A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells

Evaluation of the binding and uptake of an antibody in liver non-parenchymal cells (NPC), including liver sinusoidal endothelial cells, is important for revealing its pharmacokinetic (PK) behavior, since NPC has important roles in eliminating an antibody from the blood via the Fc fragment of IgG receptor IIB (FcγRIIB). However, there is currently no in vitro quantitative assay using NPC. This study reports on the development of a cell-based assay for evaluating the binding and uptake of such an antibody using liver NPC of mice and monkeys. In mice, the FcγRIIB-expressing cells were identified in the CD146-positive and CD45-negative fraction by flow cytometry. A titration assay was performed to determine the PK parameters, and the obtained parameter was comparable to that determined by the fitting of the in vivo PK. This approach was also extended to NPC from monkeys. The concentration-dependent binding and uptake was measured to determine the PK parameters using monkey NPC, the FcγRIIB-expressing fraction of which was identified by CD31 and CD45. The findings presented herein demonstrate that the in vitro liver NPC assay using flow cytometry is a useful tool to determine the binding and uptake of biologics and to predict the PK.

Ultrastructural Characteristics of Rat Hepatic Oval Cells and Their Intercellular Contacts in the Model of Biliary Fibrosis: New Insights into Experimental Liver Fibrogenesis

Purpose. Recently, it has been emphasized that hepatic progenitor/oval cells (HPCs) are significantly involved in liver fibrogenesis. We evaluated the multipotential population of HPCs by transmission electron microscope (TEM), including relations with adherent hepatic nonparenchymal cells (NPCs) in rats with biliary fibrosis induced by bile duct ligation (BDL). Methods. The study used 6-week-old Wistar Crl: WI(Han) rats after BDL for 1, 6, and 8 weeks. Results. Current ultrastructural analysis showed considerable proliferation of HPCs in experimental intensive biliary fibrosis. HPCs formed proliferating bile ductules and were scattered in periportal connective tissue. We distinguished 4 main types of HPCs: 0, I, II (bile duct-like cells; most common), and III (hepatocyte-like cells). We observed, very seldom presented in literature, cellular interactions between HPCs and adjacent NPCs, especially commonly found transitional hepatic stellate cells (T-HSCs) and Kupffer cells/macrophages. We showed the phenomenon of penetration of the basement membrane of proliferating bile ductules by cytoplasmic processes sent by T-HSCs and the formation of direct cell-cell contact with ductular epithelial cells related to HPCs. Conclusions. HPC proliferation induced by BDL evidently promotes portal fibrogenesis. Better understanding of the complex cellular interactions between HPCs and adjacent NPCs, especially T-HSCs, may help develop antifibrotic therapies in the future.

The Value of Circulating Nogo-B for Evaluating Hepatic Functional Reserve in Patients with Cirrhosis

Objective. To examine Nogo-B in liver tissues and plasma of patients with liver cirrhosis and associate them with various clinical parameters.Materials and Methods. Nogo-B protein expression was examined by immunohistochemistry in 24 human fibrotic/cirrhotic liver specimens and 10 healthy controls. We determined plasma Nogo-B levels by enzyme-linked immunosorbent assay in 301 patients with liver cirrhosis and 153 healthy controls, and then analyzed various clinical parameters.Results. Nogo-B was mainly expressed in nonparenchymal cells in the liver and was marked increased in liver with significant fibrosis/cirrhosis compared to controls. Moreover, Metavir F4 showed a higher level of expression than F2. Plasma Nogo-B levels were significantly higher in cirrhotic patients than in healthy controls and were the highest in Child-Pugh class C patients. Plasma Nogo-B levels were positively correlated with Child-Pugh scores. However, there was no relationship between plasma Nogo-B levels and etiology of liver diseases, ALT, AST, platelet counts, and the severity of esophagogastric varices.Conclusions. Nogo-B is mainly expressed in hepatic nonparenchymal cells and is present in plasma. Abnormally high plasma levels of Nogo-B are associated with hepatic cirrhosis and Child-Pugh score, but not correlated with the grade of liver inflammation or portal hypertension. Plasma Nogo-B may be a novel surrogate marker to reflect liver function reserve.