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en: Secretion dynamics of cytotoxic granules in the cytotoxicity of the human NK-like cultured cells KHYG-1

ja: ヒトNK様培養細胞KHYG‐1の細胞傷害における細胞傷害性顆粒の分泌動態

en: Shikoku Acta Medica

ja: 四国医学雑誌


en: Evaluation of NK cell function using human NK-like cultured cell, KHYG-1

ja: ヒトNK様培養細胞KHYG-1を用いたNK細胞機能の評価

en: 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.


en: ABO genotyping of various hematopoietic cell lines to select model cells for research purposes

en: Human Immunology

ja: Human Immunology


en: Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

en: 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. The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. 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 Ca(2+) concentration ([Ca(2+)]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. There was a positive correlation between [Ca(2+)]i and platelet aggregation induced by thrombin. Aspirin (5.6-560 µM) and cilostazol (5-10 µM) significantly inhibited thrombin-induced increases in [Ca(2+)]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 [Ca(2+)]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 [Ca(2+)]i. 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 [Ca(2+)]i is applicable to the evaluation of the effects of various drugs on platelets.

en: Drug Design, Development and Therapy

ja: Drug Design, Development and Therapy


en: Flow Cytometric Evaluation of Surface CD56 Expression on Activated Natural Killer Cells as Functional Marker.

en: 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°C. 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- mRNA gene expression was quantified by real-time PCR. The expression level of surface CD56 on NK cells, cytotoxicity, and IFN- mRNA 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- mRNA gene expression. We revealed that the quantification of surface CD56 expression was applicable to the evaluation of cytotoxicity and IFN- production 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. J. Med. Invest. 63: 199-203, August, 2016.

en: The Journal of Medical Investigation : JMI


en: SNPs rs4656317 and rs12071048 located within an enhancer in FCGR3A are in strong linkage disequilibrium with rs396991 and influence NK cell-mediated ADCC by transcriptional regulation.

en: CD16 receptors are mainly expresses on the surface of NK cells and mediate antibody-dependent cellular cytotoxicity (ADCC). The authors previously reported that NK cell-mediated ADCC is influenced by the single nucleotide polymorphism (SNP) rs396991 (T>G; F158V), and the structure and expression levels of CD16 differed among these genotypes. The authors examined haplotype frequency distributions among rs396991 and other SNPs, rs10917571 (G>T), rs4656317 (C>G), and rs12071048 (G>A), located in an enhancer of the FCGR3A gene. A total of 101 healthy Japanese were genotyped for the presence of these SNPs. The authors also measured ADCC activity, FCGR3A transcript levels, and surface CD16 expression on NK cells. We found that the regulatory SNPs (rSNPs) rs4656317 and rs12071048 were in strong linkage disequilibrium with rs396991. These two SNPs with major alleles had higher ADCC activity than those with minor alleles. In addition, FCGR3A transcript levels and surface CD16 expression levels were regulated by these SNPs. These findings suggest that NK cell-mediated ADCC could be influenced by transcriptional regulation of these rSNPs. These findings help to clarify our understanding of the linkage disequilibrium among functional SNPs in the FCGR3A gene, and provide a resource for investigating the roles of functional SNPs in NK cell-mediated ADCC.


en: The influence of NK cell-mediated ADCC: Structure and expression of the CD16 molecule differ among FcRIIIa-V158F genotypes in healthy Japanese subjects

en: NK cells express the CD16 (FcRIIIa) receptor, which mediates antibody-dependent cellular cytotoxicity (ADCC), on their cell surface. Therefore, ADCC activity may be influenced by qualitative or quantitative changes in the CD16 molecule on NK cells. Responses to NK cell-mediated ADCC have been shown to depend on single nucleotide polymorphisms (SNPs) at FcRIIIa amino acid position 158. However, a consensus has not yet been reached regarding differences in the structure and expression levels of the CD16 molecule among FcRIIIa-V158F genotypes, which have not yet been adequately investigated in healthy Japanese individuals. We herein examined the influence of the FcRIIIa polymorphism on ADCC, binding affinity of CD16 to the Fc region, FCGR3A gene expression, and cell-surface CD16 expression in healthy Japanese subjects. FcRIIIa-V158F genotyping was performed for 101 subjects. The results obtained showed that all parameters analyzed increased in the order of V/V>V/F>F/F and were significantly higher in V/V subjects than in F/F subjects. Moreover, a positive correlation was observed between ADCC activity and binding affinity, FCGR3A transcript levels, and surface CD16 expression levels. These results suggest that the structure and expression of the CD16 molecule differs among FcRIIIa-V158F genotypes, and the FcRIIIa-V158F polymorphism may be represent a haplotype with other SNPs in regulatory regions in Japanese subjects.


en: Direct Determination of ABO Blood Group Genotypes from Whole Blood Using PCR-Amplification of Specific Alleles Method

en: American Journal of BioScience

ja: American Journal of BioScience


en: Effects of lavender and grapefruit essential oil on the autonomic nervous system and immune system

ja: ラベンダー精油とグレープフルーツ精油による自律神経系および免疫系に及ぼす影響

en: アロマテラピー学雑誌

ja: アロマテラピー学雑誌


en: The evaluation of histo-blood group ABO typing by flow cytometric and PCR Amplification of Specific Alleles analyses and their application in clinical laboratories

en: 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 cisA(2)B(3) 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.

ja: The Journal of Medical Investigation : JMI


en: Preparation method for a specific anti-H lectin isolated from goya seeds and its application in clinical laboratories

ja: ゴーヤ種子抽出抗Hレクチンの臨床検査への応用

en: After the discovery of the phenomenon of aggregation of red blood cells with castor beanextract by Peter Hermann Stillmark in1888, various plant hemagglutinins were discovered fromleguminous 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-groupspecificity are important for blood-group typing and antigen recognition. In recent years, Ulexlectin(anti-H lectin)extracted from the Ulex europaeus seed was used to subdivide blood of typeO, and the subgroup of ABO, but Ulex europaeus is now difficult to obtain, so it is necessary to buyexpensive reagents.On the other hand, Momordica charantias (goya)seed extract(goya lectin)was recentlyrevealed to show higher anti-H activity than Ulex lectin. Goya is readily available and inexpensiveto 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 fromgoya seeds, and confirm its stability against long-term storage and temperature and usefulness asan anti-H lectin. We expect this method to be used in clinical practice in the future.

en: Shikoku Acta Medica