Shiro Fukuhara

Detection of monocyte-derived microparticles in patients with Type II diabetes mellitus

Aims/hypothesis. The role of plasma monocyte-derived microparticles (MDMPs) and platelet-activation markers (platelet-derived microparticle [PDMP], platelet-bound CD62P [plt-CD62P], and platelet-bound CD63 [plt-CD63]) in diabetic vascular complications is not clear. We measured and compared plasma concentrations of MDMPs and the platelet-activation markers to investigate their possible contribution to diabetic vascular complications. Methods. Activated platelets and microparticles (PDMP and MDMP) were analysed by flow cytometry. Concentrations of serum sE-selectin were measured with enzyme-linked immunosorbent assay. Results. The concentration of MDMPs in diabetic patients was higher than in normal subjects. We found no differences in the binding of anti-GPIIb/IIIa and anti-GPIb monoclonal antibodies between groups. There were differences, however, in the concentrations of PDMPs, plt-CD62P, and plt-CD63 between Type II (non-insulin-dependent) diabetes mellitus patients and control subjects (PDMPs: 585 ± 25 vs 263 ± 9, p < 0.01; plt-CD62P: 28.1 % ± 1.4 % vs 9.4 % ± 0.6 %, p < 0.001; plt-CD63: 28.1 % ± 1.4 % vs 8.6 % ± 0.5 %, p < 0.001). Amounts of MDMPs correlated positively with these platelet activation markers, and the relation between PDMP and MDMP was the most significant. The concentration of MDMP in patients who had diabetes complicated with nephropathy, retinopathy, or neuropathy was higher than in those without diabetes-related complications. The increase in MDMP was particularly significant in patients with nephropathy. Concentrations of sE-selectin were higher in Type II diabetes patients than in control subjects, and correlated with MDMP, PDMP, plt-CD62P, and plt-CD63 levels in nephropathy patients. Conclusion/interpretation. In Type II diabetes patients, we detected increased activation of monocytes, which could have been stimulated by activated platelets and PDMPs. Because the activation of monocytes is associated with vascular endothelial damage, high concentrations of MDMPs could indicate vascular complications in diabetes patients, especially those who have diabetes-related nephropathy. [Diabetologia (2002) 45: ▪–▪]

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.

State-of-the-Art Review : Effect of Sarpogrelate Hydrochloride on Platelet-Derived Microparticles and Various Soluble Adhesion Molecules in Diabetes Mellitus

We measured platelet-derived microparticles, ac tivated platelets, and various adhesion molecules in 48 patients with diabetes mellitus. We also performed a comparative study of these parameters before and after administration of sarpo grelate hydrochloride. The numbers of platelet-derived micro particles and activated platelets were increased significantly in diabetic patients, and CD63-positive platelets were increased in patients with diabetic complications and poorly controlled blood glucose. Soluble adhesion molecules and thrombomodu lin were also increased significantly. After administration of sarpogrelate hydrochloride, not only CD62p- and CD63- positive platelets, but also platelet-derived microparticles were decreased significantly. Soluble adhesion molecules and throm bomodulin were also significantly decreased after the treat ment. These data suggest that (a) in patients with diabetes, antiplatelet therapy with sarpogrelate hydrochloride is a useful antithrombin therapy because it suppresses the production of intrinsic coagulants by activated platelets; and (b) sarpogrelate hydrochloride decreases endothelial cell damage via adhesion molecules.

Clinical Evaluation of Transfusion of Prestorage-Leukoreduced Apheresis Platelets

With increased use of platelet concentrate (PC) in recent years, adverse reactions to PC transfusion have received much clinical attention. Most of these reactions stem from white blood cells (WBC) contaminating the transfused PC. Several are thought to be preventable by removing WBC before PC storage. Methods: We routinely filtered apheresis PC either during collection or immediately afterwards and monitored various indicators of platelet quality during storage. After transfusion to patients, transfusion efficacy and the type, severity, and frequency of posttransfusion side effects were compared with those of a control group in which PC was filtered at the bedside. Results: Prestorage-filtered PC contained an average of 3.1±0.7 × 1011 platelets and 0.9±1.2 × 106 contaminating leukocytes. Measurement of platelet function and metabolic indicators revealed normal values throughout the storage period. CD62 measurement revealed no undue platelet activation after filtration or during the storage period. Cytokine, histamine, bradykinin, and complement levels showed no significant increase after filtration or during storage. Transfusion efficacy and overall side effect incidence rates were similar for the prestorage- and bedside-filtered groups, but reactions of the bedside-filtered group included serious reactions such as breathing difficulties and shock. No serious reactions were noted in the prestorage-filtered group. Conclusion: Filtering PC before storage has no adverse effect on PC quality and may reduce the severity of post transfusion side effects.

Diffuse large B-cell lymphoma showing an interfollicular pattern of proliferation: a study of the Osaka Lymphoma Study Group

Aims:  Diffuse large B‐cell lymphoma (DLBCL) usually proliferates effacing lymph follicles. In occasional cases, tumour cells show an interfollicular pattern of proliferation preserving lymph follicles. The aim was to analyse clinicopathological findings in DLBCL showing an interfollicular pattern of proliferation to determine whether this type of lymphoma is a distinct entity of DLBCL.

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.

Expression of prothrombinase activity and CD9 antigen on the surface of small vesicles from stimulated human endothelial cells

We employed flow cytometry and monoclonal antibodies (MoAb) to study the surface membrane protein of shed particles (small vesicles, SV) that were released from vascular endothelial cells (EC) by agonists such as a Ca ionophore (A23187) and thrombin. After stimulation of EC by A23187, CD9 antigens disappeared entirely from the EC surface in a time- and concentration-dependent manner; they subsequently moved onto the SV surface. Von Willebrand factor (vWF) and P-selectin from Weibel-Palade (W-P) bodies were expressed rapidly on the EC surface after thrombin stimulation, but not on the SV surface. P-selectin may have some effect on maintenance of hemostasis on the EC surface. We demonstrated that the surfaces of SV and EC significantly supported prothrombinase activity and confirmed that A23187-induced SV from EC express binding sites for factors IXa and Xa. These results suggest that the SV are an important factor in a novel controlling mechanism of the coagulation system on the EC surface.

A Japanese propositus with D-- phenotype characterized by the deletion of both the RHCE gene and D1S80 locus situated in chromosome 1p and the existence of a new CE-D-CE hybrid gene

AbstractIn a family study of a Japanese propositus with the D-- phenotype, the serological data of her D-- phenotype and those of her parents were discrepant. Gene analysis of the propositus showed a gross deletion of the RHCE gene and a new rearrangement of RHCE to yield the CE-D-CE hybrid. It was demonstrated that the hybrid CE-D-CE gene consisted of exon 1 from the RHCE gene, followed by exons 3 to 7 from the RHD gene and exons 8 to 10 from the RHCE gene. However, whether or not exon 2 of the RHD or the RHCE gene was contained in the CE-D-CE gene remained unclear. Moreover, spacer analysis between both RH genes and the family study suggested that the D-- gene complex from the paternal and maternal sides consisted of only the CE-D-CE hybrid gene and a single RHD gene, respectively. For the purpose of confirming the parent-child relationship, a paternity test using DNA fingerprint and polymerase chain reaction (PCR) analysis at the D1S80 locus were performed. DNA fingerprints with two kinds of DNA minisatellite probes (33.15 and 33.6) confirmed that the parent-child relationship in the D-- propositus was compatible. However, in the present case, at the D1S80 locus, the PCR product derived from the mother was lacking, thereby negating a parent-child relationship. It is probable that the RH genes and D1S80 locus exist in close proximity, because they are situated in chromosomes 1p 34.3–36.1 and 1p 36.1–36.3, respectively. These data suggested that at the stage of gametogenesis, both the RHCE gene and the D1S80 locus from the maternal side may have been deleted, thereby producing the D-- gene complex.

A Phase II Study of VEPA/FEPP Chemotherapy for Aggressive Lymphoma in Elderly Patients: Japan Clinical Oncology Group Study JCOG9203

The Lymphoma Study Group (LSG) of the Japan Clinical Oncology Group conducted a phase II trial of LSG12 therapy for 45 elderly patients with aggressive lymphoma to clarify whether LSG12 reduces severe infection without lowering the complete response (CR) rate in comparison with LSG4. LSG12, which consisted of a regimen of vincristine, cyclophosphamide, prednisolone, doxorubicin, vindesine, etoposide, and procarbazine (VEPA/FEPP), excluded bleomycin and methotrexate of LSG4 therapy, reduced the dosages of doxorubicin and cyclophosphamide, and increased etoposide and procarbazine dosages instead. Inclusion criteria consisted of a patient age of 70 to 75 years, a World Health Organization performance status of 0 to 2, and acceptable organ function. The treatment was completed in 47% of the patients and terminated early for disease progression in 20% and for toxicity in 16%.The CR rate was 60% (95% confidence interval [CI], 44%-74%).The 5-year overall survival (OS) rate was 42% (95% CI, 27% -57%), and the median OS time was 4.3 years. Leukopenia of grade 3 to 4 occurred in 98% of the patients, and severe infection occurred in 9%. Eight patients with hepatitis C virus (HCV) antibody showed no severe hepatic toxicity and had a better CR or OS rate than the 37 HCV-negative patients. Although the outcomes of LSG12 met our expectations with a reduction in severe infection and equivalent CR and OS outcomes compared with LSG4 and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone), the possibility of a regimen more beneficial than LSG12 for aggressive lymphoma in the elderly patient should be explored because of frequent hematologic toxicity and poor compliance in LSG12.

HLA Typing of a Family with Systemic Lupus Erythematosus

Although systemic lupus erythematosus (SLE) is a representative collagen disease, the etiology remains unclear. However, both genetic and environmental factors seem to be involved. Based on serological studies, associations between certain human leukocyte antigens and many autoimmune diseases have long been suggested. Regarding the genetic aspects of SLE, the HLA haplotype is regarded as a potentially important factor, although its role remains controversial. Recently, we examined three family members who had SLE with an identical HLA haplotype.