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Background: In response to the role overprescribing has played in the U.S. opioid crisis, in the past decade states have enacted four main types of laws to curb opioid prescribing: mandatory prescription drug monitoring program (PDMP) enrollment laws requiring clinicians to register with a PDMP; mandatory PDMP query laws requiring clinicians to check a PDMP prior to prescribing opioids; pill mill laws regulating pain management clinics; and opioid prescribing cap laws limiting the dose/duration of opioid prescriptions. While 47 states now have one or more of these laws in place, little is known about implementation and enforcement strategies, facilitators, and barriers., Methods: From November 2017 to February 2019, we interviewed 114 professionals involved in state opioid prescribing law implementation and enforcement in 20 states and identified common themes., Results: Implementation efforts focused on awareness campaigns and targeted training of key front-line implementers. Enforcement strategies included active, complaint-based, and automated strategies. Collaboration across agencies and stakeholders, particularly health agencies and law enforcement, was identified as an important facilitator of implementation and enforcement. Two key interrelated barriers were identified: the complexity of state opioid prescribing laws in terms of which providers, patients, and prescriptions they applied to, and IT infrastructure., Conclusion: Despite differing approaches, our findings suggest similar barriers to implementation and enforcement across state opioid prescribing laws. Strategies are needed to ease implementation and enforcement of laws that apply only to specific sub-sets of providers, patients, or prescriptions and address issues of access and data utilization of the PDMP., Competing Interests: Declaration of Competing Interests Dr. Bicket reports Axial Healthcare (past served on an advisory board, stock options) and Alosa Health (advisory board). These arrangements have been reviewed and approved by Johns Hopkins University in accordance with its conflict of interest policies. Dr. Alexander previously served as Chair of FDA’s Peripheral and Central Nervous System Advisory Committee; has served as a paid advisor to IQVIA; and is a consultant and holds equity in Monument Analytics, a health care consultancy whose clients include the life sciences industry as well as plaintiffs in opioid litigation; and is a member of OptumRx’s National P&T Committee. This arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict of interest policies. The other co-authors have no conflicts of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: This study evaluated the association of the Maryland Medicaid behavioral health home (BHH) integrated care program with cancer screening., Methods: Using administrative claims data from October 2012 to September 2016, the authors measured cancer screening among 12,176 adults in Maryland's psychiatric rehabilitation program who were eligible for cervical (N=6,811), breast (N=1,658), and colorectal (N=3,430) cancer screening. Marginal structural modeling was used to examine the association between receipt of annual cancer screening and whether participants had ever enrolled in a BHH (enrolled: N=3,298, 27%; not enrolled: N=8,878, 73%)., Results: Relative to nonenrollment, BHH enrollment was associated with increased screening for cervical and breast cancer but not for colorectal cancer. Predicted annual rates remained low, even in BHHs., Conclusions: Despite estimates of improvements in cervical and breast cancer screening after BHH implementation, cancer screening rates remained suboptimal. Broader cancer screening interventions are needed to improve cancer screening for people with mental illness.

Direct-to-physician opioid marketing by pharmaceutical companies is widespread and may contribute to opioid overprescribing, an important driver of the US opioid crisis. Using a difference-in-differences approach and Medicare Part D prescriber data, we examined the effects of academic medical centers' conflict-of-interest policies that restrict direct-to-physician marketing of all drugs on opioid prescribing by physicians at eighty-five centers in the period 2013-16. We examined restrictions on gifts and meals, speaking and consulting engagements, and industry representatives' access to academic medical centers, as well as rules requiring conflict-of-interest disclosures. Bans on sales representatives were associated with a 4.7 percent reduction in the total volume of opioids prescribed and disclosure requirements with a 2.5 percent reduction, while having all four marketing restriction policies was associated with an 8.8 percent reduction. Policies that restrict direct-to-physician pharmaceutical marketing may curb opioid prescribing, but additional patient-level research is needed to understand how such policies affect the delivery of evidence-based treatment for chronic pain.

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.)

Background: Choroideremia is an X-linked recessive condition characterized by progressive chorioretinal degeneration. Recently, peculiar scleral ectasias, termed scleral "pits" and "tunnels," have been described as a novel finding in patients with choroideremia, but little is known regarding their etiology or their evolution over time., Subjects: This is a retrospective chart review of consecutive patients with molecularly-confirmed choroideremia and related female carriers seen at a university-based tertiary referral center from January 2010 to July 2016. Multimodal imaging was evaluated for the evolution of scleral pits on fundus photography and scleral tunnels on optical coherence tomography (OCT). The presence of scleral pits and tunnels was correlated with markers of disease severity including age, visual acuity, and severity of visual field loss., Results: Thirty patients (21 affected males, 9 female carriers) were included in the study. Scleral pits were seen in 38.1% (8/21) of affected males and found to occur at insertion sites of the posterior ciliary arteries. Those with scleral pits were older, had poorer visual acuity, and more severe visual field loss than those without (p ≤ 0.05). Scleral tunnels were common (68.4%, 13/19 affected males with available OCT imaging), but no statistically-significant associations with disease severity were seen. The development of new scleral pits and tunnels was observed on longitudinal imaging in 4 and 2 affected males, respectively. No scleral pits or tunnels were visualized in any female carriers., Conclusions: Scleral pits represent degeneration around the posterior ciliary arteries and may be useful as clinical markers of disease severity in choroideremia.

Degenerative diseases affecting retinal photoreceptor cells have numerous etiologies and clinical presentations. We clinically and molecularly studied the retina of a 70-year-old patient with retinal degeneration attributed to autoimmune retinopathy. The patient was followed for 19 years for progressive peripheral visual field loss and pigmentary changes. Single-cell RNA sequencing was performed on foveal and peripheral retina from this patient and four control patients, and cell-specific gene expression differences were identified between healthy and degenerating retina. Distinct populations of glial cells, including astrocytes and Müller cells, were identified in the tissue from the retinal degeneration patient. The glial cell populations demonstrated an expression profile consistent with reactive gliosis. This report provides evidence that glial cells have a distinct transcriptome in the setting of human retinal degeneration and represents a complementary clinical and molecular investigation of a case of progressive retinal disease., Competing Interests: The authors declare no conflict of interest.

The advent of human induced pluripotent stem cells (iPSCs) provided a means for avoiding ethical concerns associated with the use of cells isolated from human embryos. The number of labs now using iPSCs to generate photoreceptor, retinal pigmented epithelial (RPE), and-more recently-choroidal endothelial cells has grown exponentially. However, for autologous cell replacement to be effective, manufacturing strategies will need to change. Many tasks carried out by hand will need simplifying and automating. In this issue of the JCI, Schaub and colleagues combined quantitative bright-field microscopy and artificial intelligence (deep neural networks and traditional machine learning) to noninvasively monitor iPSC-derived graft maturation, predict donor cell identity, and evaluate graft function prior to transplantation. This approach allowed the authors to preemptively identify and remove abnormal grafts. Notably, the method is (a) transferable, (b) cost and time effective, (c) high throughput, and (d) useful for primary product validation.

Objective: To assess whether there are differences in support for handgun purchaser licensing., Methods: We used data from four waves of online, national polling on gun policy. To estimate differences in support for licensing across groups, we categorised respondents by whether they personally owned a gun, lived in a state with handgun purchaser licensing or lived in a state regulating private sales without a licensing system., Results: Eighty-four per cent of adults living in states with licensing supported the policy compared with 74% in states without the law (p<0.001). Seventy-seven per cent of gun owners living in states with licensing supported the policy vs 59% of gun owners in states without licensing (p<0.001)., Conclusions: Support for licensing among gun owners living in states with these laws, many of whom have presumably gone through the process, was much higher than gun owners in states without such laws., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Behavioral health homes, shown to improve receipt of evidence-based medical services among people with serious mental illness in randomized clinical trials, have had limited results in real-world settings; nonetheless, these programs are spreading rapidly. To date, no studies have considered what set of policies is needed to support effective implementation of these programs. As a first step toward identifying an optimal set of policies to support behavioral health home implementation, we use the policy ecology framework to map the policies surrounding Maryland's Medicaid behavioral health home program. Results suggest that existing policies fail to address important implementation barriers.

A challenge in oncology is to rationally and effectively integrate immunotherapy with traditional modalities, including radiotherapy. Here, we demonstrate that radiotherapy induces tumor-cell ferroptosis. Ferroptosis agonists augment and ferroptosis antagonists limit radiotherapy efficacy in tumor models. Immunotherapy sensitizes tumors to radiotherapy by promoting tumor-cell ferroptosis. Mechanistically, IFNγ derived from immunotherapy-activated CD8 + T cells and radiotherapy-activated ATM independently, yet synergistically, suppresses SLC7A11, a unit of the glutamate-cystine antiporter xc - , resulting in reduced cystine uptake, enhanced tumor lipid oxidation and ferroptosis, and improved tumor control. Thus, ferroptosis is an unappreciated mechanism and focus for the development of effective combinatorial cancer therapy. SIGNIFICANCE: This article describes ferroptosis as a previously unappreciated mechanism of action for radiotherapy. Further, it shows that ferroptosis is a novel point of synergy between immunotherapy and radiotherapy. Finally, it nominates SLC7A11, a critical regulator of ferroptosis, as a mechanistic determinant of synergy between radiotherapy and immunotherapy. This article is highlighted in the In This Issue feature, p. 1631 ., (©2019 American Association for Cancer Research.)

In efforts to increase scientific literacy and enhance the preparation of learners to pursue careers in science, there are growing opportunities for students and teachers to engage in scientific research experiences, including course-based undergraduate research experiences (CUREs), undergraduate research experiences (UREs), and teacher research experiences (TREs). Prior literature reviews detail a variety of models, benefits, and challenges and call for the continued examination of program elements and associated impacts. This paper reports a comprehensive review of 307 papers published between 2007 and 2017 that include CURE, URE, and TRE programs, with a special focus on research experiences for K-12 teachers. A research-supported conceptual model of science research experiences was used to develop a coding scheme, including participant demographics, theoretical frameworks, methodology, and reported outcomes. We summarize recent reports on program impacts and identify gaps or misalignments between goals and measured outcomes. The field of biology was the predominant scientific disciplinary focus. Findings suggest a lack of studies explicitly targeting 1) participation and outcomes related to learners from underrepresented populations, 2) a theoretical framework that guides program design and analysis, and, for TREs, 3) methods for translation of research experiences into K-12 instructional practices, and 4) measurement of impact on K-12 instructional practices.

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene ( RGCC ), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The identification of >100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses, many disease-causing genes, such as ABCA4 or USH2A , are too large to fit into these vectors. One option for large gene delivery is the family of integration-deficient helper-dependent adenoviruses (HDAds), which efficiently transduce postmitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd serotype 5 (HDAd5) was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd5 was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced green fluorescent protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3 days postinjection. This reaction included vitreous infiltration of ionized calcium-binding adapter molecule 1 (Iba1)-positive monocytes and increased expression of the proinflammatory protein, intercellular adhesion molecule 1 (ICAM-1). By 7 days postinjection, most Iba1-positive infiltrates migrated into the neural retina and ICAM-1 expression was significantly increased compared with buffer-injected control eyes. At 14 days postinjection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Subretinal injection of lower doses of HDAd5 dampened the inflammatory response, but also eGFP expression. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using HDAd5.

Gun policy is a prominent topic of debate in the 2020 US election cycle. Tracking evolving public attitudes about gun policy is critical in this context. Using data from the National Survey of Gun Policy fielded in 2013, 2015, 2017, and 2019 by the Johns Hopkins Center for Gun Policy and Research, we examined trends in support for more than two dozen gun policies over time by gun ownership status and political party affiliation, and across states. Most policies that we considered had majority support across the study period. We identified increases in public support over time for licensing and universal background checks of handgun purchasers, stronger regulation of gun dealers, and extreme risk protection orders. Gun owners and non-gun owners were highly supportive of requiring tests to demonstrate safe handling before carrying a concealed weapon, but there were large differences in support for other concealed carry policies by gun ownership status. A new item included in the 2019 survey showed that 84 percent of Americans supported requiring first-time gun purchasers to take a safety course. While gun policy continues to be characterized as highly polemical, large majorities of both gun owners and non-gun owners strongly support a range of measures to strengthen US gun laws.

Individuals with serious mental illness (SMI) experience significant premature mortality due to somatic conditions but often receive sub-optimal somatic care, but little research has been done to understand how general medical clinicians' attitudes may affect care provision or health outcomes. This review describes general medical clinicians' attitudes toward people with SMI, compares these attitudes to attitudes among mental health clinicians or toward individuals without SMI, and examines the relationship between attitudes and clinical decision making. Seventeen studies were reviewed. General medical clinicians reported negative attitudes toward individuals with SMI. These attitudes were generally more negative than attitudes among mental health clinicians and were consistently more negative when compared to attitudes toward individuals without SMI. Four studies suggest that these negative attitudes have an adverse effect on clinician decision making.

Objective: To identify the genetic cause of autosomal dominant spinocerebellar ataxia and retinitis pigmentosa in a large extended pedigree., Methods: Clinical studies were done at 4 referral centers. Ten individuals in the same extended family participated in at least a portion of the study. Records were obtained from an 11th, deceased, individual. Neurologic and dermatological examinations were performed. Ophthalmologic evaluation including funduscopic examination and in some cases ocular coherence tomography were used to identify the presence of retinal disease. Whole exome sequencing (WES), in conjunction with Sanger sequencing and segregation analysis, was used to identify potential genetic mutation., Results: Affected individuals reported slowly progressive cerebellar ataxia with age at onset between 38 and 57. Imaging demonstrated cerebellar atrophy (3/3). WES identified a novel heterozygous mutation in the elongation of very long chain fatty acids 4 ( ELOVL4 ) gene (c.512T>C, p.Ile171Thr) that segregated with ataxia in 7 members tested. Four of 8 members who underwent ophthalmologic evaluation were found to have retinitis pigmentosa. No skin findings were identified or reported. Ocular movement abnormalities and pyramidal tract signs were also present with incomplete penetrance., Conclusions: We report a family with both spinocerebellar ataxia and retinal dystrophy associated with an ELOVL4 mutation. In addition, to supporting prior reports that ELOVL4 mutations can cause spinocerebellar ataxia, our findings further broaden the spectrum of clinical presentations associated with spinocerebellar ataxia 34., (Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

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.)

Objective: Social cognitive deficits are an important consequence of multiple sclerosis (MS), yet our understanding of how these deficits manifest in progressive MS is currently limited. To this end, we examined theory of mind (ToM) ability in a sample of individuals with progressive MS using an ecologically valid virtual assessment tool that allows for delineation of cognitive ToM (inferring thoughts and intentions of others) from affective ToM (inferring emotions of others)., Methods & Results: We compared 15 individuals with progressive MS and 15 healthy controls on their ToM ability using the Virtual Assessment of Mentalising Ability. We found that, relative to healthy controls, participants with progressive MS were impaired in cognitive ToM, but not in affective ToM. Furthermore, we found that the MS participants' deficits in cognitive ToM were mediated by their general cognitive ability such that poor cognitive ToM ability in MS was explained by poor performance on tests of memory and processing speed., Conclusions: Our findings demonstrate that ToM deficits in progressive MS may be limited to cognitive ToM, while affective ToM is conserved. This could be attributable to the MS-related deficits in general cognitive ability, which appear to negatively affect only the cognitive component of ToM.

Cell replacement therapies are often enhanced by utilizing polymer scaffolds to improve retention or direct cell orientation and migration. Obstacles to refinement of such polymer scaffolds often include challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization of acrylated poly(caprolactone) (PCL) could offer a means of achieving precise microstructural control of a material in a biocompatible platform. In this work, we studied the effect of various formulation and two-photon polymerization parameters on minimum laser power needed to achieve polymerization, resolution, and fidelity to a target 3D model designed to be used for retinal cell replacement. Overall, we found that increasing the concentration of crosslink-able groups decreased polymerization threshold and the size of resolvable features while increasing fidelity of the scaffold to the 3D model. In general, this improvement was achieved by increasing the number of acrylate groups per prepolymer molecule, increasing the acrylated PCL concentration, or decreasing its molecular weight. Resulting two-photon polymerized PCL scaffolds successfully supported human iPSC derived retinal progenitor cells in vitro. Sub-retinal implantation of cell free scaffolds in a porcine model of retinitis pigmentosa did not cause inflammation, infection or local or systemic toxicity after one month. In addition, comprehensive ISO 10993 testing of photopolymerized scaffolds revealed a favorable biocompatibility profile. These results represent an important step towards understanding how two-photon polymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications. STATEMENT OF SIGNIFICANCE: Inherited retinal degenerative blindness results from the death of light sensing photoreceptor cells. To restore high-acuity vision a photoreceptor cell replacement strategy will likely be necessary. Unfortunately, single cell injection typically results in poor cell survival and integration post-transplantation. Polymeric biomaterial cell delivery scaffolds can be used to promote donor cell viability, control cellular polarity and increase packing density. A challenge faced in this endeavor has been developing methods suitable for generating scaffolds that can be used to deliver stem cell derived photoreceptors in an ordered columnar orientation (i.e., similar to that of the native retina). In this study we combined the biomaterial poly(caprolactone) with two-photon lithography to generate a biocompatible, clinically relevant scaffold suitable for retina cell delivery., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Subretinal delivery of stem cell-derived retinal cells as a strategy to treat retinal degenerative blindness holds great promise. Currently, two clinical trials are underway in which human fetal retinal progenitor cells (RPCs) are being delivered to patients by intravitreal or subretinal injection to preserve or restore vision, respectively. With the advent of the induced pluripotent stem cell (iPSC), and in turn three-dimensional derivation of retinal tissue, it is now possible to generate autologous RPCs for cell replacement. The purpose of this study was to evaluate the effect of commonly used cell isolation and surgical manipulation strategies on donor cell viability. iPSC-RPCs were subjected to various conditions, including different dissociation and isolation methods, injection cannula sizes, and preinjection storage temperatures and times. The effects of commonly used surgical techniques on both host and donor cell viability were evaluated in Yucatan mini-pigs (n = 61 eyes). We found a significant increase in cell viability when papain was used for RPC isolation. In addition, a significant decrease in cell viability was detected when using the 41G cannula compared with 31G and at storage times of 4 hours compared with 30 minutes. Although 96.4% of all eyes demonstrated spontaneous retinal reattachment following injection, retinal pigment epithelium (RPE) abnormalities were seen more frequently in eyes receiving injections via a 31G cannula; interestingly, eyes that received cell suspensions were relatively protected against such RPE changes. These findings indicate that optimization of donor cell isolation and delivery parameters should be considered when developing a subretinal cell replacement strategy. Stem Cells Translational Medicine 2019;8:797&809., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Purpose: To investigate receptor and post-receptor function in KCNV2 retinopathy [cone dystrophy with supernormal rod electroretinogram (ERG)], using the pupillary light reflex (PLR) and the ERG., Methods: Two unrelated patients (1 male and 1 female) with molecularly confirmed KCNV2 retinopathy underwent full-field two-color pupillometry testing in one eye, with monitoring of the stimulated eye by an infrared digital camera. Pupillometry stimuli consisted of 1-s duration, short-wavelength (465-nm, blue) and long-wavelength (642-nm, red) stimuli. Pupillometry intensity series were performed under both a dark-adapted condition and a light-adapted condition (on a 0.76-log cd m -2 blue background). The transient PLR, defined as the maximum constriction following flash onset, was measured under all conditions. The melanopsin-mediated sustained constriction was measured 5-7 s following flash offset for the highest flash luminance presented in the dark. Both patients were also tested in one eye with the full-field ERG, including a dark-adapted intensity series and ISCEV standard stimuli., Results: Dark-adapted PLRs were markedly attenuated or extinguished for low-luminance stimuli, but the responses to higher-luminance blue stimuli were within normal limits. Light-adapted PLRs to blue stimuli were generally within normal limits, exceeding the responses to photopically matched red stimuli. Thus, light-adapted responses were consistent with either rod or S-cone mediation of the PLR. Melanopsin-mediated sustained PLRs were within normal limits. ERG showed the characteristic findings previously reported in this condition. Cone-mediated ERG responses were markedly decreased in amplitude. Rod-mediated ERG responses were absent for low-luminance stimuli (- 3 log cd s m -2 ), but had normal amplitude for stimuli of - 2 log cd s m -2 and above (although none were "supernormal"). The b-wave for the dark-adapted ISCEV standard - 2 log cd s m -2 stimulus was markedly delayed, whereas the b-wave timing was generally normal for higher flash luminances., Conclusions: The abnormalities measured by pupillometry have a similar pattern to the outer-retinal abnormalities measured by ERG in KCNV2 retinopathy. These findings as well as the normal sustained PLR suggest that inner-retinal function may be preserved in KCNV2 retinopathy and highlight the potential for therapies designed to restore outer-retinal function in these individuals.

In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A , are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable ( i.e. , they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 ( i.e. , potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR , we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties ( i.e. , analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.

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.)

Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculae from controls (n = 99), early AMD (n = 35), or clinically diagnosed with geographic atrophy (GA; n = 9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross-sectional area of capillary lumens divided by length) and of vascular lumen/stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni-corrected P = 0.024) with greater loss in GA (Bonferroni-corrected P < 10 -9 ), even in areas of intact retinal pigment epithelium. In contrast, changes in lumen/stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P > 0.05), suggesting choriocapillaris changes are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration, predominantly in the microvasculature, contributes to dry AMD progression. Addressing capillary loss in AMD remains an important translational target., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. 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.)

Importance: Mutations in the myocilin (MYOC) gene are the most common molecularly defined cause of primary open-angle glaucoma that typically occurs in patients with high intraocular pressures (IOP). One MYOC mutation, p.Gln368Ter, has been associated with as many as 1.6% of primary open-angle glaucoma cases that had a mean maximum recorded IOP of 30 mm Hg. However, to our knowledge, the role of the p.Gln368Ter mutation in patients with normal-tension glaucoma (NTG) with an IOP of 21 mm Hg or lower has not been investigated., Objective: To evaluate the role of the p.Gln368Ter MYOC mutation in patients with NTG., Design, Setting, and Participants: In this case-control study of the prevalence of the p.Gln368Ter mutation in patients with NTG, cohort 1 was composed of 772 patients with NTG and 2152 controls from the United States (Iowa, Minnesota, and New York) and England and cohort 2 was composed of 561 patients with NTG and 2606 controls from the Massachusetts Eye and Ear Infirmary and the NEIGHBORHOOD consortium. Genotyping was conducted using real-time polymerase chain reaction that was confirmed with Sanger sequencing, the imputation of genome-wide association study data, or an analysis of whole-exome sequence data. Data analysis occurred between April 2007 and April 2018., Main Outcomes and Measures: Comparison of the frequency of the p.Gln368Ter MYOC mutation between NTG cases and controls with the Fisher exact test., Results: Of 6091 total participants, 3346 (54.9%) were women and 5799 (95.2%) were white. We detected the p.Gln368Ter mutation in 7 of 772 patients with NTG (0.91%) and 7 of 2152 controls (0.33%) in cohort 1 (P = .03). In cohort 2, we detected the p.Gln368Ter mutation in 4 of 561 patients with NTG (0.71%) and 10 of 2606 controls (0.38%; P = .15). When the cohorts were analyzed as a group, the p.Gln368Ter mutation was associated with NTG (odds ratio, 2.3; 95% CI, 0.98-5.3; P = .04)., Conclusions and Relevance: In cohorts 1 and 2, the p.Gln368Ter mutation in MYOC was found in patients with IOPs that were 21 mm Hg or lower (NTG), although at a frequency that is lower than previously detected in patients with higher IOP. These data suggest that the p.Gln368Ter mutation may be associated with glaucoma in patients with normal IOPs as well as in patients with IOPs that are greater than 21 mm Hg.

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.)

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3 . Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient's iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient's c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

Purpose: To examine the association between sequence variants in genetic risk factors for age-related macular degeneration (AMD) and delayed rod-mediated dark adaptation (RMDA), the first functional biomarker for incident AMD, in older adults with normal macular health and early AMD., Design: Cross-sectional., Participants: Adults 60 years of age or older showing normal macular health (defined as both eyes at step 1 on the Age-Related Eye Disease Study 9-step AMD classification system) and those with AMD in one or both eyes (defined as steps 2-9)., Methods: Single nucleotide polymorphisms were genotyped in the complement factor H (CFH) and ARMS2 genes using a Taqman assay. Rod-mediated dark adaptation was assessed in 1 eye after photobleach with targets centered at 5° on the inferior vertical meridian. Rate of dark adaptation was defined by rod intercept time (RIT), duration (in minutes) required for sensitivity to reach a criterion sensitivity level in the latter half of the second component of rod recovery. Associations between CFH and ARMS2 polymorphisms and RMDA were adjusted for age and smoking., Main Outcome Measure: Rod intercept time., Results: The sample consisted of 543 participants having both genotype and RIT determination; 408 showed normal macular health and 135 demonstrated AMD, most having early AMD (124 of 135). For the combined sample, higher RIT (slower RMDA) was observed for both the A69S variant in ARMS2 and the Y402H variant in CFH (adjusted P = 0.0001 and P = 0.0023, respectively). For healthy participants, the A69S variant in ARMS2 was associated with higher RIT (adjusted P = 0.0011), whereas the Y402H variant in CFH was not (adjusted P = 0.2175). For AMD patients, the A69S variant of ARMS2 and the Y402H variant of CFH were associated with higher RIT (adjusted P = 0.0182 and P = 0.0222, respectively). Those with a larger number of high-risk ARMS2 and CFH alleles showed higher RIT, in both healthy and AMD groups (adjusted P = 0.0002 and P < 0.0001, respectively)., Conclusions: We report a novel association wherein older adults with high-risk ARMS2 and CFH genotypes are more likely to demonstrate delayed RMDA, the first functional biomarker for incident early AMD. Before the AMD clinical phenotype is present, those showing normal macular health with the ARMS2 A69S allele demonstrate delayed RMDA. Understanding ARMS2 function is a research priority., (Copyright © 2018 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: Retinal vascular and structural changes, particularly outside of the central macula, are not well characterized in X-linked retinoschisis (XLRS). We aim to describe wide-field swept-source OCT (SS-OCT) and swept-source OCT angiography (SS-OCTA) findings in XLRS., Design: Retrospective, cross-sectional study at a tertiary referral center., Participants: Nine consecutive male patients with molecularly confirmed XLRS., Methods: All patients underwent complete ophthalmic examination with multimodal imaging, including SS-OCT with SS-OCTA (PLEX Elite 9000; Carl-Zeiss Meditec Inc., Dublin, CA). Images were then reviewed by 2 retinal specialists as independent graders to determine the frequency and distribution of retinal structural and vascular abnormalities., Main Outcome Measures: Structural and vascular abnormalities seen on SS-OCT and SS-OCTA in patients with XLRS, with attention to the retinal layers involved, the regional distribution of schitic spaces in the posterior pole, and vascular abnormalities within the superficial and deep capillary plexuses., Results: Eighteen eyes from 9 male patients (mean age, 20 years; range 9-40) with molecularly confirmed XLRS were included. Median best-corrected visual acuity measured 20/63 (range, 20/25-10/300). A total of 17 of 18 eyes (94.4%) were noted to have schitic spaces on SS-OCT, and these were observed to be predominantly within the inner nuclear layer in all 17 eyes. A regional variation in the distribution of cysts was noted, with schitic spaces within the ganglion cell layer (13/17 eyes; 76.5%) observed to be perifoveal and those within the outer nuclear layer (8/17 eyes, 47.1%) observed to be mostly extramacular. All eyes had vascular abnormalities on SS-OCTA, including an irregular foveal avascular zone and flow loss within the deep capillary plexus corresponding to the distribution of the schisis., Conclusions: Wide-field SS-OCT and SS-OCTA provide detailed visualization of structural and vascular changes in XLRS and may be helpful for monitoring disease progression or treatment response in clinical trials for the disease., (Copyright © 2018 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10-15% of RP cases are caused by mutations in the RPGR gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR 1-19 and RPGR ORF15 ) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR 1-19 isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR 1-19 that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR 1-19 isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR 1-19 isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants., Competing Interests: The authors declare no conflict of interest.

Objectives: Behavioral health home (BHH) models have been developed to integrate physical and mental health care and address medical comorbidities for individuals with serious mental illnesses. Previous studies identified population health management capacity and coordination with primary care providers as key barriers to BHH implementation. This study examines the BHH leaders' perceptions of and organizational capacity to conduct these functions within the community mental health programs implementing BHHs in Maryland., Methods: Interviews and surveys were conducted with 72 implementation leaders and 627 front-line staff from 46 of 48 Maryland BHH programs. In-depth coding of the population health management and primary care coordination themes identified subthemes related to these topics., Results: BHH staff described cultures supportive of evidence-based practices, but limited ability to effectively perform population health management or primary care coordination. Tension between population health management and direct, clinical care, lack of experience, and state regulations for service delivery were identified as key challenges for population health management. Engaging primary care providers was the primary barrier to care coordination. Health information technology and staffing were barriers to both functions., Conclusions: BHHs face a number of barriers to effective implementation of core program elements. To improve programs' ability to conduct effective population health management and care coordination and meaningfully impact health outcomes for individuals with serious mental illness, multiple strategies are needed, including formalized protocols, training for staff, changes to financing mechanisms, and health information technology improvements.

Purpose: We correlate optical coherence tomography (OCT) retinal layer thickness measurements with histology in wild-type and retinal degenerative pigs., Methods: OCT scans were obtained using the Bioptigen Envisu R2200. In normal pigs, three eyes were imaged in vivo, and three eyes were imaged after enucleation. In the Pro23His retinal degeneration pigs (P23H), one eye was imaged in vivo and four eyes were imaged after enucleation. All eyes were fixed in 4% paraformaldehyde and processed for histology. Corresponding retinal locations on OCT and histology were identified using anatomic landmarks (optic nerve, retinal vessels, visual streak). Individual retinal layer thicknesses were measured by two independent, masked graders, and intraclass correlation coefficients were used to determine agreement. OCT and histologic retinal thickness measurements were averaged and compared., Results: OCT and histologic measurements correlated highly in normal and diseased eyes ( R 2 = 0.91 and 0.92, respectively), and scans performed in vivo and ex vivo did not differ significantly. Despite good overall correlation, certain individual retinal layers (e.g., retinal nerve fiber layer [NFL], inner [INL] and outer [ONL] nuclear layers) appeared thicker on OCT compared to histology, while other layers (e.g., retinal pigment epithelium) appeared thinner. No statistically significant difference was found between OCT and histology for any retinal layer thickness measurement., Conclusions: Retinal layer thickness measurements correlate well with histology in pig eyes, but differences in individual retinal layers may be seen., Translational Relevance: OCT may be used in pigs to measure retinal thicknesses with good overall correlation to histologic measurements.

Background: There is a considerable body of evidence demonstrating a link between the membrane attack complex (MAC) and age-related macular degeneration (AMD), and between C-reactive protein (CRP) and AMD. Both the MAC and the monomeric form of CRP (mCRP) accumulate within the choriocapillaris in AMD. However, the precise contribution of these species to AMD pathophysiology has not been fully elucidated., Methods: We sought to directly assess CRP and MAC levels between human serum and ocular tissues from the same CFH Y402H genotyped donors using ELISA of serum and RPE/choroid proteins., Results: The Y402H polymorphism was associated with significantly increased MAC in RPE/choroid samples, but not in the serum, in a previously unstudied cohort. While MAC levels in the choroid were independent of circulating levels, choroidal CRP was correlated to serum levels., Conclusions: These data provide further evidence for local activation of complement within the choriocapillaris in AMD.

Degradable polymers are integral components in many biomedical polymer applications. The ability of these materials to decompose in situ has become a critical component for tissue engineering, allowing scaffolds to guide cell and tissue growth while facilitating gradual regeneration of native tissue. The objective of this work is to understand the role of prepolymer molecular weight and functionality of photocurable poly(caprolactone) (PCL) in determining reaction kinetics, mechanical properties, polymer degradation, biocompatibility, and suitability for stereolithography. PCL, a degradable polymer used in a number of biomedical applications, was functionalized with acrylate groups to enable photopolymerization and three-dimensional printing via stereolithography. PCL prepolymers with different molecular weights and functionalities were studied to understand the role of molecular structure in reaction kinetics, mechanical properties, and degradation rates. The mechanical properties of photocured PCL were dependent on cross-link density and directly related to the molecular weight and functionality of the prepolymers. High-molecular weight, low-functionality PCLDA prepolymers exhibited a lower modulus and a higher strain at break, while low-molecular weight, high-functionality PCLTA prepolymers exhibited a lower strain at break and a higher modulus. Additionally, degradation profiles of cross-linked PCL followed a similar trend, with low cross-link density leading to degradation times up to 2.5 times shorter than those of more highly cross-linked polymers. Furthermore, photopolymerized PCL showed biocompatibility both in vitro and in vivo, causing no observed detrimental effects on seeded murine-induced pluripotent stem cells or when implanted into pig retinas. Finally, the ability to create three-dimensional PCL structures is shown by fabrication of simple structures using digital light projection stereolithography. Low-molecular weight, high-functionality PCLTA prepolymers printed objects with feature sizes near the hardware resolution limit of 50 μm. This work lays the foundation for future work in fabricating microscale PCL structures for a wide range of tissue regeneration applications.

Age-related macular degeneration (AMD) is a common, blinding disease associated with increased complement system activity. Eyes with AMD show elevated accumulation of the membrane attack complex (MAC) in the choriocapillaris and degeneration of macular choriocapillaris endothelial cells (ECs). Thus, one could reasonably conclude that the endothelial cell death that occurs in AMD is due to injury by the MAC. We therefore sought to identify strategies for protecting ECs against MAC lysis. RF/6A endothelial cells were pre-incubated with a library of FDA-approved small molecules, followed by incubation with complement intact human serum quantification of cell death. Two closely related molecules identified in the screen, econazole nitrate and miconazole nitrate, were followed in validation and mechanistic studies. Both compounds reduced lysis of choroidal ECs treated with complement-intact serum, across a range of doses from 1 to 100 µM. Cell rescue was confirmed in mouse primary choroidal ECs. Both exosome release and cell surface roughness (assessed using a Holomonitor system) were reduced by drug pretreatment in RF/6A cells, whereas endosome formation increased with both drugs, consistent with imidazole-mediated alterations of cell surface dynamics. The results in the current study provide further proof of principle that small molecules can protect choroidal ECs from MAC-induced cell death and suggest that FDA approved compounds may be beneficial in reducing vascular loss and progression of AMD.

Objective: Mutations in myocilin ( MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation., Design: Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes., Subjects: An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51-66) with no known ocular disease were examined., Methods: Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork., Main Outcome Measure: Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects., Results: Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient's eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient's eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes., Conclusions: This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.

Objective: This study measured Americans' willingness to pay an additional $50 in taxes to improve health care and social services for individuals with serious mental illness., Methods: A nationally representative online survey was conducted with 1,010 respondents. Analysis examined how respondents' demographic characteristics and attitudes toward individuals with serious mental illness correlated with their willingness to pay additional taxes to improve health care and social services for this vulnerable population., Results: A majority of respondents expressed willingness to pay $50 in additional taxes to improve health care services (60%) and social services (58%) for individuals with serious mental illness. Those with more negative attitudes toward individuals with serious mental illness were less willing to pay additional taxes to improve either service type., Conclusions: Many Americans are willing to pay additional taxes to improve health care and social services for individuals with serious mental illness.

Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: GUCY2D has been associated with autosomal recessive Leber congenital amaurosis and autosomal dominant cone-rod dystrophy. This report expands the phenotype of autosomal recessive mutations to congenital night blindness, which may slowly progress to mild retinitis pigmentosa., Design: Retrospective case series., Methods: Multicenter study of 5 patients (3 male, 2 female)., Results: All patients presented with night blindness since childhood. Age at referral was 9-45 years. Length of follow-up was 1-7 years. Best-corrected visual acuity at presentation ranged from 20/15 to 20/30 and at most recent visit averaged 20/25. No patient had nystagmus or high refractive error. ISCEV standard electroretinography revealed nondetectable dark-adapted dim flash responses and reduced amplitude but not electronegative dark-adapted bright flash responses with similar waveforms to the reduced-amplitude light-adapted single flash responses. The 30 Hz flicker responses were relatively preserved. Macular optical coherence tomography revealed normal lamination in 3 patients, with abnormalities in 2. Goldmann visual fields were normal at presentation in children but constricted in 1 adult. One child showed loss of midperipheral fields over time. Fundus appearance was normal in childhood; the adult had sparse bone spicule-like pigmentation. Full-field stimulus testing (FST) revealed markedly decreased retinal sensitivity to light. Dark adaptation demonstrated lack of rod-cone break. Two patients had tritanopia. All 5 had compound heterozygous mutations in GUCY2D. Three of the 5 patients harbor the Arg768Trp mutation reported in GUCY2D-associated Leber congenital amaurosis., Conclusions: Autosomal recessive GUCY2D mutations may cause congenital night blindness with normal acuity and refraction, and unique electroretinography. Progression to mild retinitis pigmentosa may occur., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.)

Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.

Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a rare progressive neurodegenerative disorder caused by mutations in CLN3 . Patients present with early-onset retinal degeneration, followed by epilepsy, progressive motor deficits, cognitive decline, and premature death. Approximately 85% of individuals with Batten disease harbor at least one allele containing a 1.02 kb genomic deletion spanning exons 7 and 8. This study demonstrates CRISPR-Cas9-based homology-dependent repair of this mutation in induced pluripotent stem cells generated from two independent patients: one homozygous and one compound heterozygous for the 1.02 kb deletion. Our strategy included delivery of a construct that carried >3 kb of DNA: wild-type CLN3 sequence and a LoxP-flanked, puromycin resistance cassette for positive selection. This strategy resulted in correction at the genomic DNA and mRNA levels in the two independent patient lines. These CRISPR-corrected isogenic cell lines will be a valuable tool for disease modeling and autologous retinal cell replacement.

Purpose: Age-related macular degeneration (AMD) is a devastating disease characterized by central vision impairment in individuals with advanced age. Neovascular AMD is a form of end-stage disease in which choroidal vessel outgrowth occurs beneath the retina. While many hypotheses have been raised as to what triggers the formation of pathological choroidal neovascular membranes, the exact mechanism for their initiation remains unresolved. Polymorphisms in the FLT1 gene have previously been associated with neovascular AMD risk, including the rs9943922 single nucleotide polymorphism (SNP). Here, we aimed to determine the association between the high-risk FLT1 genotype and FLT1 protein levels in human retina or retinal pigment epithelium (RPE)/choroid tissue., Methods: Retina and RPE/choroid tissue from 10 human donor eyes was selected from a collection of eyes genotyped for the rs9943922 SNP. Differences in soluble and membrane bound FLT1 protein levels were assessed for retina versus RPE/choroid donor tissue using ELISA and Western blotting analyses. Genotype-associated changes in FLT1 protein levels were also evaluated., Results: We found soluble FLT1 levels in the RPE/choroid tissue to be approximately three times higher than that of the retina (p < 0.001), while both samples have similar levels of the membrane bound form. When tissue with the rs9943922 SNP was compared with controls, no significant genotypic differences in FLT1 protein levels were observed., Conclusions: Based on these data, we conclude that the rs9943922 SNP in the FLT1 gene does not result in a large difference in FLT1 protein levels, regardless of whether it is the soluble or the membrane bound form.

Background: Approximately 1% of normal tension glaucoma (NTG) cases are caused by TANK-binding kinase 1 (TBK1) gene duplications and triplications. However, the precise borders and orientation of these TBK1 gene copy number variations (CNVs) on chromosome 12 are unknown., Methods: We determined the exact borders of TBK1 CNVs and the orientation of duplicated or triplicated DNA segments in 5 NTG patients with different TBK1 mutations using whole-genome sequencing., Results: Tandemly duplicated chromosome segments spanning the TBK1 gene were detected in 4 NTG patients, each with unique borders. Four of 5 CNVs had borders located within interspersed repetitive DNA sequences (Alu and long interspersed nuclear element-L1 elements), suggesting that mismatched homologous recombinations likely generated these CNVs. A fifth NTG patient had a complex rearrangement including triplication of a chromosome segment spanning the TBK1 gene., Conclusions: No specific mutation hotspots for TBK1 CNVs were detected, however, interspersed repetitive sequences (ie, Alu elements) were identified at the borders of TBK1 CNVs, which suggest that mismatch of these elements during meiosis may be the mechanism that generated TBK1 gene dosage mutations.