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Biomedical subjects

M Miyasaka

Publications and source records attributed to M Miyasaka.

At least 55 records · Page 3Linked to original sources
PubMed1999

In vivo effects of monoclonal antibodies against rat beta(2) integrins on kidney ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (IR) involves adhesion of leukocytes to the activated endothelium, leading to tissue damage. CD11/CD18 beta(2) integrins interact with their ligands on endothelial cells and may therefore represent a therapeutic target for the prevention of IR. We investigated the effects of three monoclonal antibodies (mAbs) that recognize epitopes of heavy or light chain of the beta(2) integrins on IR in kidneys. METHODS: Uninephrectomized Fischer rats were subjected to 45 or 60 min of renal ischemia, treated with intravenously anti-beta(2) integrin monoclonal antibodies (anti-CD11a, anti-CD11b, and anti-CD18) 5 min prior to reperfusion, and compared to a nontreated group. Serum creatinine, blood urea nitrogen (BUN), and kidney histopathological damages were assessed at 1, 2, and 7 days after ischemia. RESULTS: After 45 and 60 min of ischemia, serum creatinine and BUN were significantly higher in the control than in animals treated with anti-CD11a and anti-CD18 at 24 and 48 h. Administration of anti-CD11b had a beneficial effect on renal function after 45 min but not after 60 min of ischemia. Histologic and immunostaining studies demonstrated mild tubular necrosis and less leukocyte infiltration in the anti-CD11a- and anti-CD18-treated groups compared to the control group. CONCLUSION: These results indicate that selected antibodies to CD11a/CD18 may decrease kidney IR injury when administered prior to reperfusion.

Animals↗
PubMed1999

The effect of flow on the neutrophil-mediated Ca2+ responses in human vascular endothelial cells stimulated by endotoxin.

Leukocyte-vascular endothelial cell (EC) interactions which promote inflammatory and immune reactions involve bidirectional signaling between two cell types. We investigated the effects of flow on neutrophil-mediated changes in endothelial intracellular Ca2+ levels ([Ca2+]i). Cultured human umbilical vein ECs stimulated by endotoxin were labeled with Fura-2 and exposed to fluid flow with neutrophils. The individual changes in [Ca2+]i were monitored. The application of flow with neutrophils to stimulated ECs led to an increase in [Ca2+]i although either flow without neutrophils or neutrophils without flow rarely induced a rise in [Ca2+]i. Furthermore, flow application with neutrophils to unstimulated ECs also rarely promoted a rise in [Ca2+]i. These findings suggest that the flow might thus induce or enhance the inflammatory process by the induction of Ca2+ signaling in endotoxin-stimulated endothelium facing neutrophils in the blood flow.

Calcium↗
PubMed1999

Analysis of the mode of action of a novel immunosuppressant FTY720 in mice.

Accelerated lymphocyte homing and apoptosis have been suggested to contribute to potent immunosuppressive effects of FTY720, however, its main mechanism of action remains to be fully elucidated. Here, we examined the mode of action of FTY720 in mice. FTY720, when given at a single dose of 1 mg/kg, markedly decreased the number of peripheral blood lymphocytes (PBL) but moderately increased the lymphocyte numbers in lymph nodes (LN) and Peyer's patches (PP) in normal mice, as previously observed in rats. However, the sharp decrease in PBL numbers was also observed in aly/aly mice lacking LN and PP, indicating that this phenomenon is not explained by accelerated lymphocyte homing to LN and PP. In addition, the finding that a single administration of FTY720 did not suppress proliferative responses of T cells suggested that the PBL reduction could occur without inhibiting lymphocyte functions. However, when administered at the same dose for 2 weeks, FTY720 induced severe systemic lymphopenia, as well as marked suppression of lymphocyte proliferative responses in normal mice. The same treatment also prolonged skin allograft survival in aly/aly mice. Our results suggest that FTY720 suppresses in vivo immune functions mainly by inducing systemic lymphopenia and also by inhibiting T cell functions.

Animals↗
PubMed1999

A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis(x), an L-selectin ligand displayed by CD34.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

Acetylglucosamine↗
PubMed1999

Expression profile of active genes in mouse lymph node high endothelial cells.

High endothelial venules (HEV) allow rapid and selective lymphocyte trafficking from the blood into secondary lymphoid tissues. Here we report the expression profile of active genes in mouse high endothelial cells (HEC). HEC were first purified from mouse lymph nodes (LN) by magnetic cell sorting with MECA-79 mAb and a 3'-directed cDNA library that faithfully represents the composition of mRNA was constructed. A total of 1495 cDNA sequences were obtained from randomly selected clones. Based on their sequence identity, they were grouped into 754 different species [gene signatures (GS)] of which 335 GS were identified in GenBank. Among the previously identified genes, expression of several endothelial cell surface molecules including endoglin and ICAM-1 was detected in HEC. Comparison of the gene expression profile with that of purified CD31(+) flat endothelial cells identified several molecules, such as KC chemokine and Duffy antigen/receptor for chemokines, that are known to be selectively expressed in activated endothelial cells or post-capillary venules. Interestingly, mac25/TAF, which is known to be expressed specifically in tumor vessels and implicated in the regulation of cell adhesion, was highly and selectively expressed in HEC in mouse LN, suggesting that it may participate in regulating HEC-specific functions. Comparison with the expression profiles obtained from 35 different cell types showed at least 22 GS that were apparently specific to HEC. Our results illustrate the expression differences between HEC and CD31(+) flat endothelial cells, and will be useful for the identification and characterization of genes specific for HEC.

Animals↗
PubMed1999

Identification and characterization of ligands for L-selectin in the kidney. I. Versican, a large chondroitin sulfate proteoglycan, is a ligand for L-selectin.

Ligands for a leukocyte adhesion molecule, L-selectin, are expressed not only in the specific vascular endothelium in lymph nodes and Peyer's patches but also in the extravascular tissues such as the brain white matter, choroid plexus and the kidney distal straight tubuli. However, the biological significance of these extravascular ligands is currently unknown. We now report the purification and characterization of a novel extravascular ligand for L-selectin in the kidney using a tubule-derived cell line, ACHN. Binding of L-selectin-IgG chimera (LEC-IgG) to the isolated ligand was specifically blocked with either (i) anti-L-selectin mAb, (ii) EDTA, (iii) fucoidan, (iv) chondroitin sulfate (CS) B or CS E, or (v) treatment with chondroitinases. Partial amino acid sequencing, Western blotting and immunoprecipitation analyses showed that a major ligand for L-selectin in ACHN cells is versican of 1600 kDa. Histochemical as well as biochemical analyses verified that a versican subspecies in the kidney was indeed reactive with L-selectin. Studies with cell lines including those derived from the kidney indicated that a certain glycoform and/or splice form of versican is reactive with L-selectin. Under pathological conditions such as those induced by unilateral ureteral obstruction, versican was shed from the distal straight tubuli and became localized in the adjacent vascular bundles around which a substantial leukocyte infiltration was concomitantly observed. Possible involvement of versican in leukocyte trafficking into the kidney under diseased conditions is discussed.

Animals↗
PubMed1999

Identification and characterization of ligands for L-selectin in the kidney. III. Characterization of L-selectin reactive heparan sulfate proteoglycans.

L-Selectin, a leukocyte adhesion molecule, mediates leukocyte rolling on the endothelium and plays a critical role in leukocyte recruitment at inflammatory sites as well as in lymphocyte homing. We have previously shown that L-selectin reactive chondroitin sulfate and heparan sulfate proteoglycans (HSPGs) are both expressed in the distal tubules of the kidney and that versican is one of the chondroitin sulfate-type ligands. In the present study, we characterized the heparan sulfate-type ligand(s) in more detail. The molecular sizes of HSPGs were approximately 600 kDa with core protein sizes of 160 and 180 kDa. Western blotting analysis showed that L-selectin reactive HSPGs were neither agrin nor perlecan, major basement membrane HSPGs in the kidney. The binding to L-selectin was mediated by the lectin domain of L-selectin in a Ca2+-dependent manner and required heparan sulfate side chains, but not sialic acid. To our knowledge, this is the first biochemical characterization of the L-selectin reactive heparan sulfate proteoglycan(s) in the distal tubules of the kidney.

Animals↗
PubMed1999

An international survey of medical ethics curricula in Asia.

SETTING: Medical ethics education has become common, and the integrated ethics curriculum has been recommended in Western countries. It should be questioned whether there is one, universal method of teaching ethics applicable worldwide to medical schools, especially those in non-Western developing countries. OBJECTIVE: To characterise the medical ethics curricula at Asian medical schools. DESIGN: Mailed survey of 206 medical schools in China, Hong Kong, Taiwan, Korea, Mongolia, Philippines, Thailand, Malaysia, Singapore, Indonesia, Sri Lanka, Australia and New Zealand. PARTICIPANTS: A total of 100 medical schools responded, a response rate of 49%, ranging from 23%-100% by country. MAIN OUTCOME MEASURES: The degree of integration of the ethics programme into the formal medical curriculum was measured by lecture time; whether compulsory or elective; whether separate courses or unit of other courses; number of courses; schedule; total length, and diversity of teachers' specialties. RESULTS: A total of 89 medical schools (89%) reported offering some courses in which ethical topics were taught. Separate medical ethics courses were mostly offered in all countries, and the structure of vertical integration was divided into four patterns. Most deans reported that physicians' obligations and patients' rights were the most important topics for their students. However, the evaluation was diverse for more concrete topics. CONCLUSION: Offering formal medical ethics education is a widespread feature of medical curricula throughout the study area. However, the kinds of programmes, especially with regard to integration into clinical teaching, were greatly diverse.

Animal Experimentation↗
PubMed1999

Constitutive expression of GlyCAM-1 core protein in the rat cochlea.

Glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) is a mucin-like glycoprotein previously identified on high endothelial venules (HEV) of lymph nodes and also in lactating mammary glands. A specifically glycosilated form of GlyCAM-1 on HEV has been shown to be a ligand for a leukocyte L-selectin, which plays an important role in leukocyte rolling along the inflamed endothelium. Here we report that GlyCAM-1 is also expressed in the cochlea. Immunohistochemistry revealed the lateral wall of the cochlea, tectorial membrane, modiolus, organ of corti, and spiral modiolar vein (SMV) to be strongly stained with polyclonal anti-GlyCAM-1 antibody. Moreover, RT-PCR of the cochlear tissue by the use of specific oligonucleotide primers for rat GlyCAM-1 generated a 378 bp product which was then verified by nucleotide sequencing to represent GlyCAM-1. Electron microscopic investigation revealed the presence of GlyCAM-1 over the entire lumenal surface of the vessels, and the basolateral infoldings in stria vascularis. However, soluble L-selectin or mAb MECA-79 which recognizes a carbohydrate epitope on functional L-selectin ligands bound only to the spiral ligament, tectorial membrane and modiolus. These observations suggest that GlyCAM-1 expressed in the cochlear region is heterogenous in terms of its glycosylation.

Animals↗
PubMed1999

[Role of adhesion molecules in the pathogenesis of collagen diseases].

Adhesion molecules play important roles and involve in many aspects of cell-cell or cell-extracellular matrix interactions in collagen diseases. In the present article, we describe the role of adhesion molecules in the pathogenesis of collagen diseases. Adhesion molecules on endothelial cells participate in leukocyte recruitment in collagen diseases. Adhesion molecules on RA synoviocytes which interact with MNCs and destroy chondrocytes by pannus formation. In PSS, adhesion molecules on fibroblasts play important roles to form fibrosis by interacting with MNCs or collagen fibrils. We further mention on adhesion molecules in interaction between MNCs and muscle fibers in myositis, those in vasculitis in SLE and other diseases, and autoantibodies inducing adhesion molecules on endothelial cells in PSS and Wegener's granulomatosis.

Autoantibodies↗
PubMed1999

Retinal ischemia-reperfusion injury attenuated by blocking of adhesion molecules of vascular endothelium.

PURPOSE: To evaluate quantitatively the effects of blocking of adhesion molecules (P-selectin or intercellular adhesion molecule-1 [ICAM-1]) on leukocyte dynamics in the retinal microcirculation in vivo during ischemia-reperfusion injury and the therapeutic efficacy of the blocking of adhesion molecules on retinal ischemia-reperfusion injury. METHODS: Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic nerve. P-selectin or ICAM-1 monoclonal antibody (mAb) was administered at 5 minutes before reperfusion. At 4, 12, and 24 hours after onset of reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 or 14 days of reperfusion, retinal damage was evaluated histologically. RESULTS: P-selectin mAb significantly inhibited leukocyte rolling along the major retinal veins after reperfusion. Subsequently, the number of accumulated leukocytes decreased in the P-selectin-inhibited rats. ICAM-1 mAb also inhibited leukocyte accumulation during the reperfusion period in a more substantial manner than P-selectin mAb. Histologic examination demonstrated the protective effect of the blocking of P-selectin or ICAM-1. In accordance with a reduction in leukocyte accumulation, the protective effect of mAb on retinal ischemia-reperfusion injury was more substantial in ICAM-1-inhibited rats. CONCLUSIONS: The present study demonstrates the inhibitory effect of P-selectin and ICAM-1 mAb on leukocyte accumulation and subsequent tissue injury during retinal ischemia-reperfusion injury.

Animals↗
PubMed1998

Blood monocyte migration to acute lung inflammation involves both CD11/CD18 and very late activation antigen-4-dependent and independent pathways.

In acute lung inflammation, blood neutrophils and monocytes migrate into the lung parenchyma and bronchoalveolar space. The infiltration of the inflamed lung by monocytes is poorly understood because of difficulties in quantifying these cells in the presence of resident macrophages. Radiolabeled monocytes were used to study monocyte migration into the inflamed rat lung. Monocytes and neutrophils were purified from blood, labeled with 51Cr and (111)In, respectively, and injected i.v. into rats given an intratracheal injection of LPS. The accumulation of 51Cr-labeled monocytes increased > 10-fold in the lung parenchyma and 170-fold in the bronchoalveolar lavage (BAL) 18 h after LPS. (111)In-labeled neutrophils increased > 30-fold in the lung tissue and 500-fold in the BAL. Treatment of rats with a blocking anti-CD18 mAb inhibited monocyte accumulation in the lung and BAL by about 30%, whereas blocking very late activation Ag-4 (VLA-4) had no effect. Combined blockade of VLA-4 and CD18 inhibited approximately 30% of the migration to the lung parenchyma, but decreased the BAL by 80%. Monocyte migration to cutaneous inflammation was completely abolished by the combined mAb treatment. Neutrophil accumulation in the lung and BAL was not decreased by blocking either CD18 or VLA-4 and was only partially reduced by blocking CD18 plus VLA-4. Thus, monocyte migration to the LPS inflamed lung is substantially CD11/CD18 and VLA-4 independent, but accumulation in BAL is mediated by CD18 and VLA-4. Monocytes as well as neutrophils may use a previously unrecognized endothelial adhesion and migration pathway in the lung.

Acute Disease↗
PubMed1998

Role of the leukocyte-adhesion molecule L-selectin in experimental autoimmune encephalomyelitis.

L-selectin is an adhesion molecule expressed on T cells and monocytes. It mediates rolling--the initial step of transendothelial migration. In this study, we investigated the role of L-selectin in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. EAE was induced in Lewis rats by active sensitization with myelin basic protein (MBP-EAE), or by adoptive transfer using MBP specific T cells (AT-EAE). Treatment with HRL3, a monoclonal antibody to L-selectin, and its F(ab')2 fragments efficiently suppressed MBP-EAE, and had a mild inhibitory effect on AT-EAE. Histological examination revealed a marked reduction of inflammatory infiltrates after treatment with HRL3. Administration of the control antibody HRL4 did not significantly alter the course of the disease. HRL3 caused T-cell depletion in the draining lymph nodes and spleen and a downregulation of L-selectin expression on T cells. We conclude that L-selectin-dependent mechanisms are involved in the pathogenesis of EAE. Modulation of L-selectin in vivo by antibodies or by competitive antagonists could be a novel therapeutic approach to autoimmune diseases of the central nervous system.

Animals↗
PubMed1998

FK506 (tacrolimus) inhibits extravasation of lymphoid cells by abrogating VLA-4/VCAM-1 mediated transendothelial migration.

Extravasation is a critical process for the physiological lymphocyte traffic as well as the hematogenous spread of malignant hemopoietic cells. Here we report that abrogation of calcineurin activity leads to in vitro transendothelial migration and in vivo infiltration of human lymphoma Nalm-6 cells, which are associated with the abrogation of the VLA-4/VCAM-1 mediated pathway. Rapamycin, which can antagonize FK506 but not CsA to inhibit calcineurin, abrogates FK-506 mediated but not CsA mediated inhibition of in vitro transendothelial migration. FK506 may exert its potent immunosuppressive action partly by inhibiting VLA-4/VCAM-1 mediated transendothelial migration or insinuation of lymphoid cells to tissues.

Animals↗
PubMed1998

A time-resolved immunofluorometric assay of sialyl Lewis x-degrading alpha 2,3-sialidase activity.

We have developed an assay for alpha 2,3-sialidase (EC 3.2.1.18) which employs a biotinylated carbohydrate-polyacrylamide conjugate as substrate for the enzyme. The solution-phase sialidase reactions are followed by a selective capture of biotinylated neoglycoconjugates onto a microtitration plate coated with streptavidin. The amount of the reaction product formed is then rapidly and easily quantified using a product-specific primary antibody and europium chelate-labeled secondary antibody. This method combines the advantages of solution-phase enzymatic reaction and suitability for high-throughput screening typical of solid-phase assays. The assay gives a detectable signal with 0.4% of substrate sites desialylated. We have demonstrated the utility of the assay by measuring alpha 2,3-sialidase activity from crude lysates of cultured rat endothelial cells by using biotinylated sialyl Lewis x glycoconjugate as substrate. Endothelial sialidase(s) showed up to 250-fold higher activity toward soluble compared to immobilized substrate. Product formation detected with an anti-Lewis x antibody was linear in the range of 0.1-4 micrograms/ml of protein in endothelial cell lysate. High sensitivity of the assay was achieved by using solution-phase enzyme reaction and time-resolved fluorometric detection. The same assay format used here is easily adapted to detect activities of several different glycosidases or glycosyl-transferases by using appropriate substrates and antibodies.

Animals↗
PubMed1998

Direct antigen presentation through binding of donor intercellular adhesion molecule-1 to recipient lymphocyte function-associated antigen-1 molecules in xenograft rejection.

Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P<0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.

Animals↗