Primary cell-based phenotypic assays to pharmacologically and genetically study fibrotic diseases in vitro.

Ongoing tissue repair and formation and deposition of collagen-rich extracellular matrix in tissues and organs finally lead to fibrotic lesions and destruction of normal tissue/organ architecture and function. In the lung, scarring is observed in asthma, chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis to various degrees. At the cellular level immune cells, fibroblasts and epithelial cells are all involved in fibrotic processes. Mechanistically, fibroblast to myofibroblast transformation and epithelial to mesenchymal transition are major drivers of fibrosis. Amongst others, both processes are controlled by transforming growth factor beta-1 (TGFß-1), a growth factor upregulated in idiopathic pulmonary fibrosis lungs. Phenotypic assays with primary human cells and complex disease-relevant readouts become increasingly important in modern drug discovery processes. We describe high-content screening based phenotypic assays with primary normal human lung fibroblasts and primary human airway epithelial cells. For both cell types, TGFß-1 stimulation is used to induce fibrotic phenotypes in vitro, with alpha smooth muscle actin and collagen-I as readouts for FMT and E-cadherin as a readout for EMT. For each assay, a detailed image analysis protocols is described. Treatment of both cell types with TGFß-1 and a transforming growth factor beta receptor inhibitor verifies the suitability of the assays for pharmacological interventions. In addition, the assays are compatible for siRNA and Cas9-ribonucleoprotein transfections, and thus are useful for genetic target identification/validation by modulating gene expression.

Ultrasonographically detected gallbladder polyps: a reason for concern? A seven-year follow-up study.

BACKGROUND: The management of coincidental detected gallbladder polyps (GP) is still nebulous. There are few published data regarding their long-term growth. Objective of the present study was to investigate the prevalence and growth of gallbladder polyps in a survey of unselected subjects from the general population of a complete rural community. METHODS: A total of 2,415 subjects (1,261 women; 1,154 men) underwent ultrasound examination of the gallbladder, in November 1996 as part of a prospective study. Subjects in whom GP were detected at the initial survey underwent follow-up ultrasound examinations after 30 and 84 months. RESULTS: At the initial survey gallbladder polyps were detected in 34 subjects (1.4%; females: 1.1%, range 14 to 74 years; males: 1.7%, range 19 to 63 years). Median diameter was 5 +/- 2.1 mm (range 2 to 10 mm) at the initial survey, 5 mm +/- 2.8 mm (range 2 to 12 mm) at 30 months and 4 +/- 2.3 mm (range 2 to 9 mm) at 84 months. At the time of first follow-up no change in diameter was found in 81.0% (n = 17), reduction in diameter in 4.8% (n = 1) and increase in diameter in 14.3% (n = 3). At the time of second follow-up no increase in polyp diameter was found in 76.9% (n = 10) and reduction in diameter in 7.7% (n = 1). No evidence of malignant disease of the gallbladder was found. CONCLUSION: Over a period of seven years little change was measured in the diameter of gallbladder polyps. There was no evidence of malignant disease of the gallbladder in any subject.