Congenital tufting enteropathy (CTE) is a devastating diarrheal disease seen in infancy that is typically associated with villous changes and the appearance of epithelial tufts. We previously found mutations in epithelial cell adhesion molecule (EpCAM) to be causative in CTE. We developed a knock-down cell model of CTE through transfection of an EpCAM shRNA construct into T84 colonic epithelial cells to elucidate the in vitro role of EpCAM in barrier function and ion transport. Cells with EpCAM deficiency exhibited decreased electrical resistance, increased permeability, and decreased ion transport. Based on mutations in CTE patients, an in vivo mouse model was developed, with tamoxifen-inducible deletion of exon 4 in Epcam resulting in mutant protein with decreased expression. Tamoxifen treatment of EpcamΔ4/Δ4 mice resulted in pathological features of villous atrophy and epithelial tufts, similar to those in human CTE patients, within 4 days post induction. EpcamΔ4/Δ4 mice also showed decreased expression of tight junctional proteins, increased permeability, and decreased ion transport in the intestines. Taken together, these findings reveal mechanisms that may underlie disease in CTE.Key messagesKnock-down EpCAM cell model of congenital tufting enteropathy was developed.In vivo inducible mouse model was developed resulting in mutant EpCAM protein.Cells with EpCAM deficiency demonstrated barrier and ion transport dysfunction.Tamoxifen-treated EpcamΔ4/Δ4 mice demonstrated pathological features.EpcamΔ4/Δ4 mice showed improper barrier function and ion transport.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-014-1239-x) contains supplementary material, which is available to authorized users.
Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule ( EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers ( Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors ( Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease. NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule ( EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.
Background Foeniculum vulgare, F. vulgare, commonly known as fennel, is believed to be one of the world’s oldest medicinal herbs and has been exploited by people for centuries as a nutritional aid for digestive disorders. In many southeast Asian countries, it is ingested as an after-meal snack, mukhvas, due to its breath-freshening and digestive aid properties. F. vulgare is used in some countries, such as Iran, as a complementary and alternative treatment for inflammatory bowel disease (IBD). Methods This study investigated the effects of fennel seed extract on intestinal epithelium barrier function and the Signal Transducer and Activator of Transcription (STAT) pathway. This pathway is active in inflammatory bowel disease. To study the protective effects of fennel seed extract in vitro, monolayers derived from the T84 colonic cell line were challenged with interferon-gamma (IFN-γ) and monitored with and without fennel seed extract. To complement our in vitro studies, the dextran sodium sulfate induced murine colitis model was employed to ascertain whether the protective effect of fennel seed extract can be recapitulated in vivo. Results Fennel seed extract was shown to exert a protective effect on transepithelial electrical resistance (TEER) in both T84 and murine models and showed increases in tight junction-associated mRNA in T84 cell monolayers. Both models demonstrated significant decreases in phosphorylated STAT1 (pSTAT1), indicating reduced activation of the STAT pathway. Additionally, mice treated with fennel seed showed significantly lower ulcer indices than control mice. Conclusions We conclude barrier function of the gastrointestinal tract is improved by fennel seed extract, suggesting the potential utility of this agent as an alternative or adjunctive therapy in IBD.
Background Non-adherence to biologic therapy in inflammatory bowel disease (IBD) is associated with risk of relapse, immunogenicity, and disease complications. Significant non-adherence prevalence is reported with TNF-antagonists but the risk of non-adherence with newer biologics with better safety profiles is unknown.This study aimed to investigate if IBD patient-preferences favoring biologic discontinuation vary by biologic class and analyze factors associated with such preferences. Methods A convenience sample of 200 adults with IBD on biologic therapy treated at an academic outpatient center was surveyed using a 22-point questionnaire. Patient-preference favoring treatment discontinuation between TNF-antagonist and non-TNF-antagonist biologics [vedolizumab (VDZ)/ustekinumab (UST)] was compared using χ 2 test. Risk factors associated with a preference to discontinue biologic therapy were evaluated using univariable and multivariable logistic regression, and Spearman rank correlation analyses. Results A total of 190 questionnaires were analyzed that contained data on preferences regarding biologic discontinuation [median age 36 years, 62% were females; 63% had Crohn’s disease; 56% were receiving a TNF-antagonist, 31% VDZ and 14% UST]. Overall, 32% patients reported a preference to discontinue biologic treatment with a higher proportion among those receiving a TNF-antagonist compared with VDZ/UST (39.6% vs 21.4%; p<0.01). Current VDZ/UST use was independently associated with a reduced odds of patient-preference favoring biologic discontinuation [adjusted OR: 2.67 (1.42-5.01); p<0.01]. The most concerning factor to patients was the perceived risk of side-effects. Patients on VDZ/UST perceived their therapy to be safer than those receiving a TNF-antagonist (r=0.2, p=0.04). Conclusion Patient-preference favoring treatment discontinuation is improved with VDZ/UST compared with TNF-antagonist biologic therapy.
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