Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

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.

Cultured Human Uveal Melanocytes Express/secrete CXCL1 and CXCL2 Constitutively and Increased by Lipopolysaccharide via Activation of Toll-like Receptor 4.

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/CXCL2Conclusions: 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.

Bioluminescence for in vivo detection of cell-type-specific inflammation in a mouse model of uveitis.

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells, T cells, and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75% of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7, the ocular T cell population increases to 50% of CD45 + cells, leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible (< 1% of CD45 + cells), the ocular B cell population increases to > 4% by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models.

Ultrastructural Organization of Uveal Melanoma Stromal Cells.

Typical blood capillaries and vessels in uveal melanoma were shown and different types of uveal melanoma stromal cells were determined by electron microscopy and immunohistochemical analysis. Macrophages, fibroblasts of varying degrees of differentiation and endothelial-like cells with numerous caveolae in the cytoplasm were found in the channels of the extracellular matrix surrounding accumulations of tumor cells. The presence local structures positively stained for markers of the blood and lymphatic vessels (CD31 and podoplanin) in channels of the extracellular matrix suggests that the described endothelial-like cells can be the structural basis for blood and lymphatic vessels of the tumor.

Uveal melanocytes express high constitutive levels of MMP-8 which can be upregulated by TNF-α via the MAPK pathway.

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.

Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

Isolation and cultivation of canine uveal melanocytes.

OBJECTIVES: To establish a method for isolation and culture of canine uveal melanocytes. ANIMALS STUDIED: Uveal explants from five mixed-breed dogs. PROCEDURES: Donor globes were dissected, and the anterior uvea removed. The uveal explants were placed in trypsin solution for enzymatic digestion. Extracted cells were cultured in modified F12 media. Immunocytochemistry was performed to confirm the identity of the extracted cells. RESULTS: Melanocytes were successfully isolated from uveal explants. Contaminating cell types were not observed. Repeated passaging of the melanocytes resulted in a gradual decrease in intracellular pigment. Melanocyte cell lines could be cryopreserved, thawed, and cultures successfully reestablished. CONCLUSIONS: This extraction technique allows for generation of large populations of canine uveal melanocytes in a relatively short period of time. This technique could be a useful tool for future studies investigating both normal cellular characteristics and alterations found in melanocytes from dogs with ocular melanocytic disorders.

Constitutive and LPS-induced expression of MCP-1 and IL-8 by human uveal melanocytes in vitro and relevant signal pathways.

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)-κB 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-κB (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 µg/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-κB in nuclear extracts. Nuclear factor-κB 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.

Telocytes and stem cells in limbus and uvea of mouse eye.

The potential of stem cell (SC) therapies for eye diseases is well-recognized. However, the results remain only encouraging as little is known about the mechanisms responsible for eye renewal, regeneration and/or repair. Therefore, it is critical to gain knowledge about the specific tissue environment (niches) where the stem/progenitor cells reside in eye. A new type of interstitial cell-telocyte (TC) (www.telocytes.com) was recently identified by electron microscopy (EM). TCs have very long (tens of micrometres) and thin (below 200 nm) prolongations named telopodes (Tp) that form heterocellular networks in which SCs are embedded. We found TCs by EM and electron tomography in sclera, limbus and uvea of the mouse eye. Furthermore, EM showed that SCs were present in the anterior layer of the iris and limbus. Adhaerens and gap junctions were found to connect TCs within a network in uvea and sclera. Nanocontacts (electron-dense structures) were observed between TCs and other cells: SCs, melanocytes, nerve endings and macrophages. These intercellular 'feet' bridged the intercellular clefts (about 10 nm wide). Moreover, exosomes (extracellular vesicles with a diameter up to 100 nm) were delivered by TCs to other cells of the iris stroma. The ultrastructural nanocontacts of TCs with SCs and the TCs paracrine influence via exosomes in the epithelial and stromal SC niches suggest an important participation of TCs in eye regeneration.

Interleukin-1beta increases baseline expression and secretion of interleukin-6 by human uveal melanocytes in vitro via the p38 MAPK/NF-kappaB pathway.

PURPOSE: Melanocyte is the major cell component in the uvea. Interleukin (IL)-6 is a proinflammatory cytokine. The authors studied the expression of IL-6 in cultured human uveal melanocytes (UMs) and their modulation by IL-1ß and explored the relevant signal pathways. METHODS: Conditioned media and cells were collected from UMs cultured in medium with and without serum and were stimulated by IL-1ß. IL-6 protein and transcript were measured using an ELISA kit and RT-PCR, respectively. NF-κB in nuclear extracts and phosphorylated p38 MAPK, ERK, and JNK in cells cultured with and without IL-1ß were measured by ELISA kits. Inhibitors of p38 (SB203580), ERK (UO1026), JNK (SP600125), and NF-κB (BAY11-7082) were added to the cultures to evaluate their effects. RESULTS: Low levels of IL-6 protein were detected in the conditioned medium in UMs cultured without serum. The addition of serum increased the secretion of IL-6. IL-1ß (0.1-10 ng/mL) increased IL-6 transcript and protein levels in a dose- and time-dependent manner up to sixfold, accompanied by a significant increase of NF-κB in nuclear extracts and phosphorylated p38 MAPK in cell lysates. NF-κB and p38 inhibitors alone decreased, whereas a combination of these two inhibitors completely abolished the IL-1ß-induced expression of IL-6. CONCLUSIONS: This is the first report on the expression and secretion of IL-6 by UMs. IL-1ß augments IL-6 production from the melanocytes. The data suggest that UMs may play a role in the pathogenesis of ocular inflammatory and autoimmune diseases.

A comparative transcriptomic analysis of uveal melanoma and normal uveal melanocyte.

BACKGROUND: Uveal melanoma is the most common primary intraocular tumor in adults in western countries. It is associated with very severe visual morbidity and may lead to distant metastases even after successful treatment of the primary tumor. In order to gain better insight into molecular mechanisms related to tumorigenesis and metastasis of uveal melanoma, we used next-generation sequencing technology (SOLiD, Life Technologies) to acquire global transcriptome alteration between posterior uveal melanoma cells and normal uveal melanocyte. RESULTS: From mRNAs of the cultured uveal melanoma cells and normal uveal melanocytes, we annotated more than 3.7 × 10(7) and 2.7 × 10(7) sequencing tags based on human Ensembl databases, respectively. For detailed analysis, we chose 5155 well-annotated genes mainly involved in the MAPK signaling pathway, cell cycle, cell adhesion junction, apoptosis, and P53 signaling pathways as well as melanogenesis. In an effort to confirm the authenticity of our sequencing results, we validated twenty-one identically differentially expressed genes by using quantitative real time PCR from cultured cell lines of other posterior uveal melanoma cells and normal uveal melanocytes. CONCLUSION: We have identified a large number of potentially interesting genes for biological investigation of uveal melanoma. The expression profiling also provides useful resources for other functional genomic and transcriptome studies. These 21 potential genes could discriminate between uveal melanoma cells and normal uveal melanocyte, which may be indicative of tumorigenesis process. Our results further suggest that high-throughput sequencing technology provides a powerful tool to study mechanisms of tumogenesis in the molecular level.

Regulatory effect of gammadelta T cells on IL-17+ uveitogenic T cells.

PURPOSE: To characterize the regulatory effect of gammadelta T cells in the activation of IL-17+ uveitogenic T cells. METHODS: The authors administered the gammadelta TCR-specific antibody GL3 to B6 mice before or after antigen immunization and examined Th1- or Th17-polarized T-cell responses. The intensity of Th17 responses was also examined in responder T cells containing varying numbers of gammadelta T cells. RESULTS: GL3 treatment resulted in varying degrees of depletion of circulating gammadelta T cells, depending on when the antibody was administered. The intensity of the alphabetaTCR+IL-17+, but not the alphabetaTCR+IFN-gamma+, IRBP-specific T-cell responses was correlated to the percentage of gammadelta T cells in the responder T cells. Kinetic studies showed that early IL-17+ T cells were primarily gammadelta T cells, with a later gradual shift to alphabeta T cells. A close association was seen between the intensity of the IL-17+ autoreactive T-cell response and the percentage of gammadelta T cells in the responder T cells. Although a modest increase in gammadelta T cells among the responder T cells promoted the expansion of IL-17+ alphabetaTCR+ T cells, a higher proportion of gammadelta T cells inhibited it. CONCLUSIONS: gammadelta T cells are actively involved in the generation of alphabetaTCR+IL-17+ T cells. The number of gammadelta T cells and the alphabeta/gammadelta T-cell ratio in the responder T cells regulate the intensity of the Th17-type autoreactive T-cell response.

Comparison of eumelanin and pheomelanin content between cultured uveal melanoma cells and normal uveal melanocytes.

Levels of eumelanin (EM) and pheomelanin (PM) of uveal melanoma cells have not been measured and compared with those of normal uveal melanocytes. EM and PM amounts in four immortal human uveal melanoma cell lines were measured by chemical degradation and microanalytical high-performance liquid chromatography and compared with those from 39 normal human uveal melanocyte cell lines reported earlier by us. Uveal melanoma cells had a very low EM/PM ratio (0.41), which was very significantly lower than that from normal melanocytes isolated both from eyes with light-colored irides (1.31) or dark-colored irides (7.32). The low EM/PM ratio was caused by a low level of EM in melanoma cells, which was only 1/8 and 1/31 of that in melanocytes from eyes with light-colored irides and dark-colored irides, respectively. The PM level in uveal melanoma cells was not statistically different from normal melanocytes from eyes with light-colored irides or dark-colored irides. The total quantity of EM and PM in uveal melanoma cells was significantly less than that in normal melanocytes. This difference was because of the low level of EM in uveal melanoma cells. The results of these studies indicate that the changes of melanin content in uveal melanoma cells mainly relate to the decrease of EM content. Low melanin and EM content may make melanoma cells more susceptible to mutagenic effects of ultraviolet radiation and oxidative stress, which may enhance the proliferation of melanoma cells and accelerate progression of melanoma.

Differential turnover rates of monocyte-derived cells in varied ocular tissue microenvironments.

Monocytes of bone marrow (BM) origin are circulating precursors that replenish dendritic cells and macrophage populations in peripheral tissues during homeostasis. The eye provides a unique range of varying tissue microenvironments in which to compare the different turnover rates of monocyte-derived cells. This was investigated in the present study using radiation chimeras, whereby BM from Cx3cr1(+/gfp) mice was used to rescue myeloablated wild-type (WT) BALB/c mice (conventional chimeras). The use of Cx3cr1(+/gfp) mice as BM donors allowed the clear visualization of newly recruited monocyte-derived cells. Following BM reconstitution, mice were killed at 2, 4, 6, and 8 weeks, and wholemount ocular tissues were processed for immunohistochemistry and confocal microscopy. "Reverse" chimeras (WT into Cx3cr1(+/gfp)) were also created to act as a further method of cross-referencing cell turnover rates. In conventional chimeras, Cx3cr1(+/gfp) cells began repopulating the uveal tract (iris, ciliary body, choroid) 2 weeks post-transplantation with close to complete replenishment by 8 weeks. By contrast, the earliest recruitment of Cx3cr1(+/gfp) cells into the host retina occurred at 4 weeks. In reverse chimeras, a steady accumulation of host Cx3cr1(+/gfp) macrophages in the subretinal space of Cx3cr1(+/gfp) adult mice suggests that these cells arise from long-term resident microglia and not newly recruited WT donor cells. In summary, chimeric mouse models, in which lineage-specific cells carry a fluorescent reporter, have been used in the present study to visualize the turnover of monocyte-derived cells in different tissue compartments of the eye. These data provide valuable insights into differential monocyte turnover rates within a single complex organ.

Retinal microglia and uveal tract dendritic cells and macrophages are not CX3CR1 dependent in their recruitment and distribution in the young mouse eye.

PURPOSE: The chemokine receptor CX3CR1 is expressed by monocyte-derived dendritic cells (DCs) and macrophages. CX3CR1 mediates leukocyte migration and adhesion in homeostatic and inflammatory conditions. Mice lacking Cx3cr1 have altered distribution and function of DC subpopulations in some tissue microenvironments. The present study compares the distribution of monocyte-derived cells in the normal retina and uveal tract as a prelude to the investigation of the role of CX3CR1 in murine models of ocular disease. METHODS: Transgenic mice in which either one (Cx3cr1 gfp/+, heterozygous) or both (Cx3cr1 gfp/gfp, homozygous) copies of the Cx3cr1 gene have been replaced by the enhanced green fluorescent protein (eGFP) reporter gene were used to investigate the role of Cx3cr1 expression on macrophages and DCs in the normal uveal tract and retina. Chimeric mice were used to investigate turnover of these cells in the normal, uninflamed eye. RESULTS: Confocal analysis found no significant differences in the density, phenotype or morphology of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice in immunostained iris, ciliary body, or choroidal and retinal wholemounts. Flow cytometry also failed to detect any difference in the density or cell shape of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice. Chimeras revealed 73% turnover of monocyte-derived cells in the iris and 63% in the choroid by 6 weeks after transplantation. CONCLUSIONS: These data illustrate that homing or migration of DCs and macrophages to the uveal tract and retina in normal young mice is not Cx3cr1 dependent and provide a solid foundation for future studies of monocyte-derived cells and the role of Cx3cr1 in models of ocular disease.

Methodology for evaluation of melanin content and production of pigment cells in vitro.

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.

Prediction of chromosomal aneuploidy from gene expression data.

Chromosomal aneuploidy is commonly observed in neoplastic diseases and is an important prognostic marker. Here we examine how gene expression profiles reflect aneuploidy and whether these profiles can be used to detect changes in chromosome copy number. We developed two methods for detecting such changes in the gene expression profile of a single sample. The first method, fold-change analysis, relies on the availability of gene expression data from a large cohort of patients with the same disease. The expression profile of the sample is compared with that of the dataset. The second method, chromosomal relative expression analysis, is more general and requires the expression data from the tested sample only. We found that the relative expression values are stable among different chromosomes and exhibit little variation between different normal tissues. We exploited this novel finding to establish the set of reference values needed to detect changes in the copy number of chromosomes in a single sample on the basis of gene expression levels. We measured the accuracy of the performance of each method by applying them to two independent leukemia datasets. The second method was also applied to two solid tumor datasets. We conclude that chromosomal aneuploidy can be detected and predicted by analysis of gene expression profiles. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Uveal melanocytes do not respond to or express receptors for alpha-melanocyte-stimulating hormone.

PURPOSE: Whereas cutaneous pigmentation increases after exposure to ultraviolet (UV) irradiation, ocular pigmentation does not. This study was designed to examine the evidence that alpha-melanocyte-stimulating hormone (alpha-MSH), which is thought to be the mediator of UV response in the skin, has any role to play in uveal melanocytes. METHODS: Human uveal melanocytes derived from the choroid and the iris were cultivated by using eyes harvested from adult cadaveric donors and were assessed by Northern blot analysis for growth and melanogenic response to alpha-MSH and expression of the receptor for alpha-MSH (MC1-R). In addition, expression of alpha-MSH was evaluated in ocular tissue by immunocytochemistry. RESULTS: Uveal melanocytes, unlike cutaneous melanocytes in vitro, exhibited no stimulation of proliferation in response to alpha-MSH at dosages ranging from 0.1 to 100 muM. In addition, tyrosine hydroxylase, DOPA oxidase, and protein levels for tyrosinase, TRP-1, and TRP-2 were not influenced by alpha-MSH. Associated with the lack of alpha-MSH response in cultured uveal melanocytes was the absence of expression of the receptor for alpha-MSH (MC1-R), as assessed by Northern blot analysis. Also in contrast to the skin, pigmented ocular tissue lacked expression of the alpha-MSH ligand, as assessed by immunocytochemistry. CONCLUSIONS: In conclusion, ocular pigmentation does not appear to be regulated by melanocyte stimulating hormone.

Dopa oxidase activity in the hair, skin and ocular melanocytes is increased in the presence of stressed fibroblasts.

We previously reported that mesenchymal cells (dermal fibroblasts and dermal papilla cells) can stimulate dopa oxidase activity in the skin melanocytes. This study extends the investigation of the influence of the fibroblast in a comparative study of melanogenesis in melanocytes from the hair, the skin and the eye. Culture of melanocytes with normal proliferative dermal fibroblasts slightly increased dopa oxidase activity of the hair, skin and ocular melanocytes (by 17, 11 and 28%, respectively), but co-culture with fibroblasts recovering from storage in liquid nitrogen or growth-arrested by means of gamma radiation showed much greater effects. Most dramatic results were obtained with fibroblasts, which had been both gamma-irradiated and then frozen in liquid nitrogen, where increases in dopa oxidase activity of 125, 227 and 185% for melanocytes of the hair, the skin and the eye, respectively, were seen. Experiments by using transwell cultures of melanocytes and fibroblasts and by using fibroblast-conditioned medium showed that a large proportion of this fibroblast influence could be mediated by diffusible factors, of which a good proportion was attributable to basic Fibroblast Growth Factor (bFGF). The addition of bFGF significantly increased dopa oxidase activity of the skin melanocytes, when fibroblasts were present, but not in their absence. These data show that fibroblasts in vitro, particularly when deliberately stressed, have the ability to increase dopa oxidase activity in melanocytes of the hair, the skin and the eye and further suggest that this effect is mediated by bFGF acting in combination with some other fibroblast-derived factors.

Uveal melanocytes produce matrix metalloproteinases-2 and -9 in vitro.

The purpose of the present study was to investigate the expression of matrix metalloproteinase (MMP)-2 and MMP-9 by cultured human uveal melanocytes, and to test the effects of 12-O-tetradecanoyl-phorbol-13-acetate on the expression of these MMPs. Gelatin zymography of conditioned culture medium from four cultures of human uveal melanocytes (two cultures of iridal melanocytes and two cultures of choroidal melanocytes) detected MMP-2 (72 kDa) and a relatively small amount of MMP-9 (92 kDa), both in the latent form. RT-PCR analysis revealed the MMP-2 mRNA and MMP-9 mRNA in cultured uveal melanocytes. Addition of 12-O-tetradecanoyl-phorbol-13-acetate (10 ng/ml) to the culture medium caused an increase of production of MMP-2 and MMP-9 by cultured uveal melanocytes, and also stimulated the transcription of MMP-2 and MMP-9 of these cells.