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Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER) stress and activity of the unfolded protein response (UPR) are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2alpha-dependent manner. Inhibition of Perk-eIF2alpha signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

Summary: Intestinal epithelial cells have a defined hierarchy with stem cells located at the bottom of the crypt and differentiated cells more at the top. Epithelial cell renewal and differentiation are strictly controlled by various regulatory signals provided by epithelial as well as surrounding cells. Although there is evidence that stromal cells contribute to the intestinal stem cell niche, their markers and the soluble signals they produce have been incompletely defined. Using a number of established stromal cell markers, we phenotypically and functionally examined fibroblast populations in the colon. CD90+ fibroblasts located in close proximity to stem cells in vivo support organoid growth in vitro and express crucial stem cell growth factors, such as Grem1, Wnt2b, and R-spondin3. Moreover, we found that CD90+ fibroblasts express a family of proteins—class 3 semaphorins (Sema3)—that are required for the supportive effect of CD90+ fibroblasts on organoid growth. : Stem cell proliferation is dependent on regulatory signals from surrounding cells, including stromal cells. Karpus et al. identify a membrane marker, CD90, that specifically marks stem cell niche fibroblasts in the colon. CD90+ fibroblasts produce class 3 semaphorins that promote stem cell proliferation via the Nrp2 receptor. Keywords: colon, intestine, stem cell, niche, fibroblast, stroma, Gli1, CD90, semaphorin, Nrp2

Background & Aims: Epidemiological evidence suggests that statins prevent colorectal cancer (CRC), but the biological mechanism remains obscure. Statins induce bone morphogenetic protein (BMP) expression in bone cells. We have previously shown that BMPs act as tumor suppressors in CRC. We hypothesized that the action of statins in CRC involves the induction of BMPs. Methods: We investigated the effects of statins on CRC cell lines using immunoblotting, measurements of apoptosis and cell proliferation, and luciferase reporter assays. The effect of statins was confirmed in a xenograft mouse model. Results: CRC cell lines show widely differing sensitivities to statin treatment. Sensitive cell lines show induction of BMP2 protein levels and a BMP2 reporter construct, activation of the BMP pathway, and induction of the BMP target gene ID-2, whereas resistant cell lines do not. The addition of the specific inhibitor of BMPs, noggin, completely prevents lovastatin-induced apoptosis in sensitive cells. Sensitive cell lines express the central BMP pathway element SMAD4, whereas the resistant cell fines do not. Targeted knockout of SMAD4 leads to the loss of statin sensitivity and reconstitution with SMAD4, to the restoration of statin sensitivity. in a xenograft mouse model, tumors from sensitive and insensitive cell lines were treated with oral simvastatin. Significant inhibition of tumor growth using sensitive cells but increased tumor growth when using insensitive cells was observed. Conclusions: Statins induce apoptosis in CRC cells through induction of BMP2. Statin therapy may only be effective in SMAD4-expressing CRCs and may have adverse effects in SMAD4-negative tumors.

Background & Aims: Gastric epithelial renewal is an asymmetric process.A stem cell located halfway up the tubular unit gives rise to both a basal gland region and a luminal pit compartment, but the mechanisms responsible for the maintenance of this asymmetry are obscure.We investigated whether Sonic hedgehog (Shh), an established polarizing signal protein during development, is expressed and functional in the adult human and murine stomach. Methods: Expression of Shh and putative transcriptional targets was investigated using immunoblot and immunohistochemistry.Mice were treated with the Shh inhibitor cyclopamine and examined for expression levels of Shh targets and proliferation of gastric epithelial cells. Results: Shh was expressed in the stomach.In cyclopamine-treated mice, we observed decreased expression of HNF3β, Islet (Isl)-1 and BMP4, 3 putative Shh target genes.Inhibition of Shh markedly enhanced gastric epithelial proliferation and affected the cell cycle of gastric epithelial gland cells, whereas pit cells remained unaffected. Conclusions Shh controls the expression of at least 3 factors important for epithelial differentiation and is a negative regulator of gastric gland cell proliferation.Shh is a candidate polarizing signal in the maintenance of gastric pit–gland asymmetry. GASTROENTEROLOGY 2001;121:317-328

Background The optimal target anti-TNF drug level that correlates with control of intestinal inflammation in IBD patients is still poorly defined. We aimed to define infliximab and adalimuamb serum levels that distinguish patients with mucosal healing (MH) from those with ongoing inflammation. Methods We conducted a retrospective study of 135 IBD (107 Crohn's disease, CD, 28 ulcerative colitis, UC) patients treated with infliximab (n=75) or adalimumab (n=60) maintenance therapy, who underwent lower endoscopy at a tertiary medical center. Infliximab/adalimumab drug levels were measured in temporal relation to endoscopy, and correlated with inflammatory markers (CRP) and with endoscopic evaluation of disease activity (SES-CD and MAYO scores for CD and UC respectively). Results Median drug levels (DL) were significantly higher in patients withMH vs. patients with endoscopically active disease, for both infliximab and adalimumab therapy (4.3 vs. 1.15 μg/ml, p=0.0002, 6.2 vs. 3.1 μg/ml, p=0.044, respectively). Patients whose CRP normalized under treatment, similarly had higher median DL than those who did not (4 vs. 2 μg/ml, p=0.022 for infliximab, 5.7 vs. 2.2 μg/ml, p=0.015 for adalimumab, respectively). Infliximab DL above 5.8 μg/ml (AUC=0.75, p

P063 Identification of novel non-transcriptionally acting glucocorticoid receptor ligands that suppress T cell activation but lack adipogenic activity A. Verhaar1 *, R. Dvorsky2, M. Wildenberg3, M. Lowenberg1, M. Peppelenbosch4, D.W. Hommes5, G. van den Brink1. 1Academic Medical Center, Gastroenterology & Hepatology, Amsterdam, Netherlands, 2Max Planck Institute of Molecular Physiology, Structural Biology, Dortmund, Germany, 3Academic Medical Center, Tytgat Institute for Liver and Intestinal Research, Amsterdam, Netherlands, 4Erasmus MC, Gastroenterology and Hepatology, Rotterdam, Netherlands, 5University of California Los Angeles, Center for Inflammatory Bowel Diseases, Los Angeles, United States

G A A b st ra ct s operated for colon cancer without UC served as controls. On HE 29% vs 33% vs 54%, p=0.009). Between acute and late UC there was no difference in collagen deposition, however between UC together and controls there was more collagen I expression in the mucosa, MM and muscularis externa (50% vs 10%, p=0.02; 28% vs 10%, p=0.048; 71% vs 20%, p=0.003). In both early and long UC duration, we did not find a correlation between inflammation or collagen deposition or MM thickness. There was a negative correlation between inflammation andαSMApositivity in the submucosa (R=-0.51, p=0.003) and MM (R=-0.37, p=0.04). Conclusion In our series of colectomy specimens, there is more collagen deposition in UC than in controls. No association between disease duration and increased collagen deposition could be found. Expression of αSMA however is lower in UC and correlates with inflammation. Fibrosis in UC does not appear to increase significantly over time.

G A A b st ra ct s of anti-inflammatory M2 BMDM, determined by expression of Arg1 and Fizz1 that are classical M2 markers, was impaired in Il10rb-/mice. These Mφ also expressed significantly higher levels of pro-inflammatory cytokines following LPS stimulation, suggesting that IL10R signaling regulates TLR4-dependent responses in murine anti-inflammatory Mφ. Il10rb-/M2 Mφ also produced less IL10 and promoted less generation of Tregs in-vitro, perhaps due to decreased surface expression of PD-L1 and PD-L2. Moreover, transfer of WT but not Il10rb-/M2 Mφ ameliorated CD4+-induced colitis in Il10rb-/-Rag2-/mice. Similar to murine studies, the generation of M2 Mφ was severely impaired in human IL10R deficient patients. These Mφ also secreted significantly higher levels of TNF, IL6 and IL12 following LPS stimulation, and promoted less generation of Tregs. Conclusion: IL10R-dependent signals play a critical role in the generation and function of blood borne and tissue resident anti-inflammatory Mφ in mice and humans. Therapeutics and cell-based therapies aimed at augmenting anti-inflammatory Mφ may have clinical utility.

Introduction: Activating mutations in the Hedgehog pathway are found in basal cell carcinomas and medulloblastomas. The role of Hedgehog signaling in intestinal tumorigenesis has not yet been clarified. Hedgehog is expressed by differentiated enterocytes and signals in a paracrine manner from the epithelium to the mesenchyme. Hedgehog controls mesenchymal factors such as Bone Morphogenetic Proteins and Activins which negatively regulate the proliferation of precursor cells. Since Indian Hedgehog (Ihh) is the major Hedgehog expressed in the intestinal epithelium we decided to study the potential role of Ihh signaling in intestinal adenoma formation.Methods: In order to study the effect of loss of Ihh signaling in sporadic tumorigenesis we conditionally deleted Ihh in adult mice in the intestinal epithelium using the Cyp1a1Cre promoter. These compound mice were crossed with conditional mutant Apc mice to generate Cyp1a1Cre-Ihh-Apc mice. At 4-7 weeks of age mice were intraperitoneally injected with 80 mg/kg β-naphthoflavone for 5 days. Cyp1a1Cre-IhhApc littermates served as controls. Four months after recombination, we sacrificed and analyzed the mice (n=15). Results: Ihh expression was upregulated in polyps of Cyp1a1CreIhh-Apc mice as assessed by in situ hybridization. Deletion of Ihh resulted in a marked reduction of 90.3% in the number of polyps (17.2 ± 13.65 polyps per mouse vs. 1.7 ± 1.5, P < 0.01). The average polyp size did not differ between groups. Neither did the number of proliferating or apoptotic cells in the polyps, as assessed by immunostaining for BrdU and cleaved caspase 3. Hh responsive cells in the adenomawere restricted to themesenchyme. In those adenomas that did develop in Ihh mutant mice, we observed a remarkable loss of α-sma/desmin double positive smooth muscle cells. COX2 is expressed early in adenoma to carcinoma transition and believed to play an important role in adenoma formation. Stromal cells are known to be a source of COX2. In accordance with this, examination of Cox2 expression by Q-PCR and immunostaining showed up regulation in polyps ofCyp1a1Cre-Ihh -Apc mice in compared to normal intestinal epithelium (P < 0.01, resp. P < 0.05). In contrast, Cox2 expression in the polyps of Cyp1a1Cre-Ihh-Apc mice was not significantly up regulated. Conclusion: Surprisingly, in contrast to its role as an anti-proliferative signal in the normal epithelium, Ihh acts as a tumor promoter. Our data show that Ihh signaling is increased during intestinal adenoma formation and as in the normal intestine Ihh signaling is exclusively from the epithelium to the mesenchyme. Loss of Ihh signaling is associated with major changes in the composition of the adenoma mesenchyme and decreased Cox2 expression, which significantly impairs adenoma formation.

Background: The degree of association between homocysteine metabolism and inflammatory bowel diseases (IBD) remains unknown and the association between hyperhomocysteinemia and thrombosis and oncogenesis remains controversial in IBD. The aim of this study was to investigate the serum homocysteine levels in patients with Crohn’s disease (CD) and ulcerative colitis (UC) and the potential folate therapeutic regimen against hyperhomocysteinemia. Methods: Serum levels of homocysteine were measured in 55 patients with (IBD) (28/55 CD and 31/55 UC patients). Patients with hyperhomocysteinemia received 15mg folate/day for 2 12 months. Levels of serum homocysteine were measured during folate treatment. Results: Hyperomocysteinemia was prevalent in 28/55 (50.90%) of patients studied (13 CD and 15 UC patients). Mean homocysteine levels among the patients with hyperhomocysteinemia were 17.72mmol/L with range 14.23 29.88mmol/L (normal values 3.7 13.9mmol/L) (Table 1). Folate was administered in 24/28 patients and serum levels of homocysteine were re-measured in 17 patients. In 14/17 patients lower levels of homocysteine were observed with a mean reduction of 5.25mmol/L by folate treatment. The baseline levels of homocysteine in these patients were 17.01 mmol/L and posttreatment 11.75mmol/L.

Background: Colorectal cancer (CRC) is a major public health problem worldwide and incidence rates are increasing, especially in areas historically at low risk. Removal of cancer precursor lesions at colonoscopy is the foundation of CRC prevention, but offers far from perfect protection from CRC. CRC prevention using chemical or natural compounds (chemoprevention) represents a promising alternative or adjuvant preventative strategy. Epidemiological studies and meta-analyses have shown an association between the use of statins and a reduced incidence of CRC. Statins are generally well tolerated and with proven beneficial effects on cardiovascular outcomes improving any overall benefit/risk ratio, they are an attractive candidate for use as a chemopreventive agent in CRC. Their exact cellular targets and underlying mechanism of action have, however, remained elusive. We have shown that statins act on CRC through activation of Bone Morphogenetic Protein (BMP) signaling. However, statins have been shown by others to have a wide range of effects on many other signaling pathways. Aim & Methods: In this study, we set out to assess the influence of statins on global cell signaling in a hypothesis-free manner. We performed an array-based kinase assay using immobilized kinase substrates spanning the entire human kinome. We used HCT116 colon cancer cells with or without Statin treatment. We confirmed our findings from the array using immunoblotting with activity-specific antibodies. Subsequent experiments in different CRC cell lines using immunoblotting with activation-specific antibodies, RNAi and kinase inhibitors focused on the most consistent and pronounced changes. Experiments in xenografts and in patients treated with Statins were performed to confirm our In Vitro data In Vivo. Results: The clinically relevant concentration of 2μM Lovastatin proved to be optimal for kinome analysis. Treatment with statin led to widespread changes in cell signaling compatible with anti-tumor activity. The strongest effect was seen on the PI3K/Akt pathway. Statins induced upregulation of PTEN activity leading to downregulation of the PI3K/Akt pathway and downstream mTOR-mediated signaling. In Vitro experiments showed that the effect on PTEN is dependent on BMP signaling. Further experiments in xenografts and in patients treated with Statins confirmed the changes in BMP pathway and PTEN activity. Conclusion: Statin treatment rapidly leads to significant changes in signal transduction favoring anti-tumor activity. Statins induce downregulation of Akt/mTOR signaling through upregulation of PTEN activity in a BMP dependent manner; both In Vitro as In Vivo. These findings should ultimately facilitate more targeted use of statins in CRC either alone or in combination with other therapies.

OP03 Induction of autophagy upon DC-T cell contact is mediated by LKB1 AMPK mTOR signalling M.E. Wildenberg1 *, A.C. Vos2, M. Duijvestein2, A. Verhaar3, G. van den Brink4, D.W. Hommes5. 1Academic Medical Center, Tytgat Institute for Intestinal and Liver Research, Amsterdam, Netherlands, 2Leiden University Medical Center, Gastroenterology-Hepatalogy, Leiden, Netherlands, 3Leiden University Medical Center, Leiden, Netherlands, 4Academic Medical Center, Gastroenterology & Hepatology, Amsterdam, Netherlands, 5UCLA, Division of Digestive Diseases, Los Angeles, United States

food resuspended in physiological salt solution. Foods scoring positive in skin tests were tested. A drop was placed on the lower lip and left for 10 seconds to 2 minutes. The result was read 15 minutes late. Positive reactions in LFT can be divided into five types: (1) smoothing of the lower lip; (2) erythema under the lip; (3) contiguous rash of the cheek and chin; (4) edema of the cheek associated with rhinitis and watering eyes; and (5) systemic reaction associated with itching of the areas affected by eczema and coughing. Results: A total of 406 LFT were performed, 314 of which were positive and 92 negative, followed by positive food challenges. The foods provoking reactions were egg white (140 cases), peanut (80 cases), cow’s milk (79 cases), cod (8 cases), kiwi (4 cases), shrimp (3 cases). The LFT positivity was mostly (88.7% of cases) manifested as a labial edema with contiguous urticaria. There were systemic reactions in 4.6% of cases: generalized urticaria, hoarnseness and rapid-onset and generalized eczema. The 92 infants with negative labial food test results had positive food challenges. The reactions were in 66 cases generalized urticaria, 20 cases asthma attacks, 2 cases generalized eczema, and in 2 cases general anaphylaxis shock. The sensitivity of LFT was 78%. Conclusions: The LFT was easy to perform with children. Positive results indicate the presence of food allergy, but negative results require further investigations preferably double-blind, placebo-controlled food challenge. Further comparative analyses of the LFT and oral food challenge will better determine the value of the LFT for the exploration of cases of food allergy.