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Journal of dermatological science 02/2012; 66(2):163-5. · 3.71 Impact Factor
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ABSTRACT: Cytosolic NADP(+)-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione.
To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant.
The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked.
The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H(2)O(2) compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H(2)O(2).
This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.
Journal of dermatological science 02/2012; 65(2):118-25. · 3.71 Impact Factor
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Sang Ho Oh,
Chang Ook Park,
Wen Hao Wu, Ji Young Kim,
Shan Jin,
Dashlkhumbe Byamba,
Byung Gi Bae,
Seongmin Noh,
Beom Jin Lim,
Ji Yeon Noh,
Kwang Hoon Lee
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ABSTRACT: Corticotropin-releasing hormone (CRH) is the central regulating hormone of the hypothalamic-pituitary-adrenal axis. CRH also has diverse functional effects in the periphery and is related to the aggravation of several cutaneous diseases; however, the effect of CRH on T cells in patients with atopic dermatitis (AD) has not been well evaluated.
We investigated whether CRH directly affects peripheral T(H)1, T(H)2, and regulatory T (Treg) cells in patients with AD.
We assessed whether T cells express the CRH receptor protein and mRNA by using flow cytometry, Western blotting, immunofluorescence, immunohistochemistry, and RT-PCR. We evaluated cytokine expression using ELISA after treating the T cells extracted from patients with AD and healthy control subjects (HCs) with CRH. Flow cytometry was then used to evaluate any direct effects of CRH on T(H)1, T(H)2, and Treg cells from patients with AD and HCs.
T cells from patients with AD expressed significantly lower CRH receptor 1/2 mRNA levels than T cells from HCs. T cells from HCs reacted with different IL-4 and IFN-γ secretions to CRH treatment, whereas T cells from patients with AD did not. IL-10 production was significantly decreased in the supernatants from both the HCs and patients with AD after CRH treatment. CRH upregulated IL-4 production by T(H)2 cells and downregulated IFN-γ production by T(H)1 cells in HCs. CRH also suppressed the production of IL-10 by forkhead box protein 3-negative Treg cells in both groups, but the difference was only significant in patients with AD.
CRH-mediated suppression of IL-10 secretion from Treg cells might explain stress-related exacerbations in patients with AD.
The Journal of allergy and clinical immunology 01/2012; 129(1):151-9.e1-6. · 9.17 Impact Factor
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Journal of dermatological science 06/2011; 62(3):202-4. · 3.71 Impact Factor
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Dermatologic Surgery 10/2010; 36(10):1642-3. · 1.80 Impact Factor
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Dermatologic Surgery 09/2010; 36(9):1457-8. · 1.80 Impact Factor
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ABSTRACT: Nerve growth factor (NGF) is a potent mediator, which regulates characteristics of mast cells, but its biological function is not well characterized. This study aimed to screen proteins associated with the maturation of human mast cells-1 (HMC-1) or mouse bone marrow-derived mast cells (BMMCs) cultured with NGF, and to examine the functions of proteins involved.
NGF (10 ng/ml) was added to cell culture medium every other day for 10 days for HMC-1 or twice a week for 5 weeks for BMMCs. Granule formation was determined by electron microscopy or May-Grunwald-Giemsa staining, TNF-alpha by ELISA, expressions of various proteins by two-dimensional gel electrophoresis (2-DE), siRNA transfection by Lipofectamine 2000, and the expressions of pyruvate kinase and annexin I by immunoblotting.
After NGF treatment, granule formation and total amounts of granular mediator, TNF-alpha increased in both mast cells. This TNF-alpha was released by calcium ionophore or by antigen/antibody reaction. Expressions of pyruvate kinase and annexin I obtained by 2-DE were confirmed by immunoblotting and siRNA-transfected HMC-1 cells. Expressions of proteins, granule formation and TNF-alpha content were blocked by both the TrkA inhibitor, K252a, and the ERK inhibitor, PD98059, but not by the PI3 kinase inhibitors, LY294002 and wortmannin.
These data suggest that pyruvate kinase and annexin I expressed by NGF contribute to granule formation containing TNF-alpha as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway.
International Archives of Allergy and Immunology 02/2008; 146(4):287-97. · 2.40 Impact Factor
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ABSTRACT: We previously reported that DA-9601, ethanol herbal extract of Artemisia asiatica, inhibited histamine and leukotriene releases in guinea pig lung mast cells activated with specific antigen/antibody reaction. This study aimed to evaluate the inhibitory effect of DA-9601 on the OVA-induced airway inflammation in allergic asthma mouse model. BALB/c mice were sensitized and challenged with OVA. DA-9601 was administered orally 1 h before every local OVA-challenge. OVA-specific serum IgE was measured by ELISA, recruitment of inflammatory cells in BAL fluids and lung tissues by Diff-Quik and H&E staining, respectively, the expressions of CD40, CD40L and VCAM-1 by immunohistochemistry, goblet cell hyperplasia by PAS staining, activities of MMPs by gelatin zymography, expressions of mRNA and proteins of cytokines by RT-PCR and ELISA, activities of MAP kinases by western blot, and activity of NF-KappaB by EMSA. DA-9601 reduced IgE level, recruitment of inflammatory cells into the BAL fluid and lung tissues, expressions of CD40, CD40L and VCAM-1 molecules, goblet cell hyperplasia, MMPs activity, expressions of mRNA and productions of various cytokines, activities of MAP kinases and NK-KappaB increased from OVA-challenged mice. These data suggest that DA-9601 may be developed as a clinical therapeutic agent in allergic diseases due to suppressing the airway allergic inflammation via regulation of various cellular molecules expressed by MAP kinases/NF-KappaB pathway.
Molecules and Cells 08/2006; 22(1):104-12. · 2.18 Impact Factor
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ABSTRACT: Eupatilin, an extract from Artemisia asiatica Nakai, is known to exert anti-gastric ulcer, anticancer, and anti-inflammatory effects. The aim of this study was to elucidate whether eupatilin has antiallergic reactions in activated guinea pig lung mast cells compared to apigenin and genistein. Mast cells were purified from guinea pig lung tissues by using enzyme digestion and rough and discontinuous density Percoll gradient. The purified mast cells were sensitized with immunoglobulin (Ig) G(1) (anti-OVA antibody) and challenged with ovalbumin (OVA). Histamine was assayed using an automated fluorometric analyzer, leukotrienes by radioimmunoassay, and tyrosine phosphorylation by immunoblotting. Intracellular Ca(2+) was analyzed by confocal laser scanning microscopy, protein kinase C (PKC) activity using protein phosphorylated with [gamma-(32)P]ATP, and phopholipase D activity (PLD) and phosphatidic acid by using labeled phosphatidyl alcohol. Eupatilin, apigenin, or genistein reduced histamine release and leukotriene synthesis in a does-dependent manner. Eupatilin inhibited mediators to a greater extent than apigenin or genistein. Eupatilin, apigenin, and genistein initially blocked phosphorylation of Syk tyrosine and Ca(2+) influx, PLD activity, phosphatidic acid, and Ca(2+)-dependent PKC alpha/betaII activities during mast cell activation in a dose-dependent manner. Our data suggest that eupatilin initially inhibits Syk kinase, and then blocks downstream multisignal pathways and Ca(2+) influx during mast cell activation triggered by a specific antigen-antibody reaction. Thus, eupatilin may have use clinically as a treatment for inflammatory disorders associated with allergic diseases including asthma.
Journal of Toxicology and Environmental Health Part A 01/2006; 68(23-24):2063-80. · 1.83 Impact Factor
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ABSTRACT: Peroxynitrite (ONOO-), the product of the reaction between the superoxide anion (*O2-) and nitric oxide (NO), is produced during inflammatory disease and may be a major cytotoxic agent. No reports are available as to whether ONOO- generates or modulates inflammatory mediator release from activated guinea pig lung mast cells. In this study, we explored the modulatory role of intracellular ONOO- on inflammatory mediator release (histamine and leukotrienes) from activated mast cells.
Guinea pig lung mast cells were purified by the enzyme digestion, and by using the rough and discontinuous Percoll density gradients. Mast cells were sensitized with IgG1 (anti-ovalbumin) antibody and challenged with ovalbumin (OVA). The intracellular ROS formation was determined by following the oxidative production of 2', 7'-dichlorofluorescein diacetate (DCFH-DA), dihydrorhodamine 123 (DHR), and anti-nitrotyrosine antibody immunofluorescence. Histamine was assayed using a fluorometric analyzer, leukotrienes by radioimmunoassay, intracellular Ca2+ levels by confocal scanning microscopy, and PLA(2) activity using prelabeling of [3H]arachidonic acid.
ROS detected by DCFH-DA weakly increased in mast cells activated with OVA (1.0 g/ml), and the ROS so generated was inhibited by ebselen (50 microM). However, the ROS detected by DHR increased 3-fold under the same conditions. Peroxynitrite scavengers sL-MT, DMTU, and inhibitor FeTPPS inhibited ROS formation but the NADPH oxidase inhibitor diphenyleneiodonium (DPI) only partially inhibited this formation. Dimethyl thiourea (DMTU) and seleno-L-methionine (sL-MT) inhibited the tyrosine nitration of cytosolic proteins, the release of histamine and leukotrienes, Ca2+ influx, and the PLA(2) activity evoked by mast cell activation.
The data obtained suggests that the ROS generated by the antigen/antibody reaction activated mast cells is ONOO-, and that this modulates the release of inflammatory mediators via Ca2+ -dependent PLA(2) activity.
International Archives of Allergy and Immunology 07/2005; 137(2):104-14. · 2.40 Impact Factor
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ABSTRACT: Histamine-releasing antibodies that act against the epitope of the alpha chain of Fc(epsilon)RI (anti-Fc(epsilon)RI(alpha) antibody) that may affect pathogenesis in serum of patients with chronic urticaria. We assessed the capability of anti-Fc(epsilon)RI(alpha) antibody in sera from patients with chronic urticaria to release histamine and cytokines, and to induce the expression of endothelial cell adhesion molecules. We also assessed the release of inflammatory mediators from cultured foreskin mast cells, and expression of endothelial cell adhesion molecules on human dermal microvascular endothelial cells. Cells were pretreated with mast cell-conditioned media: culture media of mast cells treated with sera from chronic urticaria patients containing anti-Fc(epsilon)RI(alpha) antibody. Histamine release from human foreskin mast cells challenged with sera, increased after both 20 min and 16 h intervals. Leukotriene D4 release also increased at both 20 min and 16 h. Tumor necrosis factor-alpha increased significantly in foreskin mast cell culture challenged with sera of chronic urticaria patients. After the stimulation of human dermal microvascular endothelial cells with the conditioned media, the expression of intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin increased significantly. Treatment of the conditioned media with anti-tumor necrosis factor-alpha monoclonal antibody partially inhibited the expression of intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. The data suggest that sera from patients with chronic urticaria containing anti-Fc(epsilon)RI(alpha) antibody release mediators and tumor necrosis factor-alpha by activating human foreskin mast cells. This release can play a pathogenic role in chronic urticaria by activating endothelial cells, in part due to the actions of tumor necrosis factor-alpha from mast cells.
Journal of Investigative Dermatology 05/2002; 118(4):658-63. · 6.31 Impact Factor
