Toll‐like receptors (TLR) recognize bacterial and viral components, but direct interaction of receptor and ligand is unclear. Here, we demonstrate that TLR9 binds directly and sequence‐specifically to single‐stranded unmethylated CpG‐DNA containing a phosphodiester backbone. TLR9‐CpG‐DNA interaction occurs at the acidic pH (6.5–5.0) found in endosomes and lysosomes. By sequence comparison we identified a potential CpG‐DNA binding domain homologous to that described for methyl‐CpG‐DNA binding proteins. Amino acid substitutions in this region abrogated CpG‐DNA binding and led to loss of NF‐κB activation. Furthermore, chloroquine and quinacrine, therapeutic agents for autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, directly blocked TLR9‐CpG‐DNA interaction but not TLR2‐Pam3Cys binding. Our results demonstrate direct binding of TLR9 to CpG‐DNA and suggest that the therapeutic activity of chloroquine and quinacrine in autoimmune diseases may be due to its activity as a TLR9 antagonist and inhibitor of endosomal acidification.

Purpose: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well‐characterized “real life” sample that can be used as reference standard in urine clinical proteomics studies.Experimental design: We report on the generation of male and female urine samples that are extensively characterized by different platforms and methods (CE‐MS, LC‐MS, LC‐MS/MS, 1‐D gel analysis in combination with nano‐LC MS/MS (using LTQ‐FT ultra), and 2‐DE‐MS) for their proteome and peptidome. In several cases analysis involved a definition of the actual biochemical entities, i.e. proteins/peptides associated with molecular mass and detected PTMs and the relative abundance of these compounds.Results: The combination of different technologies allowed coverage of a wide mass range revealing the advantages and complementarities of the different technologies. Application of these samples in “inter‐laboratory” and “inter‐platform” data comparison is also demonstrated.Conclusions and clinical relevance: These well‐characterized urine samples are freely available upon request to enable data comparison especially in the context of biomarker discovery and validation studies. It is also expected that they will provide the basis for the comprehensive characterization of the urinary proteome.

Early detection of malignant biliary tract diseases, especially cholangiocarcinoma (CC) in patients with primary sclerosing cholangitis (PSC), is very difficult and often comes too late to give the patient a therapeutic benefit. We hypothesize that bile proteomic analysis distinguishes CC from nonmalignant lesions. We used capillary electrophoresis mass spectrometry (CE-MS) to identify disease-specific peptide patterns in patients with choledocholithiasis (n = 16), PSC (n = 18), and CC (n = 16) in a training set. A model for differentiation of choledocholithiasis from PSC and CC (PSC/CC model) and another model distinguishing CC from PSC (CC model) were subsequently validated in independent cohorts (choledocholithiasis [n = 14], PSC [n = 18] and CC [n = 25]). Peptides were characterized by sequencing. Application of the PSC/CC model in the independent test cohort resulted in correct exclusion of 12/14 bile samples from patients with choledocholithiasis and identification of 40/43 patients with PSC or CC (86% specificity, 93% sensitivity). The corresponding receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.93 (95% confidence interval [CI]: 0.82-0.98, P = 0.0001). The CC model succeeded in an accurate detection of 14/18 bile samples from patients with PSC and 21/25 samples with CC (78% specificity, 84% sensitivity) in the independent cohort, resulting in an AUC value of 0.87 (95% CI: 0.73-0.95, P = 0.0001) in ROC analysis. Eight out of 10 samples of patients with CC complicating PSC were identified. Conclusion: Bile proteomic analysis discriminates benign conditions from CC accurately. This method may become a diagnostic tool in future as it offers a new possibility to diagnose malignant bile duct disease and thus enables efficient therapy particularly in patients with PSC. (Hepatology 2010;)