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Biomedical subjects

T Osawa

Publications and source records attributed to T Osawa.

At least 271 records · Page 15Linked to original sources
PubMed1988

Homologous human macrophage hybridomas that produce a novel cytotoxic factor in their culture supernatants.

Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte-derived macrophages and a subclone of the myelomonocytic cell line, U937-F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14+, OKM5+, Mac-1+ (equivalent to OKM1 and Mol), OKT9+, HLA-DR- and CD20+. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met-Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti-tumor necrosis factor, anti-lymphotoxin and anti-interleukin 1 antisera.

Antibodies, Monoclonal↗
PubMed1988

Enhancement of antigen-presenting function of dendritic cells with culture supernatants of mouse peritoneal macrophages stimulated with certain particulate substances.

The production from murine resident peritoneal macrophages (M phi) of a soluble factor, which was capable of enhancing the antigen-presenting (AP) function of dendritic cells (DC), was examined. The supernatants of peritoneal M phi (M phi sup) were prepared by culturing peritoneal M phi with particles, i.e., zymosan A, latex, and sheep red blood cells (SRBC), or antigen-antibody (Ag-Ab) complexes such as keyhole limpet hemocyanin (KLH)-anti-KLH, ovalbumin (OVA)-anti-OVA, and SRBC-anti-SRBC complexes. When exposed to M phi sup during antigen pulsing DC induced a marked antigen-specific T cell proliferation, relative to DC treated with the supernatants from M phi cultured without stimuli (control sup). On the other hand, M phi sup-treated splenic M phi stimulated antigen-specific T cell activation to almost the same extent as did splenic M phi treated with control sup. These results indicated that peritoneal M phi elaborated a soluble factor which preferentially enhanced the AP capacity of DC when stimulated with particles or Ag-Ab complexes. Analytical gel filtration of M phi sup revealed that the factor had an apparent molecular weight of 27,000 daltons which was distinct from interleukin 1.

Animals↗
PubMed1988

Dendritic cell activating factor produced by mouse macrophage hybridoma.

A mouse macrophage (M phi) hybridoma which produces a soluble factor responsible for the cooperation between M phi and spleen dendritic cells (DC) was constructed. The antigen-presenting activity and the stimulator cell activity in the allogeneic or syngeneic mixed leukocyte reaction of DC were significantly augmented when DC were incubated with the culture supernatant of the hybridoma treated with various stimulants including latex beads. The monokine present in the culture supernatant of the hybridoma, called dendritic cell-activating factor (DCAF), augmented the production of lymphocyte-activating factor by DC while Ia expression of DCAF-treated DC was not altered. DCAF had no effect on the antigen-presenting activity of peritoneal resident M phi or B cell blasts while the antigen-presenting activity of spleen M phi was enhanced, but the degree of the enhancement was much less than that of spleen DC. DCAF was found to have the following properties: its pI value is between pH 4 and 5; it is stable at pH 2 to 10; and it loses its activity on incubation at 75 C for 30 min. When the culture supernatant of the hybridoma stimulated with latex beads was subjected to gel filtration, the DCAF activity was detected in the 20 Kd to 25 Kd, 30 Kd to 40 Kd, and 50 Kd to 60 Kd molecular weight regions. The 30 Kd to 40 Kd fraction, which is the major peak fraction, was further purified by ion-exchange chromatography followed by gel-filtration chromatography. When each fraction was subjected to SDS-PAGE, a 30 Kd band corresponding to the DCAF activity was observed and DCAF was purified to about 90% purity.

Animals↗
PubMed1988

[Radiologic detection of metastatic bone tumors].

Radiologic strategy for early detection of bone metastasis is discussed. Incidence, pathway, and radiologic manifestation of bone metastasis are briefly discussed. Bone scintigraphy is a mainstay for early detection of bone metastases, although its role as a part of staging procedure is debatable. Plain radiograph and CT scan are to be used to increase specificity of scintigraphic findings. For detection of spinal metastases CT scan is particularly useful and should be used whenever spinal metastases are suspected. MRI has become an important modality to see an extraosseous extension and marrow invasion of metastatic bone tumor.

Bone Neoplasms↗
PubMed1988

Tumoricidal activity of lymphotoxin (tumor necrosis factor beta) in vivo: its effects on macrophages.

We investigated the cytotoxic activity of recombinant human lymphotoxin (rHuLT) against Meth A sarcoma in vitro and in vivo. In direct cytotoxic assay, Meth A cells showed very low, if any, sensitivity to rHuLT. However, rHuLT was found to be strongly cytotoxic for subcutaneously implanted Meth A cells. In order to explain this discrepancy, we examined the indirect effects of rHuLT on Meth A cells particularly through macrophages. rHuLT showed macrophage chemotactic activity in vitro and also in vivo. Thus, injection of rHuLT into the mouse peritoneal cavity caused migration of macrophages to the peritoneal cavity. In addition, histologic analysis revealed that 6 h after intratumor injection of rHuLT, nonspecific esterase-positive cells started to migrate into Meth A tumor tissues, and after a further 24 h, a larger number of nonspecific esterase-positive cells had migrated into the Meth A tumor tissues. Moreover, peritoneal exudate cells induced by rHuLT showed considerably higher cytotoxic activity against Meth A cells than proteose peptone- or phosphate-buffered saline-induced peritoneal exudate cells. Furthermore, the cytotoxic activity of peritoneal exudate cells was found to reside in macrophages. Investigation of the mechanism of macrophage activation by rHuLT revealed that rHuLT can activate macrophages for cytotoxicity synergistically with mouse interferon-gamma. These results strongly suggest that rHuLT, when injected into the tumor, can induce the migration of macrophages into the tumor and activate macrophages in situ to kill tumor cells.

Animals↗
PubMed1987

Establishment of a human T-cell hybridoma that produces human macrophage activating factor for superoxide production and translation of messenger RNA of the factor in Xenopus laevis oocyte.

Human monoblast-like histiocytic lymphoma cell line U937 was induced by a macrophage activating factor for O2- production (MAF-O) to produce O2- in response to phorbol myristate acetate stimulation. A MAF-O-producing human T-cell hybridoma, F4-29-4, was established which was also found to produce macrophage activating factor for glucose consumption (MAF-G) and colony stimulating factor (CSF) when assayed against mouse bone marrow cells. MAF-O could be successfully separated from CSF but not from MAF-G by phenyl-Sepharose CL-4B chromatography (Phenyl-EG-fraction). To differentiate MAF-O from MAF-G and to explore a route for large scale production of MAF-O and its structural elucidation, total messenger RNA was extracted from a human T-cell hybridoma, clone F4-29-4. This messenger RNA was fractionated on 5-30% sucrose gradient and each fraction was microinjected into Xenopus laevis oocytes. MAF-O activity was found in the supernatant of oocytes injected with messenger RNA sedimentated at about 13.0S, while MAF-G messenger RNA was found to be about 10.5S. The MAF-O activity, synthesized from the injected messenger RNA, was not neutralized with an excess amount of anti-human IFN-gamma anti-serum, suggesting that MAF-O is antigenically different from human IFN-gamma.

Animals↗
PubMed1987

Early transmembrane events in tumor necrosis factor and lymphotoxin-induced cytotoxicity.

We have studied early transmembrane events in tumor necrosis factor (TNF) and lymphotoxin (LT)-induced cytotoxicity by means of stopped-flow fluorometry with 3 different fluorescent probes. Both TNF and LT caused an increase in the membrane fluidity of the target cells (L X P3 cells). After this event, there was a low level of calcium influx from the external medium into the target cells. On the other hand, the release of calcium from intracellular stores in the target cells was negligible. These sequential events, however, were not observed when mutant LT molecules which lacked the lytic activity were used.

Aminoquinolines↗
PubMed1987

Production of human--human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization of peripheral blood lymphocytes.

This report describes the formation of human hybridomas after in vitro immunization of peripheral blood lymphocytes (PBL) with an antigen and fusion of the stimulated lymphocytes with a HAT-sensitive human myeloma cell line, RPMI 8226. PBL were stimulated in vitro with sheep erythrocytes (SRBC) plus fresh human serum. PBL of some donors produced anti-SRBC antibody when they were cultured at 2 X 10(6) cells per well in a 24-well plate with the antigen plus fresh human serum for 7 days. Although lymphocytes of some donors were "low-responders" under the above conditions, they responded to SRBC when they were cultured with not only the antigen plus fresh human serum but also with the culture supernatant obtained after phytohemagglutinin (PHA) stimulation of a mixture of PBL from two donors (MLC-PHA sup). The cells sensitized by this procedure were fused with RPMI 8226 cells. Hybrids secreting IgM or IgG anti-SRBC antibodies were obtained. Additionally the ratio of total IgG-producing hybridomas to IgM-producing ones was higher when the MLC-PHA sup was used at the time of the in vitro immunization.

Animals↗
PubMed1987

The carbohydrate moiety of human platelet glycocalicin: the structures of the major Asn-linked sugar chains.

Glycocalicin, a predominant glycoprotein on the human platelet surface, has been purified from a platelet suspension by means of sonication, ammonium sulfate precipitation and acid treatment followed by chromatography on columns of wheat germ agglutinin-Sepharose and Mono Q. Asparagine-linked (N-linked) oligosaccharides were released by hydrazinolysis, and then N-acetylated and reduced with NaBH4 or NaB3H4. The released carbohydrate chains were found to be of the complex-type from their interaction with immobilized lectin columns. The structures of the two major oligosaccharide-alditols separated by ion-exchange chromatography on a Mono Q column were investigated by means of methylation analysis, glycosidase digestion, and Smith periodate degradation, and they were assigned as typical di- and trisialylated complex-type oligosaccharide-alditols with two and three peripheral chains consisting of Gal-GlcNAc sequences, respectively.

Amino Acids↗
PubMed1987

Protein-O-carboxylmethyltransferase from cytosol and membranes of chicken erythrocytes.

Cytosolic protein-O-carboxylmethyltransferase was purified more than 4,000-fold in specific activity and membrane-associated protein-O-carboxylmethyltransferase carboxymethylase about 900-fold from chicken erythrocytes by use of a combination of affinity chromatography on immobilized S-adenosyl-L-homocysteine and gel filtration on Sephacryl S-200 (Pharmacia), together with 3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulfonate as a detergent to solubilize the membrane-associated enzyme. The two enzymes were characterized by examining the dependence of their activity on pH and on concentration of S-adenosyl-L-methionine using fetuin as an exogenous methyl-acceptor substrate, and were found to differ somewhat. The cytosolic enzyme had a pH optimum of 6.0 with an apparent Km value of 2.1 microM for S-adenosyl-L-methionine, whereas corresponding values for the membrane-associated enzyme were 6.5 and 0.71 microM. This report deals with the biochemical differences between purified cytosolic and membrane-associated protein carboxymethylase from the same cell source.

Animals↗
PubMed1987

Carbohydrate moieties of peanut agglutinin receptors isolated from human gastric cancer cells.

Receptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4 method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Gal beta 1----3GalNAc-ol and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-beta-galactosidase digestion.

Carbohydrates↗
PubMed1987

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein.

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.

Animals↗
PubMed1987

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice.

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.

Animals↗
PubMed1987

Human macrophage-activating factors for cytotoxicity. I. Establishment of a human T-cell hybridoma that produces macrophage-activating factors for cytotoxicity.

Human T cell hybridoma, H3-E9-6, that produces macrophage activating factors for cytotoxicity (MAF-C) was prepared by somatic fusion of phytohemagglutinin-activated peripheral blood lymphocytes with emetine/actinomycin D-treated cloned human acute lymphocytic leukemia cells (CEM 11). The activities of the following were assayed: (1) macrophage-activating factor for cytotoxicity of monocytes (MAF-C 1 day), (2) macrophage-activating factor for cytotoxicity of monocyte-derived macrophages (MAF-C 6 day), (3) macrophage-activating factor for cytotoxicity of murine macrophages (MAF-Cm), (4) macrophage-activating factor for glucose consumption (MAF-G), (5) macrophage-activating factor for O2- formation (MAF-O). The culture supernatant of H3-E9-6 showed MAF-C 1 day-MAF-C 6 day, MAF-Cm, and MAF-G activities. The MAF-Cm activity was considerably enhanced by the addition of murine recombinant interferon gamma (rIFN-gamma). The MAF-C 1 day activity in the H3-E9-6 sup was not decreased by heat treatment (56 C, 30 min), by pH 2 treatment or by the addition of monoclonal anti-human IFN-gamma antibody or polymyxin B. These data suggest that MAF-C in H3-E9-6 sup is distinct from human IFN-gamma or lipopolysaccharide (LPS).

Cytotoxicity, Immunologic↗
PubMed1987

Human macrophage-activating factors for cytotoxicity. II. Synergism of two factors produced by a human T-cell hybridoma that induce the cytotoxicity of human monocytes.

Two different factors (MAF-C I and MAF-C II) were obtained by anion exchange chromatography of the culture supernatant of a human T-cell hybridoma, H3-E9-6, which produces macrophage-activating factors for cytotoxicity (MAF-C). These 2 factors induced the cytotoxicity of monocytes synergistically as a priming signal (MAF-C I) and a triggering signal (MAF-C II), respectively. On gel filtration on a column of Superose 12, MAF-C II was eluted mainly at the void volume, whereas MAF-C I was eluted in the fractions corresponding to approximate molecular weights of 30-300 K. On the other hand, gel filtration in the presence of sodium deoxycholate revealed that MAF-C II has an approximate molecular weight of 40,000, but MAF-C I was unstable under these conditions. When the activity for mouse macrophages (MAF-Cm activity) was tested, the MAF-C II fraction showed high MAF-Cm activity in the presence of murine recombinant interferon gamma (rIFN-gamma), but the MAF-C I fraction did not show MAF-Cm activity even in the presence of lipopolysaccharide (LPS). These results suggest that MAF-C I (priming lymphokine) has species specificity but MAF-C II (triggering lymphokine) does not.

Chromatography, Gel↗
PubMed1987

A factor potentiating antibody formation spontaneously produced by splenic T cells of MRL/MP-lpr/lpr mice.

Splenocytes of MRL/l mice, one of the murine lupus strains, were found to produce a factor(s) which potentiates antibody formation. This factor was produced by splenocytes of young MRL/l mice and cell-sorting experiments by flow cytometry indicated that Thy 1.2+ cells were producing the factor. The approximate molecular weight of the factor was found to be 35,000-45,000 daltons and its pI value was between 3.3 and 3.8. It was also demonstrated that this factor is distinct from interferons, interleukins (IL-1, IL-2, IL-3) and B cell differentiation factors.

Animals↗