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Biomedical subjects

S Kurosawa

Publications and source records attributed to S Kurosawa.

At least 19 recordsLinked to original sources
PubMed1998

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Amino Acid Sequence↗
PubMed1998

[The effects of midazolam on the memory of pain].

The purpose of this study was to evaluate the effects of low dose midazolam (MZ) on memories of spinal puncture. The low doses of MZ were administered to 70 patients (ASA 1-2), of whom 37 patients were premedicated with atropine sulfate 0.5 mg and pethidine hydrochloride i.m. (group P), and 33 patients received no premedication (group N). Double blind randomized trials were conducted with the doses of MZ (0, 0.03, 0.06 mg.kg-1), and MZ was administered i.v. to the patients just prior to spinal puncture. Subjective evaluation of pain was performed with pain score (PS) on postoperative phase, and objective evaluation of pain was performed with the reaction of spinal puncture. Short term memory was impaired mainly after administration of MZ. However, subjective memory of pain almost disappeared, but objective evaluation was not so good. We conclude that MZ induces impairment to recall of pain. However, it might maintain the response to the pain.

Adjuvants, Anesthesia↗
PubMed1997

Identification of functional endothelial protein C receptor in human plasma.

The endothelial cell protein C receptor (EPCR) binds protein C and facilitates activation by the thrombin-thrombomodulin complex. EPCR also binds activated protein C (APC) and inhibits APC anticoagulant activity. In this study, we detected a soluble form of EPCR in normal human plasma. Plasma EPCR appears to be approximately 43, 000 D, and circulates at approximately 100 ng/ml (98.4+/-27.8 ng/ml, n = 22). Plasma EPCR was purified from human citrated plasma using ion exchange, immunoaffinity, and protein C affinity chromatography. Flow cytometry experiments demonstrated that plasma EPCR bound APC with an affinity similar to that previously determined for recombinant soluble EPCR (Kdapp = 30 nM). Furthermore, plasma EPCR inhibited both protein C activation on an endothelial cell line and APC anticoagulant activity in a one-stage Factor Xa clotting assay. The physiological function of plasma EPCR is uncertain, but if the local concentrations are sufficiently high, particularly in disease states, the present data suggest that the soluble plasma EPCR could attenuate the membrane-bound EPCR augmentation of protein C activation and the anticoagulant function of APC.

Anticoagulants↗
PubMed1997

Protection by alpha2-macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor-1.

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, alpha2-macroglobulin (alpha2-M), and plasminogen activator inhibitor-1 (PAI-1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA. Purified alpha2-M (5.4 mg/ml) protected 16.0% or 22.4% of tPA (12.5 IU/ml) activity from inhibition by PAI-1 at 4 or 8 IU/ml in vitro. Of nine patients treated with 5-20 mega IU of tPA for MI, the plasma activity of tPA remained increased for 15-30 min after the cessation of infusion in eight; the patient who failed to exhibit a persistent increase in tPA activity had a low plasma concentration of alpha2-M. Total tPA activity, derived from the area under the activity-versus-time curve (AUC), showed a significant inverse correlation with the ratio of the plasma PAI-1 activity to the plasma alpha2-M concentration. Total tPA activity did not correlate with plasma PAI-1 activity or plasma alpha2-M concentration alone. Results suggest that alpha2-M, by binding to tPA, protects the latter against inhibition by PAI-1.

Aged↗
PubMed1996

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.

Antibodies, Monoclonal↗
PubMed1996

The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor.

Expression of the endothelial cell protein C receptor (EPCR) gene in mammalian cells imparts the capacity to bind activated protein C (APC) or protein C. Immunochemical analysis of CCD41, apparently the murine homologue of EPCR, suggested centrosomal localization, raising questions about the location of the EPCR gene product and its role in protein C binding. In this study, we express a soluble form of EPCR, demonstrate EPCR expression on the cell surface, and direct binding between soluble EPCR and protein C/APC. Affinity purified polyclonal and a monoclonal antibody against EPCR bound to the cell surface of EPCR-transfected cells but not to control cells. A 49-kDa protein, a mass similar to soluble EPCR, was immunoprecipitated from the cell surface of endothelium and cells transfected with human EPCR but not from control cells. The FLAGtrade mark antibody and APC bound to cells expressing an EPCR construct containing the FLAGtrade mark epitope located in a putative extracellular domain, whereas an EPCR construct truncated just before the putative transmembrane domain produced only soluble EPCR antigen. Soluble EPCR inhibited APC binding to EPCR expressing cells in a concentration-dependent fashion, Kd (app) = 29 nM and bound to immobilized protein C in a Ca2+-dependent fashion. Thus, EPCR is a type 1 transmembrane protein that binds directly to APC.

Animals↗
PubMed1996

The endothelial cell protein C receptor. Inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors.

A soluble form of the endothelial cell protein C receptor (EPCR) was analyzed for the ability to modulate the functional properties of protein C and activated protein C (APC). In a plasma clotting system initiated with factor Xa, EPCR blocked the anticoagulant activity of APC in a dose-dependent fashion. EPCR had no influence on clotting in the absence of APC. Consistent with the plasma results, EPCR slowed the proteolytic inactivation of factor Va by slowing both of the key proteolytic cleavages in the heavy chain of factor Va. EPCR did not prevent protein C activation by the soluble thrombin-thrombomodulin complex, did not alter the inactivation of APC by alpha1-antitrypsin or protein C inhibitor, and did not influence the kinetics of peptide paranitroanilide substrate cleavage significantly. We conclude that EPCR binds to an exosite on APC that selectively modulates the enzyme specificity in a manner reminiscent of the influence of thrombomodulin on thrombin.

Amidohydrolases↗
PubMed1996

The concurrent administration of OK432 augments the antitumor vaccination effect with tumor cells by sustaining locally infiltrating natural killer cells.

The effect of a local injection with a streptococcal preparation OK432 on the antitumor vaccination with tumor cells was investigated. Natural killer (NK) cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells after an intraperitoneal (i.p.) injection with syngeneic B16 melanoma cells. Furthermore, a concurrent i.p. injection with OK432 efficiently sustained the locally infiltrating NK cells. The OK432 treatment also sustained the augmented NK and lymphokine-activated killer activities in the peritoneal exudate cells. This treatment also increased the ability of the locally infiltrating NK cells to produce interferon gamma in response to the tumor cells. In addition, the concurrent i.p. injection with OK432 in combination with the tumor cells enhanced the capacity of the spleen cells to turn into anti-(B16 melanoma) cytotoxic T lymphocytes after in vitro restimulation. This augmenting effect of OK432 was dependent on NK cells. Moreover, the concurrent injection with OK432 at the time of anti-tumor vaccination significantly enhanced the protective immunity against B16 melanoma at the rechallenge. Taken together, these findings indicate that a concurrent local injection with OK432 in combination with tumor cells efficiently augments the antitumor vaccination effect, in part, by sustaining the locally infiltrating activated NK cells.

Adoptive Transfer↗
PubMed1996

The opposite effect of tumor-infiltrating natural killer cells on in vivo priming of tumor-specific CD8+ T cells and CD4+ T cells.

Natural killer (NK) cells, which infiltrated the tumor site, were examined for their effects on the in vivo priming of tumor-specific CD8+ and CD4+ T cells. CD8+ T cells were responsible for the activity of B16 melanoma-specific cytotoxic T lymphocytes (CTL). The in vivo depletion of NK cells with anti-NK1.1 monoclonal antibody (mAb), prior to B16-immunization, significantly decreased the capacity of the spleen cells (s.c.) to generate B16-specific CTL after in vitro restimulation. However, the CD8+ T cells of the s.c. from NK cell-depleted and subsequently B16-immunized mice increased their potential to become B16-specific CTL compared with those from the B16-immunized mice. The tumor-infiltrating NK cells showed a low but significant degree of CTL activity against B16. In addition, the disrupted B16 melanoma cells demonstrated less of an ability to in vivo prime the tumor-specific CD8+ T cells. These findings thus suggest the possibility that the quick disruption of tumor cells by tumor-infiltrating NK cells consequently interfered with the in vivo priming of the tumor-specific CD8+ T cells. On the other hand, the CD4+ T cells of the s.c. from NK cell-depleted and subsequently B16-immunized mice showed less of a capacity to induce the tumor-specific CTL compared with those from B16-immunized mice. In addition, the delayed-type hypersensitivity response against B16 was significantly diminished by the in vivo depletion of NK cells prior to B16-immunization. These findings thus suggest that NK cells have a promoting effect on the in vivo priming of CD4+ T cells. Overall, however, our findings indicate that early-appearing tumor-infiltrating NK cells have an opposite effect on the in vivo priming of CD8+ and CD4+ T cells.

Animals↗
PubMed1996

Thromboxane rather than platelet activating factor mediates pulmonary vasoconstriction after antigen challenge in rabbits.

To investigate whether thromboxane and/or platelet activating factor (PAF) mediate the pulmonary vasoconstrictive response to antigen in vivo, we intra-arterially injected human erythrocytes as antigen into sensitized rabbits after administration of putative inhibitors: a cyclooxygenase synthetase inhibitor (indomethacin, 5 mg.kg-1), a thromboxane synthetase inhibitor (OKY 046, 10 mg.kg-1 + 100 micrograms.kg-1.min-1), and a PAF blocker (CV6209, .1 mg.kg-1). Pulmonary artery and airway pressures significantly increased after the antigen challenge in sensitized rabbits, but did not in nonsensitized rabbits. Both indomethacin and OKY046 significantly inhibited the increase in pulmonary artery pressure after the antigen challenge, while CV6209 did not. CV6209 significantly attenuated the decrease in femoral artery pressure after the antigen challenge, while neither indomethacin nor OKY046 did. There were no significant differences in the increase in airway pressure among the groups. We conclude that thromboxane rather than PAF mediates the pulmonary vasoconstriction after the antigen challenge and that mediators other than thromboxane and PAF mediate bronchoconstriction after the antigen challenge in sensitized rabbits.

Anesthesia↗
PubMed1995

Natural killer cells inhibit the development of autoantibody production in (C57BL/6 x DBA/2) F1 hybrid mice injected with DBA/2 spleen cells.

We investigated the role of natural killer (NK) cells in the development of autoantibody production in which (C57BL/6 x DBA/2) F1 (BDF1) hybrid mice were injected intravenously with spleen cells (SC) from parental DBA/2 mice (treated BDF1 mice). Treated BDF1 mice began to show an increase in serum anti-dsDNA antibody 2 weeks after injection, while the NK activity of their SC transiently increased initially in the first 1 to 2 weeks after injection, but subsequently decreased dramatically. Flow cytometric analysis suggested that this sequential change in NK activity correlated with the absolute number of host-derived NK1.1+ cells in SC from treated BDF1 mice. We demonstrated that the level of anti-dsDNA in serum is directly influenced by the level of NK activity in treated BDF1 mice. Depletion of NK cells by administration of anti-NK1.1 mAb accelerated the development of autoantibody production, whereas augmentation of NK activity by administration of poly-(I:C) inhibited the development of autoantibody production. This inhibitory effect of poly(I:C) was abolished by prior depletion of NK cells. Interestingly, suppression of autoantibody production was seen only when poly(I:C) was administrated within 1 week after injection of parental SC. Last, we demonstrate that adoptive transfer of interleukin-2 (IL-2)-activated NK cells had a protective effect against the development of autoantibody production. These findings imply that NK cells may have a protective role in lupus-like disease especially in its early stage. In addition, it opens up the possibility that adoptive immunotherapy with IL-2-activated NK cells can delay or even prevent the development of autoimmune disease.

Animals↗
PubMed1995

The antitumor activity induced by the in vivo administration of activated B cells bound to anti-CD3 monoclonal antibody.

We examined the immunotherapeutic ability of activated B cells which bound to anti-CD3 monoclonal antibody (mAb) to enhance antitumor T cell immunity in vivo. A flow cytometric analysis revealed that LPS (lipopolysaccharide)-activated B cells (LPS blasts) expressed Fc receptor (FcR) which can bind to anti-CD3 mAb. LPS blasts were also stained with CTLA-4Ig, which can bind to costimulation molecules with high affinity, which suggested that LPS blasts expressed costimulation molecules on their surface. In an in vitro assay, T cells remarkably proliferated in the presence of LPS blasts and soluble anti-CD3 mAb, whereas this proliferation was blocked by the addition of CTLA-4Ig. In a model of metastasis established by the intravenous inoculation of melanoma cells, the in vivo administration of LPS blasts incubated with anti-CD3 mAb and followed by treatment with polyethylene glycol, to reinforce the binding, induced a low but significant antitumor activity against melanoma. The antitumor activity induced by the in vivo administration of LPS blasts which bound to anti-CD3 mAb was also detected in the spontaneously established model of metastasis. These results therefore suggest that the in vivo administration of activated B cells which bound to anti-CD3 mAb was able to enhance the antitumor T cell response against metastatic melanoma.

Animals↗
PubMed1995

Anti-metastatic activity induced by the in vivo activation of purified protein derivative (PPD)-recognizing Th1 type CD4+ T cells.

The Th1 type Cd4+ T cell clone (MH2), which is capable of recognizing purified protein derivative from Mycobacterium tuberculosis (PPD), was examined for its anti-metastatic activity against melanoma. In using an in vitro proliferative assay, MH2 was able to recognize PPD-derived antigen in a major histocompatibility complex class II-restricted manner. MH2 showed neither any natural killer (NK) activity nor cytolytic activity against syngeneic B16 melanoma. This clone produced interferon-gamma, tumor necrosis factor and interleukin-2, but not interleukin-4, when co-cultured with PPD and irradiated syngeneic C57BL/6 spleen cells, suggesting that this clone could thus be assigned to the Th1 subset. An intraperitoneal (i.p.) co-injection of 2 x 10(6) MH2 and 50 micrograms PPD increased the NK activity of the peritoneal exudate cells (PEC) and the percentage of NK1.1+ cells in the PEC. These activated NK cells showed a low but significantly cytolytic activity against B16 melanoma. The augmented NK activity induced by the co-injection of MH2 and PPD was maintained by the weekly additional i.p. injections of PPD alone. Using a murine metastatic model, and i.p. co-injection of MH2 and PPD-induced anti-metastatic activity against B16 melanoma. This anti-metastatic activity was then abrogated by the in vivo administration of anti-asialo GM1 serum. In addition, the NK activity in both peripheral blood and metastatic lungs was significantly augmented in the mice which were co-injected with MH2 and PPD. Taken together, these findings indicate that the in vivo activation of Th1 type CD4+ T cells augmented the NK activity in vivo and thus could potentially be an efficient immunotherapeutic weapon against metastasis of melanoma. These results also imply that adoptive immunotherapy could induce anti-metastatic activity through cytokine production but not through any direct cytolytic activity.

Adjuvants, Immunologic↗
PubMed1995

A case of Crow-Fukase syndrome with elevated soluble interleukin-6 receptor in cerebrospinal fluid. Response to double-filtration plasmapheresis and corticosteroids.

We report the case of a 33-year-old Japanese male who presented with thrombocytosis, lower limb edema, severe polyneuropathy with elevated cerebrospinal fluid (CSF) protein level and serum IgA lambda monoclonal component, fulfilling the manifestations of Crow-Fukase syndrome. A high level of soluble interleukin-6 receptor in the CSF was also found, which fluctuated in parallel with the clinical course. Initial treatment with double-filtration plasmapheresis (DFPP) reduced the serum IgA paraprotein level with improvement of the sensory component of the polyneuropathy and decrease of soluble interleukin-6 receptor in the CSF. The remaining clinical features waned off after steroid treatment. The possible role of interleukin-6 in the pathogenesis of the Crow-Fukase syndrome and the utility of DFPP treatment are discussed.

Adult↗