Lectin and immunohistochemical comparison of glycoconjugates in the conjunctiva of patients with and without exfoliation syndrome.

AIMS--The study was carried out to search for labelling similar to that of intraocular exfoliation material in the conjunctiva by light microscopy using lectin and immunohistochemistry. METHODS--Ten formalin fixed and paraffin embedded conjunctival biopsy specimens both from patients with and without exfoliation syndrome were studied with a panel of 11 lectins and with three monoclonal antibodies to the HNK-1 carbohydrate epitope, all of which react with intraocular exfoliation material. RESULTS--The lectin binding profile was essentially the same in specimens from patients with and without exfoliation syndrome. The superficial epithelium reacted similarly with Phaseolus vulgaris (PHA-E), Caragana arborescens (CAA), Helix pomatia (HPA), concanavalin A (ConA), and wheat germ (WGA) agglutinins. Binding was also detected with peanut (PNA) and Bauhinia purpurea (BPA) agglutinins, particularly in patients with exfoliation. The basement membrane generally reacted with Ricinus communis (RCA-I), PHA-E, Vicia villosa (VVA), ConA, and Lens culinaris (LCA) agglutinins. The stroma was weakly labelled with RCA-I, PHA-E, ConA, and LCA. Lectin binding to the vascular endothelium was moderate with RCA-I, PHA-E, CAA, ConA, LCA, and WGA. Inconsistent labelling was also detected with PNA, BPA, and Erythrina cristagalli agglutinin (ECA). The subendothelial region reacted weakly but consistently with PHA-E, ConA, and LCA, and inconsistently with PNA. Pretreatment with neuraminidase did not change that pattern. Antibodies to the HNK-1 epitope reacted only with myelinated stromal nerve branches. CONCLUSION--No evidence of abnormal deposits in any specimen was found. The carbohydrate composition of intraocular exfoliation material may differ from that of exfoliation-like fibres often detected in the conjunctiva by electron microscopy.     

人正常发育角膜上皮和角膜基质的凝集素化学研究

目的:研究正常人发育角膜上皮及基质中凝集素亲和糖蛋白的分布及变化,探讨糖类化合物对角膜发育的可能作用。方法:用五种生物素标记的凝集素:麦胚凝集素(Wheat Germ Agglutin WGA)、伴刀豆凝集素(Con-Canavlin A ConA)、花生凝集素(Peanut Agglutin PNA)、蓖麻凝集素(Ricinus Communis Agglutin I RCA-I)扁豆凝集素(Lens Culinaris A LCA)对人正常发育角膜进行凝集素组织化学研究。结果:1.WGA受体主要分布在角膜上皮细胞膜中,尤其在表层上皮细胞中有明显分布,在发育期间胚胎角膜的前部基质中也有WGA受体分布。2.ConA受体分布于角膜上皮细胞膜及细胞质内,在胚胎期角膜基质中有不均一分布。3.RCALCA受体主要分布在角膜上皮基底部细胞膜上。4.PNA受体在胚胎16周角膜上皮细胞中有较少量分布。结论:1.在人正常发育角膜组织中含有ConA、WGA、PNA、LCA、RCA亲和性糖蛋白,这些糖蛋白参与发育角膜构成。2.在人角膜发育成熟过程中,有凝集素亲和糖蛋白的分布变化,这种变化可能与角膜的发育成熟有关。眼科学报1996;12:178～182。     

Characterization of galactose-containing glycoconjugates in the human retina: a lectin histochemical study

Seven specimens of morphologically normal formalin-fixed and paraffin-embedded human retina were studied using a panel of fourteen biotinylated lectins, all of which react with glycoconjugates containing galactose and N-acetylgalactosamine residues. Agaricus bisporus (ABA), Bauhinia purpurea (BPA), Phaseolus vulgaris (PHA-E), peanut (PNA) and Ricinus communis (RCA-I) agglutinins labeled photoreceptor cells prior to enzymatic predigestion. BPA and PNA bound specifically to cones. The plexiform layers reacted with ABA, BPA and PHA-E, while only ABA and PHA-E labeled the nuclear layers. After pretreatment with neuraminidase to remove terminal sialic acid, all five lectins, as well as Erythrina cristagalli (ECA), Helix pomatia (HPA) and Maclura pomifera (MPA) agglutinins labeled both rods and cones. Furthermore, the plexiform layers additionally reacted with ECA, PNA and RCA-I, and the nuclear layers with BPA and RCA-I after neuraminidase pretreatment. Retinal vascular endothelial cells consistently bound ABA, ECA, PHA-E and RCA-I, but they could also bind BPA, HPA, Bandeiraea simplicifolia (BSA-I), Dolichos biflorus (DBA) and Euonymus europaeus (EEA) agglutinins in unpretreated sections, as well as MPA, PNA, soybean (SBA) and Sophora japonica (SJA) agglutinins subsequent to predigestion with neuraminidase. The nonpigmented ciliary epithelium reacted with the same lectins as photoreceptor cells, but it was also labeled by DBA. Sambucus nigra agglutinin (SNA) did not specifically bind to any intraocular structure. These findings favor the theory that, in unpretreated specimens, Gal(beta 1----3)GalNAc (BPA and PNA) is mainly responsible for labeling of cones, while Gal(beta 1----3/4)GlcNAc units, partly substituted with terminal sialic acid (PHA-E and RCA-I), explain labeling of rods. Following pretreatment with neuraminidase, further Gal(beta 1----3)GalNAc (BPA and PNA) and, especially, Gal(beta 1----3/4)GlcNAc (BPA, ECA, PHA-E, PNA and RCA-I) and alpha GalNAc units (BPA, HPA and MPA), the latter partly linked to the protein backbone, contribute to labeling of photoreceptor cells. Gal(beta 1----3/4)GlcNAc units may be mainly responsible for labeling of nuclear and plexiform layers. Finally, other related receptor sites (SBA and SJA), some of which are blood-group specific (BSA-I, DBA, EEA and HPA) are restricted to retinal vascular endothelia.     

Alterations in stromal glycoconjugates in macular corneal dystrophy

Nine biotinylated lectins were used as histochemical probes to localize the carbohydrates residues of glycoconjugates in normal corneas and in corneas with macular and granular dystrophy. The lectin binding patterns of normal corneas and of corneas with granular dystrophy were indistinguishable from one another, but were distinctly different from those found in corneas with macular dystrophy. Concanavalin A reacted weakly with normal corneal stromal matrix, but stained stromal matrix of corneas with macular dystrophy intensely. Furthermore, unlike the normal corneal matrix, stromal matrix of corneas with macular dystrophy reacted positively with wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Ulex europeus I, Dolichos biflorus, Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, and soybean and peanut lectins. This study demonstrates specific alterations in glycoconjugates which occur in the corneal matrix of patients with macular dystrophy, namely the presence of oligosaccharides with terminal alpha-fucose, beta-galactose, N-acetylglucosamine and N-acetylgalactosamine residues, and oligosaccharide chains with a beta-galactose-N-acetylgalactosamine sequence.     

Localization of lectin binding sites in human, cat, and rabbit corneas

Paraffin sections of human, cat, and rabbit corneas were stained with nine lectins, using an avidin-biotin-complex procedure to study glycoconjugates of the epithelium, keratocytes, and stromal matrix. Wheat germ agglutinin (WGA) stained plasma membranes of all epithelial cell layers of cat and human and superficial and wing cells of rabbit. Plasma membranes of superficial and wing cells of cat epithelium also stained with peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA-I). Human and cat keratocytes stained with WGA and RCA-I. Stromal matrices of all three species were stained with concanavalin A and lentil agglutinin. In neuraminidase-treated sections, the entire epithelium and keratocytes of all three species stained with PNA. Corneal sections from the three species did not stain with Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, Ulex europeus I, and Soybean agglutinin. These data suggest the presence of oligosaccharides with: N-acetylglucosamine/sialic acid residues in cell membranes of corneal epithelium of all species studied and in the keratocytes of human and cat; terminal beta-galactose residues in cat and human keratocytes, beta-galactose-galactosamine chains in cat epithelial cell membranes; and sialic acid-beta-galactose-galactosamine chains in epithelial cell membranes and keratocytes of all three species.     

Galactose-containing glycoconjugates of the iris,the aqueous outflow passages and the cornea in capsular glaucoma

Background: The aim of the study was to compare galactose-containing glycoconjugates of the iris, the aqueous outflow pas sages and the cornea with exfoliation material in capsular glaucoma. Methods: Six formalinfixed, paraffin-embedded human eyes with capsular glaucoma and six control eyes were studied by using a panel of 11 biotinylated lectins to galactose- and N-acetylgalactosamine-containing glycoconjugates. Results: The Gal ( 3) GaINAc-reactive lectins peanut agglutinin (PNA) and Bauhinia purpurea alba agglutinin (BPA) and the Gal (14)GlcNAc-reactive lectins Ricinus communis agglutinin (RCA-I) and Phaseolus vulgaris erythroagglutinin (PHA-E) gave the strongest label with exfoliation material. Lectin binding to the iris was variable. The binding of PNA, BPA, RCA-I, Erythrina cristagalli agglutinin (ECA), PHA-E and Glycine max agglutinin (SBA) to the subendothelial region of iris blood vessels closely resembled their binding to exfoliation material. RCA-I and PHA-E bound moderately to the aqueous outflow passages. The surface of the corneal epithelium showed positive reaction with most lectins studied, but the keratocytes reacted with RCA-I and PHA-E only. Neuraminidase pretreatment generally increased the reaction intensity. Conclusions: The findings suggest that the glycoconjugate composition of exfoliation material in the classical locations along the anterior and posterior chamber closely resembles that in the subendothelial region of iris blood vessels.The authors have no financial interest in any product or process mentioned herein     

Abnormal extracellular matrix in corneas with pseudophakic bullous keratopathy

The present study describes biochemical and morphological differences of pseudophakic bullous keratopathy (PBK) corneas as compared with normal corneas. At the ultrastructural level, all PBK corneas studied had abnormal fibrillar material posterior to the Descemet's membrane. In addition, two of the six PBK buttons had subepithelial fibrocellular materials disrupting the epithelial basement membrane and Bowman's layer. Aggregates of collagen fibrils with 110 nm periodicity were occasionally seen within the stroma of the PBK corneas. Isolation and purification of the collagen from the Descemet's membrane/posterior collagenous layer (DM/PCL) showed an increased amount of material with molecular weight in the range of 50-60K daltons (presumably type VIII collagen) and decreased amounts of higher molecular weight, disulfide-bonded collagenous materials (presumably type IV collagen) as compared with normals. Sugar-specific lectin studies showed an increased deposition of peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA120) in the DM/PCL of the PBK corneas. Our data suggest that the DM/PCL of PBK corneas have an increased accumulation of terminal B-galactose and B-D-galactose (1-3)-D-N-acetylgalactosamine residues and altered ratios of low and high molecular weight collagenous proteins.     

Characterization of stroma from Fuchs' endothelial dystrophy corneas

Fuchs' endothelial dystrophy is commonly regarded as an endothelial cell disorder. In the present study we compared glycoconjugates of Fuchs' and normal corneas using FITC conjugated lectins [peanut agglutinin (PNA), castor bean agglutinin (RCA120), soybean agglutinin (SBA), and wheat germ agglutinin (WGA)]. Our results showed increased staining with RCA120 and PNA in the posterior region of the Fuchs' corneas, indicating an accumulation of terminal beta-galactose and B-D-galactose (1-3)-D-N-acetylgalactosamine residues. The stromal and epithelial regions of normal and Fuchs' corneas exhibited similar staining patterns with all lectins tested. Our collagen studies showed an increased extractability and abnormal amino acid analyses of collagen from Fuchs' corneas as compared with normals. The purified collagens did have similar banding patterns by sodium dodecyl sulfate gels. However, further characterization by 125(1) two-dimensional peptide mapping revealed that Fuchs' alpha 1-sized chains contained fingerprints that were distinctly different from normal cornea stromal collagen. These data suggest that in addition to abnormal accumulation of RCA120- and PNA-specific glycoconjugates in the posterior cornea, Fuchs' corneas contained stromal collagens with altered biochemical properties. We postulate that the characteristic deterioration of endothelial function in Fuchs' dystrophy may compromise the microenvironment of the stroma and its keratocytes, and thereby lead to an altered collagenous extracellular matrix.     

Glycoconjugates in exfoliation syndrome. A lectin histochemical study of the ciliary body and lens

The binding of ten biotin-coupled lectins with different carbohydrate specificities to exfoliative material and neighbouring tissues was studied in 16 formalin-fixed and paraffin-embedded human eyes. Eight of the eyes were exfoliation positive while the rest were exfoliation negative. Exfoliative material reacted intensely with Lens culinaris (LCA), Canavalia ensiformis (ConA) and Ricinus communis (RCA-I) agglutinins. Positive reaction was also seen with wheat germ (WGA), peanut (PNA) and soybean (SBA) agglutinins. The superficial zonular lamella, zonular fibers and the non-pigmented epithelium of the ciliary body had a rather similar lectin-binding profile to exfoliative material. The lens capsule was essentially unreactive with all the lectins used. The lens epithelium reacted faintly with ConA, LCA, WGA and RCA-I. Pre-treatment with neuraminidase to remove sialic acid resulted in increased binding of PNA and SBA to exfoliative material, zonular fibers and the zonular lamella, and in decreased binding of WGA to the non-pigmented epithelium of the ciliary body, zonular fibers and the zonular lamella. The results indicate that alpha-mannosyl, beta-galactosyl, N-acetyl-D-glucosaminyl and N-acetylneuraminic acid residues are present in glycoconjugates of exfoliative material and neighbouring tissues.     

New method of ultrastructural localization of WGA-binding sites in the rabbit cornea using quick-frozen specimens followed by freeze-substitution

Wheat germ agglutinin (WGA) conjugated with apo-horseradish peroxidase-gold was used as a probe for ultrastructural localization of N-acetyl-glucosamine and N-acetyl-neuraminic acid residues of glycoconjugates in posterior stroma and Descemet's membrane of rabbit corneas. Ultra-thin sections of quick-frozen, freeze-substituted, and Epon or Lowicryl K4M-embedded tissue were used. Numerous WGA-binding sites were detected in the stromal matrix and Descemet's membrane in both embedding procedures. Labelling was more intense with low-temperature embedding in Lowicryl K4M than with Epon. Gold particles preferentially labelled the major collagen fibrils of the stroma but otherwise appeared uniformly distributed over both extracellular matrices, except in some areas of the stromal matrix that were barely labelled. Frequently, the inter-particle distance of gold particles on collagen fibrils corresponded to the banding period of type I collagen fibrils, suggesting that they point out keratan sulphate proteoglycans which contain WGA-binding sites. The combination of cryotechniques and post-embedding procedures may be very effective for lectin histochemistry of the cornea and other eye tissues.     

Enzymatic characterization of peanut agglutinin-binding components in the retinal interphotoreceptor matrix

Previous histochemical and biochemical studies have documented the presence of carbohydrate-containing molecules in the retinal interphotoreceptor matrix (IPM). The lectin peanut agglutinin (PNA), which preferentially binds galactose-containing carbohydrates, especially galactose-galactosamine linkages, selectively labels cone photoreceptor-associated domains of the IPM ('cone matrix sheaths') in a variety of vertebrate retinas. In the studies described here, the nature of these PNA-binding components was investigated by monitoring the effects of proteolytic and glycosidic enzymes on binding of the lectin in the retina and IPM. All proteolytic enzymes tested cause a marked reduction in PNA-binding to cone matrix sheaths, suggesting that proteinaceous components are important to their organization. Exposure to O-glycanase, but not N-glycanase, markedly reduces binding of PNA to cone matrix sheaths indicating that O-linked oligosaccharides are probably responsible for its binding. Galactose oxidase treatment reduces PNA-binding throughout the retina and IPM, confirming that galactose moieties are involved in its binding. beta-Galactosidase (either before or after neuraminidase treatment) does not alter the pattern of PNA binding, suggesting that neither terminal nor penultimate beta-linked galactose residues constitute a major proportion of the lectin's binding sites in the retina. Neuraminidase treatment markedly increases the density and distribution of PNA binding throughout the retina and IPM, however, this effect appears to be, at least in part, the result of the binding of the lectin to neuraminidase molecules that become associated with tissue sections in addition to binding to carbohydrate groups unmasked by desialation. Exposure to chondroitinases causes disruption of the morphological integrity of cone matrix sheaths and slight diminution of PNA binding. Other enzymes acting on common constituents of extracellular matrices do not have similar effects. Taken together, these observations suggest that PNA-binding to cone matrix sheaths is due to the presence of glycoconjugates with galactose-containing, O-linked oligosaccharide chains.     

Lectin binding patterns of the human cornea. Comparison of frozen and paraffin sections

Twelve FITC-conjugated lectins were used to establish a reaction pattern with cellular and noncellular components in fresh-frozen and formalin-fixed paraffin sections of 11 normal human corneas. In frozen sections the epithelium demonstrated the most active lectin staining; keratocytes and endothelium stained to a lesser degree. Of the noncellular components, the epithelial basement membrane and Descemet's membrane of some of the tissues were stained most by Phasolus vulgaris agglutinin (PHA), Pisum sativum agglutinin (PSA), Concanavalin A (Con-A), and Sopora japonica agglutinin (SJA). None of the lectins stained Bowman's layer or the stromal matrix. Keratocytes in paraffin sections stained the most compared with the epithelium and endothelium. In paraffin sections, the epithelial basement membrane and Bowman's layer of some of the tissue sections were stained by Con-A, Ricinus cummuni agglutinin 120 (RCA-120), Banderiraea simplicifolia lectin (BSL-I), PHA, and wheat germ agglutinin (WGA). From this study, it was observed that the reactivity of lectins to the cellular components in paraffin-embedded sections was less pronounced than in frozen sections. Sensitivity of lectin binding was considerably enhanced by the proteolytic enzyme digestion of paraffin sections.     

Effects of neuraminidase on lectin binding sites in photoreceptor cells of monkey retina

Binding of sugar-specific lectins to the monkey retina was investigated with particular reference to the effects of pretreatment with neuraminidase on the photoreceptor cells. Neuraminidase-treated or untreated retinal sections were examined with a fluorescence microscope after incubation with the following fluorescein-labeled lectins: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Ricinus communis agglutinin-1 (RCA-1) and Dolichos biflorus agglutinin (DBA). In the neuraminidase-untreated tissue, PNA, which is specific for D-galactose beta 1----3 N-acetyl-D-galactosamine (Gal beta 1----3GalNAc), was bound preferentially to the cones, and after treatment of sections with neuraminidase, PNA could bind to both cones and rods. WGA, which is specific for sialic acid and N-acetyl-D-glucosamine, was strongly bound to the rods but moderately to the cones without neuraminidase pretreatment, whereas neuraminidase-treated sections showed a weak binding. RCA-1, which is specific for D-galactose, showed patchy fluorescence on the basal and distal portions of the outer segments of the cones and rods, whereas neuraminidase-treated sections had uniform fluorescence throughout the tissues. DBA, which is specific for N-acetyl-D-galactosamine, did not bind to either cones or rods before and after neuraminidase treatment. These results suggest that there is a difference in the carbohydrate chains of glycoconjugates in the cones and rods: in the cones Gal beta 1----3GalNAc may be the terminal sugar as recognized by PNA, whereas in the rods this sugar is contained in the carbohydrate chain but masked by terminal sialyl residues so that those glycoconjugates are inaccessible to PNA without neuraminidase treatment.     

Characterization of the interphotoreceptor matrix surrounding rod photoreceptors in the human retina.

Previous studies have documented the presence of specific lectin-binding domains in the insoluble interphotoreceptor matrix (IPM) isolated from human retina. Peanut agglutinin (PNA) selectively binds to cone matrix domains whereas wheat germ agglutinin (WGA) binds to matrix domains surrounding rods. In the present study, the rod-associated WGA-binding domains are further characterized using lectin-based cytochemistry and polyacrylamide gel electrophoresis in combination with neuraminidase digestion. The lectin-binding patterns of non-neuraminidase-treated samples are similar to those described in previous reports. After neuraminidase treatment, both rod and cone matrix domains demonstrate PNA binding while the binding of WGA to rod matrix domains is reduced. However, the binding of WGA to photoreceptor outer segments is not affected by neuraminidase. Blots of IPM proteins probed with lectins indicate that the WGA-binding macromolecules are represented as a group of high molecular weight glycoproteins, whereas the PNA-binding components are represented as a group of lower molecular weight glycoproteins. The major WGA-binding glycoprotein (147 kDa) shows reduced binding affinity to WGA and increased binding affinity to PNA following neuraminidase treatment. Further, this 147-kDa glycoprotein, although similar in molecular weight to IRBP (interphotoreceptor retinol-binding protein) (141 kDa), is not recognized by the lectin, concanavalin A (Con A), or by an anti-IRBP antibody. Our data suggest that: (1) the major component of the WGA-binding domain demonstrated by polyacrylamide gel analysis is a glycoprotein with a molecular weight of 147 kDa containing galactose residues that are masked by terminal sialic acid residues; and (2) the binding of WGA to photoreceptor outer segments is resistant to neuraminidase, consistent with the earlier reports that WGA-binding domains of photoreceptor outer segments may not be sialyl-containing glycoconjugates.     

圆锥角膜组织中EGF和bFGF变化免疫组织化学的研究

目的 观察正常人及圆锥角膜中表皮生长因子(EGF)和碱性成纤维细胞生长因子（bFGF)的表达，探讨EGF和hFGF在圆锥角膜中的变化。方法 采用免疫组织化学SABC法检测角膜组织中EGF、bFGF的表达，结果 在圆锥角膜上皮层、Bowman层和内皮层有EGF表达，EGF比正常角膜的表达强；在圆锥角膜的上皮层、实质层和内皮层可见bFGF的阳性表达，圆锥角膜的基质瘢痕区bFGF有较强的阳性表达。结论 圆锥角膜组织中EGF水平增高，促进上皮细胞和基质细胞的DNA合成，从而使组织愈合。bFGF表达增强可以促进角膜上皮、基质细胞和内皮细胞的增殖。     

Histopathology of epi-LASIK in eyes with virgin corneas and eyes with previously altered corneas

PURPOSE: To perform a histological analysis of free epithelial flaps that were intentionally created with an Epi-K epikeratome (Moria S.A.) during epi-LASIK in eyes with virgin corneas and eyes with previous corneal surgery or keratoconus. SETTING: Vissum-Instituto Oftalmológico de Alicante, Alicante, Spain. METHODS: This prospective and consecutive case series comprised 18 free flaps obtained from 18 patients. Twelve patients had virgin corneas, and 6 had altered corneas from previous surgery, trauma, or keratoconus. The flaps were fixed in 4% buffered formaldehyde (pH 7) for posterior histopathological analysis. Serial cuts of each flap were performed, and the sheets were stained with hematoxylin-eosin and periodic acid-Schiff. The main outcome measure was the histopathology of the corneal flaps. RESULTS: All flaps from virgin corneas consisted entirely of epithelium without residual stromal tissue or Bowman's layer. Histopathological analysis of the flaps after epi-LASIK in patients with previously altered corneas showed varying levels of stroma in all cases. CONCLUSION: Epi-LASIK with the Epi-K epikeratome effectively cleaved the epithelium from Bowman's layer in healthy corneas; however, when the integrity of Bowman's layer is compromised, epi-LASIK should be avoided as stromal invasion will likely occur.     

Lectin receptors in the human cornea

Five different biotin labeled lectins, Concanavalin-A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA1), Ulex europaeus agglutinin (UEA1), and soybean agglutinin (SBA) were used to study lectin receptors on formalin-fixed paraffin embedded human corneas. Con A stained the cytoplasm, cell, and nuclear membranes of the epithelial cells and stained the stroma diffusely. WGA stained the superficial epithelial cells, the epithelial cell membranes, and the keratocytes of the stroma. SBA did not react with any of the corneal layers. RCA1 heavily stained the keratocytes but did not stain the epithelium. UEA1 lightly stained the epithelial cell cytoplasm and interstitial stroma. All staining reactions could be abolished by omission of the lectin or by the use of the appropriate inhibitory sugar. The lectin binding patterns reported here provide a means for further investigation of carbohydrate structures in the human cornea in both normal and disease states.     

Colocalization of fibroblast growth factor binding sites with extracellular matrix components in normal and keratoconus corneas

Basic and acidic fibroblast growth factor (bFGF, aFGF) binding sites were determined in frozen sections of normal and keratoconus corneas. After incubation with I-125 radiolabelled growth factors, corneal binding sites were revealed by autoradiography. The growth factors were localized mainly to Descemet's membrane and to the epithelial basement membrane. FGF binding sites were generally similar in normal and keratoconus corneas. The binding specificity was demonstrated by competitive inhibition experiments with an excess of unlabelled growth factors. The binding sites were sensitive to pretreatment of the corneal sections with heparitinase. We have attributed FGF's basement membrane affinity to one of its constituents, proteoheparan sulfate. Proteoheparan sulfate, laminin, collagen type IV, and fibronectin were all revealed by immunofluorescent techniques. While keratoconus cornea stroma had less laminin but more fibronectin than normal corneas the main difference lied in type IV collagen which was overexpressed in keratoconus epithelium.     

Binding sites of peanut agglutinin in mammalian retina

Histochemical studies were carried out of the binding sites of a lectin, peanut agglutinin (PNA), in the mammalian retina, using lectin labeled with either fluorescein or horseradish peroxidase. In all mammalian species examined, monkey, pig, cat and rabbit, the cones were intensely labeled with the lectin but the rods were unlabeled. The cone synaptic pedicles, the inner synaptic layer, the internal limiting membrane and the retinal vessels were also labeled. It is suggested from the specificity of the PNA binding that galactosyl and/or galactosaminyl residues are present in the mammalian cones, but not in the rods.     

圆锥角膜的共焦显微镜表现临床分级

目的 观察临床上不同阶段圆锥角膜的共焦显微镜图像，推测圆锥角膜的病理发展过程，并进行圆锥角膜的共焦显微镜分级。方法 采用共焦显微镜（Confoscan 2．0），观察24例24眼不同发展阶段（lawless分期）圆锥角膜患者的角膜共焦显微镜图像，进行分析并提出焦显微镜下的临床分分期。结果 共焦显微镜下圆锥角膜首先出现了排列规则的裂隙状暗纹，随着病情的不断进展，病变逐渐由角膜后弹力层向前发展，逐渐累及角膜的后基质和前基质，同时角膜基质细胞核拉长、排列出现紊乱，前后基质细胞失去了原有特征。急性圆锥角膜的患者角膜基质细胞出现水肿，角膜瘢痕在共焦显微镜下呈强反光的无细胞样结构。讨论 共焦显微镜下圆锥角膜的病理发展过程，是从角膜后弹力层开始，由后向前发展，逐渐累及角膜的后基质、前期基质，最后角膜破裂引起急性圆锥角膜，角膜出现水肿，愈合后遗留瘢痕。据此提出圆锥角膜共焦显微镜分期将为四个阶段：第一阶段、角膜裂隙样暗纹累及角膜后弹力层；第二阶段、角膜裂隙样暗纹累及角膜后后基质；第三阶段、角膜裂隙样暗纹累及角膜前基质；第四阶段、出现角膜基质水肿或者瘢痕。