Yukio Gibo

Three-dimensional ultrastructure of normal rat hepatocytes by quick-freezing and deep-etching method

The three‐dimensional ultrastructure of nuclei and cell organelles including rough‐surfaced endoplasmic reticulum (RER), mitochondria, and cytoskeleton were studied in normal rat hepatocytes by the quick‐freezing and deep‐etching method. Peroxisomes and mitochondria were observed as spherical structures with granular matrices. Peroxisomes were identified by their size and matrices, which were more condensed than those of mitochondria. Ribosomes were identified as granular structures and were attached to the surface of endoplasmic reticulum. Cytoskeletal filaments were identified by their differences in diameter on the replica membranes, as well as in conventional ultrathin sections. Microfilaments were mainly localized around the bile canaliculi and adjacent to sinusoids. Intermediate filaments were observed around the bile canalicular microfilaments. Only a few filaments were observed near the lateral plasma membranes. Cross‐bridges measuring 5–7 nm in diameter were localized between the lamellae of RER and the surface of mitochondria. The quick‐freezing and deep‐etching method could be used to clarify the three‐dimensional association between the cytoskeleton and membrane‐bound organelles in hepatocytes.

Ultrastructural studies of hepatocyte cytoskeleton in experimental cholestasis by quick-freezing and deep-etching method

The ultrastructural association between the cytoskeleton and other organelles was studied by the quick‐freezing and deep‐etching method in rats treated with α‐naphthylisothiocyanate (ANIT), or phalloidin, and in rats with obstructive jaundice. Cytoplasmic filaments were classified by measuring their diameters, and actin filaments were identified by specific decoration with myosin subfragment 1 (S1). S1‐positive actin filaments and S1‐negative intermediate filaments (12–14 nm in diameter) were observed to form a three‐dimensional network around bile canaliculi, and were more numerous than in controls, not only in phalloidin‐treated rats and rats with obstructive jaundice, but also in ANIT‐administered rats. In all cholestatic rats, vesicular structures were also more numerous than in controls in the pericanalicular regions, and were closely associated with the microfilaments and the intermediate filaments. Filaments of a new type were localized between the lamellae of rough‐surfaced endoplasmic reticulum and mitochondria, and between the lamellae of Golgi sacs and vesicles. Other thin filaments were also observed within the network of actin filaments. These filaments were 4–6 nm in diameter on replica membranes and were never decorated with S1. They were also directly connected with the canalicular membranes. Cytoskeletal components associated with membrane‐bound organelles, including these new filaments, were suggested to be involved in the localization and migration of organelles.

Clinical significance of e-antigen/anti-e, with special reference to HBc-antigen in the liver.

Summarye-antigen and anti-e were assayed in sera of asymptomatic HBs-Ag carriers and of patients with liver diseases. Thirteen out of 34 (38.2%) asymptomatic carriers were positive for e-antigen, which was in sharp contrast to the reports from USA and Europe. e-antigen was detected to a greater extent in patients with chronic active hepatitis, reversely anti-e in patients with chronic persistent hepatitis. However, e-antigen was found rarely in patients with cirrhosis and never in 23 cases with hepatoma positive for HBs-Ag.HBc-Ag in the liver was detected in 4 out of 8 e-antigen positive asymptomatic carriers and in 4 out of 5 patients with chronic liver diseases with e-antigen respectively, and moreover in 3 out of 14 anti-e positive cases, so that the presence of anti-e did not necessarily mean the negativity of HBc-Ag in the liver. Anti-HBc titer, however, was lower in anti-e positive sera than in e-antigen positive ones. This may implicate the decreased replication of HBV in cases with anti-e.These results emphasize that the investigation of e-antigen/anti-e is mandatory for the evaluation of the prognosis of asymptomatic carriers and of patients with chronic hepatitis.

A case of cholangiocarcinoma detected after followup for seven years for thorotrast deposition

SummaryA 73-year-old former soldier in whom a deposition of thorotrast had been detected 7 years previously was admitted to our hospital because of high fever and epigastric pain. He had been well with standard liver function tests within the normal range until 4 months before admission. Laboratory examination on admission showed marked abnormalities in the liver function tests and an elevated level of CEA. Abdominal ultrasonography and computerized tomography, which had shown no spaceoccupying lesion in the liver one year earlier, revealed an abnormal mass in the right hepatic lobe. Angiographic examination revealed low vascularity and encasement of the intrahepatic artery. The disease was diagnosed as thorotrast-induced cholangiocarcinoma. Despite chemotherapy, the patient’s condition worsened rapidly and he died on the 78th day after admission. At autopsy, the primary tumor in the right hepatic lobe and metastatic nodular tumors throughout the liver were found. The histological diagnosis was cholangiocarcinoma.Thorotrast-induced liver cancers are inclined to grow rapidly, so early diagnosis of liver tumor accompanied by thorotrastosis is very difficult, as in this case. Repeated examinations at frequent intervals are required for early diagnosis.

Serum auto‐antibodies in patients with hepatocellular carcinoma: The clinical significance of antinuclear antibody

Auto‐antibodies including antinuclear antibody (ANA), antismooth muscle antibody, antimitochondrial antibody, rheumatoid factor, lupus erythematosus factor, antimicrosomal antibody and antithyroglobulin antibody were assayed in sera from 84 patients with hepatocellular carcinoma, 70 with liver cirrhosis, 50 with chronic hepatitis, 30 with cancer of the alimentary tract and 100 normal subjects. A significantly higher incidence and higher titre of ANA was found in patients with hepatocellular carcinoma, and the patterns of nuclear fluorescence detected by the indirect immunofluorescent test using cultured baby hamster kidney cells were predominantly speckled and nucleolar. In 16 patients with this disease, serial assays of ANA were done in sera obtained before and after development of hepatoma and/or after resection of the hepatic tumour. Antinuclear antibodies evolved in six patients in conjunction with the progression from cirrhosis to hepatoma, and two of three ANA‐positive patients who had the hepatic tumour resected lost ANA from their sera after resection.

Intranuclear virus-like particles in the hepatocytes of the patients with non-A, non-B hepatitis

SummaryLiver specimens were obtained from 21 non-A, non-B hepatitis patients when their surum transaminase levels were still elevated. The specimens were investigated by transmission electron microscope in order to investigate the presence of the structures relating to this type of hepatitis. Intranuclear electron-dense aggregates of virus-like particles were observed in 2 out of 21 cases. One was an unresolved acute hepatitis case with short-incubation posttransfusion type, and the other was a chronic persistent hepatitis case without blood-transfusion. The size of these particles varied from 22 to 28 nm in diameter. The incidence of hepatocytes with the particles was approximately 2 to 3% of the cells investigated. These particles may represent an intranuclear alteration in close association with one type of non-A, non-B hepatitis.

Past history of hepatocellular carcinoma is an independent risk factor of treatment failure in patients with chronic hepatitis C virus infection receiving direct-acting antivirals

Direct‐acting antiviral (DAA) treatment can achieve a high sustained virological response (SVR) rate in patients with hepatitis C virus (HCV) infection regardless of a history of hepatocellular carcinoma (HCC [+]). We examined 838 patients (370 men, median age: 69 years) who were treated with DAAs for comparisons of clinical findings between 79 HCC (+) (9.4%) and 759 HCC (−) (90.6%) patients and associations with treatment outcome. Male frequency was significantly higher in the HCC (+) group (60.8% vs 42.4%, P = 0.006). There were significant differences between the HCC (+) and HCC (−) groups for platelet count (115 vs 152 ×109/L, P < 0.001), baseline alpha fetoprotein (AFP) (9.9 vs 4.5 ng/mL, P < 0.001) and the established fibrosis markers of FIB‐4 index (4.7 vs 3.0, P < 0.001), AST‐to‐platelet ratio index (APRI) (1.1 vs 0.7, P = 0.009), M2BPGi (3.80 vs 1.78 COI, P < 0.001) and autotaxin (1.91 vs 1.50 mg/L, P < 0.001). The overall SVR rate was 94.7% and significantly lower in the HCC (+) group (87.3 vs 95.5%, P = 0.001). Multivariate analysis revealed that a history of HCC was independently associated with DAA treatment failure (odds ratio: 3.56, 95% confidence interval: 1.32‐9.57, P = 0.01). In conclusion, patients with chronic HCV infection and prior HCC tended to exhibit more advanced disease progression at DAA commencement. HCC (+) status at the initiation of DAAs was significantly associated with adverse therapeutic outcomes. DAA treatment for HCV should therefore be started as early as possible, especially before complicating HCC.

Studies on lymphocyte subsets for the differential diagnosis between non-A, non-B viral hepatitis and drug induced liver injury, and on the role of Ia positive cell in lymphocyte stimulation test.

薬剤性肝障害の診断上,ウイルスマーカーの確立されていない非A非B型ウイルス肝炎との鑑別が問題となる.そこで,薬剤性肝障害と非A非B型ウイルス肝炎の末梢血リンパ球亜分画を測定し,その鑑別診断的意義について検討するとともに薬剤性肝障害例におけるリンパ球刺激試験(LST)の免疫学的機序を解析した.薬剤性肝障害では,急性期OKT8陽性細胞の増加によりT4/T8比が低下し,回復期正常化している.それに対し,非A非B型ウイルス肝炎では急性期より正常範囲であった.このことから,T4/T8比の測定により両者の鑑別がある程度可能であると考えられた.また,LSTにおいてIa抗原陽性細胞が抗原呈示細胞として,薬剤特異的T細胞の増殖反応に重要な役割を演じていることが示された.

Two cases of autoimmune hepatitis died of hepatic failure with fulminant clinical course.

自己免疫性肝炎の経過中劇症化し,死亡した2症例について報告する.症例1は,42歳女性.昭和60年4月,黄疸,全身倦怠感および発熱が出現.高度の肝機能異常と高γ-グロプリン血症(4.4g/dl)と肝性昏睡を認めた.抗核抗体,抗DNA抗体および抗平滑筋抗体は陽性で,LE細胞現象も陽性であった.ステロイド療法,グルカゴンインスリン療法および血漿交換療法を施行したが,肝機能は急速に増悪し肝不全にて死亡した.症例2は46歳女性.昭和60年2月,全身倦怠感,黄疸が出現.T. Bilの著しい高値,トロンボテスト値5%,高γ-グロブリン血症(3.2g/dl)と腹水が認められた.抗核抗体および抗平滑筋抗体は陽性で,LE testは陰性であった.ステロイド療法,グルカゴンインスリン療法を施行したが肝不全は進行し死亡した.いずれの症例も,肝炎ウイルス,アルコールおよび薬剤の関与は否定的で,自己免疫性肝炎の経過中劇症化した稀な症例と考えられた.

Hepatitis B virus(HBV) carriers who developed acute hepatic failure.

急性肝不全を発症し重篤な病態を呈したHepatitis B virus (HBV) carrier 7例を経験した.Hepatitis delta virus, Hepatitis A virus, Epstein Barr virus, cytomegalo virusの重感染例はなく,多量飲酒および薬剤による急性肝不全発症例もみられなかった.輸血歴のない4例では3例で発症時HBV-DNA-polymerase (HBV-DNA-p)あるいはHBV-DNAが陽性であり,HBVのactive replicationが急性肝不全の発症に関与していると考えられた.輸血歴を有する3例ではHBV-DNA-p, HBV-DNA, IgM HBc抗体陽性例はなく,非A非B型肝炎ウイルスの重感染の可能性が推察された.全例にグルカゴン-インスリン療法および血漿交換療法を施行したが救命例は1例のみであった.acute hepatic failure on chronic liver disease (acute on chronic)と考えられた4例では生存例はなく,acute on chronicの予後は不良であると考えられた.