Replenishing IRAK-M expression in retinal pigment epithelium attenuates outer retinal degeneration.

Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor-associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants of IRAK3, which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD. Irak3-knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)-expressing human IRAK3 rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration in Irak3-knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.

Long-Term Visual Outcome of Patients with Blau Syndrome.

PURPOSE: To document the long-term visual outcomes in patients with Blau syndrome. METHODS: A retrospective institutional cohort study was conducted, and 13 patients with genetically confirmed Blau syndrome were included. Demographic and clinical data were collected from standardised medical charts. Baseline was defined as the first detected uveitis and data were recorded onwards at intervals of 1, 3, 5, 10, 15 and 20 years. RESULTS: Anterior uveitis was the most common classification at baseline (57.1%). Among patients with documented uveitis lasting 10 years or more, all of them developed panuveitis. Median logMAR visual acuity at baseline was 0 (range -0.5; 0.7), 0.19 (range 0; 1.5) at year 5, and 0.7 (range 0.1 - no perception of light) at year 20, as recorded in 13, 16, and 10 eyes, respectively. All patients received treatment with topical and oral steroids, and multiple systemic immunosuppressants including biologics. Disease control, defined as having cells <1+ in both eyes and using topical steroid eye drops less than twice daily, was achieved in 14.3% to 37.5% of patients at the different time points. Cataract surgery was performed in 12 eyes of 8 patients, 3 eyes of 3 patients necessitated glaucoma surgery, and 4 eyes of 4 patients required surgery for retinal detachment. CONCLUSION: Uveitis associated with Blau syndrome commonly leads to severe, chronic panuveitis, requiring long-term systemic immunosuppression. Early diagnosis and timely initiation of biologics may prevent significant visual impairment.

Elastin Layer in Bruch's Membrane as a Target for Immunization or Tolerization to Modulate Pathology in the Mouse Model of Smoke-Induced Ocular Injury.

Age-related macular degeneration (AMD) is associated with an overactive complement system and an increase in circulating antibodies. Our search for potential neoantigens that can trigger complement activation in disease has led us to investigate elastin. A loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported in aging and AMD together with an increase of serum elastin-derived peptides and α-elastin antibodies. In the mouse model of cigarette smoke exposure (CSE), damage in BrM, loss of the EL, and vision loss are dependent on complement activation. We have examined the hypothesis that CSE generates immunogenic elastin neoepitopes that trigger an increase in α-elastin IgG and IgM antibodies, which can then bind to the neoepitopes in the target cells or membranes, triggering complement activation. Specifically, we showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin) exacerbated ocular pathology and vision loss in CSE mice. In contrast, mice receiving peptide immunotherapy (PIT) with ox-elastin did not lose vision over the smoking period and exhibited a more preserved BrM. Immunization and PIT correlated with humoral immunity and complement activation and IgG/IgM deposition in the RPE/BrM/choroid. Finally, PIT modulated immune markers IFNγ and IL-4. The data further support the hypothesis that complement activation, triggered by immune complex formation in target tissues, plays a role in ocular damage in the CSE model. As PIT with ox-elastin peptides reduces damage, we discuss the possibility that AMD progression might be preventable.

CAR-Treg cell therapies and their future potential in treating ocular autoimmune conditions.

Ophthalmic autoimmune and autoinflammatory conditions cause significant visual morbidity and require complex medical treatment complicated by significant side effects and lack of specificity. Regulatory T cells (Tregs) have key roles in immune homeostasis and in the resolution of immune responses. Polyclonal Treg therapy has shown efficacy in treating autoimmune disease. Genetic engineering approaches to produce antigen-specific Treg therapy has the potential for enhanced treatment responses and fewer systemic side effects. Cell therapy using chimeric antigen receptor modified T cell (CAR-T) therapy, has had significant success in treating haematological malignancies. By modifying Tregs specifically, a CAR-Treg approach has been efficacious in preclinical models of autoimmune conditions leading to current phase 1-2 clinical trials. This review summarises CAR structure and design, Treg cellular biology, developments in CAR-Treg therapies, and discusses future strategies to apply CAR-Treg therapy in the treatment of ophthalmic conditions.

Applications of three-dimensional printing in ophthalmology.

Three-dimensional (3D) printing is increasingly used to produce customized objects and is a promising alternative to traditional manufacturing methods in diverse fields, such as dentistry and orthopedics. Already in use in other medical specialties, adoption in ophthalmology has been limited to date. This review aims to provide an overview of 3D printing technology with respect to current and potential applications in ophthalmic practice. Medline, Embase, and Internet searches were performed with "3D printing," "ophthalmology," "dentistry," "orthopaedics" and their synonyms used as main search terms. In addition, search terms related to clinical applications such as "surgery" and "implant" were employed. 3D printing has multiple applications in ophthalmology, including in diagnosis, surgery, prosthetics, medications, and medical education. Within the past decade, researchers have produced 3D printed models of objects such as implants, prostheses, anatomical models and surgical simulators. Further development is necessary to generate optimal biomaterials for various applications, and the quality and long-term performance of 3D models needs to be validated.

Corneal Transplant Follow-up Study II: a randomised trial to determine whether HLA class II matching reduces the risk of allograft rejection in penetrating keratoplasty.

PURPOSE: A randomised trial to test the hypothesis that human leucocyte antigen (HLA) class II matching reduces the risk of allograft rejection in high-risk penetrating keratoplasty (PK). METHODS: All transplants were matched for HLA class I antigens (≤2 mismatches at the A and B loci) and corneas were allocated to patients by cohort minimisation to achieve 0, 1 or 2 HLA class II antigen mismatches. The corneal transplants (n=1133) were followed for 5 years. The primary outcome measure was time to first rejection episode. RESULTS: Cox regression analysis found no influence of HLA class II mismatching on risk of immunological rejection (HR 1.13; 95% CI 0.79 to 1.63; p=0.51). The risk of rejection in recipients older than 60 years was halved compared with recipients ≤40 years (HR 0.51; 95% CI 0.36 to 0.73; p=0.0003). Rejection was also more likely where cataract surgery had been performed after PK (HR 3.68; 95% CI 1.95 to 6.93; p<0.0001). In univariate analyses, preoperative factors including chronic glaucoma (p=0.02), vascularisation (p=0.01), inflammation (p=0.03), ocular surface disease (p=0.0007) and regrafts (p<0.001) all increased the risk of rejection. In the Cox model, however, none of these factors was individually significant but rejection was more likely where≥2 preoperative risk factors were present (HR 2.11; 95% CI 1.26 to 3.47; p<0.003). CONCLUSIONS: HLA class II matching, against a background of HLA class I matching, did not reduce the risk of allograft rejection. Younger recipient age, the presence of ≥2 preoperative risk factors and cataract surgery after PK all markedly increased the risk of allograft rejection. TRIAL REGISTRATION NUMBER: ISRCTN25094892.

Emerging therapies and their delivery for treating age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of blindness in the Western world and is characterised in its latter stages by retinal cell death and neovascularisation and earlier stages with the loss of parainflammatory homeostasis. Patients with neovascular AMD (nAMD) are treated with frequent intraocular injections of anti-vascular endothelial growth factor (VEGF) therapies, which are not only unpopular with patients but carry risks of sight-threatening complications. A minority of patients are unresponsive with no alternative treatment available, and some patients who respond initially eventually develop a tolerance to treatment. New therapeutics with improved delivery methods and sustainability of clinical effects are required, in particular for non-neovascular AMD (90% of cases and no current approved treatments). There are age-related and disease-related changes that occur which can affect ocular drug delivery. Here, we review the latest emerging therapies for AMD, their delivery routes and implications for translating to clinical practice. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Unravelling the therapeutic potential of IL-33 for atrophic AMD.

Age-related macular degeneration (AMD), a degenerative disease affecting the retinal pigment epithelium (RPE) and photoreceptors in the macula, is the leading cause of central blindness in the elderly. AMD progresses to advanced stages of the disease, atrophic AMD (aAMD), or in 15% of cases "wet" or neovascular AMD (nAMD), associated with substantial vision loss. Whilst there has been advancement in therapies treating nAMD, to date, there are no licenced effective treatments for the 85% affected by aAMD, with disease managed by changes to diet, vitamin supplements, and regular monitoring. AMD has a complex pathogenesis, involving highly integrated and common age-related disease pathways, including dysregulated complement/inflammation, impaired autophagy, and oxidative stress. The intricacy of AMD pathogenesis makes therapeutic development challenging and identifying a target that combats the converging disease pathways is essential to provide a globally effective treatment. Interleukin-33 is a cytokine, classically known for the proinflammatory role it plays in allergic disease. Recent evidence across degenerative and inflammatory disease conditions reveals a diverse immune-modulatory role for IL-33, with promising therapeutic potential. Here, we will review IL-33 function in disease and discuss the future potential for this homeostatic cytokine in treating AMD.

Peptide-based immunotherapy against oxidized elastin ameliorates pathology in mouse model of smoke-induced ocular injury.

PURPOSE: Age-related macular degeneration (AMD), the leading cause of blindness in western populations, is associated with an overactive complement system, and an increase in circulating antibodies against certain epitopes, including elastin. As loss of the elastin layer of Bruch's membrane (BrM) has been reported in aging and AMD, we previously showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin), exacerbated ocular pathology in the smoke-induced ocular pathology (SIOP) model. Here we asked whether ox-elastin peptide-based immunotherapy (PIT) ameliorates damage. METHODS: C57BL/6J mice were injected with ox-elastin peptide at two doses via weekly subcutaneous administration, while exposed to cigarette smoke for 6 months. FcγR-/- and uninjected C57BL/6J mice served as controls. Retinal morphology was assessed by electron microscopy, and complement activation, antibody deposition and mechanisms of immunological tolerance were assessed by Western blotting and ELISA. RESULTS: Elimination of Fcγ receptors, preventing antigen/antibody-dependent cytotoxicity, protected against SIOP. Mice receiving PIT with low dose ox-elastin (LD-PIT) exhibited reduced humoral immunity, reduced complement activation and IgG/IgM deposition in the RPE/choroid, and largely a preserved BrM. While there is no direct evidence of ox-elastin pathogenicity, LD-PIT reduced IFNγ and increased IL-4 within RPE/choroid. High dose PIT was not protective. CONCLUSIONS: These data further support ox-elastin role in ocular damage in part via elastin-specific antibodies, and support the corollary that PIT with ox-elastin attenuates ocular pathology. Overall, damage is associated with complement activation, antibody-dependent cell-mediated cytotoxicity, and altered cytokine signature.

Achieving Quiescence with Fluocinolone Implants.

Persistent anterior uveitis causing cystoid macular oedema may necessitate either intraocular or systemic immunosuppression. This case report highlights how a newly licenced treatment, fluocinolone acetonide (Iluvien®, Alimera Sciences Ltd., England, UK) achieves quiescence in refractory and steroid-dependent disease and in the presence of an acute relapse.

Epidemiology of Scleritis in the United Kingdom From 1997 to 2018: Population-Based Analysis of 11 Million Patients and Association Between Scleritis and Infectious and Immune-Mediated Inflammatory Disease.

OBJECTIVE: To estimate 22-year trends in the prevalence and incidence of scleritis, and the associations of scleritis with infectious and immune-mediated inflammatory diseases (I-IMIDs) in the UK. METHODS: The retrospective cross-sectional and population cohort study (1997-2018) included 10,939,823 patients (2,946 incident scleritis cases) in The Health Improvement Network, a nationally representative primary care records database. The case-control and matched cohort study (1995-2019) included 3,005 incident scleritis cases and 12,020 control patients matched by age, sex, region, and Townsend deprivation index. Data were analyzed using multivariable Poisson regression, multivariable logistic regression, and Cox proportional hazards multivariable models adjusted for age, sex, Townsend deprivation index, race/ethnicity, smoking status, nation within the UK, and body mass index. Incidence rate ratios (IRRs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: Scleritis incidence rates per 100,000 person-years declined from 4.23 (95% CI 2.16-6.31) to 2.79 (95% CI 2.19-3.39) between 1997 and 2018. The prevalence of scleritis per 100,000 person-years was 93.62 (95% CI 90.17-97.07) in 2018 (61,650 UK patients). Among 2,946 patients with incident scleritis, 1,831 (62.2%) were female, the mean ± SD age was 44.9 ± 17.6 years (range 1-93), and 1,257 (88.8%) were White. Higher risk of incident scleritis was associated with female sex (adjusted IRR 1.53 [95% CI 1.43-1.66], P < 0.001), Black race/ethnicity (adjusted IRR 1.52 [95% CI 1.14-2.01], P = 0.004 compared to White race/ethnicity), or South Asian race/ethnicity (adjusted IRR 1.50 [95% CI 1.19-1.90], P < 0.001 compared to White race/ethnicity), and older age (peak adjusted IRR 4.95 [95% CI 3.99-6.14], P < 0.001 for patients ages 51-60 years versus those ages ≤10 years). Compared to controls, scleritis patients had a 2-fold increased risk of a prior I-IMID diagnosis (17 I-IMIDs, P < 0.001) and significantly increased risk of subsequent diagnosis (13 I-IMIDs). The I-IMIDs most strongly associated with scleritis included granulomatosis with polyangiitis, Behçet's disease, and Sjögren's syndrome. CONCLUSION: From 1997 through 2018, the UK incidence of scleritis declined from 4.23 to 2.79/100,000 person-years. Incident scleritis was associated with 19 I-IMIDs, providing data for rational investigation and cross-specialty engagement.

Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.

Cellular senescence in the aging retina and developments of senotherapies for age-related macular degeneration.

Age-related macular degeneration (AMD), a degenerative disease in the central macula area of the neuroretina and the supporting retinal pigment epithelium, is the most common cause of vision loss in the elderly. Although advances have been made, treatment to prevent the progressive degeneration is lacking. Besides the association of innate immune pathway genes with AMD susceptibility, environmental stress- and cellular senescence-induced alterations in pathways such as metabolic functions and inflammatory responses are also implicated in the pathophysiology of AMD. Cellular senescence is an adaptive cell process in response to noxious stimuli in both mitotic and postmitotic cells, activated by tumor suppressor proteins and prosecuted via an inflammatory secretome. In addition to physiological roles in embryogenesis and tissue regeneration, cellular senescence is augmented with age and contributes to a variety of age-related chronic conditions. Accumulation of senescent cells accompanied by an impairment in the immune-mediated elimination mechanisms results in increased frequency of senescent cells, termed "chronic" senescence. Age-associated senescent cells exhibit abnormal metabolism, increased generation of reactive oxygen species, and a heightened senescence-associated secretory phenotype that nurture a proinflammatory milieu detrimental to neighboring cells. Senescent changes in various retinal and choroidal tissue cells including the retinal pigment epithelium, microglia, neurons, and endothelial cells, contemporaneous with systemic immune aging in both innate and adaptive cells, have emerged as important contributors to the onset and development of AMD. The repertoire of senotherapeutic strategies such as senolytics, senomorphics, cell cycle regulation, and restoring cell homeostasis targeted both at tissue and systemic levels is expanding with the potential to treat a spectrum of age-related diseases, including AMD.

Long-term efficacy and tolerability of TNFα inhibitors in the treatment of non-infectious ocular inflammation: an 8-year prospective surveillance study.

BACKGROUND/AIM: To report the efficacy and tolerability of antitumour necrosis factor-alpha therapy (TNF inhibitors [TNFi]) in the management of non-infectious ocular inflammation, including uveitis and scleritis, in adult patients over an 8-year period. MATERIALS AND METHODS: This is a prospective cohort study of infliximab and adalimumab in the treatment of non-infectious ocular inflammatory disease. 43 of 85 adult patients on TNFi (34 infliximab, 9 adalimumab) for ≥1 year with non-infectious uveitis or scleritis were followed from 2006 to 2014. Clinical assessments, medication, adverse events and history of steroid rescues were collected at 6 monthly intervals. General quality of life (Short Form Health Survey (SF-36)) and visual quality of life (Vision-related quality of life Core Measure (VCM1)) were assessed annually. Outcome measures included rate of sustained remission, rate of relapse, systemic corticosteroid reduction, adverse events, and VCM1 and SF-36 scores. RESULTS: The median time on infliximab was 3.2 years (IQR 4.3) and on adalimumab was 2.4 years (IQR 1.8). Sustained remission was induced in 39 patients (91%) (0.5 per patient year) after a median of 1.2 years on a TNFi. 22 (51%) experienced one relapse, and 5 (12%) had two relapses. 23 (54%) had at least one adverse event; serious adverse events necessitating hospitalisation or cessation of medication occurred in four (9%) patients. 10 patients (23%) switched from the initiation of TNFi, at 1.7 years after starting, to another TNFi or another class of biologic therapy. CONCLUSION: TNFi treatment is associated with long-term drug-induced remission of ocular inflammation, visual stability and corticosteroid reduction. Adverse events were common and no new safety signals occurred. Relapse of inflammation occurs in half of the treated population.

Imaging-Based Uveitis Surveillance in Juvenile Idiopathic Arthritis: Feasibility, Acceptability, and Diagnostic Performance.

OBJECTIVE: Children with juvenile idiopathic arthritis (JIA) need regular examinations for uveitis to avoid visual morbidity from the most common extraarticular manifestation of disease. This study was undertaken to investigate the feasibility, acceptability, and performance of optical coherence tomography (OCT) imaging-based diagnosis of uveitis. METHODS: This observational cross-sectional study included children with and those without uveitis. The children underwent routine clinical examinations and anterior segment OCT scanning of intraocular inflammatory cells. Acceptability of image acquisition was assessed using a visual analog scale and length of time needed to acquire images. Interobserver and intraobserver variability of manual counting of acquired images (Bland-Altman limits of agreement), correlation between imaging and routine assessment, and sensitivity and specificity of anterior segment OCT detection of active inflammation were assessed. RESULTS: Of the 26 children ages 3-15 years (median age 8 years) who underwent imaging, 12 had active inflammation. All patients rated the acceptability of image acquisition as at least 8.5 on a scale of 0-10. Time taken to acquire images ranged from 1.5 minutes to 22 minutes (median time 8 minutes). There was good positive correlation between clinical assessment and image-based cell quantification (R2 = 0.63, P = 0.002). Sensitivity of anterior segment OCT manual image cell count for diagnosis of active inflammation was 92% (95% confidence interval [95% CI] 62-99%), specificity was 86% (95% CI 58-98%), and negative predictive value (ruling out uveitis) was 92% (95% CI 65-99%). CONCLUSION: Non-contact, high-resolution imaging for JIA uveitis surveillance is feasible, acceptable to patients, and holds the promise of transforming pediatric practice. Further work is needed to determine the analytic and clinical validity of anterior segment OCT quantification of active inflammation, and the clinical utility and cost-effectiveness of imaging-based disease monitoring.

Ambient Air Pollution Associations with Retinal Morphology in the UK Biobank.

Purpose: Because air pollution has been linked to glaucoma and AMD, we characterized the relationship between pollution and retinal structure. Methods: We examined data from 51,710 UK Biobank participants aged 40 to 69 years old. Ambient air pollution measures included particulates and nitrogen oxides. SD-OCT imaging measured seven retinal layers: retinal nerve fiber layer, ganglion cell-inner plexiform layer, inner nuclear layer, outer plexiform layer + outer nuclear layer, photoreceptor inner segments, photoreceptor outer segments, and RPE. Multivariable regression was used to evaluate associations between pollutants (per interquartile range increase) and retinal thickness, adjusting for age, sex, race, Townsend deprivation index, body mass index, smoking status, and refractive error. Results: Participants exposed to greater particulate matter with an aerodynamic diameter of <2.5 µm (PM2.5) and higher nitrogen oxides were more likely to have thicker retinal nerve fiber layer (ß = 0.28 µm; 95% CI, 0.22-0.34; P = 3.3 × 10-20 and ß = 0.09 µm; 95% CI, 0.04-0.14; P = 2.4 × 10-4, respectively), and thinner ganglion cell-inner plexiform layer, inner nuclear layer, and outer plexiform layer + outer nuclear layer thicknesses (P < 0.001). Participants resident in areas of higher levels of PM2.5 absorbance were more likely to have thinner retinal nerve fiber layer, inner nuclear layer, and outer plexiform layer + outer nuclear layers (ß = -0.16 [95% CI, -0.22 to -0.10; P = 5.7 × 10-8]; ß = -0.09 [95% CI, -0.12 to -0.06; P = 2.2 × 10-12]; and ß = -0.12 [95% CI, -0.19 to -0.05; P = 8.3 × 10-4], respectively). Conclusions: Greater exposure to PM2.5, PM2.5 absorbance, and nitrogen oxides were all associated with apparently adverse retinal structural features.

Treatment of diabetic retinopathy through neuropeptide Y-mediated enhancement of neurovascular microenvironment.

Diabetic retinopathy (DR) is one of the most severe clinical manifestations of diabetes mellitus and a major cause of blindness. DR is principally a microvascular disease, although the pathogenesis also involves metabolic reactive intermediates which induce neuronal and glial activation resulting in disruption of the neurovascular unit and regulation of the microvasculature. However, the impact of neural/glial activation in DR remains controversial, notwithstanding our understanding as to when neural/glial activation occurs in the course of disease. The objective of this study was to determine a potential protective role of neuropeptide Y (NPY) using an established model of DR permissive to N-methyl-D-aspartate (NMDA)-induced excitotoxic apoptosis of retinal ganglion cells (RGC) and vascular endothelial growth factor (VEGF)-induced vascular leakage. In vitro evaluation using primary retinal endothelial cells demonstrates that NPY promotes vascular integrity, demonstrated by maintained tight junction protein expression and reduced permeability in response to VEGF treatment. Furthermore, ex vivo assessment of retinal tissue explants shows that NPY can protect RGC from excitotoxic-induced apoptosis. In vivo clinical imaging and ex vivo tissue analysis in the diabetic model permitted assessment of NPY treatment in relation to neural and endothelial changes. The neuroprotective effects of NPY were confirmed by attenuating NMDA-induced retinal neural apoptosis and able to maintain inner retinal vascular integrity. These findings could have important clinical implications and offer novel therapeutic approaches for the treatment in the early stages of DR.

Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina.

It is not currently possible to reliably visualise and track immune cells in the human central nervous system or eye. Previous work demonstrated that indocyanine green (ICG) dye could label immune cells and be imaged after a delay during disease in the mouse retina. We report a pilot study investigating if ICG can similarly label immune cells within the human retina. Twelve adult participants receiving ICG angiography as part of routine standard of care were recruited. Baseline retinal images were obtained prior to ICG administration then repeated over a period ranging from 2 hours to 9 days. Matched peripheral blood samples were obtained to examine systemic immune cell labelling and activation from ICG by flow cytometry with human macrophage cultures as positive controls. Differences between the delayed near infrared ICG imaging and 488 nm autofluorescence was observed across pathologies, likely arising from the retinal pigment epithelium (RPE). Only one subject demonstrated ICG signal on peripheral blood myeloid cells and only three distinct cell-sized signals appeared over time within the retina of three participants. No significant increase in immune cell activation markers were detected after ICG administration. ICG accumulated in the endosomes of macrophage cultures and was detectable above a minimum concentration, suggesting cell labelling is possible. ICG can label RPE and may be used as an additional biomarker for RPE health across a range of retinal disorders. Standard clinical doses of intravenous ICG do not lead to robust immune cell labelling in human blood or retina and further optimisation in dose and route are required.

Gene Therapy for Glaucoma by Ciliary Body Aquaporin 1 Disruption Using CRISPR-Cas9.

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared with untreated eyes (25 ± 5/mm2; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.

Müller Cells Stabilize Microvasculature through Hypoxic Preconditioning.

BACKGROUND/AIMS: Hypoxia of the retina is a common pathogenic drive leading to vision loss as a result of tissue ischemia, increased vascular permeability and ultimately retinal neovascularisation. Here we tested the hypothesis that Müller cells stabilize the neurovascular unit, microvasculature by suppression of HIF-1α activation as a result of hypoxic preconditioning. METHODS: Tube Formation Assay and In vitro Vascular Permeability Image Assay were used to analyze angiogenesis and vascular integrity. Seahorse XF Cell Mito Stress Test was used to measure mitochondrial respiration. Gene and protein expression were examined by qRTPCR, ELISA and western blot. RESULTS: Hypoxic insult induces a significant induction of proangiogenic factors including vascular endothelial growth factor (VEGF) and angiopoietinlike 4 (ANGPTL-4) resulting in angiogenesis and increased vascular permeability of vascular endothelial cells. Hypoxic preconditioning of a human retinal Müller glia cell line significantly attenuates HIF-1α activation through the inhibition of mTOR and concomitant induction of aerobic glycolysis, stabilizing endothelial cells. CONCLUSION: Hypoxic preconditioning of Müller cells confers a robust protection to endothelial cells, through the suppression of HIF1α activation and its downstream regulation of VEGF and ANGPTL-4.