Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.

• MeSH
• Autoimmune Diseases etiology   microbiology   MeSH
• Gastrointestinal Tract microbiology   MeSH
• Germ-Free Life * MeSH
• Immunity MeSH
• Humans MeSH
• Metagenome immunology   MeSH
• Disease Models, Animal MeSH
• Neoplasms etiology   microbiology   MeSH
• Mucous Membrane immunology   MeSH
• Inflammation etiology   microbiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

BACKGROUND AND AIMS: Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that is induced by dietary wheat gluten proteins (gliadins) in genetically predisposed individuals. The overgrowth of potentially pathogenic bacteria and infections has been suggested to contribute to CD pathogenesis. We aimed to study the effects of gliadin and various intestinal bacterial strains on mucosal barrier integrity, gliadin translocation, and cytokine production. METHODOLOGY/PRINCIPAL FINDINGS: Changes in gut mucosa were assessed in the intestinal loops of inbred Wistar-AVN rats that were reared under germ-free conditions in the presence of various intestinal bacteria (enterobacteria and bifidobacteria isolated from CD patients and healthy children, respectively) and CD-triggering agents (gliadin and IFN-γ) by histology, scanning electron microscopy, immunofluorescence, and a rat cytokine antibody array. Adhesion of the bacterial strains to the IEC-6 rat cell line was evaluated in vitro. Gliadin fragments alone or together with the proinflammatory cytokine interferon (IFN)-γ significantly decreased the number of goblet cells in the small intestine; this effect was more pronounced in the presence of Escherichia coli CBL2 and Shigella CBD8. Shigella CBD8 and IFN-γ induced the highest mucin secretion and greatest impairment in tight junctions and, consequently, translocation of gliadin fragments into the lamina propria. Shigella CBD8 and E. coli CBL2 strongly adhered to IEC-6 epithelial cells. The number of goblet cells in small intestine increased by the simultaneous incubation of Bifidobacterium bifidum IATA-ES2 with gliadin, IFN-γ and enterobacteria. B. bifidum IATA-ES2 also enhanced the production of chemotactic factors and inhibitors of metalloproteinases, which can contribute to gut mucosal protection. CONCLUSIONS: Our results suggest that the composition of the intestinal microbiota affects the permeability of the intestinal mucosa and, consequently, could be involved in the early stages of CD pathogenesis.

• MeSH
• Bacteria pathogenicity   MeSH
• Bacterial Toxins pharmacology   MeSH
• Bifidobacterium pathogenicity   MeSH
• Celiac Disease etiology   MeSH
• Cytokines biosynthesis   MeSH
• Enterobacteriaceae pathogenicity   MeSH
• Gliadin pharmacokinetics   pharmacology   MeSH
• Germ-Free Life MeSH
• Host-Pathogen Interactions drug effects   MeSH
• Interferon-gamma pharmacology   MeSH
• Rats MeSH
• Permeability MeSH
• Goblet Cells pathology   MeSH
• Intestines microbiology   pathology   MeSH
• Intestinal Mucosa drug effects   metabolism   microbiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Toxins MeSH
• Cytokines MeSH
• Gliadin MeSH
• Interferon-gamma MeSH

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.

• MeSH
• Autoimmunity immunology   MeSH
• Autoimmune Diseases etiology   immunology   MeSH
• Infections immunology   MeSH
• Humans MeSH
• Mice MeSH
• Intestinal Mucosa immunology   microbiology   MeSH
• Immunologic Deficiency Syndromes immunology   MeSH
• Inflammation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

The aim of the study was to compare the phenotype of lymphocyte subpopulations of the GALT (gut-associated lymphatic tissue) in germfree (GF) and conventionally (CV) reared rats, i.e. to analyze the effect of microbial colonization on the development of intestinal lymphocyte subsets. Surface marker characteristics were studied in cell suspensions isolated from Peyer's patches, mesenteric lymph nodes, spleen and the intraepithelial lymphocyte compartment of 2- and 12-month old inbred AVN rats. The pattern of T lymphocyte phenotypes in Peyer's patches, mesenteric lymph nodes and spleen determined by FACS analysis did not reveal differences between GF and CV rats. In contrast, a 2-month conventionalization of GF rats led to substantial changes in the composition of intestinal intraepithelial lymphocyte subsets (IELs): increase of CD4+, CD8 alpha+, CD8 beta+, TcR alpha/beta+ bearing lymphocytes was observed after colonization of rats with normal microflora. Surprisingly, the relative numbers of lymphocytes bearing TcR gamma/delta+ did not change during conventionalization. The effect of aging was also studied and differences in IELs composition of aged (GF) and (CV) rats were found to be more pronounced: 6.6% and 30% of lymphocytes bearing TcR alpha/beta were present among IELs in two-month old GF and CV rats, respectively. 30% of IELs in 2-month old GF rats, 80% of IEL from 12-month old CV rats were found to bear TcR alpha/beta. This finding demonstrates that during conventionalization and aging the TcR alpha/beta bearing population of IELs substantially expands. It suggests that mainly this lymphocyte subset responds to microflora stimuli and is probably involved in the protection of the epithelial cell layer of intestinal mucosa.

• MeSH
• Bacteria growth & development   MeSH
• Germ-Free Life MeSH
• Immunophenotyping MeSH
• Rats, Inbred Strains MeSH
• Rats MeSH
• Lymphoid Tissue immunology   MeSH
• Aging immunology   MeSH
• Intestinal Mucosa immunology   microbiology   MeSH
• T-Lymphocyte Subsets immunology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Coeliac disease is a human, genetically linked, disorder which develops in gluten-sensitive persons. The aim of this study was to investigate the effect of prolonged feeding of gliadin, a major fraction of gluten, on enzyme activities of enterocyte brush border membrane enzymes in rats, mice and pigs. Brush-border membranes were isolated from mucosal scrapings of the small intestine of 21-d-old rat pups hand-fed with formula milk diet, two-month-old nu/nu and +/+ BALB/c mice and two-month-old piglets fed three times a week starting at birth with high doses of gliadin. Activities of lactase, sucrase and dipeptidyl peptidase IV (DPP IV) were determined. Individual animal models differed in their response to gliadin feeding. In comparison with albumin fed controls the activities of DPP IV and lactase were decreased in rat pups, nu/nu BALB/c mice and piglets. DPP IV activity was mostly affected in the ileum of rats and piglets fed with gliadin starting at birth. On the other hand, lactase and sucrase activities of nu/nu BALB/c mice and piglets decreased to the largest extent in jejunum.

• MeSH
• beta-Galactosidase metabolism   MeSH
• Time Factors MeSH
• Celiac Disease enzymology   MeSH
• Dipeptidyl Peptidase 4 metabolism   MeSH
• Gliadin administration & dosage   MeSH
• Rats MeSH
• Lactase MeSH
• Humans MeSH
• Microvilli enzymology   MeSH
• Disease Models, Animal * MeSH
• Mice, Inbred BALB C MeSH
• Mice, Nude MeSH
• Mice MeSH
• Rats, Wistar MeSH
• Swine MeSH
• Sucrase metabolism   MeSH
• Intestinal Mucosa enzymology   MeSH
• Intestine, Small enzymology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Galactosidase MeSH
• Dipeptidyl Peptidase 4 MeSH
• Gliadin MeSH
• Lactase MeSH
• Sucrase MeSH