Magnetic Hyperthermia in Y79 Retinoblastoma and ARPE19 Retinal Epithelial Cells: Tumor Selective Apoptotic Activity of Iron Oxide Nanoparticle

Purpose: To evaluate selective apoptosis of Y79 retinoblastoma versus ARPE-19 retinal pigment epithelial cells by using different doses of dextran-coated iron oxide nanoparticles (DCIONs) in a magnetic hyperthermia paradigm. Methods: Y79 and ARPE-19 cells were exposed to different concentrations of DCIONs, namely, 0.25, 0.5, 0.75, and 1 mg/ml. After 2 hours of incubation, cells were exposed to a magnetic field with a frequency of 250 kHz and an amplitude of 4 kA/m for 30 minutes to raise the cellular temperature between 42 and 46°C. Y79 and ARPE-19 cells incubated with DCION without magnetic field exposure were used as controls. Cell viability and apoptosis were assessed at 4, 24, and 72 hours after hyperthermia treatment. Results: At 4 hours following magnetic hyperthermia, cell death for Y79 cells was 1%, 8%, 17%, and 17% for 0.25, 0.5, 0.75 and 1 mg/ml of DCION, respectively. Cell death increased to 47%, 59%, 70%, and 75% at 24 hours and 16%, 45%, 50%, and 56% at 72 hours for 0.25, 0.5, 0.75, and 1 mg/ml of DCIONs, respectively. Magnetic hyperthermia did not have any significant toxic effects on ARPE-19 cells at all DCION concentrations, and minimal baseline cytotoxicity of DCIONs on Y79 and ARPE-19 cells was observed without magnetic field activation. Gene expression profiling showed that genes involved in FAS and tumor necrosis factor alpha signaling pathways were activated in Y79 cells following hyperthermia. Caspase 3/7 activity in Y79 cells increased following treatment, consistent with the activation of caspase-mediated apoptosis and loss of cell viability by magnetic hyperthermia. Conclusion: Magnetic hyperthermia using DCIONs selectively kills Y79 cells at 0.5 mg/ml or higher concentrations via the activation of apoptotic pathways. Translational Relevance: Magnetic hyperthermia using DCIONs might play a role in targeted management of retinoblastoma

Selective Death of Y79 Retinoblastoma Cells by Magnetic Hyperthermia via Caspase Dependent Apoptotic Pathway

Purpose : To evaluate the selective death of Y79 retinoblastoma cells using dextran-coated iron oxide nanoparticles in a magnetic hyperthermia paradigm and assess the molecular pathways that play a role in cell death. Methods : Y79 and ARPE-19 cells were exposed to different concentrations of dextran-coated iron oxide magnetic nanoparticles: 250, 500, 750 and 1000 µg/ml. After 2 hours of incubation, cells were exposed to a magnetic field with frequency and intensity values of 250 kHz and 4 kA/m at temperature of 43-44 degreeC for 30 minutes. Hyperthermia induced apoptosis was assessed at 4, 24 and 72 hours after treatment. Transmission electron microscopy was performed to elucidate the cellular uptake and distribution of nanoparticles in Y79 and ARPE-19 cells. Gene expression profiling for apoptosis was performed using RT2 Profiler PCR arrays. Results : At 4 hours, cell death for Y79 cells was estimated to be 16, 16, 18 and 24% for 250, 500, 750 and 1000 µg/ml, respectively. At 24 hours, cell death in Y79 cells was estimated to be 46, 57, 67, and 73% at respective four concentrations. Y79 retinoblastoma cells underwent apoptosis at 72 hours with cell death estimated at 16, 48, 52 and 57% respectively with increasing nanoparticle concentrations. Magnetic hyperthermia did not have a significant toxic effect on ARPE-19 cells for all four concentrations of nanoparticles. Minimal baseline cytotoxicity of nanoparticles was observed without magnetic field activation. Transmission Electron Microscopy images showed that dextran-coated iron oxide nanoparticles were engulfed into the cytoplasm of both Y79 and ARPE-19 cells and specifically located in endosomes. Gene expression profiling showed that genes in the FAS signaling pathway and TNF alpha signaling were activated in Y79 retinoblastoma cells at 24 hours after magnetic hyperthermia. qRT-PCR results showed an increased gene expression of caspase8, caspase10, caspase14, caspase9 and cytochromeC. 48 hours after magnetic hyperthermia, we observed significantly increased caspase 3/7 activity in Y79 cells, confirming that apoptosis through intrinsic and extrinsic pathways were activated in these cells. Conclusions : Magnetic hyperthermia using dextran-coated iron oxide nanoparticles were engulfed by both Y79 and ARPE-19 cells. They satisfactorily and selectively killed Y79 cells at 750 and 1000 µg/ml concentrations via activation of two apoptotic pathways

The effect of social determinants of health on severity of microbial keratitis presentation at a tertiary eye care hospital in Southern India

Purpose: Understanding the association between social determinants of health (SDoHs) and microbial keratitis (MK) can inform underlying risk for patients and identify risk factors associated with worse disease, such as presenting visual acuity (VA) and time to initial presentation. Methods: This was a cross-sectional study was conducted with patients presenting with MK to the cornea clinic at a tertiary care hospital in Madurai, India. Patient demographics, SDoH survey responses, geographic pollution, and clinical features at presentation were collected. Descriptive statistics, univariate analysis, multi-variable linear regression models, and Poisson regression models were utilized. Results: There were 51 patients evaluated. The mean age was 51.2 years (SD = 13.3); 33.3% were female and 55% did not visit a vision center (VC) prior to presenting to the clinic. The median presenting logarithm of the minimum angle of resolution (logMAR) VA was 1.1 [Snellen 20/240, inter-quartile range (IQR) = 20/80 to 20/4000]. The median time to presentation was 7 days (IQR = 4.5 to 10). The average particulate matter 2.5 (PM2.5) concentration, a measure of air pollution, for the districts from which the patients traveled was 24.3 μg/m3 (SD = 1.6). Age- and sex-adjusted linear regression and Poisson regression results showed that higher levels of PM2.5 were associated with 0.28 worse presenting logMAR VA (Snellen 2.8 lines, P = 0.002). Patients who did not visit a VC had a 100% longer time to presentation compared to those who did (incidence rate ratio = 2.0, 95% confidence interval = 1.3–3.0, P = 0.001). Conclusion: Patient SDoH and environmental exposures can impact MK presentation. Understanding SDoH is important for public health and policy implications to mitigate eye health disparities in India

Automatic Classification of Slit-Lamp Photographs by Imaging Illumination

PURPOSEThe aim of this study was to facilitate deep learning systems in image annotations for diagnosing keratitis type by developing an automated algorithm to classify slit-lamp photographs (SLPs) based on illumination technique. METHODSSLPs were collected from patients with corneal ulcer at Kellogg Eye Center, Bascom Palmer Eye Institute, and Aravind Eye Care Systems. Illumination techniques were slit beam, diffuse white light, diffuse blue light with fluorescein, and sclerotic scatter (ScS). Images were manually labeled for illumination and randomly split into training, validation, and testing data sets (70%:15%:15%). Classification algorithms including MobileNetV2, ResNet50, LeNet, AlexNet, multilayer perceptron, and k-nearest neighborhood were trained to distinguish 4 type of illumination techniques. The algorithm performances on the test data set were evaluated with 95% confidence intervals (CIs) for accuracy, F1 score, and area under the receiver operator characteristics curve (AUC-ROC), overall and by class (one-vs-rest). RESULTSA total of 12,132 images from 409 patients were analyzed, including 41.8% (n = 5069) slit-beam photographs, 21.2% (2571) diffuse white light, 19.5% (2364) diffuse blue light, and 17.5% (2128) ScS. MobileNetV2 achieved the highest overall F1 score of 97.95% (CI, 97.94%-97.97%), AUC-ROC of 99.83% (99.72%-99.9%), and accuracy of 98.98% (98.97%-98.98%). The F1 scores for slit beam, diffuse white light, diffuse blue light, and ScS were 97.82% (97.80%-97.84%), 96.62% (96.58%-96.66%), 99.88% (99.87%-99.89%), and 97.59% (97.55%-97.62%), respectively. Slit beam and ScS were the 2 most frequently misclassified illumination. CONCLUSIONSMobileNetV2 accurately labeled illumination of SLPs using a large data set of corneal images. Effective, automatic classification of SLPs is key to integrating deep learning systems for clinical decision support into practice workflows