This study describes and validates a new method for metagenomic biomarker discovery by way of class comparison, tests of biological consistency and effect size estimation. This addresses the challenge of finding organisms, genes, or pathways that consistently explain the differences between two or more microbial communities, which is a central problem to the study of metagenomics. We extensively validate our method on several microbiomes and a convenient online interface for the method is provided at http://huttenhower.sph.harvard.edu/lefse/.

Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology. A susceptibility locus for Crohn's disease has been mapped to chromosome 16. Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes. These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region. NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.

Crohn's disease and ulcerative colitis, the two common forms of inflammatory bowel disease (IBD), affect over 2.5 million people of European ancestry, with rising prevalence in other populations. Genome-wide association studies and subsequent meta-analyses of these two diseases as separate phenotypes have implicated previously unsuspected mechanisms, such as autophagy, in their pathogenesis and showed that some IBD loci are shared with other inflammatory diseases. Here we expand on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn's disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Most loci contribute to both phenotypes, and both directional (consistently favouring one allele over the course of human history) and balancing (favouring the retention of both alleles within populations) selection effects are evident. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. Gene co-expression network analysis emphasizes this relationship, with pathways shared between host responses to mycobacteria and those predisposing to IBD.

OBJECTIVE

We conducted a systematic review to determine changes in the worldwide incidence and prevalence of ulcerative colitis (UC) and Crohn's disease (CD) in different regions and with time.

METHODS

We performed a systematic literature search of MEDLINE (1950-2010; 8103 citations) and EMBASE (1980-2010; 4975 citations) to identify studies that were population based, included data that could be used to calculate incidence and prevalence, and reported separate data on UC and/or CD in full manuscripts (n = 260). We evaluated data from 167 studies from Europe (1930-2008), 52 studies from Asia and the Middle East (1950-2008), and 27 studies from North America (1920-2004). Maps were used to present worldwide differences in the incidence and prevalence of inflammatory bowel diseases (IBDs); time trends were determined using joinpoint regression.

RESULTS

The highest annual incidence of UC was 24.3 per 100,000 person-years in Europe, 6.3 per 100,000 person-years in Asia and the Middle East, and 19.2 per 100,000 person-years in North America. The highest annual incidence of CD was 12.7 per 100,000 person-years in Europe, 5.0 person-years in Asia and the Middle East, and 20.2 per 100,000 person-years in North America. The highest reported prevalence values for IBD were in Europe (UC, 505 per 100,000 persons; CD, 322 per 100,000 persons) and North America (UC, 249 per 100,000 persons; CD, 319 per 100,000 persons). In time-trend analyses, 75% of CD studies and 60% of UC studies had an increasing incidence of statistical significance (P < .05).

CONCLUSIONS

Although there are few epidemiologic data from developing countries, the incidence and prevalence of IBD are increasing with time and in different regions around the world, indicating its emergence as a global disease.

The inflammatory bowel diseases Crohn's disease and ulcerative colitis are common, chronic disorders that cause abdominal pain, diarrhea, and gastrointestinal bleeding. To identify genetic factors that might contribute to these disorders, we performed a genome-wide association study. We found a highly significant association between Crohn's disease and the IL23R gene on chromosome 1p31, which encodes a subunit of the receptor for the proinflammatory cytokine interleukin-23. An uncommon coding variant (rs11209026, c.1142G>A, p.Arg381Gln) confers strong protection against Crohn's disease, and additional noncoding IL23R variants are independently associated. Replication studies confirmed IL23R associations in independent cohorts of patients with Crohn's disease or ulcerative colitis. These results and previous studies on the proinflammatory role of IL-23 prioritize this signaling pathway as a therapeutic target in inflammatory bowel disease.

Colitis-associated cancer (CAC) is the most serious complication of inflammatory bowel disease. Proinflammatory cytokines have been suggested to regulate preneoplastic growth during CAC tumorigenesis. Interleukin 6 (IL-6) is a multifunctional NF-kappaB-regulated cytokine that acts on epithelial and immune cells. Using genetic tools, we now demonstrate that IL-6 is a critical tumor promoter during early CAC tumorigenesis. In addition to enhancing proliferation of tumor-initiating cells, IL-6 produced by lamina propria myeloid cells protects normal and premalignant intestinal epithelial cells (IECs) from apoptosis. The proliferative and survival effects of IL-6 are largely mediated by the transcription factor Stat3, whose IEC-specific ablation has profound impact on CAC tumorigenesis. Thus, the NF-kappaB-IL-6-Stat3 cascade is an important regulator of the proliferation and survival of tumor-initiating IECs.

BACKGROUND

Infliximab, a chimeric monoclonal antibody directed against tumor necrosis factor alpha, is an established treatment for Crohn's disease but not ulcerative colitis.

METHODS

Two randomized, double-blind, placebo-controlled studies--the Active Ulcerative Colitis Trials 1 and 2 (ACT 1 and ACT 2, respectively)--evaluated the efficacy of infliximab for induction and maintenance therapy in adults with ulcerative colitis. In each study, 364 patients with moderate-to-severe active ulcerative colitis despite treatment with concurrent medications received placebo or infliximab (5 mg or 10 mg per kilogram of body weight) intravenously at weeks 0, 2, and 6 and then every eight weeks through week 46 (in ACT 1) or week 22 (in ACT 2). Patients were followed for 54 weeks in ACT 1 and 30 weeks in ACT 2.

RESULTS

In ACT 1, 69 percent of patients who received 5 mg of infliximab and 61 percent of those who received 10 mg had a clinical response at week 8, as compared with 37 percent of those who received placebo (P<0.001 for both comparisons with placebo). A response was defined as a decrease in the Mayo score of at least 3 points and at least 30 percent, with an accompanying decrease in the subscore for rectal bleeding of at least 1 point or an absolute rectal-bleeding subscore of 0 or 1. In ACT 2, 64 percent of patients who received 5 mg of infliximab and 69 percent of those who received 10 mg had a clinical response at week 8, as compared with 29 percent of those who received placebo (P<0.001 for both comparisons with placebo). In both studies, patients who received infliximab were more likely to have a clinical response at week 30 (P< or =0.002 for all comparisons). In ACT 1, more patients who received 5 mg or 10 mg of infliximab had a clinical response at week 54 (45 percent and 44 percent, respectively) than did those who received placebo (20 percent, P<0.001 for both comparisons).

CONCLUSIONS

Patients with moderate-to-severe active ulcerative colitis treated with infliximab at weeks 0, 2, and 6 and every eight weeks thereafter were more likely to have a clinical response at weeks 8, 30, and 54 than were those receiving placebo. (ClinicalTrials.gov numbers, NCT00036439 and NCT00096655.)

Acute and chronic experimental ulcerative colitis models were produced in mice by providing them with drinking water containing synthetic dextran sulfate sodium. Mice that developed acute colitis showed signs of diarrhea, gross rectal bleeding, and weight loss within 6-10 days after ingesting 3%-10% dextran sulfate sodium. On postmortem examination, multiple erosions and inflammatory changes including crypt abscesses were found on the left side of the large intestine. Mice that developed chronic colitis showed signs of erosions, prominent regenerations of the colonic mucosa including dysplasia, shortening of the large intestine, and frequent formation of lymphoid follicles after 5 administration cycles, where each cycle was composed of 7 days' consumption of drinking water containing 5% dextran sulfate sodium followed by 10 days' consumption of distilled water. The population of intestinal microflora, Bacteroides distasonis and Clostridium spp., increased significantly in mice with acute and chronic ulcerative colitis. Further, morphological studies suggest that the administered dextran sulfate sodium was partially phagocytized by macrophages in the colonic mucosa.

OBJECTIVE

Controversy surrounds the risk of colorectal cancer (CRC) in ulcerative colitis (UC). Many studies have investigated this risk and reported widely varying rates.

METHODS

A literature search using Medline with the explosion of references identified 194 studies. Of these, 116 met our inclusion criteria from which the number of patients and cancers detected could be extracted. Overall pooled estimates, with 95% confidence intervals (CI), of cancer prevalence and incidence were obtained using a random effects model on either the log odds or log incidence scale, as appropriate.

RESULTS

The overall prevalence of CRC in any UC patient, based on 116 studies, was estimated to be 3.7% (95% CI 3.2-4.2%). Of the 116 studies, 41 reported colitis duration. From these the overall incidence rate was 3/1000 person years duration (pyd), (95% CI 2/1000 to 4/1000). The overall incidence rate for any child was 6/1000 pyd (95% CI 3/1000 to 13/1000). Of the 41 studies, 19 reported results stratified into 10 year intervals of disease duration. For the first 10 years the incidence rate was 2/1000 pyd (95% CI 1/1000 to 2/1000), for the second decade the incidence rate was estimated to be 7/1000 pyd (95% CI 4/1000 to 12/1000), and in the third decade the incidence rate was 12/1000 pyd (95% CI 7/1000 to 19/1000). These incidence rates corresponded to cumulative probabilities of 2% by 10 years, 8% by 20 years, and 18% by 30 years. The worldwide cancer incidence rates varied geographically, being 5/1000 pyd in the USA, 4/1000 pyd in the UK, and 2/1000 pyd in Scandinavia and other countries. Over time the cancer risk has increased since 1955 but this finding was not significant (p=0.8).

CONCLUSIONS

Using new meta-analysis techniques we determined the risk of CRC in UC by decade of disease and defined the risk in pancolitics and children. We found a non-significant increase in risk over time and estimated how risk varies with geography.

The discovery of a series of genetic and serological markers associated with disease susceptibility and phenotype in inflammatory bowel disease has led to the prospect of an integrated classification system involving clinical, serological and genetic parameters. The Working Party has reviewed current clinical classification systems in Crohn's disease, ulcerative colitis and indeterminate colitis, and provided recommendations for clinical classification in practice. Progress with respect to integrating serological and genetic markers has been examined in detail, and the implications are discussed. While an integrated system is not proposed for clinical use at present, the introduction of a widely acceptable clinical subclassification is strongly advocated, which would allow detailed correlations among serotype, genotype and clinical phenotype to be examined and confirmed in independent cohorts of patients and, thereby, provide a vital foundation for future work.

In recent years, investigators have readdressed the complex issues involved in the classification of inflammatory bowel diseases. In 2003, a Working Party of investigators with an interest in the issues involved in disease subclassification was formed with the aim of summarising recent developments in disease classification and establishing an integrated clinical, molecular, and serological classification of inflammatory bowel disease. The results of the Working Party were reported at the 2005 Montreal World Congress of Gastroenterology. Here we highlight the key issues that have emerged from discussions of the Montreal Working Party and the relevance to clinical practice and research activities.

We assessed oral 5-aminosalicylic acid (5-ASA) prepared with a pH-sensitive polymer coating in 87 patients with mildly to moderately active ulcerative colitis in a double-blind, placebo-controlled trial. Patients were randomly assigned to receive 5-ASA at a dosage of either 4.8 or 1.6 g per day or placebo for six weeks. The outcome was monitored by flexible proctosigmoidoscopic examinations and physicians' assessments at three-week intervals and by patients' recordings of daily symptoms. Results showed 24 percent complete and 50 percent partial responses in those receiving 4.8 g of 5-ASA per day as compared with 5 percent complete and 13 percent partial responses in those receiving placebo (P less than 0.0001, rank-sum test). At a dosage of 1.6 g per day, the response was twice as good as with placebo, but the difference did not reach statistical significance (P = 0.51). Age, sex, duration of disease, duration of active symptoms, or extent of disease did not affect the clinical outcome. We conclude that oral 5-ASA administered in a dosage of 4.8 g per day is effective therapy, at least in the short term, for mildly to moderately active ulcerative colitis.

OBJECTIVE

Interleukin (IL) 17 is a cytokine which exerts strong proinflammatory activities. In this study we evaluated changes in IL-17 expression in the inflamed mucosa and in the serum of patients with inflammatory bowel disease (IBD).

METHODS

Tissue samples were obtained endoscopically or surgically from patients with ulcerative colitis (UC) (n=20), Crohn's disease (CD) (n=20), infectious colitis (n=5), ischaemic colitis (n=8), and normal colorectal tissues (n=15). IL-17 expression was evaluated by a standard immunohistochemical procedure. Serum IL-17 levels were determined by ELISA. IL-17 mRNA expression was analysed by reverse transcriptase-polymerase chain reaction.

RESULTS

IL-17 expression was not detected in samples from normal colonic mucosa, infectious colitis, or ischaemic colitis. In the inflamed mucosa of active UC and CD patients, IL-17 expression was clearly detectable in CD3(+) T cells or CD68(+) monocytes/macrophages. The average number of IL-17(+) cells was significantly increased in active UC and CD patients compared with inactive patients. IL-17 mRNA expression was not detected in normal mucosa but was detectable in the mucosa from active UC and CD patients. IL-17 was not detected in the sera from normal individuals, infectious colitis, or ischaemic colitis patients but IL-17 levels were significantly elevated in IBD patients.

CONCLUSIONS

IL-17 expression in the mucosa and serum was increased in IBD patients. It is likely that IL-17 expression in IBD may be associated with altered immune and inflammatory responses in the intestinal mucosa.

Genome-wide association studies and candidate gene studies in ulcerative colitis have identified 18 susceptibility loci. We conducted a meta-analysis of six ulcerative colitis genome-wide association study datasets, comprising 6,687 cases and 19,718 controls, and followed up the top association signals in 9,628 cases and 12,917 controls. We identified 29 additional risk loci (P < 5 × 10(-8)), increasing the number of ulcerative colitis-associated loci to 47. After annotating associated regions using GRAIL, expression quantitative trait loci data and correlations with non-synonymous SNPs, we identified many candidate genes that provide potentially important insights into disease pathogenesis, including IL1R2, IL8RA-IL8RB, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1. The total number of confirmed inflammatory bowel disease risk loci is now 99, including a minimum of 28 shared association signals between Crohn's disease and ulcerative colitis.

Inflammatory bowel disease (IBD) has been attributed to aberrant mucosal immunity to the intestinal microbiota. The transcription factor XBP1, a key component of the endoplasmic reticulum (ER) stress response, is required for development and maintenance of secretory cells and linked to JNK activation. We hypothesized that a stressful environmental milieu in a rapidly proliferating tissue might instigate a proinflammatory response. We report that Xbp1 deletion in intestinal epithelial cells (IECs) results in spontaneous enteritis and increased susceptibility to induced colitis secondary to both Paneth cell dysfunction and an epithelium that is overly reactive to inducers of IBD such as bacterial products (flagellin) and TNFalpha. An association of XBP1 variants with both forms of human IBD (Crohn's disease and ulcerative colitis) was identified and replicated (rs35873774; p value 1.6 x 10(-5)) with novel, private hypomorphic variants identified as susceptibility factors. Hence, intestinal inflammation can originate solely from XBP1 abnormalities in IECs, thus linking cell-specific ER stress to the induction of organ-specific inflammation.

We have developed a simple and reproducible rat model of chronic colonic inflammation by the intraluminal instillation of a solution containing a "barrier breaker" and a hapten. Administration of the hapten 2,4,6-trinitrobenzenesulfonic acid (5-30 mg) in 0.25 ml of 50% ethanol as the "barrier breaker" produced dose-dependent colonic ulceration and inflammation. At a dose of 30 mg, trinitrobenzenesulfonic acid/ethanol-induced ulceration and marked thickening of the bowel wall persisted for at least 8 wk. Histologically, the inflammatory response included mucosal and submucosal infiltration by polymorphonuclear leukocytes, macrophages, lymphocytes, connective tissue mast cells, and fibroblasts. Granulomas were observed in 57% of the rats killed 3 wk after induction of inflammation. Langhan's-type giant cells were also observed. Segmental ulceration and inflammation were common. The characteristics and relatively long duration of inflammation and ulceration induced in this model afford an opportunity to study the pathophysiology of colonic inflammatory disease in a specifically controlled fashion, and to evaluate new treatments potentially applicable to inflammatory bowel disease in humans.

Crohn's disease and ulcerative colitis are idiopathic, chronic, relapsing, inflammatory conditions that are immunologically mediated. Although their exact etiologies remain uncertain, results from research in animal models, human genetics, basic science and clinical trials have provided important new insights into the pathogenesis of chronic, immune-mediated, intestinal inflammation. These studies indicate that Crohn's disease and ulcerative colitis are heterogeneous diseases characterized by various genetic abnormalities that lead to overly aggressive T-cell responses to a subset of commensal enteric bacteria. The onset and reactivation of disease are triggered by environmental factors that transiently break the mucosal barrier, stimulate immune responses or alter the balance between beneficial and pathogenic enteric bacteria. Different genetic abnormalities can lead to similar disease phenotypes; these genetic changes can be broadly characterized as causing defects in mucosal barrier function, immunoregulation or bacterial clearance. These new insights will help develop better diagnostic approaches that identify clinically important subsets of patients for whom the natural history of disease and response to treatment are predictable.

Exposure to microbes during early childhood is associated with protection from immune-mediated diseases such as inflammatory bowel disease (IBD) and asthma. Here, we show that in germ-free (GF) mice, invariant natural killer T (iNKT) cells accumulate in the colonic lamina propria and lung, resulting in increased morbidity in models of IBD and allergic asthma as compared with that of specific pathogen-free mice. This was associated with increased intestinal and pulmonary expression of the chemokine ligand CXCL16, which was associated with increased mucosal iNKT cells. Colonization of neonatal-but not adult-GF mice with a conventional microbiota protected the animals from mucosal iNKT accumulation and related pathology. These results indicate that age-sensitive contact with commensal microbes is critical for establishing mucosal iNKT cell tolerance to later environmental exposures.

Infection, ischaemia, physical damage, or specific immunologic sensitivity should be excluded as far as possible before a diagnosis of non-specific inflammatory bowel disease is made. Non-specific inflammations can be subdivided on the basis of macroscopic and microscopic anatomical criteria. Macroscopic structural abnormalities can be recognized by clinical examination, endoscopy, radiology, and inspection of an operation specimen. These complementary methods of data collection combine with microscopic examinations of tissue to separate disorders that differ in prognosis and possible response to treatment. Anatomic classifications do not necessarily imply differences in aetiology and may change with advances in knowledge.

Mice deficient for interleukin-2 develop normally during the first 3-4 weeks of age. However, later on they become severely compromised, and about 50% of the animals die between 4 and 9 weeks after birth. Of the remaining mice, 100% develop an inflammatory bowel disease with striking clinical and histological similarity to ulcerative colitis in humans. The alterations of the immune system are characterized by a high number of activated T and B cells, elevated immunoglobulin secretion, anti-colon antibodies, and aberrant expression of class II major histocompatibility complex molecules. The data provide evidence for a primary role of the immune system in the etiology of ulcerative colitis and strongly suggest that the disease results from an abnormal immune response to a normal antigenic stimulus.

Crohn's disease and ulcerative colitis are two idiopathic inflammatory bowel disorders. In this paper we discuss the current diagnostic approach, their pathology, natural course, and common complications, the assessment of disease activity, extraintestinal manifestations, and medical and surgical management, and provide diagnostic and therapeutic algorithms. We critically review the evidence for established (5-aminosalicylic acid compounds, corticosteroids, immunomodulators, calcineurin inhibitors) and emerging novel therapies--including biological therapies--directed at cytokines (eg, infliximab, adalimumab, certolizumab pegol) and receptors (eg, visilizumab, abatacept) involved in T-cell activation, selective adhesion molecule blockers (eg, natalizumab, MLN-02, alicaforsen), anti-inflammatory cytokines (eg, interleukin 10), modulation of the intestinal flora (eg, antibiotics, prebiotics, probiotics), leucocyte apheresis and many more monoclonal antibodies, small molecules, recombinant growth factors, and MAP kinase inhibitors targeting various inflammatory cells and pathways. Finally, we summarise the practical aspects of standard therapies including dosing, precautions, and side-effects.