RESUMO
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.
Assuntos
Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Megacariócitos/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Tetrazóis/farmacologia , Cilostazol , Humanos , Testes de Função PlaquetáriaRESUMO
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-γ mRNA gene expression was quantified by real-time PCR. The expression level of surface CD56 on NK cells, cytotoxicity, and IFN-γ 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-γ mRNA gene expression. We revealed that the quantification of surface CD56 expression was applicable to the evaluation of cytotoxicity and IFN-γ 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.
Assuntos
Antígeno CD56/análise , Citometria de Fluxo/métodos , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Adulto , Biomarcadores , Feminino , Humanos , Interferon gama/genética , Interleucina-12/farmacologia , Interleucina-2/farmacologia , MasculinoRESUMO
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.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/genética , Elementos Facilitadores Genéticos/genética , Células Matadoras Naturais/imunologia , Desequilíbrio de Ligação , Receptores de IgG/genética , Adolescente , Adulto , Alelos , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
NK cells express the CD16 (FcγRIIIa) 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 FcγRIIIa 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 FcγRIIIa-V158F genotypes, which have not yet been adequately investigated in healthy Japanese individuals. We herein examined the influence of the FcγRIIIa polymorphism on ADCC, binding affinity of CD16 to the Fc region, FCGR3A gene expression, and cell-surface CD16 expression in healthy Japanese subjects. FcγRIIIa-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 FcγRIIIa-V158F genotypes, and the FcγRIIIa-V158F polymorphism may be represent a haplotype with other SNPs in regulatory regions in Japanese subjects.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/genética , Células Matadoras Naturais/imunologia , Receptores de IgG/genética , Adolescente , Adulto , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Feminino , Regulação da Expressão Gênica , Frequência do Gene , Genótipo , Humanos , Japão , Masculino , Ligação Proteica/genética , Receptores de IgG/química , Adulto JovemRESUMO
Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3) as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain) as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of downstream targets and lens fiber proteins.