Human lymphocyte stimulation with pouchitis flora is greater than with flora from a healthy pouch but is suppressed by metronidazole.

BACKGROUND/AIMS: The gut flora may play an important role in the pathogenesis of inflammatory bowel disease. An ileal reservoir or pouch can be created to replace the excised rectum after proctocolectomy. In patients with ulcerative colitis this is subject to inflammation and termed pouchitis. Using bacteria from patients the authors sought evidence for the presence rather than the identity of a pathogenic species in pouchitis, and for its absence in healthy pouches by the differential effect on lymphocyte proliferation. METHODS: An ex vivo cell culture assay was used in which peripheral blood mononuclear cells or lamina propria mononuclear cells were cultured with sterile sonicates of gut flora from patients with or without pouchitis in the presence of antigen presenting cells. RESULTS: Sonicated pouchitis flora produced a consistent and intense proliferation of the mononuclear cells but that produced by sonicates from healthy pouches was minimal (p = 0.012 or 0.018, peripheral blood or lamina propria mononuclear cells). Preparation of the sonicates with the antibiotic metronidazole abolished their stimulatory ability (p = 0.005, peripheral blood mononuclear cells). In separate assays neither direct addition of metronidazole nor of its hydroxy metabolite affected the mononuclear cells' proliferation with alternative stimuli. CONCLUSIONS: These results strongly support a bacterial aetiology for pouchitis.

Coeliac disease: in vivo toxicity of the putative immunodominant epitope.

BACKGROUND: Peptides from alpha-gliadins have been used to characterise the immunodominant coeliac toxic epitope. A peptide corresponding to amino acid residues 57-73 of A-gliadin causes peripheral blood mononuclear cells from coeliac patients to secrete interferon gamma (IFN-gamma); gluten specific small intestinal T cell clones proliferate in response to peptides corresponding to residues 57-68 and 62-75 of alpha-gliadins. We wished to investigate whether a peptide corresponding to residues 56-75 of alpha-gliadins exacerbates coeliac disease in vivo. METHODS: Four adults with coeliac disease, all of whom were on a gluten free diet, underwent three challenges. Peptic-tryptic gliadin (PTG 1 g) served as a positive control. The test peptide and a negative control peptide were studied on separate occasions. The peptides were instilled into the duodenum and biopsies were taken before the infusion, and two, four, and six hours after commencing the infusions, using a Quinton hydraulic multiple biopsy capsule. Biopsy specimens were assessed blindly for villus height to crypt depth ratio (VH:CD), enterocyte cell height (ECH), and intraepithelial lymphocyte (IEL) count. We used the Mann-Whitney U test, with 95% confidence intervals, for statistical analysis. RESULTS: VH:CD and ECH fell, and IEL increased significantly 4-6 hours after commencing infusions with both PTG and the test peptide in all subjects. The negative control peptide caused no significant changes to villus morphology, enterocyte height, or IEL count in any patient. CONCLUSION: We have confirmed that the putative immunodominant epitope, a peptide corresponding to residues 56-75 of alpha-gliadins, exacerbates coeliac disease in vivo.

Investigation of the putative immunodominant T cell epitopes in coeliac disease.

BACKGROUND: Coeliac disease (CD) is an enteropathy mediated by gluten specific T cells which secrete interferon gamma (IFN-gamma) when stimulated by gluten peptides presented by HLA-DQ2 or DQ8 molecules. Residues 62-75 of alpha(2) gliadin have been proposed as the immunodominant epitope in the majority of CD patients. Deamidation by tissue transglutaminase (tTG) of the glutamine (Q) at position 65 to glutamic acid (E) is essential for T cell stimulation. AIMS: To investigate the antigenicity of this peptide and to establish whether its T cell activating properties can be downregulated by the formation of altered peptide ligands. PATIENTS: Individuals with known CD. METHODS: Peptide G4 corresponding to alpha(2) gliadin residues 62-75, Q-E65 and analogues, substituting each amino acid, except E65, in turn for alanine residues, were synthesised. Small intestinal biopsies were obtained from patients. Biopsies were cultured overnight with a peptic/tryptic digest of gliadin (PTG). Lymphocytes were cultured and restimulated with tTG treated PTG. A T cell line was cloned and clones tested for stimulation and IFN-gamma production in response to G4 and its analogues. RESULTS: Some high activity clones were isolated with, for example, a stimulation index (SI) of 15 to G4 and secreting 327 pg/ml of IFN-gamma. Substitution of amino acids at several positions abolished or downregulated stimulation and IFN-gamma production. CONCLUSIONS: Peptide G4 is highly immunogenic. Certain amino acid substitutions in peptide G4 abolish T cell reactivity while others are partial agonists which may have potential in immunomodulation in this condition.

Analysis of candidate genes on chromosome 19 in coeliac disease: an association study of the KIR and LILR gene clusters.

Coeliac disease is strongly heritable, with more than half of the genetic susceptibility estimated to come from genes outside the HLA region. Several candidate regions have been suggested from genome-wide linkage studies including chromosome 19q13.4 where linkage has been replicated between populations. The natural killer (NK) cell immunoglobulin-like receptors (KIRs) and leukocyte immunoglobulin-like receptor (LILR, also known as ILT and LIR) gene clusters lie within this region in the leukocyte receptor cluster (LRC). KIR molecules are involved in cytotoxic lymphocyte function and expressed by intraepithelial T and NK cells in the duodenum. We studied 132 unrelated UK Caucasian coeliac patients and their parents together with a control group of 171 UK Caucasians. PCR-SSP for KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, LILRA3 (ILT6), LILRA3 deletion and an LILRA3 exon 3 single nucleotide polymorphism (SNP) allowed classification of KIR genotypes into five categories and determination of homozygosity or heterozygosity for the common A and B type KIR haplotypes (as defined in the text) and for the LILRA3 deletion. Case-control analysis found no association of the five KIR genotype categories, the A or B KIR haplotypes, the LILRA3 gene deletion or the LILRA3 exon 3 SNP with coeliac disease. A transmission disequilibrium test also found no association of the A and B KIR haplotypes or the LILRA3 gene deletion with coeliac disease.

Coeliac disease: follow-up linkage study provides further support for existence of a susceptibility locus on chromosome 11p11.

Susceptibility to coeliac disease has a strong genetic component. The HLA associations have been well described but it is clear that other genes outside this region must also be involved in disease development. Two previous genome-wide linkage studies using the affected sib pair method produced conflicting results. Our own family based linkage study of 16 highly informative pedigrees identified 17 possibly linked regions, each of which produced a result significant at p & 0.05 or less. We have now investigated these 17 regions in a larger set of pedigrees using more finely spaced markers. Fifty multiply affected families were studied, comprising the 16 pedigrees from the original genome screen plus 34 new highly informative pedigrees. A total of 128 microsatellite markers were genotyped with an average spacing between markers of 5 cM. Two-point and three-point linkage analysis using classical and model free methods identified five potential susceptibility loci with heterogeneity lod scores > 2.0, at 6p12, 11p11, 17q12, 18q23 and 22q13.3. The most significant was a heterogeneity lod of 2.6 at D11S914 on chromosome 11p11. This marker maps to a position implicated in one of the two previous genome scans and taken together these results provide strong support for the existence of a susceptibility locus in this region.

In vivo gluten challenge in celiac disease.

In vivo gluten challenge has been used since the early 1950s to study the role of cereal fractions in celiac disease. While early studies relied on crude indicators of celiac toxicity, the advent of jejunal biopsy and sophisticated immunohistochemical techniques has allowed accurate studies to be performed. Studies to determine the nature of the cereal component that is toxic to patients with celiac disease have concentrated on wheat because of its nutritional importance. A number of in vitro studies indicated the presence of one or more celiac-activating epitopes with the N-terminus of the A-gliadin molecule. In vivo challenge with three synthetic peptides subsequently indicated the toxicity of a peptide corresponding to amino acids 31 to 49 of A-gliadin. In vivo gluten challenge is the gold standard for the assessment of celiac toxicity; however, jejunal biopsy is a relatively invasive procedure, thus, other methods have been investigated. Direct infusion of the rectum with gluten has been shown to result in an increase in mucosal intraepithelial lymphocytes, occurring only in celiac patients. This method has been used to study the celiac toxicity of gliadin subfractions. The in vitro technique of small intestinal biopsy organ culture is also a useful tool and appears to give the same results as in vivo challenge. The importance of tiny amounts of gliadin in the diet, such as that which occurs in wheat starch, has been studied by in vivo challenge; this technique has clarified the position of oats in the gluten-free diet. Several studies suggest that this cereal may be included in the diet of most adult celiac patients. Studies of the transport of gliadin across the enterocyte following ingestion or challenge suggest that gliadin may be metabolized by a different pathway in celiac disease. This could result in an abnormal presentation to the immune system, triggering a pathogenic rather than a tolerogenic response.

Human genome search in celiac disease using gliadin cDNA as probe.

Celiac disease is a wheat gliadin-promoted disorder that displays a complex genetic susceptibility associated with HLA-DQ2, and one or more unknown factor(s), possibly gliadin-like. The presence of mammalian proteins with partial gliadin similarity was suggested by transglutaminase-independent multi-tissue reactivity of gliadin-immunopurified antibodies from celiac patients. No non-plant sequence, however, was identified in gliadin peptide epitope searches of non-redundant and EST databanks via TBLASTN, BLASTP and FASTA, even at E values as high as 20. Therefore, an alpha-gliadin cDNA screen of human cDNA and genomic libraries was undertaken, an approach in keeping with positive human Northern and Southern analyses with the same probe. Four distinct cDNA clones were obtained, the most stringent of which (3.2 and 5.1 kb) were novel, and featured potential open reading frames with high gliadin domain II and domain IV homologies (BestFit quality scores >/=295 and 322, respectively, versus random value 126-127). Both were also homologous to ESTs. An additional 5' gliadin oligonucleotide screen identified the widely distributed cytoplasmic protein acyl coA hydrolase whose homology was restricted to the oligonucleotide probe (BestFit quality=215 versus 100 for random); and achaete-scute homologous protein, which displays particularly high gliadin domain II homology (BestFit quality 316 versus 111 for random). Genomic screening uncovered 16 positives, one of which was the ALR gene, whose similarity to three of gliadin's five domains (I, II and IV; BestFit quality 322-473 versus 121-154 for random) was remarkable. More extensive was novel genomic clone 2, with fragments hybridizing to cDNA probes approximating gliadin domains I, II+IV, V and the gliadin 5' untranslated region, and mapping by FISH to 19q13.11-13. 12. Two fragments were sequenced; one was exonic, as predicted by four different programs; and test oligonucleotides suggested widespread 4 and/or 2 kb mRNA expression, even at high stringency (t(m)-8.8 deg. C). Taken together, it is apparent that several genes with partial gliadin homology exist in the human genome. Many bear gliadin-like T-cell epitopes, are expressed in intestine and, like transglutaminase, are cytoplasmic. Glutamine to glutamic acid or other mutation within such epitopes followed by injury or infection-related release could explain enhanced disease susceptibility in affected families.

Endomysial antibody production is not related to histological damage after in vitro gluten challenge.

OBJECTIVE: To determine whether in vitro induction of endomysial antibodies is an in vitro marker of coeliac toxicity. DESIGN: To determine whether in vitro endomysial antibodies induced by gliadin incubation correlate with histological damage induced by in vitro gliadin challenge. METHODS: Small-bowel organ cultures from seven patients with treated coeliac disease were incubated in an organ culture system with gliadin; histological damage was morphometrically evaluated and endomysial antibodies were measured in the organ culture supernatant by indirect immunofluorescence. RESULTS: Although incubation with gliadin caused histological damage, there was no detectable production of endomysial antibody. CONCLUSIONS: In vitro, endomysial antibody induction cannot be used as a marker of coeliac toxicity. Endomysial antibodies are not necessary for generating the histological lesion of coeliac disease.

A genome-wide family-based linkage study of coeliac disease.

The susceptibility to develop coeliac disease (CD) has a strong genetic component, which is not entirely explained by HLA associations. Two previous genome wide linkage studies have been performed to identify additional loci outside this region. These studies both used a sib-pair design and produced conflicting results. Our aim is to identify non-MHC genetic loci contributing to coeliac disease using a family based linkage study. We performed a genome wide search in 16 highly informative multiply affected pedigrees using 400 microsatellite markers with an average spacing of 10 cM. Linkage analysis was performed using lod score and model free methods. We identified two new potential susceptibility loci with lod scores of 1.9, at 10q23.1, and 16q23.3. Significant, but lower lod scores were found for 6q14 (1.2), 11p11 (1.5), and 19q13.4 (0.9), areas implicated in a previous genome wide study. Lod scores of 0.9 were obtained for both D78507, which lies 1 cM from the gammaT-cell receptor gene, and for D2S364, which lies 12 cM from the CTLA4 gene.

Tissue transglutaminase antibodies in celiac disease.

OBJECTIVE: Tissue transglutaminase is the antigen for antiendomysial antibodies, whose power in screening for celiac disease is well known. Our aim was to assess the efficacy of an ELISA assay for tissue transglutaminase antibodies. METHODS: Tissue transglutaminase antibodies were analyzed in serum from 39 untreated celiac disease patients and 61 controls. Tissue transglutaminase was used as antigen, and test sera analyzed by ELISA. Results higher than 0.6 optical density were considered positive, lower than 0.4 negative, and between 0.4 and 0.6 borderline. RESULTS: Optical density of the serum from the patients with untreated celiac disease (median: 1.41; range: 0.33-1.47) were significantly higher than the controls (median: 0.32; range: 0.17-0.68; p < 0.0001; 95% confidence interval 0.87-1.08). Thirty-three patients with untreated celiac disease were positive, 4 borderline, and 2 negative. Fifty-five controls were negative, 4 borderline, and 2 positive. If we consider borderline results to be positive, sensitivity is 94.8% and specificity 90.1%. None of the controls gave results higher than 0.7 optical density. Apart from the 2 negative patients with untreated celiac disease, the two groups overlapped only between 0.4 and 0.7 optical density. CONCLUSIONS: Because of the high sensitivity (approximately 95%) and technical simplicity, tissue transglutaminase antibodies may prove useful for the screening of celiac disease in population at low or medium risk of celiac disease. To avoid duodenal biopsies in patients without celiac disease, the specificity of the screening procedure may be increased by confirming with antiendomysial antibodies by immunofluorescence on human umbilical cord in individuals with results between 0.4 and 0.7 optical density.

A non-toxic analogue of a coeliac-activating gliadin peptide: a basis for immunomodulation?

BACKGROUND: A-gliadin residues 31-49 (peptide A) binds to HLA-DQ2 and is toxic to coeliac small bowel. Analogues of this peptide, which bind to DQ2 molecules but are non-toxic, may be a potential route to inducing tolerance to gliadin in patients with coeliac disease. METHODS: Toxicity was investigated with small bowel organ culture in six patients with untreated coeliac disease, four with treated coeliac disease and six controls. Analogue peptides comprised alanine substituted variants of peptide A at L31 (peptide D), P36 (E), P38 (F), P39 (G) and P42 (H). RESULTS: Peptides D and E were toxic in biopsies from some patients. Peptides F, G and H were not toxic. CONCLUSIONS: Peptide F, which binds to DQ2 more strongly than peptide A, is not toxic in patients with coeliac disease in-vitro; this could be an initial step towards investigation of the induction of tolerance to gliadin in patients affected by coeliac disease.

Is gliadin mispresented to the immune system in coeliac disease? A hypothesis.

The primary pathogenic trigger in coeliac disease (CD) is still unknown. We present the hypothesis that in CD the enterocytes could metabolize gliadin through an immunogenic pathway instead of a tolerogenic one. The result of this abnormal presentation of gliadin to the immune system would be the activation of lamina propria T cells, followed by the onset of enteropathy.

Targeting of gliadin peptides, CD8, alpha/beta-TCR, and gamma/delta-TCR to Golgi complexes and vacuoles within celiac disease enterocytes.

Celiac disease (CD) is characterized by autodestruction of enterocytes after exposure of genetically susceptible individuals to dietary gluten. To define the transport pathways of proteins involved in the celiac immune response, we wished to determine the subcellular compartments of the intestinal mucosa where wheat gliadin peptides colocalize with receptors of T lymphocytes, including alpha/beta-TCR, gamma/delta-TCR, and CD8. Semithin and ultrathin frozen section of jejunal biopsies from CD patients and controls were used to perform immunofluorescence and immunogold labeling as well as in situ hybridization experiments. In patients with active CD, we detected gliadin peptides in vacuoles and Golgi complexes of enterocytes. CD8, alpha/beta-TCR, and gamma/delta-TCR were found in vacuoles and Golgi complexes within these gliadin-containing enterocytes in addition to the surface of intraepithelial and mucosal T lymphocytes. In contrast, we observed that the localization of CD4, CD3, T cell-restricted intracellular antigen (TIA), and leukocyte common antigen (LCA) was restricted to lymphocytes in CD patients. We further detected labeling signals for gliadin peptides, CD8, alpha/beta-TCR, and gamma/delta-TCR at the basal membrane of enterocytes that were interdigitated by extensions of lymphocytes. In situ hybridization experiments revealed that CD8 and gamma/delta-TCR were not expressed by CD enterocytes. We conclude that CD8, alpha/beta-TCR, and gamma/delta-TCR are targeted to Golgi complexes and vacuoles of small intestinal enterocytes in active CD. The observed process may be involved in the pathogenesis of CD enterocytes. We propose a mechanism for the uptake of CD8, alpha/beta-TCR, and gamma/delta-TCR by the basolateral membrane of small intestinal enterocytes.

The detection and localization of inducible nitric oxide synthase production in the small intestine of patients with coeliac disease.

OBJECTIVES: To investigate whether there are increased numbers of inducible nitric oxide synthase (iNOS) containing cells in the small intestine of patients with coeliac disease and the localization of nitric oxide synthase production. DESIGN: Small intestinal biopsy specimens from patients with coeliac disease (11 untreated, 10 treated) and nine disease controls were studied. METHODS: Histochemical staining of sections for NADPH-diaphorase activity was performed, which gives an indication of NOS activity. iNOS protein was detected with immunohistochemistry and iNOS mRNA expression was detected using in situ hybridization with an oligonucleotide probe cocktail for iNOS. Cell phenotype was detected using monoclonal antibodies to CD3 (T-lymphocytes) and CD45 (total inflammatory cell infiltrate). RESULTS: There was significantly greater NADPH diaphorase staining in the lamina propria of patients with untreated coeliac disease (P < 0.005). The same pattern was found for immunohistochemical and in situ hybridization methods of staining for iNOS in each of the patient groups (P < 0.005) but no epithelial staining was seen with any method. The pattern of iNOS staining in the lamina propria appeared in a similar distribution to that of the inflammatory cell infiltrate. At least 80% of the significantly increased total inflammatory cell infiltrate (CD45) in the lamina propria of patients with untreated coeliac disease was lymphocytic (CD3) whilst the iNOS staining cells made up less than 15% of the total inflammatory cell infiltrate. CONCLUSIONS: There is a significant increase in the number of NOS staining cells of the inducible isoform in the lamina propria of patients with untreated coeliac disease. The lamina propria and not the epithelium is the site of iNOS production in coeliac disease. It appears that inflammatory cells other than T-lymphocytes are likely to be the cellular sources of iNOS production within the lamina propria. This is the first study to demonstrate increased numbers of iNOS producing cells in the small intestine of patients with untreated coeliac disease and suggests a role for nitric oxide in the pathogenesis of the histological changes seen in coeliac disease although it may be a non-specific inflammatory response to immune activation by gluten in susceptible individuals.

Dietary intake, smoking, and transient anti-gliadin antibodies.

BACKGROUND: The detection of IgA anti-gliadin antibodies in adults can either be helpful in the diagnosis of coeliac disease, be persistent in subjects with normal jejunal mucosa, or occur transiently. We decided to investigate the effects of smoking, alcohol consumption, and dietary intake on the development of IgA anti-gliadin antibodies. METHODS: Serum samples from subjects enrolled from a large Northern Ireland population sample (MONICA survey) were screened for IgA anti-endomysium and IgA anti-gliadin antibodies. All subjects with positive antibodies were invited for clinical assessment 3-4 years after the initial screening sample. During this follow-up a repeat serum sample was obtained and a jejunal biopsy performed. At enrollment in the MONICA survey, lifestyle information including smoking, alcohol consumption, and dietary intake was obtained. RESULTS: At follow-up 13 subjects had persistent positive serology and villous atrophy, and 9 had persistent positive serology but normal jejunal histology; in 29 the serology had returned to normal, and the jejunal histology was normal There was no difference in smoking, alcohol consumption, or dietary intake between subjects with and without coeliac disease. Subjects with transient serology findings ate significantly more soda bread than the other groups (at the time of initial screening). Analysis of gliadin content of soda bread and plain white bread showed a significantly higher amount of gliadin present in soda bread. CONCLUSIONS: Subjects with transient IgA anti-gliadin antibodies eat significantly more soda bread. The gliadin content of Irish soda bread contained a greater amount of gliadin than white bread. Eating breads with high available gliadin content may cause the appearance of anti-gliadin antibody.