Primary Human Corneal Epithelial Cells Research Articles

SARS-CoV-2 infects cells lining the blood-retinal barrier and induces a hyperinflammatory immune response in the retina via systemic exposure.

SARS-CoV-2 has been shown to cause wide-ranging ocular abnormalities and vision impairment in COVID-19 patients. However, there is limited understanding of SARS-CoV-2 in ocular transmission, tropism, and associated pathologies. The presence of viral RNA in corneal/conjunctival tissue and tears, along with the evidence of viral entry receptors on the ocular surface, has led to speculation that the eye may serve as a potential route of SARS-CoV-2 transmission. Here, we investigated the interaction of SARS-CoV-2 with cells lining the blood-retinal barrier (BRB) and the role of the eye in its transmission and tropism. The results from our study suggest that SARS-CoV-2 ocular exposure does not cause lung infection and moribund illness in K18-hACE2 mice despite the extended presence of viral remnants in various ocular tissues. In contrast, intranasal exposure not only resulted in SARS-CoV-2 spike (S) protein in different ocular tissues but also induces a hyperinflammatory immune response in the retina. Additionally, the long-term exposure to viral S-protein caused microaneurysm, retinal pigmented epithelium (RPE) mottling, retinal atrophy, and vein occlusion in mouse eyes. Notably, cells lining the BRB, the outer barrier, RPE, and the inner barrier, retinal vascular endothelium, were highly permissive to SARS-CoV-2 replication. Unexpectedly, primary human corneal epithelial cells were comparatively resistant to SARS-CoV-2 infection. The cells lining the BRB showed induced expression of viral entry receptors and increased susceptibility towards SARS-CoV-2-induced cell death. Furthermore, hyperglycemic conditions enhanced the viral entry receptor expression, infectivity, and susceptibility of SARS-CoV-2-induced cell death in the BRB cells, confirming the reported heightened pathological manifestations in comorbid populations. Collectively, our study provides the first evidence of SARS-CoV-2 ocular tropism via cells lining the BRB and that the virus can infect the retina via systemic permeation and induce retinal inflammation.

Mitochondrial DNA-Activated cGAS-STING Signaling in Environmental Dry Eye.

The cGAS-STING pathway has been shown to be an important mediator of inflammation. There is emerging evidence of the importance of this signaling cascade in a variety of inflammatory diseases settings. Here, we present evidence that the mitochondrial DNA (mtDNA) damage-mediated cGAS-STING pathway plays an important role in the induction of inflammation in environmental dry eye (DE). RT-qPCR and Western blot were used to assess the induction of the cGAS-STING pathway and inflammatory cytokines in environmental DE mouse model, primary human corneal epithelial cells (pHCECs), and patients with DE. RNA sequencing was used to determine mRNA expression patterns of high osmotic pressure (HOP)-stimulated pHCECs. mtDNA was detected with electron microscopy, flow cytometry, and immunofluorescent staining. mtDNA was isolated and transfected into pHCECs for evaluating the activation of the cGAS-STING pathway. The expression levels of cGAS, STING, TBK1, IRF3, and IFNβ were significantly increased in an environmental DE model and HOP-stimulated pHCECs. The STING inhibitor decreased the expression of inflammatory factors in DE. An upregulation of STING-mediated immune responses and IRF3 expression mediated by TBK1 were observed in the HOP group. HOP stimulation induced mitochondrial oxidative damage and the leakage of mtDNA into the cytoplasm. Then, mtDNA activated the cGAS-STING pathway and induced intracytoplasmic STING translocated to the Golgi apparatus. Finally, we also found activated cGAS-STING signaling in the human conjunctival blot cell of patients with DE. Our findings suggest that the cGAS-STING pathway is activated by recognizing cytoplasmic mtDNA leading to STING translocation, further exacerbating the development of inflammation in environmental DE.

Integrative Analysis of miRNA and circRNA Expression Profiles and Interaction Network in HSV-1-Infected Primary Corneal Epithelial Cells

Purpose Circular RNAs (circRNAs) are products of alternative splicing with roles as competitive endogenous RNAs or microRNA sponges, regulating gene expression and biological processes. However, the involvement of circRNAs in herpes simplex keratitis remains largely unexplored. Methods This study examines circRNA and miRNA expression profiles in primary human corneal epithelial cells infected with HSV-1, compared to uninfected controls, using microarray analysis. Bioinformatic analysis predicted the potential function of the dysregulated circRNAs and microRNA response elements (MREs) in these circRNAs, forming an interaction network between dysregulated circRNAs and miRNAs. Results A total of 332 circRNAs and 16 miRNAs were upregulated, while 80 circRNAs and six miRNAs were downregulated (fold change ≥2.0 and p < 0.05). Gene ontology (GO) and KEGG pathway analyses were performed on parental genes of dysregulated circRNAs to uncover potential functions in HSV-1 infection. Notably, miR-181b-5p, miR-338-3p, miR-635, and miR-222-3p emerged as pivotal miRNAs interacting with multiple dysregulated circRNAs. Conclusions This comprehensive study offers insights into differentially expressed circRNAs and miRNAs during HSV-1 infection in corneal epithelial cells, shedding light on circRNA-miRNA interactions’ potential role in herpes simplex keratitis pathogenesis.

Purple Corn Extract Improves Dry Eye Symptoms in Models Induced by Desiccating Stress and Extraorbital Lacrimal Gland Excision.

Dry eye disease (DED) occurs when there are not enough tears, and the associated symptoms-burns, itching, and a gritty feeling in the eye-can cause great discomfort. The purpose of this study was to evaluate the therapeutic effect of purple corn extract (PCE) on DED. Pretreatment with PCE prevented desiccation-stress-induced cell damage in human retinal pigment epithelial cells and primary human corneal epithelial cells. Furthermore, PCE reduced the mRNA expression of inflammatory mediators in the induction of desiccation stress. The therapeutic effects of PCE on DED were evaluated in an animal model with induced unilateral excision of the exorbital lacrimal gland. The administration of PCE was effective at recovering tear production, corneal surface irregularity, and conjunctival goblet cell density, as well as at reducing apoptotic cell death in the outer layer of the corneal epithelium. Collectively, PCE improved dry eye symptoms, and, therefore, it could be a potential agent to ameliorate and/or treat DED.

Topical Spironolactone in the Treatment of Ocular Graft-Versus-Host Disease.

This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model. Clinically, we assessed patient response to spironolactone with a retrospective observational design. Both immortalized and primary human corneal epithelial cells were stained with oil red O after 9 days of treatment with spironolactone. C57BL/6 mice were dosed thrice daily with one drop in each eye for 18 days. Corneal tissue was stained with oil red O and BODIPY™. Twenty eyes with oGVHD, as defined by the International Chronic oGVHD Consensus Group, were studied. Corneal fluorescein staining, lid margin vascularity, meibomian gland obstruction, meibum turbidity, zone A posterior lid margin vascularity, and oGVHD diagnostic criteria severity grading were compared in a pre-post study. Follow-up times ranged from 7 to 21 weeks, with a median time of 12 weeks. Statistical analysis was done with STATA 17 by fitting data to a non-parametric model. In vitroresults showed an increased number and density of oil red O staining granules in the treatment group versus control in both primary and immortalized human corneal epithelium.In vivo, results showed translation to the mouse model with increased corneal epithelial BODIPY™ signal compared to untreated control. oGVHD patients had improved lid margin vascularity (p= 0.046), corneal fluorescein staining (p= 0.021), and International oGVHD Consensus Group severity scores (p= 0.011) after treatment with topical spironolactone. Minimal adverse effects were noted, the most common being mild stinging lasting less than a minute after instillation. The improved severity scores, lid margin inflammation, and corneal fluorescein staining after weeks of treatment support the rationale that topical spironolactone may benefit oGVHD. The observed lipid production by the corneal epithelium is thought to contribute to this protective effect against ocular surface erosive disease in oGVHD. A mineralocorticoid receptor antagonist, spironolactone may offer therapeutic benefits in oGVHD while avoiding undesirable side effects of topical or systemic glucocorticoids.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures

Purpose This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. Methods Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. Results Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. Conclusions Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

Peroxisome proliferator-activated receptor-α (PPARα) regulates wound healing and mitochondrial metabolism in the cornea

Diabetes can result in impaired corneal wound healing. Mitochondrial dysfunction plays an important role in diabetic complications. However, the regulation of mitochondria function in the diabetic cornea and its impacts on wound healing remain elusive. The present study aimed to explore the molecular basis for the disturbed mitochondrial metabolism and subsequent wound healing impairment in the diabetic cornea. Seahorse analysis showed that mitochondrial oxidative phosphorylation is a major source of ATP production in human corneal epithelial cells. Live corneal biopsy punches from type 1 and type 2 diabetic mouse models showed impaired mitochondrial functions, correlating with impaired corneal wound healing, compared to nondiabetic controls. To approach the molecular basis for the impaired mitochondrial function, we found that Peroxisome Proliferator-Activated Receptor-α (PPARα) expression was downregulated in diabetic human corneas. Even without diabetes, global PPARα knockout mice and corneal epithelium-specific PPARα conditional knockout mice showed disturbed mitochondrial function and delayed wound healing in the cornea, similar to that in diabetic corneas. In contrast, fenofibrate, a PPARα agonist, ameliorated mitochondrial dysfunction and enhanced wound healing in the corneas of diabetic mice. Similarly, corneal epithelium-specific PPARα transgenic overexpression improved mitochondrial function and enhanced wound healing in the cornea. Furthermore, PPARα agonist ameliorated the mitochondrial dysfunction in primary human corneal epithelial cells exposed to diabetic stressors, which was impeded by siRNA knockdown of PPARα, suggesting a PPARα-dependent mechanism. These findings suggest that downregulation of PPARα plays an important role in the impaired mitochondrial function in the corneal epithelium and delayed corneal wound healing in diabetes.

Applications of Polyaniline-Based Molybdenum Disulfide Nanoparticles against Brain-Eating Amoebae.

Primary amoebic meningoencephalitis and granulomatous amoebic encephalitis are distressing infections of the central nervous system caused by brain-eating amoebae, namely, Naegleria fowleri and Acanthamoeba spp., respectively, and present mortality rates of over 90%. No single drug has been approved for use against these infections, and current therapy is met with an array of obstacles including high toxicity and limited specificity. Thus, the development of alternative effective chemotherapeutic agents for the management of infections due to brain-eating amoebae is a crucial requirement to avert future mortalities. In this paper, we synthesized a conducting polymer-based nanocomposite entailing polyaniline (PANI) and molybdenum disulfide (MoS2) and explored its anti-trophozoite and anti-cyst potentials against Acanthamoeba castellanii and Naegleria fowleri. The intracellular generation of reactive oxygen species (ROS) and ultrastructural appearances of amoeba were also evaluated with treatment. Throughout, treatment with the 1:2 and 1:5 ratios of PANI/MoS2 at 100 μg/mL demonstrated significant anti-amoebic effects toward A. castellanii as well as N. fowleri, appraised to be ROS mediated and effectuate physical alterations to amoeba morphology. Further, cytocompatibility toward human keratinocyte skin cells (HaCaT) and primary human corneal epithelial cells (pHCEC) was noted. For the first time, polymer-based nanocomposites such as PANI/MoS2 are reported in this study as appealing options in the drug discovery for brain-eating amoebae infections.

Thrombospondin-1 induction and VEGF reduction by proteasome inhibition

The present study focuses on investigating the expression of thrombospondin-1 (TSP-1), a natural inhibitor of neovascularization. Immunofluorescent staining was used to detect the expression of TSP-1 in rabbit corneal tissue with vascularization induced by limbectomy. TSP-1 was detected in healthy and Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) grafted rabbit corneas. TSP-1 was not detected in diseased corneas. Rabbit and human primary oral mucosal and corneal epithelial cells were cultured and treated with proteasome inhibitor (PI) in vitro. Changes in the expression of TSP-1, HIF-1 alpha and 2 alpha, VEGF-A, and VEGF receptor were analyzed by Western blotting. Neovascularization developed in rabbits’ corneas as early as 1 month after limbectomy and was stable for at least 3 months. HIF-1 alpha and VEGF-A expression was reduced in CAOMECS grafted corneas, as compared to sham corneas. While TSP-1 expression was decreased in injured corneas, it was expressed in CAOMECS grafted corneas, but still less expressed compared to healthy corneas. PI treatment, of human oral mucosal and corneal epithelial cells increased TSP-1 expression and reduced VEGF-A expression. The results showed that TSP-1 expression was lost in injured corneal surface and that CAOMECS grafting restored TSP-1 expression to certain extent. Proteasome inhibition treatment increased TSP-1 and decreased VEGF-A expression in human oral mucosal and corneal epithelial cells. The result suggests that corneal neovascularization could be managed with the inhibition of the proteasome after CAOMECS grafting and increase corneal transparency.

AMPK/MFF Activation: Role in Mitochondrial Fission and Mitophagy in Dry Eye.

To assess the role of mitochondrial morphology and adenosine monophosphate-activated protein kinase (AMPK)/mitochondrial fission factor (MFF) in dry eye and the underlying mechanisms. Immortalized human corneal epithelial cells (HCECs) and primary HCECs were cultured under high osmotic pressure (HOP). C57BL/6 female mice were injected subcutaneously with scopolamine. Quantitative real-time PCR was used to measure mRNA expression. Protein expression was assessed by western blot and immunofluorescence staining. Mitochondrial morphology was observed by confocal microscopy and transmission electron microscopy. First, HOP induced mitochondrial oxidative damage to HCECs, accompanied by mitochondrial fission and increased mitophagy. Then, AMPK/MFF pathway proteins were increased consequent to HOP-induced energy metabolism dysfunction. Interestingly, the AMPK pathway promoted mitochondrial fission and mitophagy by increasing the recruitment of dynamin-related protein 1 (DRP1) to the mitochondrial outer membrane in the HOP group. Moreover, AMPK knockdown attenuated mitochondrial fission and mitophagy due to HOP in HCECs. AMPK activation triggered mitochondrial fission and mitophagy. Mitochondrial fission of HCECs stressed by HOP was mediated via MFF phosphorylation. MFF knockdown reversed mitochondrial fragmentation and mitophagy in HCECs treated with HOP. Inhibition of MFF protected HCECs against oxidative damage, cell death, and inflammation in the presence of HOP. Finally, we detected mitochondrial fission and AMPK pathway activation in vivo. The AMPK/MFF pathway mediates the development of dry eye by positively regulating mitochondrial fission and mitophagy. Inhibition of mitochondrial fission can alleviate oxidative damage and inflammation in dry eye and may provide experimental evidence for treating dry eye.

Design, Synthesis and Evaluation of Novel Molecular Hybrids between Antiglaucoma Drugs and H2S Donors.

Glaucoma is a group of eye diseases consisting of optic nerve damage with corresponding loss of field vision and blindness. Hydrogen sulfide (H2S) is a gaseous neurotransmitter implicated in various pathophysiological processes. It is involved in the pathological mechanism of glaucomatous neuropathy and exerts promising effects in the treatment of this disease. In this work, we designed and synthetized new molecular hybrids between antiglaucoma drugs and H2S donors to combine the pharmacological effect of both moieties, providing a heightened therapy. Brinzolamide, betaxolol and brimonidine were linked to different H2S donors. The H2S-releasing properties of the new compounds were evaluated in a phosphate buffer solution by the amperometric approach, and evaluated in human primary corneal epithelial cells (HCEs) by spectrofluorometric measurements. Experimental data showed that compounds 1c, 1d and 3d were the hybrids with the best properties, characterized by a significant and long-lasting production of the gasotransmitter both in the aqueous solution (in the presence of L-cysteine) and in the intracellular environment. Because, to date, the donation of H2S by antiglaucoma H2S donor hybrids using non-immortalized corneal cells has never been reported, these results pave the way to further investigation of the potential efficacy of the newly synthesized compounds.

Preparation and Characterisation of a Cyclodextrin-Complexed Mānuka Honey Microemulsion for Eyelid Application.

Honey has been widely purported as a natural remedy due to its antimicrobial and anti-inflammatory effects. In recent years, several studies have suggested that the considerably high methylglyoxal (MGO) concentration in Mānuka honey (MH) makes it particularly effective to manage bacterial overload, such as that observed in blepharitis. However, the poor solubility, high viscosity, and osmolarity of aqueous honey solutions, especially at the high MGO concentrations studied in the literature, render the formulation of an acceptable dosage form for topical application to the eyelids challenging. Here, the antibacterial properties of raw MH and alpha-cyclodextrin (α-CD)-complexed MH were evaluated at relatively low MGO concentrations, and a liquid crystalline-forming microemulsion containing α-CD-complexed MH was formulated. After determining pH and osmolarity, ocular tolerability was assessed using human primary corneal epithelial cells and chorioallantoic membranes, while the antibacterial efficacy was further evaluated in vitro. The α-CD–MH complex had significantly greater antibacterial activity against Staphylococcus aureus than either constituent alone, which was evident even when formulated as a microemulsion. Moreover, the final formulation had a physiologically acceptable pH and osmolarity for eyelid application and was well-tolerated when diluted 1:10 with artificial tear fluid, as expected to be the case after accidental exposure to the ocular surface in the clinical setting. Thus, a safe and efficient MH dosage form was developed for topical application to the eyelids, which can potentially be used to support optimal eyelid health in the management of blepharitis.

Evaluation of the Pathogenic Potential of Escherichia coli Strains Isolated from Eye Infections.

While primarily Gram-positive bacteria cause bacterial eye infections, several Gram-negative species also pose eye health risks. Currently, few studies have tried to understand the pathogenic mechanisms involved in E. coli eye infections. Therefore, this study aimed to establish the pathogenic potential of E. coli strains isolated from eye infections. Twenty-two strains isolated between 2005 and 2019 from patients with keratitis or conjunctivitis were included and submitted to traditional polymerase chain reactions (PCR) to define their virulence profile, phylogeny, clonal relationship, and sequence type (ST). Phenotypic assays were employed to determine hemolytic activity, antimicrobial susceptibility, and adhesion to human primary corneal epithelial cells (PCS-700-010). The phylogenetic results indicated that groups B2 and ST131 were the most frequent. Twenty-five virulence genes were found among our strains, with ecp, sitA, fimA, and fyuA being the most prevalent. Two strains presented a hemolytic phenotype, and resistance to ciprofloxacin and ertapenem was found in six strains and one strain, respectively. Regarding adherence, all but one strains adhered in vitro to corneal cells. Our results indicate significant genetic and virulence variation among ocular strains and point to an ocular pathogenic potential related to multiple virulence mechanisms.

Hyperosmolarity disrupts tight junction via TNF-α/MMP pathway in primary human corneal epithelial cells.

To investigate the mechanism of the tight junction (TJ) disruption and the association between tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMPs) under hyperosmotic condition in primary human corneal epithelial cells (HCECs). The cultured HCECs were exposed to media which adding sodium chloride (NaCl) for hyperosmolar stress or adding rh-TNF-α (10 ng/mL). NF-κB inhibitor (5 µmol/L) or GM-6001 (potent and broad spectrum MMP inhibitor, 20 µmol/L) was added 1h before that treatment. The integrity of TJ proteins was determined by immunofluorescent (IF) staining. The mRNA levels of TNF-α and MMPs were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the protein expression by enzyme-linked immunosorbent assay (ELISA). TJ proteins ZO-1 and Occludin were disrupted in primary HCECs exposed to hyperosmotic medium. The mRNA expression and protein production of TNF-α increased significantly in hyperosmotic media at 500 mOsM. TNF-α mediated the expression and production of MMP-1, MMP-13, MMP-9, and MMP-3 stimulated by hyperosmotic stress. The production of MMPs in hyperosmolar media were increased through the increase of TNF-α. GM-6001 prevent the destruction of ZO-1 and Occludin in hyperosmolar stress and rh-TNF-α treated medium. TNF-α induced activation of MMPs was involved in the TJ disruption by hyperosmolarity. TJ proteins ZO-1 and Occludin are disrupted by hyperosmolar stress and TNF-α, but protected by MMP inhibitor (GM-6001). It suggests that TNF-α/MMP pathway mediates the TJ disruption in primary HCECs exposed to hyperosmotic stress.

An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells.

Corneal epithelial cells derived from human pluripotent stem cells (hPSCs) are an important cell source for preclinical models to test ophthalmic drugs. However, current differentiation protocols lack instructions regarding optimal culturing conditions, which hinders the quality of cells and limits scale-up. Here, we introduce a simplified small molecule-based corneal induction method (SSM-CI) to generate corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimizing cell-culturing time using two defined culturing media containing TGF-β, and Wnt/β-catenin pathway inhibitors, and bFGF growth factor over 25days. Compared to the conventional human corneal epithelial cell line (HCE-T) and human primary corneal epithelial cells (hPCEpCs), corneal epithelial cells generated by SSM-CI are well differentiated and express relevant maturation markers, including PAX6 and CK12. RNA-seq analysis indicated the faithful differentiation of hPSCs into corneal epithelia, with significant upregulation of corneal progenitor and adult corneal epithelial phenotypes. Furthermore, despite the initial inhibition of TGF-β and Wnt/β-catenin, upregulation of these pathway-related transcripts was observed in the later stages, indicating their necessity in the generation of mature corneal epithelial cells. Moreover, we observed a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells, involving a decrease in glycolysis and an increase in fatty acid oxidation. This was also attributed to the overexpression of metabolic enzymes and transporter-related transcripts responsible for fatty acid metabolism. Thus, SSM-CI provides a comprehensive method for the generation of functional corneal epithelial cells for use in preclinical models.

Comparative Efficiency of Lutein and Astaxanthin in the Protection of Human Corneal Epithelial Cells In Vitro from Blue-Violet Light Photo-Oxidative Damage

The aim of this study was to compare in vitro the protective and antioxidant properties of lutein and astaxanthin on human primary corneal epithelial cells (HCE-F). To this purpose, HCE-F cells were irradiated with a blue-violet light lamp (415–420 nm) at different energies (20 to 80 J/cm2). Lutein and astaxanthin (50 to 250 μM) were added to HCE-F right before blue-violet light irradiation at 50 J/cm2. Viability was evaluated by the CKK-8 assay while the production of reactive oxygen species (ROS) by the H2DCF-DA assay. Results have shown that the viability of HCE-F cells decreased at light energies from 20 J/cm2 to 80 J/cm2, while ROS production increased at 50 and 80 J/cm2. The presence of lutein or astaxanthin protected the cells from phototoxicity, with lutein slightly more efficient than astaxanthin also on the blunting of ROS, prevention of apoptotic cell death and modulation of the Nrf-2 pathway. The association of lutein and astaxanthin did not give a significant advantage over the use of lutein alone. Taken together, these results suggest that the association of lutein and astaxanthin might be useful to protect cells of the ocular surface from short (lutein) and longer (astaxanthin) wavelengths, as these are the most damaging radiations hitting the eye from many different LED screens and solar light.

Polyaniline-Conjugated Boron Nitride Nanoparticles Exhibiting Potent Effects against Pathogenic Brain-Eating Amoebae.

Free-living amoebae include Acanthamoeba castellanii and Naegleria fowleri that are opportunistic protozoa responsible for life-threatening central nervous system infections with mortality rates over 90%. The rising number of cases and high mortality rates are indicative of the critical unmet need for the development of efficient drugs in order to avert future deaths. In this study, we assess the anti-amoebic capacity of a conducting polymer nanocomposite comprising polyaniline (PANI) and hexagonal boron nitride (hBN) against A. castellanii and N. fowleri. We observed significant amoebicidal and cysticidal effects using 100 μg/mL PANI/hBN (P < 0.05). Further, the nanocomposite demonstrated negligible cytotoxicity toward HaCaT and primary human corneal epithelial cells (pHCECs). In evaluating the mode of inhibition of A. castellanii due to treatment with PANI/hBN, increased intracellular reactive oxygen species (ROS) was measured and scanning microscopy visualized the formation of pores in the amoebae. Overall, this study is suggestive of the potential of the PANI/hBN nanocomposite as a promising therapy for amoeba infections.

IL-36α/IL-36RA/IL-38 signaling mediates inflammation and barrier disruption in human corneal epithelial cells under hyperosmotic stress

To explore the distinct expression and diverse roles of IL-36 cytokines in dry eye disease using an in vitro hyperosmolarity model of human corneal epithelial cells (HCECs). Primary HCECs were cultured from fresh donor limbal explants. Hyperosmolarity model was established by switching HCECs from isosmotic (312mOsM) to hyperosmotic medium (350-500 mOsM) alone or with addition of rhIL-36RA or rhIL-38 for 2-48h. Some cultures were treated with IL-36α (1-10ng/ml) with or without rhIL-36RA or rhIL-38. Gene expression was detected by RT-qPCR; and protein production and barrier disruption were evaluated by ELISA and/or immunofluorescent staining. IL-36 cytokines were differential expressed in primary HCECs. Among 3 pro-inflammatory agonists, IL-36α, but not IL-36β and IL-36γ, was distinctly induced at osmolarity-dependent manner while two antagonist IL-36RA and IL-38 were significantly suppressed in HCECs exposed to hyperosmotic stress. IL-36α increased to 4.4-fold in mRNA and 6.9-fold at protein levels (116.0±36.33pg/ml vs 16.79±6.51pg/ml in controls) by 450 mOsM, but dramatically inhibited by addition of rhIL-36RA or rhIL-38. Exogenous rhIL-36α stimulated expression of TNF-α and IL-1β at mRNA and protein levels and disrupted tight junction proteins ZO-1 and occludin. However, rhIL-36RA or rhIL-38 suppressed TNF-α and IL-1β production and protected HCECs from barrier disruption in response to IL-36α or hyperosmolarity. Our findings demonstrate that the stimulated pro-inflammatory IL-36α with the suppressed antagonists IL-36RA and IL-38 is a novel mechanism by which hyperosmolarity induces inflammation in dry eye. IL-36RA and IL-38 may have a therapeutic potential in dry eye.

Effects of Topical Gabapentin on Ocular Pain and Tear Secretion.

Neuropathic ocular pain is a frequent occurrence in medium to severe dry eye disease (DED). Only palliative treatments, such as lubricants and anti-inflammatory drugs, are available to alleviate patients’ discomfort. Anesthetic drugs are not indicated, because they may interfere with the neural feedback between the cornea and the lacrimal gland, impairing tear production and lacrimation. Gabapentin (GBT) is a structural analog of gamma-amino butyric acid that has been used by systemic administration to provide pain relief in glaucomatous patients. We have already shown in a rabbit model system that its topic administration as eye drops has anti-inflammatory properties. We now present data on rabbits’ eyes showing that indeed GBT given topically as eye drops has analgesic but not anesthetic effects. Therefore, opposite to an anesthetic drug such as oxybuprocaine, GBT does not decrease lacrimation, but–unexpectedly–even stimulates it, apparently through the upregulation of acetylcholine and norepinephrine, and by induction of aquaporin 5 (AQP5) expression in the lacrimal gland. Moreover, data obtained in vitro on a primary human corneal epithelial cell line also show direct induction of AQP5 by GBT. This suggests that corneal cells might also contribute to the lacrimal stimulation promoted by GBT and participate with lacrimal glands in the restoration of the tear film, thus reducing friction on the ocular surface, which is a known trigger of ocular pain. In conclusion, GBT is endowed with analgesic, anti-inflammatory and secretagogue properties, all useful to treat neuropathic pain of the ocular surface, especially in case of DED.

Autophagy Activation Protects Ocular Surface from Inflammation in a Dry Eye Model In Vitro.

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases.