Kozue Okano

Longitudinal study of a health education program for Japanese women in menopause.

In this longitudinal intervention study, a 6 week health education program consisting of lectures and exercises was implemented for 39 Japanese menopausal women. The effects of the program were assessed by measuring their exercise participation, climacteric symptoms, and quality of life immediately before, 6 weeks after, and 1 year after the program. The Simplified Menopausal Index was used to assess the climacteric symptoms and the Medical Outcomes Study 36-Item Short-Form Health (SF-36) Survey was used to assess the quality of life. Significant improvements were observed in the subscale score for general health perception and the summary score for the physical component summary in the SF-36 Survey. Favorable results also were found for women without a previous exercise habit before the program but who participated in regular exercise 1 year after the program. No improvements were observed in the climacteric symptoms. We concluded that our program was effective for menopausal women in spite of the intervention period being relatively short.

Source of elevated serum mitochondrial creatine kinase activity in patients with malignancy

Mitochondrial creatine kinase (CK-mit) is increased in cancer tissues of the digestive tract. There is no difference in molecular weight, electrophoretic and kinetic properties between the isoenzymes extracted from the tumor and those from the adjacent normal tissues. High non-CK-M/total CK activity ratios in some sera from cancer patients probably reflect leakage of CK-mit from the tumor tissues.

Therapeutic effect of dendritic cells loaded with a fusion mRNA encoding tyrosinase-related protein 2 and enhanced green fluorescence protein on B16 melanoma.

Dendritic cells (DCs) loaded with messenger RNA (mRNA) have been proposed to be useful for inducing specific cytotoxic T lymphocytes against tumor antigens. It is now also apparent that tumor antigen-specific T cell tolerance limits the efficacy of active immunotherapy. To improve the efficacy of mRNA-loaded DCs, we constructed a fusion mRNA encoding tyrosinase-related protein 2 (TRP2), which has a late endosomal/lysosomal sorting signal and enhanced green fluorescence protein (EGFP), and evaluated its effect in a murine melanoma model. C57BL/6 mice were challenged subcutaneously (s.c.) with 3 × 105 B16 tumor cells, and 3 and 10 days later, 3 × 105 DCs loaded with mRNA (DC/mRNA) were injected s.c. in the vicinity of the tumor site. Treatment with DC/TRP2 mRNA or DC/TRP2-EGFP mRNA significantly inhibited tumor growth compared to DC/PBS on day 17 after B16 challenge (DC/PBS vs. DC/TRP2 mRNA, p = 0.0411; DC/PBS vs.DC/TRP2-EGFP mRNA, p = 0.0253), whereas no antitumor effect was observed in mice treated with DC/EGFP mRNA or DC/TRP2 peptide. Moreover, the survival rate in mice immunized with DC/TRP2 mRNA or DC/TRP2-EGFP mRNA was significantly improved as compared with that in mice receiving DC/PBS (DC/PBS vs. DC/TRP2 mRNA, p = 0.0228; DC/PBS vs. DC/TRP2-EGFP mRNA, p = 0.0049). Depletion of CD4+ T cells or CD8+ T cells with antibody administration completely abrogated the therapeutic effectiveness of DC/TRP2-EGFP mRNA, suggesting the induction of a T cell immune response against the B16 tumor.

Development of an improved assay system for activated platelet counts and evaluation by aspirin monitoring

Platelets represent a linkage among inflammation, thrombosis, and atherogenesis, and enhanced platelet activation is regarded as a risk for thrombotic disorders. The level of P-selectin expressed (CD62P) on the platelet surface is a useful marker of activated platelets (aPLT). Although CD62P has been measured briefly by flow cytometry using an anti-CD62P antibody, the assay remains imprecise and we tried to establish stable conditions for its measurement. The levels of aPLT are increased significantly by many factors, such as meals, sampling and keeping conditions, centrifugation, and the timing of fixation. For optimal results, sampling should be performed quickly in a K(2)-ethylenediaminetetraacetic acid (EDTA) containing a sample tube, and whole blood should be fixed with 666 mmol/L formaldehyde plus 167 mmol/L glyoxal for 5 min. After washing with phosphate buffered saline (PBS), the fixed platelets were reacted with anti-CD62P antibody for 20 min and measured by flow-cytometric detection for aPLT. The coefficient of variation of our aPLT assay was 10.4%. We also examined basic experiments to test the clinical application of our aPLT assay by monitoring aspirin therapy. The levels of aPLT after the administration of aspirin for 3 days were significantly lower than those in the group that did not receive aspirin. These results suggest that the aPLT assay is an effective analytical procedure for measuring platelet reactivity.

Evaluation of an mRNA Lipofection Procedure for Human Dendritic Cells and Induction of Cytotoxic T Lymphocytes against Enhanced Green Fluorescence Protein

We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 µl/ml, and the mRNA concentration was 6 µg/ml. Under these conditions, ELISPOT and 51Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-γ-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs.

Exploration of hematological and immunological changes associated with the severity of type 2 diabetes mellitus in Japan

It has been postulated that immune modulation and activation play an important role in the pathogenesis of type 2 diabetes mellitus (T2DM), but evidence for this has not yet been well documented. We explored the changes in peripheral immunocompetent cells in relationship to the severity of T2DM in 142 patients, and 34 healthy individuals in Japan. A severity index with 0-12 grades was derived based on the HbA1c level and the number of complications. By multiple regression analysis, the severity index was positively associated with neutrophil counts and negatively associated with platelet and CD19+ lymphocyte counts. However, we did not observe any significant changes in other lymphocyte subsets such as CD4+, CD8+, and CD56+. These results suggest that poor diabetic control may be marked by changes in some blood cell types.

Characteristics of Japanase Thalassemia

Abstract Thalassemia is relatively rare in Japan. Of 387 β-thalassemia cases of Japanese, homozygotes for β+- and β0-thalassemias were twenty-two (5.7%) and one (0.26%), respectively. The rest (94%) were of β- thalassemia trait. Ten different kinds of β-thalassemia mutations comprised about 80% of all cases. Half of these “common” mutations were unique to Japanese and another half were possibly from abroad. Sixty-nine α-thalassemia cases were analyzed, in which HbH and α- thalassemia trait comprised nineteen and fifty, respectively. Most of the αo- and α+-thalassemia chromosomes were of Southeast Asian type (--SEA) and –α3.7 type, respectively. The frequency of α+- thalassemia as well as triplication of α-globin gene was high in northern Japan. Recent increase in the number of immigrants from Southeast Asia seems to raise the number of α-thalassemia found in Japan. They comprise at least 20% of the α-thalassemia. Six mutants classified into dominant-type β-thalassemia were found. All of them exhibited moderate anemia and marked anisopoikiocytosis. Heinz body varied in degree from copious to rare or even absent. Any mutation at initiation codon demonstrated marked microcytosis and erythremia. The breakpoint determination for large deletion-type thalassemia became feasible by estimation of gene dosage and PCR. Thus, the precise breakpoints for Filipino-type αo-thalassemia (--FIL) and Japanese type-2 δβ-thalassemia were disclosed. The genetic diagnosis for these thalassemias are now readily conducted by gap PCR. The characterization of several new large deletions is being carried out.

Simultaneous assay of activated platelet count and platelet-activating capacity by P-selectin detection using K2-EDTA-treated whole blood for antiplatelet agents

It is well recognized that examinations of activated platelets (aPLTs) and platelet‐activating capacity are very important to observe and prevent embolic diseases (events) such as ischemic stroke and myocardial infarction. Previously, we reported an appropriate measurement technique of aPLT for clinical assay. In this paper, we investigated stable conditions for measurement of activating capacity of platelets.

Influencing factors in quantitative measurement using activated platelet levels and platelet-activating capacity for the assessment of thrombosis in pre-metabolic syndrome and type 2 diabetes mellitus: Informative factors for thrombosis prevention

Activated platelet levels and platelet-activating capacity are well recognized as useful index parameters for the physiological and pharmacological prediction of thrombotic events. Recently, quantitative measurements for platelet functions using a flow cytometer have been increasing gradually. However, the relation of physiological factors, such as sex, aging, and laboratory tests, to platelet functions has not been well documented. We conducted a blood analysis of people with normal/pre-metabolic syndrome and patients with type 2 diabetes mellitus to clarify the pathological factors. The levels of basal (non-stimulated)-activated, platelet-expressed P-selectin and activated platelet stimulated by agonists were measured by a flow cytometer, and ratios of platelet-activating capacity were also calculated. Statistical analyses indicated significantly high basal-activated platelet in pre-metabolic syndrome, and basal-activated platelet was positively associated with hyperlipidemia and hepatic damage. Platelet-activating capacity was significantly low in aging and hyperlipidemia, but high in hyperglycemia, and was negatively associated with hyperlipidemia and hepatic damage. Aging and high nutrient condition impaired platelet functions. Quantitative measurements of basal-activated platelet and platelet-activating capacity are precise parameters for the prediction of thrombotic events.