Vitronectin: a possible determinant of adenovirus type 19 tropism for human corneal epithelium

PURPOSE: Adenoviruses typically demonstrate specific tissue tropisms, as in the association of Ad19 with epidemic keratoconjunctivitis. We sought to determine factors that might influence the apparent tropism of Ad19 for the cornea. DESIGN: Laboratory investigation. METHODS: Adenovirus serotypes Ad2, 5, 9, 10, 11, 13, and 19 were compared for their capacity to replicate in human corneal epithelial cells (HCECs) in culture. Organotypically cultured human corneas were infected with Ad19 or Ad2, and viral titers were compared after 7 days. Replication of both viruses was compared in HCECs cultured on various extracellular matrices. Western blot analysis and immunohistochemistry were applied to human donor corneas and HCECs. RESULTS: One week after infection of HCEC monolayer cultures, Ad2 titers were significantly higher than any of the other viruses tested (P <.05). In organotypic corneal cultures, Ad19 titers were significantly higher than Ad2 (P = .0003). Ad2 replication in HCECs equaled or exceeded that of Ad19 on all extracellular matrices except vitronectin, where Ad2 replication was reduced and Ad19 replication enhanced (P <.0001). Vitronectin was detected by immunohistochemistry within the corneal epithelial basement membranes of human donor corneas. Increased alpha(v) integrin expression and greater tyrosine kinase phosphorylation in HCECs cultured on vitronectin were demonstrated by Western blot analysis. CONCLUSIONS: In vitro, vitronectin enhances growth of Ad19, possibly by up-regulation of receptor alpha(v) integrins and increased activity of tyrosine kinases necessary for adenoviral internalization. We hypothesize that differential tissue tropisms for adenoviruses may derive in part from tissue-specific extracellular matrix expression.     

Neutral and acidic glycosphingolipids in glucocorticoid-induced cataract in chick lens

Administration of glucocorticoids induces transient cataract in 15-day-old chick embryos within 48 hr, and the opaque lens again becomes clear within the subsequent 48 hr. Oxidative stress is likely to be involved in the process of cataract formation, resulting in the appearance of numerous vacuoles around the perinuclear region. Chick lens contained low amounts of glycosphingolipids, which mainly consists of GM3, GD3, sialyl-LewisX gangliosides and glucosylceramide. Most lens gangliosides were immunohistochemically detected in lens epithelia, annular pads and developing fibers, but not in perinuclear and nuclear regions. Since cell surface gangliosides, for example GM3 and sialyl-LewisX gangliosides, are involved in cell adhesion, weak cell-to-cell interactions in the perinuclear and nuclear regions may allow vacuole formation in steroid-induced cataractogenesis.     

基质金属蛋白酶抑制剂对培养的人晶状体上皮细胞移行抑制作用的研究

目的 通过体外人晶状体囊袋模型的建立,探讨基质金属蛋白酶抑制剂GM6001对白内障患者术后晶状体上皮细胞移行的抑制作用及其对细胞活性的影响,以寻找一种安全有效地防治晶状体后囊膜混浊的药物.方法 本文为实验研究.16例供体人眼行模拟白内障手术,建立人晶状体囊袋培养模型,确认赤道部晶状体上皮细胞开始移行后,将晶状体囊袋置于不同浓度(1 μmol/L、10 μmol/L和100 μmol/L)的GM6001及其阴性对照液(100 μmol/L)中培养(每组4个囊袋).相差显微镜下分别测量培养10、20及30 d时各组晶状体上皮细胞在后囊膜移行的距离;酶联免疫吸附实验测定囊袋培养液中MMP-2和MMP-9的浓度.四甲基偶氮唑盐法(MTT)测定不同实验浓度GM6001对细胞活性的影响.多组间及组间计数资料的比较,采用随机区组设计的方差分析.结果 培养的人晶状体囊袋中赤道部晶状体上皮细胞第4天开始移行.GM6001明显抑制了晶状体上皮细胞在后囊膜的移行,呈剂量依赖性(F=53.79,P<0.01):实验第20天,10μmol/L GM6001组晶状体上皮细胞移行距离较对照组减少70%(P<0.01),100μmoL/L组减少98%(P<0.01);GM6001降低了MMP-2、MMP-9表达水平,浓度越高,抑制作用越明显(F=86.59,72.96;P<0.01):实验第20天,10 μmol/L GM6001组MMP-2和MMP-9表达水平较对照组均降低70%(P<0.01),100 μmol/LGM6001组MMP-2水平降低了90%(P<0.01),MMP-9水平降低了87%(P<0.01);MTT法显示,晶状体上皮细胞在GM6001中仍保持增殖活性,各处理浓度组光密度(A)值与对照组比较差异无统计学意义(F=0.62,P>0.05).结论 MMP抑制剂有效地抑制了体外囊袋培养的人晶状体上皮细胞在后囊膜的移行,且对细胞活性无明显影响.因此,MMP抑制剂可能对预防后囊混浊有一定作用.     

Age-dependent changes in monkey lenticular gangliosides

The content, composition, and distribution of gangliosides were examined in the lenses of normal rhesus monkeys aged 6-16 years. Gangliosides were isolated by organic solvent extraction. DEAE-Sephadex ion-exchange column chromatography, and thin-layer chromatography (TLC). Ganglioside contents determined by the thiobarbituric acid method increased in the lens with aging. TLC analysis of gangliosides showed a much more complex pattern with aging, and the predominant gangliosides were tentatively identified as GM3, GM1, and GD1a. Individual lenticular gangliosides were identified by TLC-immunostaining procedures using anti-GM1 and anti-asialoGM1 antisera.     

Increase in lens gangliosides due to aging and cataract progression in human senile cataract

Gangliosides were isolated from human senile cataractous lenses by solvent extraction, DEAE-Sephadex column chromatography, and thin-layer chromatography. The content and composition of gangliosides were examined in individual lens tissues. Three predominant gangliosides, GM3, GM1, and GD1a, were tentatively identified in comparison with authentic brain gangliosides, and several unidentified gangliosides were also recognized. The increase in ganglioside content per mg of protein content in cataractous lenses was found to be influenced by two physiologic parameters: aging and cataract progression. The mature cataractous lenses showed a higher ganglioside level on a protein basis than the immature lenses compared with the same age group. On the basis of statistical analysis, an age-dependent increase in ganglioside concentration was recognized in both mature and immature lens groups. The relative increase in slow-moving polysialogangliosides on thin-layer chromatography seemed to be caused by the maturation of cataract. The sugar composition of one of the polysialogangliosides was found to be glucose, galactose, and sialic acid in the molar ratio of 2:1:4; this suggests the presence of a unique ganglioside species in human cataractous lens.     

Caracterización del cultivo primario epitelial de conjuntiva humana

ObjectiveTo evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules.MethodsOne-hundred and forty-eight human conjunctiva explants were cultured in CnT50^® supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37 °C, 5% CO₂ and 95% HR, for 3 weeks.ResultsThe typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative).ConclusionsConjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50^® supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction.     

Ganglioside composition in human cataractous nuclei

Gangliosides were isolated from human cataractous nuclei by solvent extraction, dialysis, and thin-layer chromatography and compared to gangliosides present in human whole normal and cataractous lenses. Three predominant gangliosides were tentatively identified as GM1, GM3, and GD1a, and several other resorcinol-positive components were observed in each of the sets of lens tissue. Thin-layer chromatographic patterns were similar, although some minor and possibly significant differences in band intensities were observed when chromatograms of gangliosides from cataractous nuclei and cataractous whole lenses were visually compared with those of whole normal lenses. Total ganglioside extracts were methanolyzed and the fatty acid methyl esters extracted with hexane and resolved by gas chromatography. Nervonic acid (C-24:1) content was increased in cataractous nuclei as compared to normal and cataractous whole lenses.     

Human lens epithelium in tissue culture: Biochemical and morphological aspects

Human lens epithelium is cultured and the epithelial origin of the cells is established ultrastructurally. The epithelial cells have a limited growth capacity in vitro; only in rare cases a confluent monolayer is obtained. When primary confluent cultures are split 1:1·5 the cells again form a monolayer; they do not form a monolayer when split at 1:2. The light microscopical and ultrastructural features of early (1 week) and late (older than 1 month) stage cultures are described. Moreover, the ultrastructure of human lens epithelium in situ is presented. Incubation of early and late stage cultures in a ³⁵S-methionine containing medium revealed that the crystallin synthesis in younger cultures is reduced, whereas it has completely ceased in the older ones. These late stage cultures only synthesize cytoskeletal components (43 and 57 K daltons). These observations are confirmed by the indirect immunofluoresence study.     

The alpha1 isoform of the Na+/K+ ATPase is up-regulated in dedifferentiated progenitor cells that mediate lens and retina regeneration in adult newts

Adult newts are able to regenerate their retina and lens after injury or complete removal through transdifferentiation of the pigmented epithelial tissues of the eye. This process needs to be tightly controlled, and several different mechanisms are likely to be recruited for this function. The Na⁺/K⁺ ATPase is a transmembrane protein that establishes electrochemical gradients through the transport of Na⁺ and K⁺ and has been implicated in the modulation of key cellular processes such as cell division, migration and adhesion. Even though it is expressed in all cells, its isoform composition varies with cell type and is tightly controlled during development and regeneration. In the present study we characterize the expression pattern of Na⁺/K⁺ ATPase α1 in the adult newt eye and during the process of lens and retina regeneration. We show that this isoform is up-regulated in undifferentiated cells during transdifferentiation. Such change in composition could be one of the mechanisms that newt cells utilize to modulate this process.     

In vitro differentiation of cells of the lens epithelium of human fetus

Cells dissociated from fetal human lens epithelium of approximately 9 and 15 weeks after conception were cultured as monolayers in vitro. About 25 days after inoculation, a number of transparent piles of highly swollen cells appeared on the epithelial cell sheet. Ultra-structurally, these piles are assemblages of elongated cells with typical profiles of mature lens fibers. Gamma-crystallin which is undetectable in the lens epithelium is immunologically detected in late stage cultures containing these piles. Thus, it is concluded that cells of the human fetal lens epithelium can differentiate into lens fibers in monolayer culture.     

Human fetal lens epithelial cells in culture: an in vitro model for the study of crystallin expression and lens differentiation

We have cultured and passaged human fetal lens epithelial cells. Cultured cells exhibited hexagonal, cuboidal shape typical of epithelial cells. Unlike previous observations made with cultured mammalian lens epithelial cells, indirect immunofluorescence and temporal analysis of 35S-labeled proteins demonstrated undiminished levels of alpha B-crystallin in primary, secondary, and tertiary cultures. Among the alpha-crystallins only alpha B synthesis was detected. Two dimensional gel electrophoresis indicated the presence of alpha B2 and no alpha B1. beta B2-crystallin, a fiber cell specific protein hardly detectable in primary cultures, increased significantly upon passaging. Human fetal lens epithelial cell cultures, described in this report, thus present a useful in vitro model for the study of lens epithelial cell differentiation and its pathological manifestations.     

年龄对水合氯醛诱导的小鼠可逆性晶状体混浊及Na+-K+-ATP酶表达的影响

目的：探究年龄对水合氯醛诱导的小鼠急性可逆性晶状体混浊及Na⁺-K⁺-ATP酶表达的影响。

方法：青年(3月龄)和老年(24月龄)C57BL/6小鼠各12只,4%水合氯醛(400mg/kg)腹腔注射诱导急性可逆性晶状体混浊。在注射后10、20、30、45、60、90、120、150min分别应用裂隙灯观察晶状体混浊程度,根据晶状体混浊评价系统记录混浊程度分级情况并拍照。苏木素-伊红(HE)染色观察晶状体病理改变,免疫组织化学染色检测晶状体Na⁺-K⁺-ATP酶表达。

结果：水合氯醛注射后两组小鼠晶状体混浊和消退过程相似,但青年组小鼠晶状体混浊出现早、持续时间略长,混浊厚重,呈乳白色; 老年组小鼠晶状体混浊出现晚,持续时间略短,混浊轻薄,呈薄雾状。HE染色显示水合氯醛注射后晶状体上皮细胞(LECs)下皮质聚集大量水泡,浅层晶状体纤维细胞结构紊乱。免疫组织化学染色显示LECs及纤维Na⁺-K⁺-ATP酶的表达呈现阳性,水合氯醛注射前青年组和老年组小鼠LECs中Na⁺-K⁺-ATP酶表达较弱,注射后45min LECs中Na⁺-K⁺-ATP酶的表达上调,且老年组小鼠LECs的Na⁺-K⁺-ATP酶上调更为显著。

结论：年龄影响水合氯醛诱导小鼠急性可逆性晶状体混浊,Na⁺-K⁺-ATP酶参与水合氯醛诱导的晶状体混浊。     

Serum-free Media for Culturing and Serial-Passaging of Adult Human Retinal Pigment Epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm⁻². Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9±2.7% and 47.1±4.1%, respectively) and DMEM with serum (87.2±5.6% and 52.9±10.5%, respectively) than DMEM without serum (59.2±5.6% and 33.1±6.9%, respectively;P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12±3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21±5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

Interaction with collagen IV protects lens epithelial cells from Fas-dependent apoptosis by stimulating the production of soluble survival factors

PURPOSE: To investigate the sensitivity of lens epithelial cells (LECs) to Fas-dependent apoptosis and to determine the role of interaction with the extracellular matrix (ECM) in the regulation of Fas-dependent apoptosis. METHODS: Sensitivity to Fas-mediated apoptosis of primary human LECs and HLE-B3 LECs cultured on different substrates was determined by Hoechst staining after incubation with Fas-stimulating or control IgM. Fas expression was determined by Western blot analysis. Effects of varied cell density and conditioned media from HLE-B3 cells cultured on different substrates on the Fas sensitivity of HLE-B3 cells cultured on tissue culture (TC) plastic were determined. RESULTS: Primary LECs cultured as free-floating anterior capsulotomy specimens were resistant to Fas-dependent apoptosis. The LE cell line, HLE-B3, was sensitive to Fas-dependent apoptosis when cultured on TC plastic but not on lens capsule. Culture on collagen IV, but not on laminin, rendered HLE-B3 cells resistant to Fas-dependent apoptosis, although Fas was still expressed. Primary LECs cultured on TC plastic after migration from lens capsule explants were resistant to Fas-dependent apoptosis if the lens capsule and attached cells were still present, but not if the lens capsule had been removed. Conditioned medium from LECs cultured on collagen IV, but not TC plastic, protected cells cultured on TC plastic from Fas-dependent apoptosis. The protective effect of culture on collagen IV diminished with decreasing cell density. CONCLUSIONS: LECs are protected from Fas-dependent apoptosis by interaction with collagen IV. Soluble factors released by the LECs cultured on collagen IV protect LECs from Fas-dependent apoptosis.     

Expression patterns of beta1-related alpha integrin subunits in murine lens during embryonic development and wound healing

PURPOSE: To study the expression patterns of b1-related alpha integrin subunits in murine lens epithelial cells, comparing embryonic fiber differentiation with injury-induced epithelial mesenchymal transition (EMT). METHODS: Adult mice type C57BL/6, pregnant as well as with an eye injured, were sacrificed at different time-course intervals.The embryonic and the injured eyes were obtained and deparaffinized sections of these eyes were processed for immunohistochemistry staining for detection of integrin a subunits. RESULTS: Embryonic lens epithelial cells expressed primarily a3 and a5 subunits, whereas embryonic fiber cells expressed a2, a5, and a6 subunits.Adult lens epithelial cells expressed a3, and a6 subunits,whereas injured lens cells expressed a2, a3, and a6 integrin subunits. CONCLUSIONS: The phenotypic changes of lens epithelial cells during embryonic fiber differentiation and EMT are characterized by different expression of integrin subunits as a result both of the altered extracellular matrix conditions and of the altered cell signaling pathways recruited in each process.