Effect of 5-Hydroxytryptophan on Fatigue in Quiescent Inflammatory Bowel Disease: A Randomized Controlled Trial.

BACKGROUND & AIMS: Fatigue is highly prevalent among patients with inflammatory bowel disease (IBD), and only limited treatment options are available. Based on the hypothetical link between low serum tryptophan concentrations and fatigue, we determined the effect of 5-hydroxytryptophan supplementation on fatigue in patients with inactive IBD. METHODS: A multicenter randomized controlled trial was performed at 13 Belgian hospitals, including 166 patients with IBD in remission but experiencing fatigue, defined by a fatigue visual analog scale (fVAS) score of ≥5. Patients were treated in a crossover manner with 100 mg oral 5-hydroxytryptophan or placebo twice daily for 2 consecutive periods of 8 weeks. The primary end point was the proportion of patients reaching a ≥20% reduction in fVAS after 8 weeks of intervention. Secondary outcomes included changes in serum tryptophan metabolites, Functional Assessment of Chronic Illness Therapy Fatigue scale, and scores for depression, anxiety, and stress. The effect of the intervention on the outcomes was evaluated by linear mixed modeling. RESULTS: During 5-hydroxytryptophan treatment, a significant increase in serum 5-hydroxytryptophan (estimated mean difference, 52.66 ng/mL; 95% confidence interval [CI], 39.34-65.98 ng/mL; P < .001) and serotonin (3.0 ng/mL; 95 CI, 1.97-4.03 ng/mL; P < .001) levels was observed compared with placebo. The proportion of patients reaching ≥20% reduction in fVAS was similar in placebo- (37.6%) and 5-hydroxytryptophan (35.6%)-treated patients (P = .830). The fVAS reduction (-0.18; 95% CI, -0.81 to 0.46; P = .581) and Functional Assessment of Chronic Illness Therapy Fatigue scale increase (0.68; 95% CI, -2.37 to 3.73; P = .660) were both comparable between 5-hydroxytryptophan and placebo treatment as well as changes in depression, anxiety, and stress scores. CONCLUSIONS: Despite a significant increase in serum 5-hydroxytryptophan and serotonin levels, oral 5-hydroxytryptophan did not modulate IBD-related fatigue better than placebo. (Trial Registration: Belgian Federal Agency for Medication and Health Products, EudraCT number: 2017-005059-10 and ClinicalTrials.gov: NCT03574948, https://clinicaltrials.gov/ct2/show/NCT03574948.).

Anti-CD74 IgA antibodies show diagnostic potential for axial spondyloarthritis but are not associated with microscopic gut inflammation.

OBJECTIVES: Gut inflammation commonly occurs in axial SpA (axSpA), and is linked to disease activity and outcome. Given the role of IgA in mucosal immunity, we explored the association between anti-CD74 IgA antibodies, gut inflammation and axSpA. METHODS: Anti-CD74 IgA was measured by ELISA in serum samples of axSpA patients, fulfilling the 2009 Assessment of SpondyloArthritis international Society classification criteria. A group of fibromyalgia (FM) and RA patients served as non-inflammatory and inflammatory controls. Newly diagnosed axSpA patients underwent ileocolonoscopy; mucosal biopsies were histopathologically assessed as normal, acute or chronically inflamed. Optimal anti-CD74 IgA cut-off values were determined with a receiver operating characteristics curve. RESULTS: axSpA patients (n = 281) showed higher anti-CD74 IgA levels [mean (s.d.) 18.8 (12.4) U/ml] compared with 100 FM patients [10.9 (5.0) U/ml, P < 0.001] and 34 RA patients [13.7 (9.6) U/ml, P = 0.02]. The area under the receiver operating characteristics curve for diagnosis (axSpA vs FM) was 0.70, providing a sensitivity of 60% and specificity of 87% (cut-off 15 U/ml). Antibody concentrations were not significantly different between axSpA patients with (n = 40) and without (n = 69) gut inflammation (P = 0.83), yielding an area under the receiver operating characteristics curve of 0.51. Anti-CD74 IgA levels were not associated with degree of bone marrow oedema on MRI of the sacroiliac joints, CRP or any other disease-specific feature such as the use of NSAIDs or biological treatment. CONCLUSION: Serum anti-CD74 IgA is a potentially useful diagnostic biomarker for axSpA. However, antibody levels do not correlate with any phenotypical feature, including microscopic gut inflammation, suggesting this to be a disease-specific rather than an inflammatory marker.

MRI texture analysis of T2-weighted images is preferred over magnetization transfer imaging for readily longitudinal quantification of gut fibrosis.

OBJECTIVES: To investigate the value of magnetization transfer (MT) MRI and texture analysis (TA) of T2-weighted MR images (T2WI) in the assessment of intestinal fibrosis in a mouse model. METHODS: Chronic colitis was induced in mice by cyclic administration of dextran sodium sulphate (DSS) resulting in chronic inflammation and progressive bowel fibrosis. Mice underwent 7-T MR imaging at various time points. Bowel wall MT ratio (MTR) and textural features (skewness, kurtosis, entropy), extracted by a filtration histogram technique, were correlated with histopathology. Performance of both techniques were validated using antifibrotic therapy. Finally, a retrospective study was conducted in five patients with Crohn's disease (CD) who underwent bowel surgery. RESULTS: MTR and texture entropy correlated with histopathological fibrosis (r = .85 and .81, respectively). Entropy was superior to MTR for monitoring bowel fibrosis in the presence of coexisting inflammation (linear regression R2 = .93 versus R2 = .01). Furthermore, texture entropy was able to assess antifibrotic therapy response (placebo mice versus treated mice at endpoint scan; Δmean = 0.128, p < .0001). An increase in entropy was indicative of fibrosis accumulation in human CD strictures (inflammation: 1.29; mixed strictures: 1.4 and 1.48; fibrosis: 1.73 and 1.9). CONCLUSION: MT imaging and TA of T2WI can both noninvasively detect established intestinal fibrosis in a mouse model. However, TA is especially useful for the longitudinal quantification of fibrosis in mixed inflammatory-fibrotic tissue, as well as for antifibrotic treatment response evaluation. This accessible post-processing technique merits further validation as the benefits for clinical practice as well as antifibrotic trial design would be numerous. KEY POINTS: • Magnetization transfer MRI and texture analysis of T2-weighted MR images can detect established bowel fibrosis in an animal model of gut fibrosis. • Texture entropy is able to identify and monitor bowel fibrosis progression in an inflammatory context and can assess the response to antifibrotic treatment. • A proof-of-concept study in five patients with Crohn's disease suggests that texture entropy can detect and grade fibrosis in human intestinal strictures.

Fecal Microbiota Transplantation Reduces Symptoms in Some Patients With Irritable Bowel Syndrome With Predominant Abdominal Bloating: Short- and Long-term Results From a Placebo-Controlled Randomized Trial.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with intestinal dysbiosis. Given the reported promising results of open-label fecal microbiota transplantation (FMT) therapy in patients with predominant abdominal bloating, we studied efficacy of this treatment in a randomized, placebo-controlled trial. METHODS: Patients with refractory IBS, defined as failure of ≥3 conventional therapies, were randomly assigned to single-dose nasojejunal administration of donor stools (n = 43) or autologous stools (n = 19) in a double-blind study, performed from December 2015 through October 2017, and were followed up for 1 year. IBS-related symptoms were assessed by using a daily symptom diary to determine general abdominal discomfort, abdominal bloating, abdominal pain, and flatulence on a scale of 1-6. Number of daily bowel movements, consistency of the stools, and abdominal circumference were also recorded. Patients completed the IBS-specific quality of life questionnaire. Primary endpoints were improvement of IBS symptoms and bloating at 12 weeks (response). Secondary endpoints were changes in IBS symptom scores and quality of life. Stool samples were collected for microbiota amplicon sequencing. Open-label retransplantation was offered after the trial. RESULTS: At week 12, 56% of patients given donor stool reported improvement in both primary endpoints compared with 26% of patients given placebo (P = .03). Patients given donor stool had significant improvements in level of discomfort (mean reduction, 19%; median score before FMT, 3.98; range, 2.13-6.00; median score after FMT, 3.1; range, 951.29-5.90), stool frequency (mean reduction, 13%; median score before FMT, 2.10; range, 0.57-14.29; median score after FMT 1.7; range, 0.71-4.29), urgency (mean reduction, 38%; median score before FMT, 0.61; range, 0.00-1.00; median score after FMT, 0.37; range, 0.00-1.00), abdominal pain (mean reduction, 26%; median score before FMT, 3.88; range, 1.57-5.17; median score after FMT, 2.80; range, 1.14-4.94), flatulence (mean reduction, 10%; median score before FMT, 3.42; range, 0.71-6.00; median score after FMT, 3.07; range, 0.79-4.23), and quality of life (mean increase, 16%; median score before FMT 32.6; range, 11-119; median score after FMT, 43.1; range, 32.25-99). A significantly higher proportion of women given donor stool (69%) had a response than men (29%) (P = .01). Fecal samples from responders had higher diversity of microbiomes before administration of donor material than fecal samples from nonresponders (P = .04) and distinct baseline composition (P = .04), but no specific marker taxa were associated with response. After single FMT, 21% of patients given donor stool reported effects that lasted for longer than 1 year compared with 5% of patients given placebo stool. A second FMT reduced symptoms in 67% of patients with an initial response to donor stool but not in patients with a prior nonresponse. CONCLUSIONS: In a randomized trial of patients with treatment-refractory IBS with predominant bloating, FMT relieved symptoms compared with placebo (autologous transplant), although the effects decreased over 1 year. A second FMT restored the response patients with a prior response. Response was associated with composition of the fecal microbiomes before FMT; this might be used to as a biomarker to select patients for this treatment. ClinicalTrials.gov, Number: NCT02299973.

Evolution of Endoscopic Lesions in Steroid-Refractory Acute Severe Ulcerative Colitis Responding to Infliximab or Cyclosporine.

BACKGROUND/AIMS: Few data on the evolution of endoscopic findings are available in patients with acute severe ulcerative colitis (ASUC). The aim of this study was to describe this evolution in a prospective cohort. METHODS: Patients admitted for a steroid-refractory ASUC and included in a randomized trial comparing infliximab and cyclosporine were eligible if they achieved steroid-free clinical remission at day 98. Flexible sigmoidoscopies were performed at baseline, days 7, 42 and 98. Ulcerative colitis endoscopic index of severity (UCEIS) and its sub-scores - vascular pattern, bleeding and ulceration/erosion - were post-hoc calculated. Global endoscopic remission was defined by a UCEIS of 0, and partial endoscopic remission by any UCEIS sub-score of 0. RESULTS: Among the 55 patients analyzed (29 infliximab and 26 cyclosporine), 49 (83%) had UCEIS ≥6 at baseline at baseline. Partial endoscopic remission rates were higher for bleeding than for vascular pattern and for ulcerations/erosions at day 7 (20% vs. 4% and 5% (n = 55); p = .004 and p=.04), for bleeding and ulceration/erosion than for vascular pattern at day 42 [63% and 65% vs. 33% (n=54); p<.001 for both] and at day 98 [78% and 92% vs. 56% (n = 50); p = .007 and p < .001]. Global endoscopic remission rates at day 98 were higher in patients treated with infliximab than with cyclosporine [73% vs. 25% (n = 26 and 24); p < .001]. CONCLUSION: In steroid-refractory ASUC patients responding to a second-line medical therapy, endoscopic remission process started with bleeding remission and was not achieved in half the patients at day 98 for vascular pattern. Infliximab provided a higher endoscopic remission rate than cyclosporine at day 98.

Risk of Development of More-advanced Lesions in Patients With Inflammatory Bowel Diseases and Dysplasia.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBD) have increased risks of dysplasia and colitis-associated cancer (CAC). We evaluated the risk of development of high-grade dysplasia (HGD) or CAC after diagnosis of dysplasia using data from a national cohort of patients with IBD. METHODS: We performed a multicenter retrospective analysis of data collected from 7 tertiary referral regional or academic centers in Belgium. In searches of IBD pathology databases, we identified 813 lesions (616 low-grade dysplasias [LGDs], 64 high-grade dysplasias [HGDs], and 133 CACs) in 410 patients with IBD: 299 had dysplasia (73%) and 111 had CAC (27%). The primary aim was to determine the risk of more-advanced lesions after diagnosis of LGD or HGD. RESULTS: Of the 287 patients with LGD, 21 (7%) developed more-advanced lesions (HGD or CAC) after a median time period of 86 months (interquartile range, 34-214). Of the 28 patients with HGD, 4 (14%) developed CAC after a median time period of 180 months (interquartile range, 23-444). The overall cumulative incidence of CAC at 10 years after an initial diagnosis of HGD was 24.3% and after an initial diagnosis of LGD was 8.5% (P < .05). Metachronous lesions, non-polypoid lesions, and colonic stricture were associated with risk of occurrence of more-advanced lesions after LGD (P < .05). Of the 630 dysplastic lesions identified during endoscopy, 545 (86%) were removed during the same procedure or during a follow-up endoscopy or by surgery. Of 111 patients with CAC, 95 (86%) did not have prior detection of dysplasia and 64 of these 95 patients (67%) developed CAC outside of the screening or surveillance period recommended by the European Crohn's and Colitis Organisation. CONCLUSIONS: In an analysis of pathology data from 7 medical centers in Belgium, we found a low rate of detection of more-advanced lesions following detection of LGD or HGD-taking into account that most of the lesions were removed. Main risk factors for development of more-advanced lesions after LGD were metachronous lesions, non-polypoid lesions, and colon strictures.

Increasing Infliximab Dose Based on Symptoms, Biomarkers, and Serum Drug Concentrations Does Not Increase Clinical, Endoscopic, and Corticosteroid-Free Remission in Patients With Active Luminal Crohn's Disease.

BACKGROUND & AIMS: A combination of infliximab and immunomodulators is the most efficacious treatment for Crohn's disease (CD). Patients have the best outcomes when their serum concentrations of these drugs are above a determined therapeutic threshold. We performed a prospective, randomized trial to determine whether therapeutic drug monitoring (TDM) to maintain serum levels of infliximab above 3 µg/mL produced higher rates of clinical and endoscopic remission than adapting dose based only on symptoms. METHODS: We performed a double-blind trial in which 122 biologic-naïve adult patients with active CD (71 female, median age 29.8 years) received induction treatment with infliximab in combination with an immunosuppressant, from July 2012 through September 2015 at 27 centers in Europe. At week 14 of treatment, patients were randomly assigned (1:1:1) to 3 infliximab maintenance groups: dose increases (2 maximum) in steps of 2.5 mg/kg based on clinical symptoms and biomarker analysis and/or serum infliximab concentrations (dose intensification strategy [DIS]1 group); dose increase from 5 to 10 mg/kg based on the same criteria (DIS2 group); dose increase to 10 mg/kg based on clinical symptoms alone (controls). Patients' CD activity index scores, levels of C-reactive protein, fecal levels of calprotectin, and serum concentrations of infliximab were determined at baseline and at weeks 2, 4, 6, 12, and 14 of treatment, and then every 4 weeks thereafter until week 54. The primary endpoint was sustained corticosteroid-free clinical remission (CD activity index <150) from weeks 22 through 54 with no ulcers at week 54. RESULTS: The primary endpoint was reached by 15 (33%) of 45 patients in the DIS1 group, 10 (27%) of 37 patients in the DIS2 group, and 16 (40%) of 40 patients in the control group (P = .50). CONCLUSIONS: In a prospective randomized exploratory trial of patients with active CD, we found increasing dose of infliximab based on a combination of symptoms, biomarkers, and serum drug concentrations does not lead to corticosteroid-free clinical remission in a larger proportion of patients than increasing dose based on symptoms alone. EUDRACT NUMBER: 2011-003038-14.

Analysis of Genes Associated With Monogenic Primary Immunodeficiency Identifies Rare Variants in XIAP in Patients With Crohn's Disease.

BACKGROUND & AIMS: A few rare monogenic primary immunodeficiencies (PIDs) are characterized by chronic intestinal inflammation that resembles Crohn's disease (CD). We investigated whether 23 genes associated with 10 of these monogenic disorders contain common, low-frequency, or rare variants that increase risk for CD. METHODS: Common and low frequency variants in 1 Mb loci centered on the candidate genes were analyzed using meta-data corresponding to genotypes of approximately 17,000 patients with CD or without CD (controls) in Europe. The contribution of rare variants was assessed by high-throughput sequencing of 4750 individuals, including 660 early-onset and/or familial cases among the 2390 patients with CD. Variants were expressed from vectors in SW480 or HeLa cells and functions of their products were analyzed in immunofluorescence, luciferase, immunoprecipitation, and immunoblot assays. RESULTS: We reproduced the association of the interleukin 10 locus with CD (P = .007), although none of the significantly associated variants modified the coding sequence of interleukin 10. We found XIAP to be significantly enriched for rare coding mutations in patients with CD vs controls (P = .02). We identified 4 previously unreported missense variants associated with CD. Variants in XIAP cause the PID X-linked lymphoproliferative disease type 2, yet none of the carriers of these variants had all the clinical features of X-linked lymphoproliferative disease type 2. Identified XIAP variants S123N, R233Q, and P257A were associated with an impaired activation of NOD2 signaling after muramyl dipeptide stimulation. CONCLUSIONS: In a systematic analysis of variants in 23 PID-associated genes, we confirmed the association of variants in XIAP with CD. Further screenings for CD-associated variants and analyses of their functions could increase our understanding of the relationship between PID-associated genes and CD pathogenesis.

Feasibility study using iodine quantification on dual-energy CT enterography to distinguish normal small bowel from active inflammatory Crohn's disease.

BACKGROUND: Assessment of Crohn's disease (CD) activity is important to identify patients with active inflammation for therapy management. Quantitative analysis can provide objective measurement of disease presence. PURPOSE: To evaluate the feasibility of quantitative analysis of contrast-enhanced dual-energy computed tomography (DECT) data in detection of small bowel inflammation in patients with CD with an emphasis on iodine quantification. MATERIAL AND METHODS: DECT enterography was prospectively performed in 20 patients with active CD and in 20 healthy individuals, as the control group. Iodine overlay images were created. Wall thickness, attenuation, absolute iodine density, relative iodine density, and fat fraction were measured in the terminal ileum of all patients by two radiologists. Intraclass correlation coefficients were calculated to assess inter-rater agreement. Parameters were compared between patient groups using mixed model analysis. Receiver operating characteristic (ROC) analysis was performed. RESULTS: Both absolute and relative iodine density were significantly higher in active disease than in normal small bowel (all P < 0.001). In contrast, measurement of fat fraction was not significantly different in affected terminal ileal loops compared to normal terminal ileum ( P = 0.075). ROC analysis demonstrated a similar excellent diagnostic accuracy of wall thickness, attenuation, and absolute and relative iodine density with area under the ROC curve (AUC) values in the range of 0.96 for attenuation to 1 for relative iodine density. CONCLUSION: DECT with iodine quantification can be used in distinguishing normal small bowel from active inflammatory CD. Further research should investigate the value of iodine quantification in grading CD activity and in monitoring therapeutic response.

Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor.

BACKGROUND: Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFß-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFß1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tauroursodeoxycholic acid protects bile acid homeostasis under inflammatory conditions and dampens Crohn's disease-like ileitis.

Bile acids regulate the expression of intestinal bile acid transporters and are natural ligands for nuclear receptors controlling inflammation. Accumulating evidence suggests that signaling through these receptors is impaired in inflammatory bowel disease. We investigated whether tauroursodeoxycholic acid (TUDCA), a secondary bile acid with cytoprotective properties, regulates ileal nuclear receptor and bile acid transporter expression and assessed its therapeutic potential in an experimental model of Crohn's disease (CD). Gene expression of the nuclear receptors farnesoid X receptor, pregnane X receptor and vitamin D receptor and the bile acid transporters apical sodium-dependent bile acid transporter and organic solute transporter α and ß was analyzed in Caco-2 cell monolayers exposed to tumor necrosis factor (TNF)α, in ileal tissue of TNFΔARE/WT mice and in inflamed ileal biopsies from CD patients by quantitative real-time polymerase chain reaction. TNFΔARE/WT mice and wild-type littermates were treated with TUDCA or placebo for 11 weeks and ileal histopathology and expression of the aforementioned genes were determined. Exposing Caco-2 cell monolayers to TNFα impaired the mRNA expression of nuclear receptors and bile acid transporters, whereas co-incubation with TUDCA antagonized their downregulation. TNFΔARE/WT mice displayed altered ileal bile acid homeostasis that mimicked the situation in human CD ileitis. Administration of TUDCA attenuated ileitis and alleviated the downregulation of nuclear receptors and bile acid transporters in these mice. These results show that TUDCA protects bile acid homeostasis under inflammatory conditions and suppresses CD-like ileitis. Together with previous observations showing similar efficacy in experimental colitis, we conclude that TUDCA could be a promising therapeutic agent for inflammatory bowel disease, warranting a clinical trial.

T84 monolayers are superior to Caco-2 as a model system of colonocytes.

Colonic adenocarcinoma-derived Caco-2 and T84 epithelial cell lines are frequently used as in vitro model systems of functional epithelial barriers. Both are utilised interchangeably despite evidence that differentiated Caco-2 cells are more reminiscent of small intestinal enterocytes than of colonocytes, whereas differentiated T84 cells are less well characterised. The aim of this study was, therefore, to further characterise and compare differentiated Caco-2 and T84 cells. The objectives were to (1) compare the brush border morphology, (2) measure the expression of enterocyte- and colonocyte-specific genes and (3) compare their response to butyrate, which is dependent on the monocarboxylate transporter 1 (MCT1), an apical protein expressed primarily in colonocytes. T84 microvilli were significantly shorter than those of Caco-2 cells, which is a characteristic difference between small intestinal enterocytes and colonocytes. Also, enterocyte-associated brush border enzymes expressed in differentiated Caco-2 cells were not increased during T84 maturation, whereas colonic markers such as MCT1 were more abundant in differentiated T84 cells compared to differentiated Caco-2 cells. Consequently, T84 cells displayed a dose-responsive improvement of barrier function towards butyrate, which was absent in Caco-2 cells. On the other hand, differences in epithelial toll-like receptor expression between Caco-2 and T84 monolayers did not result in a corresponding differential functional response. We conclude that differentiated Caco-2 and T84 cells have distinct morphological, biochemical and functional characteristics, suggesting that T84 cells do not acquire the biochemical signature of mature small intestinal enterocytes like Caco-2 cells, but retain much of their original colonic characteristics throughout differentiation. These findings can help investigators select the appropriate intestinal epithelial cell line for specific in vitro research purposes.

Ursodeoxycholic Acid and Its Taurine- or Glycine-Conjugated Species Reduce Colitogenic Dysbiosis and Equally Suppress Experimental Colitis in Mice.

The promising results seen in studies of secondary bile acids in experimental colitis suggest that they may represent an attractive and safe class of drugs for the treatment of inflammatory bowel diseases (IBD). However, the exact mechanism by which bile acid therapy confers protection from colitogenesis is currently unknown. Since the gut microbiota plays a crucial role in the pathogenesis of IBD, and exogenous bile acid administration may affect the community structure of the microbiota, we examined the impact of the secondary bile acid ursodeoxycholic acid (UDCA) and its taurine or glycine conjugates on the fecal microbial community structure during experimental colitis. Daily oral administration of UDCA, tauroursodeoxycholic acid (TUDCA), or glycoursodeoxycholic acid (GUDCA) equally lowered the severity of dextran sodium sulfate-induced colitis in mice, as evidenced by reduced body weight loss, colonic shortening, and expression of inflammatory cytokines. Illumina sequencing demonstrated that bile acid therapy during colitis did not restore fecal bacterial richness and diversity. However, bile acid therapy normalized the colitis-associated increased ratio of Firmicutes to Bacteroidetes Interestingly, administration of bile acids prevented the loss of Clostridium cluster XIVa and increased the abundance of Akkermansia muciniphila, bacterial species known to be particularly decreased in IBD patients. We conclude that UDCA, which is an FDA-approved drug for cholestatic liver disorders, could be an attractive treatment option to reduce dysbiosis and ameliorate inflammation in human IBD.IMPORTANCE Secondary bile acids are emerging as attractive candidates for the treatment of inflammatory bowel disease. Although bile acids may affect the intestinal microbial community structure, which significantly contributes to the course of these inflammatory disorders, the impact of bile acid therapy on the fecal microbiota during colitis has not yet been considered. Here, we studied the alterations in the fecal microbial abundance in colitic mice following the administration of secondary bile acids. Our results show that secondary bile acids reduce the severity of colitis and ameliorate colitis-associated fecal dysbiosis at the phylum level. This study indicates that secondary bile acids might act as a safe and effective drug for inflammatory bowel disease.

Prevalence of CT features of axial spondyloarthritis in patients with Crohn's disease.

Background The sacroiliac joint and spine are seen on abdominal computed tomography (CT) and may show structural lesions as part of spondyloarthritis. Purpose To determine the prevalence of CT features of spondyloarthritis in patients with Crohn's disease (CD). Material and Methods A retrospective study of structural lesions of spondyloarthritis on abdominal CT was performed. The sacroiliac joints and spine of 120 patients were studied: study group I consisted of 40 patients with known CD and inflammatory back pain, group II involved 40 patients with CD without inflammatory back pain, and group III consisted of 40 patients without known joint or inflammatory bowel disease. Recorded CT features included sclerosis, erosions or ankylosis of the sacroiliac joint, enthesopathy, spinal syndesmophytes, and costovertebral joint lesions. Results CT showed structural lesions of the sacroiliac joints in 19/40 (48%) patients with CD and inflammatory back pain (sclerosis [n = 14; 35%], erosions [n = 14; 35%], ankylosis [n = 3; 8%]), in 8/40 (8%) patients with CD without inflammatory back pain (sclerosis [n = 3; 8%], erosions [n = 4; 10%], ankylosis [n = 3; 8%]), and in 3/40 (8%) patients without known joint or bowel disease (sclerosis [n = 2; 5%], ankylosis [n = 1; 3%]). Syndesmophytes were exclusively seen in group I (n = 6; 15%). Conclusion CT of the abdomen in patients with CD and inflammatory back pain shows structural lesions of the sacroiliac joint, entheses, or spine in almost half of the patients. Awareness and knowledge of these findings may guide the referring clinician to further clinical evaluation, imaging, and biomarker evaluation of the disease.

Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

Chronic cigarette smoke exposure induces microbial and inflammatory shifts and mucin changes in the murine gut.

Inflammatory bowel diseases (IBD) are complex multifactorial diseases characterized by an inappropriate host response to an altered commensal microbiome and dysfunctional mucus barrier. Cigarette smoking is the best known environmental risk factor in IBD. Here, we studied the influence of chronic smoke exposure on the gut microbiome, mucus layer composition and immune factors in conventional mice. We compared smoke-exposed with air-exposed mice (n = 12) after a smoke exposure of 24 weeks. Both Illumina sequencing (n = 6) and denaturing gradient gel electrophoresis (n = 12) showed that bacterial activity and community structure were significantly altered in the colon due to smoke exposure. Interestingly, an increase of Lachnospiraceae sp. activity in the colon was observed. Also, the mRNA expression of Muc2 and Muc3 increased in the ileum, whereas Muc4 increased in the distal colon of smoke-exposed mice (n = 6). Furthermore, we observed increased Cxcl2 and decreased Ifn-γ in the ileum, and increased Il-6 and decreased Tgf-ß in the proximal colon. Tight junction gene expression remained unchanged. We infer that the modulating role of chronic smoke exposure as a latently present risk factor in the gut may be driven by the altered epithelial mucus profiles and changes in microbiome composition and immune factors.

Paediatric Crohn Disease: Disease Activity and Growth in the BELCRO Cohort After 3 Years Follow-up.

OBJECTIVE: The Belgian registry for paediatric Crohn disease (BELCRO) cohort is a prospective, multicentre registry for newly diagnosed paediatric patients with Crohn disease (CD) (<18 years) recruited from 2008 to 2010 to identify predictive factors for disease activity and growth. METHODS: Data from the BELCRO database were evaluated at diagnosis, 24 and 36 months follow-up. RESULTS: At month 36 (M36), data were available on 84 of the 98 patients included at diagnosis. Disease activity evolved as follows: inactive 5% to 70%, mild 19% to 24%, and moderate to severe 76% to 6%. None of the variables such as age, sex, diagnostic delay, type of treatment, disease location, disease activity at diagnosis, and growth were associated with disease activity at M36. Paediatricians studied significantly less patients with active disease at M36 compared with adult physicians. Sixty percent of the patients had biologicals as part of their treatment at M36. Adult gastroenterologists initiated biologicals significantly earlier. They were the only factor determining biologicals' initiation, not disease location or disease severity at diagnosis. Median body mass index (BMI) z score evolved from -0.97 (range -5.5-2.1) to 0.11 (range -3.4-2) and median height z score from -0.15 (range -3.4-1.6) to 0.12 (range -2.3-2.3) at M36. None of the variables mentioned above influenced growth over time. CONCLUSIONS: Present treatment strategies lead to good disease control in the BELCRO cohort after 3 years. Logistic regression analysis did not show any influence of disease location or present treatment strategy on disease activity and growth, but patients under paediatric care had significantly less severe disease at M36.

Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease.

BACKGROUND: Leukocyte cytosolic proteins (e.g., calprotectin) are emerging biomarkers for inflammatory bowel disease. Leukocyte aryl esterase activity has been commonly used for sensitive detection of leukocytes in human body fluids such as urine. Urine test strip results are generally reported in categories. As automated strip readers allow quantitative data to be reported, sensitive quantitative detection of leukocytes in body fluids has become possible. Here, we explored the use of leukocyte esterase as a potential alternative faecal biomarker for inflammatory bowel disease. METHODS: We evaluated leukocyte esterase activity in faecal extracts and compared Cobas u 411 (Roche) quantitative reflectance data with calprotectin concentration for 107 routine samples. Stability of leukocyte esterase for trypsin digestion was carried out by adding trypsin to the extract. Incubation occurred at 37 °C for 24 h or 48 h. RESULTS: Reproducibility of the reflectance signal was good (within-run imprecision: 6.1%; between-run imprecision: 6.2%). Results were linear in the range 103-106 WBC/100 mg faeces. The lower limit of detection was 4 WBC/µL and the lower limit of quantification was 5 WBC/µL. Stability of LE activity in stool and faecal matrix was good. An adequate correlation was obtained between leukocyte esterase activity and the faecal calprotectin concentration: log(y)=4.28+0.29log(x). In vitro experiments monitored the digestion of leukocyte esterase and faecal calprotectin. Leukocyte esterase activity was significantly less affected by trypsin activity than calprotectin immunoreactivity. CONCLUSIONS: Quantitative leukocyte esterase activity of faecal extracts provides information about the leukocyte count in the gut lumen. Leukocyte esterase is a promising and affordable alternative biomarker for monitoring inflammatory bowel disease.

Tauroursodeoxycholic acid inhibits experimental colitis by preventing early intestinal epithelial cell death.

Ulcerative colitis (UC) is characterized by increased epithelial cell death and subsequent breakdown of the intestinal epithelial barrier, which perpetuates chronic intestinal inflammation. Since fecal bile acid dysmetabolism is associated with UC and tauroursodeoxycholic acid (TUDCA) has been shown to improve murine colitis, we evaluated the effect of TUDCA on intestinal epithelial cell death in a mouse model of UC-like barrier dysfunction elicited by dextran sulfate sodium (DSS). We identified the prevention of colonic caspase-3 induction, a key proapoptotic marker which was also over-activated in UC, as the earliest event resulting in a clear clinical benefit. Whereas vehicle-treated mice showed a cumulative mortality of 40%, all TUDCA-treated mice survived the DSS experiment during a 14-day follow-up period. In line with a barrier protective effect, TUDCA decreased bacterial translocation to the spleen and stimulated mucin production. Similarly, TUDCA inhibited lipopolysaccharide-induced intestinal permeability and associated enterocyte apoptosis. The anti-apoptotic effect was confirmed in vitro by a dose-dependent inhibition of both receptor-dependent (using tumor necrosis factor and Fas ligand) and receptor-independent (staurosporine) caspase-3 induction in HT29 colonic epithelial cells. These data imply that caspase-3 activation is an early marker of colitis that is prevented by TUDCA treatment. These data, together with the previously reported beneficial effect in colitis, suggest that TUDCA could be an add-on strategy to current immunosuppressive treatment of UC patients.