Naoto Iwabuchi

Establishment of a human cell line (HCC-T) from a patient with hepatoma bearing no evidence of hepatitis B or A virus infection

A human hepatoma cell line, designated HCC‐T, was established. The tumor was surgically obtained from a Japanese male patient with hepatocellular carcinoma (HCC) arising in a cirrhotic liver that had supposedly developed from nonAnonB (NANB) chronic hepatitis. HCC‐T exhibited a typical morphology of epithelial cells in culture. Population doubling time was 24 hours and HCC‐T cells had characteristics of malignant cells demonstrated by the ability to grow in a soft agar medium and transplantability to nude mice. The histologic condition of the tumor transplanted to a nude mouse showed similarity to the original tumor. A chromosome analysis showed that there were ten identifiable marker chromosomes and sex chromosomes with its modal number of 64. Alpha‐fetoprotein (AFP) production was demonstrated by direct immunofluorescence study, but albumin or hepatitis B surface antigen were not detectable. The integration of hepatitis B viral DNA was not demonstrable in the genome of HCC‐T cells or the original hepatoma.

ASSESSMENT OF HISTOLOGICAL FEATURES AND OUTCOME OF INTERFERON THERAPY IN CHRONIC HEPATITIS C

The correlation between the histological features of liver biopsy specimens before interferon (IFN) treatment and the clinical effect of IFN administration on chronic hepatitis C was investigated. A study of the relation between several histological features that were graded in 60 liver biopsy specimens from chronic hepatitis C patients before IFN treatment disclosed that the grade of portal fibrosis was positively correlated with the grade of other inflammatory features, including piecemeal necrosis and portal and lobular inflammation. The degree of portal fibrosis adversely affected the rate of normalization of ALT levels in chronic hepatitis C during and after IFN treatment. We reexamined 36 liver biopsy specimens that showed a moderate degree of portal fibrosis, and found that the degree of piecemeal necrosis was inversely correlated with the extent of lymphoid follicle formation in the portal tracts. During IFN therapy, the group of chronic hepatitis C patients who showed marked piecemeal necrosis and less lymphoid follicle formation in the liver specimens had a poor response to IFN treatment, whereas another group that showed marked lymphoid follicle formation and little piece-meal necrosis in the liver specimens had a good response to IFN. These relationships gradually disappeared after the completion of IFN treatment.

Decrease of transplantability by the immunopotentiators, OK-432 and interleukin-2: experiments on a human hepatoma cell line in nude mice.

The relationship between nonspecific cytotoxic activity of spleen cells and the resistance against the graft challenge of a human hepatoma cell line (HCC-M) was investigated in nude mice. Two administrations of an immunopotentiator, OK-432 or human interleukin-2, prior to the subcutaneous inoculation of HCC-M cells, which was performed 24 h after the last administration, significantly inhibited the tumor development in terms of rate of tumor take and tumor size. This effect was abrogated by simultaneous administration of an anti-asialo GM1 (ASGM1) antiserum. There was a significant inverse correlation between tumor volume and spleen cell cytotoxicity which was determined at the time of HCC-M cell inoculation against a YAC-1 or HCC-M target. Spleen cell cytotoxicity enhanced by these immunopotentiators could not completely be abolished by in vitro treatment with ASGM1 and complement. This result suggests that effector cells of the enhanced cytotoxicity consist of heterogeneous cells including both ASGM1+ natural killer cells and other nonselective cytotoxic cells. These results suggest that nonspecific cytotoxic cells play crucial roles in the resistance against tumor cell challenge and that the total level of cytotoxic activity of these cells at the time of tumor cell challenge is a key factor which determines tumor development.

Antibody-dependent cell-mediated cytotoxicity against cell lines generated by liver-specific idiotype-bearing antibody

We produced a murine monoclonal antibody (mAb), designated H2-mAb, against a fractionated soluble phase of human liver homogenate which antibody reacted with human liver cells. A human antibody possessing the same idiotype as the H2-mAb, designated LSIA (liver-specific idiotype-bearing antibody), can be measured by a sandwich enzyme-linked immunosorbent assay, using the anti-H2 idiotype antibody. The serum level of LSIA in patients with histologically proven chronic hepatitis (CH) was significantly higher than that in healthy subjects and it was also higher than that in subjects with other diseases, including systemic lupus erythematosus. In a comparison between patients with CH type B and those with CH type C, there was no significant difference in serum levels of LSIA. It was possible to purify LSIA from the sera of patients with CH. The purified LSIA bound to the human cell lines Chang and HCC-M, derived from liver cells and a hepatoma respectively, but not to HeLa cells, a uterine carcinoma derivative. The reactivity of this mAb to HCC-M was weaker than that to Change. Moreover, the presence of LSIA caused an antibody-dependent cell-mediated cytotoxic challenge against Change cells in vitro.

Effect of interferon-β on in vitro production of the liver specific idiotype-bearing antibody in patients with chronic hepatitis B

Recently interferon (IFN) has been used for the treatment of patients with chronic hepatitis (CH) B. It is known that IFN-β has an anti-viral effect, but clinical effect of IFN-β is limited. In Japanese cases of type B CH, complete eradication of hepatitis B virus (HBV) can not be achieved, however, partial suppresion of viral replication is possible. On the other hand, IFN-β has also an immunopotentiating effect. To analyze the effect of IFN-β on immune reactions to HBV and autoimmune reactions in patients with type B CH is thought to be important.

Ammonium and α-mannosidase are key factors determining natural killer (NK) activity in patients with liver cirrhosis

Natural killer (NK) cells are a group of lymphocytes which destroy in vitro a variety of tumor cells in a nonselective way. The serum α-mannosidase activity was significantly higher in patients with liver cirrhosis (LC) than healthy subjects. α-mannosidase inhibited NK activity in vitro at low concentrations comparable to the serum level. NK activity was demonstrated to be directly proportional to serum ammonia level in patients with LC. These results suggest that α-mannosidase and ammonium are key factors determining NK activity in patients with LC.

Cellular cytotoxicity induced by liver specific idiotype-bearing antibody against Chang liver cells

Liver-specific idiotype-bearing antibody (LSIA) was capable of being purified from the sera of patients with chronic hepatitis by affinity chromatography. It was demonstrated that LSIA bound to Chang liver cells and induced ADCC. It is suggested that ADCC induced by LSIA is related to liver cell damage in patients with chronic hepatitis (CH).