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Biomedical subjects

Y Masuho

Publications and source records attributed to Y Masuho.

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

The gp116 of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope.

The glycoprotein complex gp58/116 of human cytomegalovirus (HCMV) represents a dominant antigen for the humoral immune response. We have used the human monoclonal antibody C23, which is capable of neutralizing HCMV in tissue culture without the addition of complement, to study the origin of gp116 as well as the amino acid sequence recognized by the antibody. Our results show that gp116 is derived from the same open reading frame as gp58 and that it represents the amino-terminal portion of the precursor protein. Using prokaryote-expressed beta-galactosidase-gp116 fusion proteins, the binding site of C23 was located to between amino acids 27 to 84 of the amino-terminal portion of gp116. Analyses of HCMV-positive human sera revealed that this portion of the molecule is immunogenic during natural infection.

Amino Acid Sequence↗
PubMed1990

Antibodies to the primary immunodominant domain of human immunodeficiency virus type 1 (HIV-1) glycoprotein gp41 enhance HIV-1 infection in vitro.

Previous experiments had shown that two human monoclonal antibodies (huMAbs) directed against human immunodeficiency virus type 1 (HIV-1) enhanced HIV-1 infection in vitro (Robinson et al., Proc. Natl. Acad. Sci. USA, 87:3185-3189, 1990). This complement-mediated, antibody-dependent enhancement (C'-ADE) of HIV-1 infection caused 12-fold increases in reverse transcriptase released from MT-2 cells. In the study reported here, it was demonstrated that both of these huMAbs, 86 and V10-9, bound to an immunodominant peptide in gp41 (amino acids 586 to 620). This peptide blocked C'-ADE of HIV-1 infection in vitro regardless of whether huMAb 86 or human polyclonal anti-HIV was used as the source of anti-HIV antibody. Blockade of enhanced infections was characterized by decreases in antigen synthesis, cytopathic effect, and reverse transcriptase release. The ability of the huMAbs to enhance infection was determined to be dependent upon specific peptide reactivity and not dependent upon immunoglobulin subclass, complement fixation, or gross antigen reactivity. Since the peptide to which enhancing antibodies bind is immunodominant and does not bind neutralizing antibodies, it may be worthwhile to investigate deletion of this 35-amino-acid peptide from candidate anti-HIV vaccines.

Amino Acid Sequence↗
PubMed1990

Characterization of human anti-cytomegalovirus monoclonal antibody as biologics.

Human anti-cytomegalovirus (CMV) monoclonal antibody designated C23 was purified from the culture fluid of hybridoma cells which were generated by cell fusion of human lymphocytes and mouse myeloma cells. The purified C23 was found to be identical to human gammaglobulin (HGG) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and non-reducing conditions and in gel filtration chromatography. C23 was not contaminated with either aggregated IgG molecules or mouse immunoglobulin chains. In addition, the formulated C23 preparation showed an anti-complement activity low enough to permit its use as a biologic. A virus neutralization titer of C23 was about 1,000 times higher than the titers of HGG preparations. All the tested CMV strains were susceptible to neutralization by C23 and this neutralization was not affected by addition of either beta 2 microglobulin or fresh human serum. These results suggest that human monoclonal antibody C23 is as safe as conventional HGG preparations which have been used in humans, and much more effective in providing host protection against CMV infection.

Animals↗
PubMed1989

A hybridoma producing human monoclonal antibody specific for glycoprotein 120 kDa of human immunodeficiency virus (HIV-1.

A stable hybridoma producing anti-HIV human monoclonal antibody (HMCA) was generated by fusing CD3-depleted human splenic lymphocytes from an HIV sero-positive donor with the mouse myeloma cell line P3x63AgU1. The resultant hybridoma has been secreting IgG1, lambda chain for over nine months at a rate of 2.5 micrograms/10(6)cells/day. The HMCA shows specific reactivity in ELISA using HIV-infected cell lysates. Immunofluorescence tests have indicated that this HMCA binds specifically to the surface of H9 and C3 HIV/HTLVIIIb infected cells, HIV/N1T infected CEM cells and to MoT cells infected with an HIV clinical isolate. Western blotting revealed recognition of glycoproteins 120 and 160 kDa of HIV by the HMCA. Although this HMCA demonstrated no neutralizing activity, the production of an anti-HIV HMCA specific for glycoprotein 120 kDa indicates the possibility that a neutralizing HMCA can be developed as further fusions with lymph nodes and spleens from HIV positive donors are performed.

Antibodies, Monoclonal↗
PubMed1988

Human monoclonal antibody against glycoproteins of human immunodeficiency virus.

We have established a program to make human monoclonal antibodies to the human immunodeficiency virus (HIV). Lymphocytes of lymph nodes from patients with the acquired immunodeficiency syndrome (AIDS) related complex (ARC) spontaneously produced antibodies to HIV in vitro and their antibody production was suppressed by culturing them in the presence of HIV antigens. Therefore, in vitro stimulation with HIV antigens was not done but rather, donor lymph node or spleen lymphocytes were directly fused with mouse myeloma cells. One of the hybridomas thus generated has been stably producing human monoclonal antibody (MAb) of the IgG1 isotype with a kappa chain. This antibody, MAb86, bound to the surface membrane of HIV-infected cells but not to that of uninfected cells at all. MAb86 reacted in Western blot with both viral glycoproteins of 120,000 daltons (gp120) and 41,000 daltons (gp41). While not neutralizing alone, a combination of MAb86 with another human IgG1 MAb against gp120 showed viral neutralization. Based on these data it seems likely that this approach will result in human MAbs capable of viral neutralization and antibody-dependent cytotoxicity. These may have value for the prevention and/or treatment of AIDS.

Animals↗
PubMed1988

A human monoclonal antibody to cytokeratin intermediate filament antigens derived from a tumor draining lymph node.

Human lymphocytes derived from a lymph node draining a primary breast adenocarcinoma were fused with the mouse myeloma P3X63Ag8.653 to generate human-mouse hybridomas secreting human monoclonal antibodies (MAbs) to tumor associated antigens (TAAs). One of the resulting human MAbs, YBB 190 (IgM) is described. Enzyme-linked immunosorbent assays (ELISA) employing membrane and cytosol fractions of human tissues demonstrated YBB 190 reactivity against cytosol but not membrane components of malignant and normal epithelial tissues. When tested by an indirect immunoperoxidase staining method against fresh frozen human tissue sections, YBB 190 reacted with malignant cells in 26 of 28 epithelial cancers and with normal epithelia in 11 different benign tissues. Preliminary western blot antigen characterization indicated that YBB 190 recognizes cytokeratin intermediate filaments, or a protein that is closely associated with cytokeratins. These data indicate that B cells with specificity for intermediate filaments are present in tumor draining lymph nodes. Our findings provide insights into the nature of potential autoimmune responses in cancer patients and suggest that improved tumor directed sensitization procedures may be required to more effectively utilize lymphocytes from tumor draining lymph nodes to generate therapeutically useful human MAbs to TAAs.

Adenocarcinoma↗
PubMed1987

Hybridomas producing human monoclonal antibodies against varicella-zoster virus.

Hybridomas producing human monoclonal antibodies (mAb) against varicella-zoster virus (VZV) were generated by fusing human splenic lymphocytes with mouse myeloma cells. Before cell fusion, lymphocytes were stimulated in vitro with viral antigens and pokeweed mitogen. This combination synergistically increased the generation of VZV-specific hybridomas. Five established hybridomas have been stably producing mAb for at least 9 months. These mAb, designated V1, V2, V6, V8 and V9, were of the IgG1, lambda isotype. They bound to all 6 tested VZV strains but not to other herpes viruses, with the exception that V1 bound to herpes simplex virus (HSV) as well as VZV. Immunoprecipitation analysis showed that V1, V6 and V9 recognized glycoprotein gpII, whereas V2 and V8 recognized gpI. In addition, V1 reacted with the gB glycoprotein of HSV. All these mAb neutralized viral infectivity. The neutralizations by V2 and V8 were more effective and more complement dependent than those by V1, V6 and V9. Immunofluorescence tests revealed that all these mAb bound to the surface membrane of VZV-infected cells. These results suggest that cell fusion between in vitro stimulated lymphocytes and mouse myeloma cells is a reliable method for the generation of hybridomas capable of stable production of human mAb. The human mAb thus developed may provide a new means of passive immunization of humans against VZV infection.

Antibodies, Monoclonal↗
PubMed1987

Antiviral activities of human monoclonal antibodies to herpes simplex virus.

Hybridomas producing human monoclonal antibodies (MAbs) against herpes simplex virus (HSV) were established by fusing human tonsillar lymphocytes with mouse myeloma cells. Three hybridomas have been stably producing MAbs for more than 16 months. All three MAbs--H1, H2, and H3--were of the IgG1 isotype and recognized the gB glycoprotein of HSV types 1 and 2 (HSV-1 and HSV-2). MAbs H2 and H3 not only bound to the surface membrane of HSV-infected cells but also neutralized both HSV-1 and HSV-2, whereas MAb H1 had neither activity. In mouse infection experiments, MAbs H2 and H3 showed a potent protective effect against HSV-1 infection, whereas MAB H1 was less protective. Furthermore, the development of zosteriform skin lesions in athymic nude mice was suppressed by administering MAb H2. These results suggest that human MAbs might provide passive immunization against HSV infections in humans.

Animals↗
PubMed1987

Human monoclonal antibodies neutralizing human cytomegalovirus.

Hybridomas producing human monoclonal antibodies (MAbs) against human cytomegalovirus (CMV) were generated by fusion of human spleen cells and mouse myeloma cells. Two of the six MAbs obtained neutralized viral infectivity even at concentrations lower than 1 microgram/ml. One MAb required complement for neutralization but the other did not. Both MAbs recognized viral proteins of Mr 130,000 and 55,000. Furthermore, these neutralizing MAbs bound to the surface membrane of CMV-infected cells. These results suggest that human MAbs may provide a new means of passive immunization against CMV infection in humans.

Antibodies, Monoclonal↗
PubMed1987

Immunoprotective human monoclonal antibodies against five major serotypes of Pseudomonas aeruginosa.

Human monoclonal antibodies (Mabs) against the O antigens of Pseudomonas aeruginosa lipopolysaccharides (LPS) were produced by cell fusion between human tonsillar lymphocytes and P3-X63-Ag8-U1 (P3U1) mouse myeloma cells. To obtain human Mabs efficiently, 6 d culture supernatants of pokeweed-mitogen-stimulated lymphocytes (21 cultures from peripheral blood and 76 from tonsils) were assayed by ELISA. Five tonsillar lymphocytes which produced IgG antibody specific for P. aeruginosa LPS were preselected for fusion. The human Mabs, named P1-1 (IgG2, kappa), P5-1 (IgG2, lambda), P7-1 (IgG2, lambda), P8-1 (IgG2, lambda) and P10-1 (IgG2, kappa), bound with high specificity to Homma standard serotype strains A, E, B, G and I, respectively, and recognized O antigens. Each Mab showed opsonophagocytic killing activity of the corresponding serotype strain. Four of the Mabs caused agglutination at a very low concentration; a rather higher concentration of P7-1 was required for this effect. Although all the Mabs conferred type-specific protection against peritoneal infection, the strongly agglutinating Mabs provided better protection than the moderately agglutinating P7-1. The protective activity of P8-1 was estimated in compromised mice. A low dose (PD50 0.5-0.6 microgram per mouse) of P8-1 prevented subcutaneous infection in burned mice and peritoneal infection in leucopenic mice. All the hybridomas described here could be cultured in serum-free medium, and they have continued to secrete human Mabs for more than 14 months at rates of 10-20 micrograms per 10(6) cells in 24 h. These results suggested that these five human Mabs specific for O antigens might be useful in the prophylaxis and treatment of P. aeruginosa infections.

Animals↗
PubMed1986

Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus.

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.

Antibodies, Monoclonal↗
PubMed1986

Generation of hybridomas producing human monoclonal antibodies against herpes simplex virus after in vitro stimulation.

Hybridomas producing human monoclonal antibodies against herpes simplex virus were generated by in vitro antigen stimulation before cell fusion. The cell fusion with tonsillar lymphocytes which were stimulated with antigen and/or pokeweed mitogen generated many hybridomas producing human IgG against the virus. A combination of antigen and pokeweed mitogen synergistically enhanced the generation of virus-specific hybridomas. Furthermore, the higher the antibody response of the tonsil, the more virus-specific hybridomas were generated by the cell fusion. These results suggest that cell fusion with in vitro stimulated lymphocytes can be applied to a variety of clinically relevant viruses.

Animals↗
PubMed1986

DNA-daunorubicin complexes specifically suppress in vitro spontaneous anti-DNA antibody production in lymphocytes of patients with systemic lupus erythematosus.

Elevated production of anti-DNA antibody in patients with systemic lupus erythematosus (SLE) is a central problem in the pathogenesis of tissue injury. In the present study, we attempted to manipulate anti-DNA antibody production through the antigen-cytotoxic drug conjugates, DNA-daunorubicin complexes. The effect of DNA-daunorubicin complexes was determined by examining SLE lymphocytes for spontaneous in vitro production of anti-DNA antibody. These complexes, at 2 micrograms/ml, suppressed anti-DNA antibody production, but not total IgG production, which suggests that specific suppression of anti-DNA antibody production was achieved at this concentration. We believe that the DNA-daunorubicin complexes affected mainly B cells, since such suppression was obtained by treating B cells, as well as B plus T cells. Furthermore, the complexes had no effect on the proliferative responses of SLE T cells to DNA, phytohemagglutinin, or concanavalin A. These results indicate that DNA-daunorubicin complexes may have the potential for selectively suppressing anti-DNA antibody production in patients with SLE.

Antibodies, Antinuclear↗
PubMed1985

Characterization of a human monoclonal antibody to lipopolysaccharides of Pseudomonas aeruginosa serotype 5: a possible candidate as an immunotherapeutic agent for infections with P. aeruginosa.

A human monoclonal antibody, P3D9 (IgG2, lambda type), that bound to Pseudomonas aeruginosa Homma serotype 5 cells with high specificity was produced by cell fusion between a human tonsillar lymphocyte and a mouse plasmacytoma cell, P3-X63-Ag8-U1 (P3U1). The yield of P3D9 secreted into the culture supernatant from the mouse-human hybridoma was 2-10 micrograms/ml, and this hybridoma cell line has been stably producing this antibody for six months. Antibody P3D9 alone did not cause agglutination of serotype 5 cells, but the cells were agglutinated after addition of goat antibody to human IgG. Antibody P3D9 was proven to have protective activity against infection with P. aeruginosa, and the 50% protective dose estimated for experimental peritoneal infection with P. aeruginosa was 2.4 micrograms per mouse.

Agglutination Tests↗
PubMed1985

A new common polysaccharide antigen of strains of Pseudomonas aeruginosa detected with a monoclonal antibody.

A monoclonal antibody, E87, that binds to various serotype strains of Pseudomonas aeruginosa was produced by a hybridoma cell line prepared by fusion of mouse plasmacytoma P3-X63-Ag8-U1 with spleen cells of mice immunized with P. aeruginosa strain IFO3080 (serotype M). E87 bound to approximately 80% of the tested strains of various serotypes of P. aeruginosa. The antigen recognized with E87 was found in the lipopolysaccharide and was eluted in void volume fractions of Sephadex G50 column chromatography after acetic acid treatment. This antigen was eluted in the fraction containing substances of lower molecular weight than the O side chain by Sephacryl S-300 column chromatography, and this fraction was found to be the peak fraction of hexose. This antigen was not separated from the O side chain by Sephacryl S-300 column chromatography of untreated lipopolysaccharide. Colorimetric analyses and thin-layer chromatography showed that this antigen consisted mainly of rhamnose and ribose (molar ratio, 10:1).

Acetates↗
PubMed1984

Protection against infection with Pseudomonas aeruginosa by passive transfer of monoclonal antibodies to lipopolysaccharides and outer membrane proteins.

Experimental infection with Pseudomonas aeruginosa was treated with eight different monoclonal antibodies (MCAs) produced by hybridoma cells obtained through cell fusion of mouse plasmacytoma cells and spleen cells from mice immunized with a virulent strain of P. aeruginosa (Homma serotype 7). Five MCAs bound to lipopolysaccharides (LPSs) specific to serotype 7 or serotypes 2, 7, and 13, whereas the other three MCAs bound with broad specificities to outer membrane protein (OMP) fractions. The MCAs to LPS were highly protective against infection, with 50% protective doses of 0.05-2.5 micrograms of immunoglobulin per mouse. In contrast, the MCAs to OMP were much less protective, with a 50% protective dose range of 10 to greater than 100 micrograms of immunoglobulin per mouse. Most of the MCAs to LPS agglutinated P. aeruginosa cells, but all the MCAs to OMP produced so far have not, although all the MCAs bound well to the cells. Agglutinating MCAs provided better protection than did nonagglutinating MCAs.

Agglutination Tests↗