O. Novotná

Cytokine levels in healthy and allergic mothers and their children during the first year of life.

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‐γ and TGF‐β 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‐γ. 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‐γ in sera and stool of children from the allergic group sharply increased. TGF‐β levels in the sera of both groups were high, while in the milk they were relatively low and substantially lower that in the childrens stool. TGF‐β of mammary secretions is therefore unlikely to exert a decisive regulatory influence on the childrens 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.

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 newborns 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 3H‐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.

Immunostimulatory effect of Bacillus firmus on mouse lymphocytes.

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 stimulatedin 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 ofB. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+mice. The effect ofB. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-γ 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.

Perinatal period cytokines related to increased risk of future allergy development.

Testing of cytokine levels in colostrum, cord blood and amniotic fluid of healthy and allergic mothers and their newborns (using protein microarrays and quantitative analysis by ELISA) revealed differences in the levels of IL-5, IL-10, TGF-β, TNF-α, EGF and eotaxin between healthy and allergic groups. Significantly higher concentration of IL-5 and IL-10 in the colostrum of allergic mothers and cord blood of their children and also tendency to a higher level of IL-4 found at allergic mothers and their children (but without statistical significance) indicate a bias to TH2 response in this group. The higher level of TGF-β in the colostrum of healthy mothers should be involved in beneficial immunological tuning of their children including enhanced IgA formation and better intestine maturation. In amniotic fluid, concentration of TGF-β was higher in children of allergic mothers. A significantly higher level of EGF was proved in the colostrum of healthy mothers and in cord blood of their children in comparison with allergic group. EGF deficiency in the allergic group could impair or delay intestine maturation and support thus allergy development.

Cytokine expression in cord blood cells of children of healthy and allergic mothers

To determine some early signs connected with the increased risk of future allergy development, gene expression and production of selected cytokines were tested in children of allergic mothers and compared with newborns of healthy mothers. Expression of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-13, IFN-γ, TNF-α, TGF-β and EGF was tested in cord blood cells using real-time PCR and production of these cytokines was evaluated in cord sera by ELISA. Gene expression of IL-2, IL-4, IL-8, IFN-γ, IL-1β, TNF-α and TGF-β was decreased and that of IL-10, IL-13 and EGF increased in children of allergic mothers in comparison with those of healthy mothers. Significant differences in sera of healthy and allergic groups were only in IL-10 and EGF. Different relationship among serum cytokine levels reflects the fact that the cytokines are not produced only by blood cells. Significantly decreased production of EGF in newborns of allergic mothers could negatively influence maturation of mucosal membranes of these children and support thus their easier allergization. Allergic phenotype pointing to the bias to TH2 response and to possibly impaired intestine maturation was apparent already on the level of cord blood and could serve as a predictive sign of increased allergy risk.

Role of T cells in the adjuvant effect of Bacillus firmus on the immune system of mice: Intranasal and intratracheal immunization study with ovalbumin

Functions of T cells were determined after intranasal and intratracheal immunization of mice with ovalbumin (Ova) andBacillus firmus (Bf), a Gram-positive nonpathogenic bacterium of the external environment, or delipidatedBf (dBf) as adjuvants, with the aim to elucidate the mechanism of support of Ova-specific antibody production caused byBf that had been observed in an identical experiment. NeitherBf nor dBf in a mixture with Ova stimulated Ova-specific T-cell response tested as antigen-specific blast transformation. By contrast, a mild polyclonal stimulation was observed in splenocytes from mice given dBf. In vitro incubation of splenocytes with 100 µg (but not 10 µg) ofBf or dBf led to a highly significant inhibition of proliferation below the control level in all groups of animals. Supernatants of splenocyte cultures were further tested for cytokine production. IL-10 and IFN-γ were released afterin vitro challenge with dBf and in some cases also withBf. Analysis of sera demonstrated that administration of Ova + adjuvant brought about an increase in anti-Ova IgG1, IgG2a and IgG2b whereas treatment with Ova alone caused a rise in IgG1 only. The role ofBf or dBf in the enhancement of antigen-specific antibody production could be in influencing macrophages and inducing cytokine milieu composed of IL-10, IFN-γ and other factors that leads to a bystander stimulation of specifically activated Ova-B cell receptor (Ova-BCR)-bearing cells.

Adjuvant effect of Bacillus firmus on the expression of cytokines and toll-like receptors in mouse nasopharynx-associated lymphoid tissue (NALT) after intranasal immunization with inactivated influenza virus type A

Due to the persisting threat of development of new highly pathogenic influenza A subtypes, a mucosal vaccination which would induce a potent and cross-protective reaction is desirable. We succeeded in mucosal immunization of mice with an inactivated influenza A virus by using delipidated Bacillus firmus (DBF) as adjuvant. The mechanism of adjuvant effect was followed in NALT by comparing the response after intranasal immunization by inactivated influenza virus type A (H1N1) alone, adjuvant alone (DBF), or by a mixture of virus+DBF. Expression of selected gene groups was tested via qPCR at 7 different time-points: cytokines (IL-2, IFN-γ, IL-4, IL-6, and IL-10), type I interferons (IFN-α4, IFN-α11, IFN-α12, and IFN-β), toll-like receptors (TLR2, TLR3, TLR7, and TLR9), iNOS and CCR7. Intranasally administered DBF and the mixture of virus+DBF induced an elevated expression of IFN-γ, IL-6 and IL-10 cytokines, type I interferons, iNOS, and pDC markers in NALT. Multimarker qPCR data was analyzed by relative quantification and by principal component analysis. DBF has been shown to be a very efficient adjuvant for the stimulation of innate immunity after IN immunization. DBF accelerated, increased, and prolonged the antiviral response.

IgE against food and respiratory allergens in healthy and allergic mothers and their children

IgE against mixtures of common food or respiratory allergens were determined by ELISA in healthy (n = 38) and allergic (n = 62) mothers and their children. Significantly higher level of IgE against respiratory allergens was found in sera of allergic mothers and in cord blood of their children. No correlation between antibody level in maternal and newborn’s sera was found; this argues against the transfer of IgE from mother to fetus and points rather to offspring’s intrauterine sensitization. Specific IgE level in cord blood was higher in children who developed later allergy than in children who did not. Specific IgE level in colostrum was low both in healthy and allergic mothers; there was no correlation between high concentration of IgE against respiratory allergens in sera of allergic mothers and their colostrum, which does not support the idea of IgE transport from blood to mammary gland. Only slightly increased colostral IgE was detected in allergic mothers whose children manifested allergy later. Allergy of the mother and high level of anti-allergen IgE in her serum and in cord blood are the main predictive factors of future occurrence of allergy in the offspring. A combination of several predictive factors could have higher prognostic value.

Intratracheal and intranasal immunization with ovalbumin conjugated with Bacillus firmus as a carrier in mice

InactivatedBacillus firmus (BF), G+ nonpathogenic bacterium of the external environment, was coupled to ovalbumin (OVA) and used in immunization experiments as antigen carrier. Balb/c mice were immunized thrice intra-tracheally and intra-nasally with conjugates of OVA and BF. Surprisingly, administration of OVA-BF conjugates inhibited anti-OVA IgG response in both sera and mucosal secretions if compared to an exposure to OVA alone. The suppression of antigen-specific antibody production was accompanied by promotion of TH1 phenotype.

Cytokine expression in the colostral cells of healthy and allergic mothers

There is no doubt about the beneficial effect of breastfeeding on the newborns immune system. It is not fully elucidated what the differences are between the colostrum/milk of healthy and allergic mothers and how beneficial breastfeeding by an allergic mother is. The gene expression of selected cytokines was tested in cells isolated from colostra of healthy and allergic mothers using quantitative real-time PCR. Allergic phenotype was evident in colostral cells of allergic mothers: gene expressions of IL-4, IL-13 and EGF were increased and those of IFN-gamma decreased in comparison with colostral cells of healthy mothers. The allergic phenotype of the colostral cells of allergic mothers supporting the bias to a Th2 type response was found. It remains a question if a small number of these cells could influence the immature newborn immune system.