Brooks Scull

Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation.

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

A new animal model of postsurgical bowel inflammation and fibrosis: the effect of commensal microflora

Objective: Ileocaecal resection (ICR) is common in Crohn’s disease. Inflammation and fibrosis frequently recur at the site of anastomosis or in the small intestine (SI). No animal models of postsurgical inflammation and fibrosis exist. A model of ICR was developed in interleukin 10 (IL10) null and wild-type (WT) mice to test the hypothesis that ICR promotes postsurgical inflammation and fibrosis in the SI or anastomosis of genetically susceptible IL10 null, but not WT or germ-free (GF)-IL10 null mice. Methods: GF-IL10 null mice were conventionalised (CONV) and 3 weeks later randomised to ICR, transection (T) or no treatment (NoTx). Age-matched conventionally raised (CONV) WT and GF-IL10 null mice received ICR, T or NoTx. Animals were killed 28 days later. Histological scoring, real-time PCR for tumour necrosis factor α and collagen, and immunostaining for CD3+ T cells assessed inflammation and fibrosis. Results: After ICR, CONV-IL10 null, but not CONV-WT mice, developed significant inflammation and fibrosis in the SI and inflammation in anastomosis compared with NoTx or T controls. Fibrosis occurred in the anastomosis of both CONV-IL10 null and CONV-WT mice following ICR. GF-IL10 null mice developed little or no inflammation or fibrosis in the SI or anastomosis after ICR. Conclusions: ICR in CONV-IL10 null mice provides a new animal model of postsurgical inflammation and fibrosis in the SI and anastomosis. Absence of inflammation and fibrosis in the SI of CONV-WT and GF-IL10 null mice following ICR indicates that postsurgical small bowel disease occurs only in genetically susceptible IL10 null mice and is bacteria dependent.

Biochromoendoscopy: molecular imaging with capsule endoscopy for detection of adenomas of the GI tract

BACKGROUND Current capsule endoscopy (CE) provides minimally invasive technology for GI imaging but has limited ability to discriminate different types of polyps. Near infrared fluorescent (NIRF) probes activated by biomarkers upregulated in adenomas (eg, cathepsin B) are potentially powerful tools to distinguish premalignant or malignant lesions from benign or inflammatory lesions. OBJECTIVES To examine whether CE can be integrated with NIRF probes to detect adenomas and whether cathepsin B-activated NIRF probes are activated by benign or inflammatory lesions. DESIGN Mouse models of adenomas, hyperplactic/lymphoid polyps, and acute or chronic intestinal inflammation were injected intravenously with a cathepsin B-activated probe (Prosense 680). Dissected intestine was imaged with CE under white or NIRF light. For NIRF excitation (680 nm), dichroic and emission (700 nm) filters were combined with CE when images were recorded. Prosense 680 samples with or without protease were used as positive and negative controls. CE-based imaging data were verified by using and independent imaging system (Xenogen IVIS system). MAIN OUTCOME MEASUREMENTS Proof of principal that CE integrated with NIRF probes can detect and discriminate adenomas from other lesions. RESULTS CE-based NIRF imaging with Prosense 680 readily visualized adenomas, including in the colitis model. NIRF signals of different intensities were detected. Prosense 680 was not activated by benign or inflammatory lesions. LIMITATION Optical filters external to the capsule were used. CONCLUSIONS We demonstrate proof of the principle that biochromoendoscopy-CE combined with molecular probes--provides a novel approach that differentiates adenomas from benign polyps and inflammatory lesions.

Molecular Imaging of Gastric Neoplasia with Near-Infrared Fluorescent Activatable Probes

Gastric cancer is the second leading cause of cancer mortality worldwide and is projected to rise to tenth in all-cause mortality in the near term. Early detection requires improved sensitivity and specificity of endoscopic imaging with novel methods. The objective of this study was to evaluate the utility of activatable molecular probes for the detection of gastric cancer both in vivo and ex vivo in a preclinical model. Smad4+/− mice, which develop spontaneous gastric neoplasia, were compared to normal wild-type controls. Cathepsin-activatable and matrix metalloproteinase (MMP)-activatable molecular probes were injected 24 hours and 6 hours before imaging, respectively. In vivo imaging was performed using quantitative tomographic near-infrared fluorescence (NIRF) imaging. For validation, ex vivo imaging and histologic examination were performed. Molecular imaging in vivo of Smad4+/− gastric cancer murine models revealed intense activation of both cathepsin B and MMP probes. Ex vivo imaging and histology confirmed that the detected neoplasms were adenocarcinomas and hyperplastic lesions. This study provides proof of principle that the cathepsin- and MMP-activatable molecular probes are activated in the Smad4+/− murine model of spontaneous gastric adenocarcinoma and can be imaged by both in vivo and ex vivo NIRF methods. The cathepsin probe also detects hyperplastic lesions.

Suppressor of Cytokine Signaling-2 Gene Disruption Promotes ApcMin/+ Tumorigenesis and Activator Protein-1 Activation

Epigenetic in vitro and in vivo studies suggest that suppressor of cytokine signaling-2 (SOCS2) may normally limit tumorigenesis in the intestine; however, this theory has not been directly tested. We hypothesized that SOCS2 deficiency promotes spontaneous intestinal tumorigenesis in Apc(Min/+) mice. Therefore, we quantified tumor number, size, and load in the small intestine and colon using SOCS2(+/+)/Apc(Min/+), SOCS2(+/-)/Apc(Min/+), and SOCS2(-/-)/Apc(Min/+) mice and assayed hematocrit as an indirect marker of disease severity. Biochemical and histological assays were used to assess mechanisms. Heterozygous and homozygous disruption of SOCS2 alleles promoted 166 and 441% increases in tumor load in the small intestine, respectively, accelerated development of colon tumors, and caused severe anemia. SOCS2 deletion promoted significant increases in intestinal insulin-like growth factor-I mRNA but did not affect plasma insulin-like growth factor-I. Western blots and immunohistochemical analysis demonstrated that tumor and nontumor intestinal tissue of SOCS2(-/-)/Apc(Min/+) mice had increased serine 727 phosphorylation of signal transducer and activator of transcription 3 compared with SOCS2(+/+)/Apc(Min/+) mice. Moreover, electromobility shift assays showed that SOCS2 deletion did not alter signal transducer and activator of transcription 3 DNA binding. However, tumors and small intestine from SOCS2(-/-)/Apc(Min/+) showed dramatic increases in activator protein-1 (AP-1) DNA binding, and SOCS2 overexpression in vitro reduced levels of AP-1. These studies indicate that SOCS2 deletion promotes the spontaneous development of intestinal tumors driven by mutations in the adenomatous polyposis coli/beta-catenin pathway and activates AP-1. Therefore, reduced expression or epigenetic silencing of SOCS2 may serve as a useful biomarker for colorectal cancer risk.

L-Arginine Supplementation Modulates Injury and Repair Pathways in Dextran Sulfate Sodium Colitis

Background: L-arginine (L-Arg) is the substrate for both inducible nitric oxide synthase and arginase, which are upregulated in IBD and in mouse colitis models. L-Arg uptake by cells is mediated by cationic amino acid transporter (CAT) proteins, and CAT2 is the inducible form. We have found that L-Arg supplementation enhances restitution in wound repair models In Vitro, and improves clinical parameters of weight loss, survival, and colon weight/length, but not histology, in experimental colitis in mice induced by dextran sulfate sodium (DSS). In CAT2-/mice there is exacerbation of DSS colitis, accumulation of L-Arg in serum, and favorable effects of L-Arg treatment are lost, indicating that inducible uptake of L-Arg is involved in compensatory host responses. Our aim was to define mechanisms underlying the clinical benefit of L-Arg supplementation. Methods: Male C57BL/6 mice were given 4% DSS for 6 days followed by 4 days of 1% L-Arg (DSS/L-Arg group) or regular water (DSS group); controls were given water alone for 10 days (control group), or for 6 days followed by L-Arg for 4 days (control/L-Arg group). Colon tissue from 3 mice in each group was obtained for RNA isolation and then microarray gene expression analysis. The Affymetrix® Mouse Gene 1.0 ST array containing >16,000 protein-coding genes was used. Hierarchical clustering of global gene expression patterns was performed. Differentially expressed genes (DEGs) were selected by both a p value 2. Functional features of the DEGs were evaluated by Ingenuity Pathway Analysis. Results: Hierarchical clustering comparing expression values in the 12 samples revealed that the DSS group was markedly distinct from the control or the control/L-Arg groups. Surprisingly, the DSS/L-Arg group segregated closely with the two control groups not receiving DSS, rather than with the DSS group. There were 342 DEGs with DSS versus control, but only 59 of these genes were altered in DSS/L-Arg versus control. We specifically identified 132 genes upregulated in response to DSS that were downregulated to control levels after L-Arg supplementation. There were also 27 genes that were downregulated with DSS and returned to control levels in the DSS/L-Arg. The detailed patterns were analyzed by heatmaps, that showed marked differences with DSS that reverted to a control pattern in the DSS/L-Arg group. The pathways enriched with DEGs between DSS/L-Arg and DSS included cell-to-cell signaling (leukocyte extravasation; IL-17, IL-8, and IL-10 signaling), cell migration, and cell proliferation pathways.Conclusions:Ourmicroarray gene expression analysis indicates that L-Arg supplementation dramatically alters gene expression patterns in the DSS model, and plays a substantive role in repair, mucosal integrity, and regulation of inflammation.

M1747 Cationic Amino Acid Transporter 2 Has an Important Role in Regulation of Proinflammatory Mediators in DSS Colitis

molecular insights to IBD, we find 17-AAG use profoundly suppresses development of DSSinduced colitis. In another model of colitis, adoptive transfer of CD4+CD45Rbhi cells to Rag deficient mice leads to colitis at ~50 days, and compared with DMSO-treated controls, mice treated with 17-AAG did not develop colitis, gained weight(p<0.05), and had negligible diarrhea and near normal histology. In addition, flow cytometry showed increased absolute numbers of CD4+Foxp3+ cells in the lymph nodes and increased expression of Treg related genes, CTLA-4, and IL-10. Lastly, We noted an anti-inflammatory effect on effector T cells themselves including a decrease in IL-17 expression as well as decreases in Th1 cytokines. In summary, HSP90i(17-AAG) use can regulate Foxp3-dependent functions and enhance Treg suppression with biologically important consequences. In addition, HSP90i can inhibit Th1 and Th17 cell cytokine secretion. Dissecting the complex interactions of the heat shock response including HSF1, HSP90 , HSP70 and others, presents but one of several potential ways to pharmacologically manipulate Treg cells In Vitro and In Vivo, with likely future beneficial consequences for the therapy of IBD.

654 Nitric Oxide Inhibits Helicobacter pylori-Induced Innate Immune Function of Gastric Epithelial Cells by a Heme Oxygenase-1-Dependent Pathway

Mcl-1 expression in gastric cells co-cultured with strain 7.13, consistent with the marked downregulation of miR-320. To define the effect of the H. pylori oncoprotein CagA on these events, cells were also co-cultured with a 7.13 cagAisogenic mutant. Loss of cagA attenuated both the reduction of miR-320 and the subsequent increase in Mcl-1 expression observed in response to wild-type strain 7.13. In summary,H. pylori strain 7.13 specifically downregulates miR-320 in a CagA-dependent manner, which relieves repression of Mcl-1. Upregulation of Mcl-1 by this carcinogenic strain ofH. pylori likely promotes cell survival through inhibition of apoptosis, which may heighten retention of mutagenized cells and increase the risk of subsequent gastric carcinogenesis.