Enterocyte Cell Research Articles

Probiotic Bacteria and their Supernatants Protect Enterocyte Cell Lines from Enteroinvasive Escherichia coli (EIEC) Invasion.

Probiotic microorganisms have attracted a growing interest for prevention and therapy of gastrointestinal disorders. Many probiotic strains have been shown to inhibit growth and metabolic activity of enteropathogenic bacteria as well as their adhesion and invasion to intestinal cells. In the present study, we evaluated the interference of bacteria-free supernatants (BFS) of cultures belonging to sixteen strains of lactobacilli and bifidobacteria, with invasion of enteroinvasive Escherichia coli (EIEC) strain, using human colonic adenocarcinoma cell lines, T84 and Caco2 cells. To assess invasion of Caco-2 and T84 cells by EIEC, and measure the number of pathogens inside the enterocytes, the gentamicin protection assay was conducted. In addition, three different invasion inhibition assays were designed; namely co-incubation, pre-incubation and treatment with the BFS of probiotics. Data obtained and theoretical calculation showed that the most effective assay in the prevention of pathogen invasion was treatment with BFS. Besides, co-incubation assay was more valid than pre-incubation assay in invasion prevention. The obtained results suggest that probiotics may produce some metabolites that strongly prevent invasion of enteroinvasive E.coli into the small and large intestine. Also, probiotics are able to compete with or exclude pathogen invasion.

Glucose Protection Mediated by Glycolytic Pyruvate but Not ATP against Ischemia-Induced Enterocyte Cell Death

Based on the traditional view of energy drop as a key trigger for ischemia-induced cell death, various nutritional strategies including glucose supplementation were developed to maintain epithelial barrier integrity. Our recent finding indicated that glycolytic pyruvate was responsible for the rescue of epithelial cell death in an energy-independent manner. In contrast, ATP promoted crypt proliferation but failed to protect against villous cell death. This study challenges the classical view and supports that energy depletion is not the cause but an effect of epithelial cell death under ischemic stress.

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome.

The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

Ketogenesis contributes to intestinal cell differentiation.

The intestinal epithelium undergoes a continual process of proliferation, differentiation and apoptosis. Previously, we have shown that the PI3K/Akt/mTOR pathway has a critical role in intestinal homeostasis. However, the downstream targets mediating the effects of mTOR in intestinal cells are not known. Here, we show that the ketone body β-hydroxybutyrate (βHB), an endogenous inhibitor of histone deacetylases (HDACs) induces intestinal cell differentiation as noted by the increased expression of differentiation markers (Mucin2 (MUC2), lysozyme, IAP, sucrase-isomaltase, KRT20, villin, Caudal-related homeobox transcription factor 2 (CDX2) and p21Waf1). Conversely, knockdown of the ketogenic mitochondrial enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) attenuated spontaneous differentiation in the human colon cancer cell line Caco-2. Overexpression of HMGCS2, which we found is localized specifically in the more differentiated portions of the intestinal mucosa, increased the expression of CDX2, thus further suggesting the contributory role of HMGCS2 in intestinal differentiation. In addition, mice fed a ketogenic diet demonstrated increased differentiation of intestinal cells as noted by an increase in the enterocyte, goblet and Paneth cell lineages. Moreover, we showed that either knockdown of mTOR or inhibition of mTORC1 with rapamycin increases the expression of HMGCS2 in intestinal cells in vitro and in vivo, suggesting a possible cross-talk between mTOR and HMGCS2/βHB signaling in intestinal cells. In contrast, treatment of intestinal cells with βHB or feeding mice with a ketogenic diet inhibits mTOR signaling in intestinal cells. Together, we provide evidence showing that HMGCS2/βHB contributes to intestinal cell differentiation. Our results suggest that mTOR acts cooperatively with HMGCS2/βHB to maintain intestinal homeostasis.

268 - The Epidermal Growth Factor Receptor Inhibition Is Involved in Hexameric Procyanidins Anti-Colorectal Cancer Action

Proanthocyanidins (PACs), oligomers of flavan 3-ols, are naturally abundant in fruits and vegetables. Dietary PACs consumption has been associated to a lower risk for colorectal cancer (CRC). We previously demonstrated that hexameric PACs (Hex) interacts with the enterocyte cell membrane in specialized rafts domain. In vitro Hex has antiproliferative and proapoptotic activity in human CRC cell lines. In order to identify the connection between these findings, this work investigated if the anti-CRC actions of Hex are related to its capacity to modulate the lipid raft-located epidermal growth factor (EGF) receptor (EGFR). Without affecting the EGFR localization at lipid rafts, Hex prevented the receptor dimerization upon EGF stimulation and inhibited the EGF-induced activation of the pro-proliferation and antiapoptotic pathways Raf/MEK/ERK and PI3K/Akt signaling cascades. EGFR immunolocalization studies and ELISA assays showed that Hex acted in part promoting EGFR internalization both in the absence and presence of EGF. Inhibition of the EGFR pathway by Hex was also evidenced by decreased phosphorylation at Tyr 1068, and increased Tyr 1045 phosphorylation. The latter provides a docking site for ubiquitin ligase c-Cbl and promotes EGFR degradation by the proteasome. Inhibition of EGFR activation, promotion of receptor internalization and upregulation of EGFR degradation pathways are evidences that Hex PACs can exert anti-CRC actions at the gastrointestinal tract via the downregulation of the EGFR pro-oncogenic signaling pathway.

Enhanced serotonin signaling stimulates ordered intestinal mucosal growth

BackgroundSignificant quantities of serotonin (5-hydroxytryptamine; 5-HT) are found in the intestine, and studies have demonstrated that 5-HT can stimulate enterocyte cell division, suggesting regulatory roles in mucosal homeostasis and intestinal adaptation. We hypothesized that excess enteric 5-HT signaling enhances mucosal growth without changing intestinal villous cellular makeup. MethodsMice lacking the serotonin reuptake transporter (SERT) and wild-type littermates (WTLM) were euthanized and their ileum analyzed. Villus height (VH), crypt depth (CD), and enterocyte height (EH) were measured. Enterocyte cell division was measured using Ki-67 immunofluorescence to calculate crypt proliferation index (CPI). Cellular distribution along villi was investigated by immunofluorescent staining for enterocytes, enteroendocrine cells, and goblet cells. Group measurements were compared using t-test and chi-squared test. ResultsSERT knock-out (SERTKO) mice had significantly taller villi, deeper crypts, and taller enterocytes compared with WTLM (P < 0.0001). Similarly, enterocyte proliferation was greater in SERTKO compared with WTLM (P < 0.01). For SERTKO, mean values were: VH, 255.6 μm; CD, 66.7 μm; EH, 21.2 μm; and CPI, 52.8%. For WTLM, corresponding values were: VH, 207.8 μm; CD, 56.1 μm; EH, 19.5 μm; and CPI, 31.9%. The cellular composition along villi was not significantly different between genotypes (P > 0.05). ConclusionsEnhancing 5-HT signaling in mice increases VH, CD, EH, and crypt cell proliferation in the intestinal mucosa. 5-HT-associated growth did not alter the cellular composition of the villi. Serotonin may represent an important physiologic regulator of intestinal growth and adaptation and holds promise as a target for therapies aimed at enhancing intestinal recovery after injury or mucosal surface area loss.

Congenital Sodium Diarrhea: A Form of Intractable Diarrhea, With a Link to Inflammatory Bowel Disease.

Congenital diarrheal disorders (CDDs) represent a group of challenging clinical conditions for pediatricians because of the severity of the presentation and the broad range of possible differential diagnoses. CDDs arise from alterations in the transport of nutrients and electrolytes across the intestinal mucosa, from enterocyte and enteroendocrine cell differentiation and/or polarization defects, and from the modulation of the intestinal immune response. Advances were made recently in deciphering the etiology and pathophysiology of one of these disorders, congenital sodium diarrhea (CSD). CSD refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. A syndromic form of CSD features choanal and intestinal atresias as well as recurrent corneal erosions. Small bowel histology frequently detects an epithelial "tufting" dysplasia. It is autosomal recessively inherited, and caused by SPINT2 mutations. The nonsyndromic form of CSD can be caused by dominant activating mutations in GUCY2C, encoding intestinal receptor guanylate cyclase C (GC-C), and by autosomal recessive SLC9A3 loss-of-function mutations. SLC9A3 encodes Na/H antiporter 3, the major intestinal brush border Na/H exchanger, and a downstream target of GC-C. A number of patients with GUCY2C and SLC9A3 mutations developed inflammatory bowel disease. Both the number of recognized CDD forms as well as the number of underlying disease genes are gradually increasing. Knowledge of these CDD genes enables noninvasive, next-generation gene panel-based testing to facilitate an early diagnosis in CDD. Primary Na/H antiporter 3 and GC-C malfunction is implicated as a predisposition for inflammatory bowel disease in subset of patients.

Development of particulate drug formulation against C. parvum: Formulation, characterization and in vivo efficacy

This research aims towards developing an alternative therapy against Cryptosporidium parvum using bioadhesive paromomycin and diloxanide furoate-loaded microspheres. Microspheres were prepared using chitosan and poly(vinyl alcohol) and two types of cyclodextrins (β-CD and DM-β-CD) for the potential use of treating cryptosporidiosis. This pathogen is associated with gastrointestinal illness in humans and animals. Microparticle formulations were characterized in terms of size, surface charge, drug release and morphology. In vivo bioadhesion properties of CHI/PVA microspheres were also evaluated in mice. Finally, the in vivo efficacy of CHI/PVA microspheres against C. parvum was tested in neonatal mouse model. In this work, microspheres prepared by spray-drying showed spherical shape, diameters between 6.67±0.11 and 18.78±0.07μm and positively surface charged. The bioadhesion studies demonstrated that MS remained attached at +16h (post-infection) to the intestinal cells as detected by fluorescence. This finding was crucial taking use of the fact that the parasite multiplication occurs between 16 and 20h post-infection. The efficacy of treatment was determined by calculating the number of oocysts recovered from the intestinal tract of mice after 7days of post-infection. Mice receiving orally administered microspheres with and without drug exhibited significantly lower parasite loads compared with the control mice. Ultrastructural observations by TEM bring to light the uptake of smallest particles by enterocytes associated with conspicuous changes in enterocytic cells. Completely recovery of cell morphology was detected after 24h of first inoculation with MS. CHI/PVA microspheres appear to be a safe and simple system to be used in an anticryptosporidial treatment. The distinctive features of neonatal mice requires further work to determine the suppressive effect of this particulate delivery system on C. parvum attachment in other animal models.

Patients with mild enteropathy have apoptotic injury of enterocytes similar to that in advanced enteropathy in celiac disease

BackgroundSeverity of villous atrophy in celiac disease (CeD) is the cumulative effect of enterocyte loss and cell regeneration. Gluten-free diet has been shown to benefit even in patients having a positive anti-tissue transglutaminase (tTG) antibody titre and mild enteropathy. AimWe explored the balance between mucosal apoptotic enterocyte loss and cell regeneration in mild and advanced enteropathies. MethodsDuodenal biopsies from patients with mild enteropathy (Marsh grade 0 and 1) (n=26), advanced enteropathy (Marsh grade ≥2) (n=41) and control biopsies (n=12) were subjected to immunohistochemical staining for end-apoptotic markers (M30, H2AX); markers of cell death (perforin, annexin V); and cell proliferation (Ki67). Composite H-scores based on the intensity and distribution of markers were compared. ResultsEnd-apoptotic markers and marker of cell death (perforin) were significantly up-regulated in both mild and advanced enteropathies, in comparison to controls; without any difference between mild and advanced enteropathies. Ki67 labelling index was significantly higher in crypts of mild enteropathy, in comparison to controls, suggesting maintained regenerative activity in the former. ConclusionsEven in patients with mild enteropathy, the rate of apoptosis is similar to those with advanced enteropathy. These findings suggest the necessity of reviewing the existing practice of not treating patients with mild enteropathy.

Selected Lactobacillus strains isolated from sugary and milk kefir reduce Salmonella infection of epithelial cells in vitro.

The isolation of potentially probiotic strains and the subsequent study of their properties are very important steps to gain insight in the health benefits ascribed to sugary and milk kefir. The aim of the present study was to characterise fifteen Lactobacillus strains isolated from these beverages by determining some surface properties and their ability to antagonise enterocyte cell damage after Salmonella infection in vitro. Lactobacillus surface properties were determined by hydrophobicity, autoaggregation, and coaggregation assays with Salmonella. In addition, lactobacilli adhesion to Caco-2/TC-7 cells and the effect on Salmonella invasion were evaluated. Finally, the disassembly of F-actin cytoskeleton on intestinal epithelial cells was assayed in vitro when Salmonella infection was performed in the presence of selected Lactobacillus strains. Ten out of the 15 strains showed a high adhesion capacity to Caco-2/TC-7 cells. Most of the strains were hydrophilic and non-autoaggregating. Strains isolated from sugary kefir were non-coaggregating with Salmonella, while strains Lactobacillus paracasei CIDCA 83120, 83121, 83123, 83124, 8339, 83102 isolated from milk kefir were able to coaggregate after 1 h. L. paracasei CIDCA 8339 and Lactobacillus kefiri CIDCA 83102 were able to diminish Salmonella invasion to the enterocytes. An antagonistic effect on cytoskeleton disruption elicited by the pathogen was also demonstrated. Our results suggest that both strains isolated from milk kefir could be considered as appropriate probiotic candidates.

Phytochemical Composition and Antitumor Activities of New Salad Greens: Rucola (Diplotaxis tenuifolia) and Corn Salad (Valerianella locusta).

D. tenuifolia and V. locusta, two greens, were analyzed for active compounds and antitumor actions on colorectal cancer cells. Phenolics were determined by UHPLC-Orbitrap-MS; carotenoids and glucosinolates by HPLC-MS; and sterols and fatty acids by gas-liquid chromatography (GLC). For antitumor effects, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) tests were run on HT-29 colorectal cancer cells, and in CCD-18 untransformed enterocyte cells. Six main carotenoids were identified in both vegetables, while total carotenoids accounted for 3520 and 2970μg · g(-1) dry weight in D. tenuifolia and V. locusta, respectively. Six phenolics were detected in D. tenuifolia (68,600μg · g(-1) dry weight) and five in V. locusta (139,000μg · g(-1) dry weight). Three glucosinolates (GSL) were found in D. tenuifolia (1960μg · g(-1) dry wt. total). Low-polarity extracts from V. locusta and D. tenuifolia showed IC50 ~ 150 and ~200μg · mL(-1) on HT-29 cells, while both plants lacked actions on CCD-18 cells. V. locusta inhibited HT-29 cancer cells viability more efficiently than D. tenuiofolia, but induced less cytotoxicity. This work highlights the importance of functional foods for colorectal cancer prevention.

Identification of coeliac disease triggering glutenin peptides in adults

ABSTRACTObjective: Coeliac disease affects approximately 1% of Northern American and European populations. It is caused by an inappropriate immune response to dietary gluten. Gluten comprises of two major protein fractions: gliadins and glutenins. Glutenins have recently been found to be toxic to coeliac individuals. Proliferation assays suggest in some but not all paediatric coeliac individuals there may be immunological stimulation with high molecular weight (HMW) glutenins. Less evidence pertains to low molecular weight (LMW) glutenins. The aim is to assess adaptive, T-cell driven, and innate immune response in adult coeliac individuals towards HMW glutenin peptide, glut04, and LMW glutenin peptide, glt156.Materials and methods: Coeliac patients were recruited attending endoscopy for routine monitoring. Adaptive immune response towards glut04 and glt156 was measured by proliferation assays and measurement of interferon-γ secretion in 28 T-cell lines. The innate immune response was assessed by measurement of enterocyte cell height (ECH) in coeliac small intestinal biopsies following overnight incubation in organ culture chambers in a further nine individuals.Results: There were 3/28 and 2/28 positive proliferation results using gluten-sensitive T-cells with glut04 and glt156, respectively. All coeliac biopsies tested in organ culture chambers demonstrated clear reduction in ECH with peptic-tryptic digest of whole industrial gluten, glut04 and glt156 when compared to negative control ovalbumin (p < 0.005). Three individuals had both T-cell and organ culture study data. Their proliferation assays showed no stimulation of the T-cells.Conclusions: This study demonstrates glutenin epitopes glut04 and glt156, while minor T-cell epitopes, are important in their ability to trigger the innate immune response.

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation.Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured.The mRNA expression levels of the enterocyte marker sucrase–isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3′-, 4-, and 6′-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo

Background: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known.Objective: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS.Methods: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo.Results: The 3 galactosyllactoses (3′-GL, 4-GL, and 6′-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α– and IL-1β–induced expression of IL-8, MIP-3α, and MCP-1 to 48–51% and pathogen-induced IL-8 and MCP-1 to 26–30% of positive controls (P < 0.001). GOS reduced TNF-α– and IL-1β–induced inflammatory responses to 25–26% and pathogen-induced IL-8 and MCP-1 to 36–39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01).Conclusion: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation.

GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult midgut

Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation.

Human Milk Fortification Increases Bnip3 Expression Associated With Intestinal Cell Death In Vitro.

The aim of the present study was to determine the in vitro effect(s) of a bovine-based human breast milk fortifier (HMF) on human intestinal cells. HMF increases the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein (Bnip3) and cell death; the prostaglandin analogue misoprostol will rescue this effect. Cultured intestinal cells were exposed to in vitro-digested human breast milk (BM) ± HMF. Intracellular oxidation, cell damage/cell death, and BNIP3 expression were measured after exposure. In vitro-digested BM + HMF significantly increased intracellular oxidation, cell damage, and cell death in enterocyte cell cultures compared with either saline or BM controls, an effect that was rescued by the prostaglandin analogue, misoprostol. Bnip3 transcript and Bnip3 protein levels were significantly increased in vitro after treatment with BM + HMF. We also provide evidence that transfection of enterocytes with Bnip3 increases cell death, an effect that is rescued by a nonfunctional Bnip3 splice variant. Our data support the hypothesis that HMF increases intestinal Bnip3 in vitro, and that the gene product triggers cell death. We suggest that misoprostol is a promising therapy, which may reduce intestinal cell death.

Vitamin D receptor knockout mice exhibit elongated intestinal microvilli and increased ezrin expression

In addition to its principle function as a calcium regulator, vitamin D can affect cell and tissue morphology. The intestine is an important target tissue of vitamin D, as it must ensure the efficient transport of nutrients across the epithelium while excluding the passage of harmful molecules and bacteria into the organism. These functions require a highly organized morphology, which may be modified by vitamin D deficiency. To elucidate the role of vitamin D in gut morphology and barrier function, we compared the enterocyte microstructures, gut permeability, and cytoskeletal and cell junction protein expression in vitamin D receptor (VDR) knockout (KO) and wild-type (WT) mice. We found that the duodenal epithelial cells in the VDR-KO mice had longer microvilli (+19%) than those of the WT mice (P < .05). Interestingly, microvilli elongation in the VDR-KO mice was associated with higher messenger RNA and protein expression of ezrin, which is involved in the regulation of microvillus morphogenesis. Intestinal tight junction width and permeability were assessed by measuring the fluorescein isothiocyanate dextran concentrations in plasma; the concentrations were comparable between the 2 groups of mice. We further observed a decrease in the messenger RNA and protein expression of the calcium-transporting tight junction protein claudin-2 in the VDR-KO mice compared with the WT mice (P < .05). In conclusion, the mice lacking VDR had longer enterocyte microvilli, likely as a result of increased ezrin expression. However, the morphology of the tight junctions and the intestinal permeability for large molecules were not affected.

OP‐4 ADVANCES IN TISSUE ENGINEERING TO REGENERATE THE INTESTINAL EPITHELIUM

Introduction:Irreversible intestinal failure (IF) due to anatomical or functional loss is a devastating condition is associated with significant morbidity and mortality. Patients referred for intestinal transplantation suffer low survival rates due to the high graft‐rejection rate and sepsis as a consequence long‐term immunosuppression. These clinical challenges reveal an urgent need for research into new treatments for IF. Here, we present recent advances in our work on intestinal tissue engineering.Methods:Ethical approval is in place to collect intestinal tissue from patients at Great Ormond Street Hospital. Intestinal crypts were isolated and organoids were cultured as previously described. Decellularization of whole intestinal sections was performed using 2 cycles of the Detergent Enzymatic Treatment (DET) as previously reported. Organoids were cultured on the epithelial surface of the acellular matrix in vitro for 10 days. Cell survival was analysed by histology, immunofluorescence, immunohistochemistry and electron microscopy techniques.Results:Intestinal crypts from the small intestine and colon were successfully isolated from endoscopic biopsies and cultured in order to generate 3 dimensional organoids. The presence of goblet cells and enterocyte cells were confirmed on immunohistochemistry. Whole sections of intestine of varying lengths were successfully decellularized and assessed using H&amp;E staining and scanning electron microscopy. Organoids were seeded onto the epithelial surface of the acellular intestinal matrices and allowed to engraft for a period of 10 days in vitro. The formation of a neo‐epithelium with evidence of a microvillus brush border was evident after 10 days in culture. Furthermore, the cells had successfully migrated into the crypt domains of the matrix.Conclusions:We demonstrate the potential of tissue engineering for intestinal failure by combining organoid cultures with decellularization expertise. Our results confirm human intestinal organoids to be the ideal source of progenitor cells to regenerate the intestinal epithelium, with potential clinical applications for transplantation.

Adult Autoimmune Enteropathy Involving Small Bowel and Colon

Introduction: Adult autoimmune enteropathy (AIE) is a rare cause of intractable diarrhea that has been described in the literature in only 36 adult patients. AIE is defined by chronic malabsorptive diarrhea unresponsive to dietary modification and characteristic histological findings. Enterocyte and goblet cell autoantibodies support the diagnosis. Case Report: A 64 year old man was admitted to our hospital for chronic severe diarrhea refractory to outpatient management. Nine months prior to admission, he presented to his outpatient physician with watery, nonbloody diarrhea and crampy abdominal pain. He denied any significant past medical history, medication use, and recent travel. Initial upper and lower endoscopy were performed at an outside institution. EGD with gastric and duodenal biopsies was unremarkable. Colonoscopy was normal endoscopically, but ileal and colonic biopsies showed mildly active ileitis and colitis with crypt abscesses, and the absence of crypt distortion, granulomas, or dysplasia. Three months later, his GI physician diagnosed him with Crohn's disease, and prescribed budesonide, oral mesalamine, and dicyclomine without clinical improvement. Upon referral to NYULMC for admission, the patient reported 15 watery stools daily with weight loss of 70 lbs, and was cachectic and anasarcic on exam. Laboratory results reflected chronic, severe malnutrition and steatorrhea. Diagnostic blood, stool, and imaging tests ruled out infection, neuroendocrine tumors, and lymphoma. Autoantibody screening revealed a positive ANA (1:160) and Scl-70 antibodies. Celiac panel was negative. Repeat endoscopy and colonoscopy revealed normal-appearing mucosa. Random duodenal, terminal ileal, colonic, and rectal biopsies were consistent with AIE, demonstrating villous blunting, complete loss of goblet and Paneth cells, and increased enterocyte apoptosis. Serum was positive for anti-enterocyte antibodies IgG and IgA. Treatment of AIE with prednisone and anti-diarrheals for symptomatic relief resulted in clinical improvement. Conclusion: AIE is rare, with few adult cases reported in the literature. A high clinical suspicion is necessary for diagnosis. Although typically confined to the small bowel, AIE can also affect the colon. Characteristic histological findings are crucial to the diagnosis, lending to the need for a multidisciplinary approach with an experienced GI pathologist.Figure 1Figure 2Figure 3

Crossing of the intestinal barrier by HTLV-1 infected lymphocytes

In several areas of high endemicity, HTLV-1 can be transmitted from mother to child, through prolonged breast-feeding. This way of transmission is particularly linked to the development of Adult T Cell Leukemia (ATL). In this context, we studied the mechanisms of HTLV-1 transmission through the digestive tract. We previously demonstrated in an in vitro model of compartimentalized co-culture device of human enterocytes, lymphocytes and dendritic cells, that HTLV-1 was unable to infect enterocytes, or disrupt tight cell junctions, but could rather be transported by transcytosis to infect sub-epithelial dendritic cells (Martin-Latil et al., Blood, 2012). Since HTLV-1 infection has been shown to be more efficient through cell-associated virions, we also focused on the crossing of HTLV-1 infected lymphocytes through the intestinal barrier. We thus studied the migration of HTLV-1 infected lymphocytes across the intestinal epithelium, both ex vivo (on intestinal explant model) and in vitro (on a monolayer of human enterocytic cell line cultured on Transwell devices). We demonstrate that, in both systems, infected lymphocytes are able to cross the epithelial barrier, with increased capacities compared to uninfected lymphocytes. Our work suggests that HTLV-1 infected lymphocytes efficiently cross the intestinal barrier, which could constitute an additional way for mother to child HTLV-1 transmission during breast-feeding.