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Multigene diseases Inflammatory bowel diseases (IBDs) — a broad classification that includes Crohn's disease and ulcerative colitis — are multigenic disorders that are also linked to various environmental components. Despite this complexity, considerable progress has been made in unravelling their pathogenesis. Ramnik Xavier and Daniel Podolsky review recent developments in the field. The genetics of IBDs point to roles for epithelial barrier function, innate and adaptive immunity in pathogenesis, while key environmental factors include commensal bacteria which may provoke dysregulated immune responses in the susceptible host. The advances made in understanding IBDs show what is possible in dissecting the pathogenesis of complex disorders: similar unbundling may be possible in heart disease, diabetes and other multigenic disorders. The cover shows an image of an activated human macrophage, one of several cell populations key to the pathogenesis of IBD. Recently, substantial advances in the understanding of the molecular pathogenesis of inflammatory bowel disease (IBD) have been made owing to three related lines of investigation. First, IBD has been found to be the most tractable of complex disorders for discovering susceptibility genes, and these have shown the importance of epithelial barrier function, and innate and adaptive immunity in disease pathogenesis. Second, efforts directed towards the identification of environmental factors implicate commensal bacteria (or their products), rather than conventional pathogens, as drivers of dysregulated immunity and IBD. Third, murine models, which exhibit many of the features of ulcerative colitis and seem to be bacteria-driven, have helped unravel the pathogenesis/mucosal immunopathology of IBD.

Key Points Restitution is the initial repair process of mucosa whereby epithelial cells spread and migrate across the basement membrane to re-establish surface-cell continuity. Trefoil proteins (TFFs) are essential for restitution to occur. The TFFs are 7–12-kDa protease-resistant proteins that are secreted by mucus-secreting cells of the stomach (TFF1 and TFF2) and intestine (TFF3). All three TFFs are encoded together as a contig on human chromosome 21. The trefoil domain is a motif of 38 or 39 amino acids containing six cysteine residues forming three disulphide-linked loops. In vivo , TFFs form two trefoil domains through interchain disulphide bonds (TFF1 and TFF3) or genomic duplication (TFF2). Dimer formation seems to be essential for some TFF functions but not for cell migration. Tff1 -null mice show gastric hyperproliferation and adenoma formation. Tff2 -null mice seem normal, but show increased acid secretion and are susceptible to injury by non-steroidal anti-inflammatory drugs. Tff3 -null mice also seem normal but are fatally susceptible to colonic damage. Ulcer-associated mucosal epithelia show a substantial and coordinated increase in expression of the TFF family with the spread of expression into adjacent, non-metaplastic glands. New enhancer elements within TFF1 and TFF2 promoters have been reported, including elements that confer co-ordinated expression. TFF3 goblet-cell-specific transcription is conferred through a goblet-cell response element (GCRE) and an upstream silencer inhibitor. The GCRE is also found in the promoter of the mucin MUC2 , which is consistent with the co-regulation of mucins and TFFs. TFFs cause mucosal healing through unique mechanisms. Unlike other motogens, TFFs are not mitogenic and no receptor has yet been demonstrated. However, TFF2 and TFF3 activate signal-transduction events, including the induction of epidermal growth factor (EGF) receptor and β-catenin phosphorylation, activation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and prevention of apoptotic signalling. TFF1 mRNA and protein expression is frequently reduced in gastric cancer, often associated with somatic mutations in TFF1 . Present data do not indicate that TFF2 or TFF3 have tumour-suppressor roles. TFFs show many properties that are necessary for therapeutic efficacy. TFFs are compact, stable molecules and exert a protective effect when given orally. Possible therapeutic targets include prevention of the mucositis that follows radiotherapy and chemotherapy, which results in substantial morbidity in cancer patients. Maintaining the integrity of the gastrointestinal tract, despite the continual presence of microbial flora and injurious agents, is essential. Epithelial continuity depends on a family of small, yet abundant, secreted proteins — the trefoil factors (TFFs). TFFs protect mucous epithelia from a range of insults and contribute to mucosal repair, although the signalling events that mediate these responses are only partially understood.

Crohn's disease is a chronic inflammatory bowel disease of unknown cause, which is characterized by segmental transmural inflammation and granulomatous lesions of the intestinal mucosa. The disease is complicated by the development of fistulas in approximately one third of patients. 1 Fistulas may be internal (e.g., bowel to bowel, bowel to bladder, or rectovaginal) or enterocutaneous (extending through the abdominal wall or into the perineum). These fistulas rarely heal spontaneously or as a result of drug treatment and frequently require surgery. According to anecdotal evidence, antibiotics have short-term efficacy in their treatment. Although the use of immunomodulatory agents is associated with . . .

Intestinal trefoil factor (ITF) is an essential regulator of colonic epithelial restitution, the rapid migration of colonocytes over mucosal wounds. High levels of ITF are frequently present in colorectal cancers and derived cell lines. Mucosal restitution requires the detachment of epithelium from substrate, which would be expected to induce apoptosis. However, mice deficient in ITF showed an increase in colonocyte apoptosis unaccompanied by changes in expression of receptor-related (TNFR/Fas) or stress-related (Bcl-family) cell death regulators. An ITF-expressing colonic (HT-ITF1) cell line was resistant to apoptosis induced by serum starvation and ceramide. Exogenous ITF also protected another human colonic carcinoma-derived cell line (HCT116) and a nontransformed rat intestinal epithelial cell line (IEC-6) from apoptosis. This effect was abrogated by wortmannin and tyrphostin A25, indicating the potential involvement of phosphatidylinositol 3-kinase and epidermal growth factor (EGF) receptor activation. Expression of phosphorylated Akt, which lies downstream of phosphatidylinositol 3-kinase activation, was elevated in this HT-29-ITF line. p53-dependent cell death in the AGS human gastric cancer cell line after etoposide was similarly inhibited by transient expression of ITF but not a C-terminal truncation mutant of ITF, and it required functional phosphatidylinositol 3-kinase and EGF receptor. These findings support a central role for ITF in the maintenance of intestinal mucosal continuity, and conversely demonstrate the potential for ITF expression to confer resistance of colorectal tumors to therapy.

The intestinal flora has long been thought to play a role either in initiating or in exacerbating the inflammatory bowel diseases (IBD). Host defenses, such as those mediated by the Toll-like receptors (TLR), are critical to the host/pathogen interaction and have been implicated in IBD pathophysiology. To explore the association of genetic variation in TLR pathways with susceptibility to IBD, we performed a replication study and pooled analyses of the putative IBD risk alleles in NFKB1 and TLR4, and we performed a haplotype-based screen for association to IBD in the TLR genes and a selection of their adaptor and signaling molecules. Our genotyping of 1539 cases of IBD and pooled analysis of 4805 cases of IBD validates the published association of a TLR4 allele with risk of IBD (odds ratio (OR): 1.30, 95% confidence interval (CI): 1.15–1.48; P =0.00017) and Crohn's disease (OR: 1.33, 95% CI: 1.16–1.54; P =0.000035) but not ulcerative colitis. We also describe novel suggestive evidence that TIRAP (OR: 1.16, 95% CI: 1.04–1.30; P =0.007) has a modest effect on risk of IBD. Our analysis, therefore, offers additional evidence that the TLR4 pathway – in this case, TLR4 and its signaling molecule TIRAP – plays a role in susceptibility to IBD.

The mechanisms that maintain the epithelial integrity of the gastrointestinal tract remain largely undefined. The gene encoding intestinal trefoil factor (ITF), a protein secreted throughout the small intestine and colon, was rendered nonfunctional in mice by targeted disruption. Mice lacking ITF had impaired mucosal healing and died from extensive colitis after oral administration of dextran sulfate sodium, an agent that causes mild epithelial injury in wild-type mice. ITF-deficient mice manifested poor epithelial regeneration after injury. These findings reveal a central role for ITF in the maintenance and repair of the intestinal mucosa.

Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1-fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury.

SummaryWe report the experience of 11 patients (of 60 planned patients) enrolled in a double-blind, placebo-controlled clinical trial of infliximab in patients with severe, active steroid-refractory ulcerative colitis. The study was terminated prematurely because of slow enrollment. Patients having active disease for at least 2 weeks and receiving at least 5 days of intravenous corticosteroids were eligible to receive a single intravenous infusion of infliximab at 5, 10, or 20 mg/kg body weight. The primary endpoint used in this study was treatment failure at 2 weeks after infusion. Treatment failure was defined as 1) unachieved clinical response as defined by a modified Truelove and Witts severity score, 2) increase in corticosteroid dosage, 3) addition of immunosuppressants, 4) colectomy, or 5) death. Safety evaluations included physical examination, clinical chemistry and hematology laboratory tests, and occurrence of adverse experiences. Four of 8 patients (50%) who received infliximab were considered treatment successes at 2 weeks, compared with none of 3 patients who received placebo. Improvement in erythrocyte sedimentation rates and serum concentrations of C-reactive protein and interleukin-6 correlated with the clinical response observed in patients receiving infliximab. Infusion with infliximab produced no significant adverse events. Infliximab was well tolerated and may provide clinical benefit for some patients with steroid-refractory ulcerative colitis.

The trefoil peptide family encompasses a group of small proteins that appear to assume a distinctive secondary structure that leads to intrinsic resistance to protease digestion. Induction of these peptides has been associated with response to injury in the gastrointestinal tract and related organs. Using an oligonucleotide derived from N-terminal amino acid sequencing of a transformed growth-inhibiting protein, a cDNA was cloned from rat intestinal villus epithelial cells that encodes a protein 81 amino acids in length with the characteristic trefoil peptide cysteine residue motif. Northern blot analysis demonstrates specific expression of a single transcript of 0.43 kilobase in small and large intestinal epithelium in rat and man. Indirect immunofluorescent staining with antiserum raised using a synthetic peptide based on the predicted C-terminal sequence of this protein, designated intestinal trefoil factor, demonstrated that it is primarily expressed and secreted onto the intestinal surface by goblet cells, suggesting that it may be an important component of intrinsic mechanisms for defending mucosal integrity.

Interleukin (IL) 2 signaling requires the dimerization of the IL-2 receptor beta (IL-2R beta) and common gamma (gamma c) chains. The gamma is also a component of the receptors for IL-4, IL-7, and IL-9. To assess the extent and role of the receptor signal transducing system utilizing the gamma c chain on human intestinal epithelial cells, the expression of gamma c, IL-2R beta, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by reverse transcription-coupled PCR on human intestinal epithelial cell lines and on isolated primary human intestinal epithelial cells. Caco-2, HT-29, and T-84 cells were found to express transcripts for the gamma c and IL-4R chains constitutively. IL-2R beta chain expression was demonstrated in Caco-2 and HT-29 but not in T-84 cells. None of the cell lines expressed mRNA for the IL-2R alpha chain. After stimulation with epidermal growth factor for 24 h Caco-2, HT-29, and T-84 cells expressed transcripts for IL-7R. In addition, Caco-2 and HT-29 cells expressed mRNA for the IL-9R. Receptors for IL-2, IL-4, IL-7, and IL-9 on intestinal epithelial cells lines appeared to be functional; stimulation with these cytokines caused rapid tyrosine phosphorylation of proteins. The relevance of the observations in intestinal epithelial cell lines for intestinal epithelial function in vivo was supported by the demonstration of transcripts for gamma c, IL-2R beta, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells.