The effects of dopa and oxygen on RNA concentrations in cultured chick embryonal retinal pigment epithelial cells

We measured RNA and DNA concentrations in cultured chick embryonal retinal pigment epithelial cells to investigate the effects of dopa and oxygen on DNA and RNA synthesis. RNA/DNA ratios were decreased by addition of 250 microM dopa. Decrease of RNA/DNA ratios was suppressed when the oxygen concentrations were reduced from 20% to 10%. Incubation with medium containing 100 microM dopa increased RNA/DNA ratios in 10% oxygen. Exposure of retinal pigment epithelial cells to 250 microM dopa caused the decrease of RNA concentrations in the retinal pigment epithelial cells, which was ameliorated by lowering oxygen concentrations. However, the addition of 100 microM dopa in 10% oxygen stimulated retinal pigment epithelial cells and seemed to increase RNA concentrations.     

Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

We observed the three dimensional structure of cellular aggregates formed from chick retinal cells in a floating culture system for 2 months. The aggregated cells partially mimicked the structure of the retina and showed differentiation of photoreceptor cells and Müller cells with numerous synapses. Immunohistochemical studies showed the number of anti-rhodopsin positive cells increasing over time. In the long-term culture, increasing anti-crystalline positive cells appeared late in the culture, indicative of differentiation of lens epithelial cells. Nerve, epidermal, and basic fibroblast growth factors, and co-culture with retinal pigment epithelial cells stimulated to some degree the growth of dendrites in retinal cellular aggregates. Epidermal growth factor, in particular, promoted the production of rhodopsin in photoreceptor cells. Retinal cellular aggregates in a floating culture system could be used to examine the effect of various factors on differentiation of the neuroretina.     

Feline ocular epithelial response to growth factors in vitro

OBJECTIVE: To examine the proliferative abilities of growth factors known to participate in wound healing on feline lens, iris pigment, ciliary, and retinal pigment epithelium cultured in vitro. ANIMALS: 8 clinically normal cats. PROCEDURE: Iris pigment, lens, ciliary, and retinal pigment epithelia of normal eyes of cats were isolated and cultured. Morphologic characteristics of primary cell cultures were studied by light and electron microscopy. Subcultures of epithelial cells were exposed to media supplemented with 0.5% fetal bovine serum plus various combinations of insulin and/or growth factors, including transforming growth factor-alpha, epidermal growth factor, acidic fibroblast growth factor, and basic fibroblast growth factor. Growth promoting effects were evaluated by counting with an electronic cell counter. RESULTS: Cells retained many of the morphologic characteristics of in vivo cells. Cell proliferation assays indicated that transforming growth factor-alpha stimulated lens and ciliary epithelial cell growth, and epidermal growth factor enhanced lens and iris pigment epithelial cell growth. Acidic fibroblast growth factor had proliferative effects on lens, iris pigment, and ciliary epithelium. Basic fibroblast growth factor was the most potent stimulator of all mitogens used, and caused substantial proliferation in all cell types. Insulin alone stimulated lens and ciliary epithelial proliferation but, combined with other growth factors, had a synergistic effect with those causing cell proliferation, except acidic fibroblast growth factor with iris pigment epithelium. CONCLUSION: Morphologic studies support the argument that pigment-producing cells are involved in feline ocular sarcoma. Growth factor studies indicated that ciliary epithelium has the most profound proliferative effect of all growth factors used. These data may help guide future studies in determining the cell of origin for feline ocular sarcoma.     

Expansion of the retinal pigment epithelium in experimental myopia

Expansion of the retinal pigment epithelium was studied in neonatal chicks after one or two weeks of unilateral form vision deprivation to investigate altered ocular growth mechanisms in this experimental model of myopia. The area of individual retinal pigment epithelial (RPE) cells, measured in tangential sections, was greater in myopic eyes than in contralateral control eyes at both times. The mean RPE cell area in myopic eyes increased to the same extent as the area of the retinal pigment epithelium as a whole. In control eyes between one and two weeks, RPE cell expansion occurred predominantly in the periphery; in myopic eyes, it occurred more generally across the epithelium but was less pronounced in the temporal region. Given the absence of detectable mitotic figures in control and myopic eyes, expansion of the epithelial layer is attributable to passive stretch or growth of existing cells. Whether scleral growth or stretch occurs selectively beneath the areas of more pronounced RPE cell expansion is unknown.     

Cytokines cause cultured retinal pigment epithelial cells to secrete metalloproteinases and to contract collagen gels

PURPOSE: Because retinal pigment epithelial cells in epiretinal membranes remodel and contract their surrounding extracellular matrix, investigations were performed to determine if these cells can produce matrix metalloproteinases and contract collagen gels in vitro in the presence of serum or cytokines. METHODS: Cells were grown on collagen gels and their production of metalloproteinases was measured using zymography. RESULTS: Cells grown in a three-dimensional collagen gel culture system produce several latent metalloproteinases that are secreted into the gel and the surrounding medium. These include molecules of 49, 56, 66, and 100 kD. In addition, an enzyme that is probably the active form of the 66 kD enzyme is present. When interleukin 1 beta is added to the cultures, latent 49 kD and 100 kD gelatinase production is greatly stimulated and an active form of both enzymes is also observed in the medium. In contrast, transforming growth factor beta has no stimulatory effect. The cells contract the collagen gel but this is small without cytokines; however, contraction is greatly enhanced in the presence of serum or interleukin 1 beta plus transforming growth factor beta. Contraction is unlikely to be the result of metalloproteinase action on the underlying extracellular matrix because complete inhibition of these enzymes has little effect. CONCLUSIONS: These results show that cytokines can cause cultured retinal pigment epithelial cells to produce metalloproteinases that can, when activated, degrade most collagens and other structural molecules in extracellular matrix. In addition, they can stimulate the contraction of extracellular matrix constituents but there is not a simple causal relationship between matrix remodeling and contraction.     

Novel characteristics of a myosin isolated from mammalian retinal pigment epithelial and endothelial cells

We have isolated a novel, high Mr protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 M urea eluent with apparent molecular mass of approximately 210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one protein band with an apparent molecular mass of approximately 550 kDa. "Piglet," as this molecule is tentatively named, is present in retinal pigment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluorescent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibody was used to identify a cDNA for piglet in a bovine aortic endothelial cell expression library. Sequence data indicate a high degree of identity with non-muscle myosin II heavy chain. We subsequently found that piglet had an actin-activated ATPase activity, colocalized with actin in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodies specific for myosin heavy chain isoform A, but did not react with anti-isoform B antibodies. Although piglet has several features in common with known forms of non-muscle myosin II, the distinctly unconventional features it displays suggest that it is a novel myosin.     

Basic fibroblast growth factor as one of the essential factors regulating lens transdifferentiation of pigmented epithelial cells

In vitro transdifferentiation of retinal pigmented epithelial cells of the chick embryo into lens cells can be markedly enhanced by culture in the presence of testicular hyaluronidase and phenylthiourea. Since the commercial preparations of hyaluronidase that had previously been used were very crude, a search for the actual effective molecule(s) enhancing lens transdifferentiation was conducted. First, we purified the enzyme and tested the effect of the purified hyaluronidase. Highly purified hyaluronidase itself did not enhance lens transdifferentiation. The crude hyaluronidase was then separated according to affinity with heparin, considering the possibility that the fibroblast growth factor (FGF) is contained in the crude hyaluronidase. Transdifferentiation-enhancing activity was detected in the fraction which was bound to heparin and eluted with 2 M NaCl, where no hyaluronate-degrading activity existed. Analysis of the fraction by SDS-PAGE revealed the existence of an 18 kDa protein whose NH2-terminal sequence was identical to that of basic FGF. The basic FGF derived from bovine brain also enhanced lens transdifferentiation of pigmented epithelial cells. These findings suggest that basic FGF must play a major role in enhancing transdifferentiation of pigmented epithelial cells to lens cells.     

Antibiotic susceptibility among aerobic gram-negative bacilli in intensive care units in 5 European countries. French and Portuguese ICU Study Groups

BACKGROUND: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch's membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch's membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. METHODS: In a series of three experiments, RPE cells of nine pink-eyed, 2 1/2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco's modified Eagles' medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. RESULTS: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats' eyes, a layer of slightly pigmented cells was seen on Bruch's membrane below the transplanted IPE cells, shown in light microscopy. CONCLUSION: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch's membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration.     

Phagocytosis of rod outer segments by human iris pigment epithelial cells in vitro

BACKGROUND: We set out to evaluate the growth potential of human iris pigment epithelial (hIPE) cells in vitro, to establish whether these cells acquire the ability to phagocytose rod outer segments (ROS) and to compare the phagocytic activity of hIPE to that of human retinal pigment epithelial (hRPE) cells. METHODS: hIPE and hRPE cells were isolated and cultured from human donor eyes and surgical specimens and growth characteristics were analyzed. HIPE and hRPE of an eye of a 46-year-old donor were used for the phagocytosis assay. Phagocytosis was evaluated by adding ROS isolated from porcine retina to cultures of hIPE and hRPE, which had been labeled with the pH-sensitive fluorescent dye, carboxy-SNAFL. After 4 h the number of ingested ROS was counted with a light microscope. For each cell type phagosomes in 500 cells were counted. The epithelial characteristics of the cells used in this study were evidenced by their morphology. RESULTS: Morphologically cultured hIPE are indistinguishable from the hRPE cultured from the same donor eye and show a similar pattern of cytokeratin distribution. Cultured hIPE acquire the ability to phagocytose ROS at a level slightly lower than hRPE; hIPE contained 0.76 phagosomes per cell, hRPE 0.99 phagosomes per cell. CONCLUSION: The morphology of hIPE in culture and the acquisition of the phagocytic phenotype indicate that these cells have the ability to differentiate into cells that have characteristics in common with hRPE. The acquisition of phagocytic activity suggests that it is feasible to culture hIPE from surgical iridectomies and that these cultured cells can be transplanted into the subretinal space in individuals with retinal degenerations.     

Cultured human retinal pigment epithelial cell produced and secreted epidermal growth factor: its bioactivity and clinical significance

The epidermal growth factor bioactivity from cultured primary human and monkey retinal pigment epithelial (RPE) cell systems was detected by using the radioreceptor assay. We report that the cultured human and monkey RPE cells produce and secrete the EGF bioactively to the media as demonstrated by radioreceptor binding assay. The EGF bioactivities secreted by human and monkey RPE cells were at peak of 48 hours (human RPE cells secreted 2.11 +/- 0.46 ng/ml vs monkey RPE was 1.56 +/- 0.12 ng/ml) in the serum-free media. The results indicate that the RPE is one of important sources for EGF in the eye. The RPE cells may play much important roles in the development of proliferative retinal diseases through the autocrine or paracrine mechanism. This new discovery will be helpful to elucidate the pathogenesis of proliferative retinal diseases and also provide an important basis for the treatment of such diseases.     

Repair and adhesion mechanisms of the cryotherapy lesion in experimental retinal detachment

Cryotherapy to the pigment epithelium and retina induced a proliferation and metaplasia of pigment epithelial cells, Mueller cell hypertrophy, and proliferation of astrocytes. When cryotherapy was applied to the pigment epithelium and to the retina during retinal detachment surgery, a strong adhesion developed, characterized by the occurrence of true cell junctions between pigment epithelium and retinal cells. When only the pigment epithelium was treated, the adhesion appeared weak due to the absence of microvillous interdigitations normally present between pigment epithelium and retina.     

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium

Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor.     

Multiplane transesophageal echocardiography in diagnosis of anomalous origin of the left coronary artery from the pulmonary artery: a case report

PURPOSE: To investigate whether there is a difference in the expression of adenovirus transgenes in human retinal pigment epithelial cells when the vector was exposed to the apical or basal surface, the effect of transgene expression on rod outer segment (ROS) phagocytosis and finally, the role of phagocytosis in gene transfer to RPE cells, using the Royal College of Surgeons (RCS) rat. METHODS: Monolayers of human retinal pigment epithelium (HRPE) or an RPE cell line (A407) had the apical or basal surfaces exposed to 10(7) pfu/ml of replication deficient adenovirus (Ad.RSV.betagal) carrying the beta-galactosidase marker gene, and the numbers of expressing cells were compared. Parallel cultures were infected and challenged with fluorescein-labelled bovine rod outer segments (FBROS). The fluorescence of infected versus uninfected cells was recorded for both challenged and unchallenged states, using fluorophotometric flow cytometry. Primary cultures of RCS rat RPE were established and the transgene uptake dynamics compared to control Long Evans rat RPE cells. RESULTS: The expression of transgene in HRPE and A407 cell cultures was an order of magnitude greater when the vector was exposed apically (analysis of variance p < 0.05). There was no difference in the phagocytic capacity of Ad.RSV.betagal-infected and -noninfected cells when challenged with FBROS. There was also no difference in the number of cells expressing transgene, when compared to the RCS or Long Evans control rat RPE. CONCLUSIONS: The surface of exposure in polarized retinal pigment epithelial cells affects the rate of uptake and expression of adenovirus. The defective ROS phagocytosis in RCS rat RPE cells did not lead to a decrease in transgene expression relative to the Long Evans control cells. Finally we have found that phagocytosis is not significantly altered with adenoviral transgene expression in this in vitro model.     

Advanced glycation end products in age-related macular degeneration

OBJECTIVE: To investigate the localization of N epsilon-(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration. METHODS: Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections. RESULTS: Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm. CONCLUSION: The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration. CLINICAL RELEVANCE: Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.     

Rod outer segment renewal in the retinae of deep-sea fish

Outer segment renewal involves the synthesis of disc material in the photoreceptor inner segments, the shedding of the tips of the photoreceptor outer segments, and their phagocytosis by the retinal pigment epithelial cells. It has been suggested that in the retinae of deep-sea fish no renewal of outer segments may take place. In order to assess outer segment renewal in deep-sea fish retinae we counted (i) periciliary vesicles in rod inner segments as a parameter for disc-synthesis activity and (ii) phagosomes in retinal pigment epithelial cells as a parameter of shedding and phagocytosis in 12 species of deep-sea fish with multibank or single bank retinae. We also measured the lengths of rod outer segments in order to evaluate the balance between synthesis and phagocytotic activity. In four of these species (Synaphobranchus kaupi, Nematonurus armatus, Coryphaenoides guentheri and Halosauropsis macrochir) we further recorded size-related changes of these parameters and their relation to the position of a given rod within the banks in the retina. The number of periciliary vesicles was highest in inner segments of the most vitread bank and in the periphery of the retina. Phagosomes were most abundant in retinal pigment epithelial cells of the central retina. Long rod outer segments were most frequently recorded in the peripheral retina indicating that in this region new synthesis may outbalance shedding. Vitread rod outer segments were only slightly longer than sclerad ones. Larger animals had shorter rod outer segments than small ones. We present evidence that rod outer segment renewal takes place in the retina of all deep-sea fish. Vitread rods may be more active in this respect than sclerad ones.     

Biogenesis of the purple membrane of Halobacterium halobium

In 168 eyes with preretinal membranes studied histopathologically, five major distinct types of membranes were categorized: 61 glial, 28 fibrous, 22 cortical vitreous, 16 retinal pigment epithelium, 15 fibroinflammatory, 24 combinations, and one endothelial and melanomatous membrane each. The overall prevalence of preretinal membranes was 5.5% in 2,000 cases studied. Selected cases examined by electron microscopy were supportive of the origin of the cells in the preretinal membranes in the glial, fibrous, and retinal pigment epithelial types. Glial preretinal membranes occurred as the result of defects in the internal limiting membrane, such as retinal pits or holes, and from the optic nerve head in association with posterior vitreous detachment. Fibrous preretinal membranes were associated with proliferative retinopathy and long-standing retinal detachment. Studies of the cellular origin of these membranes were inconclusive. The cortical vitreous type of preretinal membrane was hypocellular and occurred in the absence of associated ocular abnormalities. Pigment epithelial preretinal membranes occurred in eyes with rhegmatogenous retinal detachment.     

A Sexual Adjustment Questionnaire for use in therapy and research with alcoholics and their spouses

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium and Müller glial cells of the retina, where it forms complexes with endogenous 11-cis-retinoids. We examined the distribution of CRALBP in extraretinal tissues using polyclonal antibodies (pAb) and monoclonal antibodies (mAb). A protein was detected by immunoblot analysis in extracts of bovine and rat brain and optic nerve but not in several other tissues. This protein had electrophoretic, chromatographic, and retinoid-binding properties identical to those of CRALBP from bovine retina. Comparison of the masses of tryptic peptides and of partial amino acid sequences derived from brain and retinal CRALBP indicated that the two proteins are probably identical. Immunoperoxidase cytochemistry and double labeling immunofluorescence revealed CRALBP(+) cells in brain that resembled oligondendrocytes and not astrocytes, microglial cells, or pinealocytes. In 11-day-old rat brain, approximately 11% of the CRALBP(+) cells were labeled with the Rip antibody, a marker for oligodendroglia. In developing rat optic nerve, the temporal appearance of CRALBP(+) cells corresponded to that of oligodendrocytes and not that of astrocytes. In adult rat and mouse optic nerves, the CRALBP(+) somata showed the same distribution as oligodendrocytes. No endogenous retinoids were associated with CRALBP isolated from dark-dissected adult bovine brain. The results suggest that CRALBP has functions in addition to retinoid metabolism and visual pigment regeneration.