Corneal endothelium is a cellular monolayer positioned on the Descemet's membrane at the anterior cornea, and it plays a critical role in maintaining corneal clarity. Our present study examines the feasibility of utilizing our 3-dimensional (3D) corneal stromal construct, which consists of human corneal fibroblasts (HCF) and their self-assembled matrix, to observe the development and maturation of human corneal endothelial cells (HCEndoCs) in a co-culture model. Three-dimensional HCF constructs were created by growing the HCFs on Transwell membranes in Eagles' minimum essential medium (EMEM) + 10% FBS + 0.5 mM Vitamin C (VitC) for about 4 weeks. HCEndoCs, either primary (pHCEndoC) or cell line (HCEndoCL), were either seeded in chamber slides, directly on the Transwell membranes, or on the 3D HCF constructs and cultivated for 5 days or 2 weeks. The HCEndoCs that were seeded directly on the Transwell membranes were exposed indirectly to HCF by culturing the HCF on the plate beneath the membrane. Cultures were examined for morphology and ultrastructure using light and transmission electron microscopy (TEM). In addition, indirect-immunofluorescence microscopy (IF) was used to examine tight junction formation (ZO-1), maturation (ALDH1A1), basement membrane formation (Laminin), cell proliferation (Ki67), cell death (caspase-3), and fibrotic response (CTGF). As expected, both pHCEndoCs and HCEndoCLs formed monolayers on the constructs; however, the morphology of the HCEndoCLs appeared to be similar to that seen in vivo, uniform and closely packed, whereas the pHCEndoCs remained elongated. The IF data showed that laminin localization was present in the HCEndoCs' cytoplasm as cell-cell contact increased, and when they were grown in the 3D co-culture, the beginnings of what appears to be a continuous DM-like structure was observed. In addition, in co-cultures, ALDH1A1-positive HCEndoCs were present, ZO-1 expression localized within the tight junctions, minimal numbers of HCEndoCs were Ki67-or Caspase-3-positive, and CTGF was positive in both the HCEndoCs cytoplasm and the matrix of the co-culture. Also, laminin localization was stimulated in HCEndoCs upon indirect stimuli secreted by HCF. The present data suggests our 3D co-culture model is useful for studying corneal endothelium maturation in vitro since the co-culture promotes new DM-like formation, HCEndoCs develop in vivo-like characteristics, and the fibrotic response is activated. Our current findings are applicable to understanding the implications of corneal endothelial injection therapy, such as if the abnormal DM has to be removed from the patient, the newly injected endothelial cells will seed onto the wound area and deposit a new DM-like membrane. However, caution should be observed and as much of the normal DM should be left intact since removal of the DM can cause a posterior stromal fibrotic response.

In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.

PURPOSE: Pilot study to evaluate the safety and efficacy of oral guaifenesin in reducing the signs and symptoms of filamentary keratitis. METHODS: Prospective, uncontrolled open-label pilot study. Twelve patients with non-Sjögren dry eye disease (DED) and secondary filamentary keratitis received treatment with oral guaifenesin 600 mg twice a day (total dose of 1.2 g/day) for 4 weeks. Adverse events, change in the number of corneal filaments, corneal fluorescein staining (CFS; NEI grading system), and symptoms (Ocular Surface Disease Index) were assessed. RESULTS: Before starting oral guaifenesin, all patients were on topical medical therapy for their condition. At baseline, the mean number of filaments was 5.8 ± 2.9, CFS score 7.3 ± 3.2, and OSDI score 55.6 ± 25. After 4 weeks of treatment, the number of filaments was 2.1 ± 2.2 (p = 0.04 vs. baseline), CFS score 6.5 ± 3.1 (p = 0.5), and OSDI score 46.1 ± 30.9 (p = 0.2). One patient discontinued the medication due to gastrointestinal side effects. CONCLUSIONS: Oral guaifenesin was safe and generally well tolerated, and demonstrated modest efficacy in reducing the severity of filamentary keratitis. These results should be considered preliminary; however, placebo-controlled investigations would be justified to evaluate the therapeutic efficacy of oral guaifenesin as a mucolytic in treatment of filamentary keratitis.

Autoimmune uveitis is a sight-threatening ocular inflammatory condition in which the retina and uveal tissues become a target of autoreactive immune cells. While microglia have been studied extensively in autoimmune uveitis, their exact function remains uncertain. The objective of the current study was to determine whether resident microglia are necessary and sufficient to initiate and amplify retinal inflammation in autoimmune uveitis. In this study, we clearly demonstrate that microglia are essential for initiating infiltration of immune cells utilizing a murine model of experimental autoimmune uveoretinitis (EAU) and the recently identified microglia-specific marker P2ry12. Initiating disease is the primary function of microglia in EAU, since eliminating microglia during the later stages of EAU had little effect, indicating that the function of circulating leukocytes is to amplify and sustain destructive inflammation once microglia have triggered disease. In the absence of microglia, uveitis does not develop, since leukocytes cannot gain entry through the blood-retinal barrier, illustrating that microglia play a critical role in regulating infiltration of inflammatory cells into the retina.

BACKGROUND: Nasal mucosa-derived exosomes (NMDEs) harbor immunodefensive proteins and are capable of rapid interepithelial protein transfer. OBJECTIVES: We sought to determine whether mucosal exposure to inhaled pathogens stimulates a defensive swarm of microbiocidal exosomes, which also donate their antimicrobial cargo to adjacent epithelial cells. METHODS: We performed an institutional review board-approved study of healthy NMDE secretion after Toll-like receptor (TLR) 4 stimulation by LPS (12.5 μg/mL) in the presence of TLR4 inhibitors. Interepithelial transfer of exosomal nitric oxide (NO) synthase and nitric oxide was measured by using ELISAs and NO activity assays. Exosomal antimicrobial assays were performed with Pseudomonas aeruginosa. Proteomic analyses were performed by using SOMAscan. RESULTS: In vivo and in vitro LPS exposure induced a 2-fold increase in NMDE secretion along with a 2-fold increase in exosomal inducible nitric oxide synthase expression and function through TLR4 and inhibitor of nuclear factor κB kinase activation. LPS stimulation increased exosomal microbiocidal activity against P aeruginosa by almost 2 orders of magnitude. LPS-stimulated exosomes induced a 4-fold increase in NO production within autologous epithelial cells with protein transfer within 5 minutes of contact. Pathway analysis of the NMDE proteome revealed 44 additional proteins associated with NO signaling and innate immune function. CONCLUSIONS: We provide direct in vivo evidence for a novel exosome-mediated innate immunosurveillance and defense mechanism of the human upper airway. These findings have implications for lower airway innate immunity, delivery of airway therapeutics, and host microbiome regulation.

PURPOSE: To report the clinical features, severity, and management of ocular immune-related adverse events (irAEs) in the setting of immune checkpoint inhibitor therapy for metastatic malignancies. METHODS: Retrospective chart review at three tertiary ophthalmology clinics. Electronic medical records were reviewed between 2000 and 2017 for patients with new ocular symptoms while undergoing checkpoint inhibition therapy. RESULTS: Eleven patients were identified. Ocular irAEs ranged from keratoconjunctivitis sicca to Vogt-Koyanagi-Harada-like findings. Average timing of irAEs from starting checkpoint inhibitor therapy was 15.7 weeks. Ocular inflammation was successfully controlled with corticosteroids in most cases, however three patients discontinue treatment as a result of ocular inflammation with decreased visual acuity, two discontinued due to progression of metastatic disease, and one discontinued due to severe systemic irAEs. CONCLUSION: We found a wide spectrum of ocular irAEs associated with immune checkpoint inhibitors. In most cases, ocular AEs did not limit ongoing cancer treatment.

PURPOSE: To determine the frequency of receded near point of convergence (NPC) in patients with chronic concussion-related symptoms, and among those with receded NPC to enumerate the frequency of convergence insufficiency and other oculomotor disorders. STUDY: Design: Retrospective cross-sectional study METHODS: Clinic charts were retrospectively reviewed for the prior 3.5 years to identify all patients <21 years old who were >28 days post-concussion, had chronic concussion-related symptoms, had normal visual acuity, and received a comprehensive sensorimotor examination. The frequency of receded NPC and oculomotor diagnoses were determined. RESULTS: Of the 83 eligible patients, 74 (89%) had receded NPC. Of these, 70 (95%) had oculomotor disorders; 30 (41%) had disorders of accommodation only, 21 (28%) had convergence insufficiency and accommodation deficits, and 6 (8%) had convergence insufficiency only. Six (8%) had a convergence deficit other than convergence insufficiency (all with concurrent accommodative disorders), 4 (5%) had both a non-specific vergence dysfunction and accommodation deficits, 2 (3%) had convergence excess only, and 1 (1%) had both convergence excess and accommodative deficits. CONCLUSION: A receded NPC was present in the majority of young patients with chronic post-concussion symptoms. Associated with numerous underlying oculomotor dysfunctions, the clinical finding of a receded NPC is not synonymous with the diagnosis of convergence insufficiency. Because treatment options for the various oculomotor dysfunctions differ, it is prudent that these patients undergo a thorough examination of their vergence and accommodative systems so that an accurate diagnosis can be made and appropriate treatment prescribed.

PURPOSE: To assess optic nerve head (ONH) and peripapillary microvasculature in primary open-angle glaucoma (POAG) of mild to moderate severity using swept-source optical coherence tomography angiography (OCTA). MATERIALS AND METHODS: In a cross-sectional study, swept-source OCTA images were analyzed for 1 eye from each of 30 POAG patients with glaucomatous Humphrey visual field loss and 16 controls. The anatomic boundary of ONH was manually delineated based on Bruch's membrane opening and large vessels were removed from en face angiography images to measure vessel density (VD) and the integrated OCTA by ratio analysis signal (IOS), suggestive of flow, in the ONH and peripapillary region. POAG subgroup analysis was performed based on a history of disc hemorrhage (DH) matched by visual field mean deviation (MD). RESULTS: POAG (mean MD±SD, -3.3±3.0 dB) and control groups had similar demographic characteristics and intraocular pressure on the day of imaging. Groups did not differ in superficial ONH VD or flow indicated by IOS (P≥0.28). POAG eyes showed significantly lower VD (39.4%±4.0%) and flow (38.8%±5.6%) in deep ONH, peripapillary VD (37.9%±2.9%) and flow (43.6%±4.0%) compared with control eyes (44.1%±5.1%, 44.7%±6.9%, 40.7%±1.7%, 47.8%±2.5%, respectively; P≤0.007 for all). In the subgroup analysis, POAG eyes with (n=14) and without DH (n=16) had similar measured OCTA parameters (P>0.99 for all). CONCLUSIONS: The image processing methodology based on the anatomic boundary of ONH demonstrated compromised microvasculature in the deep ONH and peripapillary region in eyes with mild to moderate POAG, regardless of the history of DH.

Conjunctivochalasis (CCH) is a bilateral conjunctival condition characterized by loose, redundant conjunctival folds, typically in the inferior bulbar conjunctiva. It is a common cause of ocular irritation, especially in older age. For asymptomatic CCH, no treatment is necessary. For treatment of symptomatic CCH, however, a variety of medical and surgical approaches are currently available, which will be thoroughly appraised in this review article. The first step in the management is medical therapy, which involves enhanced lubrication and use of anti-inflammatory medications. In refractory cases, a surgical approach may be undertaken for symptom relief. Several techniques have been described for this, with varying success rates. These include conjunctival cauterization, conjunctival excision, scleral fixation of the conjunctiva, conjunctival ligation, laser conjunctivoplasty, and radiowave electrosurgery. Among these, conjunctival cauterization and excision of the redundant conjunctiva, with or without tissue grafting, have gained popularity.