The effect of a probiotic Escherichia coli strain on regulatory T-cells in six year-old children.

Probiotics are believed to prevent or reduce allergy development but the mechanism of their beneficial effect is still poorly understood. Immune characteristics of regulatory T cells (Tregs) in peripheral blood of perinatally probiotic-supplemented children of allergic mothers (51 children), non-supplemented children of allergic mothers (42 children), and non-supplemented children of healthy mothers (28 children) were compared at the age of 6-7 years. A first dose of a probiotic Escherichia coli strain (E. coli O83:K24:H31) was administered within 2 days after the birth and then 12 times during the first months of life and children were followed longitudinally. Proportion and functional properties of Tregs were estimated by flow cytometry in relation to the children's allergy status. Proportion of Tregs in the peripheral blood of children suffering from allergy tends to be higher whereas median of fluorescence intensity (MFI) of FoxP3 was significantly decreased in allergic group. Intracellular presence of regulatory cytokine interleukin (IL)-10 was also lower in allergic children. Immune functions of Tregs reflected by both MFI of FoxP3 and IL-10 in the group of probiotic-supplemented children of allergic mothers were nearly comparable with children of healthy mothers while probiotic non-supplemented children of allergic mothers have decreased immune function of Tregs. Supplementation by probiotic E. coli strain decreases allergy incidence in high-risk children. In contrast to our expectation, proportion of Tregs has not been increased in probiotic supplemented children. Beneficial effect of probiotics on newborn immature immune system could be, at least partially, explained by the modulating immune function of Tregs. In summary, we detected increased proportion of Tregs in peripheral blood of allergic children, their functional properties were decreased in comparison with the Tregs of healthy children. A unifying hypothesis for these findings is that Treg numbers in allergic children are increased in order to compensate for decreased function.

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.

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-beta, TNF-alpha, 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 T(H)2 response in this group. The higher level of TGF-beta 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-beta 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.

[Influence of oral colonization with probiotic E. coli strain after birth on frequency of recurrent infections, allergy and development of some immunologic parameters. Long-term studies]. / Vliv perorálního osídlení probiotickým kmenem E.coli po narození na výskyt opakovaných infekcí, alergií a vývoj nekterých imunologických ukazatelu. Výsledky dlouhodobého sledování.

OBJECTIVE: prevention of repeated infections and allergies in children of allergic mothers by oral colonization with probiotic E. coli strain.The development of some immunologic parameters. Long - term studies. DESIGN: Original contribution SETTING: Mother and Child Care Institute of Prague. METHODS AND RESULTS: The results of our long-term studies confirmed that orally administered probiotic E. coli strain after birth rapidly colonized the gastrontestinal tract of the newborn and remained dominant for many weeks. The long-term presence of the strain in the intestine stimulated local and serum antibody response. Early induction of secretory IgA production is important particularly in formula-fed infants. The long-term presence of the E. coli strain in the intestine decreased the numer of pathogens colonizing intestinal and other mucous membranes , the frequency of infections and reduced need for antibiotics in premature and high-risk infants. Ten years later, there was still a lower frequency of repeated infections (23%) in comparison with control children (58%). Colonization with probiotic E. coli strain in infants treated in protected (pathogen-free) environment represented effective prevention of nosocomial infections In the colonized group infections occured in 16% of infants and 130 isolates and 7 genera of pathogens were demonstrated. In the group treated in conventional environment 40% of infants had nosocomial infections, 238 isolates and 10 genera of pathogens were proved. The hospitalization period was shorter in the first group (26 versus 34 days). Intentional colonization with probiotic E. coli after birth reduced incidence of allergies after 10 and 20 years (being 12% and 16% in the colonized groups and 33 and 32% in controls). In the present long - term study (evaluated after the first year) colonization with vaccine COLINFANT after birth influenced the levels of some cytokines ( IL-4, IFN-gama,TGF-beta) and also clinical manifestation of allergy (there were no signs of allergy in colonized infants of allergic mothers, but 25% of infants of control allergic mothers had clinical manifestations of allergies). CONCLUSIONS: By replacement of the natural but incidental ( event. pathogenic ) colonization of the intestine by a targeted orally administered E. coli strain after birth we may have come upon the possibility of how to prevent nosomial infections particularly in formula-fed and high-risk infants and prevent occurence of allergies in infants of allergic mothers.

The early postnatal development of salivary antibody and immunoglobulin response in children orally colonized with a nonpathogenic, probiotic strain of E. coli.

The development of levels of secretory immunoglobulins (SIgs) in newborns' saliva was examined under physiological conditions and after artificial colonization with nonpathogenic, probiotic bacterial strain E. coli O83. Higher levels of secretory immunoglobulin M (SIgM) and secretory immunoglobulin A (SIgA) were detected in the saliva of breast-fed children when compared with those of bottle-fed infants. SIgM was found earlier than SIgA, the levels of both SIgM and SIgA decreased after weaning. Breastfeeding actively stimulates local immunity on mucosal membranes of newborn infants. Early mucosal colonization with nonpathogenic E. coli bacteria stimulates the mucosal immune system to produce specific antibodies as well as nonspecific secretory immunoglobulins.

[Effect of care in a protected environment on the occurrence of nosocomial infections, mucosal colonization of pathogenic microflora and development of indicators of immunity in premature infants]. / Vliv osetrování v chráneném prostredí na výskyt nozokomiálních infekcí, osídlení sliznic patogenní mikroflórou a vývoj nekterých ukazatelu imunity u nedonosených detí.

OBJECTIVE: Prevention of hospital infections in premature infants by treatment in protected environment and intentional colonization of the intestine by oral administration of non-enteropathogenic E. coli. DESIGN: Original article. SETTING: Institute for Care of Mother and Child, Prague. METHODS: Fifty premature infants were followed at the Intensive Care Unit. Twenty five infants were treated after birth in protected environment and colonized during the first 48 hours by oral administration of a apathogenic E. coli strain (Vaccine COLINFANT, registered and produced in the Czech Republic), 25 infants were treated in conventional environment. Bacteriological examinations: stool, nose, throat, ear, stomach end other smears were taken 2-3 times during first week and further once a week and examined by aerobic cultivation. Immunological examinations: globulin levels (IgG, IgM) were estimated in blood samples by ELISA, using specific polyclonal antibodies. As early marker of infection T and B subpopulations were measured by FACS analysis using double labelling of cells. RESULTS: Birth weight, birth length, gestational age, Apgar score, were relatively equal in both groups. Time of hospitalization was 7.3 days shorter in infants treated in protected environment. Colonization with pathogens (130), number of infections (16%) and need for antibiotics (16%) were significantly lower in infants treated in protected environment than in infants treated in the conventional one (238 isolated pathogens, nosocomial infections in 40%, need for antibiotical treatment in 48% if infants). Serum IgM levels were lower in infants treated in protected environment other immunological parameters did not differ in the two groups. CONCLUSION: Treatment of premature and high-risk infants in protected, pathogen-free environment and intentional colonization with apathogenic E. coli reduced significantly the number of isolated pathogens, number of hospital infections, need for antibiotics and shortened the time of hospitalization in comparison with infants treated in conventional environment.

Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E. coli strain Nissle 1917.

The aim of this study was to analyze the influence of oral administration of E. coli Nissle 1917 on the systemic humoral and cellular immunity in premature infants. Thirty-four premature infants were colonized with E. coli Nissle 1917 in a randomized, placebo-controlled blinded clinical trial. Stool samples of infants were analyzed repeatedly for the presence of the administered strain. The proliferative response to bacterial antigens of E. coli origin was measured in whole blood of 34 colonized infants and 27 noncolonized controls. E. coli colonization induced a significant increase in the proliferation of blood cells cultivated with bacterial components of E. coli Nissle 1917 and another E. coli strain in colonized infants as compared with noncolonized controls. Significantly higher amounts of specific anti-E. coli Nissle 1917 antibodies (Ab) of immunoglobulin (Ig)A isotype and nonspecific polyclonal IgM were found in the blood of colonized infants compared to noncolonized placebo controls. We concluded that the oral application of E. coli Nissle 1917 after birth significantly stimulates specific humoral and cellular responses and simultaneously induces nonspecific natural immunity.

In vitro immunoglobulin response of fetal B-cells is influenced by perinatal infections and antibiotic treatment: a study in preterm infants.

UNLABELLED: The aim of our study was to analyse the influence of perinatal infections and administration of antibiotics on B-cell activity in blood cell cultures of preterm infants. We studied spontaneous and Escherichia coli induced immunoglobulin (Ig) secretion in 148 infants of 24 to 36 weeks of gestation: 53 healthy infants (Group I), 40 healthy infants receiving prophylactically antibiotics (Group II), 14 infants with intra-uterine infection (Group III) and 41 with nosocomial infection (Group IV). Spontaneous Ig secretion was significantly lower in neonates with intra-uterine infection (Group III) than in healthy infants of Group I. Nosocomial infections in Group IV increased spontaneous Ig synthesis, but only in the first days after birth. E. coli stimulation of peripheral blood mononuclear cells significantly increased Ig synthesis in healthy infants of Group I, whereas induced minimal Ig production in infected infants of Groups III and IV. Antibiotics given as prevention to Group II decreased Ig production in cell cultures as compared to healthy infants (Group I). CONCLUSION: The results indicate that perinatal infections and administration of antibiotics depress immunoglobulin secretion in cell cultures. We suggest that in vivo B-cell activity in infected preterm infants, and infants prophylactically receiving antibiotics, could also be depressed and result in decreased immunoglobulin production in these infants.

Antigenic stimuli do not influence thymic B lymphocytes: a morphological and functional study in germ-free and conventionally reared piglets.

We have recently reported that thymic B lymphocytes (TBL) are the first B-cell subpopulation undergoing isotype switching to IgG and IgA during embryonic life. The aim of this study is to analyze the influence of antigenic stimulation on TBL location and activity using a germ-free (GF) newborn pig model, in which maternal antibodies and antigens do not affect B-cell development. Immunohistological analysis showed that TBL were disseminated mainly in the thymic medulla. There were no differences in the distribution of TBL, both in GF newborn piglets before and after colonization with Escherichia coli and in older conventionally reared (CONV) piglets. The number of immunoglobulin (Ig)-secreting cells measured by the ELISPOT method was not influenced by microflora and food antigens. IgM-positive cells secreting IgM and CD45RC-positive cells spontaneously producing IgM, IgG, and IgA were detected in newborn thymus. Our findings suggest that TBL differentiation and Ig switching to IgG and IgA-secreting cells is not influenced by external antigens and that the thymic microenvironment plays an important role in this process.

Probiotics and E. coli infections in man.

After oral administration of live oral vaccines COLINFANT and MUTAFLOR prepared from non-enteropathogenic E. coli strains, both strains colonized effectively the intestine in full-term and preterm infants and remained for many weeks showing, that they were capable to establish themselves as a resident strain in the infant's gut. The presence of E. coli stimulated significantly antibody production in gut, saliva and serum of colonized infants. An early induction of secretory IgA production is important particularly in formula-fed infants, where it partly replaces the lacking immunoglobulin supplied with mother milk. In full-term and premature infants the early presence of non-pathogenic E. coli strains in the intestine decreased significantly the presence of pathogenic bacterial strains in the intestine but also other mucosal surfaces of the body. The COLINFANT strain decreased the number of nosocomial infections, mortality rate in connection with infection, and the need for antibiotics. Both strains replaced successfully pathogenic strains in carriers after treatment with antibiotics.

Effect of preventive administration of a nonpathogenic Escherichia coli strain on the colonization of the intestine with microbial pathogens in newborn infants.

In a randomized, double-blind study, 27 healthy newborn infants were colonized with the nonpathogenic Escherichia coli strain Nissle 1917 (E. coli DSM 6601, Mutaflor) during the first 5 days of life by daily oral inoculation of 1 ml of a suspension with 10(8) living cells. A second group of 27 newborns, used as controls, received a placebo suspension (1 ml of phosphate-buffered saline) instead. Stool samples were taken on days 1, 2, 3, 5, and 21, and 6 months after birth. All samples were examined for the presence of the nonpathogenic E. coli strain and of pathogenic and potentially pathogenic microorganisms. The administered E: coli strain was detected in the stools of the colonized newborns from day 2 and remained present throughout the study in more than 90% of these infants. Colonization with true and potential bacterial pathogens was significantly reduced in infants receiving E. coli strain Nissle 1917 compared to the placebo group--both with respect to numbers of pathogens and to the spectrum of species.

Polyclonal immunoglobulin response of thymic, hepatic and splenic lymphocytes from fetal, germ-free and conventionally reared pigs to different B-cell activators.

Immunoglobulin (Ig) response to different polyclonal B-cell activators was measured by ELISA in cell culture media of thymocytes, splenocytes and liver cells isolated from pig fetuses, 8-d-old germ-free piglets and conventionally reared pigs. Both in fetal and in postnatal life polyclonally stimulated lymphocytes were found to produce predominantly the IgM isotype; the first IgM formation was detected in 50-d-old fetal liver (gestation in pigs lasts 114 d). Surprisingly, 73-d-old fetal thymic cells were shown to be induced to Ig synthesis and secretion. In contrast to splenocytes of the same age, which secreted exclusively IgM, fetal thymocytes produced IgM, IgG and IgA. Polyclonally stimulated splenic cells as compared with thymic cells started to produce IgA later in fetal ontogeny, whereas the IgG response was not detectable in splenic cell culture media during the whole embryonal development and appeared only after birth. The earliest and the highest Ig stimulation was found after cultivation of lymphocytes with Nocardia delipidated cell mitogen. Interestingly, the moderate stimulatory effect of 65-kDa heat shock protein (Hsp-65) in polyclonal IgM response of fetal splenocytes was observed. We showed that thymic B lymphocytes represent probably the first maturing B cell population detectable in fetal life, which is able to differentiate after polyclonal stimulation into IgM as well as IgA and IgG producing cells.

[The effect of oral colonization by non-pathogenic E. coli on the immune response in neonates and possibilities of its use in the prevention of nosocomial infections in children at risk]. / Vliv perorálního osídlení nepatogenním kmenem E. coli na imunitní odpoved' u novorozencu a moznosti jeho vyuzití pro prevenci nozokomiálních nákaz u rizikových detí.

Oral colonization with the non-pathogenic strain of E. coli 083:K24:H31 stimulated in a significant way the local antibody formation in the gut, saliva and milk of mothers of the colonized infants. Early induction of SIgA formation is important in particular in infants who are not breastfed where it replaces partially the lacking immunoglobulin supplied in breast milk. In premature and risk infants colonization had a favourable effect on reduction of the number of infections, deaths in conjunction with infection, a reduced presence of pathogenic microflora in the alimentary tract and elsewhere. In carriers the strain replaced successfully pathogenic strains and assisted the restitution of the impaired intestinal microflora.

The antibody response in breast-fed and non-breast-fed infants after artificial colonization of the intestine with Escherichia coli O83.

The local and systemic antibody response after oral administration of a nonenteropathogenic type 1 fimbriated Escherichia coli O83 strain was followed in nine breast-fed and eight formula-fed infants during their first 15 wk of life. Five breast-fed and six formula-fed infants were followed as controls. E. coli O83 was detected in the stools of colonized infants from d 2 after colonization and persisted in the intestine for up to 26 wk. The percentage of children successfully colonized with E. coli O83 was higher among breast-fed than among formula-fed colonized infants. Also, the O83 bacteria isolated from the breast-fed children had a higher capacity to attach to colonic epithelial cells of the HT-29 cell line than those isolated from bottle-fed infants. E. coli O83 IgA and IgM antibodies estimated by ELISA were significantly elevated in the saliva of colonized as compared with control infants 2-7 wk after colonization. IgA antibodies against O83 were also higher in the stool of colonized formula-fed infants than in formula-fed controls. The results suggest that the mucosal immune system of the newborn infant can be triggered early to produce specific antibodies against bacteria colonizing the intestine.

Local and systemic antibody response in infants after oral administration of inactivated enteropathogenic E. coli serotype O111 and O55.

In ten infants divided into two groups (up to one month of age and at 2-7 months of age) the dynamics and formation of different antibody isotypes produced locally in the intestine and in serum after orally administered inactivated enteropathogenic E. coli strains O111 and O55 was followed during 30 d after the first and booster dose by using an indirect immunofluorescence method. Infants up to one month of age produced antibodies of IgM isotype in stool together with the IgA isotype after the first and booster dose of the vaccine against both antigens. Serum IgG antibody increased after 2 d following the first and second dose of antigens and remained higher during 5 d. The infants aged 2-7 months expressed predominantly the IgA isotype response in stool after the first and booster dose of antigens. The serum immunoglobulin levels did not change after oral antigen administration.

The influence of artificial colonization with E. coli strain O83 on the intestinal flora in infants.

Dominant bacterial strains present in stool (with particular emphasis on E. coli strains) were examined in 4 groups of healthy infants: breast-fed and bottle-fed, colonized with E. coli O83, and control (non-colonized) breast-fed and bottle-fed newborns. The presence of fimbriae was examined by hemagglutination, the P-fimbriae-bearing strains were tested by the PPA latex test. In addition, adherence to cell line HT-29 and serotyping was performed in selected strains. The E. coli strain O83 was found to possess type 1 fimbriae. Fewer bacterial strains possessing type 1 fimbriae were found in E. coli O83-colonized infants (except the O83 serotype) than in control infants. The E. coli O83 strain colonized significantly better the breast-fed than the bottle-fed infants; its higher adherence activity was demonstrated even in cell line HT-29. Finally, colonization with E. coli O83 influenced the character of microbial intestinal flora: the frequency of positive E. coli isolates was significantly higher in colonized (both breast- and bottle-fed) than noncolonized infants.