Advancing a stem cell therapy for age-related macular degeneration

The retinal pigment epithelium (RPE) is a multifunctional monolayer located at the back of the eye required for the survival and function of the light-sensing photoreceptors. In Age-related Macular Degeneration (AMD), the loss of RPE cells leads to photoreceptor death and permanent blindness. RPE cell transplantation aims to halt or reverse vision loss by preventing the death of photoreceptor cells and is considered one of the most viable applications of stem cell therapy in the field of regenerative medicine. Proof-of-concept of RPE cell transplantation for treating retinal degenerative disease, such as AMD, has long been established in animal models and humans using primary RPE cells, while recent research has focused on the transplantation of RPE cells derived from human pluripotent stem cells (hPSC). Early results from clinical trials indicate that transplantation of hPSC-derived RPE cells is safe and can improve vision in AMD patients. Current hPSC-RPE cell production protocols used in clinical trials are nevertheless inefficient. Treatment of large numbers of AMD patients using stem cellderived products may be dependent on the ability to generate functional cells from multiple hPSC lines using robust and clinically-compliant methods. Transplantation outcomes may be improved by delivering RPE cells on a thin porous membrane for better integration into the retina, and by manipulation of the outcome through control of immune rejection and inflammatory responses

Preserving Retinal Structure and Function with the Novel Nitroxide Antioxidant, DCTEIO

Oxidative stress is a major contributor to progressive neurodegenerative disease and may be a key target for the development of novel preventative and therapeutic strategies. Nitroxides have been successfully utilised to study changes in redox status (biological probes) and modulate radical-induced oxidative stress. This study investigates the efficacy of DCTEIO (5,6-dicarboxy-1,1,3,3-tetraethyllisoindolin-2-yloxyl), a stable, kinetically-persistent, nitroxide-based antioxidant, as a retinal neuroprotectant. The preservation of retinal function following an acute ischaemic/reperfusion (I/R) insult in the presence of DCTEIO was quantified by electroretinography (ERG). Inflammatory responses in retinal glia were analysed by GFAP and IBA-1 immunohistochemistry, and retinal integrity assessed by histology. A nitroxide probe combined with flow cytometry provided a rapid technique to assess oxidative stress and the mitigation offered by antioxidant compounds in cultured 661W photoreceptor cells. DCTEIO protected the retina from I/R-induced damage, maintaining retinal function. Histological analysis showed preservation of retinal integrity with reduced disruption and disorganisation of the inner and outer nuclear layers. I/R injury upregulated GFAP expression, indicative of retinal stress, which was significantly blunted by DCTEIO. The number of ‘activated’ microglia, particularly in the outer retina, in response to cellular stress was also significantly reduced by DCTEIO, potentially suggesting reduced inflammasome activation and cell death. DCTEIO mitigated oxidative stress in 661W retinal cell cultures, in a dose-dependent fashion. Together these findings demonstrate the potential of DCTEIO as a neuroprotective therapeutic for degenerative diseases of the CNS that involve an ROS-mediated component, including those of the retina e.g. age-related macular degeneration and glaucoma.</p

In Vivo screening and discovery of novel candidate thalidomide analogs in the zebrafish embryo and chicken embryo model systems

This study was supported by a Wellcome Trust-NIH PhD Studentship to SB, WDF and NV. Grant number 098252/Z/12/Z. SB, CHC and WDF are supported by the Intramural Research Program, NCI, NIH. NHG and WL are supported by the Intramural Research Program, NIA, NIH.Peer reviewedPublisher PD

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories

Conditional müller cell ablation causes independent neuronal and vascular pathologies in a novel transgenic model

Müller cells are the major glia of the retina that serve numerous functions essential to retinal homeostasis, yet the contribution of Müller glial dysfunction to retinal diseases remains largely unknown. We have developed a transgenic model using a portion of the regulatory region of the retinaldehyde binding protein 1 gene for conditional Müller cell ablation and the consequences of primary Müller cell dysfunction have been studied in adult mice. We found that selective ablation of Müller cells led to photoreceptor apoptosis, vascular telangiectasis, blood-retinal barrier breakdown and, later, intraretinal neovascularization. These changes were accompanied by impaired retinal function and an imbalance between vascular endothelial growth factor-A (VEGF-A) and pigment epithelium-derived factor. Intravitreal injection of ciliary neurotrophic factor inhibited photoreceptor injury but had no effect on the vasculopathy. Conversely, inhibition of VEGF-A activity attenuated vascular leak but did not protect photoreceptors. Our findings show that Müller glial deficiency may be an important upstream cause of retinal neuronal and vascular pathologies in retinal diseases. Combined neuropro-tective and anti-angiogenic therapies may be required to treat Müller cell deficiency in retinal diseases and in other parts of the CNS associated with glial dysfunction

Obesity-induced metabolic disturbance drives oxidative stress and complement activation in the retinal environment

Purpose Systemic increases in reactive oxygen species, and their association with inflammation, have been proposed as an underlying mechanism linking obesity and age-related macular degeneration (AMD). Studies have found increased levels of oxidative stress biomarkers and inflammatory cytokines in obese individuals; however, the correlation between obesity and retinal inflammation has yet to be assessed. We used the leptin-deficient (ob/ob) mouse to further our understanding of the contribution of obesity to retinal oxidative stress and inflammation.This study was supported by an Australian Government Research Training Program (RTP) Scholarship

Retinal Macrophages Synthesize C3 and Activate Complement in AMD and in Models of Focal Retinal Degeneration

PURPOSE. Complement system dysregulation is strongly linked to the progression of age-related macular degeneration (AMD). Deposition of complement including C3 within the lesions in atrophic AMD is thought to contribute to lesion growth, although the contribution of local cellular sources remains unclear. We investigated the role of retinal microglia and macrophages in complement activation within atrophic lesions, in AMD and in models of focal retinal degeneration. METHODS. Human AMD donor retinas were labeled for C3 expression via in situ hybridization. Rats were subject to photo-oxidative damage, and lesion expansion was tracked over a 2- month period using optical coherence tomography (OCT). Three strategies were used to determine the contribution of local and systemic C3 in mice: total C3 genetic ablation, local C3 inhibition using intravitreally injected small interfering RNA (siRNA), and depletion of serum C3 using cobra venom factor. RESULTS. Retinal C3 was expressed by microglia/macrophages located in the outer retina in AMD eyes. In rodent photo-oxidative damage, C3-expressing microglia/macrophages and complement activation were located in regions of lesion expansion in the outer retina over 2 months. Total genetic ablation of C3 ameliorated degeneration and complement activation in retinas following damage, although systemic depletion of serum complement had no effect. In contrast, local suppression of C3 expression using siRNA inhibited complement activation and deposition, and reduced cell death. CONCLUSIONS. These findings implicate C3, produced locally by retinal microglia/macrophages, as contributing causally to retinal degeneration. Consequently, this suggests that C3-targeted gene therapy may prove valuable in slowing the progression of AMD.Supported by a grant from Retina Australia, as well as the National Health and Medical Research Council (NHMRC; APP1049990). Also supported by an Australian Government Research Training Program (RTP) Scholarshi

rAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

PURPOSE. To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45 ϩ leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye. (Invest Ophthalmol Vis Sci. 2009;50:4279 -4287

A global perspective on the trophic geography of sharks

Sharks are a diverse group of mobile predators that forage across varied spatial scales and have the potential to influence food web dynamics. The ecological consequences of recent declines in shark biomass may extend across broader geographic ranges if shark taxa display common behavioural traits. By tracking the original site of photosynthetic fixation of carbon atoms that were ultimately assimilated into muscle tissues of 5,394 sharks from 114 species, we identify globally consistent biogeographic traits in trophic interactions between sharks found in different habitats. We show that populations of shelf-dwelling sharks derive a substantial proportion of their carbon from regional pelagic sources, but contain individuals that forage within additional isotopically diverse local food webs, such as those supported by terrestrial plant sources, benthic production and macrophytes. In contrast, oceanic sharks seem to use carbon derived from between 30° and 50° of latitude. Global-scale compilations of stable isotope data combined with biogeochemical modelling generate hypotheses regarding animal behaviours that can be tested with other methodological approaches.This research was conducted as part of C.S.B.’s Ph.D dissertation, which was funded by the University of Southampton and NERC (NE/L50161X/1), and through a NERC Grant-in-Kind from the Life Sciences Mass Spectrometry Facility (LSMSF; EK267-03/16). We thank A. Bates, D. Sims, F. Neat, R. McGill and J. Newton for their analytical contributions and comments on the manuscripts.Peer reviewe

Global Spatial Risk Assessment of Sharks Under the Footprint of Fisheries

Effective ocean management and conservation of highly migratory species depends on resolving overlap between animal movements and distributions and fishing effort. Yet, this information is lacking at a global scale. Here we show, using a big-data approach combining satellite-tracked movements of pelagic sharks and global fishing fleets, that 24% of the mean monthly space used by sharks falls under the footprint of pelagic longline fisheries. Space use hotspots of commercially valuable sharks and of internationally protected species had the highest overlap with longlines (up to 76% and 64%, respectively) and were also associated with significant increases in fishing effort. We conclude that pelagic sharks have limited spatial refuge from current levels of high-seas fishing effort. Results demonstrate an urgent need for conservation and management measures at high-seas shark hotspots and highlight the potential of simultaneous satellite surveillance of megafauna and fishers as a tool for near-real time, dynamic management