Bozena Cukrowska

Oral Administration of Probiotic Escherichia coli after Birth Reduces Frequency of Allergies and Repeated Infections Later in Life (after 10 and 20 Years)

Background: The development of allergies is a complex in which both composition and influence of the intestinal flora play an important role. We observed in earlier studies that the presence of an orally administered probiotic Escherichia coli strain in the intestine stimulated both a serum and local antibody response, decreased the presence of pathogens, the number of infections and the need for antibiotics. Methods: The preventive effect of oral colonization after birth with a probiotic E. coli strain was assessed by evaluating the results of a questionnaire both 20 years (150 full-term infants) and 10 years (77 preterm infants) after colonization. Results: Differences in occurrence of allergies in colonized and control subjects were statistically significant both after 10 and 20 years (p < 0.01). Specific serum IgE antibodies confirmed the presence of allergies in 100% of 10-year-old and 91% of 20-year-old patients with clinical symptoms of allergy. Ten years after colonization, the occurrence of repeated infections was significantly lower in colonized subjects than it was in controls (p < 0.01); 20 years later, no differences were found in these groups. Conclusions: Intentional colonization of the intestine with E. coli after birth (offering the advantage of the first colonizer) was found to decrease the incidence of allergies and repeated infections in later life.

Mucosal Immunity: Its Role in Defense and Allergy

The interface between the organism and the outside world, which is the site of exchange of nutrients, export of products and waste components, must be selectively permeable and at the same time, it must constitute a barrier equipped with local defense mechanisms against environmental threats (e.g. invading pathogens). The boundaries with the environment (mucosal and skin surfaces) are therefore covered with special epithelial layers which support this barrier function. The immune system, associated with mucosal surfaces covering the largest area of the body (200–300 m2), evolved mechanisms discriminating between harmless antigens and commensal microorganisms and dangerous pathogens. The innate mucosal immune system, represented by epithelial and other mucosal cells and their products, is able to recognize the conserved pathogenic patterns on microbes by pattern recognition receptors such as Toll-like receptors, CD14 and others. As documented in experimental gnotobiotic models, highly protective colonization of mucosal surfaces by commensals has an important stimulatory effect on postnatal development of immune responses, metabolic processes (e.g. nutrition) and other host activities; these local and systemic immune responses are later replaced by inhibition, i.e. by induction of mucosal (oral) tolerance. Characteristic features of mucosal immunity distinguishing it from systemic immunity are: strongly developed mechanisms of innate defense, the existence of characteristic populations of unique types of lymphocytes, colonization of the mucosal and exocrine glands by cells originating from the mucosal organized tissues (‘common mucosal system’) and preferential induction of inhibition of the responses to nondangerous antigens (mucosal tolerance). Many chronic diseases, including allergy, may occur as a result of genetically based or environmentally induced changes in mechanisms regulating mucosal immunity and tolerance; this leads to impaired mucosal barrier function, disturbed exclusion and increased penetration of microbial, food or airborne antigens into the circulation and consequently to exaggerated and generalized immune responses to mucosally occurring antigens, allergens, superantigens and mitogens.

Oral administration of antigens from intestinal flora anaerobic bacteria reduces the severity of experimental acute colitis in BALB/c mice

Homeostasis between indigenous intestinal flora and host response may be broken in inflammatory bowel disease. The present study explores whether repeated oral administration of intestinal flora antigens can protect mice against dextran sodium sulphate (DSS)‐induced colitis. Sonicates of Gram‐positive, Gram‐negative, or anaerobic resident bacteria isolated from mouse intestinal flora were fed to BALB/c mice by gastric gavage, with or without cholera toxin. After four weekly doses of 1u2003mg of these antigen preparations (or of PBS as control), DSS colitis was induced. One week later colitis was evaluated by clinical scores and histology. Mice fed a pool of the three sonicates had decreased inflammation scores (5 (1–14); median (range)) compared with PBS‐fed control animals (15 (7–19); Pu2003<u200a0·05). Decreased inflammation was observed in mice fed anaerobic bacteria antigens (7 (6–11); Pu2003<u200a0·05 versus control), but not in mice fed a pool of Gram‐positive and ‐negative sonicates (16 (12–16)). Inflammation scores of mice fed antigens with cholera toxin were similar to those of PBS‐fed control animals. DSS‐induced colitis can be suppressed by oral administration of normal intestinal flora antigens containing anaerobes.

Colonization of germ-free mice with a mixture of three lactobacillus strains enhances the integrity of gut mucosa and ameliorates allergic sensitization.

Increasing numbers of clinical trials and animal experiments have shown that probiotic bacteria are promising tools for allergy prevention. Here, we analyzed the immunomodulatory properties of three selected lactobacillus strains and the impact of their mixture on allergic sensitization to Bet v 1 using a gnotobiotic mouse model. We showed that Lactobacillus (L.) rhamnosus LOCK0900, L. rhamnosus LOCK0908 and L. casei LOCK0919 are recognized via Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptors and stimulate bone marrow-derived dendritic cells to produce cytokines in species- and strain-dependent manners. Colonization of germ-free (GF) mice with a mixture of all three strains (Lmix) improved the intestinal barrier by strengthening the apical junctional complexes of enterocytes and restoring the structures of microfilaments extending into the terminal web. Mice colonized with Lmix and sensitized to the Bet v 1 allergen showed significantly lower levels of allergen-specific IgE, IgG1 and IgG2a and an elevated total IgA level in the sera and intestinal lavages as well as an increased transforming growth factor (TGF)-β level compared with the sensitized GF mice. Splenocytes and mesenteric lymph node cells from the Lmix-colonized mice showed the significant upregulation of TGF-β after in vitro stimulation with Bet v 1. Our results show that Lmix colonization improved the gut epithelial barrier and reduced allergic sensitization to Bet v 1. Furthermore, these findings were accompanied by the increased production of circulating and secretory IgA and the regulatory cytokine TGF-β. Thus, this mixture of three lactobacillus strains shows potential for use in the prevention of increased gut permeability and the onset of allergies in humans.

Early development of immune system in pigs

Prenatal and early postnatal immune system development has been studied in minipigs. First leukocytes were observed in the yolk sac and fetal liver (FL) on the 17th day of gestation, the majority of them being SWC3(+). The colonization of the thymus (TH) with leukocytes was observed 21 days later. Two waves of fetal TH colonization with pro-T cells were deduced from the frequency of thymocyte subsets. Thymic B cells and immunoglobulin-secreting cells (Ig-SC) were studied by flow cytometry and ELISPOT, respectively. When the total numbers of fetal Ig-SC were compared, the TH was identified as the main source of natural antibodies and the only site of IgA and IgG synthesis. In germ-free animals, the TH also represented the major site of IgG and IgA production and the number of Ig-SC was not influenced by colonization with microflora. FL and bone marrow were identified as primary B lymphopoietic sites. The phenotype of B precursors was characterized and pre-B II cells were shown to be the dominant mononuclear fraction between DG50 and DG105. In the periphery, relative proportions of lymphocyte subsets were determined. Studies in gnotobiotic piglets have revealed that the appearance of CD4(+)CD8(+) T cells and CD2(-) B cells is absolutely dependent on the contact of immune system with live viruses and bacteria, respectively.

Specific Antibody and Immunoglobulin Responses after Intestinal Colonization of Germ-Free Piglets with Non-Pathogenic Escherichia coli O86

Colonization of the gut with components of commensal microflora profoundly affects the development of the immune system. The aim of the present study was to investigate mucosal and systemic B cell responses during the first few days after intestinal association of colostrum-deprived piglets reared in germ-free (GF) conditions with non-pathogenic Escherichia coli O86. Specific intestinal anti-E. coli antibodies (Ab), among which IgA Ab prevailed, were found 4 days after colonization (72% of standard) and their amount decreased 11 days later reaching 22% of standard. In contrast to mucosal Ab, specific serum Ab remained at the level of GF animals at day 4 (less than 10% of standard) and markedly increased 15 days after colonization (156% of standard). In addition to the occurrence of specific Ab, increased amounts of total immunoglobulins (Ig) of all isotypes were detected in sera and intestinal washings. Using the ELISPOT method an increased number of IgM, IgG and IgA-secreting lymphocytes were found in spleen, mesenteric lymph nodes (MLN) and Peyers patches (PP) in colonized animals as compared to GF piglets. Contrary to cells from these lymphatic organs, B cells from thymus were not affected by E. coli stimulation. Our results show that at the onset of intestinal colonization, non-pathogenic E. coli specifically and polyclonally stimulate the mucosal and systemic humoral immunity, but relatively soon after stimulation, mucosal-specific responses in gut decreases, indicating the possible beginning of inhibition mechanisms (oral tolerance).

The gut as a lymphoepithelial organ: The role of intestinal epithelial cells in mucosal immunity

Mucosal surfaces covered by a layer of epithelial cells represent the largest and most critical interface between the organism and its environment. The barrier function of mucosal surfaces is performed by the epithelial layer and immune cells present in the mucosal compartment. As recently found, epithelial cells, apart from their participation in absorptive, digestive and secretory processes perform more than a passive barrier function and are directly involved in immune processes. Besides the well known role of epithelial cells in the transfer of polymeric immunoglobulins produced by lamina propria B lymphocytes to the luminal content of mucosals (secretory Igs), these cells were found to perform various other immunological functions, to interact with other cells of the immune system and to induce an efficient inflammatory response to microbial invasion: enzymic processing of dietary antigens, expression of class I and II MHC antigens, presentation of antigens to lymphocytes, expression of adhesive molecules mediating interaction with intraepithelial lymphocytes and components of extracellular matrix, production of cytokines and probable participation in extrathymic T cell development of intraepithelial lymphocytes. All these functions were suggested to influence substantially the mucosal immune system and its response. Under immunopathological conditions,e.g. during infections and inflammatory bowel and celiac diseases, both epithelial cells and intraepithelial lymphocytes participate substantially in inflammatory reactions. Moreover, enterocytes could become a target of mucosal immune factors. Mucosal immunosurveillance function is of crucial importance in various pathological conditions but especially in the case of the most frequent malignity occurring in the intestinal compartment,i.e. colorectal carcinoma. Proper understanding of the differentiation processes and functions of epithelial cells in interaction with other components of the mucosal immune system is therefore highly desirable.

Impact of heat-inactivated Lactobacillus casei and Lactobacillus paracasei strains on cytokine responses in whole blood cell cultures of children with atopic dermatitis

Heat-inactivated Lactobacillus casei LOCK 0900, L. casei LOCK 0908 and Lactobacillus paracasei LOCK 0919 strains, applied to blood cell cultures obtained from children with atopic dermatitis induced production of anti-allergic TH1 cytokines (interleukin-12, interleukin-18, interferon-γ, tumor necrosis factor-α) and regulatory transforming growth factor-β1), but did not stimulate pro-allergic interleukin-5. The lactobacilli-mixture remarkably enhanced the TH1 response compared to single strains. This synergistic effect was not observed for transforming growth factor-β1. In contrast, the amount of interleukin-10 was found to be considerably lower when cells were stimulated with lactobacilli-mixture compared to single strains. The mixture of Lactobacillus strains represents a probiotic bacterial preparation modulating in vitro cytokine profile of allergic children towards anti-allergic TH1 response.

Autoimmunity, immunodeficiency and mucosal infections: Chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of “molecular mimicry” (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with, genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.

Intestinal epithelium, intraepithelial lymphocytes and the gut microbiota - Key players in the pathogenesis of celiac disease

Celiac disease (CD) is a chronic immune-mediated disorder triggered by the ingestion of gluten in genetically predisposed individuals. Before activating the immune system, gluten peptides are transferred by the epithelial barrier to the mucosal lamina propria, where they are deamidated by intestinal tissue transglutaminase 2. As a result, they strongly bind to human leucocyte antigens (HLAs), especially HLA-DQ2 and HLA-DQ8, expressed on antigen-presenting cells. This induces an inflammatory response, which results in small bowel enteropathy. Although gluten is the main external trigger activating both innate and adaptive (specific) immunity, its presence in the intestinal lumen does not fully explain CD pathogenesis. It has been hypothesized that an early disruption of the gut barrier in genetically susceptible individuals, which would result in an increased intestinal permeability, could precede the onset of gluten-induced immune events. The intestinal barrier is a complex functional structure, whose functioning is dependent on intestinal microbiota homeostasis, epithelial layer integrity, and the gut-associated lymphoid tissue with its intraepithelial lymphocytes (IELs). The aim of this paper was to review the current literature and summarize the role of the gut microbiota, epithelial cells and their intercellular junctions, and IELs in CD development.