Development of an anti-human complement C6 monoclonal antibody that inhibits the assembly of membrane attack complexes.

Membrane attack complexes (MACs; C5b-9) assembled after complement activation can directly injure self-tissues, leading to various diseases. Eculizumab, a monoclonal antibody (mAb) against complement component C5, is being used in the clinic to treat diseases in which MAC-mediated tissue damage is a primary cause. However, C5 is not a selective target for MAC assembly inhibition, and some patients respond incompletely or not at all to the eculizumab treatment. Therefore, C6, the next essential component in the terminal pathway of complement activation, may be an alternative target for the selective inhibition of MAC formation. Surprisingly, few reports describe a functional blockade of C6 using a specific mAb. Here, we report the development of an anti-human C6 mAb (clone 1C9) that recognizes C6 both in free circulation and within C5b6 complexes. This mAb blocked C7 binding to C5b6 complexes and consequently inhibited MAC formation and protected affected paroxysmal nocturnal hemoglobinuria patient red blood cells from MAC-mediated damage in vitro. In addition, this mAb cross-reacts with rhesus monkey but not mouse complement C6. Finally, 1C9 significantly reduced human complement-mediated intravascular hemolysis in vivo in a mouse model. These results suggest that the anti-C6 mAb holds promise as a new therapeutic agent that selectively targets MAC for many complement-mediated pathological conditions.

Comparative effectiveness of pyruvate kinase M2 in bile, serum carbohydrate antigen 19-9, and biliary brushings in diagnosing malignant biliary strictures.

BACKGROUND: The role of M2-PK (pyruvate kinase) in bile has not been studied in comparison with brushings and carbohydrate antigen (CA) 19-9 in the diagnosis of malignant biliary strictures. AIM: To compare the diagnostic accuracy of biliary M2-PK with cytology and serum CA 19-9 METHODS: In this prospective cross-sectional study, bile was aspirated in 74 patients (discovery and validation cohort) undergoing endoscopic retrograde cholangiopancreatography. Levels of M2-PK were measured in bile and compared to brushings for cytology and CA 19-9. RESULTS: In the discovery cohort, the median bile M2-PK levels were significantly elevated in patients with malignant biliary strictures [187.9 U/l (interquartile range (IQR) 3.5, 3626.8)] compared to those with benign biliary conditions and primary sclerosing cholangitis [0 U/l (IQR 0, 15)] (P = 0.007). A M2-PK cutoff value of 109.1 U/l distinguished malignant from benign conditions with a sensitivity and specificity of 52.9 and 94.1 %, respectively, and area under curve (AUC) of 0.77. The sensitivity of CA 19-9 and brushings in diagnosing cancer was 52.9 % and 11.1 % and specificity 94.1 and 100 %, respectively. The presence of elevated M2-PK >109.1 U/l or CA 19-9 >33 U/ml or positive brushing was 88.2 % sensitive and 88.2 % specific, AUC of 0.89 in the diagnosis of malignancy. The diagnostic accuracy was confirmed in the validation cohort. CONCLUSIONS: As a stand-alone factor, none of the markers were able to distinguish benign from malignant biliary strictures with a high sensitivity. However, a combination was highly sensitive in diagnosing malignant biliary strictures.

IgG4 Levels in Bile for Distinguishing IgG4-Associated Cholangiopathy from Other Biliary Disorders: A Single Blinded Pilot Study.

BACKGROUND/AIMS: Immunoglobulin G4 (IgG4)-associated cholangiopathy (IAC) is an inflammatory disease and may mimic primary sclerosing cholangitis (PSC), cholangiocarcinoma (CCA), or pancreatic cancer on cholangiography. We investigated whether IgG4 levels in bile aspirated during endoscopic retrograde cholangiopancreatography (ERCP) can distinguish IAC from PSC, CCA, and pancreatic cancer. METHODS: Bile was aspirated directly from the common bile duct during ERCP in patients with IAC prior to steroid therapy. For control purposes, bile was obtained from patients with PSC, CCA, pancreatic cancer, and benign biliary conditions (sphincter of oddi dysfunction/choledocholithiasis). RESULTS: Biliary IgG4 levels were measured in 54 patients. The median bile IgG4 levels were markedly elevated in patients with IAC (5.5 mg/dL; interquartile range [IQR], 5.1 to 15.6) as compared to patients with benign biliary conditions (0 mg/dL; IQR, 0 to 0.1; p=0.003). The median biliary IgG4 levels in PSC, CCA, and pancreatic cancer were 1.2 (IQR, 0.2 to 3.8), 0.9 (IQR, 0.2 to 3.4), and 0.2 mg/dL (IQR, 0.1 to 0.8), respectively. A cutoff value of 3.8 mg/dL distinguished IAC from PSC and CCA patients with 100% and 76.9% sensitivity and specificity, respectively. CONCLUSIONS: The results of this pilot study suggest that measurement of biliary IgG4 levels may have clinical value in distinguishing patients with IAC from biliary disorders that can mimic IAC.

Vascular endothelial growth factor levels in bile distinguishes pancreatic cancer from other etiologies of biliary stricture: a pilot study.

BACKGROUND: Determining the benign or malignant nature of biliary strictures can be challenging. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. OBJECTIVE: The purpose of this study was to investigate whether VEGF levels in bile aspirated during endoscopic retrograde cholangiography (ERCP) can distinguish pancreatic cancer from other causes of biliary stricture. METHODS: Bile was directly aspirated in 53 consecutive patients from March 2012 to October 2012 during ERCP from the common bile duct including 15 with pancreatic cancer, 18 with primary sclerosing cholangitis (PSC), nine with cholangiocarcinoma (CCA), and 11 with benign biliary conditions (sphincter of Oddi and choledocholihiasis). Levels of VEGF in bile were measured. The diagnostic performance was then validated in a second, independent validation cohort of 18 patients (pancreatic cancer n = 10, benign n = 8). RESULTS: A total of 53 consecutive patients were recruited. The median bile VEGF levels were significantly elevated in patients with pancreatic cancer (1.9 ng/ml (interquartile range [IQR] 0.7, 2.2) compared to those with benign biliary conditions (0.3 ng/ml [IQR 0.2, 0.6]; p < 0.001), PSC (0.7 ng/ml [IQR 0.5, 0.9]; p = 0.02) or CCA (0.4 ng/ml [IQR 0.1, 0.5]; p < 0.001). A VEGF cut-off value of 0.5 ng/ml distinguished pancreatic cancer from CCA with a sensitivity and specificity of 93.3 and 88.9 %, respectively, and area under curve (AUC) of 0.93, and from benign conditions with a sensitivity and specificity of 93.3 and 72.7 %, respectively, with AUC of 0.89. The diagnostic accuracy of biliary VEGF was confirmed in the second independent validation cohort. CONCLUSIONS: This study suggests that measurement of biliary VEGF-1 levels distinguishes patients with pancreatic cancer from other etiologies of biliary stricture. This may be particularly relevant in approaching patients with indeterminate biliary stricture.

Tumor necrosis factor alpha (TNF-alpha) receptor-II is required for TNF-alpha-induced leukocyte-endothelial interaction in vivo.

Tumor necrosis factor-alpha (TNF-alpha) binds to 2 distinct cell-surface receptors: TNF-alpha receptor-I (TNFR-I: p55) and TNF-alpha receptor-II (TNFR-II: p75). TNF-alpha induces leukocyte adhesion molecules on endothelial cells (ECs), which mediate 3 defined steps of the inflammatory response; namely, leukocyte rolling, firm adhesion, and transmigration. In this study, we have investigated the role of p75 in TNF-alpha-induced leukocyte adhesion molecules using cultured ECs derived from wild-type (WT), p75-null (p75-/-), or p55-null (p55-/-) mice. We observed that p75 was essential for TNF-alpha-induced E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) expression. We also investigated the putative role of p75 in inflammation in vivo using an intravital microscopic approach with a mouse cremaster muscle model. TNF-alpha-stimulated leukocyte rolling, firm adhesion to ECs, and transmigration were dramatically reduced in p75-/- mice. Transplanted WT cremaster in p75-/- mice showed a robust leukocyte rolling and firm adhesion upon TNF-alpha activation, suggesting that the impairment in EC-leukocyte interaction in p75-/- mice is due to EC dysfunction. These results demonstrate, for the first time, that endothelial p75 is essential for TNF-alpha-induced leukocyte-endothelial-cell interaction. Our findings may contribute to the identification of novel p75-targeted therapeutic approaches for inflammatory diseases.

Cell sorting by one gravity SPLITT fractionation.

The need for innovative separative techniques suitable for the fractionation of biomaterials prompted this investigation into the performance of the gravitational split-flow thin channel (G-SPLITT) system as a cell sorter. The rigorous mathematical description of the separation mechanism allows achievement of fast separation of several million myeloma cells from healthy splenocytes using flow conditions calculated from theory. Separation in G-SPLITT is based on differences in sedimentation rate. For accurate prediction of the optimal working conditions, this parameter was directly measured by cell tracking velocimetry rather than relying on a measure of diameter (by Multisizer) and an assumed density for each cell population. We also discuss the influence of different flow conditions on the effectiveness of separation.