Inflammatory bowel disease: cause and immunobiology

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Gastroenterology 1 Inflammatory bowel disease: cause and immunobiology Daniel C Baumgart, Simon R Carding

Crohn’s disease and ulcerative colitis are idiopathic inflammatory bowel disorders. In this paper, we discuss how environmental factors (eg, geography, cigarette smoking, sanitation and hygiene), infectious microbes, ethnic origin, genetic susceptibility, and a dysregulated immune system can result in mucosal inflammation. After describing the symbiotic interaction of the commensal microbiota with the host, oral tolerance, epithelial barrier function, antigen recognition, and immunoregulation by the innate and adaptive immune system, we examine the initiating and perpetuating events of mucosal inflammation. We pay special attention to pattern-recognition receptors, such as toll-like receptors and nucleotide-binding-oligomerisation-domains (NOD), NOD-like receptors and their mutual interaction on epithelial cells and antigen-presenting cells. We also discuss the important role of dendritic cells in directing tolerance and immunity by modulation of subpopulations of effector T cells, regulatory T cells, Th17 cells, natural killer T cells, natural killer cells, and monocyte-macrophages in mucosal inflammation. Implications for novel therapies, which are discussed in detail in the second paper in this Series, are covered briefly.

Introduction Ulcerative colitis and Crohn’s disease represent the two main types of inflammatory bowel disease. In the first paper in this two-article Series, we examine current concepts of the causes and immunobiology of these disorders. The second paper in the Series focuses on clinical aspects, including current and evolving therapies for inflammatory bowel disease.1 Crohn’s disease was first seen by German surgeon Wilhelm Fabry (aka Guilhelmus Fabricius Hildanus) in 1623,2 and was later described by and named after the US physician Burril B Crohn.3 Ulcerative colitis was first described by the British physician Sir Samuel Wilks in 1859.4

Epidemiology The highest incidence rates and prevalence of ulcerative colitis and Crohn’s disease have been reported from northern Europe, the UK, and North America, where the

Search strategy and selection criteria We searched Medline, Web of Science, the Clinical Trials database, and Scopus for articles published between 2000 and 2006. We used the medical subject heading (MeSH) term “inflammatory bowel disease”, “ulcerative colitis”, “Crohn’s disease”, “pouchitis”, “indeterminate colitis”, “epidemiology”, “incidence”, “prevalence” “environmental factors”, “ethnicity”, “genetics”, “CARD15”, “gene mutations”, “polymorphisms”, “susceptibility loci”, “antigen presentation”, “antigen-presenting cells”, “T-cells, “NK-cells”, “NK-T-cells”, “dendritic cells”, B-cells, “regulatory T-cells”, “effector T-cells”, ”microbiota”, “commensals”, “pathogens”, “pattern recognition receptors”, “tissue damage”, “TLR”, “NOD”, “cytokines”, “chemokines”, “chemoattractants”, “adhesion molecules”.

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rates are beginning to stabilise. Rates continue to rise in low-incidence areas such as southern Europe, Asia, and most developing countries5 (table 16–16 ). In North America, prevalence rates of Crohn’s disease for Hispanic (4·1 per 100 000) and Asian people (5·6 per 100 000) is much lower than those for white individuals (43·6 per 100 000) and African-American people 29·8 per 100 000).17 The importance of race and ethnic origin in risk for inflammatory bowel disease are lent further support by a study showing racial differences in disease location and extraintestinal disease complications.18 Reference

Study period

Lancet 2007; 369: 1627–40 See World Report page 1591 See Series page 1641 This is the first in a Series of two papers about inflammatory bowel disease Department of Medicine, Division of Gastroenterology and Hepatology, Charité Medical Centre, Virchow Hospital, Medical School of the Humboldt-University of Berlin, 13344 Berlin, Germany (D C Baumgart MD); and University of Leeds Research Institute of Molecular and Cellular Biology, Leeds LS2 9JT, West Yorkshire, UK (Prof S R Carding PhD) Correspondence to: Dr Daniel C Baumgart [email protected]

Crohn’s disease

Ulcerative colitis

Olmsted County, MN, USA

Sedlack et al6

1935–54

1·9 (n/r)

n/r (n/r)

Olmstead County, MN, USA

Sedlack et al6

1955–64

4·0 (n/r)

n/r (n/r)

Olmstead County, MN, USA

Loftus et al7

1940–49

2·3 (n/r)

3·1 (n/r)

Olmsted County, MN, USA

Loftus et al7

1950–59

2·6 (n/r)

4·2 (n/r)

Olmsted County, MN, USA

Sedlack et al6

1976

n/r (105·7)

n/r (n/r)

Olmsted County, MN, USA

Loftus et al7

1960–69

6·5 (n/r)

9·4 (n/r)

Olmsted County, MN, USA

Loftus et al7

1970–79

7·9 (n/r)

10·1 (n/r)

Olmsted County, MN, USA

Loftus et al7

1980–89

6·8 (n/r)

8·9 (n/r)

Olmsted County, MN, USA

Loftus et al8,9

1991

n/r (133)

n/r (229)

Olmsted County, MN, USA

Loftus et al7

1990–2000

7·9 (n/r)

8·8 (n/r)

Olmsted County, MN, USA

Loftus et al7

2001

n/r (174)

n/r (214)

Copenhagen County, Denmark

Binder et al10

1962–78

2·7 (34)

8·1 (117)

Copenhagen County, Denmark

Langholz et al11

1962–87

n/r (n/r)

8·1 (161)

Copenhagen County, Denmark

Munkholm et al12

1979–87

4·1 (54)

Manitoba, Canada

Bernstein et al13

1984–95

14·6 (198)

14·3 (169·7)

Manitoba, Canada

Blanchard et al14

1987–96

15·6 (n/r)

15·6 (n/r)

Stockholm County, Sweden

Lapidus et al15

1955–89

4·6 (n/r)

n/r (n/r)

Stockholm County, Sweden

Lapidus et al16

1990–2001

8·3 (213)

n/r (n/r)

n/r (n/r)

n/r=not reported.

Table 1: Prevalence (and incidence) of inflammatory bowel disease in well studied high-incidence groups in North America and northern Europe

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Crohn’s disease is more prevalent in Jewish people than in any other ethnic group, with the phenomenon of genetic anticipation—ie, earlier onset in offspring of people with the disease being reported for North American Ashkenazi Jews, although this might not apply to patients with inflammatory bowel disease in general.19,20 Ulcerative colitis is three to five times more prevalent in Jewish people,21 and important epidemiological differences exist between Jewish people living in Israel and those living elsewhere. Prevalence equals over time with migration to other geographic areas.21,22 This change in prevalence after migration has also been shown for other ethnic groups (eg, Chinese people living in Hong Kong compared with mainland China) and, together with the rising incidence and prevalence in developing countries, suggests additional environmental and lifestyle effects on incidence and prevalence.23,24

Genetics Familial aggregation

Figure 1: Human karyotype with inflammatory bowel disease susceptibility loci Confirmed and replicated susceptibility regions represented by red circles, others by light-blue circles.30

Familial aggregation of inflammatory bowel disease was first reported in the 1930s.25 A positive family history is still the largest independent risk factor for the disease. The greatest risk to relatives is of developing the same disease as the affected relative. People with Crohn’s disease have a first-degree relative with Crohn’s disease in 2·2–16·2% of cases and with inflammatory bowel disease in 5·2–22·5% of cases. The risk of Crohn’s disease in a sibling of a Crohn’s disease proband is higher than the average risk for a first-degree relative. People with ulcerative colitis have a first-degree relative with ulcerative colitis in 5·7–15·5% of cases, and with inflammatory bowel disease in 6·6% to 15·8%. The estimated lifetime risk of developing inflammatory bowel disease for a first-degree relative of a Crohn’s disease proband is 4·8–5·2% for non-Jews and 7·8% for Jews. The equivalent figures for first-degree relatives with a ulcerative colitis proband are 1·6% for non-Jews and 5·2% for Jews.25 Concordance for disease type, disease pattern, and presence of extraintestinal disease manifestations are 75–80%, 64%, and 70%, respectively.26

Year published

Affected sibling pairs

Crohn’s disease/ Chromosomal regions of ulcerative colitis/mixed linkage

Hugot et al31

1996

112

112/0/0

16cen

Satsangi et al32

1996

186

81/64/41

12, 7q22, 3p21

Cho et al33

1998

151 (297 97/18/36 (175/26/96) relative pairs)

16cen, 1p, 3q, 4q

Hampe et al34

1999

268

129/90/49

16cen, 10q, 1q, 6p, 12, x, 22, 4q

Ma et al35

1999

65

65/0/0

14q11, 17q21, 5q33

Disease aggregation in twins

Durer et al36

2000

94

94/0/0

14q11

Rioux et al37

2000

183

116/20/47

19p13, 5q31, 3p, 6p

Williams at al38

2002

70 (187 40/13/17 relative pairs) (105/29/53)

16cen, 11p, 6p21

Paavola-Sakki et al39

2003

138

72/19/47

11p12, 2p11, 12p13, 12q23, 19q13

Vermeire et al40

2004

149

129/0/20

14q11, Xq, 1q, 6q, 20p, 4q, 10q

Barmada et al41

2004

260

108/72/80

12, 6p, 6q, 8q, 15q, 22, 2q

Duerr et al42

2006

n/r

547/n/r

1p31

The strongest evidence of genetic factors contributing to susceptibility to inflammatory bowel disease comes from concordance studies in twins.27–29 The first systematic concordance analysis27 has since been lent support by two other studies,28,29 showing a pooled concordance in monozygotic twins of 37·3% for Crohn’s disease and 10% for ulcerative colitis. This compares with a pooled concordance in dizygotic twins of 7% for Crohn’s disease and 3% for ulcerative colitis. The genetic contribution to the development of inflammatory bowel disease seems, therefore, to be more important in Crohn’s disease than in ulcerative colitis, with multiple gene products contributing to risk.30

Adapted from reference 43. n/r=not reported

Table 2: Genome-wide scans undertaken in inflammatory bowel disease, by study

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Susceptibility genes Peptidoglycan TLR6 TLR2

CD14 TLR4

TLR5

MDP TLR3 TLR7 TLR9 MyD88

IRAK

Endosome

TRAF6

Ulcerative colitis and Crohn’s disease are polygenic diseases. 12 genome-wide scans undertaken so far have identified susceptibility regions on 12 chromosomes. Consistent with the genetic heterogeneity of inflammatory bowel disease, no single locus has been reported consistently in all genome scans. According to their initial date of reporting, the regions on chromosomes 16, 12, 6, 14, 5, 19, 1, 16, and 3 have been renamed as IBD1–9, respectively. Lately, more genes have been reported with positional cloning techniques and fine mapping of identified susceptibility regions from total genome scans (figure 1 and table 244–49).30 In a few cases the gene or genes underlying the different chromosome loci that are linked to inflammatory bowel disease have been identified: CARD15 (NOD2) is the underlying

NOD2 +/–

T AK1

Panel 1: Signalling pathways of TLRs and NOD proteins RICK NOD 1

IKKγ IKKα

RICK IKKβ 1κB p50

p65

p50

p65

NFκB-binding motif

TNFα Interleukin β Interleukin 8 Interleukin 12 Interferon γ

Interleukin 4/5 Interleukin 13 Interleukin 1ra Interleukin 10 TGFβ

R702W

G908R 1007fs

CARD

NOD

LRR

Apoptosis NFκB activation

Oligomerisation

Bacterial recognition

Figure 2: Signalling pathways of TLR and NOD proteins MDP=muramyl dipeptide. MyD=myeloid differentiation primary response protein. IRAK=interleukin-1 1-receptor receptor-associated kinase. TRAF=TNFreceptor-associated factor. TAK=transforming growth factor-β-activated kinase. IKK=inhibitor of NFκB (IκB)-kinase. RICK=receptor-interacting serine/threonine kinase. Bottom figure represents schematic of CARD15 gene identifying three functionally distinct domains, CARD, NOD, and leucine-rich receptor, and location of three major mutations in inflammatory bowel disease, which involve aminoacid substitutions and frame-shift mutations. See panel 1 for further explanation of figure.

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Two types of pattern-recognition receptors are involved in the host response to microbes in the intestinal tract: membrane-associated toll-like receptors (TLR) and cytosolic nucleotide-binding-oligomerisation-domains (NOD) proteins. NOD1 and NOD2 are members of the NACHT (domain present in NAIP, CIITA, HET-E and TP1)leucine-rich repeat (LLR) family (NLR family).50,51 This protein family shares a tripartite domain structure consisting of a carboxy terminal LLR repeat domain that is involved in ligand recognition; a central NOD domain, which facilitates self-oligomerisation and has ATPase activity; and an amino terminal domain composed of a caspase-recruitment domain or domains (CARD) that interacts with downstream adapter molecules resulting in activation of the NFκB and/or caspases involved in apoptosis. NOD1 and NOD2 are distinguished by a single or double CARD domain, respectively. The LLRs repeats have been shown to confer recognition by NOD1 and NOD2 of peptides of peptidoglycan, γ-D-glutamylmesodiaminopimelic acid, and muramyl dipeptide, respectively, although no direct evidence exists that this domain binds directly to these peptides.52–54 TLR can exist as monomers, homodimers, or heterodimers with TLR2 combining with TLR1 or TLR6 to recognise its various ligands.55 By contrast with NOD proteins, TLR use a toll interleukin 1 receptor domain for downstream signalling.55 Peptidoglycan potentially activates both TLR2 and NOD2 through the generation of muramyl dipeptide (MDP).56 Stimulation of TLR2 triggers association with an adapter protein, myeloid differentiation primary response protein 88 (MyD88), and the recruitment of interleukin1-receptor-associated kinase (IRAK) proteins. TNF-receptor-associated factor 6 (TRAF6) initially binds to this complex and then dissociates to interact with transforming growth factor-β-activated kinase-1 (TAK1), which in association with other proteins leads to the activation and phosphorylation of components of the IKK (inhibitor of NFκB kinase) complex that gives rise to downstream activation of NFκB (p50 and p65 subunits). Concomitant activation of NOD2 by MDP leads to activation of receptor-interacting serine/threonine kinase (RICK) by NOD2 and the negative or positive regulation of the TLR2 signalling pathway. In addition to the MyD88dependent pathway of TLR signalling, a separate, MyD88-independent TLR-activated signalling pathway also exists, which involves the induction of interferon-regulatory factors, caspase activation, and type I interferon production. Dependent on the TLR involved, the duration and strength of signalling, the outcome of signalling interactions between TLR and NOD, and the cell type in which these events occur, the cytokines and factors produced as a result of activation of pattern-recognition receptors can help initiate or sustain host responses that are anti-inflammatory (eg, interleukin 10 and transforming growth factor β) or proinflammatory (eg, TNFα and interleukin 12).

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Panel 2: CARD15 (NOD2) gene mutations and inflammatory bowel disease Genetic studies of a large cohort of patients with Crohn’s disease identified homozygous mutations in the CARD15 gene, which encodes NOD2, and accounts for 10–15% of patients with this disease.31,59 These mutations include aminoacid changes where Arg702Trp (R702W) and Gly908Arg (G908R) or a frame shift resulting in insertion of a cysteine residue (1007fs). Since these mutations occur mainly in the leucine-rich repeat domain, they interfere with the ability of NOD2 to recognise ligand and a reduced capacity to activate NFκB in response to stimulation with muramyl dipeptide.60,61 How these mutations give rise to susceptibility to Crohn’s disease is not known, although they are thought to result in a disturbance in the normal immunological unresponsiveness of the mucosal immune system to components of the commensal intestinal microbiota. Studies in mice with mutated NOD2 genes have shown that the absence of NOD2 expression can lead to altered TLR signalling and increased production of proinflammatory cytokines by monocytes and macrophages or defective activation of NFκB and production of antimicrobial proteins (α-defensins) by intestinal epithelial cells.62 Overexpression in transgenic mice of NOD genes containing the frameshift mutation associated with Crohn’s disease (1007fs) produced a gain-of-function mutation resulting in increased interleukin 1β production by macrophages after muramyl dipeptide stimulation.63 None of these models is an exact fit with inflammatory bowel disease and they cannot fully account for all of the pathophysiological features of Crohn’s disease. This complexity is probably because of the intricacy of human inflammatory bowel disease and our incomplete understanding of the basis and outcomes of the interactions between the mucosal immune system and intestinal microbiota. Also, the effect of NOD2 gene mutations on the interaction between the commensal microbiota and mucosal immune response is dependent on other host and environmental factors, which might vary qualitatively or quantitatively between individuals, and whether the outcome is tolerance or hyper-responsiveness and the Th1-CD4+ T-cell-mediated inflammation that underlies all forms of Crohn’s disease.

gene on chromosome 16 of which 30 non-conservative polymorphisms have been identified (figure 2 [explained in panel 131]); aminoacid substitutions in the human homolog of the drosophila gene discs large homolog 5, which is a member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which are important in signal transduction and epithelial-cell integrity, underlie the chromosome 10 linkage region; the gene underlying the IBD5 locus has been identified as the novel organic cationic transporter 1 and 2 (OCTN1 and OCTN2) genes that are members of a family or transporter proteins for organic cations that might also transport carnitine, an essential cofactor of the metabolism of lipids.44–49 Not all loci were reproduced in all studies. This might indicate poor statistical power owing to sample size and phenotypic variation. The most convincing evidence for linkage to inflammatory bowel disease across all populations and diseases according to two large meta-analyses is located on chromosome 6 (IBD3), which encodes the major histocompatibility complex.38,42,57 Certain mutations in these loci might be associated with certain disease phenotypes or disease courses. Nucleotide-bindingoligomerisation-domain 2 (NOD2, CARD15) for instance, which is exclusively associated with Crohn’s disease in white populations, has been associated with stricturing small-bowel Crohn’s disease. The HLA 1630

haplotype DRB*0103 has been linked to a particularly aggressive course of ulcerative colitis and the need for surgery and colonic Crohn’s disease. Extraintestinal disease complications, such as arthropathy or uveitis have been linked to HLA-B27 or HLA-B35, and HLAB44 or HLA-DRB*0103, respectively.30,58 Other mutations, such as the recently described mutations in the IL23R gene, might protect against Crohn’s disease (figure 1 and panel 242).

Environmental factors Geographic, temporal, and seasonal variability The highest incidence rates and prevalence for ulcerative colitis and Crohn’s disease are reported from North America and northern Europe (table 1). The lowest incidence rates are reported from South America, southeast Asia, Africa (with the exception of South Africa), and Australia.5 Although these data suggest a gradient exists from north to south, they could also indicate variation in access to, and quality of, health care as well as different extents of industrialisation, sanitation, and hygiene. Many areas with low incidence rates include developing countries. Different incidence rates could also result from different genetic backgrounds of the residents of these parts of the world. More important factors, however, seem to be other environmental ones. This hypothesis is supported by increasing incidence rates among immigrants from low-incidence regions moving to developed countries and a correlation of incidence rates with industrialisation in Hong Kong and mainland China.24 Several large epidemiological studies in North America and Europe have shown an accumulation of cases of inflammatory bowel disease in urban compared with rural communities. As with the north to south gradient, this pattern probably does not implicate geographical as much as other environmental factors, such as industrialisation, sanitation and hygiene, or differences in access to specialised health care. Early epidemiological studies from the USA and Scandinavia suggested that ulcerative colitis is more common during the autumn and winter seasons.64,65 These data are contradicted by more recent studies from the USA and Italy.66,67 Since all studies examined hospital admission rates, their results are difficult to interpret and might indicate patients’ seasonality of access to health care.

Lifestyle Breastfeeding confers immunity while the child’s intestinal immune system is still developing.68 A recent meta-analysis of 17 studies of breastfeeding and the risk of developing inflammatory bowel disease showed heterogeneous results, probably because of poor design and recall bias of mothers. However, a subgroup analysis of high quality studies for Crohn’s disease (n=4) and ulcerative colitis (n=4) showed the pooled odds ratio was 0·45 (95% CI 0·26–0·79) for Crohn’s disease and 0·56 (0·38–0·81) www.thelancet.com Vol 369 May 12, 2007

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for ulcerative colitis, lending support to the notion that breastfeeding provides protection against inflammatory bowel disease in offspring.69 Breastfeeding itself does not contribute to disease exacerbation (whereas drug cessation during breastfeeding does).70 The association between excessive consumption of carbohydrates and development of inflammatory bowel disease, especially Crohn’s disease, probably indicates the differences in sugar consumption in Asia compared with western Europe and North America rather than a true contributing factor.71 Other studies have found an association between an increased intake of polyunsaturated fats or margarine with an increased risk of inflammatory bowel disease.72 Whereas in the past, lipid mediators derived from arachidonic acid were commonly perceived as proinflammatory, new data suggest otherwise.73 Most dietary studies have weak methods and poor patient compliance or recall, which makes interpretation of findings difficult. No final conclusions about the role of nutrition or dietary intervention in inflammatory bowel disease can yet be made. The traditional low incidence of inflammatory bowel disease and other chronic inflammatory disorders in developing countries, which is now on the rise, might be related to socioeconomic changes affecting hygiene.74,75 For example, a lower risk of developing inflammatory bowel disease, particularly Crohn’s disease, has been reported for lower birth rank, absence of tap water, absence of hot water, large or poor families with several children, crowded living conditions, or consumption of contaminated foods.76–78 Excessive sanitation might limit exposure to environmental antigens and impair the functional maturation of the mucosal immune system and induction of immune tolerance, which ultimately result in inappropriate immune responses when re-exposed to these antigens later in life. Psychological stress has anecdotally been reported to increase activity of inflammatory bowel disease. Adverse life events, chronic stress, and depression seem to increase the likelihood of relapse in patients with quiescent disease.79 New experimental evidence suggests involvement of direct interactions of the nervous and immune systems. Smoking cigarettes is associated with less frequent exacerbations of ulcerative colitis. By contrast, in Crohn’s disease, smoking aggravates the course of the disease, promotes formation of fistulas and strictures, increases the rates of exacerbations and the need for corticosteroids, and accelerates the need for surgery after surgically induced remission.80 Smoking cessation seems to be an effective therapeutic intervention in Crohn’s disease, but nicotine patches or enemas failed to show efficacy in the management of ulcerative colitis.81,82 Experimental studies suggest that the beneficial effects of nicotine in ulcerative colitis is due to increased mucus production, decreased production of proinflammatory cytokines and nitric oxide, and improvement of the intestinal barrier function, www.thelancet.com Vol 369 May 12, 2007

whereas nicotine’s detrimental effects in Crohn’s disease seem to be related to an increased influx of neutrophils into the intestinal mucosa.80 The data for passive smoking are contradictory, with some reports showing a reduced risk of ulcerative colitis when exposed to smoke in childhood, and others showing that such exposure results in an increased risk of both ulcerative colitis and Crohn’s disease. The resemblance of granulomatous ileitis in Crohn’s disease with paramyxovirus-mediated vasculitis or Johne’s disease caused by Mycobacterium avium subspecies paratuberculosis in cattle have given rise to speculation that Crohn’s disease might be caused by an infection. A Swedish group83 initially noted an almost 50% higher incidence rate of Crohn’s disease in people born within 3 months of large measles epidemics in Sweden. These data, however, were not lent support by subsequent studies.84 M avium paratuberculosis has been identified in tissues and blood of inflammatory bowel disease, although alternative explanations exist for these findings.85 Inflammatory bowel disease is more common after gastrointestinal infections and people with the disease generally have higher concentrations of mucosal bacteria than do healthy people. Mucosal bacteria concentrations increase progressively with the severity of disease in both the inflamed and non-inflamed colon. Adhesive bacteria might predominate, although no single species is exclusively causative.86–88 Indeed, in our opinion, the evidence does not provide unequivocal support for any microbe being a causative agent in inflammatory bowel disease. However, these data underscore the impaired handling of microbial antigens by the intestinal immune system in inflammatory bowel disease. The hypothesis that the attenuated live measles, mumps, and rubella vaccine might increase the risk of inflammatory bowel disease has now been discredited and has not been reproduced in other case-control studies.89 No convincing evidence exists that the measles vaccine or other combined vaccinations cause inflammatory bowel disease.84 Several case-control studies have reported a weak association of the disease with contraceptive use. When adjusted for confounders, such as smoking, however, the reported differences were not significant.90 Non-steroidal anti-inflammatory drugs can exacerbate inflammatory bowel disease. Epidemiological studies suggest that appendectomy might be protective against ulcerative colitis,91 with one study finding significantly reduced risks of colectomy or the need for immunosuppressive therapy in patients who had undergone appendectomy before diagnosis.92 The opposite is true for Crohn’s disease, with appendectomy being associated with an increased risk of developing strictures.93,94 Potential explanations for these contrasting findings might relate to the handling of microbes by the mucosal immune system, the hygiene hypothesis, and a failure to develop immune tolerance after appendectomy. 1631

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Panel 3: Intestinal immune system in healthy state The intestinal immune system represents a complex network of different lymphoid and non-lymphoid cell populations and humoral factors. Luminal antigens, such as commensals, pathogens, and nutrients, are sampled by professional and nonprofessional antigen-presenting cells. Although non-professional antigen-presenting cells, such as intestinal epithelial cells, might interact with naive T cells (Th0) through major histocompatibility complex II receptors, they do produce co-stimulatory signals in the absence of inflammation, thereby suppressing or inducing anergy in mucosal T cells.97 Mucosal B cells differentiate into plasma cells and secrete IgA, which covers the mucosal epithelium. Professional antigen-presenting cells, such as dendritic cells, express the entire spectrum of pattern-recognition receptors and co-stimulatory molecules. They control both the adaptive immune response, such as the balanced differentiation of naive T cells into effector T cells (Th1, Th2, Th17) required to fight off pathogens and regulatory T cells (Tr, Th3) as well as the innate immune response, such as activation of natural killer cells. In the presence of commensals and absence of inflammation, a balance between effector and regulatory immune subpopulations is maintained through a tightly controlled cytokine network. Cellular contacts and signals—not depicted in figure 3—are equally important. Secondary effector cells, such as granulocytes, mast cells, natural killer cells, natural killer T cells, and macrophages reside mostly in their respective compartments and additional intracellular inhibitory mechanisms prevent the release of inflammatory cytokines and chemoattractants and contain tissue damaging mediators.

Immunobiology Immunhomoeostasis in the healthy gut How is it then that the mucosal immune system is not in a constant uncontrolled state of inflammation when confronted with such a high antigen load? Mucosal surfaces are physical interfaces of the immune system with the outside world. The gut houses a large part of the mucosa-associated lymphoid tissue in the human body. The intestine also harbours the largest and most diverse microbiota consisting of more than 500 species of bacteria95,96 (figure 3 [explained in panel 3]).

Intestinal microbiota and oral tolerance Commensal bacteria modulate the expression of genes involved in several important intestinal functions, including nutrient absorption, mucosal barrier fortification, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturation.98 This symbiotic relation is established during the first 2–3 years of life, with human babies being sterile before birth. Primary colonisation is orderly, with aerobic species predominating first, followed by anaerobic species, with the timing and composition of these microbial successions being influenced both by the mother (vaginal vs caesarean delivery, breast vs bottle fed, and genetic factors) and the environment (hygiene). The mechanisms responsible for establishing and maintaining oral tolerance to the microbiota and foodderived antigens are incompletely understood and involve the complicated interplay of anatomical, cellular, and humoral factors that prevent or dampen immunity against antigens approaching from the intestinal lumen, which would otherwise trigger an inflammatory response, when presented to the immune system via a non-oral route.99 1632

Figure 3: Intestinal immune system in healthy state M-cell=microfold cell (a specialised epithelial cell). Th=T helper cells. Th0=naive T cell. Th, Th1, Th2, Th17=effector T cells. Tr, Th3=regulatory T cells. B=B cell. B(PC)=plasma cell. NK=natural killer cell. NKT=natural killer T cells.

During microbial colonisation, the mucosal immune system matures, and it is during this time that immune, or oral, tolerance is established.

Epithelial barrier The first line of defence of the mucosal immune system is the epithelial barrier.100 The intestinal epithelium is a www.thelancet.com Vol 369 May 12, 2007

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Panel 4: Intestinal immune system in inflammatory bowel disease In inflammatory bowel disease, the well controlled balance of the intestinal immune system is disturbed at all levels. Luminal antigens gain access to the underlying mucosal tissue via a leaky barrier. Innate and adaptive immune cells express a different profile and number of molecular pattern-recognition receptors. Microbial antigens from commensals trigger and maintain an inflammatory response through several different pathways: myeloid dendritic cells falsely recognise commensals as pathogens, enter a maturation programme with increased expression of pattern-recognition receptors, histocompatibility, and costimulatory molecules, and stop migrating. This goes along with a change in their functional status from tolerogenic to activating and promotes differentiation of naive T cells into effector T cells (Th1, Th17, and Th2) and natural killer T cells. Intestinal epithelial cells now also express costimulatory molecules. This expression enables them to function as antigen-presenting cells and further contributes to the effector-T-cell response. Animal data suggest that T-cell and B-cell activation controlled by the antigen-presenting cells also occurs in the germinal centres of mesenteric lymph nodes, where antigen-loaded dendritic cells meet naive T and B cells. In addition to the antigen-presenting-cell controlled activation of immune effector cells, T cells, natural killer T cells, granulocytes, and macrophages express their own patternrecognition receptors in the inflammatory state and might become activated via this alternative route. Overall, in active inflammatory bowel disease effector T cells (Th1, Th2) predominate over regulatory T cells (Th3, Tr). In Crohn’s disease, naive T cells (Th0) preferably differentiate into Th1 (interferon γ+, interleukin 12+) cells. In ulcerative colitis, these cells differentiate into aberrant Th2 (interleukin 5+,) cells. Interleukin 5 is also produced by mast cells. Natural killer T cells are probably the main source of interleukin 13 in ulcerative colitis. The proinflammatory cytokines secreted by activated effector T cells stimulate macrophages to secrete large amounts of tumour necrosis factor α (TNFα), interleukin 1, and interleukin 6. In addition to their failure to balance the adaptive immune response in inflammatory bowel disease, dendritic cells might also be responsible for a dysregulated innate immune response. The false recognition or processing of microbial (commensal) naked DNA, or both, by plasmacytoid dendritic cells might augment macrophage activation indirectly through stimulation by natural killer cells.128 Evidence also exists for activation of natural killer cells independent of the antigen-presenting cells through interleukin 12. Natural killer cells can contribute to tissue damage by exerting direct cytotoxic effects on their targets and secretion of inflammatory cytokines. Ultimately, numerous leucocytes enter from the mucosal vasculature and release chemokines that attract more inflammatory cells which amplify and perpetuate this vicious circle. Tissue damage results from the release of numerous noxious mediators. Evidence is gathering for a close interaction of the immune and nervous system. Both might communicate through direct interaction of enteric glia with intestinal epithelial cells, eosinophils, or mast cells or cytokine signalling with substance P, histamine, neurokinin, serotonin, vanilloids, and many others. This complex network contributes to initiation and augmentation of the inflammation, and motility disturbance in inflammatory bowel disease and mediation of pain. Figure 4: Intestinal immune system in inflammatory bowel disease MLN=mesentric lymph node. Other abbreviations spelled out in footnote of figure 3.

polarised single layer covered by mucus in which commensal microbes are embedded.87 Defects in mucus production have been reported in people with Crohn’s disease and ulcerative colitis.101–102 The apical epithelial cell surface is also covered with secretory IgA and a glycocalyx. Fluxes through the intestinal epithelium mainly proceed through a transcellular route, with specific membrane www.thelancet.com Vol 369 May 12, 2007

pumps and channels as well as a paracellular route controlled by tight junctions comprising occludin and claudin proteins. The small intestine also contains specialised epithelial cells called Paneth cells that play an important part in innate intestinal defences as regulators of microbial density and in protecting nearby stem cells via the production of various antimicrobial proteins.103 These proteins have broad activities in vitro against gram-positive and gram-negative bacteria. One of the main classes, the defensins, can be divided into two 1633

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families: α-defensins and β-defensins.104 Whereas α-defensins and cathelicidins occur only in the small intestine, β-defensins are expressed ubiquitously on all mucosal surfaces of the gastrointestinal tract. The amphipathic properties of defensins allows them to interact with and lyse bacterial membranes.105

Antigen recognition and immunoregulation Luminal antigen recognition and processing begins at the epithelial level.106 Human intestinal epithelium constitutively express several evolutionarily conserved and structurally related receptors, called patternrecognition receptors, capable of recognising specific microbial components or microbe-associated molecular patterns such as lipopolysaccharide, peptidoglycan, lipoteichoic acid, single-stranded and double-stranded RNA, and methylated DNA (CpG motifs) that are unique to microbes. One of the most important pattern-recognition receptors families in mammals are toll-like receptors (TLR), first described in the fruitfly.55,107 The ten to 15 TLR (dependent on the species) identified so far are individually specialised, but taken together, are probably capable of recognising most microbe associated molecular patterns. TLR4 is distinct from other such receptors in that it associates with a coreceptor, CD14, which is needed to signal the presence of lipopolysaccharide.108 On recognition of their specific microbe-associated molecular patterns, TLR trigger innate and adaptive antimicrobial responses in an intracellular signalling process that culminates in the activation of the transcription factor nuclear factor kappa B (NFκB) and induction of the inflammatory cytokine cascade.109,110 In the absence of pathogens, TLR interactions with commensals have been shown to contribute to intestinal homoeostasis and maintenance of an intact epithelial barrier52,53 (figure 2). In addition to TLR, cytosolic NOD proteins provide an additional defence mechanism in the intestinal mucosa (figure 2 and panel 2).50,52,53 NOD1 and NOD2 proteins are expressed in the cytosol of antigen-presenting cells exposed to micro-organisms containing peptidoglycan.56 In the intestine, expression of NOD1 and NOD2 in epithelial cells is low or absent, but is increased in response to inflammation and inflammatory cytokines.111,112 In the small intestine, the highest level of expression of NOD2 are found in Paneth cells.113,114 In models of NOD2 overexpression, stimulation of muramyl dipeptide results in NFκB activation, which together with the ability of proinflammatory cytokines (eg, TNFα) to influence NOD2 expression suggests that NOD2 contributes to the innate immune response to microbial pathogens.111,115,116 The function of NOD2 is, however, debated, with data from studies in rodents and human beings providing evidence for both a loss and a gain of function for cells with CARD15 gene mutations (panel 1). One murine model of CARD15 gene mutation has shown loss of 1634

function and overproduction of proinflammatory cytokines in response to TLR-mediated signalling in CARD15-deficient macrophages.62 Studies with human cells generally lend support to a loss of function theory with respect to cytokine production and NFκB activation in CARD15 mutants.56 Studies in mice that express a common human CARD15 variant (Leu10007fsinsC [A: correct? 1007?]; figure 2) have provided evidence of a gain of function with increased NFκB activation resulting in greater production of interleukin 1β in response to muramyl dipeptide in macrophages that express this mutant NOD2 protein.56 The function of NOD2 in epithelial cells is, however, less certain, although the reduced production of α-defensin in mice with targeted mutation of the CARD15 gene suggests that it contributes to innate immunity in the gut by regulating Paneth-cell function.117 The significance of this finding is, however, uncertain, since mice without active forms of α-defensins or Paneth cells do not spontaneously develop chronic intestinal inflammation.118,119 The function of NOD2 in any cell type therefore remains uncertain. This uncertainty is particularly the case for the intestinal epithelium, which, as the first point of contact with enteric microbes is arguably of most significance for intestinal health.

Lymphoid cells The mucosal lymphoid tissue includes T cells, B cells, granulocytes, mast cells, natural killer (NK) cells, and NK T cells, which are located in the loose connective tissue of the lamina propria (lamina propria lymphocytes). An additional highly specialised enterocyte population serves as an interface between the epithelial layer and the underlying lymphoid tissue. Villous microfold cells function as a conduit channelling antigens (including microbes) to the underlying lymphocyte clusters (Peyer’s patches in the small intestine and lymphoid follicles in the colon), where they meet antigen-presenting cells, such as dendritic cells and macrophages.120 Additionally, dendritic cells can open the tight junctions between epithelial cells, send dendrites outside the epithelium, and directly sample bacteria.121 Owing to their expression of tightjunction proteins, such as occludin, claudin-1, and zonula occludens-1, the integrity of the epithelial barrier is preserved.122 Dendritic cells are the key cells in controlling immunity against pathogens and tolerance towards commensals.Unlike epithelial cells and immune effector cells, dendritic cells express the entire spectrum of TLR and NODs, enabling them to distinguish between commensals and pathogens and to either activate or silence T-cell responses.107 Dendritic cell function is regulated by their location, number, and maturational state.123 In healthy individuals, these cells are thought to sample antigens, display an immature phenotype, and induce T-cell unresponsiveness— www.thelancet.com Vol 369 May 12, 2007

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probably by stimulating naive T-cell differentiation into regulatory CD4+ T cells, such as Th3 cells (transforming growth factor β+, interleukin 10+/–, interleukin 4–) or Tr1 (interleukin 10+), rather than effector Th1 (interferon γ+, interleukin 2+) or Th2 (interleukin 4+, interleukin 10+) cells, thus maintaining tolerance towards commensals.123 Which human dendritic cells’ sub-population induce the differentiation of naive T cells into regulatory T cells in the mucosa is not yet known. In-vitro data suggest that dendritic cells interact with certain probiotic bacterial strains to produce interleukin 10 that drives regulatory, rather than effector, Th1-cell responses.125 When dendritic cells sense danger—ie, pathogens—they mature, acquire an activated phenotype, and induce immunity.126 This process can involve remodelling of the cell’s cytoskeleton as, for example, after activation mediated by TLRs.127

Malfunction of the immune system It is now widely accepted that inflammatory bowel disease results from an inappropriate response of a defective mucosal immune system to the indigenous flora and other luminal antigens (figure 4 [explained in panel 4]). But how and why might microbial antigens induce an inappropriate inflammatory response? Experimental evidence from studies in vitro, in animals, and in human beings suggests that several, not mutually exclusive, pathways might result in inflammatory cascades.

Initiating or primary events First, the epithelial barrier is leaky in people with inflammatory bowel disease. Several studies have shown a lowered epithelial resistance and increased permeability of the inflamed and non-inflamed mucosa in Crohn’s disease and ulcerative colitis.129 The defect precedes the clinical onset of disease in individuals with a family risk.131 Permeability defects have also been reported in healthy first-degree relatives of patients with inflammatory bowel disease with a CARD15 3020insC mutation, implicating a genetic defect.132 Several mechanisms of increased permeability have been proposed, ranging from T-cellmediated disruption of tight-junction protein to enteric neuron dysfunction.129,133–136 Second, people with inflammatory bowel disease have disturbed innate immune mechanisms of the epithelial layer. In these people, mucosal epithelial cells have a different pattern of TLR expression. Whereas healthy intestinal epithelial cells constitutively express TLR3 and TLR5 basolaterally, TLR2 and TLR4 are usually barely detectable. TLR3 is significantly down regulated in active Crohn’s disease, but not in ulcerative colitis. By contrast, TLR4 is strongly upregulated in both diseases.137 Intestinal epithelial cells also express TLR9, which enables them to directly respond to bacterial DNA, resulting in secretion of interleukin 8, which is a granulocyte chemoattractant.138 Probably because of its basolateral expression, TLR5 www.thelancet.com Vol 369 May 12, 2007

signalling is generally suppressed. However, in the injured inflammatory bowel disease mucosa, flagellin—a bacterial component and potent TLR5 ligand—can engage the receptor and thereby aggravate inflammation.139 An upregulation of NOD2 in epithelial cells, which can augment itself further in a feedback loop, when the NFκB cascade is activated has also been reported,111,112 which might compromise the ability of the host to eliminate invasive and pathogenic microbes resulting in chronic inflammation. Third, antigen recognition and processing by professional antigen-presenting cells is disturbed in people with inflammatory bowel disease. Animal and in-vitro studies suggest that dendritic cells incorrectly recognise commensal bacteria and induce a Th1 and possibly Th17, proinflammatory immune responses normally directed at pathogens. This could be due to dysfunctional or exaggerated pattern-recognition receptor responses.140 Increased expression of TLR4 by myeloid dendritic cells in inflammatory bowel disease has been reported.141 Studies of inflammatory bowel disease in animal models142–144 have shown activated dendritic cells, which prolong their survival thereby maintaining inflammation. This hypothesis is indirectly supported by animal data that implicates the receptor activator of nuclear factor-κ B (RANK)—RANK ligand (RANKL) system in this process.145 Human intestinal dendritic-cell populations in inflammatory bowel disease are insufficiently characterised, mainly owing to the scarcity of highly specific antibodies and their low numbers. We and others have shown an increased frequency of mature—ie, activated—dendritic cells in the inflamed mucosa of patients with active inflammatory bowel disease.141,146 We have recently shown a scarcity of circulating, immature—ie, potentially tolerogenic—dendritic cells in patients with inflammatory bowel disease, that strikingly correlates with the extent of inflammation.147 Human dendritic cells from inflammatory bowel disease patients express gut homing markers and also showed an aberrant response to microbial surrogate stimuli like CpG-DNA and lipopolysaccharide.147 This absence of the regulatory capacity of dendritic cells might also contribute to the repeated activation of certain (gut homing) memory T cells or failure to delete these over reactive T-cell populations (absence of peripheral tolerance), thereby perpetuating inflammation.126,148 Fourth, atypical antigen-presenting cells become potent effector-T-cell activators in people with inflammatory bowel disease. Non-professional antigen-presenting cells, such as epithelial cells, which normally induce anergy in CD4+ T cells, perhaps because of their lack of B7 (CD80, CD86) molecules, acquire an activated phenotype with increased histocompatibility molecule expression in the presence of inflammatory cytokines such as interferon γ and TFNα.97 Epithelial cells might also activate T cells via non-classical major histocompatibility complex molecules, such as CD1d, if antigens gain access to this basolaterally expressed 1635

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molecule in the injured mucosa.149 Intestinal epithelial cells from patients with inflammatory bowel disease also express alternative costimulatory molecules, which might transform them into functional antigen-presenting cells.150 Furthermore, epithelial cells might directly activate CD4 T cells through the expression of lectins and other carbohydrates.151,152 Fifth, people with inflammatory bowel disease have disturbed clearance of overreactive or autoreactive T-cell populations. Due to a failure of central (thymic) and peripheral tolerance, activated T cells persist and do not undergo apoptosis. This persistence has been shown in patients with Crohn’s disease and is exploited in current biological therapies that break this activation cycle.153,154 Sixth, the balance of regulatory and effector T cells is disturbed in inflammatory bowel disease. When the disease is active, effector T cells (Th1 and Th2) predominate over regulatory T cells as a consequence of naive T cells (Th0) preferably differentiating into Th1 (in Crohn’s disease).155 The Th1 phenotype in Crohn’s disease is mediated by the transcription factor T-bet156 and the cytokine interleukin 23.157 Activated T cells in Crohn’s disease produce and release inflammatory cytokines, such as interleukin 12, interleukin 18, TNF-like 1A and interferon γ, which stimulate macrophages to release interleukin 1, TNFα, and interleukin 6.146,157,158 Also, increased numbers of activated NK T cells producing interleukin 13 and interleukin 5 have been reported in ulcerative colitis,159 which might further augment and perpetuate inflammation. The identification of T-helper cells that produce interleukin 17 (Th17) and can promote immune-mediated inflammatory responses in various tissues, including the gut emphasises the complexity and importance of maintaining immune homoeostasis. Recent studies suggest that transforming growth factor β is pivotal in determining the balance between proinflammatory (Th17) and anti-inflammatory (Tr) T-helper cell responses that normally work together to elicit or restrain intestinal inflammation.160 Endogenous or exogenous factors that alter the production of transforming growth factor β or other key cytokines (interleukin 4, interleukin 6, and interleukin 22, and interferon γ) that sustain or terminate Th17 or Tr activity will be of major importance to the development of chronic intestinal inflammation and inflammatory bowel disease.161 Seventh, psychosocial stress might trigger or augment the inflammatory cascade through neuroimmunological interaction. In the absence of stress, the nervous system, through the vagus nerve, can have a substantial inhibitory effect and rapidly attenuate systemic inflammatory responses through the cholinergic anti-inflammatory pathway. The nicotinic acetylcholine receptor α7 subunit, needed for acetylcholine inhibition of macrophage TNFα release, which is a major source of this proinflammatory cytokine in inflammatory bowel disease, was only recently identified.162 Other evidence that identifies vagal-nerve 1636

involvement in the control of inflammation comes from studies of ulcerative colitis tissue samples that show a shift from mainly cholinergic to more substance P positive innervation.163 Stress—ie, overactivation of the sympathetic nerve—has been shown in patients with ulcerative colitis and in turn, causes increased colonic paracellular permeability involving mast-cell degranulation, overproduction of interferon γ, and altered the expression of tight junction proteins.164,165

Final common or secondary events First, migration of inflammatory cells from the vasculature into the intestinal mucosa occurs in inflammatory bowel disease. Antigen recognition by professional and nonprofessional antigen-presenting cells results in cell migration from the systemic circulation to the intestinal mucosa. This is accomplished by the release of chemoattractants, such as interleukin 8, macrophage inflammatory protein 1 (α and β), RANTES (regulated on activation normal T cells as secreted), monocyte chemoattractant proteins (1, 2, and 3) which induce conformational changes in adhesion molecules on lymphocytes (ie, α4β7-integrin, chemokine receptor 9) and granulocytes (ie, L-selectins, lymphocyte functionassociated antigen). At the same time proinflammatory cytokines such as interleukin 1 and TNFα secreted by activated macrophages up-regulate the expression of adhesion molecule ligands on the vascular endothelium of the mucosal blood vessels (E-selectins and P-selectins, intercellular adhesion molecule 1, mucosal vascular addressin cell adhesion molecule 1, carcinoembryonic antigen cell adhesion molecule 1, vascular-endothelial growth factor A) promoting leucocyte adhesion and extravasation into the tissue.166,167 Second, a multitude of aggressive metabolites and mediators accumulate in the mucosa, resulting in tissue damage. Nitric oxide, oxygen radicals, prostaglandins, leukotrienes, histamine, proteases, and matrix metalloproteinases promote fibroblast growth with collagen secretion and stricture formation.168–171

Therapeutic modulation of the immune response Established and evolving therapies are discussed in detail in the second paper in this Series.1 None of these therapies are disease specific, and they generally target events downstream of the inflammatory cascade. The real challenge for the future is to develop a tailored (ie, pharmacogenomic) approach to prevention of the initiation and perpetuation of the inflammatory cascade before tissue injury occurs. This approach could involve the induction or re-establishment of immunological tolerance by recomposing the commensal microflora, introduction of engineered microbiota to induce regulatory immune responses, blocking pattern-recognition receptors and signalling proteins, or in-vivo or in-vitro generation of regulatory T cells with tolerogenic dendritic cell or even gene transfer. www.thelancet.com Vol 369 May 12, 2007

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Conflict of interest statement We declare that we have no conflict of interest. Acknowledgments DCB is supported by research grants from Eli & Edythe L Broad Foundation, CA, USA; the Fritz Bender Foundation, Munich, Germany; and a Charité Medical School, Humboldt-University of Berlin bonus research grant. SRC is supported by grants from the Wellcome Trust, UK; the Biotechnology and Biological Sciences Research Council, UK; National Institutes of Health, USA); and the European Union. References 1 Baumgart DC, Sandborn WJ. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 2007; 369: 1641–57. 2 Anonymous. Wilhelm Fabry (1560–1624): the other fabricius. JAMA 1964; 190: 933. 3 Crohn BB, Ginzburg L, Oppenheimer GD. Landmark article Oct 15, 1932: regional ileitis: a pathological and clinical entity, by Burril B Crohn, Leon Ginzburg, and Gordon D Oppenheimer. JAMA 1984; 251: 73–79. 4 Wilks S. Morbid appearances in the intestine of Miss Bankes. London Medical Times & Gazette 1859; 2: 264. 5 Loftus EV Jr. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology 2004; 126: 1504–17. 6 Sedlack RE, Whisnant J, Elveback LR, Kurland LT. Incidence of Crohn’s disease in Olmsted County, Minnesota, 1935–75. Am J Epidemiol 1980; 112: 759–63. 7 Loftus CG, Loftus EV Jr, Harmsen WS, et al. Update on the incidence and prevalence of Crohn’s disease and ulcerative colitis in Olmsted County, Minnesota, 1940–2000. Inflamm Bowel Dis 2006; published online Dec 19. DOI 10.1002/ibd.20029. 8 Loftus EV Jr, Silverstein MD, Sandborn WJ, Tremaine WJ, Harmsen WS, Zinsmeister AR. Ulcerative colitis in Olmsted County, Minnesota, 1940–1993: incidence, prevalence, and survival. Gut 2000; 46: 336–43. 9 Loftus EV Jr, Silverstein MD, Sandborn WJ, Tremaine WJ, Harmsen WS, Zinsmeister AR. Crohn’s disease in Olmsted County, Minnesota, 1940–1993: incidence, prevalence, and survival. Gastroenterology 1998; 114: 1161–68. 10 Binder V, Both H, Hansen PK, Hendriksen C, Kreiner S, Torp-Pedersen K. Incidence and prevalence of ulcerative colitis and Crohn’s disease in the County of Copenhagen, 1962 to 1978. Gastroenterology 1982; 83: 563–68. 11 Langholz E, Munkholm P, Nielsen OH, Kreiner S, Binder V. Incidence and prevalence of ulcerative colitis in Copenhagen county from 1962 to 1987. Scand J Gastroenterol 1991; 26: 1247–56. 12 Munkholm P, Langholz E, Nielsen OH, Kreiner S, Binder V. Incidence and prevalence of Crohn’s disease in the county of Copenhagen, 1962–87: a sixfold increase in incidence. Scand J Gastroenterol 1992; 27: 609–14. 13 Bernstein CN, Blanchard JF, Rawsthorne P, Wajda A. Epidemiology of Crohn’s disease and ulcerative colitis in a central Canadian province: a population-based study. Am J Epidemiol 1999; 149: 916–24. 14 Blanchard JF, Bernstein CN, Wajda A, Rawsthorne P. Small-area variations and sociodemographic correlates for the incidence of Crohn’s disease and ulcerative colitis. Am J Epidemiol 2001; 154: 328–35. 15 Lapidus A, Bernell O, Hellers G, Persson PG, Lofberg R. Incidence of Crohn’s disease in Stockholm County 1955–89. Gut 1997; 41: 480–86. 16 Lapidus A. Crohn’s disease in Stockholm County during 1990–2001: an epidemiological update. World J Gastroenterol 2006; 12: 75–81. 17 Kurata JH, Kantor-Fish S, Frankl H, Godby P, Vadheim CM. Crohn’s disease among ethnic groups in a large health maintenance organization. Gastroenterology 1992; 102: 1940–48. 18 Nguyen GC, Torres EA, Regueiro M, et al. Inflammatory bowel disease characteristics among African Americans, Hispanics, and non-Hispanic Whites: characterization of a large North American cohort. Am J Gastroenterol 2006; 101: 1012–23. 19 Heresbach D, Gulwani-Akolkar B, Lesser M, et al. Anticipation in Crohn’s disease may be influenced by gender and ethnicity of the transmitting parent. Am J Gastroenterol 1998; 93: 2368–72.

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