Optical Coherence Tomography Angiography Characteristics Serve as Retinal Vein Occlusion Therapeutic Biomarkers for Dexamethasone Intravitreal Implant.

BACKGROUND: Retinal vein occlusion (RVO) is the second most common vision-threatening retinal vascular disease. Intravitreal dexamethasone implant has been applied to treat macular edema secondary to RVO (RVO-ME). However, the alteration of morphologic features detected with optical coherence tomography angiography (OCTA) has not been fully studied in RVO-ME patients before and after the treatment. OBJECTIVE: This study is aimed at identifying potential therapeutic targets in RVO with integrative bioinformatic analysis and compares the OCTA characteristics alterations in patients with RVO-ME receiving injection of dexamethasone intravitreal implant. METHODS: Bioinformatic analysis was analyzed in GSE101398 dataset from the Gene Expression Omnibus database. Multiple functional enrichment analyses were performed, and protein-protein interaction network was constructed to visualize the key node genes. Eleven eyes with RVO-ME were examined with OCTA before and after intravitreal dexamethasone implant. The OCTA parameters, including macular thickness, vessel density, foveal avascular zone parameters, the number of hyperreflective foci (HRF), area of cystoid edema, and subretinal fluid (SRF), were compared. The correlation was analyzed between best-corrected visual acuity (BCVA) and OCTA parameters. RESULTS: A total of 79 differentially expressed genes were identified. Functional enrichment analyses revealed the enriched inflammatory events in RVO. In RVO-ME, Pearson correlation revealed that baseline BCVA was positively correlated with the area of SRF and central macular thickness, while no correlation was detected between baseline BCVA and HRF number or the area of cystoid edema. The visual acuity improved, and the central macular thickness was decreased after intravitreal dexamethasone implant injection. Besides, the number of HRF, the area of cystoid edema, and SRF were significantly alleviated after dexamethasone intravitreal injection. CONCLUSION: Retinal inflammation plays a crucial role in RVO pathogenesis. The imaging biomarkers of RVO including Müller glial intracellular edema, and retinal pigment epithelium dysfunction, could be assessed in OCTA and attenuated by intravitreal dexamethasone implant effectively.

Adenovirus-mediated down-regulation of miR-21-5p alleviates experimental autoimmune uveoretinitis in mice.

MiR-21-5p has been found to be up-regulated in the retina of experimental autoimmune uveoretinitis (EAU) mice and correlated with the pathogenesis of EAU. The objective of the present study is to explore the role of miR-21-5p in EAU. C57 mice were immunized with residue1-20 (IRBP1-20) in complete Freund's adjuvant supplemented with Mycobacterium tuberculosis H37Ra to induce EAU, and miR-21-5p was knocked down via subretinal injection of anti-miR-21-5p adenovirus. The pathological score, TUNEL positive cells and the expression of pro-inflammatory factors in the retina were reduced, and the expression of IL-10 was increased by down-regulation of miR-21-5p. Up-regulation of miR-21-5p significantly decreased the mRNA and protein levels of IL-10 in ARPE-19 cells. The binding activity of miR-21-5p on the 3'UTR of IL-10 mRNA was confirmed by luciferase reporter assay. Moreover, the miR-21-5p level in splenic lymphocytes of EAU mice was increased at the 7th day after immunization and reached its peak at the 14th day, that was in accordance with the changing trend with the Th17 cell frequency in the spleen. Besides, lentivirus-mediated down-regulation of miR-21-5p reduced the Th17 cell frequency and increased the Treg cell fraction of IRBP1-20-stimulated lymphocytes in vitro. Taken together, in situ down-regulation of miR-21-5p attenuates EAU by inhibiting inflammatory responses and reducing retinal cell apoptosis. miR-21-5p may also participate in the progress of EAU by affecting Th17/Treg balance via the regulation of IL-10. Therefore, we demonstrate that miR-21-5p can serve as a therapeutic target in the management of uveitis and other autoimmune diseases.

MicroRNA expression profile in retina and choroid in oxygen-induced retinopathy model.

BACKGROUND: Ischemic retinopathies (IRs) are leading causes of visual impairment. They are characterized by an initial phase of microvascular degeneration and a second phase of aberrant pre-retinal neovascularization (NV). microRNAs (miRNAs) regulate gene expression, and a number play a role in normal and pathological NV. But, post-transcriptional modulation of miRNAs in the eye during the development of IRs has not been systematically evaluated. AIMS & METHODS: Using Next Generation Sequencing (NGS) we profiled miRNA expression in the retina and choroid during vasodegenerative and NV phases of oxygen-induced retinopathy (OIR). RESULTS: Approximately 20% of total miRNAs exhibited altered expression (up- or down-regulation); 6% of miRNA were found highly expressed in retina and choroid of rats subjected to OIR. During OIR-induced vessel degeneration phase, miR-199a-3p, -199a-5p, -1b, -126a-3p displayed a robust decreased expression (> 85%) in the retina. While in the choroid, miR-152-3p, -142-3p, -148a-3p, -532-3p were upregulated (>200%) and miR-96-5p, -124-3p, -9a-3p, -190b-5p, -181a-1-3p, -9a-5p, -183-5p were downregulated (>70%) compared to controls. During peak pathological NV, miR-30a-5p, -30e-5p and 190b-5p were markedly reduced (>70%), and miR-30e-3p, miR-335, -30b-5p strongly augmented (by up to 300%) in the retina. Whereas in choroid, miR-let-7f-5p, miR-126a-5p and miR-101a-3p were downregulated by (>81%), and miR-125a-5p, let-7e-5p and let-7g-5p were upregulated by (>570%) during NV. Changes in miRNA observed using NGS were validated using qRT-PCR for the 24 most modulated miRNAs. In silico approach to predict miRNA target genes (using algorithms of miRSystem database) identified potential new target genes with pro-inflammatory, apoptotic and angiogenic properties. CONCLUSION: The present study is the first comprehensive description of retinal/choroidal miRNAs profiling in OIR (using NGS technology). Our results provide a valuable framework for the characterization and possible therapeutic potential of specific miRNAs involved in ocular IR-triggered inflammation, angiogenesis and degeneration.

Autoimmune Retinopathy: An Immunologic Cellular-Driven Disorder.

Autoimmune retinopathy (AIR) was often mistaken for retinitis pigmentosa (RP), due to an overlap of clinical findings, but increasingly has been recognized as a unique entity in the last decade. AIR has distinctive features: sudden onset of photopsias and scotomata in patients with no family history of RP, followed by visual field and central vision loss. Initially, retina exams are normal with no sign of pigment deposits or retinal degeneration. A family history of autoimmune diseases (all types) occurs in 60% of patients. One hallmark of AIR has been the presence of anti-retinal autoimmune antibodies (ARAs) in patients' sera, but patients can continue to have ARAs even when the disease has been quiescent for years. The accumulation of ARAs represents a breakdown of retinal immune tolerance with many different immunoreactive bands found at different reference weights in AIR patients. We began investigating cellular immunity using flow cytometry and found abnormal distributions (>2 StDev) of increased memory lymphocytes and NK cells and decreased regulatory B cell subsets in many AIR patients compared to normal controls. Culture of patient lymphocytes with small amounts (25 µg) of recoverin protein for 6 days led to significant elevations of interferon gamma (IFNγ) and in some cases tumor necrosis factor alpha (TNFα) production. We found the IFNγ/IL-10 ratio in response to recoverin was elevated in patients with more active disease (defined by visual field contraction between visits), but in some patients, there also appeared to be independent factors influencing severity, suggesting other autoimmune mechanisms were at play. These cellular immune parameters may provide improved markers for active AIR.

The effects of bevacizumab treatment in a rat model of retinal ischemia and perfusion injury.

Purpose: To create a model of an ischemic retina with temporary ischemia and reperfusion (IR) and to examine the possible antiapoptotic and neurodegenerative effects of a vascular endothelial growth factor (VEGF) antagonist. Methods: Three groups were formed. Rats were subjected to continued ischemia for 45 min, and then reperfusion was allowed for 2 days. For the first group, ischemia was induced, but an anti-VEGF agent was not administered. For the second group, 2 days before ischemia, 0.005 ml (0.125 mg) of bevacizumab was administered intravitreally, and then the ischemic model was created. The last group's intraocular pressure was not increased as in the control group, and only a cannula was introduced into the anterior chamber through the cornea. Six animals from each group were subjected to histomorphometry, and four were subjected to immunohistochemical and histopathologic examinations. For a histomorphometric examination, the number of cells in the retinal ganglion cell (RGC) layer was counted using the optical dissector method. For immunohistochemistry, the vascular endothelial growth factor receptor-2 (VEGFR-2) levels and apoptosis were examined in the retinal and choroidal tissue. Results: It was observed that in an IR injury, bevacizumab reduces the death and apoptosis of cells in the RGC layer. It was also identified that although bevacizumab is a large molecule, the agent affects the choroid and reduces the amount of VEGFR-2 in this tissue. Conclusions: IR may be used as a model of ischemic retinopathy that includes VEGF-dependent vascular permeability and neurodegeneration. Although VEGF is a neurotrophic molecule, in IR injury, treatment with bevacizumab, which is an anti-VEGF agent, decreases apoptosis, showing that excess function of this molecule can be hazardous.

Role of PU.1 Expression as an Inflammatory Marker in Experimental Autoimmune Uveoretinitis.

PURPOSE: PU.1 is an Ets family transcription factor, which is essential for the development of immune system through generation of myeloid and lymphoid lineages. In this study, we investigated PU.1 expression in the retina of mice with experimental autoimmune uveoretinitis (EAU) and the association between PU.1 expression level and inflammation in EAU. METHODS: IRBP 1-20 peptide-immunized mice were used. Quantitative PCR, ELISA analysis, cytometric bead array (CBA), assay and immunostaining were conducted using ocular tissues and lymph nodes. RESULTS: Quantitative PCR showed significant increases in mRNA levels of PU.1 in the retina at the peak of inflammation. Immunostaining of retina flat mounts revealed that most PU.1-positive cells were co-stained with anti-CD11c and anti-F4/80 antibodies. PU.1 knockdown in lymph node cells significantly suppressed IRBP-stimulated IFN-γ production measured by ELISA and IL-2 production measured by CBA. CONCLUSION: PU.1 may play crucial roles in the development and progression of inflammation in EAU.

Loss of CD40 attenuates experimental diabetes-induced retinal inflammation but does not protect mice from electroretinogram defects.

Chronic low grade inflammation is considered to contribute to the development of experimental diabetic retinopathy (DR). We recently demonstrated that lack of CD40 in mice ameliorates the upregulation of inflammatory molecules in the diabetic retina and prevented capillary degeneration, a hallmark of experimental diabetic retinopathy. Herein, we investigated the contribution of CD40 to diabetes-induced reductions in retinal function via the electroretinogram (ERG) to determine if inflammation plays a role in the development of ERG defects associated with diabetes. We demonstrate that diabetic CD40-/- mice are not protected from reduction to the ERG b-wave despite failing to upregulate inflammatory molecules in the retina. Our data therefore supports the hypothesis that retinal dysfunction found in diabetics occurs independent of the induction of inflammatory processes.

Up-regulated basigin-2 in microglia induced by hypoxia promotes retinal angiogenesis.

Retinal microglia cells contribute to vascular angiogenesis and vasculopathy induced by relative hypoxia. However, its concrete molecular mechanisms in shaping retinal angiogenesis have not been elucidated. Basigin, being involved in tumour neovasculogenesis, is explored to exert positive effects on retinal angiogenesis induced by microglia. Therefore, we set out to investigate the expression of basigin using a well-characterized mouse model of oxygen-induced retinopathy, which recapitulated hypoxia-induced aberrant neovessel growth. Our results elucidate that basigin is overexpressed in microglia, which accumulating in retinal angiogenic sprouts. In vitro, conditioned media from microglia BV2 under hypoxia treatment increase migration and tube formation of retinal capillary endothelia cells, compared with media from normoxic condition. The angiogenic capacity of BV2 is inhibited after basigin knockdown by small interfering RNAs. A new molecular mechanism for high angiogenic capacity, whereby microglia cells release basigin via up-regulation of PI3K-AKT and IGF-1 pathway to induce angiogenesis is unveiled. Collectively, our results demonstrate that basigin from hypoxic microglia plays a pivotal pro-angiogenic role, providing new insights into microglia-promoting retinal angiogenesis.

A novel bispecific molecule delivered by recombinant AAV2 suppresses ocular inflammation and choroidal neovascularization.

Elevated vascular endothelial growth factor (VEGF) and complement activation are implicated in the pathogenesis of different ocular diseases. The objective of this study was to investigate the hypothesis that dual inhibition of both VEGF and complement activation would confer better protection against ocular inflammation and neovascularization. In this study, we engineered a secreted chimeric VEGF inhibitor domain (VID), a complement inhibitor domain (CID) and a dual inhibitor (ACVP1). Vectors expressing these three inhibitors were constructed and packaged into AAV2 (sextY-F) particles. The expression and secretion of the proteins were validated by Western blot. The effects of these inhibitors expressed from AAV2 vectors were examined in endotoxin-induced uveitis (EIU), experimental autoimmune uveoretinitis (EAU) and choroidal neovascularization (CNV) mouse models. The AAV2 vectors expressing the CID- and ACVP1-attenuated inflammation in EIU and EAU model, whereas the vector expressing VID showed improved retinal structure damaged by EAU, but not affect the infiltration of inflammatory cells in EAU or EIU eyes. Both VID and CID vectors improved laser-induced retinal and choroid/RPE injuries and CNV, whereas ACVP1 vector provided significantly better protection. Our results suggest that gene therapy targeting VEGF and complement components could provide an innovative and long-term strategy for ocular inflammatory and neovascular diseases.

Hyperoxia-Induced Proliferative Retinopathy: Early Interruption of Retinal Vascular Development with Severe and Irreversible Neurovascular Disruption.

Bronchopulmonary dysplasia (BPD) is a major cause of neonatal morbidity in premature infants, occurring as a result of arrested lung development combined with multiple postnatal insults. Infants with BPD exposed to supplemental oxygen are at risk of retinopathy of prematurity as well. Thus, we studied the effects of hyperoxia on the retinal vasculature in a murine model of BPD. The retinal phenotype of this model, which we termed hyperoxia-induced proliferative retinopathy (HIPR), shows severe disruption of retinal vasculature and loss of vascular patterning, disorganized intra-retinal angiogenesis, inflammation and retinal detachment. Neonatal mice were subjected to 75% oxygen exposure from postnatal day (P)0 to P14 to model BPD, then allowed to recover in room air for 1 (P15), 7 (P21), or 14 days (P28). We quantified retinal thickness, protein levels of HIF-1α, NOX2, and VEGF, and examined the cellular locations of these proteins by immunohistochemistry. We examined the retinal blood vessel integrity and inflammatory markers, including macrophages (F4/80) and lymphocytes (CD45R). Compared to controls, normal retinal vascular development was severely disrupted and replaced by a disorganized sheet of intra-retinal angiogenesis in the HIPR mice. At all time-points, HIPR showed persistent hyaloidal vasculature and a significantly thinner central retina compared to controls. HIF-1α protein levels were increased at P15, while VEGF levels continued to increase until P21. Intra-retinal fibrinogen was observed at P21 followed by sub-retinal deposition in at P28. Inflammatory lymphocytes and macrophages were observed at P21 and P28, respectively. This model presents a severe phenotype of disrupted retinal vascular development, intra-retinal angiogenesis inflammation and retinal detachment.

Nrf2 promotes reparative angiogenesis through regulation of NADPH oxidase-2 in oxygen-induced retinopathy.

Revascularization of ischemic tissue is a highly desirable outcome in multiple diseases, including cardiovascular diseases and ischemic retinopathies. Oxidative stress and inflammation are both known to play a role in suppressing reparative angiogenesis in ischemic disease models including oxygen-induced retinopathy (OIR), but the regulatory molecules governing these pathophysiologic processes in retinal ischemia are largely unknown. Nrf2 is a major stress-response transcription factor that has been implicated in regulating ischemic angiogenesis in the retina and other tissue beds. Using Nrf2-deficient mice, we investigated the effects of Nrf2 in regulating revascularization and modulating the retinal tissue milieu during ischemia. Strikingly, Nrf2's beneficial effect on reparative angiogenesis only became manifested in the later phase of ischemia in OIR, from postnatal day 14 (P14) to P17. This was temporally associated with a reduction in both oxidative stress and inflammatory mediators in wild-type compared to Nrf2-/- mice. Nrf2-/- retinas exhibited an increase in VEGF but also induction of anti-angiogenic Dll4/Notch signaling. NADPH oxidase (NOX), and especially NOX2, is a major pathogenic molecule and a particularly important contributor to oxidative stress in multiple retinal disease processes. Nrf2-/- mice exhibited a significant exacerbation of NOX2 induction in OIR that manifested in the later phases of ischemia. Pharmacologic inhibition of NADPH oxidase abrogated the adverse effect of Nrf2 deficiency on reparative angiogenesis. Taken together, this suggests that Nrf2 is an important regulator of the retinal milieu during tissue ischemia, and that the Nrf2/NOX2 balance may play a critical role in determining the fate of ischemic revascularization.

Immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota activate retinal microglia in mouse models.

Although its actual role in the progression of degenerative processes is not fully known, the persistent activated state of retinal microglia and the concurrent secretion of inflammatory mediators may contribute to neuronal death and permanent vision loss. Our objective was to determine whether non-ocular conditions (immunosuppression and peripheral inflammation) could lead to activation of retinal microglia. Mouse models of immunosuppression induced by cyclophosphamide and/or peripheral inflammation by chemically induced sublethal colitis in C57BL/6J mice were used. Retinal microglia morphology, spatial distribution and complexity, as well as MHCII and CD11b expression levels were determined by flow cytometry and confocal immunofluorescence analysis with anti-CD11b, anti-IBA1 and anti-MHCIIRT1B antibodies. Retinas of mice with double treatment showed changes in microglial morphology, spatial distribution and expression levels of CD11b and MHCII. These effects were higher than those observed with any treatment separately. In addition, we also observed in these mice: (i) translocation of endogenous bacteria from gut to liver, and (ii) upregulation of TLR2 expression in retinal microglia. Using a mouse model of immunosuppression and gut colonization by Candida albicans, translocation of fungal cells was confirmed to occur in wild type and, to a higher extent, in TLR2 KO mice, which are more susceptible to fungal invasion; interestingly microglial changes were also higher in TLR2 KO mice. Hence, non-ocular injuries (immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota) can activate retinal microglia and therefore could affect the progression of neurodegenerative disorders and should be taken into account to improve therapeutic options.

MicroRNAs in retina during development of experimental autoimmune uveoretinitis in rats.

PURPOSE: To determine the changes in the expression profiles of microRNAs (miRNAs) in retinas during the development of experimental autoimmune uveoretinitis (EAU) in rats. METHODS: The levels of interleukin-1ß (IL-1ß) and monocyte chemoattractant protein-1 (MCP-1) were measured in aqueous humour samples and supernatants of homogenised posterior eye cups obtained from Lewis rats immunised with interphotoreceptor retinoid binding protein peptide (R14) and complete Freund's adjuvant. Microarray analysis was performed to determine the miRNA profiles in the retina of eyes with EAU on days 0 (baseline), 7, 14 and 21 after immunisation. RESULTS: The levels of IL-1ß and MCP-1 in the aqueous humour and the supernatants of posterior eye cups were significantly elevated in eyes with EAU, and the levels corresponded with the stage of the EAU. On day 14 after immunisation, the expressions of nine miRNAs (miRNA-223, 142-5p, 142-3p, 21, 146a, 146b, 1949, 1188-3p and 193) were significantly elevated, and the expressions of four miRNAs (miRNA-181a, 183*, 124* and 331) were downregulated relative to the baseline. Quantitative PCR analyses confirmed the elevation of miRNA-223 and miRNA-146 and the downregulation of miRNA-181a in retinas with EAU on day 14 after immunisation. In situ hybridisation confirmed increased expression of miR-223 and miR-146 in retinas with EAU. CONCLUSIONS: Several miRNAs were significantly increased or decreased in retinas during the course of EAU. The expression of miR-223 and miR-146a corresponded with the clinical score of the EAU and elevation of IL-1ß/MCP-1 in the eye with EAU. Further studies are required to clarify the role of miRNA in eyes with autoimmune uveoretinitis.

Necrosis retiniana aguda bilateral en el anciano por varicela zóster / Bilateral acute retinal necrosis due to varicella zóster virus in an elderly patient

CASO CLÍNICO: Presentamos a un paciente de 77 años, con un cuadro de necrosis retiniana aguda con afectación bilateral; la reacción en cadena de la polimerasa (PCR) de la muestra de humor acuoso fue positiva al virus de varicela zóster. En su evolución desarrolla vasculitis de Kyrieleis al mes de inicio del tratamiento y con el análisis por PCR negativo. DISCUSIÓN: La PCR es un método rápido, con una sensibilidad y especificidad del 97%. La actuación debe ser urgente por la rapidez de la progresión. El famciclovir oral es buena alternativa por su mejor biodisponibilidad

CASE REPORT: The case is reported of acute retinal necrosis with bilateral involvement due to Varicella Zoster virus in a 77 year-old man. Polymerase chain reaction (PCR) of aqueous humor was positive for Varicella Zoster virus (VZV). He developed a Kyrieleis’ vasculitis a month after the starting treatment, when the PCR analysis was negative. DISCUSSION: PCR is a quick and safe technique, with a high sensitivity and specificity of 97%, useful to diagnose and monitor the viral activity. The intervention must be urgent, due to the dramatically rapid evolution. Oral famciclovir oral is good alternative owing to its bioavailability

The role of high mobility group box 1 (HMGB-1) in the diabetic retinopathy inflammation and apoptosis.

Diabetic Retinopathy (DR) is one of the most common complications of the late phase diabetes, and also a common cause of blindness. High mobility group box 1 (HMGB-1) is considered to be an inflammatory mediator in the late phase that promotes inflammation and neovascularization in diabetes. Therefore, this paper discussed the role of HMGB-1 in diabetic retinopathy inflammation and neovascularization. 96 adult SD rats were randomly divided into control and diabetes group. The diabetic rat model was established by intraperitoneal injection of streptomycin (0.1 mol/L). Western blot was applied to determine HMGB-1 and its receptor RAGE and TLR2 protein expression in the serum. TUNEL was used to detect retinal apoptosis. Immunofluorescence was performed to test HMGB1 protein expression in retina. HBGM-1 and RAGE expression in diabetic rat retina was significantly higher than the control (P < 0.05), while TLR2 expression was lower (P < 0.05). TUNEL detection showed that diabetic rat retinal cells presented obviously higher apoptosis rate (P < 0.05). Immunofluorescence test revealed that HMGB1 largely expressed in the diabetic rat retinal cells (P < 0.05). HMGB1 may involve in the pathogenesis of diabetic retinopathy by binding with RAGE receptor to accelerate rat retinal cells apoptosis.

S100B Up-Regulates Macrophage Production of IL1ß and CCL22 and Influences Severity of Retinal Inflammation.

S100B is a Ca2+ binding protein and is typically associated with brain and CNS disorders. However, the role of S100B in an inflammatory situation is not clear. The aim of the study was to determine whether S100B is likely to influence inflammation through its effect on macrophages. A murine macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages were used for in vitro studies and a model of retinal inflammatory disease in which pathogenesis is highly dependent on macrophage infiltration, Experimental Autoimmune Uveoretinitis, for in vitro study. Experimental Autoimmune Uveoretinitis is a model for the human disease posterior endogenous uveoretinitis, a potentially blinding condition, with an autoimmune aetiology, that mainly affects the working age group. To date the involvement of S100B in autoimmune uveoretinitis has not been investigated. Real-time PCR array analysis on RAW 246.7 cells indicated up-regulation of gene expression for various cytokines/chemokines in response to S100B, IL-1ß and CCL22 in particular and this was confirmed by real-time PCR. In addition flow cytometry and ELISA confirmed up-regulation of protein production in response to S100B for pro-IL-1ß and CCL22 respectively. This was the case for both RAW 264.7 cells and bone marrow derived macrophages. Induction of EAU with retinal antigen in mice in which S100B had been deleted resulted in a significantly reduced level of disease compared to wild-type mice, as determined by topical endoscopic fundus imaging and histology grading. Macrophage infiltration was also significantly reduced in S100B deleted mice. Real-time PCR analysis indicated that this was associated with reduction in CCL22 and IL-1ß in retinas from S100B knock-out mice. In conclusion S100B augments the inflammatory response in uveoretinitis and this is likely to be, at least in part, via a direct effect on macrophages.

The role of lipoprotein-associated phospholipase A2 in a murine model of experimental autoimmune uveoretinitis.

Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

In vivo multi-modal imaging of experimental autoimmune uveoretinitis in transgenic reporter mice reveals the dynamic nature of inflammatory changes during disease progression.

BACKGROUND: Experimental autoimmune uveoretinitis (EAU) is a widely used experimental animal model of human endogenous posterior uveoretinitis. In the present study, we performed in vivo imaging of the retina in transgenic reporter mice to investigate dynamic changes in exogenous inflammatory cells and endogenous immune cells during the disease process. METHODS: Transgenic mice (C57Bl/6 J Cx 3 cr1 (GFP/+) , C57Bl/6 N CD11c-eYFP, and C57Bl/6 J LysM-eGFP) were used to visualize the dynamic changes of myeloid-derived cells, putative dendritic cells and neutrophils during EAU. Transgenic mice were monitored with multi-modal fundus imaging camera over five time points following disease induction with the retinal auto-antigen, interphotoreceptor retinoid binding protein (IRBP1-20). Disease severity was quantified with both clinical and histopathological grading. RESULTS: In the normal C57Bl/6 J Cx 3 cr1 (GFP/+) mouse Cx3cr1-expressing microglia were evenly distributed in the retina. In C57Bl/6 N CD11c-eYFP mice clusters of CD11c-expressing cells were noted in the retina and in C57Bl/6 J LysM-eGFP mice very low numbers of LysM-expressing neutrophils were observed in the fundus. Following immunization with IRBP1-20, fundus examination revealed accumulations of Cx3cr1-GFP(+) myeloid cells, CD11c-eYFP(+) cells and LysM-eGFP(+) myelomonocytic cells around the optic nerve head and along retinal vessels as early as day 14 post-immunization. CD11c-eYFP(+) cells appear to resolve marginally earlier (day 21 post-immunization) than Cx3cr1-GFP(+) and LysM-eGFP(+) cells. The clinical grading of EAU in transgenic mice correlated closely with histopathological grading. CONCLUSIONS: These results illustrate that in vivo fundus imaging of transgenic reporter mice allows direct visualization of various exogenously and endogenously derived leukocyte types during EAU progression. This approach acts as a valuable adjunct to other methods of studying the clinical course of EAU.

Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India.

PURPOSE: To evaluate the diagnostic value of PCR on aqueous humour for detection and genotyping of Epstein Bar Virus in patients with viral retinitis. METHODS: 70 AH samples were collected from 20 HIV positive patients with clinically suspected viral retinitis and 25 patients with serpignous choroiditis and 25 AH from patients undergoing cataract surgery. PCR was performed to screen HHV-1 to HHV-5, Mtb and Toxoplasma gondii. Genotype prevalence was confirmed by phylogenetic analysis targetig EBV. RESULTS: EBV was detected in 17 (37.7%) samples. Genotyping to subtype EBV, revealed the circulation of only one subtype (Type 1). PCR results for other infective agents were negative except for the presence of CMV in 5 (11.1%) AH. CONCLUSION: The application of PCR to detect genotypes can be used as an epidemiological tool for clinical management. To our knowledge this is the first report on genotyping of EBV performed on intra ocular samples.

Human genetic disorders of sphingolipid biosynthesis.

Monogenic defects of sphingolipid biosynthesis have been recently identified in human patients. These enzyme deficiencies affect the synthesis of sphingolipid precursors, ceramides or complex glycosphingolipids. They are transmitted as autosomal recessive or dominant traits, and their resulting phenotypes often replicate the abnormalities seen in murine models deficient for the corresponding enzymes. In quite good agreement with the known critical roles of sphingolipids in cells from the nervous system and the epidermis, these genetic defects clinically manifest as neurological disorders, including paraplegia, epilepsy or peripheral neuropathies, or present with ichthyosis. The present review summarizes the genetic alterations, biochemical changes and clinical symptoms of this new group of inherited metabolic disorders. Hypotheses regarding the molecular pathophysiology and potential treatments of these diseases are also discussed.