Immunological Effects of Probiotic Bacteria in Prevention and Treatment of Allergic Diseases in Children

Epidemiological and experimental studies suggest that changes in gut microbial balance are associated with increases in the prevalence of allergic diseases. Probiotics are proposed to provide beneficial immunoregulatory signals which aid in oral tolerance achievement and alleviation of symptoms of allergic diseases. The present study evaluates both the immunological mechanisms of probiotics in infants with allergic diseases and their preventive aspect among infants prone to allergy. Furthermore, the purpose of the study was to characterise the immunological features of cord blood mononuclear cells (CBMCs) in infants at high genetic risk for allergy. GATA-3 expression (p = 0.03), interleukin (IL) -2(p = 0.026), and IL-5 (p = 0.013) secretion of stimulated CBMCs were higher in IgE-sensitized infants at age 2 than in non-allergic, non-sensitized infants. Lactobacillus GG (LGG) treatment increased secretion of IFN-γ by PBMCs in vitro in infants with cow s milk allergy (CMA) (p = 0.006) and in infants with IgE-associated eczema (p = 0.017), when compared to levels in the placebo group. A probiotic mixture, increased secretion of IL-4 by PBMCs in vitro in infants with CMA (p = 0.028), when compared with placebo-group levels. The LGG treatment induced higher plasma C-reactive protein (CRP) (p = 0.021) and IL-6 (p = 0.036) levels in infants with IgE-associated eczema than in the placebo group. The probiotic mixture induced higher plasma IL-10 levels in infants with eczema (p = 0.016). In the prevention study of allergic dis-eases, the infants receiving the probiotic mixture had higher plasma levels of CRP (p = 0.008), total IgA (p = 0.016), total IgE (p = 0.047), and IL-10 (p = 0.002) than did infants in the placebo group. Increased CRP level at age 6 months was associated with a decreased risk for eczema at age 2 not only in the infants who received probiotics but also in the placebo group (p = 0.034). In conclusion, the priming of the GATA-3 and IL-5 pathway can occur in utero, and a primary feature of T-cells predisposing to IgE-sensitization seems to directly favour Th2 deviation. LGG treatment induced increased plasma levels of CRP and IL-6 in infants with IgE-associated eczema, suggesting an activation of innate immu-nity. The probiotic mixture, when given to allergy-prone infants, induced inflammation, detected as increased plasma CRP levels, which at age 6 months was associated with decreased risk for eczema at age 2.The probiotic-induced response in allergy prone infants was characterized by their higher plasma IL-10, total IgE, and CRP levels, without induction of an allergen-specific IgE response. In this respect, the probiotics in infancy appear to induce protective immune profiles that are characteristic for chronic low-grade inflammation, a response resembling that of helminth-like infections.

Cow's milk allergy and the development of tolerance

Aims: To gain insight on the immunological processes behind cow’s milk allergy (CMA) and the development of oral tolerance. To furthermore investigate the associations of HLA II and filaggrin genotypes with humoral responses to early oral antigens. Methods: The study population was from a cohort of 6209 healthy, full-term infants who in a double-blind randomized trial received supplementary feeding at maternity hospitals (mean duration 4 days): cow’s milk (CM) formula, extensively hydrolyzed whey formula or donor breast milk. Infants who developed CM associated symptoms that subsided during elimination diet (n=223) underwent an open oral CM challenge (at mean age 7 months). The challenge was negative in 112, and in 111 it confirmed CMA, which was IgE-mediated in 83. Patients with CMA were followed until recovery, and 94 of them participated in a follow-up study at age 8-9 years. We investigated serum samples at diagnosis (mean age 7 months, n=111), one year later (19 months, n=101) and at follow-up (8.6 years, n=85). At follow-up, also 76 children randomly selected from the original cohort and without CM associated symptoms were included. We measured CM specific IgE levels with UniCAP (Phadia, Uppsala, Sweden), and β-lactoglobulin, α-casein and ovalbumin specific IgA, IgG1, IgG4 and IgG levels with enzyme-linked immunosorbent assay in sera. We applied a microarray based immunoassay to measure the binding of IgE, IgG4 and IgA serum antibodies to sequential epitopes derived from five major CM proteins at the three time points in 11 patients with active IgE-mediated CMA at age 8-9 years and in 12 patients who had recovered from IgE-mediated CMA by age 3 years. We used bioinformatic methods to analyze the microarray data. We studied T cell expression profile in peripheral blood mononuclear cell (PBMC) samples from 57 children aged 5-12 years (median 8.3): 16 with active CMA, 20 who had recovered from CMA by age 3 years, 21 non-atopic control subjects. Following in vitro β-lactoglobulin stimulation, we measured the mRNA expression in PBMCs of 12 T-cell markers (T-bet, GATA-3, IFN-γ, CTLA4, IL-10, IL-16, TGF-β, FOXP3, Nfat-C2, TIM3, TIM4, STIM-1) with quantitative real time polymerase chain reaction, and the protein expression of CD4, CD25, CD127, FoxP3 with flow cytometry. To optimally distinguish the three study groups, we performed artificial neural networks with exhaustive search for all marker combinations. For genetic associations with specific humoral responses, we analyzed 14 HLA class II haplotypes, the PTPN22 1858 SNP (R620W allele) and 5 known filaggrin null mutations from blood samples of 87 patients with CMA and 76 control subjects (age 8.0-9.3 years). Results: High IgG and IgG4 levels to β-lactoglobulin and α-casein were associated with the HLA (DR15)-DQB1*0602 haplotype in patients with CMA, but not in control subjects. Conversely, (DR1/10)-DQB1*0501 was associated with lower IgG and IgG4 levels to these CM antigens, and to ovalbumin, most significantly among control subjects. Infants with IgE-mediated CMA had lower β -lactoglobulin and α-casein specific IgG1, IgG4 and IgG levels (p<0.05) at diagnosis than infants with non-IgE-mediated CMA or control subjects. When CMA persisted beyond age 8 years, CM specific IgE levels were higher at all three time points investigated and IgE epitope binding pattern remained stable (p<0.001) compared with recovery from CMA by age 3 years. Patients with persisting CMA at 8-9 years had lower serum IgA levels to β-lactoglobulin at diagnosis (p=0.01), and lower IgG4 levels to β-lactoglobulin (p=0.04) and α-casein (p=0.05) at follow-up compared with patients who recovered by age 3 years. In early recovery, signal of IgG4 epitope binding increased while that of IgE decreased over time, and binding patterns of IgE and IgG4 overlapped. In T cell expression profile in response to β –lactoglobulin, the combination of markers FoxP3, Nfat-C2, IL-16, GATA-3 distinguished patients with persisting CMA most accurately from patients who had become tolerant and from non-atopic subjects. FoxP3 expression at both RNA and protein level was higher in children with CMA compared with non-atopic children. Conclusions: Genetic factors (the HLA II genotype) are associated with humoral responses to early food allergens. High CM specific IgE levels predict persistence of CMA. Development of tolerance is associated with higher specific IgA and IgG4 levels and lower specific IgE levels, with decreased CM epitope binding by IgE and concurrent increase in corresponding epitope binding by IgG4. Both Th2 and Treg pathways are activated upon CM antigen stimulation in patients with CMA. In the clinical management of CMA, HLA II or filaggrin genotyping are not applicable, whereas the measurement of CM specific antibodies may assist in estimating the prognosis.

Dietary aspects of cow's milk allergy in young children

Cow s milk allergy (CMA) affects about 2-6% of infants and young children. Environmental factors during early life are suggested to play a role in the development of allergic diseases. One of these factors is likely to be maternal diet during pregnancy and lactation. The association between maternal diet and development of CMA in offspring is not well known, but diet could contain factors that facilitate development of tolerance. After an established food allergy, another issue is gaining tolerance towards an antigen that causes symptoms. The strictness of the elimination depends on the individual level of tolerance. This study aimed at validating a questionnaire used to inquire about food allergies in children, at researching associations between maternal diet during pregnancy and lactation and subsequent development of cow s milk allergy in the offspring, and at evaluating the degree of adherence to a therapeutic elimination diet of children with CMA and factors associated with the adherence and age of recovery. These research questions were addressed in a prospective birth cohort born between 1997 and 2004 at the Tampere and Oulu University Hospitals. Altogether 6753 children of the Diabetes Prediction and Prevention (DIPP) Nutrition cohort were investigated. Questionnaires regarding allergic diseases are often used in studies without validation. High-quality valid tools are therefore needed. Two validation studies were conducted here: one by comparing parentally reported food allergies with information gathered from patient records of 1122 children, and the other one by comparing parentally reported CMA with information in the reimbursement records of special infant formulae in the registers of the Social Insurance Institution for 6753 children. Both of these studies showed that the questionnaire works well and is a valid tool for measuring food allergies in children. In the first validation study, Cohen s kappa values were within 0.71-0.88 for CMA, 0.74-0.82 for cereal allergy, and 0.66-0.86 for any reported food allergy. In the second validation study, the kappa value was 0.79, sensitivity 0.958, and specificity 0.965 for reported and diagnosed CMA. To investigate the associations between maternal diet during pregnancy and lactation and CMA in offspring, 6288 children were studied. Maternal diet during pregnancy (8th month) and lactation (3rd month) was assessed by a validated, 181-item semi-quantitative food frequency questionnaire (FFQ), and as an endpoint register-based information on diagnosed CMA was obtained from the Social Insurance Institution and complemented with parental reports of CMA in their children. The associations between maternal food consumption and CMA in offspring were analyzed by logistic regression comparing the highest and lowest quarters with two middle quarters of consumption and adjusted for several potential confounding factors. High maternal intake of milk products (OR 0.56, 95% CI 0.37-0.86 p = 0.002) was associated with a lower risk of CMA in offspring. When stratified according to maternal allergic rhinitis or asthma, a protective association of high use of milk products with CMA was seen in children of allergy-free mothers (OR 0.30, 95% CI 0.13 - 0.69, p < 0.001), but not in children of allergic mothers. Moreover, low maternal consumption of fish during pregnancy was associated with a higher risk of CMA in children of mothers with allergic rhinitis or asthma (OR 1.47, 95% CI 0.96 - 2.27 for the lowest quarter, p = 0.043). In children of nonallergic mothers, this association was not seen. Maternal diet during lactation was not associated with CMA in offspring, apart from an inverse association between citrus and kiwi fruit consumption and CMA. These results imply that maternal diet during pregnancy may contain factors protective against CMA in offspring, more so than maternal diet during lactation. These results need to be confirmed in other studies before giving recommendations to the public. To evaluate the degree of adherence to a therapeutic elimination diet in children with diagnosed CMA, food records of 267 children were studied. Subsequent food records were examined to assess the age at reintroduction of milk products to the child s diet. Nine of ten families adhered to the elimination diet of the child with extreme accuracy. Older and monosensitized children had more often small amounts of cow s milk protein in their diet (p < 0.001 for both). Adherence to the diet was not related to any other sociodemographic factor studied or to the age at reintroduction of milk products to the diet. Low intakes of vitamin D, calcium, and riboflavin are of concern in children following a cow s milk-free diet. In summary, we found that the questionnaires used in the DIPP study are valid in investigating CMA in young children; that there are associations between maternal diet during pregnancy and lactation and the development of CMA in offspring; and that the therapeutic elimination diet in children with diagnosed CMA is rigorously adhered to.