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Biomedical subjects

T Shikata

Publications and source records attributed to T Shikata.

At least 91 records · Page 5Linked to original sources
PubMed1990

Expression of pre-S1, pre-S2, S and X peptides in relation to viral replication in livers with chronic hepatitis B.

The expression of large (pre-S1), middle (pre-S2), major S (S) polypeptides of the envelope (HBs) and X peptides of hepatitis B virus (HBV) was investigated in 37 liver specimens with chronic hepatitis B by indirect immunoperoxidase staining. Primary antisera utilized were polyclonal ones against HBs (poly-HBs), core (HBc) and X and monoclonal ones against pre-S1, pre-S2 and S with (particle-S) or without (peptide-S) conformational structure. The localization of HBs proteins in hepatocytes was classified into three types: diffuse, membranous and inclusion. The peptide-S and pre-S2 were expressed at nearly the same frequency as poly-HBs in all types, whereas particle-S was found less frequently (18/29 cases) in the inclusion type, and pre-S1 was recognized relatively rarely (9/33 cases) in the membranous type. As for staining intensity, peptide-S and pre-S2 were almost identical to poly-HBs which stained the most strongly among all three staining types. Particle-S was similar to poly-HBs in the membranous type, but was weak in the inclusion type in the majority. While pre-S1 was stained in a similar intensity to poly-HBs in the diffuse and inclusion types, it was weak or negative in the membranous type. Thus, envelope particles indicated by particle-S staining appeared to be located most frequently in the membranous type, but their assembly might be suppressed in the inclusion type where pre-S1 was well expressed. The X peptide was more frequently detected in the liver with serum HBe antigen and/or HBV DNA. The X peptide was stained exclusively in the cytoplasm of hepatocytes and was correlated with the cytoplasmic HBc antigen. The X peptide was not observed differently between cases with and those without cirrhosis. This suggests that the expression of X peptide tends to occur with virus replication but not with disease progression.

Adult↗
PubMed1990

Serial transmission of a putative causative virus of enterically transmitted non-A, non-B hepatitis to Macaca fascicularis and Macaca mulatta.

In order to establish an animal model and to identify a causative virus of enterically transmitted non-A, non-B hepatitis, Macaca fascicularis was inoculated with a fecal extract obtained from Myanmar patients with acute sporadic non-A, non-B hepatitis. The primates developed acute hepatitis exhibited by a transient elevation of aminotransferases in the sera and occurrence of hepatic necroinflammation between 2 and 4 weeks postinoculation. Subsequent second passage of the fecal extract made from first-passage primates into another Macaca fascicularis and Macaca mulatta induced acute hepatitis. Likewise, third passage was also successfully performed. Immune electron microscopy of the stool extract incubated with the primate serum at the acute phase of hepatitis showed an aggregation of virus-like particles. These particles consisted of full and empty round particles without an envelope, measuring approximately 27 nm in diameter. A dispersion of similar particles was found ultrastructurally in the hyaloplasm of hepatocytes surrounding the focal necrosis. This putative causative virus appears to be a new hepatitis virus.

Animals↗
PubMed1990

Occurrence and character of a putative causative virus of enterically-transmitted non-A, non-B hepatitis in bile.

The present investigation confirms the possibility that the etiological agent of enterically-transmitted non-A, non-B (ET-NANB) hepatitis (type E hepatitis), multiplied in hepatocytes, is excreted into the feces via bile. The fecal extract was inoculated into 7 cynomolgus monkeys. Bile juice was collected directly from the gallbladder by needle puncture after abdominal operation 3 to 6 times during the experimental course. All 7 monkeys developed elevated serum aminotransferases, which began gradually approximately 2 weeks postinoculation and reached a peak at 3 to 5 weeks. In parallel with this elevation, both in time and magnitude, necroinflammation was observed in the livers. The virus-like particles (VLPs) were found in the bile juice of all 7 monkeys and the serial occurrence of VLPs was typified as follows: the VLPs were negative on day 7, appeared on day 10 after inoculation, and were present until the 3rd week when the subjects were sacrificed. While the particles were individually dispersed on day 10, they started to exhibit spontaneous aggregation on and after week 2. Also, empty particles were very rare at first, but increased in ratio compared to full ones over time. Thus, the putative causative virus of ET-NANB hepatitis was demonstrated to be excreted through bile. The spontaneous aggregation of VLPs might be due to the specific antibody secreted into the bile juice and was closely correlated with hepatitis activity. The increase in empty particles might indicate an increase in disorganized assembly of the nucleic acid and protein during virus proliferation.

Animals↗
PubMed1989

Electron microscopy of ribonucleic acid in nuclear particulate aggregates of hepatitis D using nuclease-gold complexes.

The ultrastructural localization of hepatitis delta antigen (HDAg) and ribonucleic acid (RNA) was investigated by immunoperoxidase electron microscopy and by enzyme electron microscopy of RNase-gold complexes on liver biopsies from seven patients with hepatitis D. HDAg was localized mainly in the nucleus and sometimes in the cytoplasm of hepatocytes. Ultrastructurally, intranuclear HDAg was found on nuclear particulate structures measuring 20 to 30 nm in diameter. Intranuclear RNA visualized with gold particles was found in high amounts in the nucleolus, to a small extent in the chromatin area, and also on nuclear particulate structures. These findings suggest that intranuclear aggregates of irregular granular particulate structures in hepatitis D are the internal component of hepatitis delta virus (HDV) particles in blood.

Antigens, Viral↗
PubMed1989

Effect of 3'-azido-3'-deoxythymidine on replication of duck hepatitis B virus in vivo and in vitro.

3'-Azido-3'-deoxythymidine (AZT) inhibits the replication of the human immunodeficiency virus (HIV) by blocking the formation of the phosphodiester bond and has been used clinically for the treatment of HIV infection. To assess the effect of AZT on the replication of hepadnaviruses, which replicate through reverse transcription, both the liver tissue and primary cultured hepatocytes from ducklings previously infected with duck hepatitis B virus (DHBV) were examined for DHBV DNA before and after the treatment with AZT. We did not observe suppression of DHBV replication at any doses in our system as measured by viral DNA synthesis in infected duck hepatocytes. The data strongly suggest that AZT has no inhibitory effect on DHBV reverse transcriptase.

Animals↗
PubMed1989

Clonal state of human hepatocellular carcinoma and non-tumorous hepatocytes.

We determined the clonal state in specimens of hepatocellular carcinoma (HCC) and non-tumorous hepatocytes from the integration mode of hepatitis B virus (HBV) DNA. The integration mode of HBV DNA in several parts of the tumors and non-tumorous regions of the same liver, as well as in metastatic tumors, was examined using the Southern blot analysis. In 13 of the 14 cases of HCC, the liver tumors, including metastatic tumors in lymph nodes and the lungs, were monoclonal. In one case, a different HCC clone was found in one part of the liver tumor. The integration of HBV DNA was also observed in non-tumorous tissues in 38 of the 78 cases (49%) of chronic hepatitis with and without HCC; in 16 cases of chronic hepatitis in which HBV DNA was integrated, several clones of the hepatocytes that had HBV DNA integrated into their chromosomal DNA and had proliferated clonally were found in non-tumorous tissues. These clones were different from the tumor clone of the same liver. Thus HCCs were usually monoclonal. The development of different tumor clones appeared to be unusual, but the non-tumorous hepatocytes could have proliferated clonally from different multicentric clones before carcinogenesis. The clonal growth of the non-tumorous hepatocytes suggests that the integration of HBV DNA plays an important role in hepatocarcinogenesis.

Blotting, Southern↗
PubMed1989

Non-A, non-B hepatitis: visualization of virus-like particles from chimpanzee and human sera.

Virus-like particles (VLP), morphologically similar to togavirus particles, were detected in the sera of chimpanzees and humans with chronic non-A, non-B (NANB) hepatitis. Particles resembling the virus were also found in the cytoplasm of hepatocytes obtained from NANB hepatitis infected chimpanzees. Furthermore, two chimpanzees inoculated with materials containing the VLP developed NANB hepatitis. These results strongly suggest that the VLP may be closely associated with the agent of NANB hepatitis.

Animals↗
PubMed1989

Molecular cloning and sequence analysis of duck hepatitis B virus genomes of a new variant isolated from Shanghai ducks.

The genomes of duck hepatitis B virus (DHBV) from a brown duck (S5) and a white duck (S31) kept independently in Shanghai, China, were cloned and the complete nucleotide sequence of each virion DNA (DHBV-S5 and DHBV-S31) was determined. DHBV-S5 and DHBV-S31 were both 3027 bp in length and 6 bp longer than the other two DHBVs analyzed previously, DHBV16 and DHBV3. The genomes of DHBV-S5 and DHBV-S31 encoded three long overlapping open reading frames designated as P, S, and C. A possible new open reading frame was found in a complementary strand of each viral genome, as 336 bp for DHBV-S5 and 306 bp for DHBV-S31, respectively. A pair of 3-bp insertions were found in the overlapping region of pre-S2 and P and so two amino acids were inserted in this region in DHBV-S5 and DHBV-S31. The nucleotide sequence variation between DHBV-S5 and DHBV-S31 (4.9%) was similar to that between DHBV16 and DHBV3 (5.6%), and less than the variations between either of these Shanghai clones and DHBV16 or DHBV3 (9.5-10.4%). The amino acid sequence was also conserved in the two Shanghai clones but showed group difference from DHBV16 or DHBV3. Thus these two independent Shanghai clones of DHBV showed geographical characteristics of genomic structure.

Amino Acid Sequence↗
PubMed1989

Production of antibodies directed against microtubular aggregates in hepatocytes of chimpanzees with non-A, non-B hepatitis.

We have previously used Epstein-Barr virus transformation to established two clonal lymphoblastoid cell lines (48-1 and S-1) producing monoclonal antibodies against microtubular aggregates that appear in the hepatocytes of chimpanzees with non-A, non-B hepatitis (NANBH). To obtain additional antibodies directed against the same structure, the mouse hybridoma method was employed. Partially purified microtubular aggregates were prepared from liver homogenates of a chimpanzee with NANBH and used as the immunogen. Hybridoma cultures were first screened by radioimmunoassay against the partially purified antigen and secondly by immunofluorescence (IF) using liver sections from a chimpanzee with NANBH. Twenty-seven cultures exhibited positive IF reactions similar to those observed with the original antibodies, 48-1 and S-1, and were cloned by limiting dilution. The specificities of the monoclonal antibodies were tested by IF on liver biopsy specimens from chimpanzees with hepatitis A, B, D or NANBH and from normal chimpanzees. All the antibodies proved to be IgG. Immunoelectron microscopy revealed that all 27 antibodies bound to the same structure, the microtubular aggregates, in hepatocytes of chimpanzees with NANBH. To determine the size of the antigen polypeptide recognized by these antibodies, polyacrylamide gel electrophoresis and Western blot assays were performed. Nine of the 27 antibodies specifically reacted with a single polypeptide of Mr 44K (p44). The remaining 18 antibodies detected no antigen polypeptide on the filters. The anti-p44 antibodies were then tested using cross-competition assays with 125I-labelled antibodies, and were found to be classifiable into three groups. In addition, the results indicate that at least three distinct epitopes are located on p44: epitope A recognized by group 1, epitope B recognized by group 2 and epitope C recognized by group 3.

Animals↗
PubMed1989

Enterically transmitted non-A, non-B hepatitis in cynomolgus monkeys: morphology and probable mechanism of hepatocellular necrosis.

Two cynomolgus monkeys were inoculated with a stool extract originally derived from patients suffering from enterically transmitted non-A, non-B hepatitis. Subsequently, the primates developed self-limiting acute hepatitis and their liver tissues were obtained sequentially by needle biopsy or at sacrifice. Histologically, the liver tissues exhibited necroinflammation which appeared in parallel, both in time and magnitude, with elevation in serum aminotransferases. Necroinflammation was characterized by focal dropout of hepatocytes with accumulation of lymphocytes and macrophages. These lymphocytes were positive for a cytotoxic/suppressor immunophenotype. The hepatocytes surrounding these focal necroses showed depletion of glycogen granules and decrease in glucose-6-phosphatase and succinic dehydrogenase activities. Ultrastructurally, damaged hepatocytes around the focal necroses revealed marked dilatation of both rough and smooth endoplasmic reticula, swelling and disruption of the mitochondria and leakage of nuclear materials into the cytoplasm. Frequently, direct contact between the damaged hepatocytes and lymphocytes was noted. Virus-like particles measuring about 27 nm in diameter were observed singly or in small groups within the cytoplasm of damaged hepatocytes. Primary hepatocyte culture of a cynomolgus monkey, inoculated with a transmissible stool extract did not show any cytopathic change, although similar virus-like particles were recognized ultrastructurally in the cultured hepatocytes. Morphological analysis of in vitro and in vivo transmission studies in cynomolgus monkeys strongly supported the hypothesis of immune-mediated hepatocytolysis rather than a direct cytopathic effect of this hepatitis virus.

Animals↗
PubMed1989

State of hepatitis B virus DNA in hepatocytes of patients with noncarcinomatous liver disease. Its special relationship with necroinflammatory activity and the stage of disease.

By Southern blot hybridization, the state of hepatitis B virus (HBV) in the liver was investigated by utilizing needle biopsy specimens of 59 patients with hepatitis B surface antigen-positive noncarcinomatous liver disease. The tissue HBV DNA revealed a replicative form in 23 patients with chronic active hepatitis (48%) and an integrated form in 23 (48%) of these 48 patients. A liver with a replicative form was more frequently associated with hepatitis Be antigen (HBeAg), serum HBV DNA, and expression of hepatitis B core antigen than one without it. Integration of HBV DNA was more common in the anti-hepatitis Be phase or in the absence of necroinflammatory activity, and its frequency of occurrence roughly paralleled progression of liver disease: early, 21%; advanced, 56%; and cirrhotic stage, 61%.

Adult↗
PubMed1988

Influence of aflatoxin B1 intoxication on duck livers with duck hepatitis B virus infection.

In an attempt to determine the effect of aflatoxin B1 (AFB) intoxication on livers with duck hepatitis B virus (DHBV) infection, domestic ducks were given 0.1 mg of AFB/kg body weight twice a week for a maximum period of 54 weeks employing various experimental designs. The ducks were infected with DHBV by i.v. inoculation of DHBV-positive sera within 24 h posthatch. The livers were examined histologically, immunohistochemically, and ultrastructurally, and the livers and sera were examined by molecular hybridization for DHBV DNA. AFB administration induced hepatocellular necrosis and marked biliary cell proliferation of the periportal areas, and finally liver cirrhosis. On short-term administration, the hepatocytes of DHBV-infected livers revealed a marked increase in incomplete particles of DHBV by immunostaining and electron microscopy, as compared to those without its administration. Long-term AFB administration provoked frequent nodular or cirrhotic changes. There was no significant increase in frequency of these changes in DHBV-positive ducks as compared to DHBV-negative ones. AFB administration induced hepatocellular carcinoma (HCC) in one DHBV-positive duck and in two DHBV-negative ducks. The HCC and cirrhotic livers revealed extrachromosomal but no integrated form of DHBV DNA by Southern blot hybridization analysis. Immunostaining demonstrated a heterogeneous distribution of DHBV from area to area in nodular and cirrhotic livers. Thus, AFB intoxication provoked various liver disorders independent of DHBV infection, and neither a cocarcinogenic effect of AFB and DHBV nor integration of viral DNA into the genome of neoplastic and nonneoplastic tissues was observed in the present experiments. Generally speaking, DHBV infection did not appear to accelerate hepatic disorders induced by AFB intoxication. However, AFB administration altered the DHBV in the liver in terms of its amount and distribution.

Aflatoxin B1↗
PubMed1988

Geographical pathology of duck livers infected with duck hepatitis B virus from Chiba and Shimane in Japan and Shanghai in China.

In order to evaluate geographical differences in the liver pathology of ducks infected with duck hepatitis B virus (DHBV), ducks in Chiba and Shimane, Japan, and Shanghai, China, were investigated. The numbers (DHBV positive/negative) and the maximum age of the ducks examined were 18/10 at 19 mo, 15/1 at 3 yr 4 mo, and 72/27 at 18 mo, respectively. DHBV infection was induced experimentally in ducks from Chiba and Shimane but was present congenitally in those from Shanghai. Ducks were examined regarding liver function tests, conventional histology, immunohistology, electron microscopy, and molecular hybridization for DHBV DNA in the serum and liver. There was no significant difference between DHBV-positive and -negative ducks in bilirubin and transaminase and alkaline phosphatase activities in the sera. Histologically, while the livers of ducks from Chiba and Shimane did not show necroinflammatory (hepatitis) activity, those from Shanghai frequently did (52.5%). Necroinflammatory activity of the Shanghai ducks was present almost equally in both DHBV-positive and -negative livers. The livers of Shanghai ducks but not the other two areas often (8.3%) had ground-glass inclusions which corresponded ultrastructurally to numerous virus particles in the dilated cisternae of the proliferated endoplasmic reticulum. No advanced liver disease, such as cirrhosis or hepatocellular carcinoma, was observed. There was no significant difference in the amount of DHBV DNA in the sera or in its pattern in the liver tissue among ducks of the three areas. In addition, the livers of Chiba ducks frequently had amyloidosis, while those of Shanghai ducks were contaminated with parasites. In conclusion, DHBV infection did not appear to provoke significant hepatitis activity or advanced liver disease in the examined ducks of all three areas, and the DHBV-positive livers from Shanghai ducks showed a different morphological appearance from those of the other two areas. This variation might reflect the difference in the strain of ducks, subtypes of DHBV, environmental factors, or a combination of these influences.

Animals↗
PubMed1988

Frequent integration of hepatitis B virus DNA in noncancerous liver tissue from hepatocellular carcinoma patients.

To investigate the relationship between hepatocarcinogenesis and integration of hepatitis B virus (HBV) DNA in the cellular DNA of the liver, we studied the integration of HBV DNA in various noncancerous regions of the liver from 31 patients using Southern blot analysis. Of 13 patients without hepatocellular carcinoma (HCC), 4 had heterogeneously integrated HBV DNA. Of the latter four patients, two had chronic liver disease, and two had nonspecific histological changes. In contrast, integration of HBV DNA was found in noncancerous tissue from 11 of 18 patients with HCC. In eight patients, homogeneous integration was found in noncancerous tissue, and restriction fragments of integrated HBV DNA were different from those found in cancerous tissue. Moreover, integration of HBV DNA was found in all portions examined from the same liver, and homogeneously integrated HBV DNA showed different restriction patterns in different areas. These results suggest that integration of HBV DNA may occur in heterogeneous sites of cellular DNA before hepatocarcinogenesis. Subsequently, multi-focal clonal populations develop from these hepatocytes, especially frequently in the case of HCC. Integrated HBV DNA may play an important role in the clonal growth of hepatocytes, although the development of HCC requires additional factors.

Blotting, Southern↗
PubMed1988

Effective production of mouse monoclonal antibodies against influenza virus by repeated intrasplenic immunization.

Two immunization techniques that enable production of mouse monoclonal antibodies were evaluated in terms of small quantities of antigen. Various amounts of purified influenza A virus particles were applied either for in vitro sensitization in cultured splenocytes or for intrasplenic immunization, followed by hybridization of the immunized cells with mouse myeloma cells. Hybridomas producing specific antibodies for influenza viral proteins were detected by enzyme-linked immunosorbent assay when more than 50 micrograms of antigens was used for the in vitro immunization method, and at least 5 micrograms was necessary for a single intrasplenic immunization. On the other hand, as little as 60 ng of antigen administered in two intrasplenic injections was sufficient to produce specific hybridomas. Two out of six randomly selected monoclonal antibodies obtained using the repeated intrasplenic immunization method were IgG and the other four were IgM. Immunoprecipitation analyses revealed that the recognized antigens involved a viral inner protein (nucleocapsid protein), as well as an envelope glycoprotein (hemagglutinin). We conclude that immunization by two direct injections of antigen into the spleen is the most effective method for sensitization with nanogram quantities of insoluble antigen such as influenza viruses.

Animals↗
PubMed1988

Establishment and characterization of a woodchuck hepatocellular carcinoma cell line (WH44KA).

A continuous cell line was established from a hepatocellular carcinoma obtained from a woodchuck that was sero-positive for woodchuck hepatitis virus (WHV). The cell line, designated WH44KA, grows as an adhering monolayer with a doubling time of 36 hr in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells not only showed epithelial origin on light and electron microscopic examination but also possess biosynthetic markers of the latter, such as albumin and alpha-fetoprotein, which were demonstrated in cultured cells. When they were transplanted into athymic nude mice, tumors developed at the site of inoculation. These tumors were shown to be hepatocellular carcinoma, similar in morphology to the original tumor from which the WH44KA cells were derived. Chromosome analysis revealed a chromosome number ranging from 31 to 126, with a modal number of 35. Integration of WHV DNA was shown by Southern blot analysis. However, WHV surface antigen was not demonstrated in the cultured cells or supernatant medium. The WH44KA cell line appears to be a useful in vitro model for the study of virus-induced hepatocellular carcinoma.

Albumins↗