Christopher J. Layton

Optic nerve and neuroprotection strategies

AbstractBackground Experimental studies have yielded a wealth of information related to the mechanism of ganglion cell death following injury either to the mylinated ganglion cell axon or to the ganglion cell body. However, no suitable animal models exist where injury can be directed to the optic nerve head region, particularly the unmylinated ganglion cell axons. The process of relating the data from the various animal models to many different types of optic neuropathies in man must, therefore, be cautious.Results Extensive studies on the isolated optic nerve have yielded valuable information on the way white matter is affected by ischaemia and how certain types of compounds can attenuate the process. Moreover, there are now persuasive data on how ganglion cell survival is affected when the ocular blood flow is reduced in various animal models. As a consequence, the molecular mechanisms involved in ganglion cell death are fairly well understood and various pharmacological agents have been shown to blunt the process when delivered before or shortly after the insult.Conclusions A battery of agents now exist that can blunt animal ganglion cell death irrespective of whether the insult was to the ganglion cell body or the mylinated axon. Whether this information can be applied for use in patients remains a matter of debate, and major obstacles need to be overcome before the laboratory studies may be applied clinically. These include the delivery of the pharmacological agents to the site of ganglion cell injury and side effects to the patients. Moreover, it is necessary to establish whether effective neuroprotection is only possible when the drug is administered at a defined time after injury to the ganglion cells. This information is essential in order to pursue the idea that a neuroprotective strategy can be applied to a disease like glaucoma, where ganglion cell death appears to occur at different times during the lifetime of the patient.

Survey of eye practitioners' attitudes towards diagnostic tests and therapies for dry eye disease

Background:  There is a wide variation in the use of diagnostic tests for dry eye disease. The purpose of this study was to survey the attitudes of eye practitioners towards both tests and therapies available for dry eye disease.

Neuronal death in primary retinal cultures is related to nitric oxide production, and is inhibited by erythropoietin in a glucose-sensitive manner

The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mm) or high (15 mm) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA‐labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N‐nitro‐l‐arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietins neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.

Seek and destroy: targeted adeno-associated viruses for gene delivery to hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with high incidence globally. Increasing mortality and morbidity rates combined with limited treatment options available for advanced HCC press for novel and effective treatment modalities. Gene therapy represents one of the most promising therapeutic options. With the recent approval of herpes simplex virus for advanced melanoma, the field of gene therapy has received a major boost. Adeno-associated virus (AAV) is among the most widely used and effective viral vectors today with safety and efficacy demonstrated in a number of human clinical trials. This review identifies the obstacles for effective AAV based gene delivery to HCC which primarily include host immune responses and off-target effects. These drawbacks could be more pronounced for HCC because of the underlying liver dysfunction in most of the patients. We discuss approaches that could be adopted to tackle these shortcomings and manufacture HCC-targeted vectors. The combination of transductional targeting by modifying the vector capsid and transcriptional targeting using HCC-specific promoters has the potential to produce vectors which can specifically seek HCC and deliver therapeutic gene without significant side effects. Finally, the identification of novel HCC-specific ligands and promoters should facilitate and expedite this process.

Evolving systemic targeted therapy strategies in uveal melanoma and implications for ophthalmic management: a review.

Uveal melanoma (UM) is the most common primary ocular tumour in adults. Despite good local control of the primary tumour with current methods, survival after the development of metastasis has remained poor over the last 30 years. After cutaneous melanoma, UM is the most common type of melanoma, and an ongoing debate exists regarding whether these conditions should be considered separate entities, particularly in the context of targeted therapy, where many of the initial trials for patients with metatatic cutaneous melanoma excluded metastatic UM. This paper will review the recent and ongoing investigations designed to validate systemic targeted therapy and immunotherapy in patients with metastatic UM and suggests ways in which these developments may affect management of UM by ophthalmologists in the near future.

miRNA122a regulation of gene therapy vectors targeting hepatocellular cancer stem cells

In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3’-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs.

Evaluation of the Glypican 3 promoter for transcriptional targeting of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a major health problem as evidenced by its increasing incidence and high morbidity and mortality rates. Most patients with HCC have underlying liver disease and dysfunction which limits the current therapeutic options. Treatments that spare the liver and destroy the HCC are needed. Targeting transcriptional differences between HCC and liver cells may provide this therapeutic window. In this study, we examine the potential of the Glypican 3 (GPC3) promoter as a targeting strategy. GPC3 is an oncofetal protein belonging to the proteoglycan family which is normally only expressed during fetal development. However, in HCC, the expression of this protein is reactivated. Here, we show that GPC3 is expressed primarily in HCC and not in normal liver lines. We show that the GPC3 promoter can be used to drive expression of significantly more luciferase and eYFP in HCC cell lines compared to normal liver cells. Further, we show that vectors containing cytosine deaminase (CD) under GPC3 promotor control induced significantly more killing of HCC cell lines after treatment with 5-FC compared to normal liver cell lines. These data suggest that transcriptionally targeted delivery of transgene in HCC cells can be achieved using the GPC3 promoter and this targeting strategy produces limited toxicity to normal liver cells.

Prognostic implications of imaging in atrophic macular degeneration and its use in clinical practice and clinical trial design

Clinical prognostic markers in atrophic age‐related macular degeneration include the extent of existing atrophy, fundus autofluorescence (FAF) patterns and optical coherence tomography changes in the outer retina/retinal pigment epithelium interface. The prognostic implications of these findings may be used to determine not just the rate of disease progression but also influence the likelihood, magnitude and clinical relevance of therapy responses. FAF phenotypes have been extensively investigated; however, the pathophysiological mechanisms behind their appearance have not been fully elucidated. Optical coherence tomography imaging is additive to FAF imaging in atrophic age‐related macular degeneration, allowing the visualization of detail not available through FAF imaging whilst also displaying subtle changes correlating with the FAF phenotypes themselves, thereby giving clues to their histological determinates. The developing understanding of these imaging modalities and consequent development of prognostically useful classification systems have widespread implication in clinical care and clinical trial design.

Factors associated with significant ocular injury in conservatively treated orbital fractures

Purpose. To determine factors associated with the presence of significant ocular injury in subjects with orbital fractures. Subjects. A consecutive prospective cohort of 161 patients presenting to a general tertiary referral hospital with orbital fractures and undergoing initial conservative treatment was identified. Subjects were assessed at time of injury for the need for emergency surgery, and those initially treated conservatively were subsequently followed up by the Ophthalmology Department to assess for ocular injury requiring ophthalmic management at 1–7 days after injury. Associations between ocular injury and age, sex, visual acuity, presence of blowout fracture, extent of orbital involvement, and presence of distant facial fractures were assessed. Results. 142 male (average age of 32 [95% CI 30–35]) and 19 female (average age of 49 [95% CI 39–59]) subjects were identified. 17 subjects were diagnosed with significant ocular injury. Ocular injury was significantly associated with LogMAR VA worse than 0.2 (OR 49 [95% CI 11–217, P < 0.0001]), but no relationship was noted for age, sex, presence of blowout fracture, extent of fractures, or presence of distal facial fractures. LogMAR visual acuity worse than or equal to 0.2 had a 98% negative predictive value for ocular injury in the setting of orbital fractures. Conclusions. Demographic and nonophthalmic fracture characteristics were not useful predictors of ocular injury in orbital fractures. LogMAR visual acuity worse than or equal to 0.2 is a highly sensitive and useful guide of the need for ophthalmic referral in subjects with orbital fractures.

An eye for art? A challenge of ophthalmic body modification

Over the past decade episcleral tattooing has emerged as a new body modification trend in some sections of the community, but many ophthalmologists have not yet considered the possible implications of the phenomenon in patients. Therapeutic corneal tattoos have a long history dating to classical times and have been used to treat glare, intractable diplopia and corneal leucoma. Lamellar ink deposition is now achieved by microkeratome, injection of metal salts or insoluble pigments after alcohol debridement of corneal epithelium, or femtosecond laser. In some areas, the practice is still common when sight cannot be preserved. By contrast, the new trend of episcleral tattooing is a body modification procedure. Compared with the cornea, the exposed sclera is a more permeable structure, and while corneal tattoos involve injection of dyes of known components into the stroma where the particles of ink remain, migration of episcleral proprietary tattoo ink components to the Suprachoroidal space and aqueous outflow apparatus is possible. At this stage, only short-term complications have been observed, and a case of perforation and panuveitis has been reported. It is of note that there is an extensive literature of complications, especially immune complications, in dermal and periorbital tattoos, which may be useful to the profession in anticipating possible local and regional complications of cosmetic episcleral tattooing. These include evidence of granulomatous responses (including uveitis) developing from distant dermal tattoos after latencies of up to 20years and which necessitate surgical removal of tattoos. An induced chronic immune response from tattoos forms a risk factor for cellular metaplasia or dysplasia. Tattoo inks can contain compounds including copper, which, if able to migrate to intraocular structures or the outflow apparatus, could lead to chalcosis with subsequent intraocular inflammation and glaucoma. The Food and Drug Administration has also had reports of adverse reactions to newer organic inks. Nontherapeutic ink components tend to be proprietary and are unregulated by the Food and Drug Administration or Therapeutic Goods Administration. As the ink can mimic an intraocular foreign body, all the potential complications that can result from intraocular foreign bodies are also possible, particularly if the ink includes metals. While still only involving a small number of individuals, this issue has great potential implications for their long-term ocular health. Unlike dermal tattoos, episcleral tattoos are technically almost impossible to remove, and therefore, side effects may be impossible to reverse. The proprietary nature of many tattoo inks and lack of information about long-term vision problems from the procedure make informing patients prior to any procedure and monitoring their ocular health very difficult. Nonetheless, a consideration of the known ocular side effects of dermal tattoos, the anatomy of the eye and its pharmacokinetics allows the anticipation of possible side effects of the migration of ink components into the intraocular space/outflow apparatus and local immune responses to the episcleral foreign material. This approach can inform all ophthalmologists consulting patients who either have undergone the procedure or are considering it as an option.