Risk factors and biomarkers of age-related macular degeneration

A biomarker can be a substance or structure measured in body parts, fluids or products that can affect or predict disease incidence. As age-related macular degeneration (AMD) is the leading cause of blindness in the developed world, much research and effort has been invested in the identification of different biomarkers to predict disease incidence, identify at risk individuals, elucidate causative pathophysiological etiologies, guide screening, monitoring and treatment parameters, and predict disease outcomes. To date, a host of genetic, environmental, proteomic, and cellular targets have been identified as both risk factors and potential biomarkers for AMD. Despite this, their use has been confined to research settings and has not yet crossed into the clinical arena. A greater understanding of these factors and their use as potential biomarkers for AMD can guide future research and clinical practice. This article will discuss known risk factors and novel, potential biomarkers of AMD in addition to their application in both academic and clinical settings.

Effectiveness of Community versus Hospital Eye Service follow-up for patients with neovascular age-related macular degeneration with quiescent disease (ECHoES): a virtual non-inferiority trial

OBJECTIVES: To compare the ability of ophthalmologists versus optometrists to correctly classify retinal lesions due to neovascular age-related macular degeneration (nAMD).

DESIGN: Randomised balanced incomplete block trial. Optometrists in the community and ophthalmologists in the Hospital Eye Service classified lesions from vignettes comprising clinical information, colour fundus photographs and optical coherence tomographic images. Participants' classifications were validated against experts' classifications (reference standard).

SETTING: Internet-based application.

PARTICIPANTS: Ophthalmologists with experience in the age-related macular degeneration service; fully qualified optometrists not participating in nAMD shared care.

INTERVENTIONS: The trial emulated a conventional trial comparing optometrists' and ophthalmologists' decision-making, but vignettes, not patients, were assessed. Therefore, there were no interventions and the trial was virtual. Participants received training before assessing vignettes.

MAIN OUTCOME MEASURES: Primary outcome-correct classification of the activity status of a lesion based on a vignette, compared with a reference standard. Secondary outcomes-potentially sight-threatening errors, judgements about specific lesion components and participants' confidence in their decisions.

RESULTS: In total, 155 participants registered for the trial; 96 (48 in each group) completed all assessments and formed the analysis population. Optometrists and ophthalmologists achieved 1702/2016 (84.4%) and 1722/2016 (85.4%) correct classifications, respectively (OR 0.91, 95% CI 0.66 to 1.25; p=0.543). Optometrists' decision-making was non-inferior to ophthalmologists' with respect to the prespecified limit of 10% absolute difference (0.298 on the odds scale). Optometrists and ophthalmologists made similar numbers of sight-threatening errors (57/994 (5.7%) vs 62/994 (6.2%), OR 0.93, 95% CI 0.55 to 1.57; p=0.789). Ophthalmologists assessed lesion components as present less often than optometrists and were more confident about their classifications than optometrists.

CONCLUSIONS: Optometrists' ability to make nAMD retreatment decisions from vignettes is not inferior to ophthalmologists' ability. Shared care with optometrists monitoring quiescent nAMD lesions has the potential to reduce workload in hospitals.

TRIAL REGISTRATION NUMBER: ISRCTN07479761; pre-results registration.

RETINAL ANGIOMATOUS PROLIFERATION: A Quantitative Analysis of the Fundoscopic Features of the Fellow Eye

PURPOSE: To quantitatively analyze and compare the fundoscopic features between fellow eyes of retinal angiomatous proliferation and typical exudative age-related macular degeneration and to identify possible predictors of neovascularization. METHODS: Retrospective case-control study. Seventy-nine fellow eyes of unilateral retinal angiomatous proliferation (n = 40) and typical exudative age-related macular degeneration (n = 39) were included. Fundoscopic features of the fellow eyes were assessed using digital color fundus photographs taken at the time of diagnosis of neovascularization in the first affected eye. Grading was performed by two independent graders using RetmarkerAMD, a computer-assisted grading software based on the International Classification and Grading System for age-related macular degeneration. RESULTS: Baseline total number and area (square micrometers) of drusen in the central 1,000, 3,000, and 6,000 μm were considerably inferior in the fellow eyes of retinal angiomatous proliferation, with statistically significant differences (P < 0.05) observed in virtually every location (1,000, 3,000, and 6,000 μm). A soft drusen (≥125 μm) area >510,196 μm2 in the central 6,000 μm was associated with an increased risk of neovascularization (hazard ratio, 4.35; 95% confidence interval [1.56-12.15]; P = 0.005). CONCLUSION: Baseline fundoscopic features of the fellow eye differ significantly between retinal angiomatous proliferation and typical exudative age-related macular degeneration. A large area (>510,196 μm2) of soft drusen in the central 6,000 μm confers a significantly higher risk of neovascularization and should be considered as a phenotypic risk factor.

Giant cell tumor of tendon sheath: study of 64 cases and review of literature

The giant cell tumor of tendon sheath (GCTTS) is the most common benign neoplasm in the hand after the ganglion cyst. Several hypotheses were formulated about the etiological factors of these tumors, but still there is not a common opinion on etiology, prognostic factors and recurrence rate. This article presents a review of literature of the last 15 years about GCTTS to assess the demographic, clinical and histological profile. We compared the information obtained from literature with our experience of 64 cases between 2000 and 2012. Our study showed similar results to those reported in literature, except for the recurrence rate: only 3 cases (4.7%) of 64 patients reported recurrence (versus about 15% on average in literature). Among the various possible factors that predispose to recurrence, it is necessary that the surgeon ensures complete excision of the tumor and removal of any residual satellite nodules. Although the marginal excision is the treatment of choice, it is often difficult to perform due to for the location and the strict adherence of the tumor to the tendon or neurovascular bundles. We used in all cases a magnifying loupe to help a careful research of satellite lesions and to respect surrounding structures.

RETINAL ANGIOMATOUS PROLIFERATION: A Quantitative Analysis of the Fundoscopic Features of the Fellow Eye

PURPOSE: To quantitatively analyze and compare the fundoscopic features between fellow eyes of retinal angiomatous proliferation and typical exudative age-related macular degeneration and to identify possible predictors of neovascularization. METHODS: Retrospective case-control study. Seventy-nine fellow eyes of unilateral retinal angiomatous proliferation (n = 40) and typical exudative age-related macular degeneration (n = 39) were included. Fundoscopic features of the fellow eyes were assessed using digital color fundus photographs taken at the time of diagnosis of neovascularization in the first affected eye. Grading was performed by two independent graders using RetmarkerAMD, a computer-assisted grading software based on the International Classification and Grading System for age-related macular degeneration. RESULTS: Baseline total number and area (square micrometers) of drusen in the central 1,000, 3,000, and 6,000 μm were considerably inferior in the fellow eyes of retinal angiomatous proliferation, with statistically significant differences (P < 0.05) observed in virtually every location (1,000, 3,000, and 6,000 μm). A soft drusen (≥125 μm) area >510,196 μm2 in the central 6,000 μm was associated with an increased risk of neovascularization (hazard ratio, 4.35; 95% confidence interval [1.56-12.15]; P = 0.005). CONCLUSION: Baseline fundoscopic features of the fellow eye differ significantly between retinal angiomatous proliferation and typical exudative age-related macular degeneration. A large area (>510,196 μm2) of soft drusen in the central 6,000 μm confers a significantly higher risk of neovascularization and should be considered as a phenotypic risk factor.

Impaired Mesopic Visual Acuity in Eyes with Early Age-Related Macular Degeneration

Purpose.: To determine photopic and mesopic distance high-contrast visual acuity (HC-VA) and low-contrast visual acuity (LC-VA) in eyes with early age-related macular degeneration (AMD). Methods.: Measurements were made in 22 subjects with early AMD and 28 healthy control subjects. Inclusion criteria included a photopic HC-VA of 20/25 or better. Distance VA was measured using HC (96%) and LC (10%) Bailey-Lovie logMAR letter charts under photopic (85 cd/m2) and mesopic (0.1–0.2 cd/m2) luminance conditions. Results.: Mean mesopic distance HC-VA and LC-VA were significantly worse (0.1 logMAR and 0.28 logMAR, respectively) in the early AMD group than in the control group. Under mesopic conditions, the mean difference between LC-VA and HC-VA was significantly greater in the early AMD (0.45 logMAR) than the control group (0.27 logMAR). Mean differences between mesopic versus photopic HC-VA and mesopic versus photopic LC-VA were significantly greater in the early AMD than the control group (0.13 and 0.32 logMAR of difference between the means, respectively). Sensitivity and specificity were significantly greater for mesopic LC-VA than for mesopic HC-VA (Receiver Operating Characteristics, area under the curve [AUC], 0.94 ± 0.030 and 0.76 ± 0.067, respectively). AUC values for photopic HC-VA and LC-VA were below 0.70. Conclusions.: Visual acuity testing under low luminance conditions emerged as an optimal quantitative measure of retinal function in early AMD.

Retinal Macroglial Responses in Health and Disease

Due to their permanent and close proximity to neurons, glial cells perform essential tasks for the normal physiology of the retina. Astrocytes andM¨uller cells (retinal macroglia) provide physical support to neurons and supplement them with several metabolites and growth factors.Macroglia are involved in maintaining the homeostasis of extracellular ions and neurotransmitters, are essential for information processing in neural circuits, participate in retinal glucose metabolism and in removing metabolic waste products, regulate local blood flow, induce the blood-retinal barrier (BRB), play fundamental roles in local immune response, and protect neurons from oxidative damage. In response to polyetiological insults, glia cells react with a process called reactive gliosis, seeking to maintain retinal homeostasis. When malfunctioning, macroglial cells can become primary pathogenic elements. A reactive gliosis has been described in different retinal pathologies, including age-related macular degeneration (AMD), diabetes, glaucoma, retinal detachment, or retinitis pigmentosa. A better understanding of the dual, neuroprotective, or cytotoxic effect of macroglial involvement in retinal pathologies would help in treating the physiopathology of these diseases.The extensive participation of the macroglia in retinal diseases points to these cells as innovative targets for new drug therapies.

Steroid induced neuroprotection of damaged photoreceptor cells

Retinitis Pigmentosa (RP) is the name given to a group of hereditary diseases causing progressive and degenerative blindness. RP affects over 1 in 4000 individuals, making it the most prevalent inherited retinal disease worldwide, yet currently there is no cure. In 2011, our group released a paper detailing the protective effects of the synthetic progestin ‘Norgestrel’. A common component of the female oral contraceptive pill, Norgestrel was shown to protect against retinal cell death in two distinct mouse models of retinal degeneration: in the Balb/c light damage model and the Pde6brd10 (rd10) model. Little was known of the molecular workings of this compound however and thus this study aimed to elucidate the protective manner in which Norgestrel worked. To this aim, the 661W cone photoreceptor-like cell line and ex vivo retinal explanting was utilised. We found that Norgestrel induces a increase in neuroprotective basic fibroblast growth factor (bFGF) with subsequent downstream actions on the inhibition of glycogen synthase kinase 3β. Progesterone receptor expression was subsequently characterised in the C57 and rd10 retinas and in the 661W cell line. Norgestrel caused nuclear trafficking of progesterone receptor membrane complex one (PGRMC1) in 661W cells and thus Norgestrel was hypothesised to work primarily through the actions of PGRMC1. This trafficking was shown to be responsible for the critical upregulation of bFGF and PGRMC1- Norgestrel binding was proven to cause a neuroprotective bFGF-mediated increase in intracellular calcium. The protective properties of Norgestrel were further studied in the rd10 mouse model of retinitis pigmentosa. Using non-invasive diet supplementation (80mg/kg), we showed that Norgestrel gave significant retinal protection out to postnatal day 40 (P40). Overactive microglia have previously been shown to potentiate photoreceptor cell loss in the degenerating rd10 retina and thus we focussed on Norgestrel-mediated changes in photoreceptor-microglial crosstalk. Norgestrel acted to dampen pro-inflammatory microglial cell reactivity, decreasing chemokine (MCP1, MCP3, MIP-1α, MIP-1β) and subsequent damaging cytokine (TNFα, Il-1β) production. Critically, Norgestrel up-regulated photoreceptor-microglial, fractalkine-CX3CR1 signalling 1000-fold in the P20 rd10 mouse. Known to prevent microglial activation, we hypothesise that Norgestrel acts as a vital anti-inflammatory in the diseased retina, driving fractalkine-CX3CR1 signalling to delay retinal degeneration. This study stands to highlight some of the neuroprotective mechanisms utilised by Norgestrel in the prevention of photoreceptor cell death. We identify for the first time, not only a pro-survival pathway activated directly in photoreceptor cells, but also a Norgestreldriven mediation of an otherwise damaging microglial cell response. All taken, these results form the beginning of a case to bring Norgestrel to clinical trials, as a potential therapeutic for the treatment of RP.

Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Autologous Platelet Gel: Five Cases Illustrating Use On Sickle Cell Disease Ulcers.

Leg ulcers represent a particularly disabling complication in patients with sickle cell disease (SCD). Platelet gel (PG) is a novel therapeutic strategy used for accelerating wound healing of a wide range of tissues through the continuous release of platelet growth factors. Here, we describe the use of PG preparation according to Anitua's PRGF (preparations rich in growth factors) protocol for treating chronic nonhealing ulcers in patients with SCD. A positive response occurred in 3 patients with an area reduction of 85.7% to 100%, which occurred within 7 to 10 weeks, and a 35.2% and 20.5% of area reduction in 2 other patients, who however, had large ulcers. After calcium chloride addition, the platelet-rich plasmas demonstrated enhanced platelet-derived growth factors-BB (P < .001), transforming growth factor-β1 (P = .015), vascular endothelial growth factors (P = .03), and hepatocyte growth factors (nonsignificant) secretion. Furthermore, calcium chloride addition induced a significant decrease in platelet number (P = .0134) and there was no leukocyte detection in the PG product. These results demonstrate that PG treatment might impact the healing of leg ulcers in sickle cell disease, especially in patients with small ulcers.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Integrated Proteomics Identified Up-regulated Focal Adhesion-mediated Proteins In Human Squamous Cell Carcinoma In An Orthotopic Murine Model.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.