Hideo Kagawa

Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus.

We investigated the association between low-density lipoprotein (LDL), triglycerides, and platelet activation in 18 patients with hypertension age 41-64 years and 18 with diabetes mellitus aged 43-70 years. Platelet P-selectin positivity and the microparticle level (indicators of activation) were both significantly higher in the diabetics than in healthy controls (P-selectin: 28.0% +/- 7.5% vs. 7.3% +/- 4.2%, P < 0.001; microparticles: 1900 +/- 966 vs. 526 +/- 158/10(4) platelets, P < 0.01). In contrast, there was no significant increase of either parameter in the patients with hypertension. Plasma microparticle levels were also significantly greater in the diabetics with high LDL levels than in those with low LDL levels (2375 +/- 949 vs. 1519 +/- 796/10(4) platelets, P < 0.05), and in those with high rather than low triglyceride levels (2188 +/- 845 vs. 1492 +/- 783/10(4) platelets, P < 0.05). However, platelet positivity for P-selectin was not significantly different between these two subgroups. Microparticle and P-selectin levels both showed no significant difference between the hypertensive patients with high and low LDL or triglyceride levels. These results suggest that platelet-derived microparticles may participate in the development or progression of atherosclerosis in patients with diabetes mellitus.

Vitamin-D Receptor Genotype and Renal Disorder in Japanese Patients with Systemic Lupus erythematosus

Background/Aims: It is known that allelic variants of the gene encoding the vitamin-D receptor (VDR) detected by BsmI increase the risk of some advanced malignant tumors, suggesting that such variants may cause functional differences in 1,25(OH)2 vitamin D3. We examined the VDR genes of Japanese systemic lupus erythematosus (SLE) patients, to determine whether different genotypes are correlated with SLE or its criteria. Methods: VDR genotyping of 58 unrelated Japanese SLE patients was performed based on polymerase chain reaction-restriction fragment length polymorphism (RFLP). Following amplification, products were digested with BsmI. The RFLPs were coded as Bb, where the uppercase letter signifies the absence of the digested site and the lowercase letter signifies the presence of the site. Results: The frequency of the VDR BB genotype was significantly higher in SLE patients (15.5%, n = 9/58, p < 0.0001) than in controls (5.7%, n = 5/87). Furthermore, a larger proportion of bb individuals was observed among patients with nephrotic syndrome (61.5%, n = 8/13) than among SLE patients without renal dysfunction (35.7%, n = 10/28). There was a significant tendency for the population of patients with the bb genotype to be correlated with that of patients with renal dysfunction (p = 0.0304). Conclusion: These findings suggest that the BB genotype might trigger the development of SLE, and that the bb genotype is associated with lupus nephritis.

Platelet Activation Markers and Soluble Adhesion Molecules in Patients with Systemic Lupus Erythematosus

We assessed the role of platelet activation markers (PMPs, Annexin V and CD62P on activated platelets), cytokines (IL-1 β, IL-4, IL-6, IFN- γ, GM-CSF, and TNF α), and soluble factors (sIL-2R, TM, sHLA-1, β2-m sVCAM-l, sPECAM-1, sP-selectin and sE-selectin) in vascular damage related to SLE. There were differences in the levels of PMPs and platelet activation markers between the SLE patients and controls (PMPs: 493±82 vs. 328±36, p<0.05; plt-CD62P; 8.5%±1.2 % vs. 4.6%±0.7 %, p<0.05; plt-Annexin V: 11.3%±2.1 % vs. 4.9%±0.6 %, p<0.01). There were no differences in the levels of IFN- y between the groups. However, the levels of IL-11β IL-4, IL-6, GM-CSF, TNF α, and soluble factors were higher in the SLE patients than in the controls. The levels of IL-4, IL-6, p2-m, sIL-2R, sVCAM-1, sP-selectin, and sE-selectin in SLE patients with elevated sTM levels were higher than those in the SLE patients without elevated sTM levels. On the other hand, elevations of s[L-2R, sVCAM-l, and sP-selectin were not found in patients with Behcet disease or rheumatoid arthritis. The levels of platelet CD62P, platelet annexin V, and PMP were significantly elevated in high-sTM patients. These findings suggest the possibility that activated platelets and cytokines participate in the pathogenesis of SLE in patients with elevated sTM levels.

Expression of Functional Tissue Factor on Small Vesicles of Lipopolysaccharide- Stimulated Human Vascular Endothelial Cells

We examined tissue factor expression on lipopolysaccharide-stimulated endothelial cells and their small vesicles by using specific antibodies and flow cytometry. Tissue factor functional activity was also assessed by activation of factor X. Endothelial cells were stimulated with 10 microg/ml of lipopolysaccharide in M-199/bovine serum albumin. Flow cytometry showed that expression of tissue factor on endothelial cells reached a maximum at 6 hours after stimulation, whereas that on small vesicles reached a maximum after 12 hours. Factor X activation mediated by factor VIIa and tissue factor was observed over a similar time course and was inhibited by the addition of antitissue factor antibody. Immunoelectron microscopy suggested that small vesicles with expression of some tissue factor were produced from the surface of endothelial cells. Our findings thus showed that tissue factor on endothelial cells produced by lipopolysaccharide stimulation was partly released to small vesicles. This may cause disseminated intravascular coagulation and related coagulation disorders.

Alteration of peripheral blood dendritic cells in patients with primary Sjögren's syndrome.

OBJECTIVE We recently identified 3 fractions of human peripheral blood (PB) dendritic cells (DC), including the monocyte-associated fractions 1 and 2 (CD1a+,CD11c+ and CD1a-,CD11c+, respectively) and the lymphoid-associated fraction 3 (CD1a-,CD11c-). We attempted to determine whether these fractions were altered in Sjögrens syndrome (SS). METHODS We examined 23 patients with primary SS and 22 normal control subjects. DC were purified from PB and analyzed by flow cytometry. Immunohistochemical staining of labial salivary glands of SS patients was performed with monoclonal antibodies against fascin, which is known to be specific for DC. RESULTS The total numbers of PB DC and fraction 1 DC were decreased in SS. Immunohistochemical staining demonstrated that fascin+,CD11c+,HLA-DR+ mononuclear cells were present and scattered among numerous fascin-hyperfiltrating cells in SS patients. Interferon-gamma (IFNgamma)-producing Th1 cells were shown to be increased in both PB and salivary glands of patients, indicating the presence of general IFNgamma-producing Th1 polarization in SS. Furthermore, numbers of Thl cells were increased when naive T cells were cocultured with fraction 1 DC in vitro. CONCLUSION These findings suggest selective trafficking of fraction 1 DC into focal sites of inflammation and subsequent promotion of Th1 balance, suggesting a novel pathogenesis of SS.

Plasma-soluble Fas (APO-1, CD95) and soluble Fas ligand in immune thrombocytopenic purpura

Abstract: We investigated the levels of various cytokines and soluble factors in ITP patients, in order to determine the influence of these factors on the pathogenesis of ITP. We found increases in IL‐2, IL‐6, IFN‐γ, and M‐CSF levels in ITP patients compared with those in healthy individuals. On lymphocyte phenotype analysis, we found no clear difference in total T cell population (CD2+ CD19− cells) or cytotoxic T cell frequency (CD8+ CD11b− cells) between these two groups. The frequency of helper/inducer T cells (CD4+ CD8− cells) was decreased in ITP patients. There was a significant increase in activated T cells (CD3+ HLA‐DR+ cells) in ITP patients. Furthermore, frequencies of NK cells of potent activity (CD16+ CD56+ cells) were significantly elevated in ITP patients. Seventeen of the 54 ITP patients (31.5%) had elevated levels of sFas, and 11 of the 54 patients (20.4%) of sFasL. In addition, a significant increase of sFasL was observed in sFas‐positive ITP patients, and in these patients the sFasL level was correlated with that of sFas (r=0.687, p<0.01). We found significant increases in IL‐2 and sIL‐2R levels in sFas‐positive ITP patients. For other factors examined, however, there were no differences in level between sFas‐positive and‐negative ITP patients. Percentages of activated T cells (CD3+ and HLA‐DR+ cells) and NK cells (CD16+ and CD56+ cells) were significantly higher in sFas‐positive ITP patients than in sFas‐negative ITP patients. These findings suggests that the pathogenesis of ITP includes alteration of the Fas/FasL pathway.

Relationship of microparticles withβ2-glycoprotein I and P-selectin positivity to anticardiolipin antibodies in immune thrombocytopenic purpura

We investigated the association ofβ2-glycoprotein I and P-selectin with platelet-derived microparticles in 48 patients with immune thrombocytopenic purpura and 20 normal controls using two-color flow cytometric analysis. In addition, anticardiolipin antibodies were detected by an enzyme-linked immunosorbent assay. Platelet microparticles from the patients showed a higher positivity forβ2-glycoprotein I than those from the normal controls (23.1±15.4% vs. 5.3±3.1%, p<0.01), but this positivity was not related to the presence of platelet-associated IgG or to the severity of thrombocytopenia. In the 18 patients with more than 20% P-selectin-positive microparticles,β2-glycoprotein I positivity was significantly higher than in the 30 patients with less than 20% P-selectin-positive microparticles (37.1±20.5% vs. 21.5±17.3%, p<0.01). In addition, anticardiolipin antibodies were detected in eight patients, and they had a significantly higher level ofβ2-glycoprotein I-positive microparticles than the patients without such antibodies (42.0±22.9% vs. 22.6±18.9%, p<0.05). Our results suggest that anticardiolipin antibodies activate platelets in immune throm bocytopenic purpura and cause the generation of microparticles rich inβ2-glycoprotein I and P-selectin. These microparticles may then act to regulate coagulation abnormalities in patients with anticardiolipin antibodies.

Participation of αIIbβ3 in platelet microparticle generation by collagen plus thrombin

We investigated the role of αIIbβ3 in microparticle generation by normal and thrombasthenic platelets stimulated with collagen plus thrombin. Microparticle generation by normal p

Significance of Anti-oxidized LDL Antibody and Monocyte-derived Microparticles in Anti-phospholipid Antibody Syndrome

Monocytes, platelets, endothelial cells and oxidized LDL could be very important in development of vascular complication in thrombotic diseases. We measured and compared the levels of plasma monocyte-derived microparticles (MDMPs), platelet-derived microparticles (PDMPs), and anti-oxidized LDL antibody, to develop a better understanding of their potential contribution to vascular complications in antiphospholipid antibody syndrome (APS). The concentration of MDMP in APS patients was significantly higher (p<0.01) than that in normal subjects and SLE patients. When levels of PDMPs and plt-P-selectin were compared between the control and APS patients, levels of PDMPs and plt-P-selectin were significantly higher (p<0.01 for each) in APS patients than in controls. In addition, these levels of platelet activation markers correlated with MDMP in APS patients. Twenty one of the 37 APS patients (56.8%) had elevated levels of anti-oxLDL antibody. In addition, a significant increase in MDMP was observed in anti-oxLDL antibody-positive APS patients (p<0.01). These findings suggest that elevated MDMPs may be a sign of vascular complication in APS patients, particularly those who are detected anti-oxLDL antibodies.

Relationship between Platelet Activation and Cytokines in Systemic Inflammatory Response Syndrome Patients with Hematological Malignancies

We investigated the significance of platelet activation and platelet-derived microparticles (PMP) in 14 patients with systemic inflammatory response syndrome (SIRS) and hematological malignancies. In the phenotypic analysis of lymphocytes, there was a significant decrease of total and activated T cells after panipenem/betamipron (PAPM/BP) treatment (p<0.05). The percentages of helper/inducer T cells and suppressor/cytotoxic T cells were insignificantly decreased after PAPM/BP treatment. The number of natural killer (NK) cells of potent activity was significantly decreased after treatment (p<0.05). The levels of the cytokines interleukin (IL)-1beta, IL-6, and IL-8 in the patients were increased before treatment. IL-1beta concentrations were not changed after treatment. In contrast, the IL-6 and IL-8 levels were significantly decreased (p<0.05) after treatment, while tumor necrosis factor (TNF)-alpha and interferon gamma remained almost normal. We found an increase of soluble IL-2 receptor (sIL-2R) and soluble vascular cell adhesion molecule-1 (sVCAM-1) levels in the patients before treatment. After treatment, the sIL-2R concentrations tended to be decreased and sVCAM-1 levels showed a significant decrease (p<0.01). In contrast, soluble thrombomodulin (sTM) level did not change. Regarding the platelet activation markers, CD62P, CD63, and PMP levels in the patients were increased before treatment. CD62P and CD63 tended to be decreased after treatment, whereas PMP levels were significantly reduced from 1,056+/-103 to 762+/-64/10(4) platelets (p<0.05). Furthermore, CD62P, CD63, and PMP correlated with the levels of IL-6 and IL-8. These results suggest that activated platelets and PMP may be predictive markers in pre-disseminated intravascular coagulation and hypercytokine conditions related to SIRS.