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Biomedical subjects

J Chiba

Publications and source records attributed to J Chiba.

At least 145 records · Page 8Linked to original sources
PubMed1987

Improvement in the basic technology of electrofusion for generation of antibody-producing hybridomas.

In order to define the optimum conditions of electrofusion technique for the generation of antibody-producing hybridomas, mouse spleen cells or EBV-transformed human B cells were fused with mouse myeloma cells (SP2/0) or human fusion partner cells (KR-4 or KR-12), respectively, by electric field pulse under various conditions. The results confirm reports that the presence of both Ca2+ and Mg2+ in fusion medium and pretreatment of mixed cells with proteases improve hybridoma yield. Moreover, the presence of liposome or hydrophobic protein in the fusion medium greatly enhanced the yield. Under optimum conditions, hybridoma yields of mouse cells and human cells were 2.5 X 10(-4) and 1 X 10(-4), respectively. These efficiencies were about ten times higher than those obtained by the conventional polyethylene glycol technique. Microscopic observation of the fusion-process revealed that in a human cell system 20%-50% of the cells were physically fused, although only one in 5000 physically fused human cells grew as a hybridoma after hypoxanthine-aminopterin-thymidine selection.

Animals↗
PubMed1987

[Attempts to improve hybridoma technology for the production of human monoclonal antibodies].

The technology for producing hybridomas secreting human monoclonal antibodies has not been well established, contrary to the situation for mouse monoclonal antibodies. Therefore, we attempted to find better fusion partners using two EBV-transformed human B cell lines, C5TK1 and TAPC-30 (anti-SRBC IgM and anti-HBs IgG secretors, respectively; provided by Prof. Y. Ono, Nihon Univ. School of Med.). Fifteen partner cell lines available were fused with either of these lines by conventional PEG technique, and their fusion frequencies, cloning efficiencies, and levels and stabilities of specific antibody secretion were examined. Among the fusion partners tested, KR-12, SHM-D33 and 3HL3-6J lines appeared to be the best. Hybridomas with the KR-12 line were easily cloned and produced stable specific antibodies, although the level of Ig secretion was almost the same as the antibody-producing parental B-cell lines. SHM-D33 cells efficiently formed hybrids but the stability of their Ig production was low and cloning was laborious. Although the fusion frequency of 3HL3-6J cells was not so high, some hybrids were good producers of antibodies, and the levels of specific antibody reached levels greater than 10 micrograms/ml. Their cloning was relatively easy. Therefore, the 3HL3-6J line seemed to be the best fusion partner for EBV-transformed B cells. With regard to the low fusion frequency of human cells, we attempted to improve the efficiency using the electrofusion technique. Three to 10 times more hybridomas were obtained by this technique than by the conventional PEG method. Further investigations are under way.

Antibodies, Monoclonal↗
PubMed1987

New monoclonal antibodies specific for the guinea pig line 10 hepatocarcinoma.

Monoclonal antibodies reactive to line 10 (L10) hepatocarcinoma cells, but not to L2C leukemia cells, of strain 2 guinea pig were produced and characterized. Complement-mediated cytolysis assay and quantitative absorption analysis revealed that one of the monoclonal antibodies, 3C4 antibody (IgG2a), was highly specific for L10. Neither normal guinea pig tissues including adult and fetal liver, nor other hepatomas of strain 2 guinea pig, line 1 (L1) and Hepatoma III, were reactive with the 3C4 antibody. Another antibody, 2E4 (IgM), was reactive to the liver, kidney and spleen cells but not to the thymocytes, lymph node cells, brain and red blood cells of the guinea pig. The 2E4 antibody reacted to Hepatoma III but not to L1 cells. Therefore, 3C4 antibody is specific to a tumor-associated antigen of L10 hepatoma, and 2E4 antibody is cross-reactive with a certain differentiation antigen of normal tissues. The 3C4 antibody showed antibody-dependent cytotoxicity. Since 3C4 antibody was different in the isotype and the antigen recognized from a monoclonal antibody (D3) which was reported from the NIH in the USA, 3C4 is a new monoclonal antibody reactive with L10 hepatocarcinoma.

Animals↗
PubMed1985

Properties of monoclonal antibodies against glycoproteins of western equine encephalitis virus.

To analyze the biological activities of the alphavirus glycoproteins, eight different monoclonal antibodies against the two glycoproteins of western equine encephalitis virus were isolated. Five of the eight monoclonal antibodies were shown to be specific for E1 and three for E2 protein by an enzyme-linked immunosorbent assay and by radioimmunoprecipitation. Three of the five anti-E1 and all of the anti-E2 monoclonal antibodies inhibited hemagglutination by purified virions. One anti-E1 and two anti-E2 monoclonal antibodies possessed high virus-neutralizing activity.

Antibodies, Monoclonal↗
PubMed1985

Effect of islet transplantation on diabetes mellitus after subtotal pancreatectomy in rat: a quantitative analysis of islet tissue.

A reproducible model of diabetic state was produced by resecting more than 90% of the pancreatic tissue in Wistar strain male rats. Five rats with diabetic state after the resection received islet transplantation into the liver through the portal vein. All these five rats showed normoglycemia one week after the injection and it lasted about six months. A morphometric analysis using 1190 serial sections of the control rat pancreas showed that the islet distribution accorded satisfactorily with Weibull and gamma distribution, when an islet was regarded as a sphere in the three dimensional space. The estimated value of radius and number of the islets was 0.0155 mm and 131.2/mm3, respectively, whereas volume fractions of the islet tissue in the splenic and gastric segments, and parabiliary and duodenal segments were estimated at 1.45 and 0.98%, respectively. The mean half of the largest diameter of the isolated islets under dissecting microscope reached to 0.080 mm and 500 isolated islets account for about 14% of volume fraction in a whole pancreas. As the remnant pancreas possibly had approximately five percent of islet volume, the transplanted animal contained about 19% of islet volume as compared to the control animal. It could be concluded that transplantation of 500 islets controlled diabetes in the rat with subtotal pancreatectomy. But the transplanted rats eventually became diabetic about six months after the transplantation possibly because of insufficient volume of implanted islets.

Animals↗
PubMed1985

Guinea pig T lymphocyte development analyzed by enzyme histocytochemistry, monoclonal antibodies, and flow cytometry.

Studies of T (thymus-derived) lymphocyte ontogeny in the guinea pig have been hampered by the lack of suitable antigenic or other markers for various T cell subpopulations in this species. Monoclonal antibodies that recognize three distinct surface proteins of guinea pig T cells and react with all peripheral T cells have been used in combination with membrane alkaline phosphatase (AP) to characterize stages of guinea pig T cell development and to determine anatomical localization of different T cell subpopulations. Flow cytofluorographic analysis of thymus, spleen, and lymph node lymphocytes was used to characterize monoclonal antibody specificity. Cortical thymocytes in tissue sections expressed membrane AP activity and contained nuclear terminal deoxynucleotidyl transferase; medullary thymocytes reacted strongly with one of the monoclonal antibodies (8BE6), minimally with a second (5CD2), and not at all with a third (11AE3). In contrast, polyclonal rabbit antiguinea pig T cell antiserum reacted with both cortical and medullary thymocytes. Staining of tissue sections of lymph node and spleen revealed AP+ lymphocytes to be present peripheral to the mantle region of lymph node follicles and to be randomly scattered throughout the splenic red pulp. T cells reactive with monoclonal antibodies were located primarily in paracortical regions of lymph node and the central region around the periarteriolar regions of the spleen. Dual staining of frozen sections and cell suspension of guinea pig lymphoid tissues for AP activity and surface proteins unique to T cells showed that AP+ cells lacked T cell markers. Dual staining for AP activity and surface immunoglobulins or esterase activity showed that AP+ cells are not likely to be derived from either B cell or monocyte-macrophage lineages. AP+ cells in guinea pig secondary lymphoid tissue may represent a unique subset of lymphocytes of unknown function.

Animals↗
PubMed1985

Pathologically defined neuro-, vasculo-, entero-Behçet's disease.

Behçet's disease is characterized clinically by the presence of a diagnostic triad of stomatitis, genital ulceration and uveitis. Multiple other organ systems may be involved. Prognosis is related to the presence of central nervous system, vascular or intestinal involvement. This report describes the pathology of neuro-, vasculo- and entero-Behçet's disease in a single patient.

Adult↗
PubMed1984

Characterization of a monoclonal antibody specific for lipoteichoic acid from various gram-positive bacteria.

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.

Animals↗
PubMed1984

Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections.

Antibody-producing hybridomas of myeloma SP2/O and spleen cells of BALB/c mouse immunized with pertussis toxoid and pertussis toxin were selected by the binding ability of the monoclonal antibody to the subunit protein of the toxin. Two monoclonal antibodies, 1B7 and 3F10, specific for a subunit which has no binding activity to haptoglobin and sheep erythrocytes, named S1, and one antibody, 1H2, for a subunit related to the binding activity of the pertussis toxin molecule to haptoglobin or sheep erythrocytes, named S4, were examined for mouse protective activity against pertussis infection. Antibody 1B7 not only neutralized leukocytosis-promoting and islet-activating activities of the toxin but also protected mice against intracerebral and aerosol challenge with Bordetella pertussis. The antibody, furthermore, showed therapeutic effects on mice showing severe clinical signs with pertussis infection. The other two antibodies, 3F10 and 1H2, showed neither neutralizing nor protecting activity, nor significant synergistic effects on antibody 1B7.

Animals↗
PubMed1983

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens.

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.

Animals↗
PubMed1983

Properties of cloned T cells that mediate delayed-type hypersensitivity against ovalbumin in mice.

Cloned T-cell lines that mediate delayed-type hypersensitivity (DTH) against soluble protein antigen, ovalbumin (OA), were established in (C57BL/6 X DBA/2)F1 mice and their properties were examined. They induced antigen-specific delayed-type footpad reactions, characterized histologically by a predominant mononuclear cell infiltration, when transferred intravenously into syngeneic mice. Morphologically, they were medium or large lymphoblasts with granules in the cytoplasm and expressed Lyt 1 cell surface antigens. One of them proliferated antigen specifically under the presence of both C57BL/6 and F1 accessory cells, while others proliferated antigen specifically only under the presence of F1 accessory cells. They also produced macrophage-activating factor (MAF) and substances which mediate a DTH-like footpad inflammatory reaction with a maximum 6 hr after injection into the footpad of normal mice, when incubated in the presence of specific antigen and specific accessory cells in a serum-free medium for 24 hr. These results demonstrate that cloned DTH-effector T cells, established here against soluble protein antigen, are Lyt 1-positive, large lymphoblasts and that they produce MAF and footpad-reactive inflammatory substances antigen specifically under the presence of specific accessory cells.

Animals↗