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To identify optical coherence tomography (OCT) biomarkers, including thin and thick double-layer sign (DLS) for the progression from intermediate AMD (iAMD) to exudative macular neovascularization (MNV) over 24 months.Retrospective cohort study.Setting: Retina consultants of Texas.458 eyes of 458 subjects with iAMD in at least 1 eye with 24 months of follow-up data.The following biomarkers were assessed at baseline: high central drusen volume (≥0.03 mmDuring follow-up, 18.1% (83 of 458) of eyes with iAMD progressed to exudative MNV. Thick DLS, IHRF, and fellow eye exudative MNV were found to be independent predictors for the development of exudative MNV within 2 years. The baseline frequencies, odds ratios, 95% confidence intervals, and P values for these biomarkers were as follows: thick DLS (9.6%, 4.339, 2.178-8.644; P.001), IHRF (36.0%, 2.340, 1.396-3.922; P = 0.001), and fellow eye exudative MNV (35.8%, 1.694, 1.012-2.837; P = .045).Thick DLS, IHRF, and fellow eye exudative MNV were associated with an increased risk of progression from iAMD to exudative MNV. These biomarkers, which are readily identified by the review of OCT volume scans, may aid in risk prognostication for patients and for identifying patients for early intervention trials.

Purpose To identify baseline risk factors for macular atrophy (MA) development in HARBOR via a longitudinal assessment of monthly spectral-domain (SD)-OCT scans. Previous analyses of MA in HARBOR examined data from color fundus photography (CFP) and fluorescein angiography (FA). Design Retrospective, post hoc analysis of SD-OCT images from HARBOR ( ClinicalTrials.gov identifier, NCT00891735 ), a phase 3, multicenter, prospective, randomized, double-blind, active treatment–controlled clinical trial. Participants Patients (N = 1097) with subfoveal choroidal neovascularization secondary to neovascular age-related macular degeneration (nAMD) treated with intravitreal ranibizumab 0.5 mg monthly (n = 275), 0.5 mg pro re nata (PRN) after 3 loading doses (n = 275), 2.0 mg monthly (n = 274), or 2.0 mg PRN (n = 273). Methods Evaluable SD-OCT macular cube scans from patients with 24 months of follow-up (N = 941) were examined monthly from baseline to month 24 by masked reading center–trained graders. Atrophy diagnosis criteria were consistent with those proposed by the Classification of Atrophy Meetings (CAM) group: hypertransmission of light into the choroid, loss of retinal pigment epithelium, and loss of outer retinal layers. Multivariable proportional hazards regression was performed for time to atrophy development. Main Outcome Measures Risk factors for MA as determined by time to MA development over 24 months of treatment. Results Baseline risk factors for MA were confirmed from prior analyses that used CFP and FA data: absence of subretinal fluid, presence of intraretinal cysts, presence of Type 3 neovascularization, and presence of atrophy in the fellow eye. This analysis of SD-OCT data identified new baseline risk factors for MA: higher central drusen volume, lower choroidal thickness, presence of nascent atrophy, presence of reticular pseudodrusen, and increased central foveal thickness. Ranibizumab treatment regimen and dose level were not found to be risk factors for MA development. Conclusions In this analysis of a major nAMD trial using CAM atrophy criteria, new baseline risk factors for MA development were identified using an SD-OCT dataset. Risk factors for MA development identified by prior analyses were confirmed. Monthly treatment with ranibizumab 0.5 mg was not found to be a risk factor for MA development over 24 months.

To evaluate retinal vessel quantity within various retinal structural layers using optical coherence tomography angiography (OCTA). In this IRB-approved study, 22 normal eyes (from 22 subjects) were imaged using the Spectralis OCT2, with a 15 × 15 degree OCTA scan centered on fovea and two additional 15 × 5 degree OCTA scans, displaced temporally and nasally by 15 degrees along the fovea-Bruch’s membrane opening (BMO) axis. Following projection artifact removal (PAR), vessel quantity (i.e., amount of flow signal) within each retinal nuclear and plexiform layer was assessed across the scan and was plotted as a vessel quantity profile over this fovea-BMO axis. Vessel quantity was correlated against the retinal layer thickness at the corresponding locations using the Spearman correlation. For the nerve fiber layer (NFL), the vessel quantity was highest nasally and declined towards the fovea and was near zero temporal to the fovea with or without PAR. For all other retinal layers, the retinal vessel quantities were greatest in the parafoveal retina, peaking approximately 5 degrees from the foveal center. Before PAR, the parafoveal vessel quantity was highest in the inner plexiform layer (IPL). Following PAR, the vessel quantity in the IPL decreased but was relatively unchanged in the other layers. The vessel quantity correlated moderately well with retinal layer thickness (r = 0.432 to 0.511; P < 0.05 among the various layers). Retinal vessel quantity varies significantly among the various structural layers, with significant regional variability. Projection artifact can significantly impact retinal vessel quantity in the deeper layers, but the effect appears to be most pronounced in the IPL.

Purpose Previous studies of macular atrophy (MA) in HARBOR analyzed color fundus photography and fluorescein angiography image data. This study performed a longitudinal assessment of monthly spectral-domain (SD) OCT scans to determine MA prevalence, incidence, and progression in HARBOR. Design Post hoc analysis of SD OCT images from HARBOR ( ClincalTrials.gov identifier, NCT00891735 ), a phase 3 multicenter, prospective, randomized, double-blind, active treatment–controlled clinical trial. Participants Patients (n = 1097) with subfoveal choroidal neovascularization secondary to neovascular age-related macular degeneration (nAMD) treated with ranibizumab 0.5 mg monthly (n = 275), 0.5 mg pro re nata (PRN) after 3 loading doses (n = 275), 2.0 mg monthly (n = 274), or 2.0 mg PRN (n = 273). Methods Evaluable SD OCT macular cube scans from patients with 24 months of follow-up (N = 941) were examined by masked reading center–trained graders monthly from baseline to month 24. Atrophy diagnosis criteria were consistent with those proposed by the Classification of Atrophy Meetings (CAM) group: hypertransmission of light into the choroid, retinal pigment epithelium loss, and loss of outer retinal layers. Macular atrophy was considered Definite if all 3 criteria were met and Questionable if 2 were met. Study arms were compared for time to MA detection (log-rank test) and enlargement rates (time × arm interaction test). Main Outcome Measures Prevalence, incidence, and enlargement rates of MA. Results At baseline, imbalance in MA rates across ranibizumab arms was evident (0.5 mg monthly, 19.1%; 0.5 mg PRN, 16.1%; 2.0 mg monthly, 10.1%; 2.0 mg PRN, 10.5%). At month 24, new MA development rates in eyes without baseline MA were similar between ranibizumab doses (0.5 mg, 25.9%; 2.0 mg, 25.4%) and treatment regimens (monthly, 26.4%; PRN, 25.0%). No significant differences in enlargement rate of new atrophy area (P = 0.479, square-root transformed) or time to detection of new MA (P = 0.997) were evident among study arms. Conclusions In this analysis of a major nAMD trial using CAM atrophy criteria, no differences were observed in incidence or progression rates of new MA among study arms, ranibizumab doses, or treatment regimens. Monthly versus PRN treatment did not influence MA incidence or progression.

E Randy Craven,1 Vikas Chopra,2 Jeffrey L Goldberg,3 Kenneth M Marion,4 Xiaoming Chen,5 Cheng-Tao Chang,6 Michelle Y Chen6 1Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Doheny Eye Center UCLA, Pasadena, CA, USA; 3Byers Eye Institute at Stanford University, Palo Alto, CA, USA; 4Doheny Eye Institute, Los Angeles, CA, USA; 5West China Hospital Sichuan University, Sichuan, People’s Republic of China; 6Allergan, an AbbVie Company, Irvine, CA, USACorrespondence: E Randy Craven, 600 N. Wolfe Street, Baltimore, MD, 21287, USA, Tel +1 240-482-1100, Fax +1 240-482-1105, Email erandycraven@gmail.comPurpose: To quantitatively compare iridocorneal angle assessments using gonioscopy and anterior segment optical coherence tomography (AS-OCT).Patients: US and Chinese patients with open-angle glaucoma (OAG) and/or ocular hypertension (OHT).Methods: Analysis was pooled from 2 multicenter, noninterventional studies conducted in the US and China. Gonioscopy Shaffer grade and an AS-OCT method that approximates the angle width relative to local morphologic variations were compared by measuring the same iridocorneal angles. A third, separate, single-center, noninterventional study was conducted to verify results observed from the pooled analysis.Results: From the pooled studies, a total of 239 eyes were measured using Shaffer grade and AS-OCT. Of these, 6 were Shaffer grade 2, 37 in Shaffer grade 3, and 196 in Shaffer grade 4. There was a trend of increasing Shaffer grade with increasing AS-OCT angle width. Open iridocorneal angles, Shaffer grade ≥ 3, had a ∼ 98% sensitivity and 88% positive predictive value for identifying AS-OCT angle width ≥ 300 μm, using the AS-OCT method. To verify these results, a total of 28 right eyes were imaged for the third study. A trend of increasing Shaffer grade with increasing AS-OCT angle width was observed, and angles with Shaffer grade ≤ 2 had AS-OCT angle width < 300 μm.Conclusion: The AS-OCT method can determine the space in the anterior chamber and can potentially identify angles that are the appropriate size for certain glaucoma devices. Information gathered from AS-OCT can provide additional comprehensive and quantitative assessment to gonioscopy.Keywords: gonioscopy, anterior segment optical coherence tomography, angle assessment, open-angle glaucoma

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain–containing protein 3, and apoptosis-associated speck-like protein–containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA–driven diseases.

PurposeSubretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown.MethodsTo evaluate the LC for SRI in a murine model, we analyzed training data from 3 clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffer saline injection and the presence of RPE degeneration afterAluRNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. A Cumulative Sum (CUSUM) analysis was performed and plotted individually to monitor each surgeon’s simultaneous performance.ResultsDespite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364-training case as a “cutoff” point, the predicted probability of success before and after the 364thcase was 65.38% and 99.32%, respectively (P< 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage.ConclusionsThis study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique.Translational RelevanceOur prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time.

Description, Endogenous Alu cDNA triggers inflammasome-mediated cell death in age-related macular degeneration., Long interspersed nuclear element-1 (L1)–mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA–induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA–induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Purpose Obtaining slit-lamp gonioscopic images requires a high level of expertise and can be time consuming. The images typically are limited to 4 quadrants with limited resolution and can be affected by different lighting conditions. Our goal was to study the ability of the novel imaging system to quickly and easily document the full 360 degrees of the anterior chamber angle in a standardized manner. Design Cross-sectional cohort study. Subjects Eighty-four eyes from 50 subjects were included in this study, including 26 eyes from 17 normal participants and 58 eyes from 33 patients diagnosed with glaucoma. Methods A newly introduced, commercially available gonioscope (GS-1, NIDEK Co, Gamagori, Japan) was utilized to perform semiautomated imaging of the full 360-degree iridocorneal angle on participants under an institutional review board–approved study. Topical proparacaine was administered to the eye to anesthetize the eye and imaging was completed using the 16-mirror, machine-attached gonioscopic lens coupled with lubricating ophthalmic gel ointment. The first step of the 2-step image acquisition process is analogous to manual gonioscopy, whereas the second step is performed by the instrument in an automated fashion. First, the instrument is manually moved forward by the operator to allow the machine-attached lens to make contact with the patient’s corneal surface (time = 30 seconds). Second, the instrument automatically achieves fine focus on the iridocorneal angle and takes 16 sequential high-resolution photographs at multiple different focal planes (time = less than 30 seconds). Main Outcome Measures Successful qualitative imaging of anterior chamber iridocorneal angle in normal and glaucomatous eyes including visualization of various implanted devices within angle before and after surgery in less than 1 minute per eye. Results In this pilot study, the full 360 degrees of iridocorneal angle of 84 eyes from 50 normal participants or glaucoma patients were successfully imaged using NIDEK GS-1 in under 60 seconds. All eyes were able to be imaged in 1 sitting, and we were able to image the full spectrum of angle from narrow/closed to fully open. The 360-degree views of the images were able to be displayed in 3 available formats (16-section display, a circular display, or a linear display). Successful imaging was also achieved of surgical devices and postsurgical conditions including CyPass, iStent, XEN, Baerveldt tube shunt, angle recession with iridodialysis, angle neovascularization, pigment dispersion, posttrabeculectomy sclerostomy and surgical iridectomy, and post–laser peripheral iridotomy. Conclusion The newly introduced, semiautomated imaging system was able to document the full 360-degree iridocorneal angle views in normal and glaucoma eyes in less than 1 minute, which may allow longitudinal evaluation in a standardized manner. Furthermore, this system allows the ability to easily evaluate post–angle surgery changes and assess positions of implanted devices in the anterior chamber angle.

Purpose Anterior-segment OCT (AS-OCT) platforms provide noninvasive and quantifiable evaluations of anterior chamber angle (ACA) anatomy. Although these ACA measurements have been validated with high repeatability and reproducibility, there is a paucity of data previously published regarding the technique errors and artifacts (TEA) that may occur during imaging of the ACA. The purpose of this study was to report on the types and frequency of imaging TEA that are seen in ACA evaluation of a large cohort of patients with open-angle glaucoma. Design Retrospective, cross-sectional design. Participants Images were obtained with AS-OCT from 5082 eyes of 2541 patients with glaucoma and were reviewed at the Doheny Image Reading Center (DIRC). Methods Images had been captured using AS-OCT devices from 4 different manufacturers (Zeiss Cirrus [Oberkochen, Germany], Heidelberg Spectralis [Heidelberg, Germany], Optovue RTVue [Fremont, CA], or Zeiss Visante). All images were analyzed by 2 independent and certified DIRC image readers for angle opening distance under Schwalbe’s line (SL-AOD), and then re-reviewed by a DIRC-certified principal investigator to adjudicate nonconsensus measurements. Images were defined to have TEA when image readers were unable to confidently measure the SL-AOD. Main Outcome Measures Type and frequency of TEA in the AS-OCT image. Results Of 5082 eyes reviewed, 208 images (4.09%) were found to have TEA that restricted proper visualization of irido-corneal angle structures and measurements of SL-AOD. The most common TEA was that the image readers were unable to identify Schwalbe’s line (74% of TEA images). Other common TEA included poor visualization of the anterior iris surface (15%), misaligned scan location (11%), irregular corneal irregularities (5%), and illumination-related pupillary abnormalities (4%). Conclusions This large-scale study presents the types and frequency of TEA found in AS-OCT–derived angle images. More than 95% of the images reviewed were usable for the quantification of irido-corneal angle measurements. Knowing the common types of TEA may help in enhancing the training of both human image readers and in the design of automated algorithms to improve image acquisition and image interpretation parameters. This will become especially useful as AS-OCT becomes more widely used in clinical practice for anterior chamber assessment.

PURPOSE: To investigate fluorescence lifetime imaging ophthalmoscopy (FLIO) findings in preclinical Alzheimer’s disease (AD). METHODS: This prospective, observational study enrolled patients with early AD undergoing Alzheimer’s biomarker analysis and matched controls. Alzheimer-associated parameters (β-amyloid [Aβ], total tau in cerebrospinal fluid [CSF], Mini-Mental Status Examination [MMSE], etc.), risk factor-associated data (body mass index [BMI], hypertension, lipid profile, etc.), ganglion cell layer plus inner plexiform layer (GCIPL) thickness in structural optical coherence tomography (OCT), OCT angiography data, and FLIO-derived parameters (τ(m), τ(1), τ(2), and τ(3)) in short and long spectral channels (SSC and LSC) were compared and correlated between the two groups. Additional analyses were performed separately within subgroups of phakic and pseudophakic. RESULTS: A total of 28 eyes from 15 subjects (8 control and 7 AD) were included in this analysis. In FLIO parameters, τ(m) in AD group showed longer lifetimes compared to the controls in phakic subjects (593.9 ± 93.3, 454.4 ± 38.6 ps; 475.0 ± 71.6, 394.1 ± 28.2 ps in SSC and LSC of AD and control groups, respectively, p = 0.036 and 0.024). Aβ, tau in CSF, and GCIPL thickness correlated with τ(m) in the LSC for phakic subjects (r = −0.611 to 0.562, p

Subretinal injection (SRI) is a widely used technique in retinal research and can be used to deliver nucleic acids, small molecules, macromolecules, viruses, cells or biomaterials such as nanobeads. Here we describe how to undertake SRI of mice. This protocol was adapted from a technique initially described for larger animals. Although SRI is a common procedure in eye research laboratories, there is no published guidance on the best practices for determining what constitutes a 'successful' SRI. Optimal injections are required for reproducibility of the procedure and, when carried out suboptimally, can lead to erroneous conclusions. To address this issue, we propose a standardized protocol for SRI with 'procedure success' defined by follow-up examination of the retina and the retinal pigmented epithelium rather than solely via intraoperative endpoints. This protocol takes 7-14 d to complete, depending on the reagent delivered. We have found, by instituting a standardized training program, that trained ophthalmologists achieve reliable proficiency in this technique after ~350 practice injections. This technique can be used to gain insights into retinal physiology and disease pathogenesis and to test the efficacy of experimental compounds in the retina or retinal pigmented epithelium.

Nonfibrillar amyloid-β oligomers (AβOs) are a major component of drusen, the sub-retinal pigmented epithelium (RPE) extracellular deposits characteristic of age-related macular degeneration (AMD), a common cause of global blindness. We report that AβOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AβOs induce activation of the NLRP3 inflammasome in the mouse RPE in vivo and that RPE expression of the purinergic ATP receptor P2RX7, an upstream mediator of NLRP3 inflammasome activation, is required for AβO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors—nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines—both inhibit AβOs-induced RPE degeneration. These findings crystallize the importance of P2RX7 and NLRP3 in a disease-relevant model of AMD and identify inflammasome inhibitors as potential treatments for GA.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1–reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493–0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Significance Alu elements, comprising more than 10% of the human genome, propagate via retrotransposition. This genomic expansion requires enzymatic activity of L1 that reverse transcribes Alu RNA into Alu cDNA in the nucleus. We report Alu also undergoes L1-mediated reverse transcription via self-priming in the cytoplasm independent of retrotransposition, providing evidence of human DNA synthesis in this cellular compartment. This newly discovered shunt molecule in the Alu replication cycle also induces death of the retinal pigmented epithelium, a hallmark of atrophic age-related macular degeneration. A Big Data Archeology analysis of multiple health insurance databases reveals that use of FDA-approved nucleoside reverse transcriptase inhibitors is associated with protection against macular degeneration, identifying a repurposing candidate for this blinding disease.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no FDA-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 years of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo, and that it inhibits activation of the NLRP3-ASC inflammasome in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other anti-depressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies triangulate to link fluoxetine as a potential drug repurposing candidate for a major unmet medical need that causes blindness in millions of people in the United States and across the world.Significance StatementDry age-related macular degeneration (AMD) affects the vision of millions of people worldwide. There is currently no FDA-approved treatment for dry AMD. The inflammasome components NLRP3 and ASC have been implicated in the pathogenesis of dry AMD. We report that fluoxetine, which is approved for the treatment of clinical depression, directly binds the NLRP3 protein and prevents the assembly and activation of the NLRP3-ASC inflammasome. As a result, it also blocks the degeneration of retinal pigmented epithelium (RPE) cells in an animal model of dry AMD. Furthermore, we demonstrate through an analysis of health insurance databases that use of this FDA-approved anti-depressant drug is associated with reduced incidence of dry AMD. These studies identify that fluoxetine is a potential repurposing candidate for AMD, a prevalent cause of blindness.

PURPOSE: To evaluate the effect of averaging en-face optical coherence tomography angiography (OCTA) images on quantitative measurements of the retinal microvasculature and their correlation to diabetic retinopathy (DR) disease severity. DESIGN: Cross-sectional cohort study PARTICIPANTS: 105 eyes (65 patients) with 28 eyes from 19 healthy, aged-matched controls, 14 eyes from 9 diabetics without DR and 63 eyes from 37 diabetics with varying levels of DR. METHODS: Spectral-domain OCTA images with uniform illumination, good foveal centration, and no macular edema or significant motion artifact were acquired 5 times with the 3×3 mm scan pattern on the CIRRUS™ 5000 HD-OCT with AngioPlex (Carl Zeiss Meditec, Dublin, CA) software. En-face images of the superficial retinal layer (SRL) and deep retinal layer (DRL) were registered and averaged. Included eyes had a signal strength ≥7. Vessel length density (VLD), perfusion density (PD) and foveal avascular zone (FAZ) parameters were measured on averaged versus single OCTA images. Pearson correlation coefficient compared the two groups. Univariate and multivariate linear regression correlated quantitative metrics to DR severity and best corrected visual acuity (BCVA). RESULTS: 84 eyes (55 patients) met the inclusion criteria. Almost uniformly, lower VLD and PD parameters were significantly associated with worse DR severity and BCVA. Multivariable linear regression for DR severity resulted in an R(2) value of 0.82 and 0.77 for single and averaged groups, respectively. No variables remained significantly associated with DR severity in multivariate analysis with single images but in averaged images, increased superior SRL PD significantly predicted worse DR severity (coefficient 52.7, p=0.026). Multivariate linear regression for BCVA had an R(2) value of 0.42 and 0.47 for single and averaged groups, respectively. FAZ size was not associated with DR severity when single OCTA images (p=0.98) were considered, but was highly associated when using averaged images (coefficient 6.18, p

PURPOSE To evaluate corneal epithelial thickness (CET) and corneal thickness (CT) in healthy eyes using spectral domain optical coherence tomography. METHODS Thirty-six healthy eyes were imaged using the Cirrus high-definition (HD)-optical coherence tomography device. The average CET and CT were assessed using Cirrus Review Software within predefined concentric corneal ring-shaped zones. Specific regions of CET (superior, inferior, temporal, nasal, superonasal, inferotemporal, superotemporal, and inferonasal) were also assessed. The difference between zones was compared between males and females. RESULTS The average CET was 48.3, 47.1, 46.1, and 45.8 μm in the 4 concentric zones (0-2, 2-5, 5-7, and 7-9 mm), respectively (P < 0.001). The average CT was 533.5, 550.8, and 579.4 μm in the 3 zones (0-2, 2-5, and 5-7 mm), respectively (P < 0.001). There was no statistically significant correlation between CET and CT in any of the measured zones. Males had thicker corneas than did females in each of the 3 CT zones (P < 0.05), but CET did not differ significantly. The CET superonasal-inferotemporal in 2.0 to 5.0 mm and CET superotemporal-inferonasal in 5.0- to 7.0-mm zones were significantly thinner in males than in females (-1.15 vs. 0.9 μm, -3.5 vs. -1.9 μm), respectively (P < 0.05). CONCLUSIONS Optical coherence tomography-based analysis of CET reveals that it is thinner in the periphery, whereas the total corneal thickness is greater. Although total CT seems to be influenced by sex, CET is not. Regional and sex-based variations in CT may need to be considered when assessing corneal and epithelial alterations in the setting of disease.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by death of the retinal pigmented epithelium (RPE). In this disease, the RPE displays evidence of DICER1 deficiency, resultant accumulation of endogenous Alu retroelement RNA, and NLRP3 inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human cell culture and in mouse models is driven by a non-canonical inflammasome pathway that results in activation of caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β (IFN-β) production and gasdermin D-dependent interleukin-18 (IL-18) secretion. Reduction of DICER1 levelsor accumulation of Alu RNA triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, IFN-β, and cGAS levels are elevated in the RPE of human eyes with geographic atrophy. Collectively, these data highlight an unexpected role for cGAS in responding to mobile element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial damage-induced inflammasome activation, expand the immune sensing repertoire of cGAS and caspase-4 to non-infectious human disease, and identify new potential targets for treatment of a major cause of blindness.

Objective To evaluate the agreement, reproducibility, and repeatability of 2 spectral domain optical coherence tomography (SD-OCT) devices in Schwalbe's line (SL)–based anterior chamber angle parameters. Methods The inferior anterior chamber angle of 65 eyes from 65 participants (33 right eyes and 32 left eyes) were scanned twice with the Nidek RS 3000 Advanced SD-OCT and Cirrus SD-OCT. SL angle opening distance (SL-AOD) and SL trabecular-iris-space area (SL-TISA) were graded by masked certified graders at the Doheny Image Reading Center. Results The mean SL-AOD/SL-TISA was 617.3 ± 237.9 µm/0.211 ± 0.086 mm 2 for the Cirrus and 633.7 ± 219.3 µm/0.218 ± 0.080 mm 2 for the Nidek RS 3000 Advanced SD-OCT. The repeatability (intraclass correlation coefficients [ICCs] >0.936) and intergrader reproducibility (ICCs >0.915) in SL-AOD and SL-TISA with Cirrus OCT were excellent. The repeatability (ICCs >0.948) and intergrader reproducibility (ICCs >0.709) in SL-AOD and SL-TISA with the Nidek RS 3000 Advanced SD-OCT were moderate to good. Moderate agreement between the 2 devices was also documented with a mean difference of −15.3 (limits of agreement [LoA] −246.5 to 277.1) mm for SL-AOD and 0.006 (LoA −0.096 to 0.108) mm in SL-TISA. Conclusion Both devices were able to provide consistent angle measurements, but repeatability and reproducibility were better in Cirrus SD-OCT than in Nidek RS 3000 Advanced SD-OCT.

To evaluate retinal vessel quantity within various retinal structural layers using optical coherence tomography angiography (OCTA).In this IRB-approved study, 22 normal eyes (from 22 subjects) were imaged using the Spectralis OCT2, with a 15 × 15 degree OCTA scan centered on fovea and two additional 15 × 5 degree OCTA scans, displaced temporally and nasally by 15 degrees along the fovea-Bruch's membrane opening (BMO) axis. Following projection artifact removal (PAR), vessel quantity (i.e., amount of flow signal) within each retinal nuclear and plexiform layer was assessed across the scan and was plotted as a vessel quantity profile over this fovea-BMO axis. Vessel quantity was correlated against the retinal layer thickness at the corresponding locations using the Spearman correlation.For the nerve fiber layer (NFL), the vessel quantity was highest nasally and declined towards the fovea and was near zero temporal to the fovea with or without PAR. For all other retinal layers, the retinal vessel quantities were greatest in the parafoveal retina, peaking approximately 5 degrees from the foveal center. Before PAR, the parafoveal vessel quantity was highest in the inner plexiform layer (IPL). Following PAR, the vessel quantity in the IPL decreased but was relatively unchanged in the other layers. The vessel quantity correlated moderately well with retinal layer thickness (r = 0.432 to 0.511; P0.05 among the various layers).Retinal vessel quantity varies significantly among the various structural layers, with significant regional variability. Projection artifact can significantly impact retinal vessel quantity in the deeper layers, but the effect appears to be most pronounced in the IPL.

To test the effect of ambient illumination scaling on the reproducibility and reliability anterior chamber metrics using the Visante time domain optical coherence tomography (TD-OCT) instrument.The inferior irido-corneal angles of 25 normal, healthy eyes were imaged twice with the Zeiss Visante TD-OCT under five strictly controlled ambient light conditions (foot candles (fc) measured with a light meter at camera/eye interface). Each eye was imaged 10 times totaling 250 assessments. Angle opening distance (AOD500/750), trabecular iris space area (TISA500/750), and scleral spur (SS) angle were graded twice by masked, trained graders at the Doheny Imaging Reading Center using the Visante's intrinsic tools. Lighting effects on measurements, intra-/inter-grader and acquisition analyses, and Bland-Altman plots were computed using Statistical Package for Social Science (SPSS Inc. version 18.0, Armonk, NY).With a near linear relationship of angle metrics to lights levels (RThe results of this study show the high sensitivity of the anterior chamber to changes in the illumination. The slight decrease in light had a corresponding large decrease in Anterior Chamber Angle (ACA) metrics. With clinical diagnoses and treatments of eye diseases relying on these angle measurements, these findings emphasize the importance of strictly controlling light conditions in order to obtain reproducible measurements of anterior chamber geometry.

BACKGROUND Evaluate the impact of variations in ambient lighting conditions on the reproducibility/reliability of Schwalbe's Line (SL)-based anterior chamber angle (ACA) metrics using spectral domain optical coherence tomography (SD-OCT). DESIGN Images were taken at Doheny Eye Centers-UCLA clinic, which were randomized, masked and graded twice by dual reading centre graders. PARTICIPANTS Twenty-five normal/healthy participants with open angles METHODS Inferior angles were imaged using Cirrus SD-OCT under five light levels (foot-candles (fc) measured at camera-eye interface with Sper light-meter) with the instrument's corneal illumination function set to default level of 50 (CIdef) and low 5 (CIlow). Each eye was imaged 20 times, totaling 500 assessments. MAIN OUTCOME MEASURES SL-angle-opening-distance (SL-AOD) and SL-trabecular-iris-space-area (SL-TISA) measured using custom ImageJ software. Intra-/inter-grader variability analyses were completed using Statistical-Package-for-Social-Science and Bland-Altman plots demonstrated limits of agreement for comparisons. RESULTS Light level demonstrated a linear relationship with angle size, thus differences from highest to lowest light levels were compared. Decreasing light from 1.0 → 0.0fc at CIdef decreased SL-AOD from 394 µm +/- 137 µm → 356 µm +/- 137 µm (mean percent difference (MPD) = 10.71%,P

Purpose Azidothymidine (AZT), a nucleoside reverse transcriptase inhibitor, possesses anti-inflammatory and anti-angiogenic activity independent of its ability to inhibit reverse transcriptase. The aim of this study was to evaluate the efficacy of 5'-glucuronyl azidothymidine (GAZT), an antiretrovirally inert hepatic clinical metabolite of AZT, in mouse models of retinal pigment epithelium (RPE) degeneration and choroidal neovascularization (CNV), hallmark features of dry and wet age-related macular degeneration (AMD), respectively. Methods RPE degeneration was induced in wild-type (WT) C57BL/6J mice by subretinal injection of Alu RNA. RPE degeneration was assessed by fundus photography and confocal microscopy of zonula occludens-1-stained RPE flat mounts. Choroidal neovascularization was induced by laser injury in WT mice, and CNV volume was measured by confocal microscopy. AZT and GAZT were delivered by intravitreous injections. Inflammasome activation was monitored by western blotting for caspase-1 and by ELISA for IL-1β in Alu RNA-treated bone marrow-derived macrophages (BMDMs). Results GAZT inhibited Alu RNA-induced RPE degeneration and laser-induced CNV. GAZT also reduced Alu RNA-induced caspase-1 activation and IL-1β release in BMDMs. Conclusions GAZT possesses dual anti-inflammatory and anti-angiogenic properties and could be a viable treatment option for both forms of AMD.

Purpose To demonstrate the effect of averaging multiple en face optical coherence tomography angiography images on the correlation between retinal microvasculature quantitative metrics and best-corrected visual acuity (BCVA) in eyes with retinal vein occlusion. Methods A cross-sectional cohort with unilateral retinal vein occlusion was imaged in both eyes. Five 3 mm × 3-mm spectral domain optical coherence tomography angiography images were averaged, and quantitative parameters from averaged versus single images were correlated with logMAR BCVA. Regression analyses were performed to correlate quantitative metrics with BCVA. Results Ten patients (5 male, average age 64.3 years) were included. Among retinal vein occlusion eyes, vessel length density was significantly less in averaged versus a single image for both the superficial retinal layer (15.5 ± 2.5 vs. 17.8 ± 2.4/mm, P = 0.05) and deep retinal layer (16.2 ± 1.4 vs. 18.5 ± 1.6/mm, P = 0.003). Multivariate linear regression showed an increased R value with averaging (0.93 to 0.95, for single and averaged groups, respectively). Foveal avascular zone circularity was associated with BCVA on single images (coefficient = -0.96, P = 0.002), but not with averaged images (P = 0.063). Conclusion Scan averaging of en face optical coherence tomography angiography images improves the clarity of vessels and may allow for more accurate quantification of vessel metrics. Quantitative metrics are significantly associated with BCVA, and averaging does not further improve this association compared with single-scan analysis.

To investigate the effects of ambient light level variation on spectral domain anterior segment optical coherence tomography (SD-ΟCT)-derived anterior chamber angle metrics in Caucasians versus Asians.Caucasian (n = 24) and Asian participants of Chinese ancestry (n = 24) with open angles on gonioscopy had one eye imaged twice at five strictly controlled, ambient light levels. Ethnicity was self-reported. Light levels were strictly controlled using a light meter at 1.0, 0.75, 0.5, 0.25, and 0 foot candle illumination levels. SD-OCT 5-line raster scans at the inferior 270° irido-corneal angle were measured by two trained, masked graders from the Doheny Image Reading Center using customized Image-J software. Schwalbe's line-angle opening distance (SL-AOD) and SL-trabecular iris space area (SL-TISA) in different light meter readings (LMRs) between the two groups were compared.Baseline light SL-AOD and SL-TISA measured 0.464 ± 0.115mm/0.351 ± 0.110mmSL-based angle dimensions using SD-OCT are sensitive to changes in ambient illumination in participants with Caucasian and Asian ancestry. Although Caucasian eyes had larger baseline angle opening under bright light conditions, the light-to-dark change in angle dimensions was similar in the two groups.

Purpose: To compare anterior chamber angle parameters based on the location of Schwalbe line (SL) from 2 spectral domain optical coherence tomography (SD-OCT) instruments and to measure their reproducibility. Methods: Forty-two eyes from 21 normal, healthy participants underwent imaging of the inferior irido-corneal angle with the Spectralis and Cirrus SD-OCT under tightly controlled low-light conditions. SL-angle opening distance (SL-AOD) and SL-trabecular iris space area (SL-TISA) were measured by masked, certified graders at the Doheny Imaging Reading Center using customized grading software. Interinstrument and intrainstrument, as well as interobserver and intraobserver reproducibility of SL-AOD and SL-TISA measurements were evaluated by intraclass correlation coefficients (ICCs) and Bland-Altman plots with limits of agreement (LoA). Results: The mean SL-AOD was 0.662±0.191 mm in Spectralis and 0.677±0.213 mm in Cirrus. The mean SL-TISA was 0.250±0.073 mm2 in Spectralis and 0.256±0.082 mm2 in Cirrus. The agreement for intrainstrument (ICCs>0.979), intragrader (ICCs>0.992), and intergrader (ICCs>0.929) was excellent. Excellent agreement between the 2 devices was also documented with a mean difference of −0.016 (LoA −0.125 to 0.092) mm for SL-AOD and −0.007 (LoA −0.056 to 0.043) mm2 in SL-TISA. Conclusions: Both SD-OCTs provided comparable measurements and permitted calculation of SL-based angle metrics. There was excellent interinstrument and intrainstrument and intraobserver and interobserver reproducibility for Spectralis and Cirrus SD-OCTs, suggesting true interchangeability between SD-OCT devices. This has the potential to lead to development of standardized grading assessments and quantification of angle parameters that would be valid across various SD-OCT devices.

Objectives:To determine the repeatability and reproducibility of Central Corneal Thickness (CCT) measurements using two different anterior segment imaging modalities, including those obtained with the new anterior segment lens attachments for the Cirrus 5000 HD-OCT.Methods:A total of 32 eyes from 16 normal volunteers (8 male, 8 female) were enrolled in this prospective study. CCT was measured by the same examiner using the Cirrus 5000 HD-OCT and Pentacam HR. The results of CCT obtained by each method were averaged and compared using t-test analysis. The agreement between the measurement methods was evaluated. Coefficient of Repeatability (CoR) and Intra-Class Correlation Coefficient (ICC) were computed.Results:The mean measurements taken with the Cirrus OCT anterior chamber lens (CCTAC), HD cornea lens (CCTHDC) and pachymetry scans (CCTPach) were 545.35 ± 31.02, 537.87 ± 26.82, and 532.04 ± 29.82 µm, respectively. The mean CCT obtained with the Pentacam (CCTPent) was 545.51 ± 30.71 µm. CCTPentwere significantly higher than CCTHDCand CCTPach(p< 0.0001). In contrast, the CCTPentand CCTACwere similar (p=0.87). CCT, as evaluated by the two different instruments, showed excellent correlation (r> 0.98,p< 0.0001) with an ICC > 0.99 (95% CI, 0.97 – 0.99). CoR was the highest for CCTPach(3.7 ± 1.4, 95% CI (3.0- 4.6)).Conclusion:CCT measurements from the Cirrus OCT using the new anterior segment lens attachments and the Pentacam HR are highly correlated. This should allow the use of a standardized correction factor if necessary to inter-relate the measurements between the two devices.

Background First reported study to assess the effect of diurnal variation on anterior chamber angle measurements, as well as, to re-test the effects of lighting and angle-of-incidence variation on anterior chamber angle (ACA) measurements acquired by time-domain anterior segment optical coherence tomography (AS-OCT). Methods A total of 30 eyes from 15 healthy, normal subjects underwent anterior chamber imaging using a Visante time-domain AS-OCT according to an IRB-approved protocol. For each eye, the inferior angle was imaged twice in the morning (8 am – 10 am) and then again in the afternoon (3 pm – 5 pm), under light meter-controlled conditions with ambient room lighting ‘ON’ and lights ‘OFF’, and at 5° angle of incidence increments. The ACA metrics measured for each eye were: angle opening distance (AOD, measured 500 and 750 μm anterior from scleral spur), the trabecular-iris-space area (TISA, measured 500 and 750 μm anterior from scleral spur), and scleral spur angle. Measurements were performed by masked, certified Reading Center graders using the Visante’s Internal Measurement Tool. Differences in measurements between morning and afternoon, lighting variations, and angle of incidence were compared. Results Mean age of the participants was 31.2 years (range 23–58). Anterior chamber angle metrics did not differ significantly from morning to afternoon imaging, or when the angle of incidence was offset by 5° in either direction away from the inferior angle 6 o’clock position. (p-value 0.13-0.93). Angle metrics at the inferior corneal limbus, 6 o’clock position (IC270), with room lighting ‘OFF’, showed a significant decrease (p

Recombinant human thyrotropin (rhTSH; Thyrogen®) is approved for use in a 0.9 mg dose/day for 2 consecutive days for diagnosis and treatment of differentiated thyroid cancer. It is recommended that it be injected immediately after reconstitution in the distilled water diluent supplied by the manufacturer. However, Thyrogen has been used off-label in doses less than the standard 0.9 mg dose for stimulation of radioiodine uptake in the treatment of multinodular goiter. To determine whether the biologic activity of Thyrogen can be preserved after dilution, we designed experiments to assess the biologic stability of Thyrogen under different durations and temperatures of storage.rhTSH was diluted in 1% bovine serum albumin in phosphate-buffered saline to a concentration of 0.9 mg /mL and further diluted to 0.1 mg/mL. Aliquots of 0.5 mL were stored at room temperature, 4°C, -11°C, and -60°C for various lengths of time. In addition, rhTSH aliquots were also subjected to incubation for 1 hour at 50°C and to 10 cycles of freezing in dry ice alternating with thawing at 37°C. Bioassays were performed in FRTL-5 cells. rhTSH was added to the media at a final concentration of either 5 ng/mL or 20 ng/mL, and the cells were then incubated for 48 hours. Potency was assessed by measurement of ¹²⁵I-iodide uptake in comparison to cells treated with perchlorate to block iodide uptake. Samples were immunoassayed at day 185 of storage.Samples stored at 4°C, -11°C, -60°C, and room temperature retained activity after storage periods of up to 204 days. Samples subjected to 10 freeze-thaw cycles or heated to 50°C for 1 hour retained full biologic activity. Immunoassay at day 185 showed no difference in immunoactivity in relation to the storage condition.rhTSH kept at 4°C, -11°C, -60°C, and room temperature maintained good biologic potency for more than 6 months of storage when tested in vitro, indicating that the biologic activity is very stable. However, altered sialylation occurring during storage could have altered the half-life of rhTSH. Nevertheless, the data provide reassurance that storage in the cold for a few months does not result in significant loss of biologic activity.

Sunitinib malate (Sutent, Pfizer, Inc.; SU11248) is a selective, multitargeted inhibitor of receptor tyrosine kinases and has been shown to inhibit receptors for VEGF, PDGF, KIT, FLT3, and RET. The objective of this study was to determine the effects of sunitinib on signal transduction pathways and on gene expression of iodide-metabolizing proteins in papillary cancer cells with the RET/PTC1 rearrangement.We investigated the effects of sunitinib on cell growth, signal transduction pathways, and thyroid-specific gene expression in papillary thyroid cancer (PTC) cell lines that had the RET/PTC1 rearrangement.Sunitinib inhibited proliferation of RET/PTC1 subclones in a time- and dose-related manner. The mean 50% lethal concentration in the RET/PTC1 subclones was 1.81 microM. Incubation of RET/PTC1 cells with 1 microM sunitinib inhibited their migration potential and transformed their morphology. Sunitinib inhibited RET autophosphorylation at Y1062 and the activation of signal transducer and activator of transcription 3 by blocking Y705 phosphorylation. Sunitinib caused cell cycle arrest in the G0/G1 phase and dephosphorylation of retinoblastoma protein, but did not induce apoptosis. Western blot analysis of the p38, MEK/ERK, and SAPK/JNK mitogen-activated protein kinase signal transduction pathways showed that sunitinib blocked ERK 1/2 and JNK phosphorylation in the cytoplasm. Sunitinib treatment of RET/PTC1 cell lines, in combination, with forskolin induced expression of the sodium (Na)/iodide (I) symporter (NIS) and the transcription factors that bind the NIS upstream enhancer. Mechanistically, the inhibition of both MEK/ERK and SAPK/JNK cytoplasmic pathways individually and in combination caused an increase in NIS gene expression.Sunitinib appears to target the cytosolic MEK/ERK and SAPK/JNK pathways in the RET/PTC1 cell lines, suggesting that blocking these pathways is at least part of the mechanism by which sunitinib inhibits cell proliferation and causes stimulation of NIS gene expression in RET/PTC1 cells.

The lack of Na(+)/I(-) symporter (NIS) gene expression in some thyroid cancer patients has been a major hurdle that limits the efficacy of standard radioactive iodide therapy. The molecular mechanism that contributes to low NIS expression is not well understood. Activated NIS gene expression is stimulated by thyroid-stimulating hormone-mediated cAMP/protein kinase A signaling through a NIS upstream enhancer (NUE). The cAMP pathway is also stimulated by forskolin. In the current work, we studied the mechanism of transcriptional activation of NIS in normal thyroid cells and thyroid cancer cells. We identified the cAMP response element modulator (CREM) activator as a new component of the transcription complex that is important for NIS gene expression. The CREM complex is seen in the normal thyroid cells and BRAF (V600E) thyroid cancer cells (BHP 17-10) but is missing in rearranged in transformation/papillary thyroid carcinoma-1 rearrangement thyroid cancer cells (BHP 2-7). This complex is believed to be responsible for the loss of NUE activity and reduced NIS expression in the BHP 2-7 cell line. In BHP 2-7 cells, forskolin stimulated the thyroid-specific transcription factor Pax 8, but CREM activator mRNA did not increase, and this produced a small increase in NUE activity. Ectopic expression of CREM activator enhanced activity of the NUE, indicating that CREM is an essential regulator of NIS gene expression.

The aim of this study is to compare a portable spectral domain optical coherence tomography (SD-OCT) device with a non-portable SD-OCT for the identification of anterior chamber angle parameters based on location of Schwalbe's line (SL) and to measure their reproducibility. 99 eyes from 46 normal, healthy participants underwent imaging of the inferior iridocorneal angle with the iVue and Cirrus SD-OCT under well-controlled low-light conditions. SL-angle opening distance (SL-AOD) and SL-trabecular iris space area (SL-TISA) were measured by masked, certified graders at the Doheny Image Reading Center using customized Image J grading software. Inter- and intrainstrument, as well as inter- and intraobserver reproducibility of SL-AOD and SL-TISA measurements were evaluated with intraclass correlation coefficients (ICCs) and Bland-Altman plots with limits of agreement. The mean SL-AOD was 0.814 ± 0.315 mm with the iVue and 0.797 ± 0.294 mm with the Cirrus. The mean SL-TISA was 0.247 ± 0.112 mm2 with iVue and 0.259 ± 0.113 mm2 with Cirrus. Interinstrument correlation coefficients (r) were 0.93 (P 0.95), intragrader (ICCs > 0.93), and intergrader (ICCs > 0.96) was excellent. Excellent agreement between the two devices was also documented with Bland-Altman analysis. Both instruments provide consistent and reproducible measurements of anterior chamber angle metrics.

Evaluate the impact of variations in ambient lighting conditions on the reproducibility/reliability of Schwalbe's Line (SL)-based anterior chamber angle (ACA) metrics using spectral domain optical coherence tomography (SD-OCT).Images were taken at Doheny Eye Centers-UCLA clinic, which were randomized, masked and graded twice by dual reading centre graders.Twenty-five normal/healthy participants with open anglesInferior angles were imaged using Cirrus SD-OCT under five light levels (foot-candles (fc) measured at camera-eye interface with Sper light-meter) with the instrument's corneal illumination function set to default level of 50 (CIdef) and low 5 (CIlow). Each eye was imaged 20 times, totaling 500 assessments.SL-angle-opening-distance (SL-AOD) and SL-trabecular-iris-space-area (SL-TISA) measured using custom ImageJ software. Intra-/inter-grader variability analyses were completed using Statistical-Package-for-Social-Science and Bland-Altman plots demonstrated limits of agreement for comparisons.Light level demonstrated a linear relationship with angle size, thus differences from highest to lowest light levels were compared. Decreasing light from 1.0 → 0.0fc at CIdef decreased SL-AOD from 394 µm +/- 137 µm → 356 µm +/- 137 µm (mean percent difference (MPD) = 10.71%,P 0.001) and SL-TISA from 297 µm(2) +/- 114 µm(2) → 261 µm(2) +/- 109 µm(2) (MPD = 13.7%, P 0.001). Decreasing from 1.0 → 0.0fc at CIlow decreased SL-AOD from 366 µm +/- 136 µm → 329 µm +/- 122 µm (MPD = 10.9%, P 0.001) and SL-TISA from 271 µm(2) +/- 113 µm(2) → 234 µm(2) +/- 98 µm(2) (MPD = 15.8%, P 0.001). There was 7.9/11.4% (both P 0.001) difference for SL-AOD/SL-TISA between CIdef → CIlow at 1.0fc, and 7.7/9.4% (both P 0.001) difference at 0.0fc. Intra-/inter-grader results showed high reproducibility for all metrics (MPD = 0.33-4.4%; CV = 0.96-1.36; PCC = 0.93-0.95(P 0.001); R2 = 0.94-0.98). Bland-Altman plots did not demonstrate bias, with repeat-ability and agreement among measurements.Using Cirrus SD-OCT, we found that SL-based ACA morphometrics are exquisitely sensitive to changes in ambient illumination and also corneal illumination by the OCT instrument. Consistently imaging in the darkest room possible is recommended (≤0.2 fce).

Purpose. To compare the effects of physiologic versus pharmacologic pupil dilation on anterior chamber angle (ACA) measurements obtained with spectral domain optical coherence tomography (SD-OCT).Methods. Forty eyes from 20 healthy, phakic individuals with open angles underwent anterior segment OCT imaging under 3 pupillary states: (1) pupil constricted under standard room lighting, (2) physiologic mydriasis in a darkened room, and (3) postpharmacologic mydriasis. Inferior angle Schwalbe’s line-angle opening distance (SL-AOD) and SL-trabecular-iris-space area (SL-TISA) were computed for each eye and pupillary condition by masked, certified Reading Center graders using customized grading software.Results. SL-AOD and SL-TISA under pupillary constriction to room light were0.87±0.31 mm and0.33±0.14 mm2, respectively; decreased to0.75±0.29 mmP<0.01and0.29±0.13 mm2 P<0.01, respectively, under physiologic mydriasis; and increased to0.90±0.38 mmP<0.01and0.34±0.17 mm2 P=0.06under pharmacologic mydriasis compared to baseline.Conclusions. Using SD-OCT imaging, pharmacologic mydriasis yielded the widest angle opening, whereas physiologic mydriasis yielded the most angle narrowing in normal individuals with open iridocorneal angles. Accounting for the state of the pupil and standardizing the lighting condition would appear to be of importance for future studies of the angle.

Purpose: To investigate the agreement between two spectral domain optical coherence tomography (SD-OCT) instruments in Schwalbe’s line (SL) based anterior chamber angle parameters and to evaluate their repeatability and reproducibility. Methods: The inferior irido-corneal angle of 114 eyes from 59 participants (29 glaucoma and 30 normal) were scanned twice with the Optovue SD-OCT and Cirrus SD-OCT under controlled low luminance conditions. SL angle opening distance (SL-AOD) and SL trabecular-iris-space area (SL-TISA) were graded by masked certified graders at the Doheny Image Reading Center. Results: The mean SL-AOD/SL-TISA was 623±271μm/ 0.221 ± 0.106 mm2 for the Cirrus and 611 ± 267 μm/ 0.215 ± 0.112 mm2 for the RTVue. Excellent repeatability (intraclass correlation coefficient ICC>0.934), excellent intragrader reproducibility (ICC>0.957) and very good intergrader reproducibility (ICC>0.877) were observed with both instruments in SL-AOD and SL-TISA. The agreement between Cirrus and RTVue was excellent (ICC 0.943 for SL-AOD and 0.900 for SL-TISA). Conclusion: Both instruments provide consistent and reproducible measurement of SL-AOD and SL-TISA. The excellent agreement between them allows for direct comparisons of angle measurements acquired with different SDOCTs and can lead in the introduction of criteria that could be valid across different platforms

To evaluate the reproducibility of anterior chamber angle measurements obtained by the Zeiss Visante anterior segment optical coherence tomography (AS-OCT).Twenty eyes from 20 normal subjects with open anterior chamber angles were studied. The anterior chamber angle was imaged using the Visante AS-OCT. The angle-opening distance (AOD 500, AOD 750), trabercular iris space area (TISA 500, TISA 750) and scleral spur angle (SS angle) at the inferior angle location were measured. All the subjects underwent imaging in a darkened room (1 foot candles measured at the eye). Images were graded in a masked fashion by certified Doheny Image Reading Center graders. For intra-grader reproducibility assessments, images were re-graded by the same grader 1 week later after random sorting of images. For inter-grader assessments, a second masked grader independently reviewed the images. Intraclass correlation coefficients (ICC) were used to assess reproducibility.Inferior angle measurements of AOD (500, 750), TISA (500, 750) and SS angle for 20 normal eyes were calculated. The intra-observer ICC calculations showed excellent reproducibility for all measurements (AOD 500 = 0.95, AOD 750 = 0.97, TISA 500 = 0.93, TISA 750 = 0.94, SS = 0.96; p 0.001 for all). The inter-observer ICC calculations showed lower reproducibility for all measurements (AOD 500 = 0.71, p 0.001; AOD 750 = 0.82, p 0.001; TISA 500 = 0.49, p = 0.08; TISA 750 = 0.61, p = 0.02; SS = 0.75).Determination of anterior chamber angle measurements was possible with the time-domain AS-OCT, but only modest inter-observer reproducibility was found even among experienced graders.

Sunitinib, a multitargeted vascular endothelial growth factor and receptor tyrosine kinase inhibitor, causes hypothyroidism in patients who take it for treatment of cancer. Although the pathophysiologic mechanism of the hypothyroidism is unclear, it has been claimed that it is due to inhibition of iodide uptake.To evaluate the pathologic mechanism of induction of the hypothyroidism, we studied the effect of sunitinib on FRTL-5 rat thyroid cells. We measured the effect of sunitinib on cell growth, (125)I-iodide uptake and efflux, TSH receptor (TSH-R), and sodium-iodide symporter (NIS) message.At 48 hours, sunitinib caused a dose-related inhibition of growth with LC(50) of 14.6 muM, but there was no apparent inhibition of growth at 24 hours at concentrations of 0.1-25 microM. Preincubation with sunitinib did not impair the response to TSH, indicating that it did not affect the TSH-R. Incubation with sunitinib for 24 hours caused a dose-related increase of (125)I-iodide uptake and did not reduce iodide efflux or NIS mRNA expression.The data indicate that sunitinib is unlikely to cause hypothyroidism by inhibition of iodide uptake.