The significant improvement of survival times and pathological parameters by bioartificial liver with recombinant HepG2 in porcine liver failure model.

We developed a bioartificial liver (BAL) containing human hepatoblastoma cell line, HepG2, with the addition of ammonia removal activity by transfecting a glutamine synthetase (GS) gene and estimated the efficacy using pigs with ischemic liver failure. GS-HepG2 cells showed 15% ammonia removal activity of porcine hepatocytes, while unmodified HepG2 had no such activity. The established GS-HepG2 cells were grown in a circulatory flow bioreactor to 3.5-4.1 x 10(9) cells. Survival time of the animals treated with GS-HepG2 BAL was significantly prolonged compared to the cell-free control (14.52 +/- 5.24 h vs. 8.53 +/- 2.52 h) and the group treated with the BAL consisting of unmodified wild-type HepG2 (9.58 +/- 4.52 h). Comparison showed the cell-containing BAL groups to have significantly fewer incidences of increased brain pressure. Thus, the GS-HepG2 BAL treatment resulted in a significant improvement of survival time and pathological parameters in pigs with ischemic liver failure.

Double-compartment cell culture apparatus: construction and biochemical evaluation for bioartificial liver support.

Functional demands on a bioartificial liver support (BAL) device are not limited to biosynthetic activities, but must also encompass metabolic removal of potentially toxic substances. For most BALs, however, the concept and design are exclusively directed to biosynthetic support. To add the ability to metabolize and remove toxic substances, we designed a double-compartment cell culture apparatus (DCCA). Two compartments are separated from each other by a compact epithelial cell sheet spread over a synthetic microporous membrane. When a renal proximal convoluted tubular cell line that had been transduced with the human multidrug-resistant (MDR) gene, PCTL-MDR, was introduced into one of the compartments (hereafter referred to as the "inner" compartment) of the DCCA, a compact cellular monolayer was formed on the membrane. Ammonium ions passed across the membrane, but glucose and its metabolite lactate could not, indicating that the DCCA allowed selective transportation of cellular metabolites. In addition to PCTL-MDR, HepG2, a cell line of hepatic-origin, transduced with CYP3A4 (designated GS-3A4-HepG2), was seeded on the opposite side of the membrane, and the metabolism and transportation of lidocaine were studied. The lidocaine metabolite, monoethylglycinexylidide, was detected in the inner compartment across the PCTL-MDR cell layered membrane, indicating that metabolism and the selective transportation of metabolites between the two compartments occurred by cooperation of renal and hepatic cells. These results suggest that this type of DCCA represents a novel BAL that possesses biotransporting activities, as well as biosynthetic and metabolic activities.

The bioreactor with CYP3A4- and glutamine synthetase-introduced HepG2 cells: treatment of hepatic failure dog with diazepam overdosage.

A novel recombinant human hepatic cell line, CYP3A4- and glutamine synthetase (GS, an enzyme which converts ammonium ion and glutamate to glutamine)-introduced HepG2 (HepG2-GS-CYP3A4), was established. Its usefulness in a large-scale culture with a circulatory bioreactor in vitro and in dog models of ischemic hepatic failure with acute diazepam (DZP, a substrate of CYP3A4) overdosage was further examined. HepG2-GS-CYP3A4 expressed about 9 times larger amounts of CYP3A4 protein than a control. After incubation with HepG2-GS-3A4 cells in a circulatory bioreactor for 24 h, ammonia and DZP concentrations in the culture medium significantly decreased by about 40%. Furthermore, this system improved the survival time and decreased serum concentrations of DZP, ammonia, and transaminase in dogs with ischemic hepatic failure plus acute DZP overdosage. The mean survival time with bioreactor with HepG2-GS-3A4 was 42.7 +/- 3.6 h, which was significantly longer than that without reactor, with reactor (no cells), and with HepG2-GS (23.4 +/- 2.8, 22.1 +/- 2.4, and 31 +/- 3.7 h, respectively). Therefore, it is concluded that this bioartificial liver could be a good tool for the treatment of dogs with hepatic failure and that it could potentially be a bridging procedure to liver transplantation.

Oriental medicinal herb, Periploca sepium, extract inhibits growth and IL-6 production of human synovial fibroblast-like cells.

Periploca sepium (PS) has traditionally been used in oriental medicine for treatment of rheumatoid arthritis (RA). We investigated the aqueous extract of PS (PSE) in its effects on human rheumatoid arthritis-derived fibroblast-like cells. In cell culture studies, PSE inhibited the growth and IL-6 production of the cells in dose dependent manners. The extract of Glycyrrhiza glabra (GG), which has also been used to treat RA and chosen as a reference here, slightly inhibited the growth of RA cells. A study of PSE fractionation indicated that the active material inhibiting IL-6 production is filterable by ultrafiltration, suggesting that substances with low molecular weight might be involved in an inhibition of IL-6 production. These results support the view that PSE represents a rich source of growth inhibition and anti-IL 6 production.

Molecular cloning and structural characterization of the hagfish proteinase inhibitor of the alpha-2-macroglobulin family.

The "most primitive" living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human alpha2-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg833) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg833 was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.

Artificial mimicking of physiological active transport by a membrane co-cultured with two different cells: hepatic origin HepG2 and renal origin PCTL-MDR.

We designed a membrane culture unit on which 2 different cell lines were co-cultured to achieve selective and active transport of toxins. Hepatic origin HepG2 and renal origin multidrug-resistant gene-transduced proximal convoluted tubular cell line (PCTL-MDR) were cultured on the opposite sides of an expanded polytetrafluoroethylene membrane. The activity of testosterone hydroxylation by original HepG2 was very low; however, the cytochrome p450 (CYP) 3A4-transduced recombinant HepG2 metabolized the substrate efficiently. Testosterone added into the outer medium was hydrolyzed by HepG2, and the metabolites were preferentially transported to the inner medium by PCTL-MDR. [3H]-digoxin and [14C]-inulin were added to the outer medium; the digoxin was transported from the outer to inner space through the cell monolayer but the inulin was not, suggesting that the membrane actively transported only the substrate of the channel protein, MDR. The cells were irradiated (10 Gy) to prevent a membrane leak due to overgrowth. The irradiation did not induce apoptosis but resulted in long-lasting membrane function without leakage. The membrane co-cultured with hepatic and renal origin cells will enable a novel hemofiltration system with selective and active transport activities.