Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.
Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection.
PURPOSE: To determine the diagnostic capability of spectral domain optical coherence tomography peripapillary retinal volume (RV) measurements. MATERIALS AND METHODS: A total of 156 patients, 89 primary open-angle glaucoma and 67 normal subjects, were recruited. Spectral domain optical coherence tomography peripapillary RV was calculated for 4 quadrants using 3 annuli of varying scan circle diameters: outer circumpapillary annuli of circular grids 1, 2, and 3 (OCA1, OCA2, OCA3). Area under the receiver operating characteristic curves and pairwise comparisons of receiver operating characteristic (ROC) curves were performed to determine which quadrants were best for diagnosing primary open-angle glaucoma. The pairwise comparisons of the best ROC curves for RV and retinal nerve fiber layer (RNFL) were performed. The artifact rates were analyzed. RESULTS: Pairwise comparisons showed that the smaller annuli OCA1 and OCA2 had better diagnostic performance than the largest annulus OCA3 (P<0.05 for all quadrants). OCA1 and OCA2 had similar diagnostic performance, except for the inferior quadrant which was better for OCA1 (P=0.0033). The pairwise comparisons of the best ROC curves for RV and RNFL were not statistically significant. RV measurements had lower rates of artifacts at 7.4% while RNFL measurements had higher rates at 42.9%. CONCLUSIONS: Peripapillary RV measurements have excellent ability for diagnosing not only glaucoma patients but also a subset of early glaucoma patients. The inferior quadrant of peripapillary annulus OCA1 demonstrated the best diagnostic capability for both glaucoma and early glaucoma. The diagnostic ability of RV is comparable with that of RNFL parameters in glaucoma but with lower artifact rates.
Pathological neovessels growing into the normally avascular photoreceptors cause vision loss in many eye diseases, such as age-related macular degeneration and macular telangiectasia. Ocular neovascularization is strongly associated with inflammation, but the source of inflammatory signals and the mechanisms by which these signals regulate the disruption of avascular privilege in photoreceptors are unknown. In this study, we found that c-Fos, a master inflammatory regulator, was increased in photoreceptors in a model of pathological blood vessels invading photoreceptors: the very low-density lipoprotein receptor-deficient (Vldlr(-/-) ) mouse. Increased c-Fos induced inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor (TNF), leading to activation of signal transducer and activator of transcription 3 (STAT3) and increased TNFα-induced protein 3 (TNFAIP3) in Vldlr(-/-) photoreceptors. IL-6 activated the STAT3/vascular endothelial growth factor A (VEGFA) pathway directly, and elevated TNFAIP3 suppressed SOCS3 (suppressor of cytokine signaling 3)-activated STAT3/VEGFA indirectly. Inhibition of c-Fos using photoreceptor-specific AAV (adeno-associated virus)-hRK (human rhodopsin kinase)-sh_c-fos or a chemical inhibitor substantially reduced the pathological neovascularization and rescued visual function in Vldlr(-/-) mice. These findings suggested that the photoreceptor c-Fos controls blood vessel growth into the normally avascular photoreceptor layer through the inflammatory signal-induced STAT3/VEGFA pathway.
Transmembrane mucins are highly O-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin N-glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with N-acetyllactosamine, a preferred galectin ligand. In N-glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi N-acetylglucosaminyltransferase I gene (MGAT1) induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for N-glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for N-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.
Importance: Intravitreous bevacizumab (0.25 to 0.625 mg) is increasingly used to treat type 1 retinopathy of prematurity (ROP), but there remain concerns about systemic toxicity. A much lower dose may be effective while reducing systemic risk. Objective: To find a dose of intravitreous bevacizumab that was lower than previously used for severe ROP, was effective in this study, and could be tested in future larger studies. Design, Setting, and Participants: Between May 2015 and September 2016, 61 premature infants with type 1 ROP in 1 or both eyes were enrolled in a masked, multicenter, phase 1 dose de-escalation study. One eye of 10 to 14 infants received 0.25 mg of intravitreous bevacizumab. If successful, the dose was reduced for the next group of infants (to 0.125 mg, then 0.063 mg, and finally 0.031 mg). Diluted bevacizumab was delivered using 300 µL syringes with 5/16-inch, 30-gauge fixed needles. Interventions: Bevacizumab injections at 0.25 mg, 0.125 mg, 0.063 mg, and 0.031 mg. Main Outcomes and Measures: Success was defined as improvement in preinjection plus disease or zone I stage 3 ROP by 5 days after injection or sooner, and no recurrence of type 1 ROP or severe neovascularization requiring additional treatment within 4 weeks. Results: Fifty-eight of 61 enrolled infants had 4-week outcomes completed; mean birth weight was 709 g and mean gestational age was 24.9 weeks. Success was achieved in 11 of 11 eyes at 0.25 mg, 14 of 14 eyes at 0.125 mg, 21 of 24 eyes at 0.063 mg, and 9 of 9 eyes at 0.031 mg. Conclusions and Relevance: A dose of bevacizumab as low as 0.031 mg was effective in 9 of 9 eyes in this phase 1 study and warrants further investigation. Identifying a lower effective dose of bevacizumab may reduce the risk for neurodevelopmental disability or detrimental effects on other organs.
PURPOSE: To evaluate the clinical outcome of umbilical cord patch (UCP) transplantation for deep corneal ulcers with perforations and descemetoceles. METHODS: In this retrospective, noncomparative, interventional case series, 11 eyes of 11 patients with corneal perforation or descemetocele were included. The thickness and microstructure of UCP were measured. All eyes were treated with UCP and amniotic membrane transplantation for corneal reconstruction. Corneal ulcer healing, corneal thickness, anterior chamber formation, and best-corrected visual acuity (BCVA) were recorded and analyzed. RESULTS: The thickness of human UCP is 398.6 ± 102.8 μm (n = 5) with compact aligned fibers. The average age was 56.2 ± 15.8 (ranging from 22 to 75) years. The mean follow-up period was 7.1 ± 1.7 (ranging from 5 to 10) months. Four patients had descemetocele and 7 had perforation. The anterior chambers in all the 7 perforated corneas were formed at postoperative day 1. All patients regained a normal corneal thickness and smooth corneal surface within the first postoperative month. The vision improved in 10 eyes and remained unchanged in 1 eye. No recurrence nor side effects occurred during the follow-up. CONCLUSIONS: UCP can serve as an alternative material in the treatment of corneal perforations and descemetoceles. This treatment option is also beneficial in those countries with limited cornea donors and eye bank services.
PURPOSE: The purpose was to evaluate the effectiveness and safety of rituximab (RTX) for the treatment of patients with aggressive ocular cicatricial pemphigoid (OCP). METHODS: A review of patient records at a tertiary referral center with biopsy confirmed OCP who presented between 2006 and 2016. Sixty-one eyes of 32 patients with symptomatic OCP who received treatment with RTX monotherapy or RTX in combination with additional immunomodulatory treatment (IMT) were evaluated. Main outcomes included clinically evident remission of disease, the percentage of corticosteroid sparing patients, stage of OCP (Foster), best corrected visual acuity, and treatment complications. Remission was defined as absence of progressive scarring and active ocular inflammation for ≥ 2 months. Partial remission/responding was defined as disease control and clinical improvement for ≥ 2 months. RESULTS: Mean age at the initiation of RTX treatment was 59.1 years (range, 24-80 years) with a median follow-up time after RTX initiation of 32 months (range, 14 to 127 months). Twenty-six patients achieved clinical remission with an average sustained remission of 24.5 months (from 9 months to 84 months). RTX monotherapy was used in six patients, RTX in combination with intravenous immunoglobulin in 14 patients, and RTX with intravenous immunoglobulin and/or with other IMT agent in six patients. Seven eyes (11.5%) of six patients had favorable response to RTX and achieved response and partial remission, while inflammation remained active in the other seven eyes (11.5%) of four patients though there was no progressive scarring. At the last visit, three patients (9.4%) were on topical corticosteroid, three patients (9.4%) were treated with systemic corticosteroid treatments, and the other 26 patients (81.2%) achieved corticosteroid sparing therapy. Five eyes (8.2%) progressed one Foster stage. No other cicatrization progression or worsening of LogMAR visual acuity (p = 0.641) was observed during the follow-up period. Adverse events included leukopenia in three patients (9.4%), anemia in two patients (6.2%), liver enzyme elevation in three patients (9.4%) who were also on another concomitant IMT drug, and Epstein-Barr Virus infection and sinus infection in one patient each (3.1%). No other severe adverse events were noted during the follow-up period. CONCLUSIONS: These retrospective data suggest that RTX is efficacious and well tolerated when included for the treatment of OCP. Controlled studies are necessary to identify the role of this IMT agent in the therapeutic arsenal, especially its optimum dose and duration of administration.
AIMS: To evaluate the efficacy and safety of rituximab (RTX) induction and maintenance treatment for patients with scleritis and granulomatosis with polyangiitis (GPA), Wegener's. METHODS: Nine patients (12 eyes) with scleritis with GPA who did not respond to corticosteroids and more than one immunosuppressive agent who received ongoing maintenance RTX treatment were identified. Demographics and outcome measures were recorded. RESULTS: Median follow-up time of 30 months (range, 15 to 87 months). All 12 eyes achieved remission during the RTX maintenance period with a median time in remission of 14 months (range, 5-76 months), and median interval between RTX initiation and inactive disease of 5 months (range, 2-8 months). Two eyes in two patients relapsed. One received steroid eye drops, and the other received a short-term increased dose of intravenous corticosteroids. CONCLUSIONS: RTX was effective as an induction and maintenance treatment in our small cohort of patients with GPA-associated scleritis.
PURPOSE: Investigators have discovered that topical antiglaucoma drugs may induce meibomian gland dysfunction. This response may contribute to the dry eye disease commonly found in patients with glaucoma taking such medications. We hypothesize that drug action involves a direct effect on human meibomian gland epithelial cells (HMGECs). To test this hypothesis, we examined the influence of the antiglaucoma drugs, pilocarpine and timolol, on the morphology, survival, proliferation, and differentiation of HMGECs. METHODS: Immortalized (I) HMGECs (n = 2-3 wells/treatment/experiment) were cultured with multiple concentrations of pilocarpine or timolol for up to 7 days. Experiments included positive controls for proliferation (epidermal growth factor and bovine pituitary extract) and differentiation (azithromycin). Cells were enumerated using a hemocytometer and evaluated for morphology, neutral lipid staining, and lysosome accumulation. RESULTS: Our results demonstrate that pilocarpine and timolol cause a dose-dependent decrease in the survival of IHMGECs. The clinically used concentrations are toxic and lead to cell atrophy, poor adherence, or death. By contrast, drug levels that are known to accumulate within the conjunctiva, adjacent to the meibomian glands, do not influence IHMGEC survival. These latter concentrations also have no effect on IHMGEC proliferation or differentiation, and they do not interfere with the ability of azithromycin to stimulate cellular neutral lipid and lysosome accumulation. This dose of pilocarpine, though, did suppress the epidermal growth factor+bovine pituitary extract-induced proliferation of IHMGECs. CONCLUSIONS: Our results support our hypothesis and demonstrate that these antiglaucoma drugs, pilocarpine and timolol, have direct effects on HMGECs that may influence their morphology, survival, and proliferative capacity.
Purpose: To discover novel therapies that lower IOP by increasing aqueous humor outflow facility, ex vivo ocular perfusion systems provide a valuable tool. However, currently available designs are limited by their throughput. Here we report the development of a compact, scalable perfusion system with improved throughput and its validation using bovine and porcine eyes. Methods: At a fixed IOP of 6 mm Hg, flow rate was measured by flow sensors. We validated the system by measuring the outflow responses to Y-39983 (a Rho kinase inhibitor), endothelin-1 (ET-1), ambrisentan (an antagonist for endothelin receptor A [ETA]), sphigosine-1-phosphate (S1P), JTE-013 (antagonist for S1P receptor 2 [S1P2]), S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide [NO] donor), and 3-Morpholino-sydnonimine (SIN-1, another NO donor). Results: The instrument design enabled simultaneous measurements of 20 eyes with a footprint of 1 m2. Relative to vehicle control, Y-39983 increased outflow by up to 31% in calf eyes. On the contrary, ET-1 decreased outflow by up to 79%, a response that could be blocked by pretreatment with ambrisentan, indicating a role for ETA receptors. Interestingly, the effect of ET-1 was also inhibited by up to 70% to 80% by pretreatment with NO donors, SNAP and SIN-1. In addition to testing in calf eyes, similar effects of ET-1 and ambrisentan were observed in adult bovine and porcine eyes. Conclusions: The compact eye perfusion platform provides an opportunity to efficiently identify compounds that influence outflow facility and may lead to the discovery of new glaucoma therapies.