Yasutaka Inagaki

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.

Treatment of hepatitis C virus with peg-interferon and ribavirin combination therapy significantly affects lipid metabolism.

Aim:  We investigated lipid metabolism in patients with chronic hepatitis C virus (HCV), serotype 1, undergoing combination therapy with PEG‐IFN α‐2b (PEG‐IFN) and ribavirin (RBV).

CASE REPORT: Haemorrhagic colitis associated with royal jelly intake

The case report of a 53‐year‐old woman with abdominal pain and bloody diarrhoea is described. Prior to the onset of symptoms the patient had taken royal jelly for 25 days. Colonoscopy revealed that the mucosa was haemorrhagic and oedematous throughout the 20 cm long sigmoid colon. Histopathologically, mucosal haemorrhage, oedema, and infiltration of inflammatory cells were observed. Transmission electron microscopic examination revealed platelet aggregation in 30% of capillaries in the mucosal lesions. The drug‐induced lymphocyte stimulation test was slightly positive for royal jelly (847 c.p.m., SI= 147%) compared with the control (576 c.p.m.). The patients signs and symptoms disappeared within a few days after the initiation of conservative therapy, and the colonic lesions disappeared after 2 weeks of this therapy. This is the first reported case of haemorrhagic colitis associated with royal jelly intake.

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.

Microvascular disturbances in the colonic mucosa in antibiotic-associated haemorrhagic colitis: Involvement of platelet aggregation

An ultrastructural study of the colonic mucosa was performed in four patients with antibiotic‐associated haemorrhagic colitis and new findings are reported. Colonoscopy was performed and biopsy specimens were obtained within 24 h of the onset of bloody diarrhoea. Colonoscopy demonstrated diffuse oedematous and haemorrhagic mucosa with erosions and white coat. Light microscopy revealed mucosal haemorrhage and inflammatory cell infiltration. Ultrastructurally, platelet aggregation was frequently present in the lumina of colonic mucosal capillaries, causing engorgement of red blood cells in adjacent microvessels. Mild to severe damage was observed in capillary endothelial cells, including discontinuity of basement membranes, gaps between endothelial cells and the destruction of capillaries. There was no evidence of microvascular spasm. In conclusion, our findings suggest that antibiotics directly or indirectly cause microcirculatory disturbances, which result in tissue damage and haemorrhage, in the colonic mucosa.

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.

Absence of R1066X mutation in six Japanese patients with Dubin-Johnson syndrome.

The Dubin‐Johnson syndrome (DJS)is a rare autosomal recessive liver disease characterized by chronic conjugated hyperbilirubinemia. The phenotype of this syndrome is thought to be caused by the impaired expression of the canalicular multispecific organic anion transporter (cMOAT), which transports non‐bile salt organic anions into the bile. Recently, a mutation from arginine (Arg) to stop‐codon at codon 1066 in the cMOAT gone has been reported in one Caucasian patient with DJS. In this study, we investigated whether this mutation is found in Japanesei patients with DJS. Genomic DNAs were extracted from the leukocytes of six Japanese patients and the fragments spanning codon 1066 were amplified by polymerase‐chain reaction. The digest of the amplified fragments with a restriction enzyme, Taql, demonstrated that all of six patients did not exhibit an R1066X mutation. No mutation at Arg1066 was also confirmed by direct sequencing of the amplified products. These findings suggested that this R1066X mutation was not a major mutation in Japanese patients with DJS. Further investigation will be required in an attempt to search other mutations in cMOAT gene in Japanese patients with DJS.

High serum level of P-selectin in patients responded to interferon-α treatment for chronic hepatitis C

Abstract Circulating soluble P-selectin levels in sera of patients with chronic hepatitis C treated with natural interferon- α were investigated. Serum soluble P-selectin levels gradually increased after venesection when the sera were incubated at room temperature without serum separation, but the levels did not exceed more than 300 ng ml −1 during 3 h. Serum soluble P-selectin levels in patients who completely responded to interferon therapy were significantly higher than those in healthy subjects and other patients. Those levels in complete responders (CR) increased more than the base line levels 2 weeks after the initiation of the therapy and decreased to the base line levels 6 months after the end of the therapy, but the levels were still significantly higher than those in healthy subjects and non-responders (NR). The present preliminary study suggests that serum soluble P-selectin level is another useful marker for predicting response to natural interferon- α treatment in patients with chronic hepatitis C in addition to the established viral factors identified previously.

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.

A case of appendiceal mucocele showing massive mucous production with concomitant colonic cancer

SummaryA 58-year-old male patient presented with abnormal discharge of 200 ml transparent fecal mucus. Irregular protuberance of the ascending colon into the adjacent ileocecal region was observed by colonofiberscopy, and a cystic lesion in the ileocecal region was suggested by computerized axial tomography. Pale yellowish semitransparent jellied substances were observed exudating from the site of ileocecal resection. The diagnosis established was moderately differentiated colonic adenocarcinoma and a mucocele of the appendix. The mucus discharge disappeared after resection. Biochemical analysis of the mucus suggested the mucocele as a source of the discharged mucus. We report an extremely rare case of mucocele of the appendix that may be related to the cause of mucous stool.