PURPOSE: Obstructive meibomian gland dysfunction is a leading cause of ocular morbidity and its treatment remains a challenge. Meibomian gland probing was initially described in 2010. Here, the authors describe a modified technique, dynamic intraductal meibomian probing, which offers several advantages over the traditional approach including increased magnification, greater eyelid stabilization, enhanced anesthesia, and easier identification of gland orifices through the expression of meibum. METHODS: The authors conducted a retrospective chart review of 70 eyelids with treatment-resistant obstructive meibomian gland dysfunction undergoing dynamic intraductal meibomian probing between January 2013 and April 2015. RESULTS: Immediately after the procedure, 91.4% of cases experienced symptomatic improvement, and no complications were noted. CONCLUSIONS: Dynamic intraductal meibomian probing is an effective and safe treatment for obstructive meibomian gland dysfunction that is resistant to traditional therapies.

OBJECTIVE: To compare clinical outcomes of cataract surgery in eyes with and without pseudoexfoliation (PXF). DESIGN: Retrospective deidentified data analysis. PARTICIPANTS: A total of 123 PXF and 4776 non-PXF eyes of patients who underwent cataract surgery. METHODS: We compared data on visual acuity, Visual Function Questionnaire (VFQ)-based quality of life, and complications in PXF and non-PXF eyes from the Veterans Affairs (VA) Ophthalmic Surgery Outcomes Data Project across 5 VA medical centres. RESULTS: Pupillary expansion devices were used in 31 (25.2%) PXF cases and 398 (8.4%) non-PXF cases (p < 0.0001). Capsular tension rings were used in 6 (4.9%) PXF cases and 55 (1.2%) non-PXF cases (p < 0.004). The following complications occurred more frequently in PXF cases: zonular dehiscence without vitrectomy (4 [3.3%] PXF cases vs 40 [0.8%] non-PXF cases p = 0.02), persistent inflammation (28 [24.1%] vs 668 [14.5%]; p = 0.007), and persistent intraocular pressure elevation (5 [4.3%] vs 68 [1.5%]; p = 0.03). Best corrected visual acuity (BCVA) improved in both groups after 1 month, but 87 (83.7%) PXF cases achieved postoperative BCVA better than or equal to 20/40 compared to 3991 (93.8%) non-PXF cases (p = 0.0003). There was no significant difference in the postoperative composite VFQ scores between PXF (82.1 ± 16.9) and non-PXF cases (84.2 ± 16.8, p = 0.09). CONCLUSIONS: Several complications occurred more frequently in the PXF group compared to the non-PXF group, and fewer PXF cases achieved BCVA better than or equal to 20/40. Despite this, both groups experienced similar improvement in vision-related quality of life after cataract surgery.

Enterococci are ancient commensal bacteria that recently emerged as leading causes of antibiotic-resistant, hospital-acquired infection. Vancomycin-resistant enterococci (VRE) epitomize why drug-resistant enterococcal infections are a problem: VRE readily colonize the antibiotic-perturbed gastrointestinal (GI) tract where they amplify to large numbers, and from there, they infect other body sites, including the bloodstream, urinary tract, and surgical wounds. VRE are resistant to many antimicrobials and host defenses, which facilitates establishment at the site of infection and confounds therapeutic clearance. Having evolved to colonize the GI tract, VRE are comparatively ill adapted to the human bloodstream. A recent study by Honsa and colleagues (E. S. Honsa et al., mBio 8:e02124-16, 2017, https://doi.org/10.1128/mBio.02124-16) found that a strain of vancomycin-resistant Enterococcus faecium evolved antibiotic tolerance within the bloodstream of an immunocompromised host by activating the stringent response through mutation of relA Precisely how VRE colonize and infect and the selective pressures that led to the outgrowth of relA mutants are the subjects of ongoing research.

Purpose: Optic nerve head astrocytes, a subtype of white-matter astrocytes, become reactive early in the course of glaucoma. It was shown recently that in the DBA/2J mouse model of inherited glaucoma optic nerve astrocytes extend new longitudinal processes into the axon bundles before ganglion cell loss becomes apparent. The present study aims at testing whether this behavior of astrocytes is typical of early glaucomatous damage. Methods: Mice expressing green fluorescent protein in individual astrocytes were used to evaluate the early response of astrocytes in the glial lamina of the optic nerve head after increasing the IOP using the microbead occlusion method. Tissue sections from the glial lamina were imaged consecutively by confocal and electron microscopy. Results: Confocal and electron microscope images show that astrocytes close to the myelination transition zone in the hypertensive nerve heads extend new processes that follow the longitudinal axis of the optic nerve and invade axon bundles in the nerve head. Ultrastructurally, the longitudinal processes were largely devoid of subcellular organelles except for degenerating mitochondria. Conclusions: The longitudinal processes are a common feature of glaucomatous optic nerve astrocytes, whereas they are not observed after traumatic nerve injury. Thus, astrocytes appear to fine-tune their responses to the nature and/or timing of the injury to the neurons that they surround.

Purpose: Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in VEGF-induced angiogenesis. The goal of this project was to test the hypothesis that editing genomic VEGFR2 loci using the technology of clustered regularly interspaced palindromic repeats (CRISPR)-associated DNA endonuclease (Cas)9 in Streptococcus pyogenes (SpCas9) was able to block VEGF-induced activation of Akt and tube formation. Methods: Four 20 nucleotides for synthesizing single-guide RNAs based on human genomic VEGFR2 exon 3 loci were selected and cloned into a lentiCRISPR v2 vector, respectively. The DNA fragments from the genomic VEGFR2 exon 3 of transduced primary human retinal microvascular endothelial cells (HRECs) were analyzed by Sanger DNA sequencing, surveyor nuclease assay, and next-generation sequencing (NGS). In the transduced cells, expression of VEGFR2 and VEGF-stimulated signaling events (e.g., Akt phosphorylation) were determined by Western blot analyses; VEGF-induced cellular responses (proliferation, migration, and tube formation) were examined. Results: In the VEGFR2-sgRNA/SpCas9-transduced HRECs, Sanger DNA sequencing indicated that there were mutations, and NGS demonstrated that there were 83.57% insertion and deletions in the genomic VEGFR2 locus; expression of VEGFR2 was depleted in the VEGFR2-sgRNA/SpCas9-transduced HRECs. In addition, there were lower levels of Akt phosphorylation in HRECs with VEGFR2-sgRNA/SpCas9 than those with LacZ-sgRNA/SpCas9, and there was less VEGF-stimulated Akt activation, proliferation, migration, or tube formation in the VEGFR2-depleted HRECs than those treated with aflibercept or ranibizumab. Conclusions: The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis.

Purpose: To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress. Methods: Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected "hits" were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs). Results: Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels. Conclusions: Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.

Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector, for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

PURPOSE: To investigate the association between exercise and glaucoma in a South Korean population-based sample. DESIGN: Population-based, cross-sectional study. PARTICIPANTS: A total of 11,246 subjects, 40 years and older who underwent health care assessment as part of the 2008-2011 Korean National Health and Nutrition Examination Survey. METHODS: Variables regarding the duration (total minutes per week), frequency (days per week), and intensity of exercise (vigorous, moderate exercise and walking) as well as glaucoma prevalence were ascertained for 11,246 survey participants. Demographic, comorbidity, and health-related behavior information was obtained via interview. Multivariable logistic regression analyses were performed to determine the association between the exercise-related parameters and odds of a glaucoma diagnosis. MAIN OUTCOME MEASURE(S): Glaucoma defined by International Society for Geographical and Epidemiological Ophthalmology criteria. RESULTS: Overall, 336 (2.7%) subjects met diagnostic criteria for glaucomatous disease. After adjustment for potential confounding variables, subjects engaged in vigorous exercise 7 days per week had higher odds of having glaucoma compared with those exercising 3 days per week (Odds Ratio [OR] 3.33, 95% confidence interval [CI] 1.16-9.54). High intensity of exercise, as categorized by the guidelines of the American College of Sports Medicine (ACSM), was also associated with greater glaucoma prevalence compared with moderate intensity of exercise (OR 1.55, 95% CI 1.03-2.33). There was no association between other exercise parameters including frequency of moderate exercise, walking, muscle strength exercise, flexibility training, or total minutes of exercise per week, and the prevalence of glaucoma. In sub-analyses stratifying by gender, the association between frequency of vigorous exercise 7 days per week and glaucoma diagnosis remained significant in men (OR 6.05, 95% CI 1.67-21.94) but not in women (OR 0.96 95% CI: 0.23-3.97). A U-shaped association between exercise intensity and glaucoma prevalence was noted in men (OR 1.71, 95% CI 1.09-2.69 for low intensity versus moderate intensity; OR 2.19, 95% CI 1.25-3.85 for high intensity versus moderate intensity). CONCLUSION: In a South Korean population sample, daily vigorous exercise was associated with higher glaucoma prevalence. In addition, the intensity of exercise was positively associated with glaucoma diagnosis in men but not women.

Th17 cells are principal mediators of many autoimmune conditions. Recently, memory Th17 cells have been revealed as crucial in mediating the chronicity of various refractory autoimmune disorders; however, the underlying mechanisms maintaining memory Th17 cells have remained elusive. Here, using a preclinical model of ocular autoimmune disease we show that both IL-7 and IL-15 are critical for maintaining pathogenic memory Th17 cells. Neutralization of these cytokines leads to substantial reduction of memory Th17 cells; both IL-7 and IL-15 provide survival signals via activating STAT5, and IL-15 provides additional proliferation signals via activating both STAT5 and Akt. Topical neutralization of ocular IL-7 or IL-15 effectively reduces memory Th17 cells at the inflammatory site and draining lymphoid tissues, while topical neutralization of IL-17 alone, the major pathogenic cytokine secreted by Th17 cells, does not diminish memory Th17 cells at the draining lymphoid tissues. Our results suggest that the effective removal of pathogenic memory Th17 cells via abolishing environmental IL-7 or IL-15 is likely to be a novel strategy in the treatment of autoimmune diseases.

The roles of transforming growth factor (TGF)-β in extracellular matrix production and vascular remodeling, coupled with increased TGF-β expression and signaling in diabetes, suggest TGF-β as an important contributor to the microangiopathy of diabetic retinopathy and nephropathy. To investigate whether increased TGF-β signaling could be a therapeutic target for preventing retinopathy, we used a pharmacologic approach (SM16, a selective inhibitor of the type 1 TGF-β receptor activin receptor-like kinase 5, orally active) to inhibit the increased, but not the basal, Tgf-β signaling in retinal vessels of diabetic rats. At the level of vascular gene expression, 3.5 months' diabetes induced minimal changes. Diabetes + SM16 for 3 weeks caused widespread changes in gene expression poised to enhance vascular inflammation, thrombosis, leakage, and wall instability; these changes were not observed in control rats given SM16. The synergy of diabetes and SM16 in altering gene expression was not observed in the lung. At the level of vascular network morphology, 7 months' diabetes induced no detectable changes. Diabetes + SM16 for 3 weeks caused instead distorted morphology and decreased density. Thus, in diabetes, retinal vessels become dependent on a small increase in TGF-β signaling via activin receptor-like kinase 5 to maintain early integrity. The increased TGF-β signaling may protect against rapid retinopathy progression and should not be a target of inhibitory interventions.

Specialized pro-resolving mediators (SPM), e.g. Resolvin D1, Protectin D1, Lipoxin A₄, and Resolvin E1 have each shown to be active in ocular models reducing inflammation. In general, SPMs have specific agonist functions that stimulate resolution of infection and inflammation in animal disease models. The presence and quantity of SPM in human emotional tears is of interest. Here, utilizing a targeted LC-MS-MS metabololipidomics based approach we document the identification of pro-inflammatory (Prostaglandins and Leukotriene B₄) and pro-resolving lipid mediators (D-series Resolvins, Protectin D1, and Lipoxin A₄) in human emotional tears from 12 healthy individuals. SPMs from the Maresin family (Maresin 1 and Maresin 2) were not present in these samples. Principal Component Analysis (PCA) revealed gender differences in the production of specific mediators within these tear samples as the SPMs were essentially absent in these female donors. These results indicate that specific SPM signatures are present in human emotional tears at concentrations known to be bioactive. Moreover, they will help to further appreciate the mechanisms of production and action of SPMs in the eye, as well as their physiologic roles in human ocular disease resolution.

Pathological neovascularization, a leading cause of blindness, is seen in retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Using a mouse model of hypoxia-driven retinal neovascularization, we find that fibroblast growth factor 21 (FGF21) administration suppresses, and FGF21 deficiency worsens, retinal neovessel growth. The protective effect of FGF21 against neovessel growth was abolished in adiponectin (APN)-deficient mice. FGF21 administration also decreased neovascular lesions in two models of neovascular age-related macular degeneration: very-low-density lipoprotein-receptor-deficient mice with retinal angiomatous proliferation and laser-induced choroidal neovascularization. FGF21 inhibited tumor necrosis α (TNF-α) expression but did not alter Vegfa expression in neovascular eyes. These data suggest that FGF21 may be a therapeutic target for pathologic vessel growth in patients with neovascular eye diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration.

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

IMPORTANCE: To describe presenting patterns of breast cancer metastases to the orbit and eyelids BACKGROUND: To provide clinical, radiographic, and pathologic correlations of breast metastases to the orbit or eyelids and evaluate radiographic volumetric orbital changes DESIGN: Retrospective review in an academic center PARTICIPANTS: Ten female patients with periocular metastatic breast carcinoma who were seen at the Massachusetts Eye and Ear Infirmary Oculoplastics Clinic METHODS: Retrospective review of patient records, imaging and pathology findings. MAIN OUTCOME MEASURES: Presenting clinical characteristics, radiographic findings, and histopathological features were assessed and correlated to discover distinctive presenting patterns. Volumetric measurements of the tumors and orbital soft tissue structures were made on magnetic resonance imaging studies. RESULTS: The breast metastases included 9 orbital and 1 eyelid lesions. Two distinct clinical presentations were observed. The first consisted of seven patients who had either enophthalmos or euphthalmos in the setting of a retrobulbar lesion, a radiographically indistinct lesion, and a classic microscopic invasive lobular breast carcinoma (ILBC) with a prominent fibrotic stroma. The second group consisted of two proptotic patients with mass lesions on imaging and an atypical ILBC pathological subtype (pleomorphic or alveolar). One patient had diffusely indurated eyelid fullness. Volumetric analyses demonstrated variable tumor sizes with an inconsistent impact on the orbital volume and fat. CONCLUSIONS AND RELEVANCE: This correlative study provides the clinical-radiographic-histopathologic basis for separating two overarching phenotypic presentations of metastatic breast carcinoma to the orbit. Previously postulated mechanisms for the distinctive finding of tumor-induced enophthalmos are re-examined in the light of the foregoing conclusions.

Purpose: Tumor necrosis factor (TNF)-α is upregulated in eyes following corneal alkali injury and contributes to corneal and also retinal damage. Prompt TNF-α inhibition by systemic infliximab ameliorates retinal damage and improves corneal wound healing. However, systemic administration of TNF-α inhibitors carries risk of significant complications, whereas topical eye-drop delivery is hindered by poor ocular bioavailability and the need for patient adherence. This study investigates the efficacy of subconjunctival delivery of TNF-α antibodies using a polymer-based drug delivery system (DDS). Methods: The drug delivery system was prepared using porous polydimethylsiloxane/polyvinyl alcohol composite fabrication and loaded with 85 μg of infliximab. Six Dutch-belted pigmented rabbits received ocular alkali burn with NaOH. Immediately after the burn, subconjunctival implantation of anti-TNF-α DDS was performed in three rabbits while another three received sham DDS (without antibody). Rabbits were followed with photography for 3 months. Results: After 3 months, the device was found to be well tolerated by the host and the eyes exhibited less corneal damage as compared to eyes implanted with a sham DDS without drug. The low dose treatment suppressed CD45 and TNF-α expression in the burned cornea and inhibited retinal ganglion cell apoptosis and optic nerve degeneration, as compared to the sham DDS treated eyes. Immunolocalization revealed drug penetration in the conjunctiva, cornea, iris, and choroid, with residual infliximab in the DDS 3 months after implantation. Conclusions: This reduced-risk biologic DDS improves corneal wound healing and provides retinal neuroprotection, and may be applicable not only to alkali burns but also to other inflammatory surgical procedures such as penetrating keratoplasty and keratoprosthesis implantation.

Primary open-angle glaucoma (POAG), the most common optic neuropathy, is a heritable disease. Siblings of POAG cases have a ten-fold increase risk of developing the disease. Intraocular pressure (IOP) and optic nerve head characteristics are used clinically to predict POAG risk. We conducted a genome-wide association meta-analysis of IOP and optic disc parameters and validated our findings in multiple sets of POAG cases and controls. Using imputation to the 1000 genomes (1000G) reference set, we identified 9 new genomic regions associated with vertical cup disc ratio (VCDR) and 1 new region associated with IOP. Additionally, we found 5 novel loci for optic nerve cup area and 6 for disc area. Previously it was assumed that genetic variation influenced POAG either through IOP or via changes to the optic nerve head; here we present evidence that some genomic regions affect both IOP and the disc parameters. We characterized the effect of the novel loci through pathway analysis and found that pathways involved are not entirely distinct as assumed so far. Further, we identified a novel association between CDKN1A and POAG. Using a zebrafish model we show that six6b (associated with POAG and optic nerve head variation) alters the expression of cdkn1a In summary, we have identified several novel genes influencing the major clinical risk predictors of POAG and showed that genetic variation in CDKN1A is important in POAG risk.

PURPOSE: To report a novel presentation of dyskeratosis congenita masquerading as familial exudative vitreoretinopathy. METHODS: Observational case series involving single family and literature review. RESULTS: A brother and sister were diagnosed with familial exudative vitreoretinopathy at ages 4 and 2, respectively. Both patients were managed with laser photocoagulation. Eight years after the initial presentation, both siblings developed pancytopenia secondary to bone marrow failure. Laboratory work-up revealed severely shortened telomere length in both patients, and genetic testing revealed a missense mutation in the gene that encodes the reverse transcriptase component of telomerase, confirming the diagnosis of dyskeratosis congenita. The father of both children was a carrier of the same mutation, who exhibited marked retinal vascular tortuosity of the second-order vessels. CONCLUSION: Dyskeratosis congenita is a severe multisystem disorder, which should be considered in cases of pediatric exudative retinopathies with concurrent signs and/or symptoms of bone marrow failure.