Toshio Morizane

Induction of hepatic stellate cell proliferation by LPS-stimulated peripheral blood mononuclear cells from patients with liver cirrhosis.

Abstract: We studied hepatic stellate cell proliferation in vitro. Peripheral blood mononuclear cells (PBMC) from patients with chronic active hepatitis C (CAH) and liver cirrhosis (LC) were cultured for 24 h in the presence or absence of Escherichia coli lipopolysaccharides (LPS). Hepatic stellate cell proliferation induced by the culture supernatants was measured, and interleukin-1 (IL-1) and IL-6 levels in the culture supernatants were quantified. Culture supernatants of LPS-stimulated PBMC from LC patients induced rat hepatic stellate cell proliferation by almost 2.8-fold (stimulation index, 2.83 ± 1.41) compared with when the cells were cultured without addition of PBMC culture supernatants. Production of IL-1β was significantly higher in the culture supernatants of both CAH and LC patients than in those of ten healthy controls (P < 0.01 and P < 0.05, respectively). But there was no significant correlation between IL-1 production and the induction of hepatic stellate cell proliferation by the culture supernatants. Although there were no significant differences in IL-6 production by LPS-stimulated PBMC among healthy controls and CAH and LC patients, we observed a significant correlation between IL-6 production and the induction of hepatic stellate cell proliferation in the culture supernatants of LC patients. Rat hepatic stellate cells themselves produced IL-6, and treatment with IL-6 antisense oligodeoxynucleotides suppressed the cell proliferation, suggesting that IL-6 is an autocrine growth factor for hepatic stellate cells. The addition of human recombinant IL-6 (hrIL-6) augmented rat hepatic stellate cell proliferation, indicating that excessive IL-6 may further facilitate cell proliferation. These findings suggest that a cytokine cascade including IL-6 may participate in hepatic stellate cell proliferation in LC patients when they are exposed to endotoxin.

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.

A randomized, controlled trial of weekly administration of lymphoblastoid interferon in patients with chronic hepatitis C

To evaluate the efficacy of a treatment of weekly interferon administration in patients with chronic hepatitis C, 36 patients were randomly assigned to two groups. In one group lymphoblastoid interferon was given at a dose of 6 million units, intramuscularly, once per week for 24 weeks, and no treatment was given to the other. Serum alanine aminotransferase levels in the treated group were significantly lower during therapy than in the control group, although there was no significant difference between these two groups before therapy. The normalization of serum alanine aminotransferase levels at the end of therapy was observed in 50% of the treated group, and in 11.1% of the control group. This difference was statistically significant (p < 0.03). Response to interferon was better in patients with chronic persistent hepatitis or with chronic active hepatitis than in patients with chronic active hepatitis with cirrhosis. Relapse after the end of therapy was observed in 83.3% of the responders. These results indicate that the weekly administration of 6 million units of lymphoblastoid interferon is effective in decreasing serum alanine aminotransferase levels in patients with type C chronic persistent hepatitis or chronic active hepatitis.

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.

Impaired T cell function and decreased natural killer activity in patients with liver cirrhosis and their significance in the development of hepatocellular carcinoma.

SummaryIt is well known that the incidence of hepatocellular carcinoma (HCC) in patients with liver cirrhosis (LC) is very high. We investigated the immunological state in patients with LC and HCC.T cell population of peripheral blood lymphocytes (PBL) and blast transformation of PBL by phytohaemagglutinin (PHA) were significantly decreased in patients with LC. Natural killer activity against HeLa cell was also significantly decreased in these patients.These results suggest that immunological surveillance is impaired in patients with LC and this may be one of the aetiological factors in genesis of hepatocellular carcinoma in patients with LC.

Transformation of NIH/3T3 cells by DNA from a human hepatoma cell line with integrated hepatitis B virus DNA

We have studied by means of DNA-mediated gene transfer the transforming activity of the DNA of the human hepatoma cell line HCC-M, which contains genomes of hepatitis B virus (HBV) in integrated form. DNA from HCC-M induced transformed foci on transfection of NIH/3T3 cells. DNAs from primary transformants were capable of inducing secondary transformants. Most of the DNAs of these transformants were demonstrated to contain both human repetitive sequences and HBV DNA, indicating that the transformants had incorporated exogenous human DNA and HBV DNA as well. These results suggest that transformation occurs as the result of the transfer of oncogene which might be closely associated with HBV genome.

Inhibition of growth of human hepatoma cells by dual-function antisense IL-6 oligonucleotides

Hepatocellular lineage cell lines (two hepatoma and Chang liver cell lines) were found to produce interleukin-6 (IL-6). As the human hepatoma cell line, HCC-M expresses mRNA for both IL-6 and IL-6 receptor, we examined the possibility that IL-6 acted as an autocrine growth factor for HCC-M cells using two IL-6 antisense oligonucleotides (AS-1 and AS-2 oligomers) which were synthesized from different regions of an IL-6 cDNA clone. Both IL-6 antisense oligonucleotides inhibited the growth of HCC-M cells within 48 h (% inhibition by AS-1 and AS-2 oligomers was 53 and 21%, respectively). Although inhibition of HCC-M cell growth induced by AS-2 oligomer was restored by addition of exogenous recombinant IL-6 (rIL-6), the inhibition of growth induced by AS-1 oligomer was not fully restored by exogenous rIL-6, implicating the involvement of a nonantisense mechanism associated with four contiguous guanosine residues in this sequence. The inhibitory effect of AS-2 oligomer was attenuated after 72 h (% inhibition was 8%), whereas the AS-1 oligomer-induced inhibition of growth was sustained beyond 72 h (% inhibition was 38--39%). Therefore, these dual-function oligonucleotides that act via both an antisense and nonantisense mechanism may be of potential therapeutic value against hepatoma.

Elevation of serum eosinophil cationic protein in primary biliary cirrhosis

Abstract It has recently been reported that in patients with primary biliary cirrhosis (PBC), eosinophils are not only increased in the peripheral blood, but also infiltrted in the liver portal tracts. There is general agreement that eosinophils and eosinophil cationic protein (ECP) released from eosinophils contribute to cellular damage, particularly in allergic inflammation. In the present study, ECP was measured by the radioimmunoassay in sera of patients with a variety of liver diseases including PBC. Serum ECP levels were significantly higher in patients with PBC than in those with chronic viral hepatitis, in those with liver cirrhosis, and in healthy subjects. There were no significant differences in serum ECP levels between symptomatic and asymptomatic PBC. The present study suggests that high levels of serum ECP may reflect high grades of eosinophil infiltration around the septal and interlobular bile ducts characterized by chronic non-suppurative destructive cholangitis, particularly in the early stages of PBC.

New micro-glass-tube leukocyte adherence inhibition assay assessing cell adherence of mononuclear cell subpopulations defined by monoclonal antibodies

A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OKT8 and OKM1, to glass-adherent cells, fixed by glutaraldehyde or formaldehyde. An LAI reactivity to purified protein derivative of tuberculin (PPD) was detectable in donors giving a positive PPD skin test with OKT4 reagent, but not with the other two reagents.

Treatment of chronic hepatitis C with weekly administration of interferon-α

SummaryTo evaluate the efficacy of weekly administration of interferon (IFN)-α, we studied 23 anti-HCV positive patients with chronic hepatitis diagnosed by liver needle biopsy. Thirteen patients received weekly intramuscular injections of 6 MU human lymphoblastoid interferon (HLBI) for 24 weeks, and the other 10 patients were given no treatment. We examined liver-specific idiotype-bearing antibody (LSIA) in the patients’ sera. This HLBI treatment was easily tolerated by all the treated patients. In the treated group serum alanine aminotransferase (ALT) level significantly decreased during HLBI treatment. Normalization of serum ALT level by the end of treatment was observed in 7/13 (54% ) of the treated patients but in 0% of the non-treated patients. Anti-HCV was detectable in all the patients during the treatment. Those who had high LSIA levels did not respond to HLBI treatment. These results demonstrate that weekly administration of IFN is sufficient to suppress disease activity in patients with chronic hepatitis C and that patients with high LSIA levels are unlikely to respond to IFN therapy.