Minori Nagahama

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.

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.

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.

Significance of chemokines and soluble CD40 ligand in patients with autoimmune thrombocytopenic purpura

Abstract:  We investigated the levels of various chemokines and soluble CD40L (sCD40L) in ITP patients, in order to determine the influence of CD40–CD40L interaction on the pathogenesis of ITP. We found increases in MCP‐1 and RANTES levels in ITP patients compared with those in healthy individuals. Thirty‐eight of the 65 ITP patients (58.5%) had elevated levels of sCD40L. We found significant decreases in platelet counts in sCD40L‐positive ITP patients. Although the sCD40L level did not differ significantly between the control and nonimmune thrombocytopenia groups, but among ITP patients. sCD40L level was significantly higher in those with untreated ITP than in those with treated ITP. In addition, significant increases in RANTES, MCP‐1, sCD14, and sP‐selectin levels were observed in sCD40L‐positive ITP patients, although sE‐selectin levels were not increased in such patients. For other factors examined, however, there were no differences in level between sCD40L‐positive and ‐negative ITP patients. These findings suggests that there are two groups of ITP patients, one with elevated and one with normal of sCD40L. ITP cases in which sCD40L was increased appeared to involve changes in platelet counts and monocyte activation. The pathogenesis of ITP may in some patients include alterations of the CD40/CD40L pathway.

Effect of bezafibrate on soluble adhesion molecules and platelet activation markers in patients with connective tissue diseases and secondary hyperlipidemia.

We evaluated the plasma concentrations of soluble adhesion molecules, platelet activation markers, and platelet-derived microparticles (PMPs) in patients with connective tissue diseases who had secondary hyperlipidemia caused by long-term steroid administration (n = 22) before and after treatment with bezafibrate. There were differences in levels of platelet. activation markers both before and after treatment (platelet CD62p: 15.11 ± 2.03 vs 10.38 ± 8.53%. P < 0.05; ptatetet CD63: t2.i2 ± 9.17 vs 9.90 ± 7.20%, P < 0.05). There were also differences in the levels of PMPs and soluble adheSion molecules both before and after treatment (PMP: 514 ± 273 vs 401 ± 201 /104 platelet, P < 0.05; soluble vascular cell adhesion molecule-1 : 724 ± 191 vs 666 ± 157 ng/mL, P < 0.01). After 6 months of treatment, serum lipid concentrations were reduced by 9% for total cholesterol (TC) and 32% for triglyceride (TG). The level of PMPs, activated platelets, and soluble adhesion molecules were all significantly decreased after treatment with bezafibrate. These findings suggest that bezafibrate may be useful for inhibiting both PMP-dependent and -independent vascular damage in patients with connective tissue diseases complaining of secondary hyperlipidemia.

Effect of ticlopidine on platelet-derived microparticles in patients with connective tissue diseases

We evaluated the plasma concentrations of platelet activation markers and platelet-derived microparticles (PMP) in patients with connective tissue diseases complaining of peripheral circulation disorders (n = 16) and studied the effect of ticlopidine hydrochloride (ticlopidine) on PMP generation. There were significant differences in the levels of PMP and a platelet activation marker between before and after treatment with ticlopidine (PMP: 695 ± 393 vs. 354 ± 206/104 platelets, p < 0.01; platelet CD63: 9.13 ± 5.64 vs. 5.22 ± 2.74%, p < 0.05). On the other hand, markers of vascular endothelium, such as vascular endothelium-derived small vesicles and serum thrombomodulin levels, were not affected by the administration of ticlopidine. Levels of cytokines and soluble adhesion molecules remained unchanged by ticlopidine administration. These findings suggest that ticlopidine may be useful for the inhibition of PMP-dependent vascular damage in patients with connective tissue diseases complaining of peripheral circulation disorders.

Serum levels of soluble CD30 in autologous peripheral blood stem cell transplantation

Abstract High-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) has become an important method of treatment for hematological and solid tumors. We examined levels of interleukin(IL)-4, interferon (IFN)γ, soluble (s) CD30, and sIL-2 receptor (R) before and after autologous PBSCT, and compared findings for PBSCT with those for bone marrow transplantation (BMT). We found significantly higher IL-4 levels 1 and 3 weeks after PBSCT than before PBSCT, while IFNγ levels remained almost unchanged after PBSCT. IFNγ levels were increased 3 weeks after BMT, although no increase in IL-4 level was observed. The serum sCD30 level was significantly higher 3 weeks after PBSCT, but not following BMT. For 34 samples on days 0 and 21 from 17 patients undergoing PBSCT, a strong correlation was observed between sCD30 and sIL-2R levels (r = 0.43, P < 0.01). However, no significant correlation between sCD30 and sIL-2R levels was found for BMT patients. These findings suggest that sCD30 is a useful marker for evaluating immunological activity following autologous PBSCT, and that the immunological conditions after autologous PBSCT may be associated with helper-T-cell-2-type immune responses.

Effects of Nilvadipine on Cytokine-Levels and Soluble Factors in Collagen Disease Complicated with Essential Hypertension

We examined some immunological parameters, particularly cytokines and soluble factors in collagen diseases complicated with essential hypertension. We also investigated the effects of Nilvadipine on immunological parameters after treatment with this drug for six months. The frequency of helper/inducer T cells (CD4+ CD8- cells, CD4+ CD45RA- cells) decreased in the peripheral blood on a 6 month treatment with nilvadipine. There was a significant decrease of suppressor/inducer T cells (CD4+ 45RA+ cells), and an insignificant decrease of activated T cells (CD3+ HLA-DR+ cells) and memory T cells (CD45RA- CD45RO+ cells) after treatment. Before treatment with Nilvadipine, interleukin-1beta, tumor necrosis factor-a, and interleukin-6 levels increased higher in the patients than in healthy volunteers. However, interleukin-1beta and interleukin-6 concentrations tended to decrease after treatment with Nilvadipine. Besides, tumor necrosis factor-alpha decreased significantly after treatment. The soluble interleukin-2 receptor concentrations also showed a decreased tendency after treatment, although high concentrations were found in the patients before treatment. In contrast, soluble human leukocyte antigen-1 and soluble thrombomodulin levels showed no significant change after treatment. These results suggest that Nilvadipine inhibits the generation of cytokines derived from activated T lymphocytes. Nilvadipine, calcium antagonist, may be useful for inhibition of vascular complication in collagen diseases.