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ABSTRACT: Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.
Toxicological Sciences 09/2011; 124(2):359-69. · 4.65 Impact Factor
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ABSTRACT: Nickel is a potent hapten that induces contact hypersensitivity in human skin. While nickel induces the maturation of dendritic cells via NF-κB and p38 MAPK activation, it also exerts immunosuppressive effects on T cells through an unknown mechanism. To elucidate the molecular mechanisms of its effects on T cells, we examined the effects of NiCl(2) on mRNA expression in human CD3+ T cells stimulated with CD3 and CD28 antibodies. Using a DNA microarray and Gene Ontology, we identified 70 up-regulated (including IL-1β, IL-6 and IL-8) and 61 down-regulated (including IL-2, IL-4, IL-10 and IFN-γ) immune responsive genes in NiCl(2)-treated T cells. The DNA microarray results were verified using real-time PCR and a Bio-Plex(TM) suspension protein array. Suppression of IL-2 and IFN-γ gene transcription by NiCl(2) was also confirmed using Jurkat T cells transfected with IL-2 or IFN-γ luciferase reporter genes. To explore the NiCl(2)-regulated signaling pathway, we examined the binding activity of nuclear proteins to NFAT, AP-1, and NF-κB consensus sequences. NiCl(2) significantly and dose-dependently suppressed NFAT- and AP-1-binding activity, but augmented NF-κB-binding activity. Moreover, NiCl(2) decreased nuclear NFAT expression in stimulated T cells. Using Jurkat T cells stimulated with PMA/ionomycin, we demonstrated that NiCl(2) significantly suppressed stimulation-evoked cytosolic Ca(2+) increases, suggesting that NiCl(2) regulates NFAT signals by acting as a blocker of Ca(2+) release-activated Ca(2+) (CRAC) channels. These data showed that NiCl(2) decreases NFAT and increases NF-κB signaling in T cells. These results shed light on the effects of nickel on the molecular regulation of T cell signaling.
Toxicology and Applied Pharmacology 05/2011; 254(3):245-55. · 4.45 Impact Factor
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ABSTRACT: Although hydrogen peroxide (H(2)O(2)) is better known for its cytotoxic effects, in recent years it has been shown to play a crucial role in eukaryotic signal transduction. In respiratory tract epithelial cells, the dual oxidase (DUOX) proteins 1 and 2 has been identified as the cellular source of H(2)O(2). However, the expression of DUOX1 or DUOX2 has not yet been examined in keratinocytes. In this study, using a DNA microarray, we demonstrated that, of the seven NOX/DUOX family members in normal human epidermal keratinocytes (NHEK), IL-4/IL-13 treatment augments the expression of only DUOX1 mRNA. We next confirmed the IL-4/IL-13 induction of DUOX1 in NHEK at the mRNA and protein level using quantitative real-time PCR and Western blotting, respectively. In addition, we demonstrated that this augmented DUOX1 expression was accompanied by increased H(2)O(2) production, which was significantly suppressed both by diphenyleneiodonium, an inhibitor of NADPH oxidase, and by small interfering RNA against DUOX1. Finally, we demonstrated that the increased expression of DUOX1 in IL-4/IL-13-treated NHEK augments STAT6 phosphorylation via oxidative inactivation of protein tyrosine phosphatase 1B. These results revealed a novel role of IL-4/IL-13-induced DUOX1 expression in making a positive feedback loop for IL-4/IL-13 signaling in keratinocytes.
The Journal of Immunology 03/2011; 186(8):4762-70. · 5.79 Impact Factor
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ABSTRACT: Various environmental stimuli, e.g., mechanical stress, osmolarity change, oxidative stress, and microbial products trigger ATP release from cells. It is well known that ATP regulates cell growth, differentiation, terminal differentiation, and cell-to-cell communication in keratinocytes. Moreover, extracellular ATP stimulates the expression and release of IL-6 and modulates the production several chemokines by keratinocytes.
To investigate the role of ATP-stimulated keratinocytes in skin inflammation and immune response.
We identified genes whose expression is augmented in ATP-stimulated human keratinocytes by DNA microarray. These microarray data were validated by quantitative real-time RT-PCR. Furthermore, we confirmed the observed mRNA change at protein level by ELISA and Western blotting.
The statistical analysis of the microarray data revealed that, besides IL-6, the expression of several novel genes such as IL-20, CXCL1-3, and ATF3 was significantly augmented in ATP-stimulated keratinocytes. These data was validated by quantitative real-time RT-PCR. We also confirmed the augmented production of IL-6, IL-20, CXCL1 by ELISA and that of ATF3 by Western blotting. Since both IL-6 and IL-20 that can stimulate STAT3 were produced by the ATP-stimulated keratinocytes, we examined their phosphorylation of STAT3. The study demonstrated biphasic activation of STAT3 after ATP stimulation, which was composed of a first peak at 1-2 h and a second peak at 12-24 h. The latter peak was significantly suppressed by anti-IL-6 antibody.
These studies characterized (1) STAT3 activation, (2) chemotaxis for neutrophils via CXCL1-3, and (3) ATF3 activation as possible roles of ATP-stimulated keratinocytes in skin inflammation and immune response.
Journal of dermatological science 02/2010; 58(2):143-51. · 3.71 Impact Factor
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ABSTRACT: Myristic acid (MyA), which is a saturated fatty acid (C14:0) and a side chain of phorbol 12-myristate 13-acetate (PMA), was examined if MyA stimulates human polymorphonuclear leukocytes (PMNs) to release oxygen radicals comparable to PMA by applying electron paramagnetic resonance (EPR)-spin-trapping method. When MyA was added to isolated human PMNs, spin adducts of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH and DMPO-OOH were time-dependently observed. The amounts of these spin adducts were larger than those of PMNs stimulated by PMA. These results clearly show that MyA is more potent agent to prime human PMNs than PMA, in a point of view of not only O(2) (.-) but also .OH production. This fact calls attention that too much intake of MyA that is known to be contained vegetable oils can lead to crippling effect through uncontrolled production of reactive oxygen species.
Journal of Clinical Biochemistry and Nutrition 11/2009; 45(3):309-14. · 1.98 Impact Factor
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ABSTRACT: Early detection and optimal treatment are the most effective means to improve cancer mortality. Mass screening for cancer has yielded a marked reduction of cancer mortality in the United States. Simple and effective methods are expected for screening of malignancy. Hematoporphyrin derivatives (HPDs) are known to accumulate in cancer cells; thus, HPD has been used for local diagnosis and photodynamic therapy of cancer. The lymphocytes of cancer patients also demonstrate the active uptake of HPD and this phenomenon has been applied for the diagnosis of cancer. In the present study, we have developed a novel method for measurement of the chemiluminescence of HPD in peripheral blood lymphocytes. HPD is composed of hematoporphyrin and its oligomers. Seven cancer patients and seven controls were recruited for this study. The primary cancers included two prostate cancers (one without metastasis and the other with lung metastasis), a renal cancer, a lung adenocarcinoma with systemic metastasis, two gallbladder cancers with lung metastasis, and a colon cancer with liver metastasis. HPD in lymphocytes was measured using a highly sensitive chemiluminescence analyzer with laser light irradiation to detect photoemission by (1)O(2) from HPD. The intensity of chemiluminescence exhibited a linear correlation with the concentrations of HPD. In addition, the level of HPD in lymphocytes was significantly higher in cancer patients than that in healthy volunteers (p < 0.05). These results suggest that detection of the chemiluminescence of HPD in lymphocytes could be a sensitive and simple method for cancer diagnosis and screening.
The Tohoku Journal of Experimental Medicine 10/2008; 216(1):47-52. · 1.24 Impact Factor
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Takafumi Kimura,
Kohki Takahashi,
Yoko Suzuki,
Yukinaga Konishi,
Yuta Ota,
Chihiro Mori,
Takahiko Ikenaga,
Takako Takanami, Rumiko Saito,
Eiichiro Ichiishi,
Satoshi Awaji,
Kazuo Watanabe,
Atsushi Higashitani
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ABSTRACT: Magnetic resonance imaging with high static magnetic fields (SMFs) has become widely used for medical imaging purposes because SMFs cause fewer genotoxic side effects than ionizing radiation (IR). However, the effect of exposure to high SMFs on global transcription is little understood. We demonstrate that genes involved in motor activity, actin binding, cell adhesion, and cuticles are transiently and specifically induced following exposure to 3 or 5 T SMF in the experimental model metazoan Caenorhabditis elegans. In addition, transient induction of hsp12 family genes was observed after SMF exposure. The small-heat shock protein gene hsp16 was also induced but to a much lesser extent, and the LacZ-stained population of hsp-16.1::lacZ transgenic worms did not significantly increase after exposure to SMFs with or without a second stressor, mild heat shock. Several genes encoding apoptotic cell-death activators and secreted surface proteins were upregulated after IR, but were not induced by SMFs. Real-time quantitative RT-PCR analyses for 12 of these genes confirmed these expression differences between worms exposed to SMFs and IR. In contrast to IR, exposure to high SMFs did not induce DNA double-strand breaks or germline cell apoptosis during meiosis. These results suggest that the response of C. elegans to high SMFs is unique and capable of adjustment during long exposure, and that this treatment may be less hazardous than other therapeutic tools.
Bioelectromagnetics 06/2008; 29(8):605-14. · 1.84 Impact Factor
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ABSTRACT: DNA polymerase gamma and mtSSB are key components of the mtDNA replication machinery. To study the biological influences of defects in mtDNA replication, we used RNAi to deplete the gene for a putative mtSSB, par2.1, in Caenorhabditis elegans. In previous systematic RNAi screens, downregulation of this gene has not caused any clearly defective phenotypes. Here, we continuously fed a dsRNA targeting par2.1 to C. elegans over generations. Seventy-nine percent of F1 progeny produced 60-72 h after feeding grew to adulthood but were completely sterile, with an arrest of germline cell proliferation. Analyses of mtDNA copy number and cell cytology indicated that the sterile hermaphrodites had fewer mitochondria. These results indicated that par2.1 essentially functions for germline cell proliferation through mtDNA replication; we therefore termed it mtssb-1. Comprehensive transcriptional alterations including hypoxia response induction dependent on and independent of hif-1 function, occurred by RNAi depletion of mtssb-1. Treatment with ethidium bromide, which impairs mtDNA replication and transcription, caused similar transcriptional alterations. In addition, the frequency of apoptosis in the germline cells was reduced in fertile progeny with a partial RNAi effect. These suggest that RNAi depletion of C. elegans mtssb-1 is useful as a model system of mitochondrial dysfunction.
Experimental Cell Research 02/2008; 314(1):103-14. · 3.58 Impact Factor
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ABSTRACT: High-temperature stress causes abortive male reproductive development in many plant species. Here, we report a putative mechanism of high-temperature injury during anther early development in barley plants (Hordeum vulgare L). Under high-temperature conditions (30 degrees C day/25 degrees C night), cell-proliferation arrest, increased vacuolization, over-development of chloroplasts, and certain abnormalities of the mitochondria, nuclear membrane, and rough endoplasmic reticulum (RER) were observed in developing anther cells, but not in developing ovule cells. Moreover, premature degradation of tapetum cells and premature progression to meiotic prophase in pollen mother cells (PMCs) were also observed. To monitor transcriptional alterations during high-temperature injury, we performed DNA microarray analysis using the 22K Barley1 GeneChip. Expression profiles were captured at four time points during the early development of panicles, and during vegetative growth of seedlings as a control, with or without high-temperature treatment. Abiotic or biotic stress related genes were equally or more dominantly up-regulated in the seedlings exposed to high temperatures compared with the panicles. In contrast, certain genes associated with histones, DNA replication initiation, mitochondria, and ribosomes were specifically repressed in the exposed panicles. In situ hybridization studies indicated that repression locally occurred on the developing anther cells exposed to high temperatures. Microarray analysis also indicated that a series of genes, including a meiosis-specific gene Asy1 and anther-specific lipid transfer protein genes, was prematurely up-regulated at an earlier stage under high-temperature conditions. Real-time quantitative RT-PCR analyses well confirmed the expression differences of certain key genes predicted by the DNA microarrays. These results suggest that high-temperature causes premature progression of anther early development program and fate, such as progression to meiosis of PMCs, cell-proliferation arrest and degradation in anther wall cells, accompanied by comprehensive alterations in transcription.
Molecular and General Genetics 08/2007; 278(1):31-42. · 2.63 Impact Factor
