Chie Yoshimura

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.

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.

Analysis of serum ErbB-2 protein and HLA-DRB1 in Japanese patients with lung cancer.

We investigated the relationship between ErbB-2 and HLA in order to clarify the clinical and genetic factors related to Japanese patients with lung cancer. Thirty-nine of the 73 lung cancer patients (53.4%) had elevated levels of ErbB-2. Only seven of 23 (30. 4%) patients with small cell carcinoma had elevated ErbB-2 levels. The prevalence of ErbB-2 positivity was highest (23 of 32; 71.8%) in patients with adenocarcinoma, while that in patients with squamous cell carcinoma was 50% (9 of 18). The frequencies of HLA A33, B44, B62, and B75 were lower in the lung cancer patients than in the control group. HLA-DR9 was higher in frequency in lung cancer patients than in the healthy controls (P<0.05), but HLA-DR6 was lower in frequency in lung cancer patients than in controls (P<0.01). DRB1*0901 was significantly higher in frequency in lung cancer patients than in controls (P<0.05). On the other hand, DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were completely absent in lung cancer patients. The frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. However, these types of HLA were not included in significant frequencies in our group of lung cancers. Our results suggest that some HLA-antigens/alleles participate in the pathogenesis of lung cancer in Japanese patients. In addition, the relationship between HLA-associated genetic factors and ErbB-2 seems to be weak. These findings suggest that ErbB-2 is correlated with prognostic factors for lung cancer independently of HLA-associated genetic factors.

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.

Analysis of cytotoxic T lymphocytes and Fas/FasL in Japanese patients with non-small cell lung cancer associated with HLA-A2.

Abstract Purpose. In recent years, the use of immunotherapy for malignant tumors has been proposed. To explore the significance of immunotherapy for lung cancer, we examined two systems : the HLA class I and cancer-reactive CTL system, and the Fas-FasL system. Methods. HLA class I (HLA-A, -B, and -C) antigens were determined in 61 patients with lung cancer and in 30 healthy controls. The HLA class I phenotype was investigated by serological techniques. HLA-A2 alleles were investigated by polymerase chain reaction sequence-specific primer analysis. We analyzed lymphocytes isolated from 61 patients with two-color surface labeling and flow cytometry. Furthermore, we analyzed sFas and sFasL expression by enzyme-linked immunosorbent assay (ELISA). We also examined lung cancer cell line-induced apoptosis of CD8+ lymphocytes using confocal laser scanning microscopy. Results. The HLA-A2 allele was observed in 27 of 61 patients with lung cancer. There were no differences in HLA-A2 allele classifications between lung cancer patients and controls. Thirty-six of the 61 lung cancer patients (57%) had elevated levels of sFas, and 16 of the 61 patients (26.2%) had elevated levels of sFasL. The sFas level of lung cancers with HLA-A2 was significantly higher than that of cancers without HLA-A2 (P<0.01). This tendency was observed in every lung cancer tissue type, and the sFas levels of lung cancers with HLA-A2 associated significantly with the CTL levels of lung cancers with HLA-A2. Furthermore, compared to lung cancers without HLA-A2, CD8+ T-cell levels were elevated in lung cancers with HLA-A2. In contrast, sFas levels of non-small cell lung cancers without HLA-A2 were higher than those in lung cancers with HLA-A2. In an in vitro experiment using lung cancer cell lines, we observed apoptosis of CD8+ lymphocytes induced by lung cancer cells. Lung cancer-reactive CTLs are mobilized easily by restriction of HLA-A2, but this restriction is not always specific. In addition, FasL derived from lung cancer cells can induce apoptosis of CD8+ T-cells, and the frequency of this phenomenon is increased in small cell lung cancers without HLA-A2. Conclusion. Our findings suggest that the effect of immunotherapy may be insufficient for non-small cell lung cancer without HLA-A2.

Genetic Analysis of HLA- and HPA-Typing in Idiopathic (Autoimmune) Thrombocytopenic Purpura Patients Treated with Cepharanthin

We performed genetic analysis of human leukocyte antigen (HLA) and human platelet antigen (HPA) in 45 patients with cepharanthin-treated idiopathic thrombocytopenic purpura. HLA-typing was performed by the polymerase chain reaction-restriction fragment length polymorphism method, and HPA-typing by a polymerase chain reaction-sequence-specific primer method. There were 14 responders and 31 nonresponders. Responders included many patients who had already been treated with prednisolone. HLA-DRB1*0901 was significantly more common in responders than in nonresponders. In contrast, HLA-DRB1*0410 and DQB1*0401 were significantly more common in nonresponders. The a/b genotype of HPA-2a/2a (Ko(b)/Ko(b)) was significantly increased in responders. In contrast, HPA-2a/2b (Ko(b)/Ko(a)) and HPA-3a/3b (Bak(a)/Bak(b)) were significantly more common in nonresponders. These findings suggest that genetic studies of HLA and HPA can predict the response of idiopathic thrombocytopenic purpura to cepharanthin.

HLA and HPA Typing in Idiopathic Thrombocytopenic Purpura Patients Treated with Kami-kihi-to

We performed human leukocyte antigen (HLA) and human platelet antigen (HPA) in patients with Kami-kihi-to-responsive idiopathic thrombocytopenic purpura. The HLA-A2, A61 and Cw1 were significantly increased in responders compared with nonresponders, as were HLA DRB1 *0901, DRB1 *1502, and DPB1 *0501. In contrast, HLA DPB1 *0201 and DPB1 *0901 were significantly decreased in responders. The a/b genotype of HPA-2 and a/a genotype of HPA-3 were markedly increased in nonresponders, and anti-GPIb antibody was also increased. These results suggest that HLA, HPA, and anti-GP antibody studies may predict the response of idiopathic thrombocytopenic purpura to Kami-kihi-to.

Thrombopoietin Levels in Patients Undergoing Autologous Peripheral Blood Stem Cell Transplantation

Background and Objectives: High–dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) has become an important treatment for hematological and solid tumors. Several reports on endogeneous production of thrombopoietin (TPO) in patients undergoing intensive chemotherapy have been published. We measured TPO levels in patients undergoing autologous PBSCT in order to elucidate the role of TPO in megakaryopoiesis and platelet recovery following stem cell transplantation. We also examined whether PBSC from different patient groups (patients with lung cancer and malignant lymphoma) had different effects on TPO levels. Materials and Methods: Serum levels of TPO and other cytokines were determined by ELISA before and after PBSCT in 10 patients with lung cancer and 18 patients with malignant lymphoma. Results: Compared to platelet counts at 1 week after transplantation, those at 3 weeks were significantly increased (p<0.001). In contrast, serum TPO levels were significantly decreased 3 weeks after PBSCT (p<0.01). There were no significant differences in levels of other cytokines between 1 and 3 weeks after transplantation. A strong correlation was found between serum TPO levels and circulating platelet counts (r =–0.675; p<0.0001). Platelet counts exhibited a significant recovery and TPO levels also decreased significantly in malignant lymphoma patients after transplantation. There were no significant differences in TPO levels in lung cancer patients, although the same tendencies as for malignant lymphoma patients were observed for both platelet count and TPO level. On analysis of transfused PBSC, we found that the numbers of CFU–GM were similar in lung cancer and malignant lymphoma patients. However, the numbers of CD34+ cells were significantly higher in lung cancer patients than in malignant lymphoma patients. Conclusion: Our findings suggest that TPO plays a critical role in the reconstitution of megakaryopoiesis and platelet production following PBSCT, and that the type of stem cell, tumor, and preceding chemotherapy affect serum TPO levels after transplantation.