Eosinophil cationic protein in human milk is associated with development of cow's milk allergy and atopic eczema in breast-fed infants.

The precise role of leukocytes and mediators in human milk is still unresolved. Eosinophils are uncommonly detected in human milk and their presence has previously been associated with maternal atopy and development of cow's milk allergy (CMA) in the breast-fed infant. The purpose of this study was to examine the levels of eosinophil cationic protein (ECP) in human milk and to compare the levels with development of allergic diseases in breast-fed infants. Altogether 94 breast-feeding mothers (58 atopic, 36 nonatopic) with their babies were prospectively followed from birth for development of CMA or atopic dermatitis. Colostrum and mature milk samples (at 3 mo of lactation), together with mother's peripheral blood samples, were collected. Milk and blood leukocyte content was evaluated with a light microscope. ECP concentration in human milk was measured by commercial UniCAP method. By the end of a 2-y follow-up, 51 mothers had an infant with CMA, 24 had an infant with atopic dermatitis, and 19 had a healthy infant. ECP concentration in milk was under the detection limit (2 microg/L) in all the mothers with a healthy infant, whereas detectable levels were found in 27% of mothers with a CMA infant and in 42% of those with a baby with atopic dermatitis. Measurable ECP in milk was detected in 26% of the atopic and 25% of the nonatopic mothers. Presence of ECP in human milk is associated with development of CMA and atopic dermatitis in the breast-fed infant, but has no direct association with the maternal atopy.

Mutational analysis of major, sequential IgE-binding epitopes in alpha s1-casein, a major cow's milk allergen.

BACKGROUND: Allergy to cow's milk is common in early childhood, and no therapy other than avoidance exists. In murine models of peanut allergy, immunotherapy with mutated, engineered, proteins appears promising. OBJECTIVE: We sought to identify the critical amino acids (AAs) for immunoglobulin E (IgE) binding within the major B-cell epitopes of alpha(s1)-casein, a major cow's milk allergen. This will provide the necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity. METHODS: For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions. Membranes were immunolabeled with pooled sera from 15 cow's-milk-allergic patients and with 8 individual sera. RESULTS: With the pooled sera, substitution of a single AA led to complete abrogation of IgE binding to 2 of 8 peptides and diminished binding in the remainder. Substitution of multiple AAs led to an abrogation of binding in the remaining peptides. In 4 of the 8 peptides, the critical AA identified with pooled sera did not result in significant reduction of IgE binding with 1 or more individual patients. For these patients, other critical AAs were identified, indicating a more heterogeneous pattern in IgE recognition. CONCLUSION: This study indicates that single or multiple AA substitutions within IgE-binding epitopes result in reduced binding of milk-specific IgE antibodies by patients' sera. However, for future immunotherapeutic interventions with mutated peptides, critical AAs should be evaluated with individual patient sera to determine B-cell-epitope heterogeneity.

Measurement of peptide-specific IgE as an additional tool in identifying patients with clinical reactivity to peanuts.

BACKGROUND: Peanut allergy is one of the most common food allergies, often resulting in severe reactions. Diagnostic decision levels of food-specific IgE antibody concentrations have been described. However, many patients still need to undergo oral peanut challenges because their IgE levels are in the nondiagnostic level. OBJECTIVE: The aim of this study was to determine whether differences exist in IgE-binding epitope recognition between sensitized children with and without symptomatic peanut allergy. METHODS: Eight peptides representing the immunodominant sequential epitopes on Ara h 1, 2, and 3 were synthesized on SPOTs membranes. Individual patient labeling was performed with sera from 15 patients with symptomatic peanut allergy and 16 patients who were sensitized but tolerant. Ten of these 16 patients had "outgrown" their allergy. RESULTS: Regardless of their peanut-specific IgE levels, most patients with symptomatic peanut allergy showed IgE binding to the 3 immunodominant epitopes on Ara h 2. In contrast, each of these epitopes was recognized by < 10% of the tolerant patients. In addition, tolerant patients did not recognize 2 immunodominant epitopes on Ara h 1. At least 93% of symptomatic, but only 12.5% of tolerant patients, recognized 1 of these "predictive" epitopes on Ara h 1 or 2. Moreover, the cumulative IgE binding to the peanut peptides was significantly higher in patients with peanut allergy than in tolerant patients. With up to 50% of patients with peanut-specific IgE levels below diagnostic decision levels still being clinically reactive, oral food challenges could be avoided in ~90% of these patients through determination of peptide-specific IgE. CONCLUSIONS: Determination of epitope recognition provides an additional tool to diagnose symptomatic peanut allergy, especially in children with peanut-specific IgE below diagnostic decision levels.

Cholesterol precursors and plant sterols in children with food allergy.

BACKGROUND: The data on lipid metabolism in allergic children is limited. OBJECTIVE: We investigated lipid and sterol metabolism in young children whose diets were restricted because of food allergy. DESIGN: Children in group A [n = 21; mean (+/- SD) age: 1.78 +/- 0.73 y] were allergic to fish, eggs, and either cow milk or cereals; those in group B (n = 31, aged 1.45 +/- 0.58 y) were allergic to fish, eggs, and both cow milk and cereals. Cholesterol precursor and plant sterol to cholesterol ratios (10(2) x micro mol/mmol cholesterol) and apolipoprotein E phenotype distributions were analyzed in 36 subjects. The control group for cholesterol precursor and plant sterol measurements consisted of 18 healthy age-matched children. RESULTS: The mean serum cholesterol concentration was 3.6 +/- 0.6 mmol/L, and HDL cholesterol was 1.03 +/- 0.3 mmol/L in group A. Corresponding values in group B were 3.4 +/- 0.7 and 1.09 +/- 0.2 mmol/L. The daily cholesterol intake was low: 61.3 +/- 36.0 mg in group A and 50.7 +/- 48.5 mg in group B. Cholesterol precursor plant sterol concentrations were significantly higher in allergic subjects than in control subjects. CONCLUSIONS: Allergic children with restricted diets have a low intake of cholesterol and relatively low serum cholesterol concentrations. Dietary intake of plant sterols was obviously increased because of supplementation with rapeseed oil, which is rich in plant sterols, leading to elevated plant sterol concentrations. Plant sterols may have inhibited cholesterol absorption, which in turn stimulated cholesterol synthesis in compensation, also explaining the increased precursor sterol ratios in serum in our subjects.

Identification of sequential IgE-binding epitopes on bovine alpha(s2)-casein in cow's milk allergic patients.

BACKGROUND: Caseins are the major allergens responsible for cow's milk allergy (CMA). We have previously identified the IgE-binding epitopes of the major cow's milk (CM) proteins except for alpha(s2)-casein. METHODS: Overlapping decapeptides representing the entire length of alpha(s2)-casein were synthesized on a cellulose-derivatized membrane. Sera from 13 CM-allergic children, 4-15 years of age, with a median level of CM-specific IgE >100 kU/l (range 33.7 to > 100 kU/l) were used to identify IgE-binding epitopes. RESULTS: Four major and six minor sequential IgE-binding regions were identified on alpha(s2)-casein. The first major region is located in the middle of the protein at amino acids (AA) 83-100, and the other three major regions are located in the carboxy terminal portion of the protein at AA 143-158, 157-172 and 165-188. The minor IgE-binding regions were identified at AA 31-44, 43-56, 93-106, 105-114, 117-128, and 191-200. CONCLUSION: We identified 10 sequential IgE-binding regions on alpha(s2)-casein and performed the first crucial step in the development of immunotherapeutic interventions for CMA.

Leucocytes in human milk and lymphocyte subsets in cow's milk-allergic infants.

The breast-fed infant ingests an average of 108 leucocytes per day, with breast-feeding often continuing for several months. The precise role of human milk leucocytes is still unresolved. Breast-feeding has been recommended for infants at high risk of allergy to prevent or delay the development of food allergies and atopic eczema. However, studies dealing with distinct immunologic factors in the mother's milk, and their effect on health status or development of allergies in the infant, are scarce. We evaluated the relationship between the cellular composition of human milk and the development of cow's milk allergy (CMA) in the breast-fed infant. Leucocyte subsets in the breast-fed infants were also measured. The study population comprised 61 breast-feeding mothers and their infants. Thirty-nine mothers each had a cow's milk-allergic infant, 10 had an infant with atopic dermatitis without CMA, and 12 mothers had a healthy infant. Leucocyte subsets in mothers' milk were counted using a light microscope and confirmed by flow cytometry. In infants, peripheral blood lymphocyte subsets were determined by flow cytometry and were correlated with the health status of the breast-fed infant and leucocyte composition of the mother's milk. Human milk was found to be a non-homogenous morphological entity. In the milk of mothers of infants with CMA, the proportion of macrophages was significantly smaller than in the mothers with infants without CMA (p = 0.036, t-test). Mothers with high proportions of neutrophils in their milk (> 20%) had significantly more often infants with CMA than did those with low proportions of neutrophils (p = 0.02; Fischer's exact test). Eosinophils comprising > 1% of milk cells were only detected in the mothers who had infants with CMA. Furthermore, the proportions of CD4+ T cells were positively correlated with the proportion of milk macrophages and negatively with the percentage of milk neutrophils and eosinophils. The proportions of total B cells and those expressing CD23, a low-affinity immunoglobulin E (IgE) receptor, were positively correlated with the proportions of neutrophils and eosinophils in mother's milk and negatively with the percentage of milk macrophages. To conclude, the composition of breast milk in some mothers is abnormal and correlates with a diagnosis of CMA in a breast-fed infant. This may provide a new and interesting insight into the development of food allergies.

B-cell epitopes as a screening instrument for persistent cow's milk allergy.

BACKGROUND: Cow's milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE-binding epitopes of all 6 major cow's milk proteins (alpha(s1)-, alpha(s2)-, beta-, and kappa-casein; alpha-lactalbumin; and beta-lactoglobulin) and had some evidence suggesting that IgE antibodies from patients with persistent cow's milk allergy (CMA) recognize different epitopes on cow's milk proteins than do those from patients who were likely to outgrow their allergy. OBJECTIVE: In this study we sought to assess whether recognition of IgE antibodies of certain epitopes of cow's milk proteins would clearly separate the patients with life-long CMA from those who will become clinically tolerant to cow's milk. METHODS: According to the known IgE-binding regions of cow's milk proteins, 25 decapeptides of alpha(s1)-casein, alpha(s2)-casein, kappa-casein, alpha-lactalbumin, and beta-lactoglobulin, comprising the core epitopes, were synthesized on a cellulose-derivatized membrane. Sera from 10 patients with persistent CMA and 10 patients who subsequently outgrew their milk allergy were used to investigate the differences in epitope recognition. RESULTS: Five IgE-binding epitopes (2 on alpha(s1)-casein, 1 on alpha(s2)-casein, and 2 on kappa-casein) were not recognized by any of the patients with transient CMA but showed binding by the majority of the patients with persistent allergy. The presence of IgE antibodies against at least 1 of 3 epitopes (amino acid [AA] 123-132 on alpha(s1)-casein, AA 171-180 on alpha(s2)-casein, and AA 155-164 on kappa-casein) identified all patients with persistent CMA. CONCLUSIONS: The presence of IgE antibodies to distinct allergenic epitopes of cow's milk proteins can be used as a marker of persistent CMA. Prospective studies are needed to investigate the usefulness of these informative epitopes in predicting life-long CMA in young children.