Identification of tissue transglutaminase as the autoantigen of celiac disease.

Celiac disease is characterized by small intestinal damage with loss of absorptive villi and hyperplasia of the crypts, typically leading to malabsorption. In addition to nutrient deficiencies, prolonged celiac disease is associated with an increased risk for malignancy, especially intestinal T-cell lymphoma. Celiac disease is precipitated by ingestion of the protein gliadin, a component of wheat gluten, and usually resolves on its withdrawal. Gliadin initiates mucosal damage which involves an immunological process in individuals with a genetic predisposition. However, the mechanism responsible for the small intestinal damage characteristic of celiac disease is still under debate. Small intestinal biopsy with the demonstration of a flat mucosa which is reversed on a gluten-free diet is considered the main approach for diagnosis of classical celiac disease. In addition, IgA antibodies against gliadin and endomysium, a structure of the smooth muscle connective tissue, are valuable tools for the detection of patients with celiac disease and for therapy control. Incidence rates of childhood celiac disease range from 1:300 in Western Ireland to 1:4700 in other European countries, and subclinical cases detected by serological screening revealed prevalences of 3.3 and 4 per 1000 in Italy and the USA, respectively. IgA antibodies to endomysium are particularly specific indicators of celiac disease, suggesting that this structure contains one or more target autoantigens that play a role in the pathogenesis of the disease. However, the identification of the endomysial autoantigen(s) has remained elusive. We identified tissue transglutaminase as the unknown endomysial autoantigen. Interestingly, gliadin is a preferred substrate for this enzyme, giving rise to novel antigenic epitopes.

Isolation and characterization of circulating tissue transglutaminase-specific T cells in coeliac disease.

Tissue transglutaminase (TG2) was identified as the humoral autoantigen in coeliac disease, but whether it can also serve as T cell autoantigen is still unknown. We aimed, therefore, to firstly explore the presence of TG2-specific T cells in peripheral blood of ten adult patients (four active, i.e. carrying both serological and histological features of the disease; four treated, i.e. with proven mucosal recovery and disappearance of specific antibodies after an adequate period of gluten free diet; and two potential coeliacs, i.e. carrying the serological stigmata of the disease, but not the intestinal lesions), and four healthy controls (two carrying the HLA-DQ2 haplotype of susceptibility to the disease), and secondly to carry out a detailed in vitro characterization of the isolated antigen-specific T cells. T cell lines were first established by means of weekly stimulation with human recombinant TG2 followed by generation of T cell clones through distribution of T cells on plates at one cell/well limiting dilution and further rounds of stimulation. Antigen specificity and HLA-DQ2 restriction were both assessed by evaluating the proliferative response to TG2 in the absence and presence of human sera blocking HLA-DQ2 molecules, after exclusion of impurities in the antigen preparation. Immune phenotyping of T cell clones was performed by flow cytometry, and the expression of IL-1â, IL-4, IL-6, IL-10, IL-12, TGF-beta, IFN-gamma and TNF-alpha was determined by ELISA assay on the supernatants of these clones. A total of 91 T cell clones were isolated from the three HLA-DQ2-positive, active patients, but none from the other patients and controls. The immune phenotyping showed that the majority of them (85.7 percent) were CD3/CD4+ and only a small percentage (14.3 percent) were CD3/CD8+, all carried the TCR alphabeta, and had a memory phenotype. The cytokine profile showed high levels of IFN-gamma and IL-6 that, together with the absence of IL-4, placed these T cell clones in the T helper type 1-like category. Further in vitro analysis was carried out on 32/91 CD4+ clones and showed a specific and dose-dependent proliferative response towards TG2 and an HLA-DQ2 restriction. Finally, when incubating duodenal mucosal specimens of treated patients with the supernatant of TG2-specific T cell clones, characteristic disease lesions were found, indicating a role for TG2-specific cellular immune response in the pathogenesis of coeliac disease.

Participation of the intestinal microbiota in the mechanism of beneficial effect of treatment with synbiotic Syngut on experimental colitis under stress conditions.

Gut-brain axis plays a central role in the regulation of stress related diseases such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD). It is increasingly recognized that stress modulates gut microbiota community structure and activity and represents an important causal factor in dysbiosis. This study was designed to determine the effect of daily treatment with synbiotic (Syngut) containing inulin, Lactobacillus acidophilus, Bifidobacterium lactis W51, Lactobacillus plantarum W21 and Lactococcus lactis applied i.g. at a dose of 50 mg/kg i.g. on the colonic damage and colonic mucosal blood flow in rats with experimentally induced TNBS-colitis that were additionally exposed or not to acute stress (episodes of cold restraint stress every other day before colitis induction). Control rats received daily treatment with vehicle (saline, i.g.) or mesalazine (50 mg/kg-d i.g.), the standard drug recommended in therapy of IBD. At the termination of TNBS colitis, the histologic evaluation of colonic mucosa, mucosal malonyldialdehyde (MDA) level and plasma concentrations of proinflammatory cytokines (TNF-α, IL-1ß) and adipokine adiponectin were assessed. the samples of colonic mucosa not involving colonic lesions and surrounding the flared mucosa were excised for the determination of mRNA expression for proinflammatory biomarkers TNF-α, IL-1ß, IL-10 and COX-2 as well as antioxidazing factors SOD-1 and SOD-2. Finally, the gut microbial profiles were analyzed by 16S rRNA sequencing at phylum, family and genus level. Episodes of cold stress significantly aggravated the course of TNBS colitis, and significantly increased the release of proinflammatory cytokines as well as the significant increase in the MDA concentration has been observed as compared with non-stressed TNBS rats. These changes were followed by the significant fall in the CBF and plasma adiponectin levels and by the overexpression of mRNA of proinflammatory biomarkers. Synbiotic treatment with Syngut significantly reduced the area of colonic lesions observed macroscopically and microscopically in rats with TNBS colitis with or without exposure to cold stress, significantly increased the CBF, normalized plasma adiponectin levels and significantly attenuated the release and colonic expression of proinflammatory cytokines and biomarkers. the analysis of the gut microbiota showed a significant reduction of microbial diversity (Shannon index) in rats with TNBS colitis with or without exposure to stress. The therapy with Syngut failed to significantly affect the alpha diversity. At the phylum level, the significant rise in Proteobacteria has been observed in stressed rats with TNBS colitis and this effects was attenuated by treatment with Syngut. At family level, TNBS colitis alone or in combination with stress led to a significant decrease of SCFA producing bacterial taxa such as Ruminococaceae and Lachnospiraceae and Syngut counteracted this effect. We conclude that: 1) cold stress exacerbates the gastrointestinal inflammation in experimental colitis; 2) the synbiotic therapy with Syngut ameliorates the gut inflammation in rats with TNBS colitis combined with cold stress; 3) the beneficial effect of Syngut is accompanied by increase of anti-inflammatory taxa such as Ruminococaceae and Lachnospiraceae, and 4) the modulation of gut microbiota with Syngut alleviates stress-related intestinal inflammation suggesting a potential usefulness of synbiotic therapy in intestinal disorders accompanied by stress in patients with IBD.

Microbial patterns in patients with histamine intolerance.

Histamine intolerance represents a controversially discussed disorder. Besides an impaired degradation of orally supplied histamine due to diamine oxidase (DAO) deficiency, a deranged gut flora may also contribute to elevated histamine levels. Our aim was to determine the intestinal bacterial composition in patients with proven histamine intolerance in comparison to other food intolerances and healthy controls. A total of 64 participants were included in the study, encompassing 8 patients with histamine intolerance (HIT), 25 with food hypersensitivity (FH), 21 with food allergy and 10 healthy controls (HC). All participants underwent blood testing for total and food-specific immunoglobulin E, plasma histamine and DAO serum activity. Stool samples were used to analyze stool histamine and zonulin levels and bacterial composition by 16s rRNA sequencing. No significant differences in stool histamine levels were observed, but HIT patients showed elevated levels of stool zonulin. Microbiota analysis revealed increased levels of Proteobacteria (5.4%) and a significantly reduced alpha-diversity in the HIT group (P = 0.019). On family level, HC showed a significantly higher abundance of Bifidobacteriaceae compared to other study groups (P = 0.005), with lowest levels in the HIT group (P = 0.036). Also significantly reduced abundances of the genera Butyricimonas (P = 0.026) and Hespellia (P = 0.025) were observed in the HIT patients, whereas Roseburia were significantly elevated (P = 0.021). We concluded that the altered occurrence of Proteobacteria and Bifidobacteriaceae, reduced alpha-diversity as well as elevated stool zonulin levels suggest a dysbiosis and intestinal barrier dysfunction in histamine intolerant patients, which in turn may play an important role in driving disease pathogenesis.

Successful therapy of Clostridium difficile infection with fecal microbiota transplantation.

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea and represents an important burden for healthcare worldwide. Symptoms of severe CDI include watery, foul-smelling diarrhea, peripheral leucocytosis, increased C-reactive protein (CRP), acute renal failure, hypotension and pseudomembranous colitis. Recent studies indicate that the main cause of CDI is dysbiosis, an imbalance in the normal gut microbiota. The restoration of a healthy gut microbiota composition via fecal microbiota transplantation (FMT) recently became more popular. The aim of the present study was to assess the effect of FMT on the healing of CDI and to analyze the changes in the level of pro-inflammatory markers (C-reactive protein, fecal calprotectin) and pro-inflammatory cytokines. Eighteen patients with CDI were included in our study (6 males and 12 females) with recurrent and/or severe CDI. The FMT was performed in 17 patients using colonoscopy, including 16 patients receiving a one-time FMT and 1 patient who needed 2 additional FMTs. One patient was treated with a single round of FMT using push-and-pull enteroscopy. In all CDI patients, before and 3 weeks after FMT, the following parameters were analyzed: C-reactive protein, fecal calprotectin, and plasma interleukin (IL)-6, IL-8 and IL-12, and tumor necrosis factor-alpha (TNF-α). In addition, the plasma level of LL-37, a cathelicidine peptide was assessed by fluorescence-activated cell sorting (FACS) before and 3 months after FMT. Finally, in 7 patients a microbiome analysis was performed by sequencing of 16SrRNA in stool probes obtained before and 3 weeks after FMT. The healing rate of CDI was 94%. In all successfully treated patients no recurrent CDI was observed during follow-up (16 months). The serum level of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-8 and IL-12) significantly decreased after FMT. Similarly, CRP and fecal calprotectin normalized after FMT. 3 months after FMT a significant increase of LL-37 in the plasma of successfully treated patients was monitored. The sequencing analysis demonstrated an elevated abundance of beneficial bacterial species such as Lactobacillaceae, Ruminococcaceae, Desulfovibrionaceae, Sutterellaceae and Porphyromonodacea after FMT. No serious side effects were observed. We concluded that FMT represented a very effective and safe treatment of recurrent and/or severe CDI and led to favorable shifts in the composition of gut microbiome.

Complete primary structure of human collagen type XIV (undulin).

A partial cDNA sequence coding for the human extracellular matrix protein undulin has been completed. The completed sequence provides conclusive evidence for the suggested identity of undulin and collagen type XIV. Two differently sized polyproteins of 1780 and 1796 amino acids, with an overall amino acid sequence identity of 75% compared to chicken CXIV, emerge from variant 3' sequence ends encoding the C-terminal non-collagenous (NC) NC1 domain of human collagen type XIV.

Antibodies to tissue transglutaminase as serologic markers in patients with dermatitis herpetiformis.

Dermatitis herpetiformis is a gluten-sensitive disease with a symmetrically distributed blistering over extensor surfaces. The association with celiac disease is further supported by the high rate of immunoglobulin A autoantibodies to endomysium in patients with dermatitis herpetiformis, which are highly specific and sensitive indicators of celiac disease. Therefore, we determined immunoglobulin A antibodies to tissue transglutaminase, the recently discovered endomysial autoantigen in celiac disease, in patients with dermatitis herpetiformis and controls. Sera of 61 patients with dermatitis herpetiformis, as characterized by granular immunoglobulin A deposits in the subepidermal basement membrane and known endomysial antibody titers (determined by indirect immunofluorescence) as well as 84 control sera of patients with dermal or intestinal diseases unrelated to dermatitis herpetiformis, were analyzed for circulating immunoglobulin A antibodies to tissue transglutaminase by enzyme-linked immunosorbent assay. Immunoglobulin A anti-tissue transglutaminase titers in patients with dermatitis herpetiformis were significantly elevated above the controls. Furthermore, the immunoglobulin A anti-tTG titers showed a positive correlation with semiquantitative endomysial antibody data. Compared with endomysial antibodies, determination of immunoglobulin A anti-tissue transglutaminase reached a specificity and sensitivity of 97.6% and 89.1%. Patients with dermatitis herpetiformis have elevated immunoglobulin A autoantibodies to tissue transglutaminase, confirming its pathogenic relation with celiac disease and further supporting the usefulness of this novel assay for screening and therapy control.

Identification of the autoantigen of celiac disease.

Tissue transglutaminase is demonstrated to be the unknown endomysial autoantigen by means of immunoprecipitations from a fibrosarcoma cell culture. A novel hypothesis for the pathogenesis of celiac disease is formulated: The mainly intracellular tissue transglutaminase is released from cells during wound healing where it aids in stabilizing the wound area by cross-linking a small set of extracellular matrix components.

Immunoassay for detection of IgA antitissue transglutaminase in patients with celiac disease.

Celiac disease is characterized by mucosal changes in the small intestine, ranging from increased numbers of intraepithelial lymphocytes to complete villus effacement with various signs of malabsorption (1,2). Celiac disease is diagnosed by the demonstration of an altered intestinal mucosa in a jejunal biopsy specimen (3). Celiac disease patients invariably have antibodies directed against gliadin and endomysium, a structural component of the extracellular matrix: both antibodies disappear under a gluten-free diet. Therefore, these antibodies are useful tools for diagnosis, and in the dietary control of coeliac disease (3-7). Serum IgA antibodies against endomysial antibodies (EMAs) are especially considered to be sensitive and highly specific markers for celiac disease (8)(9). Previously, EMAs were detected by indirect immunofluorescence on tissue slides of monkey esophagus or human umbilical cord (10-12).

Serum antibodies in celiac disease.

Celiac disease is characterized by small intestinal damage which often leads to global malabsorption. For diagnosis an intestinal biopsy to demonstrate the mucosal changes is mandatory. However, serological tests are useful additional tools to confirm the diagnosis and to monitor therapy. These serological tests, which are based on the detection of antibodies against gliadin and autoantibodies against components of the extracellular matrix, are described in this article.

Kinetics in one-dimensional lattice gas and Ising models from time-dependent density-functional theory.

Time-dependent density-functional theory, proposed recently in the context of atomic diffusion and nonequilibrium processes in solids, is tested against Monte Carlo simulation. In order to assess the basic approximation of that theory, the representation of nonequilibrium states by a local equilibrium distribution function, we focus on one-dimensional lattice models, where all equilibrium properties can be worked exactly from the known free energy as a functional of the density. This functional determines the thermodynamic driving forces away from equilibrium. In our studies of the interfacial kinetics of atomic hopping and spin relaxation, we find excellent agreement with simulations, suggesting that the method is also useful for treating more complex problems.

Adaption of a diamond anvil cell to an automatic four-circle diffractometer for x-ray diffraction.

A diamond anvil high-pressure device for x-ray diffraction on single crystals under hydrostatic pressures up to about 100 kilobars has been adapted to the automatic four-circle x-ray diffractometer Philips PW 1100. The mechanical adaption, the centering procedure, and quick pressure calibration on the diffractometer are described. Hints to improve the peak-to-background ratio of the intensity measurements are given.

Prevalence of seropositive reactions to Brucella canis in a limited survey of domestic cats.

The prevalence of Brucella canis agglutinins was determined in 170 cats (114 from animal shelters in California and 56 from an animal hospital in Texas). Seropositive reactions in the cats from animal shelters were 5.3, 11.4, and 0%, respectively, the the rapid slide agglutination test, salt 2-mercaptoethanol tube agglutination test, salt 2-mercaptoethanol tube agglutination test, and brucellosis card test. For hospitalized cats, the respective percentages were 7.1, 8.9, and 0%. One (0.9%) of 114 cats from the animal shelters and 5 (8.9%) of 56 hospitalized cats were seropositive by the salt 2-mercaptoethanol tube agglutination test at titers greater than or equal to 1:200. Isolation of bacteria was not attempted; thus, the findings of this study may need cautious interpretation.