Ken'ichi Okano

Presence of α-fetoprotein mRNA in blood correlates with outcome in patients with hepatocellular carcinoma

Abstract Background/Aims: Since hematogenous spread of tumor cells may adversely affect the prognosis of patients with hepatocellular carcinoma, we prospectively analyzed whether the presence of alpha-fetoprotein (AFP) messenger RNA (mRNA) in blood, used as a marker of circulating hepatocellular carcinoma cells, correlates with outcome. Methods: Eighty-eight patients were enrolled between December 1993 and August 1995, and 81 were followed until the end of 1997. All patients were treated with percutaneous ethanol injection therapy and/or transarterial embolization during follow-up. The status of AFP mRNA in blood was serially determined. Cumulative metastasis-free survival and overall survival were analyzed in relation to AFP mRNA and other clinical and laboratory variables. Results: Among 81 patients followed, 54 were positive for AFP mRNA at entry and 27 were negative. Extrahepatic metastasis developed more frequently among the AFP mRNA-positive patients (13 of 54) than among the AFP mRNA-negative patients (2 of 27) ( p =0.0296). After treatment, AFP mRNA became negative in 24 of 54 patients (44%). Cumulative metastasis-free survival and overall survival were significantly better in the 24 patients whose AFP mRNA became negative after treatment than in the 30 patients with persistently positive AFP mRNA ( p =0.0001 and p Conclusions: The presence or absence of AFP mRNA in blood is a predictor of outcome in patients with hepatocellular carcinoma.

Role of macrophages in regeneration of liver

In an attempt to clarify the role of macrophages and their mediators during regeneration of the liver, the difference of liver regeneration among C3H/HeN (LPS-responsive strain) and C3H/HeJ (LPS-resistant strain) mice was investigated. After a 67% partial hepatectomy, an increase in the weight of regenerating liver was significantly delayed in the C3H/HeJ mice, as compared with C3H/HeN mice. The number of hepatocytes labeled with antibody against PCNA reached maximum levels 48 hr after partial hepatectomy, but the PCNA labeling index in C3H/HeJ mice was 20% less than that for C3H/HeN mice. In addition, TNF-α activities in serum were enhanced shortly after partial hepatectomy in C3H/HeN strain mice, but were not increased in C3H/HeJ strain mice. Serum IL-6 levels were markedly enhanced in both C3H/HeN and C3H/HeJ mice, but a bimodial peak (14 and 48 hr after partial hepatectomy) was demonstrated in C3H/HeN mice, in contrast to a single peak (at 24 hr) in C3H/HeJ mice. Suppression of Kupffer cells by previous administration of gadolinium chloride in C3H/HeN mice reduced the increase in both serum TNF-α and IL-6 concentrations, reduced PCNA labeling index of hepatocytes by 20%, and disturbed the regeneration of the liver. Previous administration of antibody against TNF-α reduced the PCNA labeling index of hepatocytes by 20% after partial hepatectomy in C3H/HeN strain mice. These results suggest that LPS-responsive macrophages in the liver and their mediators, especially TNF-α, could partly play a role in liver regeneration.

Hepatocyte Nitric Oxide Production Is Induced by Kupffer Cells

To investigate the cellular communication in theliver, nitric oxide (NO) production by sinusoidal cellsand hepatocytes by stimulation with cytokines andKupffer cell-conditioned medium was quantitatively analyzed. NO production by the cells wasmeasured by the Griess reaction, and nitric oxidesynthase (iNOS) transcription level by a competitiveRT-PCR assay using mutant iNOS mRNA as a standard. NOproduction and iNOS mRNA transcriptional levels in Kupffercells were markedly increased by stimulation withlipopolysaccharide (LPS) and interferon-γ(IFN-γ), and moderately by interleukin-1β(IL-1β). NO production by hepatocytes was notsignificantly enhanced by LPS, but was markedly enhancedby IL-1β or the combination of tumor-necrosisfactor-α (TNF-α) and IFN-γ. HepatocyteNO production and iNOS mRNA levels were markedly enhanced bythe LPS-activated Kupffer cell conditioned medium, butthese effects were reduced by heat treatment or anti-TNFantibody. Although NG-monomethyl-L-arginine acetate and dexamethasone reduced NO productionby the cells, the iNOS mRNA level was reduced bydexamethasone only. Gel-shift assay showed NF-κBactivation in hepatocytes during this activation. These data reinforce the importance of cellularcommunication between sinusoidal cells andhepatocytes.

Antigen Presentation by Kupffer Cells in the Rat

Sprague-Dawley rats were immunized with liver-specific protein (LSP), and the functions of isolated Kupffer cells, as accessory cells and antigen-presenting cells, were examined. More than 70% of the cells showed phagocytic activity for latex particles. The proliferative response of spleen lymphocytes stimulated with concanavalin A was lost in the absence of macrophages but was restored by the addition of Kupffer cells. In vitro stimulation with LSP induced binding of lymphocytes to Kupffer cells and enhanced 3H-thymidine incorporation when lymphocytes were incubated with Kupffer cells from immunized rats. These results indicated that Kupffer cells can act both as accessory cells and as antigen-presenting cells for LSP.

Chemotactic factors released from hepatocytes exposed to acetaminophen

To clarify the mechanism of neutrophil infiltration in the liver of acetaminophen-induced hepatic injury, chemotactic factor released from hepatocytes exposed to acetaminophen has been investigated. Hepatocytes exposed to acetaminophen release nondialyzable chemotactic factor, although actaminophen in itself inhibits chemotaxis of neutrophils. Chemotactic activity of the nondialyzable chemotactic factor was reduced after treatment with heat (56°C, 30 min) or trypsin. Chemotactic activity was demonstrated at the molecular weights of around 25 and 55 kDa. Chemotactic activity of the conditioned medium was not significantly reduced in the presence of antibody against rat KC/gro protein (interleukin-8-related cytokine in rodent). Chemotactic activity of a 25-kDa factor was reduced by the antibody against KC/gro protein, but that of a 55-kDa factor was not reduced. Immunoblot analysis revealed that the peptide reacted with antibody against rat KC/gro protein was demonstrated at a molecular weight of around 20–25 kDa, but not at around 55kDa, when the conditioned medium of acetaminophen-treated hepatocytes was electrophoresed. These results suggest that hepatocytes exposed to acetaminophen release two types of chemotactic factors for neutrophils and that a major part of the chemotactic factor could be different from a member of interleukin-8 family.

Growth regulation of rabbit gastric epithelial cells and protooncogene expression

We recently developed a primary culture system for gastric epithelial cells from adult rabbits that allows the investigation of growth regulation at the cellular level. In this study, we demonstrated that epidermal growth factor (EGF), insulin, and dibutyryl adenosine 3′,5′-cyclic monophosphate (dBcAMP) all stimulated cell proliferation. Insulin and dB-cAMP potentiated the stimulation of cell proliferation by EGF, while transforming growth factor-β1 (TGF-β1) inhibited it. Expression of c-fos and c-myc was induced in response to the stimulation by these growth regulators, but the degree of expression did not necessarily correlate with the effects of these agents on cell proliferation. In conclusion, EGF, insulin, and dBcAMP were positive growth regulators, while TGF-β1 was a negative regulator in gastric epithelial cells. These growth modulators may exert their effects by distinct pathways from a standpoint of the expression of c-fos and c-myc.

Role of platelet-activating factor in pathogenesis of galactosamine-lipopolysaccharide-induced liver injury

In an attempt to clarify the role of platelet-activating factor (PAF) in the pathogenesis of hepatic injury induced by galactosamine (GalN) plus lipopolysaccharide (LPS), effects of WEB 2086 (PAF receptor antagonist) on hepatic injuryin vivo as well as on neutrophil adherence to hepatic endothelial cellsin vitro have been investigated, as we have recently clarified the role of neutrophils in this experimental model of hepatic injury. Although an enhanced serum TNF-α level after GalN-LPS administration was not reduced by WEB 2086, hepatic injury and hepatic neutrophil accumulation in the liver after GalN-LPS administration were attenuated by WEB 2086. Anin vitro study revealed that an enhanced neutrophil adhesion to hepatic endothelial cells by stimulation with the sera that were collected from the GalN-LPS-treated rats, was reduced in the presence of WEB 2086 in a dose-dependent manner. In addition, LPS, TNF-α, and PAF were found to enhance the neutrophil adherence to hepatic endothelial cells, which was reduced in the presence of WEB 2086. These results suggest that PAF play an important role in the GalN-LPS induced hepatic injury and that PAF receptor antagonist reduces the neutrophil adherence to hepatic endothelial cells in the liver.

Mortality from gastric cancer in patients followed with upper gastrointestinal endoscopy

Objective. Although commonly practiced in Japan, the effectiveness of regular screening with upper gastrointestinal (UGI) endoscopy against gastric cancer has not been well evidenced. The aim of the study was to investigate if gastric cancer-related morrality can be reduced by regular endoscopy. Material and methods. The medical records of 833 patients with gastric ulcer (GU) and 2547 without ulcer (NU) were analyzed; these patients received long-term, repeated endoscopic examinations between 1969 and 2004. Gastric cancer incidence, death by gastric cancer, and overall survival were compared with those in a Japanese general population by calculating the standardized incidence ratios (SIRs) and standardized mortality ratios (SMRs). Results. The interval between UGI endoscopic examinations was 1.4±1.4 years in the GU group and 1.8±1.5 years in the NU group. During follow-up, 32 patients with GU and 61 in the NU group developed gastric cancer, showing annual incidence rates of 0.40% (95% CI: 0.24–0.56%) and 0.38% (0.28–0.48%), and SIRs of 2.21 (1.44–2.98) and 1.72 (1.29–2.15), respectively. The 5-year survival rate exceeded 80% among patients who developed gastric cancer. SMRs for gastric cancer and overall deaths were 0.50 (0.01–0.99) and 1.05 (0.87–1.23) in GU patients, and 0.45 (0.15–0.74) and 0.78 (0.69–0.88) in NU patients. There were no significant differences between the two groups in gastric cancer incidence, mortality from gastric cancer, and overall survival. Conclusions. Mortality from gastric cancer could be reduced by regular UGI endoscopy in a population with a high incidence of gastric cancer.

Endotoxin induced cellular communication in the liver: Murine models for clarification of the role of LPS‐responsive macrophages in the pathogenesis of liver diseases

In several experimental models, lipopolysaccharide (LPS) plays an important role in the pathogenesis of liver diseases. Murine models of C3H/HeN and C3H/HeJ mice have been used to elucidate the role of LPS and its responsive‐macrophages in vivo, as C3H/HeN strain mice are known to be LPS‐responsive, while C3H/HeJ strain mice are LPS‐resistant. Furthermore, release of several kinds of biologically active mediators such as interleukin‐1, tumour necrosis factor‐α, colony stimulating factor and reactive oxygen radical is not enhanced in C3H/HeJ mice even after stimulation with LPS. Thus, these murine models could be suitable for clarification of endotoxin induced cellular communication in the liver.

Bile flow in a mutant Sprague-Dawley rat with defective biliary excretion of glutathione

The Eisai hyperbilirubinemic rat is a mutant strain of Sprague-Dawley origin with hereditary defects in the biliary excretion of bilirubin glucuronide, glutathione, and several other organic anions. The correlation between bile flow and bile acid excretion rates during taurocholate infusion revealed that bile acid-independent flow was smaller in the mutant than in intact Sprague-Dawley rats (19.3 vs 56.0 μl/kg per min), while bile acid-dependent flow was similar. The correlation between bile flow and glutathione excretion rates in Sprague-Dawley rats with modified hepatic glutathione levels revealed that a certain portion of bile flow was proportional to the biliary excretion of glutathione, with a coefficient of 551 bile per 1 mol glutathione. One-third of bile acid-independent bile flow in intact Sprague-Dawley rats was accounted for by glutathione osmosis, which feature was absent in the mutant rats.