To assess the regulatory changes of immune system in children genetically pre-disposed to allergic diseases and in their mothers, we tested cytokines IL-4, IL-5, IL-6, IL-10, IL-13, IFN-gamma and TGF-beta in 21 healthy and 21 allergic mothers (serum at the time of delivery, colostrum and milk throughout the suckling period) and their children (cord blood, venous blood and stool filtrates) up to 1 yr of age. Samples were taken at the time of delivery, 4 days post-partum and then after 3, 6 and 12 months. Significant differences between the healthy and the allergic group were found in the levels of IL-4, IL-10, IL-13 and IFN-gamma. The levels of IL-4 in the allergic group were generally higher; the levels in the sera of children of allergic mothers during the post-natal life decreased, reaching levels typical for the healthy group at 1 yr of age. Allergic mothers exhibited markedly higher IL-10 levels in the serum at the time of delivery and in milk 3 months after delivery than healthy mothers while after 6 months the IL-10 levels in all samples from the allergic group were very low. Children from allergic group had lower intestinal content of IL-13 in comparison with the healthy counterparts. At 1 yr of age, the levels of IFN-gamma in sera and stool of children from the allergic group sharply increased. TGF-beta levels in the sera of both groups were high, while in the milk they were relatively low and substantially lower that in the children's stool. TGF-beta of mammary secretions is therefore unlikely to exert a decisive regulatory influence on the children's immunity. Long-term clinical monitoring of the children will be performed to evaluate the potential prognostic significance of these changes for the future development of allergies.

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The growing knowledge of the key role of microbiota in the maturation of neonatal immune system suggests that manipulation of microbiota could be exploited in hampering allergy development. In this study, Escherichia coli O83:K24:H31 (EcO83) was administered to newborns that were followed prospectively. Several immunological characteristics (cytokines, specific IgE, total T regulatory cells (Treg) and subpopulation of natural Treg (nTreg) and induced Treg (iTreg)) were tested in peripheral blood of 8-year-old children. Incidence of allergic disease was decreased in EcO83 supplemented children and significantly elevated levels of IL-10 and IFN-ɣ were detected in serum of EcO83 supplemented children. Probiotic supplementation did not influence the numbers of the total Treg population but their functional capacity (intracellular expression of IL-10) was significantly increased in children supplemented with EcO83 in comparison to nonsupplemented children. Morover, decreased proportion of iTreg was present in peripheral blood of non-supplemented in comparison to EcO83 supplemented children. Finally, stimulation of cord blood cells with EcO83 promoted both gene expression and secretion of IL-10 and IFN-ɣ suggesting that beneficial effect of EcO83 in prevention of allergy development could be mediated by promotion of regulatory responses (by IL-10) and Th1 immune response (by IFN-ɣ). Keywords: allergy r cord blood r E. coli r probiotics r Treg Additional supporting information may be found online in the Supporting Information section at the end of the article. , L. et al., Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur. J. Immunol. 2017. 47: 1584-1797. Abbreviations: CBMC: cord blood mononuclear cells · CFU: colony forming units · EcO83: Escherichia coli O83:K24:H31 · IQR: interquartile range · nTreg: natural Treg · Treg: T regulatory cells · iTreg: induced Treg

Bacillus firmus (a Gram-positive nonpathogenic and harmless bacterium), was shown to be a strong polyclonal activator of mouse B lymphocytes as estimated by ELISA testing of Ig concentrations in culture supernatants after incubation of BALB/c mouse splenocytes with inactivated bacillus. Synthesis of all main Ig classes and all IgG subclasses was stimulated in vitro, the considerable effect on IgA formation being the most interesting feature. B cell stimulation was T cell dependent, as was demonstrated by the effect of B. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+ mice. The effect of B. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-gamma and IL-10 (detected by ELISA in splenocyte culture supernatants) showed probable stimulation of Th1 and Th2 subpopulations. Considering the stimulatory effect on IgA formation and macrophage stimulation, B. firmus seems to be a prospective mucosal adjuvant and/or probiotic.