Biomedical subjects

N Wakamiya

Publications and source records attributed to N Wakamiya.

52 records · Page 3Linked to original sources

PubMed1989

Detection of tumor necrosis factor gene expression at a cellular level in human acute myeloid leukemias.

Tumor necrosis factor (TNF) is a Mr 17,000 cytokine produced by macrophages. We have recently demonstrated that TNF is also produced by transformed human epithelial cells. The present studies have examined TNF expression in human myeloid leukemic cells. We have monitored TNF expression at a cellular level using alkaline phosphatase detection of a biotinylated TNF cDNA probe in situ. Using this approach, TNF transcripts were detectable in HL-60 cells induced along the monocytic lineage by phorbol ester but not in uninduced cells. The specific detection of TNF RNA at a cellular level was supported by the absence of histochemical staining in RNase-treated cells and when using biotinylated pBR322 plasmid without insert. These studies were extended to preparations of purified acute myeloblastic leukemia cells. The results demonstrate that TNF is expressed in myeloblasts in eight of nine patients with AML. In each preparation of myeloblasts with detectable TNF RNA, transcripts were present at 89-98% of the cells. The identification of TNF RNA in situ was also associated with the detection of TNF protein in leukemic blasts by indirect immunofluorescence. Moreover, the detection of TNF protein in these preparations of myeloblasts was confirmed by immunoblotting. However, using this approach to examine AML cells before and after purification indicated that TNF expression is induced as a result of the enrichment procedures. Thus, certain populations of purified myeloid leukemic cells are capable of expressing TNF at both the RNA and protein levels.

Cell Line↗

PubMed1988

Expression of the macrophage colony-stimulating factor and c-fms genes in human acute myeloblastic leukemia cells.

Macrophage colony-stimulating factor (CSF-1; M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocytes. The effects of CSF-1 are mediated through binding to specific, high-affinity surface receptors encoded by the c-fms gene. CSF-1 and c-fms gene expression was investigated in fresh human acute myeloblastic leukemic cells by Northern blot hybridization using cDNA probes. 4.0-kb CSF-1 transcripts were detected in 10 of 17 cases of acute myeloblastic leukemia (AML), while c-fms transcripts were detected in 7 of 15. Coexpression of CSF-1 and c-fms was observed in five cases, and in five other cases neither gene was expressed. In situ hybridization demonstrated that transcripts for CSF-1 were present in 70-90% of cells in each of three cases studied while c-fms mRNA was detected in 40-70% of cells. The constitutive expression of CSF-1 transcripts was associated with production of CSF-1 protein, although detectable amounts of CSF-1 were not secreted unless the cells were exposed to phorbol ester. These results demonstrate that leukemic myeloblasts from a subset of patients with AML express transcripts for both the CSF-1 and CSF-1 receptor genes, often in the same leukemic cells in vitro.

Colony-Stimulating Factors↗

PubMed1987

Sensitization of human tumor cells to homologous complement by vaccinia virus treatment.

Although cell membranes have potent inhibitors which protect the activation of complement on the self cell membranes, some viruses have been shown to activate complement via the alternative pathway on the virus-infected cells. Tumour cells have been made reactive to homologous complement following treatment with such viruses and became highly immunogenic to syngeneic host guinea pigs and mice. Vaccinia virus (VV) made murine tumour cells highly immunogenic thus generating complement activating capacity on the infected cells. Since it has been suggested that VV can make some human tumour cells immunogenic to the cancer patients, we examined VV to see if the virus also has the capacity to make human tumour cells reactive with homologous human complement. Our present results indicate that not only is this the case but ultraviolet-treated VV also has the same effect.

Complement Activation↗

PubMed1987

Monoclonal antibodies to cowpox virus: polypeptide analysis of several major antigens.

Monoclonal antibodies directed against major antigens induced by cowpox virus (CPV) were produced. The specificities of these antibodies were established by immunoprecipitation, immunoblotting and several serological analyses, and from the cross-reactivities of these antibodies with cells infected with various other poxviruses, ectromelia virus (EV), vaccinia virus and Shope fibroma virus. The antibodies defined included ones reacting with each of the known major antigens of poxviruses, i.e. the common antigen of all poxviruses (probably NP antigen), the Orthopoxvirus-specific antigen (probably LS antigen), the haemagglutinin, the cell surface antigen, the common A-type inclusions in CPV and EV, and the antigen involved in neutralization.

Animals↗

PubMed1987

Detection of c-fms and CSF-1 RNA by in situ hybridization.

Recent studies have demonstrated by Northern blot analysis that both the c-fms proto-oncogene and the CSF-1 gene are expressed during human monocytic differentiation. In order to examine c-fms and CSF-1 expression at the cellular level, we have applied alkaline phosphatase detection of biotinylated v-fms and CSF-1 cDNA probes in situ. Using this approach, we demonstrate that c-fms and CSF-1 transcripts are detectable in HL 60 cells induced along the monocytic lineage but not in uninduced cells. The specific detection of these transcripts is further supported by the absence of histochemical staining in RNase-treated cells and when using pBR322 plasmid without insert as the biotinylated probe. Finally, the results indicate that most of the induced HL-60 cells have detectable levels of both c-fms and CSF-1 RNA. This approach should be useful for studying expression of these genes in populations of leukemic blasts and normal hematopoietic cells.

Cell Differentiation↗

PubMed1986

The augmentation of tumor-specific immunity by virus help. III. Enhanced generation of tumor-specific Lyt-1+2- T cells is responsible for augmented tumor immunity in vivo.

The role of vaccinia virus-reactive helper T cells (Th) in augmenting in vivo generation of antitumor protective immunity and the Ly phenotype mediating the enhanced in vivo tumor immunity were investigated. C3H/HeN mice were inoculated i.p. with viable vaccinia virus to generate vaccinia virus-reactive Th activity. The mice were subsequently immunized i.p. with virus-infected syngeneic X5563 and MH134 tumor cells, and spleen cells from these mice were tested for in vivo tumor neutralizing activity. Immunization of virus-primed mice with virus-uninfected tumor cells and of virus-unprimed mice with virus-infected tumor cells failed to result in in vivo protective immunity. In contrast, spleen cells from mice immunized with virus-infected tumor cells subsequent to virus-priming exhibited potent tumor-specific neutralizing activities. Such an augmented generation of in vivo protective immunity was accompanied by enhanced induction of tumor-specific cytotoxic T lymphocyte (CTL) and antibody activities in X5563 and MH134 tumor systems, respectively. However, analysis of the effector cell type responsible for in vivo tumor neutralization revealed that enhanced in vivo immunity was mediated by Lyt-1+2- T cells in both tumor systems. Moreover, the Lyt-1+2- T cells exerted their function in vivo under conditions in which anti-X5563 tumor-specific CTL or anti-MH134 tumor-specific antibody activity was not detected in recipient mice. These results indicate that augmenting the generation of a tumor-specific Lyt-1+2- T cell population is essential for enhanced tumor-specific immunity in vivo.

Animals↗

PubMed1986

Feasibility of UV-inactivated vaccinia virus in the modification of tumor cells for augmentation of their immunogenicity.

We compared the immunity induced by tumor cells modified with UV-inactivated purified vaccinia virus (UV-VV) and with live purified vaccinia virus (L-VV). C3H/HeN mice were inoculated i.p. with UV-VV or L-VV after whole-body irradiation with 150 rads of X-rays (priming). After 3 weeks the mice were immunized i.p. 3 times at weekly intervals with syngeneic X5563 or MH134 cells that had been absorbed in vitro with UV-VV or infected with L-VV and subsequently irradiated with 10(4) rads of X-rays. Then 1 week after the last immunization, the mice were challenged s.c. with X5563 viable tumor cells or challenged i.p. with MH134 viable tumor cells. The 50% lethal dose (TLD50) of X5563 in mice primed and immunized with UV-VV (UV-VV group) on s.c. challenge (10(6.06)) was the same as for mice treated with L-VV (L-VV group), whereas the TLD50 of unprimed or nonimmunized mice (control group) was 10(2.61). The TLD50 of MH134 in the UV-VV treated group on i.p. challenge (10(6.48)) was similar to that of the L-VV treated group (10(6.54)), while the TLD50 of the control group was 10(1.00). The difference between the TLD50 values of X5563 on s.c. challenge of mice primed and immunized with UV-VV or L-VV and control mice was 10(3.4). The difference between the TLD50 values of MH134 on i.p. challenge of primed and immunized mice and control mice was 10(5.5). These results indicate that the in vivo helper function of UV-VV is similar to that of L-VV and that the augmenting effect of this protocol depends on the kind of tumor.

Allantois↗

PubMed1986

Polypeptide analysis with monoclonal antibodies of A type inclusion bodies induced by cowpox virus.

The antigen in A type inclusion bodies (ATIB) induced by cowpox virus (CPV) was examined by immunofluorescent staining with monoclonal antibodies and polypeptide analysis. In the immunofluorescence (IF) test, these monoclonal antibodies reacted only with cytoplasmic inclusion bodies in cells infected with CPV. The fluorescence became detectable in the cells 6-9 hours after infection with CPV. No fluorescence was detectable in cells infected with CPV in the presence of cytosine-I-beta-D-arabinofuranosyl-HCl (Ara C) or in cells infected with other poxviruses, such as vaccinia virus (VV) or Shope fibroma virus (SFV). On Western blotting and immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), only one component with a molecular weight of about 160,000 (160 K) was detected in CPV-infected cells. This 160 K polypeptide was first detectable 12 hours after infection of cells with CPV, and was not detectable in infected cells in the presence of Ara C. The 160 K polypeptide was not detected in cells infected with VV or SFV, or in virions purified from CPV-, VV- or SFV-infected cells.

Animals↗

PubMed1986

Cross-reactivity among cowpox, ectromelia and vaccinia viruses with monoclonal antibodies recognizing distinct antigenic determinants in A-type inclusion bodies.

Several monoclonal antibodies recognizing distinct antigenic determinants in A-type inclusion bodies (ATIB) induced by cowpox virus (CPV) were obtained to examine the cross-reactivity among various strains of poxviridae, comprising CPV, ectromelia virus (EV), vaccinia virus (VV) and Shope fibroma virus (SFV). The monoclonal antibodies were classified into at least 3 groups on the basis of the results of an immunofluorescence test and immunoblotting; i. e., strain-specific, CPV and EV-specific and Orthopoxvirus (CPV, EV and VV)-specific antibodies. Differences were found between the antigenic determinants of ATIB of LB strains (LB red and LB white) and other strains (Amsterdam, 53, 58 and 60) of CPV and also between those of ATIB of CPV and EV. An interesting finding was that VV also produces the antigen analogous to that associated with ATIB in CPV- and EV-infected cells despite the absence of morphologically defined ATIB.

Antibodies, Monoclonal↗

PubMed1984

The augmentation of tumor-specific immunity by virus help. I. Demonstration of vaccinia virus-reactive helper T cell activity involved in enhanced induction of cytotoxic T lymphocyte and antibody responses.

C3H/He mice were immunized to vaccinia virus by inoculating i.p. viable virus. Their spleen cells (SC) were tested for vaccinia virus-reactive helper T cell activity capable of augmenting (a) anti-trinitrophenyl (TNP) cytotoxic T lymphocyte (CTL) response generated from unprimed C3H/He SC (responding cells) or (b) anti-TNP antibody response generated from TNP-primed C3H/He SC (responding cells) by the stimulation with syngeneic SC infected with vaccinia virus and subsequently modified with TNP (virus-self-TNP). The results demonstrate that cultures of responding cells plus 850 rds X-irradiated vaccinia virus-primed SC failed to enhance anti-TNP CTL or plaque-forming cell (PFC) responses when in vitro stimulation was provided by either virus-self or TNP-self alone. In contrast, these cultures resulted in appreciable augmentation of CTL and PFC responses when stimulated by virus-self-TNP. Such a helper activity provided by vaccinia virus-primed SC was revealed to be T cell mediated and antigen specific. These results are discussed in the context of (a) nature of virus helper antigens, (b) mechanism of help and (c) potential of virus help in augmenting CTL and antibody responses to tumor antigens.

Animals↗

PubMed1984

The augmentation of tumor-specific immunity by virus help. II. Enhanced induction of cytotoxic T lymphocyte and antibody responses to tumor antigens by vaccinia virus-reactive helper T cells.

The present study investigates the role of vaccinia virus-reactive helper T cells in causing enhanced induction of syngeneic tumor immunity. Vaccinia virus-reactive helper T cell activity capable of inducing the augmented generation of cytotoxic T lymphocyte (CTL) or antibody responses was generated in C3H/HeN mice by inoculating i.p. live virus. Immunization of these mice with vaccinia virus-infected syngeneic X5563 plasmacytoma or MH134 hapatoma cells led to augmented induction of immune resistance against the challenge with corresponding viable tumor cells when compared with the incidence of resistance observed in control mice not primed to vaccinia virus. In vitro cytotoxicity tests utilizing spleen cells and serum from mice which resulted in the augmented tumor resistance by virus help have revealed that spleen cells from C3H/HeN mice immune to the X5563 plasmacytoma exhibited appreciable anti-X5563 CTL activity, whereas serum from these mice failed to display any antibody response. In contrast, MH134-immune mice exhibited potent anti-MH134 antibody, but not CTL responses. Such an anti-tumor CTL or antibody response augmented by vaccinia virus-reactive helper T cells was found to be tumor specific. These results are discussed in the context of (a) the functional diversity of tumor antigens, and (b) mechanisms of virus help that are involved in various forms of augmented induction of syngeneic tumor immunity.

Animals↗

PubMed1984

Cross-reactivity between cowpox and vaccinia viruses with monoclonal antibodies. Brief report.

Hybridoma cell lines producing monoclonal antibodies against cowpox virus (CPV) and vaccinia virus (VV) were established to examine the specific and cross-reactive antigenic determinants of these viruses. Monoclonal antibodies against CPV (LB red strain) and VV (Lister strain and Ikeda strain) were classified into several groups on the basis of the results of immunofluorescence and haemagglutination inhibition tests. It was suggested that the groups defined above include the group of antibodies reacting with each of known major antigens of poxvirus, i.e. nucleoprotein (NP) antigen, heat labile and stable complex (LS) antigen, haemagglutinin (HA), cell surface antigen (CSA) and type A inclusion body (A).

Antibodies, Monoclonal↗

PubMed1984

Additional evidence for the augmented induction of tumor-specific resistance in vaccinia virus-primed mice by immunization with vaccinia virus-modulated syngeneic tumor cells.

The augmenting effect of vaccinia virus infection of tumor cells on induction of tumor-specific resistance was examined in mice. C3H/HeN mice were primed intraperitoneally (ip) with live vaccinia virus after whole-body irradiation with 250 rad of X-rays. Three weeks later the mice were immunized ip 3 times at weekly intervals with syngeneic murine hepatoma MH134 or spontaneous myeloma X5563 which had been infected in vitro with vaccinia virus and subsequently irradiated with 7000 rad of X-rays. One week after the third immunization, the mice were challenged with 1 X 10(5) viable cells of MH134 or X5563 ip or 1 X 10(6) tumor cells intradermally (id). On ip challenge with viable MH134 cells all mice that had not been pretreated died within 3 weeks due to ascites tumor out-growth, whereas all mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected MH134 cells survived. On ip challenge with X5563 cells, the percentage survival of vaccinia virus-primed and vaccinia virus-modified tumor-immunized mice was 80%. On id challenge with MH134 and X5563 tumor cells, in un-treated mice tumors grew to more than 5 mm in diameter within 3 weeks, whereas 90% and 60%, respectively, of the mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected tumor cells showed no tumor out-growth. Pretreatment by only immunization with vaccinia virus-infected cells or vaccinia virus-priming and immunization with virus non-infected tumor cells were not effective for preventing induction of tumor-resistance to either ip or id challenge with MH134 or X5563 tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Detection of fowlpox virus DNA by in situ hybridization using a biotinylated probe.

In situ hybridization was applied to detect fowlpox virus (FPV) DNA in formalin-fixed paraffin-embedded sections of the skin from infected chickens by using a biotinylated probe and a streptavidin-alkalinephosphatase conjugate. The immunohistochemical examination was applied to compare the distribution of the FPV DNA to that of related antigenic protein in serial sections. In the infected epithelial cells, FPV DNA was detected in cytoplasmic inclusion bodies and in the rest of cytoplasm. Likewise, immunohistochemical examination revealed the virus antigen in cytoplasm. Ultrastructurally, virions were observed in the cytoplasmic inclusion bodies, and immature virus particles were in the rest of the cytoplasm. The study proved restricted distribution of FPV DNA in the cytoplasm.

Animals↗

PubMedDate not supplied

Identification of the cross-reactive antigens between Marek's disease virus and pseudorabies virus by monoclonal antibodies.

Among the 33 monoclonal antibodies (MAbs) against pseudorabies virus (PRV) examined, three MAbs (24-17, 74-26, and 8) were found to react with cells infected with Marek's disease virus (MDV)-related viruses by immunofluorescence test. Two of the MAbs (24-17 and 74-26) reacted with the nuclei of cells infected with MDV serotype 1 (MDV1), MDV serotype 2 (MDV2), and herpesvirus of turkeys (HVT), whereas MAb 8 reacted with the cytoplasm of MDV2- and HVT-infected cells. However, none of the MAbs against MDV1, MDV2, and HVT that were examined reacted with PRV-infected cells. None of these three MAbs against PRV reactive with MDV-related viruses cross-reacted with the cells infected with other herpesviruses, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, or human herpesvirus 6. Southern-blot hybridization under stringent or less-stringent conditions showed that no significant DNA homology was detected between PRV DNA and MDV DNA.

Animals↗

PubMedDate not supplied

Antitumor efficacy of two kinds of tumor vaccine modified with vaccinia virus.

We compared antitumor effectiveness of two types of tumor vaccine modified with vaccinia virus (VV). One type is UV-inactivated VV (UV-VV) -absorbed tumor cell vaccine (UV-VV TCV) produced by tumor cells that had been absorbed in vitro with UV-VV and subsequently X-irradiated with 10(4) rads. The other type is vaccinia oncolysate vaccine (VOV) from tumor cells that had been infected in vitro with live VV and subsequently sonicated. C3H/HeN mice were inoculated i.p. with UV-VV after whole body X-irradiation with 150 rads. After 3 weeks, the two kinds of vaccine were administered i.p. 3 times at weekly intervals. One week after the last injection, mice were challenged i.p. with syngeneic MH134 or X5563 viable tumor cells at different doses. The fifty percent tumor lethal doses (TLD50) of MH134 in mice treated with UV-VV TCV and VOV were 10(6.6) and 10(5.3), respectively, whereas the TLD50 of MH134 in non-treated mice was 10(0.6). The TLD50 of X5563 in mice treated with UV-VV TCV and VOV were 10(6.5) and 10(4.4), respectively, while the TLD50 of X5563 in non-treated mice was 10(0.5). These results show that UV-VV TCV is more effective than VOV. We suggest that the complete cell structure of the vaccine is more effective for enhancing tumor immunity.

Animals↗