Conjunctival Epithelium Cells Research Articles

Pharmacogenomic Analysis of Response to Topical Tumor Necrosis Factor α Antagonist Licaminlimab (OCS-02) in Dry Eye Disease.

The purpose of this study was to evaluate the pharmacogenomics of response to topical ocular tumor necrosis factor α (TNFα) inhibitor licaminlimab in patients with DED. Three single-nucleotide polymorphisms (SNPs) associated with Sjögren syndrome, 3 in the TNFα gene and 1 in the TNF receptor 1 (TNFR1) gene, were assessed for association with response to licaminlimab in participants from a randomized, vehicle-controlled, Phase 2 study in which adults with DED and severe ocular discomfort persisting despite treatment with artificial tears received licaminlimab or vehicle for 6 weeks. Response was assessed for change from baseline in Global Ocular Discomfort score at Day 29 of treatment. The pharmacogenomic analysis was a prospectively specified exploratory objective of the study. mRNA expression for TNFα, interleukin (IL) 1β, and IL8 in conjunctival epithelium cells was determined. The relationship between SNPs and response to licaminlimab was assessed using a mixed model repeated measures analysis. SNP rs1800693 in the TNFR1 gene showed a significant effect on response to licaminlimab (P < 0.0001, initial association test); no effect was seen for any of the other SNPs tested. The CC genotype of rs1800693 was associated with much greater response to licaminlimab than the CT or TT genotypes: LS mean changes from baseline to Day 29 in Global Ocular Discomfort score were -29.5, -0.09, and -3.90, in patients with the CC, CT, and TT genotypes, respectively (P < 0.0001). No significant effect was observed in vehicle-treated patients. Improvements from baseline were seen in 3/4 licaminlimab-treated participants with the CC genotype. Conjunctival epithelium cell levels of mRNA for TNFα, IL1β, and IL8 decreased from baseline in participants with the CC genotype, but not with the CT or TT genotypes. Between-genotype differences in mRNA levels were not observed in participants receiving vehicle. The CC genotype of rs1800693, relatively common in patients with DED, was strongly associated with response to licaminlimab and decreased inflammatory cytokine gene expression in ocular surface cells during treatment. This study is one of the first to our knowledge to investigate pharmacogenomics in the treatment of DED.

The existence of senescent cells in conjunctival epithelium from elderly individuals.

The ocular surface microenvironment changes with aging. However, it remains unclear if cellular senescence influences the ocular surface. We investigated the presence of p16INK4a-expressing senescent cells in healthy human conjunctiva. Clinical and experimental. Healthy conjunctival tissue samples were obtained from middle-aged and elderly subjects. RT-qPCR was performed to assess the expression of senescence markers CDKN2A (p16INK4a) and CDKN1A (p21CIP1/WAF1) and immunostaining was performed to examine the expression of the senescence marker p16INK4a, stem cell markers Ki67 and p63, tight-junction marker ZO-1. Our study involved 19 conjunctival tissue samples (10 elderly and 9 middle-aged), mean age [elderly: 75.8 ± 3.7 years (72-81), middle-aged: 52.7 ± 7 years (38-59)], sex (elderly: 3 men, 7 women; middle-aged: 3 men, 6 women). The expression of p16INK4a was significantly increased at the RNA level in the elderly compared to middle-aged (p < 0.05). Positivity rate of p16INK4a was significantly elevated in the elderly (15.0 ± 7.8%) compared to middle-aged (0.2 ± 0.6%) (p < 0.05). Positivity rate of Ki67and p63 was significantly reduced in the elderly (1.7 ± 1.7% and 16.5 ± 9.5%) compared to middle-aged (3.9 ± 1.8% and 24.7 ± 5.7%) (p < 0.05). ZO-1 expression was reduced in tissue samples showing p16INK4a-positivity but retained in tissue samples in which p16INK4a was undetectable. Senescent cells accumulate with age in the conjunctival epithelium, accompanied by a decrease in Ki67, p63 and ZO-1 expressing cells.

Transmembrane Mucin Response in Conjunctival Epithelial Cells Exposed to Wall Shear Stresses

Human conjunctival epithelium cells (HCEC) line the inner surface of the eyelid and cover the sclera and are continuously subjected to wall shear stresses (WSS). The effects of external forces on the conjunctival epithelium are not fully known. The conjunctival epithelium contains stratified squamous cells that synthesize the membrane-spanning mucins MUC1 and MUC16, which play important roles in protecting the ocular surface. Alterations in both gel-forming and membrane-tethered mucins occur in drying ocular surface diseases. The aim of this study was to explore the mechanobiological characteristics of transmembrane mucin secretion and cellular alterations of primary HCEC exposed to airflow-induced WSS perturbations. We exposed the HCEC to a steady WSS of 0.5 dyne/cm2 for durations of 15 and 30 min. Cytoskeletal alterations and MUC1 secretions were studied using immunohistochemically fluorescent staining with specific antibodies. We investigated for the first time an in vitro model of membrane-tethered mucin secretion by HCEC in response to WSS. The exposure of HCEC to WSS increased the polymerization of F-actin, altered the cytoskeletal shape and reduced the secretion of membrane-tethered MUC1.

The effect of amniotic membrane proteins released by ocular inserts containing self‐healing hydrogels

AbstractPurpose: Growth factors found in the amniotic membrane (AM) help accelerate epithelium regeneration in the damaged cornea or conjunctiva. This study compares the effects of hydrogels releasing the AM proteins on the proliferation, migration, and time‐to‐closure (healing) of the conjunctival epithelium.Methods: Proteins extracted from the AM using sonication were incorporated into two hydrogels used as ocular inserts. The tested inserts materials were the self‐healing, dynamic hyaluronic‐acid hydrogel based on gold‐thiolate/disulfide exchange (Hydrogel A) and a physically cross‐linked hydrogel based on a combination between Eudragit S100 and hyaluronic acid with a reinforced matrix by polyvinyl alcohol, polyvinyl pyrrolidone and polyethylene glycol (Hydrogel B). In an in vitro experiment, a scratch was made on the human conjunctival epithelium cells (Chang Conjunctiva, Clone 1‐5c‐4) to mimic an incision wound. The cells were cultured with hydrogels for 48 hours to study the effect of these materials on epithelium repair. The samples were examined using a Zeiss Observer Z1 inverted microscope, and the results were evaluated employing the Image‐J program to obtain the cell migration rate (μm/h).Results: Hydrogel A increased the rate of closure at 22 h (121 ± 19 μm) in comparison with Hydrogel B (394 ± 78 μm). After 46 h, the scratches in Hydrogel A and control cultures were completely closed, while the Hydrogel B samples had an unclosed area of 54 ± 4 μm.Conclusions: Both hydrogels promoted wound healing in cultured conjunctival epithelium cells. However, a lower rate of wound closure was observed for Hydrogel B than for Hydrogel A and the control, probably because the speed of cell proliferation was slower in Hydrogel B. The wound‐healing assay showed that the Hydrogel A and B increased cell viability and significantly stimulated cell migration.

Lectin Histochemistry of the Glycoconjugates in Partridge and Quail Conjunctival Epithelia

The aim of this study was to examine the distribution of the glycoconjugates in conjunctival epithelium of partridge (Alectoris chukar) and quail (Coturnix coturnix). Eyelid samples harvested from both species were subjected to routine tissue processing. Lectin histochemistry was applied to sections in order to demonstrate the expression of glycoconjugates. Six different HRP-conjugated lectins (Con A, UEA-I, PNA, HPA, MAA and BSA I-B4) were used for this purpose. The conjunctival epithelium in both partridge and quail consisted of goblet cells and nongoblet cells. Both PNA and HPA bound, to varying degrees, to the goblet cells and nongoblet cells in conjunctival epithelium of partridge and quail. Con A reacted with nongoblet cells in both species. While partridge conjunctival goblet cells showed no reaction to Con A, quail conjunctival goblet cells had a very weak reaction to Con A. UEA-I did not bind to any cells in partridge, however, quail conjunctival goblet cells and nongoblet cells could react to UEA-I. In both species, conjunctival epithelial surfaces reacted with Con A, UEA-I, PNA and HPA. However, MAA and BSA I-B4 did not bind any cell and/or part in both partridge and quail conjunctiva. The present data suggest that composition of glycoconjugates could be different between goblet cells and nongoblet cells of these species, but it could be very similar at conjunctival epithelial surfaces on which the content of the goblet cells and nongoblet cells is released.

MiR-3175 promotes epithelial-mesenchymal transition by targeting Smad7 in human conjunctiva and pterygium.

MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of mRNAs. However, the function of miRNAs is unclear in epithelial-mesenchymal transition (EMT) of pterygium, a disease characterized by abnormal fibrovascular proliferation and invasion on the ocular surface. Since MiR-3175 is upregulated in pterygium tissues compared to normal conjunctival tissues, in this study, we investigated the role of MiR-3175 in EMT of a cultured human conjunctival epithelium cell line (CCL) and further validated the findings in pterygium tissues from patients. Our study demonstrates that MiR-3175 promotes proliferation, migration, invasion, and EMT of CCL cells by directly inhibiting Smad7. In conclusion, our data suggested that the MiR-3175-Smad7 axis may play a critical role in pathogenesis and development of pterygium.

The Relationship Between Obesity, Insulin Resistance, and Conjunctival Impression Cytology.

This study was designed to determine whether obesity causes the development of metaplasia in conjunctival epithelial cells. A total of 61 volunteer participants who had no previous history of illness or drug use were involved in this study. Of those, 20 were obese, and 41 were of normal weight. We measured the glucose and insulin values of all volunteers. We also measured the Body Mass Index (BMI) and Homeostasis Model Assessment for Insulin Resistance (HOMA IR). The impression cytology method was used to analyze the conjunctival epithelium cells, and to classify them between Grades 0 to 3 according to the Nelson criteria. There was a certain level of loss of goblet cells on the 90% level as well as squamous metaplasia (Grade 2-3) in 80% of the obese participants and impression cytology was found to be normal in only two patients. The expected results were observed in 56.1% of the control group where the squamous metaplasia rate was nearly 17% (p < 0.001). 90.9% of the grade 3 patients were obese. The variables as independent predictors were found to indicate the existence of abnormal cytology in the conjunctiva at various levels; BMI (OR: 1.24; p=0.002) and HOMA IR (OR= 28.6; p= 0.001) in a Model I multivariable regression model, and the existence of obesity (OR: 11.91; p=0.002) and HOMA IR (OR= 15.08; p < 0.001) in a Model II multivariable regression model. Obesity was found to be a disorder that causes metaplasia in the conjunctival epithelium cells for the first time.

The relationship between obesity, insulin resistance and conjunctival impression cytology

OBJECTIVE This study was designed to determine whether obesity causes the development of metaplasia in conjunctival epithelial cells. MATERIAL AND METHOD A total of 61 volunteer participants who had no previous history of illness or drug use were involved in this study. Of those, 20 were obese, and 41 were of normal weight. We measured the glucose and insulin values of all volunteers. We also measured the Body Mass Index (BMI) and Homeostasis Model Assessment for Insulin Resistance (HOMA IR). The impression cytology method was used to analyze the conjunctival epithelium cells, and to classify them between Grades 0 to 3 according to the Nelson criteria. RESULTS There was a certain level of loss of goblet cells on the 90% level as well as squamous metaplasia (Grade 2-3) in 80% of the obese participants and impression cytology was found to be normal in only two patients. The expected results were observed in 56.1% of the control group where the squamous metaplasia rate was nearly 17% (p < 0.001). 90.9% of the grade 3 patients were obese. The variables as independent predictors were found to indicate the existence of abnormal cytology in the conjunctiva at various levels; BMI (OR: 1.24; p=0.002) and HOMA IR (OR= 28.6; p= 0.001) in a Model I multivariable regression model, and the existence of obesity (OR: 11.91; p=0.002) and HOMA IR (OR= 15.08; p < 0.001) in a Model II multivariable regression model. CONCLUSION Obesity was found to be a disorder that causes metaplasia in the conjunctival epithelium cells for the first time.

Solid lipid nanoparticles for delivery of Calendula officinalis extract

Solid lipid nanoparticles (SLN) composed of long-chain fatty acids (palmitic acid, stearic acid or arachidic acid), Epikuron 200 (purified phosphatidylcholine), and bile salts (cholate, taurocholate or taurodeoxycholate) have been prepared by dilution of a microemulsion. A total of five different systems were prepared, and characterized by photon correlation spectroscopy, transmission electron microscopy, differential scanning calorimetry, and infrared spectroscopy. The SLN formulation showing optimal properties (lowest size and polydispersity index and highest zeta potential) was obtained with stearic acid and taurodeoxycholate as cosurfactant. This formulation was loaded with Calendula officinalis extract, a natural compound used on ophthalmic formulations given its anti-inflammatory, emollient, and wound repairing activity. Calendula-loaded SLN preparations were characterized in order to determine loading capacity and entrapment efficiency. In vitro cytotoxicity and wound healing efficacy of Calendula-loaded SLN compared to that of a free plant extract were evaluated on a conjunctival epithelium cell line WKD. Our results suggest that this SLN formulation is a safe and solvent-free Calendula extract delivery system which could provide a controlled therapeutic alternative for reducing disease-related symptoms and improving epithelium repair in ocular surface.

Comparison of the effects of different lens-cleaning solutions on the protein profiles of human conjunctival cells

Many contact lens wearers suffer from dry eye syndrome. Previous studies show significant changes in the protein composition of the tears depending on the lens-cleaning solution used. Therefore, the aim of this study was to detect the influence of different lens solutions on conjunctival cells, cells also involved in tear film composition. Conjunctival epithelium cells (HCjE; IOBA-NHC) were exposed to medium containing Complete® Multipurpose Solution Easy Rub® Formula (Complete) (AMO) or Opti-Free® Express Multipurpose Disinfecting Solution (Opti-Free) (Alcon) in different concentrations (0.1, 0.5, 1, 2.5%) for 12 and 24h. Apoptosis and necrosis using FACS and protein profiles of the cells using SELDI-TOF-MS and MALDI-TOF-MS were measured. Multivariate statistics were calculated to detect the most significant changes. Complex protein profiles were measured with SELDI-TOF-MS and MALDI-TOF-MS. Significant differences of protein profiles between control and treatment cells were detected after 12 and 24h, although cells incubated with Complete showed significantly fewer changes than cells incubated with Opti-Free, also showing concentration-dependent changes of some significantly changed proteins, e.g., protein at 6,736Da (p > 0.002). Cells incubated with Complete showed significantly less apoptosis or necrosis in comparison to control cells (p < 0.05), whereas cells incubated with Opti-Free showed significantly more (p < 0.05). We were able to demonstrate that Complete shows very little effect on the protein profiles of conjunctival cells in comparison to Opti-Free, where the cells showed very large protein profile changes, and apoptosis and necrosis of the cells was increased. These results are in concordance to clinical studies showing that the use of Complete solution made the tear film proteins similar to those of people not wearing contact lenses. Therefore, we believe that Complete is less aggressive and should provoke fewer side-effects, such as dry eye syndrome, for the users.

Polyunsaturated fatty acids induce modification in the lipid composition and the prostaglandin production of the conjunctival epithelium cells

This study was conducted to evaluate whether polyunsaturated fatty acids (PUFA) such as γ-linolenic acid (GLA) and eicosapentaenoic acid (EPA), as found in the diet, may affect the lipid composition of conjunctival epithelium and whether these modifications affect prostaglandin (PG) production after inflammatory stimulation. Chang and IOBA-NHC conjunctival human cells were treated with GLA and/or EPA at 5, 10, 20, 30, 40, or 50μg/ml for 72h and then were stimulated with interferon-gamma (IFN-γ) for 48h. Changes in the composition of neutral lipids and phospholipids were monitored by gas chromatography. PGE1 and PGE2 levels were measured by enzyme immunoassay. PUFA supplementations in the culture medium induced incorporation of these fatty acids and of their metabolites in neutral lipids and phospholipids of the conjunctival cells. The fatty acid composition of neutral lipids and phospholipids was not affected by stimulation with IFN-γ. The production of PGE1 and PGE2 was affected by GLA supplementation whereas it was not modified by EPA supplementation. A combined supplementation of EPA and GLA did not change the production of PGE1 but decreased the production of PGE2. These results suggest that modulation of fatty acid composition and PG production by PUFA supplementation is possible in the conjunctival epithelium, which is an important site of inflammation in dry eye syndrome.

ICAM‐1 and HLADR expression on conjunctival epithelial cells in patients with Graves’ orbitopathy and Graves’ disease ‐ preliminary report

AbstractPurpose The objective of the study was to evaluate wheather an assessment of conjunctival epithelium expression of HLA‐DR and ICAM‐1 could be helpful as an early marker of local inflamation in the eye and the orbital tissue.Methods Specimens for flow‐cytometric evaluations of ICAM‐I and HLADR expression were collected by impression of ocular surface from 8 eyes with Graves’ diaseas without signs of active Graves’ orbitopathy (CAS ≤ 4), from 8 eyes of patients with active Graves’ orbitopathy (CAS ≥ 4) and from 14 normal speciments without any ocular surface disorders.Results Decreased fluorescence of ICAM‐1 on conjunctival epithelium cells was detected in patients with active Graves orbitopathy vs. patients with Graves wihout orbitopathy. There was no correlation between the CAS and NOSPECS values and the expression of ICAM‐I and HLADR expression on conjuctival epithelium.Conclusion Results of this preliminary study confirm that conjunctival epithelial cells may abnormally express ICAM‐1 on conjunctival epithelium cells in active ocular surface disorder like Graves orbitopathy. Nethertheless, since the number of the examined patients needs to be enlarged the study needs further examination.

Tight Junction Transmembrane Protein Claudin Subtype Expression and Distribution in Human Corneal and Conjunctival Epithelium

The combination of the tight junction transmembrane protein claudin subtypes is one of the most important determinants of variations in the tightness of individual paired tight junction strands. The barrier function of corneal epithelium is much stronger than that of conjunctival epithelium. In this study, the expression and cellular distribution of claudin species in in vivo human corneal and conjunctival epithelium were investigated. Reverse transcription-polymerase chain reaction was used to reveal the claudin mRNA. Immunohistochemistry was used to determine the tissue distribution of tight junction-related proteins and MUC5AC. Transcripts for claudin-1, -2, -3, -4, -7, -9, and -14 were identified in human corneal epithelium. Transcripts for claudin-1, -2, -4, -7, -9, -10, and -14 were identified in human conjunctival epithelium. By immunohistochemistry, claudin-1, -4, and -7 were found to be localized at the membrane of human corneal and conjunctival epithelial cells. In human conjunctival epithelium, claudin-10 staining was observed at several, but not all, apical epithelial cell-to-goblet cell junctions. Claudin-1, -4, and -7 are expressed in corneal and conjunctival epithelia. Claudin-10 is prominent at several junctions between apical epithelial cells and goblet cells in conjunctival epithelium. Except for claudin-10 expression in conjunctival epithelium, the claudin subtype expressions of corneal and conjunctival epithelia are similar. Therefore, there must be a difference between these two epithelial types with regard to the specific ratio of claudin subtypes expressed or their phosphorylation status. The distribution of goblet cells in conjunctival epithelium also influences the difference in barrier function.

Clusters of Corneal Epithelial Cells Reside Ectopically in Human Conjunctival Epithelium

The ocular surface is covered by two biologically distinct epithelia: corneal and conjunctival. The expression of keratin12 (K12) is currently considered a hallmark of cornea-type differentiation. In the current study, the biological features of K12-positive cells in human bulbar conjunctival epithelium were examined. Human conjunctival tissues were subjected to investigate the K12-positive cells in conjunctiva by immunostaining, in situ hybridization, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence-activated cell sorting (FACS). Gene expression profiling of these cells was performed with introduced amplified-fragment length polymorphism (iAFLP). To determine the presence of stem- or progenitor cells, immunostaining and colony-forming assays were performed. Western blot analysis, RT-PCR revealed that K12 was expressed in conjunctival epithelium. Immunostaining analysis showed that K12-positive cells reside mainly in clusters in conjunctival epithelium. FACS analysis showed that 0.2% to 1.7% of conjunctival epithelial cells collected from the inferior bulbar conjunctiva were K12 positive. iAFLP analysis revealed that the gene expression patterns of these cells were highly similar to that of corneal epithelial cells. p63 and ABCG2 were expressed beneath the K12-positive cells. Some colony-forming cells expressed K12. The K12-positive cells appear to be ectopically residing, self-maintaining corneal epithelial cells in the conjunctival epithelium.

Ultrastructural evidence of mucus in human conjunctival epithelial cultures

PURPOSE. To demonstrate by ultrastructural techniques that human conjunctival epithelium cells in vitro can produce mucin-like secretion. METHODS. Primary cultures of human conjunctival epithelial cells were grown in different culture media. Cultures were allowed to grow and were processed after 5 days and 1, 2, 3, 4, or 5 weeks for transmission and scanning electron microscopy, according to the method of Nichols et al. modified in our laboratory. RESULTS. Marked differences were seen between primary cultures grown with or without hydrocortisone. A thick tannic acid-stained layer was observed when hydrocortisone was present in the culture medium; however, that layer was virtually absent in cultures grown with hydrocortisone-free media. Scanning electron microscopy revealed a dense deposit showing a network-like structure. Moreover, the age of the cultures clearly influenced the thickness of the tannic acid-stained deposit, which thickened as the cultures aged. CONCLUSIONS. These results strongly suggest that the layer growing in the presence of hydrocortisone is mucus. The fact that this material became more abundant as the cultures aged indicates that mucus is actively produced and secreted by conjunctival epithelial cells in vitro. This study might contribute to the knowledge of mucus-deficient pathologies of the ocular surface.