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Biomedical subjects

I Nakashima

Publications and source records attributed to I Nakashima.

At least 163 records · Page 9Linked to original sources
PubMed1992

Establishment of mouse oligodendrocyte/type-2 astrocyte lineage cell line by transfection with origin-defective simian virus 40 DNA.

A permanent glial cell line has been established from the neonatal mouse primary mixed glial cell cultures by transfection with replication origin-defective simian virus 40 DNA. This cell line, designated OS3, has morphological similarity to type-2 astrocyte and expresses an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), when cultured in the presence of 10% calf serum (CS). OS3 cells do not express the O4 antigen, galactocerebroside (GalC) and A2B5 under this culture condition. When cultured in a medium containing 2% CS or a chemically defined medium, these cells undergo morphological transformation. Some of these cells express O4 antigen and/or GalC, and the percentage of GFAP positive cells decreases under these conditions. Thus depending on the culture conditions, the OS3 cells display either type-2 astrocyte properties or immature oligodendrocyte characteristics. Furthermore, the OS3 cells show similar responses to the various growth factors as do oligodendrocyte/type-2 astrocyte (O-2A) progenitors. Therefore, the OS3 cell line is an unique mouse bipotential permanent O-2A lineage cell line which may be useful to analyze the developmental properties of these glial cells.

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PubMed1992

Immunosuppressive activity induced by nitric oxide in culture supernatant of activated rat alveolar macrophages.

Alveolar macrophages (AM) from normal rats had immunosuppressive activity to mitogen-induced proliferative responses of splenic lymphocytes. We studied the mechanism and the implication of the nitric oxide synthetase pathway in AM-mediated suppression of concanavalin A (Con A)-induced lymphocyte proliferation. The culture supernatant from AM cultures alone did not have immunosuppressive activity to Con A-induced proliferative responses of non-adherent spleen cells (n-ad SC), but the culture supernatant from co-culture of AM and autologous n-ad SC had this activity. Con A-pulsed AM also liberated the immunosuppressive factor. When AM and autologous n-ad SC were cultured separately under the condition that medium could freely communicate, the culture supernatant did not suppress the Con A-induced proliferative response of n-ad SC. This indicated that the immunosuppressive factor was liberated when AM was activated by cell-to-cell contact with n-ad SC. Further, we examined the immunosuppressive activity of the culture supernatant of co-culture of AM and autologous n-ad SC to Con A-induced responses of allogeneic n-ad SC and xenogeneic murine n-ad SC, and allogeneic mixed leucocyte reaction, and found that this culture supernatant could suppress all these proliferative responses. Nitrate (NO2-) synthesis was markedly augmented in the culture supernatants of Con A-pulsed AM and co-culture of AM and n-ad SC. NG-monomethyl-L-arginine (MMA), a specific competitive inhibitor of the nitric oxide synthetase pathway (NOSP), extinguished both NO2- synthesis by AM and AM-mediated immunosuppressive activity. These data suggest that NOSP was important in AM-mediated suppression of Con A-induced lymphocyte proliferation.

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PubMed1992

Quantitative analysis of antigen for the induction of tolerance in carcinoembryonic antigen transgenic mice.

In order to analyse the amounts of antigen in the thymus for the induction of tolerance, several carcinoembryonic antigen (CEA) transgenic lines were established which expressed human CEA antigen with different amounts. The chimeric KSN nude mice transplanted with the thymus of the B601 line (in which CEA mRNA and CEA protein could be detected in various tissues) to kidney capsule showed tolerance to human CEA. On the other hand, the chimeric KSN nude mice transplanted with the thymus of the B602 or BC60 line (in which neither CEA mRNA nor CEA protein could be detected by Northern blot analysis and flow cytometry analysis) or normal C57BL/6 (B6) did not develop the tolerance to human CEA. However, the chimeric KSN nude mice transplanted simultaneously with thymus of the B6 and spleen of the B601 line became tolerant to human CEA antigen. In the case of systemic immunization with cells which had CEA antigen, the B601 line was tolerant to human CEA. Surprisingly, the B602 and BC60 lines were also tolerant to CEA molecule. These results indicate that not only the antigen present in the thymus but also the antigen which flows from the peripheral organs to the thymus may be necessary for the induction of CEA tolerance.

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PubMed1992

Establishment and characterization of a malignant melanocytic tumor cell line expressing the ret oncogene.

We established a cell line (designated Mel-ret) from a melanocytic tumor developed in a metallothionein/ret transgenic mouse. Unlike primary melanocytic tumors, which did not show malignant features, when the Mel-ret cells were transplanted into nude mice they invaded into surrounding tissues and had metastatic ability. Although the Ret proteins were expressed at similar levels in the cell line and the primary tumors, the level of tyrosine phosphorylation in the Mel-ret cells was much higher than that in the primary tumors. In particular, an 85-kDa tyrosine-phosphorylated band was specifically detected in the Mel-ret cells. These results suggest that the increase in tyrosine phosphorylation may be responsible for malignant transformation of the Mel-ret cells. Immunofluorescence and cell fractionation studies showed that the Ret proteins and most of tyrosine-phosphorylated proteins in the Mel-ret cells localized in the membrane fraction. No activation of phosphatidyl-inositol-3 kinase (PI-3 kinase), a target protein for several tyrosine kinases, was detected in the Mel-ret cells.

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PubMed1991

Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death.

The potential role of Thy-1 in CD3/TCR complex-mediated signal delivery to murine thymocytes was studied. Ag-mimicking cross-linked anti-CD3 mAb stimulated suspension of thymocytes from adult (6 to 8 wk old) mice for a brisk free cytoplasmic calcium ion ([Ca2+]i) rise, low level of inositol phosphate production, and marginal increase in tyrosine-specific phosphorylation of 110/120-kDa and 40-kDa cellular proteins. Weak but sustained [Ca2+]i rise, low inositol phosphate production, and weak protein tyrosine phosphorylation were also induced by the cross-linked anti-Thy-1 mAb that mimicked the putative natural ligand. The signal delivered via either of these two pathways was however insufficient for definitively promoting cell death and DNA fragmentation in the adult thymocytes. Here we demonstrated that anti-Thy-1 mAb synergized with anti-CD3 mAb for inducing a long-lasting prominent [Ca2+]i rise, definite inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakiphosphate production, and extensive tyrosine-specific phosphorylation of 110/120-, 92-, 75-, and 40-kDa proteins, which resulted in marked promotion of cell death and DNA fragmentation in the adult thymocytes. This unique anti-Thy-1 antibody activity was confirmed to be directed to glycosylphosphatidylinositol-anchored Thy-1, and was distinguished from the known anti-L3T4 activity that augmented the CD3-mediated signal transduction in a different manner. The synergistic actions of anti-CD3 and anti-Thy-1 mAb obligatorily required the cross-linking of the two mAb together. The anti-CD3 and anti-Thy-1 mAb cross-linked together acted on immature thymocytes from newborn (less than 24 h after birth) mice for rather more extensive promotion of protein tyrosine phosphorylation and cell death. In addition, they affected peripheral T lymphocytes for accelerating protein tyrosine phosphorylation but not cell death. These results suggest a novel function of glycosylphosphatidylinositol-anchored Thy-1 as a possible unique intrathymic intensifier of the CD3/TCR complex-delivered signal for negative thymocyte selection.

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PubMed1991

Preferential development of pre-B lymphomas with drastically down-regulated N-myc in the E mu-ret transgenic mice.

We established one transgenic mouse line which developed pre-B leukemic lymphomas by introducing ret cDNA driven by the SV40 promoter and the mouse immunoglobulin (Ig) enhancer. Lymphomas developed not only in the lymph nodes and the spleen but also in the thymus between the ages of 7 and 21 weeks. Analyses of cell surface phenotypes and Ig gene rearrangement revealed that these tumors were surface IgM-B220+ pre-B lymphomas. The rearrangement pattern of the Ig heavy chain locus indicated that the tumor cells were mono- or oligoclonal. Northern blot analysis showed that the ret transgene was expressed at a high level not only in the tumors but also in the prelymphomatous lymphoid tissues. We found that the expression of N-myc was dramatically down-regulated in the tumor cells, while the expression of c-myc was rather stable. Further experiments demonstrated that ret gene product did not directly down-regulate the expression of N-myc in transformed pre-B cell lines by in vitro transfection assay. From these results, we conclude that under the control of Ig enhancer, the ret transgene affected B lymphocytes at the early maturation stage as a prerequisite for transformation, preferentially generating a unique maturation stage of pre-B lymphomas whose N-myc expression was developmentally down-regulated.

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PubMed1991

Aberrant melanogenesis and melanocytic tumour development in transgenic mice that carry a metallothionein/ret fusion gene.

We generated four independent transgenic mouse lines that showed severe melanosis of the whole body by introducing the ret oncogene fused to the mouse metallothionein (MT)-I promoter-enhancer (MT/ret). Whereas melanogenesis was accelerated without distinct proliferative disorders in one line, melanocytic tumours frequently developed in the other three lines. Northern hybridization and in situ hybridization analyses showed that tumour cells and non-tumorous melanin-producing cells expressed the transgene at high levels. The aberrant melanogenesis and tumour development were influenced by genetic and environmental factors. Furthermore, crossbreeding experiments between the transgenic mice and Wv mice suggested that the ret gene product can partially compensate for the defect of melanocyte development in Wv mice. This is a novel mammalian model in which melanosis and melanocytic tumours develop stepwise, triggered by a single transgene.

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PubMed1991

A new mouse model for autoimmune orchitis.

Experimental autoimmune orchitis (EAO) was induced in SMA mice (H-2nondefined) by repeated injection at intervals of 30 days of syngeneic testis homogenate (TH) together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as a potent adjuvant. EAO was not induced by repeated injection of TH alone or KO3 LPS alone. At 10 days after the secondary injection of TH + KO3 LPS, there was marked infiltration with neutrophils in the seminiferous tubules and in the interstitium of the testis accompanied by destruction of the architecture of the seminiferous tubules and hypospermatogenesis. At 20 days after the secondary injection, infiltration with neutrophils in these areas had been replaced mostly by mononuclear cells (lymphocytes, plasma cells, and macrophages). Histopathological changes of the testes became severer by further injections until the 10th injection. The EAO lesions in the terminal stage were characterized by complete destruction of the tubular architecture of the testis, fibrosis, and aspermatogenesis. Lesions in the terminal stage were not restored at all. Spermagglutinating antibody titers in the serum increased and delayed-type hypersensitivity against TH estimated by footpad swelling developed in mice injected repeatedly with TH + KO3 LPS. Using immunofluorescence, antibodies against acrosomal components and tail components of the spermatozoa were detected in serum of these mice.

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PubMed1991

Cell type and maturation stage-dependent polymorphism of N-linked oligosaccharides on murine lymphocytes and lymphoma cells.

We analyzed the overall structures of N-linked oligosaccharides on glycoproteins of various murine lymphocytic and lymphoma cells employing a newly developed method which was performed on high-performance liquid chromatography after derivatization of oligosaccharides with 2-aminopyridine. A total of 15 types of bi, tri- and tetra-antennary N-acetyllactosamine-type oligosaccharides with or without fucose and oligomannose-type oligosaccharides were identified on these cells in variable amounts depending on the type and maturation stage of the cells. It was found that all murine lymphocytic cells carry N-acetyllactosamine-type oligosaccharides with the additional alpha-linked galactose residue on the non-reducing ends. Thymocytes had exceptionally large amounts of oligosaccharides with one or even two alpha-galactose residues per molecule. In contrast, peripheral resting T cells possessed those oligosaccharides only in a small amount, although the cells produced more the oligosaccharides after stimulation with Con A. Two thymoma lines such as BW 5147 and EL-4 and one B cell lymphoma line WEHI231 contained relatively large amount of oligosaccharides with alpha-galactose residues. Significant change of the molar ratio of component carbohydrates by cell activation was observed also in oligommanose-type oligosaccharides which were few in resting T cells but were markedly increased in Con A activated cells. Molar ratio of triantennary oligosaccharides in total N-acetyllactosamine type oligosaccharides was high in thymocytes and low in resting T cells, but was increased in T cells after Con A activation. It was also very high in WEHI 231 B cell lymphoma. Although BW 5147 and EL-4 thymoma did not contain tri-antennary oligosaccharides in high proportion, they carried larger tetra-antennary oligosaccharides with an N-acetyllactosamine repeating unit in definitive amounts. It is suggested from these results that overall structures of oligosaccharides on cell surface proteins of lymphocytes are finely controlled with link to cell differentiation, activation and transformation.

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PubMed1991

Modulation of the secondary antibody response of murine lymphocytes to sheep red blood cells in vitro by neuraminidase and exoglycosidases.

The effect of digestion of lymphocytes with neuraminidase and exoglycosidases on the secondary antibody response in vitro to sheep red blood cell (SRBC) antigen was tested. Treatment of spleen cells from SRBC-primed mice with 3 micrograms/ml of neuraminidase slightly but significantly augmented their plaque-forming cell response to SRBC, whereas treatment with 100 micrograms/ml of a mixture of exoglycosidases did not. Rather unexpectedly, however, treatment of the spleen cells with the mixture of both neuraminidase and exoglycosidases greatly augmented the response. This enzyme action was substrate specific inasmuch it was ablated by addition of mucin as a neuraminidase inhibitor to the enzyme mixture. The target of the enzyme activity was not glass-adherent macrophages, but was glass-non-adherent suppressor cells in the antigen-primed cell population. Evidence was provided that the phenotype of suppressor cells whose activity was ablated by the enzyme treatment was Thy-1+. It is suggested from these results that sialylated complex type oligosaccharides on antigen-primed T cells play a critical role in their suppressor activity.

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PubMed1991

FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a.

We examined the inhibitory effect of FUT-175 on the C3/C5 convertase activity of the cobra venom factor-derived enzyme CVF,Bb by measuring C5b6-mediated reactive lysis of unsensitized guinea pig erythrocytes and by measuring directly the released fragments C3-des-Arg and C5a-des-Arg. In this study, we showed that the concentration of 4.5 X 10(-6) M of FUT-175 caused 50% inhibition of C5 convertase activity of CVF,Bb in reactive hemolysis assays, and that 4.0 X 10(-6) M FUT-175 caused 50% inhibition of the production of C3a and C5a generated by the C3/C5 convertase activity of CVF,Bb.

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PubMed1991

A possible site of in vivo action of cyclosporin A for transplantation immunity: comparative study with cyclophosphamide.

The immunosuppressive mechanisms of cyclosporin A (CsA) and cyclophosphamide (CPM) in vivo were studied by measuring the transplantation immunity for second set rejection of an allogeneic ascites tumor. The transplantation immunity in our assay system was mediated primarily by allospecific cytotoxic T lymphocytes (CTL). Our results showed that both CsA and CPM did not affect the process of inducing the immunity, but they inhibited the effector phase of that transplant immunity. The experiments using 51Cr-release assay in vitro on peritoneal exudate cells as effector cells showed that the effector phase of the transplantation immunity in vivo involved the maturation from immunological memory CTL-precursor cells to effector CTL with clonal expansion. Further, adoptive cell transfer experiments showed that CsA did not eliminate the immunological memory CTL nor induce the suppressor cells in donor mice. All the data suggested that the maturation of memory CTL precursor cells to effector CTL was inhibited by CsA which would block lymphokine production even in vivo, whereas CPM eliminated the CPM-sensitive memory CTL precursor cells. The findings that CsA and CPM act on the different sites of CTL maturation support the possibility of effectiveness of the combination therapy of the two agents for controlling the transplantation immunity.

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PubMed1991

Establishment and characterisation of human carcinoembryonic antigen transgenic mice.

We have produced human CEA transgenic mice which were found to express CEA mRNA in all tissues. By immunoblot analysis using anti-CEA polyclonal antibody, we also detected CEA protein in all tissues. However, the molecular size of CEA in the brain was different from that in other tissues, although the mRNA size was same and no deletion nor rearrangement was detected at the DNA level. Immunohistochemical analysis of the lung and the colon showed that the expression sites were the bronchial epithelial cells of the lung and the columnar epithelial cells of the colon. Interestingly, the expression of CEA protein in the transgenic mice was polarised to the luminal side of epithelial cells similar to the normal CEA expression in human tissues. We also detected cell surface expression of human CEA on thymocytes and spleen cells and CEA expression was greatly reduced by the phosphatidylinositol-specific phospholipase C (PI-PLC) treatment.

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PubMed1991

Suppression of lymphocyte signal transduction by murine mastocytoma ascites.

The lymphocyte signal transduction, as determined by intracellular free Ca2+ mobilization of concanavalin A-stimulated T lymphocytes and of anti-immunoglobulin mu chain antibody-stimulated B lymphocytes, was suppressed in spleen cells from mice injected with murine P1.HTR mastocytoma-induced ascites and in spleen cells treated with the ascites in vitro. The suppression was observed both at the peak level and in the reactive pattern of Ca2+ influx. In the suppression, the ascites were replaceable with tumor culture supernatants or tumor homogenates. Correspondingly, primary and secondary cytotoxic T lymphocyte (CTL) responses of DBA/2 mice to allogeneic antigen were also significantly suppressed by injection of the syngeneic P1.HTR tumor-derived ascites. This new finding suggested that the mechanism of the tumorous ascites or of the tumor-derived factor-mediated immunosuppression involves at least in part the suppression of the early event of the signal transduction for lymphocyte activation.

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PubMed1991

Establishment of drug resistance in human gastric and colon carcinoma xenograft lines.

We established multidrug-resistant human gastric and colon xenograft lines by means of intratumoral injections of four agents, doxorubicin (DXR), cisplatin (CDDP), 5-fluorouracil (5-FU) and mitomycin C (MMC), into subcutaneous SC1NU and SW480 tumors once a week or less. Such intermittent drug exposure is commonly used in clinical chemotherapeutic protocols. All xenograft lines acquired resistance to the injected drugs as evaluated by in vivo drug-resistance tests. Many of the drug-resistant lines showed various patterns of cross resistance to other drugs. In order to analyze the mechanism of resistance in vivo, we investigated the expression of drug resistance gene, which has been extensively studied in vitro. We used four complementary DNAs (cDNAs) for multidrug resistance (MDR1), glutathione S-transferase-pi (GST-pi), thymidylate synthase (TS) and dehydrofolate reductase (DHFR), as probes. We observed GST-pi, DHFR and TS mRNA expression at various levels, but MDR1 mRNA expression was found only in SW480/DXR by the method of poly (A+) RNA selection. Four resistant SW480 lines had higher TS mRNA expressions. Six resistant lines had stronger GST-pi mRNA expression. Five resistant lines had higher DHFR mRNA expression. Drug resistance genes related to the treated drug were also expressed in this in vivo model; MDR1 in SW480/DXR, GST-pi in SW480/CDDP and in SC1NU/CDDP and TS in SW480/5-FU. In contrast to in vitro resistant lines which have been reported as models of drug resistance, the expression of drug resistance genes in vivo was not always correlated to the acquisition of cross resistance. These resistant xenograft lines and the methods developed to induce drug resistance in vivo should be useful for studies on the mechanism of drug resistance in the clinical setting.

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PubMed1991

Characterization of the individual and cross-reactive antigens involved in the anti-tumor immunity induced by use of an H-2K-erbB recombinant gene transfectant.

The specificities of the antisera raised in the CDF1 mice that had been immunized with the P1.HTR tumor cells xenogenized by transfection with recombinant H-2Kb-erbB gene were studied. The antisera cross-reacted with a broad range of tumor cell lines maintained either in vitro or in vivo in an immunofluorescence assay. However, they did not react at all with syngeneic normal tissue cells from thymus, spleen, bone marrow and fetal liver. Even though antigens related to the murine leukemia virus and murine mammary tumor virus (MuMTV) were demonstrated in many of the tumor cell lines tested with specific antibodies, these antigens did not seem to be primarily involved in the anti-P1.HTR antibody activity. The 74 kDa molecule, which was precipitated by the anti-P1.HTR anti-serum from the surface radiolabeled cell extract of P1.HTR tumor and was discriminated from the 70 kDa molecule precipitated by the anti-MuMTV serum, was widely distributed among various tumor cell lines tested, but was absent in normal tissue cells. In contrast to the extensive cross-reaction by the antibody, the cytotoxic T lymphocyte generated in the P1.HTR immune mice were shown to be specific to the P1.HTR tumor, and the 98 kDa molecule was precipitated by the anti-P1.HTR serum from the P1.HTR tumor but not from other tumors tested. It is suggested from these results that the 98 kDa molecule is a candidate for an individual tumor-specific transplantation antigen, and is immunodominant for inducing cytotoxic T lymphocytes to coexisting intrinsic retroviral antigens and other serologically cross-reactive tumor antigens.

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PubMed1991

Augmented production of granulocyte-macrophage colony-stimulating factor and alpha/beta interferon in mice inoculated with heat-killed Corynebacterium liquefaciens.

We demonstrated that heat-killed Corynebacterium liquefaciens bacteria, as a known potent host immune activity modulator, stimulate spleen cells to produce granulocyte-macrophage (GM) colony-stimulating factor (CSF) and another CSF with similar activity, as well as alpha/beta interferon, when injected intravenously into mice. Alpha/beta interferon was shown to be produced by C. liquefaciens-activated plastic-G-10 column-adherent cells (A cells) in a thymus-independent manner. In contrast, augmented production of GM-CSF required the action of C. liquefaciens-activated T lymphocytes that collaborated with normal A cells. Non-T spleen cells from C. liquefaciens-stimulated athymic mice, however, produced an alternative CSF that partially replaced GM-CSF. Correspondingly, the numbers of GM-producing CFU developing in cultures of spleen cells from C. liquefaciens-treated euthymic or athymic mice were 10 to 30 times higher than those in cultures of spleen cells from untreated mice. These results suggest that gram-positive rods such as C. liquefaciens activate T and A cells for production of multiple cytokines and that potential cooperative actions of these cytokines underlie the known immunomodulatory action of coryneforms.

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