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Inequitable experiences of community gun violence and victimization by police use of force led to nationwide calls to "reimagine public safety" in 2020. In January 2021, we examined public support among U.S. adults for 7 policy approaches to reforming policing and investing in community gun violence prevention. Using a nationally representative sample (N = 2778), with oversampling for Black Americans, Hispanic Americans, and gun owners, we assessed support overall and by racial, ethnic, and gun owner subgroups. Overall, we found majority support for funding and implementing police and mental health co-responder models (66% and 76%, respectively), diversion from incarceration for people with symptoms of mental illness (72%), stronger laws to assure police accountability (72%), and funding for community-based and hospital-based gun violence prevention programs (69% and 60%, respectively). Support for redirecting funding from the police to social services was more variable (44% overall; White: 35%, Black: 60%, Hispanic: 43%). For all survey items, support was strongest among Black Americans. Gun owners overall reported lower support for public safety reforms and investments than respondents who did not own guns, but this distinction was found to be driven by White gun owners. The views of Black gun owners were indistinguishable from Black non-owners and were similar to White non-owners on most issues. These findings suggest that broad public support exists for innovative violence reduction strategies and public safety reforms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gun-related deaths and gun purchases were at record highs in 2020. In light of the COVID-19 pandemic, public protests against police violence, and a tense political environment, which may influence policy preferences, we aimed to understand the current state of support for gun policies in the U.S. We fielded a national public opinion survey in January 2019 and January 2021 using an online panel to measure support for 34 gun policies among U.S. adults. We compared support over time, by gun ownership status, and by political party affiliation. Most respondents supported 33 of the 34 gun regulations studied. Support for seven restrictive policies declined from 2019 to 2021, driven by reduced support among non-gun owners. Support declined for three permissive policies: allowing legal gun carriers to bring guns onto college campuses or K-12 schools and stand your ground laws. Public support for gun-related policies decreased from 2019 to 2021, driven by decreased support among Republicans and non-gun owners., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gun-related deaths and gun purchases were at record highs in 2020. In light of the COVID-19 pandemic, public protests against police violence, and a tense political environment, which may influence policy preferences, we aimed to understand the current state of support for gun policies in the U.S. We fielded a national public opinion survey in January 2019 and January 2021 using an online panel to measure support for 34 gun policies among U.S. adults. We compared support over time, by gun ownership status, and by political party affiliation. Most respondents supported 33 of the 34 gun regulations studied. Support for seven restrictive policies declined from 2019 to 2021, driven by reduced support among non-gun owners. Support declined for three permissive policies: allowing legal gun carriers to bring guns onto college campuses or K-12 schools and stand your ground laws. Public support for gun-related policies decreased from 2019 to 2021, driven by decreased support among Republicans and non-gun owners., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Single-cell RNA sequencing has revolutionized ocular gene expression studies. This technology has enabled researchers to identify expression signatures for rare cell types and characterize how gene expression changes across biological conditions, such as topographic region or disease status. However, sharing single-cell RNA sequencing results remains a major obstacle, particular for individuals without a computational background. To address these limitations, we developed Spectacle, an interactive web-based resource for exploring previously published single-cell RNA sequencing data from ocular studies. Spectacle is powered by a locally developed R package, cellcuratoR, which utilizes the Shiny framework in R to generate interactive visualizations for single-cell expression data. Spectacle contains five pre-processed ocular single-cell RNA sequencing data sets and is accessible via the web at OcularGeneExpression.org/singlecell. With Spectacle, users can interactively identify which cell types express a gene of interest, detect transcriptomic subpopulations within a cell type, and perform highly flexible differential expression analyses. The freely-available Spectacle system reduces the bioinformatic barrier for interacting with rich single-cell RNA sequencing studies from ocular tissues, making it easy to quickly identify cell types that express a gene of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The human choroidal vasculature is subject to age-related structural and gene expression changes implicated in age-related macular degeneration (AMD). In this study, we performed both bulk and single-cell RNA sequencing on infant (n = 4 for bulk experiments, n = 2 for single-cell experiments) and adult (n = 13 for bulk experiments, n = 6 for single-cell experiments) human donors to characterize how choroidal gene expression changes with age. Differential expression analysis revealed that aged choroidal samples were enriched in genes encoding pro-inflammatory transcription factors and leukocyte transendothelial cell migration adhesion proteins. Such genes were observed to be differentially expressed specifically within choroidal endothelial cells at the single-cell level. Immunohistochemistry experiments support transcriptional findings that CD34 is elevated in infant choriocapillaris endothelial cells while ICAM-1 is enriched in adults. These results suggest several potential drivers of the pro-inflammatory vascular phenotype observed with advancing age., (Copyright © 2020 Elsevier Inc. All rights reserved.)

After the school shooting at Marjory Stoneman Douglas High School in Parkland, Florida in 2018, there was an increase in gun violence prevention-related advocacy. While much of this recent political activity and engagement was led by young adults, little is known about support for specific gun policies within this age group. This study uses data from two nationally representative surveys fielded in 2017 and 2019 to compare public support for gun policies: (1) between young adults age 18-29 years and adults age 30 and older, and (2) between young adults in 2017 and young adults in 2019, before and after the Parkland shooting. Relative to adults age 30 and older, young adults had lower support for 16 of 20 gun violence prevention policies examined. Public support was largely unchanged between 2017 and 2019 among survey respondents ages 18-29; however, support for requiring a safety test for concealed carry decreased significantly among young adults between 2017 and 2019. Despite owning fewer guns and finding gun violence prevention important generally, young adults appear to have lower support for policies that regulate guns compared to older adults., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pill mill laws impose strict regulations on pain management clinics to prevent them from issuing opioid prescriptions without medical indication. To date, little is known about the implementation or effects of these laws on opioid overdose deaths. A previously untested concern is that by restricting access to prescription opioids, pill mill laws could increase overdose from heroin and synthetic opioids, like illicitly produced fentanyl. We evaluated the effects of pill mill laws on opioid overdose deaths in Ohio and Tennessee. Of the 11 total U.S. states with pill mill laws, Ohio and Tennessee were the only two where: (1) the pill mill law was the only state law designed to curb opioid prescribing implemented in a two-year period, one-year pre/post law; and (2) high-quality drug-specific overdose death data were available from CDC. We conducted synthetic control analyses examining differences in post-pill mill law trends in overdose deaths in Ohio and Tennessee compared to weighted combinations of comparison states. We also conducted qualitative interviews with 11 leaders responsible for pill mill law implementation and enforcement in Ohio and Tennessee. Pill mill law enactment had no effects on overall, prescription opioid, heroin, or synthetic opioid overdose deaths in Ohio or Tennessee. Interview results suggest that both states engaged in robust enforcement and implementation of the law. A multi-pronged policy approach, including but not limited to pill mill laws, may be required to effectively address opioid overdose deaths., (Copyright © 2019 Elsevier Inc. All rights reserved.)

News media coverage of the U.S. opioid epidemic influences Americans' knowledge of and preferences for solutions to address the crisis. From 1998 to 2012, news media coverage of the opioid epidemic focused on criminal justice-oriented solutions. We examine whether and how news coverage of solutions has shifted in the recent years of the crisis. We analyzed a random sample of 600 U.S. news stories published/aired by high circulation/viewership national and local print and television news outlets from 2013 to 2017. We examined the proportion of news stories mentioning treatment, harm reduction, prevention, criminal justice, opioid prescribing, pharmaceutical manufacturer, insurer, and other solutions. News stories were coded using a structured coding instrument, and 200 news stories were double-coded to ensure interrater reliability. Data were collected and analyzed in 2018. Treatment (mentioned in 33% of news stories), harm reduction (30%), and prevention (24%) solutions were the most frequently mentioned types of solutions. Several evidence-based public health solutions received little news coverage: medication treatment for opioid use disorder was mentioned in 9% of news stories and the harm reduction solutions syringe services programs and safe consumption sites were mentioned in 5% and 2% of news stories. While news coverage showed a promising emphasis on public health-oriented solutions, efforts to increase news coverage of examples of effective opioid use disorder treatment and harm reduction solutions are needed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Public stigma toward people who use illicit drugs impedes advancement of public health solutions to the opioid epidemic and reduces willingness to seek addiction treatment. Experimental studies show that use of certain terms, such as "addict" and "substance abuser," exacerbate stigma while alternative terms, such as "person with a substance use disorder," are less stigmatizing. We examine the frequency with which stigmatizing terms and less-stigmatizing alternatives are used in U.S. news media coverage of the opioid epidemic. We analyzed 6399 news stories about the opioid epidemic published/aired by high-circulation and high-viewership U.S. national and regional print and television news outlets from July 2008 through June 2018. We calculated the proportion of news stories mentioning terms shown to be stigmatizing, as well as terms shown to be less-stigmatizing alternatives, in randomized experiments. Data was collected during May through August 2018 and analyzed in September 2018. Over the 10-year study period, 49% of news stories about the opioid epidemic mentioned any stigmatizing term and 2% mentioned any less-stigmatizing alternative. The proportion of news stories mentioning stigmatizing terms over the 10-year study period increased from 37% in July 2008-June 2009 to 45% in July 2017-June 2018. The language included in U.S. news media coverage of the opioid epidemic may contribute to and reinforce widespread public stigma toward people with opioid use disorders. This stigma may be a barrier to implementation of evidence-based interventions to prevent opioid overdose deaths. Establishing journalistic standards to de-stigmatize the language of addiction is a public health priority., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD., (Copyright © 2018. Published by Elsevier Inc.)

We examine Americans' support for two evidence-based harm reduction strategies - safe consumption sites and syringe exchange programs - and their attitudes about individuals who use opioids. We conducted a web-based survey of a nationally representative sample of U.S. adults in July-August 2017 (N = 1004). We measured respondents' support for legalizing safe consumption sites and syringe services programs in their communities and their attitudes toward people who use opioids. We used ordered logistic regression to assess how stigmatizing attitudes toward people who use opioids, political party identification, and demographic characteristics correlated with support for the two harm reduction strategies. Twenty-nine percent of Americans supported legalizing safe consumption sites and 39% supported legalizing syringe services programs. Respondents reported high levels of stigmatizing attitudes toward people who use opioids: 16% of respondents were willing to have a person using opioids marry into their family and 28% were willing to have a person using opioids start working closely with them on a job, and 27% and 10% of respondents rated persons who use opioids as deserving (versus worthless) and strong (versus weak). Stigmatizing attitudes were associated with lower support for legalizing safe consumption sites and syringe services programs. Democrats and Independents were more likely than Republicans to support both strategies. Stigmatizing attitudes toward people who use opioids are a key modifiable barrier to garnering the public support needed to fully implement evidence-based harm reduction strategies to combat the opioid epidemic. Dissemination and evaluation of stigma reduction campaigns are a public health priority., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The complement membrane attack complex (MAC) shows increased abundance in the choriocapillaris during normal aging and is especially prevalent in age-related macular degeneration (AMD). While perivascular MAC accumulation occurs in the choroid, it is not well understood whether similar deposition occurs in other aging tissues. In this study we examined the abundance of MAC across multiple human tissues. For studies on fixed tissues, paraffin sections were obtained from six human donor eyes and a commercially available tissue array containing 19 different tissues. Immunohistochemical labeling was performed using antibodies directed against the MAC and intercellular adhesion molecule-1 (ICAM-1), as well as the lectin Ulex europaeus agglutinin-I (UEA-I). The choriocapillaris was the only tissue with high levels of the MAC, which was not detected in any of the 38 additional samples from 19 tissues. ICAM-1 was abundantly expressed in the majority of tissues evaluated, and UEA-I labeled the vasculature in all tissues. A second experiment was performed using unfixed frozen sections of RPE-choroid and 7 extraocular tissues, which confirmed the relatively limited localization of the MAC to the choriocapillaris. In comparison to other tissues assessed, the restricted accumulation of MAC in the choriocapillaris may, in part, explain the specificity of AMD to the neural retina, RPE and choroid, and the relative absence of systemic pathology in this disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Peroxisome biogenesis disorders in the Zellweger spectrum (PBD-ZSD) are a heterogeneous group of genetic disorders caused by mutations in PEX genes responsible for normal peroxisome assembly and functions. As a result of impaired peroxisomal activities, individuals with PBD-ZSD can manifest a complex spectrum of clinical phenotypes that typically result in shortened life spans. The extreme variability in disease manifestation ranging from onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults presents practical challenges in disease diagnosis and medical management. Recent advances in biochemical methods for newborn screening and genetic testing have provided unprecedented opportunities for identifying patients at the earliest possible time and defining the molecular bases for their diseases. Here, we provide an overview of current clinical approaches for the diagnosis of PBD-ZSD and provide broad guidelines for the treatment of disease in its wide variety of forms. Although we anticipate future progress in the development of more effective targeted interventions, the current guidelines are meant to provide a starting point for the management of these complex conditions in the context of personalized health care., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sub-retinal implantation of foreign materials is becoming an increasingly common feature of novel therapies for retinal dysfunction. The ultimate compatibility of implants depends not only on their in vitro chemical compatibility, but also on how well the mechanical properties of the material match those of the native tissue. In order to optimize the mechanical properties of retinal implants, the mechanical properties of the mammalian retina itself must be carefully characterized. In this study, the compressive moduli of eye tissues, especially the retina, were probed using a dynamic mechanical analysis instrument in static mode. The retinal compressive modulus was lower than that of the sclera or cornea, but higher than that of the RPE and choroid. Compressive modulus remained relatively stable with age. Conversely, apparent retinal softening occurred at an early age in mice with inherited retinal degeneration. Compressive modulus is an important consideration for the design of retinal implants. Polymer scaffolds with moduli that are substantially different than that of the native tissue in which they will ultimately reside will be less likely to aid in the differentiation and development of the appropriate cell types in vitro and will have reduced biocompatibility in vivo., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cancer has become the leading cause of death in the developed world and has remained one of the most difficult diseases to treat. One of the difficulties in treating cancer is that conventional chemotherapies often have unacceptable toxicities toward normal cells at the doses required to kill tumor cells. Thus, the demand for new and improved tumor specific therapeutics for the treatment of cancer remains high. Alterations to cellular metabolism constitute a nearly universal feature of many types of cancer cells. In particular, many tumors exhibit deficiencies in one or more amino acid synthesis or salvage pathways forcing a reliance on the extracellular pool of these amino acids to satisfy protein biosynthesis demands. Therefore, one treatment modality that satisfies the objective of developing cancer cell-selective therapeutics is the systemic depletion of that tumor-essential amino acid, which can result in tumor apoptosis with minimal side effects to normal cells. While this strategy was initially suggested over 50 years ago, it has been recently experiencing a renaissance owing to advances in protein engineering technology, and more sophisticated approaches to studying the metabolic differences between tumorigenic and normal cells. Dietary restriction is typically not sufficient to achieve a therapeutically relevant level of amino acid depletion for cancer treatment. Therefore, intravenous administration of enzymes is used to mediate the degradation of such amino acids for therapeutic purposes. Unfortunately, the human genome does not encode enzymes with the requisite catalytic or pharmacological properties necessary for therapeutic purposes. The use of heterologous enzymes has been explored extensively both in animal studies and in clinical trials. However, heterologous enzymes are immunogenic and elicit adverse responses ranging from anaphylactic shock to antibody-mediated enzyme inactivation, and therefore have had limited utility. The one notable exception is Escherichia colil-asparaginase II (EcAII), which has been FDA-approved for the treatment of childhood acute lymphoblastic leukemia. The use of engineered human enzymes, to which natural tolerance is likely to prevent recognition by the adaptive immune system, offers a novel approach for capitalizing on the promising strategy of systemic depletion of tumor-essential amino acids. In this work, we review several strategies that we have developed to: (i) reduce the immunogenicity of a nonhuman enzyme, (ii) engineer human enzymes for novel catalytic specificities, and (iii) improve the pharmacological characteristics of a human enzyme that exhibits the requisite substrate specificity for amino acid degradation but exhibits low activity and stability under physiological conditions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Data from human genetics, histopathology, and animal models reveal a major role for the complement system in the development of age-related macular degeneration (AMD). Genetic variations in the complement factor H (CFH) gene are associated with an elevated risk of AMD. In this study we sought to determine whether eyes from donors with a high-risk genotype (homozygosity for the histidine allele at codon 402) exhibit altered levels of membrane attack complex (MAC) in the choroid, compared to eyes with a low risk genotype (homozygosity for tyrosine). Proteins were extracted from the RPE/choroid of 18 donors (10 low risk and 8 high risk) and levels of MAC were assessed using an ELISA assay. Eyes from donors homozygous for the histidine allele showed 69% higher levels of MAC than those homozygous for the tyrosine allele (p < 0.05), independent of whether the eyes showed signs of early AMD. Our results provide evidence that high-risk CFH genotypes may affect AMD risk by increased deposition of MAC around the aging choriocapillaris., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Age-related macular degeneration (AMD) is a common degenerative disease resulting in injury to the retina, retinal pigment epithelium and choriocapillaris. Recent data from histopathology, animal models and genetic studies have implicated altered regulation of the complement system as a major factor in the incidence and progression of this disease. A variant in the gene SERPING1, which encodes C1INH, an inhibitor of the classical and lectin pathways of complement activation, was recently shown to be associated with AMD. In this study we sought to determine the localization of C1INH in human donor eyes. Immunofluorescence studies using a monoclonal antibody directed against C1INH revealed localization to photoreceptor cells, inner nuclear layer neurons, choriocapillaris, and choroidal extracellular matrix. Drusen did not exhibit labeling. Genotype at rs2511989 did not appear to affect C1INH abundance or localization, nor was it associated with significant molecular weight differences when evaluated by Western blot. In a small number of eyes (n = 7 AMD and n = 7 control) AMD affection status was correlated with increased abundance of choroidal C1INH. These results indicate that C1INH protein is present in the retina and choroid, where it may regulate complement activation.

Best vitelliform macular dystrophy (BMD) is an autosomal dominant inherited macular degenerative disease caused by mutations in the gene BEST1 (formerly VMD2). Prior reports indicate that BMD is characterized histopathologically by accumulation of lipofuscin in the retinal pigment epithelium (RPE). However, this accumulation has not been quantified and the chemical composition of lipofuscin in BMD has not been examined. In this study we characterize the histopathology of a donor eye from a rare individual homozygous for a mutation (W93C) in BEST1. We find that this individual's disease was not any more severe than has been described for heterozygotes. We then used this tissue to quantify lipofuscin accumulation by enriching intracellular granules from RPE cells on sucrose gradients and counting the granules in each density fraction. Granules from the homozygous donor eye as well as a donor eye from an individual heterozygous for the mutation T6R were compared with age-matched control eyes. Interestingly, the least dense fraction, representing classical lipofuscin granules was either not present or significantly diminished in the BMD donor eyes and the autoflourescence associated with lipofuscin had shifted to denser fractions. However, a substantial enrichment for granules in fractions of higher density was also noted in the BMD samples. Inspection of granules from the homozygous donor eye by electron microscopy revealed a complex abnormal multilobular structure. Analysis of granules by HPLC indicated a approximately 1.6- and approximately fourfold overall increase in A2E in the BMD eyes versus age-matched control eyes, with a shift of A2E to more dense granules in the BMD donor eyes. Despite the increase in A2E and total intracellular granules, the RPE in the homozygous donor eyes was relatively well preserved. Based on these data we conclude that the clinical and histopathologic consequences to the homozygous donor were not any more severe than has been reported previously for individuals who are established or presumptive heterozygotes. We find that A2E is a component of the lipofuscin accumulated in BMD and that it is more abundant than in control eyes suggesting that the etiology of BMD is similar to Stargardt's disease and Stargardt-like macular dystrophy. Finally, the changes we observe in the granules suggest that the histopathology and eventual vision loss associated with BMD may be due to defects in the ability of the RPE to fully degrade phagocytosed photoreceptor outer segments.

Progressive rod-cone degeneration (prcd) is a late-onset, autosomal recessive photoreceptor degeneration of dogs and a homolog for some forms of human retinitis pigmentosa (RP). Previously, the disease-relevant interval was reduced to a 106-kb region on CFA9, and a common phenotype-specific haplotype was identified in all affected dogs from several different breeds and breed varieties. Screening of a canine retinal EST library identified partial cDNAs for novel candidate genes in the disease-relevant interval. The complete cDNA of one of these, PRCD, was cloned in dog, human, and mouse. The gene codes for a 54-amino-acid (aa) protein in dog and human and a 53-aa protein in the mouse; the first 24 aa, coded for by exon 1, are highly conserved in 14 vertebrate species. A homozygous mutation (TGC --> TAC) in the second codon shows complete concordance with the disorder in 18 different dog breeds/breed varieties tested. The same homozygous mutation was identified in a human patient from Bangladesh with autosomal recessive RP. Expression studies support the predominant expression of this gene in the retina, with equal expression in the retinal pigment epithelium, photoreceptor, and ganglion cell layers. This study provides strong evidence that a mutation in the novel gene PRCD is the cause of autosomal recessive retinal degeneration in both dogs and humans.

Inappropriate activity of the complement cascade contributes to the pathophysiology of several neurodegenerative conditions. This study sought to determine if components of the complement cascade are synthesized in the retina following the development of ocular hypertension (OHT) and if complement accumulates in association with retinal ganglion cells. Toward this goal the gene expression levels of complement components 1qb (C1qb) and 3 (C3) were determined in the retina by quantitative polymerase chain reaction in human eyes with elevated intraocular pressure (IOP) and healthy retinal tissue as well as in a rat model of OHT induced by laser cauterization of the trabecular meshwork and episcleral veins. Immunohistochemical methods were employed to determine the sites of complement deposition in the retina and optic nerve head. Our data demonstrate that transcript levels for C1q and C3 are significantly elevated in retinae subjected to OHT, both in the animal model as well as in human eyes. Immunohistochemical analyses indicate that C1q and C3 accumulate specifically in the retinal ganglion cell layer and the nerve fiber layer. In addition, we demonstrate that the terminal complement complex, or membrane attack complex, is formed both in the human and rat model as a consequence of OHT. Complement activation, particularly formation of membrane attack complexes, has the potential to exacerbate ganglion cell death through bystander lysis or glial cell activation. The results show that complement activation occurs in the retina that has been subjected to elevated IOP, and may have implications in pathophysiology of glaucoma.

Best macular dystrophy (BMD) is an autosomal dominant retinopathy caused by mutations in the VMD2 gene that encodes a chloride channel in the basolateral membrane of the retinal pigment epithelium (RPE). BMD patients were studied using optical coherence tomography (OCT) to understand the disease process in the macula leading to vision loss. BMD patients (ages 5-61), representing four families with known VMD2 mutations, were included. OCT scans were recorded in the central retina and longitudinal reflectivity profiles were analysed. The central retina in BMD showed different OCT abnormalities at or near the level of the highly reflective deep retinal band termed the outer retina-choroid complex (ORCC). Two types of ORCC change were noted to occur either separately or together: (1) splitting with or without intervening hyporeflective areas; and (2) elevation. Longitudinal study of a BMD patient indicated that such abnormalities were dynamic and changed in type and degree with time. The pathogenetic sequence in BMD may begin with defective fluid transport across the RPE secondary to the channelopathy in the basolateral membrane. In the macula, this leads to an abnormal interface with adjacent structures at both apical and basal surfaces of the RPE. The disease process results in detachments of the neurosensory retina, such as in central serous chorioretinopathy, and sub-RPE pathology resembling some stages of age-related macular degeneration, with eventual loss of photoreceptors, inner retina and central vision., (Copyright 2003 Elsevier Science Ltd.)

Choroideremia is an incurable X-linked retinal degeneration caused by mutations in the gene encoding Rab escort protein-1. A group of clinically defined and genotyped patients were studied to determine: (1) the degree of rod and cone dysfunction and structural abnormality in the central retina and the level of macular pigment; and (2) the response of macular pigment and foveal vision to a 6 month trial of supplementation with oral lutein (at 20 mg per day). Rod and cone-mediated function was measured with dark-adapted static perimetry; in vivo retinal structure was determined with optical coherence tomography; and macular pigment optical density was measured with heterochromatic flicker photometry. In this cohort of patients (ages 15-65 years), both rod- and cone-mediated central function declined with age as did central retinal thickness. Macular pigment levels did not differ between patients and male control subjects. Supplementation of oral lutein in a subset of patients led to an increase in serum lutein and macular pigment levels; absolute foveal sensitivity did not change. It is concluded that macular pigment density can be augmented by oral intake of lutein in patients with choroideremia. There was no short-term change in the central vision of the patients on the supplement, but long-term influences of lutein supplementation on disease natural history warrant further study., (Copyright 2002 Elsevier Science Ltd.)

Progressive concentric (centripetal) loss of vision is one pattern of visual field loss in retinitis pigmentosa. This study provides the first clinicopathologic correlations for this form of retinitis pigmentosa. A family with autosomal dominant concentric retinitis pigmentosa was examined clinically and with visual function tests. A post-mortem eye of an affected 94 year old family member was processed for histopathology and immunocytochemistry with retinal cell specific antibodies. Unrelated simplex/multiplex patients with concentric retinitis pigmentosa were also examined. Affected family members of the eye donor and patients from the other families had prominent peripheral pigmentary retinopathy with more normal appearing central retina, good visual acuity, concentric field loss, normal or near normal rod and cone sensitivity within the preserved visual field, and reduced rod and cone electroretinograms. The eye donor, at age 90, had good acuity and function in a central island. Grossly, the central region of the donor retina appeared thinned but otherwise normal, while the far periphery contained heavy bone spicule pigment. Microscopically the central retina showed photoreceptor outer segment shortening and some photoreceptor cell loss. The mid periphery had a sharp line of demarcation where more central photoreceptors were near normal except for very short outer segments and peripheral photoreceptors were absent. Rods and cones showed abrupt loss of outer segments and cell death at this interface. It is concluded that concentric retinitis pigmentosa is a rare but recognizable phenotype with slowly progressive photoreceptor death from the far periphery toward the central retina. The disease is retina-wide but shows regional variation in severity of degeneration; photoreceptor death is severe in the peripheral retina with an abrupt edge between viable and degenerate photoreceptors. Peripheral to central gradients of unknown retinal molecule(s) may be defective or modify photoreceptor degeneration in concentric retinitis pigmentosa.

Single-strand conformation polymorphism (SSCP) analysis has proven to be a simple and effective technique for the detection of single base substitutions. We have used SSCP to analyze 29 mouse globin mutations, 27 p53 mutations, and 8 rhodopsin mutations contained within different size PCR products. Our results indicate that the type of mutation (transition versus transversion) did not play a major role in determining whether a mutation was detected by SSCP analysis. The position of the base substitution was more important than the precise base substitution in determining whether a mutation was detected. We report that SSCP sensitivity varies dramatically with the size of the DNA fragment being analyzed. The optimal size fragment for sensitive base substitution detection by SSCP is approximately 150 bp. Our results illustrate the need to keep the size of the PCR fragment small when performing SSCP to detect mutations. Larger fragments can be analyzed when screening for polymorphisms when the need to detect every sequence variation is not as critical.