Lectin binding patterns in amphibian skin epithelium.

Seven lectins (PNA, DBA, WGA, UEA-I, RCA, SBA, Con A) were used to localize glycoconjugates in the skin of 10 species of Amphibia, 7 anurans (Bufo marinus, Bufo bufo, Rana ridibunda, Rana pipiens, Hyla arborea, Pelobates syriacus and Xenopus laevis) and 3 urodeles (Salamandra salamandra, Triturus vulgaris and Ambystoma mexicanum). It was found that every lectin has a specific binding pattern in the skin of each species. No common pattern could be established, either among frogs or toads, nor for a particular lectin. Each lectin bound specifically and selectively to a particular epithelial component, which differed from one species to the other. A number of lectins showed selective binding to mitochondria-rich cells, but, again, a pattern in positivity could not be found. It is concluded that lectin histochemistry does correlate with cellular function. Our data can be applied in studies of epithelium and skin development, and of changes that occur during adaptation to the environment by amphibian species.     

Electric impedance of human embryonic stem cell-derived retinal pigment epithelium

The barrier properties of epithelium are conventionally defined by transepithelial resistance (TER). TER provides information about the tightness of the epithelium. Electrical impedance spectroscopy (EIS) provides additional information regarding cell membrane properties, such as changes in electric capacitance and possible parallel or serial pathways that may correlate with the morphology of the cell layer. This study presents EIS of retinal pigment epithelial (RPE) cell model of the putative RPE differentiated from human embryonic stem cells (hESC-RPE). The generally utilized RPE cell model, ARPE-19, was used as immature control. The measured EIS was analyzed by fitting an equivalent electrical circuit model describing the resistive and capacitive properties of the RPE. Our results indicated that TER of hESC-RPE cells was close to the values of human RPE presented in the literature. This provides evidence that the stem cell-derived RPE in vitro can reach high-barrier function. Furthermore, hESC-RPE cells produced impedance spectra that can be modeled by the equivalent circuit of one time constant. ARPE-19 cells produced low-barrier properties, that is, an impedance spectra that suggested poor maturation of ARPE-19 cells. To conclude, EIS could give us means for non-invasively estimating the functionality and maturation of differentiated-RPE cells.     

The retinal pigment epithelium in visual function

Located between vessels of the choriocapillaris and light-sensitive outer segments of the photoreceptors, the retinal pigment epithelium (RPE) closely interacts with photoreceptors in the maintenance of visual function. Increasing knowledge of the multiple functions performed by the RPE improved the understanding of many diseases leading to blindness. This review summarizes the current knowledge of RPE functions and describes how failure of these functions causes loss of visual function. Mutations in genes that are expressed in the RPE can lead to photoreceptor degeneration. On the other hand, mutations in genes expressed in photoreceptors can lead to degenerations of the RPE. Thus both tissues can be regarded as a functional unit where both interacting partners depend on each other.     

糖尿病大鼠视网膜色素上皮细胞超微结构

目的 :探讨糖尿病大鼠视网膜色素上皮细胞超微结构病变 ,为糖尿病视功能障碍提供有关的行态学依据。方法 :选择清洁级SD大鼠随机分成正常对照组、糖尿病 1月、3月和 6月。腹腔内注射链脲佐菌素 (STZ)诱导大鼠糖尿病 ,每组 1 2只 ,取视网膜制备超薄切片 ,透射电镜观察。处死前每月测体重、血糖 1次。结果 :糖网病大鼠视网膜色素上皮细胞的微绒毛和基底部的质膜内褶随血糖的增高病情加重而逐渐稀疏、低矮直至脱落、低平。胞质内细胞器以线粒体的脱嵴、水肿和内质网的扩张为主要损害 ,而相邻色素上皮细胞之间的连接复合体始终存在。结论 :糖网病大鼠视网膜色素上皮细胞的损害主要表现于微绒毛、质膜内褶及细胞器的损害 ,并无脉络膜与视网膜之间屏障功能的损害。本研究为糖网病的临床研究提供形态学依据     

Lectin binding pattern in the embryonal and early fetal human vertebral column

Summary Paraffin sections from vertebral columns of ten human embryos and fetuses ranging from stage 16 to the 12th week were stained with the FITC-coupled lectins PNA, RCA I, Con A and WGA in order to investigate changes in carbohydrate-binding sites during vertebral development. PNA revealed a specific binding site in the vertebral body blastema in the precartilaginous stage of development. Beginning with the 25-mm CRL embryo, PNA-binding sites occurred in the developing fibrous annulus and the inner zone of the intervertebral discs. The first binding sites for RCA I were seen in the extracellular matrix of vertebral bodies during the cartilaginous stage of vertebral development. During early ossification of the vertebrae, staining for RCA I-binding sites in the cytoplasm of the chondrocytes and the area around the future cartilaginous end-plates was observed. Con A bound to the chondrocyte cytoplasm, and also very strongly to notochordal cells in all developmental stages examined. WGA-binding sites appeared simultaneously with cartilage formation. Connective tissue components, e.g. ligaments, were diffusely stained by WGA. Also this lectin showed an affinity for vertebral body chondrocytes. We discuss the biochemical aspects of these lectin-binding sites, and their possible roles in the differentiation process of the human vertebral column. The results of this first lectin histochemical study on human vertebral development are compared with related results in other species.     

Taurine receptors in membranes from retinal pigment epithelium cells in culture.

[3H]Taurine-specific binding to membranes from retinal pigment epithelium was demonstrated. A single saturable system was found, with KB = 237 nM and Bmax = 2.8 pmol/mg protein. Binding to freshly prepared membranes showed partial Na(+)-dependence while in frozen/thawed membranes, binding remained unchanged in the absence or presence of this ion. A 30-40% increase in binding was observed at physiological temperature (37 degrees C) compared to 4 degrees C in fresh but not in frozen membranes. Accumulation of taurine was followed during differentiation in vitro; results showed that changes in uptake and receptor binding to frozen membranes are not parallel, discarding the possibility of an interaction with uptake sites. Pharmacology of these binding sites suggests that they could be common to other amino acids, since displacement experiments showed that glycine, beta-alanine and strychnine were as potent as taurine itself in displacing [3H]taurine. Our data open the possibility of taurine being involved in the communication between the retina and the retinal pigment epithelium through an interaction with specific receptors.     

Claudin localization in cilia of the retinal pigment epithelium

Using immunocytochemistry and confocal microscopy we demonstrate that claudin-immunoreactivity is a novel marker for retinal pigment epithelial cilia. Claudin-immunoreactivity obtained by polyclonal anti-claudin 1 antibody, which could crossreact with claudin 3, was colocalized with acetylated tubulin-immunoreactivity in cultured human retinal pigment epithelial cells. Claudin-immunoreactivity associated with the retinal pigment epithelium (RPE) cilia was more intense than was claudin-immunoreactivity in the junctional complex. Approximately two-thirds of the RPE cells in the rat contain cilia that are immunoreactive with acetylated tubulin on postnatal day 1, and a significant portion of these cilia label with the anti-claudin 1 antibody. Cilia decrease in frequency over subsequent postnatal days, and are absent by postnatal day 30. As RPE cilia decrease in number during postnatal rat development, claudin-immunoreactivity is lost earlier than acetylated tubulin, suggesting that the loss of claudin may initiate RPE cilium degeneration. Claudin-immunoreactivity was not evident in cilia of photoreceptor cells, epithelia of nasal mucosa, small intestine, or colon, suggesting that claudin may be a unique molecule in RPE cilia. These data suggest that cilia of the RPE, unlike cilia on other cell types, contain claudin, and that this molecule may play an important and specific role in the function and/or maintenance of RPE cilia.     

Ferroxidase hephaestin's cell-autonomous role in the retinal pigment epithelium

Hephaestin (Heph) is a ferroxidase protein that converts ferrous to ferric iron to facilitate cellular iron export by ferroportin. Many tissues express either Heph or its homologue, ceruloplasmin (Cp), but the retina expresses both. In mice, a combined systemic mutation of Heph and systemic knockout of Cp (Cp(-/-), Heph(sla/sla)) causes retinal iron accumulation and retinal degeneration, with features of human age-related macular degeneration; however, the role of Heph and Cp in the individual retinal cells is unclear. Herein, we used conditional knockout mice to study Heph's role in retinal pigment epithelial (RPE) and photoreceptor cells. Loss of both Heph and Cp from RPE cells alone results in RPE cell iron accumulation and degeneration. We found, however, that RPE iron accumulation in these conditional knockout mice is not as great as in systemic knockout mice. Photoreceptor-specific Heph knockout indicates that the additional iron in the RPE cells does not result from loss of ferroxidases in the photoreceptors, and Cp and Heph play minor roles in photoreceptors. Instead, loss of ferroxidases in other retinal cells causes retinal iron accumulation and transfer of iron to the RPE cells. Cp and Heph are necessary for iron export from the retina but are not essential for iron import into the retina. Thus, our studies, revise how we think about iron import and export from the retina.     

Light-evoked responses of the mouse retinal pigment epithelium

In response to light, the retinal pigment epithelium (RPE) generates a series of slow potentials that can be recorded as the c-wave, fast oscillation (FO), and light peak (LP) of the electroretinogram (ERG). As these potentials can be related to specific cellular events, they provide information about RPE function and how that may be altered by disease or experimental manipulation. In the present study we describe a noninvasive means for recording the light-evoked responses of the mouse RPE and use this to define the stimulus-response properties of the major components in three inbred strains of mice (BALBc/ByJ, C57BL/6J, and 129/SvJ) and two mouse mutants that reduce activity in the rod pathway. All of the major ERG components generated by the RPE are readily measured in the mouse. In albino strains (BALBc/ByJ and 129/SvJ) the intensity-response functions for the c-wave, FO, and LP are shifted toward lower intensities in comparison to those for C57BL/6J mice. Each of these components was markedly reduced in mice lacking transducin in which rod phototransduction is interrupted, indicating that they reflect primarily rod photoreceptor activity. All components were observed in no b-wave (nob) mutant mice, indicating that inner retinal activity does not make a major contribution to these potentials. Further studies of mutant mice will allow us to define the functional consequences of gene manipulation on RPE function and to evaluate specific hypotheses regarding the generation of ERG components.     

视网膜色素上皮腺瘤的病理形态学观察

目的　探讨视网膜色素上皮腺瘤临床及形态学特征 ,为其诊断及鉴别提供依据。方法　常规石蜡切片HE染色、组织化学PAS和VG染色、透射电镜及用SP法做免疫组化S 10 0、Cytokeratin和Vimentin检测。结果　该瘤细胞呈椭圆形、立方形 ,部分瘤细胞排列成腺管状 ,瘤细胞团周围见均匀红染的条状物质 ;上述条状物质PAS染色阳性 ,VG染色大部分呈黄色 ,少量呈红色 ;透射电镜显示细胞的紧密连接 ;免疫组化S 10 0和Cytokeratin阳性、vimentin阴性。结论　视网膜色素上皮腺瘤的超微结构及免疫组化特征均同视网膜色素上皮相似 ,可通过形态学检查确诊。     

A novel Bruch's membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells

Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3–5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.     

Adenocarcinoma of the retinal pigment epithelium in the guppy Poecilia reticulata Peters.

A single case of adenocarcinoma of the retinal pigment epithelium occurred in a guppy, Poecilia reticulata, Peters. This is the first such tumour reported from fishes. The left eye of the affected fish was severely exophthalmic because of a large intraocular tumour mass. The tumour, which displaced normal retina anteriorly, consisted mainly of melanin-containing epithelial cells. Neoplastic cells were bilayered and arranged in a tubular pattern. The tumour was confined to the orbit. Although the specimen was from a group exposed to a mixture of halogenated organic compounds, the lesion was not considered to have been chemically induced because of its rare occurrence within the group as a whole.     

地塞米松对视网膜色素上皮细胞IL—6表达的抑制作用

目的观察视网膜色素上皮(RPE)细胞中,IL-6的表达及地塞米松对IL-6表达的抑制作用.方法培养的人RPE经IL-1β(10μg/L)刺激及地塞米松作用后,采用ELASA、免疫组化和原位杂交等方法,检测培养的人RPEIL-6mRNA及其蛋白的表达.结果用IL-1β刺激后8h,RPE细胞表达的IL-6约为2000ng/L@106细胞;地塞米松可明显抑制IL-6蛋白的产生(200ng/L@106细胞),与对照组相比较差异显著(P＜0.01).免疫组化染色显示,表达的IL-6在RPE细胞胞浆中呈浓淡不一的棕黄色,地塞米松作用组染色较浅.原位杂交表明,IL-6mRNA在RPE细胞胞浆中的表达呈浓淡不一的紫蓝色,地塞米松作用组染色与对照组相同.图像分析显示,二者的吸光度(A)值无明显差别(P＞0.05).结论在IL-1β刺激下,人RPE细胞可表达IL-6的mRNA及其蛋白,地塞米松能有效地抑制RPE细胞中IL-6蛋白的表达.     

Acid phosphatase localization in normal and dystrophic retinal pigment epithelium

Summary In this study acid phosphatase (ACPase) was localized in the retinal pigment epithelium (RPE) of normal and Royal College of Surgeons (RCS) rats pink-eyed and pigmented with inherited retinal dystrophy to determine differences in staining during the post-engulfment stages of phagocytosis using two substrates, Na--glycerophosphate and cytidine-5-monophosphate. Staining was similar using either substrate and in the normal RPE the Golgi system, lysosomes and phagosomes were ACPase-positive. In the dystrophic RPE, which has a diminished capacity to phagocytose photoreceptor rod outer segments, ACPase staining was localized on melanosomes in the pigmented dystrophic and on the apical microvillous membranes in the pink-eyed dystrophic, but was not localized on phagosomes in either the pink-eyed or pigmented dystrophic RPE. Since only a few phagosomes were seen at any given time in dystrophic RPE in vivo, a tissue expiant system was used to examine the number of latex beads phagocytosed by normal and RCS RPE, as well as the number of phagosomes containing both beads and ACPase activity in the normal and mutant RPE. Our findings indicate that in the dystrophic, fewer phagosomes are ACPase-positive than in the normal, and that some enzyme may be inappropriately shunted to either the apical microvilli or to melanosomes instead of to phagolysosomes.     

早期人胚胎视网膜色素上皮细胞的体外培养及生物学特性

目的探索早期人胚视网膜色素上皮(RPE)细胞体外培养以获得高纯度的RPE细胞的方法,并进一步研究其生物学特性。方法取意外流产4h以内人胚(6～10周)眼球,通过混合酶消化法获取RPE细胞。用F12培养液体外培养并作细胞形态学、S-100和Keratin免疫组织化学鉴定;通过Trizol一步法提取总RNA,逆转录聚合酶反应(RT-PCR)分析体外培养RPE细胞酪氨酸酶基因的表达。结果用F12培养液可获得纯度高、活性好的人胚RPE细胞;体外培养的RPE细胞不表达酪氨酸酶基因。结论早期人胚RPE细胞具有在体RPE细胞的部分特征;体外培养的RPE细胞未检测到酪氨酸酶RNA表达,故随着细胞分裂增殖,色素颗粒逐渐减少,提示体内外RPE细胞在功能上存在某些差异。     

Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration

Mitochondrial dysfunctions have been implicated in the pathophysiology of several age-related diseases including age-related macular degeneration (AMD), a progressive neurodegenerative disease affecting primarily the retinal pigment epithelium (RPE). The aims of our electron microscopic and morphometric studies were to reveal qualitative and quantitative alterations of mitochondria in human RPE from AMD and from age- and sex-matched controls. With increasing age a significant decrease in number and area of mitochondria, as well as loss of cristae and matrix density were found in both AMD and control specimens. These decreases were significantly greater in AMD than in normal aging. Alterations of mitochondria were accompanied by proliferation of peroxisomes and lipofuscin granules in both AMD and control specimens, although the difference between groups was significant only for peroxisomes. Unexpectedly, morphometric data showed that the RPE alterations seen in AMD may also develop in normal aging, 10-15 years after appearing in AMD patients. These findings suggest that (i) the severity of mitochondrial and peroxisomal alterations are different between AMD and normal aging, and (ii) the timing of damage to RPE may be critical for the development of AMD. We conclude that besides the well-documented age-related changes in mitochondrial DNA, alterations of mitochondrial membranes may also play a role in the pathogenesis of AMD. These membranes could be a new target for treatment of AMD and other age-related diseases.     

Redistribution of Na-K-ATPase in the dystrophic rat retinal pigment epithelium

Summary Our previous studies have shown that a breakdown in tight junctions in the dystrophic retinal pigment epithelium (RPE) of Royal College of Surgeons' rats is accompanied by changes in intramembrane structure which suggest a redistribution of intramembrane particles. We have now investigated, using thep-nitrophenyl phosphate technique, the possibility that a specific membrane protein, Na-K-ATPase, is redistributed as tight junctions break down in the dystrophic RPE. In the normal RPE, Na-K-ATPase activity is restricted to the apical membrane. Junctional membranes and membranes around phagosomes are free of enzyme activity, suggesting a segregation of the transport enzyme from the Junctional and phagocytic membrane. In the dystrophic RPE, prior to changes in tight junctions, enzyme activity is restricted to the apical membrane. During the initial stages of Junctional breakdown, Junctional membranes and membranes around cytoplasmic inclusions are also labelled. As the breakdown progresses, Na-K-ATPase activity is often present laterally and basolaterally and is sometimes absent apically. Enzyme activity is seen basally only where RPE cells have detached from Bruch's membrane and are superimposed over each other. These changes suggest that Na-K-ATPase redistributes during junctional breakdown, but that attachments between the RPE and Bruch's membrane may restrict the redistribution. The apparent reduction of enzyme activity apically suggests that active transport across the dystrophic RPE may be reduced as the tight junctions break down.     

Lectin binding to the human retina

Formalin-fixed, paraffin-embedded tissue sections of the human retina were stained with different fluorescein isothiocyanate-conjugated lectins. The lectins used were concanavalin A (Con A), Triticum vulgaris (WGA), glycine maximum (SBA), Dolichos biflorus (DBA), Ulex europaeus (UEA I), Arachis hypogaea (PNA), and Ricinus communis (RCA I). Con A stained both the inner and outer segments of the rods and cones, whereas WGA stained the inner and outer segments of the rods and the outer segments of the cones. PNA selectively stained only the inner segments of the cones. In addition, Con A and WGA stained neuron cytoplasm and nerve fibers in different layers of the retina. The results obtained differ in some important aspects from those previously obtained in the frog and monkey retina; this finding may be due to species differences. The results of lectin staining in the normal human retina may form the basis for future studies of retinal diseases.