High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells.

A protoplast fusion method was developed to stably transfect human cells with pSV2-derived plasmids at frequencies greater than 10(-3). This procedure made it possible to test the biological effect of a hepatitis B virus (HBV) gene independent of the viral structures required for infection. A pSV2gpt+ plasmid constructed to carry a subgenomic fragment of HBV that contained the core antigen gene (HBc gene) was transfected into human cells. A human epithelial cell line was stably transfected with the HBc+ gene by selecting recipient cells for expression of guanine phosphoribosyl transferase expression. With this gpt+/HBc+ cell line it was shown that growth in serum-free medium or treatment with 5'-azacytidine stimulates the production of the HBV core antigen. A hepatocellular carcinoma carrying the entire HBV genome was stimulated to produce the HBc gene product in response to the same factors that stimulated HBcAg production in the gpt+/HBc+ cell line constructed by transfection. The temporal relation between the cytopathologic response and HBc gene expression was similar for both cell types, indicating a primary role for HBc gene expression in the cytopathology of HBV-infected human liver.

(+)-Syringaresinol-di-O-beta-D-glucoside, a phenolic compound from Acanthopanax senticosus Harms, suppresses proinflammatory mediators in SW982 human synovial sarcoma cells by inhibiting activating protein-1 and/or nuclear factor-kappaB activities.

(+)-Syringaresinol-di-O-beta-D-glucoside (SR), syringin, and isofraxidin isolated from the stem bark of Acanthopanax senticosus Harms are its major constituents. The present work was undertaken to analyze effects of these compounds on inflammatory functions in SW982 human synovial sarcoma cell system. When cells were exposed to SR, syringin, or isofraxidin, only isofraxidin had significant inhibitory effects on cell growth, although a slight inhibition was observed at the highest concentration of SR. SR suppressed the production of IL-6 at lower concentrations than syringin and isofraxidin. SR and syringin significantly suppressed the production of prostaglandin E(2), while isofraxidin suppressed only slightly. SR was more potent than syringin and isofraxidin at inhibiting the expression of IL-1beta, IL-6, cyclooxygenase (COX)-2 and matrix metalloproteinases (MMP)-1 mRNA, but was less potent than syringin at inhibiting the expression of MMP-2. We further demonstrated that SR significantly reduced MMP-1 promoter luciferase activity and DNA-binding activity of transcriptional factors AP-1 and NF-kappaB. Taken together, these results suggest that SR, an active component of the stem bark of A. senticosus, modulates the inflammatory process involved in arthritis by suppressing various gene expression through inhibiting AP-1 and/or NF-kappaB activities.

Peracute Bacterial Meningitis due to Infection with Klebsiella pneumoniae in Captive-bred Ruffed Lemurs (Varecia variegate).

We describe the development of neurological signs in four juvenile black-and-white ruffed lemurs (Varecia variegate), housed at a petting zoo in Japan. The clinical course was severe, with three lemurs dying within 1 day of the appearance of clinical signs. The other lemur was treated and survived. Pathological analyses demonstrated meningitis and the presence of gram-negative bacilli in the cerebrum, cerebellum, palatine tonsil and liver. Klebsiella pneumoniae was isolated from the brain of all of the dead lemurs. Multilocus sequence typing analysis showed that all the isolates were sequence type 86 (ST86). To our knowledge, this is the first determination of K. pneumoniae infection in ruffed lemurs of this genus. K. pneumoniae infection may represent a risk to lemurs and people who come into contact with infected animals.

Malignant transformation of rat liver cells by N-benzoyloxy-3'-methyl-4-methylaminoazobenzene and its related compound.

N-Benzoyloxy-3'-methyl-4-methylaminoazobenzene-(N-benzoyloxy-3'-Me-MAB) was highly toxic to rat liver epithelial cells (Ac2F) and induced many more chromosome aberrations and unscheduled DNA synthesis than did 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). N-Benzoyloxy-3'-Me-MAB and its related compound, N-acetoxy-4-methylaminoazobenzene, efficiently induced malignant transformation of the cells, but 3'-Me-DAB was not so effective. The cells transformed by N-acyloxy-4-methylaminoazobenzene (N-acyloxy-MAB) derivatives showed significant increases in plating efficiency in liquid medium and in the size of aggregates in rotation culture. Such increases were not seen in the 3'-Me-DAB-transformed cells. The results indicate that the N-acyloxy form of the 4-dimethylaminobenzene derivatives seems very likely to be the ultimate metabolite.

Influence of renal and hepatic failure on the pharmacokinetics of recombinant human granulocyte colony-stimulating factor (KRN8601) in the rat.

The pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was studied in rats experiencing renal and hepatic failure. The serum concentration of rhG-CSF after i.v. administration to male Sprague-Dawley rats at a dose of 10 micrograms/kg was investigated by a sandwich enzyme immunoassay. Total-body clearance of rhG-CSF was 44.48 ml/h/kg in sham-operated rats compared with 9.429 ml/h/kg in bilaterally nephrectomized rats. In sham-operated rats, the half-life (beta) of rhG-CSF was 1.512 h, and it increased to 5.333 h after nephrectomy. The volumes of distribution were identical in both rats. In rats with acute renal failure induced by uranyl nitrate, the clearance and volume of distribution were identical to those of control rats, but the half-life (beta) was slightly shorter. In partially (70%) hepatectomized rats, the clearance of rhG-CSF decreased from 42.08 ml/h/kg to 31.93 ml/h/kg. Similar half-lives were observed in rats in both the sham-operated and hepatectomized groups. However, the volume of distribution decreased after hepatectomy. In rats with hepatic failure induced by CCl4, the pharmacokinetic changes were similar to those observed in hepatectomized rats. These results suggest that renal clearance makes a major contribution to total-body clearance compared with hepatic clearance.

Prevalence of Baylisascaris Roundworm in Captive Kinkajous in Japan.

Baylisascaris potosis causes larva migrans in animals. The present study evaluated the prevalence of B. potosis in captive kinkajous ( Potos flavus ) and the ability of milbemycin to treat natural infections of B. potosis in 2 female wild-caught kinkajous. In 2012, fecal samples were collected from 16 kinkajous in 6 zoological gardens and 29 imported captive kinkajous from 4 pet traders in Japan. Although all samples from zoological gardens were negative, 8 kinkajous from traders were positive for Baylisascaris eggs, at least 4 of which were wild caught in the Republic of Guyana. No associated human illness was reported from any of the facilities. The 2 infected kinkajous received a single oral administration of Milbemycin® A Tablets, which delivers 0.69-0.89 mg/kg milbemycin oxime. Fecal examinations on days 14 and 30 were negative for Baylisascaris eggs. These results demonstrated that milbemycin oxime has possible anthelmintic efficacy against Baylisascaris roundworms in captive kinkajous. We conclude that Baylisascaris infections are highly prevalent in wild-caught kinkajous in Japan and that most of the infected kinkajous were imported from the Republic of Guyana.

Experimental infection of Mongolian gerbils with Baylisascaris potosis.

The present study evaluated the pathogenicity of Baylisascaris potosis, a newly described ascarid nematode, in Mongolian gerbils. Gerbils were infected with varying doses of either B. potosis or Baylisascaris transfuga embryonated eggs (100, 1,000, and 4,000) for 30 days postinfection (pi). Baylisascaris potosis-infected gerbils showed no clinical signs of disease; however, gerbils exposed to 1,000 and 4,000 B. transfuga eggs showed severe neurologic signs at 22-29 days and 14-15 days pi, respectively. Histopathologic examination revealed larvae and lesions in the intestine, lung, liver, and muscles of B. potosis-infected gerbils, but not in the brain, whereas B. transfuga larvae were found only in the brain and muscle. These results indicate that B. potosis larvae migrate through numerous organs and are associated with visceral larva migrans in gerbils, but less frequently migrate to the nervous system in gerbils than does B. transfuga .

Comparative pharmacokinetics of KRN8602, a new morpholino anthracycline, and adriamycin in tumor-bearing mice.

It has been reported that KRN8602 shows antitumor effects similar or superior to those of Adriamycin (ADM) against several murine and human cell lines and has been found to be effective against multidrug resistant tumor cells. We investigated the pharmacokinetics of KRN8602, a new morpholino anthracycline, in comparison with ADM in mice bearing colon26 adenocarcinoma. After intravenous administration, both drugs disappeared triexponentially from the plasma and KRN8602 was eliminated faster than ADM. The rate of elimination of KRN8602 from tissues was also faster than than of ADM. The relative order of the area under the curve (AUC) of KRN8602 was spleen > tumor > small intestine > lung > kidney > heart > liver > brain > plasma, while that of ADM was spleen > kidney > lung > liver > heart > small intestine > tumor > plasma. ADM was not detectable in the brain. The AUC of KRN8602 was higher than that of ADM in the tumor and brain, but it was lower in other tissues. The tissue-to-plasma concentration ratio (KPapp) of KRN8602 was higher than that of ADM in the tumor, spleen, small intestine and brain. KRN8602 was metabolized to several metabolites. The concentrations of M1 and M2 (glycoside-type metabolites) was relatively high in the spleen. M3 (aglycone-type metabolite) showed a very high AUC ratio in the liver (34%). In tumor, M1 and M2 concentrations were low and M3 was not detected. KRN8602 had a greater activity than ADM and M2 had a cytotoxic activity similar to KRN8602 against colon26 cells in an MTT assay. These results suggest that the strong antitumor effect of KRN8602 against colon26 is due not only to its strong cytotoxic activity but also to its marked transferability into tumors. KRN8602 shows better selective toxicity than ADM, because KRN8602 is more selective for tumors than ADM and less is transferred to normal tissues.

[Establishment and characterization of a human cholangiocellular carcinoma cell line].

A cell line, HuH-28 was established in vitro from a patient with cholangiocellular carcinoma (CCC). This cell line has been in continuous culture over 10 month period with slow growth potential. HuH-28 was composed of spindle-shaped cells as major population besides a small percentage of polygonal-shaped cells. Chromosome number of the cells were distributed near the hypotriploid region on the 3rd passage. HuH-28 cells were not transplantable into nude mice, but secreted some tumor markers including alkaline phosphatase (ALP), gamma glutamyltranspeptidase (GGT), beta 2-microglobulin (BMG), ferritin, elastase-1 and tissue polypeptide antigen (TPA). This HuH-28 cell line will represent a good model for the investigation of carcinogenesis, histogenesis+ and diagnosis of CCC.

[Culture methods of human liver cells].

Attempts to isolate and cultivate human liver cells have been described. Many viable liver cells have been obtained when dissociated with collagenase followed by dispase. The morphology and differentiated functions have been maintained for more than 3 weeks when human fetal liver cells were cultured not only in medium containing 10(-3) M hydrocortisone, but also on collagen gel substrates with 5 x 10(-7) M hydrocortisone. The colony-forming capacity of primary cultured fetal human livers has also been described in the presence of conditioned medium.

[Effects of various factors on the growth and function of a human hepatoblastoma cell line, HuH-6--an approach for serum-free cell culture].

The effects on a human hepatoblastoma cell line (HuH-6) of serum and Ca2+ were investigated. A higher concentration of serum reduced the production of alpha-fetoprotein, whereas Ca2+ enhanced that of both albumin and alpha-fetoprotein. In view of these facts, a serum-free medium using RPMI-1640 supplemented with lactalbumin hydrolysate, epidermal growth factor, hydrocortisone, insulin, chrelatoxine, glucagone and selenium was then developed. Collagen type IV was superior to collagen type I, laminin, fibronectin and plastic in supporting the growth of HuH-6 in the serum-free medium. Higher amounts of both albumin and alpha-fetoprotein were secreted in HuH-6 cultured in serum-free medium than in serum-supplemented medium.

[Studies on AFP-producing capacity and some other properties of human hepatoma cells treated with various anticancer drugs].

The effect of various anticancer drugs on alpha-fetoprotein (AFP) secretion and some other properties of human hepatoma cells was investigated in vitro with the following results. (1) There was a high correlation between AFP secretion and cell number after treatment of human hepatoma cells with anticancer drugs and the amounts of AFP secreted per 10(4) cells per 72 hours (AFP-secreting capacity) were not affected within therapeutically achievable concentrations (TAC). (2) The AFP-secreting capacity was affected with some exceptions in the cells treated with higher concentration of drugs than TAC. Furthermore, chromosomal and morphological aberrations in the similarly treated cells, were also observed, suggesting the relationship between the change of AFP-producing capacity and that of some other properties.

[Tissue culture course of a human hepatoma cell line].

A cell line, HuH-33 was cultured in vitro from a patient with hepatocellular carcinoma. This cell line has been in continuous culture over 12 month period with slow growth potential. HuH-33 was composed spindle-or polygonal-shaped cells as a major population. Chromosome number of the cells were widely distributed even in the primary culture. HuH-33 was transplantable into nude mice and secreted alpha-fetoprotein, albumin, beta 2 microglobulin, ferritin and tissue polypeptide antigen.

A mild transient decrease of peripheral red blood cell counts induced by a suprapharmacological dose of pegylated human megakaryocyte growth and development factor in rats.

Previous studies have shown that pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) at suprapharmacological dose induces a mild transient decrease of red blood cell counts according to thrombopoiesis in normal mice. To unravel the mechanism underlying this mild transient decrease of red blood cells, we have studied the effect of PEG-rHuMGDF on the circulating plasma and blood volume, and the serum biochemical parameters of anaemia and splenectomy. Also, we have performed histological studies of the bone marrow and the spleen of PEG-rHuMGDF-treated rats. PEG-rHuMGDF (300 microg kg(-1)]) or vehicle was subcutaneously administered to rats once a day for up to five days. From day 6 after the start of PEG-rHuMGDF administration, the platelet counts and plateletcrit levels were significantly increased, reaching peak values on day 10, and recovering to normal by day 20. The red blood cell counts and the haematocrit levels were significantly decreased on day 6 to 13. The decreases in red blood cell levels and haematocrit produced by PEG-rHuMGDF treatment were mild and had recovered by day 15. The plasma and blood volumes were significantly increased on day 10 in PEG-rHuMGDF-treated rats. No alteration of the serum biochemical parameters for anaemia, iron or total bilirubin, were observed on day 10. The histological examination on day 10 revealed a marked increase in megakaryocytes and a slight decrease in erythropoiesis in the bone marrow of rats that received PEG-rHuMGDF (300 microg kg(-1)). There was also a slight increase in splenic megakaryocytes and erythropoiesis. The decrease of red blood cells by PEG-rHuMGDF was not affected by splenectomy. These results suggest that the mild transient decrease of red blood cells induced by PEG-rHuMGDF treatment for up to five days is based mainly on the increases in the plasma and blood volume. These events are secondary changes due to the regulation of the excess production of megakaryocytes in the marrow and the peripheral platelets.

An attempt to eliminate fibroblast-like cells from primary cultures of fetal human livers.

The elimination of fibroblast-like cells from primary cultures of fetal human livers was studied. A fibroblast-like cell line (HuF), which was obtained by subculturing fetal human liver cells 4 or more times, was briefly treated with hydrocortisone (HC) or putrescine (PUT). The growth of HuF cells was inhibited by HC at a concentration of 10(-2) M and by PUT at a concentration higher than 10(-3) M. Long-term treatment of HuF cells with 10(-3) M HC inhibited the growth of the cells. Primary cultures of fetal human livers were made in medium containing HC or PUT, and morphological and functional examinations were made. The cultures were predominantly composed of epithelial-like cells, with few fibroblast-like cells, when the HC concentration was 10(-5)M to 10(-3) M. A high amount of albumin was secreted at these concentrations of HC. On the other hand, at 10(-3) M PUT, many epithelial-like cells were seen, but albumin was undetectable. The present results indicate that albumin-producing epithelial-like cells can be selectively maintained in medium containing HC, in primary cultures of fetal human livers.

Preparation of single cell suspensions from hepatoma cells in culture.

Fundamental examination was carried out on the liberation of single cells from hepatoma cells in culture. dRLa-74 cells derived from rat hepatoma, which hardly disperse as single cell suspensions with several proteolytic enzymes or EDTA alone, dispersed with a high yield of single cells by the combination of trypsin and EDTA. HUH-6 cells derived from human hepatoblastoma also showed similar results. The degree of cell dissociation by the combination was dependent on the incubation temperature or pH.

Effects of 3'-methyl-4-dimethylaminoazobenzene and 4-aminoazobenzene on plating efficiency, mitotic index, and macromolecular synthesis of cultured liver cells.

The response of a cultured liver cell line to azo dyes was investigated by the application of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and 4-aminoazobenzene (AB) with observation of, in particular, the changes which occurred in plating efficiency, macromolecular synthesis and mitotic indices. The results were as follows. (1) The application of 3'-Me-DAB resulted in a decrease in size of colonies and plating efficiency of both J-5-2 cells (derived from 7-day-old rat liver tissues) and cells of the colonical clone, J-5-2cl. (2) AB caused greater inhibition of plating efficiency than did 3'-Me-DAB. (3) Both 3'-Me-DAB and AB inhibited synthesis of DNA but not of RNA or of protein. (4) AB exhibited a greater inhibitory effect on mitotic indices than did 3'-Me-DAB.

Effects of laminin and collagen type I on the morphology and secretion of proteins in human hepatoblastoma and hepatoma cell lines.

The effects of laminin (LAM) and collagen type I (C-I) on human hepatoblastoma (HuH-6) and hepatoma (HuH-7) cell lines were investigated. C-I was superior to LAM in supporting the attachment of the cells, especially of HuH-6, to plastic surfaces. No effect of LAM and C-I on cellular morphology was recognizable by phase contrast microscopy. By scanning electron microscopy (SEM), much more microvilli were found on the cell surface of HuH-6 on LAM substrate than on C-I substrate. In HuH-7 cells, however, these microvilli were rarely found on either LAM substrate or C-I substrate. The gel profile of the proteins secreted by HuH-6 and HuH-7 cells was not affected by the culture substrate except for the major band, though the amount of alpha-fetoprotein (AFP) secreted was larger when the cells were cultured on LAM substrate than on C-I substrate. These results indicate that the ability of LAM or C-I to enhance attachment is different from that to enhance AFP production or microvilli expression in HuH-6 cells and probably in HuH-7 cells.

Examination of HeLa cell contamination of human cell lines derived from primary hepatomas using glucose-6-phosphate dehydrogenase and lactate dehydrogenase isozymes.

Isozyme patterns of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) in human cell lines derived from primary hepatomas were compared with those in HeLa cells. Some cell lines derived from primary hepatomas having type B G6PD showed one or two isozymes of LDH. On the other hand, HeLa cells having type A G6PD showed four LDH isozymes. These findings suggest that not only G6PD, but also LDH may be useful for the detection of HeLa cell contamination of a culture in some cases.

Effect of 3'-methyl-N,N-dimethyl-4-aminoazobenzene in the presence or absence of liver microsomes on the liver cells having low tumor-producing capacity in culture.

A cell strain having low tumor-producing capacity was exposed in culture to 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) in the presence or absence of liver microsomes, and whether or not the cells will progress to those having high tumor-producing capacity was examined. When transplanted into rats, the cells treated with 3'-Me-DAB four (Exp-I) or thirteen times (Exp-II) formed larger tumors than untreated control cells, the latter treatment being more efficient in this regard. Furthermore, the tumors formed by the cells treated with 3'-Me-DAB in the presence of liver microsomes were considerably larger than those formed by the cells treated with 3'-Me-DAB alone. The subcutaneous tumors produced by the cells treated with 3'-Me-DAB with S-15 Mix showed poorly differentiated histology compared with those produced by control and 3'-Me-DAB-treated cells. The frequency of lung metastasis tended to increase by 3'-Me-DAB with S-15 Mix. The cells treated with 3'-Me-DAB in the presence or absence of liver microsomes differed from untreated control cells in vitro in some properties, including the size of aggregates in rotation culture, plating efficiency in liquid medium and morphology. These observations suggest that cell malignancy was promoted by 3'-Me-DAB alone as well as by 3'-Me-DAB in the presence of liver microsomes.