Hanna Suomalainen

Does Low IgA in Human Milk Predispose the Infant to Development of Cow's Milk Allergy?

We sought a relationship between total and cows milk-specific IgA levels in colostrum and human milk and subsequent development of cows 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 cows 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 cows milk were measured by ELISA during the breast-feeding period. The levels of total and cows 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 cows 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 cows 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.

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 cows milk allergy (CMA) by cows milk challenge through human milk, mothers were given increasing doses of cows milk after they had been on a cows 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 cows 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 cows 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 cows 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 cows 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 mothers milk on the infants 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-Grünwald-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 × 106/mL (95% CI, 0.12 to 0.25), and those with a healthy child, 0.08 × 106/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.

Defective tumor necrosis factor-α 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‐α (TNF‐α) 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‐α and interferon‐γ (IFN‐γ) by isolated peripheral blood leukocytes was evaluated. TNF‐α and IFN‐γ 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‐α‐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.

Large number of CD19+/CD23+ B cells and small number of CD8+ T cells as early markers for cow's milk allergy (CMA)

Assessment of activation of immune mechanisms is valuable in the early diagnosis of cows milk allergy (CMA). The purpose of this study was to evaluate peripheral blood lymphocyte subclasses in children suspected of having CMA and healthy infants in order to detect an early marker for food allergy. Altogether 47 breast‐fed infants, aged from 0.4 to 10 months were followed‐up prospectively from birth because of atopic heredity. Twenty‐three of the infants were healthy and 24 infants had a strong suspicion of and later challenge‐proven cows milk allergy. Leucocyte subsets were determined from peripheral blood mononuclear cells by flow cytometry. In response to a clinical cows milk challenge, seven infants developed urticaria, 11 infants had eczema, three patients had loose stools, diarrhoea or vomiting and three infants had eczema and diarrhoea, loose stools or vomiting. The total percentage of B cells and also the proportion of B cells bearing a low‐affinity IgE receptor as a marker for activation were significantly higher, whereas the percentage of CD8+ T cells was significantly lower in infants with challenge‐proven CMA than in healthy controIs. These results imply that infants with active CMA have a defect in regulation of B‐cell function. Further, they suggest that imbalance of the ratio of suppressor and helper T cells might be an important factor in the etiopathogenesis of CMA. Our results show that large numbers of activated CD19 B cells and low numbers of CD8+ T cells could be considered as early markers for food allergy since they are already detectable in peripheral blood during the earliest symptoms of CMA.

Low frequency of CD4+, but not CD8+, T cells expressing interferon-γ is related to cow's milk allergy in infancy

Low interferon‐γ (IFN‐γ) and tumor necrosis factor‐α (TNF‐α) production in peripheral blood mononuclear cells (PBMC) from patients with atopic dermatitis and food allergy have been reported previously. However, it remains unclear whether the weak cytokine production is caused by the imbalance of specific T‐cell subsets or by dysregulation of T‐cell function. In the present study we investigated the intracellular expression of these cytokines at a single‐cell level to clarify the background of the disruption. Twelve of 27 breast‐fed infants (0.1–8.8 months of age) had challenge‐proven cows milk allergy (CMA), and 15 infants were studied as a healthy control group. PBMC were stimulated with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. The frequencies of the cells expressing intracellular IL‐4, IFN‐γ, and TNF‐α were assessed using flow cytometry. In addition, at this time‐point leucocyte subsets from the milk of mothers of these infants were evaluated using light microscopy. A lower number of CD8+ T cells and the defective capability of CD4+ T cells to express IFN‐γ in infants peripheral blood co‐existed with a lower number of macrophages in their mothers milk.

Immunologic disturbances in cow's milk allergy, 1: Delayed maturation of suppressor activity.

To assist in identifying pathogenetic mechanisms in different subtypes of cows milk allergy (CMA), the function of immunoregulatory T‐lymphocytes was studied. The study population consisted of 23 patients, mean [95% confidence interval] age of 25. 6 [19. 5, 33. 6] months, who had challenge‐proven cows milk allergy manifested with either skin (n=9) or gastrointestinal (n=14) symptoms; in addition, 13 age‐matched disease controls were studied. Patients with challenge‐proven CMA were rechallenged to establish whether they had acquired clinical tolerance to cows milk. The suppressor activity of isolated lymphocytes was measured in vitro by a cell coculture at rechallenge and in 10/23 patients at diagnosis. At diagnosis, patients with CMA (n=10) showed a decreased mean [95% CI] suppressor activity, induced by either Concanavalin A, 7[‐2, 15]%, or cows milk, 3[‐8, 14]% as compared with disease controls (n = 13), 19[15, 24]% and 24[17, 31]%; F = 7. 1, p = 0.004 and F = 6. 7, p = 0.005, respectively. At rechallenge the suppressor activity, induced both by Concanavalin A and cows milk, reached the level of disease controls only in patients who had acquired clinical tolerance to cows milk (n = 13/23), but not in those retaining CMA (n = 10/23). Our results indicate that the maturation of suppressor function is delayed in CMA, which might be of primary importance in the etiopathogenesis of CMA.

T-cell signal transduction in children with cow's milk allergy – increased MAP kinase activation in patients with acute symptoms of cow's milk allergy

The precise immune mechanisms behind cows milk allergy (CMA) are still unknown. Previously, the production of the cytokines TNF‐α and IFN‐γ in T cells from children with CMA has been shown to be decreased, and the production of IL‐4 has been shown to be increased when compared to healthy children. As these aberrations in cytokine production may be associated with disturbances in cellular function, we investigated whether T‐cell signal transduction is abnormal in children with CMA. For this purpose we evaluated the activation of the MAP kinase Erk2. Thirty‐nine infants were included in the study. Of those with CMA, 13 had acute symptoms and 9 were free of symptoms due to a successful elimination diet at the time of the study. To activate T cells and to stimulate MAP kinase phosphorylation, peripheral blood mononuclear cells (PBMC) were incubated with Concanavalin A (ConA). The change in MAP kinase phosphorylation was measured by Western blotting. The increase in MAP kinase phosphorylation after stimulation with ConA for 5 min was significantly higher in cells from patients with acute symptoms of CMA than in cells from CMA patients free of symptoms or cells from healthy children. A time‐course experiment showed that the change in MAP kinase phosphorylation was still increasing after 10 min incubation in cells from patients with acute symptoms of CMA. The increased MAP kinase activation was found to correlate positively with non‐IgE mediated CMA in patients with acute symptoms of CMA.

Sensitisation through breast milk

The benefit of breast feeding to prevent or delay the development of food allergy in infants is somewhat controversial. Although prolonged breast feeding has widely been recommended for infants at high risk of allergy, studies concerning the composition of human milk in atopic mothers are scarce. Human breast milk is not a stabile concept. We have previously found that the cellular composition of human milk is significantly different in mothers with a food allergic infant than in those with a healthy child. The number of monocytes, lymphocytes or eosinophils, was greatly elevated whereas the number of activated macrophages was decreased in the breast milk of mothers with a severely allergic infant. The high numbers of eosinophils could easily increase the permeability of the gut thereby increasing the absorption of dietary antigens, and thus enhance the development of food allergies. Breast feeding provide many advantages to the suckling infant. However, infants developing food allergic symptoms on exclusive breast feeding may benefit from changing to formula feeding to protect adequate growth and to prevent the development of a more severe allergic symptomatology.

Bronchoalveolar Lavage Findings from Persons Exposed to Moulds in Water-Damaged Houses

Bronchoalveolar lavage (BAL) samples were taken in 47 patients whose symptoms were attributable to mould exposure and had visited the Clinic for Indoor Air Health Problems at the Helsinki University Central Hospital for a diagnosis of possible interstitial pulmonary disease. Tests showed that the total number of lymphocytes was elevated in 19 of these patients (40%). The percentage of lymphocytes in the cells from the lavage fluid ranged from 2 to 63% with a median of 19%. One patient had slight neutropenia.