- Email: [email protected]
869 Up-Regulation of Delta1 Expression is Required for Goblet Cell Differentiation in Human Intestinal Epithelial Cells
866
ring at the pylorus. A day later, bilateral cords of stained cells appear along the lesser curvature of the stomach, and by E18.5, these structures have extended anteriorly to the level of the esophagus. These structures are superficial and may represent tendons or nervous tissue. In sectioned tissue, we confirmed that staining is mesenchymal and located exterior to the inner circular muscle of the pylorus. The pattern of Gata3 expression at the pylorus is similar to that of the homeodomain transcription factor Nkx2-5. Thus, we obtained Nkx25(lacZ/+) reporter mice and analyzed Nkx2-5 expression by whole-mount X-gal staining of dissected foregut. The staining pattern was nearly identical to Gata3, with a few notable exceptions: pyloric expression is first observed at E9.5, the ring at the pylorus is complete, and expression of Nkx2-5 (but not Gata3) is detected in the spleen beginning at E12.5. Finally, to investigate a potential role for Gata3 in morphogenesis of the pylorus, we analyzed Gata3-null [Gata3(lacZ/lacZ)] mice at E18.5. The shape of the pylorus was abnormal in Gata3-deficient embryos, causing the antrum and duodenum to intersect at an obtuse angle that was apparent in both the gross specimen and in histological sections. Also, the diameter of the pyloric lumen was larger in Gata3-null than wild type embryos. These data establish for the first time that Gata3 is essential for morphogenesis of the pylorus and raise the possibility that it functionally interacts with Nkx2-5.
AGA Abstracts
Aberrant Intestinal Crypt Morphology and Cell Positioning in Mice With Intestine-Specific Deletion of the KrüPpel-Like Factor 5 Gene Beth B. McConnell, Samuel S. Kim, Ryozo Nagai, Vincent W. Yang BACKGROUND: Krüppel-like factor 5 (KLF5) is a pro-proliferative transcription factor that is highly expressed in dividing epithelial cells at the crypt base of the intestinal epithelium. KLF5 promotes proliferation In Vitro and In Vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in maintaining intestinal homeostasis, we characterized the morphology of mice with conditional deletion of Klf5 in the gut using the villin-Cre (VilCre) recombinase system. METHODS: Mice carrying floxed alleles of Klf5 (Klf5fl/fl) were crossed with Vil-Cre;Klf5fl/+ mice to generate Vil-Cre;Klf5fl/fl mice. Klf5fl/fl littermates served as controls. Mice were sacrificed at 8 weeks of age and the small intestines and colon removed for histological examination. Immunohistochemical staining was used to examine the expression of Klf5, the proliferation marker, Ki67 and various markers of differentiation. RESULTS: Immunohistochemical staining of Klf5 in the small and large bowels of the VilCre;Klf5fl/fl mice showed that deletion of the floxed Klf5 alleles was variable, with the most complete deletion occurring in the distal small intestine and proximal colon. Initial characterization of the mice indicated that the Vil-Cre;Klf5fl/fl mice were leaner than the Klf5fl/fl littermates, weighing 18% less than gender- and age-matched controls (n=5, P = 0.01). Histological staining of intestinal and colonic tissues from Vil-Cre;Klf5fl/fl mice revealed a disruption of normal crypt architecture and a regenerative phenotype, particularly in the colon. Staining with the proliferation marker, Ki67, showed proliferating cells scattered throughout the epithelia of the small and large intestines, with a phenotype similar to that of EphB2/EphB3-null mice. Paneth cells likewise migrated upward from the crypt base. Immunohistochemical staining for EphB2 and EphB3 in Klf5-deleted tissues revealed reduced expression of both receptors and loss of a normal expression gradient within the crypts. Reduction of EphB2 and EphB3 correlated with reduced expression of the differentiation markers intestinal alkaline phosphatase and carbonic anhydrase I. CONCLUSION: Deletion of Klf5 in the gut epithelium results in dramatic changes in morphology that correlate with reduced expression of Wnt targets, EphB2 and EphB3. The boundary between proliferating and differentiation zones are lost, resulting in scattering of proliferating cells throughout the epithelium and reduced differentiation. Thus, KLF5 is critical for maintenance of normal crypt architecture in the intestinal mucosa due to its effects on epithelial cell positioning and differentiation.
868 E-Cadherin is Essential for Intestinal Epithelial Morphogenesis in Mice Benjamin J. Bondow, Mary L. Faber, Michele A. Battle Background & Aims: E-cadherin is the primary cadherin adhesion protein expressed in the intestinal epithelium. Several studies in cell culture systems have implicated E-cadherin as required for cellular junction formation, regulation of cellular polarity, cellular migration, and proliferation. Both over-expression of E-cadherin protein and disruption of E-cadherin mediated adhesion through expression of a dominant-negative N-cadherin protein lacking a functional extracellular domain demonstrated a key role for E-cadherin in the maintenance of normal intestinal epithelial homeostasis. These studies led us to propose that loss of Ecadherin itself from the developing mouse intestinal epithelium would result in disruption of intestinal epithelial morphogenesis. Methods: Because E-cadherin null mouse embryos die at 3.5-4.5 days post-coitus due to failure of trophectoderm epithelium formation, we employed a conditional knockout approach to eliminate E-cadherin specifically in the intestinal epithelium during embryonic development. To generate control and experimental animals, we crossed mice homozygous for the E-cadherin conditional allele (Cdh1loxP/loxP) with heterozygous Cdh1loxP/+Villin-Cre animals. Results: We found that E-cadherin conditional knockout mice failed to survive to weaning age of 3 weeks. Of 98 weanlings, only one animal with the Cdh1loxP/loxPVillin-Cre genotype survived, and immunohistochemical staining for Ecadherin protein showed normal levels of E-cadherin in its intestine reflecting inefficiency of Cre recombinase in this animal. E-cadherin conditional knockout mice die perinatally within the first 24 hours of birth. Analysis of intestinal tissue sections harvested from control and E-cadherin conditional knockout embryos at 18.5 days post-coitus show that loss of E-cadherin severely disrupted intestinal morphogenesis. We observed significant changes in epithelial cell shape as well as marked blunting of the villi. Consistent with a role for Ecadherin in barrier morphogenesis, the presence of blood cells in the intestinal lumen of mutant animals suggests compromised integrity of the intestinal epithelium. Using RT-PCR, we found that the Snail transcript was up-regulated in mutant intestinal tissue compared with control tissue suggesting that E-cadherin null intestinal epithelial cells are undergoing increased epithelial to mesenchymal transition (EMT). Conclusions: We conclude that intestinal E-cadherin expression is required for the formation of a functional intestinal epithelium in mice.
867 An Alternative Splicing/Translation Variant Fine-Tunes the Activity of the Homeotic Transcription Factor CDx2 in the Gut Marie Vanier, Elisabeth Martin, Claire Domon-Dell, Isabelle Gross, Isabelle Duluc, JeanNoel Freund The homeobox gene Cdx2 encodes a transcription factor that plays multiple developmental functions including the specification of intestinal identity. At the adult stage, it is involved in the homeostasis of the intestinal epithelium. We have identified by Northern blot and/ or RT-PCR a splicing variant of Cdx2 expressed in the human and mouse intestine and in human intestinal cell lines cultured In Vitro. Sequencing revealed that this splicing variant encodes a truncated protein, miniCDX2, in which the 181-amino acid Nter-region of CDX2 responsible for its transcriptional activity is replaced by a short 12-amino acid peptide. Translation of miniCDX2 uses an alternative start site compared to CDX2. A specific antibody raised against miniCDX2 showed that miniCDX2 is nuclear, and expressed at a higher level in proliferative than in differentiated colon cancer cells cultured In Vitro, in contrast to CDX2. In the mouse colon, miniCDX2 accumulates in cells located at the crypt bottom, whereas CDX2 is expressed in all epithelial cells. Luciferase assays conducted with promoter targets of CDX2 revealed a dominant-negative effect exerted by miniCDX2 over the DNAbinding dependent function of CDX2. Indeed, miniCDX2 competes with CDX2 for its DNA-binding sites, as shown by chromatin immuno-precipitation. Beside its DNA-binding dependent effect, CDX2 also exerts non-DNA-binding dependent functions including the inhibition of the Wnt pathway and the inhibition of the Notch pathway. Unlike CDX2, miniCDX2 has no effect on the Wnt pathway, but like CDX2 it inhibits the Notch pathway. Co-immunoprecipitation experiments showed that the inhibition of the Wnt pathway by CDX2 and of the Notch pathway by CDX2 and by miniCDX2 correlates with protein-protein interactions, respectively with β-catenin and NICD. In Vivo, overexpressing miniCDX2 in the anterior gut of transgenic mice perturbs the area encompassing the Brunner's glands. In conclusion, in addition to the transcriptional and post-translational levels of regulation of CDX2 that have already been documented, we showed here that the function of CDX2 is also regulated at the post-transcriptional level in the gut epithelium by a splicing / translation variant.
869 Up−Regulation of Delta1 Expression is Required for Goblet Cell Differentiation in Human Intestinal Epithelial Cells Junko Akiyama, Ryuichi Okamoto, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe Background & Aims:We have previously shown that forced activation of the Notch signaling pathway inhibits goblet cell differentiation, whereas forced inactivation of the same pathway conversely promotes goblet cell differentiation of human intestinal epithelial cells(IECs). Studies have shown that activation of the Notch signaling pathway In Vivo is initiated by its ligands, Delta(DLL1, DLL3, and DLL4) and Jagged(JAG1 and JAG2). However, precise role of Notch ligands upon goblet cell differentiation in the human intestine remain mostly unknown. Methods:Expression of Notch ligands in biopsy specimens or in human IECderived cell lines was analyzed by RT-PCR. Expression of Notch ligand within the human intestinal tissue was further examined by immunohistochemistry. Analysis of Notch ligand expression upon forced activation or inhibition of the Notch-Hes1 pathway in human IECderived cell lines was performed by quantitative RT-PCR. Tetracycline-dependent expression system was used for over-expression of activated Notch1(NICD1) or Hes1, whereas a γsecretase inhibitor(GSI) was used to inhibit endogenous activation of Notch in IECs. siRNAmediated gene silencing was used to inhibit expression of DLL1 in IECs. Results:mRNA expression of DLL1, DLL4, JAG1 and JAG2 was detected both in human intestinal tissue specimens and in various human IEC-derived cell lines. Immunostaining of a human colonic tissue revealed that Delta ligands are expressed in predominant population of goblet cells. Forced expression of NICD1 or Hes1 significantly down-regulated both DLL1 and DLL4 expression, which leaded to significant suppression of goblet cell differentiation in LS174T cells. Conversely, inhibition of endogenous Notch activation by GSI treatment significantly down-regulated Hes1 expression, but up-regulated both DLL1 and DLL4 expression, resulting in significant promotion of goblet cell differentiation, in LS174T and HT29 cells. In contrast, such a response to intracellular Notch activity was not observed in expression of JAG1 or JAG2. Differentiation towards goblet cells appeared to be dependent not only upon Notch inactivation but also upon up-regulation of DLL1 expression, as siRNA-mediated reduction of DLL1 expression completely abrogated the promotion of goblet cell differentiation by GSI treatment, in LS174T cells. Conclusion:Up-regulation of DLL1 expression is required for goblet cell differentiation, in addition to inactivation of Notch signaling in human IECs. Present results suggest that DLL1 might not only act as an extra-cellular ligand for Notch,
867a GATA3 is Essential for Proper Morphogenesis of the Pylorus Aaron M. Udager, Ajay Prakash, James D. Engel, Kim-Chew Lim, Deborah L. Gumucio The pylorus plays an important role in gastrointestinal function by regulating the passage of food between the stomach and small intestine. Malfunction or malformation of the pylorus can lead to infantile hypertrophic pyloric stenosis (IHPS). However, despite its important role in digestion, little is known about pyloric development. Tissues that constitute the adult pylorus include: epithelium (gastric and intestinal types), and mesenchyme (muscle, vasculature, nerves), but how these cell populations interact to generate proper pyloric structure and function is unclear. Thus, understanding the mechanisms involved in pylorus morphogenesis could provide new therapeutic insights into pyloric pathologies, including IHPS. In a recent study (Li and Udager et al., 2009), we identified Gata3, a zinc-finger transcription factor, as a novel pyloric gene, with mesenchymal expression at embryonic day (E) 14.5 in the mouse. To further investigate this pattern during embryogenesis, we obtained Gata3(lacZ/+) reporter mice and analyzed Gata3 expression by whole-mount Xgal staining of dissected foregut. Beginning at E12.5, staining is detected in an incomplete
AGA Abstracts
S-120
were enrolled into the younger group and 19 (mean age 68.9 yrs, M/F 11/8) into the older group. Older patients had a significantly higher % total as well as recumbent time pH<4 than younger patients (14.7 vs. 8.6 and 6.1 vs. 4.5, respectively, P<0.05). Both groups had a similar total number of awakenings during sleep (52), but the older group had significantly more mean awakenings per patient that were associated with acid reflux events as compared to the younger group (2.38 vs. 1.92, P<0.05). The older group also had a significantly higher total number of acid reflux events during awakenings as compared to the younger group (121 vs. 95, P<0.05). Mean duration of an acid reflux event during awakening was significantly higher in the older group as compared to the younger group (75.9 sec vs. 51.2 sec, P<0.05). However, younger patients had a significantly greater percentage of awakenings with acid reflux that were associated with symptoms as compared to the older group (47.8% vs. 19%, P<0.05). Conclusions: Older patients demonstrated significantly greater acid reflux during the sleep period as compared to younger patients with GERD. However, asymptomatic awakenings from sleep due to acid reflux are significantly more common in older versus younger patients.
870 Cooperative Effect of Hnf-1α With CDX-2 and GATA-4 Triggers Initiation of the Enterocytic Differentiation Program in Human Normal Progenitor Cells Yannick D. Benoit, Manon Lepage, Eric Tremblay, Fabrice Escaffit, Jean-Francois Beaulieu In the intestinal epithelium, the Cdx, GATA and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx-2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx-2, GATA-4 and HNF-1α using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx-2 or HNF-1α, alone and in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1α and GATA-4 together induced morphological modifications of the cells towards polarization resulting in the appearance of functional features such as microvilli. HNF-1α was also sufficient to induce the expression of cadherins and dipeptidylpeptidase while in combination with Cdx-2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large scale analysis of gene expression confirmed the cooperative effects of these factors. Finally, while DCAMKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi-1 and Lgr5, were unaffected. These observations show that, cooperatively with Cdx2, HNF-1α acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 initiates the establishment of cell polarity.
873 Mechanical Properties of the Esophageal Body in Patients With Eosinophilic Esophagitis (EoE): A Pilot Study Using the Functional Luminal Imaging Probe (Endoflip®) Monika A. Kwiatek, Daniel Luger, Ikuo Hirano, Peter J. Kahrilas, John E. Pandolfino Background and Aim: Although the dominant symptoms of EoE are related to mechanical abnormalities of the esophagus, there are no studies that have assessed compliance of the esophageal wall in EoE. The aim of the current study was to analyze the mechanical properties of the esophageal body in EoE using the functional luminal imaging probe (EndoFLIP®, Crospon Medical Devices, Galway, Ireland). Methods: 14 patients (4F, 23 - 67 yr) with EoE were included (normal n = 1, furrows n = 3, rings n = 4, rings with focal stricture n = 6). Subjects were evaluated during endoscopy with the EndoFLIP® probe spanning 3-11 cm above the squamocolumnar junction. The probe comprised an infinitely compliant cylindrical bag (maximal diameter 25 mm) mounted on a 3 mm catheter with 16 impedance planimetry segments. Stepwise bag distensions from 2 to 40 ml were conducted and the associated intrabag pressure and intraluminal geometry were analyzed. Intrabag pressure and smallest diameter at the impedance planimetry segment in the esophagus in the absence of peristalsis were plotted to assess compliance. Results: The EndoFLIP® clearly displayed the tubular esophageal lumen geometry and was able to localize strictures (Figure 1). Stepwise distension increased intrabag pressure eliciting a progressive esophageal opening that eventually reached a plateau value substantially lower than the upper limit of the bag diameter despite an escalation of pressure (Figure 2). Distal esophageal compliance was similar in EoE patients with a normal endoscopy or furrows, but diminished in the presence of rings (p < 0.01). There was a further decrease in compliance in patients with a focal stricture (p < 0.001). Conclusion: The current pilot data support that the EndoFLIP® can be utilized to assess intraluminal esophageal geometry and mechanics in patients with EoE. Compliance levels in EoE patients were below the compliance limit of the bag, particularly in patients with rings and strictures documented endoscopically.
871 Eosinophilic Esophagitis (EoE)-Associated Eosinophil Products and Cytokines Reduce Neurogenic Esophageal Smooth Muscle Contractility Florian Rieder, Ilche T. Nonevski, Jie Ma, Zhufeng Ouyang, Gail West, Anja Schirbel, John R. Goldblum, Tracey L. Bonfield, Gary W. Falk, Piero Biancani, Claudio Fiocchi BACKGROUND: EoE is associated with marked motility abnormalities whose pathogenesis is unknown. The EoE immune response involves the enhanced secretion of eosinophil degranulation products, Th2 cytokines and other inflammatory mediators. We investigated which soluble mediators are actively secreted ex vivo in mucosal biopsies of EoE and noninflammatory control patients, and tested the impact of eosinophil products, selective Th2cytokines, and IL-6 and TGF-β1 on ex vivo esophageal motility. METHODS: Endoscopic biopsies were obtained from the upper, middle and lower esophageal segments of EoE and control subjects, organ-cultured, and secreted cytokines were measured in the undernatants by Luminex and ELISA. Sonicates of the human eosinophil cell line AML14.3D10, and human recombinant IL-5, IL-6, IL-13, eotaxin-1, eotaxin-3 and TGF-β1 were tested in esophageal muscle contraction assays assessing neurogenic and acetylcholine (ACh)-induced contraction. RESULTS: Cytokine assessment in the undernatants revealed that IL-5 and eotaxin-1 were produced exclusively in EoE biopsies (p<0.03); IL-6 and TGF-β1 were produced in all biopsies, but at significantly higher levels in EoE compared to control biopsies (p<0.03 and p<0.01, respectively); IL-13 was also detected in both EoE and control biopsies (5±2 vs. 2±1 pg/ml/mg). Increased cytokine levels were uniformly distributed throughout all segments of the esophagus in EoE patients. Eosinophil sonicates significantly (p<0.04) reduced electrical field-induced contraction of muscle strips by 50%. IL-6 (1nM, 52% reduction, p<0.003), IL-13 (3nM, 73% reduction, p<0.001) and TGF-β1 (1ng/ml, 50% reduction, p<0.01) also significantly reduced esophageal muscle contraction. Interestingly, decreased contraction was also induced by eotaxin-3 (1nM, 36% reduction, p<0.02), but not eotaxin-1 (1nM). None of the sonicates or cytokines influenced ACh-induced contraction, indicating that they may primarily affect neurotransmitter release, without directly affecting muscle contraction. CONCLUSIONS: Active and uniformly increased secretion of Th2 and inflammatory mediators, including IL-5, IL-6, IL-13, eotaxin-1 and TGF-β1, was detected in the mucosa of EoE compared to control patients. Eosinophil sonicates as well as IL6, IL-13, eotaxin-3, and TGF-β1 reduced neurally mediated esophageal smooth muscle contraction. These results suggest a direct functional link between secretion of eosinophil products, Th2 cytokines, and inflammatory mediators with EoE-associated esophageal muscle contraction abnormalities.
874 Esophageal and Laryngeal Dilated Intracellular Space in GERD and Chronic Laryngitis: A Prospective Blinded Assessment Marion Goutte, W. Gray Jerome, Kay Washington, James C. Slaughter, C. Gaelyn Garrett, Michael F. Vaezi
872 Asymptomatic Awakenings From Sleep Due to Acid Reflux are Significantly More Common in Older Versus Younger Patients With GERD Larissa M. Allen, Benjamin H. Levy, Choo Hean Poh, Anita Gasiorowska, Tomas NavarroRodriguez, Jeannette Powers, Stuart F. Quan, Marcia R. Willis, Nicole Ashpole, Isaac B. Malagon, Tiberiu Hershcovici, Ronnie Fass
Objectives Reports suggest that dilated intracellular space (DIS) is a sensitive marker of GERD. There are currently no data on esophageal or laryngeal tissue DIS measurements in pts with suspected GERD-related chronic laryngitis. In this prospective study we aimed: 1) to determine, if microscopic findings (histology or EM) assessed blinded to pt groups could differentiate chronic laryngitis, GERD or controls, and 2) to determine if tissue changes (if any) are predictor of PPI response. Methods Three groups studied: controls, GERD and chronic laryngitis. Controls= EGD for reasons other than GERD (diarrhea, anemia) without a hx of HB/reg. and no prior acid suppressive tx; GERD= pts with HB/reg. with either esophagitis or abnormal pH; Chronic Laryngitis= pts with chronic throat sxs. Off PPI tx, pts underwent EGD with 2 bx's each from distal esophagus (5cm above SCJ) and from postcricoid area. Biopsies were sent for H&E and EM analysis performed by two separate pathologists both blinded to pt group status. GERD and larynigits pts next tx'ed with BID PPI's for 3 mo and EGD repeated with bx's from same regions. Specimens were evaluated and scored based on modified Chadwick scoring system (normal=0, mild=1, moderate=2 and severe=3). EM measurements were from the suprabasal region of the epithelium and based on published protocol. Results 51 pts underwent study: controls (n=15; 61%F, mean
Background: Older patients appear to have much more GERD-related mucosal damage than younger patients with GERD but with lower severity and intensity of symptoms. As compared to daytime, nighttime gastroesophageal reflux has been shown to be more commonly associated with aggressive and complicated GERD. Aim: To compare the frequency of awakenings that are associated with acid reflux during sleep and their relationship with symptoms between younger and older patients with GERD Methods: Patients with heartburn at least 3 times a week for the last 3 months were enrolled. All patients were evaluated by the demographics and GERD Symptoms Checklist questionnaires. Patients underwent pH testing concomitantly with actigraphy. FRIM© analysis was used to superimpose simultaneously recorded raw actigraphy over pH data, resulting in more accurate assessment of acid reflux during the recumbent awake, recumbent asleep, and awakening periods. This technique allowed better evaluation of the relationship between symptoms and acid reflux events in the aforementioned periods. Results: A total of 20 patients (mean age 46.8 yrs, M/F 15/5)
S-121
AGA Abstracts
AGA Abstracts
but also have some additional intra-cellular function to promote goblet cell differentiation of human IECs.
ring at the pylorus. A day later, bilateral cords of stained cells appear along the lesser curvature of the stomach, and by E18.5, these structures have extended anteriorly to the level of the esophagus. These structures are superficial and may represent tendons or nervous tissue. In sectioned tissue, we confirmed that staining is mesenchymal and located exterior to the inner circular muscle of the pylorus. The pattern of Gata3 expression at the pylorus is similar to that of the homeodomain transcription factor Nkx2-5. Thus, we obtained Nkx25(lacZ/+) reporter mice and analyzed Nkx2-5 expression by whole-mount X-gal staining of dissected foregut. The staining pattern was nearly identical to Gata3, with a few notable exceptions: pyloric expression is first observed at E9.5, the ring at the pylorus is complete, and expression of Nkx2-5 (but not Gata3) is detected in the spleen beginning at E12.5. Finally, to investigate a potential role for Gata3 in morphogenesis of the pylorus, we analyzed Gata3-null [Gata3(lacZ/lacZ)] mice at E18.5. The shape of the pylorus was abnormal in Gata3-deficient embryos, causing the antrum and duodenum to intersect at an obtuse angle that was apparent in both the gross specimen and in histological sections. Also, the diameter of the pyloric lumen was larger in Gata3-null than wild type embryos. These data establish for the first time that Gata3 is essential for morphogenesis of the pylorus and raise the possibility that it functionally interacts with Nkx2-5.
AGA Abstracts
Aberrant Intestinal Crypt Morphology and Cell Positioning in Mice With Intestine-Specific Deletion of the KrüPpel-Like Factor 5 Gene Beth B. McConnell, Samuel S. Kim, Ryozo Nagai, Vincent W. Yang BACKGROUND: Krüppel-like factor 5 (KLF5) is a pro-proliferative transcription factor that is highly expressed in dividing epithelial cells at the crypt base of the intestinal epithelium. KLF5 promotes proliferation In Vitro and In Vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in maintaining intestinal homeostasis, we characterized the morphology of mice with conditional deletion of Klf5 in the gut using the villin-Cre (VilCre) recombinase system. METHODS: Mice carrying floxed alleles of Klf5 (Klf5fl/fl) were crossed with Vil-Cre;Klf5fl/+ mice to generate Vil-Cre;Klf5fl/fl mice. Klf5fl/fl littermates served as controls. Mice were sacrificed at 8 weeks of age and the small intestines and colon removed for histological examination. Immunohistochemical staining was used to examine the expression of Klf5, the proliferation marker, Ki67 and various markers of differentiation. RESULTS: Immunohistochemical staining of Klf5 in the small and large bowels of the VilCre;Klf5fl/fl mice showed that deletion of the floxed Klf5 alleles was variable, with the most complete deletion occurring in the distal small intestine and proximal colon. Initial characterization of the mice indicated that the Vil-Cre;Klf5fl/fl mice were leaner than the Klf5fl/fl littermates, weighing 18% less than gender- and age-matched controls (n=5, P = 0.01). Histological staining of intestinal and colonic tissues from Vil-Cre;Klf5fl/fl mice revealed a disruption of normal crypt architecture and a regenerative phenotype, particularly in the colon. Staining with the proliferation marker, Ki67, showed proliferating cells scattered throughout the epithelia of the small and large intestines, with a phenotype similar to that of EphB2/EphB3-null mice. Paneth cells likewise migrated upward from the crypt base. Immunohistochemical staining for EphB2 and EphB3 in Klf5-deleted tissues revealed reduced expression of both receptors and loss of a normal expression gradient within the crypts. Reduction of EphB2 and EphB3 correlated with reduced expression of the differentiation markers intestinal alkaline phosphatase and carbonic anhydrase I. CONCLUSION: Deletion of Klf5 in the gut epithelium results in dramatic changes in morphology that correlate with reduced expression of Wnt targets, EphB2 and EphB3. The boundary between proliferating and differentiation zones are lost, resulting in scattering of proliferating cells throughout the epithelium and reduced differentiation. Thus, KLF5 is critical for maintenance of normal crypt architecture in the intestinal mucosa due to its effects on epithelial cell positioning and differentiation.
868 E-Cadherin is Essential for Intestinal Epithelial Morphogenesis in Mice Benjamin J. Bondow, Mary L. Faber, Michele A. Battle Background & Aims: E-cadherin is the primary cadherin adhesion protein expressed in the intestinal epithelium. Several studies in cell culture systems have implicated E-cadherin as required for cellular junction formation, regulation of cellular polarity, cellular migration, and proliferation. Both over-expression of E-cadherin protein and disruption of E-cadherin mediated adhesion through expression of a dominant-negative N-cadherin protein lacking a functional extracellular domain demonstrated a key role for E-cadherin in the maintenance of normal intestinal epithelial homeostasis. These studies led us to propose that loss of Ecadherin itself from the developing mouse intestinal epithelium would result in disruption of intestinal epithelial morphogenesis. Methods: Because E-cadherin null mouse embryos die at 3.5-4.5 days post-coitus due to failure of trophectoderm epithelium formation, we employed a conditional knockout approach to eliminate E-cadherin specifically in the intestinal epithelium during embryonic development. To generate control and experimental animals, we crossed mice homozygous for the E-cadherin conditional allele (Cdh1loxP/loxP) with heterozygous Cdh1loxP/+Villin-Cre animals. Results: We found that E-cadherin conditional knockout mice failed to survive to weaning age of 3 weeks. Of 98 weanlings, only one animal with the Cdh1loxP/loxPVillin-Cre genotype survived, and immunohistochemical staining for Ecadherin protein showed normal levels of E-cadherin in its intestine reflecting inefficiency of Cre recombinase in this animal. E-cadherin conditional knockout mice die perinatally within the first 24 hours of birth. Analysis of intestinal tissue sections harvested from control and E-cadherin conditional knockout embryos at 18.5 days post-coitus show that loss of E-cadherin severely disrupted intestinal morphogenesis. We observed significant changes in epithelial cell shape as well as marked blunting of the villi. Consistent with a role for Ecadherin in barrier morphogenesis, the presence of blood cells in the intestinal lumen of mutant animals suggests compromised integrity of the intestinal epithelium. Using RT-PCR, we found that the Snail transcript was up-regulated in mutant intestinal tissue compared with control tissue suggesting that E-cadherin null intestinal epithelial cells are undergoing increased epithelial to mesenchymal transition (EMT). Conclusions: We conclude that intestinal E-cadherin expression is required for the formation of a functional intestinal epithelium in mice.
867 An Alternative Splicing/Translation Variant Fine-Tunes the Activity of the Homeotic Transcription Factor CDx2 in the Gut Marie Vanier, Elisabeth Martin, Claire Domon-Dell, Isabelle Gross, Isabelle Duluc, JeanNoel Freund The homeobox gene Cdx2 encodes a transcription factor that plays multiple developmental functions including the specification of intestinal identity. At the adult stage, it is involved in the homeostasis of the intestinal epithelium. We have identified by Northern blot and/ or RT-PCR a splicing variant of Cdx2 expressed in the human and mouse intestine and in human intestinal cell lines cultured In Vitro. Sequencing revealed that this splicing variant encodes a truncated protein, miniCDX2, in which the 181-amino acid Nter-region of CDX2 responsible for its transcriptional activity is replaced by a short 12-amino acid peptide. Translation of miniCDX2 uses an alternative start site compared to CDX2. A specific antibody raised against miniCDX2 showed that miniCDX2 is nuclear, and expressed at a higher level in proliferative than in differentiated colon cancer cells cultured In Vitro, in contrast to CDX2. In the mouse colon, miniCDX2 accumulates in cells located at the crypt bottom, whereas CDX2 is expressed in all epithelial cells. Luciferase assays conducted with promoter targets of CDX2 revealed a dominant-negative effect exerted by miniCDX2 over the DNAbinding dependent function of CDX2. Indeed, miniCDX2 competes with CDX2 for its DNA-binding sites, as shown by chromatin immuno-precipitation. Beside its DNA-binding dependent effect, CDX2 also exerts non-DNA-binding dependent functions including the inhibition of the Wnt pathway and the inhibition of the Notch pathway. Unlike CDX2, miniCDX2 has no effect on the Wnt pathway, but like CDX2 it inhibits the Notch pathway. Co-immunoprecipitation experiments showed that the inhibition of the Wnt pathway by CDX2 and of the Notch pathway by CDX2 and by miniCDX2 correlates with protein-protein interactions, respectively with β-catenin and NICD. In Vivo, overexpressing miniCDX2 in the anterior gut of transgenic mice perturbs the area encompassing the Brunner's glands. In conclusion, in addition to the transcriptional and post-translational levels of regulation of CDX2 that have already been documented, we showed here that the function of CDX2 is also regulated at the post-transcriptional level in the gut epithelium by a splicing / translation variant.
869 Up−Regulation of Delta1 Expression is Required for Goblet Cell Differentiation in Human Intestinal Epithelial Cells Junko Akiyama, Ryuichi Okamoto, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe Background & Aims:We have previously shown that forced activation of the Notch signaling pathway inhibits goblet cell differentiation, whereas forced inactivation of the same pathway conversely promotes goblet cell differentiation of human intestinal epithelial cells(IECs). Studies have shown that activation of the Notch signaling pathway In Vivo is initiated by its ligands, Delta(DLL1, DLL3, and DLL4) and Jagged(JAG1 and JAG2). However, precise role of Notch ligands upon goblet cell differentiation in the human intestine remain mostly unknown. Methods:Expression of Notch ligands in biopsy specimens or in human IECderived cell lines was analyzed by RT-PCR. Expression of Notch ligand within the human intestinal tissue was further examined by immunohistochemistry. Analysis of Notch ligand expression upon forced activation or inhibition of the Notch-Hes1 pathway in human IECderived cell lines was performed by quantitative RT-PCR. Tetracycline-dependent expression system was used for over-expression of activated Notch1(NICD1) or Hes1, whereas a γsecretase inhibitor(GSI) was used to inhibit endogenous activation of Notch in IECs. siRNAmediated gene silencing was used to inhibit expression of DLL1 in IECs. Results:mRNA expression of DLL1, DLL4, JAG1 and JAG2 was detected both in human intestinal tissue specimens and in various human IEC-derived cell lines. Immunostaining of a human colonic tissue revealed that Delta ligands are expressed in predominant population of goblet cells. Forced expression of NICD1 or Hes1 significantly down-regulated both DLL1 and DLL4 expression, which leaded to significant suppression of goblet cell differentiation in LS174T cells. Conversely, inhibition of endogenous Notch activation by GSI treatment significantly down-regulated Hes1 expression, but up-regulated both DLL1 and DLL4 expression, resulting in significant promotion of goblet cell differentiation, in LS174T and HT29 cells. In contrast, such a response to intracellular Notch activity was not observed in expression of JAG1 or JAG2. Differentiation towards goblet cells appeared to be dependent not only upon Notch inactivation but also upon up-regulation of DLL1 expression, as siRNA-mediated reduction of DLL1 expression completely abrogated the promotion of goblet cell differentiation by GSI treatment, in LS174T cells. Conclusion:Up-regulation of DLL1 expression is required for goblet cell differentiation, in addition to inactivation of Notch signaling in human IECs. Present results suggest that DLL1 might not only act as an extra-cellular ligand for Notch,
867a GATA3 is Essential for Proper Morphogenesis of the Pylorus Aaron M. Udager, Ajay Prakash, James D. Engel, Kim-Chew Lim, Deborah L. Gumucio The pylorus plays an important role in gastrointestinal function by regulating the passage of food between the stomach and small intestine. Malfunction or malformation of the pylorus can lead to infantile hypertrophic pyloric stenosis (IHPS). However, despite its important role in digestion, little is known about pyloric development. Tissues that constitute the adult pylorus include: epithelium (gastric and intestinal types), and mesenchyme (muscle, vasculature, nerves), but how these cell populations interact to generate proper pyloric structure and function is unclear. Thus, understanding the mechanisms involved in pylorus morphogenesis could provide new therapeutic insights into pyloric pathologies, including IHPS. In a recent study (Li and Udager et al., 2009), we identified Gata3, a zinc-finger transcription factor, as a novel pyloric gene, with mesenchymal expression at embryonic day (E) 14.5 in the mouse. To further investigate this pattern during embryogenesis, we obtained Gata3(lacZ/+) reporter mice and analyzed Gata3 expression by whole-mount Xgal staining of dissected foregut. Beginning at E12.5, staining is detected in an incomplete
AGA Abstracts
S-120
were enrolled into the younger group and 19 (mean age 68.9 yrs, M/F 11/8) into the older group. Older patients had a significantly higher % total as well as recumbent time pH<4 than younger patients (14.7 vs. 8.6 and 6.1 vs. 4.5, respectively, P<0.05). Both groups had a similar total number of awakenings during sleep (52), but the older group had significantly more mean awakenings per patient that were associated with acid reflux events as compared to the younger group (2.38 vs. 1.92, P<0.05). The older group also had a significantly higher total number of acid reflux events during awakenings as compared to the younger group (121 vs. 95, P<0.05). Mean duration of an acid reflux event during awakening was significantly higher in the older group as compared to the younger group (75.9 sec vs. 51.2 sec, P<0.05). However, younger patients had a significantly greater percentage of awakenings with acid reflux that were associated with symptoms as compared to the older group (47.8% vs. 19%, P<0.05). Conclusions: Older patients demonstrated significantly greater acid reflux during the sleep period as compared to younger patients with GERD. However, asymptomatic awakenings from sleep due to acid reflux are significantly more common in older versus younger patients.
870 Cooperative Effect of Hnf-1α With CDX-2 and GATA-4 Triggers Initiation of the Enterocytic Differentiation Program in Human Normal Progenitor Cells Yannick D. Benoit, Manon Lepage, Eric Tremblay, Fabrice Escaffit, Jean-Francois Beaulieu In the intestinal epithelium, the Cdx, GATA and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx-2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx-2, GATA-4 and HNF-1α using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx-2 or HNF-1α, alone and in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1α and GATA-4 together induced morphological modifications of the cells towards polarization resulting in the appearance of functional features such as microvilli. HNF-1α was also sufficient to induce the expression of cadherins and dipeptidylpeptidase while in combination with Cdx-2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large scale analysis of gene expression confirmed the cooperative effects of these factors. Finally, while DCAMKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi-1 and Lgr5, were unaffected. These observations show that, cooperatively with Cdx2, HNF-1α acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 initiates the establishment of cell polarity.
873 Mechanical Properties of the Esophageal Body in Patients With Eosinophilic Esophagitis (EoE): A Pilot Study Using the Functional Luminal Imaging Probe (Endoflip®) Monika A. Kwiatek, Daniel Luger, Ikuo Hirano, Peter J. Kahrilas, John E. Pandolfino Background and Aim: Although the dominant symptoms of EoE are related to mechanical abnormalities of the esophagus, there are no studies that have assessed compliance of the esophageal wall in EoE. The aim of the current study was to analyze the mechanical properties of the esophageal body in EoE using the functional luminal imaging probe (EndoFLIP®, Crospon Medical Devices, Galway, Ireland). Methods: 14 patients (4F, 23 - 67 yr) with EoE were included (normal n = 1, furrows n = 3, rings n = 4, rings with focal stricture n = 6). Subjects were evaluated during endoscopy with the EndoFLIP® probe spanning 3-11 cm above the squamocolumnar junction. The probe comprised an infinitely compliant cylindrical bag (maximal diameter 25 mm) mounted on a 3 mm catheter with 16 impedance planimetry segments. Stepwise bag distensions from 2 to 40 ml were conducted and the associated intrabag pressure and intraluminal geometry were analyzed. Intrabag pressure and smallest diameter at the impedance planimetry segment in the esophagus in the absence of peristalsis were plotted to assess compliance. Results: The EndoFLIP® clearly displayed the tubular esophageal lumen geometry and was able to localize strictures (Figure 1). Stepwise distension increased intrabag pressure eliciting a progressive esophageal opening that eventually reached a plateau value substantially lower than the upper limit of the bag diameter despite an escalation of pressure (Figure 2). Distal esophageal compliance was similar in EoE patients with a normal endoscopy or furrows, but diminished in the presence of rings (p < 0.01). There was a further decrease in compliance in patients with a focal stricture (p < 0.001). Conclusion: The current pilot data support that the EndoFLIP® can be utilized to assess intraluminal esophageal geometry and mechanics in patients with EoE. Compliance levels in EoE patients were below the compliance limit of the bag, particularly in patients with rings and strictures documented endoscopically.
871 Eosinophilic Esophagitis (EoE)-Associated Eosinophil Products and Cytokines Reduce Neurogenic Esophageal Smooth Muscle Contractility Florian Rieder, Ilche T. Nonevski, Jie Ma, Zhufeng Ouyang, Gail West, Anja Schirbel, John R. Goldblum, Tracey L. Bonfield, Gary W. Falk, Piero Biancani, Claudio Fiocchi BACKGROUND: EoE is associated with marked motility abnormalities whose pathogenesis is unknown. The EoE immune response involves the enhanced secretion of eosinophil degranulation products, Th2 cytokines and other inflammatory mediators. We investigated which soluble mediators are actively secreted ex vivo in mucosal biopsies of EoE and noninflammatory control patients, and tested the impact of eosinophil products, selective Th2cytokines, and IL-6 and TGF-β1 on ex vivo esophageal motility. METHODS: Endoscopic biopsies were obtained from the upper, middle and lower esophageal segments of EoE and control subjects, organ-cultured, and secreted cytokines were measured in the undernatants by Luminex and ELISA. Sonicates of the human eosinophil cell line AML14.3D10, and human recombinant IL-5, IL-6, IL-13, eotaxin-1, eotaxin-3 and TGF-β1 were tested in esophageal muscle contraction assays assessing neurogenic and acetylcholine (ACh)-induced contraction. RESULTS: Cytokine assessment in the undernatants revealed that IL-5 and eotaxin-1 were produced exclusively in EoE biopsies (p<0.03); IL-6 and TGF-β1 were produced in all biopsies, but at significantly higher levels in EoE compared to control biopsies (p<0.03 and p<0.01, respectively); IL-13 was also detected in both EoE and control biopsies (5±2 vs. 2±1 pg/ml/mg). Increased cytokine levels were uniformly distributed throughout all segments of the esophagus in EoE patients. Eosinophil sonicates significantly (p<0.04) reduced electrical field-induced contraction of muscle strips by 50%. IL-6 (1nM, 52% reduction, p<0.003), IL-13 (3nM, 73% reduction, p<0.001) and TGF-β1 (1ng/ml, 50% reduction, p<0.01) also significantly reduced esophageal muscle contraction. Interestingly, decreased contraction was also induced by eotaxin-3 (1nM, 36% reduction, p<0.02), but not eotaxin-1 (1nM). None of the sonicates or cytokines influenced ACh-induced contraction, indicating that they may primarily affect neurotransmitter release, without directly affecting muscle contraction. CONCLUSIONS: Active and uniformly increased secretion of Th2 and inflammatory mediators, including IL-5, IL-6, IL-13, eotaxin-1 and TGF-β1, was detected in the mucosa of EoE compared to control patients. Eosinophil sonicates as well as IL6, IL-13, eotaxin-3, and TGF-β1 reduced neurally mediated esophageal smooth muscle contraction. These results suggest a direct functional link between secretion of eosinophil products, Th2 cytokines, and inflammatory mediators with EoE-associated esophageal muscle contraction abnormalities.
874 Esophageal and Laryngeal Dilated Intracellular Space in GERD and Chronic Laryngitis: A Prospective Blinded Assessment Marion Goutte, W. Gray Jerome, Kay Washington, James C. Slaughter, C. Gaelyn Garrett, Michael F. Vaezi
872 Asymptomatic Awakenings From Sleep Due to Acid Reflux are Significantly More Common in Older Versus Younger Patients With GERD Larissa M. Allen, Benjamin H. Levy, Choo Hean Poh, Anita Gasiorowska, Tomas NavarroRodriguez, Jeannette Powers, Stuart F. Quan, Marcia R. Willis, Nicole Ashpole, Isaac B. Malagon, Tiberiu Hershcovici, Ronnie Fass
Objectives Reports suggest that dilated intracellular space (DIS) is a sensitive marker of GERD. There are currently no data on esophageal or laryngeal tissue DIS measurements in pts with suspected GERD-related chronic laryngitis. In this prospective study we aimed: 1) to determine, if microscopic findings (histology or EM) assessed blinded to pt groups could differentiate chronic laryngitis, GERD or controls, and 2) to determine if tissue changes (if any) are predictor of PPI response. Methods Three groups studied: controls, GERD and chronic laryngitis. Controls= EGD for reasons other than GERD (diarrhea, anemia) without a hx of HB/reg. and no prior acid suppressive tx; GERD= pts with HB/reg. with either esophagitis or abnormal pH; Chronic Laryngitis= pts with chronic throat sxs. Off PPI tx, pts underwent EGD with 2 bx's each from distal esophagus (5cm above SCJ) and from postcricoid area. Biopsies were sent for H&E and EM analysis performed by two separate pathologists both blinded to pt group status. GERD and larynigits pts next tx'ed with BID PPI's for 3 mo and EGD repeated with bx's from same regions. Specimens were evaluated and scored based on modified Chadwick scoring system (normal=0, mild=1, moderate=2 and severe=3). EM measurements were from the suprabasal region of the epithelium and based on published protocol. Results 51 pts underwent study: controls (n=15; 61%F, mean
Background: Older patients appear to have much more GERD-related mucosal damage than younger patients with GERD but with lower severity and intensity of symptoms. As compared to daytime, nighttime gastroesophageal reflux has been shown to be more commonly associated with aggressive and complicated GERD. Aim: To compare the frequency of awakenings that are associated with acid reflux during sleep and their relationship with symptoms between younger and older patients with GERD Methods: Patients with heartburn at least 3 times a week for the last 3 months were enrolled. All patients were evaluated by the demographics and GERD Symptoms Checklist questionnaires. Patients underwent pH testing concomitantly with actigraphy. FRIM© analysis was used to superimpose simultaneously recorded raw actigraphy over pH data, resulting in more accurate assessment of acid reflux during the recumbent awake, recumbent asleep, and awakening periods. This technique allowed better evaluation of the relationship between symptoms and acid reflux events in the aforementioned periods. Results: A total of 20 patients (mean age 46.8 yrs, M/F 15/5)
S-121
AGA Abstracts
AGA Abstracts
but also have some additional intra-cellular function to promote goblet cell differentiation of human IECs.