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To establish the most proper method of in situ hybridization in detection of HCV-RNA in the liver, various detailed procedures were examined using frozen as well as paraffin-embedded sections of tissue derived from patients. In frozen sections of the liver from hepatitis C patients obtained at autopsy or surgery, HCV-RNA was detectable by in situ hybridization using thymine-thymine dimerized oligonucleotide DNA probes when the sections were treated with ethanol-acetic acid at first, then 0.2 N hydrochloric acid, proteinase K (0.02 u/ml) and DNase. When the paraffin-embedded liver sections were used, more intense proteinase K treatment (0.2-2 u/ml) was required to expose viral RNA and even after that, the positive HCV-RNA signals were less than those in frozen sections, because the cytoplasmic RNA in the routine paraffin-embedded sections was preserved unevenly and less than in frozen sections. These findings indicate that in situ hybridization of HCV-RNA is useful for diagnosing HCV infection and should be a potent tool for monitoring the state of virus activities during therapy. However, the liver biopsy method should be modified so that RNA is retained properly to utilize biopsies more effectively for the routine diagnosis of HCV infection.

The present experiment was undertaken to study what types of human cancers are responsive to the antiproliferative effects of suramin. The human malignant cells used were as follows: cervical cancer (HeLa), mammary cancer (MCF-7), bladder cancer (EJ), hepatoma (HuH-7, PLC/PRF/5), embryonal carcinoma (PA-1), in vitro transformed fibroblasts (KMST-6, SUSM-1, VA-13), five myeloma cell lines (KMM-1, KMS-5, KMS-11, KMS-12, RPMI 8226), Burkitt's lymphoma (Raji), acute promyelocytic leukemia (HL-60), chronic myelocytic leukemia (K562), Epstein-Barr virus nuclear antigen positive lymphoblastoid cells (KMS-9). The cells were treated with 25 to 100 micrograms/ml suramin for 72h. Proliferation of HuH-7 and two human myeloma cells (KMS-11 and KMS-12) was remarkably inhibited, and that of PA-1, PLC/PRF/5, KMST-6, two other myeloma cell lines (KMM-1 and KMS-5), Raji and HL-60, was moderately inhibited. In order to confirm part of the results obtained from in vitro experiments, in vivo experiments were also undertaken. The growth of HuH-7 cells transplanted subcutaneously into nude mice was significantly suppressed by intravenous injection of suramin. We discussed the possibility that certain types of human cancers, the growth of which seemed to be more or less dependent on polypeptide growth factors, might be sensitive to the antiproliferative effects of suramin.

Since detection of hepatitis C virus RNA by the polymerase chain reaction (PCR) showed that there existed anti-C100-3 (anti-HCV) antibody negative patients infected with HCV, we attempted to find out whether there were any clinical or viral genomic differences between the anti-HCV antibody positive and negative groups. One hundred and fifty-nine patients with chronic liver diseases with hepatitis C virus RNA in their sera were selected. Anti-HCV antibody was tested for anti-C100-3 antibody by an enzyme linked immunosorbent assay. The incidence of anti-HCV antibody was 129/159. The concentration of serum gamma-globulin, the titier of ZTT, and the positive rate of the PCR with the primers of the NS3/4 region (NS3/4PCR) were significantly higher in the anti-HCV antibody positive group than in the negative group. However, the other data such as alanine aminotransferase activity or past history were not significantly different. Nucleotide sequence of the cDNA fragments of NS3/4 region amplified by the PCR did not differ significantly between isolates from anti-HCV antibody positive and negative sera. The sequences observed in the present study did not differ significantly from those reported previously. Although there remains the possibility that the variation of viral genomic sequences may cause the absence of anti-HCV antibody, these results suggested that the individual clinical backgrounds or immunoreactivity of the patients might influence the antibody development.

1. When chicken sarcoma virus is serially inoculated on the mouse brain, it loses its carcinogenecity, but when it is inoculated on young chicken, granuloma develops in the liver and lung. When this granuloma is transplanted on adult chicken, a transplantable fibrosarcoma is obtained. 2. According to literature, the originaltumor of the Brown-Pearce cancer is a basal cell cancer, but that imported to Japan in 1953 presented a histological picture of carcinosarcoma. The metastasized tumor of the eye presents a purely cancer tissue, but when this is inoculated on the testis, carcinosarcoma is reproduced. It is therefore considered that the mother cell of the sarcoma is of host origin. 3. MY sarcoma is not a sarcoma, but is a spindle cell cancer. It might be a sarcoma which transformed into a cancer during serial transplantation, but perhaps it was originally a cancer but had been erroneously diagnosed as sarcoma. 4. The tumors we obtained by means of the feeding tests of Yoshida tumor all developed at organs other than those of the digestive tract. They are chiefly reticulo-sarcoma, but others which develop are malignant granuloma in the liver and lung, malignant adenoma in the kidney, papilloma of pelvis, and ependymoma in the cerebral ventricle. Since the discovery of the Yoshida tumor in 1943, serial transplantation has been conducted for 19 years with this tumor not only in Japan but also in foreign countries, but there has been no report to this date that a transformed strain has developed by cell transplantation. It therefore must be considered that the carcinogenesis observed in our feeding tests is a carcinogenesis due to a mechanism completely unlike that of cell transplantation. It has been confirmed by electron microscopy that in the early stage of transplantation of this tumor into the abdominal cavity there was an additional tumor growth due to the anaplastic proliferation of serous cells. 5. During the serial transplantation of viral tumors and/or virus dependent tumors, the tumor sometimes undergoes a morphological change. Though the cause of this is not yet sufficiently elucidated, it is suspected that there is some relationship with virus in the wide sense.

There are many electron microscopic observations of the cells infected with measles virus (1-6), and all of them appear to be concerned mainly with observation on the inclusion bodies and not any seems to have described the morphology of mature virus particles located within the infected cell.

<P>The mitochondrial, the microsomal, and the supernatant fractions were prepared from the cell homogenate of tumors induced by viruses, such as adenovirus type 12, SV 40, and Rous sarcoma virus, etc. and the antigenicities of these fractions were investigated. In the virus-induced tumors, there existed no antigenicity common to the mitochondrial and the microsomal fractions as in the tumors induced by chemical carcinogens, and the highest antigenicity was recognized in the mitochondrial fraction. Therefore, the properties of the tumor cell mitochondria were precisely investigated with virus-induced tumor mitochondria. 1. The mitochondria of tumors induced by viruses have clearly the specific antigenicity. 2. This specific antigenicity of virus.induced tumor mitochondria IS common to all the virus-induced tumors used in the present study. 3. This tumor mitochondria.specific antigenicity is found commonly in all the tumor mitochondria in the present experiments. 4. The specific cancer antigenicity of tumor cell mitochondria does not exist in normal organ mitochondria, but the regenerating organ mitochondria exhibit a slight antigenicity common to cancer cell mitochondria.

As a step towards the elimination of Japanese encephalitis virus in natural surroundings, we inoculated pigs, rabbits and chicks with inactivated Japanese encephalitis vaccine supplemented with complete or incomplete Freund's adjuvant twice at one-week interval. Subsequently, we compared HI antibody titers of the groups inoculated with vaccine containing complete Freund's adjuvant (pigs, rabbits, chicks), of the group inoculated with vaccine containing incomplete adjuvant (rabbits), ar;d of the groups inoculated with vaccine containing no adjuvant (pigs, rabbits, chicks), and also observations on changes in the antibody titers due to natural infection. In a certain portion of these animals neutralizing antibody titers were also determined. The results of this study are briefly summarized as follows. 1. In the groups of pigs and rabbits inoculated with vaccine containing complete Freund's adjuvant, titers of HI antibody and neutralizing antibody were higher than those inoculated with vaccine containing no adjuvant and their high titers persisted. Further, in the group of chicks inoculated with inactivated Japanese encephalitis vaccine containing complete Freund's adjuvant, HI antibody titers were higher and persistent as compared with the antibody titers in the chicks inoculated with inactivated Japanese encephalitis vaccine alone. 2. In the rabbits inoculated with inactivated Japanese encephalitis vaccine contammg incomplete adjuvant, HI antibody titers were lower than in those receiving the vaccine with complete adjuvant, but it has been demonstrated clearly that vaccination of inactivated Japanese encephalitis vaccine supplemented with incomplete adjuvant brings about less sideeffects. Hence such a method of vaccination can be applied as the vaccination with least side-effects. 3. With respect to natural infection of swine, on August 27 when the pigs were thought to have been infected, there was observed a rise in antibody titers. And on being infected with Japanese encephalitis, the antibodies formed in those pigs inoculated with inactivated Japanese ence- phalitis vaccine with or without complete adjuvant proved to be all 2-ME resistant type, whereas the antibodies produced in the control groups not receiving such a vaccination were 2-ME sensitive antibody.

A series of experiments was conducted to study the base composition of DNA in AVl2-induced tumor and host cells by paper chromatography, and it was found that DNA per cent. guanine-cystosine contents were around 42 % in both of them. The base composition of DNA of AV12 itself differs considerably from that of AVl2-induced tumor cells, while the DNA of tumor cells shows the property similar to that of host cell DNA. The genetical relationship among virus, host cells and tumor cells was discussed.

In the immunofluorescent study it has been revealed that rabbit sera immunized with transformed cells induced by SV-40 DNA, produce circulating antibody capable of re:lcting with intranuclear antigens synthesized by SV-40 complyte virus transforming process, In addition, the result confirmed that SV-40 DNA replicates DNA-containing viruses in the host cell and that also the genome coding for the synthesis of SV-40 tumor antigen is resposible for viral DNA.

An electron microscope study was performed on the ultrastructure and developmental process of the Mukai strain of Japanese B encephalitis virus propagated in vitro on porcine kidney stable cells. The virus particle of Japanese B encephalitis is hexagonal in sections and approximately 40 mμ in the maximum diameter, composed of an outer membrane, 20Å thick, viroplasm, 30 Å thick and an electron-dense nucleoid, 25 mμ in diameter. The virus particles develop by a budding process on the wall of the cytoplasmic vacuole. Thereafter, virus particles are densely packed in the vacuole usually in random arrangement and rarely in crystalline arrays. The vacuole containing virus particles gradually moves toward the cell surface and liberates the virus particles to the exterior of the cells through a narrow canaliculus. A structure suggestive of incomplete virus particles was also observed.

Interference of oncogenic N-nitrosourea in intraocular tumor induction by human adenovirus type 12 in rats was examined. Transplacental administration of methylnitrosourea to rat embryo reduced significantly the latent period of the intraocular adenovirus tumor in each animal whereas in groups preadministered with ethylnitrosourea the decrease in the latent period showed marked individual variation. Preadministration of N-nitrosourea caused little change in the morphology and incidence of adenovirus tumors. The histological picture of adenovirus induced intraocular tumors which developed in each group of rats was that of retinoblastoma-like tumor identical to the tumor induced by single virus injection.

The oncogenicity of xenotropic pseudotype Kirsten murine sarcoma virus (MSV) was investigated in Sprague-Dawley rats. When fetal or newborn rats were inoculated intracerebrally with xenotropic pseudotype MSV, brain tumors developed after about one month. Tumors were induced both in the cerebrum and the cerebellum. Histologically, the tumors were predominantly glioblastoma multiforme and hemangioendotheliomas. In cerebellar lesions, malignant transformation of vascular endothelial cells, polycystic areas and numerous giant cells were noted. Proliferation of Purkinje cells was also observed in some of the cerebellar tumors. Inoculation of the same virus by other routes, such as s.c., i.p. and i.m., also caused cerebral and cerebellar tumors. Brain tumors thus induced were transplantable subcutaneously into suckling rats.

A blood recipient, aged 66, was found to have positive adult T-cell leukemia-associated antigens (ATLA), approximately half a year after a transfusion. The donor's ATLA-antibody titer was 1: 640. Routine screening of blood donors for ATLA antibody was proposed.

Electron microscopy of four human T-cell lines revealed the production of type C virus particles in two T-cell lines: one derived from acute lymphoblastic leukemia and the other from a leukemic T-lymphoid malignancy. Virus particles isolated from these cells had reverse transcriptase activity and the major internal structural protein of 30,000 daltons (p30). The indirect immunofluorescence test of these virus-producing cells with sera of patients with adult T-cell leukemia (ATL) was negative. The data indicate that these retroviruses are different from adult T-cell leukemia virus (ATLV).

We investigated the restriction of the host range to infectivity of MSV by helper leukemia virus in vivo. When newborn SD-rats were inoculated intracerebrally, subcutaneously, intraperitoneally or intramuscularly with xenotropic pseudotype Kirsten MSV, Ki-MSV(BV2), either brain tumors or myogenic sarcomas were induced, depending upon the route of inoculation. However, no tumors developed in SW-Icr mice inoculated with Ki-MSV(BV2) either intracerebrally or intramuscularly at birth. Ecotropic Ki-MSV(Ki-MuLV) induced myogenic sarcomas in mice when inoculated intramuscularly and also induced brain tumors and myogenic sarcomas in rats when inoculated intracerebrally and intramuscularly, respectively. Thus, the host range of pseudotype MSV appeared to depend on a helper leukemia virus.

The susceptibility of Rous sarcoma virus (RSV) genomes integrated in mouse ascites sarcoma cells (SR-C3H/He cells) to DNase I and DNase II was investigated. Approximately half of the viral sequences were sensitive to DNase I and DNase II when 17% and 7.4% of the chromatin DNA was rendered acid soluble, respectively. The results suggest that newly acquired exogenous proviral sequences are integrated into both transcriptionally active and inactive regions of chromatin in cells lacking related endogenous viral sequences.

The localization of both the large T and small t tumor (T) antigens in cultured cells (Vn 12 cells) of hamster brain tumors induced with BK virus (BKV), a new human papovavirus, was studied by an enzyme labelled antibody method at both the light and electron microscopic levels. Under the light microscope, BKV T antigen was observed in the nucleus, except for the nucleoli, of cells in interphase, and under the electron microscope it was observed in the nucleus except for the nucleoli and nuclear membrane. BKV T antigen appears to be closely associated with nuclear chromatin as previously reported for simian virus 40 tumor antigen (SV40 T antigen). The intracellular localization of BKV T antigen was the same as that of SV40 T antigen. In metaphase, BKV T antigen seems to be distributed diffusely throughout the cytoplasm except for the chromosomes. In telophase, BKV T antigen transfers from the cytoplasm to the nucleus. The migration of BKV T antigen during the cell cycle is thought to be related to the function of T antigen.

Glucuronide formations by mouse liver homogenate in several liver impairments were studied by using 4-methyl umbelliferone as a glucuronide receptor. The results were as follows : 1. Subcutaneous or intraperitoneal administration of carbon tetrachloride to the mouse produced a significant increase in the liver glucuronyl transferase activity 12 or 24 hours after the treatment regardless of histological and enzymatic evidences of liver-cell necrosis. This increase was not attributed to the increase in the 'activator' of glucuronide formation but to the increase in the enzyme activity itself. 2. In Ectromelia virus mouse hepatitis, the glucuronyl transferase activity of the liver tissue was markedly reduced in severe cases. In moderate or milder cases, a slight increase in the activity was observed in a few of them in the early stage of the disease, and the activity was significantly decreased on the recovery in all of the cases which survived. 3. In the early stage of carbon tetrachloride injury when the glucuronyl transferase activity of whole mouse liver was increased and the decomposition of uridine diphosphate glucuronic acid by the liver tissue was also enhanced, the glucuronide formation in vivo was rather increased. It was thus considered that the whole liver glucuronyl transferase activity rather than the uridine diphosphate glucuronic acid content was responsible for the glucuronide formation in vivo as a rate-limiting factor.

The authors have succeeded in isolating a biologically-active leukemia virus from leukemic tissues of AKR mice with a fluorocarbon. From the chemical analysis of the biologically-active virus fraction it has been clarified that the AKR leukemia virus is of the RNA type.

We conducted Myanmar-Japan cooperation studies on hepatitis B and hepatitis C virus markers in patients with thalassemias and those with liver diseases. Among the 102 patients with liver diseases, 92% had a history of hepatitis B virus infection (antibody to hepatitis B core antigen positive), 35% were hepatitis B surface antigen positive, 39% were positive for anti-HCV. Among 28 patients with hepatocellular carcinoma, 46% had hepatitis B surface antigen, 21.4% had antibody to hepatitis C virus, and 7% were positive for both hepatitis B surface antigen and anti hepatitis C virus. The history of HCV infection among blood recipients at the Haematology Department of the Yangon General Hospital and at the Yangon Children's Hospital was found to be 55.5% and 46.7%, respectively, which is comparable to the history of hepatitis B infection (66.7% and 46.7%, respectively). This preliminary survey also encountered 2 cases positive for anti-HCV among 34 voluntary blood donors. This survey is the first one to report that hepatitis C is at the epidemic stage in Myanmar. As there is no effective treatment for hepatitis C in this country, a screening program for blood used in transfusion should be started immediately.