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.

Role of maternal elimination diets and human milk IgA in the development of cow's milk allergy in the infants.

BACKGROUND: The role of maternal avoidance diets in the prevention of food allergies is currently under debate. Little is known regarding the effects of such diets on human milk (HM) composition or induction of infant humoral responses. OBJECTIVE: To assess the association of maternal cow's milk (CM) avoidance during breastfeeding with specific IgA levels in HM and development of cow's milk allergy (CMA) in infants. METHODS: We utilized HM and infant serum samples from a prospective birth cohort of 145 dyads. Maternal serum and HM samples were assessed for casein and beta-lactoglobulin (BLG)-specific IgA and IgG by ELISA; 21 mothers prophylactically initiated a strict maternal CM avoidance diet due to a sibling's history of food allergy and 16 due to atopic eczema or regurgitation/vomiting seen in their infants within the first 3 months of life. Infants' sera were assessed for casein and BLG-specific IgG, IgA and IgE; CMA was confirmed by an oral food challenge. The impact of HM on BLG uptake was assessed in transcytosis assays utilizing Caco-2 intestinal epithelial cell line. RESULTS: Mothers avoiding CM had lower casein- and BLG-specific IgA in HM than mothers with no CM restriction (P = 0.019 and P = 0.047). Their infants had lower serum casein- and BLG-specific IgG(1) (P = 0.025 and P < 0.001) and BLG-specific IgG(4) levels (P = 0.037), and their casein- and BLG-specific IgA levels were less often detectable than those with no CM elimination diet (P = 0.003 and P = 0.007). Lower CM-specific IgG4 and IgA levels in turn were associated with infant CMA. Transcytosis of BLG was impaired by HM with high, but not low levels of specific IgA. CONCLUSIONS: Maternal CM avoidance was associated with lower levels of mucosal-specific IgA levels and the development of CMA in infants. CLINICAL RELEVANCE: HM IgA may play a role in preventing excessive, uncontrolled food antigen uptake in the gut lumen and thereby in the prevention of CMA.

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.

Identification of amino acids critical for IgE-binding to sequential epitopes of bovine kappa-casein and the similarity of these epitopes to the corresponding human kappa-casein sequence.

BACKGROUND: The delineation of allergenic (i.e. IgE-binding) epitopes in cow's milk proteins and the amino acids (AAs) critical for IgE-binding is necessary to understand better the structural properties of an allergen and to develop more efficacious immunotherapeutic reagents. Furthermore, this information may enable us to understand better cross-sensitivity between different allergens. METHODS: Eleven peptides, 10-14 AAs in length, representing the IgE-binding epitopes of kappa-casein were synthesized on a derivatized cellulose membrane with single AA substitutions at each position. Membranes were incubated with pooled sera from 15 milk-allergic patients and individual sera from 10 of the patients included in the pool. RESULTS: For 10/11 allergenic peptides, one to five different single AA substitutions resulted in elimination of IgE-binding of pooled patient sera. Overall at least one mutated peptide could be found for these 10 IgE-binding sites that resulted in a reduction of IgE-binding in at least 80% of the patients who recognized the native protein. Furthermore, the IgE-binding region at AA104-112 on bovine kappa-casein showed a high degree of similarity with the human kappa-casein, respectively, including the AAs critical for IgE-binding. CONCLUSION: This finding suggests that critical AAs should be assessed with both pooled and individual patient sera to account for the B-cell epitope heterogeneity between patients, with cow's milk allergy. In addition, we identified two potentially cross-reactive peptides between bovine and human caseins of unknown clinical relevance.

Specificity of IgE antibodies to sequential epitopes of hen's egg ovomucoid as a marker for persistence of egg allergy.

BACKGROUND: Approximately two-thirds of egg-allergic infants become tolerant within the first 5 years of life. OBJECTIVE: We sought (1) to compare the recognition of sequential (linear) and conformational binding sites of ovomucoid, ovalbumin and ovotransferrin, by IgE antibodies of children with persistent and transient egg allergy, (2) to identify immunodominant IgE-and IgG-binding epitopes of ovomucoid, and (3) to compare epitope-specificity of IgE antibodies between patients with differing natural histories of egg allergy. METHODS: Using immunodot-blots or ImmunoCAPs, IgE-antibodies against conformational (native) and sequential (reduced and alkylated) egg proteins were determined at the time of clinical reactivity in patients who retained their allergy and in those who developed clinical tolerance. IgE- and IgG-binding epitopes were mapped for ovomucoid using overlapping decapeptides on SPOTs membranes. Recognition of the major IgE-binding epitopes were compared between patients with differing natural histories of egg allergy. RESULTS: The patients with long-lasting egg allergy had a higher concentrations of IgE antibodies against sequential and native ovomucoid and ovalbumin than the children who subsequently gained tolerance (P < 0.01). Four major IgE-binding epitopes were identified in ovomucoid at amino acid 1-10, 9-20, 47-56, and 113-124. IgE antibodies of all seven patients with persistent egg allergy recognized these epitopes whereas none of the 11 children who outgrew their egg allergy did so. CONCLUSIONS: Patients with persistent egg allergy develop IgE antibodies against more sequential and conformational epitopes of ovomucoid and ovalbumin. The presence of serum IgE antibodies to specific sequential epitopes of ovomucoid may be used as a screening instrument for persistent egg allergy.

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.

Expression of intercellular adhesion molecules on circulating lymphocytes in relation to different manifestations of cow's milk allergy.

BACKGROUND: The complex interactions between immune cells are partly mediated by different adhesion molecules, but little is known about their role in the systemic immunoinflammatory process following sensitization to food antigens in early infancy. OBJECTIVE: The aim of this study was to investigate the expression of intercellular adhesion molecule-1 (ICAM-1or CD54) and the alpha subunits of its ligands' lymphocyte function-associated antigen-1 (LFA-1) (alphaL subunit or CD11a) and Mac-1 (alphaM subunit or CD11b) on peripheral blood leucocytes in infants with cow's milk allergy (CMA) and in healthy controls. METHODS: Thirty-nine breastfed infants, aged from 0.6 to 8.3 months, and their lactating mothers were included in the study from delivery onwards. During follow-up, 25 infants developed CMA and 14 remained healthy. Expressions of CD54 and CD11b on peripheral blood leucocytes were evaluated by flow cytometry. In addition, the expression of CD11a on peripheral blood leucocytes was analysed by immunocytochemistry. Mothers' milk samples were collected and their leucocyte content was evaluated using a light microscope. RESULTS: The frequency of ICAM-1 expressing peripheral blood lymphocytes was significantly higher in patients with CMA than in healthy infants (P=0.03, Mann-Whitney U-test). Furthermore, the high proportion of ICAM-1-expressing cells was associated with gastrointestinal and multiorgan symptoms in the CMA infants. There was no significant difference in the expression of Mac-1 alphaM on lymphocytes in our study groups, but the LFA-1 alphaL expression seemed to be higher in the IgE-mediated CMA. CONCLUSION: We suggest that the high expression of ICAM-1 on peripheral blood lymphocytes may reflect enhanced stimulation of T cells in vivo and their migration to the effector tissues in an early-phase of developing CMA. Furthermore, high ICAM-1 expression may be associated with the presence of multiorgan manifestations of CMA, whereas high LFA-1 expression may reflect the IgE-mediated disease.

Concurrent cereal allergy in children with cow's milk allergy manifested with atopic dermatitis.

BACKGROUND: There is increasing consensus about the significance of food allergens in the pathogenesis of atopic dermatitis (AD) in infancy and childhood, with cow's milk and egg accounting for most of the reactions. Previous studies have indicated that multiple food sensitization, such as cereals, is very common in patients with cow's milk allergy (CMA). Evidence is lacking, however, as to its clinical relevance. OBJECTIVE: The purpose of this study was to determine the concurrent occurrence of cereal allergy among children with challenge-proven CMA who have residual symptoms, such as AD and/or gastrointestinal symptoms, during cow's milk elimination diet. Further, we sought to evaluate the utility of patch testing in prescreening foods other than cow's milk behind allergic symptoms in children. METHODS: The study population comprised 90 children, aged from 2.5 to 36 months (mean 1.1 years), with challenge-proven CMA. As a result of residual symptoms during meticulous cow's milk elimination diet (AD: n=80, and gastrointestinal: n=10), the children were put on a cereal elimination diet (oats, wheat, rye, and barley) and skin prick tests (SPT) and patch testing with cereals were performed. Open cereal challenge was performed to confirm cereal allergy. RESULTS: Cereal challenge was positive in 66 (73%) of the children with CMA. Of them, 17% reacted with immediate reactions and delayed-onset reactions were seen in 83% of the children. SPT was positive in 23%, patch test in 67%, and either SPT or patch test was positive in 73% of the children with cereal allergy. SPT gave the best positive predictive value, whereas SPT together with patch test gave the best negative predictive value. CONCLUSIONS: Residual symptoms, such as eczema or gastrointestinal symptoms in CMA children may be a sign of undetected allergy to other food antigens. SPT with cereals aids in diagnosing cereal allergy in small children, especially when used together with patch testing.

IgE and IgG binding epitopes on alpha-lactalbumin and beta-lactoglobulin in 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 and IgG binding epitopes for alpha(s1)-casein, a major cow's milk allergen, and found an association between recognition of certain epitopes and clinical symptoms of cow's milk allergy (CMA). Since alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) are suspected to be significant allergens in cow's milk, we sought to determine the structure of sequential epitopes recognized by IgE antibodies to these proteins. We further sought to assess the pattern of epitope recognition in association with the clinical outcome of CMA. METHODS: According to the known amino acid sequence of ALA and BLG, 57 and 77 overlapping decapeptides (offset by two amino acids), respectively, were synthesized on a cellulose derivatized membrane. Sera from 11 patients 4-18 years of age with persistent CMA (IgE to cow's milk >100 kU(A)/l) were used to identify IgE binding epitopes. In addition, 8 patients < 3 years of age and likely to outgrow their milk allergy (IgE to cow's milk < 30 kU(A)/l) were used to investigate the differences in epitope recognition between patients with 'persistent' and those with 'transient' CMA. Seven patients 4-18 years of age were used for assessing the IgG binding regions. RESULTS: In patients with persistent allergy, four IgE binding and three IgG binding regions were identified on ALA, and seven IgE and six IgG binding epitopes were detected on BLG. The younger patients that are likely to outgrow their allergy recognized only three of these IgE binding epitopes on BLG and none on ALA. CONCLUSIONS: The presence of IgE antibodies to multiple linear allergenic epitopes may be a marker of persistent CMA. The usefulness of IgE binding to distinct epitopes on whey proteins in defining the patients that would have a lifelong CMA needs to be investigated in further studies.

Role of conformational and linear epitopes in the achievement of tolerance in cow's milk allergy.

BACKGROUND: Cow's milk (CM) is one of the leading causes of food allergy in children. However, approximately 85% of milk-allergic children become clinically tolerant to CM within the first 3 years of life. The mechanisms involved in the achievement of tolerance remain unknown. OBJECTIVE: To study whether IgE antibodies from children with persistent cow's milk allergy (CMA) differ from children who become clinically tolerant in their ability to recognize linear and conformational epitopes of alpha(s1)- and beta-casein. METHODS: Thirty-six milk-allergic children were included in the study: 11 of the children became clinically tolerant, and 25 had persistent CMA. Blood was obtained from all patients during the time they showed clinical reactions to milk challenge. Six non-milk-allergic children served as controls. Specific IgE antibodies against linear (denatured) as well as conformational (native) milk proteins were determined by probing dot-blots with patients' sera. In addition, selected decapeptides from alpha(s1)- and beta-casein, previously found to be suggestive of persistent CMA, were synthesized on a cellulose-derivatized membrane and probed with individual sera from 10 patients who outgrew CMA and from 10 patients with persistent CMA. RESULTS: Analysis of immunodot-blots showed that, in comparison to tolerant patients, milk-allergic children with persistent symptoms had a significantly higher ratio of specific IgE antibodies to linearized than to native alpha- and beta-casein (P < 0.005 and P < 0.02, respectively). Comparing the selected decapeptides, six of the 10 patients with persistent allergy recognized the peptide corresponding to amino acids 69-78 from alpha(s1)-casein while none of the patients who outgrew CMA had IgE binding to this epitope. CONCLUSION: Patients with persistent milk allergy possess higher detectable levels of IgE antibodies to linear epitopes from alpha(s1)- and beta-casein than children who have achieved tolerance. Specific IgE binding to particular linear epitopes in alpha(s1)-casein may be a predictive factor for persistence of CMA.

Identification of IgE and IgG binding epitopes on beta- and kappa-casein in cow's milk allergic patients.

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children aged less than 2 years of age. Although beta- and kappa-casein are considered among the major allergens responsible for CMA, no data are available on their allergenic epitopes in humans. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on beta- and kappa-casein and to determine whether the pattern of epitope recognition is associated with the natural history of CMA. METHODS: Overlapping decapeptides representing the entire length of beta- and kappa-casein, respectively, were synthesized on a cellulose-derivatized membrane. Sera from 15 milk-allergic children, 4-18 years of age, with high levels of specific IgE antibodies to cow's milk were used to identify IgE- and IgG-binding epitopes. In addition, IgE epitopes were screened with pooled or individual sera from younger patients aged less than 3 years and who had low levels of specific serum IgE, who are likely to outgrow CMA. RESULTS: Six major and three minor IgE-binding epitopes, as well as eight major and one minor IgG binding regions, were identified on beta-casein. Eight major IgE-binding epitopes, as well as two major and two minor IgG-binding epitopes, were detected on kappa-casein. Three of the IgE binding regions on beta-casein and six on kappa-casein were recognized by the majority of patients in the older age group, but not by the younger patients. CONCLUSION: Information regarding the immunodominant epitopes in beta- and kappa-casein may be important for understanding the pathophysiology and natural history of CMA. Differences in epitope recognition may be useful in identifying children who will have persistent milk hypersensitivity.

Identification of IgE- and IgG-binding epitopes on alpha(s1)-casein: differences in patients with persistent and transient cow's milk allergy.

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children less than 2 years of age, but about 80% become clinically tolerant within the first 3 years of life. Casein is one of the major allergens responsible for CMA and seems to play an important role in persistent allergy. Previous studies on egg allergy suggested that linear epitopes are associated with long-lasting food allergy. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on alpha(s1)-casein and to determine whether the patterns of epitope recognition are associated with the natural history of CMA. METHODS: According to the known amino acid (AA) sequence, 96 overlapping decapeptides representing the entire length of alpha(s1)-casein were synthesized on a cellulose-derived membrane. Sera from 24 children with milk allergy were used to identify IgE- and IgG-binding epitopes. RESULTS: Six major and 3 minor IgE-binding, as well as 5 major and 1 minor IgG-binding, regions on alpha(s1)-casein were identified. Two IgE-binding regions (AA 69-78 and AA 173-194) were recognized by the majority of patients over 9 years of age with persistent allergy (67% and 100%, respectively) but by none of the children less than 3 years of age who are likely to outgrow CMA. No differences in IgG binding between the groups were observed. CONCLUSION: There appears to be a difference in epitope recognition between patients with different natural histories of CMA. Screening for IgE antibodies to these epitopes may be useful in identifying children who will have persistent milk hypersensitivity.

Does low IgA in human milk predispose the infant to development of cow's milk allergy?

We sought a relationship between total and cow's milk-specific IgA levels in colostrum and human milk and subsequent development of cow's milk allergy (CMA) in the breast-fed infant. The study included 87 nursing mothers and their infants (age, 2 d to 7 mo), followed prospectively up to 1 y. At 1 y, 48 mothers (69% with an atopic constitution) had an infant with CMA, verified by clinical cow's milk challenge, eight (38% with an atopic constitution) had a baby who had had protracted infantile colic but no CMA (disease control group), and 31 (23% with an atopic constitution) had a healthy infant. Total breast-milk IgA was measured by radial immunodiffusion, and IgA antibodies to cow's milk were measured by ELISA during the breast-feeding period. The levels of total and cow's milk-specific IgA antibodies in colostrum and human milk were significantly lower in the mothers whose baby later developed CMA [estimated third day value, 0.38 g/L (95% confidence interval, 0. 24-0.82)] than in the ones whose infant remained healthy or had had infantile colic but not CMA [0.82 g/L (95% confidence interval, 0. 99-1.51); p < 0.05]. The infants developed CMA significantly more often if the concentration of total IgA antibodies in milk was <0.25 g/L, when measured between 6 d and 4 wk postpartum [sensitivity, 0. 55; specificity, 0.92; odds ratio, 14.7 (95% confidence interval, 3. 1-70.2); p < 0.001]. The levels of cow's milk-specific IgA positively correlated with the levels of total IgA but not with the development of CMA in the infant. The levels of total or cow's milk-specific IgA did not correlate with maternal atopy. IgA antibodies in colostrum and human milk may prevent antigen entry at the intestinal surface of the breast-fed infant. A low IgA content in human milk may lead to defective exclusion of food antigens and thus predispose an offspring to develop food allergies.

Defective tumour necrosis factor-alpha production in mother's milk is related to cow's milk allergy in suckling infants.

BACKGROUND: The precise role of leucocytes in human milk is still unresolved. OBJECTIVE: To assist in clarifying the immune mechanisms involved in the development of CMA in suckling infants, we studied the role of immunoregulatory leucocytes and their mediators in human breast milk. METHODS: The study population consisted of 43 lactating mothers and their infants, aged 0.25-8.0 months, followed-up prospectively from birth. Of these mothers, 27 had an infant with challenge-proven cow's milk allergy manifested with either skin (n = 23), gastrointestinal (n = 2) or skin and gastrointestinal symptoms (n = 3). Sixteen mothers with a healthy infant served as controls. We evaluated the spontaneous and mitogen-induced tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) production of human milk leucocytes and isolated peripheral blood lymphocytes in vitro with a commercial ELISA kit. RESULTS: TNFalpha production of breast milk leucocytes was significantly lower in the mothers with a cow's milk-allergic infant, whereas IFNgamma production of these cells was comparable in the two groups. CONCLUSION: Our results suggest that in the breast milk of mothers having an infant with cow's milk allergy, the number and function of TNFalpha-producing cells is defective. This might lead to a disturbance in the development of oral tolerance and thereby to the development of CMA in suckling infants. These novel results may help in clarifying the etiopathogenesis of CMA.

Defective tumor necrosis factor-alpha production in infants with cow's milk allergy.

As an aid to clarifying the role of immune mechanisms in the development of cow's milk allergy (CMA) in suckling infants, we studied the capacity of peripheral blood mononuclear cells (PBMC) to produce tumor necrosis factor-alpha(TNF-alpha) in vitro. The study population consisted of 43 infants, aged 0.12-11.2 months; of these, 31 had challenge-proven cow's milk allergy manifested with either skin or gastrointestinal symptoms or both. In addition, 12 healthy infants were studied as controls. The spontaneous, unstimulated and mitogen-induced production of TNF-alpha and interferon-gamma (IFN-gamma) by isolated peripheral blood leukocytes was evaluated. TNF-alpha and IFN-gamma production of PBMC was significantly lower in infants with cow's milk allergy than in healthy children. Our results indicate that, in infants with CMA, the function of TNF-alpha-producing cells is defective. This might disturb the development of oral tolerance and thereby lead to cow's milk allergy. These results may help to clarify the etiopathology of CMA.

Cow's milk challenge through human milk evokes immune responses in infants with cow's milk allergy.

OBJECTIVES: In order to measure the immune response evoked in breast-fed infants with cow's milk allergy (CMA) by cow's milk challenge through human milk, mothers were given increasing doses of cow's milk after they had been on a cow's milk elimination diet. Another objective was to study the secretion of beta-lactoglobulin (BLG) into human milk before and during milk challenge in relation to the appearance of symptoms in infants. STUDY DESIGN: Seventeen asymptomatic mothers who had infants with challenge-proven CMA and 10 asymptomatic mothers who had healthy infants were recruited. Infants ranged in age from 1.8 to 9.4 months. A solid-phase enzyme-linked immunoassay (ELISPOT) was used to assess the total number of immunoglobulin-secreting and specific antibody-secreting cells. Flow cytometry was used to enumerate different lymphocyte subpopulations among peripheral blood lymphocytes primed during provocation by cow's milk antigens. BLG levels were assessed in human milk before the challenge and 1, 2, 3, and 4 hours after the commencement of the challenge. RESULTS: All but one of the infants with CMA showed symptoms of CMA during cow's milk challenge through human milk. There was a significant rise in the total number of immunoglobulin-secreting cells in the IgA and IgG classes associated with a positive cow's milk challenge response, but the proportions of peripheral blood B cells bearing CD19, CD23, CD19 and 23, CD5, or CD19 and CD5 were comparable. BLG levels were comparable in both study groups. CONCLUSIONS: Most of the infants with CMA reacted to cow's milk challenge through human milk. Hypersensitivity reactions to food antigens through human milk may be more common than previously thought.

Relation between weak HLA-DR expression on human breast milk macrophages and cow milk allergy (CMA) in suckling infants.

The precise role of breast milk leukocytes is unknown. We therefore studied the cellular composition of breast milk and the activation of breast milk macrophages in mothers with a cow milk-allergic infant and in those with a healthy infant. Further, we sought to determine the influence of the cellular composition of mother's milk on the infant's risk of developing cow milk allergy. Thirty-six asymptomatic mothers (26 with atopic constitution) whose babies had challenge-proven cow milk allergy and 24 asymptomatic mothers (17 with atopic constitution) with healthy infants were recruited. Geometric mean ages of the infants were 3.2 mo (95% confidence interval [CI], 2.3 to 4.4) and 2.4 mo (95% CI, 1.6 to 3.7), respectively. After separation of the fat layer, breast milk cells were incubated with fluorescein-labeled MAb to CD antigens (CD14, 45, 3, 4, 8, 19, 23, and HLA-DR) and analyzed by flow cytometry. Breast milk samples, collected with a breast pump, were processed immediately. Cytospin preparations of milk samples, made after separation of the fat layer, were stained with May-Grunwald-Giemsa and examined with a light microscope. HLA-DR expression on breast milk macrophages was significantly lower in the mothers whose infant was allergic to cow milk, 58.3% (95% CI, 44.9 to 75.6), than in the mothers of a healthy infant, 86.9% (95% CI, 78.7 to 96.1), p=0.012 (ANOVA). There was also a significant difference in the total number of breast milk leukocytes between the mothers with an allergic child, 0.17 x 10(6)/mL (95% CI, 0.12 to 0.25), and those with a healthy child, 0.08 x 10(6)/mL (95% CI, 0.05 to 0.14), p=0.019 (Mann-Whitney U test). These results suggest impaired function of breast milk macrophages in mothers whose infants had cow milk allergy. They may also reflect decreased antigen presentation to the inexperienced T cells in the gut or on other mucosal surfaces of the suckling infant, leading to subsequent development of food or respiratory allergies, e.g. asthma.