Ca2+ Mobilization and Aggregation in Platelets

Platelets play an important role in physiological hemostatic mechanisms. In contrast, platelet activation has been implicated in pathological conditions, such as atherosclerosis, angiogenesis, and inflammation. Thrombin is considered to be of particular pathological importance as a platelet-activating substance, and thrombin-activated platelets are detected in the blood of patients with advanced occlusive arterial disease. Ca2+ acts as a second messenger in platelet activation, and the regulation of intracellular Ca2+ concentrations ([Ca2+]i) is important for controlling platelet functions. However, changes in [Ca2+]i by antiplatelet agents remain unclear. Therefore, we herein investigated the relationship between [Ca2+]i and the intensity of platelet aggregation after a thrombin stimulation, the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents, and the effects of antiplatelet agents on thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. Fura2-loaded platelets were treated with phosphate-buffered saline or a low concentration of thrombin (0.005 U/mL), followed by antiplatelet agents (aspirin or cilostazol), and changes in [Ca2+]i and the intensity of platelet aggregation by the thrombin stimulation were measured using fluorescence spectrophotometry. Changes in [Ca2+]i and the intensity of platelet aggregation after the thrombin stimulation as well as the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents indicated that cilostazol exerted stronger antiplatelet effects than aspirin and also that antiplatelet effects may be attenuated in thrombin-activated platelets. The present results also suggest the utility of thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. These results may contribute to future drug development for antiplatelet therapy

The evaluation of histo-blood group ABO typing by flow cytometric and PCR-amplification of specific alleles analyses and their application in clinical laboratories

ABO antigens are oligosaccharide antigens, and are widely distributed on red blood and tissue cells as well as in saliva and body fluid. Therefore, these antigens are important not only for blood transfusion, but also for tissue cell and organ transplantations. Also, blood, hair, and seminal fluid are important sources of evidence at crime scenes, and these antigens are some of the most important markers for personal identification in forensic investigations. Here, we describe the development and use of quantitative analysis of A, B, and H antigens on red blood cells by employing flow cytometric analysis and the ABO genotyping method based on PCR-amplification of specific alleles (PASA) within DNA, especially from blood and saliva. In this study, flow cytometric analysis could be used to compare the differences between the expression of A and/or B and H antigens on red blood cells with various phenotypes, and the PASA method was able to determine the genotype of the type cisA2B3 pedigree using only DNA extracted from saliva. These analysis methods are simple and useful for judging the ABO blood group system and genotyping, and are used widely throughout research and clinical laboratories and forensic fields

ゴーヤ シュシ チュウシュツ コウ H レクチン ノ リンショウ ケンサ エノ オウヨウ

After the discovery of the phenomenon of aggregation of red blood cells with castor bean extract by Peter Hermann Stillmark in１８８８, various plant hemagglutinins were discovered from leguminous plant seed extracts. These plant hemagglutinins were called“lectins”by William C. Boyd et al . Lectins are sugar-binding proteins that are highly specific for their sugar moieties, physiological activity, and hemagglutinating activity. In particular, lectins displaying blood-group specificity are important for blood-group typing and antigen recognition. In recent years, Ulex lectin（anti-H lectin）extracted from the Ulex europaeus seed was used to subdivide blood of type O, and the subgroup of ABO, but Ulex europaeus is now difficult to obtain, so it is necessary to buy expensive reagents. On the other hand, Momordica charantias （goya）seed extract（goya lectin）was recently revealed to show higher anti-H activity than Ulex lectin. Goya is readily available and inexpensive to obtain. Therefore, we examined a simple method for the preparation of goya lectin. As a result, we were able to establish a simple method for preparing goya lectin isolated from goya seeds, and confirm its stability against long-term storage and temperature and usefulness as an anti-H lectin. We expect this method to be used in clinical practice in the future

CD56抗原を指標としたNK様培養細胞の活性評価

NK cells play an important role in the elimination of viral infection and tumors. In recent years, immune cell therapy using activated NK cells has attracted attention, and the search for activators and evaluation of NK cell activity have become important. We previously reported a positive correlation between CD５６antigen expression, which is the major cell membrane antigen of human NK cells, and NK cell activity or cytotoxicity, and demonstrated that it is possible to evaluate NK cell function using the CD５６ antigen as an index. This simple evaluation method is useful for functional evaluation of NK cells and the search for activators. However, it requires blood sampling and preparation of NK cells because it uses human NK cells. Therefore, in this study, we examined whether the CD５６ antigen functions as an activation index using KHYG‐１ human NK-like cultured cells as a substitute for NK cells. As a result, the CD５６antigen on the KHYG‐１cell membrane was increased in a concentration-dependent manner by IL‐２ stimulation, as was the cytotoxicity. This suggests that the CD５６ antigen on the KHYG‐１ cell membrane can be used as an evaluation index of NK activity as in human NK cells

KHYG‐１における細胞傷害性と細胞傷害性顆粒の変化

Three major therapies, “surgery”, “chemotherapy”, and “radiotherapy”, have been used to treat cancer. Recently, “immunotherapy” has attracted attention as the fourth treatment. We previously performed fundamental studies using NK cells, one cell type that has attracted attention in immunotherapy, and revealed that the surface CD５６ antigen on the KHYG‐１ human NK cell-like cultured cells as a substitute for human NK cells can be used as an evaluation index of NK cell activity as in human NK cells. We also reported that the cytotoxicity of KHYG‐１increases by IL‐２ stimulation. In this study, we improved the conventional cytotoxicity measurement method to evaluate the effects of a small amount of activator on NK cells. As a result, the cytotoxicity rate and measurement sensitivity at low-concentration IL‐２ stimulation were increased, and it became possible to evaluate the effects of a smaller amount of the activator. In the dynamic observation of KHYG‐１ intracellular granules, it was possible to observe in real time how the target cells were damaged after the influx of granules. Furthermore, the relationship between the activation of KHYG‐１and the change in the intracellular expression level of granzyme B by IL‐２stimulation was clarified, and future research tasks were shown

Effects of various drugs on platelet functions

Background: The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. Objective: The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. Materials and methods: Human erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. Results: There was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6–560 µM) and cilostazol (5–10 µM) significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8–200 µM) and sodium valproate (50–1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca2+]i. Conclusion: It is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions, and the quantification of the inhibition of thrombin-induced increases in [Ca2+]i is applicable to the evaluation of the effects of various drugs on platelets

Evaluation of Surface CD56 Expression

Surface CD56 is the most important cell marker for defining NK cells. However, the relationship between the expression of surface CD56 and NK cell activity has not yet been elucidated in detail. Thirteen healthy volunteers were enrolled in the present study. Peripheral blood mononuclear cells (PBMCs) were stimulated with rIL-2 or rIL-12 (1, 10, 100 U/mL) for 18 h at 37 ℃. After incubation, surface CD56 expression on NK cells was evaluated using a flow cytometric analysis. A colorimetric-based lactate dehydrogenase (LDH) assay was used for experiments on cytotoxicity. IFN-γmRNA gene expression was quantified by real-time PCR. The expression level of surface CD56 on NK cells, cytotoxicity, and IFN-γmRNA gene expression were significantly increased by the rIL-2 and rIL-12 stimulations. In addition, a positive correlation was found between surface CD56 expression and cytotoxic activity or IFN-γmRNA gene expression. We revealed that the quantification of surface CD56 expression was applicable to the evaluation of cytotoxicity and IFN-γproduction in activated NK cells. These results suggest that the measurement of surface CD56 expression represent an easy and rapidly reproducible technique to evaluate the activated state of NK cells and monitor NK cell activity in immunotherapy

Possibility for Prevention of Type 2 Diabetes Mellitus and Dementia Using Three Kinds of Brown Rice Blends after High-Pressure Treatment

As it has been reported that type 2 diabetes mellitus increases the risk of Alzheimer’s disease, we investigated how to prevent type 2 diabetes and dementia using biofunctional boiled rice. We adopted unpolished super-hard rice (SHBR) for diabetes and wax-free unpolished black rice (WFBBR) for dementia and blended those with ordinary non-polished rice (KBR) (blending ratio 4:4:2), adding 2.5% waxy black rice bran (WBB) and 0.3% rice oil after high-pressure treatment (HPT) (WFBSK) to improve its palatability. This boiled rice is rich in dietary fiber, anthocyanin, free ferulic acid and β-secretase inhibitory activity. A randomized, parallel-group comparison study was conducted for 12 weeks with 24 subjects, using Cognitrax to evaluate their cognitive function primarily. Furthermore, as the secondary purpose, we performed a single-dose test for postprandial blood glucose and insulin secretion at the end of the human intervention test. After 12 weeks, consumers of the WFBSK rice exhibited significant improvement in language memory by cognitive test battery compared with those who consumed the control white rice (p < 0.05). Moreover, subjects who consumed the WFBSK rice had lower insulin secretion levels than those who consumed the control polished rice (p < 0.05)