Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques

Conjunctival biopsies from patients with cicatricial pemphigoid affecting the conjunctiva and patients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva.     

Protein components of rat lens and their age-related changes observed with two-dimensional polyacrylamide gel electrophoresis

The protein components of rat lens crystallins were analyzed by two-dimensional urea-sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The following age-related changes were observed. (i) In alpha-crystallin, one or two spots at the anodic sides of alpha A2 and alpha B2 spots were observed in the lens cortex after the 8 months. beta-Crystallin, consisting of seven components designated as beta-1 to beta-7 components in the case of 1.5-month-old rats, showed marked changes during ageing. Component beta-3 was not detectable in neonatal lens. Component beta-5' located at the cathodic side of component beta-5 was observed in the lens older than 8 months. Components at the anodic sides of beta-4, beta-6 and beta-7 spots were detected in 8-month and especially 19- and 47-month-old lenses. The amount of gamma-crystallin decreased in dependence on age. (ii) The electrophoretogram of the nucleus of 8-month-old lens showed additional spots in the area with the lower molecular weight than alpha A2. gamma-Crystallin was accumulated in the nucleus. The electrophoretogram of gamma-crystallin of the 8-month-old nucleus was more similar to that of 19-month-old lens cortex than that of neonatal lens or young lens cortex.     

干眼病患者结膜损害的观察研究

目的 探讨干眼病患者结膜损害的观察方法.方法 选择2006年6月至2007年10月在我院门诊确诊的47例(94只眼)干眼病患者,对其94只眼用荧光素染色后分别用钴蓝光和蓝光滤过片观察眼表结构的改变情况.结果 在眼表上皮细胞方面,钴蓝光观察94只眼中有85只眼染色有异常,其中染色累及睑裂部角结膜有75只眼,染色累及下方结膜有10只眼;而用蓝光滤过片观察后,发现染色有异常的有90只眼,其中染色累及睑裂部角结膜有25只眼,染色累及下方结膜46只眼,染色累及上方结膜19只眼,Kappa一致性检验,二者有差异;染色累及上方结膜19只眼的干眼病患者泪膜破裂时间为均值4.07 s,标准差0.14 s;与正常眼泪膜破裂时间至少5 s相比有统计学差异.结论 眼表结构荧光染色后的的蓝光滤过片观察方法简便、特异性、敏感性高,尤其是在结膜上皮的损害观察方面优势明显.所有上方结膜损害的干眼病患者的泪膜稳定性差,主观症状中异物感症状重.     

蛋白质组学技术分析眼优势柱可塑性相关蛋白

目的利用二维电泳-质谱(2-DE/MS)技术分析Long Evans大鼠初级视皮层中与眼优势柱可塑性相关的蛋白质。方法取22d龄Long Evans大鼠通过向其初级视皮层中灌注放线菌酮建立幼年眼优势柱不移动的动物模型,然后分别提取22d龄大鼠、100d龄大鼠和22d龄放线菌酮灌注大鼠的初级视皮层灰质总蛋白,利用2-DE/MS技术分析与眼优势柱可塑性相关的蛋白质。结果通过2-DE/MS技术鉴定了与眼优势柱终止相关的5种蛋白质,这5种蛋白质与蛋白质的更新相关,分别为proteasome protein p45/sug、Malate dehydrogenase、Proteasome alpha1subunits、adolase A和septin4。结论二维电泳-质谱(2-DE/MS)技术可以有效地分离和鉴定视皮层中的特殊蛋白质。随着蛋白质组学技术的进步和完善,必将在视觉可塑性研究中占有重要地位。     

Isolation by polyacrylamide gel electrophoresis of a light-sensitive vitamin A-protein complex from the retina of the honeybee drone

Polyacrylamide gel electrophoresis was performed on retinal homogenates from honeybee drones previously treated with tritiated vitamin A. After a 6-hr exposure to tritiated vitamin A in vivo or a 1-hr exposure in vitro, radioactivity appears as a single band in the gel of Davis and Ornstein for soluble proteins. No radioactivity is recovered in the SDS gel of Weber and Osborn for insoluble proteins. When labeled retinal homogenates are irradiated with white light, the radioactivity recovered in the gel of Davis and Ornstein diminishes proportionally with the time of illumination. In the presence of hydroxylamine, illumination removes all radioactivity from the protein band. Conversely, sodium borohydride added to the labeled homogenates prevents the radioactivity to be removed by illumination. These results indicate that the drone retina contains a soluble, photosensitive vitamin A-protein complex, which may represent a precursor of the visual pigment in the eye.     

Lectin-cytochemical study on epithelial mucus glycoprotein of conjunctiva and pterygium

The epithelium of pterygium and conjunctiva was studied with reference to cytochemical reactivity to six fluorescein-labeled lectins that recognize a certain carbohydrate residue(s) of cellular membrane-bound or secretory glycoprotein: Ulex europaeus agglutinin-1 (UEA-1, specific for fucose); Dolichos biflorus agglutinin (DBA, specific for N-acetylgalactosamine); peanut agglutinin (PNA, specific for galactose-beta 1-3N-acetylgalactosamine): wheat germ agglutinin (WGA, specific for N-acetylglucosamine and N-acetylneuraminic acid); Concanavalia ensiformis (Con A, specific for mannose); Ricinus communis agglutinin-1 (RCA-1, specific for galactose). Non-goblet epithelial cells of pterygium were labeled with UEA-1, DBA and PNA, while those of conjunctiva were not. Distribution density of goblet cells was larger in pterygium than in conjunctiva, but there was no distinct difference in lectin reactivity between the two tissues, with marked label with WGA, PNA and RCA-1. Con A did not bind to either pterygium or conjunctiva. The observations suggest the presence of anomalous mucus glycoproteins secreted from pterygium.     

Isolation and preliminary characterization of tear prealbumin from human ocular mucus

Tear prealbumin was purified from crude tear prealbumin previously isolated from the saline soluble human ocular mucus. Purification was achieved by further column chromatographies on DEAE Sephadex A-25 and Sephadex G-75. Preliminary characterization included amino acid analysis, gel electrophoresis, and isoelectric focusing. Unlike serum prealbumin, the purified tear prealbumin showed a predominance of acidic residues and a trace amount of tryptophan. It exhibited polymorphic nature, with pI values of 4.8 and 4.9. The possibility of a tear prealbumin/retinol complex was also examined. The protein was found to incorporate with 3H retinol. The 3H retinol-incorporated tear prealbumin did not exhibit the characteristic UV spectrum of retinol; however, it did display emission and excitation fluorescence spectra at high concentrations similar to serum retinol-binding protein.     

Fractionation and partial characterization of macromolecular components from human ocular mucus

Crude human ocular mucus was extracted with 0·154 m-NaCl to separate soluble protein components from mucus. Small amounts of lipoglycoprotein of high molecular weight, as well as twelve plasma proteins, were detected in the soluble extract by gel filtration and immunodiffusion studies. After the NaCl extraction, the remaining mucus residue was further extracted with 6 m-urea-0·2 m-Tris-phosphoric acid buffer. From this portion of soluble extract, a relatively larger amount of lipoglycoprotein of high molecular weight, as well as a lower molecular weight fraction containing eight detectable plasma proteins, were both isolated by gel filtration. The glycoprotein moieties of the lipoglycoproteins of high molecular weight had similar chemical composition. Both contained approximately 40–43% protein and 57–60% carbohydrate, giving a carbohydrate-protein ratio of 1·30 to 1·48. Fucose, galactose, N-acetylhexosamine and N-acetylneuraminic acid comprised about 423–516 residues per 1000 amino acid residues, while serine and threonine constituted about 285–299. All analyses indicated mucin-like character in the lipoglycoproteins of high molecular weight.Plasma proteins constituted approximately three-fifths of the macromolecular components in ocular mucus. These proteins also appeared to be in complexes with lipids, but to a much lesser extent than the high molecular weight fractions. The relevance of present findings to the structure and composition of precorneal tear film is discussed.     

Effects of individual premedication on ocular hemo- and hydrodynamics in patients with cataracts

Relationship between psychoemotional status of patients and ocular hemo- and hydrodynamics has been studied. Individual premedication prescribed with due consideration for patient's psychological reactions to preoperative stress promoted a decrease of ophthalmic tone and blood filling of ocular vessels.     

High-resolution genome profiling differentiated Staphylococcus epidermidis isolated from patients with ocular infections and normal individuals

PURPOSE: To investigate the potential phenotypic and genetic differences among the Staphylococcus epidermidis isolates obtained from control subjects (lower conjunctival sac; n = 14) with those from patients with keratitis (corneal scrapings; n = 18) or endophthalmitis (vitreous; n = 24). METHODS: Biofilm-forming capacity was detected by PCR for the icaAB gene and phenotyping by microtiter plate assay and congo red agar plate. Genotyping was performed by using fluorescence-amplified fragment length polymorphism (FAFLP) and in silico analysis of the FAFLP profiles. RESULTS: Biofilm phenotyping (congo red agar/microtiter plate) differentiated disease-causing strains from control subjects. PCR assays (mecA, icaAB) were not useful in differentiating disease-causing strains from that of control subjects. The biofilm-forming capability appeared more critical in the pathogenesis of keratitis than in that of endophthalmitis. Cluster analysis of FAFLP data generated 11 clusters comprising 4 major clusters (I, II, III, and V) and 7 minor ones. FAFLP analysis clearly showed clustering of most of the commensal isolates in cluster I, separate from keratitis and endophthalmitis isolates. In silico analysis mapped signature bands to genes such as ebh, tagD, ptsI, and sepA, which might have a significant role in transforming less virulent populations of S. epidermidis to more virulent ones. CONCLUSIONS: The population dynamics of S. epidermidis revealed that there are significant genetic variations that can be detected through FAFLP between ocular disease causing isolates and the commensal population.     

老年性白内障与正常晶状体蛋白质双向电泳的差异

目的:应用双向电泳技术对比研究老年性白内障与正常晶状体,寻找差异表达的蛋白质。方法:分别收集老年性白内障和正常晶状体,采用固相pH梯度(IPG)等电聚焦双向电泳技术进行蛋白质分离,使用ImageMaster图像分析软件分析电泳图像,对比分析蛋白质表达的差异。结果:大部分晶状体蛋白质分布在Mr14000～97000,等电点(pI)5～9的范围内;高丰度蛋白质斑点分布在Mr20000～31000、等电点(pI)6～8的区域内。经软件分析正常组共识别出133±7个蛋白质斑点,白内障组共识别出136±8个斑点,两组之间的匹配率为87%(115个),其中有8个点蛋白质表达量增加了300%以上,其中44号增加了480%,有6个点蛋白质表达量减少了300%以上,其中125号减少了336%。结论:老年性白内障与正常晶状体存在差异表达的蛋白质。     

正常人与Sjogren''s Syndrome患者球结膜免疫细胞的研究

目的 研究正常人与干燥综合征(Sjogren’s Syndrome,SS)患者球结膜HLA-DR+细胞、朗格罕斯细胞(LC)、T淋巴细胞亚群的变化特征。方法 采用链菌素-过氧化物酶法检测正常人与SS患者球结膜组织。结果 (1)SS患者球结膜大量T淋巴细胞浸润,抑制性T淋巴细胞(CD~(+8))较辅助性T淋巴细胞(CD~(+4))为多。CD~(+4)/CD~(+8)比值升高。(2)SS球结膜中LC数量减少,且鳞状上皮化生明显。(3)SS球结膜中HLA-DR~+细胞较正常人明显增加。结论 SS患者球结膜有显著的病理改变和免疫细胞异常。     

Analysis of UVA-related alterations upon aging of eye lens proteins by mini two-dimensional polyacrylamide gel electrophoresis

This study is a first approach to identify UVA-related alterations in situ of bovine eye lens proteins from the water-soluble and urea-soluble fractions upon aging. The fractions were obtained from irradiated long-term organ culture lenses and analyzed by mini two-dimensional gel electrophoresis. This micropreparative method followed by computer analysis allows high resolution and separation of microgram quantities of proteins and to detect spots which arose as a consequence of irradiation. To facilitate the analysis we first separated the water-soluble fraction into the major crystallin classes by gel filtration. Moreover, we immunoblotted the gel of the urea-soluble fraction with a specific antibody against the intermediate filament protein vimentin. Upon irradiation of young and adult lenses, alphaA-crystallin and vimentin showed obvious modifications. During aging the susceptibility to irradiation increased when vimentin started to degrade, whereas deamidation of alphaA-crystallin seems to occur.     

Differences in basement membrane zone components of normal conjunctiva, conjunctiva in glaucoma and normal skin

Purpose: To describe the distribution of basement membrane zone (BMZ) components in normal conjunctiva, conjunctiva of patients with glaucoma and normal skin. Methods: Thirty-five normal conjunctival biopsies and 16 conjunctival biopsies of patients with glaucoma under topical anti-glaucomatous therapy were examined by immunohistochemistry. Antibodies were directed against laminin α chains, laminin subchains α1, α2, α3, β1, β3, γ1, γ2, γ3, kalinin, β4-integrin, and collagens IV and VII. Results were compared to the antigen distribution at the BMZ in normal skin. Results: The BMZ of skin stained positive for all antibodies tested. In contrast to skin, the BMZ of normal conjunctiva was negative for laminin subunits α2, β1, β3, γ2 and γ3 in most or all specimens. Positive findings at the conjunctival BMZ of patients with glaucoma were comparable to normal conjunctiva for laminin α, α1, β3, γ1, γ3, laminin 5, β4-integrin, collagen IV and collagen VII. However, staining of the BMZ with antibodies to laminin β1 (p?=?0.002) and γ2 (p?=?0.017) was seen in a significantly higher rate in glaucoma compared to controls. Conclusion: Characteristic differences exist in the antigenicity of the BMZ in normal skin, normal conjunctiva and conjunctiva from patients with glaucoma, especially for laminin subchains. These differences may explain the variable ocular involvement in diseases of the BMZ. Moreover, they may explain the susceptibility of patients with glaucoma to develop mucous membrane pemphigoid-like disease. Alterations in conjunctival BMZ in glaucoma may be induced by long-term topical anti-glaucoma medications including various preservation agents.     

Cytokeratin 15 can be used to identify the limbal phenotype in normal and diseased ocular surfaces

PURPOSE: To elucidate the expression pattern of K15, K19, K14, and K12 in human and mouse ocular surface epithelium as putative markers of epithelial phenotype. METHODS: Immunohistochemical staining with specific antibodies for K15, K19, K14, and K12 was performed in human donor cornea tissue and normal ICR mouse corneas, with emphasis on localization of immunopositive cells. Immunohistochemistry was performed in a limbus-deficient mouse model as well as in clinical samples of pannus surgically removed from a thermal burn and a patient with Saltzmann's dystrophy. Staining patterns were classified as limited to the most basal layer (K(bas)), basal and suprabasal layers (K(bas-sup)), predominantly in suprabasal layers (K(sup)) and negative staining (K(-)). RESULTS: In human conjunctival epithelium, strong expression of K15 was observed in basal cells, whereas K19 was expressed in both basal and suprabasal layers (K15(bas)/K19(bas-sup)/K12(-)). Limbal epithelial cells were K15(bas-sup)/K19(bas-sup)/K12(sup), whereas epithelial cells in the central cornea were K15(-)/K19(bas-sup)/K12(bas-sup). In contrast, the mouse ocular surface demonstrated a different expression pattern of K15 and K19 than did the human tissue in the conjunctiva (K15(bas-sup)/K19(bas)/K12(-)) and the limbus (K15(bas-sup)/K19(bas)/K12(sup)). Neither K15 nor K19 was expressed in the central mouse cornea (K15(-)/K19(-)/K12(bas-sup)). Similar cytokeratin expression was observed in conjunctivalized corneas in mice and in surgically removed pannus tissue. CONCLUSIONS: Although the expression of K15 and K19 differ in humans and mice, specific staining patterns can be used to characterize the epithelial phenotype in normal and diseased ocular surface.     

Human mast cell subtypes in conjunctiva of patients with atopic keratoconjunctivitis,ocular cicatricial pemphigoid and Stevens-Johnson syndrome

Purpose: Investigate mast cell (MC S ) subtypes in atopic keratoconjunctivitis (AKC), ocular cicatrical pemphigoid (OCP), and Stevens-Johnson Syndrome (SJS). Methods: MC S subtypes were determined by immunohistochemistry of conjunctiva (obtained from 34 patients – 9 AKC, 9 OCP, 9 SJS and 7 normal) using monoclonal antibodies directed against chymase (MC C ) and tryptase (MC T ). Double staining was used to distinguish MC S as positive for both chymase and tryptase (MC TC ). Results: The number of MC S was significantly increased in AKC, OCP and SJS patients, compared to normal subjects. MC C were especially high in AKC, and moderately high in OCP. MC T and MC TC were similar in each disease group. Conclusions: While the AKC findings were not surprising, the result in OCP and SJS suggests that MC S play an underappreciated role in the inflammatory process of these disorders. Disparate proportions of MC S subtypes in these diseases may imply differential functions of MC S in these disorders.