Metabolome and inflammasome in inflammatory bowel disease

IN-DEPTH REVIEW: INFLAMMATORY BOWEL DISEASE Metabolome and inflammasome in inflammatory bowel disease RADU M. NANAU, and MANUELA G. NEUMAN TORONTO, ONTARIO, CANADA

Inflammatory bowel disease (IBD) encompasses several chronic inflammatory disorders leading to the damage of the gastrointestinal tract. The 2 principal forms of these disorders are ulcerative colitis (UC) and Crohn’s disease (CD). Bacteria are involved in the etiology of IBD. Many microorganisms have been put forward as causative factors in IBD, but the primary etiologic agents are still not known. The underlying genetic, environmental, and lifestyle issues can affect the individual’s predisposition to these diseases. Immune factors identified in IBD are: dysregulation of the innate and adaptive immune system directed against luminal bacteria or their products found in the intestinal lumen and inappropriate immune responses to organisms in the intestine that normally do not elicit a response, possibly because of intrinsic alterations in mucosal barrier function. However, recent advances in basic science research revealed new insights into the role of specific immune cells and their mediators in intestinal inflammation. The inflammatory mediators known as ‘‘inflammasome’’ are a consequence of the metabolic products (metabolom) of cells and commensal or pathogenic bacteria. Elucidation of inflammasome and metabolom has led to the development of biomarkers specific for each disease that are involved into management strategies targeted at altering specific pathogenic mechanisms that have the potential to modify or change the natural course of these disease entities. The review discusses the potential role of biomarkers in monitoring the inflammasome and therefore the severity of intestinal damage. The microbial ecosystem in the human gut in different microhabitats and metabolic niches contribute to the bowel metabolome.In addition, this review will focus on our expanding understanding of microbial factors associated with both the initiation and maintenance of IBD. New insights acquired from murine genetic models of inflammatory bowel disease will also be discussed. (Translational Research 2012;160:1–28) Abbreviations: ACCA ¼ antichitobioside carbohydrate antibodies; ALCA ¼ antilaminaribioside carbohydrate antibodies; AMCA ¼ antimannobioside carbohydrate antibodies; anti-OmpC ¼ antibodies against porin protein C of Escherichia coli; AT1R ¼ angiotensin II type 1 receptor; CAM ¼ cell adhesion molecule; CD ¼ Crohn’s disease; CI ¼ confidence interval; CXCL16 ¼ chemokine (C-X-C motif) ligand 16; DSS ¼ dextran sodium sulfate; ELISA ¼ enzyme-linked immunoFrom the In Vitro Drug Safety and Biotechnology; Department of Pharmacology and Toxicology, University of Toronto, Toronto, Canada.

MaRS Discovery District, 101 College St., South Tower, Laboratory 351, Toronto, Ontario, Canada M2R 1W6; e-mail: m_neuman@ rogers.com.

Submitted for publication June 3, 2011; revision submitted August 22, 2011; accepted for publication August 22, 2011.

1931-5244/$ - see front matter

Reprint requests: Manuela G. Neuman, PhD, Department of Pharmacology and Toxicology, In Vitro Drug Safety and Biotechnology,

doi:10.1016/j.trsl.2011.08.006

Ó 2012 Mosby, Inc. All rights reserved.

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Nanau and Neuman

sorbent assay; ER ¼ endoplasmic reticulum; GAB ¼ autoantibodies to intestinal goblet cells; gASCA ¼ anti-Saccharomyces cerevisiae antibodies; GI ¼ gastrointestinal; GM-CSF ¼ granulocyte-macrophage colony-stimulating factor; GM-CSF Ab ¼ neutralizing antibodies directed against granulocyte-macrophage colony-stimulating factor; HIMEC ¼ human intestinal microvascular endothelial cells; IBD ¼ inflammatory bowel disease; IBS ¼ irritable bowel syndrome; IC ¼ indeterminate colitis; ICAM ¼ intercellular adhesion molecule; IFN ¼ interferon; Ig ¼ immunoglobulin; IL ¼ interleukin; IL12R ¼ interleukin 12 receptor; IRF ¼ IFN-regulatory factor; IRGM ¼ immunity-related guanosine triphosphatase; LPS ¼ lipopolysaccharide; MAdCAM ¼ mucosal vascular addressin cell adhesion molecule; MAPK ¼ mitogen-activated protein kinase; mRNA ¼ messenger RNA; NF ¼ nuclear factor; NHE ¼ Na1/H1 exchanger; NOD1 ¼ nucleotidebinding oligomerization domain-containing protein 1; PAB ¼ autoantibodies to exocrine pancreas; pANCA ¼ perinuclear antineutrophil cytoplasmic antibodies; PBMC ¼ polymorphonuclear cell; PECAM ¼ platelet/endothelial cell adhesion molecule; PPV ¼ positive predictive value; RANTES ¼ regulated on activation, normal T cell expressed and secretedCCL5; RT-PCR ¼ reverse transcriptase-polymerase chain reaction; siRNA ¼ small interfering RNA; SNP ¼ single nucleotide polymorphism; SOCS ¼ suppressor of cytokine signaling; sRAGE ¼ soluble receptor for advanced glycation end products; STAT3 ¼ signal transducers and activator of transcription, member 3; TGF ¼ transforming growth factor; Th ¼ Thelper response; TLR ¼ Tolllike receptor; DNBS ¼ dinitrobenzene sulfonic acid; TNF ¼ tumor necrosis factor; UC ¼ ulcerative colitis; VCAM ¼ vascular cell adhesion molecule

Inflammatory bowel disease (IBD) is an idiopathic chronic disease of the gastrointestinal (GI) tract characterized by intermittent periods of chronic inflammation and remission.1 IBD progression is accompanied by an increase in granulomas and activated monocytes, which results in a significant release of eicosanoids and cytokines. Transmural inflammation characterized by lymphoid hyperplasia, submucosal edema, ulcerative lesions, and fibrosis are typically observed in affected areas.1,2 Ulcerative colitis (UC) and Crohn’s disease (CD) are the most common variants of IBD. UC can be distinguished from CD histologically by the presence of localized inflammation in the superficial layer of the colon mucosa.1,2 CD, which is characterized by discontinuous, transmural lesions of the gut wall, can affect the whole length of the intestine, yet the most affected portions are the lower ileum and the colon. The pathology of CD is characterized by the T helper (Th) 1 response, which is mediated by high levels of tumor necrosis factor (TNF)-a, interferon (IFN)-g, and tissue-infiltrating Th17 cells. In contrast, UC, which is characterized by continuous and superficial lesion, is confined to the lining of the colon and the rectum. The pathology of UC is characterized by the atypical Th2 response, mediated by high levels of Th17 cells.3,4 Worldwide, the incidence of CD varies from 0.7 to 14.6 individuals per 100,000 inhabitants, whereas the incidence of UC varies from 1.5 to 24.5 individuals per 100,000 inhabitants, depending on the region.5-8 The prevalence of UC seems to increase with age.7-9 Both genetic factors and environmental factors were shown to play an important role in IBD.10,11 Polymorphic genes associated with IBD are involved in bacterial recognition, the early immune response, and the innate immune response.4

The pathogenesis of IBD is, thus, thought to involve a dysregulation of the early mucosal immune response to commensal microorganisms in genetically predisposed individuals under the action of genetic factors, environmental factors, and immunologic factors, which are mediated by excess helper T cell-induced tissue inflammation.2,4,12,13 BIOMARKERS IN IBD PATIENTS

Disease-specific biomarkers in serum, stool, or urine, as well as the expression profiles of proteins characteristic of disease, can be indicative of early disease development and can be used to differentiate either IBD from irritable bowel syndrome (IBS), CD from UC, or active IBD from inactive IBD. These biomarkers represent unique fingerprints that depict specific cellular processes (Table I). Autoantibodies. The presence of several anticarbohydrate antibodies [antibodies against mannan and mannan residues, antichitobioside carbohydrate immunoglobulin A (IgA) antibodies (ACCA), antilaminaribioside carbohydrate IgG antibodies (ALCA), antimannobioside carbohydrate IgG antibodies (AMCA), and antichitin IgA and antilaminarin IgA], anti-Saccharomyces cerevisiae IgA and IgG antibodies (gASCA), perinuclear antineutrophil cytoplasmic IgG antibodies (pANCA), antinuclear associated antieutrophil IgG antibodies (atypical pANCA), serum IgA and IgG autoantibodies to intestinal goblet cells (GABs), serum IgG and a combination of IgG and IgA autoantibodies to exocrine pancreas (PAB), and IgA antibodies against porin protein C of Escherichia coli (anti-OmpC) were assessed as potential serological diagnostic markers in various cohorts of IBD patients.

Study population

Settings

Biomarker

128 IBD patients (72 CD; 56 UC) Tel Aviv, Israel and 41 healthy controls 1 validation cohort of 230 IBD patients (124 CD; 106 UC), 61 with other GI diseases (27 celiac disease, 20 IBS, 14 colonic polyps) and 40 healthy controls

Serum ACCA, ALCA, AMCA, and gASCA

187 IBD patients (109 CD and 78 Marseilles, Paris, Montpellier, UC, both children and adults), 45 Strasbourg, Lyon, and Dijon, celiac disease (29 with active France, and Luxembourg disease, 16 in remission); 50 healthy donors

Serum gASCA, atypical pANCA, and PAB

Brescia, Italy

Serum pANCA and gASCA

653 IBD patients (558 CD; 95 UC) and 100 healthy controls

Multiple centers in Hungary

Serum gASCA, pANCA, and anti-OmpC

Sensitivity, specificity, PPV, and NPV of biomarker

Ref

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14 The presence of gASCA differentiate CD from UC (66.1% sensitivity, 85.8% specificity, 84.5% PPV, and 68.4% NPV). The presence of at least 1 of ALCA, ACCA, or gASCA differentiate CD from UC (77.4% sensitivity, 90.6% specificity, 90.6% PPV, and 77.4% NPV). The presence of at least 2 of ALCA, ACCA, or gASCA differentiate CD from UC (26.6% sensitivity, 99.1% specificity, 97.1% PPV, and 53.6% NPV). The presence of all 3 of ALCA, ACCA, and gASCA differentiate CD from UC (3.2% sensitivity, 100.0% specificity, 100.0% PPV, and 46.9% NPV). 15 The prevalence of gASCA (both IgA Being positive for gASCA and negative for atypical pANCA was and IgG) was significantly higher associated with CD (46.2% in CD compared with UC or sensitivity, 97.6% specificity, healthy donors (P , 0.001). 94.2% PPV, 68.7% NPV). The prevalence of atypical pANCA Being positive for PAB was was significantly higher in UC associated with CD (36.7% compared with CD or healthy sensitivity, 94.5% specificity, donors (P , 0.001). 85.1% PPV, and 63.7% NPV). Positive for pANCA and negative for 16 gASCA was more prevalent in gASCA was associated with UC CD patients compared with UC (70% sensitivity, 86% specificity, patients and healthy controls. 82% PPV, 76% NPV, and 78% pANCA was more prevalent in UC diagnostic accuracy). Positive for patients compared with CD gASCA and negative for pANCA; patients and healthy controls. was associated with CD (86% sensitivity, 93% specificity, 75% PPV, 96% NPV, and 91% diagnostic accuracy). Significantly more CD positive for Positive for gASCA and negative for 17 all of gASCA (both IgA and IgG) pANCA; was associated with CD and anti-OmpC than UC (52% sensitivity, 92% specificity, 86% PPV, 66% NPV) compared (Continued ) Antibodies against mannan and mannan residues were more prevalent in CD compared with UC patients (P , 0.001). ALCA was more prevalent in CD compared with UC (P , 0.0001), healthy controls (P , 0.0001), and patients with other GI diseases (P , 0.05). ACCA was prevalent in CD compared with UC (P , 0.0001). gASCA was prevalent in CD compared with any of the other 3 groups (P , 0.0001). No significant difference in serum anticarbohydrate antibodies was found between UC compared with CD or controls.

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47 IBD patients (14 CD and 33 UC; age 2 to 18 years) and 22 patients with infective colitis or other types of colitis

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Table I. Biomarkers as a diagnostic tool

Settings

Biomarker

Main findings

patients (P , 0.0001) and healthy controls (P , 0.0001).

301 early IBD patients (197 CD, 76 Glasgow, Scotland UC, 28 IC) 76 healthy controls

Serum gASCA

gASCA was more prevalent in CD compared with UC patients.

526 IBD patients (357 UC; 169 CD) Multiple centers in Norway and 135 geographical areamatched healthy donors

Serum pANCA and gASCA

gASCA was more prevalent in CD compared with UC patients and healthy controls. pANCA was more prevalent in UC compared with CD patients and healthy controls.

200 IBD patients (116 CD; 84 UC) and 72 healthy controls

Serum ACCA, ALCA, AMCA, and gASCA

gASCA prevalence was higher in CD than it was in UC patients.

Prague, Czech Republic

818 IBD patients (517 CD patients and 301 UC patients)

Toronto Canada

with UC. Positive for pANCA and negative for gASCA; was associated with UC (36% sensitivity, 93% specificity, 85% PPV, 59% NPV) compared with CD. The presence of gASCA IgG can be used to differentiate CD from UC (41.6% sensitivity and 88.6% specificity). The presence of gASCA can be used to differentiate CD from UC (27% sensitivity, 95% specificity, and 83% PPV). The presence of pANCA can be used to differentiate UC from CD (31% sensitivity, 86% specificity, and 82% PPV). gASCA can be used to predict overall CD compared with healthy controls (67% sensitivity, 94% specificity, and 95% PPV) and compared with UC (67% sensitivity, 86% specificity, and 86% PPV). The presence of gASCA can be used to predict colonic CD only compared with healthy controls (52% sensitivity, 94% specificity and 84% PPV) and compared with UC (52% sensitivity, 86% specificity and 64% PPV). gASCA can be used to differentiate CD from UC (63.1% sensitivity, 85.2% specificity, 77.1% PPV, and 74.6% NPV).

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21 Serum ACCA, ALCA, AMCA, All of ACCA, ALCA, AMCA, antichitin, antilaminarin, gASCA, antichitin, antilaminarin, gASCA, and pANCA and pANCA were more prevalent in CD patients compared with the control groups combined (P , 0.001). Serum ACCA, ALCA, AMCA, All of ACCA, ALCA, AMCA, The presence of gASCA IgG can be 22 antichitin, antilaminarin and antichitin, antilaminarin and used to differentiate CD from UC gASCA gASCA were more prevalent in (60.74% sensitivity, 85.38% specificity, 87.71% PPV and

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503 IBD patients (363 CD; 130 UC), Regensburg, Germany 74 with other GI disorders and 257 with noninflammatory GI disorders

Sensitivity, specificity, PPV, and NPV of biomarker

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Study population

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Table I. (Continued )

55.87% NPV). The presence of gASCA IgA can differentiate CD from UC (47.00% sensitivity, 94.02% specificity, 93.10% PPV and 50.81% NPV) 23 .95% specificity and .91% PPV pANCA was detected more for all 3 biomarkers with respect frequently among UC patients to the type of IBD that they than among healthy controls predict. (P , 0.001). GAB was detected more frequently Testing positive for pANCA or positive for GAB and negative for among UC patients than among PAB was strongly associated healthy controls (P , 0.001). with UC rather than CD or PAB was detected exclusively healthy controls (.95% among CD patients. specificity, .92% PPV, and .87% NPV). Associated with UC Not discussed 24

Serum pANCA, GAB, and PAB

197 IBD patients

Multiple centers in United States 39 IBD patients (27 CD, 10 UC and Stockholm and €ster 2 IC patients, median age 13.8) Va as, Sweden and 12 patients with no signs of colonic inflammation or IBD

Serum atypical pANCA Fecal levels of calprotectin

Higher in IBD patients compared with controls (P , 0.001).

59 IBD patients (32 CD and 27 UC), Essen, Germany 24 IBS, 88 infectious gastroenteritis (65 bacterial enteritis and 23 viral enteritis), and 24 healthy controls

Fecal levels of S100A12

Higher in IBD patients compared with IBS patients (P , 0.001) or healthy controls (P , 0.001). Higher in IBD patients with active disease compared with IBD patients with inactive disease (P , 0.001).

31 IBD patients [(30 CD, 1 UC) Sidney, Australia mean age 11.9) and 30 non-IBD patients]

Fecal levels of S100A12

Higher in IBD patients in general compared with non-IBD patients (P , 0.0001). Higher in IBD compared with non-IBD patients (P , 0.0001) .

Fecal levels of calprotectin

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25 Predictive cut-off of 50.0 mg/g for the development of microscopic inflammation (93% sensitivity, 73% specificity, 90% PPV. and 80% NPV). The maximal sum between sensitivity and specificity was achieved with a fecal calprotectin concentration of 85.7 mg/g for the development of microscopic inflammation (93% sensitivity, 82% specificity, 93% PPV, and 82% NPV). Predictive cut-off of 0.8 mg/g for 26 active CD (100% specificity and 81% sensitivity) and active UC (100% specificity and 81% sensitivity) compared with healthy controls. Predictive cut-off of 0.8 mg/g for active IBD (96% specificity and 86% sensitivity) compared with IBS. Predictive cut-off of 10 mg/g for IBD 27 (97% sensitivity, 97% specificity, 97% PPV, and 97% NPV). Predictive cut-off of 50 mg/g for IBD (100% sensitivity, 67% (Continued )

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71 IBD patients (43 CD patients and Maribor, Slovenia 28 UC patients) and 41 healthy controls

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CD patients compared with UC patients (P , 0.0015)

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Table I. (Continued ) Settings

Biomarker

Serum levels of S100A12 32 IBD patients (11 CD, 21 UC) and Milwaukee Wisconsin 34 healthy controls (5 to 20 years of age)

Fecal levels of calprotectin

73 IBD patients (32 CD, 41 UC, age Rome, Italy #18). 32 presented with relapse at last endoscopy (14 CD, 18 UC) and 41 in remission (18 CD and 23 UC)

Fecal levels of calprotectin

23 IBD patients (22 CD, 1 UC; mean Sidney, Australia age 11.0) and 25 controls

Fecal levels of S100A12

39 IBD patients (29 CD, 4 UC and 6 Sidney, Australia IC; mean age 9.7 years old), 16 controls (celic disease) and 33 non-IBD controls (normal endoscopy, histology and serum markers inflammation)

Serum and mucosal levels of S100A12

Serum and mucosal levels of calprotectin

Larissa, Greece

Serum levels of S100A12

Sensitivity, specificity, PPV, and NPV of biomarker

specificity, 74% PPV, and 95% NPV). Not different between IBD and non- Poor diagnosis tool. IBD patients. Predictive cut-off of $400 mg/g for Higher in IBD patients in general CD relapse within 9 months of (P , 0.0001) compared with baseline (CI, 30% to 80%, controls. P 5 0.03). Higher in IBD with active disease compared with IBD patients with disease in remission (P , 0.0001) or controls (P , 0.0001). Significantly higher in IBD patients Predictive cut-off of $275 mg/g for with disease in relapse IBD (97% sensitivity, 85% compared with IBD patients with specificity, 85% PPV, and 97% disease in remission. NPV), for CD (100% sensitivity, 71% specificity, 78% PPV, and 100% NPV), and for UC (94% sensitivity, 95% specificity, 94% PPV and 95% NPV). Higher in IBD patients at the time of Predictive cut-off of 10 mg/g for IBD diagnosis compared with (96% sensitivity and 92% controls (P , 0.0001). specificity). Mucosal S100A12 (79% sensitivity Serum levels higher in CD and 69% specificity) for IBD (P , 0.01) and in UC (P , 0.01) compared with non-IBD. compared with controls. Mucosal levels higher in CD (P , 0.001), UC (P , 0.01), IC (P , 0.01), and celiac patients (P , 0.05) compared with controls. Serum levels higher in CD Mucosal calprotectin (91% sensitivity and 71% specificity) (P , 0.001) and in UC (P , 0.01) for IBD compared with non-IBD. compared with controls. Mucosal levels higher in CD (P , 0.001), UC (P , 0.01), IC (P , 0.001), and celiac (P , 0.01) compared with controls. Higher in CD (70.0 ng/mL) and in Predictive cut-off of 55.0 ng/mL for UC (68.2 ng/mL) compared CD (64.1% sensitivity and 64.4% with IBS patients (43.3 ng/mL) specificity) compared with IBS. (P 5 0.001).

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128 IBD patients (64 CD, 64 UC) and 73 IBS patients as controls

Main findings

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Study population

Intestinal release of S100A12

Intestinal release of calprotectin Mucosal release of S100A12

31 active CD, 23 inactive CD (age range 4 to 20) and 37 non-IBD patients

Indianapolis, Indiana

Mucosal release of calprotectin Fecal levels of lactoferrin

Fecal levels of lactoferrin

230 IBD (126 UC and 104 CD), 137 Sheffield, England IBS and 98 controls

Fecal levels of lactoferrin

36 IBD patients (24 CD and 12 UC), Bern and Lucerne 20 IBS, 18 with other forms of Switzerland colitis (8 with infectious colitis, 5

Fecal levels of calprotectin

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77 IBD (42 active UC, 17 inactive Shanghai, China UC, 13 active CD, 5 inactive CD), 41 bacterial infectious bowel disease, 25 IBS, 34 healthy volunteers

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60 IBD patients (30 CD and 30 UC), Essen, Germany 30 IBS patients and 30 patients without intestinal inflammation

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No difference between CD and UC Predictive cut-off of 54.4 ng/mL for patients. UC (68.8% sensitivity and 64.4% specificity) compared with IBS. Not discussed 33 Higher in IBD compared with IBS (P , 0.0001) or healthy controls (P , 0.0001). Higher in inflamed tissue compared with noninflamed tissue (P , 0.0001). Higher in inflamed tissue compared Not discussed. with noninflamed tissue (P , 0.0001). Higher in active CD and active UC Not discussed. compared with inactive IBD, IBS, and healthy controls. Not significantly different between Not discussed. active CD and inactive CD. Higher in CD (280 mg/g in active CD Predictive cut-off of 60 mg/g (84% 34 and 22 mg/g in inactive CD) sensitivity, 74% specificity, 81% compared with non-IBD PPV and 77% NPV) for active (0.56 mg/g) (P , 0.001). CD. Higher in active CD (280 mg/g) vs inactive CD (22 mg/g) (P , 0.001). Predictive cut-off of 324 mg/g (90% 35 Higher in IBD than they were in sensitivity and 88% specificity) bacterial infectious bowel for the diagnosis of active UC. disease, IBS patients or healthy volunteers (P , 0.01). Predictive cut-off of 240 mg/g (92% Higher in active UC than in inactive sensitivity and 80% specificity) UC (P , 0.01). Higher in active for the diagnosis of active CD. CD than in inactive CD (P , 0.01). 36 Predictive of active IBD over IBS Higher levels in IBD (mean 69.5 (67% sensitivity, 96% specificity, mg/g in UC and 41.4 mg/g in CD) 92% PPV, and 80% NPV). compared with either IBS (mean 1.4 mg/g) (P , 0.001) or controls Predictive of active UC over IBS (78% sensitivity, 96% specificity, (mean 2.4 mg/g) (P , 0.001). 86% PPV, and 92% NPV). Trend for higher fecal lactoferrin Predictive of active CD over IBS levels in UC than in CD (P 5 (58% sensitivity, 96% specificity, 0.051). 83% PPV, and 86% NPV). Higher in active IBD than in inactive IBD (P , 0.001 for UC and P 5 0.002 for CD). Higher in IBD and other forms of Predictive cut-off of 50 mg/g (79% 37 colitis compared with IBS sensitivity, 100% specificity, (P , 0.0001). Higher in active 100% PPV, 83% NPV, and 90% IBD compared with IBD in (Continued )

Settings

Biomarker

with ischemic colitis and 5 with medication-induced colitis)

Fecal levels of lactoferrin

491 IBD (373 CD, 118 UC) and 76 healthy volunteers

Multiple centers Germany

Serum levels of soluble TNF-a receptor TNF-RI

Serum levels of soluble TNF-a receptor TNF-RII

130 IBD patients (84 CD and 46 UC)

Mannheim, Germany

Serum levels of IL-8

remission (P 5 0.0007). Higher in moderately active UC compared with slightly active UC (P 5 0.0045). Higher in UC with moderate to severe inflammation versus UC with low inflammation (P 5 0.0335). Higher in CD moderate to severe endoscopic activity vs CD in remission (P 5 0.0001). Higher in IBD and patients with other forms of colitis compared with IBS patients (P , 0.0001). Higher in active IBD compared with IBD in remission (P 5 0.0016). Higher in CD with moderate to severe endoscopically-assessed activity compared with CD in remission (P 5 0.0008). Higher in CD in remission (P 5 0.0055), active CD (P , 0.0001), chronically active CD (P , 0.0001), UC in remission (P 5 0.0002), active UC (P , 0.0001), and chronically active UC (P 5 0.0003) compared with controls. Higher in CD in remission (P 5 0.0004), active CD (P , 0.0001), and UC in remission (P 5 0.01) compared with controls. Higher in UC and CD patients compared with historical data in a control group. Higher in UC compared with CD patients. Higher in CD and UC compared with historical data in a control group. Higher in CD compared with UC.

Sensitivity, specificity, PPV, and NPV of biomarker

Ref

accuracy) for CD in remission compared with IBS.

Predictive cut-off of 7 mg/g (79% sensitivity, 100% specificity, 100% PPV, 83% NPV, and 90% accuracy) for CD in remission compared with IBS.

Not discussed.

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Not discussed.

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Not discussed.

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Not discussed.

Not discussed.

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Serum levels of IL-18

Main findings

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Table I. (Continued )

Cleveland, Ohio and Milwaukee, Wisconsin

126 IBD patients (68 CD, 58 UC) and 71 healthy controls

Prague, Czech Republic

Expression of IL-4, IL-10 in mucosal T cell mRNA expression profiles of IL12Rb2, IL12p40 in mucosal biopsies Serum levels of galectin-3

139 IBD patients (61 active CD, 38 Vienna, Austria patients with CD in remission, 17 active UC, and 23 patients with UC in remission) and 97 controls (28 infectious enterocolitis, 32 lactose and/or fructose intolerant, and 37 healthy donors)

CD64 ex-pressed/ neutrophil

353 IBD patients (239 CD, 114 UC) Munich, Germany and 144 controls

Serum levels of CXCL16

Serum levels of C-reactive protein

Bahrain

Expression of RANTES

102 IBD (60 UC and 42 CD), 16 with other forms of colitis, 12 colorectal cancer patients and 40 normal subjects

Japan

Serum levels of growthrelated oncogene-a

Not discussed.

40

Not discussed.

41

Predictive cut-off of 800 CD64 molecules per PBMC neutrophil (88% sensitivity and 93% specificity) for active IBD compared with lactose and/or fructose intolerance. Predictive cut-off of 10,000 CD64 molecules per PBMC neutrophil (96% sensitivity and 97% specificity) for bacterial enterocolitis compared with active IBD. Not discussed.

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43

Not discussed.

Not discussed.

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Not discussed.

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(Continued )

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40 IBD patients (21 CD and 19 UC), 3 patients with colonic inflammatory lesions other than IBD and 15 controls with normal colonic biopsy results or noninflammatory lesions

Higher in late CD compared with early CD (P 5 0.001 for and IL-4 levels and P 5 0.002 for IL-10) Higher in early CD compared with late CD patients (P , 0.032 for and IL12Rb2 and P , 0.033 IL12p40). Higher in UC active, UC in remission, CD active and CD in remission vs controls (P , 0.001). Higher percentage of neutrophils expressing CD64 in IBD compared with healthy donors (P , 0.0001). Higher percentage of neutrophils expressing CD64 in active IBD compared with IBD in remission (P , 0.0001). Higher percentage of neutrophils expressing CD64 in bacterial enterocolitis compared with IBD (P , 0.0001). Higher in CD compared with UC or controls (P , 0.001). Higher in UC compared with healthy controls (P , 0.001). Higher in UC (P , 0.05) and CD (P , 0.01) with inactive disease compared with controls. Higher in UC (P , 0.001) and CD (P , 0.001) with active disease compared with controls. Epithelial expression higher in CD (P , 0.05) and UC (P , 0.05) compared with controls. Lymphocytes or histiocytes expression higher in CD (P , 0.05) and UC (P , 0.05) compared with controls and higher in UC compared with CD (P 5 0.04). Higher in active IBD compared with inactive IBD (P , 0.0001 for UC; P 5 0.0447 for CD).

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20 pediatric CD, 10 CD with longstanding CD, 4 with infectious colitis, 2 patients with eosinophilic colitis and 7 normal subjects without intestinal inflammation

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Table I. (Continued ) Settings

93 patients with inactive IBD (49 Berlin, Germany CD; 44 UC), 35 with active IBD (18 CD, 17 UC), and 37 age and body mass index-matched healthy controls

Biomarker

Serum levels of resistin Serum levels of visfatin Serum levels of retinolbinding protein-4 Levels of adiponectin Serum levels of insulin

10 with active IBD (4 CD, 6 UC), 10 Veldhoven, Nether lands IBD in remission (5 CD, 5 UC) and 10 controls

Urine levels of claudin-3

354 IBD (82 adult (57 CD and 25 UC) and 272 pediatric patients (229 CD; 43 UC) and 108 (88 adult; 20 pediatric) healthy controls

Serum levels of GM-CSF Ab

United States

Higher in inactive IBD compared with normal subjects (P , 0.0001 for UC; P 5 0.0067 for CD). Higher in UC with active disease compared with CD with active disease (P , 0.0084). Higher in active IBD patients compared with healthy controls. Higher in active UC patients compared with healthy controls. Higher in all IBD patients compared with healthy controls. Lower in all IBD patients compared with healthy controls. Higher in all IBD patients compared with healthy controls. Higher in IBD with active disease compared with IBD in remission (P 5 0.001) and healthy controls (P 5 0.001). Higher in IBD with either ileal or ileocolonic involvement compared with IBD with only colon involvement. Higher in CD with ileal or ileocolonic involvement compared with controls (median 2.4 mg/mL vs 0.4 mg/mL (P , 0.001) pediatric; median 11.7 mg/mL compared with 0.4 mg/mL (P , 0.001) for adults. Higher in CD carrying the allele rs13361189 compared with those no carrying SNPs in IRGM. Higher in adult CD compared with pediatric CD. Elevated GM-CSF Ab levels positively associated with gASCA seropositivity in CD and negatively associated with ANCA seropositivity in colonic CD.

Sensitivity, specificity, PPV, and NPV of biomarker

Not discussed.

Ref

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Not discussed. Not discussed. Not discussed. Not discussed. Not discussed.

47

Predictive cut-off of 0.08 mg/mL for 48 the development of stricturing/ penetrating behavior 108 months after CD diagnosis (71% PPV and 73% NPV) The presence of gASCA IgA (72% PPV and 59% NPV), gASCA IgG (74% PPV and 59% NPV), ANCA (61% PPV and 72% NPV), and anti-OmpC (76% PPV and 56% NPV) for the development of stricturing/penetrating behavior 108 months after CD diagnosis. Translational Research July 2012

Abbreviations: NPV, negative predictive value; PPV, positive predictive value.

Main findings

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Study population

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Population characteristics and study settings are presented in Table I. gASCA (both IgA and IgG) was the biomarker with the highest capacity to discriminate between CD and non-CD, as gASCA positivity was significantly more prevalent among CD patients than it was among UC patients, indeterminate colitis (IC) patients, or healthy controls.14-21 gASCA (both IgA and IgG) was also the biomarker with the highest capacity to discriminate between CD and UC.14-16,18-20,22 One study found that there was a 7 times higher likelihood that patients testing positive for gASCA would suffer from CD rather than UC.18 In contrast, pANCA seropositivity in IBD patients was associated with a diagnostic of UC.16,17,19,23 As such, pANCA was detected in a significantly higher proportion of UC patients compared with control subjects16,17,19,23 and to CD patients.16,19 Other study reports that pANCA was detected in a significantly higher proportion of CD patients than healthy donors,19 while a subsequent study failed to find a difference between these two groups.23 The prevalence of atypical pANCA was also significantly higher in UC patients than it was in CD patients or healthy donors.15,24 Several of these autoantibodies can be tested at once to obtain greater diagnostic power. Both gASCA for CD and pANCA for UC had lower power to differentiate between CD and UC as single predictors of disease type than a combination of the 2 (84% PPV).19 The combination of gASCA and pANCA produced the highest discrimination between CD and UC.21 An improvement in disease diagnosis based on gASCA IgG antibodies and pANCA was obtained when either ACCA (P 5 0.03), ALCA (P 5 0.04), antichitin (P 5 0.03), antilaminarin (P , 0.0001), and a combination of antichitin and antilaminarin (P 5 0.0001) were used in combination with gASCA.22 gASCA IgG (P , 0.001) and antilaminarin (P , 0.001) were the only biomarkers that could differentiate between isolated inflammatory colonic CD and UC.22 Another example involves the combination of pANCA, PAB, and GAB for the diagnosis of UC. pANCA and GAB were detected more frequently among UC patients than among healthy controls, with no difference between CD patients and healthy controls.23 PAB was associated with a diagnosis of CD.15,23 As such, pANCA and GAB can be used for the diagnosis of UC, and PAB can be used for the diagnosis of CD.23 Testing positive for either pANCA or GAB and negative for PAB (ie, pANCA1 or GAB1/PAB-) was associated strongly with UC compared with CD or healthy controls.23 Malickova et al20 found that whereas none of the serum anticarbohydrate antibodies assayed were useful in differentiating CD from UC on their own, they can be

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used to improve to the power of gASCA and pANCA.20 Adding antichitin and antilaminarin increased the power of this model to differentiate between CD cases and non-CD cases compared with gASCA alone (P 5 0.0002) and between CD cases and UC cases compared with gASCA and pANCA alone (P 5 0.006).21 Studies did identify significantly higher levels of ACCA, ALCA, AMCA, antichitin, and antilaminarin in CD patients compared with non-IBD patients and healthy controls,21 and even compared with UC patients.22 In a separate study, only antibodies against mannan and mannan residues, ACCA and ALCA were significantly higher in CD patients than they were in UC patients, thus showing a satisfactory discriminatory potential between the 2 conditions (P , 0.01).14 Also, significantly more CD patients were positive for antiOmpC compared with UC patients and healthy controls.17 In a sample of 818 IBD patients (517 CD patients and 301 UC patients), 73% of CD patients were positive for at least 1 of ACCA, ALCA, AMCA, antichitin, antilaminarin and gASCA (both IgG and IgA), whereas 72% of UC patients were negative for all 7 of these antibodies.22 Following gASCA (both IgA and IgG), antilaminarin, AMCA, and ALCA were the biomarkers with the highest capacity to discriminate between CD and UC.22 Several interesting links between the presence of certain serum autoantibodies biomarkers and disease onset, behavior, or localization were discovered. For example, the presence of gASCA seems to be associated with a younger age at disease onset among CD patients (P 5 0.01 to P , 0.0001).15,17,22 The levels of gASCA were higher in CD patients diagnosed prior to the age of 40 (P 5 0.06), particularly prior to the age of 17 (P , 0.001), compared with CD patients diagnosed after the age of 40.19,20 As a positive correlation was found between gASCA levels and the duration of CD (P , 0.05), it is unclear whether gASCA levels truly correlate with age at onset.20,22 Higher levels of PAB (P , 0.05)15 and anti-OmpC (P 5 0.025)17 were associated with younger age at onset among CD patients, whereas higher pANCA levels were associated with disease after the age of 40 rather than before the age of 40 among UC patients (P 5 0.001).19 Antilaminarin had the strongest association with CD complications. In addition, gASCA and AMCA were also associated with CD complications in a multivariate analysis.21 gASCA, AMCA, and antilaminarin were associated with a previous occurrence of disease complications.21 Furthermore, gASCA was associated with a higher prevalence of CD behavior progression during a 10-year period (P 5 0.001).19 Higher levels of gASCA (both IgA and IgG) were associated strongly with ileal

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involvement in CD patients (P 5 0.01 to P , 0.0001).14,17,20-22 Higher levels of gASCA (both IgA and IgG) at CD diagnosis were associated with either stricturing and/or penetrating behavior compared with nonstricturing, nonpenetrating behaviors (P , 0.05 to P , 0.0001).14,17,19,20,22 In contrast, gASCA was associated negatively with nonstricturing and nonpenetrating phenotype (P , 0.05)22 and with inflammatory disease (P 5 0.02 to P , 0.0001).17,19 AMCA, ACCA, antilaminarin, and antichitin were also associated positively with penetrating phenotype (P 5 0.01) and associated negatively with the nonstricturing and nonpenetrating phenotypes (P , 0.05).22 Anti-OmpC in CD patients was associated with penetrating behavior (P 5 0.001) and noninflammatory behavior (P , 0.0001).17 gASCA IgA positivity was associated with perianal disease among CD patients (P 5 0.04 to 0.002),17,18,22 especially perianal abscess, anal fissure, and anal fistula (P , 0.001).15 AMCA, antilaminarin, and antichitin were also associated with perianal disease (P , 0.01).22 Being positive for gASCA (both IgA and IgG) at CD diagnosis was associated with higher odds of requiring abdominal surgery (P 5 0.05 to P 5 0.001),17-19,22 especially during a follow-up period of 10 years (P 5 0.001).19 ACCA, ALCA, antilaminarin, antichitin,21,22 and anti-OmpC (P 5 0.05 to P , 0.0001)17 were also associated with disease requiring surgery. Among CD patients, gASCA (P 5 0.02) 18 and antiOmpC (P 5 0.019)17 were associated with the presence of extraintestinal manifestations. Overall, gASCA can be used as a diagnostic tool to help predict the presence of CD and to help differentiate CD from UC and other GI complications. Serum anticarbohydrate antibodies, although not particularly useful on their own, can be used to improve the power of gASCA,20 whereas the presence of gASCA, ACCA, ALCA, AMCA, antichitin, and antilaminarin can help predict disease complications and the need for surgery in CD patients.21 It is unclear whether an association between gASCA positivity and the presence of single-nucleotide polymorphisms (SNP) in the IBD susceptibility genes caspase recruitment domain family, member 15/nucleotide-binding oligomerization domain-containing protein 2 (CARD15/NOD2) (R702W, G908R, and 1007finsC) and drosophila discs large homolog 5 (the 113A variant or the TC haplotype) exists, as a gene dosage effect was observed in 1 study in which higher proportions of patients testing positive for either gASCA (P , 0.0001) or anti-OmpC (P 5 0.007) possessed more CARD15/NOD2 variant alleles.17 However, such observations were not confirmed by subsequent studies.18,19

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Phagosome-derived S100 proteins. Phagocyte-derived S100 proteins are endogenous activators of innate immune responses. One such protein, the S100A8/ S100A9 complex (calprotectin), is a ligand for Tolllike receptor (TLR)4.33 In contrast, the neutrophil activation marker S100A12 is a calcium-binding, proinflammatory protein secreted by granulocytes that binds to the receptor for advanced glycation endproducts and that has been associated with different inflammatory diseases, among them IBD.32,33 The levels of fecal calprotectin were significantly higher in IBD patients in general than they were in IBS patients or in healthy controls.25-28,29 The levels of fecal calprotectin were significantly higher in IBD patients (both CD patients and UC patients) with disease in relapse at the last endoscopic analysis than they were in the same population at baseline or in IBD patients with disease in remission.26,28,29 Despite fecal calprotectin levels not being an indicator of the time lapse between baseline and when relapse would occur in patients who relapsed within 2 years of baseline, a significantly higher proportion of IBD patients who relapsed within 3 years of baseline had fecal calprotectin levels in excess of 275 mg/g than the proportion of IBD patients who did not relapse.29 Levels of fecal calprotectin correlated with inflammation, as they differed significantly between patients with inflamed mucosa and patients with noninflamed mucosa among asymptomatic IBD patients.25,28 The levels of fecal calprotectin correlated with both microscopic [the severity of changes in crypts, enterocytes, and cellularity of the lamina propria (mononuclear cells and neutrophils) were graded in each biopsy specimen] and macroscopic (the macroscopic appearance of the colonic mucosa, subdivided into cecum, ascending colon, right flexure, transverse colon, left flexure, descending colon, sigmoid, and rectum, was evaluated) scores of inflammation.25 Higher microscopic and macroscopic scores were indicators of disease severity. In terms of microscopic damage, levels of fecal calprotectin correlated with all of changes in crypts (P , 0.001), enterocytes (P , 0.001), mononuclear cells (P , 0.001) and neutrophils (P , 0.001). A cutoff level of 50 mg/g for fecal calprotectin was used a predictive tool for the development of microscopic inflammation in this pediatric IBD population. The maximal sum between sensitivity and specificity was achieved with a fecal calprotectin concentration of 85.7 mg/g. This result suggests that this concentration would be optimal to discriminate between inflamed and noninflamed mucosa in pediatric IBD patients.25 The levels of fecal S100A12 were also significantly higher in IBD patients at the time of diagnosis compared with non-IBD subjects, including IBS patients.26,27,30

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No significant differences were found between CD patients and UC patients. Fecal S100A12 levels were significantly higher in IBD patients with active disease compared with IBD patients with inactive disease. Age and sex had no significant influence of fecal S100A12 levels.26 Although 0.8 mg/g has been used to distinguish active CD and active UC from healthy controls and active IBD from IBS,26 a fecal S100A12 level of 10 mg/g is a more reliable diagnostic tool to differentiate patients with IBD at diagnosis from healthy controls.27,30 After treatment with an exclusive enteral nutrition with a polymeric formula, significantly lower levels of fecal S100A12 were observed in patients who achieved remission (P , 0.05).30 Higher C-reactive protein levels (P , 0.001) and erythrocyte sedimentation rate (P 5 0.003) were other biomarkers distinguishing active IBD from IBS.26 Infiltrating cells in inflamed areas of the gut were shown by immunostaining to express S100A12. S100A12 was absent from the epithelial layer, but it was present on granulocytes in the subepithelial layer. S100A12 was also observed in the extracellular vicinity of S100A12positive cells, showing that this granulocyte-derived proinflammatory protein gets secreted during a state of inflammation. S100A12 staining was found to correlate with the histology inflammation score (P , 0.05) and with fecal levels of S100A12 (P 5 0.05) but not with the clinical activity scores.26 The levels of serum calprotectin were significantly higher in IBD patients (CD patients and UC patients, but not in IC patients) compared with controls.1 The levels of serum calprotectin were significantly correlated with levels of C-reactive protein (P , 0.05) and with the Pediatric Crohn’s Disease Activity Index (P , 0.05).31 The levels of serum S100A12 were correlated significantly with levels of fecal S100A12 (P , 0.05)30 and serum calprotectin in IBD patients (P , 0.0001) and non-IBD controls (P , 0.01) but not in celic disease patients.31,27 Serum S100A12 levels were associated significantly with the inflammatory markers C-reactive protein levels (P , 0.001) and serum amyloid A levels (P 5 0.018).32 Serum S100A12 levels seemed to be higher in active IBD patients than in inactive IBD patients. However, this biomarker cannot be used as a predictor of disease activity as this difference was not significant.32 Furthermore, serum S100A12 levels could not be use to predict the extent of disease in UC patients, the location and behavior of disease in CD patients, the duration of disease in IBD patients, or the presence of extraintestinal symptoms in IBD patients. No significant fluctuations were found in serum S100A12 levels with respect to sex, age, smoking status, or treatment modalities in IBD patients.32

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The mucosal release of calprotectin was not significantly different between active CD cases and inactive CD cases. The mucosal release of calprotectin was not significantly different between active ileal CD cases and inactive CD cases.33 The mucosal release of S100A12 was higher in active ileal CD than it was in inactive CD (P , 0.05).33 A significantly higher intestinal release of calprotectin and S100A12 was observed in biopsy specimen collected from IBD patients compared with IBS patients and healthy controls.33 The release of phagocytic S100 proteins was location specific, with significantly less S100A12 release (P , 0.01) and calprotectin release (P , 0.05) observed from inflamed ileal sites compared with inflamed colonic sites.33 A significant correlation between both S100A12 tissue release and calprotectin tissue release, and tissue inflammation was observed in IBD patients. A positive correlation between supernatant S100A12 levels and calprotectin levels was observed.33 The release of both S100A12 and calprotectin was attenuated by treatment in active IBD cases. Staining for S100A12 revealed that there was strong subepithelial staining, suggesting that the significant infiltration of S100A12-expressing granulocytes.33 S100A8, S100A9, and S100A12 were detected in the lamina propria but not in the epithelium in biopsies obtained from noninflamed patients. S100A8, S100A9, and S100A12 were more abundant in biopsies obtained from inflamed patients, and they were detected in both the lamina propria and throughout the epithelium. S100A8, S100A9, and S100A12 expression was detected on CD151 neutrophils and CD681 macrophages.31 However, the levels of serum soluble receptor for advanced glycation end products (sRAGE) (a putative S100 ligand) were not different between groups, and levels of mucosal sRAGE were undetectable by enzyme-linked immunosorbent assay (ELISA; lower limit of detection was 78 pg/mL).31 Lactoferrin. Activated leukocytes infiltrate the intestinal mucosa during the course of IBD and can be subsequently detected in feces as they are shed into the intestinal lumen. Lactoferrin is an iron-binding glucoprotein that can reflect the activity of neutrophil leukocytes and can be excreted in its iron-free form into most exocrine fluids. Lactoferrin levels have been shown to increase dramatically in body fluids during the course of inflammation. In particular, fecal lactoferrin has been used as a specific and sensitive noninvasive biomarker of intestinal inflammation.34 The levels of fecal lactoferrin were significantly higher in IBD patients, especially CD patients, than they were in non-IBD patients (bacterial infectious bowel disease patients, IBS patients, or healthy volunteers).34-36

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There was also a nonsignificant trend for higher fecal lactoferin levels in UC patients than in CD patients.36 The levels of fecal lactoferrin were significantly higher in active IBD patients than they were in inactive IBD patients.34-36 The levels of fecal lactoferin were significantly higher in patients with inactive IBD than in patients with IBS (P , 0.001 for inactive UC and P 5 0.002 for inactive CD).36 Based on these findings, fecal lactoferrin can be used to diagnose disease activity in IBD patients36 with cut-off values between 60 mg/g34 and 240 mg/g for active CD and 324 mg/g for active UC.35 The levels of fecal calprotectin and the levels of fecal lactoferrin were increased significantly both in IBD patients and patients with other forms of colitis than they were in IBS patients. These parameters were not useful in discriminating IBD from other forms of colitis.37 Higher levels of both fecal calprotectin and fecal lactoferrin were observed in patients with active IBD compared with IBD in remission. Higher levels of fecal calprotectin but not fecal lactoferrin were observed in patients with UC characterized as moderately active compared with UC characterized as slightly active. Higher levels of both fecal calprotectin and fecal lactoferrin were observed in CD patients with moderate to severe endoscopically assessed activity compared with CD patients in remission.37 Proinflammatory cytokines. The levels of TNF-a receptors TNF-RI and TNF-RII, which are TNF-a antagonists that can inhibit TNF-a-mediated proinflammatory effects, usually cell surface-bound but can be released by proteolytic cleavage, were measured as potential biomarkers for IBD.38 Significantly increased levels of soluble TNF-RI in serum samples from CD patients in remission (P 5 0.0055), active CD patients (P , 0.0001), chronically active CD patients (P , 0.0001), UC patients in remission (P 5 0.0002), active UC patients (P , 0.0001), and chronically active UC patients (P 5 0.0003) were compared with controls.38 Significantly increased levels of soluble TNF-RII were measured in CD patients in remission (P 5 0.0004), active CD patients (P , 0.0001), chronically active CD patients (P , 0.0001), and UC patients in remission (P 5 0.01) compared with controls. Because of the significantly higher levels of TNF-RII in CD patients compared with UC patients, this biomarker has the potential to discriminate between these two types of IBD.38 The cytokines and markers of mucosal damage were analyzed in biopsies collected from both affected areas and unaffected areas in 42 IBD patients (26 UC patients and 16 CD patients).49 Messenger RNA (mRNA) levels of extracellular matrix-remodeling molecule-3 (P 5 0.018) and its physiologic inhibitor TIMP-1 (P 5 0.001) were in-

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creased in the affected areas from UC patients compared with controls. No changes in the mRNA levels of extracellular matrix-remodeling molecule-3 were observed between CD patients and controls, whereas the mRNA levels of TIMP-1 were increased in both affected areas from CD patients (P 5 0.043) and unaffected areas from CD patients (P 5 0.047) compared with controls. The mRNA levels of inducible nitric oxide synthase, which is a key inflammatory mediator that increases nitric oxide levels (P , 0.001), and apoptosis-related molecule granzyme B (P 5 0.006), which is a protein crucial for the rapid induction of apoptosis by cytotoxic T cells in the context of cell-mediated immune response, were increased in the affected areas from UC patients compared with the unaffected areas from UC patients. The mRNA levels of inducible nitric oxide synthase were increased in both the affected areas from CD patients (P , 0.001) and the unaffected areas from CD patients (P 5 0.019) compared with controls. The mRNA levels of granzyme B were increased in the affected areas from CD patients only (P 5 0.009) compared with controls.49 No correlation was observed between the mRNA levels of either of these molecules involved in tissue damage and histologic score of mucosal damage in affected areas from either UC patients or CD patients. Moreover, cytokine mRNA levels did not correlate with histologic damage.49 The mRNA levels of the proinflammatory cytokines TNF-a and IFN-g are increased in IBD patients compared with controls. The protein levels of IFN-g were increased in the affected areas (P 5 0.033) and unaffected areas (P 5 0.041) from UC patients, but not from CD patients, compared with controls.49 The mRNA levels of interleukin (IL)-6 were increased in the affected areas (P , 0.001) and unaffected areas (P 5 0.002) in UC patients and in the affected areas (P 5 0.01) and unaffected areas (P 5 0.004) in CD patients compared with controls. The IL-15 mRNA levels were increased in both the affected areas (P 5 0.003) and unaffected areas (P 5 0.001) in UC patients, but not in CD patients, compared with controls. IL-15 was expressed in all IBD samples at the protein level but not in controls. IL-18 mRNA levels were increased in the affected areas (P 5 0.002) and unaffected areas (P , 0.001) in UC patients and in the affected areas (P 5 0.026) in CD patients compared with controls. IL-18 was expressed in all IBD samples at the protein level and only sporadically in controls.49 IL-23 (p19) mRNA levels were increased in both the affected areas (P 5 0.012) and unaffected areas (P 5 0.02) in CD patients, but not in UC patients, compared with controls. IL-1b mRNA levels were increased in the affected areas (P 5 0.009) and unaffected areas (P 5 0.008) in UC patients and in the affected areas (P 5 0.017) and

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unaffected areas (P 5 0.004) in CD patients compared with controls. IL-4 mRNA levels were increased in affected areas in UC patients compared with both controls (P 5 0.009) and unaffected areas (P 5 0.056). IL-6 levels are increased in all patient samples compared with controls, with the higher levels being observed in affected areas in UC patients (P 5 0.015). IL-12 (p40) mRNA were increased in affected areas in UC patients compared with both controls (P 5 0.023) and unaffected areas in UC patients (P 5 0.015). No changes in IL-12 (p40) mRNA levels were observed in CD patients. IL-12 (p35) mRNA were increased in the affected areas in UC patients compared with controls (P 5 0.044). IL-27 (p28) mRNA levels were increased in affected areas in UC patients compared with both controls (P 5 0.01) and unaffected areas in UC patients (P 5 0.044) and in affected areas in CD patients compared with controls (P 5 0.027).49 The levels of IL-1b, IL-4, IL-6, IL-12 (both p35 and p40), and IL23 correlate significantly with the levels of mucosal damage biomarkers nitric oxide synthase and granzyme B in the affected areas in UC patients. These correlations were not significant in the affected areas in CD patients. The levels of IL-12 (p40) (P 5 0.002) and IL-15 (P 5 0.026) were increased in the affected areas in UC patients compared with the affected areas in CD patients.49 The suppressor of cytokine signaling (SOCS)-2 mRNA, which is a protein belonging to the cytokineinducible negative regulators of cytokine signaling family, was increased in the affected areas (P 5 0.003) and unaffected areas in UC patients (P 5 0.041) compared with controls. No changes were observed in CD patients. The levels of SOCS-3 mRNA were increased in the affected areas in UC patients (P , 0.001 compared with control and P , 0.001 compared with the unaffected areas in UC patients), unaffected areas in UC patients compared with controls (P 5 0.027), and affected areas in CD patients compared with controls (P , 0.001). No changes were observed in the mRNA levels of SOCS-4, SOCS-5, transforming growth factor (TGF)-b, and IL10 between patients and controls.49 The levels of IL-8 and IL-18 were above normal ranges in both CD patients and UC patients compared with historical data in a control group.39 IL-8 levels were higher in UC patients than they were in CD patients, whereas IL-18 levels were higher in CD patients than they were in UC patients. IL-12p70 and IFN-g levels were within normal limits in both CD patients and UC patients. Both IL-8 levels and IL-18 levels were deemed appropriate biomarkers of disease activity (assessed by the Crohn’s disease activity index for CD and the clinical activity index for UC) in IBD patients.39

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The immunoregulatory events that give rise to IBD symptoms, the T helper response profile (Th1, Th2, or Th17), and the different cytokine expression profiles associated with different stages throughout the evolution of these events in pediatric patients were investigated in mucosal biopsies and T cell clones derived from CD patients.40 Pediatric CD patients with a duration of disease of 0 to 6 months since diagnosis were used to examine the early mechanisms of gut inflammation before masking or modification by disease evolution or therapy could occur. The IL-2 and IFN-g expression profiles in mucosal T cell clones were nonsignificantly different between groups. The IL-4 levels (P 5 0.001) and IL-10 levels (P 5 0.002) were significantly higher in children with late CD compared with children with early CD. IFN-g expression was enhanced significantly in cells from children with acute colitis induced by Campylobacter or Aeromonas incubated with IL-12 (P 5 0.0004) but not with IL-4, compared with cells incubated with IL-2. IFN-g expression was significantly enhanced in cells from children with early CD incubated with IL-12 (P 5 0.0001) but not with IL-4, compared with cells incubated with IL-2, mimicking the response observed in acute colitis. No significant modulation in IFN-g expression was observed when cells from children with early CD were stimulated. These results confirm the involvement of Th1 in the pathogenesis of CD and point toward a disease stage-dependent mucosal T cell response.40 Based on this, T cell clones from early CD patients with upregulated IFN-g expression should also express the high-affinity IL-12 receptor (IL12R). In these cells, IL-12 responsiveness was associated with the expression of the IL12Rb2 chain at the mRNA level, which is expressed exclusively by the Th1 response, assessed by reverse transcriptasepolymerase chain reaction (RT-PCR). Increased IL12Rb2 expression in response to increased IFN-g expression was observed in all T cell clones with upregulated IFN-g expression, regardless of whether they originated from acute colitis patients (P 5 0.03 in acute colitis vs normal) or early CD patients (P , 0.05 in early CD vs normal and P , 0.05 in early CD vs late CD).40 The mRNA expression of IL12Rb2 chain and IL12p40 chain were quantified by real-time PCR in mucosal biopsies belonging to 15 early CD patients and 6 late CD patients. The levels of both IL12p40 chain (P 5 0.033) and IL12Rb2 chain (P 5 0.032) were significantly higher in early CD patients than they were in late CD patients.40 Other cytokines whose expression was modulated differently between early CD and late CD include IL-4, whose expression was increased by IL-4 stimulation (P 5 0.0025) but not by IL-12 stimulation, and IL-10, whose expression was increased by

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IL-12 stimulation (P , 0.0001) but not by IL-4 stimulation in early CD only.40 Mast cell activation has been linked with IBD. No increase in the number of mast cells was observed between 15 inactive IBD patients and 7 controls. However, a greater fraction of these cells are activated in IBD patients than in controls.50 Other biomarkers in humans. Members of the galectin family, particularly b-galactoside termini of glycan chains, are known to be involved in cell adhesion and growth. Galectin-3 is important for its function as an immune system mediator. The serum concentrations of galectin-3 were measured in IBD patients and healthy controls. Patients deemed to be in the remission stage of the disease showed no signs of disease activity at the time of sampling.41 Serum galectin-3 levels were significantly higher in UC patients with active disease, UC patients in remission, CD patients with active disease, and CD patients in remission than they were in healthy controls. Galectin-3 expression, which is assayed by immunohistochemistry with a panel of monoclonal antibodies for distinct cell surface determinants used to classify reactive cells, was observed in CD14-positive cells in IBD tissue samples, whereas the expression on enterocytes was minimal. Moreover, CD14 expression was higher in tissue samples from IBD patients with active disease than it was in IBD patients in remission, whereas no CD14-positive cells were observed in healthy controls. Fluorescein isothiocyanate labeling of nonpathogenic Escherichia coli O83, used as a measurement of bacterial adhesion, revealed colocalization and overlap of these signals with galectin-3 expression. The binding sites for bacterial adhesion were observed on CD14 cells from IBD patients, but not from healthy controls. Furthermore, co-localization of bacterial signals and the potent glycoprotein ligand asialofetuin was observed.41 Similar results were reported in a dextran sodium sulfate (DSS)-induced colitis mouse model. The cells with binding sites for fluorescein isothiocyanate-labeled bacteria were identified as CD14 cells. Fluorescein isothiocyanate-labeled bacteria were identified only in DSS-treated animals. Furthermore, the presence of galectin-3 was shown to coincide with asialofetuin reactivity.41 Overall, this study showed that galectin-3 is present in serum collected from IBD patients, either during active disease or remission, galectin-3 is expressed primarily in CD14 cells in IBD patients and DSS-treated mice, and galectin-3 expression coincides with bacterial binding. Upregulation of galectin-3 expression in intestinal tissue samples points to the potential of using galectin-3 as a drug target.41

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A higher percentage of polymorphonuclear cells (PBMCs), which are neutrophils expressing CD64, was found in IBD patients than in healthy donors (P , 0.0001), in patients with active IBD than in patients with IBD in remission (P , 0.0001), and in patients with bacterial enterocolitis than in IBD patients (P , 0.0001) when the association between the quantitative expression of the high-affinity IgG1 and IgG3 receptor FcRI (CD64) on PBMC neutrophils and the presence of IBD was analyzed.42 Numerous PBMC neutrophils expressing CD64 were found in the lamina propria or adjacent to the epithelium in patients with active CD, suggesting that PBMC neutrophils expressing CD64 infiltrate the intestinal mucosa in inflamed colonic tissue.42 The quantitative expression of CD64 was significantly higher in patients with active IBD compared with patients with IBD in remission (P , 0.0001) and in both in patients with active IBD (P , 0.0001) and in patients with IBD in remission (P 5 0.003) compared with healthy donors.42 Higher CD64 expression on PBMC neutrophils was associated with higher Crohn ’s disease activity index (P , 0.0001), higher colitis activity index (P , 0.0001), and higher levels of C-reactive protein in both CD patients (P , 0.0001) and UC patients (P , 0.0001).42 To discriminate between patients with different intestinal diseases, the CD64 levels in IBD patients with active disease were lower than CD64 levels in infectious enterocolitis (P , 0.0001) but were higher than CD64 levels in lactose or fructose intolerant (noninflammatory intestinal conditions) patients (P , 0.0001).42 Serum levels of soluble chemokine (C-X-C motif) ligand 16 (CXCL16), a small protein combining the functionality of a pattern recognition receptor with the properties of an inflammatory chemokine, were significantly higher in CD patients compared with UC patients and healthy controls (P , 0.001).43 Also, the serum levels of soluble CXCL16 were significantly higher in UC patients compared with healthy controls (P , 0.001). There were no significant differences in serum levels of soluble CXCL16 between active cases of IBD and inactive cases of IBD.43 Serum levels of C-reactive protein were higher in both UC patients (P , 0.05) and CD patients (P , 0.01) with inactive disease and even higher in both UC patients (P , 0.001) and CD patients (P , 0.001) with active disease compared with controls. A significant correlation was found between higher levels of soluble CXCL16 and higher levels of C-reactive protein (P , 0.05) in a multivariate analysis taking into account age, sex, body mass index, white blood cell count, resistin, and C-reactive protein. This relationship lost statistical significance when disease activity was added into the model (P 5 0.078).43

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Higher serum levels of soluble CXCL16 were associated with CD (P , 0.001), particularly active CD. Higher levels of C-reactive protein were also observed in CD patients, yet this proved a less powerful test, especially in patients with inactive CD. Higher serum levels of soluble CXCL16 were associated with UC (P , 0.01), with no difference between cases of active UC and inactive UC. Higher levels of C-reactive protein were also observed in UC patients, and C-reactive protein proved to be a better biomarker for UC than CXCL16. Overall, this study showed that CXCL16 may play a proinflammatory role in IBD patients, particularly CD patients.43 Regulated on activation, normal T cell expressed, and secreted (RANTES) expression was detected in crypt epithelial cells and inflammatory cells (especially lymphocytes and histiocytes) in mucosal biopsies belonging to IBD patients and controls with normal colonic biopsy results or noninflammatory lesions.44 Both epithelial and lymphocytes or histiocytes RANTES expression were higher in CD patients and UC patients compared with controls (P , 0.05). RANTES expression was higher in UC patients than it was in CD patients (P 5 0.04), showcasing the suitability of this biomarker as potential diagnostic tool to differentiate between CD patients and UC.44 Growth-related oncogene-a, measured by ELISA, was found in sera belonging to IBD patients, patients with other forms of colitis, colorectal cancer patients, and normal subjects. Higher levels of oncogenea were found in IBD patients, patients with other forms of colitis and patients with colorectal cancer, compared with normal subjects.45 Higher levels of oncogenea were measured in UC patients (P , 0.0001) and CD patients (P 5 0.0447) with active disease compared with respective IBD patients with inactive disease, in UC patients with active disease compared with CD patients with active disease (P , 0.0084), and in UC patients (P , 0.0001) and CD patients (P 5 0.0067) with inactive disease compared with normal subjects.45 Serum oncogene-a levels (P 5 0.0015 in UC patients and P 5 0.0076 in CD patients), serum C-reactive protein levels (P 5 0.0022 in UC patients and P 5 0.0022 in CD patients), and serum IL-6 levels (P 5 0.0128 in UC patients and P 5 0.0029 in CD patients) decreased after therapy initiation.45 Significant associations between oncogene-a levels and C-reactive protein (P , 0.005 in UC patients and P , 0.05 CD patients) and between oncogene-a levels and IL6 levels (P , 0.001 in UC patients and P , 0.005 in CD patients) were found. In light of these findings, the serum levels of oncogene-a were proposed as a potential diagnostic tool for predicting disease activity in IBD patients.45

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Most adipokines, fat-derived hormones, and cytokines with immune-modulating and metabolic properties are associated with insulin resistance. Circulating serum levels of adipokines (leptin, resistin, visfatin, retinol-binding protein-4 and adiponectin), glucose homeostasis parameters (glucose and insulin levels), and inflammatory parameters were measured in IBD patients and healthy controls.46 Most IBD patients had increased levels of total, saturated, and monounsaturated fatty acids compared with healthy controls. Increased levels of polyunsaturated fatty acids compared with controls were observed only in patients with inactive CD. Inflammatory markers were increased in patients with IBD compared with controls, yet these levels were within normal ranges for more than 70% of patients with inactive IBD.46 Circulating levels of leptin, which is a protein hormone that plays a key role in regulating energy intake and energy expenditure, were similar between controls and IBD patients, and they correlated well with fat mass in all 3 groups of subjects, with no differences between CD patients and UC patients. Higher leptin concentrations were observed in UC patients who relapsed within 3 months compared with UC patients who did not relapse during this time period (P 5 0.026). In CD patients, no associations between leptin levels and inflammatory parameters or actual disease activity and relapse were observed.46 The levels of resistin, which is a cytokine associated with obesity and type II diabetes mellitus, were higher in active IBD patients compared with controls. No differences were observed between patients with inactive IBD and controls. Resistin levels were correlated with Crohn’s Disease Activity Index, Colitis Activity Index, and inflammatory markers with the exception of IL-6. Resistin levels were not correlated with either body fat mass or plasma fatty acids.46 The levels of visfatin, which is a protein that mimics the effects of insulin, were higher in active UC patients compared with healthy controls but were comparable between active CD patients, inactive IBD patients, and controls. Visfatin was not associated with inflammatory parameters or body composition.46 The levels of retinol-binding protein-4, which is an adipokine that induces insulin resistance, were increased in all IBD patients, regardless of the stage of disease. A positive correlation was observed between retinol-binding protein-4 levels and circulating fatty acids levels (P , 0.001). The levels of retinol-binding protein-4 were not correlated with inflammatory parameters or body composition. Higher levels of retinolbinding protein-4 were observed in CD patients compared with UC patients, regardless of disease activity (P 5 0.033).46

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Significantly lower levels of adiponectin, which is a protein hormone that regulates the metabolism of lipids and glucose, were observed in all IBD patients compared with controls, regardless of disease type (CD or UC) and state (active or inactive). Significantly negative correlations were observed between adiponectin levels and retinol-binding protein-4 (P , 0.001), patient body mass index (P , 0.005), and patient body fat mass in women only (P 5 0.034).46 Whereas serum glucose levels were comparable among all patients, increased levels of insulin were observed in all IBD patients compared with controls. The homeostasis model assessment test was employed: insulin [mU/mL (1 pg of insulin equals 1/34.2 mU)] glucose (milligrams per 100 mL)/405]. Values of ,1 are considered normal and values of .2.5 indicate a high probability of insulin resistance. A positive correlation between insulin concentrations and body fat mass (P , 0.001) and plasma fatty acids (P 5 0.008) was observed in IBD patients. Furthermore, hyperinsulinemia was associated with increased levels of IL-6. A negative correlation between insulin concentrations and adiponectin was observed in both IBD patients (P , 0.001) and controls (P 5 0.037). Adiponectin levels were normal in patients with insulin levels #180 pg/mL.46 Higher levels of insulin (P 5 0.006) and higher values in the homeostasis model assessment test (P 5 0.017) were associated with a decreased risk of IBD relapse, yet these were not associated with adipokine levels or inflammatory parameters. The protective role of hyperinsulinemia was more pronounced in CD patients, whom were observed to relapse less frequently. In multivariate analysis, homeostasis model assessment test values between 1 and 2.5 were found to be more protective than values of .2.5 (P , 0.005).46 IBD is primarily a disease of the GI tract, yet other organs such as the skeleton are often affected as well. Treatment-naive children offer the best opportunity to study the effects of IBD on the skeleton, as the observed low bone density and bone mass loss at diagnosis is likely to be caused by the disease only, with minimal confounding contributions from drugs such as corticosteroids.9 Possible explanations for these symptoms include compromised nutritional intake, malabsorption, delayed puberty, growth retardation, vitamin D deficiency, and factors that inhibit osteoblasts as observed in vitro in serum belonging to pediatric IBD patients. Bone abnormalities in pediatric IBD patients were analyzed at the tissue level in newly diagnosed IBD children.9 The average height for this sample was normal. However, mean weight was slightly low (P 5 0.05). Lumbar spine areal bone mineral density (P 5 0.002) and bone mineral apparent density (P 5 0.04), total body bone mineral content (P , 0.001), total body

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lean mass (P , 0.001), and height were significantly lower than the results expected for healthy subjects in the respective age and sex groups, whereas the ratio between total body BMC and lean mass was normal for the respective age group.9 Serum levels of calcium, phosphorus, parathyroid hormone, and creatinine were within normal levels for all participants. No significant correlation between the 25-OH vitamin D status and total body lean mass was observed. The ratio between the bone resorption marker urinary cross-linked N-telopeptide of type I collagen and either creatine (P , 0.01) or the carboxy-terminal propeptide of type I collagen (P , 0.01) were below normal ranges for the respective age groups, indicating low bone metabolism.9 The average cortical width of ileac bone was below the mean value for the respective age group (P , 0.001). A mineralization defect was diagnosed from a reduced osteoid thickness. Interestingly, both osteoid thickness and osteoid volume were low, yet resorption parameters were not increased, as a low osteoclast surface was observed in conjunction with a large eroded surface.9 The suitability of using urine levels of claudin-3 as a noninvasive technique for the detection of early intestinal tight junction breakdown, which is an early indication of gut wall integrity loss and IBD development, was tested in rats after exposure to hemorrhagic shock.47 Claudin-3 is one of the many proteins making up tight junctions, and it is expressed in high quantities in the intestine. A significant loss of claudin-3 was observed in the colon of rats 60 min after being exposed to hemorrhagic shock compared with control rats. A similar loss of claudin-3 was observed in inflamed colonic tissue samples collected from patients with active IBD and patients with IBD in remission. These data indicate a claudin-3 loss after intestinal injury.47 Significant increases of claudin-3 were observed in the urine of rats 90 min after exposure to hemorrhagic shock (P 5 0.05). Similar findings were observed in human patients, with significantly higher concentration of claudin-3 in the urine of IBD patients in the relapse stage compared with healthy volunteers (P 5 0.001) and IBD patients in the remission stage (P 5 0.001). Increased urine concentrations of claudin-3 in both the rat model and in human IBD patients in the relapse stage correlated with reduced intestinal immunostaining.47 Plasma levels of C-reactive protein, higher levels of which were used as an indicator of inflammation and IBD severity, were measured in patients. These were found to be higher in patients with active IBD than patients with IBD in remission (P , 0.05). This shows that a strong correlation exists between urinary concentration of claudin-3 and severity of IBD, as measured by C-reactive protein concentrations (P 5 0.0003).47

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The role played by serum-neutralizing antibodies directed against granulocyte-macrophage colony-stimulating factor (GM-CSF Ab) in regulating neutrophil function in CD patients was analyzed.48 Higher levels of serum glycosylated GM-CSF-binding GMCSF Ab, as well as unglycosylated GM-CSF-binding GM-CSF Ab were observed in samples collected from IBD patients with either ileal or ileocolonic involvement compared with IBD patients with only colon involvement or healthy age-matched controls. Higher levels of serum GM-CSF Ab were observed in CD patients with ileal or ileocolonic involvement compared with controls [median 2.4 mg/mL compared with 0.4 mg/mL (P , 0.001) for pediatric patients and median 11.7 mg/mL compared with 0.4 mg/mL (P , 0.001) for adult patients].48 Higher serum GM-CSF Ab levels were associated with higher odds of developing ileal disease (P 5 0.002), more aggressive stricturing/penetrating disease (P 5 0.002), disease requiring surgery (P 5 0.006), and more rapidly progressing disease (P 5 0.005).48 Susceptibility SNPs in either CARD15/NOD2 or autophagy related 16-like 1 gene had no influence on serum GM-CSF Ab level. However, the immunityrelated guanosine triphosphatase (IRGM) susceptibility allele rs13361189 was associated with significantly higher serum GM-CSF Ab level in CD patients harboring this mutation (median 6.6 mg/mL) compared with CD patients harboring wild-type alleles (median 1.3 mg/mL) (P , 0.04). Median serum GM-CSF Ab levels were higher in adult-onset CD compared with pediatric-onset CD, with no additional effect brought about by duration of disease.48 ASCA seropositivity was increased significantly (approximately 5-fold) in CD patients with elevated serum GM-CSF Ab levels. ANCA seropositivity was significantly decreased (approximately 4-fold) only in colonic CD patients with elevated serum GM-CSF Ab levels.48 The neutrophil neutralizing effect of GMCSF Ab was assessed using the GM-CSF-dependent upregulation of cell surface CD11b on neutrophils. GM-CSFdependent CD11b activation on neutrophils was lower in CD patients with increased serum CSF Ab levels than in CD patients with low serum GM-CSF Ab levels. Neutrophil phagocytic capacity was related inversely with serum GM-CSF Ab levels, such that it was low in CD patients with increased serum GM-CSFAb levels. Interestingly, serum GM-CSF Ab levels did not have any influence on neutrophil mean oxidative burst.48 The frequency of neutrophils with tyrosine-phosphorylated STAT3 was increased in ileal sections from CD patients with increased serum GM-CSF Ab levels. Dysregulated STAT3 activation may exert a proinflammatory effect by promoting leukocyte survival in

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CD. Increased levels of serum GM-CSF Ab led to a higher overall frequency of neutrophils and thus a higher frequency of tyrosine-phosphorylated STAT3 neutrophils in ileal sections from CD patients.48 Significantly higher circulating GM-CSF Ab levels were observed after the administration of GM-CSF Ab in mice compared with isotype-treated mice (P 5 0.007). Administration of the GM-CSF Ab led to almost complete inhibition of CD11b activation. Compared with wild-type controls, GM-CSF-null mice, NOD2null mice, wild-type mice pretreated with GM-CSF Ab, and NOD2-null mice pretreated with GM-CSF Ab experienced increased ileal paracellular permeability only, with no discernable differences in colonic paracellular permeability between groups. Consequently, GMCSF-null mice, NOD2-null mice, and wild-type mice pretreated with GM-CSF Ab experienced higher bacterial translocation to mesenteric lymph nodes than wildtype controls. The finding that bacterial translocation to mesenteric lymph nodes was higher in NOD2-null mice pretreated with GM-CSF Ab than it was in NOD2-null mice not exposed to GMCSF Ab suggests that a loss of NOD2 and exposure to GM-CSF Ab have an additive effect with respect to membrane disruption and bacterial translocation.48 No differences in the intestinal epithelial architecture or lamina propria cellularity were observed between GM-CSF-null mice and NOD2-null mice and wildtype mice, either in the presence or in the absence of GM-CSF Ab treatment. Exposure to the nonsteroidal anti-inflammatory drug piroxicam led to modest ileal inflammation in wild-type and NOD2-null mice, a greater degree of transmural ileal inflammation and more extensive ulceration in GM-CSF-null mice and increased ileal injury in NOD2-nul mice pretreated with GM-CSF Ab. Colonic inflammation was only modest after piroxicam exposure, with no differences between groups. Taken together, these data point toward a risk of ileal injury after treatment with GM-CSF Ab.48 Differential protein expression profiles. Differential protein expression profiles in IBD patients were compared in the hope of identifying new protein targets for therapy using purified intestinal epithelial cells obtained from 12 IBD patients (ileal tissue from 6 CD patients and colonic tissue from 6 UC patients) and 6 control colonic cancer patients.51 Inflammation in IBD patients and lack of inflammation in colonic cancer controls was confirmed by a histologic analysis. Four different proteins whose expression profiles showed at least a 2-fold statistically significant chance over steadystate values were identified in both CD and UC patients, namely L-lactate dehydrogenase, carbonyl reductase (NADPH prostaglandin E2 reductase), keratin 19, and the Rho-GDP dissociation inhibitor a.

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Increases in Rho-GDP dissociation inhibitor a were shown to be caused by inflammation in intestinal epithelial cells, rather than IBD-specific events.51 Using intestinal epithelial cells from both noninflamed and inflamed sections of tissue from 2 UC patients, significant increases were found in the expression of annexin A2 (7.77-fold increase) and the programmed cell death protein 8 (7.49-fold increase) in inflamed tissue compared with noninflamed tissue.51 New transcription factors involved in pediatric IBD were identified ex vivo in inflamed tissue sample and noninflamed tissue sample collected from 43 IBD patients (28 CD patients and 15 UC patients) and 20 healthy controls from Rome, Italy.52 In a microarray analysis of 3 CD patients and 3 healthy controls, 40 genes were found to be upregulated in inflamed tissue samples collected from CD patients compared with control tissue samples (P , 0.01). Of these, 17 remained upregulated in noninflamed tissue samples collected from CD patients compared with control tissue samples (P , 0.01). STAT1 and ATF3 were the most upregulated genes in inflamed tissue. STAT1 was also upregulated in noninflamed tissue, whereas ATF3 did not show a similar pattern.52 STAT1, ATF3, SMAD9, IFNregulatory factor-1 (IRF1), HIF1a, C/EBPb, ETS2, E2F6, FOXA2, and JUND were all found to be upregulated by RT-PCR in inflamed colonic and ileal mucosa from 28 CD patients compared with 20 healthy controls (P , 0.05). STAT1, IRF1, HIF1a, and SMAD9 were also upregulated in noninflamed in mucosal tissue from CD patients compared with healthy controls (P , 0.05). The colonic expression of these 10 transcription factors was not different from ileal expression. ATF3, HIF1a, FOXA2, and STAT1 were upregulated in inflamed colonic mucosa from 15 UC patients compared with 20 healthy controls. HIF1a and STAT1 were upregulated in noninflamed colonic mucosa from UC patients compared with healthy controls.52 Moreover, ATF3 and HIF1a were upregulated at the protein level in inflamed colonic and ileal mucosa from CD patients compared with healthy controls (P , 0.05). HIF1a was upregulated in noninflamed mucosal tissue from CD patients compared with healthy controls (P , 0.05). These results support findings from RT-PCR.52 The effects of ATF3 and HIF1a upregulation at the protein level were tested in the human intestinal barrier cell line Caco2 treated with either cytomix (TNF- a and IFN-g) or cobalt chloride (CoCl2) to induce hypoxia. ATF3 expression was noted 3 h after cytokine stimulation and remained detectable even after 24 h. HIF1a expression was noted only 24 h after cytokine stimulation. ATF3 expression was noted only 24 h after hypoxia was induced. HIF1a expression was noted 3 h after hypoxia was induced, and it peaked after 6 h. Cytokine stimula-

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tion and hypoxia had additive effects. ATF3 expression increased progressively, peaking after 24 h of treatment. HIF1a expression peaked after 3 h, followed by gradual decrease.52 BIOMARKERS OF INFLAMMATION IN CELL CULTURE AND ANIMAL MODELS OF COLITIS The link between the proinflammatory cytokine TNF-a and Na/H ion-exchanger. Cell cultures and animals are

often employed for a better understanding of the mechanisms that underlie the pathogenesis of human diseases. With Na1 absorption compromised in IBD, the effects of TNF-a treatment on the epithelial ion exchanger Na1/H1 exchanger (NHE) was examined in intestinal epithelial C2BBe1 cells. An NHE catalyzes the transfer of an extracellular Na1 ion for an intracellular H1 ion.53 A significant repression of NHE2 mRNA was observed by real-time RT-PCR after treatment with 5 ng/mL, 10 ng/mL, and 20 ng/mL doses of TNF-a for 6 h, for all TNF-a doses. NHE2 repression was time dependent, peaking 8 h after TNFa treatment. Amin et al53 found that TNF-a impacts NHE2 mRNA transcription rather than NHE2 mRNA stability. This decrease in NHE2 mRNA translates into a reduction of NHE2 protein level, which reached approximately 50%, 6 h after TNF-a treatment. Protein activity was also reduced, by about 55% for NHE2 and about 70% for NHE3. Taken together, these findings show that TNF-a inhibits NHE2 mRNA translation at the promoter region, and it leads to decreased protein expression and activity.53 TNF-a leads to the degradation of cytoplasmic IkB-a in a time-dependent manner, which leads to the activation and the nuclear translocation of the transcription factor nuclear factor (NF)-kB. The p50 and p65 subunit of NF-kB were shown to interact with the NF-kB binding site on the NHE2 promoter. NF-kB binding was shown to be essential for TNF-amediated NHE2 repression, as an overexpression of NF-kB subunits p50 and p65 was enough to repress NHE2 expression, with the addition of TNF-a not leading to subsequent repression. The addition of NF-kB led to a lack of response to TNF-a treatment.53 Overall, this study establishes the link between the proinflammatory cytokine TNF-a and Na/H ion-exchanger isoform NHE2, which are mediated by the transcription factor NF-kB.53 Cytokines as immunoregulators in models of colitis. IL10 is an important immunoregulator in the intestinal tract. IL-10 downregulates inflammation by mediating the growth, differentiation, and activation of cells under the influence of proinflammatory cytokines.54 In an IL-10 knockout (IL-10 -/-) mouse model, uncontrolled inflammation is expected to ensue as

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result of the loss of the suppressive effect of IL-10 on the Th1 response, a phenomenon that has been linked to the development of IBD.54 IL-10 -/- mice will thus develop achronic colonic inflammation that mimics human IBD, marked by an immune inflammatory response against normal commensal intestinal microbiota.54,55 This chronic process seems to be mediated by endoplasmic reticulum (ER). An ER-mediated stress response in intestinal epithelial cells is marked by energy deficiency and alterations in oxidative metabolism in the affected tissue.54 Inflammation developed gradually and started to become apparent in the colon and cecum of IL-10 -/- 129(B6)-IL10tm1Cgn/J mice at week 8, and it continued through week 16 and week 24. A histologic analysis revealed infiltration of inflammatory cells such as lymphocytes and PBMC in the mucosal and submucosal layers of the affected area. Inflammation was more pronounced in older animals. The damage was mild, as it was limited to hyperproliferation and irregular crypts.54 Serum amyloid A and soluble TNF II were measurable in the blood of wild-type mice and IL-10 -/- mice starting at the age of 1 week. The levels of soluble TNF II continued to increase until week 8, and they remained unchanged until week 24 in both groups of animals. However, they were higher in IL-10 -/- mice than in wild-type mice starting at week 8. Serum amyloid A levels were also higher in IL-10 -/- mice than in wildtype mice starting at week 8, and the difference between groups continued to increase at both week 16 and week 24 without reaching statistical significance.54 Metabolic profiles in models of colitis. Proton nuclear magnetic resonance revealed the presence of lipoproteins and lipids in plasma, as well as, low-molecularweight molecules such as glucose, ketone bodies (3-D-hydroxybutyrate and acetoacetate), amino acids, and organic acids such as acetate, lactate, succinate, pyruvate, and citrate. IL-10 -/- mice showed lower levels of lipoproteins and lipids, choline, phosphocholine, and glycerophosphocholine, whereas the levels of lactate, alanine, and glutamine were increased, compared with wild-type mice.54 Time-dependent changes in metabolic profiles between the 2 groups were observed, as differences became more apparent with time, peaking at week 24. Time-dependent changes were linear in wild-type animals and nonlinear in IL-10 -/- animals. Colitis development was associated with increased levels of phospholipids-rich lipoprotein particles, glycosylated proteins, polyunsaturated fatty acids, pyruvate and alanine, and reduced levels of triglycerides rich lipoproteins and glutamine. Compared with controls at week 1, age-matched IL-10 -/- mice exhibited higher levels of citrate, glutamine, and fumarate, as well as lower

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levels of glucose and dimethylglycine. Variations in lipoproteins, namely increased levels of polyunsaturated lipids, high-density lipoprotein, and low-densitylipoprotein and decreased levels of very low-density lipoprotein concentrations, were also observed in IL-10 -/- mice.54 Higher levels of isoleucine, citrate, pyruvate, lactate, phenylalanine, and choline in phospholipids, glycoproteins, and lower levels of glutamine, leucine, tyrosine, methionine, creatine, dimethylglycine, trimethylamine, and glucose were observed in IL-10 -/mice at week 16.54 IL-10 -/- animals showed increased levels of alanine, arginine, isoleucine, and phenylalanine, as well as decreased levels of trimethylamine at week 24, compared with wild-type animals. Also at week 24, higher levels of high-density lipoprotein, low-density lipoprotein, certain polyunsaturated fatty acids, and lower concentrations of very low-density lipoprotein were observed in IL-10 -/- animals.54 This study shows that gradual development of colitis, on a dysfunctional IL-10 -/- background, is multifactorial and involves disruptions in normal metabolism.54 Urine metabolite analysis was performed in IL-10 gene-deficient 129/SvEv mice, and the profile was compared with that obtained from wild-type 129/SvEv mice.56 Several differences between IL-10 gene-deficient animals and wild-type animals were uncovered. For example, significantly higher levels of trimethylamine and fucose were identified in IL-10 gene-deficient mice, starting between week 8 and week 12 and increasing with age. Trimethylamine-N-oxide, dimethylamine, xylose, and valine were also increased in IL-10 gene-deficient mice. N-Isovaleroylglycine was decreased in IL-10 gene-deficient mice, beginning at week 16.56 In mice 12 weeks or older, fucose (P , 0.0001), trimethylamine (P , 0.0001), dimethylamine (P , 0.0001), fumarate (P , 0.0001), N-isovaleroylglycine (P , 0.0001), 2-oxoglutarate (P 5 0.0008), trimethylamine-N-oxide (P 5 0.0011), butyrate (P 5 0.0025), citrate (P 5 0.0027), succinate (P 5 0.0035), 3indoxylsulfate (P 5 0.0036), valine (P 5 0.0058), and phenylacetylglycine (P 5 0.0087) were significantly different between animals developing IBD and animals not developing IBD, indicating intestinal disease progression.56 Also, a urine protein analysis was performed to identify protein profiles that were different between initially germ-free wild-type mice and IL-10 -/- mice colonized with Enterococcus fecalis OG1RF strain for 14 weeks to induce colitis, wild-type controls, and germ-free IL-10 -/- controls.57 After 14 weeks of colonization, severe inflammation was observed in the distal colon of IL-10 -/mice but not in wild-type mice.57 Glucose-regulated protein (grp)-78, which is a molecular chaperone

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involved in inflammation and apoptosis, was upregulated in experimental colitis IL-10 -/- animals. A clear upregulation of grp-78 was observed in inflamed tissue (IL-10 -/-) compared with noninflamed tissue (wild type), supporting the theory that this protein is highly expressed under conditions of chronic inflammation. Endoplasmic reticulum stress. Similar results are observed in tissue samples from CD patients, UC patients, sigmoid diverticulitis patients (inflamed), and colonic cancer patients (noninflamed), which suggests that grp-78 upregulation and ER stress are features of chronic inflammation in general, rather than IBDspecific. This was confirmed by grp-78 upregulation in inflamed sections only, rather than noninflamed sections of tissue belonging to the same IBD patients.57 IL-10 triggered p38 mitogen-activated protein kinase (MAPK) and STAT3 phosphorylation in the murine intestine-derived epithelial cell line Mode-K reconstituted with an IL-10 receptor. p38 phosphorylation was IL-10 receptor dependent, IL-10 dose dependent, and time dependent. p38 phosphorylation occurred after 3 h of stimulation with 50 ng/mL IL-10 and grp-78 degradation occurred after 6 h of stimulation in IL-10 receptor reconstituted Mode-K cells. In addition, IL-10 also triggered a time-dependent degradation of steady state grp-78 mRNA, stating after 12 h. As expected, phospho-p38 was observed in intestinal epithelial cells from wild-type mice but not in IL-10 -/- mice. Of note, the IL-10 receptor was present in equal amounts in wild-type animals and in IL-10 -/- animals. IL-10 -/- mice also revealed a higher expression of cleaved caspase-3 and a lower level of E-cadherin.57 TNF-a treatment induces the translocation of grp-78 from the ER lumen into the cytoplasm of IL-10 receptor in reconstituted Mode-K cells in as little as 1 h. However, at this point, TNF-a had no effects on grp-78 synthesis. IL-10 treatment for 24 h led to decreases in grp-78 mRNA particularly in TNF-a-treated cells but also in TNF-a-untreated cells. This was shown to occur by the IL-10-mediated inhibition of histone acetylation of the grp-78 promoter. IL-10 also inhibited the recruitment of grp-78 into the IkB-a kinase complex, and it blocked TNF-a-induced RelA phosphorylation.57 A change in the homeostasis between intestinal bacteria and host signaling, at the level of intestinal epithelial cells, may lead to the development of functional and immune disturbances, which can translate into chronic inflammation. TLRs and NOD receptors are important for innate immune response bacterial recognition pathways.55 TLR2 and TLR4 recognize components of gram-negative bacteria cell walls. The activation of TLR leads to the recruitment of a set of adaptor proteins, which in turn activate MAPK proteins capable of activating the IkB/NF-kB transcriptional system, which in

Translational Research July 2012

turn results in the induction of proinflammatory cytokines, chemokines, and costimulatory proteins.55 The responses to inflamed and noninflamed conditions were assessed in TLR2 -/- mice, TLR4 -/- mice, and IL-10 -/- mice, as well as the cross-breeds IL10XTLR2 -/- mice and IL-10XTLR4 -/- mice.55 IL-10 -/- mice showed signs of moderate colon inflammation at week 8 and week 16. In contrast, no histologic evidence of colitis was present in TLR2 -/- mice or TLR4 -/- mice. IL-10XTLR4 -/- mice were protected against colonic inflammation, whereas IL-10XTLR2 -/- mice developed signs of chronic colonic inflammation and tissue pathology.55 A total of 74 differently regulated proteins were identified, with 41 in noninflamed conditions (wild-type mice compared with TLR2 -/mice and TLR4 -/- mice) and 33 in inflamed conditions (IL-10 -/- mice compared with IL-10XTLR2 -/- mice and IL-10XTLR4 -/- mice).55 Two stress-related proteins, oxidative stress-related peroxiredoxin 6 and the ER stress-associated chaperone grp-78, were different between wild-type mice and TLR2 -/- mice. Two mitochondrial stress-response proteins, chaperonin 60 and grp-75, were different between wild-type mice and TLR4 -/- mice. Most of the other different protein expression profiles between wild-type mice and TLR2 -/- mice and TLR4 -/- mice were related to proteins involved in energy metabolism and cell cytoskeleton.55 Most of the other protein expression profiles between IL-10 -/- mice and IL-10XTLR2 -/- mice and IL-10XTLR4 -/- mice were related to proteins involved in energy metabolism and protein folding.55 The induction of C/EBP homologous protein, which is a major regulator of ER stress-induced apoptosis, was only detectable in TLR4 -/- mice but not in TLR2 -/- mice or wild-type mice under noninflamed conditions at both week 8 and week 16. Interestingly however, apoptosis-related cleavage of caspase-3 was only observed in IL-10 -/- mice and IL-10XTLR2 -/- mice, showing an uncoupling between grp-78 induction and apoptosis in IL-10XTLR4 -/- mice. These findings could be interpreted as the development of adaptive ER stress responses that maintain the function of the intestinal epithelial cells lacking TLR4.55 Antimicrobial activities. Serum amyloid A is a protein expressed primarily in the liver, but also in other tissues, including the intestinal epithelium, with major acute phase antimicrobial activities.58 Colitis was induced by 3% DSS administered in the drinking water for 1 week in a serum amyloid A 1/serum amyloid A 2 double knockout mouse model. The antibacterial effects of this enzyme were tested in vitro in mouse colonic epithelium cells exposed to live E. coli.58 Significant levels of serum amyloid A 1, serumamyloid A 2, and to a lesser extent, serum

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amyloid A 3 were detected by in situ hybridization and immunohistochemistry. The overexpression of both serum amyloid A 1 and serum amyloid A 2 decreased the viability of cocultured E. coli in cultured cells.58 The results from in vivo studies revealed that serum amyloid A is expressed in the intestinal epithelium and is secreted into the gut lumen, with the highest concentrations being observed at the tip of villi. The strongest expression was detected in the colon, with weaker expression in the ileum, indicating that there is an interaction between bacterial concentration and serum amyloid A expression. However, expression seems to be constitutive in the colon, as colonic results obtained from infected mice and germ-free mice were comparable, whereas the ileum of germ-free mice was virtually devoid of serum amyloid A expression. In addition, serum amyloid A 3 is inducible by bacterial lipopolysaccharide (LPS), whereas serum amyloid A 1 and serum amyloid A 2 are high in the presence of bacteria, regardless of the actual LPS levels in cultured CMT93 mouse rectal epithelial cells.58 Serum amyloid A overexpression in cultured cells treated with E. coli led to decreased viability of these gram-negative bacteria.58 Compared with wild-type mice, serum amyloid A 1/serum amyloid A 2 double knockout mice lost significantly more blood (decrease in hematocrit) and showed significantly increased colon shortening compared with controls, following colitis initiation by DSS. Double knockout mice also expressed significantly higher levels of proinflammatory cytokine TNF-a and osteopontin mRNA than control mice.58 In CD patients, inflamed tissue samples had higher expression of serum amyloid A 1 and serum amyloid A 2 than noninvolved tissue samples or tissue samples from non-IBD controls. Taken together, these findings suggest that serum amyloid A is involved in IBD.58 Vasopressin, which is a peptide neurohormone synthesized in the GI tract, acts through the 3 G protein-coupled receptors V1a, V1b/V3, and V2. Gut vasopressin receptors can be activated via either locally synthesized vasopressin or circulating vasopressin.59 The Vb1 receptor was detected by immunohistochemistry in healthy human colon, UC patients, and CD patients in enterocytes, both at the membrane level and at the cytoplasm level. No difference in staining intensity and localization was observed between healthy tissue and inflamed tissue. Similar results were found in rat tissue.59 Colitis was induced by dinitrobenzene sulfonic acid (DNBS) treatment in rats. Treatment with the selective Vb1 antagonist SSR149415 (3 mg/kg) did not reverse the decreases in weight gain observed between animals treated with DNBS and untreated animals. Use of 10 mg/kg and 30 mg/kg decreased macroscopic

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damage induced by DNBS treatment. In addition, animals treated with SSR149415 at doses of 10 mg/kg only also exhibited significantly lower myeloperoxidase activity (P 5 0.01).59 Macroscopic damage associated with high myeloperoxidase activity and weight loss after 2 days of DNBS treatment were significantly lower in Vb1 -/- knokout mice compared with wild-type mice (P , 0.05), suggesting that vasopressin, acting through the Vb1 receptor, is associated strongly with early mucosal damage. The specificity of the antagonist was shown, as cotreatment with SSR149415 in wild-type mice led to results similar to those observed in Vb1 -/- knockout mice, and no additional effects of cotreatment with SSR149415 were observed. SSR149415 was ineffective in DSS-induced colitis.59 Drastic mortality in treated mice was observed after an increase in circulating vasopressin levels, brought about by water deprivation. SSR149415 reversed this effect, particularly in waterdeprived animals.59 The Vb1 receptor was identified as a participant in gut inflammation via mast cell activation, mediated by vasopressin.59 CELL ADHESION MOLECULES

Cell adhesion molecules (CAM) regulate the interaction between inflammatory cells and target tissues. In the process, they mediate the development of disease, ranging from cancer metastasis to inflammatory conditions in a variety of organs.60 Differences in the expression patterns of cell adhesion molecules exist in peripheral blood and isolated gut lamina propria lymphocytes between IBD patients (UC and CD) and normal controls (Table II). Activation of the endothelial layer in patients with IBD leads to enhanced leukocyte and platelet recruitment, the acquisition of a procoagulant phenotype, and angiogenesis. Leukocyte infiltration is a tightly regulated process, comprising of capture, rolling, arrest, adhesion, spreading, and paracellular or transcellular migration of the leukocyte. Numerous molecules, among them selectins, integrins, chemokines, and other members of the immunoglobulin superfamily, mediate these steps.64 Selectins are expressed on the surface of endothelial cells, leukocytes, or platelets. They are involved in mediating the rolling of leukocytes on the surface of the endothelium through interactions with their respective ligands. The endothelium expresses E-selectin and P-selectin.61 Integrins are heterodimeric proteins composed of an a chain and a b chain. Integrins a1b2, a2b2, a3b2, a4b1, and a4b7 are the major modulators of leukocyte–endothelial cell adhesion. a4b7 integrin specifically

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mediates leukocyte migration to gut inflammatory sites. a4b7 integrin recognizes both mucosal vascular addressin cell adhesion molecule (MAdCAM)-1 and vascular cell adhesion molecule (VCAM)-1. Integrin interaction with VCAM-1 mediates adherence of leukocytes to the activated endothelium, whereas its interaction with MAdCAM-1 mediates colonization of the gutassociated lymphoid tissue with leukocytes.64 Integrin adhesion molecules consist of pairs of CAMs and complementary target addressins. Examples of such pairs include (1) the aLb2 integrin (CD11a/CD18-LFA-1), expressed on all leukocytes and its pair intercellular adhesion molecule (ICAM)-1 or ICAM-2, (2) a4b1 integrin (VLA-4), expressed on lymphocytes, eosinophils, and monocytes and its pair VCAM-1 or extracellular matrix molecules, and (3) a4b7 integrin (CD49d/Beta7), expressed on lymphocytes and its pair the mucosal addressin MAdCAM-1 or VCAM-1. MAdCAM-1 is especially important for intestinal health, as this addressin is involved in mucosal-specific homing of lymphocytes to the gut.60 Monoclonal antibodies are the current form of biologic antiadhesion therapy.60 Epithelial barrier dysfunction, which is caused by the reduction of tight junction strands in UC and tight junction strand breaks in CD, is a common feature of IBD. Apoptosis also contributes to epithelial barrier dysfunction.65 Tight junction disruptions are believed to play an important role in the uptake of food antigens and bacterial LPS. Although no changes in the expression profiles of claudin-1 and claudin-4 were observed in CD patients, occludin, claudin-5, and claudin-8 were found to be downregulated, with the cation channel-former claudin-2 being upregulated. Furthermore, an immunohistochemistry analysis reveals that claudin-5 and claudin-8 were distributed away from tight junctions. Epithelial erosions were not present in CD patients, offering a possible explanation as to why epithelial barrier disruptions were not as pronounced in CD patients as they were in UC patients.65 Claudin-2 was upregulated in UC patients as well. Furthermore, a higher rate of apoptosis was observed throughout the epithelium in UC. Higher rates of the proapoptotic cytokine IL-13 were observed in HT-29/B6 cells, which is a UC cell culture model.65 It is believed that immune tolerance in healthy individuals is mediated in part by a low rate of antigen uptake because of the presence of tight junctions. As tight junctions become damaged, an increased rate of antigen uptake occurs, leading to a state of inflammation. Water can leak in the other direction, leading to diarrhea.65 Aside from a loss of tight junctions, the early stages of UC are mediated by apoptotic foci, while the later stages of UC are mediated by ulcerative, erosive le-

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sions.65 Antigens can also travel between cells via endocytotic uptake and transcytosis. This mechanism of antigen trafficking seems to be intensified in CD.65 IBD pathology is mediated by continuous leukocyte infiltration into the intestine wall and leukocyte migration is mediated by CAMs.61 The gene expression of 69 leukocyte/endothelial CAMs (13 leukocyte CAMs, 14 endothelial CAMs, 4 leukocyte and endothelial CAMs, and 38 chemokines/ chemokine receptors) and E-cadherin was measured in a biopsy specimen collected from 61 IBD patients (24 UC patients, 19 CD (colon localization) patients, and 18 CD (ileus localization) patients) before and after the first dose of the drug infliximab and in 12 normal controls. The initial results obtained by microarray were confirmed by quantitative RT-PCR, immunohistochemistry, and Western blotting.61 There were no genes whose expression was different between UC patients and CD (colon localization) patients, either at baseline or after the first dose of infliximab. The expression pattern of the epithelial CAM E-cadherin was significantly decreased in active colonic IBD samples (UC patients and CD (colon localization) patients together) compared with control samples.61 The expression of most CAMs that were found to be upregulated in colonic IBD samples decreased significantly in responsive patients after infliximab treatment compared with before treatment baseline levels. The expression patterns of chemokines CCL20 and CXCL1-2, although significantly decreased in responsive colonic IBD patients treated with infliximab compared with before treatment baseline, remained significantly higher than they were in healthy controls. No changes in gene expression patterns were observed in nonresponsive colonic IBD patients treated with infliximab compared with before treatment baseline, and the expression patterns of these genes remained dysregulated compared with healthy controls.61 The expression patterns of the chemokines/chemokine receptors CCL5 (RANTES), and CCL25 (TECK) mRNA were significantly increased, whereas the expression patterns of the chemokines/chemokine receptors CCL28 (MEC) and CXCL1 (MGSAa) were significantly downregulated in CD patients with ileitis compared with colitis CD patients at baseline. The expression pattern of the endothelial CAMs MAdCAM-1 and THY-1, the leukocyte and endothelial CAMs PECAM-1, and the chemokines/chemokine receptors CCL28, CXCL1, CXCL2, CXCL5, CXCL6, CXCL11, and IL8 were upregulated in active ileitis CD patients compared with control samples. The expression pattern of the endothelial ACM CD58 was downregulated in active ileitis CD patients compared with control samples.61

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Table II. Cell Adhesion Molecules Study population

Settings

Gene and SNAP

61 IBD patients [24 UC, 19 CD (colitis), 18 CD (ileitis)] before and after the first dose of infliximab; 12 normal controls

Leuven, Belgium

Leukocyte CAMs CD2, ITGA4, ITGAL, ITGAM, ITGB2, SELL, and SELPLG Endothelial CAMs ICAM-1, ICAM2, JAM2, MaDCAM-1, SELE, SELP, THY1 and VCAM-1 Leukocyte, endothelial CAMs JAM3 and PECAM-1 Chemokines: CCL2, CCL3, CCL4, CCL11, CCL19, CCL20, CX3CL1, CXCL1, CXCL2, CXCL5, CXCL6, CXCL9, CXCL10, CXCL11, CXCL13, IL8, Receptors: CCR5, CCR7, CXCR2, CXCR4 E-cadherin

Human intestinal microvascular endothelial cells Murine cell lines SVEC and MJC-1 AT1R -/- mouse colitis model

n/a

VCAM-1 expression

n/a

MAdCAM-1 expression

n/a

MAdCAM-1 expression

Compared with normal mucosa, untreated IBD mucosa showed increased expression of MAdCAM-1 in blood vessel endothelium by immunohistochemistry. MAdCAM-1 expression decreased in infliximabtreated responsive mucosal samples. In normal mucosa, PECAM-1 was detected in endothelial cells. In normal mucosa, CXCL1 was expressed mainly in crypts but not in the surface epithelium. Compared with normal mucosa, untreated IBD mucosa showed increased expression of CXCL1 in the epithelium and in inflammatory cells. CXCL1 expression decreased slightly in infliximab-treated responsive mucosal samples. Western blotting confirmed that CCL25 (TECK) mRNA was present in ileitis CD patients only.61 The inhibition of leukocyte a4 integrin binding to intestinal endothelial CAM ligands has shown therapeutic benefits in IBD. The mechanisms of a4 integrin ligand VCAM-1 expression were assessed in human intestinal microvascular endothelial cells (HIMEC). The effects of curcumin, phosphatidylinositol 3-kinase (PI 3kinase)/protein kinase B (Akt) inhibitors, and MAPK inhibitors on VCAM-1 expression and function were investigated.62 TNF-a and LPS were shown to increase ICAM-1 expression, E-selectin expression, and VCAM-1 expression in HIMEC. VCAM-1 mRNA expression after TNF-a/LPS stimulation was shown to be time dependent, with maximum levels observed after 4 h and baseline levels observed after 24 h. VCAM-1

Main findings

Upregulated in active colonic IBD compared with control

Ref

61

Upregulated in active colonic IBD compared with control Upregulated in active colonic IBD compared with control Upregulated in active colonic IBD compared with control

Downregulated in active colonic IBD compared with control Inhibited by curcumin Prevented by AT1R selective inhibitor candesartan Lower MAdCAM-1 expression, lower disease activity index, and milder histologic damage in AT1R -/- animals

62 63

protein expression was also shown to be upregulated after TNF-a/LPS stimulation, reaching maximum levels between 12 h and 24 h and returning to baseline after 48 h.62 TNF-a/LPS stimulation resulted in a timedependent phosphorylation of protein kinase B. In the unstimulated cells, no phosphorylation of protein kinase B was observed. Pretreatment for 30 before TNF-a/LPS stimulation with either curcumin (10 mmol/L) or PI 3-kinase inhibitor LY-294002 (10 mmol/L) led to the complete inhibition of protein kinase B phosphorylation. SN-50, which is an NF-kB inhibitor, had no such effect. The activity of phosphorylated protein kinase B was shown through activation of GSK-3 fusion protein as substrate. As expected, protein kinase B isolated from HIMEC pretreated with curcumin or LY-294002 could not activate GSK-3.62 The inhibition of PI 3-kinase has been shown to lead to VCAM-1 expression downregulation and loss of monocyte-endothelial cell adhesion in animal models. Additional attention was provided to the PI 3-kinase/ protein kinase B signaling pathway, as it is involved in cellular differentiation and was previously shown to play a critical role in a4-integrin-expressing leukocyte binding through MAdCAM-1 expression. The significant inhibition of VCAM-1 mRNA, protein, cell surface expression, and leukocyte binding was inhibited by all of curcumin, PI 3-kinase/protein kinase B inhibitor LY-294002, p38 MAPK inhibitor SB-203580, NF-kB

26

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inhibitor SN-50, and vitamin E, but not by c-Jun NH2terminal kinase inhibitor SP-600125 and only partially by p44/p42 MAPK inhibitor PD-098059. This suggests that protein kinase B/p38 MAPK/NF-kB and possibly p44/p42 MAPK signaling is involved in VCAM-1 expression in HIMEC, and curcumin is a potent inhibitor of all of these inflammatory pathways.62 The involvement of the PI 3-kinase/protein kinase B signaling pathway in MAdCAM-1 expression after TNF-a/LPS stimulation was demonstrated previously. As this pathway is involved in both VCAM-1 expression and MAdCAM-1 expression, curcumin was shown to inhibit both VCAM-1 expression and MAdCAM-1 expression in HIMEC.62 To confirm whether the effects on VCAM-1 expression were linked to protein kinase B inhibition, Aktspecific small interfering RNA (siRNA) was transfected into human intestinal microvascular endothelial cells. The involvement of NF-kB activation in E-selectin and ICAM-1 expression after TNF-a/LPS stimulation in human intestinal microvascular endothelial cells has been demonstrated previously.62 Treatment with an antibody directed against the MAdCAM-1 ligand integrin a4b7 has shown promise in the treatment of IBD. Although not fully understood, MAdCAM-1 regulation seems to involve the angiotensin II type 1 receptor (AT1R). The role of AT1R in MAdCAM-1 expression after cytokine stimulation was assessed in the 2 murine cell lines SVEC (high endothelial venule cell line) and MJC-1 (colonic endothelial cell line). The effects of AT1R in the pathogenesis of IBD were evaluated even more in an immune-mediated AT1R -/- mouse colitis model.63 The involvement of AT1R in MAdCAM-1 expression was shown as candesartan (10 mmol/L to 50 mmol/L), a selective inhibitor of AT1R, was able to prevent MAdCAM-1 expression in SVEC cells (P , 0.05) and MJC-1 cells (P , 0.05) treated with TNF-a (20 ng/mL for 24 h). Candesartan inhibited TNF-a–induced MAdCAM-1 expression in a dose-dependent manner, both at the mRNA level confirmed by real-time RT-PCR and at the protein level confirmed by microarray.63 Candesartan 50 mmol/L inhibited ICAM-1 (P , 0.05) and VCAM-1 (P , 0.05) protein expression in SVEC cells treated with TNFa (20 ng/mL for 6 h).63 These effects were confirmed in a model in which SVEC and MJC-1 cells were treated with TNFa (20 ng/mL for 24 h) 36 h after transfer siRNA transfection. TNF-a–induced ICAM-1 (P , 0.05) and VCAM-1 (P , 0.05) protein expression in SVEC cells was prevented significantly by AT1R knockdown.63 The mechanism by which angiotensin II type 1 receptor blocker candesartan inhibits MAdCAM-1 expression was found to be independent of p38 MAPK phosphory-

lation and IkB phosphorylation and degradation. TNFa–induced transient phosphorylation of p38 MAPK and IkB produced the degradation of IkB. Candesartan could not reverse the phosphorylation of p38 MAPK and the phosphorylation and degradation of IkB in both cell types.63 In vivo, colitis induction by DSS was ameliorated in AT1R -/- mice compared with wild-type mice. Taken together, these results suggest that AT1R might be a potential drug target in IBD patients. Lower disease activity index and milder histological damage in AT1R -/mice support this argument.63 CONCLUSION

A specific link between IBD and metabolome and inflamasome was found. Abnormalities of the mucosal immune system have been suggested in several studies. Two basic arguments in the pathogenesis of IBD emerge: (1) dysregulation of the innate and adaptive immune system directed against luminal bacteria or their toxins found in the intestinal lumen; and (2) inappropriate immune responses to organisms in the intestine that normally do not elicit a response, possibly because of intrinsic alterations in mucosal barrier function. In addition, the immune response to intestinal microbial flora is important in the pathogenesis of IBD. Using the specific biomarkers involved in gut metabolism and inflammation, one can monitor the evolution in time of the disease as well as the possible repair of intestinal damage.

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