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Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell–specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate inMG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a “citrullination bunker” that initiates and sustains citrullination in retinal degeneration.

Brolucizumab (Beovu®) is an anti-vascular endothelial growth factor (anti-VEGF) agent approved for the treatment of neovascular age-related macular degeneration (nvAMD). Brolucizumab was marketed for its noninferiority to aflibercept and its potential for greater durability. However, post-marketing utilization has been tempered by safety concerns.ImportanceBrolucizumab (Beovu®) is an anti-vascular endothelial growth factor (anti-VEGF) agent approved for the treatment of neovascular age-related macular degeneration (nvAMD). Brolucizumab was marketed for its noninferiority to aflibercept and its potential for greater durability. However, post-marketing utilization has been tempered by safety concerns.We evaluate the visual and anatomic efficacy of brolucizumab, examine changes in treatment intervals after switching to brolucizumab, and estimate the incidence of drug-related adverse events in the real world.ObjectiveWe evaluate the visual and anatomic efficacy of brolucizumab, examine changes in treatment intervals after switching to brolucizumab, and estimate the incidence of drug-related adverse events in the real world.This was a retrospective consecutive case series of 626 eyes (543 patients) with nvAMD treated with 1438 brolucizumab injections at a single retina practice between 10/1/2019 and 5/15/2020.Design Setting and ParticipantsThis was a retrospective consecutive case series of 626 eyes (543 patients) with nvAMD treated with 1438 brolucizumab injections at a single retina practice between 10/1/2019 and 5/15/2020.Changes in visual acuity (VA); anatomic outcomes assessed by optical coherence tomography (OCT) including central subfield thickness (CST), macular volume (MV), presence of intraretinal fluid (IRF), subretinal fluid (SRF), and serous pigment epithelial detachment (sPED) on foveal line scans; treatment intervals before and after receiving brolucizumab; and the incidence of brolucizumab-related adverse events.Main Outcomes and MeasuresChanges in visual acuity (VA); anatomic outcomes assessed by optical coherence tomography (OCT) including central subfield thickness (CST), macular volume (MV), presence of intraretinal fluid (IRF), subretinal fluid (SRF), and serous pigment epithelial detachment (sPED) on foveal line scans; treatment intervals before and after receiving brolucizumab; and the incidence of brolucizumab-related adverse events.The majority of eyes (N = 531, 89.7%) had received prior anti-VEGF therapy with aflibercept, ranibizumab, and/or bevacizumab. VA improved in treatment-naïve eyes (+3.7 letters, p = 0.04), and was maintained in previously treated eyes. There were significant improvements in all anatomic outcomes in both groups (p < 0.001). We observed a 4.8% incidence of intraocular inflammation (IOI) and a 0.6% incidence of retinal vasculitis. The average treatment interval increased from 6.3 to 6.8 weeks (p = 0.001).ResultsThe majority of eyes (N = 531, 89.7%) had received prior anti-VEGF therapy with aflibercept, ranibizumab, and/or bevacizumab. VA improved in treatment-naïve eyes (+3.7 letters, p = 0.04), and was maintained in previously treated eyes. There were significant improvements in all anatomic outcomes in both groups (p < 0.001). We observed a 4.8% incidence of intraocular inflammation (IOI) and a 0.6% incidence of retinal vasculitis. The average treatment interval increased from 6.3 to 6.8 weeks (p = 0.001).Brolucizumab treatment was associated with VA improvement in naïve eyes and maintenance of VA in previously treated eyes. Switching to brolucizumab was associated with improved anatomic outcomes and extended treatment intervals in most eyes. We observed a similar incidence of IOI and a lower incidence of retinal vasculitis compared to the Safety Review Committee's analysis of HAWK and HARRIER.Conclusions and RelevanceBrolucizumab treatment was associated with VA improvement in naïve eyes and maintenance of VA in previously treated eyes. Switching to brolucizumab was associated with improved anatomic outcomes and extended treatment intervals in most eyes. We observed a similar incidence of IOI and a lower incidence of retinal vasculitis compared to the Safety Review Committee's analysis of HAWK and HARRIER.

Pathologic vascular growth causes vision impairment in several vascular eye diseases. Specifically targeting molecular signatures distinguishing pathologic neovascularization from normal vessels will allow targeted treatment options. This study demonstrates that miR-150 was specifically enriched in normal retinal vessels and down-regulated in pathologic neovessels in a mouse model of proliferative retinopathy. MiR-150 suppressed pathologic ocular neovascularization in mice with decreased expression of angiogenic target genes and inhibited endothelial cell function in vitro. Loss of miR-150 also promoted vascular sprouting in ex vivo aortic and choroidal assays and laser-induced choroidal neovascularization in mice. These data suggest that endothelial miR-150 is an endogenous suppressor of ocular neovascularization and a drug target for vascular eye diseases. Pathologic ocular neovascularization commonly causes blindness. It is critical to identify the factors altered in pathologically proliferating versus normally quiescent vessels to develop effective targeted therapeutics. MicroRNAs regulate both physiological and pathological angiogenesis through modulating expression of gene targets at the posttranscriptional level. However, it is not completely understood if specific microRNAs are altered in pathologic ocular blood vessels, influencing vascular eye diseases. Here we investigated the potential role of a specific microRNA, miR-150 , in regulating ocular neovascularization. We found that miR-150 was highly expressed in normal quiescent retinal blood vessels and significantly suppressed in pathologic neovessels in a mouse model of oxygen-induced proliferative retinopathy. MiR-150 substantially decreased endothelial cell function including cell proliferation, migration, and tubular formation and specifically suppressed the expression of multiple angiogenic regulators, CXCR4, DLL4 , and FZD4 , in endothelial cells. Intravitreal injection of miR-150 mimic significantly decreased pathologic retinal neovascularization in vivo in both wild-type and miR-150 knockout mice. Loss of miR-150 significantly promoted angiogenesis in aortic rings and choroidal explants ex vivo and laser-induced choroidal neovascularization in vivo. In conclusion, miR-150 is specifically enriched in quiescent normal vessels and functions as an endothelium-specific endogenous inhibitor of pathologic ocular neovascularization.

To study the effects of intravitreal injection (IVI) of anti-VEGF (vascular endothelial growth factor) agents on intraocular pressure (IOP) and find associations with acute pressure spikes. This was a three-month, prospective study of patients receiving outpatient IVI of anti-VEGF agents for diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinal vein occlusion (RVO) at the Acuity Eye Group Medical Centers. IOP was measured pre- and post-injection at 10-minute intervals up to 50 minutes after injection with a handheld tonometer. Patients with an IOP greater than 35 mmHg at 30 minutes received an anterior chamber paracentesis (ACP), while patients below 35 mmHg were monitored without intervention. A total of 617 patients (51% female, 49% male) received IVI for DR (n = 199), AMD (n = 355), and RVO (n = 63). ACP was performed in 17 patients. Average pre-injection IOP was 16 ± 4 compared to 24 ± 7 mmHg for the non-ACP vs ACP group, respectively (mean ± standard deviation), p < 0.0001. IOP returned to baseline in 98% of patients at 50 minutes. A diagnosis of glaucoma and glaucoma suspect was more prevalent in the ACP group compared to the non-ACP group, 82.3% vs 14.2% and 17.6% vs 9.0%, respectively, p < 0.0001 and p > 0.05. Patients with a pre-injection IOP >25 mmHg and a history of glaucoma had a 58.3% rate of ACP. A 31-gauge needle had a higher mean increase in IOP from baseline compared to 30-gauge needle, p < 0.0001. IOP spikes are most significant in the first 10 minutes after IVI but typically resolve within the first hour. However, utilizing a smaller 31-gauge IVI in patients with a glaucoma history and pre-injection IOP >25 mmHg may be associated with significant IOP spikes lasting longer than 30 minutes.

The mouse model of laser-induced choroidal neovascularization (CNV) has been used in studies of the exudative form of age-related macular degeneration using both the conventional slit lamp and a new image-guided laser system. A standardized protocol is needed for consistent results using this model, which has been lacking. We optimized details of laser-induced CNV using the image-guided laser photocoagulation system. Four lesions with similar size were consistently applied per eye at approximately double the disc diameter away from the optic nerve, using different laser power levels, and mice of various ages and genders. After 7 days, the mice were sacrificed and retinal pigment epithelium/choroid/sclera was flat-mounted, stained with Isolectin B4, and imaged. Quantification of the area of the laser-induced lesions was performed using an established and constant threshold. Exclusion criteria are described that were necessary for reliable data analysis of the laser-induced CNV lesions. The CNV lesion area was proportional to the laser power levels. Mice at 12-16 weeks of age developed more severe CNV than those at 6-8 weeks of age, and the gender difference was only significant in mice at 12-16 weeks of age, but not in those at 6-8 weeks of age. Dietary intake of omega-3 long-chain polyunsaturated fatty acid reduced laser-induced CNV in mice. Taken together, laser-induced CNV lesions can be easily and consistently applied using the image-guided laser platform. Mice at 6-8 weeks of age are ideal for the laser-induced CNV model.

Maternal decidual CD8 + T cells must integrate the antithetical demands of providing immunity to infection while maintaining immune tolerance for fetal and placental antigens. Human decidual CD8 + T cells were shown to be highly differentiated memory T cells with mixed signatures of dysfunction, activation, and effector function. However, no information is present on how specificity for microbial or fetal antigens relates to their function or dysfunction. In addition, a key question, whether decidual CD8 + T cells include unique tissue-resident memory T cells (Trm) or also effector memory T cell (Tem) types shared with peripheral blood populations, is unknown. Here, high-dimensional flow cytometry of decidual and blood CD8 + T cells identified 2 Tem populations shared in blood and decidua and 9 functionally distinct Trm clusters uniquely found in decidua. Interestingly, fetus- and virus-specific decidual CD8 + Trm cells had similar features of inhibition and cytotoxicity, with no significant differences in their expression of activation, inhibitory, and cytotoxic molecules, suggesting that not all fetus-specific CD8 + T cell responses are suppressed at the maternal-fetal interface. Understanding how decidual CD8 + T cell specificity relates to their function and tissue residency is crucial in advancing understanding of their contribution to placental inflammation and control of congenital infections.

IgE-mediated food allergies in infants are a significant health concern, with peanut allergy being of particular interest due to its prevalence and severity. Among individuals who produce peanut-specific IgE some experience no adverse reaction on peanut consumption. This asymptomatic phenotype is known as sensitized tolerance. To elucidate the immune environment of peanut sensitized tolerant and clinically allergic one-year-olds, high-dimensional mass cytometry was conducted as part of the HealthNuts study. The resulting data includes peripheral blood mononuclear cells from 36 participants encompassing non-allergic, peanut sensitized with tolerance, and clinically peanut allergic infants. The raw mass cytometry data is described here and freely available for reuse through the Immunology Database and Analysis Portal (ImmPort). Additional allergy information and serum vitamin D levels of the participants were measured and are also included in the data upload. These high-dimensional mass cytometry data, when combined with clinical information, offer a broad immune profile of peanut allergic and sensitized tolerant infants. Measurement(s) expression profiling Technology Type(s) cytometry time of flight assay Sample Characteristic - Organism Homo sapiens Sample Characteristic - Location Australia

Retinal neovascularization, as occurs in age-related macular degeneration, may result from an increase in VEGFA levels due to dysregulated lipid and glucose metabolism within photoreceptors. Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine 1 . Current dogma suggests that high-energy–consuming photoreceptors depend on glucose 2 , 3 . Here we show that the retina also uses fatty acid β-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors 4 and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid 5 , 6 . In the retinas of Vldlr −/− mice with low fatty acid uptake 6 but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr −/− photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr −/− retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD) 7 , which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases.

Emerging viruses pose an ongoing threat to human health. While certain viral families are common sources of outbreaks, predicting the specific virus within a family that will cause the next outbreak or pandemic is not possible, creating an urgent need for broad spectrum antiviral drugs that are effective against an array of related viral pathogens. However, broad spectrum drug development is hindered by the lack of detailed knowledge of compound binding sites that are structurally and functionally conserved between viral family members and are essential for virus replication. To overcome this limitation, we developed an in silico approach that combines AI-driven protein structure prediction, computational fragment soaking, multiple sequence alignment, and protein stability calculations to identify highly conserved target sites that are both solvent-accessible and conserved. We applied this approach to the Togaviridae family, which includes emerging pandemic disease threats such as chikungunya and Venezuelan equine encephalitis virus for which there are currently no approved antiviral therapies. Our analysis identified multiple solvent accessible and structurally conserved pockets in the alphavirus non-structural protein 2 (nsP2) protease domain, which is essential for processing of the viral replicase proteins. Mutagenesis of key solvent accessible and conserved residues identified novel pockets that are essential for protease activity and the replication of multiple alphaviruses, validating these pockets as potential antiviral target sites for nsP2 inhibitors. These findings highlight the potential of artificial intelligence-informed modeling for revealing functionally conserved, accessible pockets as a means of identifying potential target binding sites for broadly active direct acting antivirals. Here we present a novel integrative computational approach to identify novel target sites for broadly acting antiviral drugs. We used this technique to identify multiple functionally and structurally conserved protein surface pockets within the alphavirus nsP2 protease and methyl-transferase-like domain. Mutagenesis of these pockets identified that they are essential for protease activity and replication of a genetically diverse group of alphaviruses, validating these sites as potential targets for broadly active small molecule alphavirus inhibitors. This integrative AI-driven approach thus provides an important tool in developing antivirals essential for pandemic preparedness.

Background Patients co-infected with HIV and HCV represent a unique subpopulation with specific high-risk characteristics including increased transmission efficiency of HCV, higher HCV viral load and more rapid progression of liver disease when compared with mono-infected patients. Although virologic failure is rare in the direct acting antiviral (DAA) era, we have anecdotally observed a high rate of failure in our patients who are co-infected and have cirrhosis. Our objective was to evaluate the impact of cirrhosis on co-infected patients compared with co-infection without cirrhosis and mono-infected patients with cirrhosis as it relates to cure of HCV treated with DAAs. Methods A retrospective chart review was performed. Patients from UConn Health Infectious Diseases and Gastroenterology clinics and Hartford Hospital Comprehensive Liver Center treated January 1, 2014 through December 31, 2017 were included. Patients were grouped as follows: (1) HCV/HIV coinfected without cirrhosis, (2) HCV/HIV coinfected with cirrhosis, (3) HCV infected with cirrhosis. Data were analyzed in SAS, variables were compared by chi square analysis and Fishers Exact test to determine statistical significance. Results No differences in baseline characteristics were noted (Table 1). Cirrhotic patients were 63% of the total cohort. There was no statistical difference in the rates of sustained virologic response (SVR) among the 3 groups. The overall rate of SVR was 95%. SVR for patients with cirrhosis (co- and mono-infected) was 92%. All treatment failures (n = 3) in this cohort had cirrhosis. Among the 38 cirrhotic patients, 3 (8%) had treatment experience with DAAs. In contrast, none of the non-cirrhotic patients had prior DAAs. The use of protease inhibitors or ribavirin had no impact on cure; ribavirin was evenly distributed between the two groups with cirrhosis. SVR rates were lower with genotypes 2–4 as compared with genotype 1. No immunologic or virologic factors were correlated with SVR. Conclusion We found no differences in rates of SVR in coinfected patients with or without cirrhosis. However, all treatment failures were noted in patients with cirrhosis, and cirrhotic patients tended to have treatment experience with DAAs. Whether coinfected patients with cirrhosis should be managed differently will require additional study. Disclosures All authors: No reported disclosures.