Effect of Contact Lens Wear on the Conjunctival Mucous System

Biopsy specimens from the upper tarsal conjunctiva of 20 contact lens wearers with a clinically evident increase in mucus and ten non-lens wearing subjects were examined by light microscopy and scanning and transmission electron microscopy to determine the effect of contact lens wear on the mucous cell system(s). Three types of crypts associated with mucous secretion were found in all specimens: those with intracellular openings (type I, 0.1 to 0.2 μm) associated with non-goblet mucous secretory cells; those with small intercellular openings (type II, 1 to 2 μm) usually associated with goblet cells; and those with intraepithelial and intrastromal crypts with large intercellular openings (type III, 10 to 60 μm) lined with goblet and non-goblet mucous secretory cells. Contact lens wearers had increased numbers of non-goblet cells with mucous secretory vesicles lining the surface of the conjunctiva and the epithelial infoldings of type III crypts than did the normal subjects. We conclude that increased mucous secretion in contact lens wearers is associated with an increased number of cells and number of secretory vesicles involved in the non-goblet cell mucous system.     

Detection of mucus glycoconjugates in human conjunctiva by using the lectin colloidal gold technique in TEM. I. A quantitative study in normal subjects

We applied a specific cytochemical reaction to characterize the glycoconjugates produced by goblet and non-goblet epithelial cells of normal human conjunctiva. For this purpose we utilized the lectins, proteins of vegetal origin, which are extremely sensitive in binding glycosidic residues. In particular, we used WGA, PNA, SBA and ConA conjugated with colloidal gold as ultrastructural marker for Transmission Electron Microscopy. This technique allowed us also to perform a quantitative analysis, by counting colloidal gold particles present on mucus granules. In this way we analyzed the content both of goblet and non-goblet epithelial cells. In the former, WGA, PNA, SBA and ConA receptors, here reported in decreasing density, were present. In the latter WGA was always positive, SBA and PNA sometimes were negative, ConA was always negative. We speculate the different contribution to mucus production by these two sources may be important in evaluating tear film stability alterations occurring in those diseases in which non-goblet epithelial cell vesicles increase.     

Detection of mucus glycoconjugates in human conjunctiva by using the lectin-colloidal gold technique in TEM. III. A quantitative study in asymptomatic contact lens wearers

We characterized the mucus glycoconjugates produced by goblet and non-goblet epithelial cells in asymptomatic contact lens (CL) wearers. We employed four lectins (proteins of vegetal origin which specifically recognize glycosidic residues: WGA, PNA, SBA and ConA) conjugated with colloidal gold as ultrastructural marker, at Transmission Electron Microscopy. A computerized quantitative analysis was carried out in order to compare the results from the CL wearers to those from the control patients. Goblet cells produce different amount of glycosidic residues, in particular, a significant decrease in the distribution of sialic acid, N-acetylglucosamine, N-acetylgalactosamine, galactose-N-acetylgalactosamine and mannose was observed. The content of glycosidic residues in the mucus vesicles of the non-goblet epithelial cells appeared unchanged as to the normal situation. We speculate that the CL could possibly contribute to the failure of the tear film stability by altering the production of mucus.     

On the nature and the role of the subsurface vesicles in the outer epithelial cells of the conjunctiva.

The layer of the tear film in contact with the conjunctiva is mucus. This mucus comes from two sources, the conjunctival goblet cells and the subsurface vesicles. These vesicles are found just below the surface of the conjunctival cells. They contain long chain mucus glycoprotein molecules that are joined to the vesicle membrane. The vesicles fuse with the surface membrane of the conjunctival cells and expose their mucus glycoprotein chains to the overlying mucus. Chemical and physical bonds between the two types of mucus help to bind the mucus layer to the conjunctiva. The vesicle membrane becomes incorporated in the cell membrane and supplies the membranes for the microvilli that cover the exposed surface.     

Expansion of conjunctival epithelial progenitor cells on amniotic membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells. Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture. After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase. Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.     

Impression cytology as a noninvasive method of conjunctival biopsy and its results

Impression cytology is a non-invasive, repeatable, and uncomplicated method of examining the conjunctiva. Cellulose-acetate filters are used to remove the superficial layers of the conjunctiva. This technique causes no subjective discomfort to the patient. The method combines the advantages of a biopsy with those of a "H?utchenpr?parat." The authors used 3 different stains for the conjunctival imprints: PAS-hematoxylin, Alcian blue and a combination of Alcian blue and PAS. From the different staining characteristics of the goblet cells it was possible to draw conclusions about different functional cell conditions. The metachromatic properties of a few cells among the large number of epithelial cells led to speculation on the existence of different cell types and their possible correlation with the various cell types described by Rohen for the conjunctiva of monkeys. In patients with keratoconjunctivitis sicca or Sj?gren's syndrome pathologic nuclear changes occur that lead to chromatin taking on a snake-like appearance, the so-called "snakes." To the authors' surprise, similar chromatin changes were found in a great number of cells of contact lens wearers, with frequency depending on the type of contact lens. Impression cytology is a special method of non-invasive biopsy providing new possibilities for diagnosis, differential diagnosis, and therapeutic control of conjunctival disorders.     

MUC19 expression in human ocular surface and lacrimal gland and its alteration in Sjögren syndrome patients

This study investigated the expression of MUC19, a newly discovered gel-forming mucin gene, in normal human lacrimal functional unit components and its alteration in Sj?gren syndrome patients. Real-time PCR and immunohistochemistry were performed to determine the expression of MUC19 and MUC5AC in human cornea, conjunctiva, and lacrimal gland tissues. Conjunctival impression cytology specimens were collected from normal control subjects and Sj?gren syndrome patients for Real-time PCR, PAS staining, and immunohistochemistry assays. In addition, conjunctiva biopsy specimens from both groups were examined for the expression differences of MUC19 and MUC5AC at both mRNA and protein level. The MUC19 mRNA was found to be present in cornea, conjunctiva and lacrimal gland tissues. The immunohistochemical staining of mucins showed that MUC19 was expressed in epithelial cells from corneal, conjunctival, and lacrimal gland tissues. In contrast, MUC5AC mRNA was only present in conjunctiva and lacrimal gland tissues, but not in cornea. Immunostaining demonstrates the co-staining of MUC19 and MUC5AC in conjunctival goblet cells. Consistent with the significant decrease of mucous secretion, both MUC19 and MUC5AC were decreased in conjunctiva of Sj?gren syndrome patients compared to normal subjects. Considering the contribution of gel-forming mucins to the homeostasis of the ocular surface, the decreased expression of MUC19 and MUC5AC in Sj?gren syndrome patients suggested that these mucins may be involved in the disruption of the ocular surface homeostasis in this disease.     

Collagen gel-embedding culture of conjunctival epithelial cells

Background: Collagen has effects on cell morphology, differentiation characteristics and function. Using collagen gel culture, several studies about cell differentiation were reported. In this study, the differentiation of rabbit conjunctival epithelial cells in a collagen gel-embedding culture system was investigated by electron microscope and lectin labeling. Methods: Rabbit bulbar conjunctival epithelial cells were cultured in type I collagen gel. After I and 2 weeks of culture, some of these cells were stained with PAS and seven kinds of lectins, and others were examined by transmission electron microscopy. Results: The conjunctival epithelial cells cultured within collagen gel formed stratified cell layers and globules with cavities. The inner layer cells facing the cavities showed PAS and lectin staining patterns similar to those of conjunctival goblet cells in vivo, whereas the staining patterns of the outer layer cells on the collagen matrices resembled the patterns of non-goblet epithelial cells. Microvilli on the surface of the innermost cells, basement membranes beneath the outermost cells, tight junctions, adherent junctions, interdigitating folds and desmosomes between cells were identified on electron microscopic examination. Conclusion: These results indicate that cell junction structures of the conjunctival epithelial cells are well developed in collagen gel-embedding culture systems, and that the inner layer cells have carbohydrates similar to those of conjunctival goblet cells. Culture of conjunctival epithelial cells within collagen gel is a useful model for examining differentiation of these cells.     

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.     

维生素A对角膜移植排斥反应后结膜杯状细胞的影响

目的:探讨维生素A对角膜移植排斥反应后结膜杯状细胞的影响。方法:建立大鼠角膜移植排斥反应模型,随机分3组:A、B、C组为SD-Wistar大鼠行同种异体角膜移植术,SD鼠为受体,Wistar鼠为供体,其中A组为空白对照组,B组为(维生素A)诺沛凝胶滴眼液组,C组为0.1%地塞米松滴眼液组,另设D组为正常眼组。术后不同时间裂隙灯显微镜记录及比较各组角膜移植排斥指数(RejectionIndex,RI)。通过结膜组织学切片HE、PAS染色,并运用显微图像分析系统对结膜上皮杯状细胞数量及形态进行分析。结果:观察期间检测结膜杯状细胞发现,A、B、C组杯状细胞数量均少于D组(P<0.01),A、B、C组内部比较,以C组数量为多,其次为B组,有差异有显著性(P<0.05)。结论:维生素A具有保护角膜移植引起的结膜杯状细胞减少的作用。     

10 g?

目的　评估聚维酮碘对大鼠眼表结构和泪膜稳定性的影响。方法　30只SD大鼠左眼使用生理盐水滴眼为A组,右眼使用10 g?L^-1聚维酮碘滴眼为B组,均为每天2次,分别在干预前和干预后2 d、4 d、6 d、8 d、10 d、12 d、14 d用裂隙灯观察角膜形态,并计算泪膜破裂时间（BUT）、泪液分泌量、角膜荧光素染色评分和炎症指数以评估干眼的临床指标改变,使用共聚焦显微镜观察两组角膜组织结构,干预后14 d完成临床指标记录后,取眼球组织行病理学检查,HE染色观察角膜各层结构,PAS染色观察结膜杯状细胞情况,PMN/ED1双重免疫组织化学染色观察中性粒细胞/巨噬细胞在角膜组织层间的浸润情况。结果　裂隙灯检查结果显示,干预后14 d B组大鼠角膜上皮粗糙,荧光素染色阳性,呈点片状着染。干预后6 d、8 d、10 d、12 d、14 d,与A组相比,B组BUT均明显缩短、泪液分泌量均明显下降,角膜荧光素染色评分及炎症指数均明显升高,差异均有统计学意义（均为P<0.05）。角膜共聚焦显微镜检查结果显示,B组角膜上皮层及基质层可见炎症细胞浸润。HE染色结果显示,B组角膜上皮细胞层数增多,上皮厚度增加,基底细胞呈现“空泡样”结构。PAS染色结果显示,B组结膜PAS染色阳性细胞数量显著降低,结膜上皮增厚,细胞层次增加。PMN/ED1双重免疫组织化学染色结果显示,B组角膜上皮层及基质层可见炎症细胞聚集。结论　10 g?L^-1聚维酮碘局部滴眼可引起实验大鼠眼表结构损伤和泪膜稳定性下降,聚维酮碘的眼表毒性有必要引起临床医师的高度重视。     

Dry eye before and after therapy with hydroxypropyl methylcellulose. Ultrastructural and cytochemical study in 20 patients

We have utilized hydroxypropyl methylcellulose in the therapy of 20 patients suffering from dry eye in the initial stage. The effects of the substance have been evaluated both from a clinical and a morphological standpoint. In particular, transmission and scanning electron microscopy were applied to investigate the ultrastructural features of the conjunctival epithelium before and after treatment. In addition, lectin cytochemistry was carried out to analyse semiquantitatively the presence of glycoconjugates in the conjunctival mucus produced by goblet and non-goblet epithelial cells. Hydroxypropyl methylcellulose did not improve the tear film stability as demonstrated by the clinical tests. The normal surface of the conjunctival epithelium did not appear recovered as well. Only a minimal increase in the density of the second mucus system vesicles was observed. From a cytochemical point of view, the content of glycosidic residues appeared altered in dry-eye patients both before and after treatment. This alteration can lead to changes in the physical and functional properties of the mucus, impairing its role as a surfactant. We conclude that the common treatment by means of lubrificant-like substances palliates the dryness condition. The search for a drug which reintegrates the correct composition of the conjunctival mucus should be seriously considered as a valid alternative.     

Assessing the human lid margin epithelium using impression cytology

Purpose: To establish if impression cytology combined with histochemical and immunocytochemical staining can be used to assess epithelium of the human upper lid margin. Methods: Following an initial eye examination of 40 healthy subjects (19 soft contact lens wearers and 21 non‐contact lens wearers, aged 18–57 years), lid margin staining was assessed with lissamine green using slit lamp biomicroscopy and graded (grade 0–3). Impression cytology of the upper lid margin of both eyes was collected, fixed and stained with periodic acid Schiff (PAS) and haematoxylin for cell morphology analysis (Nelson grade) or for immunocytochemistry (keratinization‐related proteins: filaggrin, transglutaminase1 (TGase1) and cytokeratin 1/10). Results: In 57% of all subjects, grade 0 lissamine green staining showed a thin line (the Marx line), just posterior to the meibomian gland ducts. Grade 2 or 3 lissamine green staining was observed in 17% of all subjects. There was no difference between contact lens and non‐contact lens wearers for lid margin staining or Nelson grade (p = 0.4, Fisher’s exact test). PAS/haematoxylin staining and immunocytochemistry showed transition in epithelial cell morphology, with marginal conjunctival epithelium, mucocutaneous junction and squamous epithelium, adjacent to meibomian gland ducts. This transition in epithelium was associated with differential expression of keratinization‐related proteins (filaggrin, cytokeratin 1/10 and TGase1). Conclusion: Lid margin epithelium can be successfully sampled using impression cytology and further characterized using histochemistry and immunocytochemistry staining techniques. This approach can be applied to assess lid margin changes in conditions such as dry eye and meibomian gland dysfunction.     

Extended contact lens wear enhances Pseudomonas aeruginosa adherence to human corneal epithelium.

Extended wear of soft contact lenses is associated with an increased risk of Pseudomonas aeruginosa infection of the cornea. To assess the role of bacterial adherence in the pathogenesis of these infections, superficial corneal epithelial cells and leukocytes from ten patients who use extended-wear soft lenses and ten control eyes were compared for their propensity to attach P. aeruginosa in vitro. Cells were washed from the cornea by saline irrigation, incubated with a 10-ml solution containing 10(7) colony-forming units/ml of bacteria at 35 degrees C for 30 min, collected on a filter, and prepared using a modified acridine orange staining method. Fluorescence microscopy showed bacterial adherence to corneal epithelial cells, leukocytes, and ocular mucus. The mean number of bacteria adhering to epithelial cells was 2.6 for control eyes and 6.6 for the lens-wearing eyes (P = 0.002). The percentage of epithelial cells attaching greater than or equal to four bacteria was higher for lens-wearing eyes than control eyes (57.4% versus 26.0%, P = 0.0005). There was no significant difference between contact lens-wearing eyes and control eyes in the number of leukocytes collected or in the number of bacteria attached to these cells. These results show that P. aeruginosa adherence to epithelial cells is enhanced in those who use extended-wear soft contact lenses, and this may contribute to the increased incidence of P. aeruginosa keratitis for this population.     

microRNA146a在干眼中的表达及作用

目的　研究microRNA146a在干眼小鼠模型中的表达及作用。方法　5周龄BALB/c雄性小鼠20只,左眼为正常对照组（20眼）,右眼为干眼组（20眼）。用2 g·L^-1苯扎氯铵诱导小鼠建立中重度干眼模型。造模完成3 d后,采用泪膜破裂时间（break-up time,BUT）和角膜荧光素钠染色（fluorescein staining,FLS）对小鼠进行眼表相关检查;随后随机选取10只小鼠,采用HE染色观察正常对照组和干眼组角膜和结膜的组织结构差异,结膜PAS染色后计数杯状细胞的量并对比;剩余10只小鼠分别提取角膜与结膜组织的总RNA,采用实时荧光定量PCR检测microRNA146a、白细胞介素（interleukin,IL）-1、IL-6、IL-8以及IL-1受体相关激酶1（IL-1 receptor-associated kinase 1,IRAK1）和肿瘤坏死因子受体相关因子6（tumor necrosis factor receptor associated factor-6,TRAF6）的相对表达量。结果　完成干眼造模3 d后,干眼组BUT为 （3.640±0.493）s,明显低于正常对照组的（10.645±0.583）s,差异有统计学意义（P<0.05）;干眼组角膜FLS评分为（14.650±0.860）分,明显高于正常对照组的（1.233±0.927）分,差异有统计学意义（P<0.05）。小鼠角膜和结膜组织HE染色结果显示:与正常对照组相比,干眼组角膜上皮欠平整,细胞数量增多,角膜基质层胶原纤维排列紊乱,成纤维细胞活化;干眼组小鼠结膜上皮细胞数量增多,排列欠规整,并伴有缺损。小鼠结膜PAS染色结果显示:干眼组结膜杯状细胞数量为（9.500±4.506）个,相较于正常对照组的［（29.667±8.756）个］明显降低,差异有统计学意义（P<0.05）。实时荧光定量PCR结果显示:干眼组角膜和结膜中microRNA146a、IL-1、IL-6、IL-8的相对表达量相较于正常对照组均升高,差异均有统计学意义（均为P<0.05）。相较于正常对照组,干眼组角膜中IRAK1的相对表达量降低,TRAF6的相对表达量升高,而干眼组结膜中IRAK1的相对表达量升高,TRAF6的相对表达量降低,差异均有统计学意义（均为P<0.05）。结论　干眼时机体可通过增加microRNA146a的表达,以及其对炎症中靶点的调控作用,从而形成对干眼炎症反应的负反馈调节机制。     

Morphological differentiation of the conjunctival goblet cells in the chick (Gallus domesticus)

Background: These is no consensus in the literature regarding the differentiation of conjunctival goblet cells in vertebrates. Method: The conjunctival epithelium of the chick was studied before and after hatching in order to demonstrate the morphological evolution of the goblet cells. The entire conjunctiva was processed for light microscopy either on semithin sections stained with toluidine blue-pironine or on traditional sections stained with Alcian blue pH 2.5-PAS. Results: It was possible to demonstrate that goblet cells underwent remarkable changes in their secretory activity. At 12 h after hatching, isolated Alcian blue-positive cells were present in the fornix. At 24 h after hatching, cells positive for both Alcian blue and PAS were scattered among epithelial cells. Two days after hatching, cells which reacted positively only to PAS were also present. Conclusion: It is suggested that the differentiation of conjunctival goblet cells occurs first in the fornix, probably due to the particular vascular environment of this region, and then spreads all over the conjunctiva.     

Secretory immune system of rabbit ocular adnexa.

A distinct system of immunity in a variety of animals is located subjacent to epithelial surfaces and is typified by the predominance of immunoglobulin A (IgA) and secretory component (SC) in various external secretions, including tears. The present study examined normal rabbit lacrimal gland, conjunctiva, and cornea for the presence of immunoglobulin and SC. IgA-staining plasma cells predominated within lacrimal gland and conjunctival stroma, and SC was found in the epithelial cells of both these tissues but not within corneal epithelium. These observations are consistent with findings for other secretory sites in both rabbits and humans and establish lacrimal gland and conjunctiva as integral parts of the rabbit secretory immune system.     

P2Y(2) receptor elicits PAS-positive glycoprotein secretion from rabbit conjunctival goblet cells in vivo.

We investigated in vivo whether UTP and ATP increased periodic acid and Schiff's reagent (PAS)-positive glycoprotein release from rabbit conjunctival goblet cells. Fifty microL of UTP or ATP at the concentrations of 0.003, 0.03, 0.3, 3.0, 8.5% (54 microM-154 mM) or saline were applied to rabbit eyes. Impression cytology was performed on the upper nasal bulbar conjunctiva and the cells were stained with PAS. To clarify purinergic receptor-mediated involvement in this response, suramin (1%; 7 mM), P2Y(2) antagonist and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 0.01%; 167 microM), P2Y(1) antagonist were applied in the rabbit conjunctival sac for 10 min. before UTP or ATP application. Images of the specimens were taken with a digital camera mounted on a microscope and the PAS staining area was measured using an image analyzing system. UTP or ATP eye drop instillation transiently decreased the PAS staining area in a dose-dependent manner, but it gradually recovered after another 30 min. Saline instillation had no effect until 60 min. later. All of the agonists-induced declines were inhibited by pretreatment with 1% (7 mM) suramin but not 0.01% (167 microM) PPADS. UTP and ATP stimulate PAS-positive glycoprotein secretion via P2Y(2) receptor on goblet cells in the rabbit bulbar conjunctiva in vivo.     

Cytological changes of the conjunctiva in immunoglobulin-producing dyscrasias

The conjunctivae of 15 patients with confirmed immunoglobulin-producing dyscrasias were examined by cytological techniques. Of 13 patients with no gross ocular symptoms, 10 showed distinct epithelial cell changes and/or the presence of periodic acid-Schiff (PAS)-positive cells. Two other patients with gross abnormalities of the conjunctiva revealed the presence of PAS-positive cells, Dutcher bodies, and lymphocytoid-plasma cells. Lymphomatous deposition may develop in the conjunctiva and present as conjunctival nodules or as a diffuse infiltration. It is concluded that the cytological preparation of conjunctival smears may serve as a sensitive indicator of systemic immunoglobulin-producing dyscrasias.