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Biomedical subjects

T Nishimura

Publications and source records attributed to T Nishimura.

At least 217 records · Page 12Linked to original sources
PubMed1999

Self-priming cell culture system for monitoring genetically-controlled spontaneous cytokine-producing ability in mice.

To monitor genetically-controlled cytokine-producing ability in mice in vitro, we developed a high-density cell culture system, which is preferable for inducing CD4+ T cell-dependent self-priming responses without any antigenic stimulation. When BALB/c spleen cells were cultured at high density (over 1.0 x 10(7) cells/well) in 12-well culture plate, they spontaneously produced cytokines including IFN-gamma, IL-2, IL-3, IL-5 and IL-6. The spontaneous cytokine production in this self-priming cell culture (SPCC) system was totally dependent on MHC class II-restricted CD4+ T cells. It was demonstrated that Th2-type BALB/c background mice exhibited higher levels of spontaneous cytokine production in SPCC culture compared with Th1-type C57BL/6 mice. Moreover, using BALB/c x C57BL/6 F1 mice and B10D2 congenic mice, it was demonstrated that highly spontaneous cytokine-producing ability in BALB/c background is genetically dominant and it is controlled by non-MHC genes. Unexpectedly, BALB/c mice spontaneously produced higher levels of IL-2 and IFN-gamma than C57BL/6 mice. However, BALB/c mice revealed lower levels of CTL and NK cell-generation in SPCC system compared with C57BL/6 mice. These results suggested that genetically-controlled predisposition of BALB/c mice toward Th2 immunity appeared not to be derived from their poor IFN-gamma-producing ability but rather derived from their poor responsiveness to IFN-gamma.

Animals↗
PubMed1999

Different neural substrates for Kanji and Kana writing: a PET study.

To investigate the neural substrate underlying the mechanisms of Kanji and Kana writing, we conducted a PET activation study during mental writing task in eight right-handed normal Japanese subjects. During scans subjects were required to mentally write a Kanji or three Kana letters with their right hand, for each stimulant word presented auditorily. The direct comparisons between Kanji writing and Kana writing revealed that the left posterior inferior temporal gyrus was activated in Kanji writing while the left angular gyrus was activated in Kana writing. In addition, more extensive areas were activated in Kanji writing compared with Kana writing. These results suggest that different respective neural substrates are involved in Kanji and Kana writing respectively.

Adolescent↗
PubMed1999

The influence of dietary protein antigen on Th1/Th2 balance and cellular immunity.

Dietary administration of ovalbumin (OVA) antigen (Ag) into OVA-specific T cell receptor alphabeta-transgenic (TCR-Tg) mice resulted in the induction of activated CD4+ Th cells expressing CD69 early activation Ag. However, the number of CD4+ Th cells rather decreased by dietary administration of OVA antigen. The production of Th1-cytokines such as IFN-gamma and IL-2 markedly reduced in spleen of OVA-fed mice compared to mice fed with normal diet. In sharp contrast, the production of Th2-cytokine, IL-4 greatly increased in spleen of OVA-fed mice though the number of CD4+ T cells decreased to less than 10% of control mouse spleen. The decrease of IFN-gamma production and the increase of IL-4 production by CD4+ T cells was demonstrated at a single cell level by intracellular cytokine staining analysis. Moreover, such a polarized cytokine production pattern was also demonstrated using highly purified CD4+ T cells obtained from mice fed with OVA. In addition to the decrease of Th1-cytokine production, TCR-Tg mice fed with OVA-containing diet showed greatly reduced in vivo generation of NK cells, LAK cells and CTL. These results suggested that dietary protein antigen caused the polarization of Th1/Th2 balance into Th2-dominant immunity and inhibited cellular immunity.

Animals↗
PubMed1999

Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo.

The role of T helper type 1 (Th1) and Th2 cells in tumor immunity was investigated using Th cells induced from ovalbumin (OVA)-specific T cell receptor transgenic mice. Although Th1 cells exhibited stronger cytotoxicity than Th2 cells, both cell types completely eradicated tumors when transferred into mice bearing A20 tumor cells transfected with the OVA gene (A20-OVA). Th1 cells eradicated the tumor mass by inducing cellular immunity, whereas Th2 cells destroyed the tumor by inducing tumor necrosis. Both Th1 and Th2 cells required CD8(+) T cells to eliminate tumors, and neither of these cells were able to completely eliminate A20-OVA tumors from T and B cell-deficient RAG2(-/-) mice. Mice cured from tumors by Th1 and Th2 cell therapy rejected A20-OVA upon rechallenge, but CD8(+) cytotoxic T lymphocytes were induced only from spleen cells prepared from cured mice by Th1 cell therapy. Moreover, we demonstrated that Th1 and Th2 cells used distinct adhesion mechanisms during tumor eradication: the leukocyte function-associated antigen (LFA)-1-dependent cell-cell adhesion step was essential for Th1 cell therapy, but not for Th2 cell therapy. These findings demonstrated for the first time the distinct role of antigen-specific Th1 and Th2 cells during eradication of established tumors in vivo.

Animals↗
PubMed1999

Chemosensitivity of micrometastases and circulating tumor cells to uracil and tegafur as evaluated using LacZ gene-tagged Lewis lung carcinoma cell.

The chemosensitivity of the sequence of steps responsible for metastasis formation including circulating tumor cells and micrometastases to a 5-fluorouracil derivative (UFT) was examined with a novel micrometastasis model featuring Lewis lung carcinoma cells tagged with the bacterial LacZ gene and hygromycinR gene (hygR). Metastases in the lung could be specifically detected at the single-cell level by X-Gal staining after inoculation of LacZ-transfected tumor cells. Spontaneous metastasis in mice bearing subcutaneous primary tumors was significantly inhibited by UFT at doses of 15-20 mg/kg when it was orally administered from day 14, during the early stage of micrometastasis formation, but not by late administration from day 22. Experimental pulmonary metastasis was also inhibited without significant toxic side effects by oral administration of UFT at a daily dose of 20 mg/kg from day 4, when the tumor cells start new growth in the lung, but not by daily treatment from 8 days after intravenous injection. Oral administration of UFT had no effect on tumor cell arrest in the lung as detected by X-Gal staining. Furthermore, PCR analysis revealed that circulating tumor cells in the peripheral blood of mice bearing primary tumors after subcutaneous injection of hygR-tagged tumor cells were significantly reduced by the oral administration of UFT for 7 days in a dose-dependent manner. These results indicate that circulating tumor cells in the peripheral blood and micrometastases in the initial growth phase in the lungs are sensitive to this chemotherapeutic agent, and suggest that micrometastasis formation can be effectively inhibited by long-term oral administration of anticancer agents with minimal toxic side effects.

Administration, Oral↗
PubMed1999

Intracellular sodium accumulation during ischemia as the substrate for reperfusion injury.

To elucidate the role of intracellular Na+ kinetics during ischemia and reperfusion in postischemic contractile dysfunction, intracellular Na+ concentration ([Na+]i) was measured in isolated perfused rat hearts using 23Na nuclear magnetic resonance spectroscopy. The extension of the ischemic period from 9 minutes to 15, 21, and 27 minutes (at 37 degrees C) increased [Na+]i at the end of ischemia from 270.0+/-10.4% of preischemic level (mean+/-SE, n=5) to 348.4+/-12.0% (n=5), 491.0+/-34.0% (n=7), and 505.3+/-12.1% (n=5), respectively, whereas the recovery of developed pressure worsened with the prolongation of the ischemic period (95.1+/-4.2%, 84.3+/-1. 2%, 52.8+/-13.7%, and 16.9+/-6.4% of preischemic level). The kinetics of [Na+]i recovery during reperfusion was analyzed by the fitting of a monoexponential function. When the hearts were reperfused with low-[Ca]o (0.15 mmol/L) solution, the time constants of the recovery (tau) after 15-minute (8.07+/-0.85 minutes, n=5) and 21-minute ischemia (6.44+/-0.90, n=5) were significantly extended, with better functional recovery (98.5+/-1.4% for 15-minute [P<0.05]; 98.0+/-1.0% for 21-minute [P<0.05]) compared with standard reperfusion ([Ca]o=2.0 mmol/L, tau=3.58+/-0.28 minutes for 15-minute [P<0.0001]; tau=3.02+/-0.20 for 21-minute [P<0.0001]). A selective inhibitor of Na+/Ca2+ exchanger also decelerated the [Na+]i recovery, which suggests that the recovery reflects the Na+/Ca2+ exchange activity. In contrast, high-[Ca]o reperfusion (5 mmol/L) accelerated the [Na+]i recovery after 9-minute ischemia (tau=2.48+/-0.11 minute, n=5 [P<0.0001]) and 15-minute ischemia (tau=2.10+/-0.07, n=6 [P<0. 05]), but functional recovery deteriorated only in the hearts with 15-minute ischemia (29.8+/-9.4% [P<0.05]). [Na+]i recovery after 27-minute ischemia was incomplete and decelerated by low-[Ca]o reperfusion, with limited improvement of functional recovery (42. 5+/-7.9%, n=5 [P<0.05]). These results indicate that intracellular Na+ accumulation during ischemia is the substrate for reperfusion injury and that the [Na+]i kinetics during reperfusion, which is coupled with Ca2+ influx, also determines the degree of injury.

Animals↗
PubMed1999

Crystallization and preliminary X-ray studies of the Ia component of Clostridium perfringens iota toxin complexed with NADPH.

A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent. The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ). The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit. Native data were collected at 2.4-A resolution. The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference. The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit.

ADP Ribose Transferases↗
PubMed1999

A critical role for antigen-specific Th1 cells in acute liver injury in mice.

A novel liver injury model was established in mice by targeting of OVA-containing liposomes into the liver, followed by adoptive transfer of OVA-specific Th1 cells. Combined treatment of mice with OVA-containing liposomes and Th1 cell transfer caused an increase in serum transaminase activity that was paralleled with an elevation of serum IFN-gamma levels. In sharp contrast, OVA-specific Th2 cell transfer resulted in an increase of serum IL-4 levels, but did not induce liver injury. Neither NK, NK T, nor CD8+ T cells were required for the Th1-induced liver injury. The liver injury was blocked by anti-IFN-gamma mAb and anti-TNF-alpha mAb, but not by anti-Fas ligand mAb. The Fas/Fas ligand independency was also demonstrated using Fas-deficient lpr mice. These findings indicate that Th1 cells are the major effector cells in acute liver injury.

Acute Disease↗
PubMed1999

Synthesis and biological activities of topoisomerase I inhibitors, 6-N-amino analogues of NB-506.

6-N-Amino analogues of NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D-glucopyranosyl) -5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione] (3b) were synthesized and tested with respect to topoisomerase inhibition, cytotoxicity and anticancer effects. Among them, a 1,3-dihydroxypropane analogue (J-109,404, 5t) showed more than ten times more potent anticancer activity in MKN-45 human stomach cancer cells implanted in mice than NB-506.

Animals↗
PubMed1999

In vitro metabolism of chlorotriazines: characterization of simazine, atrazine, and propazine metabolism using liver microsomes from rats treated with various cytochrome P450 inducers.

The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ), and propazine (PRZ) was studied using control, 3-methylcholanthrene-, phenobarbital-, pyridine-, dexamethasone-, and clofibrate-treated rat liver microsomes. The metabolites were determined by HPLC. The principal reactions by cytochrome P450 (P450) system were N-monodealkylation and isopropylhydroxylation in all rat liver microsomes. As a result, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3, 5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M3) (ATZ-M3 for ATZ), and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazi ne (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N-bidealkylation and 2-hydroxylation were not found in this system. The formation rates of SIZ-M1, ATZ-M1, ATZ-M2, and PRZ-M2 were markedly induced by 3-methylcholanthrene, phenobarbital, and pyridine. On the other hand, the formation rates of ATZ-M3 and PRZ-M4 were significantly induced by phenobarbital, pyridine, and/or clofibrate, but not by 3-methylcholanthrene. The enzyme kinetics of chlorotriazine metabolism were examined by mean of Eadie-Hofstee analyses. Although there was no remarkable difference of Km for the products in chlorotriazine metabolism among the microsomes tested, the Vmax and Clint (Vmax/Km) for the products in chlorotriazine metabolism are affected by P450 inducers, except for dexamethasone. The formation rates of SIZ-M1, ATZ-M1, ATZ-M2, and PRZ-M2 were significantly correlated with 7-ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, 7-ethoxycoumarin O-deethylase, 4-nitrophenol 2-hydroxylase, and testosterone 7alpha-hydroxylase activities and CYP1A1/2 level, whereas the formation rates of ATZ-M3 and PRZ-M4 were significantly correlated with testosterone 16beta-hydroxylase, bufuralol 1'-hydroxylase, and 4-nitrophenol 2-hydroxylase activities and CYP2B1/2 level. These results suggest that the inducibility in metabolism of SIZ, ATZ, and PRZ is different between N-monodealkylation and isopropylhydroxylation and that the N-monodealkylation and isopropylhydroxylation are induced by CYP1A1/2, CYP2B1/2, and CYP2B1/2, respectively.

Animals↗
PubMed1999

The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells.

The natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of alpha-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by alpha-GalCer. We first established, using purified subsets of various lymphocyte populations, that alpha-GalCer selectively activates NKT cells for production of interferon (IFN)-gamma. Production of IFN-gamma by NKT cells in response to alpha-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, alpha-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Valpha14(-/-) mice. This effect of alpha-GalCer required the production of IFN-gamma by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of alpha-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-gamma production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by alpha-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.

Adjuvants, Immunologic↗
PubMed1999

Functional skewing of bone marrow-derived dendritic cells by Th1- or Th2-inducing cytokines.

To investigate the functional difference of bone marrow (BM)-derived dendritic cells (DC), BM cells were cultured under three different cytokine conditions and the induced DC were temporarily designated as DC0, DC1, or DC2 cells. DC0 were induced by culture of BM cells with granulocyte macrophage colony-stimulating factor (GMCSF) plus interleukin 3 (IL-3). DC1 were induced by culture with Th1-inducing cytokine (IL-12, gamma-interferon-IFN-gamma) in addition to GMCSF plus IL-3. DC2 were induced by culture with Th2-inducing cytokine (IL-4) in addition to GMCSF plus IL-3. Flow cytometric analysis demonstrated that almost all DC0, DC1 and DC2 cells were stained with anti-CD11c, which reacts with a marker for DC cells. However, interestingly, DC0, DC1 and DC2 cells expressed different amounts of functional molecules on their cell surface. Namely, DC1 cells expressed the highest levels of MHC class I, class II, CD40, B7.1 and B7.2 compared with DC0 and DC2 cells. In terms of IL-12 production, DC1 cells showed enhanced production, while DC2 cells showed reduced production compared with DC0 cells. Moreover, it was shown that both DC0 and DC1 supported the differentiation of IFN-gamma-producing Th1 cells, but not IL-4-producing Th2 cells from TCR-transgenic mouse naive Th cells. However, DC2 cells selectively enhanced the differentiation of IL-4-producing Th2 cells. These data strongly suggested that DC1 cells might be preferable antigen-presenting cells for application to immunotherapy.

Animals↗
PubMed1999

Comparison between the decrease of dopamine transporter and that of L-DOPA uptake for detection of early to advanced stage of Parkinson's disease in animal models.

Early diagnosis of Parkinson's disease (PD) is important for the potential application of neuroprotective therapies. The purpose of this study was to assess the detection of the early changes of PD by either imaging the dopamine transporter (DAT) or uptake of L-3,4-dihydroxyphenylalanine (L-DOPA). An early to advanced stage model of PD was induced in rats by stereotaxic injection of 1-10 microg 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta. Using adjacent sections of the same animals, the binding of [I-125]beta-CIT, which labels DAT and the uptake of [C-14]L-DOPA, were evaluated 4 weeks after induction of the lesion. Any decrease in dopaminergic neurons was evaluated by in situ hybridization histochemistry (ISH) by detection of DAT mRNA-positive neurons. In addition, the expression levels of DAT, dopa decarboxylase (DDC), and vesicular monoamine transporter (VMAT2) in each neuron were studied with ISH. Our results show a decrease in both [I-125]beta-CIT binding and [C-14]L-DOPA uptake in parallel with a decrease in DA neurons from early to advanced stage models of PD. The decrease in [C-14]L-DOPA uptake was smaller than that in [I-125]beta-CIT binding in the same animal (P < 0.0001). Expression levels of DAT, DDC, and VMAT2 mRNAs were also decreased with the progression of the disease. Although ISH failed to detect the origin of the discrepancy between [I-125]beta-CIT and [C-14]L-DOPA levels, it was concluded that [C-14]L-DOPA levels underestimated the decrease of dopaminergic neurons and that [I-125]beta-CIT levels more precisely reflected the decrease.

Animals↗
PubMed1999

Immunohistochemical expression of growth factors in subacute thyroiditis and their effects on thyroid folliculogenesis and angiogenesis in collagen gel matrix culture.

The inflammatory-mechanistic basis of subacute thyroiditis remains unclear. To elucidate the roles of vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF), transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) in the inflammatory process, their immunoexpression was examined in biopsy specimens of ten cases. At the granulomatous stage, all cases expressed VEGF, bFGF, PDGF, and TGF-beta1 in monocytes/macrophages infiltrating into follicle lumina, and in both epithelioid histiocytes and multinucleated giant cells of the granulomas. In fibroblasts and endothelial cells around the granulomas, all cases displayed VEGF, bFGF, and PDGF, but TGF-beta1 was detected only in fibroblasts in two cases. No cases expressed EGF in any of the above cell types. At the regenerative stage, all cases expressed VEGF, bFGF, and EGF in regenerating thyrocytes, whereas three and no cases displayed PDGF and TGF-beta1, respectively. Ten, seven and six cases expressed PDGF in fibroblasts, endothelial cells, and monocytes, respectively. In these cell types, all cases expressed VEGF and bFGF, whereas no cases displayed TGF-beta1 and EGF. To estimate the roles of these growth factors in thyroid tissue regeneration, their effects on thyroid folliculogenesis and angiogenesis were examined using collagen gel culture of thyrocytes and endothelial cells, respectively. Cell proliferation was also studied by bromodeoxyuridine (BrdU) uptake. EGF decreased follicle formation and TGF-beta1 drastically inhibited it, but the others had no effect. VEGF showed the greatest effect on vessel formation, although all of the others promoted it. EGF and VEGF or bFGF caused the highest BrdU uptake in thyrocytes and endothelial cells, respectively. The data suggest firstly, that at the granulomatous stage of subacute thyroiditis, growth factor-rich monocytes/macrophages infiltrating into follicle lumina trigger the granulomatous reaction, and VEGF, bFGF, PDGF, and TGF-beta1 produced by the stromal cell types tested mediate the reaction; secondly, that at the regenerative stage, EGF serves follicle regeneration through its mitogenic effect on thyrocytes, although some cofactors with EGF are involved in folliculogenesis and the decreased expression of TGF-beta1, a fibrogenic factor, contributes to thyroid tissue repair; and thirdly, that VEGF and bFGF are more responsible for the angiogenesis at both stages than the other factors studied.

Cell Culture Techniques↗
PubMed1999

The essential role of phorbol ester-sensitive protein kinase C isoforms in activation-induced cell death of Th1 cells.

Th1 and Th2 cells, which were induced from naive T cells of TCR-transgenic mice, showed differential sensitivity to activation-induced cell death (AICD) triggered by stimulation with anti-CD3 monoclonal antibody. The Th1 cells showed more rapid AICD than Th2 cells. This accelerated AICD of Th1 cells was strongly blocked by protein kinase C (PKC) inhibitors (H-7 or GF 109203X). Moreover, long-term treatment of Th1 cells with phorbol 12-myristate 13-acetate (PMA) caused the abrogation of anti-CD3-induced AICD in parallel with the disappearance of PMA-sensitive PKC isoforms such as PKC alpha, gamma, epsilon and theta. Therefore, it was clearly demonstrated that PMA-sensitive PKC isoforms are essential for AICD of Th1 cells. The different susceptibility to AICD between Th1 and Th2 cells was not due to their differential expression levels of PMA-sensitive PKC isoforms but appeared to be due to their differential requirement for PMA-sensitive isoforms in the up-regulation of Fas ligand which is involved in suicide killing of activated Th1 cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗