- Email: [email protected]
599 Human Pyloric Sphincters Bioengineered Using Autologous Pyloric Smooth Muscle Cells and Neural Progenitor Cells
AGA Abstracts
retrospective observational study was conducted in French tertiary centers from the GETAID, including all consecutive patients who received subcutaneous ustekinumab for refractory CD to conventional and anti-TNF therapy and having a follow-up of more than 3 months. The primary objective was ustekinumab clinical benefit at 3 months, defined by a significant improvement as judged by the physician leading to continue the treatment with complete steroids weaning if given at inclusion. Ustekinumab safety and clinical benefit at 6, 12 months and end of follow-up were also recorded. Results: Ninety-seven CD patients (27 males, mean age 35.1±11.6 years) received at least one subcutaneous ustekinumab injection in 16 centers. At baseline, median (IQR) disease duration was 11.9 (7.3-16.2) years; 97 (100%) patients experienced previous failure or intolerance to thiopurines or methotrexate and to at least one anti-TNF agent (infliximab or adalimumab, with 85 (87%) who received both anti-TNFs) and 59 (61%) patients underwent prior intestinal resection. Ustekinumab was given for luminal CD in 88 (91%) patients and for perianal disease in 9 (9%). Median follow-up duration was 39.2±32.8 weeks. Mean cumulative dose of ustekinumab given for induction from week 0 to 4 was 148.5 ± 65 mg (range: 45-396 mg). At inclusion, 15 patients received corticosteroids and 12 patients concomitant immunosuppressant (IS). Clinical benefit at 3 months was observed in 69 (71%) patients in the whole population and in 8/9 of patients with perianal CD. Among the primary ustekinumab responders, 78% and 86% experienced clinical benefit at 6 and 12 months, respectively. Concomitant IS and absence of corticosteroids at time of ustekinumab introduction were associated, but nonsignificantly, with clinical response to ustekinumab at 3 months (p=0.09). No serious adverse effects related to ustekinumab were reported. Conclusions: In patients with highly refractory CD, a clinical benefit to subcutaneous ustekinumab was observed in 71% at 3 months and was maintained in the majority of patients for up to 12 months. Along this, ustekinumab could be considered as a rescue therapy in CD patients who experienced prior failure or intolerance to conventional IS and to anti-TNF agents. 1 Sandborn WJ, Gasink C, Gao LL, et al. Ustekinumab induction and maintenance therapy in refractory Crohn's disease. N Engl J Med. 2012 Oct 18;367(16):1519-28.
recurrence. Two-thirds of patients underwent colonoscopy at 6 months and all underwent colonoscopy at 18 months post-operatively. Serologic markers (ANCA, pANCA, ASCA IgA/ IgG, anti-OmpC, anti-CBir1, anti-A4-Fla2, anti-Fla-X) were tested at each time point. Univariate analysis was performed for each (positive/negative) at all time points for endoscopic recurrence (Rutgeert's score ‡ i2). Antibody titre was investigated using quartile sum score (QSS) method (range 7-28) and logistic regression analysis. Results: Patients with recurrence at 18 months were more likely to be positive for anti-Fla-X than those without recurrence, when measured at baseline (64% v 45%; P=0.049), 6 months (65% v 43%, P=0.021) and 18 months (53% v 33%, P=0.038) post operatively. Patients negative for anti-Fla-X at 6 months had a lower risk of recurrence at 18 months (OR 0.38, 95%CI 0.17-0.84, P=0.018), when adjusted for age, gender, disease behaviour and smoking. A negative ANCA titre at 6 months was associated with recurrence at 6 months (ANCA -ve v +ve: recurrence 87% v 13%, P=0.002). Adjusted for baseline characteristics (age, gender disease location, smoking, pANCA) the total antibody titre for all antibodies combined was not predictive of endoscopic recurrence at 6 or 18 months. Conclusion: The presence of the serological antibody antiFla-X identifies patients at higher risk of developing early disease recurrence, while the presence of positive ANCA predicts patients at lower risk of post-operative recurrence. FlaX is an immunogenic bacterial antigen, arising from subunits of bacterial flagella (flagellin) from Clostridium subphylum cluster XIVa. Serologic screening of patients prior to surgery may assist in selecting patients at elevated risk of post-operative recurrence. The role of FlaX in relation to microbiota that may be linked to recurrence requires evaluation.
598 Selective Serotonin Reuptake Inihibitor (SSRI) Exposure In Utero and During Breastfeeding Results in Abnormal Enteric Nervous System Development and Gastrointestinal Function Korey D. Stevanovic, Virginia E. Saurman, Michael D. Gershon, Zhishan Li, Garrette Furo, Christina M. Roberts, Kara G. Margolis
596 Depression, which occurs in 14-23% of women during pregnancy, is often treated with a selective serotonin reuptake inhibitor (SSRI). Although SSRI use is associated with increased risk of congenital malformation, little is known about the effects of maternal SSRI use on enteric nervous system (ENS) development. The serotonin reuptake transporter (SERT), which SSRIs inhibit, terminates 5-HT's action. Enteric neuronal 5-HT is an essential ENS growth factor; therefore, SERT inhibition due to SSRI ingestion during pregnancy might be expected to affect fetal ENS development and particularly that of neurons that follow 5-HT during the sequence of enteric neurogenesis. To test these hypotheses in mice, an SSRI (fluoxetine;7.5 mg/kg) or vehicle (control) was administered to dams from gestational day E1 through weaning (P21). Pups were allowed to mature without further treatment until they were euthanized at P42-70. Total numbers of enteric neurons in both plexuses were quantified immunocytochemically in fluoxetine- and vehicle-exposed mice using antibodies to Hu. Late-born neuronal markers included calcitonin gene related peptide (CGRP), GABA, and tyrosine hydroxylase (TH; dopamine). Both plexuses of fluoxetine-treated mice were hyperplastic, containing significantly more total, CGRP-expressing, GABAergic and dopaminergic neurons than controls. Significant changes in intestinal motility accompanied ENS hyperplasia. Both the frequency and velocity of colonic migrating motor complexes (CMMCs) were significantly increased in isolated colons of fluoxetine-exposed mice. Paradoxically, total GI, as well as intestinal and colonic transit, was significantly slower in fluoxetineexposed than control animals. Chemical sympathectomy with 6-hydroxydopamine (100 mg/ kg) normalized in vivo transit time in fluoxetine-exposed animals; therefore, fluoxetine exposure enhances sympathetic outflow, which slows in vivo motility. Sympathetic input is eliminated when gut is isolated. Additional intestinal abnormalities found in fluoxetineexposed mice included significant increases in villus height and crypt depth, increased permeability of the mucosa to fluorescein-labeled dextran, and decreased severity of colitis (both clinical scores and cytokine expression) induced either with trinitrobenzene sulfonic acid or dextran sulfate sodium. Increased mucosal growth and intestinal permeability are seen also in mice that lack SERT, while intestinal inflammation is potentiated in mice that lack neuronal 5-HT. Observations are compatible with the idea that SSRI-exposure during development potentiates developmental actions of 5-HT, which leads to long-lasting abnormalities of the ENS and the GI functions it controls. These studies provide insight into the potential pathogenesis of some congenital GI disorders and suggest caution in the use of an SSRI during pregnancy.
Clinical Outcomes of Inflammatory Bowel Disease Patients With Antibody Deficiencies: A 5-Year Prospective Study Jorge D. Machicado, Toufic Kabbani, Claudia Ramos Rivers, Arthur Barrie, Douglas J. Hartman, Miguel Regueiro, Jason M. Swoger, Marc Schwartz, Leonard Baidoo, Jana G. Hashash, Michael A. Dunn, David G. Binion Background: Previous studies have advocated a primary pathogenic role of immunodeficiency disorders in the development of inflammatory bowel disease (IBD). However, the implication of B-cell disorders in the outcomes of patients with IBD has not been previously studied. We aimed to determine the role of antibody deficiencies in the clinical course of patients with IBD. Methods: We used a prospective, longitudinal IBD registry of patients followed in a tertiary referral center between 2009-2013. Patients who were tested for immunoglobulin (Ig) A, G, or M, levels comprised the study population. We defined IgA deficiency as serum levels <81 mg/dL; IgG deficiency <750mg/dL; and IgM deficiency <40mg/dL. The presence of other normal serum antibodies was required for the diagnosis of selective immunodeficiencies. Presumptive common variable immunodeficiency (CVID) was defined as decreased IgG level and decrease in at least one of the isotypes IgM or IgA. Normal levels of IgG4 were considered between 7-79 mg/dL. Controls were patients with IBD without serum antibody abnormalities. The clinical course of patients with IBD was measured with patterns of medication use, healthcare utilization, disease activity scores and health related quality of life. Results: Out of 616 subjects (CD 62.6%, females 52.1%, mean age 39.5), 12.4% had any serum immunoglobulin deficiency. Selective IgG deficiency (7.7%) was the most frequent B-cell disorder, followed by IgA deficiency (3.2%), common variable immunodeficiency (3.2%), and selective IgM deficiency (2.8%). IgG4 level was abnormal in 28.2% individuals tested (low, 24.9% vs. high, 3.3%). Patients with selective IgG deficiency had significantly poorer scores of quality of life (p<0.001), required more hospitalizations (p=0.004), and ED visits (p= 0.04), compared to those with normal immunoglobulin levels. Using multivariate regression controlling for demographic factors, IBD disease, and medication use, selective IgG deficiency was only significantly associated with poorer scores of quality of life (p=0.03). Patients with low IgG4 levels required more clinic visits (p=0.02); while patients with elevated IgG4 required significantly more ED visits (p=0.04). Presumptive CVID was associated with lower scores of quality of life (p=0.01). After multivariate analysis, these associations were not longer statistically significant (p>0.05). Neither of the evaluated antibody abnormalities was associated with use of biologics, immunomodulators, steroids, disease activity, CRP levels, surgeries, or phone calls (p>0.05). Conclusion: A significant proportion of patients with IBD have antibody deficiencies. Low IgG level is independently associated with lower quality of life scores in patients with IBD. This may represent a therapeutic target in a subgroup of patients with B-cell mediated dysfunction and IBD.
599 Human Pyloric Sphincters Bioengineered Using Autologous Pyloric Smooth Muscle Cells and Neural Progenitor Cells Stephen L. Rego, Elie Zakhem, Giuseppe Orlando, Khalil N. Bitar Background: Pyloric stenosis and gastroparesis lead to inadequate emptying of the stomach. Appropriate long term treatments for these diseases may require pyloric sphincter tissue replacements. Here we bioengineer functional innervated human pylorus tissues utilizing human pyloric sphincter smooth muscle cells (SMCs) and enteric human neural progenitor cells (ENPCs). Methods: SMCs were isolated from human donor pyloric sphincter tissues and expanded in vitro. ENPCs were isolated from human donor duodenal tissues and expanded as neurosphere-like bodies. Pyloric sphincter constructs were bioengineered with human pyloric sphincter SMCs, innervated with human ENPCs and cultured for ten days. Results: SMCs within bioengineered human pylorus constructs aligned circumferentially and maintained a contractile phenotype as demonstrated by expression of smoothelin, a smooth muscle actin (SMA), caldesmon and calponin. Bioengineered human pyloric sphincters also expressed Ras homolog gene family, member A (RhoA), Rho-associated protein kinase (ROCKII), C-kinase potentiated Protein phosphatase-1 Inhibitor (CPI-17) and phospho-CPI-17. Physiologic analysis of bioengineered human pyloric sphincters demonstrated the generation of spontaneous basal tones and contraction in response to potassium chloride. The presence of differentiated neurons within bioengineered constructs was indicated by expression of bIII-tubulin, neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT). Bioengineered innervated human pyloric sphincters responded appropriately to physiologically relevant neurotransmitters including acetylcholine (ACh) and vasoactive
597 Serological Antibodies for the Prediction of Post-Operative Recurrent Crohn's Disease Results From the POCER Study Amy L. Hamilton, Michael A. Kamm, Fabiyola Selvaraj, Fred Princen, Peter De Cruz, Emily K. Wright, Kathryn J. Ritchie, Efrosinia O. Krejany, Alexandra Gorelik, Danny Liew, Lani Prideaux, Ian C. Lawrance, Jane M. Andrews, Peter A. Bampton, Miles Sparrow, Timothy Florin, Peter R. Gibson, Henry Debinski, Richard B. Gearry, Finlay A. Macrae, Rupert W. Leong, Ian Kronborg, Graham L. Radford-Smith, Warwick Selby, Michael J. Johnston, Rodney Woods, Peter R. Elliott, Sally Bell, Steven J. Brown, William Connell, Paul V. Desmond, Sharat Singh Background: Disease recurrence after Crohn's disease resection occurs in up to up to 80% of patients, with two thirds ultimately requiring further surgery. Although clinical risk factors help in assessing the risk of relapse, a test that identifies patients at higher risk of recurrence would be clinically valuable. We investigated the value of serological antimicrobial antibodies to predict disease recurrence after surgery in a large prospective cohort of patients. Methods: 171 patients had 525 samples tested peri-operatively, and 6, 12 and 18 months postoperatively as part of a structured study ("POCER") designed to diminish post-operative
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intestinal peptide (VIP), and neural network integrity was demonstrated by neurotoxin sensitive responses to electrical field stimulation (EFS). Conclusion: This is the first demonstration of a bioengineered innervated human pyloric sphincter. Bioengineered pyloric sphincters maintained appropriate myogenic and neurogenic functionality. These bioengineered tissues have the potential for use as (1) functional replacement organs and (2) physiologically relevant models to investigate human pyloric sphincter disorders. This work was supported by Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0052; GU 7 and Wake Forest School of Medicine Internal Funds.
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Background. Most of the inflammatory and immune responses in inflammatory bowel disease and irritable bowel syndrome are mediated by NF kB signaling. NFkB in intestinal epithelial cells plays a critical role in protecting mucosal integrity during acute inflammation, whereas, NFkB in immune cells and smooth muscle cells mediates the inflammatory responses during chronic inflammation. Little is known about the role of NF kB signaling in enteric nervous system. Aim. To assess the effects of NFkB signaling within enteric neuronal cells (ENCs) on gastrointestinal motility in the presence or absence of intestinal inflammation. Methods. A conditional stop-floxed mouse line carrying a constitutively active form of IKK2 (IKK2CA) was crossbred sequentially with Rosa26-tdTomato reporter mice and with Calretinin-Cre-ERT2 mice to generate Calretinin-Cre-ERT2-IKK2CA/tdTomato transgenic (TG) mice. At the age of 2 months old, the animals were treated with tamoxifen (i.p. 100 mg/kg/day, 3 days), allowing ENC-specific IKK2CA expression and continuous NFkB activation. At 1 month after induction,, the colonic motility (bead exclusion assay), water/ food intake and general phenotypes were evaluated before 2.5% dextran sulfate sodium (DSS)-induced colitis was induced. The course of inflammation was monitored by daily disease activity index, end macroscopic observation and histopathologic examination. Spontaneous and acetylcholine (10-9 to 10-3 M)-stimulated contraction of longitudinal colonic muscle strips as well as sodium nitroprusside-induced relaxation were measured using 16 organ-baths with a PowerLab data acquisition system. Muscle strip whole mount and transverse sections of colon tissues were used for multilabeled immunohistochemistry. Results. At one month after accumulated tamoxifen induction, 50% of ENCs were labeled with the tdTomato reporter. There was no significant difference in food/water intake and colonic motility between TG and littermate wild-type (WT) mice. After DSS-induced acute colitis, the weight loss, bleeding, colon shortening and mucosa damage were less in TG than that in WT mice. Consistent with previous reports, DSS-colitis induced significant inhibition of acetylcholine-stimulated colonic muscle contraction and promotion of nitric oxide-stimulated colonic relaxation in WT mice. However, ENC-specific activation of NFkB signaling had no significant effect on acetylcholine-stimulated colonic muscle contraction under both physiological and DSS-challenge conditions, but significantly induced partial recovery of colonic relaxation after DSS challenge. Conclusion. Enteric neuronal NFkB overactivation in adult animals protects against acute colitis and inhibits colonic relaxation. This study highlights the importance of enteric neuronal NF kB signaling in regulating enteric neuronal plasticity and neurogastrointestinal motility.
600 In Situ Implantation of Autologous BiosphincterTM Re-Instates Continence in a Large Animal Model of Passive Fecal Incontinence Khalil N. Bitar, Jaime Bohl, John E. Fortunato, Sita Somara, Shreya Raghavan, Elie Zakhem, Stephen L. Rego, Kenneth L. Koch The Internal Anal Sphincter (IAS) is primarily responsible in maintaining anorectal continence. Sphincter dysfunction can lead to fecal incontinence (FI). A regenerative medicine approach to restore impaired IAS function offers a safe and sustained solution. Objective: In the present study, we bioengineered autologous innervated IAS BioSphinctersTM from cells isolated from small intestine and anal sphincter biopsies. The objective of this research was to develop a rabbit FI model, and surgically implant autologous constructs to remedy FI. Materials and Methods: IAS injury was produced in the rabbit by partial IAS sphincterectomy. The excised sphincteric tissue was used as source of IAS smooth muscle cells. Jejunal biopsies were taken to obtain tissue for the isolation of enteric neural progenitor cells. BioSphinctersTM were bioengineered from these two cell lines. Bioengineered constructs were implanted in situ six weeks post Sphincterectomy. Anorectal manometry was used to monitor anal canal pressures. Results: Sufficient smooth muscle cells were isolated from a semi-lunar IAS biopsy to bioengineer 4-6 BioSphincterTM constructs. Bioengineered intrinsically innervated sphincters mimicked native sphincter architecture and function. BioSphincterTM constructs demonstrated presence of contractile smooth muscle and mature neuronal populations. A deterioration in fecal hygiene and decreased basal tone (55% decrease) and RAIR (58% decrease) were observed in anorectal manometry post sphincterectomy. Autologous IAS constructs were successfully implanted into the donor rabbit without complications. The in situ implanted construct was neovascularized and showed no signs of inflammation or infection. Restoration of basal tone and RAIR were observed following implantation with anorectal manometry. To date we have successfully implanted 3 rabbits with end point of 3 months. Conclusions: A large animal model of FI has been developed and validated deterioration of fecal hygiene and loss of basal tone and RAIR. Bioengineering and implantation of autologous cells is technically feasible in an in vivo FI model. This provides proof of concept of safety and efficacy of BioSphincterTM constructs and restoration of IAS integrity and function and fecal continence. This regenerative medicine approach can be used as a therapeutic option to treat FI. This work was supported by Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH13-2-0052; GU 7 and NIH/NIDDK R01DK071614
603 Helicobacter pylori Activates Gastric Epithelial Stem Cell Through Direct Colonization of the Gastric Glands Michael Sigal, Manuel R. Amieva INTRODUCTION: Helicobacter pylori is a bacterial pathogen that colonizes the human stomach and is the main risk factor for gastro-duodenal ulcers and gastric cancer. H. pylori is found in close proximity to the surface of the stomach epithelium either as a free-swimming population in the gastric mucus or adhered to epithelial cells. The attached bacteria are known to alter cell signaling and behavior though different virulence factors. AIMS & METHODS: While the effects of attachment have been studied extensively in vitro, we aimed to study the localization and pathological relevance of the direct interaction of bacteria with the gastric epithelium, and in particular with gastric stem cells, in vivo. We utilized a murine model of H. pylori infection. We developed novel techniques to visualize and quantify H. pylori in mouse stomachs using 3D confocal microscopy. Lgr5eGFP mice were used to study the interaction of H. pylori with gastric stem cells. Lgr5eGFPCreER; RosadtTomato compound heterozygous mice were used for lineage tracing experiments. RESULTS: We discovered that H. pylori colonize the epithelial surface deep in the gastric glands where they grow as distinct microcolonies associated with the epithelial junctions. Using proliferation markers, we find that the gland-associated H. pylori directly colonize the surface of replicating progenitor cells. In addition, a proportion of bacteria can be found in the base of glands, where long-lived epithelial stem cells reside. We hypothesized that direct colonization of precursor/ stem cells may drive pathological responses. Using quantitative microscopy we mapped the distribution of bacteria in the glands of the antrum versus the corpus. We found that the location of bacteria in the glands correlates with hyperplastic lesions. Using Lgr5eGFP mice, we observed a direct interaction of H. pylori with Lgr5 expressing stem cells in the stomach antrum. Infection induced an expansion of the stem cell compartment. In addition, lineage tracing experiments revealed a significantly higher number of daughter cells being generated from Lgr5 expressing stem cell in infected animals compared to uninfected controls. The accelerated tracing tightly correlated with the bacteria in the gastric glands. Infection with a bacterial chemotaxis mutant that is able to colonize the mucus but is unable to penetrate deep into the glands did not induce an activation of stem cells. Moreover, a bacterial mutant that is unable to deliver the main virulence factor CagA into the epithelium induced diminished activation of stem cells and caused less hyperplasia. CONCLUSION: Taken together our data reveals that H. pylori directly interacts with progenitor and stem cells deep in the gastric glands. This gland-associated subpopulation of H. pylori alters stem cell homeostasis of the colonized glands, leading to gland hyperplasia.
601 The Appendix As a Viable Source of Neural Progenitor Cells to Functionally Innervate Bioengineered Gastrointestinal Smooth Muscle Tissues Elie Zakhem, Stephen L. Rego, Shreya Raghavan, Khalil N. Bitar Background: Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, however, utilizing these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix differentiate into functional enteric neural subsets similar to NPCs isolated from the small intestine. Methods: (1) NPCs were isolated from the appendix and small intestines (SI) of rabbits. (2) IAS smooth muscle cells were isolated from rabbit internal anal sphincter (IAS) tissue. (3) Bioengineered (IAS) constructs were developed, using the same source of smooth muscle, and innervated with NPCs derived from either the appendix or SI. (4) Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry. (5) Physiological functionality was assessed through force generation studies. Results: (1) NPCs from both the appendix and the SI expressed enteric neural stem cell markers (p75-NTR, SOX2 and nestin). (2) Immunostaining analysis demonstrated expression of neuronal differentiation marker bIII tubulin in both bioengineered constructs, indicating that neural progenitor cells derived from either the appendix or the SI have the capability to differentiate into neurons. (3) Western blot analysis demonstrated the expression of enteric neuronal protein (excitatory and inhibitory motor neurons) and glial protein (GFAP) confirming neural and glial differentiation in both types of constructs. (4) The addition of 1 µM acetylcholine (Ach) caused a robust contraction in appendix and SI constructs of magnitudes 279.3 ± 20.01 µN and 294.8 ± 17.85 µN, respectively. The contraction was significantly decreased by pretreatment with neural inhibitor, tetrodotoxin (TTX). (5) Electrical field stimulation, EFS, (5 Hz, 0.5 ms) caused relaxation of -257.7 ± 7.410 µN and -229.3 ± 13.53 µN in the appendix and SI constructs, respectively. The relaxation was completely abolished in the presence of TTX. (6) EFS-induced relaxation was significantly reduced upon pre-treatment with nitric oxide synthase inhibitor (L-NAME) in both constructs. Conclusion: The appendix can be accessed using minimally invasive surgery. This study indicates that NPCs isolated from the appendix have the potential to differentiate into functional neurons in a similar manner as NPCs isolated from the small intestine. The neurons were also capable of responding to physiological stimuli. This study demonstrates the potential use of the appendix in human as a source of neural progenitor cells. This work was supported by Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0052; GU 7 and NIH/NIDDK R01DK071614.
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Cre/LoxP-Mediated Inducible Activation of IKK2 in Adult Enteric Neuronal Cells Protects Against DSS-Induced Colitis in Transgenic Mice Yonggang Zhang, Wensheng Yang, Alan S. Braverman, Fang Li, Henry P. Parkman, K. S. Murthy, Michael R. Ruggieri, Wenhui Hu
retrospective observational study was conducted in French tertiary centers from the GETAID, including all consecutive patients who received subcutaneous ustekinumab for refractory CD to conventional and anti-TNF therapy and having a follow-up of more than 3 months. The primary objective was ustekinumab clinical benefit at 3 months, defined by a significant improvement as judged by the physician leading to continue the treatment with complete steroids weaning if given at inclusion. Ustekinumab safety and clinical benefit at 6, 12 months and end of follow-up were also recorded. Results: Ninety-seven CD patients (27 males, mean age 35.1±11.6 years) received at least one subcutaneous ustekinumab injection in 16 centers. At baseline, median (IQR) disease duration was 11.9 (7.3-16.2) years; 97 (100%) patients experienced previous failure or intolerance to thiopurines or methotrexate and to at least one anti-TNF agent (infliximab or adalimumab, with 85 (87%) who received both anti-TNFs) and 59 (61%) patients underwent prior intestinal resection. Ustekinumab was given for luminal CD in 88 (91%) patients and for perianal disease in 9 (9%). Median follow-up duration was 39.2±32.8 weeks. Mean cumulative dose of ustekinumab given for induction from week 0 to 4 was 148.5 ± 65 mg (range: 45-396 mg). At inclusion, 15 patients received corticosteroids and 12 patients concomitant immunosuppressant (IS). Clinical benefit at 3 months was observed in 69 (71%) patients in the whole population and in 8/9 of patients with perianal CD. Among the primary ustekinumab responders, 78% and 86% experienced clinical benefit at 6 and 12 months, respectively. Concomitant IS and absence of corticosteroids at time of ustekinumab introduction were associated, but nonsignificantly, with clinical response to ustekinumab at 3 months (p=0.09). No serious adverse effects related to ustekinumab were reported. Conclusions: In patients with highly refractory CD, a clinical benefit to subcutaneous ustekinumab was observed in 71% at 3 months and was maintained in the majority of patients for up to 12 months. Along this, ustekinumab could be considered as a rescue therapy in CD patients who experienced prior failure or intolerance to conventional IS and to anti-TNF agents. 1 Sandborn WJ, Gasink C, Gao LL, et al. Ustekinumab induction and maintenance therapy in refractory Crohn's disease. N Engl J Med. 2012 Oct 18;367(16):1519-28.
recurrence. Two-thirds of patients underwent colonoscopy at 6 months and all underwent colonoscopy at 18 months post-operatively. Serologic markers (ANCA, pANCA, ASCA IgA/ IgG, anti-OmpC, anti-CBir1, anti-A4-Fla2, anti-Fla-X) were tested at each time point. Univariate analysis was performed for each (positive/negative) at all time points for endoscopic recurrence (Rutgeert's score ‡ i2). Antibody titre was investigated using quartile sum score (QSS) method (range 7-28) and logistic regression analysis. Results: Patients with recurrence at 18 months were more likely to be positive for anti-Fla-X than those without recurrence, when measured at baseline (64% v 45%; P=0.049), 6 months (65% v 43%, P=0.021) and 18 months (53% v 33%, P=0.038) post operatively. Patients negative for anti-Fla-X at 6 months had a lower risk of recurrence at 18 months (OR 0.38, 95%CI 0.17-0.84, P=0.018), when adjusted for age, gender, disease behaviour and smoking. A negative ANCA titre at 6 months was associated with recurrence at 6 months (ANCA -ve v +ve: recurrence 87% v 13%, P=0.002). Adjusted for baseline characteristics (age, gender disease location, smoking, pANCA) the total antibody titre for all antibodies combined was not predictive of endoscopic recurrence at 6 or 18 months. Conclusion: The presence of the serological antibody antiFla-X identifies patients at higher risk of developing early disease recurrence, while the presence of positive ANCA predicts patients at lower risk of post-operative recurrence. FlaX is an immunogenic bacterial antigen, arising from subunits of bacterial flagella (flagellin) from Clostridium subphylum cluster XIVa. Serologic screening of patients prior to surgery may assist in selecting patients at elevated risk of post-operative recurrence. The role of FlaX in relation to microbiota that may be linked to recurrence requires evaluation.
598 Selective Serotonin Reuptake Inihibitor (SSRI) Exposure In Utero and During Breastfeeding Results in Abnormal Enteric Nervous System Development and Gastrointestinal Function Korey D. Stevanovic, Virginia E. Saurman, Michael D. Gershon, Zhishan Li, Garrette Furo, Christina M. Roberts, Kara G. Margolis
596 Depression, which occurs in 14-23% of women during pregnancy, is often treated with a selective serotonin reuptake inhibitor (SSRI). Although SSRI use is associated with increased risk of congenital malformation, little is known about the effects of maternal SSRI use on enteric nervous system (ENS) development. The serotonin reuptake transporter (SERT), which SSRIs inhibit, terminates 5-HT's action. Enteric neuronal 5-HT is an essential ENS growth factor; therefore, SERT inhibition due to SSRI ingestion during pregnancy might be expected to affect fetal ENS development and particularly that of neurons that follow 5-HT during the sequence of enteric neurogenesis. To test these hypotheses in mice, an SSRI (fluoxetine;7.5 mg/kg) or vehicle (control) was administered to dams from gestational day E1 through weaning (P21). Pups were allowed to mature without further treatment until they were euthanized at P42-70. Total numbers of enteric neurons in both plexuses were quantified immunocytochemically in fluoxetine- and vehicle-exposed mice using antibodies to Hu. Late-born neuronal markers included calcitonin gene related peptide (CGRP), GABA, and tyrosine hydroxylase (TH; dopamine). Both plexuses of fluoxetine-treated mice were hyperplastic, containing significantly more total, CGRP-expressing, GABAergic and dopaminergic neurons than controls. Significant changes in intestinal motility accompanied ENS hyperplasia. Both the frequency and velocity of colonic migrating motor complexes (CMMCs) were significantly increased in isolated colons of fluoxetine-exposed mice. Paradoxically, total GI, as well as intestinal and colonic transit, was significantly slower in fluoxetineexposed than control animals. Chemical sympathectomy with 6-hydroxydopamine (100 mg/ kg) normalized in vivo transit time in fluoxetine-exposed animals; therefore, fluoxetine exposure enhances sympathetic outflow, which slows in vivo motility. Sympathetic input is eliminated when gut is isolated. Additional intestinal abnormalities found in fluoxetineexposed mice included significant increases in villus height and crypt depth, increased permeability of the mucosa to fluorescein-labeled dextran, and decreased severity of colitis (both clinical scores and cytokine expression) induced either with trinitrobenzene sulfonic acid or dextran sulfate sodium. Increased mucosal growth and intestinal permeability are seen also in mice that lack SERT, while intestinal inflammation is potentiated in mice that lack neuronal 5-HT. Observations are compatible with the idea that SSRI-exposure during development potentiates developmental actions of 5-HT, which leads to long-lasting abnormalities of the ENS and the GI functions it controls. These studies provide insight into the potential pathogenesis of some congenital GI disorders and suggest caution in the use of an SSRI during pregnancy.
Clinical Outcomes of Inflammatory Bowel Disease Patients With Antibody Deficiencies: A 5-Year Prospective Study Jorge D. Machicado, Toufic Kabbani, Claudia Ramos Rivers, Arthur Barrie, Douglas J. Hartman, Miguel Regueiro, Jason M. Swoger, Marc Schwartz, Leonard Baidoo, Jana G. Hashash, Michael A. Dunn, David G. Binion Background: Previous studies have advocated a primary pathogenic role of immunodeficiency disorders in the development of inflammatory bowel disease (IBD). However, the implication of B-cell disorders in the outcomes of patients with IBD has not been previously studied. We aimed to determine the role of antibody deficiencies in the clinical course of patients with IBD. Methods: We used a prospective, longitudinal IBD registry of patients followed in a tertiary referral center between 2009-2013. Patients who were tested for immunoglobulin (Ig) A, G, or M, levels comprised the study population. We defined IgA deficiency as serum levels <81 mg/dL; IgG deficiency <750mg/dL; and IgM deficiency <40mg/dL. The presence of other normal serum antibodies was required for the diagnosis of selective immunodeficiencies. Presumptive common variable immunodeficiency (CVID) was defined as decreased IgG level and decrease in at least one of the isotypes IgM or IgA. Normal levels of IgG4 were considered between 7-79 mg/dL. Controls were patients with IBD without serum antibody abnormalities. The clinical course of patients with IBD was measured with patterns of medication use, healthcare utilization, disease activity scores and health related quality of life. Results: Out of 616 subjects (CD 62.6%, females 52.1%, mean age 39.5), 12.4% had any serum immunoglobulin deficiency. Selective IgG deficiency (7.7%) was the most frequent B-cell disorder, followed by IgA deficiency (3.2%), common variable immunodeficiency (3.2%), and selective IgM deficiency (2.8%). IgG4 level was abnormal in 28.2% individuals tested (low, 24.9% vs. high, 3.3%). Patients with selective IgG deficiency had significantly poorer scores of quality of life (p<0.001), required more hospitalizations (p=0.004), and ED visits (p= 0.04), compared to those with normal immunoglobulin levels. Using multivariate regression controlling for demographic factors, IBD disease, and medication use, selective IgG deficiency was only significantly associated with poorer scores of quality of life (p=0.03). Patients with low IgG4 levels required more clinic visits (p=0.02); while patients with elevated IgG4 required significantly more ED visits (p=0.04). Presumptive CVID was associated with lower scores of quality of life (p=0.01). After multivariate analysis, these associations were not longer statistically significant (p>0.05). Neither of the evaluated antibody abnormalities was associated with use of biologics, immunomodulators, steroids, disease activity, CRP levels, surgeries, or phone calls (p>0.05). Conclusion: A significant proportion of patients with IBD have antibody deficiencies. Low IgG level is independently associated with lower quality of life scores in patients with IBD. This may represent a therapeutic target in a subgroup of patients with B-cell mediated dysfunction and IBD.
599 Human Pyloric Sphincters Bioengineered Using Autologous Pyloric Smooth Muscle Cells and Neural Progenitor Cells Stephen L. Rego, Elie Zakhem, Giuseppe Orlando, Khalil N. Bitar Background: Pyloric stenosis and gastroparesis lead to inadequate emptying of the stomach. Appropriate long term treatments for these diseases may require pyloric sphincter tissue replacements. Here we bioengineer functional innervated human pylorus tissues utilizing human pyloric sphincter smooth muscle cells (SMCs) and enteric human neural progenitor cells (ENPCs). Methods: SMCs were isolated from human donor pyloric sphincter tissues and expanded in vitro. ENPCs were isolated from human donor duodenal tissues and expanded as neurosphere-like bodies. Pyloric sphincter constructs were bioengineered with human pyloric sphincter SMCs, innervated with human ENPCs and cultured for ten days. Results: SMCs within bioengineered human pylorus constructs aligned circumferentially and maintained a contractile phenotype as demonstrated by expression of smoothelin, a smooth muscle actin (SMA), caldesmon and calponin. Bioengineered human pyloric sphincters also expressed Ras homolog gene family, member A (RhoA), Rho-associated protein kinase (ROCKII), C-kinase potentiated Protein phosphatase-1 Inhibitor (CPI-17) and phospho-CPI-17. Physiologic analysis of bioengineered human pyloric sphincters demonstrated the generation of spontaneous basal tones and contraction in response to potassium chloride. The presence of differentiated neurons within bioengineered constructs was indicated by expression of bIII-tubulin, neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT). Bioengineered innervated human pyloric sphincters responded appropriately to physiologically relevant neurotransmitters including acetylcholine (ACh) and vasoactive
597 Serological Antibodies for the Prediction of Post-Operative Recurrent Crohn's Disease Results From the POCER Study Amy L. Hamilton, Michael A. Kamm, Fabiyola Selvaraj, Fred Princen, Peter De Cruz, Emily K. Wright, Kathryn J. Ritchie, Efrosinia O. Krejany, Alexandra Gorelik, Danny Liew, Lani Prideaux, Ian C. Lawrance, Jane M. Andrews, Peter A. Bampton, Miles Sparrow, Timothy Florin, Peter R. Gibson, Henry Debinski, Richard B. Gearry, Finlay A. Macrae, Rupert W. Leong, Ian Kronborg, Graham L. Radford-Smith, Warwick Selby, Michael J. Johnston, Rodney Woods, Peter R. Elliott, Sally Bell, Steven J. Brown, William Connell, Paul V. Desmond, Sharat Singh Background: Disease recurrence after Crohn's disease resection occurs in up to up to 80% of patients, with two thirds ultimately requiring further surgery. Although clinical risk factors help in assessing the risk of relapse, a test that identifies patients at higher risk of recurrence would be clinically valuable. We investigated the value of serological antimicrobial antibodies to predict disease recurrence after surgery in a large prospective cohort of patients. Methods: 171 patients had 525 samples tested peri-operatively, and 6, 12 and 18 months postoperatively as part of a structured study ("POCER") designed to diminish post-operative
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intestinal peptide (VIP), and neural network integrity was demonstrated by neurotoxin sensitive responses to electrical field stimulation (EFS). Conclusion: This is the first demonstration of a bioengineered innervated human pyloric sphincter. Bioengineered pyloric sphincters maintained appropriate myogenic and neurogenic functionality. These bioengineered tissues have the potential for use as (1) functional replacement organs and (2) physiologically relevant models to investigate human pyloric sphincter disorders. This work was supported by Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0052; GU 7 and Wake Forest School of Medicine Internal Funds.
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Background. Most of the inflammatory and immune responses in inflammatory bowel disease and irritable bowel syndrome are mediated by NF kB signaling. NFkB in intestinal epithelial cells plays a critical role in protecting mucosal integrity during acute inflammation, whereas, NFkB in immune cells and smooth muscle cells mediates the inflammatory responses during chronic inflammation. Little is known about the role of NF kB signaling in enteric nervous system. Aim. To assess the effects of NFkB signaling within enteric neuronal cells (ENCs) on gastrointestinal motility in the presence or absence of intestinal inflammation. Methods. A conditional stop-floxed mouse line carrying a constitutively active form of IKK2 (IKK2CA) was crossbred sequentially with Rosa26-tdTomato reporter mice and with Calretinin-Cre-ERT2 mice to generate Calretinin-Cre-ERT2-IKK2CA/tdTomato transgenic (TG) mice. At the age of 2 months old, the animals were treated with tamoxifen (i.p. 100 mg/kg/day, 3 days), allowing ENC-specific IKK2CA expression and continuous NFkB activation. At 1 month after induction,, the colonic motility (bead exclusion assay), water/ food intake and general phenotypes were evaluated before 2.5% dextran sulfate sodium (DSS)-induced colitis was induced. The course of inflammation was monitored by daily disease activity index, end macroscopic observation and histopathologic examination. Spontaneous and acetylcholine (10-9 to 10-3 M)-stimulated contraction of longitudinal colonic muscle strips as well as sodium nitroprusside-induced relaxation were measured using 16 organ-baths with a PowerLab data acquisition system. Muscle strip whole mount and transverse sections of colon tissues were used for multilabeled immunohistochemistry. Results. At one month after accumulated tamoxifen induction, 50% of ENCs were labeled with the tdTomato reporter. There was no significant difference in food/water intake and colonic motility between TG and littermate wild-type (WT) mice. After DSS-induced acute colitis, the weight loss, bleeding, colon shortening and mucosa damage were less in TG than that in WT mice. Consistent with previous reports, DSS-colitis induced significant inhibition of acetylcholine-stimulated colonic muscle contraction and promotion of nitric oxide-stimulated colonic relaxation in WT mice. However, ENC-specific activation of NFkB signaling had no significant effect on acetylcholine-stimulated colonic muscle contraction under both physiological and DSS-challenge conditions, but significantly induced partial recovery of colonic relaxation after DSS challenge. Conclusion. Enteric neuronal NFkB overactivation in adult animals protects against acute colitis and inhibits colonic relaxation. This study highlights the importance of enteric neuronal NF kB signaling in regulating enteric neuronal plasticity and neurogastrointestinal motility.
600 In Situ Implantation of Autologous BiosphincterTM Re-Instates Continence in a Large Animal Model of Passive Fecal Incontinence Khalil N. Bitar, Jaime Bohl, John E. Fortunato, Sita Somara, Shreya Raghavan, Elie Zakhem, Stephen L. Rego, Kenneth L. Koch The Internal Anal Sphincter (IAS) is primarily responsible in maintaining anorectal continence. Sphincter dysfunction can lead to fecal incontinence (FI). A regenerative medicine approach to restore impaired IAS function offers a safe and sustained solution. Objective: In the present study, we bioengineered autologous innervated IAS BioSphinctersTM from cells isolated from small intestine and anal sphincter biopsies. The objective of this research was to develop a rabbit FI model, and surgically implant autologous constructs to remedy FI. Materials and Methods: IAS injury was produced in the rabbit by partial IAS sphincterectomy. The excised sphincteric tissue was used as source of IAS smooth muscle cells. Jejunal biopsies were taken to obtain tissue for the isolation of enteric neural progenitor cells. BioSphinctersTM were bioengineered from these two cell lines. Bioengineered constructs were implanted in situ six weeks post Sphincterectomy. Anorectal manometry was used to monitor anal canal pressures. Results: Sufficient smooth muscle cells were isolated from a semi-lunar IAS biopsy to bioengineer 4-6 BioSphincterTM constructs. Bioengineered intrinsically innervated sphincters mimicked native sphincter architecture and function. BioSphincterTM constructs demonstrated presence of contractile smooth muscle and mature neuronal populations. A deterioration in fecal hygiene and decreased basal tone (55% decrease) and RAIR (58% decrease) were observed in anorectal manometry post sphincterectomy. Autologous IAS constructs were successfully implanted into the donor rabbit without complications. The in situ implanted construct was neovascularized and showed no signs of inflammation or infection. Restoration of basal tone and RAIR were observed following implantation with anorectal manometry. To date we have successfully implanted 3 rabbits with end point of 3 months. Conclusions: A large animal model of FI has been developed and validated deterioration of fecal hygiene and loss of basal tone and RAIR. Bioengineering and implantation of autologous cells is technically feasible in an in vivo FI model. This provides proof of concept of safety and efficacy of BioSphincterTM constructs and restoration of IAS integrity and function and fecal continence. This regenerative medicine approach can be used as a therapeutic option to treat FI. This work was supported by Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH13-2-0052; GU 7 and NIH/NIDDK R01DK071614
603 Helicobacter pylori Activates Gastric Epithelial Stem Cell Through Direct Colonization of the Gastric Glands Michael Sigal, Manuel R. Amieva INTRODUCTION: Helicobacter pylori is a bacterial pathogen that colonizes the human stomach and is the main risk factor for gastro-duodenal ulcers and gastric cancer. H. pylori is found in close proximity to the surface of the stomach epithelium either as a free-swimming population in the gastric mucus or adhered to epithelial cells. The attached bacteria are known to alter cell signaling and behavior though different virulence factors. AIMS & METHODS: While the effects of attachment have been studied extensively in vitro, we aimed to study the localization and pathological relevance of the direct interaction of bacteria with the gastric epithelium, and in particular with gastric stem cells, in vivo. We utilized a murine model of H. pylori infection. We developed novel techniques to visualize and quantify H. pylori in mouse stomachs using 3D confocal microscopy. Lgr5eGFP mice were used to study the interaction of H. pylori with gastric stem cells. Lgr5eGFPCreER; RosadtTomato compound heterozygous mice were used for lineage tracing experiments. RESULTS: We discovered that H. pylori colonize the epithelial surface deep in the gastric glands where they grow as distinct microcolonies associated with the epithelial junctions. Using proliferation markers, we find that the gland-associated H. pylori directly colonize the surface of replicating progenitor cells. In addition, a proportion of bacteria can be found in the base of glands, where long-lived epithelial stem cells reside. We hypothesized that direct colonization of precursor/ stem cells may drive pathological responses. Using quantitative microscopy we mapped the distribution of bacteria in the glands of the antrum versus the corpus. We found that the location of bacteria in the glands correlates with hyperplastic lesions. Using Lgr5eGFP mice, we observed a direct interaction of H. pylori with Lgr5 expressing stem cells in the stomach antrum. Infection induced an expansion of the stem cell compartment. In addition, lineage tracing experiments revealed a significantly higher number of daughter cells being generated from Lgr5 expressing stem cell in infected animals compared to uninfected controls. The accelerated tracing tightly correlated with the bacteria in the gastric glands. Infection with a bacterial chemotaxis mutant that is able to colonize the mucus but is unable to penetrate deep into the glands did not induce an activation of stem cells. Moreover, a bacterial mutant that is unable to deliver the main virulence factor CagA into the epithelium induced diminished activation of stem cells and caused less hyperplasia. CONCLUSION: Taken together our data reveals that H. pylori directly interacts with progenitor and stem cells deep in the gastric glands. This gland-associated subpopulation of H. pylori alters stem cell homeostasis of the colonized glands, leading to gland hyperplasia.
601 The Appendix As a Viable Source of Neural Progenitor Cells to Functionally Innervate Bioengineered Gastrointestinal Smooth Muscle Tissues Elie Zakhem, Stephen L. Rego, Shreya Raghavan, Khalil N. Bitar Background: Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, however, utilizing these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix differentiate into functional enteric neural subsets similar to NPCs isolated from the small intestine. Methods: (1) NPCs were isolated from the appendix and small intestines (SI) of rabbits. (2) IAS smooth muscle cells were isolated from rabbit internal anal sphincter (IAS) tissue. (3) Bioengineered (IAS) constructs were developed, using the same source of smooth muscle, and innervated with NPCs derived from either the appendix or SI. (4) Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry. (5) Physiological functionality was assessed through force generation studies. Results: (1) NPCs from both the appendix and the SI expressed enteric neural stem cell markers (p75-NTR, SOX2 and nestin). (2) Immunostaining analysis demonstrated expression of neuronal differentiation marker bIII tubulin in both bioengineered constructs, indicating that neural progenitor cells derived from either the appendix or the SI have the capability to differentiate into neurons. (3) Western blot analysis demonstrated the expression of enteric neuronal protein (excitatory and inhibitory motor neurons) and glial protein (GFAP) confirming neural and glial differentiation in both types of constructs. (4) The addition of 1 µM acetylcholine (Ach) caused a robust contraction in appendix and SI constructs of magnitudes 279.3 ± 20.01 µN and 294.8 ± 17.85 µN, respectively. The contraction was significantly decreased by pretreatment with neural inhibitor, tetrodotoxin (TTX). (5) Electrical field stimulation, EFS, (5 Hz, 0.5 ms) caused relaxation of -257.7 ± 7.410 µN and -229.3 ± 13.53 µN in the appendix and SI constructs, respectively. The relaxation was completely abolished in the presence of TTX. (6) EFS-induced relaxation was significantly reduced upon pre-treatment with nitric oxide synthase inhibitor (L-NAME) in both constructs. Conclusion: The appendix can be accessed using minimally invasive surgery. This study indicates that NPCs isolated from the appendix have the potential to differentiate into functional neurons in a similar manner as NPCs isolated from the small intestine. The neurons were also capable of responding to physiological stimuli. This study demonstrates the potential use of the appendix in human as a source of neural progenitor cells. This work was supported by Army, Navy, NIH, Air Force, VA and Health Affairs to support the AFIRM II effort, under Award No. W81XWH-13-2-0052; GU 7 and NIH/NIDDK R01DK071614.
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Cre/LoxP-Mediated Inducible Activation of IKK2 in Adult Enteric Neuronal Cells Protects Against DSS-Induced Colitis in Transgenic Mice Yonggang Zhang, Wensheng Yang, Alan S. Braverman, Fang Li, Henry P. Parkman, K. S. Murthy, Michael R. Ruggieri, Wenhui Hu