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One of the characteristic features of celiac disease is an increase in anti-gliadin antibodies (Abs). Recently we found that some of the monoclonal Abs to gliadin cross-react with molecules on rat enterocytes. One of these cross-reacting molecules was identified as rat calreticulin. This study shows that the levels of serum IgA Abs to gliadin, rat, and human enterocytes; purified enterocyte antigens; and calreticulin in sera from patients with active disease were significantly higher than in patients on a gluten-free diet and healthy controls (P < 0.001). Anti-gliadin Abs were isolated by affinity chromatography from the sera of six active celiac patients. The reactivity of these anti-gliadin Abs was demonstrated to be significantly higher (P < 0.05) with human enterocytes and human calreticulin than with other antigens tested. Furthermore, using isolated patients' anti-gliadin Abs bound to Sepharose 2B, two main proteins of molecular mass 62 and 66 kDa were purified from a lysate of human enterocytes. The 62-kDa enterocyte antigen was identified as human calreticulin. These findings suggest that anti-gliadin Abs may play a pathogenic role in celiac disease by cross-reacting with enterocytes. Calreticulin in enterocytes may be one of the putative targets for autoimmune reactions.

The growing interest in the composition and effects of microbiota raised the question how drug pharmacokinetics could be influenced by concomitant application of probiotics. The aim of this study was to find whether probiotic E. coli strain Nissle 1917 (EcN) influences the pharmacokinetics of concomitantly taken antiarrhythmic drug amiodarone (AMI). Live bacterial suspension of probiotic EcN (or non-probiotic E. coli strain ATCC 25922) was applied orally to male Wistar rats for seven days, while a control group of rats was treated with a saline solution. On the eighth day, the amiodarone hydrochloride was administered as one single oral dose (50 mg/kg) to all rats (N = 60). After 0, 1, 2, 3, 4, 5.5, 7, 9, 14, 22, and 30 hours, blood samples were taken from the rat abdominal aorta. The plasma level of AMI and its metabolite N-desethylamiodarone (DEA) was determined using the HPLC with UV detection. Administration of EcN led to a 43% increase of AMI AUC0-30 in comparison with control samples. However, this effect was not observed if EcN was replaced by a reference non-probiotic E. coli strain. Thus, EcN administration was most probably responsible for better drug absorption from the gastrointestinal tract. Plasma levels of DEA were also increased in plasma samples from animals treated with EcN. This change was again not found in the experiment with the reference non-probiotic strain. Higher DEA levels in samples from EcN-treated rats may be explained either by better absorption of AMI and/or by an increased activity of CYP2C forms, known to participate in metabolism of this drug, after EcN administration. In this paper, it is documented that concomitantly taken probiotic EcN may modulate pharmacokinetics of a drug; in this case, it led to an increased bioavailability of AMI.

Background Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model. Methods Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor-associated kinase M (IRAK-M)-deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and β-glucuronidase activity was measured in intestinal content by fluorescence assay. Results ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased β-glucuronidase activity. However, the β-glucuronidase activity was not as low as in germ-free mice. IRAK-M-deficient mice not only developed invasive tumors, but ATB-induced decrease in β-glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M-deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue. Conclusions We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M-negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.

Nocardia opaca , a Gram-positive bacterium, is a potent source of immunostimulatory substances. Screening of sera of adult human donors revealed that all sera tested contained antibodies reactive with isolated Nocardia fractions (Nocardia delipidated cell mitogen, NDCM; Nocardia lysozyme digest, NLD; Nocardia water-soluble mitogen, NWSM; and fraction B). The respective values of reciprocal titres for IgM and IgG were in the range of 100 to 12 800, and 10 to 320 for IgA antibody isotypes, when NLD or fraction B were used as antigens in enzyme-linked immunosorbent assay (ELISA) tests. The level of antibodies directed to NDCM, a potent polyclonal B cell activator, was found to be the lowest. In ritro spontaneous as well as NDCM-induced production of antibodies to NDCM by human peripheral blood lymphocytes involved mainly the IgM class. Western-blot analysis demonstrated that antibodies in normal human sera react with nocardial antigens of molecular mass ∼60, 40, 20 and 15–10 kDa. The same antigens were also recognized by rabbit and mouse hyperimmune sera, also confirming the immundominancy of these nocardial antigens in other species. The presence of anti-nocardia antibodies in human sera and their production by both stimulated and non-stimulated lymphocytes points to the natural sensitization of humans either by ubiquitous nocardial components or by cross-reactive bacterial or food antigens.

SUMMARY The Gram-positive bacterium, Nocardia opaca, is a source of substances with adjuvant effect, ability to stimulate macrophages and natural killer cells for enhanced cytotoxity and cytokine production and B lymphocytes for polyclonal immunoglobulin secretion. We determined the immunogenicity of isolated N. opaca fractions and prepared MoAbs against immunogenic water-soluble mitogen (NWSM). Two main proteins of molecular mass 15 and 56 kD were detected in Western blot analysis and isolated by affinity chromatography using anti-NWSM MoAb B7/7. Both these isolated nocardial antigens were found to stimulate mouse peritoneal macrophage NOS. The effect of 5 μg NWSM was comparable to that of 5 μg lipopolysaccharide (LPS) or 20 U of interferon-gamma (IFN-γ) added to cell cultures. The MoAb B7/7 decreased NO−2 production induced by NWSM or by isolated nocardial antigens, but did not significantly influence the production elicited by LPS or IFN-γ. On the other hand, NOS activation by NWSM was not affected by anti-IFN-γ MoAb. The possible independent pathway for IFN-γ and NWSM macrophage activation is discussed.

To analyze the possible involvement of natural killer (NK) cell activity in the pathogenetic mechanism of coeliac disease (CD) we measured the spontaneous cytotoxic cell activity of peripheral blood mononuclear cells (PMNC) from patients with CD and from healthy donors. No significant differences were found between the NK cell activity of PMNC from healthy donors and from patients with CD using a standard 51 Cr release assay. However, a 30-min treatment of PMNC with gliadin inhibited NK cell activity in patients with CD. On the other hand, a 1-d incubation with gliadin induced cytotoxic cell activity of PMNC against the NK-resistant target cells such as the epithelial HT-29 and the lymphoblastoid RAJI cell lines, suggesting that activation of PMNC by cultivation with gliadin can occur.

Monoclonal, hyperimmune rabbit and human serum anti-gliadin antibodies were analyzed by ELISA and immunoblotting techniques. In Western blotting the difference in reactivity between monoclonal and human antibodies was quantitative rather than qualitative. Rabbit antisera differed in reactivity according to the protein used for immunization. The rabbits immunized by the peptic-tryptic pancreatic digest of gliadin reacted similarly to the patients. In ELISA, significantly higher reactivity with crude, A-, glyc-gli, alpha-, beta- and omega-gliadins was found in the patients' sera than in controls.

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.

Structural similarities between external antigen and self components are believed to be one of the possible causes of autoimmunity. This study describes the presence of similar structures shared by gliadin and enterocyte surface molecules recognized by antigliadin mAbs. The reactivity of mAbs to gliadin was followed by ELISA using fixed enterocytes, their brush-border membranes, or purified enterocyte antigen. The specificity of reaction was confirmed by ELISA inhibition studies and by immunohistochemical staining of rat tissue sections using biotin-avidin-peroxidase technique. Immunoprecipitation analysis of 125I-labeled intestinal epithelial cells using antigliadin mAb revealed the presence of two main cross-reactive molecules of 28 and 62 kDa. The 62-kDa and an associated 66-kDa protein were isolated by affinity chromatography. Immunoblotting analysis showed that a 28-kDa protein detected by immunoprecipitation also reacted with IgA of celiac disease patient sera.

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.

Background Ankylosing enthesopathy (ANKENT) is an animal model of human ankylosing spondylitis. ANKENT is an inflammatory disease affecting the ankle and tarsal joints of the hind limbs in susceptible mouse strains. In the disease, the participation of intestinal microbiota components was suggested. Therefore, we attempted to increase the incidence of ANKENT by systemic administration of lipopolysaccharide (LPS), which is a component of bacterial cellular walls and stimulates inflammatory processes. Methods ANKENT occurrence, serum cytokine profiles, spleen cellular composition and in vitro cytokine response to LPS were analysed in LPS-treated and control LPS-untreated B10.BR male mice. Results Contrary to expectations, LPS treatment decreased the incidence of ANKENT in LPS-treated group compared to control LPS-untreated group. Flow cytometry analysis of splenocytes showed an increased percentage of macrophages, dendritic cells and neutrophils and a decreased percentage of B cells, T cells and T helper cells in LPS-treated males following LPS administration. In addition, LPS-treated males had significantly elevated IL-6 and IL-10 serum levels. At 20–22 weeks after the final LPS application, splenocytes from LPS-treated mice were more susceptible to in vitro LPS stimulation than those of the controls and produced significantly higher levels of TNFα and IL-6. Conclusions Repeated systemic stimulation with microbial component lipopolysaccharide in early adulthood significantly reduced the incidence of ANKENT in B10.BR mice and this finding can support the “hygiene hypothesis”. In LPS-treated mice, the innate immunity parameters and the level of anti-inflammatory IL-10 cytokine were significantly increased. Nevertheless, the immunological mechanism of the LPS protective effect remains unclear.

AIM: To study the coincidence of celiac disease, we tested its serological markers in patients with various liver diseases. METHODS: Large-scale screening of serum antibodies against tissue transglutaminase (tTG), and deamidated gliadin using enzyme-linked immunosorbent assay and serum antibodies against endomysium using immunohistochemistry, in patients with various liver diseases (n = 962) and patients who underwent liver transplantation (OLTx, n = 523) was performed. The expression of tTG in liver tissue samples of patients simultaneously suffering from celiac disease and from various liver diseases using immunohistochemistry was carried out. The final diagnosis of celiac disease was confirmed by histological analysis of small-intestinal biopsy. RESULTS: We found that 29 of 962 patients (3%) with liver diseases and 5 of 523 patients (0.8%) who underwent OLTx were seropositive for IgA and IgG anti-tTG antibodies. However, celiac disease was biopsy-diagnosed in 16 patients: 4 with autoimmune hepatitis type I, 3 with Wilson's disease, 3 with celiac hepatitis, 2 with primary sclerosing cholangitis, 1 with primary biliary cirrhosis, 1 with Budd-Chiari syndrome, 1 with toxic hepatitis, and 1 with non-alcoholic steatohepatitis. Unexpectedly, the highest prevalence of celiac disease was found in patients with Wilson's disease (9.7%), with which it is only rarely associated. On the other hand, no OLTx patients were diagnosed with celiac disease in our study. A pilot study of the expression of tTG in liver tissue using immunohistochemistry documented the overexpression of this molecule in endothelial cells and periportal hepatocytes of patients simultaneously suffering from celiac disease and toxic hepatitis, primary sclerosing cholangitis or autoimmune hepatitis type I. CONCLUSION: We suggest that screening for celiac disease may be beneficial not only in patients with associated liver diseases, but also in patients with Wilson's disease.

Summary One of the promising approaches in the therapy of ulcerative colitis is administration of butyrate, an energy source for colonocytes, into the lumen of the colon. This study investigates the effect of butyrate producing bacterium Clostridium tyrobutyricum on dextran sodium sulphate (DSS)-induced colitis in mice. Immunocompetent BALB/c and immunodeficient severe combined immunodeficiency (SCID) mice reared in specific-pathogen-free (SPF) conditions were treated intrarectally with C. tyrobutyricum 1 week prior to the induction of DSS colitis and during oral DSS treatment. Administration of DSS without C. tyrobutyricum treatment led to an appearance of clinical symptoms – bleeding, rectal prolapses and colitis-induced increase in the antigen CD11b, a marker of infiltrating inflammatory cells in the lamina propria. The severity of colitis was similar in BALB/c and SCID mice as judged by the histological damage score and colon shortening after 7 days of DSS treatment. Both strains of mice also showed a similar reduction in tight junction (TJ) protein zonula occludens (ZO)-1 expression and of MUC-2 mucin depression. Highly elevated levels of cytokine tumour necrosis factor (TNF)-α in the colon of SCID mice and of interleukin (IL)-18 in BALB/c mice were observed. Intrarectal administration of C. tyrobutyricum prevented appearance of clinical symptoms of DSS-colitis, restored normal MUC-2 production, unaltered expression of TJ protein ZO-1 and decreased levels of TNF-α and IL-18 in the descending colon of SCID and BALB/c mice, respectively. Some of these features can be ascribed to the increased production of butyrate in the lumen of the colon and its role in protection of barrier functions and regulation of IL-18 expression.

Background & Aims The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. Methods We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc +/Min and Apc CKO/CKO /Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. Results We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer–derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli ( APC ) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. Conclusions TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.

BackgroundProbiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.Methodology/principal findingsThe preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4(+)FoxP3(+) regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4(+)FoxP3(+) regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer's patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.Conclusion/significanceOur study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.

The most important trigger for immune system development is the exposure to microbial components immediately after birth. Moreover, targeted manipulation of the microbiota can be used to change host susceptibility to immune-mediated diseases. Our aim was to analyze how differences in early gut colonization patterns change the composition of the resident microbiota and future immune system reactivity. Germ-free (GF) mice were either inoculated by single oral gavage of caecal content or let colonized by co-housing with specific pathogen-free (SPF) mice at different time points in the postnatal period. The microbiota composition was analyzed by denaturing gradient gel electrophoresis for 16S rRNA gene followed by principal component analysis. Furthermore, immune functions and cytokine concentrations were analyzed using flow cytometry, ELISA or multiplex bead assay. We found that a single oral inoculation of GF mice at three weeks of age permanently changed the gut microbiota composition, which was not possible to achieve at one week of age. Interestingly, the ex-GF mice inoculated at three weeks of age were also the only mice with an increased pro-inflammatory immune response. In contrast, the composition of the gut microbiota of ex-GF mice that were co-housed with SPF mice at different time points was similar to the gut microbiota in the barrier maintained SPF mice. The existence of a short GF postnatal period permanently changed levels of systemic regulatory T cells, NK and NKT cells, and cytokine production. In conclusion, a time window exists that enables the artificial colonization of GF mice by a single oral dose of caecal content, which may modify the future immune phenotype of the host. Moreover, delayed microbial colonization of the gut causes permanent changes in the immune system.

The aim of the study was to find whether probiotic Lactobacillus casei influences the expression or the activity of cytochromes P450 (CYP) and whether it has an influence on the level of CYP mRNA in male rats.Live bacterial suspension of L. casei was administered orally (gavage) to healthy male Wistar rats daily for 7 days. Control group of rats was treated with the saline solution. Sections of the duodenum, jejunum, ileum, caecum and colon were dissected from each experimental animal. In all individual samples, the expression of selected CYPs was determined by Western blotting. The levels of expression of CYPs were also evaluated by mRNA using the real-time PCR method.There were changes observed in the expression of CYP enzymes and in the CYP mRNA levels along the intestine after application of L. casei. The expression of CYP1A1 enzyme was found to be decreased in the proximal part of the jejunum and colon, CYP1A1 mRNA level was decreased in the distal part of the jejunum, ileum and caecum. Thus, the changes in CYP1A1 protein or mRNA were observed along the intestine of male rats. Similarly, a decreased expression of the caecal CYP2E1 mRNA and of the duodenal CYP3A9 mRNA after treatment of rats with L. casei was found.Probiotic L. casei might be able to contribute to prevention against colorectal cancer by decreasing levels of certain forms of xenobiotic-metabolizing enzymes; moreover, in general, there is a possibility of interactions with concomitantly taken pharmacotherapeutic agents.

The therapeutic effect of probiotics has been studied in many clinical and experimental studies but no data exist concerning the influence of probiotics on pharmacokinetics of contemporary administered drugs. In this paper, we describe the influence of indomethacin-induced gastrointestinal lesions and Escherichia Coli Nissle 1917 medication on absorption of 5-aminosalicylic acid and its metabolite N-acetyl-5-aminosalicylic acid in rat.5-aminosalicylic acid (5-ASA) was given orally to rat using gastric probe as a suspension (25 mg/kg). The plasma time profiles of 5-ASA and its metabolite were compared between Group A (animals medicated with a suspension of Escherichia coli Nissle 1917 [EcN] in dose of 5 × 108 CFUs/day for 14 consecutive days), Group B (animals with indomethacin [IND]-induced gastrointestinal lesions; single dose of 25 mg/kg of IND), Group C (simultaneous administration of EcN and IND), and Group D (control animals without any medication). The blood samples for HPLC analysis has been taken from incannulated vena jugularis in time 30, 60, 90, 120, 180, 240, 360 min after 5-ASA administration to rat.The pharmacokinetics of 5-ASA was not significantly changed by EcN medication (Group A) in comparison to control animals (Group D). The significantly elevated absorption (AUC and cmax) of 5-ASA was found in animals with induced gastro-enteropathy with concurrently medicated with EcN (Group C) when compred to controls. In the case of metabolite N-acetyl-5-ASA, statistically no-significant differences were found between groups.Simultaneous probiotics (EcN) medication did not affect absorption 5-ASA from intestinal tract (the main site of ASAs action).

International audience; Gliadins, and primarily α-gliadins containing several sequences such as aa 31-49, aa 56-88 (33-mer), aa 57-68, and aa 69-82, are critical in the induction of immune response or toxic reaction leading to the development of celiac disease (CLD). The role of IgA anti-gliadin antibodies (IgA AGA) is unknown. To this end, we prepared several humanized monoclonal IgA AGA using transgenic α1KI mice. Employing Pepscan with overlapping decapeptides of α-gliadin we observed a robust similarity between the specificity of humanized mouse monoclonal IgA AGA and IgA AGA from patients with florid CLD. The common immunodominant region included several sequential epitopes localized in the N-terminal part of α-gliadin (QFQGQQQPFPPQQPYPQPQPFP, aa 29-50, and QPFPSQQPYLQL, aa 47-58). Notably, IgA AGA produced by clones 8D12, 15B9, 9D12, and 18E2 had significant reactivity against sequences localized in the 33-mer, LQLQPFPQPQ (aa 56-65) and PQLPYPQPQPFL (aa 69-80). Humanized mouse monoclonal IgA AGA that have a known specificity are suitable as standard in ELISAs to detect serum IgA AGA of CLD patients and for studying the AGA pathogenic role in CLD, especially for analyzing the translocation of complex of specific IgA antibodies and individual gliadin peptides through enterocyte barrier.

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.

Summary Commensal bacteria have been shown to modulate the host mucosal immune system. Here, we report that oral treatment of BALB/c mice with components from the commensal, Parabacteroides distasonis, significantly reduces the severity of intestinal inflammation in murine models of acute and chronic colitis induced by dextran sulphate sodium (DSS). The membranous fraction of P. distasonis (mPd) prevented DSS-induced increases in several proinflammatory cytokines, increased mPd-specific serum antibodies and stabilized the intestinal microbial ecology. The anti-colitic effect of oral mPd was not observed in severe combined immunodeficient mice and probably involved induction of specific antibody responses and stabilization of the intestinal microbiota. Our results suggest that specific bacterial components derived from the commensal bacterium, P. distasonis, may be useful in the development of new therapeutic strategies for chronic inflammatory disorders such as inflammatory bowel disease.

Different probiotic strains used in clinical trials have shown prophylactic properties in different inflammatory diseases of the gastrointestinal tract. This study was aimed to investigate the influence of Escherichia coli strain Nissle 1917 (EcN) components on the integrity of the Caco-2 cell monolayer (human adenocarcinoma cell line).The effect of supernatant of EcN suspension and lipopolysaccharide (LPS) isolated from EcN (in concentrations from 0.001 to 1 000 µg/ml) on paracellular transport of 14C-mannitol marker through epithelial cell monolayer was estimated.Both LPS and EcN supernatant exerted almost the same effect; whereas no effect was shown using high concentrations (100 and 1 000 µg/ml), low concentrations (0.001, 0.1 and 1 µg/ml) significantly decreased permeability of 14C-mannitol. Concentration (0.001 µg/ml) decreased 14C-mannitol permeability approximately about 20% (LPS) and 30% (EcN supernatant). To elucidate the observed changes in monolayer permeability ("tighter monolayer") induced by concentrations of LPS or supernatant, media able to open epithelial intercellular junctions were used. The effects of Ca2+-free transport medium and of medium containing 5, 10, 20, 50, and 100% of Ca2+ on the 14C-mannitol transport in the presence of the lowest (0.001 µg/ml) and high (100 µg/ml) concentrations of LPS were studied. Using Ca2+-free medium both concentrations of LPS significantly decreased apparent permeability coefficient (Papp) of 14C-mannitol indicating that changes of 14C-mannitol permeability are independent of dimensions of paracellular spaces.The decrease of 14C-mannitol permeability caused by EcN LPS indicates the ability of components of probiotic EcN strain to restore disrupted epithelial barrier.

The aim of the study was to find whether probiotic Escherichia coli Nissle 1917 O6:K5:H1 (EcN) influences the expression of cytochromes P450 (CYP) in the rat intestine.Live bacterial suspension of EcN was administered to healthy male Wistar rats daily for 7 days. Control group of rats was stressed by oral application of the saline solution daily for 7 days as well. Sections of the duodenum, jejunum, ileum, caecum and colon have been taken from each experimental animal. With all individual samples, microsomal fraction has been prepared and expression of selected CYPs was determined by Western blotting. The levels of expression of CYPs were also evaluated by mRNA using real-time PCR.It was found that there are changes in expression of CYP enzymes studied along the intestine. CYP1A1, 2B1/2 and 2E1 are present mainly in the duodenum and jejunum; on the other hand, CYP2C6 is expressed mainly in the caecum and colon. CYP3A was found all over the rat intestine. The results show that there are no prominent differences between control samples and samples with EcN, only the expression of CYP3A protein in the duodenum appears to exhibit a clear tendency to decrease. In the case of the colon, a significant increase in the expression of CYP3A (most likely CYP3A1) after treatment of rats with EcN was found.This in vivo study revealed that the levels of colon CYP3A could be significantly increased in rats treated with probiotic EcN. On the contrary, the expression of CYP3A in the duodenum decreased. However, the changes in the expression of CYP enzymes are probably not as extensive to be clinically important in man; hence, most likely the probiotic EcN has little influence on the intestinal drug metabolism by CYP enzymes.

Background Oral thiopurines are effective and widely used in treatment of inflammatory bowel disease (IBD) in humans, although their use is limited due the development of adverse events. Here, we examine the efficacy and toxicity of oral treatment with 6-tioguanine (6-TG) and azathioprine (AZA) in a murine model of IBD. Methods We induced acute or chronic colitis in BALB/c mice by one or four cycles of 3% dextran sulphate sodium (DSS), respectively. Mice were treated by daily gavages of various dosages of 6-tioguanine, azathioprine, or by phosphate buffered saline (PBS) starting the first day of DSS or after two cycles of DSS, respectively. We monitored the efficacy and toxicity by measuring the weight change and serum alanine aminotransferase (ALT) activity and by disease severity and histology, at the end of the experiment. Moreover, we measured cytokine production after colon fragment cultivation by enzyme-linked immunoabsorbent assay and numbers of apoptotic cells in the spleen by flow cytometry. Results 6-TG is effective in the treatment of acute DSS-induced colitis in a dose-dependent manner and 40 μg of 6-TG is significantly more effective in the treatment of acute colitis than both AZA and PBS. This effect is accompanied by decrease of IL-6 and IFN-γ production in colon. We did not observe histological abnormalities in liver samples from control (PBS) or 6-TG treated mice. However, liver samples from most mice treated with AZA showed mild, yet distinct signs of hepatotoxicity. In chronic colitis, all thiopurine derivatives improved colitis, 20 μg of 6-TG per dose was superior. High doses of 6-TG led to significant weight loss at the end of the therapy, but none of the thiopurine derivatives increased levels of serum ALT. Both thiopurine derivatives reduced the proportion of apoptotic T helper cells, but a high production of both IL-6 and TGF-β was observed only in colon of AZA-treated mice. Conclusions Use of 6-TG in the treatment of experimental colitis in mice appears superior to AZA administration and placebo. In contrast to 6-TG, the use of AZA resulted in histological liver abnormalities.

Background T1DM is a T-cell-mediated autoimmune disease targeting insulin-producing beta-cells. Multiple factors may contribute to the development of T1DM. Among these, the metabolic state of beta-cells and pro-inflammatory cytokines, produced by infiltrating immune cells, have been implicated in the precipitation of T1DM. Methods and Results In this study, confocal immunofluorescence microscopy of human pancreata revealed a distinct subset of beta-cells expressing the innate LPS co-receptor CD14. Human islets expressed fully functional CD14 as LPS stimulation led to a dose-dependent secretion of tumour necrosis factor (TNFα), interleukin (IL)-1β and IL-8, which were substantially inhibited by a blocking anti-CD14 mAb. In addition, LPS stimulation impaired the glucose-mediated insulin secretion in rat islets. β-GalCer and sulfatide, glycolipids that are related to insulin processing and secretion, are possibly interacting with the CD14 receptor complex. β-GalCer had an LPS-like, serum- and CD14-dependent effect on the induction of pro-inflammatory cytokines in a human monocyte cell line. In contrast, the LPS-mediated cytokine production was inhibited by sulfatide. Human islets also responded to β-GalCer (10 µg/mL) by secreting TNFα, IL-1β and IL-8, whereas sulfatide partly inhibited the effect of LPS. Conclusions A subset of human beta-cells expresses functional CD14 receptor and thus is able to recognize both exogenous bacterial (LPS) as well as endogenous ligands (e.g. glycolipids of beta-cell origin). The CD14 expression on a subset of human beta-cells may play a role in the innate surveillance of the endocrine environment but may also contribute to innate immune mechanisms in the early stages of beta-cell aggression. Copyright © 2010 John Wiley & Sons, Ltd.

The intestinal environment is considered to play an important role both in colorectal tumor development and in the evolution and modulation of mucosal immunity. Studies in animals reared in germ-free (GF, without any intestinal microflora) versus conventional (CV, with regular microflora in bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and anti-cancer immune responses in situ. The lower incidence of colon cancers and better immunological parameters in GF animals versus CV ones after chemically-induced carcinogenesis raises questions about specific characteristics of the immunological networks in each respective condition. Different levels of tolerance/regulatory mechanisms in the GF versus CV animals may influence the development of immune responses not only at the level of mucosal, but also at the systemic, immunity. We hypothesize that GF animals can better recognize and respond to evolving neoplasias in the bowel as a consequence of their less-tolerogenic immunity (i.e., due to their more limited exposure to antigens to become tolerated against at the intestinal level). In this paper, we review the role of bacteria in modulating gut environment and mucosal immunity, their importance in cancer development, and aspects of immune regulation (both at local and systemic level) that can be modified by bacterial microflora. Lastly, the use of GF animals in comparison with conventionally-raised animals is proposed as a suitable and potent model for understanding the inflammatory network and its effect on cancer immunity especially during colorectal cancer development.

The aim of the study was to find, whether probiotic Escherichia coli Nissle 1917 O6:K5:H1 (EcN) influence the amount and activity of cytochromes P450 (CYP) in rat liver.Live bacterial suspension of EcN was applied to the female Wistar rats in single dose or for 14 days consecutively. The bacterial lipopolysaccharide (LPS) isolated by phenol extraction from the EcN was given to the rats for 14 days as well. Control rats were treated with the saline solution daily for 14 days. Relative amount of CYP2C6, CYP2C9 (corresponding to rat CYP2C11), CYP3A1 and CYP1A2 protein expression in rat liver microsomes was determined by Western blotting. For the determination of six CYP activities (corresponding to human CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2C9 and CYP2D6) fluorescence, luminescence or absorbance detection was used.The data presented show that the changes of the total content of the CYP enzymes in rat liver are not significant after administration of the probiotic for 1 or 14 days as well as of the LPS. Western blots revealed a slight increase in CYP2C6 protein expression; level of another rat CYP2C protein (readings with anti-human CYP2C9 antibody corresponding to the rat CYP2C11) as well as of CYP1A2 was elevated after administration of LPS; a small decrease was observed with CYP3A1 protein. Changes in activities of CYP forms are not significant, only the activity of rat CYP2C forms in liver microsomal samples of rats given free LPS appeared to exhibit a small, but significant tendency to increase.The results show that the p.o. administration of probiotics to rat does not markedly influence the rat hepatic CYP enzymes.

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.

BackgroundMammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harbored by the distal intestine. The gut microbiota outnumber ~10 times the total number of our somatic and germ cells. The host-microbiota relationship has evolved to become mutually beneficial. Studies in germ-free mice have shown that gut microbiota play a crucial role in the development of the immune system. The principal aim of the present study was to elucidate whether the presence of gut microbiota and the quality of a sterile diet containing various amounts of bacterial contaminants, measured by lipopolysaccharide (LPS) content, can influence maturation of the immune system in gnotobiotic mice.ResultsWe have found that the presence of gut microbiota and to a lesser extent also the LPS-rich sterile diet drive the expansion of B and T cells in Peyer's patches and mesenteric lymph nodes. The most prominent was the expansion of CD4+ T cells including Foxp3-expressing T cells in mesenteric lymph nodes. Further, we have observed that both the presence of gut microbiota and the LPS-rich sterile diet influencein vitrocytokine profile of spleen cells. Both gut microbiota and LPS-rich diet increase the production of interleukin-12 and decrease the production of interleukin-4. In addition, the presence of gut microbiota increases the production of interleukin-10 and interferon-γ.ConclusionOur data clearly show that not only live gut microbiota but also microbial components (LPS) contained in sterile diet stimulate the development, expansion and function of the immune system. Finally, we would like to emphasize that the composition of diet should be regularly tested especially in all gnotobiotic models as the LPS content and other microbial components present in the diet may significantly alter the outcome of experiments.

It is an open question whether multifunctional galectin-3 can be a serum marker in inflammatory bowel disease. Western blots and commercial ELISA detected and quantitated the lectin immunocytochemistry using double labeling localized it in tissue sections. Serum concentrations were significantly increased in specimen of patients with active and remission-stage ulcerative colitis and Crohn’s disease, associated with emerging positivity of CD14+ cells. Enhanced concentration of galectin-3 in serum reflects presence of disease and points to its involvement in the pathogenesis.

Summary Refractory coeliac disease (RCD) is a very rare and dangerous form of CD, in which gluten-free diet loses its therapeutic effect and the damage of intestinal mucosa persists. Because of the adherence to the diet, serological markers of CD [immunoglobulin A (IgA) antibodies against gliadin, tissue transglutaminase (tTG) and endomysium] are often missing in RCD patients. We found substantially elevated levels of IgA anti-calreticulin (CRT) antibodies in the sera of almost all RCD patients tested. These sera were negative for IgA antibodies to gliadin and tTG and only some of them showed IgA antibodies to enterocytes. Analysis of patients' IgA reactivity to CRT fragments (quarters and halves) by Western blotting revealed differences in the specificity of IgA antibodies between RCD and CD patients. We therefore used the Pepscan technique with synthetic overlapping decapeptides of CRT to characterize antigenic epitopes recognized by serum IgA antibodies of RCD patients. Employing this method we demonstrated several dominant antigenic epitopes recognized by IgA antibodies of RCD patients on the CRT molecule. Epitope GVTKAAEKQMKD was recognized predominantly by serum IgA of RCD patients. Our results suggest that testing for serum IgA antibodies against CRT and its selected peptide could be a very useful tool in RCD differential diagnosis.

Nasal polyps (NP), edematous projections of nasal mucosa (NM), are characterized by an inflammatory cellular infiltrate, however, little is known about etiopathogenesis of NP. Both innate immune mechanisms leading to activation of NF-kappaB and homeostasis of epithelial cells were implicated in the pathogenesis of NP. In this study we investigated the expression of insulin-like growth factor-1 receptor (IGF-1R) and inducible nitric-oxide synthase (iNOS) in NP compared to healthy NM in both the epithelial and stromal compartments. Using immunohistochemistry, frozen tissue sections of NP from 18 patients, and mucosal biopsy specimens of the inferior turbinate from 17 subjects were stained for IGF-1R and iNOS markers. Fluorescence microscopy and computerized image analysis revealed low numbers of IGF-1R-positive cells in all specimens. However, substantially increased numbers of IGF-1R-positive cells were found in NP compared to NM both within the epithelium (1.63 vs. 0.43) and stroma (3.27 vs. 1.03). Positivity for iNOS was detected within the epithelium of NP compared with NM. Numbers of iNOS-positive single cells were highly increased in NP vs. NM in both epithelial (3.83 vs. 1.08) and stromal (4.96 vs. 2.67) compartments. An increased iNOS expression within the epithelial layer as well as increased number of iNOS- and IGF-1R-positive cells in NP was observed. This suggests that innate immune mechanism, and to a lesser extent also growth and homeostasis of epithelial cells, may play a role in formation of NP.

Dysregulation of innate and adaptive intestinal immune responses to bacterial microbiota is supposed to be involved in pathogenetic mechanisms of inflammatory bowel diseases (IBDs). We investigated expression of Toll-like receptor 2 (TLR2), TLR4, and their transmembrane coreceptor CD14 in biopsy samples from patients with IBD and in non-inflamed gut mucosa from controls. Small intestine and colon samples were obtained by colonoscopy from patients with Crohn's disease (CD), ulcerative colitis (UC), and controls. Immunohistochemical analysis of cryostat sections using polyclonal and monoclonal antibodies specific for TLR2, TLR4, and CD14 showed a significant increase in TLR2 expression in the terminal ileum of patients with inactive and active UC against controls. Significant upregulation of TLR4 expression relative to controls was found in the terminal ileum and rectum of UC patients in remission and in the terminal ileum of CD patients with active disease. CD14 expression was upregulated in the terminal ileum of CD patients in remission and with active disease, in the cecum of UC patients in remission and with active disease, and in rectum of UC patients with active disease. Hence, dysregulation of TLR2, TLR4, and CD14 expression in different parts of the intestinal mucosa may be crucial in IBD pathogenesis. (J Histochem Cytochem 56:267–274, 2008)

The indigenous microflora plays an integrative role in the maintenance of immunological homeostasis. Several studies reported that immunological tolerance is dependent on microbial colonization of the gut. In the present study, we investigated whether the absence of the microflora influences the sensitization process to an allergen as well as the ability to develop mucosal tolerance in a mouse model of birch pollen allergy. Germ-free or conventional BALB/c mice were intranasally or intragastrically pre-treated with the major birch pollen allergen Bet v 1 prior to sensitization with this allergen. Both germ-free and conventional mice displayed comparable Th2 biased immune responses after allergic sensitization. Oral as well as intranasal tolerization led to suppression of allergen-specific serum antibodies (IgG1, IgE, IgA) as well as cytokine production by splenocytes (IL-5, IFN-gamma) in both germ-free and conventional animals. Peyer's patches of germ-free animals were approximately 20 times smaller than in conventional animals, but the relative distribution of lymphocyte subpopulations was equal. We conclude that the absence of the microflora does not influence the ability to mount Th2 responses nor to establish tolerance towards the aeroallergen Bet v 1. Our findings may challenge the view that the commensal microflora is a key factor for breakdown of physiological tolerance and allergy development.

Background Environmental factors such as nutrition or exposure to infections play a substantial role in the pathogenesis of type 1 diabetes (T1D). We have previously shown that gluten-free, non-purified diet largely prevented diabetes in non-obese diabetic (NOD) mice. In this study we tested hypothesis that early introduction of gluten-enriched (gluten+) diet may increase diabetes incidence in NOD mice. Methods Standard, gluten-free, gluten+ modified Altromin diets and hydrolysed-casein-based Pregestimil diet were fed to NOD females and diabetes incidence was followed for 310 days. Insulitis score and numbers of gut mucosal lymphocytes were determined in non-diabetic animals. Results A significantly lower diabetes incidence (p < 0.0001) was observed in NOD mice fed gluten-free diet (5.9%, n = 34) and Pregestimil diet (10%, n = 30) compared to mice on the standard Altromin diet (60.6%, n = 33). Surprisingly, gluten+ diet also prevented diabetes incidence, even at the level found with the gluten-free diet (p < 0.0001, 5.9%, n = 34). The minority of mice, which developed diabetes on all the three diabetes-protective (gluten+, gluten-free, Pregestimil) diets, did that slightly later compared to those on the standard diet. Lower insulitis score compared to control mice was found in non-diabetic NOD mice on the gluten-free, and to a lesser extent also gluten+ and Pregestimil diets. No substantial differences in the number of CD3+, TCR-γδ+, and IgA+ cells in the small intestine were documented. Conclusions Gluten+ diet prevents diabetes in NOD mice at the level found with the non-purified gluten-free diet. Possible mechanisms of the enigmatic, dual effect of dietary gluten on the development of T1D are discussed. Copyright © 2007 John Wiley & Sons, Ltd.

Continuous, reciprocal interaction between the organism and the environment is a basic feature of the life. Starting from first hours after delivery from sterile uterine environment microorganisms colonizes most of the mucosal surfaces and skin. Number of autochtonous bacteria exceeds the number of cells forming the human body. Mucosal microbiota belongs to a complex of innate, natural mechanisms that safeguard resistance of organism against pathogenic microorganisms. Immune system associated with mucosal surfaces covering the largest area of the body (200-300m) evolved mechanisms discriminating between harmless antigens, autochtonous microorganisms and dangerous pathogens. Numerous chronic diseases may occur as a result of changes in mechanisms regulating mucosal immunity and tolerance, which leads to impaired barrier function of the mucosa and increased penetration of microbial antigens into circulation and consequently to exaggerated and generalized immune responses. Participation of pathologically increased immunological activity to autologous microbiota and/or their components in induction of inflammatory and autoimmune processes was demonstrated. Regulation of microflora composition offers the possibility to develop new approaches in prevention and therapy. Bacteria in Human Health and Disease: From Commensalism to Pathogenicity

Initial events and effector mechanisms of most inflammatory and autoimmune diseases remain largely unknown. Dysfunction of the innate and adaptive immune systems associated with mucosae (the major interface between the organism and its environment, e.g., microbiota, food) can conceivably cause impairment of mucosal barrier function and development of localized or systemic inflammatory and autoimmune processes. Animal models help in elucidating the etiology and pathogenetic mechanisms of human diseases, such as the inflammatory bowel diseases, Crohn's disease and ulcerative colitis, severe chronic diseases affecting the gut. To study the role of innate immunity and gut microbiota in intestinal inflammation, colitis was induced by dextran sulfate sodium (DSS) in mice with severe combined immunodeficiency (SCID). Conventionally reared (microflora-colonized) SCID mice displayed severe inflammation like that seen in immunocompetent Balb/c mice, whereas only minor changes appeared in the intestinal mucosa of DSS-fed gnotobiotic germ-free SCID mice. The presence of microflora facilitates the inflammation in DSS-induced colitis that develops in immunodeficient SCID mice, that is, in the absence of T and B lymphocytes. Celiac disease, a chronic autoimmune small bowel disorder, afflicts genetically susceptible individuals with wheat gluten intolerance. We showed that, in contrast with any other food proteins, wheat gliadin and its peptic fragments activate mouse macrophages and human monocytes to produce proinflammatory cytokines through the nuclear factor-kappaB signaling pathway. Activation of innate immunity cells by food proteins or components from gut microbiota thus could participate in the impairment of intestinal mucosa and the development of intestinal and/or systemic inflammation.

This study was designed to investigate the effect of monoassociation of germ-free piglets with Escherichia coli strains on the development of intestinal brush-border enzyme activities. Piglets were delivered by hysterectomy, reared for seven days under germ-free conditions and fed milk formula diet. One group was maintained germ-free, the other four groups were monoassociated on day eight with one of four E. coli strains: non-pathogenic O86 or O83 and G58-1, or pathogenic 933D. The development of brush-border digestive enzyme functions in the small intestine was evaluated after 15 days. Germ-free controls exhibited slower developmental declines of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and delayed increases of sucrase and glucoamylase compared to conventionally grown animals. Association of germ-free piglets with the non-pathogenic E. coli strains O86 and O83 resulted in increased enterocyte differentiation along the length of the small intestine, accompanied by declining activities of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and elevated activities of maturational markers such as sucrase and glucoamylase. Maturational changes also occurred along the villus-crypt axis, as revealed by histochemical localization of aminopeptidase N on the villi tips in piglets colonized with E. coli O83. Interestingly, colonization with the pathogenic E. coli strain 933D stimulated changes in the main differentiation enzyme markers lactase, sucrase and glucoamylase to an extent comparable with those produced by the non-pathogenic and probiotic E. coli strains. In conclusion, germ-free piglets represent a valuable tool to study the consequences of colonization of the immature sterile gut with defined strains of bacteria.

This chapter discusses the role of mucosal microbiota in the development, maintenance, and pathologies of the mucosal immune system. Most mucosal sites of lymphoid tissue—respiratory tract, adenoids, salivary glands, and urogenital tract—in healthy mammals are in a quiescent state and generally resemble the status of lymphoid areas in spleen and most peripheral lymph nodes (PLN). The intestinal tract, palatine tonsils, and occasionally the nasal-associated lymphoid tissue (NALT) are the exceptions. These mucosal lymphoid tissues are in a “physiologically normal state of inflammation.” The chapter focuses on how the intestinal microbes drive the development of gut-associated lymphoid tissue (GALT) during neonatal life and act to maintain its physiologically normal steady state of inflammation. Specific and adaptive, “natural” and semi-specific, and aspecific elements of the mucosal immune systems may benefit and be activated by host interactions with environmental antigens (Ags). To provide the experimental rationale for implicating intestinal or oral/nasal microflora in the development of GALT, palatine tonsil, and sometimes NALT and their steady state of inflammation, the chapter briefly contrasts the status of systemic lymphoid tissue in healthy mammals—spleen, PLN—with GALT, palatine tonsils, and NALT.

Calreticulin (CRT) was identified as a frequent target of serum autoantibodies (Ab) in various diseases, but anti-CRT Ab of IgA isotype were described only in coeliac (CLD) and some hepatic diseases. Employing ELISA with recombinant CRT we found significantly higher (P0.001) levels of IgA anti-CRT Ab in sera of patients with primary biliary cirrhosis (PBC) (77.6+/-8.9 AU/mean+/-SE), autoimmune hepatitis (AIH) (105.1+/-9.2 AU) and alcoholic liver cirrhosis (ALC) (193.5+/-21.0 AU) relative to healthy controls (38.6+/-2.0 AU). The levels of IgG anti-CRT Ab in sera of patients with PBC (59.5+/-3.4 AU), AIH (89.7+/-7.9 AU) and ALC (86.4+/-6.2 AU) were also significantly increased (P0.001) when compared with controls (38.5+/-2.1 AU). Pepscan technique with decapeptides of CRT (each overlapping by eight amino acids) revealed antigenic epitopes of this molecule recognised by IgA Ab of almost all tested patients-KGKNVLINKD and QVKSGTIFDNFL. We also identified disease specific antigenic epitopes on CRT molecule, predominantly recognised by IgA Ab of patients suffering from a particular disease: GGYVKLFPNS and YVKLFPNSLD in AIH (83%, 92% of patients), GLQTSQDARF and EQRLKEEEED in CLD (both 75%) and ASKPEDWDER in ALC (67%). Identification of disease specific CRT epitopes contributes to clarification of autoreactivity against this molecule.

SUMMARY Latent autoimmune diabetes of adults (LADA) manifested after the age of 35 is characterized by the presence of disease-specific autoantibodies (anti-glutamate decarboxylase GADAb, anti-IA2Ab). However, autoimmunity in Type 1 diabetes mellitus is not targeted only to pancreatic beta-cells. No data have so far been published concerning the antibodies associated with other autoimmune disease in LADA patients. The presence of anti-thyroglobulin (TGAb), anti-thyroid peroxidase (TPOAb), anti-gliadin IgA (AGAAb) and IgG (AGGAb) and endomysial antibodies (EMAb) in sera of 68 diabetics typed as LADA was compared with the antibody presence in sera of 85 patients with Type 2 diabetes. We found a significantly higher occurrence of gliadin antibodies in LADA patients: the rate of AGGAb was 19·1% in comparison with 3·5% in the T2DM group (P = 0·0026), the rate of AGAAb was 13·2% in comparison with 3·5% (P = 0·035). The prevalence of EMAb was very low in both groups (1·5% and 0). The two groups differed significantly in the TPOAb rate: 22·1% in LADA compared to 9·4% in T2DM (P = 0·04), whereas no significant difference was found in the presence of TGAb (8·8% and 3·5%, P = 0·187). In comparison with T2DM patients, LADA patients were found to express higher antibody activity against gluten-related antigens and against TPO.

Background Inflammatory processes play an important role in development of nasal polyps (NP), but the etiology and to a great degree also the pathogenesis of NP is not known. Several cytokines and chemokines such as eotaxin, IL-3, IL-5, IL-6, IL-8, RANTES may influence development of NP by regulation of migration, activation and survival of the chronic inflammatory cellular infiltrate. Methods In this study we investigated expression of selected chemokine receptors in human NP and non-affected human nasal mucosa and carried out a comparison with NP from allergic patients with aspirin intolerance. Biopsies of NP were obtained from 20 patients and 4 patients with NP and aspirin intolerance. Mucosal biopsy specimens of the inferior turbinate were obtained from 12 NP patients and 4 healthy controls. Using indirect imunohistochemistry, frozen tissue sections were stained for CCR1, CCR3, CCR4, CCR5, CCR8 and CXCR3. Results Numbers of infiltrating cells expressing CCR3, CCR8 and to a lesser extend also CCR1 were significantly higher in biopsies of NP compared to healthy nasal mucosa. Only a slight increase in CCR5 expressing cells was detected in NP compared to nasal mucosa. No differences in expression of CCR4 and CXCR3 were found in NP compared to nasal mucosa. There were no significant differences between NP of patients with or without aspirin intolerance. Conclusions We documented an increased expression of selected chemokine receptors within the cellular infiltrate of NP that may play an important part in the inflammatory pathogenesis of NP. Supported by grant NS10054 from the IGA MZ, Czech Republic.

Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs). However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail. We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins. Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs. Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs. No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 μg/ml. Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs. Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 μg/ml) by interleukin 8 release. Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14. As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS. The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.

Calreticulin (CRT), a high-affintiy calcium binding protein and chaperone, was recently identified as one of the targets of autoantibodies in coeliac disease. We evaluated the level of IgA and IgG antibodies to CRT in sera from patients with coeliac disease and various autoimmune diseases. The level of antibodies to gliadin (shown previously to cross-react with CTR), isolated enterocytes and tissue transglutaminase were determined for comparison. The mean level of IgA antibodies to CRT was significantly higher ( P 0.001) in sera from coeliac patients with active disease (139.9±11.2 AU/±SE) than in healthy controls (20.9±1.7 AU). In sera of patients with systemic lupus erythematosus (SLE), insulin dependent diabetes mellitus (IDDM), multiple sclerosis (MS) and autoimmune thyroiditis (AT) or inflammatory bowel disease (IBD) the mean level (25.8±3.7 to 38.1±5.6 AU) did not exceed the cut-off value. A low level of these antibodies, however, was detected in some sera of patients with MS and IBD. The level of IgG anti-CRT antibodies was increased in coeliac patients (mean 125.4±8.0 AU, P 0.001) when compared to that in healthy controls (33.9±2.3 AU). The IgG anti-CRT antibodies were also detected in about 30% of SLE patients sera (54.1±3.6 AU, P 0.001), but the mean level reached only half that detected in coeliac patients.

Macrophages play an important role in effector mechanisms of various chronic inflammatory diseases. We studied the effect of gluten, the agent inducing celiac disease, and other food antigens on the activation of macrophages. Nitric oxide (NO) and cytokine production were followed as markers of activation, using cultured murine peritoneal macrophages. None of the food antigens tested caused direct inducible nitric oxide synthase (iNOS) activation in macrophages. Unlike other food antigens gluten, gliadin, and their proteolytic fragments significantly enhanced NO production when applied together with interferon-γ (IFN-γ), the most efficient being fragments originating from 25-to 45-min peptic digestion. The activation pathway was mediated via direct stimulation of tumor necrosis factor α (TNF-α) secretion. The NO-enhancing effect was confirmed at the level of iNOS mRNA transcription. In case of sustained local inflammatory reaction connected with increase of IFN-γ, gluten and its proteolytic fragments may thus elevate NO production. Increased NO level could consequently participate in the development of mucosal lesions in the gut of celiac patients. J. Leukoc. Biol. 67: 312–318; 2000.

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.