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2016 Glenn A. Fry Award Lecture: Mechanisms and Potential Treatments of Early Age-Related Macular Degeneration

Fletcher, Erica Lucy PhD, MScOptom*

doi: 10.1097/OPX.0000000000001124
Feature Articles – Public Access

ABSTRACT Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in those older than 80 years. Understanding the mechanisms that cause this condition or its progression is critical for developing novel treatments. Here we summarize our studies evaluating the role of purine, adenosine triphosphate (ATP), in early AMD as well as photoreceptor loss and have also provided some insights to our investigations of a new laser treatment for those with early AMD. One of the receptors that are activated by ATP, P2X7, is expressed by neurons and immune cells and has a different function in each cell type. In neurons, P2X7 receptors form a ligand-gated ion channel, whereas on immune cells P2X7 receptors act as a scavenger receptor. These distinct functions have provided new insights to the mechanisms of AMD. On the one hand, high concentrations of ATP can cause photoreceptor death, most likely via stimulation of P2X7 receptors localized on photoreceptor terminals. On the other hand, P2X7 receptors mediate removal of dead and dying cells by monocytes. By understanding the fundamental cell biological changes that occur in patients and animal models of disease, we have uncovered mechanisms that may help us manage and treat patients in the future.

Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Victoria, Australia *

Submitted: May 4, 2017

Accepted: July 7, 2017

Funding/Support: NH&MRC grant no.APP1061419 (to Erica Lucy Fletcher).

Conflict of Interest Disclosure: The author reports no conflict of interest.

Author Contributions and Acknowledgments: Conceptualization, ELF; Data curation, ELF; Formal analysis, ELF; Funding acquisition, ELF; Investigation, ELF; Methodology, ELF; Project administration, ELF; Supervision, ELF; Writing – original draft, ELF; Writing – review & editing, ELF.

I have been very fortunate to have worked with a number of very talented PhD students and postdocs, all of whom have contributed in various ways to the work I have presented here. Drs Theresa Puthussery, Andrew Jobling, Una Greferath and Joanna Phipps have all contributed in immeasurable ways over many years to the research efforts of my group and I extremely grateful to them all. I would also like to thank Dr. Kirstan Vessey who has also contributed to my research activity over many years and also generously created the figures for this article.

I have been extremely fortunate to have been mentored for three giants of vision science. I would like to thank Prof Suzanne Fleiszig (University of California, Berkeley), Prof Michael Kalloniatis (University of New south Wales) and Prof Dr Heinz Wassle for teaching me the importance of the “3 Fs in research” -have Fun, Focus on an answering an important problem and communicate with Finesse. Over my career, I have tried to emulate these values! In addition, I have worked with dedicated research staff and students and had a great deal of support from collaborators, too numerous to mention here. For many years, my research has been well funded by the National Health and Medical Research Council of Australia, the Australian Research Council as well as a range of charities and commercial organizations and to these organizations I would like to extend my sincere appreciation. Lastly, I would like to thank my family, my husband Richard Lindsay and three children, Paul, Charlotte and Hanna. Without a great deal of support and patience, it wouldn't have been possible to have had a research career.



Age-related macular degeneration is responsible for almost half the cases of irreversible vision loss in the Western world.1,2 There are two types of advanced age-related macular degeneration referred to as “dry” or “wet” age-related macular degeneration, depending on the presence of chronic atrophic changes (dry age-related macular degeneration) or pathological growth of blood vessels into the retina (wet age-related macular degeneration). Despite the emergence of anti–vascular endothelial growth factor agents to treat wet age-related macular degeneration, there are currently no specific treatments for preventing the development of age-related macular degeneration or slowing its progression. Understanding the mechanisms that occur in the early stages of age-related macular degeneration is crucial for developing treatment strategies that prevent or slow progression of age-related macular degeneration to the vision-threatening forms of the disease.3

A guiding principle for my research since starting my PhD in 1992 has been that to unravel the mechanisms of disease one has to understand and apply knowledge of how the retina functions under normal circumstances. I became interested in how the retina functions as an undergraduate optometry student, sitting in lectures from doyens of the field, Professors David Vaney and Rachel Wong. I was intrigued by the way the neurons of the retina communicate with one another. In particular, I was fascinated by how precise connections of what I thought were a relatively small number of neurons could lead to the richness and complexity of vision that we perceive.

Much of my research, which is summarized in the sections that follow, has concentrated on the class of neuromodulators called purines, which include adenosine triphosphate. Our initial studies focused on how purines mediate communication between the various retinal cell types. This information formed a basis for understanding disease mechanisms, especially age-related macular degeneration. With reference to age-related macular degeneration, we have addressed several questions: First, does excessive extracellular adenosine triphosphate cause overstimulation of photoreceptors and ultimately their death? Second, do purines contribute to the development of early age-related macular degeneration by modulating the innate immune system? Finally, can our understanding of the pathogenesis of age-related macular degeneration be used as a foundation for evaluating novel treatments designed to reduce signs of the disease or its progression? As documented in the following sections, our work has shed light on the highly complex etiology of age-related macular degeneration and has provided the first steps in developing treatments that target the early stages of disease.

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Setting the Scene: How Age-Related Macular Degeneration Influences Vision

Large epidemiology studies, such as the Beaver Dam Eye Study, have shown that the prevalence of all forms of age-related macular degeneration is greater than 30% in patients older than 75 years4 and accounts for 46% of cases of irreversible severe vision loss in those older than 40 years in the United States.5 Of the two types of advanced age-related macular degeneration (dry and wet age-related macular degeneration), the dry atrophic is far more common and associated with progressive loss of vision due to gradual loss of retinal pigment epithelial cells and photoreceptors. By contrast, wet age-related macular degeneration, which afflicts approximately 15% of those with advanced age-related macular degeneration, leads to sudden and severe loss of vision due to choroidal neovascularization. Even with the use of anti–vascular endothelial growth factor agents to treat those with neovascular age-related macular degeneration, gains in visual acuity are lost within 7 years in a significant number of patients.6 Vision loss associated with advanced disease is primarily caused by photoreceptor death, either secondary to anomalies in the function of the retinal pigment epithelial cells or because of direct loss due to hemorrhage associated with neovascularization and/or scarring.

Regardless of the type of age-related macular degeneration, there are common characteristics that occur in the early stages of the disease. Drusen, accumulations that occur between the retinal pigment epithelial cells and Bruch's membrane, are a visible biomarker of disease onset.1,7 Large size of drusen (>125 μm) in combination with pigmentary changes is associated with the highest risk of progression to advanced stages of the disease.8 In addition to the presence of drusen, thickening of Bruch's membrane, a semipermeable membrane that lies between the retinal pigment epithelial cells and choroid, also occurs. Functional changes manifest in early stages of the disease and are detected by losses in both rod- or cone-mediated function prior to the loss in visual acuity (e.g., flicker sensitivity, cone threshold, rod adaptation).9,10 Although age-related macular degeneration affects the cone-rich macula, rod functional abnormalities appear to exceed cone abnormalities in the earliest stages of disease and are consistent with histological evidence indicating a reduction in rods in the perifoveal area.9,11,12 These findings highlight that photoreceptor dysfunction and death occur from an early stage of age-related macular degeneration most likely secondary to defects in the retinal pigment epithelial cells dysfunction and/or Bruch’s membrane. An additional type of deposit, called reticular pseudodrusen, has been recently characterized and represents the development of deposits within the subretinal space between the photoreceptor outer segment and retinal pigment epithelial cells.13 The presence of reticular pseudodrusen has been associated with high rate of progression.14,15

Over the last two decades, there has been an exponential increase in our understanding of pathogenesis of age-related macular degeneration.1,16,17 However, the underlying cause for the development of the disease and why it progresses in some people remain to be determined. The multifactorial nature of the etiology of age-related macular degeneration is well accepted, and factors including advancing age, environmental stress, and genetic factors have all been implicated.1 More recently, the importance of the innate immune system has been proposed.18 Analysis of the protein and lipid components on drusen composition has revealed a large number of immune fragments within drusen including complement fragments.19 In addition, some 45% to 75% of cases of age-related macular degeneration are associated with genetic risk factors, many of which involve genes affecting components of the innate immune system including complement factor H.20,21 Further evidence for anomalies in innate immunity comes from a series of animal studies highlighting the association of defects in chemokine or cytokine signaling with changes in the outer retina.22–24 What is not clear from these studies is what triggers the innate immune system to initiate disease, or how the cells of the innate immune system contribute to disease pathogenesis and progression.

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Purines and Their Role in the Retinal Neurotransmission

Purine, adenosine triphosphate, is well known for its role as an intracellular molecule mediating energy metabolism. Purines including adenosine triphosphate can also exert effects on a range of cell types when located extracellularly, by activating P2X or P2Y receptors.25–27 P2X receptors consist of seven isoforms of ligand-gated ion channels (Fig. 1A). In contrast, P2Y receptors are seven transmembrane spanning receptors that elicit cellular responses by activating G-protein cascades.27 One the most interesting features of purine signaling that has relevance to eye disease is the multiple cellular functions that purines can elicit depending on which cell type is activated by adenosine triphosphate. In the context of our work, we have focused on two functions—the role of purines in modulating neural function and the role of purines in immune cell function.28,29



Our work implicates purines in neural signaling within both the inner and outer retina.25,26 Using a range of high-resolution microscopy methods, we have shown that P2X and P2Y receptors are localized at synaptic sites in both the inner and outer retina,25 enzymatic mechanisms that degrade extracellular adenosine triphosphate are present within both synaptic layers of the retina,30,31 and proteins involved in packaging of adenosine triphosphate into synaptic vesicles are expressed in some retinal neurons.32 These findings imply that purines contribute to processing of visual information within the retina. Most of the seven P2X receptor isoforms are expressed in retina; however, P2X7 and P2X4 are the receptors most relevant to understanding the mechanisms of age-related macular degeneration. Indeed, P2X7 has been implicated in neuronal death in a range of regions in the retina and central nervous system.26,33 P2X7 and P2X4 receptors are expressed by several neuronal subtypes within the retina including ganglion cells, amacrine cells, and also cells of the outer retina.31,34,35 Double-labeling experiments using known markers of neuronal subtypes have revealed immunolabeling of P2X7 within or close to photoreceptor terminals.31,35 At the ultrastructure level, P2X7 receptors have been localized presynaptically to rod and cone pedicles. Evidence for a role of P2X7 in modulation of photoreceptor function comes from studies of retinal function in the presence of the P2X7 agonist, BzATP. Following intravitreal injection of BzATP, the amplitude of the a-wave of the electroretinogram is increased, suggesting that activation of P2X7 receptors on photoreceptors leads to depolarization of these cells.35

P2X7 receptors are highly permeable to calcium (Fig. 1A) and have been evaluated in several regions of the central nervous system for a potential role in neurodegeneration.33,36 In particular, following death or damage, neurons can release high levels of adenosine triphosphate into the extracellular space, leading to death of neighboring neurons via a mechanisms involving excessive activation of P2X7 receptors expressed on neighboring neurons.33,36 In addition, adenosine triphosphate can be released from glial cells, important support cells within the retina.37 Our work has shown that intravitreal injection of high concentrations of adenosine triphosphate leads to rapid loss of photoreceptors.38–40 Fig. 1 shows an example of this in the rat retina. Figs. 1B and C show vertical sections of rat retinae 7 days after receiving an intravitreal injection of sterile saline or adenosine triphosphate. The outer nuclear layer is significantly reduced following treatment with adenosine triphosphate. In order to establish that P2X receptors were involved in the adenosine triphosphate–induced photoreceptor death, coinjection of adenosine triphosphate with the P2X antagonist, pyridoxalphosphate-6-azophenyl-2',4' -disulfonic acid, was evaluated. In this case, photoreceptor loss was reduced by approximately 60%.39

Evidence for a potential role for adenosine triphosphate in modulating photoreceptor integrity in patients with age-related macular degeneration is demonstrated by an increase in adenosine triphosphate concentration in the vitreous of patients presenting with subretinal hemorrhage, compared with those presenting with macula hole or retinal detachment.41 Hemorrhages are a source of high concentrations of adenosine triphosphate, owing to its high concentration in blood. Using an animal model of subretinal hemorrhage, Notomi and colleagues41 showed that treating animals with an antagonist to P2X7 receptors prevented photoreceptor death associated with exposure to hemorrhage. We have evaluated whether dying photoreceptors could be a source of adenosine triphosphate and evaluated whether blockade of P2X receptors, with the nonspecific P2X antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, reduced photoreceptor death in an animal model of inherited retinal degeneration. Our results showed that blockade of P2X receptors reduced photoreceptor death by approximately 30%.39 Taken together, these results suggest that adenosine triphosphate, released in abnormal amounts either from dying photoreceptors or from another source such as hemorrhages, can cause death of neighboring but normal photoreceptors. Based on our immunolocalization studies, we propose that purine-induced photoreceptor death occurs most likely via a mechanism involving activation of P2X7 receptors localized on photoreceptors.

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P2X7 Receptors on Immune Cells: A Possible Role for Abnormal Phagocytosis in Early Age-Related Macular Degeneration

Purinergic receptors, including P2X7 receptors, are not only localized to neuronal synapses but are also expressed by other cell types including resident immune cells in the retina called microglia.42 Indeed, P2X7 and P2X4 receptors are expressed by microglia as well as monocytes within the vasculature.42 Microglia are resident immune cells of the nervous system that are known to be rapidly activated in response to disease and play important roles in phagocytizing or removing dead and dying cells that accumulate during disease.43

Retinal microglia and infiltrating macrophages/monocytes of the choroid are thought to migrate toward and engulf insoluble material found in the subretinal space and in Bruch's membrane, respectively. Consequently, anomalies in macrophage and microglial function are thought to contribute to the development of age-related macular degeneration. Microglia accumulate in the subretinal space with age and display altered motility and dynamic behavior.44–46 In addition, activated microglia accumulate near drusen and subretinal deposits in tissues isolated from those with age-related macular degeneration.47 Furthermore, genetic association studies have shown an increased risk of developing advanced age-related macular degeneration in those carrying a loss-of-function single-nucleotide polymorphism in the gene encoding Cx3cr1, a chemokine receptor, important in regulating the function of microglia. Thus, changes in microglia/macrophage function have been proposed to lead to a local, chronic inflammatory environment, where the deposition of extracellular debris in the subretinal space and/or Bruch's membrane may not be cleared effectively, resulting in some of the early signs of age-related macular degeneration.

Phagocytosis of cellular debris by macrophages and monocytes involves recognition by “scavenger receptors” of moieties exposed on the surface of extracellular debris or on dead or dying cells. Indeed, recent work by our collaborators has shown that specific parts of the membrane of dying cells can attach to monocytes via P2X7 receptors, leading to their phagocytosis.48 In monocytes, P2X7 receptors are tightly associated with nonmuscle myosin H chain, the adenosine triphosphatase that provides the energy for cytoskeletal rearrangement needed for engulfment.49,50 A unique feature of the scavenger role of P2X7 receptors is that moieties on the surface of extracellular debris or dying cells are recognized by six cysteine residues that are in the extracellular domain of P2X7 receptors (Fig. 2A).29 Our recent work has shown that retinal microglia and choroidal monocytes/macrophages express P2X7 receptors42 and that P2X7 receptors are likely to act as a scavenger receptor–mediating phagocytosis.



We have tested whether anomalies in P2X7 receptor function are associated with the development of early signs of age-related macular degeneration in a mouse where P2X7 receptor expression is genetically inactivated.51 We reasoned that if the scavenger role of P2X7 receptors was impaired or absent Bruch's membrane would thicken. Our analysis showed that with age P2X7-null mice develop a thicker Bruch's membrane compared with age-matched wild-type control mice. We correlated the changes in retinal pathology with phagocytic function of both blood monocytes and retinal microglia. A schematic diagram of how phagocytosis is measured is shown in Fig. 2B. Briefly, monocytes or microglia are isolated from the retina or blood and incubated with fluorescent beads. Uptake of fluorescent beads by cells is measured and quantified over time. As shown in Fig. 2C, monocytes isolated from P2X7-null mice show reduced phagocytosis compared with controls.51

We next evaluated whether changes in P2X7 receptor function were associated with an increased risk of developing age-related macular degeneration. We performed a case-control study in 744 patients with advanced age-related macular degeneration (geographic atrophy or choroidal neovascularization) and 557 age-matched controls and evaluated the frequency of inheritance of 12 single-nucleotide polymorphisms in P2X7 and one in P2X4.42 The frequency of inheritance was no different in those with age-related macular degeneration compared with controls for any of the 12 P2X7 receptor single-nucleotide polymorphisms. However, inheritance of a P2X4 single-nucleotide polymorphism increased risk of advanced disease, and inheritance of a combination of P2X7 and P2X4 further increased the risk of developing advanced age-related macular degeneration (odds ratio, 4.05; P = .026).42 We confirmed that inheritance of these single-nucleotide polymorphisms was associated with changes in monocyte phagocytosis by quantifying the ability of monocytes isolated from patients to take up fluorescently-labeled beads (YG beads; Fig. 2B). Phagocytic function of peripheral blood monocytes isolated from patients with these rare P2X7 and P2X4 single-nucleotide polymorphisms showed that inheritance of the loss-of-function single-nucleotide polymorphism in P2X7 alone reduced macrophage phagocytosis, but when coinherited with a P2X4 single-nucleotide polymorphism, phagocytosis was virtually abolished.42 In order to understand the interaction between P2X7 and P2X4 receptors and how this combination might influence phagocytosis, we next transfected HEK293 cells with the human variants of these different forms of P2X7 and P2X4 receptors. HEK293 cells do not normally show any form of phagocytic ability. However, when transfected with P2X7 receptors, they become phagocytic cells. In contrast, HEK293 cells that expressed P2X4 receptors showed very little phagocytic capacity. The differing effects that P2X7 and P2X4 have on a cell's phagocytic capacity are highly significant. It is likely that the P2X4 single-nucleotide polymorphism attenuates phagocytosis by interfering with the six-cysteine residues of the P2X7 receptor that confers scavenger activity on cells, thereby increasing the risk of age-related macular degeneration.

Overall, our work has shown that purines contribute to how the retina functions in two different ways depending on the cell types activated. P2X7 receptors are expressed by a number of neuronal subtypes within the retina and most likely modulate neural communication within specific circuits. In addition, excessive stimulation of neurons by adenosine triphosphate is associated with neural death, especially photoreceptors. Beyond a role in neurotransmission, our work has also identified P2X7 receptors as a potential scavenger receptor. Building on in vitro studies examining the mechanisms by which P2X7 receptors on monocytes mediate phagocytosis, our results highlight a potential role for anomalies in P2X7 receptor function in the development of age-related macular degeneration.

A central question to be answered now is whether altering phagocytosis can reduce the development of early age-related macular degeneration or attenuate its progression to advanced disease. Our ongoing studies are exploring pharmacological agents that enhance monocyte phagocytosis and whether these have potential in the treatment of age-related macular degeneration.

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Novel Treatments of Targeting Early Age-Related Macular Degeneration

Over the last decade, there have been tremendous advances in the treatment of wet age-related macular degeneration, with the emergence of anti–vascular endothelial growth factor agents that reduce acute vision loss in those with choroidal neovascularization.52–54 However, there remains no commercially available treatment for those with early-stage disease, and management revolves around careful monitoring and discussion of risk factors including age, genetics, smoking, and dietary supplements.

A common theme for those managing patients with early age-related macular degeneration has been that treatments that lead to the reduction in drusen may reduce disease burden and progression. Reduction in drusen following treatment of the retina with a continuous wave ophthalmic laser was observed many decades ago by Gass.55 These initial observations lead to a number of clinical trials where prophylactic treatment with a continuous wave laser was evaluated as a means of reducing drusen load and progression of disease.56 However, a number of studies showed that prophylactic laser treatment leads to an acceleration of choroidal neovascularization. Consequently, prophylactic laser treatment as a means to reduce progression to choroidal neovascularization in those with early age-related macular degeneration has been abandoned because of the potential risk of worsening patient outcomes.

A new class of ophthalmic laser is under investigation that has the potential to reduce drusen load and progression of age-related macular degeneration, without the risks of continuous wave lasers. Nanosecond lasers deliver short nanosecond pulses of laser energy to the posterior eye.57,58 The pulsed nature of laser delivery reduces energy absorption by at least 1/500th compared with conventional continuous wave.57 This reduction in energy absorption reduces the bystander damage that commonly occurs with treatment with continuous wave lasers. The retinal pigment epithelial cell is the main cell type that absorbs laser energy. When a continuous wave laser is applied, thermal damage extends into the overlying neural retina, leading to the formation of small retinal scars. In contrast, our work has shown that in both human and mouse retinae application of nanosecond laser spots selectively ablates the retinal pigment epithelial cells without inducing damage in the overlying retina. Moreover, treatment with a nanosecond laser reduces the proportion of eyes with drusen over a 12- or 24-month period.58,59 Thus, unlike continuous wave lasers, treatment with a nanosecond laser provides a means of delivering laser pulses to the posterior eye that reduces drusen, but does this in a manner that does not affect the overlying retina.

We next evaluated whether treatment with a nanosecond laser can reduce signs of early age-related macular degeneration in a mouse known to exhibit a thickened Bruch's membrane. Laser treatment of 9-month-old apoE-null was associated with a reduction in Bruch's membrane thickness when examined 4 months after treatment (Fig. 3). In order to understand how nanosecond laser treatment leads to a reduction in Bruch's membrane thickness, we evaluated gene expressional changes in the retinal pigment epithelial cells isolated from laser- and non–laser-treated wild-type and apoE-null mice. Our results showed that genes associated with formation and turnover of extracellular matrix including matrix metalloproteinases were modified in the retinal pigment epithelial cells of laser-treated eyes compared with the retinal pigment epithelial cells of eyes isolated from untreated animals. Thus, nanosecond laser treatment induces changes in the retinal pigment epithelial cells that are critical for how the retinal pigment epithelial cells control the formation and turnover of the components of Bruch's membrane.



A particularly interesting observation was that untreated fellow eyes showed similar gene expressional changes to those that had received laser treatment, emphasizing that treatment with a laser can induce distant effects, even in the fellow (non–laser-treated) eye. The mechanisms by which nanosecond lasers induce distant effects, whether in the laser-treated eye or the non–laser-treated fellow eye, remain to be explored. Our early observations are that immune cells, either within the retina or within the choroid, could be instrumental. Notably, they are the first cell type to respond following laser application, extending processes into the subretinal space. We propose that immune cell function could be altered by laser treatment, leading to changes in retinal pigment epithelial cell function and potentially altered phagocytic ability.

Overall, our work demonstrates that treatment with a nanosecond laser modifies the posterior eye in a manner that is unique. It reduces the thickness of Bruch's membrane in mice and reduces drusen in patients with early age-related macular degeneration in the absence of any retinal damage. Further work is necessary to determine whether this laser type is beneficial in reducing the progression of age-related macular degeneration.

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In conclusion, our work has shown that purinergic receptors, including P2X7, play important roles in both health and disease. P2X receptors also modulate immune cell function and by virtues of their cellular expression pattern can contribute to retinal disease in distinct ways. Indeed, when P2X7 receptors are excessively activated, photoreceptor death ensues. P2X7 receptors on immune cells act as a scavenger receptor, binding to moieties on dead or dying cells. Our work has shown that signs of age-related macular degeneration, especially thickening of Bruch's membrane, occurs to a greater extent in P2X7-null mice and is associated with reduced phagocytosis. Moreover, inheritance of a rare P2X7 and P2X4 single-nucleotide polymorphism increases risk of advanced age-related macular degeneration four-fold. Overall, these results suggest that phagocytosis could be an important determinant in the development of age-related macular degeneration and/or its progression. Future work is now needed to examine whether restoring phagocytosis can reduce the signs of early age-related macular degeneration. Our studies to date have evaluated a novel laser nanosecond laser treatment that induces the resolution of drusen in patients and thins Bruch's membrane in a mouse model with features of age-related macular degeneration. This novel laser treatment also triggers an early change in immune cells. An evaluation of the impact that nanosecond laser treatment has on immune cell phagocytosis is the topic of future exploration.

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