Antioxidant pattern in uveal melanocytes and melanoma cell cultures.
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PURPOSE: To investigate the antioxidant status of cultured uveal melanocytes from patients with uveal melanoma and uveal melanoma cells to characterize some of the biochemical properties of these cells in respect to the normal cutaneous melanocytes. METHODS: The fatty acid pattern of membrane phospholipids, intracellular vitamin E level, and superoxide dismutase (SOD) and catalase activities were studied in uveal melanocytes (n = 10) and uveal melanoma cell (n = 10) cultures, by gas chromatography mass spectrometry or by spectrophotometer. RESULTS: Among the uveal melanocyte cultures, two groups were differentiated, according to catalase activity: group A with catalase values comparable to those of cutaneous ones and higher SOD activity and group B with catalase values 2 SD lower (P<0.001) and lower SOD activity. Vitamin E concentration was not significantly different between melanoma cells and melanocytes, whereas a significantly higher percentage of polyunsaturated fatty acids was found in melanoma cells and the B group of melanocytes (P = 0.022). In uveal melanoma cells SOD activity was significantly lower than that detected in uveal melanocytes (P< 0.005). CONCLUSIONS: These results show a different pattern of antioxidants in uveal melanocytes with respect to cutaneous ones, possibly related to the anatomic distribution. However, as in cutaneous melanocytes, two subgroups were identified on the basis of the antioxidant pattern that could be the expression of a constitutional increased susceptibility to oxidative stress in some subjects. Moreover, an imbalance of the antioxidants was observed in melanoma cells, possibly related to the disease status and progression. (+info)
Localization of myocilin/trabecular meshwork--inducible glucocorticoid response protein in the human eye.
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PURPOSE: To study distribution and cellular localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein (TIGR) in the human eye. METHODS: A peptide antibody against a portion of the myosin-like domain of myocilin/TIGR was developed. Different ocular tissues from three human donors were investigated by one- and two-dimensional gel electrophoresis and Western blot analysis. Immunohistochemistry was performed on 25 human eyes enucleated because of posterior choroidal melanoma and on 7 normal human donor eyes. RESULTS: By Western blot analysis, a band at approximately 57 kDa was visualized in cornea, trabecular meshwork, lamina cribrosa, optic nerve, retina, iris, ciliary body, and vitreous humor. By immunohistochemistry, immunoreactivity for myocilin/TIGR was observed in cells of the corneal epi- and endothelium and extracellularly in the corneal stroma and sclera. In the trabecular meshwork, cells of the uveal and corneoscleral meshwork were stained, as was the cribriform area directly adjacent to Schlemm's canal. Positive staining was seen in cells of the ciliary epithelium, ciliary muscle, lens epithelium, and in stromal and smooth muscle cells of the iris. Throughout the entire vitreous body, fine filamentous material was positively labeled. In the retina, staining was seen along the outer surface of rods and cones, in neurons of the inner and outer nuclear layer, and in the axons of optic nerve ganglion cells. Optic nerve axons were stained in the prelaminar, laminar, and postlaminar parts of the nerve. In the region of the lamina cribrosa, astrocytes in the glial columns and cribriform plates were positively labeled. CONCLUSIONS: Myocilin TIGR is expressed in almost every ocular tissue. Depending on the respective tissue, it is observed extra- or intracellularly. The presence of myocilin/TIGR in optic nerve axons and lamina cribrosa astrocytes indicates that the trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked open-angle glaucoma. (+info)
Effect of local corticosteroids on antibody-forming cells in the eye and draining lymph nodes.
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Significant numbers of antibody-forming cells (AFC) have been found in the cornea, uveal tract, and draining lymph nodes after the intracorneal injection of bovine gamma-globulin (BGG). To study the effect of locally administered corticosteroids on these antibody-forming tissues, we made unilateral intracorneal injections of rabbit eyes with BGG. These we followed immediately with subconjunctival injections of 10 mg. of triamcinolone suspension, and then with a second round of 10 mg. injections seven days later. A control group of animals received the BGG injections followed by two subconjunctival saline injections. We killed the animals on postinjection days 6, 9, 12, 15, and 21, and tested the draining lymph nodes, homolateral uveal tissue, and homolateral cornea for AFC by a modification of the Jerne placque technique. The local steroids had no effect on the number of AFC produced in the draining lymph nodes or on the circulating antibody response, but they reduced the number of AFC in the homolateral uveal tracts and corneas. Clinically there was less inflammatory response in the steroid-treated eyes than in the control eyes. The possible mechanisms by which corticosteroids achieve their anti-immunologic and anti-inflammatory benefits are discussed. (+info)
Identification of Th2-type suppressor T cells among in vivo expanded ocular T cells in mice with experimental autoimmune uveoretinitis.
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Experimental autoimmune uveoretinitis (EAU), which is a T cell mediated organ specific autoimmune disease, is induced by immunization with interphotoreceptor retinoid binding protein (IRBP) in susceptible strains of mice. It has been found that IRBP-derived peptide 518-529 (p518-529) generates Th2-type responses and inhibits IRBP-induced EAU, indicating that the p518-529 might be an epitope for suppressor T cells in IRBP-induced EAU. First, we observed that there were T cells producing the Th2 type cytokines such as IL-4 and IL-10 in late phase of EAU. Furthermore, to examine whether p518-529-reactive T cells expand in the eye during EAU, T cell receptor (TCR) of ocular T cells was compared with that of p518-529 reactive T cells in spleen from mice with EAU by PCR-single strand conformation polymorphism (PCR-SSCP) and nucleotide sequence analysis. SSCP and sequence analyses indicated that p518-529 reactive TCR BV10+ T cells bearing amino acid motif(PWG) and TCR BV13+ T cells bearing amino acid motif(PGLGGY) in their complementary-determining region 3 (CDR3) region were clonally expanding in ocular tissues on day 28 after immunization, although these T cells were not detected on day 14. These findings demonstrate that p518-529 reactive Th2-type T cells expand oligoclonally in the uveitic eyes in the late stage of EAU and may function as Th2-type suppressor T cells for improvement of the disease. (+info)
Local antibody formation within the eye: a study of immunoglobulin class and antibody specificity.
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Primary immunogenic uveitis was induced in the rabbit eye with a single injection of antigen into the vitreous, and secondary booster uveitis responses were induced two months later by intravenous administration of the same antigen. The distribution of immunoglobulin classes and the specificity of the antibodies produced were assessed early and late in the primary response and early and late in the secondary response, and were compared with the analogous responses in the spleen and regional lymph nodes. At each of these stages of intraocular antibody response, IgG formation was higher and IgM formation lower than that seen in organized lymphoid tissues, while the proportion of IgA-forming cells was similar to the low levels usually found in the spleen. A significant proportion of IgA-forming cells was found in the perilimbal conjunctiva, and even greater levels in the lacrimal glands. At each stage of the response, the proportion of immunoglobulin-forming cells making antibody specific for the inciting ovalbumin antigen was surprisingly low, reaching only seven per cent during the late primary reaction and 18 per cent during the late secondary reaction. (+info)
Visualization of uveal perfusion by contrast-enhanced harmonic ultrasonography at a low mechanical index: a pilot animal study.
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OBJECTIVE: To evaluate contrast-enhanced harmonic ultrasonography at a low mechanical index for its usefulness in visualizing uveal perfusion. METHODS: The study was performed with 9 rabbits, 6 intact and 3 with focal impaired blood flow in the uvea. Ultrasonography was performed by harmonic imaging (transmit, 5 MHz; receive, 10 MHz) with a contrast agent. The agent was administered at a dose of 50 microL/kg. Transmission power was at a mechanical index of 0.2, which is below the US Food and Drug Administration guideline. The images were compared between the impaired and intact eyes. For uveal measurements, video signal intensity-versus-time plots were generated in all cases. The plots were analyzed to obtain the rate of signal intensity increase and peak signal intensity. RESULTS: A clear increase of signal intensity was observed after contrast agent administration. The signal intensity of the uvea was lower in the impaired eye than in the intact eye. In the impaired eye, the intensity was lower on the side with impaired flow than on the other side. The differences were significant. CONCLUSIONS: Our findings suggest that uveal perfusion can be visualized by contrast-enhanced harmonic ultrasonography in the harmonic imaging mode at a low mechanical index. (+info)
Identification of the mouse uveoscleral outflow pathway using fluorescent dextran.
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PURPOSE: This study was undertaken to assess directly whether there is uveoscleral outflow in the mouse eye by monitoring the movement of intracamerally injected fluorescent dextran. METHODS: After anesthesia, NIH Swiss mice received intracameral injection of 1.5 microL of 0.2 pg/microL 70-kDa dextran conjugated to tetramethyl-rhodamine and to lysine. After survival times of 10, 20, 60, and 120 minutes, the experiments were terminated by transcardial perfusion with 2% paraformaldehyde. The eyes were enucleated and embedded in paraffin, and sections were prepared. These sections were then analyzed by fluorescence microscopy. RESULTS: Fluorescent tracer in the eyes of animals that survived for 10 minutes was prominent in the iris root and ciliary processes and was of moderate intensity in the adjacent sclera. Moderate intensity fluorescence also was observed in the trabecular meshwork and adjacent cornea. At 20 minutes, intense fluorescence was observed in the ciliary processes and the ciliary muscle. This fluorescence in the ciliary muscle extended from the posterior edge of the ciliary muscle's tail into the anterior choroid. At 60 minutes, the fluorescence in the choroid extended to the equator and adjacent sclera. The intensity of the fluorescence within the ciliary processes of these eyes was substantially reduced when compared with the 20-minute-survival eyes. At 120 minutes, label was observed only within trabecular meshwork and Schlemm's canal. CONCLUSIONS: These results indicate that at least a portion of aqueous outflow in the mouse eye is through the uveoscleral outflow pathway. (+info)
Innervation of the uvea by galanin and somatostatin immunoreactive axons in macaques and baboons.
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The neuropeptide galanin has not been localized previously in the primate uvea, and the neuropeptide somatostatin has not been localized in the uvea of any mammal. Here, the distribution of galanin-like and somatostatin-like immunoreactive axons in the iris, ciliary body and choroid of macaques and baboons using double and triple immunofluorescence labeling techniques and confocal microscopy was reported. In the ciliary body, galanin-like immunoreactive axons innervated blood vessels and the ciliary processes, particularly at their bases. In the iris, the majority of these axons was associated with the loose connective tissue in the stroma. Somatostatin-like immunoreactive axons were found in many of the same areas of the uvea supplied by cholinergic nerves. In the ciliary body, there were labelled axons within the ciliary processes and ciliary muscle. They were also found alongside blood vessels in the ciliary stroma. In the iris, somatostatin-like immunoreactive axons were abundant in the sphincter muscle and less so in the dilator muscle. A unilateral sympathectomy had no effect on the distribution of somatostatin-like or galanin-like immunoreactive axons, and these axons did not contain the sympathetic marker tyrosine hydroxylase. They did not contain the parasympathetic marker choline acetyltransferase, either. The galanin-like immunoreactive axons contained other neuropeptides found in sensory nerves, including calcitonin gene-related peptide, substance P and cholecystokinin. Somatostatin-like immunoreactive axons did not contain any of these sensory neuropeptides or galanin-like immunoreactivity, and they were neither labelled with an antibody to 200kDa neurofilament protein, nor did they bind isolectin-IB(4). Nevertheless, they are likely to be of sensory origin because somatostatin-like immunoreactive perikarya have previously been localized in the trigeminal ganglion of primates. Taken together, these findings indicate galanin and somatostatin are present in two different subsets of sensory axons in primate uvea. (+info)