Effects of tumor necrosis factor-α and interleukin-1ß on human retinal endothelial cells.

Uveitis, or intraocular inflammation, is a potentially blinding condition that mostly affects the working-age population. The cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, play a role in the pathogenesis of non-infectious uveitis and have been linked to the breakdown of the inner blood-retinal barrier, composed mainly of retinal endothelial cells, leading to macular oedema and vascular leakage. However, the effects of TNF-α and IL-1ß on human retinal endothelial function are not fully understood. In this work, we investigated the impact of TNF-α and IL-1ß on several aspects of human retinal endothelial cell biology. Through a real-time biosensor, the impact of TNF-α and IL-1ß on formation of a retinal endothelial cell barrier was analyzed. Changes in junctional components were assessed via RT-qPCR and immunolabelling. Cell survival, necrosis and apoptosis were appraised via cell proliferation and flow cytometric studies. Tumor necrosis factor-α and IL-1ß impaired the electrical resistance of the retinal endothelial cell barrier, while the addition of a potentially barrier-impairing cytokine, IL-6, did not enhance the effect of TNF-α and IL-1ß. Level of the gene transcript encoding zonula occludens (ZO)-1 was diminished, while ZO-1 protein configuration was changed by TNF-α and IL-1ß. Both cytokines affected human retinal endothelial cell proliferation and viability, while only TNF-α increased rates of necrosis. These results indicate that TNF-α and IL-1ß are important drivers of retinal endothelial dysfunction in non-infectious uveitis, suggesting that targeting these cytokines is critical when treating complications of uveitis, such as macular oedema and vascular leakage.

Disease profiles in the Indigenous Australian population are suggestive of a common complement control haplotype.

Aboriginal and Torres Strait Islander People (respectfully referred to as Indigenous Australians herein) are disparately burdened by many infectious and chronic diseases relative to Australians with European genetic ancestry. Some of these diseases are described in other populations to be influenced by the inherited profile of complement genes. These include complement factor B, H, I and complement factor H-related (CFHR) genes that can contribute to a polygenic complotype. Here the focus is on the combined deletion of CFHR1 and 3 to form a common haplotype (CFHR3-1Δ). The prevalence of CFHR3-1Δ is high in people with Nigerian and African American genetic ancestry and correlates to a higher frequency and severity of systemic lupus erythematosus (SLE) but a lower prevalence of age-related macular degeneration (AMD) and IgA-nephropathy (IgAN). This pattern of disease is similarly observed among Indigenous Australian communities. Additionally, the CFHR3-1Δ complotype is also associated with increased susceptibility to infection with pathogens, such as Neisseria meningitidis and Streptococcus pyogenes, which also have high incidences in Indigenous Australian communities. The prevalence of these diseases, while likely influenced by social, political, environmental and biological factors, including variants in other components of the complement system, may also be suggestive of the CFHR3-1Δ haplotype in Indigenous Australians. These data highlight a need to define the Indigenous Australian complotypes, which may lead to the discovery of new risk factors for common diseases and progress towards precision medicines for treating complement-associated diseases in Indigenous and non-Indigenous populations. Herein, the disease profiles suggestive of a common complement CFHR3-1Δ control haplotype are examined.

Human retinal endothelial cells express functional interleukin-6 receptor.

BACKGROUND: Interleukin (IL)-6 is an inflammatory cytokine present in the eye during non-infectious uveitis, where it contributes to the progression of inflammation. There are two major IL-6 signaling pathways: classic signaling and trans-signaling. Classic signaling requires cellular expression of the IL-6 receptor (IL-6R), which exists in membrane-bound (mIL-6R) and soluble (sIL-6R) forms. Prevailing dogma is that vascular endothelial cells do not produce IL-6R, relying on trans-signaling during inflammation. However, the literature is inconsistent, including with respect to human retinal endothelial cells. FINDINGS: We examined IL-6R transcript and protein expression in multiple primary human retinal endothelial cell isolates, and assessed the effect of IL-6 on the transcellular electrical resistance of monolayers. Using reverse transcription-polymerase chain reaction, IL-6R, mIL-6R and sIL-6R transcripts were amplified in 6  primary human retinal endothelial isolates. Flow cytometry on 5 primary human retinal endothelial cell isolates under non-permeabilizing conditions and following permeabilization demonstrated intracellular stores of IL-6R and the presence of mIL-6R. When measured in real-time, transcellular electrical resistance of an expanded human retinal endothelial cell isolate, also shown to express IL-6R, decreased significantly on treatment with recombinant IL-6 in comparison to non-treated cells across 5 independent experiments. CONCLUSIONS: Our findings indicate that human retinal endothelial cells produce IL-6R transcript and functional IL-6R protein. The potential for classic signaling in human retinal endothelial cells has implications for the development of therapeutics targeted against IL-6-mediated pathology in non-infectious uveitis.

Selective Transcription Factor Blockade Reduces Human Retinal Endothelial Cell Expression of Intercellular Adhesion Molecule-1 and Leukocyte Binding.

The interaction between leukocytes and cytokine-activated retinal endothelium is an initiating step in non-infectious uveitis involving the posterior eye, mediated by cell adhesion molecules. However, because cell adhesion molecules are required for immune surveillance, therapeutic interventions would ideally be employed indirectly. Using 28 primary human retinal endothelial cell isolates, this study sought to identify transcription factor targets for reducing levels of the key retinal endothelial cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, and limiting leukocyte binding to the retinal endothelium. Five candidate transcription factors-C2CD4B, EGR3, FOSB, IRF1, and JUNB-were identified by differential expression analysis of a transcriptome generated from IL-1ß- or TNF-α-stimulated human retinal endothelial cells, interpreted in the context of the published literature. Further filtering involved molecular studies: of the five candidates, C2CD4B and IRF1 consistently demonstrated extended induction in IL-1ß- or TNF-α-activated retinal endothelial cells and demonstrated a significant decrease in both ICAM-1 transcript and ICAM-1 membrane-bound protein expression by cytokine-activated retinal endothelial cells following treatment with small interfering RNA. RNA interference of C2CD4B or IRF1 significantly reduced leukocyte binding in a majority of human retinal endothelial cell isolates stimulated by IL-1ß or TNF-α. Our observations suggest that the transcription factors C2CD4B and IRF1 may be potential drug targets for limiting leukocyte-retinal endothelial cell interactions in non-infectious uveitis involving the posterior eye.

Expression of Long Non-Coding RNAs in Activated Human Retinal Vascular Endothelial Cells.

PURPOSE: Retinal endothelial cell activation is a central event in non-infectious posterior uveitis. There is recent interest in long non-coding (lnc)RNA-targeted therapeutics for retinal diseases. We aimed to identify human retinal endothelial cell lncRNAs that might be involved in activation. METHODS: Eleven candidate lncRNAs were identified: GAS5, KCNQ1OT1, LINC00294, MALAT1, MEG3, MIR155HG, NEAT1, NORAD, OIP5-AS1, SENCR, TUG1. Expression was assessed by RT-PCR in human retinal endothelial cells, at baseline and following activation with interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. RESULTS: IL-1ß significantly upregulated MEG3 and SENCR at 4 and 24 hours; LINC00294, NORAD, OIP5-AS1 and TUG1 at 24 hours; and MIR155HG at 4, 24 and 48 hours; but downregulated GAS5 at 24 and 48 hours. TNF-α significantly upregulated KCNQ1OT1, LINC00294, MEG3, NORAD and SENCR at 4 hours; SENCR and TUG1 at 24 hours; and MIR155HG at all time points. CONCLUSIONS: Future studies involving manipulation of MIR155HG may be warranted to explore potential therapeutic applications for non-infectious posterior uveitis.

Transcriptomic Responses of Human Retinal Vascular Endothelial Cells to Inflammatory Cytokines.

Purpose: Molecular profiling of human retinal endothelial cells provides opportunities to understand the roles of this cell population in maintenance of the blood-ocular barrier, and its involvements in diverse retinal vasculopathies. We aimed to generate a transcriptome of human retinal endothelial cells in the unstimulated state, and following treatment with inflammatory cytokines linked to cell dysfunction. Methods: Endothelial cells were isolated from retinae of five human cadaveric donors, and treated for 60 minutes and 24 hours with interleukin-1ß or tumor necrosis factor-α, or exposed to medium alone for the same intervals. Expression of intercellular adhesion molecule-1 was measured by RT-qPCR to confirm cytokine-induced activation of the cells. RNA was sequenced on the Illumina NovaSeq 6000 platform. Reads were aligned to the human GRCh38 genome, and reads that aligned to Ensembl-annotated genes were counted. Quality control of sequencing was performed with FastQC, and sequences were classified by Kraken. Results: A human retinal endothelial cell RNA-sequencing dataset with mean of 99% reads aligned to the human genome was produced as raw RNA sequence data (FASTQ files) and processed read data (XLSX files). Multidimensional scaling analysis showed a strong donor effect, which was readily controlled by ComBat. Conclusions: Our dataset may be useful for human retinal endothelial cell transcriptomic assemblies, functional gene annotating and/or gene expression and enrichment analyses, as well as cross-dataset harmonization. Translational Relevance: The molecular profile of the human retinal endothelium is a source of candidate biologic targets for retinal vasculopathies.

Infection of Human Retinal Pigment Epithelial Cells with Dengue Virus Strains Isolated during Outbreaks in Singapore.

Prevalence of dengue retinopathy varies across epidemics, with the disease linked to circulation of dengue virus serotype 1 (DENV-1). The retinal pigment epithelium has been implicated in the pathology. We investigated infectivity, molecular response, and barrier function of epithelial cells inoculated with DENV strains from different outbreaks in Singapore. Monolayers of human retinal pigment epithelial cells (multiple primary cell isolates and the ARPE-19 cell line) were inoculated with six DENV strains, at multiplicity of infection of 10; uninfected and recombinant strain-infected controls were included where relevant. Infectivity and cell response were assessed primarily by RT-qPCR on total cellular RNA, and barrier function was evaluated as electrical resistance across monolayers. Higher viral RNA loads were measured in human retinal pigment epithelial cells infected with DENV-1 strains from the 2005 Singapore epidemic, when retinopathy was prevalent, versus DENV-1 strains from the 2007 Singapore epidemic, when retinopathy was not observed. Type I interferon (IFN) transcripts (IFN-ß and multiple IFN-stimulated genes) were up-regulated, and impact on barrier function was more pronounced, for cells infected with DENV-1 strains from the 2005 versus the 2007 Singapore epidemics. Aside from serotype, strain of DENV may determine the potential to induce retinal pathology. Identification of molecular markers of disease-associated DENV strains may provide insights into the pathogenesis of dengue retinopathy.

Zika Virus Infection of Human Iris Pigment Epithelial Cells.

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.

Lamb as a potential source of Toxoplasma gondii infection for Australians.

OBJECTIVE: Toxoplasmosis may follow consumption of undercooked meat containing Toxoplasma gondii cysts. Lamb is considered to pose the highest risk for contamination across meats. Red meat is often served undercooked, yet there are no current data on T. gondii contamination of Australian sourced and retailed lamb. We sought to address this gap in public health knowledge. METHODS: Lamb mincemeat was purchased at the supermarket counter three times weekly for six months. T. gondii was detected by real-time polymerase chain reaction (PCR) of DNA extracted from the meat following homogenisation. Purchases were also tested for common foodborne bacterial pathogens. RESULTS: Conservative interpretation of PCR testing (i.e. parasite DNA detected in three of four tests) gave a probability of 43% (95% confidence interval, 32%-54%) that lamb mincemeat was contaminated with T. gondii. None of the purchases were contaminated with Campylobacter jejuni, Salmonella species or S. enterica serovar Typhimurium, indicating sanitary meat processing. CONCLUSIONS: Australian lamb is commonly contaminated with T. gondii. Future studies should be directed at testing a range of red meats and meat cuts. Implications for public health: Consuming undercooked Australian lamb has potential to result in toxoplasmosis. There may be value in health education around this risk.

Neutrophil Activities in Human Ocular Toxoplasmosis: An In Vitro Study With Human Cells.

Purpose: Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation. Methods: Human neutrophils were isolated from peripheral blood. Migration to disease-relevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPE-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays. Results: Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6) and IL-18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells. Conclusions: Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.

Expression of microRNA in human retinal pigment epithelial cells following infection with Zaire ebolavirus.

OBJECTIVE: Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation-or uveitis-which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection. RESULTS: Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors.

Expression of Long Non-Coding RNAs by Human Retinal Müller Glial Cells Infected with Clonal and Exotic Virulent Toxoplasma gondii.

Retinal infection with Toxoplasma gondii-ocular toxoplasmosis-is a common cause of vision impairment worldwide. Pathology combines parasite-induced retinal cell death and reactive intraocular inflammation. Müller glial cells, which represent the supporting cell population of the retina, are relatively susceptible to infection with T. gondii. We investigated expression of long non-coding RNAs (lncRNAs) with immunologic regulatory activity in Müller cells infected with virulent T. gondii strains-GT1 (haplogroup 1, type I) and GPHT (haplogroup 6). We first confirmed expression of 33 lncRNA in primary cell isolates. MIO-M1 human retinal Müller cell monolayers were infected with T. gondii tachyzoites (multiplicity of infection = 5) and harvested at 4, 12, 24, and 36 h post-infection, with infection being tracked by the expression of parasite surface antigen 1 (SAG1). Significant fold-changes were observed for 31 lncRNAs at one or more time intervals. Similar changes between strains were measured for BANCR, CYTOR, FOXD3-AS1, GAS5, GSTT1-AS1, LINC-ROR, LUCAT1, MALAT1, MIR22HG, MIR143HG, PVT1, RMRP, SNHG15, and SOCS2-AS1. Changes differing between strains were measured for APTR, FIRRE, HOTAIR, HOXD-AS1, KCNQ1OT1, LINC00968, LINC01105, lnc-SGK1, MEG3, MHRT, MIAT, MIR17HG, MIR155HG, NEAT1, NeST, NRON, and PACER. Our findings suggest roles for lncRNAs in regulating retinal Müller cell immune responses to T. gondii, and encourage future studies on lncRNA as biomarkers and/or drug targets in ocular toxoplasmosis.

Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii.

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome-with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)-but very limited changes in the small RNA transcriptome-totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.

Selection of Reference Genes for Studies of Human Retinal Endothelial Cell Gene Expression by Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction.

BACKGROUND: Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. METHODS: Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) less than 0.2 and coefficient of variation (CV-value) less than 0.1. RESULTS: Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11-21 (41-78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. CONCLUSIONS: We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.

ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells.

OBJECTIVE: Regulation of intercellular adhesion molecule (ICAM)-1 in retinal endothelial cells is a promising druggable target for retinal vascular diseases. The ICAM-1-related (ICR) long non-coding RNA stabilizes ICAM-1 transcript, increasing protein expression. However, studies of ICR involvement in disease have been limited as the promoter is uncharacterized. To address this issue, we undertook a comprehensive in silico analysis of the human ICR gene promoter region. RESULTS: We used genomic evolutionary rate profiling to identify a 115 base pair (bp) sequence within 500 bp upstream of the transcription start site of the annotated human ICR gene that was conserved across 25 eutherian genomes. A second constrained sequence upstream of the orthologous mouse gene (68 bp; conserved across 27 Eutherian genomes including human) was also discovered. Searching these elements identified 33 matrices predictive of binding sites for transcription factors known to be responsive to a broad range of pathological stimuli, including hypoxia, and metabolic and inflammatory proteins. Five phenotype-associated single nucleotide polymorphisms (SNPs) in the immediate vicinity of these elements included four SNPs (i.e. rs2569693, rs281439, rs281440 and rs11575074) predicted to impact binding motifs of transcription factors, and thus the expression of ICR and ICAM-1 genes, with potential to influence disease susceptibility. We verified that human retinal endothelial cells expressed ICR, and observed induction of expression by tumor necrosis factor-α.

Emerging diagnostic tests for vitreoretinal lymphoma: a review.

Vitreoretinal lymphoma, which most commonly is diffuse large B-cell non-Hodgkin in type, is a rare cancer with high morbidity and high mortality. Making a tissue diagnosis of vitreoretinal lymphoma is a major challenge for clinicians due to biological and technical factors. Yet, the delay in start of treatment may have vision- and life-threatening consequences, and there is considerable interest in the application of molecular assays to improve the accuracy of the diagnostic process: detection of a clonal immunoglobulin heavy-chain rearrangements in lymphoma cells by polymerase chain reaction; measurement of vitreous or aqueous interleukin-10 protein levels in ocular fluids; and identification of mutations in the myeloid differentiation primary response gene 88 in tumour cells. In this article, we review the historical development and current application of each of these molecular methods. We also discuss future opportunities for the molecular diagnosis of vitreoretinal lymphoma through next-generation sequencing technologies.

Angiogenic and Immunologic Proteins Identified by Deep Proteomic Profiling of Human Retinal and Choroidal Vascular Endothelial Cells: Potential Targets for New Biologic Drugs.

PURPOSE: Diseases that involve retinal or choroidal vascular endothelial cells are leading causes of vision loss: age-related macular degeneration, retinal ischemic vasculopathies, and noninfectious posterior uveitis. Proteins differentially expressed by these endothelial cell populations are potential drug targets. We used deep proteomic profiling to define the molecular phenotype of human retinal and choroidal endothelial cells at the protein level. METHODS: Retinal and choroidal vascular endothelial cells were separately isolated from 5 human eye pairs by selection on CD31. Total protein was extracted and digested, and peptide fractions were analyzed by reverse-phase liquid chromatography tandem mass spectrometry. Peptide sequences were assigned to fragment ion spectra, and proteins were inferred from openly accessible protein databases. Protein abundance was determined by spectral counting. Publicly available software packages were used to identify proteins that were differentially expressed between human retinal and choroidal endothelial cells, and to classify proteins that were highly abundant in each endothelial cell population. RESULTS: Human retinal and/or choroidal vascular endothelial cells expressed 5042 nonredundant proteins. Setting the differential expression false discovery rate at 0.05, 498 proteins of 3454 quantifiable proteins (14.4%) with minimum mean spectral counts of 2.5 were differentially abundant in the 2 cell populations. Retinal and choroidal endothelial cells were enriched in angiogenic proteins, and retinal endothelial cells were also enriched in immunologic proteins. CONCLUSIONS: This work describes the different protein expression profiles of human retinal and choroidal vascular endothelial cells, and provides multiple candidates for further study as novel treatments or drug targets for posterior eye diseases. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells.

BACKGROUND: Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. METHODS: Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 µM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. RESULTS: Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. CONCLUSIONS: Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.

Erratum to "Molecular Responses of Human Retinal Cells to Infection with Dengue Virus".

[This corrects the article DOI: 10.1155/2017/3164375.].

Retinal Pigment Epithelial Cells are a Potential Reservoir for Ebola Virus in the Human Eye.

PURPOSE: Success of Ebola virus (EBOV) as a human pathogen relates at the molecular level primarily to blockade the host cell type I interferon (IFN) antiviral response. Most individuals who survive Ebola virus disease (EVD) develop a chronic disease syndrome: approximately one-quarter of survivors suffer from uveitis, which has been associated with presence of EBOV within the eye. Clinical observations of post-Ebola uveitis indicate involvement of retinal pigment epithelial cells. METHODS: We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site, and network analyses. We measured infection-induced changes of selected transcripts by reverse transcription-quantitative polymerase chain reaction. RESULTS: Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 upregulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response. Following EBOV infection, cells continued to express multiple immunomodulatory molecules linked to ocular immune privilege. CONCLUSIONS: Human retinal pigment epithelial cells may serve as an intraocular reservoir for EBOV, and the molecular response of infected cells may contribute to the persistence of live EBOV within the human eye. TRANSLATIONAL RELEVANCE: This bedside-to-bench research links ophthalmic findings in survivors of EVD who suffer from uveitis with interactions between retinal pigment epithelial cells and EBOV.