Structure-function studies of bovine rhodopsin

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1994.Includes bibliographical references (leaves 209-216).Shalesh Kaushal.Ph.D

Ophthalmology Education in Medical School Curriculum Design: Assessing the Home Front

Background:As medical education moves towards integrated programs, ophthalmology is being increasingly pushed towards the sidelines. It is important for all future physicians, and especially those going into primary care, to have competency in examining the eye and identifying basic pathology in order to better serve their patients. Objectives:The purpose of this study was to assess the self-perceived competence in the basic eye exam by medical students at the University of Massachusetts Medical School. The secondary purpose was to compare this assessment to medical education content from nationwide medical schools. Methods:The study sample consisted of 273 University of Massachusetts Medical School students divided into groups by graduating class (50 entering first year students, 67 entering second year students, 81 entering third year students, and 75 entering fourth year students). Online surveys were distributed in July 2009 with the following questions (based on a 5-point Likert scale ranging from 1- “Not confident at all” to 5- “Very confident”) : “I can test visual acuity,” “I can use a direct ophthalmoscope,” and “I can perform a dilated eye exam.” For the nationwide medical school data collection, online surveys were distributed to 152 medical deans from US accredited allopathic and osteopathic medical schools. The deans were instructed to forward the survey to the appropriate person in charge of designing the medical curriculum if they were not able to answer the questions themselves. These surveys were distributed from August 2009-March 2010 and consisted of the following yes/no statements: “Students learn how to perform visual acuity testing,” “Students are evaluated on performing visual acuity testing,” “Students learn how to use a direct ophthalmoscope,” “Students are evaluated on direct ophthalmoscopy,” and “Students perform a dilated eye exam.” Results:Response rates ranged from 40-81% of medical students by class group and 26% of medical deans (n=40). Wilcoxon-Mann-Whitney non-parametric tests using SPSS were used to compare Likert scores between medical student classes. By the time students entered their final year of medical school, 4% were not confident at all testing visual acuity, 5.3% were not confident at all using the ophthalmoscope, and 74.3% were not confident at all performing a dilated eye exam. In terms of education, 97.5% of schools report teaching students how to perform visual acuity testing and 52.5% state that they evaluate their students on performing this skill. 100% of schools teach students how to use a direct ophthalmoscope and 82.5% evaluate their students on this. 57.5% of medical schools report teaching their students how to perform a dilated eye exam. Conclusion:Current ophthalmology education at the University of Massachusetts Medical School provides opportunities for students to build confidence in performing visual acuity tests and in the basic ophthalmoscope exam, but inadequate training in performing a dilated eye exam. This appears to fit well with the national data, in which most schools taught their students visual acuity testing and direct ophthalmoscopy, but nearly half did not teach the dilated eye exam. Increasing rates of evaluation of student skills would be an effective way to build confidence and self-efficacy in these tasks. Presented as part of the Senior Scholars Program at the University of Massachusetts Medical School, May 3, 2010

Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood–Onset Retinal Dystrophy

PurposeTo provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations.DesignNonrandomized, multicenter clinical trial.ParticipantsEight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood–onset retinal degeneration (SECORD).MethodsPatients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment.Main Outcome MeasuresThe primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire.ResultsAll patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area.ConclusionsTreatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns

Exosomes as a tumor immune escape mechanism: possible therapeutic implications

Advances in cancer therapy have been substantial in terms of molecular understanding of disease mechanisms, however these advances have not translated into increased survival in the majority of cancer types. One unsolved problem in current cancer therapeutics is the substantial immune suppression seen in patients. Conventionally, investigations in this area have focused on antigen-nonspecific immune suppressive molecules such as cytokines and T cell apoptosis inducing molecules such as Fas ligand. More recently, studies have demonstrated nanovesicle particles termed exosomes are involved not only in stimulation but also inhibition of immunity in physiological conditions. Interestingly, exosomes secreted by cancer cells have been demonstrated to express tumor antigens, as well as immune suppressive molecules such as PD-1L and FasL. Concentrations of exosomes from plasma of cancer patients have been associated with spontaneous T cell apoptosis, which is associated in some situations with shortened survival. In this paper we place the "exosome-immune suppression" concept in perspective of other tumor immune evasion mechanisms. We conclude by discussing a novel therapeutic approach to cancer immune suppression by extracorporeal removal of exosomes using hollow fiber filtration technolog

Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics

The RPE65 gene encodes the isomerase of the retinoid cycle, the enzymatic pathway that underlies mammalian vision. Mutations in RPE65 disrupt the retinoid cycle and cause a congenital human blindness known as Leber congenital amaurosis (LCA). We used adeno-associated virus-2-based RPE65 gene replacement therapy to treat three young adults with RPE65-LCA and measured their vision before and up to 90 days after the intervention. All three patients showed a statistically significant increase in visual sensitivity at 30 days after treatment localized to retinal areas that had received the vector. There were no changes in the effect between 30 and 90 days. Both cone- and rod-photoreceptor-based vision could be demonstrated in treated areas. For cones, there were increases of up to 1.7 log units (i.e., 50 fold); and for rods, there were gains of up to 4.8 log units (i.e., 63,000 fold). To assess what fraction of full vision potential was restored by gene therapy, we related the degree of light sensitivity to the level of remaining photoreceptors within the treatment area. We found that the intervention could overcome nearly all of the loss of light sensitivity resulting from the biochemical blockade. However, this reconstituted retinoid cycle was not completely normal. Resensitization kinetics of the newly treated rods were remarkably slow and required 8 h or more for the attainment of full sensitivity, compared with \u3c1 h in normal eyes. Cone-sensitivity recovery time was rapid. These results demonstrate\u3edramatic, albeit imperfect, recovery of rod- and cone-photoreceptor-based vision after RPE65 gene therapy

Safety in Nonhuman Primates of Ocular AAV2-\u3cem\u3eRPE65\u3c/em\u3e, a Candidate Treatment for Blindness in Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV- RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2. RPE65 in RPE65 -mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vector-related toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2. RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2. RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina

Vitreous substitutes: a comprehensive review

Vitreoretinal disorders constitute a significant portion of treatable ocular disease. Advances in vitreoretinal surgery have included the development and characterization of suitable substitutes for the vitreous. Air, balanced salt solutions, perfluorocarbons, expansile gases, and silicone oil serve integral roles in modern vitreoretinal surgery. Vitreous substitutes vary widely in their properties, serve different clinical functions, and present different shortcomings. Permanent vitreous replacement has been attempted with collagen, hyaluronic acid, hydroxypropylmethylcellulose, and natural hydrogel polymers. None, however, have proven to be clinically viable. A long-term vitreous substitute remains to be found, and recent research suggests promise in the area of synthetic polymers. Here we review the currently available vitreous substitutes, as well those in the experimental phase. We classify these compounds based on their functionality, composition, and properties. We also discuss the clinical use, advantages, and shortcomings of the various substitutes. In addition we define the ideal vitreous substitute and highlight the need for a permanent substitute with long-term viability and compatibility. Finally, we attempt to define the future role of biomaterials research and the various functions they may serve in the area of vitreous substitutes