Combined VEGF/PDGF inhibition using axitinib induces αSMA expression and a pro-fibrotic phenotype in human pericytes.

PURPOSE: Large trials on anti-VEGF/PDGF (vascular endothelial/platelet-derived growth factor) combination therapy have been established to improve management of neovascular activity in age-related macular degeneration. Targeting pericytes, PDGF is thought to induce vessel regression and reduce fibrovascular scarring. The fate of pericytes exposed to anti-VEGF/PDGF combination therapy is not clear. Therefore, this study was designed to study the influence of anti-VEGF/PDGF on pericyte phenotype and cellular behavior. METHODS: Human pericytes from placenta (hPC-PL) were treated with axitinib, a tyrosine kinase inhibitor targeting VEGFR1-3 and PDGFR. Toxic effects were excluded using live/dead staining. Phenotypic changes were evaluated using phalloidin staining for actin cytoskeleton and the expression of stress fibers. MRNA and protein expression levels of α-smooth muscle actin (αSMA) as a marker of proto-myofibroblastic transition were evaluated with real-time PCR and Western blotting. Influences of fibrotic cellular mechanisms were evaluated with a scratch wound migration and a collagen gel contraction assay. RESULTS: Treatment with 0.5, 1, and 2.5 µg/ml axitinib strongly induced a proto-myofibroblast-like actin cytoskeleton with a marked increase in stress fibers. Quantitative real-time PCR and Western blotting revealed these changes to be linked to dose-dependent increases in αSMA mRNA and protein expression. However, fibrotic cellular mechanisms were significantly reduced in the presence of axitinib (scratch wound closure: up to - 78.4%, collagen gel contraction: up to - 37.4%). CONCLUSIONS: Combined anti-VEGF/PDGF inhibition seems to induce a proto-myofibroblast-like phenotype in human pericytes in vitro, but reduce profibrotic cellular mechanisms due to prolonged anti-PDGF inhibition.

Combined VEGF and PDGF inhibition for neovascular AMD: anti-angiogenic properties of axitinib on human endothelial cells and pericytes in vitro.

PURPOSE: Drugs currently approved for neovascular age-related macular degeneration (nAMD) offer anti-VEGF monotherapy only. Platelet-derived growth factor (PDGF) signaling is pivotal to pericyte-induced stabilization of choroidal neovascularizations (CNV), and causes partial anti-VEGF resistance. No combination therapy for VEGF and PDGF has been approved yet. Axitinib is a tyrosine kinase inhibitor interfering with VEGF and PDGF signaling, and has been approved for the treatment of renal cell carcinoma. This study evaluates anti-angiogenic properties of axitinib in an in-vitro model of choroidal neovascularizations in nAMD. METHODS: Human endothelial cells (HUVEC) and human pericytes (hPC-PL) were treated with axitinib doses ranging from 1.0 ng/ml to 10 µg/ml. Cellular viability and proliferation were assessed with a modified MTT assay. VEGF- and PDGF-stimulated migration was observed in modified Boyden chambers. Formation of capillary structures was evaluated on Cultrex basement membrane. RESULTS: Proliferation was significantly inhibited in both cell lines in a dose-dependent manner. VEGF and PDGF significantly induced, whereas simultaneous axitinib normalized cellular migration in HUVEC and pericytes. On growth-factor-reduced Cultrex, VEGF induced the formation of capillary structures, while axitinib significantly reverted this effect. Axitinib reduced the amount of vessel associated tissue on full growth factor Cultrex. All effects on both cell lines were observed in non-toxic concentrations of axitinib. CONCLUSIONS: Axitinib inhibits angiogenesis in endothelial cells and pericytes via VEGFR and PDGFR modulation in vitro. Further studies are needed to elucidate whether axitinib may also improve therapy of CNV in AMD in vivo by interference with pericyte stabilization of pathological vessels.

A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

PURPOSE: To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. METHODS: Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. RESULTS: Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. CONCLUSIONS: This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

EGFR inhibitor Gefitinib attenuates posterior capsule opacification in vitro and in the ex vivo human capsular bag model.

PURPOSE: Posterior capsule opacification (PCO) occurs as a common complication after cataract surgery. Gefitinib is a selective inhibitor of the epidermal growth factor receptor (EGFR) which represents a potential pharmacological target for PCO prevention. In this in vitro study, we assessed the effect and biocompatibility of Gefitinib in PCO prophylaxis. METHODS: The effect of Gefitinib on the key pathological features of PCO was assessed in vitro. We determined growth in the human capsular bag model, prepared from sixteen cadaver eyes that underwent sham cataract surgery. Furthermore, two lens epithelial cell lines, HLE-B3 and FHL-124, were used to determine concentration-based effects on cell proliferation. In addition, cell-migration, matrix-contraction, and cell spreading were investigated. To exclude toxic concentrations, Gefitinib was assessed for its biocompatibility on six different human ocular cell types from the anterior and posterior segment of the eye. RESULTS: Gefitinib significantly increased the time until confluence of the capsular bag compared to controls (p < 0.001)). In both human lens epithelial cell lines (HLE-B3 and FHL-124), proliferation decreased significantly and as equally strong after incubation with Gefitinib (p < 0.001), as did chemotactic migration (p = 0.004), matrix contraction (p = 0.001), and cell-spreading (p = 0.001). At the IC50 concentration, Gefitinib was well tolerated by six different human ocular cell types of the anterior and posterior segment. CONCLUSION: The specific EGFR inhibitor Gefitinib might become of clinical relevance in PCO prophylaxis as it attenuated cellular growth and other pathological PCO factors in the ex vivo human capsular bag model and in two human lens epithelial cell lines, while showing good biocompatibility in vitro.

Pharmacological drug screening to inhibit uveal melanoma metastatic cells either via EGF-R, MAPK, mTOR or PI3K.

AIM: To screen five potential pharmacological substances specifically targeting EGF-R, MAPK, mTOR, or PI3K for their antiproliferative effects, possible impact on cell viability, as well as cell death rates on three different uveal melanoma metastasis cell lines in vitro. METHODS: Three different uveal melanoma metastasis cell lines (OMM2.5, OMM2.3, and OMM1), that originated from human hepatic and subcutaneous metastasis, were exposed to inhibitors of different targets: erlotinib (EGF-R), everolimus (mTOR), selumetinib (MAPK), trametinib (MAPK) or the alkylphosphocholine erufosine (PI3K). Cell viability was assessed with a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) dye reduction assay after 24h of treatment. Antiproliferative effects were evaluated separately after a 72-hour incubation of the cells with the pharmacological substance. Subsequently, the IC50 was calculated. Tumor cell death was investigated using a double stain apoptosis detection assay. RESULTS: Selumetinib, trametinib, and erufosine significantly decreased cell viability of all OMM cell lines (P<0.04). In addition, selumetinib and trametinib showed a significant inhibition of cell proliferation (P<0.05). Everolimus and erlotinib solely inhibited cell proliferation at the used concentrations (P<0.05). Besides an increase of necrotic cells after erufosine treatment (P<0.001), no changes in the number of dead cells for the other substances were observed. CONCLUSION: The preliminary drug screening demonstrates five new candidates, successfully targeting the canonical MAPK/ERK and PI3K/AKT/mTOR pathways in uveal melanoma metastasis cells in vitro. Hence, these findings provide an experimental basis to explore future single or combined therapy strategies for metastatic uveal melanoma.

The safety profile of alkylphosphocholines in the model of the isolated perfused vertebrate retina.

BACKGROUND: Alkylphosphocholines (APCs) are synthetic phospholipid derivatives, and have been demonstrated to inhibit ocular cell proliferation in vitro and in vivo. Currently, they are applied clinically for their antitumoral and antiparasitic properties, but have not yet been implemented for clinical use in proliferative ophthalmic disorders. The purpose of this study was to assess the safety of APC in the ex vivo model of the isolated perfused vertebrate retina. METHODS: Bovine retina preparations were perfused with an oxygen pre-equilibrated standard solution. The electroretinogram (ERG) was recorded using Ag/AgCl-electrodes. After recording stable b-wave amplitudes, an APC was applied at the following concentrations to the nutrient solution: 0.25 microM, 2.5 microM and 25 microM. To investigate the effects of APC on photoreceptor function, a test series at the same concentrations was performed to evaluate the effects of APC on the a-wave amplitude. Aspartate at a concentration of 1 mM was added to the nutrient solution to obtain stable a-wave amplitudes. Thereafter, APC was applied at the same concentrations to the nutrient solution. The recovery of the ERG amplitudes was followed up for 75 minutes. RESULTS: No reduction of the a- and b-wave amplitude was found at the end of the exposure time with APC added in each test series. No differences were found between the ERG amplitudes before and after application of APC at the end of the washout. CONCLUSIONS: In the ex vivo model of the isolated perfused vertebrate retina, APC has proved to be a safe compound in the concentrations applied. Thus, APCs should further be considered as promising candidates for future clinical applications in ophthalmology.

Supercritical fluid technology for the development of innovative ophthalmic medical devices: Drug loaded intraocular lenses to mitigate posterior capsule opacification.

Supercritical impregnation technology was applied to load acrylic intraocular lenses (IOLs) with methotrexate to produce a sustained drug delivery device to mitigate posterior capsule opacification. Drug release kinetics were studied in vitro and used to determine the drug loading. Loaded IOLs and control IOLs treated under the same operating conditions, but without drug, were implanted ex vivo in human donor capsular bags. The typical cell growth was observed and immunofluorescence staining of three common fibrosis markers, fibronectin, F-actin and α-smooth muscle actin was carried out. Transparent IOLs presenting a sustained release of methotrexate for more than 80 days were produced. Drug loading varying between 0.43 and 0.75 ± 0.03 µgdrug·mg-1IOL were obtained when varying the supercritical impregnation pressure (8 and 25 MPa) and duration (30 and 240 min) at 308 K. The use of ethanol (5 mol%) as a co-solvent did not influence the impregnation efficiency and was even unfavorable at certain conditions. Even if the implantation of methotrexate loaded IOLs did not lead to a statistically significant variation in the duration required for a full cell coverage of the posterior capsule in the human capsular bag model, it was shown to reduce fibrosis by inhibiting epithelial-mesenchymal transformation. The innovative application presented has the potential to gain clinical relevance.

BILATERAL VISUAL FIELD DEFECTS IN A PATIENT TREATED WITH THE MEK AND BRAF INHIBITORS TRAMETINIB AND DABRAFENIB FOR MELANOMA OF UNKNOWN ORIGIN.

INTRODUCTION: Although the introduction of BRAF and MEK inhibitors has greatly enhanced treatment possibilities in advanced BRAFV600-mutated melanoma, class-related toxicities are rather frequent and often involve the eye. Ophthalmologic side effects most commonly include central/diffuse serous retinopathy and retinal vein occlusion. Affection of the optic nerve head however has not been described clinically. CASE REPORT: A 29-year-old man presented in our eye clinic with bilateral blurred vision. Seventeen days earlier, he had been started on trametinib and dabrafenib combination therapy for metastasized melanoma of unknown origin. Visual field testing revealed diffuse bilateral defects, which regressed spontaneously on pause of MEK and BRAF inhibitor treatment. DISCUSSION: In addition to the widely known class-related retinal toxicity, MEK and BRAF inhibitor-associated adverse events may also involve the optic nerve head, causing visual field defects probably regressing spontaneously after discontinuation of targeted oncologic therapy. In such cases, repeat brain imaging and exclusion of melanoma-associated retinopathy is recommended. Reinitiation of treatment and subsequent dose escalation seem to be feasible, but should be monitored by an ophthalmologist.

Poly(lactic-co-glycolic) Acid as a Slow-Release Drug-Carrying Matrix for Methotrexate Coated onto Intraocular Lenses to Conquer Posterior Capsule Opacification.

PURPOSE: Posterior capsule opacification (PCO) still represents the main long-term complication of cataract surgery. Research into pharmacologic PCO prophylaxis is extensive. One promising candidate drug is methotrexate (MTX). Our aim is to determine the in vitro feasibility of MTX-loaded poly(lactic-co-glycolic) (PLGA) biomatrices sprayed on intraocular lenses (IOLs) as a drug-delivery implant. METHODS: Hydrophilic and hydrophobic acrylic IOLs were spray-coated with MTX-loaded PLGA. Unsprayed, solvent only, and solvent-PLGA-sprayed IOLs served as controls. All IOLs were evaluated for their growth-inhibiting properties in an in vitro anterior segment model and the ex vivo human capsular bag. The release kinetics of MTX from the IOLs was determined. The toxicity of MTX on corneal endothelial cells was evaluated by using a dye reduction colorimetric assay. MTX was also used in a scratch assay. RESULTS: MTX-PLGA-IOL showed a significant difference in cell proliferation and migration compared with all controls in the anterior segment model (p < 0.001) and in the human capsular bag model (p = 0.04). No difference in viability was observed on corneal endothelial cells (p = 0.43; p = 0.61). MTX significantly inhibited cells in the scratch assay (p = 0.02). At all measured points, the released MTX dose remained above EC50 and below the toxic dose for the endothelium. CONCLUSIONS: In view of the strong inhibition of PCO in vitro with the lack of toxic effects on a corneal cell line, MTX encapsulating microspheres seem to be a promising method for modifying IOL.

The intraocular lens as a drug delivery device for an epidermal growth factor-Receptor inhibitor for prophylaxis of posterior capsule opacification.

PURPOSE: Posterior capsule opacification (PCO) occurs as a common complication after cataract surgery. Erlotinib is an inhibitor of the epidermal growth factor-Receptor and reduces critical cellular events leading to PCO. In this in vitro study, Erlotinib-modified intraocular lenses (IOLs) employed as a drug delivery device have been evaluated for PCO prevention. METHODS: The IC50 concentration of Erlotinib was determined by using FHL-124 cells. For the human capsular bag model, 40 cadaver eyes underwent sham cataract surgery. Sixteen capsular bags were exposed to the IC50 of Erlotinib. Intraocular lens (IOL) of three different materials was pharmacologically modified and tested in the anterior segment model and implanted into 24 capsular bags. To test for corneal toxicity, pairs of human cornea were exposed to high concentrations of Erlotinib and corneal endothelial cells (CEC) were exposed to the modified IOL. Release kinetics of Erlotinib from the IOL was measured. RESULTS: IC50 of Erlotinib was determined to be 10 µm. Erlotinib alone (p = 0.002) and when soaked into IOLs (p < 0.001) significantly increased the number of days needed until total cell coverage of the capsular bags in comparison with the control. Modified IOLs mitigated cell growth in the anterior segment model (p < 0.001). No short-term corneal toxicity was observed up to a concentration of 100 µm, and IOLs did not show toxicity on CEC. Erlotinib was released constantly from IOL. CONCLUSION: Erlotinib might be of clinical relevance in PCO prophylaxis, as its short-term application induces a long-term deceleration of cellular growth. Erlotinib can be introduced into the eye via soaked IOLs.

Effect of femtosecond laser-assisted lens surgery on posterior capsule opacification in the human capsular bag in vitro.

PURPOSE: To compare posterior capsule opacification (PCO) by observing lens epithelial cell growth in the human capsular bag in vitro between conventional lens surgery using phacoemulsification (Phaco) technique and femtosecond laser-assisted lens surgery (FLACS). METHODS: For the in vitro human capsular bag model, 18 cadaver eyes from nine human donors underwent three types of lens surgery. Three groups consisting of six capsular bags were established, that is FLACS, Phaco and extracapsular lens extraction (ECCE). The capsular bag was transferred into equal cell culture conditions after using one of the defined surgical approaches. Cellular growth of lens epithelial cells was observed and photo-documented. The time until full cell-coverage of the capsular bag was measured. RESULTS: The human capsular bag model can be successfully prepared using FLACS. There was no statistically significant difference in time until cell-coverage of the human donor capsular bag in vitro in all three surgical settings (ECCE versus Phaco p = 0.6; ECCE versus FLACS p = 1.0; Phaco versus FLACS p = 1.0). CONCLUSIONS: In our in vitro human capsular bag model, we could not observe a statistically significant difference in PCO formation using different surgical approaches of lens extraction. Therefore, PCO formation might not be attributed to the type of surgery. Furthermore, this study shows that FLACS can be used for the human capsular bag model preparation and validates the human capsular bag model for future research.

Incidence of secondary glaucoma after treatment of uveal melanoma with robotic radiosurgery versus brachytherapy.

PURPOSE: Different modalities of radiation therapy nowadays allow for effective treatment of uveal melanoma combined with the advantage of eye preservation. However, this advantage can secondarily be impaired by radiation-related side effects. After local recurrence, secondary glaucoma (SG) has been described as second most frequent complication leading to need of enucleation. This study compares the incidence of SG after conventional Ruthenium (Ru)-106 brachytherapy (BT) versus CyberKnife robotic radiosurgery (RRS) which has been gaining importance lately as an efficient treatment option offering improved patient comfort. METHODS: Medical records of all patients diagnosed with uveal melanoma in the Eye Clinic of the Ludwig-Maximilians-University Munich between 2007 and 2013 were reviewed. A total of 268 eyes of 268 patients treated with Ru-106 BT or CyberKnife-RRS as monotherapy were entered in this retrospective cohort study. Incidence of SG was correlated with treatment modality and baseline tumour characteristics. RESULTS: Fifty-three patients (19.8%) developed SG. At 5 years, SG was significantly more frequent after RRS (46.7%) than BT (11.1%); however, tumour thickness (maximum apical height) as a marker of tumour progress was more pronounced in the RRS group. Subgroup analysis of 178 patients for tumours amenable to both BT and RRS (thickness ≤6 mm) revealed comparable results at 3 years (RRS: 13.8 versus BT: 11.2%), but a trend towards increased incidence after RRS beyond year three. However, this difference was not significant at 5 years (28.2% versus 11.2%, p = 0.138). Tumour thickness was significantly associated with incidence of SG. CONCLUSION: In tumours ≤6 mm thickness, RRS and BT seem to offer a comparable safety profile in terms of SG. Beyond year three, SG was tendentially, but not significantly more frequent after RRS. Increasing tumour thickness is associated with risk of SG.

The Intraocular Lens as a Drug Delivery Device: In Vitro Screening of Pharmacologic Substances for the Prophylaxis of Posterior Capsule Opacification.

Purpose: Numerous pharmacologic substances have been proposed for preventing posterior capsule opacification (PCO). The following trial was to compare those drugs to find more suitable options. IOL should then be modified by the pharmaceuticals as a drug-delivery device. Methods: A systematic literature search was performed to identify published substances. FHL-124 was used to determine cell proliferation and toxicity using a dye reduction test (XTT). Prescreened substances showing a reduction on cell growth without being toxic were soaked into an IOL. Those IOL were tested for their effect on PCO in an anterior-segment model and the human ex vivo capsular bag model. Toxicity on a corneal endothelial cell line (CEC-SV40) was determined. Release kinetics of methotrexate from the IOL was measured. Toxicity testing in both cell lines was done in serum-free conditions. All growth assays were exposed to 10% fetal calf serum (FCS)-supplemented medium. Results: The substances inhibited cell growth at the following EC50: caffeic acid phenethyl ester 1.6 ± 0.9 nM, disulfiram 359 ± 33 nM, methotrexate 98.0 ± 29.7 nM, rapamycin 70.2 ± 14.0 pM, and retinoic acid 1.1 ± 0.12 nM. All but disulfiram showed an effect in the anterior segment model when soaked into an IOL. Long-term inhibitory effects in the human capsular bag model were observed for caffeic acid phenethyl ester and methotrexate IOLs. Only methotrexate and disulfiram did not show any toxicity on endothelial cells. Methotrexate was released constantly from the hydrophilic IOL for 2 weeks. Conclusions: We could identify caffeic acid phenethyl ester and methotrexate in vitro as potential candidates for IOL modification for PCO prophylaxis.

Robotic radiosurgery for the treatment of medium and large uveal melanoma.

The aim of the study was to analyze the local efficacy and eye retention rate after frameless, image-guided robotic radiosurgery against uveal melanoma. A total of 217 patients, mostly with medium and large unilateral uveal melanomas (3% small, 62% medium, and 35% large) were treated. The median age was 64 years (range 21-95 years). All patients underwent a single-session procedure beginning with retrobulbar anesthesia, followed by MRI and computerized tomography scanning to generate the treatment plan. The tumor dose was 18-22 Gy (mean, 20.3 Gy) prescribed to the 70% isodose line. Follow-up occurred at 3, 6, 12, and 18 months and yearly thereafter with clinical, ultrasound, and MRI studies. The median follow-up time was 26.4 months. All patients were treated in the frameless setup within 3 h. The actuarial 3- and 5-year eye retention rates were 86.7 and 73%, respectively. Local control at 3 and 5 years was 87.4 and 70.8%, respectively. Serviceable vision was maintained in 30.9% of patients at last follow-up. Treatment-induced glaucoma developed in 33 patients at a median 20.8 months (range, 5.8-54.0 months). Other adverse effects were hemorrhage (26 patients) and macular edema (seven patients). Frameless, single-session, image-guided robotic radiosurgery is an effective and straightforward treatment option for patients with medium and large uveal melanoma that are otherwise difficult to treat.

Erufosine, a phosphoinositide-3-kinase inhibitor, to mitigate posterior capsule opacification in the human capsular bag model.

PURPOSE: To determine whether erufosine alone or erufosine-loaded intraocular lenses (IOLs) can inhibit growth of human lens epithelial cells after a single administration in the human capsular bag model. SETTING: Laboratory for Cell Biology, Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Sixteen human cadaver eyes had sham cataract surgery. The capsular bag was transferred into cell culture. The tissue was exposed to the half maximum inhibitory concentrations of erufosine alone for 72 hours; solvent-only tissue served as a control. Erufosine is a potent inhibitor of phosphoinositide-3-kinase, a downstream kinase with major implications in posterior capsule opacification (PCO) pathogenesis. The IOLs were soaked with erufosine and implanted in the capsular bags; unsoaked IOLs served as controls. For both settings, the time until confluence of the capsular bag was measured. Cell growth was observed and photodocumented. RESULTS: Erufosine as a single therapeutic agent increased the time until confluence of the capsular bag, but not significantly compared with the control. When IOLs were soaked with erufosine, a long-term prophylactic effect was observed in this organ model for PCO, which is known to closely reflect the clinical situation. CONCLUSION: Erufosine-soaked IOLs effectively inhibited PCO formation as seen in long-term organ culture and might become of clinical relevance. FINANCIAL DISCLOSURES: Drs. Kampik and Eibl-Lindner are inventors of IOLs treated with alkylphosphocholines for pharmacological after-cataract prophylaxis, patent international application PCT/EP2010/051490. No other author has a financial or proprietary interest in any material or method mentioned.

Hemianopia and visual loss due to progressive multifocal leukoencephalopathy in natalizumab-treated multiple sclerosis.

This case describes typical ophthalmic findings as a key feature for diagnosis of progressive multifocal leukoencephalopathy (PML) and its possible differential diagnosis. A 58-year-old female patient with relapsing-remitting multiple sclerosis on immunotherapy with natalizumab developed visual disturbance, reading problems, and visual field defects due to PML. PML is a reactivation of latent infection with the John Cunningham virus, which is a type of polyomavirus acquired in childhood or adolescence and is quite common in the general population. PML so far has been mostly associated with other immunodeficiency disorders, such as acquired immunodeficiency syndrome, but is also gaining importance in association with the increasing use and duration of treatment with natalizumab in patients suffering from multiple sclerosis. Natalizumab is a highly specific α4-integrin antagonist approved for treatment of patients with active relapsing-remitting multiple sclerosis.

Inhibition of TLR3-mediated proinflammatory effects by Alkylphosphocholines in human retinal pigment epithelial cells.

PURPOSE. To elucidate the role of Toll-like receptor 3 (TLR3) in the pathogenesis of age-related macular degeneration (AMD) and to investigate the effect of alkylphosphocholines (APCs) on the TLR3-mediated expression of cytokines and growth factors in human retinal pigment epithelial (RPE) cells. METHODS. Confluent cultures of human RPE cells (ARPE-19) were stimulated with poly (I:C) RNA as a well-established ligand for TLR3. Cytokine profiles were determined by RT-PCR on the activation of TLR3. RPE cells were transfected with siRNA specific for TLR3 and RIG-1 to determine the receptors involved. The effect of preincubation of RPE cells with APCs on the expression level of target genes was assessed. RESULTS. Poly (I:C) RNA stimulation led to a dose-dependent increase in the expression of TLR3 and RIG-I. A significant increase in expression levels of IL-6, TNF-α, IL-8, MCP-1, ICAM-1, and BFGF was observed after poly (I:C) RNA stimulation (P < 0.05). This effect was time and dose dependent. No effect on PEDG or VEGF expression was seen. Transfection of RPE cells with siRNA specific for TLR3 reduced poly (I:C) RNA-induced mRNA expression of the genes (P < 0.05). Preincubation of RPE cells with APCs significantly reduced the poly (I:C) RNA-induced expression of the target genes (P < 0.05). CONCLUSIONS. The authors demonstrate that the expression of proinflammatory cytokines and chemokines in RPE cells depends on the activation of TLR3. The induction of downstream gene expression is blocked by siRNA specific for TLR3 and alkylphosphocholines. Therefore, TLR3 should be considered a novel target in AMD therapy.