Molecular And Immunopathology Of Experimental And Clinical Ocular Diseases

我们在患者标本和动物组织中识别和定位炎性细胞、退行性细胞和恶性细胞及其产物和相关分子。我们主要通过常规病理学、免疫组织化学和分子病理学对这些细胞及其产物进行分析。尖端技术的应用，如显微解剖与分子技术(聚合酶链式反应、逆转录聚合酶链式反应、基因分型等)相结合。使我们能够提供更准确的病理诊断(评估)和了解疾病的发病机制。这些技术和发现将指导我们为患者选择最有针对性的治疗方法。我们还从不同的动物模型研究了不同眼病的发病机制。利用动物模型，我们可以评估新的治疗药物对各种眼病的疗效。2014财年，我们在研究中完成了以下工作： 1.老年性黄斑变性的分子病理学： AMD是世界上导致老年人严重中心视力丧失的主要原因。据预测，到2020年，AMD将影响300万50岁以上的美国人。AMD的病理特征是黄斑软性玻璃体积聚、视网膜色素上皮(RPE)和感光细胞变性、地理性萎缩和/或渗出伴脉络膜新生血管。虽然包括年龄、种族、吸烟、饮食、氧化应激和炎症在内的几个危险因素与AMD有关，但这种疾病的病因和发病机制在很大程度上仍不清楚。目前的知识表明，AMD的发育受到遗传因素的强烈影响。新生血管性AMD的治疗选择包括玻璃体内注射抗血管内皮生长因子(VEGF)；然而，对于地理性萎缩AMD的治疗选择极其有限。 2014财年，我们报道了与AMD相关的TIM3/SYN3、RAD51B和FBN2基因的几个单核苷酸多态性。我们发现在AMD患者的黄斑中存在白介素17A(IL17A)及其受体IL17RC的异常表达。在体外，IL17A诱导RPE细胞死亡，其特征是胞浆脂质和自噬小体积聚，随后激活促凋亡的Caspase-3和Caspase-9。这种病理通过IL17RC的siRNA敲除而减轻。我们研究了AMD中巨噬细胞、炎症体和IL-17之间的联系。我们还发现了编码可溶性IL-17受体、辣椒上皮源性因子(PEDF)和血小板衍生生长因子-C(PDGF-C)的腺相关病毒载体对我们的双缺陷CCL2和CX3CR1在Crb1rd8背景小鼠(AMD模型)上的治疗效果。虽然在小鼠模型中的发现不一定转化为在人类中的发现，但它们仍然可以为我们理解AMD的发病机制和研究潜在的治疗靶点提供有用的信息。 2.眼部淋巴瘤： 原发性玻璃体视网膜淋巴瘤(PVRL)，以前称为原发性眼内淋巴瘤(PIOL)，是一种罕见且致命的眼部恶性肿瘤。PVRL的诊断通常具有挑战性，因为它可以伪装成慢性葡萄膜炎。我们发表了一篇文章，描述了一种通过玻璃体中miRNA-155的水平来区分PVRL和葡萄膜炎的方法，而PVRL中的miRNA-155水平显著低于PVRL。在人类组织中，miRNA-155调节许多转录因子，如FOXP3、NFKB1、TGFB1和AKT1。MicroRNA-155与这些转录因子的相互作用在先天免疫反应和肿瘤发生中起着重要作用。但在PVRL的诊断中，玻璃体miRNA水平并不优于IL-10/IL-6水平的比值。 我们回顾了2004-2012年间NIH葡萄膜炎诊所的853例患者，发现21例(2.5%)患有肿瘤伪装综合征。他们更有可能是年龄较大的非非洲裔美国人，他们表现为单侧后葡萄膜炎。我们还报告了诊断PVRL的困难，这通常需要医生高度的临床怀疑和不止一种侵入性程序(NIH的27名PVRL患者中的63%)才能做出诊断。我们还报道了自体荧光颗粒模式(超自体荧光点)与活动性PVRL之间的关系。 3.眼科疾病的新病理学和发病机制： 在NEI的H.Nida Sen博士、西北大学的Deborah Goldstein和巴斯科姆帕尔默研究所的Janet Davis的合作下，我们编辑了一期关于性别和葡萄膜炎的特刊。本期特刊包括10篇文章，试图确定感染性和非感染性自身免疫性葡萄膜炎的性别和性别差异，例如，女性多发性硬化症，女孩幼年型特发性关节炎，以及艾滋病毒感染男性患者的梅毒葡萄膜炎。在某些葡萄膜炎中，临床表现和病程可能因性别不同而不同。我们提出了在von Hippel-Lindau(VHL)相关性视网膜血管母细胞瘤中HIF表达的相关性。我们在交感性葡萄膜炎、眼结节病和病毒性视网膜炎中提供了独特和新的分子病理学。

We identify and topographically localize inflammatory, degenerative, and malignant cells, as well as their products and related molecules, in patient specimens and animal tissues. We analyze these cells and their products mainly by routine pathology, immunohistochemistry, and molecular pathology. The application of cutting-edge technology, such as microdissection combined with molecular techniques (PCR, RT-PCR, genotyping, etc.) allows us to provide a more accurate pathological diagnosis (assessment) and understand the pathogenesis of the disease. These technologies and findings will guide us in selecting the most targeted treatments for patients. We also study the mechanisms of different ocular diseases from various animal models. Using animal models, we can assess the efficacy of novel therapeutic agents for various ocular diseases. In FY2014, we accomplished the following in our research: 1. Molecular Pathology of Age-Related Macular Degeneration (AMD): AMD is the leading cause of irreversible severe central vision loss among the elderly in the world. It is projected that AMD will affect 3 million Americans over the age of 50 by the year 2020. The pathology of AMD is characterized by the accumulation of soft drusen, retinal pigment epithelium (RPE) and photoreceptor degeneration, geographic atrophy, and/or exudation with choroidal neovascularization in the macula. While several risk factors, including age, race, smoking, diet, oxidative stress and inflammation have been linked to AMD, the etiology and pathogenesis of the disease remain largely unclear. Current knowledge has shown that AMD development is strongly influenced by genetic factors. Treatment options for neovascular AMD include intravitreal injections of anti-vascular endothelial growth factor (VEGF); however, treatment options for geographic atrophy AMD are extremely limited. In FY2014, we reported several single nucleotide polymorphisms in the TIM3/SYN3, RAD51B, and FBN2 genes associated with AMD. We documented aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. We investigate the link among macrophage, inflammasome, and IL-17 in AMD. We also found therapeutic effectiveness of adeno-associated virus vector encoding soluble IL-17 receptor, pimento epithelium-derived factor (PEDF), and platelet-derived growth factor-C (PDGF-C) on our double deficient Ccl2 and Cx3cr1 on Crb1rd8 background mice, an AMD model. Although the findings in the mouse model may not necessarily translate to findings in humans, they could still provide useful information for our understanding AMD pathogenesis and investigating potential therapeutic targets. 2. Ocular Lymphoma: Primary vitreoretinal lymphoma (PVRL), previously called primary intraocular lymphoma (PIOL), is a rare and fatal ocular malignancy. The diagnosis of PVRL is often challenging as it can masquerade as chronic uveitis. We published an article describing a method to differentiate PVRL from uveitis by using the level of miRNA-155 in the vitreous, which was significantly lower in PVRL. In human tissues, miRNA-155 regulates numerous transcriptional factors such as FOXP3, NFKB1, TGFB1, and AKT1. The interaction of microRNA-155 and those transcriptional factors plays an important role in innate immune responses and tumorigenesis. However, vitreal miRNA level does not offer an advantage to the ratio of IL-10 and IL-6 levels for the diagnosis of PVRL. We reviewed 853 patients at the NIH Uveitis clinic between 2004-2012 and found 21 (2.5%) with neoplastic masquerade syndrome. They were more likely to be older, non-African Americans who presented with unilateral, posterior uveitis. We also reported the difficulty in diagnosing PVRL, which often requires a high degree of clinical suspicion by physician and more than one invasive procedure (63% of the 27 PVRL patients at NIH) to make the diagnosis. We also reported an association between autofluorescent granularity patterns (hyperautofluorescent spots) with active PVRL. 3. New Pathology and Pathogenesis of Ocular Diseases: In collaboration with Drs. H. Nida Sen of NEI, Deborah Goldstein of Northwestern University, and Janet Davis of Bascom Palmer Institute, we edited a Special Issue on Gender and Uveitis. This special issue includes 10 articles and attempts to identify gender- and sex-based differences in infectious and noninfectious autoimmune uveitis, for example, multiple sclerosis in women, juvenile idiopathic arthritis in girls, and syphilitic uveitis in HIV-infected male patients. Clinical manifestations and courses may appear differently between genders in certain uveitides. We raised the relevance of HIF expression in von Hippel-Lindau (VHL) associated retinal hemangioblastoma. We provided unique and new molecular pathology in the eyes with sympathetic uveitis, ocular sarcoidosis, and viral retinitis.