Cytokines in breast milk from allergic and nonallergic mothers

The allergy-preventing effect of breast-feeding remains controversial, possibly because of individual variations in the composition of the breast milk. The aim of this study was to investigate the concentrations of cytokines involved in allergic reactions and IgA antibody production in breast milk from allergic and nonallergic mothers. The cytokine concentrations were determined in colostrum and 1-mo. milk samples from 24 mothers with, and 25 mothers without, atopic symptoms, using commercial ELISA kits. The immunosuppressive cytokine transforming growth factor-beta was predominant and was detectable in all milk samples. IL-6 was detected in the majority of colostral and mature milk samples, whereas the other cytokines were less commonly detected. The concentrations of IL-6, IL-10, and transforming growth factor-beta, which are all involved in IgA synthesis, correlated with each other and with total IgA concentrations in colostrum. The concentrations of IL-4 were higher in colostrum from allergic than nonallergic mothers, and similar trends were seen for IL-5 and IL-13. In conclusion, transforming growth factor-beta and IL-6 were the predominant cytokines in human milk. The correlation between the concentrations of cytokines involved in IgA synthesis, i.e. IL-10, IL-6, and transforming growth factor-beta, may explain the stimulatory effect on IgA production in breast-fed babies. Varying concentrations of IL-4, IL-5, and IL-13 may explain some of the controversy regarding the possible allergy-preventive effect of breast-feeding.     

Effects of breast milk from allergic and non-allergic mothers on mitogen- and allergen-induced cytokine production

Breast milk contains several components that provide specific immunity and affect the maturation of the infant's immune system. The aim of this study was to analyze the effects of breast milk, on mitogen- and allergen-induced cytokine production from cord blood mononuclear cells (CBMC), and if those effects differ between allergic and non-allergic mothers. The cells were incubated for 96 h with phytohemagglutinin (PHA), ovalbumin or cat dander in the presence of various dilutions of colostrum. Colostrum inhibited both mitogen- and cat-induced IFN-γ and mitogen-induced interleukin-4 (IL-4) production. The inhibition on IFN-γ production was to some extent caused by TGF-β, as the effect was modified when an anti-TGF-β antibody was added to the cultures. In contrast, colostrum enhanced allergen-induced production of the Th2-like cytokines IL-5 and IL-13, and this was accompanied with increased production of IL-10. No differences were found between allergic and non-allergic mothers. The inhibitory effect of breast milk on IFN-γ production, which was partly due to the high levels of TGF-β, together with the enhancing effect on IL-10 secretion, confirm that breast milk is anti-inflammatory. Although the production of IL-5 and IL-13 was enhanced by colostrum, this was accompanied with an increased production of IL-10. Together with the high levels of TGF-β in breast milk and inhibitory effect of colostrum on IL-4 production, this suggests a possible mechanism whereby breast-feeding may protect against the development of allergy. Despite differences in the composition of breast milk between allergic and non-allergic mothers, the effects of breast milk on cytokine production from CBMC were independent of the atopic status of the mothers.     

Fatty acids in breast milk of allergic and non-allergic mothers: The PIAMA birth cohort study

Fatty acid composition was studied in breast milk of allergic and non-allergic mothers, focusing in particular on concentrations of the n-6 and n-3 long-chain polyunsaturates (LCP) in relation to maternal allergy. Milk samples were obtained from 168 mothers with asthma or inhalant allergies and 107 mothers without asthma and inhalant allergies, between 2 and 35 weeks after delivery. Mean values of fatty acid concentration (weight% of total fatty acids) were estimated for individual fatty acids, for groups of fatty acids and for the metabolic index (the ratio between the sum of n-6 polyunsaturates and linoleic acid). For the most relevant fatty acids, the association with maternal allergy was subsequently analyzed in more detail using multiple regression analysis. The metabolic index in breast milk was significantly lower in the allergic than in the non-allergic mothers, but no significant differences were observed in the concentrations of any of the n-6 fatty acids. Also concentrations of the n-3 fatty acids and nearly all other fatty acids were similar in allergic and non-allergic mothers' breast milk. No differences in fatty acid concentrations were observed between mothers with asthma and mothers with single or with multiple inhalant allergies. Our data suggest that differences in fatty acid composition between breast milk of allergic and non-allergic mothers are small and are unlikely to be responsible for the differential effects of breastfeeding by allergic and non-allergic mothers that have been observed in some studies.     

Effect of breast milk of healthy and allergic mothers on in vitro stimulation of cord blood lymphocytes

Maternal milk has beneficial effects on the development and function of the newborn's immune system. Whether the milk of allergic mother has the same effects as the milk of healthy mothers is not yet quite clear. To contribute to the characterization of its immunomodulatory action, we tested the effect of milk of healthy and allergic mothers on the proliferation and immunoglobulin formation in cultures of cord blood mononuclear leucocytes (CBML) of newborns of healthy and allergic mothers. CBML proliferation was tested by (3)H-thymidine incorporation, IgM, IgG and IgA production by reverse ELISPOT. CBML response was examined in unstimulated cultures and after stimulation with polyclonal activators in the presence or absence of colostrum or milk. The cells of children of allergic mothers have a significantly higher proliferative activity than those of children of healthy mothers. Maternal colostrum/milk in high doses markedly suppresses cell proliferation after stimulation with polyclonal activators, whereas lower milk doses in the cultures have no such effect and exert a rather stimulatory action. Immunoglobulin production by cord blood lymphocytes is also different in the two groups of children. Low basal immunoglobulin formation is increased after stimulation with a strong polyclonal activator of B cells--Bacillus firmus, CBML of children of allergic mothers produce more IgA than those of children of healthy mothers. The stimulated production of all immunoglobulin classes in cells of children of healthy mothers is still enhanced by colostrum/milk. Children of allergic mothers show a markedly increased production of only IgM and IgA. The effect of healthy and allergic colostrum and milk on cell proliferation and immunoglobulin production is similar. The lymphocytes of children of allergic mothers differ from the lymphocytes of children of healthy mothers in their proliferative activity and the ability to form immunoglobulin already at birth.     

Immunological factors in milk from Brazilian mothers delivering small-for-date term neonates

Breast milk samples from three groups of Brazilian women were evaluated: G1, mothers delivering term babies of low birth weight (n=16); G2, mothers delivering preterm babies of appropriate birth weight (n = 20); G3, mothers delivering term babies of appropriate birth weight ( n = 30). Milk samples were obtained at 48 h and on the 7th, 15th, 30th and 60th days after delivery and they were analyzed for lysozyme and total IgA levels and for the presence of specific antibodies against Poliovirus types I, II, III, Rotavirus, Herpes simplex virus, Varicella zoster and Cytomegalovirus. The groups were not statistically different in relation to mother's age, parity, type of delivery or socio-economic levels. IgA levels were higher in both low-birth-weight groups (G1 & G2) compared to the control group (G3) throughout the study period. Lysozyme levels decreased up to the 15th day, increasing thereafter up to the 60th day in all groups. Specific antibodies were detected throughout the study period, with no differences among groups. We conclude that breast milk composition of mothers delivering low-birth-weight babies (G1 & G2) was similar despite the different gestational ages.     

Transmission of cytomegalovirus from mothers to preterm infants by breast milk

OBJECTIVES: To assess the risk of transmission of cytomegalovirus (CMV) by breast milk from CMV-seropositive mothers to their breast-fed preterm infants and to evaluate their outcome. PATIENTS AND METHODS: The study population comprised breast-fed preterm infants with a birth weight of <1,500 g and gestational age of <35 weeks. Venous blood samples from the mothers and infants were tested for CMV IgG and IgM antibodies on the 5th and 30th day after birth. Breast milk was obtained for CMV DNA detection by polymerase chain reaction and viral culture on the 5th day and on the 3rd, 6th and 12th week. Urine samples of the babies were collected at the same time for CMV culture. Neurodevelopmental assessment was done at 6 months of age, corrected for preterm birth. RESULTS: Thirty-eight mothers and 42 infants (including 4 sets of twins) were enrolled in the study. A mother-infant pair was excluded because of inadequate breast milk collection. Thirty-six mothers (97.3%) were CMV-seropositive. CMV DNA of breast milk was detected in 35 seropositive mothers. Six infants of 5 mothers were infected (infected group) at a mean of 77 days after birth, and 34 infants of 31 mothers were not (noninfected group). In all the mothers of the infected group, CMV virus could be cultured from the milk whey. The average maternal CMV IgG on day 5 after delivery was higher in the infected than in the noninfected group. Sepsis-like symptoms and hyperbilirubinemia were more frequently noted in the infected infants than in the noninfected, but the difference was not statistically significant. Neurodevelopmental outcome did not significantly differ between the 2 groups. CONCLUSIONS: The risk of CMV infection in breast-fed premature infants was highest when the mothers shed viable virus in their breast milk. These mothers had high CMV IgG, which may help identify those mother-infant pairs at risk. Inactivation of the virus in milk by freezing may be a way of reducing the transmission of this virus via breast milk.     

IgE receptor-bearing lymphocytes in allergic and nonallergic children

Using a monoclonal anti-human IgE receptor (Fc epsilon R) antibody, the percentage of Fc epsilon R(+) cells among peripheral blood lymphocytes in children with or without allergic disorders was determined. The percentage of Fc epsilon R(+) cells in 63 nonallergic children was 4.3 +/- 1.5%, which did not vary with age and was equal to that of adults (4.2 +/- 1.2%). Allergic younger children (0-2 yr) showed a significantly higher percentage of Fc epsilon R(+) cells (7.7 +/- 3.0%) than nonallergic younger children (0-2 yr) (4.0 +/- 1.3%, p less than 0.001). Similarly, in allergic younger children, serum IgE levels (geometric mean = 58.9 IU/ml) were also significantly higher than those of nonallergic younger children (geometric mean = 2.0 IU/ml) (p less than 0.01). A positive correlation between the percentages of Fc epsilon R(+) cells and serum IgE levels was observed (Spearman rank = 0.88, p less than 0.01] in eight allergic younger children (0-2 yr) with serum IgE levels higher than 100 IU/ml. The increase in the percentage of Fc epsilon R(+) cells in allergic younger children (0-2 yr) was not a secondary phenomenon caused by serum IgE because serum IgE levels in these children were much lower than the concentration at which IgE enhance Fc epsilon R expression on lymphocytes. In conclusion, Fc epsilon R(+) lymphocytes may play a regulatory role in IgE synthesis in allergic younger children (0-2 yr).     

Transfer of antibodies across the placenta and in breast milk from mothers on intravenous immunoglobulin

We studied the levels of immunoglobulins in colostrum, milk and sera from two common variable immunodeficiency (CVID) mothers (M1 and M2), and in sera from their newborn infants. During pregnancy they continued intravenous immunoglobulin therapy (IVIG). Antibody levels from maternal and cord blood collected at delivery and colostrum and milk, collected on the 3rd and 7th post‐partum days, respectively, were analyzed. Although cord/maternal blood ratios of total immunoglobulins and subclasses, as well as specific antibodies differed between M1 and M2, both showed good placental transfer of anti‐protein and anti‐polysaccharide antibodies, despite lower cord/maternal blood ratios in M2. Anti‐Streptococcus pneumoniae antibody avidity indexes were similar between paired maternal and cord serum. Both mothers’ colostrum and milk samples showed only traces of IgA, and IgM and IgG levels in colostrum were within normal range in M1, whereas M2 presented elevated IgG and low IgM levels, when compared with healthy mothers. The study of colostrum and milk activity showed that they strongly inhibited enteropathogenic Escherichia coli adhesion in vitro. CVID patients must be informed about the relevance of regular IVIG administration during pregnancy, not only for their own health but also for their immune immature offspring. Breast‐feeding should be encouraged as colostra from these CVID patients strongly inhibited E. coli adhesion to human epithelial cells thus providing immunological protection plus nutritional and psychological benefits for the infant.     

Humoral factors of immunity in maternal milk. Comparison of their levels in the breast milk of mothers with premature and at-term delivery

Eight proteins (Immunoglobulin A, G, M, C3 and C4 fractions of complement, alpha 1-glycoprotein, lactoferrin and alpha 1- antitrypsin) were measured by immuno-diffusion or laser nephelometry in 50 milk samples. Thirty nine were heated thrice at 62 degrees C for 20 minutes. Twenty six came from mothers who delivered prematurely (less than 37 weeks) and 13 from mothers who delivered at term. Eleven samples were, used to determine the effect of the heating process. There was no significative difference of the concentrations of the eight proteins between the breast milk obtained at term or prematurely, even when the comparisons were made between colostral milks or transitional milks. The heating process reduced the concentration by 47 % for IgA, more than 88 % for IgG and IgM, 41 to 74 % for the other proteins; only orosomucoid seemed little affected (-16 %). These data suggest that the heating process impairs the immunologic effect of breast milk. This effect must be particularly considered in regard to the absence of any significant difference between the milks obtained at term or prematurely.     

Immunoglobulin concentrations and bacterial antibody titres in breast milk from mothers of 'preterm' and 'term' infants

IgA, IgM and IgG concentrations and their bacterial antibodies to E. coli, group B streptococci and Brucella abortus were measured in human breast milk collected from the 1st to 10th day post-partum from mothers delivered of 'preterm' infants (Premature Breast Milk or PBM) and from mothers delivered of term infants (Term Breast Milk or TBM). Reverse passive haemagglutination tests (RPH), rocket immuno-electrophoresis and mixed reverse passive antiglobulin haemagglutination tests (MRPAH) were employed. PBM at 2-5 days post-partum (though not beyond this period) contained higher IgA levels than did TBM, and this difference persisted even when total IgA was expressed as a proportion of total milk protein: in contrast the IgM and IgG contents of PBM and TBM were the same at both these postnatal ages. The titre of IgA antibody to E. coli, which was absorbable only by the corresponding bacteria, showed no significant difference between PBM and TBM, whereas the titres of IgA reacting with Br. abortus and, to a lesser extent group B streptococci, were higher in PBM than those in TBM. However the IgA which reacted with Br. abortus and group B streptococci was not specific to those organisms but was absorbed by all three bacteria studied. It is speculated that the high IgA content of early preterm milk and perhaps the presence of especially high titres of what appears to be a non-specific or cross-reacting bacterial IgA in such milk, may be immunologically advantageous to low birthweight infants fed on their own mother's milk.     

Vitamin B6- and protein concentrations in breast milk from mothers of preterm and term infants

The vitamin B6 and protein composition of milk obtained during the first 75 days postpartum from 16 mothers delivering preterm and 24 mothers delivering at term was determined. Both groups of mothers had supplemental intake of 5 mg vitamin B6 beginning with the 2nd trimester of gestation but not during lactation. Milk from mothers delivering preterm contained significantly higher concentrations of vitamin B6 and protein than milk from mothers delivering at term. Vitamin B6-protein relationship, however, was significantly lower in milk from mothers delivering preterm than in milk from mothers delivering term. Both, concentrations of vitamin B6 and protein, and vitamin B6-protein ratio decreased with increasing time of lactation. Supplemental vitamin B6 intake during pregnancy is made responsible for this phenomenon. Milk of mothers with preterm infants is adequate to meet the maintainance requirements for vitamin B6 and protein for a premature infant.     

Macronutrient and energy content of breast milk of mothers delivering prematurely

The protein, lactose, fat and energy contents of the fore-milk of mother with term (n=23) and preterm (n=29) infants were estimated on postpartum days 3, 7, 14 and 21. During the first 4 weeks of lactation, the mean (±SD) energy (Kcal/dl), protein (g/dl), fat (g/dl) and lactose (g/dl) levels of the preterm milk were: 56.39 (±7.99), 2.17 (±0.66), 2.30 (±0.48) and 5.78 (±0.99), respectively. The same for term milk were: 59.39 (±8.30), 1.99 (±0.70), 2.48 (±0.53) and 6.24 (±1.08), respectively. The differences in composition between the term and preterm milk were not significant. The composition of breast milk showed changes over the first 3 weeks of lactation. With increasing post-partum days, there was a decline in protein content while fat, lactose and energy contents increased. These trends were more pronounced for preterm milk than term milk. The macronutrient composition and energy estimates of preterm breast milk of Indian mothers in this study may be useful for calculation of nutritional intake by premature neonates fed on expressed breast milk.     

Reactivity of secretory IgA antibodies in breast milk from 107 Japanese mothers to 20 environmental antigens

The secretory IgA (sIgA) antibody response to 20 environmental antigens, including microorganisms, toxins, food, and inhaled allergens, was evaluated in the breast milk from 107 Japanese mothers 1-10 days after delivery. Specific sIgA antibody responses were detected in most milk samples against almost all of the antigens tested, although there was a wide variation in the specific sIgA antibody profiles of each individual's milk. With regard to twelve bacterial antigens, highly specific sIgA antibody responses were detected against Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa. With regard to eight nonbacterial antigens, highly specific sIgA antibody responses were detected against rotavirus, cholera, and pertussis toxins. Similar sIgA antibody profiles were obtained when the 107 milk specimens were divided into colostrum (milk 1-5 days after delivery, n = 36) and transitional milk (milk 6-10 days after delivery, n = 71). This study provides information on the possible protective role of human milk sIgA antibodies and will serve as a baseline for future studies.     

Breast milk fatty acids,eicosanoids, and cytokines in mothers with and without allergic disease

Allergic disease (AD), including atopic eczema, asthma, allergic rhinitis, and food allergy, is characterized by an imbalance between cytokines produced by distinct T-helper cell subtypes. Whether this imbalance can be transferred from mother to breast milk remains to be established. The objective was to investigate the concentrations and interactions of nutritional and inflammatory factors in breast milk. Breast milk samples were collected from mothers with AD (n = 43) and without AD (n = 51). The concentrations of transforming growth factor (TGF)-beta2, tumor necrosis factor-alpha, IL-4, IL-10, prostaglandin E2, and cysteinyl leukotrienes were measured by immunoassays and fatty acid composition by gas chromatography. Mothers with AD had a lower concentration of TGF-beta2 in breast milk [median (interquartile range), 420 (278-701) ng/L] compared with those without AD [539 (378-1108) ng/L; p = 0.003], whereas other cytokines, prostaglandin E2, and cysteinyl leukotriene concentrations or fatty acid composition were not significantly different between the groups. The breast milk inflammatory factors and fatty acid composition were shown to be related. A positive association was observed between TGF-beta2 and the proportion of polyunsaturated fatty acids (p = 0.038) and a negative association between TGF-beta2 and the proportion of saturated fatty acids (p = 0.029) in breast milk. The reduced TGF-beta2 concentration in the breast milk of mothers with AD may interfere with the development of the mucosal immune system of the breast-fed infant. The observed associations between nutritional and inflammatory factors in breast milk suggest that it may be possible to influence the immunologic properties of breast milk by dietary intervention of the mother.     

Day-to-day variations in iron, zinc and copper in breast milk of Guatemalan mothers

OBJECTIVE: To assess the within-subject and between-subject coefficients of variation (CV) of iron, zinc and copper concentrations in the milk of Guatemalan mothers. METHODS: We performed a cross-sectional study in lactating women who had delivered a healthy infant 1 to 6 months previously in two low-income peri-urban areas (San Bartolome Milpas Altas and Ciudad Peronia) and a low-income rural area (San Juan Chamelco) in Guatemala. Women infested with Ascaris lumbricoides or Trichuris trichiura received a single dose of albendazole (400 mg) or placebo. Two weeks after treatment, milk samples were collected on 3 or 4 consecutive days. Trace element concentrations in milk were measured by inductively coupled plasma/atomic emission spectrometry. RESULTS: The instrumental error of the inductively coupled plasma/atomic emission spectrometry method, expressed as SD, was 0.04, 0.27 and 0.02 mg/L for iron, zinc and copper, respectively. Concentrations in milk samples collected from 47 mothers on 3 or 4 consecutive days, expressed as mean +/- SD, were 0.28 +/- 0.13, 2.03 +/- 0.37 and 0.29 +/- 0.07 mg/L for iron, zinc and copper, respectively. The within-subject CV was 46.1%, 18.2%, and 22.8% and the between-subject CV was 61.2%, 48.3% and 31.7% for iron, zinc and copper, respectively. Stage of lactation, infestation with intestinal parasites and residential area had a significant influence on milk zinc, copper and iron concentrations. CONCLUSIONS: One sample of milk is sufficient to give a reliable estimate of the zinc concentration in milk. Two samples taken on consecutive days are required for a reliable estimate of iron and copper concentrations.