Liver sections (5 µm) were incubated with 25 ng

of each oligonucleotide added to 50 μL of hybridization buffer containing 20% formamide for 90 minutes at 46°C before washing with the same stringency. Signal specificity was demonstrated by comparing to the nonrelated Cy3-labeled control NONEUB-338 oligonucleotide. Paraffine liver sections from patients with PSC (n = 18), autoimmune hepatitis (AIH; n = 5), nonalcoholic steatohepatitis (NASH; n = 5), ASH (n = 3), and PBC (n = 2) were stained for IL-17A with antihuman IL-17A antibodies (Abs) (IL-17A [H-132]: sc-7927; Santa Cruz Biotechnology, GS 1101 Heidelberg, Germany) and an Envision kit (EnVision+ System-HRP; Dako, Hamburg, Germany), according to the manufacturer’s instructions. Comparison between groups was performed with one-way analysis of variance followed by Bonferroni’s multiple comparison or by Dunn’s multiple comparison test, depending on whether or not variables were normally distributed. Normal distribution was assessed by Kolmogorov-Smirnov’s test. Significance is indicated as P < 0.05. All Selleckchem EX 527 horizontal bars represent the median. Because inflammation in PSC is centered around bile ducts, we first investigated whether bile of PSC patients may be colonized with microbes. Therefore, bile was obtained during ERCP from 58 PSC patients. Microbial

cultures could be grown from 41 of 58 individual bile specimens. In 19 of 41 cases, more than one microbial species was detectable (Fig. 1). Staphylococci (coagulase negative: 13×; S. aureus: 5×), streptococci (enterococci: 12×; α-hemolytic: 7×), and C. albicans (12×) were the main isolates detected. Because previous ERCP may be a risk factor for biliary bacterial colonization, we determined the rate of biliary interventions before bile sampling. In 23 of 41 cases of positive microbial bile cultures, ERCP had been performed previously. However, 24% of the analyzed selleck compound patients with PSC had positive microbial cultures without previous manipulation of the biliary tract. To investigate whether bacteria can

be found not only in bile fluid, but also in liver tissue, liver sections from 6 PSC, 5 hepatitis C virus (HCV), and 4 AIH patients were stained for bacterial 16S rRNA using FISH. All 6 PSC patients showed bacterial 16S rRNA within portal tracts, whereas none of the 5 HCV patients and only 1 of 4 AIH patients showed positive staining (Fig. 2). To exclude an effect of previous endoscopic intervention on these findings, another set of liver sections obtained from 7 patients with PSC in whom previous ERCP could be excluded was investigated, where 6 of 7 stained positive for bacterial 16S rRNA. These findings confirm previous reports that bile of patients with PSC is frequently colonized with pathogens, including Candida, even in the absence of earlier endoscopic intervention.