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A. Palotie on työryhmän Int IBD Genetics Consortium jäsen. One goal of human genetics is to understand the genetic basis of disease, a challenge for diseases of complex inheritance because risk alleles are few relative to the vast set of benign variants. Risk variants are often sought by association studies in which allele frequencies in case subjects are contrasted with those from population-based samples used as control subjects. In an ideal world we would know population-level allele frequencies, releasing researchers to focus on case subjects. We argue this ideal is possible, at least theoretically, and we outline a path to achieving it in reality. If such a resource were to exist, it would yield ample savings and would facilitate the effective use of data repositories by removing administrative and technical barriers. We call this concept the Universal Control Repository Network (UNICORN), a means to perform association analyses without necessitating direct access to individual-level control data. Our approach to UNICORN uses existing genetic resources and various statistical tools to analyze these data, including hierarchical clustering with spectral analysis of ancestry; and empirical Bayesian analysis along with Gaussian spatial processes to estimate ancestry-specific allele frequencies. We demonstrate our approach using tens of thousands of control subjects from studies of Crohn disease, showing how it controls false positives, provides power similar to that achieved when all control data are directly accessible, and enhances power when control data are limiting or even imperfectly matched ancestrally. These results highlight how UNICORN can enable reliable, powerful, and convenient genetic association analyses without access to the individual-level data.

The relationship between the gross connective tissue and inflammatory changes in ileal Crohn's disease remains unclear. This study investigated 20 patients undergoing ileal resection for Crohn's disease and 20 normal controls. The specimens were blocked in 1 cm serial sections and fully examined, including fresh morphometry and documentation of a range of pathological features. Pathological features of disease showed uniform distributions within affected segments, although specimens showed different patterns and severity of affliction. Serosal fat wrapping (FW) was present in all cases and was significantly greater than normals [mean 63.5% (SD 27. 8) vs. 21.0% (6.4), p

Serum levels of complement components Clq, C4, C3, and Properdin factor B, from the classical and alternative pathways of complement activation, have been estimated in patients with ulcerative colitis and Crohn's disease. C3, factor B, and to some extent C4 concentrations all increased when the disease was active. In remission the levels of these components did not differ from hospital control patients. There was no evidence for the preferential consumption of the proteins of either pathway of activation, even in those patients with evidence of circulating immune complexes.

Colonic resection leads to insulin resistance, but the mechanisms are unknown. We used an integrated approach to examine adipose tissue and skeletal muscle metabolism in patients lacking a colon. Ten healthy colectomized patients having undergone surgery for ulcerative colitis and 10 matched control subjects were studied with a hyperinsulinemic-euglycemic clamp to measure insulin sensitivity, an arteriovenous sampling meal tolerance study to measure postprandial substrate flux across adipose tissue and skeletal muscle, and adipose tissue and skeletal muscle biopsies to quantify the expression of genes involved in glucose and lipid metabolism. Colectomized subjects exhibited lower insulin sensitivity (homeostatic model assessment model, 33% reduction, P = 0.03; minimal model, 29% reduction, P = 0.05), elevated aldosterone (9-fold, P = 0.003), leptin (2.2-fold, P = 0.03), and an increased rate of nonesterified fatty acid and glycerol release from adipose tissue (P = 0.02) especially in the late postprandial period. The uptake of fatty acids into muscle was also significantly increased (P = 0.007), as were muscle CD36 and LPL mRNA expression compared with controls. In adipose tissue, hormone-sensitive lipase mRNA expression was increased (P = 0.015), whereas peroxisome proliferator-activated receptor-gamma expression was decreased (P = 0.02), as was that of CD36 (P = 0.001). In this study, alterations in fatty acid metabolism after colonic resection altered may have contributed to the impairment of insulin sensitivity.

Summary Background : We performed an audit of methotrexate for ulcerative colitis, because efficacy is unclear. Aim : To investigate the role of methotrexate in the management of ulcerative colitis. Methods : Patients with ulcerative colitis treated with oral methotrexate at the inflammatory bowel disease clinics of Oxford and Wycombe General Hospital, UK, were evaluated. Efficacy was defined by remission (complete steroid withdrawal for >3 months) and response (good, partial or nil, proportionate reduction of steroids). Results : There were 50 patients (42 ulcerative colitis alone; eight had rheumatoid arthritis associated with ulcerative colitis and were analysed separately). Indications for methotrexate in ulcerative colitis alone were azathioprine intolerance (31 of 42) and lack of benefit from azathioprine (11 of 42). The mean dose of methotrexate in ulcerative colitis alone was 19.9 mg/week for a median of 30 weeks (range: 7–395). Remission occurred in 42%. The response was good in 54% and partial in 18%. Side-effects occurred in 23%; 10% stopped treatment because of side-effects. Of those treated with methotrexate because of treatment failure with azathioprine, three of 11 achieved remission, but four came to colectomy within 90 days of starting methotrexate. The colitis remained in remission in seven of eight of those with RA treated with methotrexate and ulcerative colitis (mean dose 15.0 mg/week). Conclusion : Oral methotrexate (approximately 20 mg/week) is well-tolerated and moderately effective in steroid-dependent or steroid-refractory patients with ulcerative colitis.

'Cap polyposis' is a poorly recognised condition with distinct clinical, sigmoidoscopic, and pathological features that may be confused with other inflammatory conditions of the large intestine including pseudomembranous colitis and idiopathic chronic inflammatory bowel disease. The pathogenesis is unknown but on the basis of the characteristic histological appearances, which are similar to those seen in situations where mucosal prolapse is the underlying mechanism, it has been suggested that the latter may be an important aetiological factor. Two cases are described. Histological features in the first (presence of intramucosal elastin) and clinical features in the second (rectal prolapse) support the above hypothesis.

The genetic revolution has been with us for over a decade now. We have yet to see this impacting the care of patients except in a few rare examples. However, progress has been made in the field of inflammatory bowel disease (IBD) that could soon be translated to the bedside, both in terms of predicting the disease course as well as in the response to therapy. IBD traditionally has been classified as ulcerative colitis and Crohn's disease, with 10% of patients classified as having indeterminate colitis on the basis of clinical, radiologic, endoscopic, and histologic findings. However, this traditional view is now being challenged. Developments in genetics and serological markers, as well as an appreciation of the disease course, have led to an understanding that IBD is a heterogeneous group of diseases with some common genetic and environmental factors but different clinical manifestations in terms of disease behavior, location, and response to treatment. Data are now emerging that may allow us to more objectively select the correct therapy for the correct patient, rather than the current approach, which is based on clinical experience backed up by a less-than-perfect evidence base. In this article, we will review the evidence for this.

Inflammatory bowel disease (IBD), encompassing Crohn’s disease and ulcerative colitis, is an immune-mediated condition of unknown aetiology. It is possible that a single infectious agent is responsible for these diseases, however, it is generally believed that chronic intestinal inflammation results from an inability to down-regulate the host immune response against enteric bacteria following an initial breach of the intestinal mucosal barrier. It is likely that a range of environmental and genetic factors also result in this host immune dysfunction. Complex combinations of such factors may give rise to chronic intestinal inflammation, which is subsequently characterised clinically as Crohn’s disease or ulcerative colitis (Figure 20.1). Therefore, characterising disease heterogeneity within IBD patients is important in our understanding of the aetiology and pathogenesis of this condition.

Summary: Objective: To determine the effectiveness of medical therapy (including metronidazole, bismuth carbomer enemas, oral probiotic bacteria, butyrate suppositories, and glutamine suppositories) for inducing a response or for maintaining remission in pouchitis. Search strategy: Studies were selected using the MEDLINE data base (1966-December 1997), abstracts from major gastrointestinal meetings, and references from published articles and reviews. Selection criteria: Four randomized controlled trials of medical therapy in adult patients with pouchitis were identified: two placebo controlled trials in active chronic pouchitis; one maintenance of remission trial comparing two active agents in chronic pouchitis; and one placebo-controlled maintenance of remission trial for chronic pouchitis. A single patient "n-of-l" trial for active chronic pouchitis was excluded. Data collection and analysis: Data were extracted by three independent observers based on the intention to treat principle. Extracted data were converted to 2 x 2 tables (response versus no response and medical therapy versus placebo or medical therapy versus medical therapy) and an odds ratio with 95% confidence intervals (0) were determined as described by Cochrane and Mantel and Haenszel. In addition, the absolute risk reduction, relative risk reduction, and number needed to treat were determined. Main results: The odds ratios of inducing a response using oral metronidazole or bismuth carbomer foam enemas compared with placebo in active chronic pouchitis were 12.34 (95% Cl 2.34-64.95) and 1.00 (95% Cl 0.29-3.42), respectively. The odds ratio of maintaining remission in chronic pouchitis for oral probiotic bacteria (VSL-3) compared with placebo was 15.33 (95% Cl 4.5152.14). There was no difference in the odds ratio of inducing symptomatic remission and then maintaining symptomatic remission after discontinuing suppressive medical therapy for chronic pouchitis with glutamine suppositories compared with butyrate suppositories, 2.75 (95% CI 0.48-15.94). Conclusions: Metronidazole is an effective therapy for active chronic pouchitis. Bismuth carbomer foam enemas are not effective therapy for active chronic pouchitis. Oral probiotic therapy with VSL-3 is an effective therapy for maintaining remission in patients with chronic pouchitis in remission. There is no difference in maintenance of symptomatic remission in patients with chronic pouchitis treated with glutamine versus butyrate suppositories, and it is unknown whether glutamine and butyrate are equally effective or ineffective. Additional randomized, double-blind, placebo-controlled, dose-ranging clinical trials are needed to determine the efficacy of empiric medical therapies currently being used in patients with pouchitis. Key Words: Pouchitis-Ulcerative colitis.

A single dose of a synthetic peptide of A gliadin (residues 206-217) sharing homology with the E1b protein of adenovirus 12 was instilled intraduodenally in two treated coeliac patients. Biopsy specimens were taken before and repeatedly up to 24 h after the instillation by means of a Quinton hydraulic multiple biopsy instrument and processed for histology, morphometry (intraepithelial lymphocyte counts, crypt-to-villus ratio), immunocytochemistry, electron microscopy, and disaccharidase assays. Two subjects with irritable bowel syndrome served as controls. In the coeliac group disaccharidase activities decreased at 24 h, and abnormalities were seen on light and electron microscopy and in morphometric measurements. The lamina propria became infiltrated with mononuclear cells after 2 h, and there was also a rise in IgA-containing cells in one patient. No such abnormalities were seen in the control group. The serum concentrations of C3, C4, and C1 esterase inhibitor remained unchanged. Thus, the dodecapeptide may be one epitope of gliadin mediating the pathogenesis of coeliac disease.

Well-designed studies that help guide physicians to apply the optimal therapeutic strategy for the management of pyoderma gangrenosum are lacking in the literature. A multidisciplinary approach is paramount for the effective management of this condition, with close involvement of a wound-care specialist and a microbiologist. Treatment should be stepwise in nature. Local wound care, avoidance of trauma and the application of local steroid or tacrolimus ointment are the first-line treatments. Steroid therapy is the most widely published effective therapy for achieving resolution of pyoderma gangrenosum, although there is growing evidence for the efficacy of infliximab in refractory cases. Other therapies such as dapsone and clofazamine should be left as third-line agents for refractory pyoderma gangrenosum, while novel treatments such as granulocyte apheresis should only be used under trial conditions, to gain an objective evaluation of their efficacy. This article reviews the published treatment strategies in current use, and aims to guide the effective management of pyoderma gangrenosum.

A prospective double blind controlled trial was undertaken to examine the role of metronidazole as an adjunct to corticosteroids in the management of severe ulcerative colitis. Thirty nine patients with severe ulcerative colitis were randomised on admission to hospital to receive either intravenous metronidazole 500 mg eight hourly (19 patients) or an identical intravenous placebo (20 patients). The two groups were similar with respect to age, sex, and the extent of colitis. In addition all patients received a standard intravenous regimen consisting of methyl prednisolone 16 mg six hourly and parenteral nutrition together with a twice daily hydrocortisone 100 mg enema. Treatment was continued for five days when the patients were formally assessed. Fourteen of 19 patients (74%) receiving metronidazole and 14/20 (70%) receiving placebo were substantially improved, or in remission at the end of five days. Five patients treated with metronidazole and six with placebo had no improvement and all proceeded to urgent colectomy with no operative mortality. There were three late deaths, one in the metronidazole and two in the placebo group. These results do not support the routine use of intravenous metronidazole in the treatment of severe ulcerative colitis.

Fluticasone propionate is a new corticosteroid with low systemic bioavailability. This study reports the outcome of a double blind clinical trial comparing oral fluticasone propionate (5 mg four times daily) with placebo for the treatment of active distal ulcerative colitis. Sixty patients were treated for four weeks, with assessments at two and four weeks. One patient was withdrawn when she was found to have amoebiasis. Thus, results are presented for 29 patients who received placebo and 30 who received fluticasone propionate. The two groups were well matched for age, sex, length of history, and extent of disease. After four weeks of therapy the clinical, sigmoidoscopic, and histological responses were similar in the two groups. It is concluded that fluticasone propionate (5 mg four times daily) is not effective treatment for active distal ulcerative colitis.

Splenic phagocytic function has been assessed in patients with inflammatory bowel disease. No Howell-Jolly bodies were found on blood film examination. Slow clearance times of heat damaged red cells were found in 13 of 16 patients with ulcerative colitis compared with 4 of 17 patients with Crohn's disease (P less than 0.01). For ulcerative colitis prolonged clearance times were strongly related to the length of history (r = 0.90; P less than 0.01). The prolongation of half-clearance times correlated poorly with splenic size suggesting a functional impairment of the reticuloendothelial system.

The IL-6 gene maps to an area of chromosome 7 known to be significant for susceptibility to inflammatory bowel disease. The functional effects of interleukin-6 (IL-6) polymorphisms in the 4th intron and in the 3' flanking region of IL-6 gene were studied in 192 inflammatory bowel disease patients and healthy subjects. A polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to determine a G to A polymorphism (* at position 4470 in intron 4 of IL-6 gene). Four alleles in the 3' flanking region were studied using a variable number of tandem repeats PCR (VNTR-PCR) amplification. Production of IL-6 was measured in lipopolysaccharide (LPS) stimulated whole blood samples by an enzyme-linked immunosorbent assay (ELISA). A modest increase in the frequency of the IL-6*G allele was noted in Crohn's disease (CD) patients (50%) and ulcerative colitis (UC) patients (46.1%) as compared to controls (39.8%, P = 0.025). We were unable to find any significant functional effect of the IL-6 polymorphisms tested on IL-6 protein production. We postulate that the IL-6 polymorphisms investigated here may be in linkage disequilibrium with a susceptibility gene and that they may be utilised as genetic markers.

To evaluate the optimum dose of olsalazine for maintaining remission in ulcerative colitis, 198 patients in remission for more than three months were randomly assigned to receive 0.5 g, 1.0 g, or 2.0 g/day for 12 months. A dose-ranging effect was detected in the per protocol analysis, with remission rates of 60% (0.5 g), 70% (1.0 g), and 78% (2.0 g) (p = 0.03, trend in proportions). The higher dose was most effective in patients with proctitis (90% remission on 2 g/day, p = 0.03) or those in remission for less than 12 months before the trial (88% remission on 2 g/day, p = 0.0006). There was little dose-ranging effect in patients with extensive colitis or those in remission for more than 12 months. Diarrhoea necessitated treatment withdrawal in 12%. The optimal dose of olsalazine for maintaining remission in ulcerative colitis is 1 g/day. For patients with proctitis or recent relapse, 2 g/day may be preferable, although the dose seems to be less important in patients with more extensive disease or those in long term remission.

Epithelial histocompatibility locus antigen (HLA) class II expression was studied to evaluate its induction by mucosal mononuclear cells in inflammatory bowel disease and to characterise the responsible cytokine. Unstimulated cells of the HT-29 epithelial cell line did not produce class II molecules. After being stimulated with the mitogenic lectin phytohaemagglutinin mucosal mononuclear cells released a cytokine that induced epithelial HLA-DR expression. The cytokine had the physicochemical and immunological characteristics of interferon-gamma, and no additional cytokines were detected.

Distal ulcerative colitis can be treated with oral or rectal mesalazine, or both. A foam enema preparation has been developed and its efficacy investigated. The aim of this study was to evaluate the efficacy and safety of mesalazine foam enemas compared with prednisolone foam enemas in the treatment of patients with acute distal ulcerative colitis. Patients aged over 18 years presenting with a relapse of distal ulcerative colitis were randomly allocated treatment with mesalazine foam enema (n = 149 evaluable patients) and prednisolone foam enema (n = 146 evaluable patients) for four weeks. A randomised multicentre investigator blind parallel group trial was conducted. It was found that after four weeks of treatment, clinical remission was achieved by 52% of mesalazine treated patients and 31% of patients treated with prednisolone (p < 0.001). There was a trend in favour of more patients in the mesalazine group achieving sigmoidoscopic remission (40% v 31%, p = 0.10). Histological remission was achieved by 27% and 21% of patients receiving mesalazine and prednisolone respectively. Symptoms improved in both treatment groups. Significantly more mesalazine patients had no blood in their stools after four weeks of treatment (67% v 40%, p < 0.001). Prednisolone treated patients had significantly fewer days with liquid stools than mesalazine patients, with a median of 0 and 1 days respectively by week 4 (p = 0.001). In this study mesalazine foam enema was superior to prednisolone foam enema with regards to clinical remission, this was supported by favourable trends in sigmoidoscopic and histological remission rates. Both treatments were well tolerated.

Two patients are described with Crohn's disease and selective IgA deficiency. Serum IgA was undetectable in each case, and immunoperoxidase studies of the lamina propria showed a gross diminution of IgA-bearing plasma cells. Peripheral blood lymphocytes, however, showed normal numbers of IgA-bearing lymphocytes. The typical clinical course and histology in these two patients suggest that IgA-mediated responses in the mucosa are not involved in the pathogenesis of Crohn's disease.

Background: Genomewide linkage studies identified chromosome 3p21 as an IBD locus. Genomewide association studies have supported this locus and the Wellcome Trust Case Control Consortium (WTCCC) study narrowed it to a 0.6 Mb region. Our objectives were to perform a 2-stage candidate gene association study of the 3p locus and to identify linkage disequilibrium (LD) between significant single-nucleotide polymorphisms (SNPs) and an Oxfordshire subset (n = 282) of the WTCCC as well as the HapMap SNPs. Methods: A total of 197 SNPs in 53 genes from the 3p locus were genotyped on the Illumina platform in a screening cohort of 469 Crohn's disease (CD) patients and 461 controls. Significant associations were then genotyped on the iPLEX platform in the original as well as a second cohort of 139 CD patients, 670 ulcerative colitis (UC) patients, and 1131 controls. All cases and controls were Caucasian and from the Oxfordshire region of the UK. Results: An intronic SNP rs1128535 in the TRAIP gene was associated with CD in the screening and validation cohorts (combined [n = 608] P = 0.0004 [corrected 0.002], odds ratio [OR] 0.77, 95% confidence interval [CI], 0.67–0.89]). No association was seen for UC. Epistasis was seen with the common CARD15 mutations (P = 0.00003 [corrected 0.0006], OR 0.48, 95% CI, 0.34–0.68). No LD was demonstrated with the WTCCC SNPs. Strong LD was demonstrated with 2 nonsynonymous HapMap SNPs in the MST1R gene in an adjacent LD block to the peak WTCCC association, suggesting a distinct association signal. Conclusions: The LD with these functional MST1R variants implicate this gene as having a possible role in CD pathogenesis. (Inflamm Bowel Dis 2008)

The colonic faecal and mucosal associated bacterial populations of five patients with ulcerative colitis and four control patients were studied in detail to assess their ability to produce IgA1-proteases. A total of 330 bacterial strains were isolated from the patients with ulcerative colitis and IgA1-protease activity was unable to be reliably shown in any. It is therefore unlikely that such enzyme production by colonic bacteria plays a significant role in the pathogenesis of ulcerative colitis.

Targeted delivery of 5-aminosalicylic acid to the small intestine and colon by controlled-release or azo-bonded compounds potentially offers treatment for ileal Crohn's disease as well as ulcerative colitis. The pharmacokinetics of sulphasalazine and aminosalicylate derivatives have been discussed and potential modes of action reviewed. These include actions on epithelial cell-surface receptors, cellular events and barrier function. Evidence for an influence of salicylates on circulating and tissue inflammatory cells is presented, as well as actions on adhesion molecules, chemotactic peptides, eicosanoids, cytokines and reactive oxygen metabolites. The precise mechanism remains unknown, but a pluripotential mode of action is an advantage when influencing the network of events that constitutes chronic inflammation. Controlled clinical trials of salicylates in ulcerative colitis and Crohn's disease have been reviewed. Their main role remains as maintenance therapy for ulcerative colitis, but relatively high doses of controlled-release preparations benefit patients with ileal Crohn's disease, following resection, or those who have recently relapsed. Finally, issues of clinical relevance have been addressed, including the choice of salicylate and safety, indications for initiating therapy, dose and duration of treatment, role in managing refractory colitis and future developments.

Random motility and chemotaxis of peripheral blood neutrophils and monocytes from patients with Crohn's disease and ulcerative colitis have been measured using a modified Boyden Chamber filter assay. Increased random motility and chemotaxis of monocytes were found in patients with active ulcerative colitis. Monocyte motility was normal in Crohn's disease and no abnormality of neutrophil motility or chemotaxis was found in either disease. Drug therapy with prednisolone or sulphasalazine received in vivo was found to have no effect on the motility of the washed neutrophils and monocytes in vitro. This work adds to the evidence that monocytes are activated in ulcerative colitis but does not support the hypothesis that Crohn's disease is due to an inherent defect in phagocyte motility.

HLA-DR+ gut epithelial cells may present antigen to intraepithelial lymphocytes (IEL). This study aimed to isolate an IEL population from the human colon to activate CD3 + IEL by a human colonic epithelial cell line (HT-29), bearing different concentrations of class II antigen (HLA-DR). IEL were isolated by a mechanical method from six patients with ulcerative colitis (UC) and from 14 control patients. IEL were cocultured with HT-29 which had been induced to express class II molecules by gamma-interferon (IFN-gamma) in a dose dependent manner. The phenotype and the subsequent expression of activation markers by the IEL were determined to two colour flow cytometry. The IEL population had a CD4/CD8 ratio similar to that seen in tissue sections. In the mixed cell culture, the degree of IEL activation showed a positive correlation with the degree of HLA-DR expression by the HT29 cells and the IEL secreted a IFN-gamma like factor that in turn stimulated the HT-29. Thus, depending on their expression of HLA molecules, colonic epithelial cells are able to activate CD3+CD8+IEL.

The objectives of this study were to activate human colonic intraepithelial lymphocytes at the transcriptional level with HLA-DR+ human colonic epithelial cell line (HT29) in synergy with CD3 monoclonal antibody and to investigate the molecular mechanism for the therapeutic effects of 5-aminosalicylic acid. Lymphocytes were isolated by a mechanical method from resected colon of 22 cases and then cocultured on 10 ng/ml CD3mAb immobilized plates with HT29 which had been induced to express MHC class II molecules by interferon gamma. Flow cytometry analysis suggested that the lymphocyte population had a CD4/CD8 ratio similar to that observed in intact tissue sections and that there was no HT29 contamination of the lymphocytes isolated again from cocultured cells. The activation of intraepithelial lymphocytes showed the gene transcription of interferon gamma and tumor necrosis factor alpha, as measured by means of the reverse-transcriptase polymerase chain reaction, and this activation was antagonized by 5-aminosalicylic acid. Thus, epithelial cells bearing HLA-DR are capable of enhancing CD3-induced activation of human colonic intraepithelial lymphocytes and subject to inhibition by 5-aminosalicylic acid, the active moiety of salicylates used in inflammatory bowel disease.

Several risk factors for Crohn's disease have been identified in recent genome-wide association studies. To advance gene discovery further, we combined data from three studies on Crohn's disease ( a total of 3,230 cases and 4,829 controls) and carried out replication in 3,664 independent cases with a mixture of population-based and family-based controls. The results strongly confirm 11 previously reported loci and provide genome-wide significant evidence for 21 additional loci, including the regions containing STAT3, JAK2, ICOSLG, CDKAL1 and ITLN1. The expanded molecular understanding of the basis of this disease offers promise for informed therapeutic development.

The relative distribution of lymphocyte subpopulations in the blood and liver of patients with primary sclerosing cholangitis (PSC) and related diseases has been studied using immunoenzyme techniques. The peripheral blood CD4/CD8 T lymphocyte ratio was significantly higher in active ulcerative colitis (UC) and in PSC with inactive UC than in inactive UC alone. In contrast, no relationship with disease activity was seen in Crohn's disease. The portal tract t lymphocyte count per high power field (mean +/- S.D.) was higher in pre-cirrhotic PSC (173 +/- 105) and primary biliary cirrhosis (PBC: 210 +/- 110) than in histologically normal liver (42 +/- 27). However, the overall portal tract CD4/CD8 ratio was similar in PSC (1.49), PBC (1.89) and normal controls (1.63). The results are consistent with immunological involvement in the pathogenesis of PSC, but argue against the hypothesis that changes in the peripheral blood T cell subsets are due to sequestration at the site of tissue inflammation.

BACKGROUND: Current understanding of T cell epitopes in coeliac disease (CD) largely derives from intestinal T cell clones in vitro. T cell clones allow identification of gluten peptides that stimulate T cells but do not quantify their contribution to the overall gluten specific T cell response in individuals with CD when exposed to gluten in vivo. AIMS: To determine the contribution of a putative dominant T cell epitope to the overall gliadin T cell response in HLA-DQ2 CD in vivo. PATIENTS: HLA-DQ2+ individuals with CD and healthy controls. METHODS: Subjects consumed 20 g of gluten daily for three days. Interferon gamma (IFN-gamma) ELISPOT was performed using peripheral blood mononuclear cells (PBMC) to enumerate and characterise peptide and gliadin specific T cells before and after gluten challenge. RESULTS: In 50/59 CD subjects, irrespective of homo- or heterozygosity for HLA-DQ2, IFN-gamma ELISPOT responses for an optimal concentration of A-gliadin 57-73 Q-E65 were between 10 and 1500 per million PBMC, equivalent to a median 51% of the response for a "near optimal" concentration of deamidated gliadin. Whole deamidated gliadin and gliadin epitope specific T cells induced in peripheral blood expressed an intestinal homing integrin (alpha4beta7) and were HLA-DQ2 restricted. Peripheral blood T cells specific for A-gliadin 57-73 Q-E65 are rare in untreated CD but can be predictably induced two weeks after gluten exclusion. CONCLUSION: In vivo gluten challenge is a simple safe method that allows relevant T cells to be analysed and quantified in peripheral blood by ELISPOT, and should permit comprehensive high throughput mapping of gluten T cell epitopes in large numbers of individuals with CD.

A method is described for the isolation of parietal cells from the gastric mucosa of the guinea pig by enzymatic digestion with collagenase. A suspension was obtained that contained 70-80% parietal cells. About 80% of the cells were viable immediately after incubation, but viability dropped sharply after one hour. Parietal cells were identified by their morphology on light and electron microscopy, by their uptake of neutral red, by immunofluorescent staining and by carbonic anhydrase activity. Antibodies to four distinct parietal-cell antigens were obtained from rabbits immunized with the isolated parietal cells or fractions thereof. These antibodies were directed against the microsomal fraction of the parietal-cell cytoplasm, the plasma and nuclear membranes, the soluble proteins, and Castle's intrinsic factor. The antibody against the microsomal fraction, though reacting in the same way as the antibody to parietal cell canaliculi found in the serum of patients with pernicious anaemia, showed greater species specificity.

To investigate the nature of the morphological changes that occur in ileal pouches, 26 biopsy specimens from patients with functioning ileo-anal pouches (eight with pouchitis) were studied. Normal ileum (n = 10) was used as a control. Mucosal morphometry (using linear measurements), crypt cell proliferation (CCP) (using the monoclonal antibody Ki67), and epithelial HLA-DR expression (monoclonal antibody CR3/43) were assessed. CCP (expressed as the percentage of Ki67 positive nuclei for each crypt) was significantly higher in pouches with pouchitis, compared with those without, and in pouches without pouchitis compared with normal ileum. CCP values in some pouches without pouchitis approached values found in those with pouchitis. CCP was related inversely to villous height and an index of villous atrophy (VH/TMT), and directly to crypt depth. In the presence of pouchitis there was intense epithelial HLA-DR expression that extended into the crypts. In some pouches with high CCP values, but without clinically important inflammation, surface epithelial HLA-DR expression was weak and patchy. It is concluded that villous atrophy and crypt hyperplasia in ileal pouches are associated with high CCP values. These may be increased even in the absence of active inflammation, and this increase may occur as a response to the new luminal environment.

PURPOSE: Cyclosporin is used in severe ulcerative colitis that is refractory to intravenous steroids. Cyclosporin is a potent immunosuppressant and can cause side effects such as opportunistic infections. This study aimed to investigate the incidence of perioperative complications in patients treated with intravenous cyclosporin and steroids compared with patients treated with intravenous steroids alone. METHODS: We retrospectively reviewed the case notes of 44 patients with severe ulcerative colitis who underwent total abdominal colectomy and ileostomy. Twenty-five patients were treated with intravenous steroids and 19 patients were treated with intravenous cyclosporin and steroids. Details were recorded with respect to age, length of illness, extent of disease, Truelove and Witt's criteria, hemoglobin and albumin at surgery, surgical procedure, and perioperative morbidity. RESULTS: Twenty-four percent of patients treated with intravenous steroids alone and 15.8 percent of patients treated with intravenous cyclosporin and steroids had major surgical complications. Sixteen percent of patients treated with intravenous steroids alone and 5.2 percent of patients treated with intravenous cyclosporin and steroids had minor surgical complications. Eight percent of patients treated with intravenous steroids alone and 10.5 percent of patients treated with intravenous cyclosporin and steroids had major medical complications. There was no mortality in either group. CONCLUSIONS: There is no increased incidence of perioperative complications associated with the use of intravenous cyclosporin in addition to steroids in acute severe ulcerative colitis provided cyclosporin treatment is for a defined period and surgery is not delayed.

This study aimed to investigate the degree of colonic metaplasia in ileo - anal pouches. Biopsy specimens from 25 patients with functioning pouches, eight of whom had pouchitis, were studied using routine histology, mucosal morphometry, mucin histochemistry, and immunoperoxidase staining with monoclonal antibodies directed towards a 40kD colonic protein and a small bowel specific disaccharidase-sucrase isomaltase. Thirteen patients (including all eight with pouchitis) had subtotal or total villous atrophy and crypt hyperplasia. In this group, nine had colorectal type sulphomucin and the 40kD colonic protein was detected in two. These changes were not observed in patients with less severe villous abnormalities. Sucrase-isomaltase activity was, however, present in all 25 pouch specimens. We conclude that although some ileal pouches acquire certain colonic characteristics, complete colonic metaplasia does not occur.

Thirty-nine patients with ulcerative colitis and 35 with Crohn's disease have been investigated for evidence of reaginic hypersensitivity and compared with control subjects. There was no difference in the frequency of a personal or family history of atopy or in serum IgE levels. Similarly, no overall difference was noted in prick test responses to 21 allergens. However, further analysis of prick test responses showed that patients with inflammatory bowel disease responded more frequently to food allergens. This was highly significant when compared with healthy controls (p less than 0.001). The relevance of this finding to the aetiology of inflammatory bowel disease is discussed.

BACKGROUND: Despite strong evidence implicating immune dysfunction and genetic predisposition in the pathogenesis of the chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis, the importance of the genes of the major histocompatibility complex remains uncertain. We have investigated the contribution of HLA DRB1 and DQB genes by the strategies of non-parametric linkage analysis (affected sibling pair method) as well as association study. The relation between genotype and phenotype was examined in detail. METHODS: For linkage analysis 74 families in whom two or more siblings had inflammatory bowel disease were studied. A total of 83 affected sibling pairs were involved: in 42 pairs both siblings had Crohn's disease; in 29 both had ulcerative colitis; in 12 one sibling had Crohn's disease, the other ulcerative colitis. For the association study there were 175 patients with ulcerative colitis, 173 with Crohn's disease, and 472 controls. Details of sex, age of onset, disease extent, and family history were analysed. 24 patients with ulcerative colitis and 92 with Crohn's disease required surgery for refractory disease. HLA DRB1 and DQB1 gene-typing was performed by polymerase chain reaction with sequence-specific primers. FINDINGS: In ulcerative colitis, the sharing of alleles among affected sibling pairs provided evidence for linkage with DRB1 locus (p = 0.017, chi2 = 5.32). Of 29 affected sibling pairs studied, only one pair shared no DRB1 DQB haplotypes. 15 shared two DRB DQB haplotypes. In contrast, no linkage was noted for Crohn's disease (42 sibling pairs; p = 0.30, chi2 = 0.16) or for inflammatory bowel disease overall (83 sibling pairs, p = 0.16, chi2 = 2.28). In the association study the rare DRB1*103 (8.3% vs 3.2% in controls) and DRB1*12 (8.6% vs 2.1% in controls) alleles were associated with ulcerative colitis (p = 0.0074, chi2 = 7.22, odds ratio OR = 2.9 [95% CI 1.3-6.4] and p = 0.0056, chi2 = 12.63, OR = 4.33 [1.8-11.0] respectively). No association with alleles representing DR2 (p = 0.55, chi2 = 0.34) was noted. No overall association was seen in Crohn's disease. In ulcerative colitis, the frequency of DRB1*0301 DQB*0201 (DR3 DQ2) was reduced in females (9.8% vs 26.3% in controls, p = 0.037, chi2 = 8.39 OR = 0.34 [0.15-0.71]), particularly in those with distal disease (2.3%, p = 0.001 vs controls, chi2 = 11.35, OR = 0.07 [0.00-0.39]). In both males and females, the DR3 DQ2 haplotype was predictive of extensive ulcerative colitis (32.9% vs 10.7% in distal disease, p < 0.01, chi2 = 10.94, OR 4.09 [1.70-10.6]) but not of need for surgery (p = 0.93, chi2 = 0.01). INTERPRETATION: These data provide strong evidence for genetic heterogeneity in inflammatory bowel disease. Genes of the major histocompatibility complex are implicated as important inherited determinants of susceptibility to ulcerative colitis and may also influence the pattern of disease. In Crohn's disease, important susceptibility genes are likely to exist outside the HLA region.

Summary MHC class I chain gene A (MICA) is a non-classical Class I gene which is expressed on the surface of epithelia without β2-microglobulin. The gene is found in the major histocompatibility complex (MHC) in tight linkage disequilibrium with human leucocyte antigen-B (HLA-B). Its precise function is unknown, but it interacts with γδ T cells of the intestinal immune system. This region of the MHC has been implicated in inflammatory bowel disease (IBD) pathogenesis by recent association mapping studies and this study was performed to examine the prevalence of MICA gene polymorphisms in IBD, in particular in type 2 peripheral arthropathy (PeA), which also has a strong HLA–B association. An assessment of the prevalence of MICA polymorphisms in IBD was made. Blood from 50 ulcerative colitis (UC) and 50 Crohn's disease controls was taken and MICA status determined using allele-specific PCR for 16 known alleles of MICA. A further 91 UC patients were recruited to confirm the results of this stage, and then the polymorphisms were studied in 52 type 1 and 45 type 2 PeA patients. The MICA status of these groups was compared with 118 blood and organ donor controls with appropriate correction for multiple comparisons. UC overall was associated with possession of MICA*007 in 32% compared to 11% of controls (Pc = 0·017). This association was confirmed in a second cohort of 91 patients (23% versus 11%, P= 0·02). These were independent of HLA class I status. Type 2 IBD PeA was associated with MICA*008 in 98% compared to 73% of controls (P = 0·0001). MICA*007 is associated with susceptibility to UC in our population and MICA*008 with type 2 IBD PeA. Further work is now required to assess the distribution and expression of MICA throughout the gut in health and disease.

Crohn's disease is a heterogeneous entity. Previous attempts of classification have been based primarily on anatomic location and behavior of disease. However, no uniform definition of patient subgroups has yet achieved broad acceptance. The aim of this international Working Party was to develop a simple classification of Crohn's disease based on objective variables. Eight outcome-related variables relevant to Crohn's disease were identified and stepwise evaluated in 413 consecutive cases, a database survey, and by clinical considerations. Allocation of variables was conducted with well-defined Crohn's disease populations from Europe and North America. Cross-table analyses were performed by chi-square testing. Three variables were finally elected: Age at Diagnosis [below 40 years (A1), equal to or above 40 years (A2)], Location [terminal ileum (L1), colon (L2), ileocolon (L3), upper gastrointestinal (L4)], and Behavior [nonstricturing nonpenetrating (B1), stricturing (B2), penetrating (B3)]. The allocation of patients to these 24 subgroups proved feasible and resulted in specific disease clusters. Cross-table analyses revealed associations between Age at Diagnosis and Location, and between Behavior and Location (all p < 0.001). The Vienna classification of Crohn's disease provides distinct definitions to categorize Crohn's patients into 24 subgroups. Operational guidelines should be used for the characterization of patients in clinical trials as well as for correlation of particular phenotypes with putative biologic markers or environmental factors.

HLA class II beta-chain polymorphism was investigated in the haplotype HLA-DR3 to determine if patients with HLA-DR3-associated diseases express normal or variant class II polymorphisms. Analysis was carried out by two-dimensional gel electrophoresis of immunoprecipitated HLA class II molecules, DNA hybridization with DR beta and DQ beta gene probes on Taq 1, Bam H1, or Rsa 1 digests, and mixed lymphocyte culture. Two subtypes of HLA-DR3 were identified in normal homozygous DR3 individuals on the basis of polymorphism in one of two DR beta chains detected, corresponding to differences in DR beta restriction fragment patterns. These polymorphisms exhibited significant linkage disequilibrium with the A1,B8,DR3 and B18,DR3 haplotypes, respectively. In proliferative experiments, cells with the B18,DR3-associated polymorphism strongly stimulated cells from donors with the B8,DR3-related polymorphism, suggesting that a T-cell epitope recognized by B8,DR3 cells lies on the B18,DR3-associated DR beta chain. In seven HLA-DR3 homozygous patients with celiac disease and three HLA-DR3-homozygous patients with idiopathic membranous nephropathy, only the normal patterns of HLA class II molecules were displayed, the B8,DR3 type occurring in all patients and the B18,DR3 type in one patient. These data suggest that celiac disease and idiopathic membranous nephropathy are not related to disease-specific HLA-DR beta or -DQ beta gene variants within the DR3 population that are revealed by these methods.

Using an autologous system, suppressor cell function to a range of mycobacterial antigens and Kunin antigen of peripheral blood mononuclear cells and lamina propria lymphocytes has been investigated in normal subjects and patients with inflammatory bowel disease. In the peripheral blood there was reduced antigen induced suppression in patients with Crohn's disease in remission to Mycobacterium paratuberculosis, purified protein derivative (PPD), M fortuitum, and Kunin antigens (p less than 0.05). In patients with ulcerative colitis in remission there was reduced antigen induced suppression in the peripheral blood to Kunin antigen (p less than 0.001), M avium (p less than 0.01), M nonchromogenecin, and M fortuitum (p less than 0.05). The phenomenon of antigen induced suppression was largely CD8 dependent, as depleting CD8+ cells reduced the effect and the concentration of soluble CD8 in the culture supernatant was directly related to the suppressor index (r = 0.25, p less than 0.05). These results are likely to be a true reflection of the cell mediated response to antigen as patients with a positive Mantoux skin test have a significantly higher suppressor index to PPD than Mantoux negative subjects (p less than 0.05). These findings may have significance in the aetiopathogenesis of inflammatory bowel disease. However, a similar effect could not be shown in the lamina propria lymphocytes of patients having colectomy for active disease.

Linkage and association studies implicate the human leucocyte antigen (HLA) region in genetic susceptibility to ulcerative colitis (UC). However, associations with specific variants have been inconsistent, even within defined ethnic groups. A genetic basis for the disease heterogeneity of UC may account for these discrepant findings from studies in unselected populations. Here, we examine the contribution of the HLA region to the clinical phenotype of UC. We studied 321 accurately phenotyped patients recruited from a single UK centre, with a median follow-up time of 15 years. Individuals were genotyped for 340 polymorphisms constructed into 25 gene-specific allelic haplotypes between HLA-A and Tapasin. Data were analysed with respect to age of onset, disease extent and severity. Strongest association with overall susceptibility was identified with HLA-DRB1 alleles replicating previous studies (DRB1*0103, DRB1*1502 and DRB1*0401). We report a novel association with homozygosity of a tumour necrosis factor (TNF) promoter haplotype (TNF-1031T, -863C, -857C, -380G, -308G and -238G) and distal disease extent that does not extend with time (distal vs total 40.9 vs 25.7%; RR = 2.0; 95% CI 1.23-3.24). We confirm the association of DRB1*0103 with total disease and/or disease requiring colectomy and further demonstrate that DRB1*0103 is associated with shorter time to surgery. Genes in the HLA play a role in modifying disease phenotype. Further studies are required to dissect how these genes functionally interact with each other and with environmental factors to determine clinical patterns of disease