Akihiko Sugiyama

Interleukin-10 inhibits hepatic injury and tumor necrosis factor-α and interferon-γ mRNA expression induced by staphylococcal enterotoxin B or lipopolysaccharide in galactosamine-sensitized mice

BACKGROUND/AIMS Proinflammatory cytokines including tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) play a critical role in the pathogenesis of liver injury induced by lipopolysaccharide (LPS) or staphylococcal enterotoxin B (SEB) in D-galactosamine (GalN)-sensitized mice. The aim of this study was to examine the ability of interleukin-10 (IL-10), a recently characterized, highly potent anti-inflammatory mediator, to protect sensitized mice against hepatotoxicity induced by SEB or LPS. METHODS IL-10 was injected at various concentrations into BALB/c mice treated by GalN/SEB or GalN/LPS. Liver injury was assessed biochemically and histologically. Serum levels of TNF-alpha and IFN-gamma were measured and the expressions of TNF-alpha and IFN-gamma mRNA in the liver and spleen were determined by reverse-transcription polymerase chain reaction. RESULTS Treatment with IL-10 markedly reduced serum transaminase activities in a dose-dependent manner and reduced hemorrhagic liver damage in sensitized mice exposed to either toxin. IL-10 also inhibited increases in serum TNF-alpha and IFN-gamma concentrations with either toxin. Treatment with IL-10 significantly reduced TNF-alpha mRNA and IFN-gamma mRNA expression in the liver and spleen after administration of either toxin to sensitized mice. CONCLUSIONS These findings suggest that IL-10 is capable of regulating both T cell- and macrophage-mediated hepatic injury in vivo and that this cytokine might be useful in the treatment of acute liver failure.

Lethal hepatic apoptosis mediated by tumor necrosis factor receptor, unlike Fas-mediated apoptosis, requires hepatocyte sensitization in mice

BACKGROUND/AIMS Tumor necrosis factor a (TNF-alpha) and Fas ligand are apoptotic cell-death mediators that act by binding to their responsive receptors. The aims of this study were to assess the differences between liver cell deaths induced by TNF-alpha and anti-Fas antibody, and to investigate the mechanism by which GalN sensitizes the hepatocyte to injury by TNF-alpha. METHODS TNF-alpha or anti-Fas antibody was injected into BALB/c mice sensitized or unsensitized by D-galactosamine (GalN). Liver injury was assessed biochemically and histologically. The expressions of TNF receptor (TNFR)1 and TNFR2 mRNA in the liver were determined by Northern blot analysis. Nuclear factor-kappaB (NF-kappaB) DNA binding activity was determined by gel shift assay. RESULTS In GalN-sensitized mice, hepatocyte apoptosis and liver failure were observed after TNF-alpha injection, but neither occurred in unsensitized mice. Microscopically, GalN preceding TNF-alpha caused massive hemorrhagic liver damage with fragmented hepatocyte nuclei resembling effects of anti-Fas antibody, but GalN largely failed to sensitize to injury by this antibody. TNFR1 mRNA expression in the liver was upregulated within 3 h after GalN administration, and anti-TNFR1 antibody protected GalN-sensitized mice from hepatotoxic effects of TNF-alpha. GalN treatment failed to affect TNF-alpha-induced NF-kappaB activation. CONCLUSIONS Unlike Fas-related apoptosis, TNFR-mediated apoptosis requires hepatocyte sensitization involving TNFR1 upregulation.

Nocturnal branched-chain amino acid administration improves protein metabolism in patients with liver cirrhosis: Comparison with daytime administration

BACKGROUND In an attempt to optimize oral branched-chain amino acid (BCAA) administration to improve serum albumin in cirrhotic patients, we compared the effects of nocturnal and daytime BCAA administration on protein metabolism in cirrhotic patients. METHODS Twelve cirrhotic patients were enrolled in a short-term study. Patients were administered either conventional daytime BCAA granule or nocturnal BCAA for a week, and metabolic analyses were performed, followed by a crossover study in the next week. Another 12 patients, who showed no improvement of serum albumin level with previous daytime BCAA administration, were randomly assigned to either a nocturnal or a daytime BCAA administration group in a long-term study. RESULTS Low Fischers ratio, reduced respiratory quotient, and low serum albumin were observed at entry in cirrhotic patients. Whereas daytime BCAA administration improved nitrogen balance and Fischers ratio, these 2 were further significantly improved after nocturnal BCAA administration. There were no changes in parameters of energy metabolism throughout the study. In the 3-month follow-up, a significant increase in serum albumin was observed in patients administered nocturnal BCAA but not in those administered daytime BCAA. CONCLUSIONS Nocturnal BCAA administration improved serum albumin in cirrhotic patients who showed no improvement in serum albumin level with daytime BCAA administration. This effect could be partly caused by the improved protein sparing with this administration method.

Development and characterization of a hybrid bioartificial liver using primary hepatocytes entrapped in a basement membrane matrix

For the development of a bioartificial liver (BAL) support device, it is most important to establish highly differentiated liver cells cultured at high density. When rat hepatocytes were cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, their rates of albumin secretion were very high, as measured by ELISA, and these high rates were maintained for more than three weeks of culturing. This level of activity greatly exceeded that of hepatocytes cultured on a plastic substratum, poly-N-p-vinylbenzyl-d-lactonamide (PVLA), on a single layer of collagen, or in a collagen sandwich culture. In an in vitro perfusion experiment, rat hepatocytes rapidly and completely removed ammonia from Eagles MEM supplemented with 0.2 mM NH4Cl, although ammonia levels of the medium serially increased in modules containing HepG2 cells. A hybrid liver support system was developed and consisted of plasma perfusion through porous hollow fiber modules inoculated with 10 billion porcine hepatocytes entrapped in EHS gel. This system was applied to pigs with ischemic liver failure 8 hr after creation of a portocaval shunt and hepatic devascularization. In animals treated with the BAL support system, blood bicarbonate levels were increased immediately after treatment, and hemodynamic stability was improved. In control pigs, on the other hand, blood bicarbonate levels and blood pressure remained low. Plasma levels of ammonia and lactate decreased in pigs treated with the BAL device, but not in control animals. These results indicate that primary hepatocytes outperform HepG2 cells as a source of biotransformation functions in a BAL system and that the use of a BAL support device in combination with a hollow fiber module and hepatocytes entrapped in EHS gel has potential advantages for clinical use in patients with fulminant hepatic failure.

Control of cyclins, cyclin-dependent kinase inhibitors, p21 and p27, and cell cycle progression in rat hepatocytes by extracellular matrix

BACKGROUND/AIMS The extracellular matrix plays an essential role in the regulation of cell proliferation in different cell types. However, the regulation of cell cycle control in hepatocytes in response to growth factors and extracellular matrix signals is not well understood. The aims of this study were to investigate the expression of key cell cycle control elements, including cyclins, A and D1, and cyclin-dependent kinase inhibitors, p21 and p27, in rat hepatocytes in primary culture on dried collagen or Engelbreth-Holm-Swarm in the presence of epidermal growth factor. METHODS Hepatocytes prepared from Wistar rats were cultured on various extracellular matrix in Williams medium E in the presence or absence of 20 ng/ ml epidermal growth factor. DNA synthesis was measured by [3H]thymidine uptake and mRNA expression of cell cycle-related genes was determined by reverse transcription polymerase chain reaction. RESULTS Cyclins D1 and A mRNA levels were high at the G1/S boundary in epidermal growth factor-stimulated hepatocytes cultured on dried collagen. In contrast to spread cells, hepatocytes cultured on an Engelbreth-Holm-Swarm gel that were prevented from spreading failed to progress through the G1 phase and enter the S phase. This shape-dependent blockage of cell cycle progression correlated with the up-regulation of the cell cycle inhibitors p21 and p27. CONCLUSIONS Changes in hepatocyte-extracellular matrix interactions may control hepatocyte growth within the local microenvironment by modulating cell shape and regulating cyclins and the cyclin-dependent kinase inhibitors p21 and p27.

Endoscopic papillary balloon dilatation for common bile duct stones: efficacy of combination with extracorporeal shockwave lithotripsy for large stones.

Background Endoscopic papillary balloon dilatation (EPBD) is generally considered a safe and effective technique for removal of common bile duct (CBD) stones. However, some reports have prompted concern about the risk of pancreatitis following the procedure, and it seems to be more difficult and to require adjunctive procedures more frequently in patients with large stones. Aims To analyse the factors influencing pancreatitis after the procedure, and to examine which is the more suitable adjunct for treating large stones, mechanical lithotripsy (ML) or extracorporeal Shockwave lithotripsy (ESWL). Patients and methods EPBD was performed in 92 patients, including 40 with large stones (≥ 12 mm). These 40 patients were randomly assigned to two groups receiving ML or ESWL to fragment stones (20 patients each). Results Complete ductal clearance was obtained in all 92 patients. Significant elevation of the serum amylase level compared with the prior value (> 300 IU/I) was observed in 26 (28%), and eight (8.7%) developed clinical pancreatitis. To assess the influence of various factors on the amylase level, multivariate analysis was used. The number of stones and the time required for treatment had a significant influence on the incidence of increased amylase level (P < 0.05), and ML also significantly increased it (P < 0.05). On the other hand, the amylase level remained low in the ESWL group. ML caused elevation of amylase level in 11 patients (55%), while three (15%) had elevation after ESWL. Conclusions In patients with multiple stones, elevation of the amylase level is more frequent This seems to be because repeated cannulation and much time is required for treatment. In patients with large stones, the rate was also high if ML was used, but was low when ESWL was used. ESWL may reduce the incidence of pancreatitis.

Combination of proton pump inhibitor and rebamipide, a free radical scavenger, promotes artificial ulcer healing after endoscopic submucosal dissection with dissection size >40 mm

In our previous study, the healing effect of proton pump inhibitor plus rebamipide for endoscopic submucosal dissection-related artificial ulcer smaller than 40 mm showed statistical significance. However, such effect of the combination was not yet clear for ulcers with dissected diameter more than 40 mm. The aim of this present study was to resolve this problem under sufficient statistical power, with adequate sample size. We conducted a randomized controlled study. Either the proton pump inhibitor mono-therapy or the combination therapy was prescribed for 28 days after endoscopic submucosal dissection. Eighty-seven patients were eligible for outcome evaluation. Combination therapy was significantly superior to mono-therapy, 27.8% vs 0% reached healing stage (scar stage) in cases with ulcers of dissection diameter more than 40 mm. In conclusion, the combination therapy with rebamipide was favorable regimen in patients with larger artificial ulcer after endoscopic submucosal dissection.

Effects of Cytokines on the Binding of Leukocytes to Cultured Rat Hepatocytes and on the Expression of ICAM-1 by Hepatocytes

Adhesions of leukocytes to hepatocytes andsinusoidal endothelial cells mediates the induction andprogression of hepatic injury. However, in contrast toendothelial cells, information regarding the regulation of interactions between leukocytes andhepatocytes is limited. In the present study, weinvestigated the effect of inflammatory mediatorsincluding lipopolysaccharide (LPS), staphylococcalenterotoxin B (SEB), interferon-γ (IFN-γ), tumornecrosis factor-α (TNF-α), andinterleukin-1β (IL-1β) on the adhesion ofpolymorphonuclear leukocytes or lymphocytes to primarycultured rat hepatocytes, and on the expression of intercellular adhesionmolecule-1 (ICAM-1) gene in hepatocytes. Bothpolymorphonuclear leukocyte and lymphocyte adhesion tohepatocytes were enhanced after exposure of hepatocytes to IFN-γ and TNF-α, but not afterexposure to LPS, SEB or IL-1β. The adhesion inducedby either IFN-γ or TNF-α was inhibited bymonoclonal antibodies against ICAM-1 or lymphocytefunction-associated antigen-1 (LFA-1). Nonstimulated hepatocytesexpressed faintly ICAM-1 mRNA, which increased slightlyduring the culture period. ICAM-1 mRNA expression wasup-regulated to a greater extent by incubating hepatocytes with IFN-γ or TNF-α,and peaked after 12 hr of incubation with TNF-αand after 24 hr with IFN-γ. These results indicatethat IFN-γ and TNF-α induce the expressionof ICAM-1 on parenchymal hepatocytes and that theLFA-1-ICAM-1 pathway plays an important role in theinteraction between hepatocytes and neutrophils orlymphocytes.

Hemostatic Radiotherapy Used Twice for Inoperable Progressive Gastric Cancer with Bleeding

Both men and women suffer from high morbidity as a result of gastric cancer in Asian countries, including Japan. The first choice of treatment of gastric cancer without distant metastasis is surgery, and adjuvant chemotherapy is performed in stage II and stage III. Radiotherapy as an adjuvant therapy of the surgery is not provided for standard treatment in Japan. Reports on the effect of radiotherapy performed to achieve hemostasis in gastric cancer exist, but few are prospective [1–3]. Additionally, to our knowledge, only one patient in Japan has been irradiated twice so far [4]. Herein, we report a case of gastric cancer with bleeding during the management of which we administered radiotherapy twice and successfully achieved hemostasis. Case Report