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Pam3CSK4 activates Toll-like receptors 2 and 1 (TLR1/2), which recognize mainly molecules from gram-positive pathogens. The effect of Pam3CSK4 on various cytokine and chemokine expression in cultured human uveal melanocytes (UM) has not been studied systematically. The purpose of this study was to investigate the mechanistic expressions of seven cytokines and chemokines of interleukin- (IL-) 6, IL-10, MCP-1 (CCL-2), CXCL-1 (GRO-α), CXCL-8 (IL-8), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in UM. These cytokines are reported to be increased in intraocular fluids or tissues of the patients with endophthalmitis and non-infectious uveitis, as well as in various experimental animal uveitic models in the literature. Flow cytometry was used to measure the effects of Pam3CSK4 on the expression of TLR1/2 in UM. ELISA and Real-time PCR analysis were used to estimate the ability of Pam3CSK4 to elevate these cytokines and chemokines levels in conditioned media and cell lysates of UM, respectively. Flow cytometry measured and compared the phosphorylated MAPK pathway and activated NF-κB signals pathway in UM, treated with and without Pam3CSK4. ELISA analysis tested the effect of various signal inhibitors (ERK1/2, JNK1/2, p38 and NF-κB) on Pam3CSK4-induced IL-6 levels in cultured UM. The role of TLR2 in Pam3CSK4-induced acute anterior uveitis in experimental mouse model was tested in TLR2 knockout (TLR2 KO) mice and their wild-type C57Bl/6 controls. Pam3CSK4 increased the expression of TLR1/2 proteins in cultured UM. Pam3CSK4 significantly elevated the IL-6, MCP-1, CXCL-1, CXCL-8 protein, and mRNA levels in cultured UM, but not IL-10, TNF-α, or IFN-γ. Pam3CSK4 activated NF-κB, ERK, JNK, and p38 expression. Pam3CSK4-induced expression of IL-6 was decreased by NF-κB, ERK, INK, and p38 inhibitors; especially the NF-κB inhibitor, which can completely block the IL-6 stimulation. Intravitreal injection of Pam3CSK4 induced acute anterior uveitis in C57Bl/6 mice, this effect was significantly reduced in TLR2 KO mice. TLR1/2 plays an important role against invading pathogens, especially gram-positive bacteria; but an excessive reaction to molecules from gram-positive bacteria may promote non-infectious uveitis. UM can produce IL-6, MCP-1, CXCL-1, and CXCL-8, and are one of the target cells of TNF-α and IFN-γ. TLR-2 inhibitors might have a beneficial effect in the treatment of certain types of uveitis and other ocular inflammatory-related diseases and warrant further investigation. • Pam3CSK4 (TLR1/2 agonist) activates TLR1/TLR2 in cultured uveal melanocytes (UM). • Pam3CSK4 increases IL6, MCP1, CXCL1/8 expression via MAPK and NF-κB pathways in UM. • TLR2 knock out inhibits Pam3CSK4-induced uveitis in mouse. • UM participate in immune response against gram-positive bacterial infection. • Excess activation of TLR1/2 may cause inflammation, such as non-infectious uveitis. [ABSTRACT FROM AUTHOR]

Purpose: Lipopolysaccharide (LPS) can activate Toll-like receptor 4 (TLR4) and increase the expression of CXCL1 and CXCL2, the potent neutrophils chemoattractants, in various cell types. These effects have not been previously reported in the uveal melanocytes. This study was designed to investigate the effects of LPS on the activation of TLR4 and expression of CXCL1/CXCL2 in cultured human uveal melanocytes and the relevant signal pathways. Methods: Effects of LPS on the expression of TLR4 were tested using real-time PCR, flow cytometry and fluorescence immunostaining. Effects of LPS-induced expression/secretion of CXCL1/CXCL2 were studied using real-time PCR in cell lysates and ELISA in conditioned media of cultured uveal melanocytes. Activated NF-κB and phosphorylated MAPK signals were tested in cells with and without LPS treatment using flow cytometry. Effects of various signal inhibitors on p38, ERK1/2, JNK1/2 and NF-κB on the secretion of CXCL1/CXCL2 were tested by ELISA. The effects of neutralized antibodies of CXCL1/CXCL2 on the severity of LPS-induced uveitis were tested in a mouse model. Results: LPS stimulation increased the expression of TLR4 mRNA and protein in culture uveal melanocytes. Constitutive secretion of CXCL1/CXCL2 was detected in uveal melanocytes and was significantly increased dose- and time-dependently by LPS stimulation. LPS mainly increased the activated NF-κB and phosphorylated JNK1/2. LPS-induced expression of CXCL1/CXCL2 was blocked by NF-κB and JNK1/2 inhibitors. The severity of LPS-induced uveitis was significantly inhibited by neutralizing antibody to CXCL1/CXCL2 Conclusions: This is the first report on the LPS-induced expression of CXCL1 and CXCL2 by uveal melanocytes via the activation of TLR4. These results suggest that uveal melanocytes may play a role in the immune reaction that eliminates the invading pathogens. Conversely, an excessive LPS-induced inflammatory reaction may also lead to the development of inflammatory ocular disorders, such as non-infectious uveitis. [ABSTRACT FROM AUTHOR]

While fibroblasts constitute the main cell component of the sclera, the purpose of the present study was to investigate the cell densities of melanocytes at different regions of the sclera, and to compare them with associated scleral fibroblast densities in human donor eye sections.. Paraffin-embedded sections of sclera from 21 human eyes were stained with hematoxylin-eosin (H&E) and immunohistochemical staining (S-100/AEC). Scleral melanocyte and fibroblast numbers were counted in different regions of the sclera. The relationship between the melanocyte density and iris pigmentation was also analyzed.. Melanocytes were found in the posterior region of the sclera, especially around the vessels and nerves in emmissarial canals, whereas no or rare melanocytes were found in equatorial and anterior regions. In H&E sections, melanocyte densities in eyes with light-colored irides were significantly less than in eyes with medium or dark-colored irides (P <.05). In S-100-stained sections, more melanocytes could be detected than those in the H&E sections in light-colored eyes (P <.05), but not in medium or dark-colored eyes (P >.05). The numbers of scleral fibroblasts were relatively stable in different regions. In the posterior scleral region, the numbers of fibroblasts were slightly higher than the number of melanocytes, however, this differences were not statistically significant (P >.05).. Notable numbers of melanocytes were present in the posterior sclera suggesting that these cells may play a role in ocular physiology and in the pathogenesis of various disorders of the sclera. [ABSTRACT FROM AUTHOR]

Butein is a chalcone, a flavonoid that is widely biosynthesized in plants. Butein has been identified to possess varied pharmacological activity and is extractable from traditional Chinese medicinal herbs, therefore applicable for disease treatment. Recently, in vitro and in vivo studies have shown that butein may induce apoptotic cell death in various human cancer cells. In this study we investigated the apoptotic effect of butein and the underlying mechanisms in human cervical cancer cells. Two cell lines, C-33A and SiHa cells, were treated with butein at different dosages for different durations. The effect of butein on cell viability was assessed by MTT assay, which revealed that butein exerted cytotoxicity in both cervical cancer cells in a dose- and time-dependent fashion. Apoptotic pathway-related factors in the butein-treated cervical cancer cells were then examined. JC-1 flow cytometry, cytochrome c assay, and caspase activity assays demonstrated that butein disturbed mitochondrial transmembrane potential, and increased cytosolic cytochrome c levels and caspase activities in both cervical cancer cells. Western blot analysis revealed that butein downregulated anti-apoptotic protein Bcl-xL and led to proteolytic cleavage of poly (ADP-ribose) polymerase. In addition, butein decreased expressions of the inhibitor of apoptosis (IAP) proteins, including X-linked IAP, survivin, and cellular IAP-1. The findings of this study suggest that butein can decrease cervical cancer cell viability via a pro-apoptotic effect, which involves inhibition of the IAP proteins and activation of both extrinsic and intrinsic pro-apoptotic pathways. Therefore, butein may be applicable for cervical cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract Matrix metalloproteinase (MMP)-8 is the most potent MMP for degrading collagen type-1 and plays an important role in inflammatory reactions and tissue remolding processes. MMP-8 is expressed mainly by polymorphonuclear leukocytes and is not expressed constitutively by most non-leukocytes. We studied the constitutive and TNF-α-induced expression of MMP-8 in cultured human uveal melanocytes (UM) and the relevant signal pathways involved. Conditioned media and cells were collected from UM and other cell types. MMP-8 proteins and mRNA were measured using ELISA kit, western blot and real time RT-PCR, respectively. Phosphorylated p38 MAPK, ERK1/2, and JNK1/2 were measured by ELISA kit and western blot. Very high levels of MMP-8 proteins and mRNA were detected in the conditioned media and cell lysates in 11 UM cell lines and three uveal melanoma cell lines cultured without serum, but not in media and cell lysates from other ocular resident cells or 12 malignant cell lines from other tissues, with exception of cutaneous melanoma cells. TNF-α moderately increased MMP- 8 mRNA and protein levels in a dose- and time-dependent manner, accompanied by a significant increase of phosphorylated JNK1/2 and ERK1/2 in cell lysates. ERK1/2 (U0126) and JNK1/2 (SP600125) inhibitors significantly blocked TNF-α-induced and constitutive expression of MMP-8 in UM. This is the first report on the expression and secretion of MMP-8 by UM and uveal melanoma cells. The data suggest that UM may play a role in the remolding process and pathogenesis of inflammatory-related diseases in the eye via secretion of MMP-8. Highlights • Cultured uveal melanocytes constitutively express high levels of MMP-8. • Other ocular cells and cancers from other tissues do not express MMP-8. • TNF-α significantly increases MMP-8 levels via JNK1/2 and ERK pathways. • Melanocyte may participate remolding and inflammatory process via MMP-8. [ABSTRACT FROM AUTHOR]

Purpose. To identify an effective method to prevent myopia progression by characterizing the regulation of matrix metalloproteinase- (MMP-) 2 expression and its secretion from scleral fibroblasts and retinal pigment epithelium (RPE) cells by miR-29a. Methods. The effects of miR-29a on the growth of scleral fibroblasts and RPE cells were assessed using the cell counting kit-8. The changes in MMP-2 mRNA levels in scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor were measured by quantitative PCR. Enzyme-linked immunosorbent assays were used to determine the changes in MMP-2 secretion from scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor. Results. The miR-29a mimics or inhibitor did not significantly alter the growth of scleral fibroblasts or RPE cells at 24, 48, or 72 hours after transfection. MMP-2 mRNA levels were significantly decreased in scleral fibroblasts and RPE cells transfected with the miR-29a mimics. The secretion of MMP-2 by scleral fibroblasts and RPE cells was significantly decreased in cells transfected with the miR-29a mimics. Conclusions. Suppression of scleral fibroblast and RPE cell expression and secretion of MMP-2 by miR-29a can be used as a therapeutic target for the prevention and treatment of myopia. [ABSTRACT FROM AUTHOR]

Purpose: To study the relationships between matrix metalloproteinases (MMP)-2, MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3 aqueous humor levels in patients with high myopia or cataract. Materials and Methods: MMPs and TIMPs protein levels in 65 aqueous humor samples collected from patients with high myopia or cataract during cataract or clear lens extraction surgery were measured with the Luminex xMAP Technology. The relationship between MMPs and TIMPs levels was analyzed with Spearman’s correlation test. Results: MMP-2 levels, but not MMP-3 levels, were increased in the aqueous humor from high-myopia patients. Levels of TIMP-1, -2, and -3 were positively and very significantly correlated with the MMP-2 levels (TIMP-1:r=0.626,p< 0.001; TIMP-2:r= 0.545,p< 0.001; TIMP-3:r= 0.439,p< 0.001). TIMP-2 and-3 levels did not significantly correlate with MMP-3 levels (TIMP-2:r= 0.175,p> 0.05; TIMP-3:r= 0.127,p> 0.05) and TIMP-1 levels only marginally correlated with MMP-3 levels (r= 0.278, 0.01

Background Hypoxia is the most important stimulus leading to up-regulation of vascular endothelial growth factor (VEGF) in the retina via elevation of hypoxia-inducible factors-1α (HIF-1α) protein. The purpose of this study was to test the effects of melatonin on the expression of VEGF and HIF-1α in the cultured human retinal pigment epithelial (RPE) cells under normoxia and hypoxia. Method An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2) to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without melatonin under normoxia and hypoxia. The protein and RNA levels of VEGF and HIF-1α were measured by ELISA kits and RT-PCR, respectively. Result Hypoxia induced a significant increase of expression and secretion of VEGF and accumulation of HIF-1α protein in RPE cells ( P < 0.05). Melatonin at 10−5 to 10−8 M significantly inhibited hypoxia-induced expression, the secretion of VEGF and the accumulation of HIF-1α protein ( P < 0.05), but not affected expression of VEGF and HIF-1α under normoxia ( P > 0.05). Conclusion This study suggests that melatonin may have potential value in the prevention and treatment of various retinal diseases associated with increase of VEGF, vascular leakage and angiogenesis. [ABSTRACT FROM AUTHOR]

Lutein, zeaxanthin, and meso-zeaxanthin are xanthophyll carotenoids found within the retina and throughout the visual system. The retina is one of the most metabolically active tissues in the body. The highest concentration of xanthophylls is found within the retina, and this selective presence has generated many theories regarding their role in supporting retinal function. Subsequently, the effect of xanthophylls in the prevention and treatment of various eye diseases has been examined through epidemiological studies, animal studies, and clinical trials. This paper attempts to review the epidemiological studies and clinical trials investigating the effects of xanthophylls on the incidence and progression of various eye diseases. Observational studies have reported that increased dietary intake and higher serum levels of lutein and zeaxanthin are associated with lower risk of age-related macular degeneration (AMD), especially late AMD. Randomized, placebo-controlled clinical trials have demonstrated that xanthophyll supplementation increases macular pigment levels, improves visual function, and decreases the risk of progression to late AMD, especially neovascular AMD. Current publications on the preventive and therapeutic effects of lutein and zeaxanthin on cataracts, diabetic retinopathy, and retinopathy of prematurity have reported encouraging results. [ABSTRACT FROM AUTHOR]

Uveal melanoma cells were inoculated into the choroid of nude mice and treated with or without intraocular injection of zeaxanthin. After 21 days, mice were sacrificed and the eyes enucleated. Histopathological analysis was performed in hematoxylin and eosin stained frozen sections. Melanoma developed rapidly in the control group (without treatment of zeaxanthin). Tumor-bearing eye mass and tumor mass in the control group were significantly greater than those in zeaxanthin treated group. Melanoma in the controlled eyes occupied a large part of the eye, was epithelioid in morphology, and was with numerous mitotic figures. Scleral perforation and extraocular extension were observed in half of the eyes. Melanomas in zeaxanthin treated eyes were significantly smaller with many necrosis and apoptosis areas and no extraocular extension could be found. Quantitative image analysis revealed that the tumor size was reduced by 56% in eyes treated with low dosages of zeaxanthin and 92% in eyes treatment with high dosages of zeaxanthin, as compared to the controls. This study demonstrated that zeaxanthin significantly inhibits the growth and invasion of human uveal melanoma in nude mice, suggesting that zeaxanthin may be a promising agent to be explored for the prevention and treatment of uveal melanoma. [ABSTRACT FROM AUTHOR]

Objective: To determine the nationwide incidence of non-traumatic subconjunctival hemorrhage (NTSCH) in Taiwan from 2000-2011 and to analyze the risk factors for NTSCH using a case-control analysis. Methods: This is a population-based cohort administrative database study. Randomly selected 1,000,000 residents from the Taiwan National Health Insurance Research Database in 2000 and followed for 12 years to determine the population incidence of NTSCH. Individuals with the first diagnosis of subconjunctival hemorrhage were identified by the corresponding International Classification of Diseases code (ICD) 372.72. Traumatic subconjunctival hemorrhages (ICD-9 codes 921, 871, 850-854 and 959.01) were excluded. The association of NTSCH with various demographic factors, comorbidities and use of medications was studied by a population based case-control analysis using data of 2008-2011. Results: A total of 67,720 patients with a first-time diagnosis of NTSCH were identified during the 12 years period. The mean annual incidence was 65 per 10,000 individuals (crude incidence) and 60 per 10,000 individuals (age- and sex-standardized incidence). The incidence rate of NTSCH was higher in women than in men, [men-women ratio: 0.80 (95% confidence interval: 0.78-0.81)]. The age-specific incidence decreased from childhood to the group of teenagers, after which it increased to a maximal value of 136.2 per 10,000 people in the age group of 60-69 years. Case-control analysis showed that comorbidities of hypertension, purpura and thrombocytopenia, and the use of aspirin were significantly associated with the risk of NTSCH. Conclusions: This study indicates that NTSCH is a common eye disease that occurs once in 167 individuals in a general East Asian population per year. It occurs more often in women than in men and the age-specific incidence peaked in the age group of 60-69 years. Hypertension, purpura and thrombocytopenia, and the use of aspirin are the major risk factors for NTSCH. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis. • FSL-1 (TLR2/6 agonist) activates TLR2 and TLR6 in cultured uveal melanocytes (UM). • FSL-1 increases CXCL1/2 expression via JNK and NF-κB signal pathways in UM. • Invitreal injection of FSL-1 induces acute noninfectious uveitis in the mouse. • TLR2 antagonist inhibits FSL-1-induced mouse uveitis. • UM may participate in immune response against gram-positive bacterial infection. [ABSTRACT FROM AUTHOR]

Antrodia camphorata is a Chinese herb indigenous to Taiwan. Previous reports demonstrated that it could induce apoptosis in some cancer cells. The purpose of this study was to investigate the apoptotic effect of the crude extract of A. camphorata in cervical cancer cells. Two human cervical cancer cell lines, and C-33A, were treated with extract of A. camphorata (10-1000 μg/mL). We found that A. camphorata extract was cytotoxic to both cervical cancer cells in a dose- and time-dependent manner as examined by MTT assay. Treatment with A. camphorata extract at 400 μg/mL induced a 2.3- and 4.4-fold increase in oligonucleosome formation from the cleaved chromosomal DNA in and C-33A cells, respectively. A. camphorata extract also activated caspase-3, -8, and -9 activities and increased the cytosolic level of cytochrome c in both cell lines as the dosage increased. Furthermore, A. camphorata extract increased expressions of Bak, Bad and Bim, while decreasing expressions of Bcl-2 and Bcl-xL of the Bcl-2 family proteins in and C-33A cells. The expression of IAP proteins, XIAP and survivin, was also decreased in both cervical cancer cells after treatment with A. camphorata. Our in vitro study suggests that A. camphorata is cytotoxic to cervical cancer cells through both extrinsic and intrinsic apoptotic mechanisms. It could be used as a novel phytotherapeutic agent or auxiliary therapy in the treatment of cervical cancer. [ABSTRACT FROM AUTHOR]

Oxidative stress and autoimmune reaction are involved in the pathogenesis of vitiligo. Levels of hydrogen peroxide (HO) and interleukin-6 (IL-6), a proinflammation cytokine and a key factor in the pathogenesis of autoimmune diseases, have been reported to be elevated in vitiligo lesions. The objective of this study was to evaluate the effects of subtoxic levels of HO on the expression of IL-6 by cultured human epidermal melanocytes and to explore the relevant signal pathways. Cultured human melanocytes were stimulated with of HO at subtoxic levels. Levels of IL-6 protein in the medium and IL-6 mRNA in the cells were measured by IL-6 ELISA analysis and RT-PCR, respectively. NF-κB and phosphorylated p38 mitogen-activated protein kinase (MAPK), ERK and JNK in cells cultured with and without HO were measured by relevant ELISA kits. In cultured melanocytes, subtoxic levels of HO (30-300 μM) significantly increased the IL-6 mRNA and protein levels in a dose-dependent manner. NF-κB in nuclear extracts and phosphorylated p38 MAPK levels in cell lysates were significantly increased in HO treated cells. Pretreatment of cells with inhibitors of p38 MAPK (SB203580) and NF-κB (BAY11-7082), but not inhibitors of ERK (UO1026) and JNK (SP600125), abolished HO-induced expression of IL-6. HO-induced overexpression of IL-6 by melanocytes may be a molecular linkage for the oxidative stress and inflammatory/autoimmune reactions in vitiligo and may provide a novel target for the treatment of vitiligo. [ABSTRACT FROM AUTHOR]

Abstract: Increased tear osmolarity is an essential feature of dry eye disease. Curcumin, a natural polyphenol extracted from herb turmeric, has recently been reported to have anti-inflammatory effects. However, its anti-inflammatory effects have not been investigated in dry eye disease. It has been reported that elevated osmolarity achieved by adding sodium chloride to the culture medium of corneal epithelial cells increased the production of IL-1β, a proinflammation cytokine. This in vitro dry eye model was used to test the anti-inflammatory effects of curcumin. In the present study, a 450 mOsM hyperosmotic medium was produced by adding sodium chloride to the culture medium to reach a final concentration of 90mM. Human corneal epithelial cells cultured in this hyperosmotic medium for 24h showed an increase of IL-1β, IL-6 and TNF-α levels in the conditioned medium. IL-1β was also upregulated at mRNA levels. Activation of p38 MAP kinase (p38), JNK MAP kinase (JNK) and NF-κB in cultured corneal epithelial cells were also induced by hyperosmotic conditions. Curcumin at concentrations of 1–30μM did not affect the cell viability of cultured corneal epithelial cells. Pretreatment of curcumin (5μM) completely abolished the increased production of IL-1β induced by the hyperosmotic medium. Increased phosphorylation of p38 caused by high osmolarity was also completely abolished by curcumin, whereas the phosphorylation of JNK was only partially inhibited. SB 203580 (p38 inhibitor), but not SP 600125 (JNK inhibitor), completely suppressed hyperosmoticity-induced IL-1β production, indicating that the inhibition of production of IL-1β by curcumin may be achieved through the p38 signal pathway. Curcumin completely abolished a hyperosmoticity-induced increase of NF-κB p65. NF-κB inhibitor suppressed hyperosmoticity-induced IL-1β production. p38 inhibitor suppressed hyperosmoticity-induced NF-κB activation, indicating that NF-κB activation was dependent on p38 activation. The present study suggests that curcumin might have therapeutic potential for treating dry eye disease. [Copyright &y& Elsevier]

Purpose: To determine whether postoperative oral 13-cis-retinoic acid (RA) treatment could improve the outcome of vitreoretinal surgery with silicone oil for the management of proliferative vitreoretinopathy (PVR). Design: Prospective controlled randomized interventional case series. Methods: This study included 35 eyes of 35 patients with primary rhegmatogenous retinal detachment and PVR. All patients underwent surgical repair by similar procedures. The RA group consisted of 16 patients who received 10 mg oral RA twice daily for eight weeks postoperatively. The control group included 19 patients without taking RA. The outcome measure included the rate of retinal attachment, macular pucker formation, ambulatory vision, and RA–related side effects. Results: At last follow-up (at least one year postoperatively), 15 of 16 eyes (93.8%) in the RA group and 12 of 19 eyes (63.2%) in the control group maintained retinal attachment (P = .047). The rate of macular pucker formation was significantly lower in the RA group (18.8% vs 78.9% in the control group; P = .001). A higher rate of ambulatory vision was achieved in the RA group as compared to the control group (56.3% vs 10.5%; P = .009). Conclusions: Postoperative administration with oral moderate dosage of RA for eight weeks appears to maintain retinal attachment, decrease the macular pucker, and improve vision after surgical repair for eyes with PVR. [Copyright &y& Elsevier]

Melanin has a photo-screening, a biophysical/biochemical and a cosmetic effect. Melanin content of cultured pigmented cells can be measured by spectrophotometry and expressed either as melanin content per cell or melanin content per culture (area). Melanin production can be calculated from melanin content and cell number at the beginning and at the end of a culture using various formulas and expressed as melanin production per cell per day or melanin production per culture per day. Melanin content or production per cell have been used widely to compare melanin content in various cell lines or to compare the melanin content during different stages in the culture ( e.g. growing stage and senescent stage). For the evaluation of changes in melanin content and production in a given pigment cell line after treatment with a special chemical, physical or biological stimulator or inhibitor, different parameters used for the evaluation of experimental data can lead to conflicting results. Melanin content per area is determined by melanin content per cell and the number of cells in this area. The biological and cosmetic effects of melanin in vivo are determined mainly by melanin content per area, not melanin content per cell. For example, if melanin content per cell is the same, but the number of cells in a given area is increased after the treatment, then the melanin content per area is also increased. Under this circumstance, the color of skin turns darker and the total antioxidant activity provided by melanin in this area is increased even though the melanin content per cell measured remains the same; therefore, melanin content or production per culture is more important than melanin content or production per cell under this circumstance. [ABSTRACT FROM AUTHOR]

Purpose: To investigate racial and ethnic differences in the incidence of conjunctival melanoma in a large population-based study. Design: Observational cross-sectional study. Methods: Using data from 1992 through 2003 provided by the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) program, we calculated age-adjusted incidence rates of conjunctival melanoma in various racial and ethnic groups (Black, American Indian, Asian and Pacific Islander, Hispanic, and non-Hispanic White). In addition, we calculated the standard incidence ratios (risk ratios) and 95% confidence intervals to describe the differences within these racial and ethnic groups. Results: From 1992 through 2003, there were a total of 168 conjunctival melanomas diagnosed in 13 SEER registries with known racial and ethnic groups. The annual age-adjusted incidence rates (per million population) of conjunctival melanoma was 0.18 (Blacks), 0.17 (American Indians), 0.15 (Asians), 0.33 (Hispanics), and 0.49 (non-Hispanic Whites). The difference in the incidence of conjunctival melanoma between Whites and Blacks or Asians was statistically significant, but was not significant between Blacks and Asians. Conclusions: The overall White-to-Black incidence ratio in conjunctival melanoma was 2.6:1, which is much less than that of uveal melanoma (18:1) and cutaneous melanoma (13:1 to 26:1), but is similar to that of mucosal melanoma (2.2:1 to 2.3:1). The cause and significance of this difference of racial and ethnic incidence in various melanomas are discussed. [Copyright &y& Elsevier]

Abstract: The purpose of the present study was to develop methods for isolation, purification and cultivation of human conjunctival melanocytes. Conjunctiva excised from donor eyes or corneal rims was subjected with various enzyme digestion methods or by the enzyme-microdissection method. Cells were cultured with F12 medium supplemented by fetal bovine serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin. Contaminant cells were eliminated by a selective cytotoxic agent, geneticin. Both trypsin digestion and dispase-microdissection methods provided pure conjunctival melanocyte cultures with high cell yields, good viability and rapid growth rate. Melanocytes isolated with dispase-microdissection method showed better viability and growth capacity. Cells grew well, could be passaged for 5–10 generations and divided 20 times in vitro. They maintained a constant melanin content per cell and produced measurable amounts of melanin in vitro. Melanogenesis correlated with the degree of pigmentation of the eyes (iris color). This method provides a valuable source of large numbers of human conjunctival melanocytes, which can be used to study their biological behavior, to compare with the epidermal and uveal melanocytes; and to compare them to their malignant counterparts in the exploration of the pathogenesis of conjunctival melanoma. [Copyright &y& Elsevier]

It has been reported that retinoic acid (RA) may inhibit the growth of RPE and be used in the treatment of proliferative vitreoretinopathy (PVR). However, previous reports in this field have been conflicting. The main reason for these contradictory findings is that different methods for evaluating the effects of RA on RPE from different species have been used. In human specimens, only RPE from the donor eye (stationary) but not RPE from the PVR membrane (already at active proliferation status) have been tested. This study tested the effects of RA on the growth of RPE using a novel in vitro model: RPE from the PVR membranes, which simulates the in vivo situation of PVR patients better than RPE from the donor eyes. This study also used various methods to solve the conflicting results reported previously. We found that both all transretinoic acid (all-RA) and 13-cis-retinoic acid (cis-RA) can promptly (though not completely) inhibit proliferation of RPE (inhibition rate of 89%-90%) over a very wide range of concentrations (10-9-10-5 M) and various lengths of periods (2-12 days) in a dosedependent and time-dependent manner and without evident cytotoxic effects. Previously reported disadvantages discovered from the study of RPE from donor eyes, e.g., the absence of inhibitory effects on the early passages of cultured cells and inhibition occurring only after long-term treatment, do not present in RPE cells from the PVR membrane. The proliferation of RPE recovered from the inhibition by RA rapidly after the discontinuation of treatment, indicating that a continuous supply of the drug over a long period, i.e., until the breakdown of the blood-retinal barrier has been repaired, is essential for the success of drug treatment of PVR. [ABSTRACT FROM AUTHOR]

Uveal melanocytes and the ocular pigment epithelium are located in the middle and inner layers of the eye. Müller cells (a type of glial cell) are located in the neural retina. Melanocytes, retinal pigment epithelium (RPE), and Müller cells do not participate directly in the detection or transfer of visual information, but they have various functions that support the neural retina and are essential for the maintenance of vision. Methods for the isolation and cultivation of melanocytes, RPE, and Müller cells have been established by us and other investigators. These cultured cells can be used as in vitro model systems for studying the toxicology of visible light, ultraviolet (UV) radiation, drugs, and other potentially toxic agents. Toxic effects on these cells may give rise to altered retinal function and result in impaired vision. Both melanocytes and pigment epithelium contain melanin, which has the ability to bind organic amines and metal ions. This results in the accumulation of these substances in the eye. Melanin may protect cells from chemical stress by binding toxic chemicals; but in chronic exposure, increased and lengthy binding may cause damage to these cells. Two different types of melanin are found in the eye: eumelanin and pheomelanin, which may have photoprotective and phototoxic effects, respectively. Pigment epithelium contains mainly eumelanin, whereas melanocytes contain both eumelanin and pheomelanin. Melanin is an antioxidant and with age, the antioxidant properties may diminish to the point that it may even become a prooxidant. There are also other functions of pigment epithelium and uveal melanocytes not related to melanin and there are also several functions of Müller cells that play a role in the toxicological aspects of the eye. Cultured uveal melanocytes, pigment epithelial cells, and Müller cells can be used to study the toxicology of these cells in vitro. [ABSTRACT FROM AUTHOR]

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 ± 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 ± 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow. [ABSTRACT FROM AUTHOR]

Purpose: To investigate racial/ethnic differences in the development of uveal malignant melanoma in a large population-based study. Design: Observational cross-sectional study. Methods: With the 1992 to 2000 data that was provided by the National Cancer Institute’s Surveillance, Epidemiology, and End Results Program, we calculated age-adjusted incidence rates of uveal melanoma in various racial/ethnic groups (black, Asian and Pacific Islander, Hispanic, and non-Hispanic white). In addition, we calculated the standard incidence ratio (risk ratio) and 95% confidence interval to describe the differences within these racial/ethnic groups. Results: From 1992 to 2000, there were a total of 1352 uveal melanomas that were diagnosed in 11 Surveillance, Epidemiology, and End Results registries with known racial/ethnic groups. The annual age-adjusted incidence (per million population) of uveal melanoma was 0.31 (black), 0.38 (Asian), 1.67 (Hispanic), and 6.02 (non-Hispanic white). The difference in the incidence of uveal melanoma between each racial/ethnic group was highly statistically significant, with the exception of the black versus the Asian population in which there was no statistically significant difference. Conclusion: The relative risk of uveal melanoma was 1.2 for Asian and Pacific Islander patients, 5.4 for Hispanic patients, and 19.2 for non-Hispanic white patients as compared with the black patients. If the non-Hispanic white population and the Hispanic population were combined, then the overall white:black ratio was 18:1. [Copyright &y& Elsevier]

Purpose: To study the cytotoxic effects and related signaling pathways of butein on human uveal melanoma cells in vitro. Materials and Methods: Three human uveal melanoma cell lines (M17, SP6.5, and C918), retinal pigment epithelial (RPE) cells and scleral fibroblasts were treated with butein at different dosages. The effects of butein on cell viability were assessed by using the MTT assay. Cell apoptosis was determined using annexin V-FITC/ethidium homodimer III flow cytometry. Mitochondrial transmembrane potential changes were assessed by using the JC-1 fluorescent reader, cytosol cytochrome c levels, and the activities of caspase-3, -8, and -9 were measured by using an enzyme-linked immunosorbent assay or colorimetric assay. Results: Butein reduced the cell viability of cultured human uveal melanoma cells in a dose-dependent manner (10, 30, and 100 μM), with IC50 at 13.3 μM and 15.8 μM in SP6.5 and M17 cell lines, respectively. Similar effects were also found in a highly aggressive and metastatic C918 cell line (IC50 16.7 μM). Butein at lower concentrations (10-30 μM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of RPE cells and fibroblasts. Butein-induced apoptosis of melanoma cells, increased mitochondrial permeability and the level of cytosol cytochrome c, caspase-9 and -3 activities (but not caspase-8) in a dose-dependent manner. Conclusions: Butein has selectively potent pro-apoptotic effects on cultured human uveal melanoma cells via the intrinsic mitochondrial pathway. [ABSTRACT FROM AUTHOR]

Purpose: To study the cytotoxic effects and related signaling pathways of curcumin on human uveal melanoma cells in vitro. Materials and Methods: Two human uveal melanoma cell lines (M21 and SP6.5), scleral fibroblasts, and choroidal melanocytes were treated with curcumin. The effects of curcumin on cell viability were assessed by using the MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay. Cytosol cytochrome c levels and the activities of caspase-9 and caspase-3 were measured by using an enzyme-linked immunosorbent assay. Results: Curcumin induced cell death of cultured human uveal melanoma cells in a dose-dependent manner (10, 30, and 100 μM) and time-dependent manner (3–48 hr), with IC50 at 19.05 μM and 22.39 μM in M21 and SP6.5 cell lines, respectively. Curcumin at lower concentrations (10–30 μM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of fibroblasts and choroidal melanocytes. Curcumin significantly increased the level of cytosol cytochrome c (2-fold greater than the controls after 2 hr treatment), caspase-9 and caspase-3 activities (approximately 4.5- and 6-fold greater than the controls after 2–6 hr treatment, respectively) in a dose-dependent manner. Conclusions: Curcumin has selectively potent cytotoxic effects on cultured human uveal melanoma cells. This effect is associated with the release of cytochrome c from the mitochondria and the activation of caspase-9 and caspase-3 in uveal melanoma cells after treatment with curcumin. [ABSTRACT FROM AUTHOR]

Abstract: All-trans retinoic acid (atRA) has been reported to inhibit the proliferation of retinal pigment epithelial (RPE) cells and used in treatment of proliferative vitreoretinopathy (PVR) in animal model. This study aimed at examining the effectiveness of atRA in inhibiting the extracellular matrix (ECM) biosynthesis by RPE cells and the RPE cell-mediated collagen gel contraction. Cultured RPE cells were treated with atRA and the expression of four ECM proteins (collagen types I, III, IV and laminin β1) was profiled. The results indicated that atRA treatment up-regulated de novo synthesis of collagen type I, but decreased that of laminin β1 in a dose-dependent manner. Moreover, the effect of atRA on RPE cell contraction was evaluated by measuring the area of collagen gel where RPE cells populated. Treatment with atRA significantly inhibited RPE cell-mediated collagen gel contraction. Addition of exogenous laminin nonapeptide into gels promoted RPE cell contraction, while atRA reversed the laminin-enhanced contractility. atRA treatment significantly suppressed the gene expression of integrin β3 but not αV subunit, and effectively inhibited the tyrosine phosphorylation of integrin β3 at residue 747 in RPE cells grown on laminin-coated dish, suggesting that atRA may suppress the RPE contractility through either inhibiting integrin β3 expression or abrogating the integrin β3-mediated signaling. In conclusion, atRA pharmacologically possesses a tissue-remodeling capacity and inhibits contractility of RPE cells. Therefore, atRA might be potentially a therapeutic agent for certain ocular disorders such as PVR. [Copyright &y& Elsevier]

The effects of various antiproliferative drugs have been tested in numerous cell types in vitro, but a comparison of effects of these drugs on cultured human retinal pigment epithelium (RPE) has not been reported. We studied the effects of four most widely used antiproliferative drugs (5-FU, daunomycin, mitomycin and dexamethasone) in a new in vitro model system (cultured human RPE). Various concentrations and exposure periods were tested in four human RPE cell lines. 5-FU showed a dose-dependant growth inhibition, with an ID[sub 50] of 5.35 ± 2.38 × 10[sup -7] M after 4 days culture. A decrease of cell number occurred relatively later, the slope of the dose-response curve was less steep than that of others. Mitomycin C showed an immediate and strong growth inhibition and cytotoxic effects, with an ID[sub 50] of 3.73 ± 0.71 × 10[sup -9] M. RPE cultured with daunomycin showed an abrupt decrease of cell number from 10[sup -9] M to 10[sup -8] M, the ID value was 1.07 ± 0.23 × 10[sup -8] M. Dexamethasone showed a biphasic effect; it stimulated cell growth at 10[sup -7] to 10[sup -6] M and inhibited cell growth at 10[sup -4] M or higher, with an ID[sub 50] of 6.05 ± 1.61 × 10[sup -3] M. The advantages and disadvantages of these drugs and the prospective clinical application of these drugs for management of proliferative vitreoretinopathy (PVR) were discussed. Development of an in vitro model using cultured human RPE to study the effects of various antiproliferative drugs can provide a rapid, safe and inexpensive method for selection of drugs used for management of proliferative vitreoretinopathy. [ABSTRACT FROM AUTHOR]

The article describes the case of a 22-year-old woman with progressive white spots on her face. She was diagnosed to have vitiligo with repigmentation of leukotrichia. It also discusses the transplantation of autologous cultured melanocytes in liquid media, retrograde migration with the use of a fibrin matrix, and the effectiveness of hair follicle grafting.

N6‐methyladenosine (m6A) is a novel epitranscriptomic marker that contributes to regulating diverse biological processes through controlling messenger RNA metabolism. However, it is unknown if m6A RNA methylation affects uveal melanoma (UM) development. To address this question, we probed its function and molecular mechanism in UM. Initially, we demonstrated that global RNA m6A methylation levels were dramatically elevated in both UM cell lines and clinical specimens. Meanwhile, we found that METTL3, a main m6A regulatory enzyme, was significantly increased in UM cells and specimens. Subsequently, cycloleucine (Cyc) or METTL3 targeted small interfering RNA was used to block m6A methylation in UM cells. We found that Cyc or silencing METTL3 significantly suppressed UM cell proliferation and colony formation through cell cycle G1 arrest, as well as migration and invasion by functional analysis. On the other hand, overexpression of METTL3 had the opposite effects. Furthermore, bioinformatics and methylated RNA immunoprecipitation‐quantitative polymerase chain reaction identified c‐Met as a direct target of m6A methylation in UM cells. In addition, western blot analysis showed that Cyc or knockdown of METTL3 downregulated c‐Met, p‐Akt, and cell cycle‐related protein levels in UM cells. Taken together, our results demonstrate that METTL3‐mediated m6A RNA methylation modulates UM cell proliferation, migration, and invasion by targeting c‐Met. Such a modification acts as a critical oncogenic regulator in UM development. [ABSTRACT FROM AUTHOR]

Purpose Understanding the association of iris colour and myopia may provide further insights into the role of the wavelength of lights in the pathophysiology of myopia. We aim to assess the association of iris colour and myopia in a school-based sample of Chinese students. Methods Two thousand three hundred and forty-six Year 7 students from 10 middle schools (93.5% response rate) aged 13-14 years in Mojiang, a small county located in Southwestern China, participated in the study. We obtained standardised slit lamp photographs and developed a grading system assessing iris colour (higher grade denoting a darker iris). Refractive error was measured after cycloplegia using an autorefractor by optometrists or trained technicians. An IOLMaster () was used to measure ocular biometric parameters including axial length ( AL). Results Of all the study participants, 693 (29.5%) were affected by myopia with the prevalence estimates being higher in girls (36.8%; 95% confidence interval [ CI]: 34.0, 39.6) than in boys (22.8%; 95% CI: 20.4, 25.1) ( p < 0.001). After adjusting for gender, height, parental history of myopia, time spent on computer, time spent watching TV, time spent outdoors, and time spent reading and writing, participants with a darker iris colour tended to have a higher prevalence of myopia, a more myopic refraction and a longer AL. Dose-response relationships were observed in all regression models ( p for trend <0.05). Conclusions Darker iris colour was associated with more myopic refractive errors and longer ALs among Chinese school-aged children and this association was independent of other known myopia-related risk factors. [ABSTRACT FROM AUTHOR]

Purpose: Previous studies of the relationship between visual acuity (VA) and ocular dominance have produced conflicting results. We hypothesized that (1) the discrepancies were related mostly to sample size and interocular visual acuity difference (IOVAD); (2) in large samples of individuals with marked IOVADs, the eye with the better uncorrected distance visual acuity (UDVA) would be dominant. These hypotheses were tested in a large group of myopic patients. Methods: This prospective study of cycloplegic refraction involved 2045 myopic refractive surgery candidates. Patients with amblyopia or strabismus were excluded. Ocular dominance was assessed using the hole-in-the-card test. Results: In 2045 patients, the dominant eye had significantly better UDVA (p= 0.028) and was less astigmatic (p= 0.000) than the nondominant eye. In 426 patients with marked interocular difference in the UDVA (≥0.2 logMAR), the dominant eye not only had significant UDVA (p= 0.022) but also significantly less myopic (p= 0.028) and had a shorter axial length (AL;p= 0.001). In patients with smaller differences in UDVA (0.1 logMAR,n= 411) or no difference (n= 1208), the dominant and nondominant eyes did not differ significantly with respect to UDVA, myopic power, and AL (p> 0.05). Conclusions: The present study showed that the dominant eyes had significantly better UDVA than the nondominant eyes, especially in individuals with marked differences in UDVA. These results supported our hypothesis regarding the relationship between better VA and ocular dominance. [ABSTRACT FROM PUBLISHER]

As increases in hepatocyte growth factor/scatter factor (HGF/SF) induce retinal pigment epithelial (RPE) migration and proliferation into the vitreous cavity and contribute to proliferative vitreoretinopathy (PVR) development, we determined if changes in miR-182 expression affect such behavioral changes. We found that miR-182 expression was less in PVR clinical samples than in primary RPE cells whereas c-Met was upregulated. Ectopic miR-182 inhibited RPE cell proliferation, cell cycle, and migration. Bioinformatic analysis identified c-Met as a miR-182 target, which was confirmed with the luciferase reporter assay. Transfection of miR-182 into RPE cells induced c-Met downregulation, which led to reduced cell proliferation and migration through declines in p-Akt formation. MiR-182 downregulation along with c-Met upregulation in PVR tissues suggest that these two opposing effects play important roles in PVR development. As ectopic miR-182 expression suppressed RPE cell proliferation and migration, strategies to selectively upregulate miR-182 expression in a clinical setting may provide a novel option to treat this disease. [ABSTRACT FROM AUTHOR]

Dry eye is a common disorder characterized by deficiency of tear. Hyperosmoticity of tear stimulates inflammation and damage of ocular surface tissues and plays an essential role in the pathogenesis of dry eye. Cultured human corneal epithelial (CE) cells were used for the study of effects of lutein and hyperosmoticity on the secretion of IL-6 by CE cells. Cell viability of CE cells was not affected by lutein at 1–10 μM as determined by MTT assay. Hyperosmoticity significantly elevated the secretion of IL-6 by CE cells as measured by ELISA analysis. The constitutive secretion of IL-6 was not affected by lutein. Lutein significantly and dose-dependently inhibited hyperosmoticity-induced secretion of IL-6. Phosphorylated- (p)- p38 MAPK, p-JNK levels in cell lysates and NF-κB levels in cell nuclear extracts were increased by being exposed to hyperosmotic medium. JNK, p38, and NF-κB inhibitors decreased hyperosmoticity-induced secretion of IL-6. Lutein significantly inhibited hyperosmoticity-induced elevation of NF-κB, p38, and p-JNK levels. We demonstrated that lutein inhibited hyperosmoticity-induced secretion of IL-6 in CE cells through the deactivation of p38, JNK, and NF-κB pathways. Lutein may be a promising agent to be explored for the treatment of dry eye. [ABSTRACT FROM AUTHOR]

Zeaxanthin and lutein are two carotenoid pigments that concentrated in the retina, especially in the macula. The effects of lutein and zeaxanthin on the prevention and treatment of various eye diseases, including age-related macular degeneration, diabetic retinopathy and cataract, ischemic/hypoxia induced retinopathy, light damage of the retina, retinitis pigmentosa, retinal detachment, and uveitis, have been studied in different experimental animal models. In these animal models, lutein and zeaxanthin have been reported to have beneficial effects in protecting ocular tissues and cells (especially the retinal neurons) against damage caused by different etiological factors. The mechanisms responsible for these effects of lutein and zeaxanthin include prevention of phototoxic damage by absorption of blue light, reduction of oxidative stress through antioxidant activity and free radical scavenging, and their anti-inflammatory and antiangiogenic properties. The results of these experimental animal studies may provide new preventive and therapeutic procedures for clinical management of various vision-threatening diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α (HIF-1α) protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1α expression in the primary culture of human retinal pigment epithelial (RPE) cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2) to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1α protein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1α in RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1α protein caused by hypoxia but did not affect expression of VEGF and HIF-1α under normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1α levels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization. [ABSTRACT FROM AUTHOR]

Fullerol is a fullerene derivative that is extensively hydroxylated [nano-C60(OH)24] and this makes it water-soluble. These fullerene derivatives have shown promise as drug carriers that bypass ocular barriers but fullerols are also potentially phototoxic to human lens and retinal tissues. Fluorescence imaging is a powerful and non-invasive means of probing nanoparticles in biological systems. However, fullerol nanoparticles have a very low level of fluorescence and have not as yet been imaged in vitroand in vivo. Using specialized measurements including time-correlated single photon counting (TCSPC), fullerol fluorescence was determined in aqueous solutions and detected in both human lens and retinal pigment epithelial cells. Time-resolved fluorescence of fullerol (5–200 μM) was characterized in aqueous environment, where the fluorescence decay is best fitted with three lifetimes (3 ns, 0.7–0.9 ns and 0.2 ns). Time-resolved microspectrofluorimetry and time-gated fluorescence imaging were performed on both human lens and retinal pigment epithelial cells incubated with increasing fullerol doses (5–500 μM and 5–50 μM, respectively). Upon increasing concentration, we observe some shortening of the lifetimes, a reduction in the relative amplitude of the shortest-living component and a corresponding increase in the weight of the intermediate-living species. Time-gated imaging of fullerol fluorescence provided information on its intracellular distribution that correlates with progressive cell damage. Therefore time-gated imaging may potentially be used as a means to investigate fullerol distribution and toxicity in the human lens and retina in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Involvement of activities of mitogen-activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs) remains unsolved in norcantharidin-associated breast cancer cell apoptosis. This study investigated the anti-cancer effect of norcantharidin and its underlying mechanism in two human breast cancer cell lines, estrogen receptor (ER)− HS-578T and ER+ MCF-7 cells. Norcantharidin induced potent cytotoxicity and arrested cell growth through increasing phosphorylation of Chk1, Chk2 and total p21Waf1/Cip1 and reducing cyclin B and cdc25c expression. It also induced apoptosis through extrinsic death receptor and intrinsic mitochondrial pathways by cytochrome c release, caspase activation, oligonucleosome appearance, PARP cleavage, and aberration of Bcl-2 family protein expression and phosphorylation. Although norcantharidin did not affect STAT1, STAT3, and STAT5 protein expression, it suppressed STAT3 and STAT5 phosphorylation in HS-578T cells, whereas it up-regulated STAT1 phosphorylation and down-regulated STAT5 phosphorylation in MCF-7 cells. Moreover, norcantharidin activated MAPK family member proteins, extracellular signal-regulated kinase (ERK), p38MAPK and c-Jun N-terminal kinase (JNK), were all phosphorylated by treatment. Pretreatment with selective kinase inhibitors significantly attenuated the norcantharidin-induced cytotoxicity in breast cancer cells. These findings suggest the potential involvement of MAPK and STAT pathways in norcantharidin-induced apoptogenesis. Norcantharidin may be an effective anti-cancer drug against breast cancer. [Copyright &y& Elsevier]

Uveal melanoma, the most common primary intraocular malignancy in adults, is highly resistant to most chemotherapeutic drugs. Arsenic trioxide (ATO) is known to inhibit ocular melanoma cell growth. However, the effects of ATO on human uveal melanoma cells are poorly understood. Therefore, this study evaluated the mechanisms of ATO and its inhibiting effects on a human uveal melanoma cell line (SP6.5). An MTT assay indicated that, compared to human fibroblasts, ATO had a stronger inhibiting effect on SP6.5 cell proliferation in a dose- and time-dependent manner. The apoptosis ratio in SP6.5 cells, which was indicated by cell DNA fragmentation, was 4.1- to 7.7-fold higher after ATO-treatment. The ATO treatment substantially increased the activities of caspase-3 and caspase-9, but not of caspase-8. These findings were consistent with the protein expression observed by Western blots. ATO also significantly enhanced expression of Bax and cytochrome c proteins but suppressed those of Bcl-2. Therefore, ATO-induced apoptosis in uveal melanoma cells occurs mainly through the mitochondrial pathway rather than through the death receptor pathway. This report is the first to evaluate the complete mitochondria-dependent apoptotic pathway of ATO in uveal melanoma cells. These results can be used to improve the clinical effectiveness of ATO treatment for uveal melanoma. [ABSTRACT FROM AUTHOR]

Abstract: Norcantharidin exhibits cytotoxicity in many cancer cell lines, including colorectal cancer (CRC) cells. Its cytotoxic potency on primary CRC cells and other normal constituent cells of the human body remains elusive. This study investigates whether norcantharidin differentially exhibits cytotoxicity on primarily isolated CRC cells and dermal fibroblasts. The in vitro chemosensitivity of norcantharidin was measured using a MTT tetrazolium assay and compared with 73 primary tumor cells from surgically excised colorectal tumors, six human CRC cell lines and dermal fibroblasts. Observations of cytotoxicity to primary tumor cells reveal significant differences among genders and histological types; however, drug-induced chemosensitivity was not correlated with age or clinical stages of CRC patients. Norcantharidin had a similar cytotoxic effect on primary tumor cells and CRC cell lines in a dose-dependent manner. In contrast, normal fibroblasts were more resistant to norcantharidin-induced cytotoxicity than CRC cells. DAPI staining results demonstrated that norcantharidin caused CRC cell apoptosis by nuclear fragmentation and chromatin condensation. The release of cytochrome c and the triggering of caspase-3, -8 and -9 activation mediated apoptotic induction. Conversely, pretreatment with caspases or mitogen-activated protein kinase (MAPK) inhibitors significantly suppressed norcantharidin-induced CRC cytotoxicity. These in vitro results suggest that norcantharidin may be a safe and effective anti-cancer drug for CRC. [Copyright &y& Elsevier]

Abstract: The water-soluble nanoparticle hydroxylated fullerene [fullerol, nano-C60(OH)22–26] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have previously found that fullerol is both cytotoxic and phototoxic to human lens epithelial cells (HLE B-3) and that the endogenous antioxidant lutein blocked some of this phototoxicity. In the present study we have found that fullerol induces cytotoxic and phototoxic damage to human retinal pigment epithelial cells. Accumulation of nano-C60(OH)22–26 in the cells was confirmed spectrophotometrically at 405 nm, and cell viability, cell metabolism and membrane permeability were estimated using trypan blue, MTS and LDH assays, respectively. Fullerol was cytotoxic toward hRPE cells maintained in the dark at concentrations higher than 10 μM. Exposure to an 8.5 J·cm−2 dose of visible light in the presence of >5 μM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 μM lutein offered some protection against fullerol photodamage. Using time resolved photophysical techniques, we have now confirmed that fullerol produces singlet oxygen with a quantum yield of Φ =0.05 in D2O and with a range of 0.002–0.139 in various solvents. As our previous studies have shown that fullerol also produces superoxide in the presence of light, retinal phototoxic damage may occur through both type I (free radical) and type II (singlet oxygen) mechanisms. In conclusion, ocular exposure to fullerol, particularly in the presence of sunlight, may lead to retinal damage. [Copyright &y& Elsevier]

Abstract: Oxidative damage from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration, in which the retinal pigment epithelium (RPE) is considered a primary target. The aim of this study was to determine whether erythropoietin (EPO) protects cultured human RPE cells against oxidative damage and to identify the pathways that may mediate protection. EPO (1 IU/ml) significantly increased the viability of oxidant-treated RPE cells, decreased the release of the inflammatory cytokines tumor necrosis factor-α and interleukin-1β, recovered the RPE cells'' barrier integrity disrupted by oxidative stress, prevented oxidant-induced cell DNA fragmentation and membrane phosphatidylserine exposure, and also reduced the levels of oxidant-induced intracellular ROS and restored cellular antioxidant potential, total antioxidant capacity, glutathione peroxidase, and superoxide dismutase and decreased malondialdehyde, the end product of lipid peroxidation. EPO inhibited caspase-3-like activity. Protection by EPO was partly dependent on the activation of Akt1 and the maintenance of the mitochondrial membrane potential. No enhanced or synergistic protection was observed during application of Z-DEVD-FMK (caspase-3 inhibitor) combined with EPO compared with cultures exposed to EPO and H2O2 alone. Together, these results suggest that EPO could protect against oxidative injury-induced cell death and mitochondrial dysfunction in RPE cells through modulation of Akt1 phosphorylation, mitochondrial membrane potential, and cysteine protease activity. [Copyright &y& Elsevier]

St. John’s wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges. In vivo studies have determined that hypericin is phototoxic to skin and our previous in vitro studies with lens tissues have determined that it is potentially phototoxic to the human lens. To determine if hypericin might also be phototoxic to the human retina, we exposed human retinal pigment epithelial (hRPE) cells to 10−7 to 10−5 M hypericin. Fluorescence emission detected from the cells ( λex = 488 nm; λem = 505 nm) confirmed hypericin uptake by human RPE. Neither hypericin exposure alone nor visible light exposure alone reduced cell viability. However when irradiated with 0.7 J cm−2 of visible light (λ > 400 nm) there was loss of cell viability as measured by MTS and lactate dehydrogenase assays. The presence of hypericin in irradiated hRPE cells significantly changed the redox equilibrium of glutathione and a decrease in the activity of glutathione reductase. Increased lipid peroxidation as measured by the thiobarbituric acid reactive substances assay correlated to hypericin concentration in hRPE cells and visible light radiation. Thus, ingested SJW is potentially phototoxic to the retina and could contribute to retinal or early macular degeneration. [ABSTRACT FROM AUTHOR]

Purpose: Latanoprost, the active principle of XalatanR eye drops, has been shown to cause increased iridial pigmentation as a side-effect in some patients. The purpose of the present study was to investigate whether latanoprost affects tyrosinase, the rate limiting enzyme in melanogenesis, at the gene transcription level in the iridial melanocytes. Methods: Four cynomolgus monkeys were treated unilaterally with 3 or 11 μg latanoprost once daily for 10 days. The contralateral eye received the vehicle only. Tyrosinase mRNA was visualized by in situ hybridization using radiolabelled riboprobes. The transcription of tyrosinase was also studied in vitro using cultivated human iridial melanocytes. Tyrosinase RNA was quantified by Northern blotting. Results: In the monkeys transcription of tyrosinase was found to be increased in iridial melanocytes of the treated eyes compared to the control eyes. Increased transcription of tyrosinase was in addition found in the iridial pigment epithelium and in melanocytes of the anterior choroid. Latanoprost was also found to increase the transcription of tyrosinase in melanocytes isolated from at least one human eye. Conclusions: Although the tyrosinase enzyme has to undergo complex post-translational modification to become biologically active, which we have not studied, it appears that latanoprost treatment may increase the transcription of the tyrosinase gene in some individuals, consistent with increased melanogenesis in the iridial melanocytes leading to darker eye colour. [ABSTRACT FROM AUTHOR]

The new glaucoma drugs latanoprost, isopropyl unoprostone, travoprost, and bimatoprost cause increased pigmentation of the iris in some patients. The purpose of the present article is to survey the available preclinical and clinical data on prostaglandin-induced iris pigmentation and to assess the phenomenon from a clinical perspective. Most of the data have been obtained with latanoprost, and it appears that there is a predisposition to latanoprost-induced iris pigmentation in individuals with hazel or heterochromic eye color. As latanoprost and travoprost are selective agonists for the prostaglandin F2α receptor, it is likely that the phenomenon is mediated by this receptor. Several studies indicate that latanoprost stimulates melanogenesis in iridial melanocytes, and transcription of the tyrosinase gene is upregulated. The safety aspects of latanoprost-induced iris pigmentation have been addressed in histopathologic studies, and no evidence of harmful consequences of the side effect has been found. Although a final assessment of the clinical significance of prostaglandin-induced iris pigmentation currently is impossible to make, it appears that the only clear-cut disadvantage is a potential heterochromia between the eyes in unilaterally treated patients because the heterochromia is likely to be permanent, or very slowly reversible. [Copyright &y& Elsevier]