Application and Comparison of Supervised Learning Strategies to Classify Polarity of Epithelial Cell Spheroids in 3D Culture.

Three-dimensional culture systems that allow generation of monolayered epithelial cell spheroids are widely used to study epithelial function in vitro. Epithelial spheroid formation is applied to address cellular consequences of (mono)-genetic disorders, that is, ciliopathies, in toxicity testing, or to develop treatment options aimed to restore proper epithelial cell characteristics and function. With the potential of a high-throughput method, the main obstacle to efficient application of the spheroid formation assay so far is the laborious, time-consuming, and bias-prone analysis of spheroid images by individuals. Hundredths of multidimensional fluorescence images are blinded, rated by three persons, and subsequently, differences in ratings are compared and discussed. Here, we apply supervised learning and compare strategies based on machine learning versus deep learning. While deep learning approaches can directly process raw image data, machine learning requires transformed data of features extracted from fluorescence images. We verify the accuracy of both strategies on a validation data set, analyse an experimental data set, and observe that different strategies can be very accurate. Deep learning, however, is less sensitive to overfitting and experimental batch-to-batch variations, thus providing a rather powerful and easily adjustable classification tool.

Flotillin-2 expression in the human gut: from a cell model to human tissue in health and inflammatory bowel diseases.

BACKGROUND AND AIMS: The etiopathogenesis of inflammatory bowel diseases (IBD) remains largely unexplained. Flotillins (flotillin-1 and flotillin-2) are ubiquitous proteins which have been linked to inflammation and regeneration. We hypothesized that alterations in the expression of flotillin-2 in enterocytes may be related to the pathogenesis of IBD as a classical example of an inflammatory disorder of mostly unknown origin. METHODS: Cell and tissue localization of flotillin-2 (and -1) were investigated by immunofluorescent staining in 1. polarized and unpolarized CaCo-2w cells as a model of human enterocytes (native and after TNFα stimulation) and 2. intestinal biopsies from controls, patients with ulcerative colitis (UC) and patients with Crohn's disease (CD). For quantification of flotillin-2, we analyzed its expression in ileal and colonic biopsies from controls, UC patients and CD patients using real-time RT-PCR, Western blot and indirect immunofluorescence. RESULTS: In polarized CaCo-2w cells and human enterocytes in biopsies, flotillins were localized at the basolateral membrane and on subapical vesicles, but not in the apical membrane. Flotillin-2 expression did not differ between UC patients, CD patients and controls. However, it was significantly higher in colonic biopsies compared to ileal biopsies in all groups. CONCLUSIONS: By virtue of its abundant expression in enterocytes, flotillin-2 must have an essential function in intestinal physiology, especially in the colon. Yet our data could not link flotillin-2 to the pathogenesis of IBD.

Alterations of phospholipid concentration and species composition of the intestinal mucus barrier in ulcerative colitis: a clue to pathogenesis.

BACKGROUND: Phospholipids are essential for the normal function of the intestinal mucus barrier. The objective of this study was to systematically investigate phospholipids in the intestinal mucus of humans suffering from inflammatory bowel diseases, where a barrier defect is strongly supposed to be pathogenetic. METHODS: Optimal mucus recovery was first validated in healthy mice and the method was then transferred to the endoscopic acquisition of ileal and colonic mucus from 21 patients with ulcerative colitis (UC), 10 patients with Crohn's disease (CD), and 29 healthy controls. Nano-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to determine phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in lipid extracts of mucus specimens. RESULTS: Human and rodent mucus contained very similar phospholipid species. In the ileal and colonic mucus from patients suffering from UC, the concentration of PC was highly significantly lower (607 +/- 147 pmol/100 microg protein and 745 +/- 148 pmol/100 microg protein) compared to that of patients with CD (3223 +/- 1519 pmol/100 microg protein and 2450 +/- 431 pmol/100 microg protein) and to controls (3870 +/- 760 pmol/100 microg protein and 2790 +/- 354 pmol/100 microg protein); overall, P = 0.0002 for ileal specimens and P < 0.0001 for colonic specimens. Independent of disease activity, patients suffering from UC showed an increased saturation grade of PC fatty acid residues and a higher LPC-to-PC ratio. CONCLUSIONS: The intestinal mucus barrier of patients with UC is significantly altered concerning its phospholipid concentration and species composition. These alterations may be very important for the pathogenesis of this disease and underline new therapeutic strategies.