Publications (14)29.1 Total impact
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Article: Direct flowcytometric analysis of eosinophils using a whole blood staining technique
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ABSTRACT: Background: Flowcytometric analysis of purified eosinophils requires several hours to accomplish due mainly to purification of cells; moreover, it requires more than 20 mL blood and expensive reagents. The aim of the present study was to develop a method of direct flowcytometric analysis for eosinophils using whole blood.Methods: Peripheral blood obtained from five healthy individuals (mean age 42 years) and 10 patients with eosinophilia (mean age 40.3 years) were used for analysis. We stained antigens (CD9 or CD16) and fixed cells with parabenzoquinone (PBQ) or paraformaldehyde (PFA) after hemolyzation followed by treatment with N-octyl-β-glucopyranoside (OG).Results: On comparison of forward scatter with side scatter dot plots among samples treated with hemolyzation alone, PBQ fixation and PFA fixation, PBQ fixation showed the best results in discriminating eosinophils from other leukocyte fractions on the cytogram. Following fixation and permeabilization of cells, EG2, a secretory form of eosinophil cationic protein, was stained as an intracellular antigen. Flowcytometric analysis for EG2 showed a high positivity rate only in the eosinophil fraction. There were no differences in EG2 positivity or mean fluorescence intensity (MFI) between heparinized and EDTA-treated blood. Comparison of samples treated with OG at 6.0 and 7.4 mg/mL showed that the latter had a higher MFI for EG2 without significant change in the positivity rate.Conclusions: The findings show that intra- and extracellular properties of eosinophils can be analyzed with whole blood using PBQ fixation and OG treatment at a concentration of 7.4 mg/mL. Direct flowcytometric analysis of eosinophils saves time and requires only a small amount of blood, both of which are advantageous for patients and laboratory workers.Allergology International 06/2008; 50(4):319 - 324. -
Article: C-reactive protein levels in the serum of asthmatic patients.
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ABSTRACT: Asthma is a chronic airway inflammatory disease caused by immune cells such as T lymphocytes and eosinophils. Recently, highly sensitive C-reactive protein (hs-CRP) assays have become available for detecting small changes in CRP levels within the reference range, allowing for the evaluation of clinical inflammation. To investigate the relationship between hs-CRP levels and bronchial asthma. We collected blood samples from 109 patients with bronchial asthma, with or without attacks, and measured serum eosinophil cationic protein levels, pulmonary function, and serum CRP levels using an hs-CRP assay. Mean serum hs-CRP levels were significantly higher in patients without attacks (0.473 mg/L) and with attacks (0.908 mg/L) (P < .001 for both) than in controls (0.262 mg/L). Serum hs-CRP levels were inversely correlated with forced expiratory volume in 1 second/forced vital capacity in asthmatic patients (r = -0.4915; P < .01). Serum hs-CRP levels may be related to the state of asthma exacerbation and allergic inflammation.Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology 08/2007; 99(1):48-53. · 2.83 Impact Factor -
Article: RANTES production from mononuclear cells in response to the specific allergen in asthma patients.
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ABSTRACT: Eosinophils are considered to be the major inflammatory cells in asthma. Since regulated on activation, normal T expressed and secreted (RANTES) is a potent chemoattractant for various important inflammatory cells such as eosinophils as well as memory T cells potentially recruiting these cells to an inflamed focus, RANTES has been considered to play a key role in various allergic disorders such as asthma. To extend our understanding of the participation of eosinophils and T cells in relation to the production of RANTES in response to the specific allergen in asthma, we examined the production of RANTES from peripheral blood mononuclear cells cultured with specific allergen in atopic asthma patients by a sandwich enzyme-linked immunosorbent assay. It was revealed that mononuclear cells produced RANTES but not eotaxin in response to the specific allergen in asthma. RANTES production from mononuclear cells of asthma patients with eosinophilia was greater than that of asthma patients without eosinophilia. Moreover, in this study, no differences in RANTES production between CD4 negative cells and CD8 negative cells were observed. Taken together, these findings may suggest that mononuclear cells play a crucial role in the pathogenesis, particular in eosinophil and T lymphocyte recruitment into the inflamed focus of asthma through RANTES production in response to the specific allergen.Allergology International 10/2006; 55(3):253-9. -
Article: Theophylline and dexamethasone induce peroxisome proliferator-activated receptor-gamma expression in human eosinophils.
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ABSTRACT: Eosinophils are major effector cells in allergic diseases including asthma. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates immune reaction. We have previously demonstrated that human eosinophils express PPARgamma and that stimulation with a synthetic agonist for PPARgamma attenuated the factor-induced eosinophil survival and chemotaxis. However, the modulator of the eosinophil PPARgamma expression has not yet been studied. In this study, we investigated the effect of theophylline and dexamethasone (widely used drugs in the treatment of asthma) on PPARgamma expression in eosinophils. Purified human peripheral blood eosinophils were cultured, and therapeutic concentrations of theophylline and dexamethasone were added. Subsequently, PPARgamma was measured using quantitative real-time RT-PCR and flow cytometry. Theophylline and dexamethasone markedly enhanced both mRNA and protein levels of PPARgamma. These findings suggest that the increase in PPARgamma expression on eosinophils may play a role in the anti-inflammatory effects of theophylline and dexamethasone.Pharmacology 02/2006; 77(1):33-7. · 1.79 Impact Factor -
Article: Procaterol upregulates peroxisome proliferator-activated receptor-gamma expression in human eosinophils.
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ABSTRACT: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates immune reaction. We have previously demonstrated that human eosinophils express PPARgamma and that stimulation with a synthetic agonist for PPARgamma attenuated the factor-induced eosinophil activations. However, the modulator of PPARgamma expression in eosinophils has not yet been studied. In this study, we investigated the effect of procaterol, the synthetic beta2-adrenoceptor agonist widely used as bronchodilators in asthma, on the PPARgamma expression in eosinophils. Purified human peripheral blood eosinophil and the eosinophilic cell line EoL-1 were cultured with procaterol. This was followed by PPARgamma measurement using flow cytometer and quantitative real-time RT-PCR. We observed that PPARgamma was constitutively expressed by EoL-1 and the purified eosinophils and that the therapeutic concentration (10(-9)M) of procaterol markedly enhanced PPARgamma protein expression, which was reversed by the selective beta2-adrenoceptor antagonist ICI-118551. The PPARgamma mRNA expression in EoL-1 and eosinophils was also induced by procaterol. These findings suggest that procaterol could modulate the eosinophil function by increasing the expression of PPARgamma.International Archives of Allergy and Immunology 02/2006; 140 Suppl 1:35-41. · 2.40 Impact Factor -
Article: Prostaglandin D2 induces IL-8 and GM-CSF by bronchial epithelial cells in a CRTH2-independent pathway.
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ABSTRACT: Prostaglandin D(2) (PGD(2)), a major prostanoid produced by activated mast cells, has long been implicated in allergic diseases. PGD(2) demonstrates its effects through two G-protein-coupled receptors, DP and CRTH2. The PGD(2)/CRTH2 system mediates chemotaxis of eosinophils, basophils, and Th2 cells, which are involved in the induction of allergic inflammation. Although recent studies have shown that the specific receptors for PGD(2), DP, and CRTH2 are expressed in various human tissues, the role of PGD(2) is unknown in human bronchial epithelial cells. In this study, we investigated the expression and function of CRTH2/DP on NCI-H(292) and NHBE cells. The CRTH2/DP expression was examined by RT-PCR and flow-cytometric analysis. NCI-H(292) and NHBE cells were cultured in the presence of various stimulants. The resulting supernatants were measured by ELISA. We demonstrated that PGD(2) induced production of IL-8 and GM-CSF in NCI-H(292) and NHBE cells. DK-PGD(2) (CRTH2 agonist) and latanoprost (FP, a prostaglandin F receptor, agonist) failed to augment the production of these cytokines. Pretreatment with ramatroban (CRTH2 antagonist) and AL8810 (FP antagonist) did not reduce the production of these cytokines. The PGD(2)-induced cytokine production was inhibited by pertussis toxin or specific inhibitors for MAP/ERK kinase (PD98059) and p38 MAP kinase (SB202190). These results suggest that PGD(2) is a potent inducer of IL-8 and GM-CSF production with MAP/ERK and p38 MAP kinase activation, but this is independent of CRTH2 activation.International Archives of Allergy and Immunology 02/2006; 141(3):300-7. · 2.40 Impact Factor -
Article: Physiological levels of 15-deoxy-Delta12,14-prostaglandin J2 prime eotaxin-induced chemotaxis on human eosinophils through peroxisome proliferator-activated receptor-gamma ligation.
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ABSTRACT: 15-Deoxy-Delta12,14-PGJ2 (15d-PGJ2), mainly produced by mast cells, is known as a potent lipid mediator derived from PGD2 in vivo. 15d-PGJ2 was thought to exert its effects on cells exclusively through peroxisome proliferator-activated receptor-gamma (PPARgamma) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), which are both expressed on human eosinophils. However, the physiological role of 15d-PGJ2 remains unclear, because the concentration generated in vivo is generally much lower than that required for its biological functions. In the present study we found that low concentrations (picomolar to low nanomolar) of 15d-PGJ2 and a synthetic PPARgamma agonist markedly enhanced the eosinophil chemotaxis toward eotaxin, and the effect was decreased in a dose-dependent manner. Moreover, at a low concentration (10(-10) M), 15d-PGJ2 and troglitazone primed eotaxin-induced shape change and actin polymerization. These priming effects were completely reversed by a specific PPARgamma antagonist, but were not mimicked by CRTH2 agonist 13,14-dihydro-15-keto-PGD2, suggesting that the effects were mediated through PPARgamma ligation. The effect exerted by 15d-PGJ2 parallels the enhancement of Ca2+ influx, but is not associated with the ERK, p38 MAPK, and NF-kappaB pathways. Furthermore, the time course and treatment of eosinophils with actinomycin D, an inhibitor of gene transcription, indicated that the transcription-independent pathway had a role in this process. PPARgamma might interact with an eotaxin-induced cytosolic signaling pathway, because PPARgamma is located in the eosinophil cytosol. Taken together with current findings, these results suggest that under physiological conditions, 15d-PGJ2 contributes to allergic inflammation through PPARgamma, which plays a role as a biphasic regulator of immune response.The Journal of Immunology 12/2005; 175(9):5744-50. · 5.79 Impact Factor -
Article: RANTES and eotaxin enhance CD11b and CD18 expression on eosinophils from allergic patients with eosinophilia in the application of whole Blood flow cytometry analysis.
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ABSTRACT: C-C chemokines and adhesion molecules expressed on eosinophils play an important role in the pathology of allergic inflammatory disease. C-C chemokines such as eotaxin or RANTES are involved in beta(2) integrin expression on purified eosinophils; so far we have no data on unpurified eosinophils in the peripheral blood. We measured beta(1) and beta(2) integrin activation after stimulation with eotaxin or RANTES in vitro using whole-blood flow-cytometric analysis. Heparinized whole blood obtained from allergic patients with eosinophilia or normal subjects was diluted with the same volume of RPMI 1640, and then cells were incubated in the presence or absence of PMA/ionomycin or chemokines for 45 min at 37 degrees C. After hemolyzation with lysing solution, expression of CD11b, CD11a, CD18 and CD49d on eosinophils was measured using flow cytometry. The expression of CD11b, CD11a and CD18 in allergic patients was significantly higher than that in normal subjects. CD11b and CD18 expression showed a significant increase after stimulation with C-C chemokines, which was remarkable in allergic patients. Eosinophils in the blood of allergic patients exhibited a higher expression of beta(2) integrins and were more sensitive to RANTES and eotaxin than those of normal subjects.International Archives of Allergy and Immunology 02/2005; 137 Suppl 1:12-6. · 2.40 Impact Factor -
Article: [Hospital infection control reduced medical care costs by saving dispensable antibiotics].
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ABSTRACT: Medical costs associated with hospital-acquired infection is a critical problem for hospital management. The introduction of the Diagnosis Procedure Combination (DPC) system requires the re-evaluation of the cost-effectiveness of any medical procedure including the usage of antibiotics. Achieving high cost-effectiveness and quality of medical service are essential for hospitals to survive the current changes in medical systems. Inappropriate use of antimicrobial agents results in unnecessary exposure to medication, persistent or progressive infection, emergence of resistance, and increased costs. We undertook an observational pre and post-intervention study to assess whether a comprehensive antimicrobial management program developed by the new ICT installed 2002 in Akita University Hospital could reduce the use of antibiotics. Annual total amounts of antibiotics and anti-MRSA antibiotics, and the number of patients undergoing long-term treatment with antibiotics, fell dramatically. This ICT approach thus reduced antibiotic costs, contributed to infection control, and improved the quality of antibiotic prescription.Rinsho byori. The Japanese journal of clinical pathology 01/2005; 52(12):1001-6. -
Article: Peroxisome proliferator-activated receptor gamma regulates eosinophil functions: a new therapeutic target for allergic airway inflammation.
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ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates lipid metabolism and glucose homeostasis. PPARgamma is not only highly expressed in adipose tissue but also in cells involved in the immune system, and it exerts anti-inflammatory activities. We showed that eosinophils, a major inflammatory cell in allergic inflammation, express PPARgamma. PPARgamma negatively modulates eosinophil functions, such as survival, chemotaxis, antibody-dependent cellular cytotoxicity and degranulation. Recently, three independent groups have demonstrated that PPARgamma agonists inhibit airway inflammation in an animal model of asthma. This evidence suggests that PPARgamma agonists may be a new therapeutic modality for the treatment of allergic diseases including asthma.International Archives of Allergy and Immunology 07/2004; 134 Suppl 1:30-6. · 2.40 Impact Factor -
Article: Effect of ketotifen on the production of reactive oxygen species from human eosinophils primed by eotaxin.
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ABSTRACT: Ketotifen is an antiallergic drug and may have direct inhibitory effects on eosinophils. To investigate the anti-eosinophilic effect of ketotifen, we examined the effect of ketotifen on the production of reactive oxygen species (ROS) from eotaxin-primed human eosinophils. Ketotifen at 10(-10)-10(-6) mol/l significantly reduced the production of ROS evoked by A23187 from eosinophils primed by eotaxin. In contrast, ketotifen at 10(-5) mol/l significantly augmented the production in the absence of eotaxin. We demonstrated that appropriate concentrations of ketotifen may have direct inhibitory effects on eosinophil oxidative metabolism primed by eotaxin. Ketotifen may contribute to the treatment of allergic disease through its anti-eosinophilic effects.Pharmacology 12/2003; 69(3):138-41. · 1.79 Impact Factor -
Article: [Progress in the study of allergy and collagen disease in the last 100 years: Drug allergy].
Nihon Naika Gakkai Zasshi 10/2002; 91(9):2688-92. -
Article: The role of mitogen-activated protein kinases in eotaxin-induced cytokine production from bronchial epithelial cells.
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ABSTRACT: Eotaxin is a critical chemokine eliciting migration of eosinophils and basophils in the pathogenesis of bronchial asthma. Recent studies have shown that the specific receptor for eotaxin, CCR3, is expressed in bronchial epithelial cells. Although mitogen-activated protein (MAP) kinases are involved in diverse cell functions of bronchial epithelial cells, their role in eotaxin signaling is unknown. In this study, we studied the activation and functional relevance of MAP kinases in bronchial epithelial cells stimulated with eotaxin. Eotaxin (1-100 nM) induced tyrosine/threonine phosphorylation and activation of extracellular regulated kinase (ERK) 1/2 and p38 in NCI-H(292) cells and normal human bronchial epithelial cells. The phosphorylation of these MAP kinases was detectable after 30 s, and peaked at 5 min. Eotaxin stimulated production of interleukin-8 and granulocyte macrophage colony-stimulating factor. Pretreatment of Compound X (a specific CCR3 antagonist), pertussis toxin, genistein, and wortmannin reduced the MAP kinase phosphorylation and cytokine production. The eotaxin-induced cytokine production was inhibited by specific inhibitors for MAP/ERK kinase (PD98059) and p38 MAP kinase (SB202190). These results suggest that both ERK1/2 and p38 MAP kinase activated by eotaxin have a critical role in the pathogenesis of asthma.American Journal of Respiratory Cell and Molecular Biology 10/2002; 27(3):329-35. · 5.13 Impact Factor -
Article: EG2 expressed by eosinophils as a clinically useful indicator of asthma.
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ABSTRACT: The monitoring of airway inflammation is mandatory for the improved control of bronchial asthma. We previously reported that intracellular EG2 levels of eosinophils, a marker of bronchial asthma increased in asthma patients. In this study, we hypothesized that eosinophil EG2(+) expression increases during airway inflammation in asthmatic individuals. Eosinophil EG2(+) and percentage eosinophil EG2(+) with whole blood flow cytometry, eosinophil counts, serum total IgE, serum eosinophil cationic protein, eosinophil-derived neurotoxin, and percent of forced expiratory volume in 1 second/force vital capacity (FEV(1)/FVC) were measured in 33 asthmatic patients and 22 healthy volunteers. The relationships between these markers were evaluated. Comparisons were made on EG2(+) expression between attack and asymptomatic periods in six asthmatic patients. EG2(+) expression was significantly greater in the asthmatic patients than in healthy subjects. Furthermore, the EG2(+) expression showed a significant increase during attacks. EG2(+) expression inversely correlated with the FEV(1)/FVC. These results suggest that EG2(+) expression may be a useful clinical marker of airway inflammation in asthma.Allergy and Asthma Proceedings 29(6):609-13. · 2.17 Impact Factor
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2002–2007
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Akita University Hospital
Akita, Akita-ken, Japan
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