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Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo. Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098–106. ©2018 AACR.

Supplementary Methods from Cyclin D2–Cyclin-Dependent Kinase 4/6 Is Required for Efficient Proliferation and Tumorigenesis following Apc Loss

Supplementary Figure Legends 1-4 from Cyclin D2–Cyclin-Dependent Kinase 4/6 Is Required for Efficient Proliferation and Tumorigenesis following Apc Loss

Supplementary Figures 1-4 from Cyclin D2–Cyclin-Dependent Kinase 4/6 Is Required for Efficient Proliferation and Tumorigenesis following Apc Loss

Inactivation of the Apc gene is recognized as the key early event in the development of sporadic colorectal cancer (CRC), where its loss leads to constitutive activation of β-catenin/T-cell factor 4 signaling and hence transcription of Wnt target genes such as c-Myc. Our and other previous studies have shown that although cyclin D1 is required for adenoma formation, it is not immediately upregulated following Apc loss within the intestine, suggesting that proliferation following acute Apc loss may be dependent on another D-type cyclin. In this study, we investigated the expression and functional relevance of cyclin D2 following Apc loss in the intestinal epithelium. Cyclin D2 is upregulated immediately following Apc loss, which corresponded with a significant increase in cyclin-dependent kinase 4 (CDK4) and hyperphosphorylated Rb levels. Deficiency of cyclin D2 resulted in a reduction in enterocyte proliferation and crypt size within Apc-deficient intestinal epithelium. Moreover, cyclin D2 dramatically reduced tumor growth and development in ApcMin/+ mice. Importantly, cyclin D2 knockout did not affect proliferation of normal enterocytes, and furthermore, CDK4/6 inhibition also suppressed the proliferation of adenomatous cells and not normal cells from ApcMin/+ mice. Taken together, these results indicate that cyclin D–CDK4/6 complexes are required for the efficient proliferation of cells with deregulated Wnt signaling, and inhibiting this complex may be an effective chemopreventative strategy in CRC. Cancer Res; 70(20); 8149–58. ©2010 AACR.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc−/−Ihh−/− double mutant mice. We found that recombinant EREG, BTC, and NRG1 but not Lgr5 ligand R-Spondin promoted growth and proliferation of Apc double mutant colonic organoids. Thus, the loss of Ihh enhances Apc-driven colonic adenomagenesis via upregulation of ErbB pathway family members in colonic stromal cells. Our findings highlight the critical role of epithelium-derived Indian Hedgehog as a stromal tumor suppressor in the intestine.

Background & Aims Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model. Methods We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage. Results Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-α–induced damage. Conclusions ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair., Graphical abstract

Supplemental Digital Content is available in the text., Background Patient reported outcomes are important in Crohn’s disease. In this prospective cohort, we investigated the performance of the Bristol Stool Form Scale (BSFS) and a visual analog scale (VAS) for abdominal pain as outcome measures in Crohn’s disease. Methods Patients with active Crohn’s disease starting glucocorticoids or anti-tumor necrosis factor were included. Before treatment and 10 weeks later we collected: clinical activity [Harvey Bradshaw Index (HBI) and Crohn’s-Disease-Activity-Index (CDAI)], serum C-reactive protein (CRP) and fecal calprotectin, and BSFS (1–7) and a 100-mm VAS based on a 7-day diary. Clinical response was defined as a reduction by at least 3 and at least 100 of HBI and CDAI, respectively. Fecal calprotectin-response and CRP-response were defined as reduction of at least 50%. Results Thirty-eight patients completed follow-up. At baseline, BSFS-parameters correlated more strongly with clinical activity (range: rs: 0.31–0.74) than with CRP (rs: −0.01 to 0.16) and fecal calprotectin (rs: 0.14–0.26). VAS scores correlated very weakly to moderately with clinical activity (rs: 0.18–0.45), and weakly to moderately with CRP (rs: 0.24–0.34) and fecal calprotectin (rs: 0.35–0.43). Changes in VAS scores correlated moderately to strongly (rs: 0.55–0.71) with changes in clinical activity, and weakly with changes in CRP and fecal calprotectin (rs: 0.21–0.35). Changes in BSFS parameters correlated weakly to moderately (rs: 0.23–0.53) with changes in clinical activity, and very weakly to weakly (rs: 0.01–0.35) with changes in CRP and fecal calprotectin. Responsiveness of VAS and BSFS was moderate to high (Guyatt’s statistic 0.41–2.17) and highly dependent on the definition of response. Conclusions The BSFS and a VAS appear to be responsive with moderate-to-strong construct validity to monitor patients with Crohn’s disease.

Background & Aims Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking. Methods We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration. Results We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies. Conclusions In conclusion, we established and validated an in vitro damage–repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518., Graphical abstract

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

Background and aims Rectal resection in inflammatory bowel disease [IBD] is frequently complicated by disturbed perineal wound healing. Close rectal dissection, where the mesorectum remains in situ, is hypothesized to reduce complications by minimizing dead space, compared to total mesorectal excision. The aim of this study was to analyse post-operative outcomes of both techniques. In addition, immune activity in mesorectal tissue was assessed. Methods Perineal complications and healing were retrospectively assessed in a series of 74 IBD patients undergoing proctectomy using close rectal dissection or total mesorectal excision. The mesorectums of 15 patients were analysed by fluorescence-activated cell sorting, immunofluorescence and in situ hybridization. Based on the clinical and in vitro findings, a novel surgical approach for Crohn's disease patients with disturbed perineal healing after proctectomy was developed. Results In Crohn's disease, perineal complications were more frequent after close rectal dissection than after total mesorectal excision [59.5% vs 17.6%; p = 0.007] with lower healing rates [51.4% vs 88.2%; p = 0.014]. No differences were observed in ulcerative colitis. The mesorectal tissue in Crohn's disease contained enhanced numbers of tumour necrosis factor α-producing CD14+ macrophages, with less expression of the wound-healing marker CD206. Based on these findings, mesorectal excision with omentoplasty was performed in eight patients with perineal complications after close rectal dissection, resulting in complete perineal wound closure in six. Pro-inflammatory characteristics remained present in the mesorectum after close rectal dissection in these patients. Conclusions In Crohn's disease, close rectal dissection resulted in more perineal complications, associated with a pro-inflammatory immune status of the mesorectal tissue. Excision of this pro-inflammatory mesenteric tissue resulted in improved perineal healing rates.

Deregulated global mRNA translation is an emerging feature of cancer cells. Oncogenic transformation in colorectal cancer (CRC) is driven by mutations in APC, KRAS, SMAD4, and TP53, known as the adenoma-carcinoma sequence (ACS). Here we introduce each of these driver mutations into intestinal organoids to show that they are modulators of global translational capacity in intestinal epithelial cells. Increased global translation resulting from loss of Apc expression was potentiated by the presence of oncogenic Kras(G12D). Knockdown of Smad4 further enhanced global translation efficiency and was associated with a lower 4E-BP1-to-eIF4E ratio. Quadruple mutant cells with additional P53 loss displayed the highest global translational capacity, paralleled by high proliferation and growth rates, indicating that the proteome is heavily geared toward cell division. Transcriptional reprogramming facilitating global translation included elevated ribogenesis and activation of mTORC1 signaling. Accordingly, interfering with the mTORC1/4E-BP/eIF4E axis inhibited the growth potential endowed by accumulation of multiple drivers. In conclusion, the ACS is characterized by a strongly altered global translational landscape in epithelial cells, exposing a therapeutic potential for direct targeting of the translational apparatus.

International audience; Intestinal stem cells (ISCs) fuel the lifelong self-renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA-binding protein is crucial for the maintenance of the Lgr5 + ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator-activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5 + stem cell signature. Furthermore, we identify PPARc activity as a molecular intermediate of MEX3A-mediated regulation. We also show that high PPARc signalling impairs Lgr5 + ISC function, thus uncovering a new layer of post-transcriptional regulation that critically contributes to intestinal homeostasis.

Background and Aims Although several endoscopic and histopathologic indices are available for evaluating the severity of inflammation in mouse models of colitis, the reliability of these scoring instruments is unknown. Our aim was to evaluate the reliability of the individual items in the existing indices and develop new scoring systems by selection of the most reliable index items. Methods Two observers scored the histological slides [n = 224] and endoscopy videos [n = 201] from treated and untreated Interleukin[IL]-10 knock-out and T-cell transferred SCID mice. Intra-rater and inter-rater reliability for endoscopy and histology scores, and each individual item, were measured using intraclass correlation coefficients [ICCs]. The Mouse Colitis Histology Index [MCHI] and Mouse Colitis Endoscopy Index [MCEI] were developed using the most reliable items. Both were correlated to the colon density and to each other and were evaluated for their ability to detect changes in pathobiology. Results The intraclass correlation coefficients (ICCs) for inter-rater agreement (95% CIs) for the total histology and endoscopy scores were 0.90 [0.87–0.92] and 0.80 [0.76–0.84], respectively. The MCHI and MCEI were highly correlated with colon density, with a Spearman Rho = 0.81[0.75–0.85] and 0.73 [0.66–0.79], respectively, and with each other, Spearman Rho = 0.71 [0.63–0.77]. The MCHI and MCEI were able to distinguish between the experimental groups within the models, with pairwise differences between the treated and untreated groups being statistically significant [p < 0.001]. Conclusions These histological and endoscopic indices are valid and reliable measures of intestinal inflammation in mice, and they are responsive to treatment effects in pre-clinical studies.

BACKGROUND Several factors influencing the pharmacokinetics of infliximab (IFX) in inflammatory bowel disease (IBD) have been identified. We studied the impact of patient, disease, and treatment characteristics on clearance and immunogenicity of IFX in a real-world patient-with-IBD cohort. METHODS Serum concentrations of IFX and antibodies to IFX (ATIs) were measured in patients with IBD at a single center using an enzyme-linked immunosorbent assay and radioimmunoassay. Patient, disease, and treatment characteristics were retrospectively collected along with laboratory values. Pharmacokinetics and ATI titer were analyzed simultaneously by nonlinear mixed-effects modeling. RESULTS Nine hundred ninety-seven IFX concentrations and 756 ATI measurements from 332 patients with IBD (253 Crohn's disease and 79 ulcerative colitis) were included. Mean (SD) IFX dose was 5.47 ± 1.33 mg/kg. ATIs were detected in 75/332 (23%) patients; insufficient exposure below an IFX trough level of 3 μg/mL was the most predictive factor of developing ATI and resulted in a 4-fold increased risk of ATI development. ATI titer was a better predictor of IFX clearance than ATI as a dichotomous parameter. ATI titers >30 AU/mL were consistently associated with undetectable IFX concentrations. IFX clearance was affected by body weight (40-149 kg) ranging from 0.27 to 0.53 L/d, serum albumin (2-5.4 g/dL) from 0.93 to 0.24 L/d, and titers of ATIs (0-53,000 AU/mL) from 0.36 L/d to 15.93 L/d (P < 0.001). Previously biologic-treated patients exhibited a higher clearance of IFX. CONCLUSIONS IFX exposure below 3 μg/mL increases risk of ATIs. Identification of influential pharmacokinetics and ATI factors improves prediction of IFX levels, potentially allowing individualized dosing and cost reduction.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc

Intestinal stem cells (ISCs) fuel the lifelong self‐renewal of the intestinal tract and are paramount for epithelial repair. In this context, the Wnt pathway component LGR5 is the most consensual ISC marker to date. Still, the effort to better understand ISC identity and regulation remains a challenge. We have generated a Mex3a knockout mouse model and show that this RNA‐binding protein is crucial for the maintenance of the Lgr5 + ISC pool, as its absence disrupts epithelial turnover during postnatal development and stereotypical organoid maturation ex vivo. Transcriptomic profiling of intestinal crypts reveals that Mex3a deletion induces the peroxisome proliferator‐activated receptor (PPAR) pathway, along with a decrease in Wnt signalling and loss of the Lgr5 + stem cell signature. Furthermore, we identify PPARγ activity as a molecular intermediate of MEX3A‐mediated regulation. We also show that high PPARγ signalling impairs Lgr5 + ISC function, thus uncovering a new layer of post‐transcriptional regulation that critically contributes to intestinal homeostasis., The RNA‐binding protein MEX3A is expressed at the base of intestinal crypts and is critical for the maintenance of Lgr5 + ISCs during postnatal development. MEX3A regulates the PPARγ pathway, thereby modulating ISC identity and tissue homeostasis.

Background: The appropriate location for biopsy procurement relative to an ulcer in active Crohn's disease is unknown. Aim: To explore the relationship between biopsy location, histological disease activity, proinflammatory gene expression and the presence of inflammatory cells. Methods: Fifty-one patients with Crohn's disease and ulcers >0.5 cm diameter in the colon and/or ileum were prospectively enrolled at three centres. Biopsies were obtained from 0 mm, 7 to 8 mm and 21 to 24 mm from the edge of the largest ulcer. Histological activity was blindly assessed with the Global Histological Disease Activity Score, the Robarts Histopathology and Nancy Histological indices. Messenger ribonucleic acid (mRNA) levels for interleukins-6, -8 and -23 (p19 and p40 subunits), CD31 and S100A9 were measured using quantitative polymerase chain reaction. The number of CD3+, CD68+ and myeloperoxidase-positive cells was quantified by immunohistochemistry. Data were analysed using mixed models with location and segment as fixed effects and patients as random effect to account for correlation among segments within a patient. Results: Histological disease activity scores (P

The unfolded protein response (UPR) acts through its downstream branches, PERK-eIF2α signaling, IRE1α-XBP1 signaling and ATF6 signaling. In the intestine, activation of the UPR through the kinase PERK results in differentiation of intestinal epithelial stem cells and colon cancer stem cells, whereas deletion of XBP1 results in increased stemness and adenomagenesis. How downstream activation of XBP1 and ATF6 influences intestinal stemness and proliferation remains largely unknown. We generated colorectal cancer cells (LS174T) that harbor doxycycline inducible expression of the active forms of either XBP1(s) or ATF61-373. Activation of either XBP1 or ATF6 resulted in reduced cellular proliferation and reduced expression of markers of intestinal epithelial stemness. Moreover, XBP1 and ATF6 activation reduced global protein synthesis and lowered the threshold for UPR activation. XBP1-mediated loss of stemness and proliferation resulted from crossactivation of PERK-eIF2α signaling and could be rescued by constitutive expression of eIF2α phosphatase GADD34. We thus find that enforced activation of XBP1 and ATF6 results in reduction of stemness and proliferation. We expose a novel interaction between XBP1 and PERK-eIF2α signaling.

During the suckling‐to‐weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium‐derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivo . In vitro postnatal development of the fetal‐derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo . Together, our data show that organoids derived from fetal epithelium undergo suckling‐to‐weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

BACKGROUND The inflammatory burden influences therapeutic decisions in patients with ulcerative colitis (UC). We aimed to study which commonly used markers of disease activity correlate best with inflammatory burden in patients with UC using leukocyte scintigraphy (single-photon emission computed tomography [SPECT-CT]) as the gold standard. METHODS Patients with different severity of UC underwent colonoscopy with biopsies and leukocyte SPECT-CT scintigraphy. Serum C-reactive protein (CRP), fecal calprotectin, and clinical questionnaires were collected. The maximum uptake of technetium-labeled leukocytes was calculated as a SPECT score for each colon segment and a summed activity score for 5 colonic segments combined. RESULTS Thirty patients with UC were included; 14 of 30 (47%) had left-sided colitis, and 16 of 30 (53%) had pancolitis. One patient (3%) had inactive UC, 5 of 30 (17%) had mild, 11 of 30 (37%) had moderate, and 13 of 30 (43%) had severe disease activity based on the endoscopic Mayo score. The endoscopic Mayo score correlated better with the SPECT score than with the ulcerative colitis endoscopic index of severity (UCEIS) (r = 0.50; P < 0.01 and r = 0.32; P = 0.08, respectively). The Geboes UC histologic score correlated equally well as the Mayo score (r = 0.50; P < 0.01). We found a significant correlation between scintigraphy and fecal calprotectin (r = 0.44; P = 0.02) but not with serum CRP (r = 0.25; P = 0.18). Fecal calprotectin reflected inflammatory burden significantly better in left-sided colitis (r = 0.80; P = 0.001) than in pancolitis (r = 0.22; P = 0.41). CONCLUSIONS The inflammatory burden in patients with UC, measured by SPECT-CT, is better reflected by the endoscopic Mayo score and the Geboes histologic score than by the UCEIS. Fecal calprotectin is a more accurate inflammatory marker than CRP, predominantly in patients with left-sided colitis. REGISTRATION This study was approved by the Ethics Committee Review at October 22, 2012 and, in accordance with Dutch legislation, prospectively registered at the CCMO (Dutch central commission for human research) https://www.toetsingonline.nl with NL39801.018.12.

Objective Infliximab maintenance treatment for Crohn's disease (CD) consists of intravenous infusions that are usually given at 6-8-week intervals. We aimed to evaluate whether home-based infliximab infusions could offer a useful and safe alternative for the management of CD patients. Methods Adult CD patients receiving infliximab maintenance treatment at the Academic Medical Center in Amsterdam were invited to receive their infusions at home for the duration of 1 year. Patients had to be in clinical remission and should have had no adverse events during previous infusions. Patient satisfaction and experience were studied. Costs were analyzed and compared with hospital-based infliximab infusions. Results Twenty-nine patients were invited, of whom 13 (45%) wanted to participate. Of the participants, 54% were female, and the median age was 33 years. In total, 59 infliximab infusions were administered at home at a median dose of 360 mg. The median rating of patient satisfaction was 8 on a scale from 1 to 10 for both home and hospital treatment settings. An important observation was that patients' willingness to participate would have been 70% if the possibility of receiving infusions at home outside office hours had been offered. Costs of infliximab infusions at home were €229 per infusion compared with €284 at the infusion clinic (excluding drug costs). Conclusion Home-based infliximab infusions were associated with a cost saving of €55 per infusion. Most participants were satisfied and would recommend home-based infusions to others. Infliximab treatment at home might be recommended as routine care for CD patients.

Background and Aims: Ciclosporin A [CsA] and infliximab [IFX] are similarly effective in preventing short-term colectomy in ulcerative colitis [UC] patients, but long-term data are scarce. We aimed to compare short- and long-term efficacy of CsA and IFX by analysing colectomy rates and failure of remission-induction treatment as outcome parameters for treatment success. Methods: We retrospectively studied hospitalised UC patients who received CsA or IFX for moderate-to-severe UC, between January 2000 and April 2014. The primary endpoint was time to colectomy, and treatment failure [defined as colectomy or another remission-induction treatment with corticosteroids, CsA, or IFX] was used as secondary endpoint. Variables possibly affecting colectomy outcomes were analysed. Results: A total of 55 patients were studied for colectomy outcome and 58 patients for treatment failure. A significantly longer follow-up duration was available for CsA-treated patients [ p < 0.001, both subcohorts]. Patients showed comparable patient- and disease-specific characteristics. Colectomy rates did not differ significantly at 3, 12, and 36 months: 36% versus 29%, 58% versus 48%, and 64% versus 67% for CsA- and IFX-treated patients, respectively. Multivariate Cox regression analysis revealed the lowest hazard ratio [HR] for colectomy in patients concomitantly using thiopurines: HR 0.28 (confidence interval [CI] 0.13–0.64), p = 0.002. Treatment failure rates were not significantly different at 3, 12 and 36 months: 35% versus 48%, 51% versus 68%, and 62% versus 83% for CsA- and IFX-treated patients, respectively. Conclusions: Treatment with CsA and IFX is similarly effective in preventing short- and long-term colectomy in hospitalised UC patients. Furthermore, failure rates of these remission-induction treatments were comparable.

Inflammation-associated pathways are active in intestinal epithelial cells (IECs) and contribute to the pathogenesis of colorectal cancer (CRC). Calcineurin, a phosphatase required for the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors, shows increased expression in CRC. We therefore investigated the role of calcineurin in intestinal tumor development. We demonstrate that calcineurin and NFAT factors are constitutively expressed by primary IECs and selectively activated in intestinal tumors as a result of impaired stratification of the tumor-associated microbiota and toll-like receptor signaling. Epithelial calcineurin supports the survival and proliferation of cancer stem cells in an NFAT-dependent manner and promotes the development of intestinal tumors in mice. Moreover, somatic mutations that have been identified in human CRC are associated with constitutive activation of calcineurin, whereas nuclear translocation of NFAT is associated with increased death from CRC. These findings highlight an epithelial cell-intrinsic pathway that integrates signals derived from the commensal microbiota to promote intestinal tumor development.

Introduction Pre-pouch ileitis (PI) is a complication that can occur after panproctocolectomy and ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). It is characterised by inflammation of pre-pouch ileum in the afferent limb of the pouch. Our aims were to assess the prevalence of PI as well as to identify predictive factors and investigate the medications needed for its management. Method Data on 546 patients who underwent IPAA for UC was retrospectively collected from three tertiary inflammatory bowel disease (IBD) referral centres. Data was collected from sites in the Netherlands (Academic Medical Centre, Amsterdam), Belgium (Leuven University Hospital) and England (University College London Hospital). PI was considered present if there was endoscopic, as well as histological inflammation in the afferent limb. Results PI was present in 33/546 (6%) UC patients, all of these had concurrent pouchitis. 144 (26%) patients had pouchitis without PI. 369 (68%) patients did not have any inflammatory pouch problems. Rates of requiring potent immunosupressive treatment were higher amongst patients with PI than those with pouchitis alone. Patients who went on to develop PI were significantly younger at the time of their UC diagnosis. PSC was significantly more common in patients with PI than those with pouchitis alone. Conclusion PI is a much less common and more treatment refractory condition than pouchitis alone. Pouchoscopy should be considered in any patient with symptoms of pouchitis. This should include careful endoscopic evaluation of the afferent pouch limb as well as biopsies of the pre-pouch ileum. Once a diagnosis of PI is made, clinicians should commence immunomodulatory therapy early in the disease course and consider escalating to an anti-TNF if this proves ineffective. Disclosure of interest None Declared.

Several anti-tumour necrosis factor [TNF] blocking strategies have been evaluated in patients with Crohn's disease. Compounds that have been tested included the full monoclonal IgG1 antibodies infliximab and adalimumab, the pegylated anti-TNF F[ab']2 fragment certolizumab, an IgG4 anti-TNF CDP571 with reduced affinity for the Fc receptor, the soluble TNF receptor I onercept, and the TNF receptor II-Fc fusion protein etanercept. The endpoints of these studies suggest that not all methods of blocking TNF are equal. Here we will review the differences in the clinical, biochemical, and endoscopic endpoints of the major clinical studies. Collectively the data suggest that only IgG1 monoclonal antibodies have the ability to induce complete clinical, biochemical, and endoscopic remission. We discuss the potential multiple modes of action that may contribute to the response to full IgG1 anti-TNFs, focusing on the rapid induction of lamina propria T cell apoptosis and Fc receptor-dependent induction of M2-type wound-healing macrophages. We discuss how novel insights into the mechanism of action of anti-TNFs in Crohn's disease may contribute to the development of novel anti-TNFs with improved efficacy.

ObjectiveAfter prophylactic colectomy, adenomas continue to develop in the remaining intestine of patients with familial adenomatous polyposis (FAP). There is a lack of standard clinical recommendation for chemoprevention in patients with FAP. Because of promising in vivo studies, the aim of this pilot study was to investigate the safety of sirolimus and its effect on progression of intestinal adenomas.DesignPatients with FAP with InSiGHT Polyposis Staging System 3 of the retained rectum or pouch received sirolimus for 6 months, dosed at plasma concentration levels of 5–8 µg/L. Primary outcomes were safety and change in marked polyp size. Secondary outcomes were change in number of polyps and effect on proliferation and apoptosis assessed by immunohistochemistry.ResultsEach of the included four patients reported 4 to 18 adverse events (toxicity grades 1–3). One patient prematurely terminated the study because of adverse events. Marked polyp size decreased in 16 (80%)/20 and remained the same in 4 (20%)/20 patients. The number of polyps decreased in all patients (MD −25.75, p=0.13). Three out of four patients showed substantial induction of apoptosis or inhibition of proliferation.ConclusionSix months of sirolimus treatment in four patients with FAP showed promising effects especially on the number of polyps in the rectal remnant and ileal pouch, although at the cost of numerous adverse events.Trial registration numberClinicalTrials.gov ID NCT03095703.

CD103+ dendritic cells (DC) are crucial for regulation of intestinal tolerance in humans. However, upon infection of the lamina propria this tolerogenic response is converted to an inflammatory response. Here we show that immunoglobulin A (IgA) immune complexes (IgA-IC), which are present after bacterial infection of the lamina propria, are important for the induction of inflammation by the human CD103+SIRPα+ DC subset. IgA-IC, by recognition through FcαRI, selectively amplify the production of proinflammatory cytokines TNF, IL-1β and IL-23 by human CD103+ DCs. These cells then enhance inflammation by promoting Th17 responses and activating human intestinal innate lymphoid cells 3. Moreover, FcαRI-induced cytokine production is orchestrated via upregulation of cytokine translation and caspase-1 activation, which is dependent on glycolytic reprogramming mediated by kinases Syk, PI3K and TBK1-IKKε. Our data suggest that the formation of IgA-IC in the human intestine provides an environmental cue for the conversion of a tolerogenic to an inflammatory response., Dendritic cells (DC) are important for maintaining immune homeostasis in the gut, but how they promote intestinal inflammation upon bacterial infection is still unclear. Here the authors show that IgA immune complexes induce proinflammatory cytokine production by metabolic reprogramming of otherwise tolerogenic human CD103+ DCs.

Introduction Overt fibrostenotic disease is a relative contraindication for anti-TNF therapy in Crohn’s disease. We hypothesized that subclinical fibrosis may also contribute to an incomplete response to anti-TNF therapy before the onset of symptomatic stenosis. Methods In a previous trial, patients with ileocecal Crohn’s disease were randomized to either immediate ileocecal resection or medical treatment with Infliximab. In case of insufficient response to Infliximab, the latter underwent secondary ileocecal resection. We compared specimens from those patients undergoing immediate resection (Infliximab naïve, n = 20) to those who failed Infliximab therapy (n = 20). Results Infliximab naïve and Infliximab failure patients had similar severity of inflammation when assessed by CRP levels (median 14 vs 9 mg/L) and histology (Geboes-D’Haens-score, median 10 vs 11 points). On immunohistochemistry, collagen-III and fibronectin depositions were increased in patients previously exposed to Infliximab compared to patients naïve to Infliximab. On mRNA level, procollagen peptidase showed significantly more mucosal mRNA expression in Crohn’s disease patients who failed Infliximab. Infliximab responders showed no increase of this marker after 4 weeks of successful Infliximab treatment. Discussion Failure to Infliximab therapy is associated with subclinical fibrosis in Crohn’s disease.

BACKGROUND AND AIMS: We have recently shown that the mode of action of IgG1 anti-tumour necrosis factor [TNF] antibodies in inflammatory bowel disease [IBD] requires Fcγ-receptor [FcγR] engagement on macrophages. Here we examine the effect of Fcγ-receptor signalling by anti-TNF on macrophage IL-12/IL-23 secretion. METHODS: Cytokine production by human inflammatory macrophages was assessed at the level of RNA and protein. TNF-anti-TNF immune complex formation was determined by size-exclusion chromatography and signalling visualized by immunofluorescence. IL-12/IL-23p40 was measured in CD14+ lamina propria cells from IBD patients. RESULTS: Infliximab and adalimumab potently suppressed IL-12/IL-23 production by inflammatory macrophages, but Fab' fragment certolizumab did not. IL-12/IL-23 suppression depended on Syk activity and was mediated at the level of IL-12/IL-23p40 mRNA. Etanercept, a soluble TNF receptor fused to an Fc-region, did not inhibit IL-12/L-23 secretion, suggesting that the presence of an Fc-region was not sufficient. Infliximab and adalimumab formed immune complexes with soluble TNF whereas etanercept did not, suggesting that FcγR-mediated suppression of IL-12/IL-23 required the formation of immune complexes. Indeed, non-specific IgG1 immune complexes, but not uncomplexed IgG1, similarly suppressed IL-12/IL-23 secretion. Finally, infliximab significantly decreased IL-12/IL-23p40 production in myeloid cells isolated from the lamina propria of IBD patients. CONCLUSIONS: TNF-anti-TNF antibody immune complexes potently inhibit IL-12/IL-23 expression by inflammatory macrophages. Our data suggest that anti-TNFs and antibodies against IL-12/IL-23 may therefore have partially overlapping modes of action in patients with IBD. ispartof: JOURNAL OF CROHNS & COLITIS vol:12 issue:9 pages:1122-1130 ispartof: location:England status: published

The mechanisms that orchestrate the development of a multicellular organism from a single common ancestor cell have fascinated people for centuries. We are still a long way from truly understanding the robust programs that allow building of our specialized organs that regulate digestion, respiration, and hemodynamics and that guard us from invasion by other unsolicited organisms. However, in the past three decades, many of the genes that play a role in patterning our bodies have been identified. We have learned much about the mechanisms involved in development by characterizing the nature of these patterning genes and the way they work together. In this chapter, we will discuss patterning mechanisms, introduce the Hedgehog pathway and then summarize our current understanding of the role of Hedgehog signaling in development and maintenance of the gastrointestinal (GI) tract.

The large randomized placebo controlled trials of the Women’s Health Initiative have shown that the combination of estrogen and progestin medroxyprogesterone acetate (MPA) protects from colorectal cancer in postmenopausal women. No effect was observed in women treated with estrogen alone. This suggests that progesterone, or more specifically the progestin MPA may have chemopreventive activity. The effect of MPA on colorectal carcinogenesis has been difficult to study in animal models. Most models are not affected by either depleting female hormones by ovariectomy or treatment with MPA. Importantly, an ovariectomy fails to reproduce one of the hall marks of the postmenopausal state in women with intact ovaries. That is, the continued production of androgens by the atrophic postmenopausal ovaries. Here we show that adenoma incidence is increased in the vinyl cylcohexene diepoxide (VCD) mouse model of the menopause compared to age matched fertile female mice. Treatment with MPA protected VCD treated mice from adenomagenesis, but had no effect on adenoma numbers in age-matched fertile female mice. Our data show that the protective effect of MPA depends on the postmenopausal state and suggest that MPA monotherapy may be studied as a chemopreventive agent in postmenopausal women.

The esophagus is a relatively simple organ that evolved to transport food and liquids through the thoracic cavity. It is the only part of the gastrointestinal tract that lacks any metabolic, digestive, or absorptive function. The mucosa of the adult esophagus is covered by a multilayered squamous epithelium with a remarkable similarity to the epithelium of the skin despite the fact that these tissues originate from two different germ layers. Here we review the developmental pathways involved in the establishment of the esophagus and the way these pathways regulate gut-airway separation. We summarize current knowledge of the mechanisms that maintain homeostasis in esophageal epithelial renewal in the adult and the molecular mechanism of the development of Barrett's metaplasia, the precursor lesion to esophageal adenocarcinoma. Finally, we examine the ongoing debate on the hierarchy of esophageal epithelial precursor cells and on the presence or absence of a specific esophageal stem cell population. Together the recent insights into esophageal development and homeostasis suggest that the pathways that establish the esophagus during development also play a role in the maintenance of the adult epithelium. We are beginning to understand how reflux of gastric content and the resulting chronic inflammation can transform the squamous esophageal epithelium to columnar intestinal type metaplasia in Barrett's esophagus.

Background: Intestinal fibrosis is a process driven by chronic inflammation leading to increased presence of myofibroblasts and collagen deposition. Although strictures are rarely seen in ulcerative colitis [UC], longstanding disease is believed to cause fibrosis resulting in altered bowel function. Methods: The presence of fibrosis was studied in colectomy specimens from patients with recent-onset UC refractory to medical treatment [ n = 13] and longstanding UC [ n = 16], and colon cancer patients without UC [ n = 7] as controls. Severity of inflammation was scored according to the Geboes score on haematoxylin and eosin stainings. Immunohistochemistry was performed to detect α-smooth muscle actin, fibronectin and collagen I and III. Results: Colectomy specimens from patients with acute UC showed significantly more inflammation than those with longstanding disease [19 vs 9 points, p = 0.01]. Both acute and longstanding UC showed a thicker muscularis mucosa than controls [0.10 vs 0.10 vs 0.05mm, respectively, p = 0.019]. An increase in collagen I and III deposition in the mucosa was observed in UC compared with controls (40% [30–75] vs 25% [10–25], p = 0.033), but this did not differ significantly among acute and longstanding UC patients. Conclusions: Collagen deposition is enhanced in UC compared with controls. However, UC collagen deposition does not increase significantly over time and does not seem to aggravate the entire fibrotic process.