Periodontitis and Outer Retinal Thickness: a Cross-Sectional Analysis of the United Kingdom Biobank Cohort.

Purpose: Periodontitis, a ubiquitous severe gum disease affecting the teeth and surrounding alveolar bone, can heighten systemic inflammation. We investigated the association between very severe periodontitis and early biomarkers of age-related macular degeneration (AMD), in individuals with no eye disease. Design: Cross-sectional analysis of the prospective community-based cohort United Kingdom (UK) Biobank. Participants: Sixty-seven thousand three hundred eleven UK residents aged 40 to 70 years recruited between 2006 and 2010 underwent retinal imaging. Methods: Macular-centered OCT images acquired at the baseline visit were segmented for retinal sublayer thicknesses. Very severe periodontitis was ascertained through a touchscreen questionnaire. Linear mixed effects regression modeled the association between very severe periodontitis and retinal sublayer thicknesses, adjusting for age, sex, ethnicity, socioeconomic status, alcohol consumption, smoking status, diabetes mellitus, hypertension, refractive error, and previous cataract surgery. Main Outcome Measures: Photoreceptor layer (PRL) and retinal pigment epithelium-Bruch's membrane (RPE-BM) thicknesses. Results: Among 36 897 participants included in the analysis, 1571 (4.3%) reported very severe periodontitis. Affected individuals were older, lived in areas of greater socioeconomic deprivation, and were more likely to be hypertensive, diabetic, and current smokers (all P < 0.001). On average, those with very severe periodontitis were hyperopic (0.05 ± 2.27 diopters) while those unaffected were myopic (-0.29 ± 2.40 diopters, P < 0.001). Following adjusted analysis, very severe periodontitis was associated with thinner PRL (-0.55 µm, 95% confidence interval [CI], -0.97 to -0.12; P = 0.022) but there was no difference in RPE-BM thickness (0.00 µm, 95% CI, -0.12 to 0.13; P = 0.97). The association between PRL thickness and very severe periodontitis was modified by age (P < 0.001). Stratifying individuals by age, thinner PRL was seen among those aged 60 to 69 years with disease (-1.19 µm, 95% CI, -1.85 to -0.53; P < 0.001) but not among those aged < 60 years. Conclusions: Among those with no known eye disease, very severe periodontitis is statistically associated with a thinner PRL, consistent with incipient AMD. Optimizing oral hygiene may hold additional relevance for people at risk of degenerative retinal disease. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Adeno-associated virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome.

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03-mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9-mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches.

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.

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

Intraoperative Complications and Visual Outcomes of Cataract Surgery in Diabetes Mellitus: A Multicenter Database Study.

PURPOSE: To compare the visual outcome and the rate of intraoperative complications in eyes of diabetic and nondiabetic patients undergoing phacoemulsification over 15 years. DESIGN: Retrospective clinical cohort study. METHODS: Data of 179,159 eyes that underwent phacoemulsification at 8 centers were classified based on the presence or absence of diabetes mellitus. Visual acuity (VA) was defined as the best value of uncorrected or corrected distance measure available. For the VA analysis, eyes with co-pathologies or combined surgical procedures were further excluded, leaving a subset of 90,729 eyes. Main outcome measures were logarithm of the minimum angle of resolution (logMAR) VA at 4-12 weeks postoperatively, and rate of intraoperative complications. RESULTS: Cataract surgery in eyes of diabetic patients was associated with an improvement in mean VA of 0.48 logMAR (5 Snellen lines). Mean postoperative VA was slightly worse in diabetic compared to nondiabetic group (logMAR 0.23 vs 0.13; Snellen 20/30 vs 20/25; P < .0001) and the proportions of eyes achieving a visual gain of ≥3 Snellen lines (≥0.3 logMAR) was lower in the diabetic group (56.6% vs 63.5%; P < .0001). There was a linear relationship between diabetic retinopathy severity and worse postoperative visual acuity (ß coefficient 0.098 to 0.288; P < .0001). We observed higher rates of posterior capsule rupture (2.3% vs 1.6%; P < .001) and dropped nuclear fragments (0.3% vs 0.2%; P < .001) in the diabetic group. CONCLUSIONS: Postoperative VA negatively correlated with diabetes and diabetic retinopathy severity. Eyes of diabetic subjects had higher risks of posterior capsule rupture.

Intraocular Pressure Reduction After Real-world Cataract Surgery.

PRéCIS:: A large cohort undergoing cataract extraction was retrospectively analyzed to ascertain the degree of real-world intraocular pressure (IOP) reduction in normal eyes and those with glaucoma, and a predictive formula was developed. PURPOSE: The purpose of this study was to define the real-world degree of IOP reduction after cataract extraction to guide its role as an isolated intervention for glaucoma. MATERIALS AND METHODS: A retrospective analysis was carried out of clinical data collected in 8 clinical sites in the United Kingdom from an electronic medical record system between January 2006 and May 2015. A total of 20,508 eyes without known pathology and 2251 eyes from patients with glaucoma undergoing phacoemulsification and intraocular lens insertion were included. Eyes with intraoperative complications, undergoing additional procedures, axial lengths outside 22 to 26.5 mm, preoperative IOP under 6 mm Hg or over 30 mm Hg, and copathology, except for amblyopia or glaucoma, were excluded. The main outcome measure was the change in preoperative IOP compared with the next recorded visit for up to 12 weeks. RESULTS: In eyes without pathology, the mean reduction in IOP was 1.40 mm Hg (±3.74) compared with 1.03 (±5.02), P-value <0.001, in eyes with a diagnosis of glaucoma. A multiple linear regression model identified preoperative IOP, a glaucoma diagnosis, preoperative corrected visual acuity, age, and axial length as determinants of IOP reduction. The model was validated against an independent cohort. CONCLUSIONS: We quantify mean IOP reduction achieved in a real-world setting from cataract surgery alone. In glaucomatous eyes where angle closure is not differentiated, phacoemulsification alone yields only a modest reduction of IOP.

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.

A Database Study of Visual Outcomes and Intraoperative Complications of Postvitrectomy Cataract Surgery.

PURPOSE: To analyze the visual outcomes and rate of intraoperative complications of phacoemulsification surgery after prior pars plana vitrectomy (PPV). DESIGN: Retrospective, multicenter database study. PARTICIPANTS: Eyes that underwent phacoemulsification between June 2005 and March 2015 at 8 sites in the United Kingdom. METHODS: Study eyes were classified as vitrectomized (prior PPV group) or nonvitrectomized (reference group) depending on the vitreous state at the time of cataract surgery. Eyes with multiple intraocular surgeries or history of ocular diseases known to cause cataract progression or increased risk of intraoperative complications during phacoemulsification were excluded. MAIN OUTCOME MEASURES: Logarithm of the minimum angle of resolution (logMAR) visual acuity (VA), rate of intraoperative complications, and time interval to cataract surgery. RESULTS: Eyes in the prior PPV group (n = 2221) had worse preoperative logMAR VA (0.96±0.60 vs. 0.62±0.52, P < 0.0001), were from younger patients, and had longer axial lengths than the nonvitrectomized group (n = 136 533). At all postoperative time points measured up to 24 weeks, mean vision was poorer in the prior PPV group (0.41±0.47 vs. 0.17±0.29 at 4-12 weeks, P < 0.0001) and a smaller proportion of eyes achieved postoperative VA ≤0.30 logMAR (Snellen, ≥20/40) (60.8% vs. 86.5% at 4-12 weeks, P < 0.0001). The rate of posterior capsular rupture was not different between the prior PPV (1.5%) and the nonvitrectomized (1.7%) groups, but the incidences of zonular dialysis (1.3% vs. 0.6%) and dropped nuclear fragments (0.6% vs. 0.2%) were higher in the prior PPV group (P < 0.0001). The mean time interval between PPV and cataract surgery was 399 days. CONCLUSIONS: We found a significant improvement in VA with postvitrectomy cataract surgery. However, compared with eyes without prior PPV, there was a worse mean postoperative vision of 0.2 logMAR units, a higher rate of zonular dialysis and dropped nuclear fragments, and a similar rate of posterior capsule rupture.

Cataract Surgery Outcomes in Eyes With Primary Epiretinal Membrane.

Importance: Primary epiretinal membrane (ERM) is a common retinal disorder with a prevalence of 4% to 18.5%. Although ERM and cataracts commonly occur together, to our knowledge, no studies have investigated the outcome of cataract surgery alone in this setting. Objective: To analyze the visual outcome and cystoid macular edema risk with cataract surgery in eyes with primary ERM. Design, Setting, and Participants: In this retrospective clinical database study, data were collected from July 2003 to March 2015 from 8 locations in the United Kingdom. Cataract surgery data of 217 557 eyes were extracted from the electronic medical record of the UK National Health Service. After exclusion of 57 561 eyes with combined surgery, prior vitrectomy, copathology, and complications, 812 eyes with primary ERM and 159 184 reference eyes were analyzed. Main Outcomes and Measures: We report on visual acuity (VA), the incidence of cystoid macular edema, and the need for ERM surgery. Results: The mean (SD) age of patients in the ERM group was 73.7 (9.23) years, and 395 of 812 were men (46.8%). The mean (SD) age of patients in the reference group was 74.4 (12.19) years, and 65 265 of 159 184 were men (41%). Epiretinal membrane eyes assessed at 4 to 12 weeks postoperatively gained 0.27 (0.32) logMAR (approximately 3 Snellen lines), with 200 of 448 (44.6%) improving by 0.30 logMAR or more (≥3 Snellen lines) and 32 of 448 (7.1%) worsening by 0.30 logMAR or more. Reference eyes gained a mean (SD) of 0.44 (0.26) logMAR (approximately 4 Snellen lines), with 48 583 of 77 408 (62.8%) improving by 0.30 logMAR or more and 2125 of 77 408 (2.7%) worsening by 0.30 logMAR or more. Although all eyes with preoperative VA of 20/40 or less improved, only reference eyes with preoperative VA of more than 20/40 showed improvement. Cystoid macular edema developed in 57 of 663 ERM eyes (8.6%) (95% CI, 6.69-10.98) and 1731 of 125 435 reference eyes (1.38%) (95% CI, 1.32-1.45) (P < .001). Epiretinal membrane surgery was performed in 43 of 663 (6.5%) ERM eyes. Conclusions and Relevance: On average, VA improved 0.27 logMAR (approximately 3 Snellen lines) in eyes with ERM. Eyes with ERM and VA of 20/40 or less showed more benefit after cataract surgery than those with better preoperative vision. However, compared with eyes without ERM, higher rates of cystoid macular edema and a lower postoperative VA gain were noted.

Cataract surgery in uveitis: a multicentre database study.

BACKGROUND/AIMS: Cataract is an important source of visual loss in patients with uveitis. Whether or not outcomes of cataract surgery in eyes with uveitis are worse compared with non-uveitic eyes have to date been compromised by lack of reliable estimates of benefit and harm, which require data from large cohorts. METHODS: Electronic medical record data were extracted from eight independent UK clinical sites for eyes undergoing cataract extraction between January 2010 and December 2014. 1173 eyes with a recorded diagnosis of uveitis were compared with a reference group of 95 573 eyes from the same dataset. RESULTS: Uveitic eyes represented 1.2% of all eyes undergoing cataract surgery. Eyes in the uveitic group had worse preoperative visual acuity (0.87 vs 0.65 logarithm of the minimum angle of resolution (logMAR) units), were from younger patients and had shorter axial lengths and a higher incidence of ocular copathology including glaucoma. A greater number had documented small pupils, required additional surgical procedures, developed more intraoperative complications and had poorer postoperative visual acuity at all time points measured up to 6 months (0.41 vs 0.27 logMAR units at 12-24 weeks). CONCLUSIONS: This large study cohort of eyes with a diagnosis of uveitis undergoing cataract surgery highlights more precisely the complex surgical demands, copathology and worse visual outcomes in this group. These data will allow more accurate preoperative counselling and planning. Although improvement in visual acuity is achieved in most cases, prognosis should be guarded, so that patient expectations are met. Compared with the non-uveitic population, the mean postoperative visual acuity is between one and two lines worse at all time points.

Risk Factors and Incidence of Macular Edema after Cataract Surgery: A Database Study of 81984 Eyes.

PURPOSE: To define the incidence of pseudophakic macular edema (PME) after cataract surgery and to identify contributory risk factors. DESIGN: Retrospective database study of electronic medical records (EMRs). PARTICIPANTS: A total of 81984 eyes undergoing cataract surgery between December 2010 and December 2014 from 8 independent United Kingdom clinical sites. METHODS: Structured clinical data mandated by the EMR were anonymized and extracted for each eye undergoing cataract surgery including: perioperative visual acuity, copathologic features, simultaneous surgical procedures, and the presence or absence of a specified list of intraoperative complications. Diabetic status with matched Early Treatment Diabetic Retinopathy Study (ETDRS) grading also was mandated by the EMR. Eyes receiving prophylactic nonsteroidal anti-inflammatory drugs were excluded. MAIN OUTCOME MEASURE: Diagnosis of cystoid macular edema or new-onset macular edema in patients with diabetes, recorded by a healthcare professional within 90 days of surgery. RESULTS: Baseline incidence of PME in eyes without operative complications, diabetes, or risk factors was 1.17%. Eyes in which PME developed were more likely to be male, older, and to demonstrate risk factors. The relative risk (RR) was increased in eyes with capsule rupture with or without vitreous loss (RR, 2.61; 95% confidence interval [CI], 1.57-4.34), a previous diagnosis of epiretinal membrane (RR, 5.60; 95% CI, 3.45-9.07), uveitis (RR, 2.88; 95% CI, 1.50-5.51), retinal vein occlusion (RR, 4.47; 95% CI, 2.56-5.92), or retinal detachment repair (RR, 3.93; 95% CI, 2.60-5.92). High myopia, age-related macular degeneration, or prostaglandin analog use were not shown to increase risk. Eyes with PME on average had poorer postoperative visual acuity, which persisted to the latest time point assessed, up to 24 weeks. Eyes from patients with diabetes, even in the absence of retinopathy, had an increased RR (RR, 1.80; 95% CI, 1.36-2.36) of new macular edema after surgery. The risk was higher in the presence of any diabetic retinopathy (DR; RR, 6.23; 95% CI, 5.12-7.58) and rose proportionately with increasing severity of DR. CONCLUSIONS: Pseudophakic macular edema occurs commonly after phacoemulsification cataract surgery, even in the absence of complications and risk factors. This large retrospective study using structured EMR data quantified the RRs of PME and the risk with increasing ETDRS severity of DR. It highlights the need for prophylactic therapy, especially in those groups of eyes with the highest RRs.

TNFα Regulates SIRT1 Cleavage during Ocular Autoimmune Disease.

Elevated tumor necrosis factor (TNF) α levels are associated with chronic autoimmune diseases in which effects of TNFα on immune cells are multiple and complex. Analysis of uveitis in mice exhibiting severe autoimmune inflammation, resulting in a destructive subtotal loss of photoreceptors, revealed the presence of high plasma levels of TNFα and a significant population of CD4(+)TNFα(+) cells in the periphery and the eye at peak disease (TNFα(hi)). We have shown previously by pharmacological activation that the deacetylase Sirtuin 1 (SIRT1) has an anti-inflammatory role in a less severe, TNFα(lo) model of uveitis. We now show that SIRT1 activation fails to clinically suppress severe TNFα(hi) disease, whereas glucocorticoid treatment is successful. TNFα has been reported to mediate cleavage and inactivation of SIRT1 during inflammation, and at peak disease we observed both full-length and cleaved SIRT1 in draining lymph node cells. In vivo systemic TNFα blockade suppressed severe ocular disease and restricted SIRT1 cleavage in the periphery, maintaining full-length active SIRT1 protein. When combining a suboptimal TNFα blockade with SIRT1 activation, a synergistic suppression of severe disease compared with TNFα blockade alone occurred. Our data suggest a new role for TNFα in exacerbating the severity of autoimmune disease by regulating SIRT1 cleavage in draining lymph node effector cells. SIRT1 activation may be effective as an adjunctive treatment for inflammatory conditions not fully controlled by TNFα inhibitors.

Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.