infants a possible risk group for vitamin D deficiency.

Resumo Objetivo: Verificar se lactentes com alergia à proteína do leite de vaca (APLV) apresentam níveis inadequados de vitamina D. Métodos: Estudo transversal, envolveu 120 crianças de até dois anos, um grupo com APLV e outro de comparação, captadas dos ambulatórios de Gastroenterologia Pediátrica, Alergologia Pediátrica e Puericultura de um hospital universitário, no Nordeste brasileiro. Foi aplicado um formulário e foram coletadas amostras sanguíneas para a análise da vitamina D, foram considerados inadequados os níveis < 30 ng/mL. Níveis de vitamina D foram expressos em média e desvio padrão e a frequência dos graus de suficiência e demais variáveis, em proporções. Resultados: Lactentes com APLV, quando comparados com os saudáveis, apresentaram uma menor média do nível da vitamina D (30,93 vs. 35,29 ng/mL) (p = 0,041) e maior frequência de deficiência (20,3% vs. 8,2) (p = 0,049). Maior frequência de níveis inadequados de vitamina D foi observada nas crianças com APLV que estavam em aleitamento materno exclusivo/predominante (p = 0,002). Independentemente do período de exposição solar, a frequência de um status inadequado de vitamina D foi semelhante entre os grupos (p = 0,972). Conclusões: Menores níveis de vitamina D foram observados em lactentes com APLV, especialmente naqueles em aleitamento materno exclusivo/predominante, que configura esse como um possível grupo de risco para essa deficiência.

Multiple epitope presentation and surface density control enabled by chemoselective immobilization lead to enhanced performance in IgE-binding fingerprinting on peptide microarrays.

Multiple ligand presentation is a powerful strategy to enhance the affinity of a probe for its corresponding target. A promising application of this concept lies in the analytical field, where surface immobilized probes interact with their corresponding targets in the context of complex biological samples. Here we investigate the effect of multiple epitope presentation (MEP) in the challenging context of IgE-detection in serum samples using peptide microarrays, and evaluate the influence of probes surface density on the assay results. Using the milk allergen alpha-lactalbumin as a model, we have synthesized three immunoreactive epitope sequences in a linear, branched and tandem form and exploited a chemoselective click strategy (CuAAC) for their immobilization on the surface of two biosensors, a microarray and an SPR chip both modified with the same clickable polymeric coating. We first demonstrated that a fine tuning of the surface peptide density plays a crucial role to fully exploit the potential of oriented and multiple peptide display. We then compared the three multiple epitope presentations in a microarray assay using sera samples from milk allergic patients, confirming that a multiple presentation, in particular that of the tandem construct, allows for a more efficient characterization of IgE-binding fingerprints at a statistically significant level. To gain insights on the binding parameters that characterize antibody/epitopes affinity, we selected the most reactive epitope of the series (LAC1) and performed a Surface Plasmon Resonance Imaging (SPRi) analysis comparing different epitope architectures (linear versus branched versus tandem). We demonstrated that the tandem peptide provides an approximately twofold increased binding capacity with respect to the linear and branched peptides, that could be attributed to a lower rate of dissociation (Kd).

Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

BACKGROUND: We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. OBJECTIVE: We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). METHODS: We measured specific IgA to a series of overlapping peptides in major CM allergens (αs1 -, αs2 -, ß- and κ-caseins and ß-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. RESULTS: There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in αs1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3 months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. CONCLUSION & CLINICAL RELEVANCE: These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated.

Changes in biomarkers during a six-month oral immunotherapy intervention for cow's milk allergy.

AIM: Oral immunotherapy (OIT) is a promising but still experimental method to treat children with cow's milk (CM) allergy (CMA). We evaluated changes in allergic, immunological and inflammatory parameters, which happened during the six-month OIT for CMA. METHODS: We treated 28 school-aged children with CMA using OIT with a double-blind placebo-controlled design. After the controlled study finished, the placebo group was treated with the same but open-label OIT protocol. Sixteen immune variables were tested before and after the six-month OIT. RESULTS: Before OIT, the median serum CM-specific immunoglobulin (Ig) E was 18.0kIU/L in the intervention group and 9.4kIU/L in the placebo group (p = 0.46). At six months, interleukin (IL)-6 and IL-10 were significantly higher in the intervention group. When the changes during the blinded and open OIT were analysed together for both groups, blood eosinophils and serum total IgE decreased and milk-specific IgG and IgG4, serum IL-4 and IL-6, and serum leptin and resistin increased significantly. CONCLUSION: Preliminary evidence was found that markers of allergy such as blood eosinophils and serum IgE decreased and milk-specific IgG and IgG4 increased during OIT. Adipokines, leptin and resistin, which functionally are cytokines linked to Th1-type response, increased during OIT.

High IgE levels to α-lactalbumin, ß-lactoglobulin and casein predict less successful cow's milk oral immunotherapy.

BACKGROUND: A new treatment option for persistent cow's milk allergy (CMA) is oral immunotherapy (OIT). Not all patients develop tolerance during therapy, and markers to identify those who will benefit from it are needed. The objective was to study the IgE and IgG4 antibody profiles to milk and milk proteins before and after OIT in relation to clinical outcome. METHODS: Seventy-six children (5-17 years) with challenge-verified CMA were subjected to a 6-month OIT protocol. The treatment aimed at reaching a maintenance dose of 200 ml CM (high dose = HD). Those who did not reach target were analysed as a low-dose (LD) group. Sera were characterized before and after OIT regarding serum levels of IgE and IgG4 to milk and five milk allergen components evaluated together with clinical CMA symptoms and outcome of OIT. RESULTS: Fifty-five (72%) patients reached the maintenance dose (HD) during therapy. High specific IgE levels towards the milk allergens α-lactalbumin (P = 0.048), ß-lactoglobulin (P = 0.006) and casein (P = 0.015) before OIT start were associated with lower maintenance dose reached. Patients who developed desensitization had a larger increase in IgG4 levels to α-lactalbumin (P = 0.034), ß-lactoglobulin (P = 0.010), casein (P = 0.047) and lactoferrin (P = 0.030) during treatment than those who failed. CONCLUSIONS: Component-resolved diagnostics before OIT can help to identify children with lower probability of a successful OIT outcome, as high IgE levels to α-lactalbumin, ß-lactoglobulin and casein are associated with lower maintenance dose reached. An increase in the IgG4 concentration to milk components during treatment indicated effective desensitization.

Influence of early-life exposures on food sensitization and food allergy in an inner-city birth cohort.

OBJECTIVE: Previous data suggest that food allergy (FA) might be more common in inner-city children; however, these studies have not collected data on both sensitization and clinical reactivity or early-life exposures. METHODS: Children in the Urban Environment and Childhood Asthma birth cohort were followed through age 5 years. Household exposures, diet, clinical history, and physical examinations were assessed yearly; levels of specific IgE to milk, egg, and peanut were measured at 1, 2, 3, and 5 years of age. On the basis of sensitization (IgE ≥0.35 kU/L) and clinical history over the 5-year period, children were classified as having FA or being possibly allergic, sensitized but tolerant, or not allergic/not sensitized. RESULTS: Five hundred sixteen children were included. Overall, 55.4% were sensitized (milk, 46.7%; egg, 31.0%; and peanut, 20.9%), whereas 9.9% were categorized as having FA (peanut, 6.0%; egg, 4.3%; and milk, 2.7%; 2.5% to >1 food). The remaining children were categorized as possibly allergic (17.0%), sensitized but tolerant (28.5%), and not sensitized (44.6%). Eighteen (3.5%) reported reactions to foods for which IgE levels were not measured. Food-specific IgE levels were similar in children with FA versus sensitized but tolerant children, except for egg, levels of which were higher in patients with FA at ages 1 and 2 years. FA was associated with recurrent wheeze, eczema, aeroallergen sensitization, male sex, breast-feeding, and lower endotoxin exposure in year 1 but not with race/ethnicity, income, tobacco exposure, maternal stress, or early introduction of solid foods. CONCLUSIONS: Even given that this was designed to be a high-risk cohort, the cumulative incidence of FA is extremely high, especially considering the strict definition of FA that was applied and that only 3 common allergens were included.

Serum concentrations of sclerostin and bone turnover markers in children with cow's milk allergy.

THE AIM: of this study was to assess concentrations of sclerostin and biochemical markers of bone metabolism in children with cow's milk allergy. MATERIAL AND METHODS: The study included 45 children (age range 2-6 years) with diagnosed cow`s milk allergy, who were on a dairy-free diet and under systematic medical and dietary control at the Institute of Mother and Child in Warsaw. The control group consisted of 40 healthy children (2-6 years), who did not have any symptoms of cow`s milk allergy nor any diseases influencing bone metabolism. Their diets included milk and dairy products. Dietary intake of macro- and micronutrients was assessed based on 3-day records using the Dietetyk2® nutritional program. In the serum samples, we measured concentrations of calcium, phosphate and total alkaline phosphatase by standard methods, 25-hydroxyvitamin D3 by chemiluminescence method and bone metabolism markers by immunoenzymatic methods. The Statistica (version 10.0) computer software was used for statistical analysis. RESULTS: The nutritional status of studied children based on BMI value was normal. In all patients, the average daily value of dietary energy and percentage of energy from protein, fat and carbohydrates were consistent with the recommended values. The intake of calcium in the diets of all children was deficient, however, the intake of vitamin D was consistent with recommendations in the children with allergy, while in the healthy children it was below the recommended values. Mean serum concentrations of calcium, phosphate, alkaline phosphatase, 25-hydroxyvitamin D3, osteocalcin and C-terminal telopeptide of type I collagen were similar in both studied groups. We observed significantly lower sclerostin levels in children with cow`s milk allergy (0.295±0.116 ng/ml) than in the healthy children (0.353±0.126 ng/ml) (p<0.05). The ratio of cytokines RANKL/OPG (receptor activator of nuclear factor κB ligand/osteoprotegerin) was significantly higher in children with allergy compared with their healthy counterparts (p<0.05). CONCLUSIONS: Basic laboratory parameters related to bone turnover in children with cow`s milk allergy, who were under medical and nutritional care, were normal. Reduced levels of sclerostin and increased ratio of cytokines RANKL/OPG may suggest disturbances in the balance between bone formation and bone resorption in these patients. Further research is needed on bone metabolism in children with food allergy, who due to the use an elimination diet may be at risk of developing abnormalities in the skeletal system.

Serum and tissue CD23, IL-15, and FasL in cow's-milk protein-sensitive enteropathy and in coeliac disease.

OBJECTIVES: The aim of the study was to explore pathogenesis and find new serum markers for cow's-milk-sensitive enteropathy (CMSE) and coeliac disease (CD). We assessed the intestinal expression and serum concentration of CD23, IL-15, and FasL. We hypothesised that the serum levels of CD23, a protein expressed in the lymphoid follicles, would be associated with lymphonodular hyperplasia (LNH), a feature characteristic of CMSE. We also presumed that interleukin (IL)-15 and FasL, functionally connected with proliferation and apoptosis of the intraepithelial lymphocytes (IELs), would relate with the increased numbers of IELs present in both CMSE and CD. METHODS: Twenty-three children with CMSE, 20 with untreated CD, and 14 controls were studied for CD3, α/ß- and γ/δ-expressing IELs, and for duodenal and ileal expression of CD23, FasL, and IL-15 by immunohistochemistry, and for serum concentration of sCD23, sFasL, and sIL-15 by enzyme-linked immunosorbent assay. RESULTS: There was a trend for increase in sCD23 serum levels in untreated CMSE and in CD (P = 0.074; P = 0.077). CD23 was expressed in the mucosal germinal centres, but sCD23 was not related to presence of LNH. In CMSE, there was a trend for increase in serum sFasL (P = 0.07) and high levels associated with LNH (P = 0.025) and correlated with the IEL numbers (P < 0.05). Mucosal high endothelial venules adjacent to lymphoid follicles showed an intensive FasL expression. CONCLUSIONS: Serum sCD23 shows a trend of increment in CMSE and CD, and in the latter, sCD23 level may provide information about the severity of villous atrophy. In CMSE, high serum sFasL indicates both LNH and an increase of IELs, suggesting importance of FasL-mediated mechanisms in the pathogenesis of these features characteristic of CMSE. Further studies are necessary to evaluate whether intensive FasL expression in mucosal high endothelial venules presents a regulatory element in mucosal immunity.

Presence of functional, autoreactive human milk-specific IgE in infants with cow's milk allergy.

BACKGROUND: Occasionally, exclusively breastfed infants with cow's milk allergy (CMA) remain symptomatic despite strict maternal milk avoidance. OBJECTIVE: To determine whether or not persistence of symptoms could be due to sensitization against endogenous human milk proteins with a high degree of similarity to bovine allergens. METHODS: Ten peptides representing known bovine milk IgE-binding epitopes [α-lactalbumin (ALA), ß- and κ-casein] and the corresponding, highly homologous human milk peptides were labelled with sera from 15 breastfed infants with CMA, aged 3 weeks to 12 months, and peptide (epitope)-specific IgE antibodies were assessed. Nine of the 15 breastfed infants became asymptomatic during strict maternal avoidance of milk and other major food allergens; six infants remained symptomatic until weaned. Ten older children, aged 5-15 years, with CMA were also assessed. The functional capacity of specific IgE antibodies was assessed by measuring ß-hexosaminidase release from rat basophilic leukaemia cells passively sensitized and stimulated with human and bovine ALA. RESULTS: A minimum of one human milk peptide was recognized by IgE antibodies from 9 of 15 (60%) milk-allergic infants, and the majority of older children with CMA. Genuine sensitization to human milk peptides in the absence of IgE to bovine milk was occasionally seen. There was a trend towards specific IgE being detected to more human milk peptides in those infants who did not respond to the maternal milk elimination diet than in those who did (P = 0.099). Functional IgE antibody to human ALA was only detected in infants not responding to the maternal diet. CONCLUSIONS AND CLINICAL RELEVANCE: Endogenous human milk epitopes are recognized by specific IgE from the majority of infants and children with CMA. Such autoreactive, human milk-specific IgE antibodies appear to have functional properties in vitro. Their role in provoking allergic symptoms in infants exclusively breastfed by mothers strictly avoiding dietary milk remains unclear.

The safety and efficacy of sublingual and oral immunotherapy for milk allergy.

BACKGROUND: Oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) are potential therapies for food allergy, but the optimal method of administration, mechanism of action, and duration of response remain unknown. OBJECTIVE: We sought to explore the safety and efficacy of OIT and SLIT for the treatment of cow's milk (CM) allergy. METHODS: We randomized children with CM allergy to SLIT alone or SLIT followed by OIT. After screening double-blind, placebo-controlled food challenges and initial SLIT escalation, subjects either continued SLIT escalation to 7 mg daily or began OIT to either 1000 mg (the OITB group) or 2000 mg (the OITA group) of milk protein. They were challenged with 8 g of milk protein after 12 and 60 weeks of maintenance. If they passed the 60-week challenge, therapy was withdrawn, with challenges repeated 1 and 6 weeks later. Mechanistic correlates included end point titration skin prick testing and measurement of CM-specific IgE and IgG(4) levels, basophil histamine release, constitutive CD63 expression, CD203c expression, and intracellular spleen tyrosine kinase levels. RESULTS: Thirty subjects with CM allergy aged 6 to 17 years were enrolled. After therapy, 1 of 10 subjects in the SLIT group, 6 of 10 subjects in the SLIT/OITB group, and 8 of 10 subjects in the OITA group passed the 8-g challenge (P = .002, SLIT vs OIT). After avoidance, 6 of 15 subjects (3 of 6 subjects in the OITB group and 3 of 8 subjects in the OITA group) regained reactivity, 2 after only 1 week. Although the overall reaction rate was similar, systemic reactions were more common during OIT than during SLIT. By the end of therapy, titrated CM skin prick test results and CD63 and CD203c expression decreased and CM-specific IgG(4) levels increased in all groups, whereas CM-specific IgE and spontaneous histamine release values decreased in only the OIT group. CONCLUSION: OIT was more efficacious for desensitization to CM than SLIT alone but was accompanied by more systemic side effects. Clinical desensitization was lost in some cases within 1 week off therapy.

Selectively high level of serum interleukin 5 in a newborn infant with cow's milk allergy.

Cow's milk allergy (CMA) in the neonatal period is thought to include several clinical conditions, yet the pathophysiology remains unclear. We report here the case of a term newborn infant who showed hematochezia 36 hours after the first feeding with cow's milk formula. His serum immunoglobulin E levels were not elevated, although eosinophils were detected in the stool. Elimination of cow's milk formula resolved the symptoms, and from the clinical course and laboratory data the infant was diagnosed with CMA. The serum interleukin 5 (IL-5) (125 pg/mL) level in this patient was selectively elevated. However, serum levels of other T-helper 2 (Th2) cytokines (including IL-4 and IL-13), Th1 cytokines (including interferon γ), and proinflammatory cytokines (including tumor necrosis factor α) were not elevated. These findings suggest that, for this patient, IL-5 and eosinophils might have played a role in the development of neonatal CMA. Although this finding is reported from only 1 case, it highlights the need for serum IL-5 to be determined in more neonatal patients with CMA to further clarify the pathophysiology of this condition in the neonatal period.

Allergic enterocolitis presenting as recurrent necrotizing enterocolitis in preterm neonates.

An uncommon clinical entity mimicking necrotizing enterocolitis (NEC) is allergic enterocolitis secondary to cow's milk protein allergy. Although milk protein allergy is the most common food allergy among infants and young children, the incidence and prevalence of this disease entity presenting as enterocolitis in neonates is not well documented. We report this case of milk protein-associated allergic enterocolitis to highlight the unusual recurrent presentation as NEC, (with recurrent pneumatosis, bloody stools) managed successfully with modification of milk formula.

Mammalian milk allergy: clinical suspicion, cross-reactivities and diagnosis.

PURPOSE OF REVIEW: Cow's milk allergy affects 2-3% of young children, the economic impact of which necessitates search for simple diagnostic tools and affordable milk substitutes. This review examines recent studies on the diagnosis of cow's milk allergy as well as on the allergenicity of milk from other mammalian species. RECENT FINDINGS: Resolution of symptoms during strict milk avoidance and their re-appearance during the double-blind, placebo-controlled milk challenge remains the gold standard for the diagnosis of cow's milk allergy. Allergic eosinophilic esophagitis/gastroenteritis requires confirmatory endoscopic biopsy. There are increasing data in various populations on cut-off points based on positive predictive values for skin prick test and milk-specific IgE measurements to aid in the diagnosis of cow's milk allergy and to decrease the number of unnecessary food challenges. For non-IgE-mediated manifestations, noninvasive diagnostic tests are still largely lacking. The significant homology between milk from cow, sheep and goat results in clinical cross-reactivity. However, mare's or donkey's milk may be tolerated by some individuals. SUMMARY: Data have been accumulating on the utility of diagnostic tools for mostly IgE-mediated milk allergy and allergenicity of milk from other mammalian species, although further studies are sought.

Mutational analysis of immunoglobulin E-binding epitopes of beta-casein and beta-lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera.

BACKGROUND: For immunotherapeutic approaches, 'critical' amino acids (AAs) within allergenic epitopes are replaced with alternate AAs to eliminate IgE antibody binding. OBJECTIVE: To determine the critical AAs for IgE binding in beta-casein and beta-lactoglobulin (BLG). METHODS: Peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with a pool of sera from 15 cow's milk-allergic patients and individual sera from six of the 15 patients. In cases where no decrease in binding occurred with a single AA substitution, peptides with two AA substitutions were generated and labelled. RESULTS: Using pooled patient sera, single AA substitutions led to complete elimination of binding to six of 11 peptides for beta-casein and to all six peptides for BLG. Substituting two AAs led to an elimination of binding to four of the remaining five beta-casein epitopes. However, in three of the 11 modified beta-casein peptides and five of the six BLG peptides, no decrease in IgE binding occurred in at least one individual patient. For these patients, critical AAs other than those defined by the patient serum pool were identified, indicating a heterogeneous pattern of IgE recognition. CONCLUSION: These results indicate that AAs critical for IgE binding are more heterogeneous than initially defined by pooled milk-allergic patient sera. For future immunotherapeutic interventions with mutated peptides, critical AAs should also be identified with individual patient sera to account for heterogeneity of IgE binding between patients.

In vitro methods for specific IgE detection in cow's milk allergy.

BACKGROUND: A new method for determining serum specific IgE (IMMULITE 2000 3gAllergy) has recently become available. OBJECTIVE: To evaluate the clinical performance of IMMULITE 2000 in the diagnosis of cow's milk allergy compared with that of UniCAP. Additionally, we verified the behavior of both methods at two diagnostic decision points proposed by other authors. METHODS: The study population consisted of 31 children with cow's milk allergy (group A) and a control group of 19 atopic children without food allergy (group B). A blood sample from each child was tested using both methods and the results were compared. RESULTS: In group A, the values for cow's milk IgE ranged from 0.35 kU/L (the lowest common detection limit) to above 100 kU/L. In group B, the values were less than 1.1 kU/L for IMMULITE 2000 and less than 1.6 kU/L for UniCAP. An agreement of 90% in IgE classes was obtained. Both methods demonstrated exactly the same diagnostic performance (sensitivity: 100%; specificity: 78.9%; negative predictive value: 100%; positive predictive value: 84.6%; efficiency: 90.2%). The evaluation of the two methods at the two different decision points proposed in the literature showed a better positive predictive value with UniCAP, but we obtained equivalent performance with IMMULITE 2000 by choosing higher cutoff values. CONCLUSIONS: We conclude that IMMULITE 2000 is as effective as UniCAP in the diagnosis of cow's milk allergy. Both methods can be used to obtain site-specific decision points that are population, age and disease dependent.