PURPOSE. Canonical Wnt signaling has emerged as a critical regulator of aqueous outflow facility and intraocular pressure (IOP). In this study, we examine the role of canonical Wnt signaling on extracellular matrix (ECM) expression in the trabecular meshwork (TM) and explore the molecular mechanisms involved. METHODS. β-catenin localization in human TM tissue was examined using immunofluorescent staining. Primary human TM cells were incubated with lithium chloride (LiCl) and the effect on active β-catenin expression was assessed by immunoblot. Adenovirus expressing a dominant-negative TCF4 mutant that lacks a β-catenin binding domain was used. Changes in the levels of the microRNA-29 (miR-29) family and ECM proteins were determined by real-time quantitative PCR and immunoblot analysis, respectively. RESULTS. β-catenin was expressed throughout the TM, with localization primarily to the plasma membrane. Incubation of TM cells with lithium chloride increased the expression of active β-catenin. Lithium chloride treatment upregulated miR-29b expression, and suppressed the levels of various ECM proteins under both basal and TGF-β2 stimulatory conditions. Infection of TM cells with a dominant-negative TCF4 mutant induced ECM levels without a significant change in the expression of the miR-29 family. CONCLUSIONS. Collectively, our data identify the canonical Wnt signaling pathway as an important modulator of ECM expression in the TM and provide a mechanistic framework for its regulation of outflow facility and IOP.

Purpose: To investigate bilateral tear cytokine levels in patients with unilateral bacterial keratitis (BK) as associated with in vivo confocal microscopic (IVCM) alterations in corneal nerves and dendritiform immune cells (DCs). Methods: Fifty-four (13 BK, 13 contralateral, 28 healthy controls) tear samples were collected prospectively and analyzed by multiplex microbeads assay. IVCM of the central cornea was performed on the same day and assessed for corneal nerve and DC alterations Results: Interleukin (IL)-1β, IL-6, and IL-8 were significantly elevated only in affected eyes (66.6±26.8 ρg/ml, 7,174±2,430, 810±315, P=0.04, P<0.001 and P<0.001), compared to healthy controls (13.0±4.0 ρg/ml, 171.8±32.1, 56.5±33.8). CCL-2, IL-10 and IL-17a were elevated only in contralateral eyes (813±478 ρg/ml, 86.7±38.3, 3,350±881, P=0.02, P=0.01, P=0.04), compared to controls (73.7±25.3 ρg/ml, 17.5 ±4.9, 1,350±337). Triggering receptor expressed on myeloid cells (TREM)-1 was significantly elevated in both affected (551±231 ρg/ml, P=0.02) and contralateral unaffected eyes (545±298 ρg/ml, P=0.03), compared to controls (31.3±12.4 ρg/ml). The density of DCs was significantly increased in both affected (226.9±37.3 cells/mm2, P<0.001) and unaffected eyes (122.3±23.7 cells/mm2, P<0.001) compared to controls (22.7±5.9 cells/mm2). Subbasal nerve density significantly decreased in affected eyes (3,337±1,615 μm/mm2, P<0.001) and contralateral eyes (13,230±1,635 μm/mm2, P<0.001) compared to controls (21,200±545 μm/mm2). IL-1β, IL-6 and IL-8 were significantly correlated with DC density (R=0.40, R=0.55 and R=0.31, all P<0.02) and nerve density (R=-0.30, R=-0.53 and R=-0.39, all P<0.01). Conclusions: Pro-inflammatory tear cytokines are elevated bilaterally in patients with unilateral BK, and are correlated strongly with alterations in DCs and nerve density as detected by IVCM.

PURPOSE: To investigate the effect of host immunity (allospecific) and surgical manipulation (non-allospecific) on corneal endothelial cells (CECs) in corneal transplantation. METHODS: Draining lymph nodes and grafted C57BL/6 corneas were harvested from syngeneic recipients, allograft acceptors, and allograft rejectors (BALB/c) 1, 3, and 8 weeks after transplantation. We analyzed CEC apoptosis using an ex vivo cornea-in-the-cup assay, and visualized cell-to-cell junctions using immunohistochemical staining (ZO-1). Automatic cell analysis using Confoscan software was used to measure CEC density as well as changes in CEC morphology by quantifying the coefficient of variation in cell size (polymegethism) and shape (pleomorphism). RESULTS: The cornea-in-the-cup assay showed that allogeneic acceptor T cells and to an even greater extent rejector T cells (but not syngeneic T cells) induced CEC apoptosis. CEC density after corneal transplantation was significantly reduced in allogeneic acceptors compared with syngeneic grafts (P<0.001), and CEC density was even further reduced in the allo-rejector group compared with the allo-acceptor group. Allogeneic grafts showed a greater increase in the coefficient of variation in cell size (polymegethism) when compared with syngeneic grafts 1 week after transplantation (P=P<0.001). However, pleomorphism was not significantly different between syngeneic and allo-acceptor grafts, indicating that polymegethism (but not pleomorphism or cell density) is a sensitive indicator of the effect of alloimmunity on CECs. CONCLUSIONS: Our data demonstrate that host alloimmunity rather than surgical manipulation alone is the major cause of CEC damage in corneal transplantation, and such morphologic changes of CECs can be detected before the clinically visible onset of allograft rejection.

There is currently considerable controversy about existence and classification of "lymphatic vessels" in the eye. Some of the confusion is certainly caused by inappropriate use (or nonuse) of the correct immunohistochemical markers. Many experts in the field expressed the need for a consensus statement, and, in this perspective, authors offer arguments and solutions to reliably continue with immunohistochemical ocular lymphatic research.

The intestinal microbial ecosystem is complex, and few of the principles that contribute to homeostasis in health are well understood. Pham et al. (2014) show that a network including the epithelial interleukin-22 receptor protects against infection with the opportunistic pathogen Enterococcus faecalis through promotion of host-microbiota mutualism.

PURPOSE: Quantitative fundus autofluorescence (qAF) and spectral-domain optical coherence tomography (SD OCT) were performed in patients with bull's-eye maculopathy (BEM) to identify phenotypic markers that can aid in the differentiation of ABCA4-associated and non-ABCA4-associated disease. DESIGN: Prospective cross-sectional study at an academic referral center. SUBJECTS: Thirty-seven BEM patients (age range, 8-60 years) were studied. All patients exhibited a localized macular lesion exhibiting a smooth contour and qualitatively normal-appearing surrounding retina without flecks. Control values consisted of previously published data from 277 healthy subjects (374 eyes; age range, 5-60 years) without a family history of retinal dystrophy. METHODS: Autofluorescence (AF) images (30°, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The grey levels (GLs) from 8 circularly arranged segments positioned at an eccentricity of approximately 7° to 9° in each image were calibrated to the reference (0 GL), magnification, and normative optical media density to yield qAF. In addition, horizontal SD OCT images through the fovea were obtained. All patients were screened for ABCA4 mutations using the ABCR600 microarray, next-generation sequencing, or both. MAIN OUTCOME MEASURES: Quantitative AF, correlations between AF and SD OCT, and genotyping for ABCA4 variants. RESULTS: ABCA4 mutations were identified in 22 patients, who tended to be younger (mean age, 21.9±8.3 years) than patients without ABCA4 mutations (mean age, 42.1±14.9 years). Whereas phenotypic differences were not obvious on the basis of qualitative fundus AF and SD OCT imaging, with qAF, the 2 groups of patients were clearly distinguishable. In the ABCA4-positive group, 37 of 41 eyes (19 of 22 patients) had qAF8 of more than the 95% confidence interval for age. Conversely, in the ABCA4-negative group, 22 of 26 eyes (13 of 15 patients) had qAF8 within the normal range. CONCLUSIONS: The qAF method can differentiate between ABCA4-associated and non-ABCA4-associated BEM and may guide clinical diagnosis and genetic testing.

PURPOSE: To report the visual outcomes of prosthetic replacement of the ocular surface ecosystem (PROSE) treatment in patients with ocular surface disease related to Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). DESIGN: Retrospective cohort study. SUBJECTS: We included 86 patients (167 eyes) with history of SJS/TEN who underwent PROSE treatment from January 1, 2006, to January 1, 2011. METHODS: Etiology, previous interventions, change in visual acuity, change in visual function, and duration of follow-up are reported. Paired t test and Friedman test with Dunn's post hoc test for multiple comparisons were used for statistical analysis. MAIN OUTCOME MEASURES: Visual acuity at last follow-up and visual function based on the National Eye Institute 25-item Visual Functioning Questionnaire (NEI VFQ-25) at 6 months. RESULTS: We treated 35 males and 51 females with a history of SJS/TENS; median age was 36 years. The most common reported etiologies for SJS/TENS were antibiotics (n = 25), ibuprofen (n = 15), and lamotrigine (n = 11). The median visual acuity at the initial visit was 20/60 (range, 20/400-20/25; 0.48 logarithm of the minimum angle of resolution [logMAR]), and the visual acuity at completion of customization was 20/25 (range, 20/200-20/20; 0.096 logMAR; P < 0.001), with no decline in median acuity at the end of follow-up. Median duration of follow-up was 16 months. There was a significant improvement in the visual function of the patients based on the NEI VFQ-25 questionnaire (mean of 48 points at baseline vs. mean of 72 points at 6 months; P < 0.001). In addition, there was also an improvement in the self-reported general health of the patients (mean of 57 points at baseline vs. mean of 65 points at 6 months; P < 0.01). CONCLUSIONS: In a large cohort of patients with chronic ocular surface disease related to SJS/TEN, PROSE treatment offers sustained and significant large improvement in visual function and acuity.

Age-related macular degeneration (AMD) is a multifactorial degeneration of photoreceptors and retinal pigment epithelium. The societal impact is significant, with more than 2 million individuals in the United States alone affected by advanced stages of AMD. Recent progress in our understanding of this complex disease and parallel developments in therapeutics and imaging have translated into new management paradigms in recent years. However, there are many unanswered questions, and diagnostic and prognostic precision and treatment outcomes can still be improved. In this article, we discuss the clinical features of AMD, provide correlations with modern imaging and histopathology, and present an overview of treatment strategies.

Complex traits can be triggered by environmental factors in genetically predisposed individuals. The lysyl oxidase-like 1 gene (LOXL1) variants associated with exfoliation syndrome (XFS) are detected in >90% of cases that have been genotyped from sites around the world. Remarkably, roughly 80% of people without XFS also possess these same variants in all populations that have been tested. Nonetheless, the prevalence of XFS varies from ≤0.4% to >20%. These data suggest that other genetic variants, epigenetic modifications, or environmental factors also contribute to XFS. Furthermore, it is possible that environmental factors modify the association between LOXL1 and XFS. Interactions between LOXL1 variants and environmental factors could explain the varying prevalence of XFS seen throughout the world. At the very least, the discovery of the association between LOXL1 variants and XFS has opened the door to the discovery of environmental risk factors for this condition. Candidate gene-environment interactions in XFS will be discussed.

Variants in LOXL1 are significantly associated with exfoliation syndrome (XFS), however the impact of the associated variants on disease development is not yet understood. Initially the associated missense changes, R141L and G153D, were considered to be pathogenic alleles. Flipping of the risk allele in certain populations for both missense variants provided strong evidence that these missense changes are not biologically significant and suggest that other LOXL1 variant(s), in linkage disequilibrium with these missense variants, predispose to exfoliation syndrome by affecting gene expression or protein function. Several lines of evidence support dysregulation of LOXL1 gene expression as a contributing factor to disease development. First, in the German population the R141L (rs1048661) risk allele reduced LOXL1 expression by 20%. Second, haplotype analysis identified a risk haplotype that includes including R141L, G153D, as well as a LOXL1 promoter region variant previously shown to reduce gene expression (rs16958477). Third, the LOXL1 risk haplotype influences gene expression induced by disease-associated factors TGF-B1, oxidative stress, UV light and hypoxia. Finally, a LOXL1 null mouse has some features of XFS suggesting that decreased enzyme activity contributes to predisposition to the disease. Collectively, these results suggest that dysregulation of LOXL1 expression is a contributing factor to exfoliation disease development.

Elevated intraocular pressure (IOP) is an important risk factor in developing glaucoma, and variability in IOP might herald glaucomatous development or progression. We report the results of a genome-wide association study meta-analysis of 18 population cohorts from the International Glaucoma Genetics Consortium (IGGC), comprising 35,296 multi-ancestry participants for IOP. We confirm genetic association of known loci for IOP and primary open-angle glaucoma (POAG) and identify four new IOP-associated loci located on chromosome 3q25.31 within the FNDC3B gene (P = 4.19 × 10(-8) for rs6445055), two on chromosome 9 (P = 2.80 × 10(-11) for rs2472493 near ABCA1 and P = 6.39 × 10(-11) for rs8176693 within ABO) and one on chromosome 11p11.2 (best P = 1.04 × 10(-11) for rs747782). Separate meta-analyses of 4 independent POAG cohorts, totaling 4,284 cases and 95,560 controls, showed that 3 of these loci for IOP were also associated with POAG.

Primary open-angle glaucoma (POAG) is a major cause of irreversible blindness worldwide. We performed a genome-wide association study in an Australian discovery cohort comprising 1,155 cases with advanced POAG and 1,992 controls. We investigated the association of the top SNPs from the discovery stage in two Australian replication cohorts (932 cases and 6,862 controls total) and two US replication cohorts (2,616 cases and 2,634 controls total). Meta-analysis of all cohorts identified three loci newly associated with development of POAG. These loci are located upstream of ABCA1 (rs2472493[G], odds ratio (OR) = 1.31, P = 2.1 × 10(-19)), within AFAP1 (rs4619890[G], OR = 1.20, P = 7.0 × 10(-10)) and within GMDS (rs11969985[G], OR = 1.31, P = 7.7 × 10(-10)). Using RT-PCR and immunolabeling, we show that these genes are expressed within human retina, optic nerve and trabecular meshwork and that ABCA1 and AFAP1 are also expressed in retinal ganglion cells.

Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue tropism, and limited viral host response has made it an attractive vector system for gene therapy. The viral protein capsid, the primary interface with the host, is the main determinant for these phenotypes, is highly variable, and is most subject to pressures during replication. Here, we explore the evolutionary path of AAV and other parvoviruses in respect to these phenotypes, as well as directed evolution and engineering strategies that have exploited the lessons learned from natural selection in order to address remaining limitations of AAV as a therapeutic gene transfer platform.