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Up to 10% of uveitis cases occur in children, with notable implications due to the risk of chronicity and vision loss. It can result from infections, autoimmune and autoinflammatory diseases, trauma, or masquerade syndromes. Primary care providers are vital in early detection, symptom management, and timely specialist referral. Depending on the etiology, symptoms may include redness, pain, photophobia, vision changes, and sometimes more severe vision loss or intense pain. Conservative initial treatment may include pain relievers and corticosteroid eye drops. Specialist care involves a thorough ocular examination and diagnostic tests to identify the underlying cause. Treatment aims to control inflammation and preserve vision, often necessitating long-term medication, with compliance being crucial. In low-resource settings, telemedicine and generic drugs may be useful to manage costs. Preventative strategies focus on prenatal care to avoid congenital infections, and distinguishing uveitis from conditions like conjunctivitis is essential for proper treatment. Multidisciplinary care ensures comprehensive management of the child’s health. In summary, early intervention and a collaborative approach among healthcare providers are key to mitigating the impact of uveitis on a child’s life.

Abstract Ocular syphilis is a re-emerging inflammatory eye disease with a clear gender imbalance, disproportionately affecting men. We investigated the impact of gender on the presentation, management practices and clinical outcomes of this condition. Data generated from a study of patients consecutively diagnosed with ocular syphilis who attended a subspecialist uveitis service at one of four hospitals in Brazil over a 30-month period were disaggregated for analysis by gender. Two-hundred and fourteen eyes (161 men and 53 women) of 127 patients (96 men and 31 women) were included. Posterior uveitis was the most common presentation in both men and women (80.1% vs. 66.7%, p > 0.05), but men were significantly more likely to have vitritis as a feature of their disease (49.4% versus 28.8%, p = 0.019). Three eyes of women had nodular anterior scleritis (p = 0.015). Men were more likely to undergo a lumbar puncture to assess for neurosyphilis (71.9% vs. 51.6%, p = 0.048), but men and women undergoing a lumbar puncture were equally likely to have a cerebrospinal fluid abnormality (36.2% vs. 25.0%, p = 0.393). All patients were treated with aqueous penicillin G or ceftriaxone, and there was a trend towards more men receiving adjunctive systemic corticosteroid treatment as part of their management (65.2% vs. 46.7%, p = 0.071). There were no significant differences in the age of presentation, bilaterality of disease, anatomical classification of uveitis, initial or final visual acuity, and rates of ocular complications between men and women. Our findings indicate that ocular syphilis has comparable outcomes in men and women, but that there are differences in the type of ocular inflammation and management practices between the genders.

As a key host protein involved in cellular infection by the severe acute respiratory syndrome coronavirus (SARS-CoV-2), angiotensin converting enzyme (ACE)2 is an ideal target for antiviral drugs. Manipulation of transcription provides opportunity for graduated blockade that preserves physiological functions. We sought to develop a model system for evaluating manipulation of ACE2 gene transcription using human retinal pigment epithelium. Retinal pigment epithelial cell isolates were prepared from human posterior eyecups (n = 11 individual isolates). The cells expressed ACE2 transcript and protein, and expression was not induced by hypoxia mimetic dimethyloxaloylglycine, or inflammatory cytokine IL-1β. ACE2 gene transcription factors were predicted in silico and cross-referenced with the human retinal pigment epithelial cell transcriptome, and five candidate transcription factors were identified: ETS proto-oncogene 1 transcription factor (ETS1), nuclear factor I C (NFIC), nuclear receptor subfamily 2 group C member 1 (NR2C1), TEA domain transcription factor 1 (TEAD1), and zinc finger protein 384 (ZNF384). The candidates were individually targeted in cells by transfection with small interfering (si)RNA. Knockdowns reduced mean cellular expression of all the transcription factors in comparison to expression in cells transfected with control non-targeted siRNA. Mean cellular ACE2 transcript was reduced under the condition of NR2C1 knockdown, but not for ETS1, NFIC, TEAD1, and ZNF384 knockdowns. Our findings build on previous work demonstrating the potential for drugging gene transcription. Importantly, we show the value of human retinal pigment epithelium as a system for evaluating ACE2 transcriptional blockade, a possible approach for treating SARS-CoV-2 infection. Brief Research Report.

Toxoplasma gondii is an Apicomplexan parasite that is estimated to infect at least one-third of the global human population. T. gondii infection may be transmitted horizontally or vertically. The main risk factors for transmission to humans are related to diet, especially the consumption of undercooked meat, along with soil contact. In immunocompetent persons, the acute infection may go undetected as it typically produces minor, non-specific symptoms that are self-limited. After infection is established, recurrent retinochoroiditis is the most common clinical disease. In contrast, severe systemic or cerebral toxoplasmosis may be life-threatening for immunocompromised individuals. Furthermore, congenital toxoplasmosis acquired in utero may have devastating consequences if not recognized and promptly treated. A growing body of research has identified associations between latent T. gondii infection, and personality traits and risk-taking behaviors. Other studies have documented associations between latent infection and psychiatric conditions that include schizophrenia and bipolar affective disorder. With no current treatment regimens being curative of T. gondii infection, effective prevention measures at both the public health and individual levels are vitally important.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Abstract Multiple studies have showed negative impact of non-infectious uveitis on quality of life (QoL). Less is understood regarding life experiences in patients with infectious uveitis. We investigated vision-related QoL in individuals who had recovered from ocular syphilis. 32 adults treated for ocular syphilis at a uveitis service in Brazil completed the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25), and a comprehensive ophthalmic examination was performed. Medical records were reviewed to confirm resolution of ocular inflammation for 3 months pre-enrolment, and collect clinical data. The NEI VFQ-25 composite score was low overall (75.5 ± 19.8, mean ± standard deviation), and subscale scores varied from relative lows of 59.1 ± 39.6 (driving) and 60.9 ± 24.5 (mental health), to relative highs of 84.8 ± 21.8 (ocular) and 89.1 ± 21.0 (color vision). Adults aged over 40 years and those with a final visual acuity of 20/50 or worse had significantly lower mean composite and subscale scores. Other clinical characteristics—including gender, HIV co-infection, and type of uveitis—did not significantly influence scores. Our findings, taken in context with previous observations that prompt recognition achieves better vision outcomes, suggest early treatment may improve QoL after recovery from ocular syphilis.

Abstract 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.

Abstract Background To report a case of bilateral intermediate uveitis without cystoid macular edema secondary to paclitaxel therapy, and its successful management with oral corticosteroids. Case presentation A 66-year-old female developed bilateral intermediate uveitis with reduced best corrected visual acuity to 20/40 right and 20/200 left, following 12 cycles of paclitaxel therapy for breast carcinoma. Optical coherence tomography demonstrated no cystoid macular edema in either eye, and fundus fluorescein angiography showed localized retinal vascular leakage. Resolution of uveitis and improvement of visual acuity followed treatment with oral prednisolone for two months. Fourteen months after presentation, right and left visual acuities had returned to 20/32 and 20/40, respectively, and there was no recurrence of the uveitis. Conclusions This is the first reported case of bilateral intermediate uveitis in a patient treated with paclitaxel. Drug-induced uveitis should be considered in patients with visual symptoms in the setting of taxane chemotherapy, and oral corticosteroids are a safe and effective treatment.

Abstract Toxoplasmic retinochoroiditis is a common, potentially blinding parasitic infection. We sought to define the spectrum and frequency of signs of active toxoplasmic retinochoroiditis by spectral domain optical coherence tomography (SD-OCT), and to identify clinical associations. Ninety eyes of 90 individuals presenting consecutively to a tertiary referral uveitis service with active toxoplasmic retinochoroiditis and gradable SD-OCT scans were evaluated prospectively. SD-OCT features were collated, and associations with lesion location, primary versus recurrent episode, serological status, human immunodeficiency virus infection and best-corrected Snellen visual acuity were explored. Active toxoplasmic retinochoroiditis presented with thickened (65%) and hyperreflective (61%) retina, choroidal thickening (55%) and hyporeflectivity (61%), hyperreflective vitreous dots (80%) and deposits (36%), and posterior hyaloid thickening (35%) on SD-OCT. Most signs occurred with similar frequency across clinical groups. Retinal hyporeflectivity (17%) was significantly associated with a visual acuity of 20/200 or worse at resolution. Our observations demonstrate that active toxoplasmic retinochoroiditis has diverse SD-OCT signs and that none are universally present. Retinal hyporeflectivity—suggesting liquefactive necrosis—predicts poor visual outcome.

Macular edema is the pathological accumulation of fluid in the central retina. It is a complication of many retinal diseases, including diabetic retinopathy, retinal vascular occlusions and uveitis, among others. Macular edema causes decreased visual acuity and, when chronic or refractory, can cause severe and permanent visual impairment and blindness. In most instances, it develops due to dysregulation of the blood-retinal barrier which permits infiltration of the retinal tissue by proteins and other solutes that are normally retained in the blood. The increase in osmotic pressure in the tissue drives fluid accumulation. Current treatments include vascular endothelial growth factor blockers, corticosteroids, and non-steroidal anti-inflammatory drugs. These treatments target vasoactive and inflammatory mediators that cause disruption to the blood-retinal barrier. In this review, a clinical overview of macular edema is provided, mechanisms of disease are discussed, highlighting processes targeted by current treatments, and areas of opportunity for future research are identified.

Diabetic retinopathy is the retinal disease associated with hyperglycemia in patients who suffer from type 1 or type 2 diabetes. It includes maculopathy, involving the central retina and characterized by ischemia and/or edema, and peripheral retinopathy that progresses to a proliferative stage with neovascularization. Approximately 10% of the global population is estimated to suffer from diabetes, and around one in 5 of these individuals have diabetic retinopathy. One of the major effects of hyperglycemia is oxidative stress, the pathological state in which elevated production of reactive oxygen species damages tissues, cells, and macromolecules. The retina is relatively prone to oxidative stress due to its high metabolic activity. This review provides a summary of the role of oxidative stress in diabetic retinopathy, including a description of the retinal cell players and the molecular mechanisms. It discusses pathological processes, including the formation and effects of advanced glycation end-products, the impact of metabolic memory, and involvements of non-coding RNA. The opportunities for the therapeutic blockade of oxidative stress in diabetic retinopathy are also considered.

Retinopathy is a recently recognized complication of dengue, affecting up to 10% of hospitalized patients. Research on the pathogenesis has focused largely on effects of dengue virus (DENV) at the blood–retinal barrier. Involvement of retinal Müller glial cells has received little attention, although this cell population contributes to the pathology of other intraocular infections. The goal of our work was to establish the susceptibility of Müller cells to infection with DENV and to identify characteristics of the cellular antiviral, inflammatory, and immunomodulatory responses to DENV infection in vitro. Primary human Müller cell isolates and the MIO-M1 human Müller cell line were infected with the laboratory-adapted Mon601 strain and DENV serotype 1 and 2 field isolates, and cell–DENV interactions were investigated by immunolabelling and quantitative real-time polymerase chain reaction. Müller cells were susceptible to DENV infection, but experiments involving primary cell isolates indicated inter-individual variation. Viral infection induced an inflammatory response (including tumour necrosis factor-α, interleukin [IL]-1β, and IL-6) and an immunomodulatory response (including programmed death-ligand [PD-L]1 and PD-L2). The type I interferon response was muted in the Müller cell line compared to primary cell isolates. The highest infectivity and cell responses were observed in the laboratory-adapted strain, and overall, infectivity and cell responses were stronger in DENV2 strains. This work demonstrates that Müller cells mount an antiviral and immune response to DENV infection, and that this response varies across cell isolates and DENV strain. The research provides a direction for future efforts to understand the role of human retinal Müller glial cells in dengue retinopathy.

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein in the immunoglobulin superfamily expressed on the surface of multiple cell populations and upregulated by inflammatory stimuli. It mediates cellular adhesive interactions by binding to the β2 integrins macrophage antigen 1 and leukocyte function-associated antigen 1, as well as other ligands. It has important roles in the immune system, including in leukocyte adhesion to the endothelium and transendothelial migration, and at the immunological synapse formed between lymphocytes and antigen-presenting cells. ICAM-1 has also been implicated in the pathophysiology of diverse diseases from cardiovascular diseases to autoimmune disorders, certain infections, and cancer. In this review, we summarize the current understanding of the structure and regulation of the ICAM1 gene and the ICAM-1 protein. We discuss the roles of ICAM-1 in the normal immune system and a selection of diseases to highlight the breadth and often double-edged nature of its functions. Finally, we discuss current therapeutics and opportunities for advancements.

Abstract Ocular toxoplasmosis is the leading cause of posterior uveitis worldwide. We conducted an observational study of 262 consecutive individuals (n = 344 eyes) with ocular toxoplasmosis who were followed over a 34-month period. Most subjects were T. gondii IgG + /IgM- (n = 242; 92.4%; 317 eyes), and 140 eyes (40.7%) had active lesions. For eyes in which retinal lesions were active at recruitment and best-corrected visual acuity (BCVA) could be measured (n = 133), 21.0% (n = 28) remained blind (BCVA below 20/400) after inflammation resolved. In these eyes, atypical ocular toxoplasmosis (OR 4.99; 95% CI 1.14–22.85; p = 0.0330), macular lesion (OR 9.95; 95% CI 2.45–47.15; p = 0.0019) and any complication (OR 10.26; 95% CI 3.82–30.67; p

Uveitis is a common manifestation of post-Ebola syndrome, associated with persistence of Ebola virus (EBOV; Zaire ebolavirus) inside the eye. The iris and retinal pigment epithelia are key components of the blood-ocular barriers, but have the capacity to act as hosts for microorganisms. We investigated the ability of EBOV to productively infect these cell populations. Donor-matched human iris and retinal pigment epithelial isolates (n = 5) were infected with EBOV at a multiplicity of infection of 1 for up to 72 hours. Parallel cultures were infected with Reston virus (RESTV; Reston ebolavirus) or Zika virus (ZIKV), or held uninfected under the same conditions. Viral transcript expression by RT-qPCR on total cellular RNA, cytoimmunofluorescence, and assays of 50% tissue culture infected dose of culture supernatant showed that both iris and retinal pigment epithelial isolates were permissive to infection, and supported replication and release of EBOV, as well as RESTV and ZIKV. However, in comparison to cells isolated from iris, those from retina demonstrated obvious EBOV-induced cytopathic effect, had higher intracellular EBOV nucleoprotein transcript, expressed intracellular EBOV protein more widely, and released EBOV at higher titer. Comparable results were obtained for isolates infected with RESTV and ZIKV. Consistent with observations of retinal pigment epithelial scars in Ebola survivors, our results suggest that an early event in post-Ebola uveitis is infection of the retinal pigment epithelium. Relative susceptibility of retinal pigment epithelial cells to infection with RESTV and ZIKV, as well as EBOV, implies this phenomenon may relate to a cell-specific attribute, such as high phagocytic activity.

How mutations in the microbial receptor NOD2 induce Blau syndrome in humans and related uveitis is unclear. Here the authors show, using Nod2-deficient mice and experimental uveitis, that Nod2 negatively regulates T cell activation and transcription of autoimmunity-related genes to suppress Th17 responses and uveitis.

Abstract 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.

Abstract 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.

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.

Background: Retinopathy is a common adverse event with mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors. Little is known about the pathophysiology of MEK inhibitor-associated retinopathy (MEKAR). Since MEKAR has many similarities to central serous chorioretinopathy (CSCR), they may share common risk factors. The aim of this study was to evaluate the association between baseline characteristics and MEKAR in melanoma patients initiating MEK inhibitor treatment. Methods: Data from patients treated with cobimetinib and vemurafenib for advanced melanoma in the coBRIM trial were subjected to secondary analysis. Consistent with CSCR risk factors, assessed baseline characteristics included: age, gender, past ocular disease, weight, hypertension, diabetes, dyslipidemia, glomerular filtration rate (eGFR) and corticosteroid use. Associations between characteristics and retinopathy events (any grade and symptomatic) were evaluated using univariate logistic regression and represented as odds ratios (OR). Results: A total of 247 patients were treated with cobimetinib and vemurafenib, of whom 72 (29%) had retinopathy of any grade and 33 (13%) had symptomatic retinopathy. Patients with a history of ocular disease were at significantly higher risk of retinopathy (any grade, 44%; symptomatic, 22%) than those with no ocular disease history (any grade, 22%; symptomatic, 10%). Individuals with a history of ocular inflammation or infection were at highest risk: 4 of 5 developed symptomatic retinopathy during MEK inhibitor therapy. Increased age was associated with a higher risk of any grade retinopathy {decade increase OR [95% confidence interval (CI)] = 1.03 (1.01–1.05); p = 0.009}, while increasing eGFR was significantly associated with a decreased risk of any grade retinopathy [0.98 (0.96–0.99); p = 0.004]; the associations were not statistically significant with symptomatic retinopathy. Other assessed CSCR risk factors were not significantly associated with MEKAR. Conclusion: Age, glomerular filtration rate and history of ocular disease (particularly inflammatory eye disease) were associated with a risk of MEK inhibitor induced retinopathy. Patients who are at increased risk of MEKAR may benefit from more regular ophthalmologic assessment during treatment.

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-β 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.

Abstract 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.

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.

Recent clinical reports indicate that infection with dengue virus (DENV) commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin-β1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

When a person becomes infected with Toxoplasma gondii, ocular toxoplasmosis is the most common clinical presentation. The medical literature describes retinitis with surrounding hyperpigmentation secondary to proliferative changes in the retinal pigment epithelium, which is sufficiently characteristic that investigation often is not needed to make the diagnosis. We aimed to establish the frequency of “typical” ocular toxoplasmosis and delineate its molecular basis. Among 263 patients presenting consecutively with ocular toxoplasmosis to Ribeirão Preto General Hospital in Brazil, where T. gondii infection is endemic, 94.2% of 345 eyes had retinal hyperpigmentation. In ARPE-19 and primary human retinal pigment epithelial cell monolayers exposed to minimal numbers of T. gondii tachyzoites, the proliferation marker−KI-67−was increased in uninfected cells, which also were rendered more susceptible to infection. RT-qPCR and ELISA detected increased expression of vascular endothelial growth factor A (VEGF) and insulin-like growth factor (IGF)1, and decreased expression of thrombospondin (TSP)1 by infected cells. Blockade of VEGF and IGF1—or supplementation of TSP1—reversed the proliferation phenotype in uninfected cells. Our findings confirm that hyperpigmentation is a characteristic feature of retinitis in ocular toxoplasmosis, and demonstrate that T. gondii-infected human retinal pigment epithelial cells secrete VEGF and IGF1, and reduce production of TSP1, to promote proliferation of adjacent uninfected cells and create this disease-specific appearance.

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.

Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1) mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1), in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α), and identified putative binding sites for NF-κB1 within the ICAM-1 regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (si)RNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-α but did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-α but did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.