- Email: [email protected]
Su2020 Autoimmunity to Vinculin in Humans May Be Important in the Pathophysiology of IBS
AGA Abstracts
tissue myeloperoxidase (MPO) activity and qPCR quantification of TLR4 gene expression, as markers for immune cell infiltration and host-gut flora interactions, respectively (6 samples/ mouse). Results: Our studies showed a strong, yet conditional, correlation between stereomicroscopy and histology, and treatment effect. For ileitis, there was a strong exponential correlation (y=6.7216*e0.1889x; R2=0.8919), which was explained by a plateau effect of histological scores between (10-12/18) as the severity of 3D-steromicroscopic abnormalities in SAMP increased. Such correlation differed from that of other studies with ileitis-free mice (R2<0.3), indicating that histological scores need to be validated or adjusted for a diverse categories of intestinal pathologies. For colitis, the correlation between stereomicroscopy and histology was strong as it correlated with the high histological scores observed in DSScolitis, and intermediate or low after dexamethasone or placebo treatments (y=8.03x+13.369; R2=0.908). The variability observed between the two methods, was explained by nonrandom segmental distribution of 3-D abnormal pathologies, not captured by the integer histological scoring system. Of immunological and statistical relevance, the ability for MPO and TLR4 to differentiate the groups was significantly enhanced when the analysis was stratified by the degree of 3-D mucosal abnormalities (p<0.01). Conclusion: Stereomicroscopy is a very cost effective visualization tool that can be used as a complementary research and diagnostic tool in studies of therapeutic and intestinal pathology.
chromoendoscopy (DC), magnification (M) and high definition (HD) had shown excellent results for the diagnosis of Barret esophagus (BE) and esophagitis. However, not all patients are investigated with this kind of technology, where UE results are considered as absence of organic lesions, thus diagnosed as FD. Based on the hypothesis that HD UE associated to DC and M can detect more mucosal details than standard UE, we evaluate the effectiveness of i-Scan™ (HD UE+DC+M) in patients with functional dyspepsia for the identification of organic esophageal lesions. Patients and Methods After approval by the ethics committee and signing of an informed consent, a prospective study was performed in consecutive patients undergoing for UE from Nov 2012 to June 2013. Inclusion criteria: Criteria of FD in accordance to ROMA III criteria, normal standard UE in the last 3 months previous to the inclusion in this study. Exclusion criteria: age<18; pregnancy, history of: gastritis, GERD, gastrointestinal cancer, H pylori infection, pancreatic disease, choledocolitiasis, alcohol or smoke abuse, use of medications (IBP, NSAIDs, Antibiotics). HD UE+DC and M was performed using the EPK-i processors with i-Scan™ from Pentax. Under sedation patient underwent to HD UE, analyzing all the mucosa aspects using initially white light (WL), with especial regard in the Z-line at the level of the cardia. Them DC was performed using i-Scan. Any alteration in the mucosa pattern (color, pitt or vascular pattern) was analyzed and them classified as inflammation or BE using Los Angeles and Prague classifications respectively. Finally acetic acid was performed and a target biopsy was done as the gold standard method to confirm i-Scan findings. Results 491 patients where included. 48% were men with a mean age of 47 (ranges: 18-87). 151/491 patients (30.7%) had an organic esophageal lesion detected at i-Scan. 45/151 patients were detected initially by HD-UE-WL. Biopsy confirm the esophageal lesions in 125 cases. i-Scan detect 94 cases of short BE (C<1,M<1), 25 cases of esophagitis (Grade A), and 6 cases where considered to have a mixed disease (BE and esophagitis). The accuracy to predict BE for i-Scan was 95% and 100% for esophagitis. Conclusion HD UE+M+DC (i-Scan™) could detect an important number of organic esophageal lesions as BE and esophagitis in patients initially overdiagnosed as a functional disease.
Su2017 In Vivo Performance of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transport Diana Vivian, Kunrong Cheng, Sandeep Khurana, Su Xu, Edwin H. Kriel, Paul Dawson, James Polli, Jean-Pierre Raufman Background: Emerging evidence suggests that dysregulated bile acid synthesis and transport may be the cause of diarrhea in more than one-third of patients misdiagnosed with IBS. However, current in vivo approaches to assess intestinal bile acid uptake by the ileal bile acid transporter, ASBT, transport into the enterohepatic circulation and accumulation in the gallbladder are limited. To address this limitation we designed and synthesized a cholic acid derivative, CA-lys-TFA, containing three atoms/molecule of naturally-occurring fluorine (19F) suitable for dual 1H/19F magnetic resonance imaging (MRI). Objectives: To compare the in vivo imaging performance of CA-lys-TFA with direct measurement of CA-lys-TFA in gallbladders harvested from C57BL/6J WT and Asbt-deficient (Slc10a2-/-) mice. Methods: In mice anesthetized by ketamine/xylazine injection, small-animal MRI (7 Tesla) was performed 2, 7 and 48 h after gavage with CA-lys-TFA . Following imaging, CA-lys-TFA was measured in harvested gallbladders by liquid chromatography/tandem-mass spectrometry (LC/MS/MS). To assess the ability of the novel imaging agent to detect impaired bile acid transport, we measured differences in gallbladder bile [CA-lys-TFA] after gavaging WT and Asbt-deficient mouse littermates with the test agent (n=4 mice/genotype). Results: Mice were successfully imaged in vivo. In WT mice, CA-lys-TFA MRI signals were detected in the gallbladder following oral gavage of either a single 150 mg/kg dose (Figure) or following one week of once-daily gavage with 50 mg/kg. At all time points, CA-lys-TFA concentrations determined by MRI were higher with the single 150 mg/kg dose than with repetitive dosing of 50 mg/ kg, likely reflecting bacterial hydrolysis of CA-lys-TFA in the gut as predicted by our in vitro studies. Optimum and reproducible gallbladder CA-lys-TFA concentrations were detected using MRI at 7 h after gavage with the single 150-mg/kg dose. In Asbt-deficient mice, after gavage with a single 150 mg/kg dose CA-lys-TFA was undetectable with MRI (Figure) and gallbladder bile concentrations of CA-lys-TFA were 30-fold lower than in WT mice (0.3 ± 0.0 vs. 9.8 ± 2.1 mM, P=0.004). In both WT and Asbt-deficient mice, liver and plasma CA-lys-TFA concentrations were orders of magnitude lower than those observed in the gallbladder (0.70-22.97 μM in liver and 0.02-16.56 μM in plasma) and were too low to generate 19F MRI signals. Conclusions: To our knowledge, this represents the first report that a drug administered orally can be imaged in vivo by 19F MRI. CA-lys-TFA, a novel fluorine-labeled imaging agent, has great potential as a tool to measure in vivo bile acid transport. Using 19F MRI gallbladder imaging, a methodology involving no ionizing radiation, also has potential as a clinical test to detect bile acid malabsorption.
Su2019 Functional Characterization of a Novel RAD21 Mutation in Familial Chronic Intestinal Pseudo-Obstruction (CIPO) Elena Bonora, Francesca Bianco, Mike Bamshad, Dustin Dowless, Ludmila Francescatto, Nicholas Katsanis, Gregory Cooper, Daniel Savic, Vincenzo Stanghellini, Zeynel Mungan, Kivanc Cefle, Rosanna Cogliandro, Elisa Boschetti, Claudio Graziano, Marco Seri, Giovanni Romeo, Roberto De Giorgio Objectives CIPO is characterized by a severe impairment of gut motility mimicking a mechanical sub-occlusion in the absence of occluding causes. Although most cases are sporadic, some CIPO cases show a familial clustering suggesting a genetic origin. Using whole exome analysis, we identified a novel homozygous mutation c.1864 (p.622 Ala>Thr) G>A in RAD21, member of the cohesin complex, in a Turkish family in which we previously mapped a locus for CIPO to an interval on chr8q23-q24. The aim of this work was to establish whether the RAD21 mutation exerts a functional role in CIPO pathophysiology. Methods cDNA and proteins derived from lymphobastoid cell line (LCLs) from Turkish family members and relative controls were used for a number of assays: qRT-PCR, western blot, chromatin immunoprecipitation and sequencing (ChIP-seq), transcriptome analysis (RNA-seq), and electromobility shift assays. For in vivo analysis a morpholino rescue phenotyping in zebrafish was performed. Results The mutant RAD21 mRNA and protein were expressed in comparable amount to wild-type LCLs. Co-immunoprecipitation experiments showed that the mutant protein still binds to SMC1, suggesting that the formation of the cohesin complex is not impaired in the affected members. However, the expression of RAD21 major target, RUNX1, showed a significant decrease in the patient's cell line (P<0.05). The RAD21 knock-out zebrafish model showed severe reduction of the gut lumen resulting in a delayed transit. Runx1 expression analysis showed an absent or incomplete expression pattern in the knock-out animals vs. controls. Wild-type cDNA, but not the one carrying the mutant RAD21 allele, rescued Runx1 expression and a normal gut physiology. Based on the role of RAD21 in both gene expression and higher-order chromatin architecture, ChIP-seq and RNA-seq experimentation in LCLs from patient and parents is currently ongoing, and will aid in identifying additional potential genomic abnormalities. Conclusions We identified a novel mutation of RAD21 in a familial syndromic form of CIPO and provide evidence that the RAD21 mutation exerts a pathogenetic role in this syndrome. Our findings contribute to a better understanding of the genetic mechanisms involved in CIPO pathophysiology. Su2020 Autoimmunity to Vinculin in Humans May Be Important in the Pathophysiology of IBS Mark Pimentel, Constantinos Brikos, Venkata B. Pokkunuri, Walter Morales, Stacy Weitsman, Shanthi Srinivasan, Gillian M. Barlow, Zachary Marsh, Emily Marsh, Gene Kim, Christopher Chang
Comparison of 19F MRI in WT and Asbt-deficient mice gavaged with 150 mg/kg CA-lysTFA. Arrowheads and arrow indicate 19F CA-lys-TFA signals emanating from phantoms and gallbladder, respectively. A 19F gallbladder signal is seen only in MRI of the WT mouse.
In a C. jejuni rat model of post-infectious IBS, rats develop altered bowel habits, small intestinal bacterial overgrowth (SIBO) and a reduction of interstitial cells of Cajal (ICC) predictive of IBS. After infection with mutant C. jejuni lacking cytolethal distending toxin B (CdtB), these sequelae were mitigated. Later experiments determined that antibodies to CdtB predict SIBO and through molecular mimicry have affinity for ICC and enteric ganglia. In this translational study, we investigate the human antigen to which anti-CdtB binds. Methods: First, non-IBS human full thickness ileal tissue (from right hemicolectomy) was obtained. Sections were probed with purified rabbit antibodies to CdtB. Colocalization studies were performed with anti-c-kit (specific for ICC), S-100 (specific for neurons) and PGP 9.5 (specific for ganglia). Next, a lysate of enteric neuronal cells (Emory University) was passed through an anti-CdtB affinity column. Anti-CdtB adherent protein was eluted and two western blots were performed. One was incubated with anti-CdtB and the other with anti-CdtB pre-incubated with CdtB protein (blocking peptide). An 117kDa protein was purified and identified by mass spectroscopy as human vinculin. An aliquot of 0.4 ug of commercial vinculin was coated per well in 96 well plates. Anti-CdtB was added to one
Su2018 Pentax I-SCAN™ With Magnification for the Identification of Underdiagnosis Organic Esophageal Lesions (Barret Esophagus and Esophagitis) in Patients With Functional Dyspepsia: A Prospective Study Carlos Robles-Medranda, Raquel S. Del Valle, Miguel Soria ALcívar, Gladys Bravo Velez, Francisco Abarca Rendon, Carlos A. Robles-Jara Background Functional dyspepsia (FD) is a highly prevalent gastrointestinal disorder characterized by symptoms originating from the gastroduodenal region in the absence of underlying organic disease as defined by Roma III criteria. Upper endoscopy (UE) associated to digital
AGA Abstracts
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AGA Abstracts
series of wells, and anti-CdtB mixed with whole CdtB protein (blocking peptide) to another. Results: In full-thickness human ileum, anti-CdtB was specific for ICC and ganglia, based on colocalization of anti-CdtB with anti-c-kit, PGP 9.5 and S-100 (see figure). Thus antiCdtB appeared to interact with a human protein on ICCs and ganglia. Based on immunoprecipitation, a protein band was identified at 117kDa. Using mass spectroscopy this protein was identified as human vinculin. Subsequently, human vinculin was obtained commercially and by ELISA, anti-CdtB had a high affinity for human vinculin but not the control peptide. Binding to vinculin was blocked by the CdtB peptide. Conclusions: In the pathophysiology of post-infectious IBS, subjects develop antibodies to CdtB which have cross reactivity through molecular mimicry to vinculin, a cell membrane cytoskeletal protein important in neural cell migration and adherence. Given our emerging data of reduced vinculin levels in post-infectious rats and circulating anti-vinculin antibodies discriminating IBS from IBD, molecular mimicry to vinculin may be important to the cause of IBS through effects on ICC and ganglia.
Su2021 Table 2. Post-receptor signaling pathways linked to UTP-Ca2+ oscillations
UTP Evokes CA2+ Oscillations in Human Enterochromaffin Cells via Multiple Intracellular Signaling and Ionic Mechanisms Andromeda Linan Rico, Fernando Ochoa-Cortes, Mazin A. Alhaj, Josh Enneking, Emily Bitticker, Bradley J. Needleman, Alan Harzman, Iveta Grants, Fievos L. Christofi Background/Aims: Enterochromaffin cells (EC) are sensory cells in gut mucosa releasing 5-HT and ATP to initiate gut reflexes or pain transmission. Alterations in 5-HT signaling are implicated in IBD and IBS. However, mechanisms modulating 5-HT release are not well understood. Purines like ATP act in an autocrine or paracrine manner to modulate Ca2+dependent 5-HT release in a human BON (EC) cell model or EC cells in intact human mucosa {IBD, 2013}. UTP elicits Ca2+ oscillations or single transients in EC cells and our pharmacological analysis supports a P2Y4R. We used imaging and electrophysiological approaches to elucidate post-receptor signaling pathways and ionic mechanisms linked to UTP-induced Ca2+ transients and 5-HT release. Experimental Design: 5-HT release was monitored in BON cells or EC cells isolated from human Roux-en-Y surgical tissues by percoll or nycodenz-gradient separation (IRB-2012H0231). Ca2+ responses and post-receptor mechanisms were studied in BON by laser confocal fluo-4/AM-Ca2+imaging, and patchclamp recordings of Ca2+ currents, IK+ currents and membrane potential depolarization. IHC and WB identified P2Y4R. Data is summarized in Tables 1 (MP,IK+,ICa2+currents) & Table 2 (Ca2+ responses) . Results: P2Y4 agonists UTP and UTPγS elicit Ca2+ oscillations in nearly 70% of responsive BON cells. Remaining cells display single Ca2+ transients. Chelating intracellular free Ca2+(BAPTA-AM) prevented Ca2+ responses. PLC inhibition by U73122 blocked Ca2+ responses in 60% of cells or had no effect in the remaining 40% of cells. An IP3R antagonist (2APB) that also inhibits SOCa2+ release, blocked responses as did La3+ in 100% of cells. Thapsigargin prevented Ca2+ oscillations by blocking the SERCA Ca2+-pump. Zero-Ca2+ buffer augmented responses in 39% cells. Inhibitors for L-type Ca2+channels, Ryanodine Ca2+-pools, PI3K, protein kinase C, SRC-Kinase or TRPC-channels had no effect. UTP (or UTPγS) inhibited IK+, stimulated an increase in voltage-sensitive Ca2+currents and depolarized cells. PLC inhibition blocked depolarization to UTP, whereas PKC inhibition, La3+, thapsigargin or zero Ca2+ buffer had no effect. UTP > ATP in stimulating 5-HT release in BON or isolated human EC cells from surgical-specimens (N=3). WB identified P2Y4R-bands in isolated human EC and BON cells. P2Y4R-ir was found in 5HT+(EC) cells of human mucosa (N=3). Conclusions: UTP activates a P2Y4R to elicit Ca2+ oscillations in most EC cells via PLC/IP3/IP3R/ SERCA Ca2+-pump signaling and SOC channels, and stimulate 5-HT release. Membrane depolarization depends on PLC signaling, IK+ and VOCCs, but is not linked to Ca2+ oscillations. P2Y4 is involved in fine-tune modulation of 5-HT secretion. The P2Y4 signal transduction pathways are worth exploring as a suitable model to evaluate potential changes in Ca2+ or ionic mechanisms in IBD or IBS (NIH DK093499;OSUWMC-funds). Table 1. UTP-induced alterations in MP, K-type IK+ and ICa2+ currents and post receptor signaling
Su2022 Cytokine Modulation of Enteric Neural Stem Cell Proliferation Laren Becker, Aida Habtezion Background: Interleukin 22 (IL-22), a cytokine produced by a variety of hematopoietic cells, has been found to have cytoprotective effects in various tissues including lung, liver, pancreas, and colon. IL-22 signaling occurs through the IL-22 receptor (IL-22R), which is expressed on non-hematopoietic cells. Recently, IL-22R was demonstrated on intestinal stem cells and IL-22 deficiency caused stem cell loss. We evaluated whether IL-22R is expressed on enteric neural stem cells (ENSCs) and whether modulation with IL-22 affects proliferation. Methods: We performed immunofluorescence with antibodies to IL-22R and nestin on longitudinal muscle and myenteric plexus (LMMP) whole mounts and ENSCs cultured as neurospheres. Utilizing Wnt1-cre;tdTomato (tdT) mice which fluorescently marks neural crestderived cells, we performed flow cytometry with an antibody to CD49 (ENSC marker) and subsequently performed RT-PCR analysis on sorted cells. We assessed for IL-22 signaling by Western blot analysis using antibodies to pSTAT-3 and STAT-3 with both in vitro organotypic culturing of LMMP and in vivo injection of mice with rIL-22. Finally, we performed EdU uptake assays using organotypic cultures of LMMP and ENSC cultures in the presence or absence of rIL-22. Results: IL-22R expression was detected in Nestin+ cells in enteric ganglia and cultured neurospheres. Compared to spleen cells that were double negative, LMMP cells that co-expressed tdT (neural crest) and CD49b-APC (ENSC marker) demonstrated over 5-fold higher expression of IL-22R by RT-PCR. These cells also expressed 1,000-fold higher nestin compared to corresponding cells from spleen as would be expected for ENSCs. Organotypic cultures of LMMP incubated with IL-22 demonstrated increase in pSTAT3:STAT3 expression in a dose dependent manner. Similar increase in pSTAT3:STAT3 expression was detected in LMMP 1 h following IP injection of rIL-22 compared to sham. Organotypic cultures of LMMP in the presence EdU with rIL-22 (10 ng/ml) for 40h resulted in increased EdU uptake of neural crest cells (tdT+) compared with control (42% versus 32%). Addition of rIL-22 (10 ng/ml) to cultures of ENSCs resulted in a similar increase in
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AGA Abstracts
tissue myeloperoxidase (MPO) activity and qPCR quantification of TLR4 gene expression, as markers for immune cell infiltration and host-gut flora interactions, respectively (6 samples/ mouse). Results: Our studies showed a strong, yet conditional, correlation between stereomicroscopy and histology, and treatment effect. For ileitis, there was a strong exponential correlation (y=6.7216*e0.1889x; R2=0.8919), which was explained by a plateau effect of histological scores between (10-12/18) as the severity of 3D-steromicroscopic abnormalities in SAMP increased. Such correlation differed from that of other studies with ileitis-free mice (R2<0.3), indicating that histological scores need to be validated or adjusted for a diverse categories of intestinal pathologies. For colitis, the correlation between stereomicroscopy and histology was strong as it correlated with the high histological scores observed in DSScolitis, and intermediate or low after dexamethasone or placebo treatments (y=8.03x+13.369; R2=0.908). The variability observed between the two methods, was explained by nonrandom segmental distribution of 3-D abnormal pathologies, not captured by the integer histological scoring system. Of immunological and statistical relevance, the ability for MPO and TLR4 to differentiate the groups was significantly enhanced when the analysis was stratified by the degree of 3-D mucosal abnormalities (p<0.01). Conclusion: Stereomicroscopy is a very cost effective visualization tool that can be used as a complementary research and diagnostic tool in studies of therapeutic and intestinal pathology.
chromoendoscopy (DC), magnification (M) and high definition (HD) had shown excellent results for the diagnosis of Barret esophagus (BE) and esophagitis. However, not all patients are investigated with this kind of technology, where UE results are considered as absence of organic lesions, thus diagnosed as FD. Based on the hypothesis that HD UE associated to DC and M can detect more mucosal details than standard UE, we evaluate the effectiveness of i-Scan™ (HD UE+DC+M) in patients with functional dyspepsia for the identification of organic esophageal lesions. Patients and Methods After approval by the ethics committee and signing of an informed consent, a prospective study was performed in consecutive patients undergoing for UE from Nov 2012 to June 2013. Inclusion criteria: Criteria of FD in accordance to ROMA III criteria, normal standard UE in the last 3 months previous to the inclusion in this study. Exclusion criteria: age<18; pregnancy, history of: gastritis, GERD, gastrointestinal cancer, H pylori infection, pancreatic disease, choledocolitiasis, alcohol or smoke abuse, use of medications (IBP, NSAIDs, Antibiotics). HD UE+DC and M was performed using the EPK-i processors with i-Scan™ from Pentax. Under sedation patient underwent to HD UE, analyzing all the mucosa aspects using initially white light (WL), with especial regard in the Z-line at the level of the cardia. Them DC was performed using i-Scan. Any alteration in the mucosa pattern (color, pitt or vascular pattern) was analyzed and them classified as inflammation or BE using Los Angeles and Prague classifications respectively. Finally acetic acid was performed and a target biopsy was done as the gold standard method to confirm i-Scan findings. Results 491 patients where included. 48% were men with a mean age of 47 (ranges: 18-87). 151/491 patients (30.7%) had an organic esophageal lesion detected at i-Scan. 45/151 patients were detected initially by HD-UE-WL. Biopsy confirm the esophageal lesions in 125 cases. i-Scan detect 94 cases of short BE (C<1,M<1), 25 cases of esophagitis (Grade A), and 6 cases where considered to have a mixed disease (BE and esophagitis). The accuracy to predict BE for i-Scan was 95% and 100% for esophagitis. Conclusion HD UE+M+DC (i-Scan™) could detect an important number of organic esophageal lesions as BE and esophagitis in patients initially overdiagnosed as a functional disease.
Su2017 In Vivo Performance of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transport Diana Vivian, Kunrong Cheng, Sandeep Khurana, Su Xu, Edwin H. Kriel, Paul Dawson, James Polli, Jean-Pierre Raufman Background: Emerging evidence suggests that dysregulated bile acid synthesis and transport may be the cause of diarrhea in more than one-third of patients misdiagnosed with IBS. However, current in vivo approaches to assess intestinal bile acid uptake by the ileal bile acid transporter, ASBT, transport into the enterohepatic circulation and accumulation in the gallbladder are limited. To address this limitation we designed and synthesized a cholic acid derivative, CA-lys-TFA, containing three atoms/molecule of naturally-occurring fluorine (19F) suitable for dual 1H/19F magnetic resonance imaging (MRI). Objectives: To compare the in vivo imaging performance of CA-lys-TFA with direct measurement of CA-lys-TFA in gallbladders harvested from C57BL/6J WT and Asbt-deficient (Slc10a2-/-) mice. Methods: In mice anesthetized by ketamine/xylazine injection, small-animal MRI (7 Tesla) was performed 2, 7 and 48 h after gavage with CA-lys-TFA . Following imaging, CA-lys-TFA was measured in harvested gallbladders by liquid chromatography/tandem-mass spectrometry (LC/MS/MS). To assess the ability of the novel imaging agent to detect impaired bile acid transport, we measured differences in gallbladder bile [CA-lys-TFA] after gavaging WT and Asbt-deficient mouse littermates with the test agent (n=4 mice/genotype). Results: Mice were successfully imaged in vivo. In WT mice, CA-lys-TFA MRI signals were detected in the gallbladder following oral gavage of either a single 150 mg/kg dose (Figure) or following one week of once-daily gavage with 50 mg/kg. At all time points, CA-lys-TFA concentrations determined by MRI were higher with the single 150 mg/kg dose than with repetitive dosing of 50 mg/ kg, likely reflecting bacterial hydrolysis of CA-lys-TFA in the gut as predicted by our in vitro studies. Optimum and reproducible gallbladder CA-lys-TFA concentrations were detected using MRI at 7 h after gavage with the single 150-mg/kg dose. In Asbt-deficient mice, after gavage with a single 150 mg/kg dose CA-lys-TFA was undetectable with MRI (Figure) and gallbladder bile concentrations of CA-lys-TFA were 30-fold lower than in WT mice (0.3 ± 0.0 vs. 9.8 ± 2.1 mM, P=0.004). In both WT and Asbt-deficient mice, liver and plasma CA-lys-TFA concentrations were orders of magnitude lower than those observed in the gallbladder (0.70-22.97 μM in liver and 0.02-16.56 μM in plasma) and were too low to generate 19F MRI signals. Conclusions: To our knowledge, this represents the first report that a drug administered orally can be imaged in vivo by 19F MRI. CA-lys-TFA, a novel fluorine-labeled imaging agent, has great potential as a tool to measure in vivo bile acid transport. Using 19F MRI gallbladder imaging, a methodology involving no ionizing radiation, also has potential as a clinical test to detect bile acid malabsorption.
Su2019 Functional Characterization of a Novel RAD21 Mutation in Familial Chronic Intestinal Pseudo-Obstruction (CIPO) Elena Bonora, Francesca Bianco, Mike Bamshad, Dustin Dowless, Ludmila Francescatto, Nicholas Katsanis, Gregory Cooper, Daniel Savic, Vincenzo Stanghellini, Zeynel Mungan, Kivanc Cefle, Rosanna Cogliandro, Elisa Boschetti, Claudio Graziano, Marco Seri, Giovanni Romeo, Roberto De Giorgio Objectives CIPO is characterized by a severe impairment of gut motility mimicking a mechanical sub-occlusion in the absence of occluding causes. Although most cases are sporadic, some CIPO cases show a familial clustering suggesting a genetic origin. Using whole exome analysis, we identified a novel homozygous mutation c.1864 (p.622 Ala>Thr) G>A in RAD21, member of the cohesin complex, in a Turkish family in which we previously mapped a locus for CIPO to an interval on chr8q23-q24. The aim of this work was to establish whether the RAD21 mutation exerts a functional role in CIPO pathophysiology. Methods cDNA and proteins derived from lymphobastoid cell line (LCLs) from Turkish family members and relative controls were used for a number of assays: qRT-PCR, western blot, chromatin immunoprecipitation and sequencing (ChIP-seq), transcriptome analysis (RNA-seq), and electromobility shift assays. For in vivo analysis a morpholino rescue phenotyping in zebrafish was performed. Results The mutant RAD21 mRNA and protein were expressed in comparable amount to wild-type LCLs. Co-immunoprecipitation experiments showed that the mutant protein still binds to SMC1, suggesting that the formation of the cohesin complex is not impaired in the affected members. However, the expression of RAD21 major target, RUNX1, showed a significant decrease in the patient's cell line (P<0.05). The RAD21 knock-out zebrafish model showed severe reduction of the gut lumen resulting in a delayed transit. Runx1 expression analysis showed an absent or incomplete expression pattern in the knock-out animals vs. controls. Wild-type cDNA, but not the one carrying the mutant RAD21 allele, rescued Runx1 expression and a normal gut physiology. Based on the role of RAD21 in both gene expression and higher-order chromatin architecture, ChIP-seq and RNA-seq experimentation in LCLs from patient and parents is currently ongoing, and will aid in identifying additional potential genomic abnormalities. Conclusions We identified a novel mutation of RAD21 in a familial syndromic form of CIPO and provide evidence that the RAD21 mutation exerts a pathogenetic role in this syndrome. Our findings contribute to a better understanding of the genetic mechanisms involved in CIPO pathophysiology. Su2020 Autoimmunity to Vinculin in Humans May Be Important in the Pathophysiology of IBS Mark Pimentel, Constantinos Brikos, Venkata B. Pokkunuri, Walter Morales, Stacy Weitsman, Shanthi Srinivasan, Gillian M. Barlow, Zachary Marsh, Emily Marsh, Gene Kim, Christopher Chang
Comparison of 19F MRI in WT and Asbt-deficient mice gavaged with 150 mg/kg CA-lysTFA. Arrowheads and arrow indicate 19F CA-lys-TFA signals emanating from phantoms and gallbladder, respectively. A 19F gallbladder signal is seen only in MRI of the WT mouse.
In a C. jejuni rat model of post-infectious IBS, rats develop altered bowel habits, small intestinal bacterial overgrowth (SIBO) and a reduction of interstitial cells of Cajal (ICC) predictive of IBS. After infection with mutant C. jejuni lacking cytolethal distending toxin B (CdtB), these sequelae were mitigated. Later experiments determined that antibodies to CdtB predict SIBO and through molecular mimicry have affinity for ICC and enteric ganglia. In this translational study, we investigate the human antigen to which anti-CdtB binds. Methods: First, non-IBS human full thickness ileal tissue (from right hemicolectomy) was obtained. Sections were probed with purified rabbit antibodies to CdtB. Colocalization studies were performed with anti-c-kit (specific for ICC), S-100 (specific for neurons) and PGP 9.5 (specific for ganglia). Next, a lysate of enteric neuronal cells (Emory University) was passed through an anti-CdtB affinity column. Anti-CdtB adherent protein was eluted and two western blots were performed. One was incubated with anti-CdtB and the other with anti-CdtB pre-incubated with CdtB protein (blocking peptide). An 117kDa protein was purified and identified by mass spectroscopy as human vinculin. An aliquot of 0.4 ug of commercial vinculin was coated per well in 96 well plates. Anti-CdtB was added to one
Su2018 Pentax I-SCAN™ With Magnification for the Identification of Underdiagnosis Organic Esophageal Lesions (Barret Esophagus and Esophagitis) in Patients With Functional Dyspepsia: A Prospective Study Carlos Robles-Medranda, Raquel S. Del Valle, Miguel Soria ALcívar, Gladys Bravo Velez, Francisco Abarca Rendon, Carlos A. Robles-Jara Background Functional dyspepsia (FD) is a highly prevalent gastrointestinal disorder characterized by symptoms originating from the gastroduodenal region in the absence of underlying organic disease as defined by Roma III criteria. Upper endoscopy (UE) associated to digital
AGA Abstracts
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AGA Abstracts
series of wells, and anti-CdtB mixed with whole CdtB protein (blocking peptide) to another. Results: In full-thickness human ileum, anti-CdtB was specific for ICC and ganglia, based on colocalization of anti-CdtB with anti-c-kit, PGP 9.5 and S-100 (see figure). Thus antiCdtB appeared to interact with a human protein on ICCs and ganglia. Based on immunoprecipitation, a protein band was identified at 117kDa. Using mass spectroscopy this protein was identified as human vinculin. Subsequently, human vinculin was obtained commercially and by ELISA, anti-CdtB had a high affinity for human vinculin but not the control peptide. Binding to vinculin was blocked by the CdtB peptide. Conclusions: In the pathophysiology of post-infectious IBS, subjects develop antibodies to CdtB which have cross reactivity through molecular mimicry to vinculin, a cell membrane cytoskeletal protein important in neural cell migration and adherence. Given our emerging data of reduced vinculin levels in post-infectious rats and circulating anti-vinculin antibodies discriminating IBS from IBD, molecular mimicry to vinculin may be important to the cause of IBS through effects on ICC and ganglia.
Su2021 Table 2. Post-receptor signaling pathways linked to UTP-Ca2+ oscillations
UTP Evokes CA2+ Oscillations in Human Enterochromaffin Cells via Multiple Intracellular Signaling and Ionic Mechanisms Andromeda Linan Rico, Fernando Ochoa-Cortes, Mazin A. Alhaj, Josh Enneking, Emily Bitticker, Bradley J. Needleman, Alan Harzman, Iveta Grants, Fievos L. Christofi Background/Aims: Enterochromaffin cells (EC) are sensory cells in gut mucosa releasing 5-HT and ATP to initiate gut reflexes or pain transmission. Alterations in 5-HT signaling are implicated in IBD and IBS. However, mechanisms modulating 5-HT release are not well understood. Purines like ATP act in an autocrine or paracrine manner to modulate Ca2+dependent 5-HT release in a human BON (EC) cell model or EC cells in intact human mucosa {IBD, 2013}. UTP elicits Ca2+ oscillations or single transients in EC cells and our pharmacological analysis supports a P2Y4R. We used imaging and electrophysiological approaches to elucidate post-receptor signaling pathways and ionic mechanisms linked to UTP-induced Ca2+ transients and 5-HT release. Experimental Design: 5-HT release was monitored in BON cells or EC cells isolated from human Roux-en-Y surgical tissues by percoll or nycodenz-gradient separation (IRB-2012H0231). Ca2+ responses and post-receptor mechanisms were studied in BON by laser confocal fluo-4/AM-Ca2+imaging, and patchclamp recordings of Ca2+ currents, IK+ currents and membrane potential depolarization. IHC and WB identified P2Y4R. Data is summarized in Tables 1 (MP,IK+,ICa2+currents) & Table 2 (Ca2+ responses) . Results: P2Y4 agonists UTP and UTPγS elicit Ca2+ oscillations in nearly 70% of responsive BON cells. Remaining cells display single Ca2+ transients. Chelating intracellular free Ca2+(BAPTA-AM) prevented Ca2+ responses. PLC inhibition by U73122 blocked Ca2+ responses in 60% of cells or had no effect in the remaining 40% of cells. An IP3R antagonist (2APB) that also inhibits SOCa2+ release, blocked responses as did La3+ in 100% of cells. Thapsigargin prevented Ca2+ oscillations by blocking the SERCA Ca2+-pump. Zero-Ca2+ buffer augmented responses in 39% cells. Inhibitors for L-type Ca2+channels, Ryanodine Ca2+-pools, PI3K, protein kinase C, SRC-Kinase or TRPC-channels had no effect. UTP (or UTPγS) inhibited IK+, stimulated an increase in voltage-sensitive Ca2+currents and depolarized cells. PLC inhibition blocked depolarization to UTP, whereas PKC inhibition, La3+, thapsigargin or zero Ca2+ buffer had no effect. UTP > ATP in stimulating 5-HT release in BON or isolated human EC cells from surgical-specimens (N=3). WB identified P2Y4R-bands in isolated human EC and BON cells. P2Y4R-ir was found in 5HT+(EC) cells of human mucosa (N=3). Conclusions: UTP activates a P2Y4R to elicit Ca2+ oscillations in most EC cells via PLC/IP3/IP3R/ SERCA Ca2+-pump signaling and SOC channels, and stimulate 5-HT release. Membrane depolarization depends on PLC signaling, IK+ and VOCCs, but is not linked to Ca2+ oscillations. P2Y4 is involved in fine-tune modulation of 5-HT secretion. The P2Y4 signal transduction pathways are worth exploring as a suitable model to evaluate potential changes in Ca2+ or ionic mechanisms in IBD or IBS (NIH DK093499;OSUWMC-funds). Table 1. UTP-induced alterations in MP, K-type IK+ and ICa2+ currents and post receptor signaling
Su2022 Cytokine Modulation of Enteric Neural Stem Cell Proliferation Laren Becker, Aida Habtezion Background: Interleukin 22 (IL-22), a cytokine produced by a variety of hematopoietic cells, has been found to have cytoprotective effects in various tissues including lung, liver, pancreas, and colon. IL-22 signaling occurs through the IL-22 receptor (IL-22R), which is expressed on non-hematopoietic cells. Recently, IL-22R was demonstrated on intestinal stem cells and IL-22 deficiency caused stem cell loss. We evaluated whether IL-22R is expressed on enteric neural stem cells (ENSCs) and whether modulation with IL-22 affects proliferation. Methods: We performed immunofluorescence with antibodies to IL-22R and nestin on longitudinal muscle and myenteric plexus (LMMP) whole mounts and ENSCs cultured as neurospheres. Utilizing Wnt1-cre;tdTomato (tdT) mice which fluorescently marks neural crestderived cells, we performed flow cytometry with an antibody to CD49 (ENSC marker) and subsequently performed RT-PCR analysis on sorted cells. We assessed for IL-22 signaling by Western blot analysis using antibodies to pSTAT-3 and STAT-3 with both in vitro organotypic culturing of LMMP and in vivo injection of mice with rIL-22. Finally, we performed EdU uptake assays using organotypic cultures of LMMP and ENSC cultures in the presence or absence of rIL-22. Results: IL-22R expression was detected in Nestin+ cells in enteric ganglia and cultured neurospheres. Compared to spleen cells that were double negative, LMMP cells that co-expressed tdT (neural crest) and CD49b-APC (ENSC marker) demonstrated over 5-fold higher expression of IL-22R by RT-PCR. These cells also expressed 1,000-fold higher nestin compared to corresponding cells from spleen as would be expected for ENSCs. Organotypic cultures of LMMP incubated with IL-22 demonstrated increase in pSTAT3:STAT3 expression in a dose dependent manner. Similar increase in pSTAT3:STAT3 expression was detected in LMMP 1 h following IP injection of rIL-22 compared to sham. Organotypic cultures of LMMP in the presence EdU with rIL-22 (10 ng/ml) for 40h resulted in increased EdU uptake of neural crest cells (tdT+) compared with control (42% versus 32%). Addition of rIL-22 (10 ng/ml) to cultures of ENSCs resulted in a similar increase in
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AGA Abstracts