2A: A linear regression analysis of intestinal length vs body length. Red circles represent zebrafish that have been allowed to grow in their adult stage, while blue triangles are controls. The dotted lines represent the 95% confidence interval. 2B: T-tests were performed between the two groups on their body length (B’) and intestinal length (B’’). The p-values are <0.0001 and 0.0029 respectively, indicating a statistically significant change in lengths between experimental and control groups. 2C: A linear regression analysis of enteric neuron number vs intestinal length. Red circles represent zebrafish that underwent adult-phase growth, while blue triangles are controls. The dotted lines represent the 95% confidence interval. 2D: T-tests were performed between the two groups on their total enteric neuron counts. The p-value is 0.0178, indicating a statistically significant difference in neuron number between experimental and control groups. Su1240 EXPANSION OF INTESTINAL STEM CELLS THROUGH CRYPT FISSION DURING POSTNATAL GROWTH OF THE SMALL INTESTINE Zenab M. Dudhwala, Paul D. Hammond, Gordon S. Howarth, Adrian G. Cummins Introduction: The small intestine (SI) is important for absorption and digestion of nutrients throughout life. The SI develops postnatally by increasing in length and width, driven by crypt fission. Intestinal crypt fission is an under-appreciated and relatively ‘hidden’ mechanism of intestinal growth. A more complete understanding of the mechanisms underpinning the process of postnatal small intestinal growth is vital, as an immature intestine arising from pathologies such as prematurity and short bowel syndrome can result in impaired nutrient absorption and poor tolerance to feeding. This can lead to significant health burden, evidenced by increased morbidity and mortality, duration of hospitalization and associated economic costs. Objective: To investigate the age dependence of crypt fission using a tissue microdissec- tion technique in humans and rats; and secondly, the relationship of crypt fission to expansion of intestinal stem cells during postnatal growth Methods: Duodenal biopsies were obtained from 15 infants, children and teenagers, during endoscopy intended to assess severity of reflux oesophagitis, which would not affect the duodenum. Crypt fission was assessed using microdissection of 100 whole crypts and the percentage of bifid crypts was recorded. Morphological analyses included villous area, crypt area, mitotic counts and apoptotic counts. Intestinal Lqr5 stem cells were identified by in situ hybridisation. Results: Crypt fission peaked broadly during infancy (27%) and declined after 3-4 y (8.8%), p<0.0001. Crypt area was significantly increased during infancy, p=0.01. Occasional mitotic figures were evident in early bifid crypt fission during infancy, whereas apoptotic figures began to increase after 8 y, p<0.0001. Numbers of intestinal stem cells and intensity of expression also peaked broadly during infancy. Clustering of Paneth cells was detected at the base of crypts in the vicinity of stem cells. LGR5 assessment for stem cells declined after infancy, p<0.0001, following a similar time-course to crypt fission. Conclusion: Crypt fission peaked during infancy but was relatively ‘cloaked’ by the slow proliferation of stem cells and rapid dispersal of cell progeny to the transit amplifying zone where further proliferation and differentiation occurs. Understanding the complex mechanisms underlying crypt fission could provide a practical and simple means of developing growth-stimulatory molecules to promote more rapid recovery from short bowel syndrome in infants with intestinal failure. Su1241 INTESTINAL INFLAMMATION ALTERS THE EXPRESSION OF HEPATIC BILE ACID RECEPTORS CAUSING LIVER DAMAGE Noemi Fiaschini, Anna Negroni, Vincenzo Cesi, Francesca Palone, Salvatore Cucchiara, Marina Aloi, Danilo Rossetti, Laura Stronati Background and aims: Gut and liver are anatomically connected by portal circulation, and their functional unit realizes the gut-liver axis (GLA), via bile acid (BA) metabolism. The regulatory functions of BAs are predominantly mediated by the bile receptors, such as the nuclear receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR) and the G protein coupled receptor TGR5 We aimed to assess in vivo and in vitro the impact of gut inflammation on liver health by focusing on the modulation of BA receptors FXR, PXR and S-555 AGA Abstracts TGR5. The strategy to improve liver health by reducing gut inflammation was also considered. Methods: 10 C57BL/6 female mice (age: 8-9 weeks) were treated with 3% dextran sodium sulphate (DSS) in drinking water for 7 days; 10 mice received regular drinking water and served as controls. Histology and analysis of mRNA expression of FXR, PXR, TGR5, TNF IL-6 and IL-1 by RT-PCR were performed in the colon and liver of animals. mRNA expression of iNOS and COX2 was analysed in the liver. Co-cultures of intestinal epithelial cells, (Caco2, above) and hepatics cells, (HepG2, below) were set up for in vitro experiments. Full confluence of Caco2 to form a whole intestinal barrier was proven by the trans epithelial electric resistance (TEER) assay. Caco2 were inflamed by TNF + INF or adherent invasive Escherichia coli (AIEC) LF82, and the mRNA expression of FXR, PXR, TGR5, IL-8 and IL- 1 was analysed in HepG2 by RT-PCR. The anti-inflammatory agent dipotassium glycyrrhi- zate (DPG) was used (150 and 300uM) to reduce gut inflammation. Results: Histological extensive inflammatory infiltration was seen in the gut, and, interestingly, although to a lesser extent, also in the liver. Similarly, L-6, and IL-1 levels strongly increased in intestinal and liver samples. FXR and PXR mRNA expression was decreased in the inflamed mice colon, while TGR5 was increased consistent with its typically high expression in macrophages; in the liver, FXR, PXR and TGR5 were found significantly decreased. Intriguingly, mRNA levels of nitric oxide synthase (iNOS) and cyclooxygenase (COX2) were also found increased in the mice liver. The exposure of only Caco2 to TNF + INF or LF82 (24, 48 hours) induced inflammation in HepG2 cells, as shown by increased IL-8 and IL1- , and decreased BA receptor expression. Co-exposing Caco2 to TNF + INF and DPG strongly reduced IL-8 and significantly increased FXR and PXR expression. Conclusions: gut inflammation, featured by decreased intestinal FXR and PXR expression and by a parallel increased TGR5 due to recruitment of macrophages, affects hepatic cells by altering BA receptor levels and by increasing production of pro-inflammatory cytokines and oxidative stress. Hence, reducing gut inflammation is needed non only to improve the intestinal disease but also to protect the liver. Su1242 A TWO DIMENSIONAL ENTEROID MODEL TO STUDY THE ROLE OF ASBT IN NECROTIZING ENTEROCOLITIS Christine M. Calton, Melissa D. Halpern Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Patients who develop NEC suffer from hemorrhagic, necrotic inflammation of the distal ileum that can require surgical repair if the bowel sustains significant damage. Despite intensive research, the molecular mechanisms that cause intestinal damage in NEC remain poorly characterized. Our lab has previously demonstrated that the apical sodium dependent bile acid transporter (ASBT) plays a role in the development of NEC. ASBT is an apically expressed protein that transports bile acids from the intestinal lumen into the cytosol of enterocytes. Much of the work to characterize ASBT has relied on immortalized cell culture, such as the Caco-2 line. However, other groups have reported low and inconsistent expression of ASBT in Caco-2 cells. Furthermore, these cell lines are frequently derived from adult tumors and thus do not fully recapitulate the physiology of the neonatal small intestine. The development of methods to culture intestinal epithelial organoids (enteroids) now allows primary cells to replicate and differentiate in a convenient in vitro system. Furthermore, enteroids can be derived from mice at different ontogenetic stages, which may be particularly useful for the study of developmentally related diseases such as NEC. While enteroids are commonly cultured in 3D, this technique poses challenges for the study of apically expressed proteins, which face the lumen of the enteroid. To overcome this difficulty, we adapted a 2D enteroid culture system established by Thorne et al. The 2D enteroid system makes apical proteins easily accessible within the culture dish. To examine the feasibility of using 2D enteroids to study the role of ASBT in NEC, crypts were isolated from the distal ileum of neonatal mice, expanded in 3D culture and plated in 2D for experiments. Cells grown in 2D expressed ASBT protein that was readily detectable by both immunofluorescence microscopy and western blot. Treatment of 2D enteroids with bile acids resulted in a dose dependent decrease in ASBT expression that could be detected as early as 12 hours post- treatment. Intriguingly, expression of high molecular weight ASBT multimers decreased to a greater extent than the monomeric form of ASBT. Our results indicate 2D enteroids reproduce the ASBT response to bile acids observed in previously characterized systems, making them a promising in vitro model to study how bile acids contribute to NEC pathogen- esis. Su1243 BENCH TO BED-SIDE AND BACK TRANSLATION IN CF USING PATIENT- DERIVED STEM-CELL BASED MODEL SYSTEMS Kavisha Arora, Fanmuyi Yang, John Brewington, Alexander R. Cortez, Nambirajan Sundaram, Yashaswini Ramananda, Ogden Herbert, Michael Helmrath, Anjaparavanda P. Naren Cystic fibrosis is a multi-organ disorder caused by loss of function mutations in gene that codes for cystic fibrosis transmembrane conductance regulator chloride/bicarbonate ion channel in the epithelial cells. More than 2000 mutations have been identified in the CFTR gene to date with the majority as extremely rare. Considering the phenotypic variation due to CFTR mutations in the patients and impracticality of clinical trials with a limited patient pool, it is becoming increasingly accepted that individualized therapy based on testing in patient-derived stem cells could be the most effective route for CF therapy especially for rare mutation patients. In this study, we present report of a case of rare CF mutation demonstrating a direct translational outcome of stem-cell based preclinical models to clinical intervention of CF modulator Ivacaftor (also known as Kalydeco, VX-770) previously undocu- mented for the mutation type in this patient. For the first time, we demonstrate that the clinical outcome could be recapitulated in the human intestinal organoids (HIOs) derived from patient’s induced pluripotent stem cells (iPSCs). Hence in this study we provide an elegant demonstration of bench side to bedside application and back. AGA Abstracts