Host cell-type and pathogen-specific immunomodulatory functions of macrophage migration inhibitory factor (MIF) in infectious keratitis.

Therapeutic management of inflammation in infectious keratitis (IK) requires new strategy and targets for selective immunomodulation. Targeting host cell-type specific inflammatory responses might be a viable strategy to curtail unnecessary inflammation and reduce tissue damage without affecting pathogen clearance. This study explores the possibility of pathogen and host cell-type dependent differences in the inflammatory pathways relevant in the pathogenesis of IK. Human corneal epithelial cell line (HCEC) and phorbol 12-myristate-13 acetate (PMA) differentiated THP-1 macrophage line were infected with either Aspergillus flavus conidia or Acanthamoeba castellanii trophozoites and the elicited inflammatory responses were studied in terms of gene expression and secretion of proinflammatory factors interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and an upstream inflammatory regulator and mediator protein-the Macrophage Migration Inhibitory Factor (MIF). Given the pleotropic mode of MIF function in diverse cell types relevant in many human diseases, we tested if MIF driven responses to infection is different in HCECs and THP-1 macrophages by studying its expression, secretion and involvement in inflammation by siRNA mediated knockdown. We also examined IK patient tear samples for MIF levels. Infection with A. flavus or A. castellanii induced IL-8 and TNF-α responses in HCECs and THP-1 macrophages but to different levels. Our preliminary human data showed that the level of secreted MIF protein was elevated in IK patient tear, however, MIF secretion by the two cell types were strikingly different in-vitro, under both normal and infected conditions. We found that HCECs released MIF constitutively, which was significantly inhibited with infection, whereas THP-1 macrophages were stimulated to release MIF during infection. MIF gene expression remained largely unaffected by infection in both the cell lines. Although MIF in HCECs appeared to be intracellularly captured during infection, MIF knockdown in HCECs associated with a partial reduction of the IL-8 and TNF-α expression produced by either of the pathogens, suggesting a pro-inflammatory role for MIF in HCECs, independent of its canonical cytokine like function. In contrast, MIF knockdown in THP-1 macrophages accompanied a dramatic increase in IL-8 and TNF-α expression during A. castellanii infection, while the responses to A. flavus infection remained unchanged. These data imply a host cell-type and pathogen specific distinction in the MIF- related inflammatory signaling and MIF as a potential selective immunomodulatory target in infectious keratitis.

Association between Fluoxetine Use and Overall Survival among Patients with Cancer Treated with PD-1/L1 Immunotherapy.

Checkpoint inhibitors can be a highly effective antitumor therapy but only to a subset of patients, presumably due to immunotherapy resistance. Fluoxetine was recently revealed to inhibit the NLRP3 inflammasome, and NLRP3 inhibition could serve as a target for immunotherapy resistance. Therefore, we evaluated the overall survival (OS) in patients with cancer receiving checkpoint inhibitors combined with fluoxetine. A cohort study was conducted among patients diagnosed with lung, throat (pharynx or larynx), skin, or kidney/urinary cancer treated with checkpoint inhibitor therapy. Utilizing the Veterans Affairs Informatics and Computing Infrastructure, patients were retrospectively evaluated during the period from October 2015 to June 2021. The primary outcome was overall survival (OS). Patients were followed until death or the end of the study period. There were 2316 patients evaluated, including 34 patients who were exposed to checkpoint inhibitors and fluoxetine. Propensity score weighted Cox proportional hazards demonstrated a better OS in fluoxetine-exposed patients than unexposed (HR: 0.59, 95% CI 0.371-0.936). This cohort study among cancer patients treated with checkpoint inhibitor therapy showed a significant improvement in the OS when fluoxetine was used. Because of this study's potential for selection bias, randomized trials are needed to assess the efficacy of the association of fluoxetine or another anti-NLRP3 drug to checkpoint inhibitor therapy.

Outcomes of cataract surgery in patients with Human Immunodeficiency Virus infection in a developing country.

PURPOSE: To report the outcomes of cataract surgery in patients with Human Immunodeficiency Virus (HIV) infection. Setting Tertiary care ophthalmic hospital DESIGN: Retrospective study METHODS: This study included all eyes of patients with known HIV infection undergoing cataract surgery with a minimum follow-up of 6 months between January 2017 and December 2020. Patients who underwent combined surgeries and pediatric patients were excluded from analysis. Data were retrieved from electronic medical records and we documented demographics, history, detailed anterior and posterior segment examination, pre-operative grade and type of cataract, type of surgery done, its complication and post-operative course. All these parameters were recorded at the baseline visit and at 1 month and 6 months postoperatively. RESULTS: One hundred and twenty nine eyes of 107 HIV infected patients that underwent cataract surgery were evaluated. Mature cataract was seen in 31% of the eyes. Features of HIV related uveitis/retinitis were seen in 21 (16.2%) eyes. Phacoemulsification was performed in 44 (34.1%) eyes while manual small incision cataract surgery (MSICS) was done in 85 (65.9%) eyes. Intra-operative complications were encountered in 4 (3.1%) eyes. At the final follow-up, there was a significant improvement in median corrected distance visual acuity (CDVA) from LogMAR 1.08 (5/60) at baseline to LogMAR 0 (6/6) at 6 months follow-up. CONCLUSION: Patients with HIV infection usually present early and with advanced cataracts. Visual outcomes after cataract surgery are generally good but affected by presence of prior HIV related uveitis or retinitis.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

Purpose: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. Methods: To evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA 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. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance. Results: Despite 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 cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (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. Conclusions: This 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 Relevance: Our 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, to the best of our knowledge.

PHACOSIT: A sitting phacoemulsification technique for patients unable to lie down flat during cataract surgery.

Phacoemulsification is routinely performed with the patient lying supine on the surgical table with his or her head flat and facing the overhead microscope. This routine technique can be a challenge in medical conditions such as kyphosis, scoliosis, orthopnea, Meniere's disease, and CNS abnormality. Some cardiovascular and respiratory conditions make the patients breathless when they lie down, whereas other neurological and spinal problem patients are also equally uncomfortable. The only reasonable solution to conduct surgery on a patient who cannot lie down flat on the operating table is to position them face to face in a sitting position. We describe an innovative phacoemulsification technique in a sitting position called "phacosit" in an 80-year-old wheelchair-bound female patient who was denied cataract surgery by other eye surgeons owing to her medical condition.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular 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.

Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy.

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

A Clinical Metabolite of Azidothymidine Inhibits Experimental Choroidal Neovascularization and Retinal Pigmented Epithelium Degeneration.

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-1ß 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-1ß 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.

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice.

Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

Low Fluence Photodynamic Therapy Versus Graded Subthreshold Transpupillary Thermotherapy for Chronic Central Serous Chorioretinopathy: Results From a Prospective Study.

BACKGROUND AND OBJECTIVE: The authors compare low fluence photodynamic therapy (PDT) against graded subthreshold transpupillary thermotherapy (TTT) for chronic central serous chorioretinopathy (CSC). PATIENTS AND METHODS: Forty-two eyes of 42 patients with chronic CSC were included in this prospective, nonrandomized, interventional trial. All patients were offered PDT as an initial intervention. If they declined PDT, TTT was offered. RESULTS: Twenty patients underwent PDT and 22 underwent TTT. The difference in best-corrected visual acuity between the TTT and PDT groups at baseline and at 6 months after treatment was not significant (P = .728). The mean foveal thickness decreased significantly in both groups (P = .001). However, patients in the TTT group required more treatments (P = .013) and longer time for resolution of the CSC (P = .013). CONCLUSIONS: Both PDT and TTT may be equally effective in the treatment of chronic CSC. Graded subthreshold TTT may be a cost-effective and safe alternative for PDT. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:334-338.].

Purtscher-like retinopathy: A rare complication of peribulbar anesthesia.

Purtscher and Purtscher-like retinopathy is a distinctive retinal syndrome characterized by ischemic retinal whitening in a peripapillary pattern. We report a case of Purtscher-like retinopathy in a healthy 64-year-old man after a routine peribulbar anesthetic injection for cataract surgery. Although peribulbar anesthesia is considered to be a safer alternative to retrobulbar anesthesia, it has been associated with unusual but grave complications including central retinal artery occlusion.