Shuji Nambu

Interleukin‐6 functions as an autocrine growth factor in a cholangiocarcinoma cell line

Abstract The tumour cells of a human cholangiocarcinoma cell line, HuCC‐T1, were found to express mRNA of interleukin‐6 (IL‐6) and to secrete a large amount of biologically active IL‐6 in the culture medium at the concentration of 22.6 ng/mL. Interleukin‐6 was demonstrated in the cytoplasm of the cells by immunohistochemical staining. Furthermore, these cells showed the presence of receptors for IL‐6 on the surface, and DNA synthesis of the cells was stimulated by the exogenous addition of recombinant human IL‐6 into the culture medium. The cell growth was significantly inhibited in the presence of anti‐human IL‐6 antibody in the culture medium. These findings indicate that IL‐6 is one of the autocrine growth factors of this cell line in vitro.

Evaluation of transcatheter arterial embolization with epirubicin-lipiodol emulsion for hepatocellular carcinoma*

SummaryA total of 18 patients with hepatocellular carcinoma (HCC) were treated by transcatheter arterial embolization (TAE) with a 4′-epi-doxorubicin (EDX)-lipiodol emulsion. Infusion of the EDX-lipiodol emulsion (EDX-L) via the hepatic artery was followed by the injection of gelatin sponge in 12 cases. The response and survival of these 12 patients following EDX-L treatment were compared with those of 42 subjects treated with a doxorubicinlipiodol emulsion (DX-L) and those of 23 patients treated by TAE with gelatin sponge (GS) only. In the group treated with EDX-L, nine cases were AFP-positive in sera and four showed a decrease in serum AFP values to less than 10% of the pretreatment level. Seven cases showed a partial response, and nine cases showed no change in the size of the tumor. In the group treated with EDX-L, nine cases are alive, and the oldest has survived for more than 431 days since the treatment. The half-year survival value was 57%, and the 1-year survival value was 49%. These values did not differ significantly from those calculated for the group treated with DX-L. The 1-year survival value determined for patients treated with a lipiodol emulsion (EDX-L or DX-L) followed by GS was 65%, and the 2-year survival value was 39%. These results rates are significantly better than those obtained in patients treated with GS only (1-year survival, 39%; 2-year survival, 13%).

Effects of an infusion of branched-chain amino acids on neurophysiological and psychometric testings in cirrhotic patients with mild hepatic encephalopathy

Abstract Psychotropic action of a branched‐chain‐enriched amino acid solution (Aminoleban) was quantitatively and visually examined in six cirrhotic patients with mild hepatic encephalopathy (grades I and II) using electrophysiological and psychometric methods. Neurophysiological effects of the amino acid solution were observed by comparing topographic spectrum analyses of electroencephalography (EEG) before and immediately after an intravenous 3 h infusion of the solution. The delta wave in the frontal region diminished from 61 ± 13 to 12 ± 4% (P < 0.01) and the alpha wave in the occipital region increased from 11 ± 3 to 51 ± 11% (P < 0.01). Latencies of the P3 wave in visual evoked potentials, which were topographically recorded in the occipital region, shortened from 220 ± 32 to 148 ± 19 ms (P < 0.01). Latencies of the P300 wave in event‐related potentials, which were topographically recorded in the centro‐temporal region, shortened from 493 ± 81 to 360 ± 93 ms (P < 0.05). Topographic reaction pattern of P300 was irregular toward the occipital or parietal region in cirrhotic patients. The EEG frequency power spectrum, illustrated by the colour density spectral array of computer‐aided polysomnography analysis, clearly showed a gradual increase of the alpha wave spectrum and a gradual decrease of the delta wave spectrum after initiation of the infusion. These immediate neurophysiological changes were confirmed by improvement of quantitative psychometric tests including number connection test, reaction time to sound, and digit symbol and block design tests of Wechsler Adult Intelligence Scale.

Analysis of T-cell receptor Vβ repertoire in liver-infiltrating lymphocytes in chronic hepatitis C

Abstract Background/Aims: To examine the T-cell repertoire which is involved in the immunopathogenesis of chronic hepatitis, we analyzed the T-cell receptor Vβ gene usage in liver-infiltrating lymphocytes by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical technique. Methods: Complementary DNA was synthesized from RNA which was extracted from 26 liver biopsy specimens and from peripheral blood lymphocytes from eight subjects, and amplified by RT-PCR. Radioactivity of each amplified product using 32 P-labeled primers was measured and the percentage of each Vβ expression was calculated. Results: The mean frequency of Vβ5.1 (11.1%) in liver-infiltrating lymphocytes of chronic hepatitis C was highest among those of all Vβ regions, and was significantly higher than that in both peripheral blood lymphocytes of chronic hepatitis C and liver-infiltrating lymphocytes of chronic hepatitis B. In the immunohistochemical analysis, Vβ5.1-positive cells were mostly observed in portal areas where inflammatory reactions occurred. The sequence of the complementarity determining region (CDR)3 on T-cell receptor expressing Vβ5.1 were examined in six patients with chronic hepatitis C. The sequences were similar to each other and all had one common amino acid (valine) irrespective of different HLA haplotype. Conclusions: These data suggest that Vβ5.1-positive cells are preferentially accumulated in the liver of chronic hepatitis C and are involved in the immunopathogenesis of the disease. Sequence analysis showed that Vβ5.1-positive cells recognize a common conventional antigen and valine recognized at the same position of the CDR3 may be a key residue in determining an antigen/major histocompatibility complex contact point.

Hypomethylation of the c-myc oncogene in liver cirrhosis and chronic hepatitis

SummaryIn order to investigate how chronic liver diseases, including liver cirrhosis and chronic hepatitis, are associate with hepatocarcinogenesis in terms of gene alteration, the methylation states of the c-myc and c-Ki-ras genes were examined in 34 liver tissues from patients with chronic liver disease without hepatocellular carcinoma (HCC), 34 non-tumor liver tissues from patients with HCC, 18 HCC tissues and 31 control liver tissues. The methylation states were analyzed by the Southern hybridization method using the restriction endonuclease isoschizomersMspI andHpaII. The CCGG sites at the second exon of the c-myc gene tended to be more extensively hypomethylated both in chronic liver disease and in non-tumor tissues than in control livers. Whereas the CCGG sites of the c-Ki-ras, and the third exon of the c-myc gene tended to be hypomethylated only in HCC tissues in comparison with other tissue groups. These results suggest that chronic liver disease may be situated between normal liver and HCC based on the state of DNA methylation and associated with the development of HCC through hypomethylation of the c-myc and /or c-Ki-ras gene.

α-Fetoprotein messenger RNA in peripheral blood as a marker of circulating hepatocellular carcinoma cells

Abstract AFP mRNA was detected in the peripheral blood of patients with hepatocellular carcinoma (HCC) by two-step polymerase chain reaction (PCR) preceded by reverse transcription to elucidate whether the existence of AFP mRNA in the blood could be helpful for clinical detection of metastasis of HCC. AFP mRNA in the blood was positive in eight of 21 patients with HCC (38.1%). The three HCC patients with distant metastasis were among the eight patients with detectable mRNA. Serum AFP level was more than 400 ng/ml in five of the eight HCC patients with mRNA in the blood, but less than 100 ng/ml in 11 of the 13 without detectable mRNA. In contrast, AFP mRNA in the blood was detected in only one of 32 patients without HCC (3.1%); these latter consisted of 10 patients with chronic hepatitis, six with liver cirrhosis and 16 with non-liver disease. The AFP mRNA-positive patient without HCC had chronic hepatitis type C, whose serum AFP concentration was 4 ng/ml. These data suggest that AFP mRNA may derive from AFP-producing HCC cells in the blood and that its existence may be helpful for clinical detection of metatasis of HCC. However, further study is necessary to determine what the AFP mRNA detected in the blood of the chronic hepatitis patient is derived from.

Analyses of proliferating cell nuclear antigen‐positive cells in hepatocellular carcinoma: Comparisons with clinical findings

Abstract Proliferating tumour cells in 92 patients with hepatocellular carcinoma (HCC) were identified by an immunohistochemical method using a monoclonal antibody against proliferating cell nuclear antigen (PCNA). The rate of PCNA‐positive cells in HCC tissues was positively correlated with histological grade and the tumour size and T factor of the tumour.

CASE REPORT: Intrahepatic portal‐hepatic venous shunts associated with a huge pelvic leiomyoma

We present a case of portal‐systemic encephalopathy due to intrahepatic multiple portalhepatic venous shunts. A 71‐year‐old woman was admitted to our hospital because of recurrent episodes of disturbed consciousness. She showed no clinical signs of portal hypertension. Liver function was normal, except for an indocyanine green retention rate of 34% at 15 min and blood ammonia level of 282 μg/dL. Portal venography revealed dilatation of the portal vein and multiple portal‐hepatic venous shunts, and a liver biopsy specimen revealed almost normal liver. Further clinical examination revealed a huge pelvic tumour. At laparotomy, two dilated veins were seen to arise from the pelvic tumour with blood flow into the mesentery. The tumour was resected successfully and a histological diagnosis of leiomyoma was made. The blood ammonia concentration decreased to the normal range postoperatively. A follow‐up portal venogram demonstrated decreased portal vein dilatation and minor portalhepatic venous shunts, considered to be congenital in origin. It is concluded that liepatic encephalopathy was produced in this patient due to an excess portal blood flow from the huge pelvic leiomyoma via the mesentery, with portosystemic shunting through pre‐existent (probably congenital) intrahepatic anastomoses.

Induction of allogeneic tumour‐ and lymphokine‐activated lymphocytes against hepatocellular carcinoma

For clinical application of adoptive immunotherapy against hepatocellular carcinoma (HCC), it is not easy to prepare tumour specific effector cells such as cytotoxic T lymphocytes (CTL). To induce potent and broad‐spectrum effectors, allogeneic cultured hepatoma cell lines (JHH‐4 and HuH‐6) were used as stimulators of peripheral blood lymphocytes (PBL) instead of autologous HCC cells. Allogeneic tumour‐ and lymphokine‐activated killer cells (ATLAK) were generated by a mixed culture of lymphocytes and allogeneic cultured tumour cells with recombinant interleukin‐2 (rIL‐2). The tumour‐killing activity of ATLAK induced by HuH‐6 was confirmed against HuH‐6 and other different HCC cell lines (JHH‐2, HuH‐7 and PLC). These activated lymphocytes were significantly more potent than lymphokine‐activated killer cells (LAK) in [51Cr]‐releasing assay. The JHH‐4 stimulated ATLAK was reactive not only with JHH‐4 but also with JHH‐2. The lysis of allogeneic targets could be partially inhibited by anti‐CD8 and anti‐CD3 but not by anti‐CD4. Anti‐tumour cytotoxicity in these cultures might be mediated by CD3+CD56‐ and CD3+CD56+ effectors. These results imply that adoptive immunotherapy for HCC with ATLAK may be more feasible than that with LAK.

Plasma PIVKA-II values as an indicator of AFP mRNA in the circulation in patients with advanced hepatocellular carcinoma

We examined the relationship between the values of plasma protein induced by vitamin K absence or antagonist II (PIVKA-II) and serum α-fetoprotein (AFP) and the presence of AFP mRNA in the mononuclear cells of the peripheral blood in 50 patients with hepatocellular carcinoma (HCC) . AFP mRNA in the blood was detected in 26 of 50 patients with HCC (52.0%). The frequency of positive cases in each TNM stage was: none of 5 in stage I, 3 of 14 (21.4%) in stage II, 6 of 10 (60.0%) in stage III and 17 of 21 (81.0%) in stage IV. AFP mRNA was detected more frequently in patients with high serum AFP level (400 ng/ml and more than 400 ng/ml) (12/16, 75.0%) than in those with low AFP level (less than 400 ng/ml) (14/34, 41.2%) (P<0.05). However, there was no significant difference between the frequencies of AFP mRNA positivity in patients with high and low serum AFP level in stage III or IV, to which most of AFP mRNA-positive cases belonged. On the contrary, the mRNA was more frequently detected in patients with positive level of plasma PIVKA-II (0.1 AU/ml and more than 0.1 AU/ml) (19/24, 79.2%) than in those with the negative level (less than 0.1 AU/ml) (7/26, 26.9%) (P<0.005). In addition, AFP mRNA was more frequently detectable in PIVKA-II-positive patients (16/17, 94.1%) than in PIVKA-II-negative ones (1/4, 25.0%) in stage IV (P<0.05), although there was no significant difference in the other stages. Furthermore, all 17 patients with plasma PIVKA-II levels more than 1.0 AU/ml in stages III and IV were positive for the mRNA in the blood. These data suggested that plasma PIVKA-II value may be a good indicator of AFP mRNA in the blood of patients with HCC in advanced stage.