Seishi Nagamori

Mother-to-child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region

BACKGROUND/AIMS We have analyzed the molecular basis of mother-to-child transmission of hepatitis C virus. METHODS/RESULTS Healthy pregnant women were screened for anti-HCV antibody and babies born to hepatitis C virus carrier mothers were prospectively investigated. Among the 35 pairs studied, the hepatitis C virus genome was detectable in only one baby, who did not show any significant symptoms of hepatitis. The viral load in the blood of the mother was one of the highest of the 35, and the population of the hepatitis C virus genome was heterogeneous. Furthermore, she was found to have a mixed infection with type 1a and type 1b hepatitis C virus. However, the hepatitis C virus genome obtained from the baby was only from type 1b, less heterogeneous and composed of the clones which were detected in the blood of the mother. The selected hepatitis C virus had a 12-nucleotide insertion in the amino-terminus of the E2 hypervariable region of the genome. CONCLUSIONS The incidence of mother-to-child transmission of hepatitis C virus from carrier mothers was shown by this prospective study to be low. The presence of selection pressure during transmission was suggested. The biological significance of the virus with 12-nucleotide insertion has to be determined.

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT

Abstract Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.

Persistence of hepatitis C virus RNA in established human hepatocellular carcinoma cell lines.

The persistence of the viral RNA of hepatitis C virus (HCV) was examined in 13 hepatocellular carcinoma (HCC) and two hepatoblastoma cell lines by reverse transcription followed by the polymerase chain reaction (RT‐PCR). HCV RNA was detected in three HCC lines (JHH‐1, JHH‐4, and JHH‐6) and negative‐strand viral RNA was found in JHH‐4, indicating that there is a putative replicative intermediate of HCV in JHH‐4 cells. To rule out the possibility of contamination, the partial nucleotide sequences of HCV‐specific PCR products of these three cell lines were determined. The clone from JHH‐1 belonged to genotype 1 (1a or 1b), and the clones from JHH‐4 and JHH‐6 belonged to genotype 2b, but their sequences differed from each other. These cell lines may be useful for studies related to HCV.

Involvement of insulin-like growth factor binding protein-3 in the retinoic acid receptor-α-mediated inhibition of hepatocellular carcinoma cell proliferation

We examined the relationship between the expression of retinoic acid receptor-alpha (RAR-alpha) and upregulation of insulin-like growth factor binding protein-3 (IGFBP-3) in the retinoid-induced inhibition of hepatocellular carcinoma (HCC) cell proliferation. HCC cell lines showed a marked expression of RAR-alpha, whereas the expression levels of RAR-beta and RAR-gamma were relatively lower. An RAR-alpha agonist significantly inhibited the HCC cell proliferation both in vitro and in vivo. The RAR-alpha expression closely related to the upregulation of IGFBP-3 as compared with RAR-beta or RAR-alpha expressions. RAR-alpha agonist would be beneficial to inhibit the growth of HCC.

Effects of nerve growth factor and glucocorticoid on cultured human pheochromocytoma cells

Primary cell cultures of two human pheochromocytomas (PC) that were associated with high serum levels of adrenaline and noradrenaline were developed to study the effects of nerve growth factor (NGF) and dexamethasone on the morphology and function of PC cells in vitro. By phase-contrast microscopy, cultured cells were small and hyperchromatic on the first day of culture; neurite-like processes that extended to other cells developed several days later and were maintained for more than 3 months. NGF (100ng/ml), dexamethasone (10−5M), or NGF + dexamethasone were added to the culture media 2 weeks after the cultured cells had stabilized. Catecholamine concentrations in the medium were maintained at higher levels after addition of NGF, dexamethasone, or NGF + dexamethasone as compared to control cells. In the presence of NGF, extension of neurite-like processes was clearly accelerated, while high levels of dexamethasone inhibited growth of processes. These in vitro studies showed that the addition of NGF or the removal of dexamethasone induces differentiation of adrenal neurons present in pheochromocytomas, suggesting that adrenocortical steroid hormones influence the morphological control of adrenal medullary cells.

Genotyping of hepatitis C virus by a simple ELISA method.

BACKGROUND Hepatitis C virus (HCV) has been classified into five distinct types by nucleotide sequence analysis of the genome. The correlation between genotypes of HCV and sensitivity to treatment or prognosis is still controversial. OBJECTIVES We tried to establish a simple antibody assay to determine the HCV serotype instead of genotype determined by PCR or nucleotide sequence. STUDY DESIGN We made an enzyme-linked immunosorbent assay (ELISA) system by using synthetic oligopeptides of NS4 regions of types 1 (CH) and 2 (KN) HCV, and examined sera of various stages of hepatitis C patients. RESULTS Among 13 HCV RNA-positive sera, serotyping was consistent with genotyping. Four sera of type 1 and 7 sera of type 2 were positive to anti-CH and anti-KN antibodies, respectively. Two sera of patients mixedly infected with type 1 and 2 HCV were positive to both antibodies. The number of type 1 and 2 in patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma was 27 and 18, 28 and 15, and 46 and 12 respectively. CONCLUSIONS Our result suggests that this simple ELISA method is useful for typing of HCV, and there is no significant relationship between HCV type and liver failure.

Morphological changes in human gall bladder carcinoma cell line-NOZ due to epirubicin and doxorubicin.

Epirubicin is widely used in various cancer therapies because its side effects and less than those of doxorubicin. We compared the direct effects of doxorubicin and epirubicin to cellsin vitro. In this study, we used the human gall bladder carcinoma cell line-NOZ. We evaluated the cytocidal effects using a DNA fluorimetric assay with fluorochrome Hoechst 33342. The addition of epirubicin in a culture medium showed stronger cytocidal effects than the addition of doxorubicin. Morphologically, after treatments with the two drugs, cells were enlarged, large vacuoles appeared in the cytoplasm, and concentrated microvillus-like structures due to doxorubicin were observed by transmission electron microscopy.

Effects of the combination of hyperthermia and adriamycin on cultured human liver cancer cells.

著者らは抗癌剤の併用による温熱の効果を,著者らの教室にて樹立しえたアルブミン高産生性のヒト肝細胞癌JHH-4株を用い,in vitroにおいて検討を行った.ペトリ皿に付着増殖した肝癌細胞を,抗癌剤として0～20μg/mlのAdriamycinを含む培養液にて,従来のコロニー形成法とは異なり,温度勾配培養装置を用い,37～43℃で2日間培養を行い,生細胞数の算定のみならず機能的,形態的にも判定を行った.温度の上昇に伴い生細胞数の減少,培養上清中のアルブミン濃度の低下,3Hラベルのサイミジン,ウリジン,ロイシンの取込みの低下,付着細胞の形態的変化がみられた.温熱単独でみられた肝癌細胞に対するin vitroにおけるこれらの効果は,Adriamycinを併用することにより増強が認められた.

Characterization of alpha-fetoprotein secreted from cultured reuber H-35 hepatoma cells

SummaryReuber H-35 hepatoma cells were examined for their ability to synthesize protein in vitro, especially to produce alpha-fetoprotein (AFP). The presence of AFP in the culture supernatant solution was determined immunologically by the micro-Ouchterlony method. Charge heterogeneity of AFP was examined electrophoretically in continuous gradient polyacrylamide microgels. With regard to the duration of culture, there was no remarkable change in the ratio of two peaks of AFP, and which came out as a major combined peak and a similar peak by PAS staining on the condition of added SDS. These findings indicated that Reuber H-35 hepatoma cells had potential to produce two charge variants of AFP in vitro.