Biomedical subjects

Y Oshimi

Publications and source records attributed to Y Oshimi.

At least 19 recordsLinked to original sources

PubMed2001

Exercise intensity based on heart rate while walking in spastic cerebral palsy.

We examined the heart rate (HR) of subjects with spastic cerebral palsy (CP) in order to estimate exercise intensity while walking. The subjects were 17 subjects with CP (14.0 +/- 3.7 years of age) containing 7 subjects rated as level 1, 4 subjects rated as level 2, and 6 subjects rated as level 3 by the Gross Motor Function Classification System, and 7 normal subjects (12.4 +/- 2.8 years of age) were used as a controls. Even in subjects whose gross motor function was excellent (rated as level 1), the HR significantly increased while walking when compared to normal subjects (p < 0.05), although the walking speed between the groups was not different. According to the HR, the exercise intensity while walking was adapted from weakly to moderately and thought to be appropriate for exercise. On the other hand, walking speed was significantly reduced in the subjects rated as level 2 and 3 (p < 0.05), and the HR increased significantly (p < 0.05). Seven of the ten subjects rated as either level 2 or 3 showed a high HR of over 150 beats/min while walking. The HR while walking of the two subjects rated as level 3 continued to increase although the walking speed was kept constant. The walking exercise would be too strong and become detrimental to such subjects.

Adolescent↗

PubMed1999

ATP-induced Ca2+ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells to macrophages.

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i). We measured changes in [Ca2+]i using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1-2 days. A transient rise in [Ca2+]i (duration 3-4 min) occurred in 10-15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo-osmotic treatment. Ca2+ transients were also evoked by ATP in a dose-dependent manner between 0.1 and 100 microm. The ATP-induced [Ca2+]i rise was reduced to less than one-quarter in Ca2+-free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P2U purinoceptor agonist) was more potent than ATP or 2-chloro-ATP (P2Y agonist). Oxidized ATP (P2Z antagonist) had no inhibitory effect. Both cell lysate- and ATP-induced Ca2+ responses were inhibited by Reactive Blue 2 (P2Y and P2U antagonist) to the same extent, but were not affected by PPADS (P2X antagonist). Sequential stimuli by cell lysate and ATP underwent long-lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+]i rise via P2U and P2Y receptors.

Adenosine Triphosphate↗

PubMed1996

Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells.

The cytotoxicity of NK cells has been thought to be mediated mainly by the perforin-dependent pathway. We investigated the involvement of Fas-mediated pathway in the killing activity of purified human CD3-, CD16+ NK cells. Fas ligand mRNA was expressed in freshly isolated NK cells. Apoptosis, which was identified by the fragmented chromatin in individual cells, was induced in the cells that expressed high levels of Fas via direct NK-to-target cell interaction or Ab-dependent cell-mediated cytotoxicity, even in Ca(2+)-free medium, in which perforin pores are known not to be formed. Apoptosis in both the presence and absence of external Ca2+ was inhibited by Fab of an anti-Fas mAb. Transfection of the Fas gene in target cells facilitated the induction of apoptosis, compared with the parental cell line. The function of the Fas-mediated pathway in the coexistence of the perforin-dependent pathway was examined in 10 cell lines expressing different levels of Fas by Ca2+ imaging and morphologic observation of single cells. With a certain boundary level, low or high levels of Fas expression in target cells were correlated to a great degree with either acute necrosis due to severe membrane damage after NK-target cell contact or apoptosis at a later period, respectively. We concluded that Fas ligand/Fas interaction is present and plays a significant role in the human NK cell-induced apoptosis.

Antibodies, Monoclonal↗

PubMed1996

Necrosis and apoptosis associated with distinct Ca2+ response patterns in target cells attacked by human natural killer cells.

1. During the process of cell death, rises in cytosolic Ca2+ concentration ([Ca2+]i) together with structural changes were investigated in target cells attacked by purified CD3-,CD16+ human natural killer (NK) cells. 2. In the target cell line K562, a rapid [Ca2+]i rise to 1-2 microM occurred a few minutes after NK cell-target cell contact, immediately followed by leakage of the Ca2+ indicator dye fura-2 from the cell. Cells were permeabilized, but their chromatin was not fragmented. The changes were basically consistent with those seen in necrosis induced by activated complement. 3. In the target cell line MOLT-4, which expressed the apoptosis-inducing surface antigen Fas much more strongly than K562, the majority of attacked cells displayed a [Ca2+]i rise to 0.7-1 microM followed by a slow decline, often associated with diminishing [Ca2+]i oscillations. As a whole, [Ca2+]i remained higher than 150 nM for at least 1.5-3 h (approximately 100 nM in control cells). 4. MOLT-4 cells attacked by NK cells became bubble shaped within 20 min of the main [Ca2+]i rise reaching its peak, and then both the cell and chromatin were fragmented into small pieces. These findings were basically consistent with those in apoptosis induced by a monoclonal antibody against the surface antigen Fas. 5. NK cells induced both necrosis and apoptosis in cell lines insensitive to NK cells in the presence of an antibody against the major histocompatibility complex class I (antibody-dependent cell-mediated cytotoxicity, ADCC). The distinct Ca2+ responses patterns described above corresponded to necrosis or apoptosis in different cells stimulated by the common ADCC pathway. 6. Human NK cells were found to be capable of inducing necrosis (membrane damage) or apoptosis (nuclear damage) depending on the target cell types. The characteristic Ca2+ response profile was a good indicator for distinguishing between the modes of cell death induced by the cytotoxicity of the killer cells.

Antibodies, Monoclonal↗

PubMed1995

Early activation signal transduction pathways of Th1 and Th2 cell clones stimulated with anti-CD3. Roles of protein tyrosine kinases in the signal for IL-2 and IL-4 production.

In the present experiments, TCR-CD3-associated early activation signal transduction pathways were examined in Th1 and Th2 clones by the stimulation with soluble monovalent anti-CD3 which resulted in efficient production of IL-2 and IL-4 in Th1 and Th2 cells, respectively. Although protein tyrosine kinases such as Fyn and ZAP-70 were activated in Th1 clones shortly after stimulation, these kinases in Th2 clones were not activated; but, their activity in resting conditions was shown to be decreased by the stimulation. In accordance with these findings, neither phospholipase C-gamma 1 activation nor phosphatidyl inositol-4,5-bisphosphate breakdown was induced in Th2 clones, in contrast to positive responses in Th1 clones. The oscillation of intracellular free Ca2+ concentration ([Ca2+]i) was a common signal for the activation of both Th1 and Th2 clones; however, the [Ca2+]i elevation in Th1 clones was herbimycin A sensitive, whereas that in Th2 was clone resistant, suggesting that the mechanism of the [Ca2+]i elevation in Th2 cells is different from that in Th1 cells in terms of the participation of protein tyrosine kinases. The anti-CD3 stimulation did not cause Lck activation in either the Th1 or Th2 clone, although remarkable activation was induced in both clones following anti-CD4 stimulation, indicating that Lck activation was not required for either IL-2 or IL-4 production of Th cells. Taken together, these results indicate that Th1 and Th2 cells are different from each other in early activation signal transduction pathways, especially in the role of protein tyrosine kinases.

Animals↗

PubMed1995

Fas antigen-mediated DNA fragmentation and apoptotic morphologic changes are regulated by elevated cytosolic Ca2+ level.

More than 80% of cells of the human B cell line FMO, which expresses the Fas Ag, underwent apoptosis within 2 h after addition of an anti-Fas mAb. Rises in cytosolic Ca2+ concentration ([Ca2+]i) and their effects were investigated by imaging individual cells continuously and introducing Ca2+ chelators to the outside and/or inside of the cell. The typical Ca2+ response consisted of 1) an early [Ca2+]i rise (basal [Ca2+]i, 85 to 110 nM; peak, 140 to 200 nM; duration, 15 to 20 min), 2) sustained elevation of [Ca2+]i at 140 to 150 nM, 3) a second Ca2+ rise (200 to 500 nM, 15 to 30 min) between 1.5 and 2 h, and 4) a large [Ca2+]i rise (1.2 to 2.0 microM) between 2 and 4 h after addition of mAb. Responses 1, 3, and 4 were mainly caused by Ca2+ entry, and response 2 involved intracellular Ca2+ release from stores. Apoptosis could be induced by mAb even in Ca(2+)-deprived external medium. Chelating cytosolic Ca2+ revealed that the [Ca2+]i rise is a prerequisite for fragmentation of DNA and chromatin, and is also necessary for fragmentation of cells. The critical [Ca2+]i was 140 to 150 nM and a sustained [Ca2+]i rise was more effective. A [Ca2+]i rise itself (without mAb) was ineffective. About 20% of mAb-treated cells showed chromatin condensation at the periphery of the nucleus (possibly an earlier stage of nuclear change) and a bubble-like cell shape even when [Ca2+]i was held below 100 nM. Thus, Ca2+ is mobilized immediately after Fas stimulation and functions as a key factor causing advanced apoptotic changes. Response 4 was related to secondary necrosis.

Antibodies, Monoclonal↗

PubMed1994

Ultrastructural changes in glycerol-extracted skeletal muscle fibers after chemical modification of myosin heads with p-phenylenedimaleimide.

We examined the structural changes in relaxed glycerinated rabbit psoas muscle fibers induced by modification of the myosin heads with p-phenylenedimaleimide (p-PDM), which reacts with sulfhydryls on the myosin head to cause its loss of ability to combine with actin and to hydrolyse ATP. In the longitudinal sections of both chemically fixed and quickly frozen muscle fibers, ladder-like structures, interpreted as the myosin heads extending nearly at right angles with the thick filaments, were more prominent in the p-PDM-modified fibers than in the control fibers. Fourier transform diffractograms of the longitudinal sections exhibited a distinct 14.3-nm meridional reflection, which arises from axial spacing of the myosin heads on the thick filament, in the p-PDM-modified fibers, but not in the control fibers. These results indicate that the p-PDM-modification of the myosin heads causes an increase in the regularity of myosin head arrangement on the thick filament.

Animals↗

PubMed1993

Ca2+ response in single human T cells induced by stimulation of CD4 or CD8 and interference with CD3 stimulation.

A Ca2+ imaging method has been used to demonstrate simultaneously the magnitude and time course of the increase in the intracellular Ca2+ concentration ([Ca2+]i) in 10-30 individual human peripheral T cells following stimulation by anti-CD4 or anti-CD8 monoclonal antibody (MAb) as well as anti-CD3 MAb. The rise in [Ca2+]i began within 10 s of the introduction of the MAb and reached a peak of 240 nM (mean of 73 cells) in 20-40 s. The peak was followed by a slow decrease persisting for 6-8 min. Comparing Ca2+ responses in the presence and absence of external Ca2+, the rise in [Ca2+]i was found to be caused by both transient intracellular Ca2+ release and a long-lasting Ca2+ influx from outside the cell. Cross-linking of CD4 or CD8 using anti-IgG antibody augmented the response in individual cells, as seen in the higher peak (365-390 nM) and the longer duration (over 10 min). Simultaneous stimulation of CD3 and CD4 did not cause a summation of Ca2+ responses but caused a suppression in the CD3-mediated Ca2+ response. The results support the view that CD4 and CD8 play a role in signal transduction for T cell activation and that the CD4-derived signal interferes with the CD3-derived signal at some stage in the signalling pathway causing the Ca2+ response.

Antibodies, Monoclonal↗

PubMed1993

Cross-bridge angle distribution and thin filament stiffness in frog skeletal muscle fibers as studied by quick-freeze deep-etch electron microscopy.

To give information about changes in orientation of myosin heads (cross-bridges) during contraction, mechanically skinned frog muscle fibers were rapidly frozen in various states, and cross-bridge angles were measured on the freeze-etch replicas. Histograms of cross-bridge angle distribution showed a peak around 90 degrees in relaxed, contracting and rigor states. The proportion of cross-bridges taking angles around 90 degrees decreased when rigor fibers were stretched or released before freezing. These results are explained by assuming the stretch-induced tilting of cross-bridges due to elastic recoil of the thin filaments in the I-band. As a matter of fact, the axial spacing of actin monomers in the thin filament increased with increasing rigor force before freezing. The stiffness times unit length of the thin filament was estimated to be about 1.8 x 10(4) pN.

Actin Cytoskeleton↗

PubMed1993

Freeze-fracture studies on the cross-bridge angle distribution at various states and the thin filament stiffness in single skinned frog muscle fibers.

To give information about the changes of cross-bridge (myosin head) orientation during muscle contraction, mechanically skinned frog muscle fibers were rapidly frozen at various states, and the cross-bridges were observed on freeze-etch replicas. The number of cross-bridges per unit length of thick filament in relaxed state was less than one-third of that in contracting and rigor states. The interval between adjacent cross-bridges was maximum around 35 nm, a value close to the crossover repeat of actin helix. The cross-bridge angle distribution, as measured with a digital image processor, showed a peak around 90 degrees in all the states examined. The proportion of cross-bridges with angles around 90 degrees decreased either after stretch or after release of fibers in rigor state. The axial spacing of actin monomers in the thin filament was found to increase with increasing rigor force, to give the thin filament stiffness (times unit length) of about 1.8 x 10(4) pN. These results are discussed in connection with the mechanical properties of cross-bridges.

Actins↗

PubMed1992

Differential expression of non-major histocompatibility complex-restricted cytotoxicity in patients with granular lymphocyte-proliferative disorders associated with or unassociated with severe anemia.

Of 27 patients with granular lymphocyte-proliferative disorders (GLPD), 18 patients had CD3+ T-cell-lineage GLPD (T-GLPD), and 9 patients had CD3-CD16+ natural killer (NK) cell-lineage GLPD (NK-GLPD). In 9 of the 18 patients with T-GLPD, severe anemia of less than or equal to 7.5 g/dl hemoglobin (mean 5.4 g/dl) and erythroid hypoplasia in the bone marrow developed, while the remaining 9 patients with T-GLPD and 9 patients with NK-GLPD exhibited hemoglobin levels of greater than or equal to 10.0 g/dl, and erythroid hypoplasia was not found. The number of leukocytes, neutrophils, lymphocytes or granular lymphocytes in the peripheral blood, or the percentage of lymphocytes in the bone marrow did not differ significantly between the patients with T-GLPD associated with severe anemia and those with T-GLPD not associated with severe anemia, and the immunophenotypes of peripheral blood mononuclear cells (PBMC) were not significantly different either. However, when non-major histocompatibility complex (MHC)-restricted cytotoxicity was assayed with PBMC, T-GLPD patients with severe anemia and NK-GLPD patients exhibited significantly higher levels of non-MHC-restricted cytotoxicity than T-GLPD patients without severe anemia. Because PBMC obtained from T-GLPD patients with severe anemia were shown not to lyse erythroblasts directly, the possibility that patient PBMC lyse erythroblasts in the bone marrow and thus cause anemia seems unlikely. The pathogenesis of anemia in GLPD was discussed.

Anemia↗

PubMed1991

Increased lysis of patient CD10-positive leukemic cells by T cells coated with anti-CD3 Fab' antibody cross-linked to anti-CD10 Fab' antibody.

An anti-CD3 Fab' x anti-CD10 Fab' bispecific hybrid F(ab')2 antibody (Ab) was generated. This bispecific Ab had a molecular mass of 100 to 110 Kd, and the capacity to react with both CD3+ T cells and CD10+ acute lymphoblastic leukemia (ALL) cells. We studied whether cytotoxic T lymphocytes (CTLs) could lyse patient CD10+ ALL cells after addition of the bispecific Ab. As effector CTLs, interleukin-2 (IL-2)-stimulated peripheral blood mononuclear cells (PBMCs) and CTL clones were used. When IL-2-stimulated PBMCs were assayed for cytotoxicity to 61Cr-labeled CD10+ ALL cells, their activity was shown to be markedly enhanced by the addition of the bispecific Ab. Most of the CTL clones established lacked cytotoxicity for CD10+ ALL cells, but addition of the bispecific Ab induced a significant level of cytotoxicity. CTLs derived from ALL patients also showed significant cytotoxicity for autologous CD10+ ALL cells after addition of the bispecific Ab. However, this Ab did not affect the cytotoxicity of CTLs when CD10- leukemic cells were used as the targets. These findings suggest that the bispecific Ab can be used for immunotherapy in patients with CD10+ ALL.

Antigens, CD↗

PubMed1990

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes.

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1990

Perforin gene expression in granular lymphocyte proliferative disorders.

By Northern blot analysis using a cDNA clone of the perforin gene, we studied the levels of perforin mRNA in peripheral blood mononuclear cells from 11 cases of granular lymphocyte-proliferative disorders (GLPDs). The granular lymphocytes studied were characterized by morphologic, immunophenotypic, and immunogenotypic analyses. Cytolytic functions of the lymphocytes assayed included nonmajor histocompatibility complex-requiring cytotoxicity, anti-CD3-redirected cytotoxicity, antibody-dependent cellular cytotoxicity, and lectin-dependent cellular cytotoxicity. The results showed that in lymphocytes with strong cytolytic functions high levels of perforin mRNA existed, whereas in lymphocytes with weak or undetectable levels of cytolytic functions, low levels of perforin mRNA existed. Because the levels of perforin mRNA correlated with those of cytolytic functions, perforin is probably a mediator in cytolytic functions of granular lymphocytes in patients with GLPDs. When the lymphocytes were cultured for 1 day, however, the levels of cytolytic activity were increased, and those of perforin mRNA were decreased. Therefore, we cannot rule out the possibility that factors other than perforin protein are involved in the cytolytic functions of granular lymphocytes.

Antigens, CD↗

PubMed1989

Electron microscopic studies on the stretch-induced disordering of the myofilament lattice in tetanized frog skeletal muscle fibers.

To study the effect of stretch on the hexagonal myofilament lattice in frog skeletal muscle fibers, the fibers were fixed at rest and during the isometric tetanus with or without stretch, and their cross sections were examined electron microscopically. The degree of disorder of the myofilament lattice as estimated by the Fourier transform and rotation methods in the digital image analysis was found to be largest during the isometeric tetanus with stretch and smallest during the isometeric tetanus without stretch, supporting the view that the stretch-induced force enhancement results from the disordering of the myofilament lattice.

Actin Cytoskeleton↗

PubMed1988

Cytotoxicity of interleukin-2-activated lymphocytes for autologous normal blood mononuclear cells.

When peripheral blood mononuclear cells (PBMC) were cultured for 5 days with 1000 U/ml of recombinant interleukin-2 (IL-2), lymphokine-activated killer (LAK) cytotoxicity for normal autologous and allogeneic PBMC developed. Autologous PBMC lytic activity was detected in media containing autologous serum and was dependent on the concentration of IL-2 added to the cultures. When purified T lymphocytes, B lymphocytes, monocytes and large granular lymphocytes were compared for their susceptibilities to lysis by autologous LAK, monocytes and B lymphocytes were found to be the most susceptible. In addition, mitogen-induced blasts were more susceptible than unstimulated lymphocytes. Since PBMC cultured in IL-2-containing medium consisted mainly of CD3-positive T lymphocytes and CD16-positive natural killer cells, and since treatment with anti-CD3 monoclonal antibody, but not anti-CD16 monoclonal antibody, reduced LAK activity for normal target cells, T cells appeared to be the effectors for LAK activity in the LAK normal target lysis system.

Cytotoxicity, Immunologic↗

PubMed1988

Characterization of interleukin 2-expanded human peripheral blood lymphocytes: not only NKH-1+-NK cells but also NKH-1+ and NKH-1- T cells are LAK effectors.

In vitro culture of human peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the expansion of lymphocytes including lymphokine-activated killer (LAK) cells. Using flow cytometry, studies were undertaken to determine the phenotype and LAK activity of each subset of lymphocytes expanded in vitro as a result of incubation for 2 weeks with 2500 U/ml of recombinant IL-2. Such expanded PBMC, when examined by two-color staining with various combinations of anti-CD3, 4, 8, 16, and NKH-1 monoclonal antibodies, consisted of the following six subgroups of cells: (1) CD3+4+8-, (2) CD3+4-8+, (3) CD3+4-8-, (4) CD3-16+NKH-1+, (5) CD3-16-NKH-1+, and (6) CD3-16-NKH-1-. Of the six subgroups, all five subgroups that could be tested, i.e., CD3+ T cells (CD3+4+8-, CD3+4-8+, CD3+4-8-), CD16+ natural killer (NK) cells (CD3-16+NKH-1+), and CD3-16-NKH-1- non-T non-NK cells, possessed LAK activity. Both NKH-1- as well as NKH-1+ T and non-T cells possessed LAK activity.

Antibodies, Monoclonal↗

PubMed1988

Role of T-cell antigens in the cytolytic activities of large granular lymphocytes (LGLs) in patients with LGL lymphocytosis.

By analyzing surface antigens and cytolytic functions of proliferating large granular lymphocytes (LGLs), three types of T cell LGL lymphocytosis were delineated. The first, most commonly encountered type exhibited CD3+4-8+16+, WT31+ phenotype, low or undetectable non-major histocompatibility complex (MHC)-restricted cytotoxicity, and moderate to strong antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). Because these LGLs carried T cell antigen receptor (Ti) recognized by WT31 monoclonal antibody (MoAb), and treatment with anti-Ti, anti-CD3 MoAbs and phytohemagglutinin elicited non-MHC-restricted cytotoxicity, they may have developed from populations of in vivo primed cytotoxic T lymphocytes with unknown antigen specificity. The second, rare type of LGL lymphocytosis exhibited CD3+4-8-16+, WT31 phenotype, and strong non-MHC-restricted, ADCC and LDCC cytotoxicities. These cells were probably derived from the lymphocytes of the same phenotype found in small numbers in normal peripheral blood. Because anti-CD3 MoAb inhibited non-MHC-restricted cytotoxicity of the LGLs, a Ti not detected by WT31 MoAb, but putatively present seemed to serve as a specific receptor for target tumor cell recognition. The third type of LGL lymphocytosis showed CD3+4+8-16+, WT31+ phenotype, and lacked cytolytic activities and parallel tubular arrays. These LGLs probably evolved from cells with the same characteristics selectively located in the germinal centers of lymphoid tissues. Taken together, in patients with LGL lymphocytosis, T cell-associated antigens expressed on LGLs were shown to be involved in the regulation of LGL-mediated cytolytic activities. In addition, studies of surface antigens and the effects of MoAbs and lectins on cytolytic activities may be useful in clarifying the normal counterpart of LGLs from which leukemic or reactively proliferating LGLs originate.

Adult↗