The pathogenesis of fulminant hepatitis B virus (HBV) infection is not well understood. The aim of this study was to investigate whether there is an association between specific viral variants and a fulminant disease course. The entire HBV genomes from the serum of eight patients with fulminant HBV infection and one patient with fulminant hepatitis during reinfection after liver transplantation were investigated. After isolation and amplification of viral DNA by polymerase chain reaction (PCR), plus and minus strands were directly sequenced. Sequence data were analyzed by comparative sequence alignments with 35 and 2 complete HBV genome sequences from patients without and with fulminant hepatitis, respectively. Several point mutations were present in all regions of the genomes. Many nucleotide changes had never or rarely been found in the reported HBV isolates from patients without fulminant hepatitis. A distinct mutation present in all genomes was not identified. Clusters of rare and unique mutations were observed in the enhancer II core promoter region. Mutations previously suggested to be associated with fulminant HBV infection were not consistently found. A precore stop codon mutation at nucleotide position 1896 or an A-to-T mutation at nucleotide position 1762 and a G-to-A mutation at nucleotide position 1764 in the core promoter region were present in four and three cases, respectively. Fulminant HBV infection does not appear to be caused by a specific genomic mutation. However, various mutations clustering in the enhancer II core promoter region may contribute to a fulminant disease course.
Our laboratory has investigated heterotopic hepatocyte transplantation on biodegradable polymer matrices as an experimental treatment for end-stage liver disease. One of the limitations has been survival of sufficient cell mass after transplantation. We hypothesize that in vitro conditioning of cells within polymer matrices prior to implantation may increase hepatocyte survival and function. In this preliminary study we investigated the effect of continuous flow on hepatocytes and sinusoidal endothelial cells on poly-L-lactic acid (PLLA) discs in vitro. Highly porous PLLA discs were manufactured measuring 18 mm diameter by 1 mm thickness using previously described techniques. Hepatocytes were isolated from adult, male Lewis rats (200-300 g) using a two-step collagenase digestion. Sinusoidal endothelial cells were isolated using a two-step collagenase digestion, differential sedimentation, Percoll gradient centrifugation, and selective adherence. PLLA discs were seeded with hepatocytes alone or with co-cultures of hepatocytes and sinusoidal endothelial cells. Seeded discs were then secured within a flow bioreactor chamber and exposed to continuous flow of culture media at a rate of 20 ml/minute through the chamber. Seeded discs placed in static culture conditions served as controls. Specimens seeded with only hepatocytes were harvested at 24 hours, 48 hours, and 168 hours after seeding. Co-culture specimens were harvested after 168 hours. Specimens were viewed under phase-contrast microscopy and then formalin-fixed and prepared for histologic sectioning. Sections were stained with Hematoxylin and Eosin and then analyzed with light microscopy. Hepatocytes under flow conditions formed spheroidal aggregates of cells of 50 to 200 microns in diameter by 24 hours in culture. Hepatocytes in static conditions showed decreased aggregation of cells and spheroid formation was absent. Co-cultured specimens under flow also showed spheroid formation with endothelial cells lining the outside of hepatocyte spheroids. Co-cultured specimens in static culture showed no spheroid formation and no organization between sinusoidal endothelial cells and hepatocytes. These results suggest that continuous flow increases organization of hepatocytes cultured within biodegradable polymer matrices.
Deroofing in combination with an omental transposition flap is a safe and effective therapy for symptomatic solitary liver cysts and can be performed using minimal-access surgical techniques.
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