Human recombinant activated MMP-1 and -2 were used for calibration. Extraction procedures were performed in the presence of EDTA-free concerning complete protease inhibitor (Roche) to prevent ex vivo MMP activation (37). Substrate gel zymography. Substrate gel zymography was performed as outlined (37). Briefly, liver extracts (see above) or cell lysates at 20 ��g protein/lane were separated on 10% polyacrylamide gels copolymerized with 1 mg/ml gelatin, incubated in activity buffer for 24 h, stained with 0.25% Coomassie Blue, and destained in acetic acid/methanol/H2O (10:1:89). Proteolytic bands corresponding to MMP-2 and MMP-9 were identified and quantified as described (37). Substrate in situ zymography. Substrate in situ zymography was performed as detailed (37). Briefly, liver sections were dried and overlaid with 0.

1 mg/ml DQ-gelatin in MMP activity buffer supplemented with 0.5% low-melt agarose, covered with coverslips, gelled at 4��C for 1 h, and incubated at room temperature for 2�C16 h. Images were documented on a Nikon E800 photodocumentation microscope. Live cell matrix-degrading activities. Live cell matrix-degrading activities were determined as follows. Freshly isolated rat peritoneal macrophages or cell lines representing other liver cell types were plated in 96-well culture plates at constant density (3 �� 104 cells/well) in phenol red and serum-free DMEM (1% penicillin/streptomycin). Gelatinase and collagenase activities were measured as an absolute increase in fluorescence after addition of self-quenched fluorogenic substrates DQ-gelatin or DQ-collagen (0.

02 mg/ml), respectively, as previously described (37). These substrates were added to cells for 1�C16 h, either alone or immediately before addition of the apoptotic cholangiocytes prepared as described above. Statistical Analyses Data are expressed as means �� SE, and statistical analyses were performed using Microsoft excel and GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA). Multiple comparisons were performed by one-way ANOVA. Two planned comparisons were performed to each of the control groups, healthy nonfibrotic rats (sham) and rats at peak of fibrosis (BDL) using the Dunnett’s posttest. Differences among selected experimental groups were compared using the Tukey posttest. P values >0.05 Dacomitinib were considered significant. RESULTS Advanced Secondary Biliary Fibrosis Reverses After Bilio-digestive Anastomosis At week 4 after BDL (peak of fibrosis), serum markers of cholestasis and inflammation were significantly increased (Supplemental Table S2). After RY-anastomosis these parameters rapidly decreased to near normal on day 3 and remained at this level throughout the following 12 wk (Supplemental Table S2).