摘要:

Purpose. The vertebrate eye contains both melanocytes and retinal pigment epithelial (RPE) cells. Little is known of the pigmentary behaviour of these embryologically dissimilar cells. The aim of this study was to examine aspects of the pigmentary properties of both cell typesin vitroandex vivoto learn more of the function of these cells.Methods. Sections of normal adult human eye were stained for tyrosinase related protein 1(TRP1), and cultures of RPE cells and choroidal melanocytes were examined immunocytochemi-cally for TRP1 and 2 and enzymatically for tyrosinase activity (by assaying dopa oxidase activity).Results. Over half of the choroidal melanocytes expressed TRP1ex vivo; in contrast, a very small percentage of RPE cells were TRP1 positive.In vitro, passage 1 to 3 ocular melanocytes expressed TRP1 and TRP2 and had tyrosinase activity, which was influenced by the choice of substrate on which the cells were grown. Tyrosinase activity was highest when cells were grown on fibronectin and plastic, intermediate on laminin and lowest on vitreous extracellular matrix (ECM) containing pigment to which they attached and spread out poorly. In contrast, passage 3 RPE cells (which were unpigmented) showed little evidence of tyrosinase activity in short-term culture, irrespective of the substrate on which they were grown, and failed to express TRP1 and TRP2. When cells were grown on plastic for greater than 3 weeks in culture, a very low percentage of cells (<0.1%) became TRP1 positive and this percentage was increased threefold if cells were cultured on laminin in the presence of bFGF. A few cells were also seen to contain pigment but cultures failed to show any tyrosinase activity. In contrast, RPE cells (but not melanocytes) showed a marked ability to take up pigment granulesin vitro.Conclusions. The data suggest that normal human ocular melanocytes retain the capacity to produce pigment throughout adult life, and this can be demonstrated bothex vivoandin vitro. In contrast, we were unable to confirm that the majority of RPE cells play any significant role in active pigment production in the adult.

ISSN:0271-3683    

DOI:10.3109/02713689608995139

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. This study was designed to determine the effect of overnight eye closure on the rate and composition of protein deposition on high water content ionic matrix soft contact lenses (Group IV SCLs) and to extrapolate from this data information on the probable change in the rate of reflex-type tear secretion associated with eye closure.Methods. Group IV SCLs were temporally sampled after equivalent periods of wear under closed eye (C) or open eye (O) conditions. Lenses were rinsed in saline and the majority of the tightly bound protein extracted at 90d`C in 40% urea, containing 1% SDS, 1 mM DTT, 100 mM Tris-HC1 (pH 8.00). Residual protein was determined by Coomassie staining of the extracted lenses and densitometric analysis. Extracted protein was quantitated and separated by SDS-PAGE. Gels were either stained with Coomassie blue or reversibly stained with imidazole-zinc and blotted. Blots were PAS stained, or lectin and antibody probed for glycoproteins, secretory IgA (sIgA), IgG, lysozyme and complement C3. Laboratory simulated deposition studies were carried out on unworn lenses exposed to HPLC purified lysozyme.Results. The protein in the saline rinse, to a large degree mirrored the composition of tear fluid in which the lens had been residing (O or C). This would suggest that the saline wash consists of residual tear fluid and loosely adherent protein. In contrast, the urea extracts were highly homogeneous consisting primarily of lysozyme and to a lesser extent lysozyme dimer. This supports the contention that the Group IV SCL functions in the eye much as cationic exchange resin selectively absorbing lysozyme. C extracts also proved relatively enriched in trace amounts of sIgA, IgG and complement C3 and its breakdown products. High levels of C3 and C3 breakdown products were specifically recovered only in the C worn lens extracts from a subject experiencing unilateral contact lens associated corneal infiltrates from the affected eye. In all subjects, markedly less protein (lysozyme) was recovered in urea extracts of lenses exposed to 7-8 h of closed eye as compared to open eye wear (0.20±. 08 versus 0.79±. 15 mg/lens (n = 6)). Temporal studies further revealed that deposition was linearly related to duration of wear during the initial phase of conditioning film formation giving rise to rate constants for lysozyme deposition of 2.2±0.29 (n = 5) and 0.20±0.06μg/min (n = 4) under open and closed eye conditions respectively. With further wear, deposition eventually reached a steady state. Under laboratory conditions, lysozyme was much rapidly and quantitatively removed from solution in a manner following a hyperbolic plot. This suggests that during the initial phase of deposition the rate of deposition is limited by the capacity of the tear fluid to deliver lysozyme to the lens surface under these two extremes of conditions.Conclusions. Eye closure profoundly affects the rate of lysozyme deposition on Group IV hydrogels and the composition of minor biofilm constituents in a manner that could affect biocompati-bility. Findings support the contention that eye closure results in a>90% reduction in the rate of reflex-type tear secretion.

ISSN:0271-3683    

DOI:10.3109/02713689608995140

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To examine the distribution of proteoglycans in the exfoliation materials in order to investigate the nature of the materials.Methods. The anterior parts of two eyes with exfoliation syndrome were examined by electron microscopy after staining with cupromeronic blue (cmb). Some specimens were treated with enzymes and/or nitrous acid prior to staining. The effects of the enzymes were evaluated statistically by counting the density of the cmb-positive filaments in the exfoliation materials, using a computer. One eye with exfoliation syndrome stained with alcian blue was observed with light microscopy.Results. Exfoliation materials were observed along the epithelial cells of the iris and ciliary body, and in the trabecular meshwork and zonules. In tissue specimens treated with cmb, electron-dense filaments were seen associated with the exfoliation materials. Microfibrils in the trabecular meshwork and iris, and zonular fibrils themselves were free of any filament staining, while the exfoliation materials located closely to the fibrils contained the electron-dense filaments. In the tissue specimens treated with chondroitinase AC, chondroitinase B, chondroitinase ABC or nitrous acid before cmb staining, the amount of the filament associated with exfoliation materials decreased in comparison to the controls. Digestion with keratinase did not demonstrate any significant changes in staining. A combination treatment with chondroitinase ABC and nitrous acid eliminated almost all filaments associated with the exfoliation materials. In the eye stained with alcian blue, the zonules that did not stain for the dye demonstrated an accumulation of exfoliation materials that stained strongly for alcian blue.Conclusions. Exfoliation materials contain chondroitin sulfate, dermatan sulfate, heparan sulfate proteoglycans. Depositions of proteoglycans on the microfibrils may be closely associated with the formation of exfoliation materials.

ISSN:0271-3683    

DOI:10.3109/02713689608995141

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Aclacinomycin A or aclarubicin is an anthracycline that, by contrast with daunomycin, lacks carcinogenicity and is less toxic to the retina. We investigated the toxicity and antiproliferative effect of aclacinomycin A on retinal pigment epithelial cells that are known to play a mayor role in the pathogenesis of proliferative vitreoretinopathy.Methods. In 3 experimental set-ups, RPE cells from pig eyes were incubated with aclacinomycin A at different concentrations (0.5–15μg/ml) and for various lengths of time (1–10 min). Cells were counted on day 3 after exposure to evaluate toxicity, subcul-tured, and counted once more on day 15 to test for the antiproliferative effect. Data were analyzed using the Tukey's Student-ized Range (HSD) Test. Furthermore, RPE cells were examined by light microscopy.Results. Cell numbers on day 3 after treatment were reduced significantly (p≤0.05) already at the lowest dosage tested (1μg/ml for 1 min). Higher doses, up to 15μg/ml for 5 min, did not lower cell numbers below 20% of those of control cultures. Logarithms of cell numbers on day 15 were inversely correlated to drug concentration as well as to incubation time. Cells that had been treated with 5μg/ml aclacinomycin A for 5 min were not able to start a new culture when subcultured 3 days after drug exposure.Conclusions. Aclacinomycin A applied intraocularly during vitreoretinal surgery may be an alternative to daunomycin in the treatment of proliferative vitreoretinopathy.

ISSN:0271-3683    

DOI:10.3109/02713689608995142

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Kallistatin is a serine proteinase inhibitor, which binds to tissue kallikrein and inhibits its proteolytic activity. This study is to determine the expression, cellular localization and the potential function of kallistatin in the eye.Methods. Tissue kallikrein-kallistatin complex formation was performed to detect the kallikrein-binding activity in ocular tissues. Immunoreactive kallistatin was detected and quantified by an enzyme-linked immunosorbent assay using polyclonal antibody specific to human kallistatin.In situhybridization histo-chemistry was employed to localize the kallistatin mRNA in human eyes using an antisense riboprobe of kallistatin.Results. We have identified active kallistatin in the cornea, ciliary body, sclera, choroid, optic nerve, retina, vitreous and aqueous fluids. Kallistatin binds to tissue kallikrein and forms an SDS-stable complex. Immunoreactive kallistatin was identified in these tissues. Linear dose-dependent curves of the tissue extracts of the retina and choroid are parallel to that of purified human kallistatin, suggesting their immunological identity. The kallistatin mRNA was identified in the ciliary muscle, lens epithelial cells, all the layers of retina cells, optic nerve, choroid and vascular endothelial cells. These cells were not stained by the sense riboprobe under the same conditions, indicating the specificity of the hybridization. We also compared immunoreactive kallistatin levels in vitreous fluids from 18 patients with diabetic retinopathy and 17 non-diabetic subjects. The results show that diabetic subjects have significantly lower kallistatin levels (233.0±14.6 ng/mg protein) compared to non-diabetic subjects (334.1±26.9 ng/mg protein).Conclusions. Kallistatin is produced endogenously in the eye and the decrease in the vitreous kallistatin levels may be involved in diabetic retinopathy.

ISSN:0271-3683    

DOI:10.3109/02713689608995143

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. Keratoconus is characterized by thinning and scarring of the central portion of the cornea. This study was performed on keratoconus corneas to examine the expression of proteins related to wound healing including vimentin, an intermediate filament protein, and tenascin, an extracellular matrix protein. The expression of stress-related cytokines, heat shock proteins and ubiquitin was also investigated.Methods. Corneal buttons were collected from patients with keratoconus, normal subjects and patients with other corneal diseases such as pseudophakic bullous keratopathy. Immunofluo-rescence staining was performed on frozen sections for vimentin and tenascin, and immunoperoxidase staining was carried out on paraffin sections for cytokines, heat shock proteins and ubiquitin.Results. To varying degrees, all proteins examined, except tenascin and heat shock protein 90, were found to be expressed in normal human corneas. The expression of vimentin, tenascin, transforming growth factor-β, interleukin-1, heat shock protein 27, and ubiquitin was enhanced in keratoconus corneas. A similar enhancement however was also observed in other diseased corneas.Conclusions. Altered expression of several wound healing or stress-related proteins was noted in keratoconus corneas. The alterations appear to be nonspecific injury or wound responses in association with corneal diseases.

ISSN:0271-3683    

DOI:10.3109/02713689608995144

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. This study investigates the role of insulin in retinal function by examining the effects of insulin on the a- and b-waves of the electroretinogram (ERG) recorded from anin vitrobovine retina eye-cup preparation.Methods. Bovine eyes were enucleated immediately after exsan-guination, hemisected to form an eye cup, and transported to the laboratory in oxygenated medium. Eye cups were then transferred to a perfusion system that provided constant superfusion of oxygenated perfusate solution to the retina, enabling it to remain in a functional statein vitro. The ERG was recorded, as the retinal response to photic stimulation, from electrodes mounted within the perfusion system. The effects of insulin on the ERG were investigated by adding insulin to the perfusate solution.Results. Application of insulin to thein vitroretina preparation decreased the amplitudes of the a- and b-waves of the ERG in a dose dependent manner with a maximal effect at doses of 0.1 U/ml and above. These effects were reversible.Conclusion. The reduction of ERG amplitudes may result from the hyperpolarising effect of insulin reported in other tissues. The findings suggest that insulin may have a regulatory role in retinal activity; however extrapolation of these results to the intact organism is dependent on the presence of insulin in retina.

ISSN:0271-3683    

DOI:10.3109/02713689608995145

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. The effects of embryonic development on lipid composition in the retina were studied in 7, 11, 15, and 18-day-old chick embryos and newly hatched chicks.Methods. The proportions of phospholipids, free and esterified cholesterol, diacylglycerides, and free fatty acids were determined using the Iatroscan TLC/FID procedure. Gas chromatog-raphy and mass spectrometry were used to determine the fatty acid composition.Results. The major phospholipid species were phosphatidyl-ethanolamine, phosphatidylcholine, phosphatidylserine, phos-phatidylinositol, lysophosphatidylcholine, and sphingomyelin. Concentrations of the analyzed components have been related to the chronology of concrete stages of retinal development. The fatty acid composition of the total lipids, (n-6):(n-3) and saturated:unsaturated fatty acid ratios, and other parameters are reported. The proportions of total saturated and total monoun-saturated fatty acids decreased very little from day 7 to hatching, whereas total polyunsaturated fatty acids nearly doubled over the same period. The increase in C18:2(n-6) from day 11 onwards was not followed by a similar increase in C20:4(n-6), hence the C20:4 to C18:2 ratio decreased with age.Conclusions. The cholesterol: phospholipid ratio decreased from day 7 to day 15 and increased from day 15 to hatching. High proportions of esterified cholesterol, very probably originating in the retinal pigment epithelium, were also recorded. Total saturated and monounsaturated fatty acids decreased, while polyunsaturated fatty acids increased during the period of initial retinal growth.

ISSN:0271-3683    

DOI:10.3109/02713689608995146

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Purpose. To examine whether transforming growth factor-β2 (TGF-β2) induces the expression ofα-smooth muscle actin (SMA), a biochemical marker of myofibroblasts, in cultured bovine retinal pigment epithelial (RPE) cells.Methods. Bovine RPE cells were cultured in F-12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), with or without TGF-β2 (0.01–10 ng/ml) for 6 days. During this culture period, cells did not reach a confluence. Alpha-SMA was detected immunocytochemically with a mouse monoclonal antibody, and the ratio of the number ofα-SMA positive cells to the total number of cells was calculated.Results. About 10% of the cells in control cultures with only FBS were positive forα-SMA. TGF-β2 increased the ratio of positive cells dose-dependently (p<0.0001), while a neutralizing antibody against TGF-β2 blocked this effect.Conclusions. TGF-β2 induced expression ofα-SMA in bovine RPE cells.

ISSN:0271-3683    

DOI:10.3109/02713689608995147

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

ISSN:0271-3683    

DOI:10.3109/02713689608995148

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor