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PURPOSE: Validated and accurate prognostic testing is critical for precision medicine in uveal melanoma (UM). Our aims were to (1) prospectively validate an integrated prognostic classifier combining a 15-gene expression profile (15-GEP) and PRAME RNA expression and (2) identify clinical variables that enhance the prognostic accuracy of the 15-GEP/PRAME classifier. MATERIALS AND METHODS: This study included 1,577 patients with UM of the choroid and/or ciliary body who were enrolled in the Collaborative Ocular Oncology Group Study Number 2 (COOG2) and prospectively monitored across 26 North American centers. Test results for 15-GEP (class 1 or class 2) and PRAME expression status (negative or positive) were available for all patients. The primary end point was metastasis-free survival (MFS). RESULTS: 15-GEP was class 1 in 1,082 (68.6%) and class 2 in 495 (31.4%) patients. PRAME status was negative in 1,106 (70.1%) and positive in 471 (29.9%) patients. Five-year MFS was 95.6% (95% CI, 93.9 to 97.4) for class 1/PRAME(-), 80.6% (95% CI, 73.9 to 87.9) for class 1/PRAME(+), 58.3% (95% CI, 51.1 to 66.4) for class 2/PRAME(-), and 44.8% (95% CI, 37.9 to 52.8) for class 2/PRAME(+). By multivariable Cox proportional hazards analysis, 15-GEP was the most important independent predictor of MFS (hazard ratio [HR], 5.95 [95% CI, 4.43 to 7.99]; P < .001), followed by PRAME status (HR, 1.82 [95% CI, 1.42 to 2.33]; P < .001). The only clinical variable demonstrating additional prognostic value was tumor diameter. CONCLUSION: In the largest prospective multicenter prognostic biomarker study performed to date in UM to our knowledge, the COOG2 study validated the superior prognostic accuracy of the integrated 15-GEP/PRAME classifier over 15-GEP alone and clinical prognostic variables. Tumor diameter was found to be the only clinical variable to provide additional prognostic information. This prognostic classifier provides an advanced resource for risk-adjusted metastatic surveillance and adjuvant trial stratification in patients with UM.
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AIM: To evaluate differences in microparticle profiles in vitreous samples between diabetic and non-diabetic eyes undergoing vitrectomy. METHODS: Un-masked cross-sectional series of 34 eyes undergoing vitrectomy. Vitreous specimens were collected and processed to evaluate for membrane integrity (DAPI), apoptosis (Annexin-V), and endothelial-cell origin (V-Cadherin). A BD LSR II flow cytometer was used for analysis and standardized sub-micron-sized beads were used for size comparison. RESULTS: Thirty-four specimens underwent analysis. Greater levels of Annexin-V were found on microparticles from specimens in which blood had entered the vitreous (n=12) compared to those without blood (n=22; 52.3%±30.7% vs 19.6%±27.2%, P=0.002). Patients with diabetes having surgery with hemorrhage (n=7) had greater expression of Annexin-V than those without hemorrhage (n=8; 62.1%±31.7% vs 18.9%±20.9%, P=0.009). However, in patients with non-diabetic vitreous hemorrhage, the level of Annexin-V expression was not significantly different compared to other disease processes (38.6%±25.7%, n=5 vs 20.0%±30.9%, n=14, P=0.087). CONCLUSION: Increased expression of the apoptotic marker, Annexin-V is detected on vitreous microparticles in diabetes-related vitreous hemorrhage. When evaluating vitreous hemorrhage in patients without diabetes, the apoptotic signal is not significantly different. Vitrectomy in patients with diabetes, and improvement in visual outcomes, may be related to the removal of a serum-derived, pro-apoptotic vitreous. Further investigation is warranted in order to identify the molecular characteristics of microparticles that regulate disease.
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Metabolic reprogramming has been shown to occur in uveal melanoma (UM), the most common intraocular tumor in adults. Mechanisms driving metabolic reprogramming in UM are poorly understood. Elucidation of these mechanisms could inform development of new therapeutic strategies for metastatic UM, which has poor prognosis because existing therapies are ineffective. Here, we determined whether metabolic reprogramming is driven by constitutively active mutant α-subunits of the heterotrimeric G proteins Gq or G11 (Gq/11), the oncogenic drivers in â¼90% of UM patients. Using PET-computed tomography imaging, microphysiometry, and GC/MS, we found that inhibition of oncogenic Gq/11 with the small molecule FR900359 (FR) attenuated glucose uptake by UM cells in vivo and in vitro, blunted glycolysis and mitochondrial respiration in UM cell lines and tumor cells isolated from patients, and reduced levels of several glycolytic and tricarboxylic acid cycle intermediates. FR acutely inhibited glycolysis and respiration and chronically attenuated expression of genes in both metabolic processes. UM therefore differs from other melanomas that exhibit a classic Warburg effect. Metabolic reprogramming in UM cell lines and patient samples involved protein kinase C and extracellular signal-regulated protein kinase 1/2 signaling downstream of oncogenic Gq/11. Chronic administration of FR upregulated expression of genes involved in metabolite scavenging and redox homeostasis, potentially as an adaptive mechanism explaining why FR does not efficiently kill UM tumor cells or regress UM tumor xenografts. These results establish that oncogenic Gq/11 signaling is a crucial driver of metabolic reprogramming in UM and lay a foundation for studies aimed at targeting metabolic reprogramming for therapeutic development.
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Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Subunidades alfa de la Proteína de Unión al GTP , Melanoma , Neoplasias de la Úvea , Carcinogénesis , Línea Celular Tumoral , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patología , Neoplasias de la Úvea/metabolismoRESUMEN
Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking. A novel therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins. Here, we have addressed the therapeutic potential of FR for UM. We found that FR inhibited all oncogenic Gq/11 mutants reported in UM. FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few. Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors. FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential. FR administered systemically below its LD50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models and reduced blood pressure transiently. FR did not regress xenografted UM tumors or significantly affect heart rate, liver function, hematopoiesis, or behavior. These results indicated the existence of a therapeutic window in which FR can be explored for treating UM and potentially other diseases caused by constitutively active Gq/11.
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Depsipéptidos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Péptidos Cíclicos/farmacología , Neoplasias de la Úvea/tratamiento farmacológico , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Metástasis de la Neoplasia , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Retinal screening examinations can prevent vision loss resulting from diabetes but are costly and highly underused. We hypothesized that artificial intelligence-assisted nonmydriatic point-of-care screening administered during primary care visits would increase the adherence to recommendations for follow-up eye care in patients with diabetes. DESIGN: Prospective cohort study. PARTICIPANTS: Adults 18 years of age or older with a clinical diagnosis of diabetes being cared for in a metropolitan primary care practice for low-income patients. METHODS: All participants underwent nonmydriatic fundus photography followed by automated retinal image analysis with human supervision. Patients with positive or inconclusive screening results were referred for comprehensive ophthalmic evaluation. Adherence to referral recommendations was recorded and compared with the historical adherence rate from the same clinic. MAIN OUTCOME MEASURE: Rate of adherence to eye screening recommendations. RESULTS: By automated screening, 8.3% of the 180 study participants had referable diabetic eye disease, 13.3% had vision-threatening disease, and 29.4% showed inconclusive results. The remaining 48.9% showed negative screening results, confirmed by human overread, and were not referred for follow-up ophthalmic evaluation. Overall, the automated platform showed a sensitivity of 100% (confidence interval, 92.3%-100%) in detecting an abnormal screening results, whereas its specificity was 65.7% (confidence interval, 57.0%-73.7%). Among patients referred for follow-up ophthalmic evaluation, the adherence rate was 55.4% at 1 year compared with the historical adherence rate of 18.7% (P < 0.0001, Fisher exact test). CONCLUSIONS: Implementation of an automated diabetic retinopathy screening system in a primary care clinic serving a low-income metropolitan patient population improved adherence to follow-up eye care recommendations while reducing referrals for patients with low-risk features.
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Instituciones de Atención Ambulatoria , Inteligencia Artificial , Retinopatía Diabética/diagnóstico , Procesamiento de Imagen Asistido por Computador/métodos , Tamizaje Masivo/métodos , Atención Primaria de Salud/métodos , Retina/diagnóstico por imagen , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
In humans, midget and parasol ganglion cells account for most of the input from the eyes to the brain. Yet, how they encode visual information is unknown. Here, we perform large-scale multi-electrode array recordings from retinas of treatment-naive patients who underwent enucleation surgery for choroidal malignant melanomas. We identify robust differences in the function of midget and parasol ganglion cells, consistent asymmetries between their ON and OFF types (that signal light increments and decrements, respectively) and divergence in the function of human versus non-human primate retinas. Our computational analyses reveal that the receptive fields of human midget and parasol ganglion cells divide naturalistic movies into adjacent spatiotemporal frequency domains with equal stimulus power, while the asymmetric response functions of their ON and OFF types simultaneously maximize stimulus coverage and information transmission and minimize metabolic cost. Thus, midget and parasol ganglion cells in the human retina efficiently encode our visual environment.
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Potenciales de Acción/fisiología , Células Ganglionares de la Retina/fisiología , Vías Visuales/fisiología , Neoplasias de la Coroides/fisiopatología , Neoplasias de la Coroides/cirugía , Dendritas/fisiología , Humanos , Melanoma/fisiopatología , Melanoma/cirugíaAsunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Cámara Anterior/diagnóstico por imagen , Degeneración Macular/tratamiento farmacológico , Femenino , Humanos , Presión Intraocular/fisiología , Inyecciones Intravítreas , Degeneración Macular/diagnóstico , Degeneración Macular/fisiopatología , Masculino , Persona de Mediana Edad , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
BACKGROUND AND OBJECTIVE: To evaluate the vitreomacular interface and its relation to treatment burden for diabetic macular edema (DME) in patients without overt vitreomacular traction (VMT). PATIENTS AND METHODS: A retrospective cohort study of 494 eyes from 274 patients who had macular spectral-domain optical coherence tomography (SD-OCT) and did not have proliferative diabetic retinopathy, DME, or VMT at the initial visit. Posterior vitreous detachment (PVD) was categorized at the initial visit into five stages (0-4) using SD-OCT parameters alone. RESULTS: Two of 34 eyes (6.9%) presenting with a complete PVD required DME treatment during follow-up, whereas 144 of 460 eyes (31.3%) without a complete PVD at baseline required treatment (P = .001, Chi-squared). After adjusting for age, ethnicity, gender, and HbA1c, complete PVD at baseline was associated with a significant reduction in risk of DME therapy (hazard ratio: 0.18; 95% confidence interval, 0.05-0.73; P = .02). CONCLUSION: Complete PVD is independently associated with a reduced need for DME treatment. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:e266-e273.].
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Inhibidores de la Angiogénesis/uso terapéutico , Retinopatía Diabética/tratamiento farmacológico , Edema Macular/tratamiento farmacológico , Desprendimiento del Vítreo/patología , Anciano , Anciano de 80 o más Años , Humanos , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodosAsunto(s)
Antirreumáticos/toxicidad , Hidroxicloroquina/toxicidad , Lupus Eritematoso Sistémico/tratamiento farmacológico , Retina/efectos de los fármacos , Enfermedades de la Retina/inducido químicamente , Adulto , Femenino , Angiografía con Fluoresceína , Humanos , Retina/diagnóstico por imagen , Enfermedades de la Retina/diagnóstico por imagen , Tomografía de Coherencia Óptica , Agudeza Visual/efectos de los fármacosRESUMEN
PURPOSE: With new compounding pharmacy laws, the only economically feasible approach to using lissamine is through dye-impregnated strips. This research aims to determine the concentration of lissamine that can be obtained using a single commercially available lissamine strip. With the optimal vital staining requiring 1% concentration of lissamine, we sought to obtain this concentration using supplies in an ordinary ophthalmology clinic. METHODS: A standard curve was generated using compounded lissamine green 1% solution. Serial dilutions were made with 3 different diluents and measured using a spectrophotometer at a wavelength of 633 µm. Combinations of the number of strips, amount of solvent, and absorption time were performed to obtain a 1% solution. Cost analyses were performed to select the most economical method. RESULTS: Single lissamine strips wetted with any of the diluents produced 0.17% ± 0.05% (95% confidence interval) lissamine solution, a 5-fold weaker concentration than the optimal for vital staining. Combinations of 4 strips in 200 µL (4 drops) for 1 minute and 2 strips in 200 µL for 5 minutes were found to reach concentrations of 1%. Cost analysis showed that the 2 strip/4 drops/5 minutes method costs $0.67 and the 4 strips/4 drops/1 minute method $1.27. CONCLUSIONS: Use of a single lissamine strip leads to suboptimal concentrations for vital staining. With only the addition of disposable microcentrifuge tubes to the clinical setting, ophthalmologists can make 1% solutions of lissamine. This solution is both more economical and in compliance with both state and national compounding laws.
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Colorantes/análisis , Composición de Medicamentos , Colorantes Verde de Lisamina/análisis , Tiras Reactivas/química , Técnicas de Diagnóstico Oftalmológico , Humanos , Preparaciones Farmacéuticas/químicaRESUMEN
BACKGROUND: Giant cell arteritis is a granulomatous vasculitis of the aorta and its branches that causes blindness, stroke, and aortic aneurysm. CD4 T cells are key pathogenic regulators, instructed by vessel wall dendritic cells to differentiate into vasculitic T cells. The unique pathways driving this dendritic cell-T-cell interaction are incompletely understood, but may provide novel therapeutic targets for a disease in which the only established therapy is long-term treatment with high doses of corticosteroids. METHODS AND RESULTS: Immunohistochemical and gene expression analyses of giant cell arteritis-affected temporal arteries revealed abundant expression of the NOTCH receptor and its ligands, Jagged1 and Delta1. Cleavage of the NOTCH intracellular domain in wall-infiltrating T cells indicated ongoing NOTCH pathway activation in large-vessel vasculitis. NOTCH activation did not occur in small-vessel vasculitis affecting branches of the vasa vasorum tree. We devised 2 strategies to block NOTCH pathway activation: γ-secretase inhibitor treatment, preventing nuclear translocation of the NOTCH intracellular domain, and competing for receptor-ligand interactions through excess soluble ligand, Jagged1-Fc. In a humanized mouse model, NOTCH pathway disruption had strong immunosuppressive effects, inhibiting T-cell activation in the early and established phases of vascular inflammation. NOTCH inhibition was particularly effective in downregulating Th17 responses, but also markedly suppressed Th1 responses. CONCLUSIONS: Blocking NOTCH signaling depleted T cells from the vascular infiltrates, implicating NOTCH- NOTCH ligand interactions in regulating T-cell retention and survival in vessel wall inflammation. Modulating the NOTCH signaling cascade emerges as a promising new strategy for immunosuppressive therapy of large-vessel vasculitis.
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Proteínas de Unión al Calcio , Dipéptidos/farmacología , Arteritis de Células Gigantes , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/metabolismo , Receptor Notch1 , Transducción de Señal/efectos de los fármacos , Traslado Adoptivo , Animales , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Células Dendríticas/inmunología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Arteritis de Células Gigantes/tratamiento farmacológico , Arteritis de Células Gigantes/inmunología , Arteritis de Células Gigantes/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteína Jagged-1 , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones SCID , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/inmunología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Quimera por Trasplante , Trasplante HeterólogoRESUMEN
Immune-mediated damage to medium-sized arteries results in wall remodeling with intimal hyperplasia, luminal stenosis and tissue ischemia. In the case of the aorta, vasculitis may result in dissection, aneurysm or rupture. The response-to-injury program of the blood vessel is a concerted action between the immune system and wall-resident cells, involving the release of growth and angiogenic factors from macrophages and giant cells and the migration and hyperproliferation of vascular smooth muscle cells. Innate immune cells, specifically, dendritic cells (DC) positioned in the vessel wall, have been implicated in the earliest steps of vasculitis. Pathogen-derived molecular patterns are capable of activating vascular DC and initiating adaptive immune responses. The pattern of the emerging vessel wall inflammation is ultimately determined by the initial insult. Ligands to toll-like receptor (TLR) 4, such as lipopolysaccharides, facilitate the recruitment of CD4 T cells that invade deep into the wall and distribute in a panarteritic pattern. Conversely, ligands for TLR5 condition vascular DC to support perivasculitic infiltrates. In essence, both innate and adaptive immune reactions collaborate to render the arterial wall susceptible to inflammatory damage. Unique features of the tissue microenvironment, including specialized DC, shape the course of the inflammatory response. Differences in vascular damage pattern encountered in different patients may relate to distinct instigators of vasculitis.
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Vasos Sanguíneos/inmunología , Células Dendríticas/inmunología , Arteritis de Células Gigantes/inmunología , Inflamación/inmunología , Miocitos del Músculo Liso/inmunología , Inmunidad Adaptativa/inmunología , Animales , Presentación de Antígeno/inmunología , Vasos Sanguíneos/metabolismo , Células Dendríticas/metabolismo , Arteritis de Células Gigantes/metabolismo , Humanos , Tolerancia Inmunológica/inmunología , Inflamación/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
Dendritic cells (DCs) shape T-cell response patterns and determine early, intermediate, and late outcomes of immune recognition events. They either facilitate immunostimulation or induce tolerance, possibly determined by initial DC activation signals, such as binding Toll-like receptor (TLR) ligands. Here, we report that DC stimulation through the TLR3 ligand dsRNA [poly(I:C)] limits CD4 T-cell proliferation, curtailing adaptive immune responses. CD4+ T cells instructed by either lipopolysaccharide (LPS) or poly(I:C)-conditioned DCs promptly upregulated the activation marker CD69. Whereas LPS-pretreated DCs subsequently sustained T-cell clonal expansion, proliferation of CD4+ T cells exposed to poly(I:C)-pretreated DCs was markedly suppressed. This proliferative defect required DC-T cell contact, was independent of IFN-alpha, and was overcome by exogenous IL-2, indicating T-cell anergy. Coinciding with the downregulation, CD4+ T cells expressed the inhibitory receptor PD-1. Antibodies blocking the PD-1 ligand PD-L1 restored proliferation. dsRNA-stimulated DCs preferentially induced PD-L1, whereas poly(I:C) and LPS both upregulated the costimulatory molecule CD86 to a comparable extent. Poly(dA-dT), a ligand targeting the cytoplasmic RNA helicase pattern-recognition pathway, failed to selectively induce PD-L1 upregulation, assigning this effect to the TLR3 pathway. Poly(I:C)-conditioned DCs promoted accumulation of phosphorylated SHP-2, the intracellular phosphatase mediating PD-1 inhibitory effects. The ability of dsRNA to bias DC differentiation toward providing inhibitory signals to interacting CD4+ T cells may be instrumental in viral immune evasion. Conversely, TLR3 ligands may have therapeutic value in silencing pathogenic immune responses.