None

of the IAC patients showed signs of malignant disease Selleck Pifithrin-�� (hematological, pancreatico biliary, or other) observed to date (mean follow-up, 16 months; range, 8-20 months). From all newly diagnosed patients, peripheral blood was drawn before the start of treatment with prednisolone (median, 40 mg/day; range, 20-40 mg/day). After 4 and 8 weeks, additional blood samples were collected. Two patients underwent endoscopic retrograde cholangio pancreatography for stent replacement, which allowed the collection of a duodenal papilla biopsy (ampulla of Vater), and paired peripheral blood. Patients included in the PSC control group were selected based on a diagnosis of PSC compliant with the current European Association for the Study of the Liver guidelines16 and including a history of colitis; one patient with elevated serum IgG4 underwent additional short-term prednisolone treatment without biochemical response. Patients included in the malignancy control group had a histologically proven hepatobiliary malignancy (pancreatic cancer or bile duct cancer) (Table 2). (An extended overview of clinical characteristics of all analyzed patients is provided Regorafenib chemical structure in Supporting Table 2.) Anonymous healthy individuals were age- and sex-matched to the IAC patient group. The study was performed according to the Declaration of Helsinki and was approved

by the local medical ethical committee of the Academic Medical Center (METC10/007). All patients provided written informed consent prior to inclusion in the study. Peripheral blood was collected and stored using PAXGene Blood RNA 上海皓元医药股份有限公司 tubes according

to the manufacturer’s instructions (catalog #762165, PreAnalytiX, Breda, The Netherlands). Isolation of total RNA was performed using the PAXGene Blood RNA isolation kit (catalog #762174, Qiagen, Venlo, The Netherlands). Biopsies of the duodenal papilla (ampulla of Vater) during endoscopic retrograde cholangio pancreatography were immediately preserved in RNAlater reagent (Qiagen) and stored at −80°C until use. Total RNA was isolated using polytron homogenizer in the presence of STAT60 reagent as described.17 cDNA was synthesized with 250 ng total RNA input using Superscript III RT (Invitrogen Life Technologies, Carlsbad, CA). The linear amplification used in this study was based on the protocol used for T cells and B cells in previous studies.17, 18 In the first step of the protocol, a linear amplification of the complete immunoglobulin repertoire was performed using a primer set covering all functional Vheavy genes of the BCR (Supporting Fig. 4) (primer sequences available on request). The Vheavy-primers contained a primer B sequence required for Amplicon sequencing according to the 454 titanium protocol (version 2010; Roche Diagnostics, Mannheim, Germany).