[Show abstract][Hide abstract] ABSTRACT: Hepatic progenitor cells (HPC) play important roles in both liver regeneration and carcinogenesis. Combined hepatocellular-cholangiocarcinoma (CHC), a malignant primary liver tumor with poor prognosis, is thought to be of HPC origin. However, the prognostic significance of this etiology is not well defined. Therefore, we retrospectively investigated the relationship of HPC-related pathological features and long-term outcome in patients with CHC in our department. In a cohort of 80 patients identified between 1997 and 2003, including 70 patients who underwent resection with curative intent, overall survival (OS) and disease-free survival (DFS) were correlated with the proliferative activity of nontumor ductular reaction (DR) and the expression levels of HPC and biliary markers including α-fetoprotein (AFP), keratin 7 (K7), keratin 19 (K19), oval cell (OV)-6, epithelial cell adhesion molecule (EpCAM), and c-Kit in both tumor and nontumor liver. We found that nontumor ductular reactions (DRs), specifically the proliferating cell nuclear antigen (PCNA) labeling index of the ductular reaction (PI-DR), a surrogate for transit-amplifying compartments, was an independent prognostic factor for both OS and DFS. By contrast, intratumoral expression of only one marker, absence of AFP, was associated with OS. PI-DR was also independently associated with synchronous "multicentric occurrence" in hepatocellular carcinoma components, a feature of CHC that may predispose to metachronous multifocal tumorigenesis. Conclusion: Proliferative ductular reaction related to HPC activation is associated with recurrence of CHC. Background HPC activation is strongly associated with multifocal occurrence and related tumor recurrence, highlighting the critical role of background liver disease, a "field effect," in the recurrence of CHC. (Hepatology 2012).
[Show abstract][Hide abstract] ABSTRACT: Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application.
Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3(550-558) and a truncated GPC3(368-548) fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC(50) ranging from 5.0-110.9 nM were identified.
Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized.
[Show abstract][Hide abstract] ABSTRACT: While RNA interference (RNAi) has been widely used to study rotavirus gene function in vitro, the potential therapeutic role for RNAi in vivo has not been explored. To this end, we constructed two recombinant lentiviral vectors containing short hairpin RNA (shRNA) against non-structural protein-4 (NSP4) of bovine rotavirus (BRV), RNAi-351 and RNAi-492. RNAi-351 and RNAi-492 strongly suppressed the transient expression of a FLAG-tagged NSP4 fusion protein in 293T cells. In BRV-susceptible MA104 cells, RNAi-492 more potently silenced NSP4 mRNA than RNAi-351 and combination of the two shRNAs almost completely silenced viral NSP4 gene expression. While 100% of suckling mice exposed to BRV and control shRNA developed severe diarrhoea, no suckling mice exposed to BRV in the presence of RNAi-492 or a combination of RNAi-492/RNAi-351 developed severe diarrhoea, and only 20 and 3.3% developed mild diarrhoea, respectively. In addition, RNAi-492 and RNAi-351 markedly abrogated rotaviral replication in MA104 cells and significantly inhibited BRV replication in mouse pups. These results indicated that shRNAs silencing NSP4 gene had substantial antiviral properties and inhibited replication of BRV in a sequence-specific manner that may have clinical application.
Journal of General Virology 12/2010; 92(Pt 4):945-51. · 3.53 Impact Factor