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Tu2016 NHE8 Is Expressed in the Intestinal Goblet Cells and Its Expression Is Transcriptionally Regulated by TNF-α
Tu2015 Par1a/B Regulates Jag1 Expression and Bile Duct Development in Mice Alex Koral, Nadira Ramkellawan, Kimberly Reidy, Zhongfang Du, Oleh Akchurin Background: Partitioning defective (Par) 1a/b are members of a family apico-basal polarity proteins. Par1a/b contribute to epithelial cell-cell adhesion and morphogenesis in vitro. They are functionally redundant serine-threonine kinases that may compensate for each other in vivo. Complete loss of Par1 a/b leads to death early in embryogenesis, prior to liver development, while mice with only one out of four alleles, Par 1a -/-: Par 1b +/- (Par1a/b KH) and Par 1a +/-: Par 1b -/- (Par 1a/b HK) die postnatally. Par 1a/b HK and HK kidneys are hypoplastic with abnormal proximal tubules associated with decreased Jagged-1 expression. Alterations in signaling in the Jagged-1/Notch-2 pathways have been associated with several types of biliary tract abnormalities, including Alagelle's Syndrome and biliary atresia. Hypothesis: Par1a/b contributes to hepatic bile duct epithelial development. Results: We examined the time-course of Par 1a/b in embryonic and adult livers using western blotting and immunostaining. Par 1a/b are expressed throughout liver development (E16-E18) and are expressed in adult bile ducts. Next, to examine the role of Par1a/b in liver epithelial development, Par 1a/b HH mice underwent timed matings. Bile duct development was examined in E18 and newborn mice by light microscopy and immunostaining with cytokeratin 8 and 19 and Rhodamine DBA. While littermates had readily identifiable bile ducts around portal veins, Par1a/b KH livers had decreased bile ducts, and Par1a/b HK mice lacked bile duct development. Decreased Jag1 expression was identified in Par1a/b KH and HK livers. Conclusions: Par 1a/b are expressed in the developing liver and are required for bile duct development in mice, likely via effects on Jag1 expression. Ongoing experiments are examining the interaction of Par1a/b and Notch signaling in the developing liver.
Tu2018 The Developing Gastrointestinal Microbiota of Healthy Infants Over the First Year of Life Juliana Durack, Michael Cabana, Michelle McKean, Susan Lynch The gut microbiome is emerging as a critical contributor to human health. Several studies have indicated that early life exposures, particularly those known to impact microbiome composition, are associated with childhood disease development. Despite this, little is known of the features that characterize healthy microbiome assembly in this critical neo-natal phase of development. We examined gut bacterial community development, using 16S rRNA sequencing on the Illumina MiSeq platform, of 28 healthy, full-term infants using fecal samples collected from birth, 1, 3, 6, 9 and 12 months of age. Consistent with previous reports, community complexity as measured by richness (r=0.7393, p<0.0001) and phylogenic diversity (r=0.7478, p<0.0001) expanded significantly over the first year of life and microbial community composition was associated with age. We show that microbial communities present at birth are compositionally distinct, and that the bacterial burden of the initial inoculum does not influence the rate of phylogenic expansion (p=0.224). This rate of diversification varied across infants, most likely reflecting personal environmental exposures. Interpersonal variation in community composition was higher in younger infants and decreased considerably following introduction of solid foods at 6 months of age. Despite diversity accumulation with age, intra-individual community composition similarity was high across all time points sampled. In addition, a core community, defined as OTUs present in all samples from the same infant at 1 month through to 12 month of age was identified in each infant. These core communities consisted on average of 6.3% (2.0%-11.8%) of the total number of OTU's detected at 12 months, in an individual infant. No core microbiome was detected across all participants. This strongly suggests that personal environmental exposures begin to shape the microbial community shortly after birth and that though the complexity and diversity increase with infant's age, each individual develops a personal microbiota of which the core constituents remain present across the first year of life. In conclusion this study highlights the heterogeneity within microbial assemblages found in healthy infants, strongly supporting the view that personal environmental exposures shortly after birth, much more than the initial microbial inoculum, influence gut microbiome development during this crucial period.
Tu2016 NHE8 Is Expressed in the Intestinal Goblet Cells and Its Expression Is Transcriptionally Regulated by TNF-α Hua Xu, Yang Zhao, Jing Li, Fayez K. Ghishan Introduction: Although the intestine plays an important role in digestion and absorption, the mucus lining the epithelium represents a pivotal function in mucosal protection. Goblet cells lie scattered in both the crypts and among enterocytes, and participate in mucosal protection. We have previously showed that NHE8 is expressed on the apical membrane of intestinal epithelial cells and functions as sodium/hydrogen exchanger. We also noted that loss of NHE8 function resulted in reduced mucin production and increased bacterial adhesion. In this current study, we explored the expression of NHE8 in goblet cells and the mechanism of TNF-α regulation on NHE8 in mucin-producing cells. Method: Human mucous-secreting goblet cells (HT29-MTX) was used. NHE8 localization in HT29-MTX was detected by immunochemical labelling. To test the effect of TNF-α on NHE8 expression, various concentrations of human recombinant TNF-α and duration were used to treat HT29MTX cells. RNA and cell lysate were prepared from treated and non-treated cells for PCR and western blotting detection. Promoter assay was used to measure NHE8 gene promoter activity. Result: Immunochemical labelling indicated that NHE8 was expressed at the plasma membrane in HT29-MTX cells. In the presence of TNF-α, the expression of NHE8 was significantly reduced at both mRNA and protein levels. TNF-α mediated NHE8 mRNA expression inhibition could be blocked by actinomycin D, a transcription inhibitor. Promoter reporter assay indicated that NHE8 promoter activity was indeed reduced by TNF-α. Conclusion: NHE8 is a membrane expressing protein in the human mucous-secreting goblet cells (HT29-MTX). Proinflammatory cytokine, TNF-α, is capable of impairing NHE8 function by inhibiting NHE8 expression at gene expression level. The mechanism of TNF- α regulation on NHE8 involves gene transcriptional inhibition.
Tu2019 Special Considerations for Fecal Microbiota Transplantation in Pediatric Recurrent Clostridium difficile Infection Richard Kellermayer, Emily B. Hollister, Dorottya Nagy-Szakal, Faith D. Ihekweazu, Abria Haynes, Milena Pitashny, Claire E. Bocchini, Ruth A. Luna, James Versalovic BACKGROUND: Fecal microbiota transplantation (FMT) is the most effective treatment for recurrent (antibiotic refractory) Clostridium difficile infection (rCDI). Higher asymptomatic carriage rates and more commonly existing underlying clinical conditions compared to adults provide special considerations for FMT in pediatric rCDI. We performed metagenomic analyses to evaluate the microbial mechanism of action of FMT with the incorporation of these pediatric considerations in a cohort of rCDI patients. METHODS: Nine rCDI patients (1.5-16 year old) received filtered, frozen-thawed fecal preparation from screened, selfdesignated or universal donors through colonoscopy under an IRB approved protocol, followed by enema FMT, if clinically indicated. Two patients had inflammatory bowel disease (1 ulcerative colitis [UC], 1 Crohn disease [CD]), 1 had heart transplant, and 2 had significant neurologic impairment as underlying conditions. All patients provided a stool sample 1 day prior and 8 weeks after the first FMT. Select patients gave additional samples to be analyzed. The fecal microbiomes were studied by 454 pyrosequencing of the bacterial 16S rRNA gene. RESULTS: All 4 patients without underlying disease had resolution of their symptoms for more than 2 months following a single FMT. One of these patients was found to be positive for C. difficile 4 months after FMT during an upper respiratory infection, but without gastrointestinal complaints. He was considered as an asymptomatic carrier. Metagenomic analyses indicated that FMT induced convergence of the recipient microbiomes to the donor's microbiome. Bacteroidetes specifically increased in abundance following FMT. Interestingly, asymptomatic carriage of C. difficile did not significantly modify microbiome composition. Out of the 5 patients with complicating clinical conditions, only 2 had obvious clinical
Tu2017 Distinct Role of Notch Ligands, DLL1 and Dll4, in Normal and in Tumor Intestinal Epithelium Toru Nakata, Hiromichi Shimizu, Kohei Suzuki, Satoru Fujii, Go Ito, Kiichiro Tsuchiya, Tetsuya Nakamura, Ryuichi Okamoto, Katsuto Hozumi, Mamoru Watanabe Background & Aims: Studies have shown that the Notch ligands, Dll1 and Dll4, are indispensable for the maintenance of the integrity of the intestinal epithelium. We have previously shown that Dll1 and Dll4 are expressed by distinct populations of intestinal epithelial cells in the normal mouse intestine. However, the precise expression pattern of
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AGA Abstracts
AGA Abstracts
those ligands in intestinal tumors has never been shown. Furthermore, whether an intestinal stem cells (ISCs) that are deficient in both ligands can maintain its function in normal or in tumor tissues remains uncertain. Therefore, we analyzed the effect of ISCs-specific deletion of both Dll1 and Dll4 in the normal mouse intestine, and also in adenomas in APC-deficient mice. Methods: Dll1 and Dll4 were deleted in the normal intestine by crossing Dll1-floxed mice and Dll4-floxed mice with LGR5EGFP-Ires-CreERT2 mice. Those mice were further crossed with APC-floxed mice, to analyze adenoma formation by Dll1 and Dll4 deficient LGR5+ve cells. LGR5EGFP-Ires-CreERT2 mice or LGR5EGFP-Ires-CreERT2/APCflox/flox mice served as controls. Results: The number of secretory lineage cells in the small intestinal villi was significantly increased in Dll1 and Dll4-deleted mouse at Day 20 accompanied by a substantial loss of Ki67 and Hes1 expression in the crypts. The lower part of those crypts were mostly dominated by lysozyme+ve (LYZ+ve) cells, accompanied with a certain number of remaining Lgr5+ve cells, suggesting that these ligands are indispensable for the maintenance of the normal stem cell niche. In APC-deficient mice, LGR5+ve cells clearly maintained the expression of Hes1, but gave rise to LYZ+ve cells to form a stem cell niche with adenoma development by Day 21 in both small and large intestine. Yet in those tumors, Dll1 expression was rarely observed, whereas Dll4 expression was highly heterogenous between tumors. Dll1 and Dll4/APCdeficient mice developed adenomas at an equivalent size and number, as compared to the control mice. Also, the number or the distribution of LGR5+ve cells and LYZ+ve cells remained mostly unchanged, suggesting a formation of the functional tumor stem cell niche. Interestingly, Hes1 expression was clearly maintained in those tumors, suggesting that an alternative pathway may become dominant in the maintenance of Hes1 expression under an APCdeficient background. Conclusions: Loss of APC function in LGR5+ve cells may predispose those cells to lose dependency on Dll1 and Dll4, and thereby assure formation of the tumor stem cell niche.
Tu2018 The Developing Gastrointestinal Microbiota of Healthy Infants Over the First Year of Life Juliana Durack, Michael Cabana, Michelle McKean, Susan Lynch The gut microbiome is emerging as a critical contributor to human health. Several studies have indicated that early life exposures, particularly those known to impact microbiome composition, are associated with childhood disease development. Despite this, little is known of the features that characterize healthy microbiome assembly in this critical neo-natal phase of development. We examined gut bacterial community development, using 16S rRNA sequencing on the Illumina MiSeq platform, of 28 healthy, full-term infants using fecal samples collected from birth, 1, 3, 6, 9 and 12 months of age. Consistent with previous reports, community complexity as measured by richness (r=0.7393, p<0.0001) and phylogenic diversity (r=0.7478, p<0.0001) expanded significantly over the first year of life and microbial community composition was associated with age. We show that microbial communities present at birth are compositionally distinct, and that the bacterial burden of the initial inoculum does not influence the rate of phylogenic expansion (p=0.224). This rate of diversification varied across infants, most likely reflecting personal environmental exposures. Interpersonal variation in community composition was higher in younger infants and decreased considerably following introduction of solid foods at 6 months of age. Despite diversity accumulation with age, intra-individual community composition similarity was high across all time points sampled. In addition, a core community, defined as OTUs present in all samples from the same infant at 1 month through to 12 month of age was identified in each infant. These core communities consisted on average of 6.3% (2.0%-11.8%) of the total number of OTU's detected at 12 months, in an individual infant. No core microbiome was detected across all participants. This strongly suggests that personal environmental exposures begin to shape the microbial community shortly after birth and that though the complexity and diversity increase with infant's age, each individual develops a personal microbiota of which the core constituents remain present across the first year of life. In conclusion this study highlights the heterogeneity within microbial assemblages found in healthy infants, strongly supporting the view that personal environmental exposures shortly after birth, much more than the initial microbial inoculum, influence gut microbiome development during this crucial period.
Tu2016 NHE8 Is Expressed in the Intestinal Goblet Cells and Its Expression Is Transcriptionally Regulated by TNF-α Hua Xu, Yang Zhao, Jing Li, Fayez K. Ghishan Introduction: Although the intestine plays an important role in digestion and absorption, the mucus lining the epithelium represents a pivotal function in mucosal protection. Goblet cells lie scattered in both the crypts and among enterocytes, and participate in mucosal protection. We have previously showed that NHE8 is expressed on the apical membrane of intestinal epithelial cells and functions as sodium/hydrogen exchanger. We also noted that loss of NHE8 function resulted in reduced mucin production and increased bacterial adhesion. In this current study, we explored the expression of NHE8 in goblet cells and the mechanism of TNF-α regulation on NHE8 in mucin-producing cells. Method: Human mucous-secreting goblet cells (HT29-MTX) was used. NHE8 localization in HT29-MTX was detected by immunochemical labelling. To test the effect of TNF-α on NHE8 expression, various concentrations of human recombinant TNF-α and duration were used to treat HT29MTX cells. RNA and cell lysate were prepared from treated and non-treated cells for PCR and western blotting detection. Promoter assay was used to measure NHE8 gene promoter activity. Result: Immunochemical labelling indicated that NHE8 was expressed at the plasma membrane in HT29-MTX cells. In the presence of TNF-α, the expression of NHE8 was significantly reduced at both mRNA and protein levels. TNF-α mediated NHE8 mRNA expression inhibition could be blocked by actinomycin D, a transcription inhibitor. Promoter reporter assay indicated that NHE8 promoter activity was indeed reduced by TNF-α. Conclusion: NHE8 is a membrane expressing protein in the human mucous-secreting goblet cells (HT29-MTX). Proinflammatory cytokine, TNF-α, is capable of impairing NHE8 function by inhibiting NHE8 expression at gene expression level. The mechanism of TNF- α regulation on NHE8 involves gene transcriptional inhibition.
Tu2019 Special Considerations for Fecal Microbiota Transplantation in Pediatric Recurrent Clostridium difficile Infection Richard Kellermayer, Emily B. Hollister, Dorottya Nagy-Szakal, Faith D. Ihekweazu, Abria Haynes, Milena Pitashny, Claire E. Bocchini, Ruth A. Luna, James Versalovic BACKGROUND: Fecal microbiota transplantation (FMT) is the most effective treatment for recurrent (antibiotic refractory) Clostridium difficile infection (rCDI). Higher asymptomatic carriage rates and more commonly existing underlying clinical conditions compared to adults provide special considerations for FMT in pediatric rCDI. We performed metagenomic analyses to evaluate the microbial mechanism of action of FMT with the incorporation of these pediatric considerations in a cohort of rCDI patients. METHODS: Nine rCDI patients (1.5-16 year old) received filtered, frozen-thawed fecal preparation from screened, selfdesignated or universal donors through colonoscopy under an IRB approved protocol, followed by enema FMT, if clinically indicated. Two patients had inflammatory bowel disease (1 ulcerative colitis [UC], 1 Crohn disease [CD]), 1 had heart transplant, and 2 had significant neurologic impairment as underlying conditions. All patients provided a stool sample 1 day prior and 8 weeks after the first FMT. Select patients gave additional samples to be analyzed. The fecal microbiomes were studied by 454 pyrosequencing of the bacterial 16S rRNA gene. RESULTS: All 4 patients without underlying disease had resolution of their symptoms for more than 2 months following a single FMT. One of these patients was found to be positive for C. difficile 4 months after FMT during an upper respiratory infection, but without gastrointestinal complaints. He was considered as an asymptomatic carrier. Metagenomic analyses indicated that FMT induced convergence of the recipient microbiomes to the donor's microbiome. Bacteroidetes specifically increased in abundance following FMT. Interestingly, asymptomatic carriage of C. difficile did not significantly modify microbiome composition. Out of the 5 patients with complicating clinical conditions, only 2 had obvious clinical
Tu2017 Distinct Role of Notch Ligands, DLL1 and Dll4, in Normal and in Tumor Intestinal Epithelium Toru Nakata, Hiromichi Shimizu, Kohei Suzuki, Satoru Fujii, Go Ito, Kiichiro Tsuchiya, Tetsuya Nakamura, Ryuichi Okamoto, Katsuto Hozumi, Mamoru Watanabe Background & Aims: Studies have shown that the Notch ligands, Dll1 and Dll4, are indispensable for the maintenance of the integrity of the intestinal epithelium. We have previously shown that Dll1 and Dll4 are expressed by distinct populations of intestinal epithelial cells in the normal mouse intestine. However, the precise expression pattern of
S-961
AGA Abstracts
AGA Abstracts
those ligands in intestinal tumors has never been shown. Furthermore, whether an intestinal stem cells (ISCs) that are deficient in both ligands can maintain its function in normal or in tumor tissues remains uncertain. Therefore, we analyzed the effect of ISCs-specific deletion of both Dll1 and Dll4 in the normal mouse intestine, and also in adenomas in APC-deficient mice. Methods: Dll1 and Dll4 were deleted in the normal intestine by crossing Dll1-floxed mice and Dll4-floxed mice with LGR5EGFP-Ires-CreERT2 mice. Those mice were further crossed with APC-floxed mice, to analyze adenoma formation by Dll1 and Dll4 deficient LGR5+ve cells. LGR5EGFP-Ires-CreERT2 mice or LGR5EGFP-Ires-CreERT2/APCflox/flox mice served as controls. Results: The number of secretory lineage cells in the small intestinal villi was significantly increased in Dll1 and Dll4-deleted mouse at Day 20 accompanied by a substantial loss of Ki67 and Hes1 expression in the crypts. The lower part of those crypts were mostly dominated by lysozyme+ve (LYZ+ve) cells, accompanied with a certain number of remaining Lgr5+ve cells, suggesting that these ligands are indispensable for the maintenance of the normal stem cell niche. In APC-deficient mice, LGR5+ve cells clearly maintained the expression of Hes1, but gave rise to LYZ+ve cells to form a stem cell niche with adenoma development by Day 21 in both small and large intestine. Yet in those tumors, Dll1 expression was rarely observed, whereas Dll4 expression was highly heterogenous between tumors. Dll1 and Dll4/APCdeficient mice developed adenomas at an equivalent size and number, as compared to the control mice. Also, the number or the distribution of LGR5+ve cells and LYZ+ve cells remained mostly unchanged, suggesting a formation of the functional tumor stem cell niche. Interestingly, Hes1 expression was clearly maintained in those tumors, suggesting that an alternative pathway may become dominant in the maintenance of Hes1 expression under an APCdeficient background. Conclusions: Loss of APC function in LGR5+ve cells may predispose those cells to lose dependency on Dll1 and Dll4, and thereby assure formation of the tumor stem cell niche.