- Email: [email protected]
128 Evidence for Two Functionally Distinct Types of Cell Division in the Early Intestinal Epithelium
AGA Abstracts
within 3 months of surgery. There were seven major complications in NI patients (shock (n=1), abscess or other infection (n=5), obstruction (n=1)). There were five major INF group complications; four patients experienced early major complications and one experienced a delayed complication. Early complications included anastomotic leak, tubular necrosis requiring dialysis, bacteremia, and intra-abdominal phlegmon treated with antibiotics. The delayed complication was a laparoscopic lysis of adhesions three years following an open ileocecectomy. There was no significant difference in postoperative length of stay between groups. Conclusions: Infliximab use prior to surgery is not associated with an increased number of complications in our pediatric CD population following a major operation. As this therapy continues to be used for management CD, further large-volume and multicenter studies will be beneficial to understanding the interaction of infliximab with major surgical intervention.
comparison of the SIFT-MS results of the obese group to the lean group revealed differences in concentration of more than 50 compounds. Discriminant analysis via stepwise variable selection of mass scanning ion peak data demonstrated four ion peaks that identified subjects with overweight/obesity with an accuracy of 92% (-2 log likelihood 0.172; Wilks' Lambda 0.145 (p , 0.0001)). Further analysis revealed that breath isoprene, 1-octene, ammonia and hydrogen sulfide were significantly higher in the obese group compared to lean group; p value , 0.005 for all (Table). Conclusion: Obese children have a unique pattern of exhaled VOCs compared to lean controls. Changes in VOCs observed in this study may help to gain insight into pathophysiological processes and pathways leading to the development of childhood obesity and its complications. Future studies are needed to validate our findings. Mean Volatile Organic Compound Levels Adjusted For Age, Height and Race
Table 1: Demographics and Outcome Data
126 Altered Gut Microbial Energy and Metabolism in Children With Non Alcoholic Fatty Liver Disease Sonia Michail, Mark R. Frey, Rob Fanter, Daniel Wai, John G. Cleary, Brian Hilbush Background: Obesity is becoming the new pediatric epidemic of the century, with more children becoming obese than ever before. The gut microbiome is the major metabolic organ and determines how ingested calories are processed for use or waste, serving as a caloric gate. Non-alcoholic fatty liver disease (NAFLD) is frequently seen in obese children, and has become the most common cause of liver disease in older children in the United States. The microbiome has potential to contribute towards the pathogenesis of fatty liver through the production of metabolites that promote deposition of fat in the liver. Objective: The goal of this study is to examine the fecal gut microbial profile in children with NAFLD using combined metagenomic and proteomic approaches. Design/Methods: Human fecal samples were obtained from obese children with fatty liver and healthy lean children. Specimens were subjected to shotgun sequencing detection using Ion Torrent sequencing technology (n=7 NAFLD, 12 healthy). Metaproteomes were sequenced by LC-MS/MS, proteins were identified and quantitated by Proteome Discoverer and Scaffold software, and biological pathways were mapped using the databases of Ingenuity Pathway Analysis and Clusters of Orthologous Groups (n=3 for both groups). Results: Children with fatty liver had increased protein abundance for energy production including NAD-dependent aldolase dehydrogenase (9 versus 0.3, a 27 fold increase) and F0F1-type ATP synthase (2.3 vs undetectable). There was a significant reduction in carbohydrate and amino acid metabolism pathways as well as urea cycle and urea transport systems. For example, urea transporter utp gene had a relative abundance of 20.4 in NAFLD and 96.0 in healthy children (4.7 fold). The urea permease protein B, urtB was 0.003 in NAFLD and 0.929 in healthy children (281 fold difference). The metaproteome and metagenome showed similar findings and confirmed changes in energy, carbohydrate and amino acid metabolism. Conclusions: In summary, the metagenomic and proteomic profiles of the gut microbiome in pediatric NAFLD were found to be distinctly different from those of non-obese children. NAFLD patient profiles showed a shift away from carbohydrate and amino acid metabolism to favor energy production, which may contribute to the development of disease.
SD = standard deviation; Dx = diagnosis; LOS = length of stay 124 Brain Processing of Rectal Sensation in Children With Chronic Functional Constipation Suzanne M. Mugie, Maartje M. van den Berg, Paul Groot, Liesbeth Reneman, Aart J. Nederveen, Marc A. Benninga Background and Hypothesis: The pathophysiologic mechanisms underlying chronic defecation disorders in children without obvious organic etiology are poorly understood. Children with functional constipation (FC) often report loss of defecatory urge sensation. However, a clear relation between lack of sensation and anorectal parameters has never been identified. Functional magnetic resonance imaging studies (fMRI) have been used to unravel the brain processing of visceral sensation in adults with functional gastrointestinal disorders. However, fMRI in combination with rectal distension has never been performed in the pediatric patients with constipation. The hypothesis of this study is that cerebral activity in response to rectal distension in children with chronic constipation is impaired. Patients and Methods: 10 patients between 12-18 years old (6 male, median age 14.5 years), who suffered for at least 2 years from FC defined by the ROME III criteria. All patients underwent colonic transit time measurement and rectal barostat. A stepwise pressure-controlled intermittent distension protocol was performed to determine rectal sensation. The rectal pressure at which urge to defecate was felt was used, during acquisition of blood oxygenation level-dependent (BOLD) fMRI. Subjects received two sessions of 5 stimulations consisting of repetitions of 30 seconds of rectal stimulation followed by 30 seconds of rest. Functional and structural images were acquired on a 3Tesla MRI scanner equipped with an 8-channel SENSE head receive coil. A T2*-weighted echo planar imaging (EPI) sequence was acquired with the following parameters: TR/TE=3000/30 ms, slice thickness = 3.0 mm, voxel size = 1.72 x 1.72 x 3 mm, with 40 axial slices, in ascending mode covering the whole brain. In addition, a structural T1weighted 3D anatomical image of the whole brain was obtained. Whole brain fMRI BOLD signal differences between rectal stimulation and non-stimulation sessions were analyzed using SPM8 implemented in Matlab version 7.14, thresholded at p ,0.01. Results: All patients had a delayed colonic transit time ( . 62 hours, median 100.8 hrs). Sensation of urge to defecate was reported at a median pressure of 28.5 mmHg (range 18-33 mmHg). There were no significant activated brain areas associated with sensation of urge to defecate during rectal distension. Conclusion: Our preliminary results suggest that there is no responsiveness of the central nervous system of the defecatory urge sensation in hyposensitive children with severe functional constipation. In future research it is important to compare these results with a healthy pediatric control group and to specify regions of interest for a more detailed analysis.
127 Kruppel-Like Factor 5 Controls Villus Formation and Initiation of Cytodifferentiation in the Embryonic Intestinal Epithelium Sheila M. Bell, Liqian Zhang, Yan Xu, Valerie Besnard, Susan E. Wert, Noah F. Shroyer, Jeffrey A. Whitsett Kruppel-like factor 5 (Klf5), a transcription factor expressed by the embryonic endoderm, gives rise to epithelium lining the gastrointestinal tract. Shh-Cre;Klf5fl/fl mice were generated in which Klf5 was efficiently deleted from the intestinal epithelium as early as E14.5 resulting in the inhibition of villus morphogenesis and epithelial differentiation. While proliferation of the intestinal epithelium was maintained, loss of KLF5 inhibited the expression of Elf3, Pparγ, Atoh1, Neurog3, Hnf4α, and other genes associated with epithelial cell differentiation and blocked the normal down regulation in expression of FoxA1 and Sox9 that accompanies villus morphogenesis. The failure to form villi was not attributable to the absence of HH or PDGF signaling, known mediators of this developmental process. Genetic network analysis identified KLF5 as a key regulator of genes involved in transcriptional regulation of terminal differentiation and cell-cell adhesion. These studies indicate a novel requirement of KLF5 to initiate morphogenesis of the early endoderm into a compartmentalized intestinal epithelium comprised of villi. KLF5 orchestrates this morphogenetic event in part by repression of FoxA1 expression. Transfection assays demonstrated that KLF5 directly inhibited activity of the FoxA1 promoter and the loss of Elf3 expression was associated with persistent expression of FoxA1 in the Klf5-deficient intestine.
125 Breathprints of Childhood Obesity: Changes in Volatile Organic Compounds in Obese Children and Adolescents Compared to Healthy Controls Naim Alkhouri, Katharine Eng, Frank Cikach, Nishaben Patel, Chen Yan, Allison Brindle, Ellen S. Rome, Ibrahim A. Hanouneh, David Grove, Stanley L. Hazen, Raed A. Dweik Exhaled breath has hundreds of volatile organic compounds (VOCs) that reflect the metabolic condition of an individual. The aim of this study was to investigate changes in VOCs measured by mass spectrometry in overweight/ obese children compared to their lean counterparts. Methods: Overweight and obese children were recruited from the Pediatric Preventive Cardiology and Metabolic Clinic. Healthy controls were recruited from the General Pediatric Clinic during routine well-child visits. Single exhaled breath was collected and analyzed per protocol using selective ion flow tube (SIFT-MS). Results: 60 overweight/ obese children and 55 lean controls were included in the study. Compared to the lean group, the obese group was significantly older (14.1 ± 2.8 vs. 12.1 ± 3.0 years), taller (164.8 ± 10.9 vs. 153.3 ± 17.1 cm), and more likely to be Caucasian (60% vs. 35.2%); p , 0.05 for all. Single exhaled breath testing was feasible and tolerated well by all participants. A
AGA Abstracts
128 Evidence for Two Functionally Distinct Types of Cell Division in the Early Intestinal Epithelium Andrew M. Freddo, Kenichiro Taniguchi, Ann S. Grosse, Deborah L. Gumucio Intestinal villi provide an enormous surface area for nutrient absorption. Intestinal failure occurs when the amount of surface area is compromised such that impaired functionality results. Though short-term treatments for intestinal failure are available, all patients need life-long monitoring for growth and nutritional status, and would benefit from treatments
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AGA Abstracts
increasing intestinal surface area. In mice, a large increase in surface area is initiated with villus development at embryonic day (E)14.5, when the thick pseudostratified epithelium with a flat luminal surface is converted to a columnar epithelium covering a regularly patterned field of emerging villi. Though it has long been thought that epithelial remodeling occurs by formation and fusion of secondary lumina, recent work in our laboratory showed that secondary lumina do not exist (Grosse et al., Development 138:4423, 2011). Seeking an alternative mechanism for luminal expansion, we found that a unique type of cell division occurs only at E14.5, which we have named an e-division (lumen extending division). Functionally and cell biologically, e-divisions are distinct from cell divisions that occur before E14.5, called g-divisions (girth increasing divisions). Midbodies (spindle remnants) are bent in g-divisions and the cytokinetic plane is perpendicular to the lumen. In e-divisions, the midbody is straight and nascent apical surface separates the two daughters; the cytokinetic plane is parallel to the extending lumen. These e-divisions are both temporally and spatially patterned, and this pattern is perturbed in Wnt5a-/- mice, which have notoriously short guts. Our working model is that g-divisions promote proliferation of epithelial progenitors and drive the growth of intestinal girth, while e-divisions function to carve out the villi at E14.5. Mice lacking Ezrin appear to exhibit stochastic failure of the e-division. This is being modeled in an in vitro organoid assay. Further understanding of this process of villus development will improve in vitro bioengineering of intestinal surface, potentially yielding a new avenue for treatment of intestinal failure. 129 Figure 1 GSI treatment reduces proliferation and partially rescues Mtgr1-/- secretory lineage depletion. Quantification of A) PH3 B) Goblet C) Enterendocrine and D) Paneth cells per ileal crypt. One way anova statistical analysis: # WT Control vs WT GSI P ,0.05; Φ WT Control vs Mtgr1-/- Control P,0.05; *** Mtgr1-/- Control vs Mtgr1-/- GSI P,0.001.
Cell Division Cycle 42 Is Essential for Intestinal Crypt Homeostasis and Brush Border Morphogenesis Ryotaro Sakamori, Nan Gao BACKGROUND & AIMS: Malabsorptive and maldigestive diseases are the major causes of morbidity and mortality on a global scale, causing millions of death per year. Abnormal morphogenesis of the intestinal enterocytes can cause severe nutrient deprivation particularly in infants in the first year of life. The mammalian intestinal villus mucosa is continuously replenished by stem cells in the crypts, following a well-staged program composed of transient amplification, polarization, and terminal differentiation. We aimed to investigate the hypothesis that intestinal stem cell defects may contribute to abnormal enterocyte morphogenesis and function that underlie the ontogenesis of microvillus diseases. METHODS: A major regulator of cell polarity, cell division cycle 42 (Cdc42), was inactivated globally in the mouse intestinal epithelium, or specifically in the intestinal stem cell population. The resulting phenotypes were characterized at histological and molecular levels. RESULTS: Intestine-specific Cdc42 inactivation in mice led to severe accumulations of microvillus bodies within the mutant enterocytes. The inner surfaces of these inclusion bodies were lined by typical brush borders that normally appear at apical surfaces of the enterocytes. This phenotype recapitulates the microvillus inclusion human disease counterpart. Cdc42deficient intestinal stem cells underwent defective cell division and polarization, and did not give rise to Paneth cells. The affected mice demonstrated severely reduced epithelial uptake of glucose, amino acids and peptides. CONCLUSIONS: Stem cell division and polarization are tightly coupled to enterocyte development and brush border morphogenesis. Molecular defects targeting the stem cell compartment may contribute to the ontogenesis of microvillus diseases.
131 RNA-Binding Protein HuR Regulates MicroRNA Processing by Altering Drosha Expression in Intestinal Epithelial Cells Ran Zhuang, Rao N. Jaladanki, Tongtong Zou, Lan Xiao, Natasha Z. Hansraj, Myriam Gorospe, Jian-Ying Wang microRNAs (miRNAs) are small noncoding RNAs and regulate gene expression by triggering translational repression or mRNA degradation. miRNAs have emerged as master regulators of GI mucosal growth and are linked to several GI diseases recently. Tissue miRNA levels are tightly regulated and their maturation is controlled by Drosha that is responsible for the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is the first step of miRNA maturation and influences cellular levels of mature miRNAs. However, the exact mechanisms that control Drosha expression remain largely unknown. The RNAbinding protein HuR primarily binds to AU-rich elements located in the 3'-untranslated regions (UTRs) of target transcripts and regulates mRNA stability and translation. Since there are several predicted HuR-hit motifs in the Drosha mRNA, this study determined if HuR regulates miRNA processing by modulating Drosha expression. Methods: Studies were conducted in IEC-6 cells derived from rat intestinal crypts. miRNA Levels were examined by real time-PCR analysis; HuR interaction with the Drosha mRNA was detected by ribonucleoprotein/IP and biotin pull-down assays. HuR function was investigated by HuR silencing and its ectopic overexpression. Drosha translation was measured by polysomal profile analysis and chimeric Drosha 3'-UTR luciferase reporter assays. Results: HuR silencing by transfection with specific siRNA targeting HuR (siHuR) inhibited Drosha expression and repressed maturation of several miRNAs, including miR-195, miR-542, and miR-34c, as indicated by decreased levels of mature miRNAs, along with increased content of primary miRNAs. HuR was found to bind the Drosha mRNA via its 3'-UTR rather than 5'-UTR and coding region. HuR silencing by siHuR reduced [HuR/Drosha mRNA] complex, inhibited Drosha translation, and decreased its protein levels. In contrast, ectopic HuR overexpression enhanced HuR association with the Drosha mRNA and stimulated Drosha translation. Interestingly, HuR silencing also reduced Drosha mRNA levels, whereas HuR overexpression increased Drosha mRNA abundance; neither intervention changed the stability of Drosha mRNA. Further studies showed that increased HuR also enhanced Drosha gene transcription as indicated by an increase in Drosha-promoter activity. This induction in Drosha transcription by HuR resulted predominantly from induced c-Myc transcription factor, since HuR enhanced c-Myc expression and because increased Drosha transcription was prevented by c-Myc silencing in cells overexpressing HuR. Conclusions: These results indicate that 1) HuR regulates miRNA maturation by altering Drosha levels and 2) the mechanism underlying Drosha expression by HuR operates at the levels of transcription and translation.
130 MTGR1 Is Required for Gsi-Induced Paneth Cell Differentiation Bobak Parang, Vishruth K. Reddy, Daniel Rosenblatt, Amanda Williams, Aubrey Hunt, Frank Revetta, Yuan-hung Lo, Kay Washington, Michael Engel, Scott W. Hiebert, Noah F. Shroyer, Christopher S. Williams Background: The specification of cell fate in the intestinal epithelium is determined by Notch signaling. High Notch activity drives progenitors toward enterocyte differentiation while low Notch expression triggers secretory cell differentiation. Myeloid translocation gene related-1 (Mtgr1) is a transcriptional co-repressor in the MTG/ETO family. Mtgr1-/- mice display loss of intestinal epithelial secretory cells and MTG family members interact with Notch family intracellular domains (ICD). We hypothesized that Mtgr1 suppresses Notch signaling and that Notch inhibition could rescue the lineage allocation deficit seen in Mtgr1-/small intestine. Methods: 8-12 week old Wild-type (WT) and Mtgr1-/- mice were treated intraperitoneally with vehicle or γ-secretase inhibitor-20 (GSI-20) at 10 umol/L/kg twice a day for 5 consecutive days. Mice were sacrificed, and the small intestine and thymus were harvested. Thymocyte subsets were determined by flow cytometry. Crypt architecture and secretory cells were analyzed by histology and immunohistochemistry (IHC). Proliferation was measured using anti-phospho-histone H3 (PH3) in IHC. Co-immunoprecipitation was performed by transfecting HA-epitope tagged Mtgr1 and Myc-epitope tagged CSL in COS7L cells. Cell lysates were immunoprecipitated with anti-Myc monoclonal antibody 9E10 and immune complexes were collected on Protein-G sepharose beads. Hes1 promoter activity was assessed by transiently expressing Mtgr1 in NIH3T3 cells with or without N1-ICD coexpression, along with the transcriptional reporter Hes1-Lux. Results: As predicted, GSI treatment decreased thymus weight and cellularity and increased thymic CD4-CD8- cells in both WT and Mtgr1-/- mice. GSI increased Goblet cells per crypt (0.97 ±0.3 to 6.1 ±0.8 P,0.001) and EE cells per crypt (0.035 ±0.02 to 0.36 ±0.06 P ,0.001) in Mtgr1-/- mice. Consistent with Notch-inhibition, GSI also reduced the number of PH3 positive cells per crypt in the Mtgr1-/- mice (2.7 ±0.2 to 1.2 ±0.1 P ,0.001). GSI treatment did not increase Paneth cells, suggesting Mtgr1 is required for the effects of Notch-inhibition on Paneth cells. To determine how Mtgr1 modifies Notch signaling, we tested for Mtgr1 associations with Notch signaling family members. We discovered that Mtgr1 immunopurified with CSL, the DNA-binding partner of Notch intracellular domains. Moreover, Mtgr1 expression suppressed N1-ICD transcriptional activation of the Hes1-promoter. Conclusions: Overall, inhibition of Notch signaling using GSI restored Goblet and EE cells and reduced proliferation in the Mtgr1-/-- mice. GSI did not have an effect on Paneth cells in these mice, suggesting that Mtgr1 is required for the effects of Notch inhibition on Paneth cell differentiation. Mtgr1 interacts with CSL and suppresses N1-ICD transcriptional activation of Hes1.
132 BMP Signaling Promotes Basal Cell Differentiation Through Activation of NRF2 Signaling in the Adult Mouse Esophagus Ming Jiang, Jeffrey H. Peters, Jianwen Que Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells and involves intricate signaling regulatory networks. We previously reported Bone morphogenetic protein (BMP) signaling is important for the epithelial morphogenesis of the embryonic esophagus. However, it remains unclear whether/how this signaling is involved in the maintenance of adult esophagus. Here, we utilize multiple mouse genetic lines to address the issue. We found that BMP signaling is specifically activated in the differentiated suprabasal cells but not in the undifferentiated basal progenitor cells. When treated with Bmp4 basal progenitor cells differentiate towards a squamous cell fate. Consistent with the in vitro assay, ectopic BMP signaling in the esophageal basal cells of the Krt5-CreER; R26RCABMPR1A mice leads to premature differentiation, resulting in epithelial hyperkeratosis. Interestingly, BMP activation induces intracellular oxidative stress and enhances the nuclear accumulation of Nrf2 and its activity. Suppression of Nrf2 by the negative regulator, Keap1 inhibits Bmp4-induced basal cell differentiation. Together, our study demonstrates a novel role of BMP signaling promoting squamous differentiation of basal progenitor cells mediated through activation of intracellular oxidant stress and Nrf2 signaling.
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AGA Abstracts
within 3 months of surgery. There were seven major complications in NI patients (shock (n=1), abscess or other infection (n=5), obstruction (n=1)). There were five major INF group complications; four patients experienced early major complications and one experienced a delayed complication. Early complications included anastomotic leak, tubular necrosis requiring dialysis, bacteremia, and intra-abdominal phlegmon treated with antibiotics. The delayed complication was a laparoscopic lysis of adhesions three years following an open ileocecectomy. There was no significant difference in postoperative length of stay between groups. Conclusions: Infliximab use prior to surgery is not associated with an increased number of complications in our pediatric CD population following a major operation. As this therapy continues to be used for management CD, further large-volume and multicenter studies will be beneficial to understanding the interaction of infliximab with major surgical intervention.
comparison of the SIFT-MS results of the obese group to the lean group revealed differences in concentration of more than 50 compounds. Discriminant analysis via stepwise variable selection of mass scanning ion peak data demonstrated four ion peaks that identified subjects with overweight/obesity with an accuracy of 92% (-2 log likelihood 0.172; Wilks' Lambda 0.145 (p , 0.0001)). Further analysis revealed that breath isoprene, 1-octene, ammonia and hydrogen sulfide were significantly higher in the obese group compared to lean group; p value , 0.005 for all (Table). Conclusion: Obese children have a unique pattern of exhaled VOCs compared to lean controls. Changes in VOCs observed in this study may help to gain insight into pathophysiological processes and pathways leading to the development of childhood obesity and its complications. Future studies are needed to validate our findings. Mean Volatile Organic Compound Levels Adjusted For Age, Height and Race
Table 1: Demographics and Outcome Data
126 Altered Gut Microbial Energy and Metabolism in Children With Non Alcoholic Fatty Liver Disease Sonia Michail, Mark R. Frey, Rob Fanter, Daniel Wai, John G. Cleary, Brian Hilbush Background: Obesity is becoming the new pediatric epidemic of the century, with more children becoming obese than ever before. The gut microbiome is the major metabolic organ and determines how ingested calories are processed for use or waste, serving as a caloric gate. Non-alcoholic fatty liver disease (NAFLD) is frequently seen in obese children, and has become the most common cause of liver disease in older children in the United States. The microbiome has potential to contribute towards the pathogenesis of fatty liver through the production of metabolites that promote deposition of fat in the liver. Objective: The goal of this study is to examine the fecal gut microbial profile in children with NAFLD using combined metagenomic and proteomic approaches. Design/Methods: Human fecal samples were obtained from obese children with fatty liver and healthy lean children. Specimens were subjected to shotgun sequencing detection using Ion Torrent sequencing technology (n=7 NAFLD, 12 healthy). Metaproteomes were sequenced by LC-MS/MS, proteins were identified and quantitated by Proteome Discoverer and Scaffold software, and biological pathways were mapped using the databases of Ingenuity Pathway Analysis and Clusters of Orthologous Groups (n=3 for both groups). Results: Children with fatty liver had increased protein abundance for energy production including NAD-dependent aldolase dehydrogenase (9 versus 0.3, a 27 fold increase) and F0F1-type ATP synthase (2.3 vs undetectable). There was a significant reduction in carbohydrate and amino acid metabolism pathways as well as urea cycle and urea transport systems. For example, urea transporter utp gene had a relative abundance of 20.4 in NAFLD and 96.0 in healthy children (4.7 fold). The urea permease protein B, urtB was 0.003 in NAFLD and 0.929 in healthy children (281 fold difference). The metaproteome and metagenome showed similar findings and confirmed changes in energy, carbohydrate and amino acid metabolism. Conclusions: In summary, the metagenomic and proteomic profiles of the gut microbiome in pediatric NAFLD were found to be distinctly different from those of non-obese children. NAFLD patient profiles showed a shift away from carbohydrate and amino acid metabolism to favor energy production, which may contribute to the development of disease.
SD = standard deviation; Dx = diagnosis; LOS = length of stay 124 Brain Processing of Rectal Sensation in Children With Chronic Functional Constipation Suzanne M. Mugie, Maartje M. van den Berg, Paul Groot, Liesbeth Reneman, Aart J. Nederveen, Marc A. Benninga Background and Hypothesis: The pathophysiologic mechanisms underlying chronic defecation disorders in children without obvious organic etiology are poorly understood. Children with functional constipation (FC) often report loss of defecatory urge sensation. However, a clear relation between lack of sensation and anorectal parameters has never been identified. Functional magnetic resonance imaging studies (fMRI) have been used to unravel the brain processing of visceral sensation in adults with functional gastrointestinal disorders. However, fMRI in combination with rectal distension has never been performed in the pediatric patients with constipation. The hypothesis of this study is that cerebral activity in response to rectal distension in children with chronic constipation is impaired. Patients and Methods: 10 patients between 12-18 years old (6 male, median age 14.5 years), who suffered for at least 2 years from FC defined by the ROME III criteria. All patients underwent colonic transit time measurement and rectal barostat. A stepwise pressure-controlled intermittent distension protocol was performed to determine rectal sensation. The rectal pressure at which urge to defecate was felt was used, during acquisition of blood oxygenation level-dependent (BOLD) fMRI. Subjects received two sessions of 5 stimulations consisting of repetitions of 30 seconds of rectal stimulation followed by 30 seconds of rest. Functional and structural images were acquired on a 3Tesla MRI scanner equipped with an 8-channel SENSE head receive coil. A T2*-weighted echo planar imaging (EPI) sequence was acquired with the following parameters: TR/TE=3000/30 ms, slice thickness = 3.0 mm, voxel size = 1.72 x 1.72 x 3 mm, with 40 axial slices, in ascending mode covering the whole brain. In addition, a structural T1weighted 3D anatomical image of the whole brain was obtained. Whole brain fMRI BOLD signal differences between rectal stimulation and non-stimulation sessions were analyzed using SPM8 implemented in Matlab version 7.14, thresholded at p ,0.01. Results: All patients had a delayed colonic transit time ( . 62 hours, median 100.8 hrs). Sensation of urge to defecate was reported at a median pressure of 28.5 mmHg (range 18-33 mmHg). There were no significant activated brain areas associated with sensation of urge to defecate during rectal distension. Conclusion: Our preliminary results suggest that there is no responsiveness of the central nervous system of the defecatory urge sensation in hyposensitive children with severe functional constipation. In future research it is important to compare these results with a healthy pediatric control group and to specify regions of interest for a more detailed analysis.
127 Kruppel-Like Factor 5 Controls Villus Formation and Initiation of Cytodifferentiation in the Embryonic Intestinal Epithelium Sheila M. Bell, Liqian Zhang, Yan Xu, Valerie Besnard, Susan E. Wert, Noah F. Shroyer, Jeffrey A. Whitsett Kruppel-like factor 5 (Klf5), a transcription factor expressed by the embryonic endoderm, gives rise to epithelium lining the gastrointestinal tract. Shh-Cre;Klf5fl/fl mice were generated in which Klf5 was efficiently deleted from the intestinal epithelium as early as E14.5 resulting in the inhibition of villus morphogenesis and epithelial differentiation. While proliferation of the intestinal epithelium was maintained, loss of KLF5 inhibited the expression of Elf3, Pparγ, Atoh1, Neurog3, Hnf4α, and other genes associated with epithelial cell differentiation and blocked the normal down regulation in expression of FoxA1 and Sox9 that accompanies villus morphogenesis. The failure to form villi was not attributable to the absence of HH or PDGF signaling, known mediators of this developmental process. Genetic network analysis identified KLF5 as a key regulator of genes involved in transcriptional regulation of terminal differentiation and cell-cell adhesion. These studies indicate a novel requirement of KLF5 to initiate morphogenesis of the early endoderm into a compartmentalized intestinal epithelium comprised of villi. KLF5 orchestrates this morphogenetic event in part by repression of FoxA1 expression. Transfection assays demonstrated that KLF5 directly inhibited activity of the FoxA1 promoter and the loss of Elf3 expression was associated with persistent expression of FoxA1 in the Klf5-deficient intestine.
125 Breathprints of Childhood Obesity: Changes in Volatile Organic Compounds in Obese Children and Adolescents Compared to Healthy Controls Naim Alkhouri, Katharine Eng, Frank Cikach, Nishaben Patel, Chen Yan, Allison Brindle, Ellen S. Rome, Ibrahim A. Hanouneh, David Grove, Stanley L. Hazen, Raed A. Dweik Exhaled breath has hundreds of volatile organic compounds (VOCs) that reflect the metabolic condition of an individual. The aim of this study was to investigate changes in VOCs measured by mass spectrometry in overweight/ obese children compared to their lean counterparts. Methods: Overweight and obese children were recruited from the Pediatric Preventive Cardiology and Metabolic Clinic. Healthy controls were recruited from the General Pediatric Clinic during routine well-child visits. Single exhaled breath was collected and analyzed per protocol using selective ion flow tube (SIFT-MS). Results: 60 overweight/ obese children and 55 lean controls were included in the study. Compared to the lean group, the obese group was significantly older (14.1 ± 2.8 vs. 12.1 ± 3.0 years), taller (164.8 ± 10.9 vs. 153.3 ± 17.1 cm), and more likely to be Caucasian (60% vs. 35.2%); p , 0.05 for all. Single exhaled breath testing was feasible and tolerated well by all participants. A
AGA Abstracts
128 Evidence for Two Functionally Distinct Types of Cell Division in the Early Intestinal Epithelium Andrew M. Freddo, Kenichiro Taniguchi, Ann S. Grosse, Deborah L. Gumucio Intestinal villi provide an enormous surface area for nutrient absorption. Intestinal failure occurs when the amount of surface area is compromised such that impaired functionality results. Though short-term treatments for intestinal failure are available, all patients need life-long monitoring for growth and nutritional status, and would benefit from treatments
S-30
AGA Abstracts
increasing intestinal surface area. In mice, a large increase in surface area is initiated with villus development at embryonic day (E)14.5, when the thick pseudostratified epithelium with a flat luminal surface is converted to a columnar epithelium covering a regularly patterned field of emerging villi. Though it has long been thought that epithelial remodeling occurs by formation and fusion of secondary lumina, recent work in our laboratory showed that secondary lumina do not exist (Grosse et al., Development 138:4423, 2011). Seeking an alternative mechanism for luminal expansion, we found that a unique type of cell division occurs only at E14.5, which we have named an e-division (lumen extending division). Functionally and cell biologically, e-divisions are distinct from cell divisions that occur before E14.5, called g-divisions (girth increasing divisions). Midbodies (spindle remnants) are bent in g-divisions and the cytokinetic plane is perpendicular to the lumen. In e-divisions, the midbody is straight and nascent apical surface separates the two daughters; the cytokinetic plane is parallel to the extending lumen. These e-divisions are both temporally and spatially patterned, and this pattern is perturbed in Wnt5a-/- mice, which have notoriously short guts. Our working model is that g-divisions promote proliferation of epithelial progenitors and drive the growth of intestinal girth, while e-divisions function to carve out the villi at E14.5. Mice lacking Ezrin appear to exhibit stochastic failure of the e-division. This is being modeled in an in vitro organoid assay. Further understanding of this process of villus development will improve in vitro bioengineering of intestinal surface, potentially yielding a new avenue for treatment of intestinal failure. 129 Figure 1 GSI treatment reduces proliferation and partially rescues Mtgr1-/- secretory lineage depletion. Quantification of A) PH3 B) Goblet C) Enterendocrine and D) Paneth cells per ileal crypt. One way anova statistical analysis: # WT Control vs WT GSI P ,0.05; Φ WT Control vs Mtgr1-/- Control P,0.05; *** Mtgr1-/- Control vs Mtgr1-/- GSI P,0.001.
Cell Division Cycle 42 Is Essential for Intestinal Crypt Homeostasis and Brush Border Morphogenesis Ryotaro Sakamori, Nan Gao BACKGROUND & AIMS: Malabsorptive and maldigestive diseases are the major causes of morbidity and mortality on a global scale, causing millions of death per year. Abnormal morphogenesis of the intestinal enterocytes can cause severe nutrient deprivation particularly in infants in the first year of life. The mammalian intestinal villus mucosa is continuously replenished by stem cells in the crypts, following a well-staged program composed of transient amplification, polarization, and terminal differentiation. We aimed to investigate the hypothesis that intestinal stem cell defects may contribute to abnormal enterocyte morphogenesis and function that underlie the ontogenesis of microvillus diseases. METHODS: A major regulator of cell polarity, cell division cycle 42 (Cdc42), was inactivated globally in the mouse intestinal epithelium, or specifically in the intestinal stem cell population. The resulting phenotypes were characterized at histological and molecular levels. RESULTS: Intestine-specific Cdc42 inactivation in mice led to severe accumulations of microvillus bodies within the mutant enterocytes. The inner surfaces of these inclusion bodies were lined by typical brush borders that normally appear at apical surfaces of the enterocytes. This phenotype recapitulates the microvillus inclusion human disease counterpart. Cdc42deficient intestinal stem cells underwent defective cell division and polarization, and did not give rise to Paneth cells. The affected mice demonstrated severely reduced epithelial uptake of glucose, amino acids and peptides. CONCLUSIONS: Stem cell division and polarization are tightly coupled to enterocyte development and brush border morphogenesis. Molecular defects targeting the stem cell compartment may contribute to the ontogenesis of microvillus diseases.
131 RNA-Binding Protein HuR Regulates MicroRNA Processing by Altering Drosha Expression in Intestinal Epithelial Cells Ran Zhuang, Rao N. Jaladanki, Tongtong Zou, Lan Xiao, Natasha Z. Hansraj, Myriam Gorospe, Jian-Ying Wang microRNAs (miRNAs) are small noncoding RNAs and regulate gene expression by triggering translational repression or mRNA degradation. miRNAs have emerged as master regulators of GI mucosal growth and are linked to several GI diseases recently. Tissue miRNA levels are tightly regulated and their maturation is controlled by Drosha that is responsible for the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is the first step of miRNA maturation and influences cellular levels of mature miRNAs. However, the exact mechanisms that control Drosha expression remain largely unknown. The RNAbinding protein HuR primarily binds to AU-rich elements located in the 3'-untranslated regions (UTRs) of target transcripts and regulates mRNA stability and translation. Since there are several predicted HuR-hit motifs in the Drosha mRNA, this study determined if HuR regulates miRNA processing by modulating Drosha expression. Methods: Studies were conducted in IEC-6 cells derived from rat intestinal crypts. miRNA Levels were examined by real time-PCR analysis; HuR interaction with the Drosha mRNA was detected by ribonucleoprotein/IP and biotin pull-down assays. HuR function was investigated by HuR silencing and its ectopic overexpression. Drosha translation was measured by polysomal profile analysis and chimeric Drosha 3'-UTR luciferase reporter assays. Results: HuR silencing by transfection with specific siRNA targeting HuR (siHuR) inhibited Drosha expression and repressed maturation of several miRNAs, including miR-195, miR-542, and miR-34c, as indicated by decreased levels of mature miRNAs, along with increased content of primary miRNAs. HuR was found to bind the Drosha mRNA via its 3'-UTR rather than 5'-UTR and coding region. HuR silencing by siHuR reduced [HuR/Drosha mRNA] complex, inhibited Drosha translation, and decreased its protein levels. In contrast, ectopic HuR overexpression enhanced HuR association with the Drosha mRNA and stimulated Drosha translation. Interestingly, HuR silencing also reduced Drosha mRNA levels, whereas HuR overexpression increased Drosha mRNA abundance; neither intervention changed the stability of Drosha mRNA. Further studies showed that increased HuR also enhanced Drosha gene transcription as indicated by an increase in Drosha-promoter activity. This induction in Drosha transcription by HuR resulted predominantly from induced c-Myc transcription factor, since HuR enhanced c-Myc expression and because increased Drosha transcription was prevented by c-Myc silencing in cells overexpressing HuR. Conclusions: These results indicate that 1) HuR regulates miRNA maturation by altering Drosha levels and 2) the mechanism underlying Drosha expression by HuR operates at the levels of transcription and translation.
130 MTGR1 Is Required for Gsi-Induced Paneth Cell Differentiation Bobak Parang, Vishruth K. Reddy, Daniel Rosenblatt, Amanda Williams, Aubrey Hunt, Frank Revetta, Yuan-hung Lo, Kay Washington, Michael Engel, Scott W. Hiebert, Noah F. Shroyer, Christopher S. Williams Background: The specification of cell fate in the intestinal epithelium is determined by Notch signaling. High Notch activity drives progenitors toward enterocyte differentiation while low Notch expression triggers secretory cell differentiation. Myeloid translocation gene related-1 (Mtgr1) is a transcriptional co-repressor in the MTG/ETO family. Mtgr1-/- mice display loss of intestinal epithelial secretory cells and MTG family members interact with Notch family intracellular domains (ICD). We hypothesized that Mtgr1 suppresses Notch signaling and that Notch inhibition could rescue the lineage allocation deficit seen in Mtgr1-/small intestine. Methods: 8-12 week old Wild-type (WT) and Mtgr1-/- mice were treated intraperitoneally with vehicle or γ-secretase inhibitor-20 (GSI-20) at 10 umol/L/kg twice a day for 5 consecutive days. Mice were sacrificed, and the small intestine and thymus were harvested. Thymocyte subsets were determined by flow cytometry. Crypt architecture and secretory cells were analyzed by histology and immunohistochemistry (IHC). Proliferation was measured using anti-phospho-histone H3 (PH3) in IHC. Co-immunoprecipitation was performed by transfecting HA-epitope tagged Mtgr1 and Myc-epitope tagged CSL in COS7L cells. Cell lysates were immunoprecipitated with anti-Myc monoclonal antibody 9E10 and immune complexes were collected on Protein-G sepharose beads. Hes1 promoter activity was assessed by transiently expressing Mtgr1 in NIH3T3 cells with or without N1-ICD coexpression, along with the transcriptional reporter Hes1-Lux. Results: As predicted, GSI treatment decreased thymus weight and cellularity and increased thymic CD4-CD8- cells in both WT and Mtgr1-/- mice. GSI increased Goblet cells per crypt (0.97 ±0.3 to 6.1 ±0.8 P,0.001) and EE cells per crypt (0.035 ±0.02 to 0.36 ±0.06 P ,0.001) in Mtgr1-/- mice. Consistent with Notch-inhibition, GSI also reduced the number of PH3 positive cells per crypt in the Mtgr1-/- mice (2.7 ±0.2 to 1.2 ±0.1 P ,0.001). GSI treatment did not increase Paneth cells, suggesting Mtgr1 is required for the effects of Notch-inhibition on Paneth cells. To determine how Mtgr1 modifies Notch signaling, we tested for Mtgr1 associations with Notch signaling family members. We discovered that Mtgr1 immunopurified with CSL, the DNA-binding partner of Notch intracellular domains. Moreover, Mtgr1 expression suppressed N1-ICD transcriptional activation of the Hes1-promoter. Conclusions: Overall, inhibition of Notch signaling using GSI restored Goblet and EE cells and reduced proliferation in the Mtgr1-/-- mice. GSI did not have an effect on Paneth cells in these mice, suggesting that Mtgr1 is required for the effects of Notch inhibition on Paneth cell differentiation. Mtgr1 interacts with CSL and suppresses N1-ICD transcriptional activation of Hes1.
132 BMP Signaling Promotes Basal Cell Differentiation Through Activation of NRF2 Signaling in the Adult Mouse Esophagus Ming Jiang, Jeffrey H. Peters, Jianwen Que Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells and involves intricate signaling regulatory networks. We previously reported Bone morphogenetic protein (BMP) signaling is important for the epithelial morphogenesis of the embryonic esophagus. However, it remains unclear whether/how this signaling is involved in the maintenance of adult esophagus. Here, we utilize multiple mouse genetic lines to address the issue. We found that BMP signaling is specifically activated in the differentiated suprabasal cells but not in the undifferentiated basal progenitor cells. When treated with Bmp4 basal progenitor cells differentiate towards a squamous cell fate. Consistent with the in vitro assay, ectopic BMP signaling in the esophageal basal cells of the Krt5-CreER; R26RCABMPR1A mice leads to premature differentiation, resulting in epithelial hyperkeratosis. Interestingly, BMP activation induces intracellular oxidative stress and enhances the nuclear accumulation of Nrf2 and its activity. Suppression of Nrf2 by the negative regulator, Keap1 inhibits Bmp4-induced basal cell differentiation. Together, our study demonstrates a novel role of BMP signaling promoting squamous differentiation of basal progenitor cells mediated through activation of intracellular oxidant stress and Nrf2 signaling.
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AGA Abstracts