Bile proteomic profiles differentiate cholangiocarcinoma from primary sclerosing cholangitis and choledocholithiasis.

UNLABELLED: 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.

Lipopolysaccharide-mediated mast cell activation induces IFN-gamma secretion by NK cells.

Mast cells (MCs) that are well known for their important effector function in IgE-associated immune responses play a key role in innate immune defenses. In this study, we investigate the interaction between MCs and NK cells in vitro and in vivo. We show that mouse bone marrow-derived cultured MCs activated with LPS, polyinosinic-polycytidylic acid, or CpG can stimulate NK cells to secrete increasing concentrations of IFN-gamma. MCs induce a 20-fold increase in IFN-gamma release from NK cells after LPS stimulation. This enhancement of IFN-gamma secretion is cell contact dependent and TNF-alpha independent. Furthermore, we show that this interaction is in part mediated by OX40 ligand on MCs. NK cell-mediated cytotoxicity was not affected by the presence of MCs. Intracellular IFN-gamma levels in splenic NK cells are significantly decreased after i.p. injection of LPS in mast cell-deficient (C57BL/6 Kit(wsh/wsh)) mice in comparison with wild-type mice. In conclusion, our data show for the first time a direct mast cell-dependent NK cell activation. This interaction might play an important role in innate immune defense, as it is dependent on the presence of stimulators relevant in innate immune responses.

The nuclear migration protein NUDF/LIS1 forms a complex with NUDC and BNFA at spindle pole bodies.

Nuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC in Aspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (for binding to NUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.

Role of gastrointestinal eosinophils in inflammatory bowel disease and intestinal tumours.

Eosinophils have been implicated in the pathogenesis of inflammatory bowel disease (IBD). Immunohistopathological studies have revealed accumulation and activation of eosinophils in actively inflamed intestinal mucosa of Crohn's disease and ulcerative colitis patients. Elevated levels of chemokines relevant for eosinophil chemotaxis and mediator release from eosinophils can be detected in serum and faeces of patients with active IBD. Animal studies have revealed that abrogation of chemokines (such as eotaxin) promoting eosinophil chemotaxis and circulation results in decreased severity of murine experimental colitis, suggesting a pro-inflammatory role for eosinophils in IBD. Furthermore, selective deletion of certain eosinophil-specific granule products results in attenuation of experimental intestinal inflammation. Shortly after their initial discovery by Ehrlich, eosinophils have been associated with intestinal tumours. However, as only very few studies have addressed the role of eosinophils in intestinal cancerogenesis, their impact on intestinal tumour development remains obscure; in particular, functional data are missing.