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NK cells, which contribute to immune defense against certain viral infections and neoplasia, are emerging as modifiers of chronic immunologic diseases including transplant rejection and autoimmune diseases. Immunobiology and genetic studies have implicated NK cells as a modifier of Crohn’s disease, a condition often treated with thiopurine agents such as 6-mercaptopurine (6-MP). Here, we demonstrate that thiopurines mediate NK cell apoptosis via a caspase 3 and 9 inclusive pathway, and that this process is triggered by thiopurine-mediated inhibition of Rac1. We also show that CD patients in clinical remission maintained on 6-MP have decreased NK cell Rac1 activity, and decreased NK cell numbers in their intestinal biopsies. These observations suggest that thiopurine targeting of NK cells may be a previously unappreciated therapeutic action of these agents in IBD.

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.

Crohn's disease and ulcerative colitis are chronic inflammatory disorders caused by a disruptive interaction between the immune system and gut luminal factors. Although the exact aetiology of IBD remains unclear, accumulating data, including genome-wide association studies (GWAS), have advanced our understanding of the immunopathogenesis. This review highlights the role in gut homeostasis and IBD pathogenesis. It focuses on past and recent advances in our understanding of IBD, including genetics and immunobiology. Recently published GWAS have confirmed earlier findings related to the NOD2 gene and the IBD5 locus. In addition, over 30 novel loci have been identified. Several promising associations between Crohn's disease and gene variants have been identified and replicated, the two most widely replicated being variants in the IL23R and ATG16L1 genes. These findings highlight and further support the importance of the immune system and its interactions with the intestinal flora in the pathogenesis of inflammatory bowel disease.

Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressant agents. Unfortunately, they also produce serious side effects that limit their usage. This discrepancy is the driving force for the intensive search for novel GC receptor ligands with a better benefit-risk ratio as compared to conventional GCs. A better understanding of the molecular mode of GC action might result in the identification of novel drug targets. Genomic GC effects are mediated by transrepression or transactivation, the latter being largely responsible for GC side effects. We here discuss novel GC receptor ligands, such as selective glucocorticoid receptor agonists (SEGRAs), which might optimize genomic GC effects as they preferentially induce transrepression with little or no transactivating activity. In addition to genomic GC effects, GCs also produce rapid genomic-independent activities, termed nongenomic, and we here review the possible implications of a recently reported mechanism underlying nongenomic GC-induced immunosuppression in T cells. It was shown that the synthetic GC dexamethasone targets membrane-bound GC receptors leading to impaired T cell receptor signaling. As a consequence, membrane-linked GC receptors might be a potential candidate target for GC therapy. The ultimate goal is to convert these molecular insights into new GC receptor modulators with an improved therapeutic index.

BACKGROUND & AIMS: To evaluate the safety and biological activity of visilizumab (a humanized anti-CD3 monoclonal antibody) and to determine a maximum tolerated dose in patients with severe ulcerative colitis that had not responded to 5 days of treatment with intravenous corticosteroids. METHODS: In this open-label phase 1 study, 32 subjects received visilizumab at a dose of 10 or 15 microg/kg, administered intravenously on 2 consecutive days. Clinical response was defined as a Modified Truelove and Witts Severity Index 30 days in 2 of 8 patients), the dose was reduced to 10 microg/kg in 24 patients. On day 30, 84% of patients demonstrated a clinical response, 41% achieved clinical remission, and 44% achieved endoscopic remission. Forty-five percent of patients did not require salvage therapies or colectomy during the first year postdose. Mild to moderate symptoms of cytokine release occurred in 100% and 83% of patients in the 15- and 10-microg/kg dose groups, respectively. All patients exhibited a rapid decrease in circulating CD4(+) T-cell counts, which returned to baseline values by day 30 in 26 of 30 evaluable patients (86%). There were no serious infections. CONCLUSIONS: Visilizumab had an acceptable safety profile at the 10-microg/kg dose level and may be clinically beneficial in patients with severe intravenous corticosteroid-refractory ulcerative colitis.

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.

SummaryHow the development of antibacterial T helper 17 (Th17) cells is selectively promoted by antigen-presenting dendritic cells (DCs) is unclear. We showed that bacteria, but not viruses, primed human DCs to promote IL-17 production in memory Th cells through the nucleotide oligomerization domain 2 (NOD2)-ligand muramyldipeptide (MDP), a derivative of bacterial peptidoglycan. MDP enhanced obligate bacterial Toll-like receptor (TLR) agonist induction of IL-23 and IL-1, which promoted IL-17 expression in T cells. The role of NOD2 in this IL-23-IL-1-IL-17 axis could be confirmed in NOD2-deficient DCs, such as DCs from selected Crohn's disease patients. Thus, antibacterial Th17-mediated immunity in humans is orchestrated by DCs upon sensing bacterial NOD2-ligand MDP.

Background & Aims: The use of living, genetically modified bacteria is an effective approach for topical delivery of immunomodulatory proteins. This strategy circumvents systemic side effects and allows long-term treatment of chronic diseases. However, treatment of patients with a living, genetically modified bacterium raises questions about the safety for human subjects per se and the biologic containment of the transgene. Methods: We treated Crohn's disease patients with genetically modified Lactococcus lactis ( LL-Thy12 ) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human interleukin-10. Ten patients were included in a placebo-uncontrolled trial. Patients were assessed daily for the presence of potential adverse effects by direct questioning and assessment of disease activity. We evaluated the presence and kinetics of LL-Thy12 release in the stool of patients by conventional culturing and quantitative polymerase chain reaction of LL-Thy12 gene sequences. Results: Treatment with LL-Thy12 was safe because only minor adverse events were present, and a decrease in disease activity was observed. Moreover, fecally recovered LL-Thy12 bacteria were dependent on thymidine for growth and interleukin-10 production, indicating that the containment strategy was effective. Conclusions: Here we show that the use of genetically modified bacteria for mucosal delivery of proteins is a feasible strategy in human beings. This novel strategy avoids systemic side effects and is biologically contained; therefore it is suitable as maintenance treatment for chronic intestinal disease.

BACKGROUND & AIMS: The use of azathioprine (AZA) in inflammatory bowel disease (IBD) patients is limited by toxicity, which occurs in up to 20% of treated patients. Mutations in the thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) genes have been associated with the occurrence of AZA-related toxicity. The aim of our study was to determine the relative contribution of ITPA and TPMT mutations to the development of toxicity induced by AZA treatment in IBD patients. METHODS: ITPA(94C>A, IVS2+21A>C) and TPMT (238G>C, 460G>A, and 719A>G) genotypes were assessed in 262 IBD patients (159 females, 103 males; 67 patients with ulcerative colitis, 195 patients with Crohn's disease) treated with AZA and were correlated with the development of leukopenia and hepatotoxicity. RESULTS: Leukopenia (leukocyte count, A and TPMT alleles were significantly higher in the leukopenic population compared with patients without leukopenia (16.7% and 5.4%, respectively, for ITPA 94C>A, and 20.8% and 4%, respectively, for TPMT). Moreover, the ITPA 94C>A and TPMT mutations predicted leukopenia: ITPA 94C>A odds ratio, 3.504; 95% confidence interval, 1.119-10.971 (P = .046); TPMT odds ratio, 6.316; 95% confidence interval, 2.141-18.634 (P = .004). Neither TPMT nor ITPA genotype predicted hepatotoxicity. CONCLUSIONS: ITPA 94C>A and TPMT polymorphisms are associated with AZA-related leukopenia in IBD patients

Summary CD4 + T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4 + T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1 −/− hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1 −/− mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Autologous hematopoietic stem cell transplantation (HSCT) and mesenchymal stromal cell therapy have been proposed for patients with refractory Crohn's disease (CD) and fistulizing CD, respectively. Will these highly advanced techniques be available only for select patients, at specialized centers, or is further clinical development justified, with the aim of offering widespread, more definitive therapeutic options for often very difficult to treat disease? Patients with CD who are eligible for HSCT have typically been failed by most approved therapies, have undergone multiple surgeries, and have coped with years of disease activity and poor quality of life. The objective of HSCT is to immediately shut down the immune response and allow the transplanted stem cells to develop into self-tolerant lymphocytes. For patients with fistulizing CD, mesenchymal stromal cell therapy deposits MSCs locally, into fistulizing tracts, to down-regulate the local immune response and induce wound healing. Recent trials have produced promising results for HSCT and mesenchymal stromal cell therapy as alternatives to systemic therapies and antibiotics for patients with inflammatory bowel diseases, but are these immunotherapies ready for prime time?

Background: Identification of a population with cirrhosis is challenging given manual data collection is time-intensive and laborious, while use of International Classification of Diseases Ninth Revision (ICD-9) codes can be inaccurate. Natural language processing (NLP), a novel computerized approach to analyzing electronic free text, has been used to automatically identify other patient cohorts with gastrointestinal pathologies such as inflammatory bowel disease and intraductal papillary mucinous neoplasms. Aim: To utilize NLP as a supplement to ICD-9 codes and laboratory values to better define and risk-stratify patients with cirrhosis. Methods: A cohort of patients with ICD-9 codes for chronic liver disease was identified during March 2013 to September 2014 from an academic medical center's administrative data. Patients with cirrhosis were further characterized using an algorithm incorporating NLP of radiology reports, ICD-9 codes and laboratory data. Patients who met any of the inclusion criteria were determined to have cirrhosis. Charts were manually reviewed at random to confirm cases of cirrhosis. The reviewer was blinded to the algorithm's results. Positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity were calculated. Results: Of 4292 patients with chronic liver disease, the algorithm yielded 801 patients with cirrhosis. Of the 174 manually reviewed charts, 97 patients had cirrhosis. The algorithm had a PPV of 0.76, NPV of 0.97, sensitivity of 0.97 and specificity of 0.77. Conclusion: Combining NLP, ICD-9 codes and laboratory data is a powerful and sensitive way to detect patients with cirrhosis within a large population. Our algorithm mimics clinicians' manual review process, and therefore shows promise as a tool for automated identification of patients with cirrhosis for both clinical and research use.

is cardiovascular disease for which antiplatelets and anticoagulants are prescribed. Thus, baby boomers are at risk of gastrointestinal bleeding (GIB) related to both pharmacologic exposure and advancing age. In 2012, the cost of GIB care was estimated at >$2.5 billion; half of which was billed to Medicare. Quantifying health care utilization of current baby boomers with GI bleeding will assist policy makers to forecast impact of this generation on future health care resource needs. Methods: A retrospective cohort study using 5 years of the Nationwide Inpatient Sample (2007-2011) was conducted to identify temporal trends in non-variceal, upperand lower-GIB to assess impact of age, co-morbidity, early vs. late endoscopy, transfer status, and disposition on the outcomes of hospital length of stay, 30day mortality and economic outcomes (charge). Temporal trends were evaluated using the Cochrane-Armitage test. The Chi-square test and multivariable linear regression models were used to quantify the impact of exposures of interest and potential effect modifiers on hospital length of stay and charge. Results: From 2007 to 2011 there were 1,322,122 hospital visits associated with GIB in 18,259,654 patients >50 years. Three-quarters of admissions were emergent, 19% occurred on the weekend and 51% were lower GIB. Overall prevalence was 7.2%, with an average length of stay (LOS) of 5.5 days (SD: 6.1) in 2007 that decreased to 5.1 days (SD: 5.7) by 2011 (p

The small intestine and colon differ greatly in both function and exposure to microbial products. Specific studies are needed to assess the response of microbial signaling in the small intestine versus the colon. Double-stranded RNAs (ds-RNA) are primarily viral products, but can also be derived from bacteria. Acute enteric viruses usually impact the small intestine, but viruses have also been investigated in inflammatory bowel disease and in post-infective irritable bowel syndrome and viruses are frequently found in colon cancer tissue. As such, understanding differences in the response of epithelial cells from the small intestine and colon to exposure to viral products will be important for dissecting the impact of infection at the different anatomical locations. Using newly developed culture techniques for growing primary enteroids and colonoids, we sought to compare the responses of enteroids versus colonoids to stimulation with the synthetic ds-RNA polyinosinic-polycytidylic acid (poly I:C). Murine crypts from jejunum or colon were plated in Matrigel for two days prior to the addition of poly I:C for the duration of culture. Stimulation of enteroids with poly I:C significantly altered the surface area but decreased the number of surviving enteroids compared to unstimulated. In colonoids, stimulation with poly I:C resulted in a significant decrease in bud count, but did not impact the area or survival of the organoids. Gene expression measured by the probe-based Nanostring technology in enteroids following poly I:C stimulation showed significantly decreased expression of stem cell markers including: Sox9, Lgr5, Dclk1, Tert and Lrig1. Additionally, differentiation markers Sis and Muc2 were significantly decreased. In contrast, fewer genes were significantly impacted by poly I:C stimulation in colonoids. Stem cell markers were not altered by poly I:C stimulation in colonoids. As expected, in both enteroids and colonoids inflammatory markers were significantly increased in response to poly I:C stimulation. Additionally, pro-apoptotic genes in both enteroids and colonoids were decreased following poly I:C stimulation. Comparing the response of enteroids and colonoids to poly I:C stimulation we found differences in the magnitude or direction of change in several classes of genes. These distinctions may derive frombaseline differences in expression levels. Enteroids had significantly increased expression of stem cell markers at baseline compared to colonoids, while colonoids had increased levels of inflammatory markers and toll-like receptors. Together, these data indicate that important functional differences exist between enteroids and colonoids at baseline and after viral product stimulation providing evidence which may help explain the different anatomical responses to viral infection throughout the intestinal tract.

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

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.