Whole exome sequencing of 491 individuals with inherited retinal diseases reveals a large spectrum of variants and identification of novel candidate genes.

BACKGROUND: Inherited retinal diseases (IRDs) include a range of vision loss conditions caused by variants in different genes. The clinical and genetic heterogeneity make identification of the genetic cause challenging. Here, a cohort of 491 unsolved cases from our cohort of Israeli and Palestinian families with IRDs underwent whole exome sequencing (WES), including detection of CNVs as well as single nucleotide variants (SNVs). METHODS: All participants underwent clinical examinations. Following WES on DNA samples by 3 billion, initial SNV analysis was performed by 3 billion and SNV and CNV analysis by Franklin Genoox. The CNVs indicated by the programme were confirmed by PCR followed by gel electrophoresis. RESULTS: WES of 491 IRD cases revealed the genetic cause of disease in 51% of cases, of which 11% were due wholly or in part to CNVs. In two cases, we clarified previously incorrect or unclear clinical diagnoses. This analysis also identified ESRRB and DNM1 as potential novel genes. CONCLUSION: This analysis is the most extensive one to include CNVs to examine IRD causing genes in the Israeli and Palestinian populations. It has allowed us to identify the causative variant of many patients with IRDs including ones with unclear diagnoses and potential novel genes.

An automated process for bulk downloading optical coherence tomography scans.

OBJECTIVE: To develop an automated method for efficiently downloading a large number of optical coherence tomography (OCT) scans obtained using the Heidelberg Spectralis (Heidelberg Engineering, Heidelberg, Germany) platform. METHODS: The electronic medical records and OCT scans were extracted for all patients with age-related macular degeneration treated at the Hadassah University Hospital Retina Clinic between 2010 and 2021. A macro was created using Visual Basic for Applications (VBA) and Microsoft Excel to automate the export process and anonymize the OCT scans in accordance with hospital policy. OCT scans were extracted as proprietary Heidelberg E2E files. RESULTS: The VBA macro was used to export a total of 94,789 E2E files from 2807 patient records, with an average processing time of 4.32 min per volume scan (SD: 3.57 min). The entire export process took a total of approximately 202 h to complete over a period of 24 days. In a smaller sample, using the macro to download the scans was significantly faster than manually downloading the scans, averaging 3.88 vs. 11.08 min/file, respectively (t = 8.59, p < 0.001). Finally, we found that exporting the files during both off-clinic and working hours resulted in significantly faster processing times compared to exporting the files solely during working hours (t = 5.77, p < 0.001). CONCLUSIONS: This study demonstrates the feasibility of using VBA and Excel to automate the process for bulk downloading data from a specific medical imaging platform. The specific steps and techniques will likely vary depending on the software used and hospital constraints and should be determined for each application.

The pseudohypopyon stage in adult-onset foveomacular vitelliform dystrophy.

PURPOSE: To gain insight into the pathogenesis of adult-onset foveomacular vitelliform dystrophy (AFVD) via assessment of its pseudohypopyon stage (PHS). METHODS: Retrospectively, data were collected in a tertiary center from established cohorts of a genetically evaluated AFVD and best vitelliform macular dystrophy (BVMD) eyes in the pseudohypopyon stage. Best-corrected visual acuity (BCVA, LogMAR), lesion characterization, including lesion dimensions, liquefaction areas and patterns (altitudinal or lateral), and ellipsoid zone integrity were analyzed from spectral-domain optical coherence tomography images. RESULTS: Out of 167 eyes of 90 AFVD patients and 56 eyes of 28 BVMD patients, 8 eyes of six AFVD patients and five eyes of four BVMD patients were at the PHS were included. The mean LogMAR BCVA ± SD was 0.21 ± 0.20 and 0.41 ± 0.10 in AFVD and BVMD diseases, respectively (p = 0.13). Seven AFVD eyes (87.5%) demonstrated lateral liquefaction, while all BVMD eyes demonstrated an altitudinal pattern (p = 0.005). Maximal horizontal lesion diameters were 1.41 ± 0.46 mm and 2.64 ± 0.77 mm in AFVD and BVMD, respectively (p = 0.02). AFVD patients were older (69 ± 14) than BVMD patients (22 ± 13; p = 0.009). CONCLUSION: The pseudohypopyon stage in AFVD is often characterized by a lateral liquefaction pattern, unlike the altitudinal pattern characterizing BVMD. Age, lesion size, or pathogenesis pathways may underline the different pseudohypopyon stage patterns in AFVD and BVMD.

Relatively mild blue cone monochromacy phenotype caused by various haplotypes in the L- and M-cone opsin genes.

Purpose: Blue cone monochromacy (BCM) is an X-linked retinopathy caused by mutations in the red and green cone opsin genes. The aim of this study was to establish the clinical, genetic, and electrophysiological characteristics of a specific form of BCM. Methods: Patients harboring mutations in the OPN1LW/OPN1MW genes underwent a full clinical examination, including ocular examination, color vision, full-field electroretinography, color fundus and autofluorescence photography, and optical coherence tomography. Genetic analysis was performed using whole-exome sequencing, duplex PCR, PCR/restriction fragment length polymorphism, and Sanger sequencing. IBM SPSS Statistics v. 21.0 was used for the data analysis. Results: Twenty-five patients harboring various haplotypes in exon 3 of the OPN1LW/OPN1MW genes were recruited. They showed a milder incomplete phenotype of BCM than the typical BCM control group. They presented significantly better visual acuity (logarithm of the minimum angle of resolution [logMAR] 0.48 ± 0.26 vs. 1.10 ± 0.54; p < 0.0001) and a highly myopic refraction (-7.81 ± 5.81 D vs. -4.78 ± 5.27 D; p = 0.0222) compared with the BCM control group. The study group had higher 30-Hz cone flicker responses (28.60 ± 15.02 µv; n = 24), whereas the BCM group had none (0.66 ± 2.12 µv; n = 21; p < 0.0001). The Lanthony 15-HUE desaturated test was variable for the exon 3 haplotype group, with a tendency toward the deutan-protan axis. Conclusions: The present study included genetic and clinical data from the largest cohort of patients with exon 3 haplotypes that were previously shown to cause missplicing of the OPN1LW and OPN1MW genes. Analysis of the clinical data revealed better best-corrected visual acuity, more severe myopia, and higher 30-Hz cone flicker responses in the patients with exon 3 haplotypes than in those with typical BCM.

The detrimental effects of delayed intravitreal anti-VEGF therapy for treating retinal pathology: lessons from a forced test-case.

PURPOSE: Determine the anatomical consequences of delaying intravitreal injection (IVI) therapy with anti-vascular endothelial growth factor (anti-VEGF) in patients using treat-and-extend (T&E) protocol. METHODS: Retrospective medical record review of consecutive patients receiving intravitreal anti-VEGF therapy using T&E protocol prior to and during the COVID-19 pandemic. RESULTS: The study included 923 eyes of 691patients; 58.8% (543 eyes), 25% (231 eyes), and 16.2% (149 eyes) had nvAMD, DME, and RVO, respectively. Mean (± SD) patient age was 74.5 ± 11.7 years. Overall, 56.3% of cases had a delay in therapy of ≥ 7 days; specifically, 56.2%, 61.5%, and 49.0% of nvAMD, DME, and RVO cases, respectively, had a delay. The median delay in days, among cases ≥ 7 days late was 21 (IQR 7 to 42) days, with 21(IQR 7 to 45), 22.5(IQR 8 to 42), and 14(IQR 7 to 33.5) days of delay among patients with nvAMD, DME, and RVO, respectively. Delaying therapy by ≥ 7 days resulted in increased CST in 47.5%, 58.5%, and 58.9% of nvAMD, DME, and RVO cases, respectively, with a significant correlation between the length of treatment delay and the increase in CST (Spearman's rho: 0.196; p < 0.001). CONCLUSIONS: Delayed IVI treatment in eyes treated with T&E protocol was associated with increased macular thickness with potential consequences with respect to visual outcome.

Autoimmune retinopathy: clinical, electrophysiological, and immunological features in nine patients with long-term follow-up.

PURPOSE: We aim to report on the clinical, imaging, immunological, and electrophysiological features of patients with autoimmune retinopathy (AIR) with long-term follow-up. METHODS: Single-center, retrospective study of a consecutive group of AIR patients treated in a tertiary academic medical center. RESULTS: Included were nine patients with a mean ± SD age at presentation of 65 ± 13 years and a median follow-up of 63 months (range 18-120). Five patients were known to have cancer. Median interval between onset of ocular symptoms and diagnosis of AIR was 36 months. Mean baseline and final LogMAR visual acuity were 0.72 ± 0.9 and 1.1 ± 1.2, respectively (p = 0.17). The most common funduscopic findings included optic atrophy and bone-spicule-like pigmentation. Thinning of the nerve fiber layer was the most frequent optical coherence tomographic abnormality. Electroretinographic (ERG) recordings demonstrated variably reduced cone- and rod-derived amplitudes in the majority of eyes at presentation. The most commonly detected anti-retinal antibody was anti-α-enolase. Treatment included immunomodulatory therapy and plasmapheresis. ERG tests showed stability in 64% of eyes throughout the treatment period. CONCLUSION: This study highlights the importance of maintaining a high index of suspicion of AIR, particularly in late middle-aged and elderly patients with "unexplained" visual loss, in light of the non-specific posterior segment signs and the inconsistency of the routinely used ancillary tests.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY FINDINGS IN PIGMENTED PARAVENOUS CHORIORETINAL ATROPHY.

PURPOSE: To analyze the retino-choroidal vascular characteristics of patients affected by pigmented paravenous chorio-retinal atrophy by means of optical coherence tomography (OCT) angiography. METHODS: This study was designed as an observational, cross-sectional case series. Multimodal imaging included fundus autofluorescence, structural OCT, and OCT angiography. The quantitative OCT angiography analyses included the calculation of the vessel density and choriocapillaris porosity. RESULTS: Overall, 12 patients (24 eyes) affected by pigmented paravenous chorio-retinal atrophy were recruited. Structural OCT of the areas involved by pigmented paravenous chorio-retinal atrophy as visualized on the fundus autofluorescence showed a complete ellipsoid zone and external limiting membrane absence, with thinning of ganglion cell complex, outer nuclear layer, and outer plexiform layer, but associated with the optical partial preservation of the retinal pigment epithelium. Optical coherence tomography angiography quantitative assessment of the retinal regions affected by PPRCA, as visualized by fundus autofluorescence, was characterized by normal vessel density at the level of superficial capillary plexus but significantly altered vessel density of deep capillary plexus and choriocapillaris, with higher choriocapillaris porosity. The presence of macular atrophy was significantly correlated with worse deep capillary plexus and choriocapillaris vessel density values. Furthermore, a statistically significant correlation between the fundus autofluorescence patterns and the retinal vascular status was found. CONCLUSION: Optical coherence tomography angiography quantitative analyses in pigmented paravenous chorio-retinal atrophy demonstrate a specific impairment at the level of the deep capillary plexus, which could in turn bring about a thinning of ganglion cell complex and outer nuclear layer. The alterations at the level of the choriocapillaris and the choroid, in general, could then represent a secondary effect.

Ophthalmic Emergency Visits in the Wake of the COVID 19 Pandemic: Our Experience at a Tertiary Hospital in Israel.

BACKGROUND: In response to the coronavirus disease-2019 (COVID-19) pandemic, routine clinical visits to the ophthalmic emergency department (OED) were deferred, while emergency cases continued to be seen. OBJECTIVES: To assess the consequences of the COVID-19 pandemic for ophthalmic emergencies. METHODS: A retrospective chart analysis of patients who presented to the OED during the peak of the COVID-19 pandemic was conducted. The proportions of traumatic, non-traumatic-urgent, and non-traumatic-non-urgent presentations in 2020 were compared to those of the same time period in 2019. Duration of chief complains and best-corrected visual acuity were also assessed. RESULTS: There were 144 OED visits in 2020 compared to 327 OED visits during the same 3-week-period in 2019. Lower mean age of OED patients was present in 2020. Logarithmic expression (LogMAR) best corrected visual acuity (BVCA) was similar in both years. In 2020 there was a reduction in traumatic, non-traumatic-urgent, and non-traumatic-non-urgent cases compared to 2019 (15.4% reduction, P = 0.038; 57.6% reduction, P = 0.002; 74.6% reduction, P = 0.005, respectively). There was a higher proportion of same-day presentations at commencement of symptoms in 2020 compared with 2019 (52.8% vs. 38.8%, respectively P = 0.006). CONCLUSIONS: During the COVID-19 pandemic, the number of OED visits at a tertiary hospital dropped by more than half. Although the drop in visits was mostly due to decrease in non-traumatic-non-urgent cases, there was also decrease in non-traumatic-urgent presentations with possible important visual consequences. Additional studies should elucidate what happened to these patients.

Anti-tumor necrosis factor alpha reduces the proangiogenic effects of activated macrophages derived from patients with age-related macular degeneration.

Purpose: Macrophages are believed to promote choroidal neovascularization (CNV) in neovascular age-related macular degeneration (nvAMD); however, the underlying proangiogenic mechanism is poorly understood. Therefore, we examined this mechanism in proinflammatory macrophages derived from patients with nvAMD. Methods: Monocytes were isolated from patients with nvAMD and polarized to form an M1 proangiogenic phenotype. We then screened for the role of proangiogenic cytokines expressed by these macrophages, including TNF-α, VEGF, IL-6, IL-8, and IL-1ß, using an ex vivo choroid sprouting assay and an in vivo rodent model of laser-induced CNV (LI-CNV). We also examined the value of inhibiting TNF-α inhibition with respect to reducing the proangiogenic effects of M1 macrophages. Finally, we analyzed the macrophage cytokine expression database to evaluate the feasibility of modulating the expression of TNF-α. Results: The cytokines above are expressed at high levels in patient-derived M1 macrophages. However, among the cytokines tested only TNF-α significantly increased choroid sprouting. Moreover, adoptive intravitreal transfer of M1 macrophages significantly increased LI-CNV, and blocking TNF-α abolished the proangiogenic effects of M1 macrophages in both models. An analysis of cytokine expression revealed that >50% of TNF-α expression is determined by modifiable factors. Conclusions: Blocking TNF-α can reduce the proangiogenic effects of M1 macrophages in nvAMD. Thus, activated macrophages may represent a potential therapeutic target for altering TNF-α expression in nvAMD.

FULL-THICKNESS MACULAR HOLE IN AGE-RELATED MACULAR DEGENERATION PATIENTS WITH TWO DISTINCT ENTITIES: A Multicenter Study.

PURPOSE: To describe optical coherence tomography characteristics of full-thickness macular holes (FTMHs) in age-related macular degeneration patients. METHODS: A multicenter, retrospective, observational case series of patients diagnosed with age-related macular degeneration and FTMHs seen between January 1, 2009, and January 3, 2020. Clinical charts and spectral-domain optical coherence tomography images were reviewed. Optical coherence tomography findings included FTMH-inverted trapezoid or hourglass appearance, central macular thickness (CMT), complete retinal pigment epithelium and complete retinal outer retinal atrophy, and presence of pigment epithelium detachment and epiretinal membrane. The mean outcome was the morphologic and functional characterization of different subtypes of FTMHs. RESULTS: A total of 86 eyes of 85 consecutive patients, with mean age of 80.31 ± 8.06 and mean best-corrected visual acuity of 1.17 ± 0.58 logarithm of the minimal angle of resolution. Two different subtypes of FTMHs were identified: tractional and degenerative. Fifty (58%) degenerative FTMHs characterized with inverted trapezoid appearance and 36 (42%) tractional FTMHs characterized with hourglass appearance. Degenerative FTMHs presented with 66% of CMT < 240 µm, 14% of CMT > 320, and 70% of complete retinal outer retinal atrophy, in comparison with 41% of CMT < 240 µm, 42.9% of CMT > 320%, and 20% of complete retinal outer retinal atrophy in the tractional FTMH group (P = 0.002, 0.003, <0.001, respectively). The presence of epiretinal membrane and pigment epithelium detachment where significantly higher in tractional FTMHs (P = 0.02, 0.03, respectively). CONCLUSION: Degenerative and tractional FTMHs may be two distinct clinical entities. Discerning degenerative from tractional FTMHs is possible by using optical coherence tomography features including shape of the FTMHs, CMT, internal-external ratio of FTMHs, and presence of complete retinal outer retinal atrophy, pigment epithelium detachment, and epiretinal membrane.

Risk of SARS-CoV-2 transmission to medical staff and patients from an exposure to a COVID-19-positive ophthalmologist.

PURPOSE: To evaluate the risk of transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after exposure to a COVID-19+ physician in a retina clinic. METHODS: A retrospective observational study. Records of 142 patients and 11 staff members from a single retina clinic that were exposed to a COVID-19+ ophthalmologist were reviewed. All 153 individuals were placed in quarantine for 14 days. They were contacted after the quarantine period to inquire about symptoms consistent with COVID-19, and the results of diagnostic test for SARS-CoV-2 when performed. RESULTS: All patients (n = 142) were contacted successfully. The mean age was 72.8 ± 13.6 years; 54.2% (n = 77) were females. Twenty-three patients (16.2%) were exposed during an ophthalmic exam, 111 (78.2%) during intraocular injection, 4 (2.8%) underwent exam and injection, 3 (2.1%) underwent surgery, and one patient (0.7%) had laser photocoagulation. Half of the patients (50%; n = 71) were in contact with the COVID-19+ physician while he was symptomatic. Forty-four patients (31%) wore a mask on the day of their visit. 11.3% (n = 16) of the patients, and all involved staff had been tested for the virus and all were negative. One patient (0.7%) reported transient cough and sore throat, and the remaining 141 (99.3%) patients and 11 (100%) staff did not develop symptoms. CONCLUSIONS: Low risk for SARS-CoV-2 transmission in the ophthalmic setting was observed when universal safety measures such as social distancing, meticulous hand hygiene, enlarged breath shields, and mask wear during procedures were taken.

Evaluation of antioxidant treatments for the modulation of macrophage function in the context of retinal degeneration.

Purpose: Oxidative stress and macrophages have been implicated in the pathogenesis of atrophic and neovascular age-related macular degeneration (aAMD and nvAMD). It is unclear whether oxidative injury mediates macrophage involvement in AMD. We aimed to investigate the effect of antioxidant treatments on human monocyte-derived macrophages (hMDMs) from patients with AMD in models for the disease. Methods: Four antioxidant treatments were evaluated (G1: lutein + zeaxanthin, G2: lutein + zeaxanthin and zinc, G3: lutein + zeaxanthin, zinc, Lyc-O-Mato, and carnosic acid, G4: lutein + zeaxanthin, carnosic acid, and beta-carotene, G5: olive oil as vehicle control). The compounds were added to the culture medium of M1 (interferon-gamma [IFN-Ɣ] and lipopolysaccharide [LPS]) and M2a (interleukin-13 [IL-13] and IL-4) hMDMs from patients with AMD (n=7 and n=8, respectively). Mouse choroidal tissue was cultured with supernatants from treated M1/M2a hMDMs, to evaluate the effect of treatments on the angiogenic properties of macrophages with choroidal sprouting assay (CSA). Mouse retinal explants were cultured with treated hMDMs for 18 h, and evaluated for photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling. Adult BALB/c mice (n=8) were exposed to 8,000 lux bright light for 3 h, and treated orally with antioxidant supplements for 7 days that preceded light injury and following it. Oxidative stress was assessed using an anti-4 hydroxynonenal (4-HNE) antibody. Retinal function and the thickness of the outer nuclear layer were evaluated with electroretinography (ERG) and histological analysis, respectively. Results: The G3 treatment reduced M2a hMDMs-associated sprouting in the CSA compared to the untreated group (n=7, -1.52-fold, p=0.05). Conversely, the G2 treatment was associated with an increased neurotoxic effect of M2a hMDMs in the retinal explant assay compared to the control group (n=7, 1.37-fold, p=0.047), as well as compared to the G3 treatment group (1.46-fold, p=0.01). The G4 treatment was also associated with increased cytotoxicity compared to the control group (1.48-fold, p=0.004), and compared to the G3 treatment group (1.58-fold, p=0.001). In the in vivo light damage model, mice (n=8) supplemented with G2, G3, and G4 had decreased levels of oxidative injury assessed using 4-HNE labeling (-2.32-fold, -2.17-fold, and -2.18-fold, respectively, p<0.05 for all comparisons). None of the treatments were associated with reduced photoreceptor cell loss, as shown with histology and ERG. Conclusions: Antioxidant treatment modulates M2a hMDMs at the functional level. In particular, we found that the G3 combination has a beneficial effect on M2a macrophages in reducing their angiogenic and neurotoxic capacity ex vivo. In addition, antioxidant treatments considerably reduced the oxidative stress level in light-damaged retinas. Further research is required to assess whether such therapies may curb macrophage-driven photoreceptor loss and neovascularization in AMD.

A novel intronic mutation of PDE6B is a major cause of autosomal recessive retinitis pigmentosa among Caucasus Jews.

Purpose: To identify the genetic basis for retinitis pigmentosa (RP) in a cohort of Jewish patients from Caucasia. Methods: Patients underwent a detailed ophthalmic evaluation, including funduscopic examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potentials (VEP). Genetic analysis was performed with a combination of whole exome sequencing (WES) and Sanger sequencing. Bioinformatic analysis of the WES results was performed via a customized pipeline. Pathogenicity of the identified intronic variant was evaluated in silico using the web tool Human Splicing Finder, and in vitro, using a minigene-based splicing assay. Linkage disequilibrium (LD) analysis was used to demonstrate a founder effect, and the decay of LD over generations around the mutation in Caucasus Jewish chromosomes was modeled to estimate the age of the most recent common ancestor. Results: In eight patients with RP from six unrelated families, all of Caucasus Jewish ancestry, we identified a novel homozygous intronic variant, located at position -9 of PDE6B intron 15. The c.1921-9C>G variant was predicted to generate a novel acceptor splice site, nine bases upstream of the original splice site of intron 15. In vitro splicing assay demonstrated that this novel acceptor splice site is used instead of the wild-type site, leading to an 8-bp insertion into exon 16, which is predicted to cause a frameshift. The presence of a common ancestral haplotype in mutation-bearing chromosomes was compatible with a founder effect. Conclusions: The PDE6B c.1921-9C>G intronic mutation is a founder mutation that accounts for at least 40% (6/15 families) of autosomal recessive RP among Caucasus Jews. This result is highly important for molecular diagnosis, carrier screening, and genetic counseling in this population.

MICROPERIMETRY IN BEST VITELLIFORM MACULAR DYSTROPHY.

PURPOSE: To investigate retinal sensitivity in eyes with all the clinical stages of Best vitelliform macular dystrophy (VMD). METHODS: Thirty-two patients affected by VMD in subclinical, vitelliform, pseudohypopyon, vitelliruptive, and atrophic stages were enrolled in this prospective cross-sectional study. Patients underwent a complete ophthalmologic examination, including determination of best-corrected visual acuity (BCVA), staging of the disease (Gass's classification), and microperimetry by means of the macular integrity assessment microperimeter. The primary outcome measure was to describe the alterations in the retinal sensitivity of eyes affected by VMD in different stages. Secondary outcome measures included correlations between retinal sensitivity and best-corrected visual acuity and the correlation between the VMD stage and the specific microperimetry pattern. RESULTS: Mean retinal sensitivity was reduced in all the VMD stages. Nevertheless, vitelliform, pseudohypopyon, and vitelliruptive stages turned out to be very similar, especially within 10°. Fixation was classified as stable in 27 eyes (44.2%), relatively unstable in 16 eyes (26.2%), and unstable in 18 eyes (29.5%). Fixation stability correlated both with the disease stage and best-corrected visual acuity. CONCLUSION: VMD is characterized by complex microperimetric abnormalities, involving the whole macular area. Microperimetry may contribute to the global clinical assessment of patients affected by VMD and could be used in future therapeutic approaches.

Characterizing the effect of supplements on the phenotype of cultured macrophages from patients with age-related macular degeneration.

Purpose: Oral vitamin and mineral supplements reduce the risk of visual loss in age-related macular degeneration (AMD). However, the pathways that mediate this beneficial effect are poorly understood. Macrophages may exert oxidative, inflammatory, and angiogenic effects in the context of AMD. We aim to assess if oral supplements can modulate the macrophage phenotype in this disease. Methods: Monocytes were isolated from patients with neovascular AMD (nvAMD), cultured, matured to macrophages, and polarized to classical [M1 (stimulated by IFNγ and lipopolysaccharide (LPS))] and alternative [M2 (stimulated with IL-4 and IL-13)] phenotypes. Combinations of antioxidants including lutein+zeaxanthin (1 µM; 0.2 µM), zinc (10 µM), carnosic acid (2 µM), beta-carotene (2 µM), and standardized tomato extract containing lycopene and other tomato phytonutrients were added to the culture media. Levels of anti-inflammatory, antioxidant, and pro-angiogenic gene and protein expression were then evaluated. Results: Combinations of lutein and carnosic acid with zinc and standardized tomato extract or with beta-carotene yielded an antioxidative, anti-inflammatory, and antiangiogenic effect in M1 and M2 macrophages. These effects manifested in the upregulation of antioxidative genes (HMOX1, SOD1) and the downregulation of pro-angiogenic genes and pro-inflammatory genes (SDF-1, TNF-alpha, IL-6, MCP-1). Lutein monotherapy or a combination of lutein and zinc had less effect on the expression of these genes. Conclusions: Combinations of supplements can modify the expression of genes and proteins that may be relevant for the involvement of macrophages in the pathogenesis of AMD. Further studies are required to evaluate if the modulation of the macrophage phenotype partially accounts for the beneficial effect of oral supplements in AMD and if modification of the AREDS formula can improve its effect on macrophages.

Retinal Phenotype following Combined Deletion of the Chemokine Receptor CCR2 and the Chemokine CX3CL1 in Mice.

PURPOSE: Conflicting data were reported with respect to the retinal phenotype of mice with dual perturbation of the CCL2 and CX3CR1 genes. We report the generation and retinal phenotype of mice with a reverse CCR2/CX3CL1 gene deficiency as a suggested model for age-related macular degeneration (AMD). METHODS: Crossing of single-deficient mice generated CCR2/CX3CL1 DKO mice. DKO mice were compared with age-matched C57BL6J mice. Evaluation included color fundus photographs, electroretinography (ERG), histology and morphometric analysis. Immunohistochemistry for CD11b in retinal cross-sections and retinal pigment epithelium (RPE)-choroid flat mounts was performed to assess microglia and macrophage recruitment. RESULTS: A minority of DKO mice showed yellowish subretinal deposits at 10 months. ERG recordings showed reduced cone sensitivity in young, but not older DKO mice. Compared to wild-type mice, DKO mice exhibited 11% reduction in the number of outer nuclear layer nuclei. Old DKO mice had an increased number of CD11b-positive cells across the retina, and on RPE-choroid flat mounts. CONCLUSIONS: In the absence of the rd8 allele, deficiency of CCR2 and CX3CL1 in mice leads to a mild form of retinal degeneration which is associated with the recruitment of macrophages, particularly to the subretinal space. This model enables to assess consequences of perturbed chemokine signaling, but it does not recapitulate cardinal AMD features.

Bevacizumab treatment for neovascular age-related macular degeneration in the setting of a clinic: "real life" long-term outcome.

BACKGROUND: To evaluate the long-term outcome of bevacizumab therapy for neovascular age related macular degeneration (NVAMD) in the setting of a clinic. METHODS: Consecutive group of NVAMD patients who were treated in a single 3(rd) referral center with bevacizumab using a loading dosage of 3 monthly injections followed by variable dosing for at least 48 months were retrospectively evaluated. Genotyping was performed for CFH (rs1061170), HTRA1 (rs1200638), and C3 (rs2230199). Main outcome measures included functional and morphological treatment outcomes as well as their risk allele associations. RESULTS: Out of 128 patients who started bevacizumab treatment over 4 years before the study endpoint [mean (± SD): 60 ± 10.9 months], 75 eyes of 67 (52.3%) patients, were still followed. Mean best corrected visual acuity (BCVA) (LogMAR ± SEM) improved from 0.66 ± 0.07 at baseline to 0.48 ± 0.05 (p = 0.012) at 1 year, but deteriorated from the 3(rd) year on and at the final exam reduced to 0.69 ± 0.07 (p = 0.6, compared with initial BCVA). Macular thickness mirrored visual acuity (VA) changes showing initial thinning followed by thickening from the 3(rd) year on. Individuals carrying the CFH risk -allele had a mean thickening (microns ± SEM) of 66.9 ± 70.4 versus a mean thinning of 76.8 ± 22 in non-carriers (p = 0.015). CONCLUSIONS: Bevacizumab therapy for NVAMD using a flexible treatment algorithm in a "real life" clinical setting initially obtained VA gain and thinning of the macula that were maintained for two years, but were lost later on.

The role of monocytes and macrophages in age-related macular degeneration.

White blood cells, particularly monocytes and their descendants, macrophages, have been implicated in age-related macular degeneration (AMD) pathology. In this minireview, we describe the current knowledge of monocyte and macrophage involvement in AMD. Chemokine receptors present on these cells such as CCR1, CCR2, and CX3CR1, and their roles in monocyte/macrophage recruitment to sites of injury and inflammation in the context of AMD will be reviewed. Mice models for perturbation of chemokine receptors that recapitulate some of the features of AMD are also described. The body of evidence from human and rodent studies at this point in time suggests that monocyte and macrophages may modulate the course of AMD.

The importance of vision care for displaced populations: lessons from Israeli evacuees.

BACKGROUND: This study explores vision care priorities and coping mechanisms for Israeli evacuees and following the October 7th, 2023, attack by Hamas, which displaced 150,000 individuals, with about 15,000 being evacuated to the Dead Sea area. Faced with minimal health care infrastructure in the Dead Sea area and often lacking personal belongings, including eyeglasses and ocular medicine, these evacuees confronted significant vision care challenges. This context sets the stage for investigating the emergency vision care needs and solutions for populations affected by conflict and displacement. METHODS: In response to this crisis, a consortium led by Hadassah Academic College's Department of Optometry and the Dept. of Ophthalmology at Hadassah Medical Center established ophthalmic clinics in the Dead Sea region. These clinics offered comprehensive vision care services, including refractive and vision examinations, ophthalmological assessments, ocular imaging, and provision of free glasses. The setup included multiple stations for different vision tests, staffed by an interdisciplinary team of professionals. The study analyzes the effectiveness of these clinics, patient flow challenges, and the psychological impact of vision care in a crisis setting. RESULTS: Approximately 800 evacuees received examinations, with around 700 pairs of glasses distributed. Notable cases included emergency referrals for serious conditions and instances where glasses served as psychological support. The operation highlighted the necessity of vision care during crises and its potential psychological and social implications. The clinics successfully provided immediate vision care, but challenges in patient flow and insufficient electronic medical record integration were noted. The experience underscores the importance of prepared eye care interventions in crises. Recommendations for health policy decision-makers include establishing a national emergency vision care network, developing standardized treatment protocols, training local health workers, and raising public awareness about eye health in emergencies. CONCLUSIONS: The consortium's effort in providing urgent vision care to evacuees from the Hamas attack on Israel demonstrates the critical role of rapid, organized eye care in crisis situations. Vision care, along with hearing and mobility, is often overlooked during evacuations but is vital for the well-being and survival of evacuees, especially under trying circumstances. This project serves as a model for future humanitarian interventions, emphasizing the importance of addressing overlooked healthcare issues once the immediate crisis has passed, and the need for strategic planning in health care policy for similar emergency scenarios.

Anomaly Detection and Biomarkers Localization in Retinal Images.

Background: To design a novel anomaly detection and localization approach using artificial intelligence methods using optical coherence tomography (OCT) scans for retinal diseases. Methods: High-resolution OCT scans from the publicly available Kaggle dataset and a local dataset were used by four state-of-the-art self-supervised frameworks. The backbone model of all the frameworks was a pre-trained convolutional neural network (CNN), which enabled the extraction of meaningful features from OCT images. Anomalous images included choroidal neovascularization (CNV), diabetic macular edema (DME), and the presence of drusen. Anomaly detectors were evaluated by commonly accepted performance metrics, including area under the receiver operating characteristic curve, F1 score, and accuracy. Results: A total of 25,315 high-resolution retinal OCT slabs were used for training. Test and validation sets consisted of 968 and 4000 slabs, respectively. The best performing across all anomaly detectors had an area under the receiver operating characteristic of 0.99. All frameworks were shown to achieve high performance and generalize well for the different retinal diseases. Heat maps were generated to visualize the quality of the frameworks' ability to localize anomalous areas of the image. Conclusions: This study shows that with the use of pre-trained feature extractors, the frameworks tested can generalize to the domain of retinal OCT scans and achieve high image-level ROC-AUC scores. The localization results of these frameworks are promising and successfully capture areas that indicate the presence of retinal pathology. Moreover, such frameworks have the potential to uncover new biomarkers that are difficult for the human eye to detect. Frameworks for anomaly detection and localization can potentially be integrated into clinical decision support and automatic screening systems that will aid ophthalmologists in patient diagnosis, follow-up, and treatment design. This work establishes a solid basis for further development of automated anomaly detection frameworks for clinical use.