Determinants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as -reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed , as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both and , suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy. Adeno-associated virus (AAV) is a nonpathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies with this vector.

PURPOSE: To evaluate the presence of atopy in patients with ocular cicatricial pemphigoid (OCP). METHOD: Patient encounters between August 2005 and November 2016 at the Massachusetts Eye Research and Surgery Institute (MERSI) were searched to identify those with biopsy-proven OCP who had concurrent evidence of atopy. RESULTS: There were 230 patients with biopsy-proven OCP. Thirty-three of them were found to have clinical symptoms of atopy (asthma, hay fever, and eczema) and of these, 23 had evidence of atopy in their conjunctival biopsy specimens. All patients were administered immunomodulatory therapy for treatment of their OCP with 20 patients requiring additional antiallergy treatment to control residual atopic ocular symptoms. Among patients who used antiallergy medications, 80% showed improvement in residual symptoms. Rituximab and/or intravenous immunoglobulin is a preferred OCP medication for patients with OCP with some evidence of atopy. CONCLUSIONS: Clinicians should consider the coexistence of atopy in patients with OCP, especially in those with persistent symptoms after initiation of immunomodulatory therapy.

Macrophages are crucial drivers of inflammatory corneal neovascularization and thus are potential targets for immunomodulatory therapies. We hypothesized that therapeutic use of cornea-derived mesenchymal stromal cells (cMSCs) may alter the function of macrophages. We found that cMSCs can modulate the phenotype and angiogenic function of macrophages. In vitro, cMSCs induce apoptosis of macrophages while preferentially promoting a distinct CD14 CD16 CD163 CD206 immunophenotype that has significantly reduced angiogenic effects based on in vitro angiogenesis assays. In vivo, application of cMSCs to murine corneas after injury leads to reduced macrophage infiltration and higher expression of CD206 in macrophages. Macrophages cocultured ("educated") by cMSCs express significantly higher levels of anti-angiogenic and anti-inflammatory factors compared with control macrophages. In vivo, injured corneas treated with cMSC-educated macrophages demonstrate significantly less neovascularization compared with corneas treated with control macrophages. Knocking down the expression of pigment epithelial derived factor (PEDF) in cMSCs significantly abrogates its modulating effects on macrophages, as shown by the reduced rate of apoptosis, decreased expression of sFLT-1/PEDF, and increased expression of vascular endothelial growth factor-A in the cocultured macrophages. Similarly, cMSCs isolated from PEDF knockout mice are less effective compared with wild-type cMSCs at inhibiting macrophage infiltration when applied to wild-type corneas after injury. Overall, these results demonstrate that cMSCs therapeutically suppress the angiogenic capacity of macrophages and highlight the role of cMSC secreted PEDF in the modulation of macrophage phenotype and function. Stem Cells 2018;36:775-784.

PURPOSE: To report a case of Ahmed glaucoma valve-induced hypotony that was successfully managed with postoperative intraluminal stenting of the aqueous shunt tube. PATIENT AND METHODS: We describe a 68-year-old man with advanced uveitic glaucoma with an intraocular pressure (IOP) of 25 mm Hg in the left eye. The patient initially responded well to an Ahmed glaucoma valve implant, but at 10 weeks postimplantation, the patient underwent cataract surgery and developed persistent hypotony, choroidal folds, and decreased vision. RESULTS: Before partial occlusion of the aqueous shunt tube, the patient had an IOP of 3 mm Hg and a best-corrected visual acuity (BCVA) of 20/60. Following intraluminal stenting of the aqueous shunt tube with 4-0 polypropylene suture (Prolene; Ethican), IOP rose from 7 to 10 mm Hg, BCVA improved to 20/30, and the choroidal folds resolved; IOP and BCVA remained stable through 1 year of follow-up and no additional surgical or pharmacological interventions were required. CONCLUSIONS: Aqueous shunt-induced hypotony can be successfully managed with intraluminal stenting and should be considered before tubal ligation or shunt removal.

The design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment epithelium (RPE) and Bruch's membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in an AMD, we generated abnormal extracellular matrix (ECM) using CRISPR-edited ARPE-19 cells. We introduced to these cells the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The abnormal ECM binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. Human fetal RPE (hfRPE) cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 activity. The local cleavage of C3 via convertase-independent mechanisms can be a new therapeutic target for early AMD.

PURPOSE: To describe the distribution, clinical findings, visual outcomes, treatment, and complications of children with uveitis at a tertiary referral ophthalmic center. METHODS: Retrospective cohort study. We reviewed the medical records of all patients ≤16 years with uveitis referred to Massachusetts Eye Research and Surgery Institution from March 2005 to July 2016. RESULTS: Of 286 included children, 62.24% were female. Mean age of onset was 8.4 years. The uveitis was mainly anterior (61.9%), recurrent (68.53%), bilateral (81.82%), and noninfectious (96.5%). Idiopathic cases accounted for 51.4%. The most frequent systemic association was juvenile idiopathic arthritis (34.96%). The majority of patients (78.32%) experienced complications. All patients, except one, needed systemic therapy. CONCLUSION: Pediatric uveitis is challenging to diagnose and manage, with frequent and potentially severe complications. Most cases were bilateral, recurrent, and idiopathic. Prompt referral to uveitis-specialized centers and an appropriate systemic therapy are mandatory for good visual outcomes.

Purpose: Drivers with homonymous hemianopia (HH) were previously found to have impaired detection of blind-side hazards, yet in many jurisdictions they may obtain a license. We evaluated whether oblique 57Δ peripheral prisms (p-prisms) and perceptual-motor training improved blind-side detection rates. Methods: Patients with HH (n = 11) wore p-prisms for 2 weeks and then received perceptual-motor training (six visits) detecting and touching stimuli in the prism-expanded vision. In a driving simulator, patients drove and pressed the horn upon detection of pedestrians who ran toward the roadway (26 from each side): (1) without p-prisms at baseline; (2) with p-prisms after 2 weeks acclimation but before training; (3) with p-prisms after training; and (4) 3 months later. Results: P-prisms improved blind-side detection from 42% to 56%, which further improved after training to 72% (all P < 0.001). Blind-side timely responses (adequate time to have stopped) improved from 31% without to 44% with p-prisms (P < 0.001) and further improved with training to 55% (P = 0.02). At the 3-month follow-up, improvements from training were maintained for detection (65%; P = 0.02) but not timely responses (P = 0.725). There was wide between-subject variability in baseline detection performance and response to p-prisms. There were no negative effects of p-prisms on vehicle control or seeing-side performance. Conclusions: P-prisms improved detection with no negative effects, and training may provide additional benefit. Translational Relevance: In jurisdictions where people with HH are legally driving, these data aid in clinical decision making by providing evidence that p-prisms improve performance without negative effects.

Purpose: Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics. In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/β-catenin-mediated transcriptional program. Methods: We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression. In vivo efficacy of ICG-001 was evaluated in a UM xenograft model. Results: ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration. Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition. Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling. Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC. Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome. Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice. Conclusions: Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell. Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.

PURPOSE: To determine the factors that influence the sensitivity and specificity of laser-scanning in vivo confocal microscopy (IVCM) for diagnosing Acanthamoeba keratitis (AK). METHODS: This retrospective, controlled study included 28 eyes of 27 patients with AK and 34 eyes of 34 patients with bacterial keratitis (as the control group). All patients had undergone corneal imaging with a laser-scanning IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module). The IVCM images were independently evaluated by 2 experienced and 2 inexperienced masked observers. Sensitivity and specificity of IVCM for diagnosing AK and the effects of various clinical and imaging parameters on the sensitivity were then investigated. RESULTS: Overall, IVCM had average sensitivity and specificity of 69.7% ± 2.5% and 97.1% ± 4.2% for experienced observers and 59.0% ± 7.6% and 92.7% ± 10.4% for inexperienced observers, respectively. However, the sensitivity did not show any significant association with the duration of disease, size of ulcer, depth of involvement, culture results, or cyst morphology. Although interobserver agreement was good (κ = 0.60, P < 0.001) for the experienced observers, it was only at a moderate level (κ = 0.48, P < 0.001) for the inexperienced observers. CONCLUSIONS: IVCM has a moderate sensitivity and a high specificity for diagnosis of AK. Although clinical parameters do not affect this diagnostic accuracy, a higher sensitivity is seen when images are interpreted by experienced observers.

PURPOSE: To assess the relationship of forward and backward scattering and corneal higher-order aberrations (HOAs) with corrected distance visual acuity (CDVA) after penetrating keratoplasty (PK). METHODS: This retrospective study comprised 25 eyes of 25 consecutive patients who underwent PK using the VisuMax femtosecond laser system and age-matched 25 eyes of 25 healthy subjects. We quantitatively assessed objective scattering index (OSI) using the double-pass instrument (OQAS II, Visiometrics), corneal densitometry (CD) and corneal HOAs with the Scheimpflug rotating camera (Pentacam HR, Oculus) 1 year postoperatively. RESULTS: The OSI, CD, and corneal HOAs were significantly larger in the PK group than those in the control group (p ≤ 0.011). We found significant correlations of logMAR CDVA with the OSI (r = 0.477, p = 0.016), and with the anterior, posterior, and total corneal HOAs of the central 4-mm zone (anterior: r = 0.573, p = 0.003, posterior: r = 0.596, p = 0.002, total: r = 0.472, p = 0.017), but no significant association with the CD of the 0-2 mm zone at any layers (anterior: r = 0.236, p = 0.257, center: r = 0.139, p = 0.506, posterior: r = 0.073, p = 0.728, total: r = 0.212, p = 0.308). Similar results were obtained when the analysis was repeated with corneal HOAs of the central 6-mm zone and CDs in 2-6 mm zone. CONCLUSIONS: Our pilot study demonstrated that the postoperative CDVA was significantly correlated with OSI and corneal HOAs, but not with backward scattering in post-PK eyes, suggesting that OSI as well as corneal HOAs plays an essential role in postoperative visual performance after PK.