Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology.

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl- channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl- measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

Plasminogen Activator Inhibitor 1 Is a Novel Faecal Biomarker for Monitoring Disease Activity and Therapeutic Response in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Crohn's disease [CD] and ulcerative colitis [UC] require lifelong treatment and patient monitoring. Current biomarkers have several limitations; therefore, there is an unmet need to identify novel biomarkers in inflammatory bowel disease [IBD]. Previously, the role of plasminogen activator inhibitor 1 [PAI-1] was established in the pathogenesis of IBD and suggested as a potential biomarker. Therefore, we aimed to comprehensively analyse the selectivity of PAI-1 in IBD, its correlation with disease activity, and its potential to predict therapeutic response. METHODS: Blood, colon biopsy, organoid cultures [OC], and faecal samples were used from active and inactive IBD patients and control subjects. Serpin E1 gene expressions and PAI-1 protein levels and localisation in serum, biopsy, and faecal samples were evaluated by qRT-PCR, ELISA, and immunostaining, respectively. RESULTS: The study population comprised 132 IBD patients [56 CD and 76 UC] and 40 non-IBD patients. We demonstrated that the serum, mucosal, and faecal PAI-1 concentrations are elevated in IBD patients, showing clinical and endoscopic activity. In responders [decrease of eMayo ≥3 in UC; or SES-CD  50% in CD], the initial PAI-1 level decreased significantly upon successful therapy. OCs derived from active IBD patients produced higher concentrations of PAI-1 than the controls, suggesting that epithelial cells could be a source of PAI-1. Moreover, faecal PAI-1 selectively increases in active IBD but not in other organic gastrointestinal diseases. CONCLUSIONS: The serum, mucosal, and faecal PAI-1 concentration correlates with disease activity and therapeutic response in IBD, suggesting that PAI-1 could be used as a novel, non-invasive, disease-specific, faecal biomarker in patient follow-up.

Effects of bowel cleansing on the composition of the gut microbiota in inflammatory bowel disease patients and healthy controls.

Background: In patients with inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC), numerous cases of exacerbations could be observed after colonoscopy, raising the possible pathogenetic effect of colonic microbiota alterations in IBD flare. Objectives: We aimed to investigate the changes in the fecal microbiota composition in IBD patients influenced by the bowel preparation with sodium picosulfate. Design: We enrolled patients with IBD undergoing bowel preparation for colonoscopy in the prospective cohort study. The control group (Con) comprised non-IBD patients who underwent colonoscopy. Clinical data, blood, and stool samples were collected before colonoscopy (timepoint A), 3 days later (timepoint B), and 4 weeks later (timepoint C). Methods: Disease activity and gut microbiota changes were assessed at each timepoint. Fecal microbiota structure - at family level - was determined by sequencing the V4 region of the 16S rRNA gene. Statistical analysis included differential abundance analysis and Mann-Whitney tests. Results: Forty-one patients (9 CD, 13 UC, and 19 Con) were included. After bowel preparation, alpha diversity was lower in the CD group than in the UC (p = 0.01) and Con (p = 0.02) groups at timepoint B. Alpha diversity was significantly higher in the UC group than in the CD and Con (p = 0.03) groups at timepoint C. Beta diversity difference differed between the IBD and Con (p = 0.001) groups. Based on the differential abundance analysis, the Clostridiales family was increased, whereas the Bifidobacteriaceae family was decreased in CD patients compared to the Con at timepoint B. Conclusions: Bowel preparation may change the fecal microbial composition in IBD patients, which may have a potential role in disease exacerbation after bowel cleansing.

Orai1 calcium channel inhibition prevents progression of chronic pancreatitis.

Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.

Advances in the optimization of therapeutic drug monitoring using serum, tissue and faecal anti-tumour necrosis factor concentration in patients with inflammatory bowel disease treated with TNF-α antagonists.

INTRODUCTION: The relationship between clinical outcomes and serum anti-TNF levels is controversial. The aim of this study was to perform simultaneous analyses of serum, mucosal, and fecal anti-TNF-α levels. METHODS: Consecutive IBD patients who received maintenance anti-TNF-α therapy were enrolled. The number of TNF-α positive cells in the mucosa was detected using immunofluorescent labeling on biopsy samples. Serum, mucosal and fecal anti-TNF-α, serum anti-drug antibody, and fecal calprotectin levels were determined using ELISA. Each patient underwent body composition analysis as well. RESULTS: Data of 50 patients were analyzed. The number TNF-α positive cells was significantly higher in the inflamed part of the colon than in the un-inflamed part of the colon. Tissue and fecal drug levels did not show any association with serum drug levels; moreover, serum anti-TNF concentration did not correlate with endoscopic activity. Mucosal anti-TNF levels were higher only in IFX-treated patients in remission and IFX-treated patients with detectable fecal anti-TNF had lower tissue drug levels. Presence of the drug in the feces was significantly different according to disease activity. CONCLUSION: Fecal drug concentration is suggested to be a better predictor of endoscopic activity and loss of response, and fecal drug monitoring may improve the estimation accuracy of tissue drug levels.