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Liu X.,Jilin University | Ye F.,Mount Sinai School of Medicine | Xiong H.,Mount Sinai School of Medicine | Hu D.-N.,Tissue Culture Center | And 12 more authors.
Experimental Cell Research

IL-6 plays an important role in various inflammatory ocular diseases, including diabetic retinopathy. Müller cells are the major source of inflammatory mediators, including IL-6, in the retina. However, the mechanism of regulating IL-6 production in these cells remains unclear. Examination of signaling pathways in human retinal Müller cells (MIO-M1 cell line) cultured with IL-1β, TNF-α, IL-6, IL-8, VEGF, IFN-γ, glucose or mannitol showed that IL-1β was the most potent stimulator of IL-6 production. In addition, IL-1 β also increased NF-κB p50 protein level and phosphorylation of p38 MAPK, ERK1/2 and c-Jun. Induction of IL-6 production by IL-1β was significantly reduced by addition of p38 MAPK (SB203580), MEK1/2 (U0126) or NF-κB (BAY11-7082) inhibitors, with the highest effect being observed with SB203580. To explore the specific elements in IL-6 promoter responsible for IL-1β-induction of IL-6 expression, a series of plasmids bearing various IL-6 promoter mutations were transiently expressed in MIO-MI cells cultured in the presence or absence of IL-1β (10. ng/ml) and/or SB203580 (10. μM). Results showed that IL-6 promoter activity of the parent pIL-6-Luc651 was significantly enhanced by IL-1β, but the level was significantly attenuated by SB203580. Furthermore, the IL-6 promoter activity was also reduced upon deletion of NF-κB, AP-1 or C/EBP binding sites, with NF-κB deletion being the greatest. These results are the first demonstration that IL-1β induces IL-6 production in Müller cells by activation of IL-6 promoter activity predominantly through the p38 MAPK/NF-κB pathway. © 2014 Elsevier Inc. Source

Hong W.S.,Hangzhou Institute of Dermatology and Venereology | Hu D.N.,Tissue Culture Center | Qian G.P.,Hangzhou Institute of Dermatology and Venereology | McCormick S.A.,Tissue Culture Center | Xu A.E.,Hangzhou Institute of Dermatology and Venereology
Journal of the European Academy of Dermatology and Venereology

Background Transplantation of autologous cultured pure melanocytes is a well-established procedure for the treatment of refractory and stabilized vitiligo. However, there was no report specifically comparing the efficacy with the regard to defined age groups (children-adolescence-adult). Objective We analysed the efficacy of this procedure in the treatment of vitiligo in children and adolescents and compare it with the results in adults treated during the same period and using identical procedures. Methods Melanocytes were isolated from the roof of suction blister, cultured and expanded with Hu16 medium in vitro, and transplanted to laser-denuded receipt area. A total of 12 children (8-12 years), 20 adolescents (13-17 years) and 70 adults with vitiligo were treated using this procedure. Results The patients obtained satisfactory results (repigmentation of 50% or more) results in children, adolescents and adults were 83.3%, 95.0% and 84.0% respectively. The mean extent of repigmentation in children, adolescents and adults was 80.7%, 78.9% and 76.6% respectively. There was no statistical difference in repigmentation among these three groups. After adjusting for all factors (gender, type of vitiligo, period of stability, location of the lesion or transplanted cell density) individually or totally using multiple regression analysis, age still did not correlate to the extent of repigmentation. Conclusions The satisfactory results obtained in the treatment of vitiligo in children and adolescents by transplantation of cultured autologous pure melanocytes are comparable with the results in adults. Therefore, this procedure can be considered in refractory and stable vitiligo in children and adolescents, especially in patients with large vitiliginous lesions. © 2010 European Academy of Dermatology and Venereology. Source

Hu D.-N.,Tissue Culture Center | Hu D.-N.,New York Eye and Ear Infirmary at Mount Sinai Health Center | Bi M.,Tissue Culture Center | Bi M.,New York Medical College | And 5 more authors.
Investigative Ophthalmology and Visual Science

PURPOSE. Melanocytes are one of the major cellular components in the uvea. Interleukin-8/ CXCL8 and monocyte chemoattractant protein-1 (MCP-1/CCL2) are the two most important proinflammatory chemokines. We studied the constitutive and lipopolysaccharide (LPS)-induced expression of IL-8 and MCP-1 in cultured human uveal melanocytes (UM) and explored the relevant signal pathways.METHODS. Conditioned media and cells were collected from UM cultured in medium with and without stimulation of LPS. Interleukin-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Nuclear factor (NF)-jB in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases1/2 (ERK1/2), and c-Jun N-terminal kinase1/2 (JNK1/2) in cells cultured with and without LPS were measured by ELISA kits. Inhibitors of p38 (SB203580), ERK1/2 (UO1026), JNK1/2 (SP600125), and NF-jB (BAY11-7082) were added to the cultures to evaluate their effects.RESULTS. Low levels of IL-8 and MCP-1 proteins were detected in the conditioned media in UM cultured without serum. Lipopolysaccharide (0.01-1 lg/mL) increased IL-8 and MCP-1 mRNAs and proteins levels in a dose-and time-dependent manner, accompanied by a significant increase of phosphorylated JNK1/2 in cell lysates and NF-jB in nuclear extracts. Nuclear factor-jB and JNK1/2 inhibitors significantly blocked LPS-induced expression of IL-8 and MCP-1.CONCLUSIONS. This is the first report on the expression and secretion of chemokines by UM. The data suggest that UM may play a role in the pathogenesis of ocular inflammatory diseases. © 2014 The Association for Research in Vision and Ophthalmology, Inc. Source

Hong W.S.,Hangzhou Institute of Dermatology and Venereology | Hu D.N.,Tissue Culture Center | Qian G.P.,Hangzhou Institute of Dermatology and Venereology | McCormick S.A.,Tissue Culture Center | Xu A.E.,Hangzhou Institute of Dermatology and Venereology
British Journal of Dermatology

Summary Background Autologous melanocytes can be expanded in vitro, allowing the treatment of large lesions of vitiligo in one session. Theoretically, this procedure could provide a higher donor/recipient size ratio (DR ratio) compared with that in noncultured cell transplantation (with a DR ratio < 1: 10). However, the exact DR ratio obtained from this procedure has not been reported. Objectives To study whether transplantation of cultured pure melanocytes at a high DR ratio is as efficient as that at a low DR ratio. Methods One hundred and two patients with vitiligo were treated by transplantation of cultured pure melanocytes and were divided into two groups: a low DR ratio group, including patients with DR ratio ≤ 1: 10 (mean 1: 8, 35 cases) and a high DR ratio group with DR ratio > 1: 10 (mean 1: 27, 67 cases). The extent of repigmentation between these two groups was compared. Results There was no significant difference in repigmentation between the low DR ratio group (mean ± SD 77·4 ± 22·5%) and the high DR ratio group (77·6 ± 24·8%). Multiple regression analysis showed that even after adjustment for age, sex, type of vitiligo and transplanted cell density, there was no significant correlation between the extent of repigmentation and the DR ratio, indicating that patients treated with high DR ratio obtained a satisfactory result and showed no difference from the low DR ratio group. Conclusions Various surgical procedures for the treatment of vitiligo which involve melanocyte transplantation or skin grafts have different inherent DR ratios. Transplantation of cultured pure melanocytes is an expensive and complicated procedure; however, it provides the highest DR ratio (> 1: 10 and up to 1: 60). Surgeons can select one of these methods for the treatment of vitiligo based on their experience and skill, on the size of lesions, and the availability of laboratory support. © 2011 British Association of Dermatologists. Source

Yang P.-Y.,Show Chwan Memorial Hospital | Hu D.-N.,Changhua Christian Hospital | Lin I.-C.,Chung Shan Medical University | Liu F.-S.,Tissue Culture Center
American Journal of Chinese Medicine

Butein is a polyphenol, one of the compounds of chalcones, which are flavonoids that are widely biosynthesized in plants, and exhibits different pharmacological activities. Plants containing butein have been used in Chinese traditional medicine. Recently, it has been reported that butein suppresses proliferation and triggers apoptosis in various human cancer cells in vitro and in vivo. The aim of this study was to investigate its pro-apoptotic effect and mechanisms in two cultured human ovarian cancer cells (ES-2 and TOV-21G). The effects of butein on cell viability were assessed by a MTT assay at 3, 10, 30, and 100 μ/M. The apoptotic pathway related factors, including the mitochondrial transmembrane potential (MTP), cytochrome c, caspase cascade, and Bcl-2 family proteins, were examined. MTT assay revealed that butein was cytotoxic to both ovarian cancer cells in a dose-and time-dependent manner. JC-1 flow cytometry, cytochrome c, and caspase activity assays revealed that butein damaged the MTP, increased the level of cytosol cytochrome c and the activities of caspase-3,-8, and-9 in the two ovarian cancer cells. Western blot analysis revealed that butein down-regulated the anti-apoptotic proteins Bcl-2 and Bcl-xL and increased the pro-apoptotic proteins Bax and Bad. These findings suggest that butein-induced apoptosis in ovarian cancer cells via the activation of both extrinsic and intrinsic pathways. In addition, butein also down-regulated the expressions of the inhibitor of apoptosis (IAP) proteins, XIAP, survivin, CIAP-1, and CIAP-2. This indicates that the inhibition of IAP proteins was also involved in butein-induced apoptosis. The results of our study suggest that butein may be a promising anticancer agent in treating ovarian cancer. © 2015 World Scientific Publishing Company & Institute for Advanced Research in Asian Science and Medicine. Source

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