Molecular And Immunopathology Of Experimental And Clinical Ocular Diseases

通过常规病理学、病理生理学、免疫组织化学和分子病理学分析患者标本和动物样本中眼部炎症、变性和肿瘤细胞及其产物的身份和地形位置。 显微切割等尖端技术的应用与PCR、RT-PCR、RFLP等分子技术相结合，使我们能够对疾病进行更准确的病理诊断（评估），并指导我们为患者选择合适的治疗方法。 我们生成和/或应用我们从各种眼部疾病动物模型中学到的知识，因此我们可以了解不同眼部疾病的机制。 使用这些动物模型，我们还可以评估不同治疗药物对各种眼部疾病的疗效。 这有助于我们更好地了解疾病的发病机制，并更好地选择针对特定疾病的治疗方法。 于2008财政年度，我们继续并完成了以下研究： 1.眼部淋巴瘤和附件肿瘤： 我们继续证实眼内原发性B细胞淋巴瘤和眼B细胞淋巴瘤细胞IgH基因重排患者眼液中白细胞介素-10升高。 高剂量甲氨蝶呤的化疗方案被推荐用于原发性CNS淋巴瘤。 眼内化疗具有副作用有限的优点，并已被证明是有用的原发性眼内淋巴瘤患者。 我们发现，甲氨蝶呤跨细胞膜转运的改变可能有助于反复玻璃体内注射后的耐药性。 绝大多数原发性眼内淋巴瘤是恶性B细胞，而眼内T细胞淋巴瘤是罕见的。 我们报告了一例转移性T细胞淋巴瘤的眼睛，并证明了免疫分型和分子分析的效用，使用显微切割和PCR，作为关键的后续研究，在患者的伪装综合征。 此病例可见玻璃体视网膜受累，但无葡萄膜受累，与文献中许多眼转移性T细胞淋巴瘤相似。这是有趣的，因为葡萄膜，而不是视网膜，是大多数转移性肿瘤（包括系统性B细胞淋巴瘤）中侵袭的典型眼部组织。 我们还探讨了感染性微生物（人乳头状瘤病毒、HIV、人单纯疱疹病毒-8、幽门螺杆菌、衣原体和丙型肝炎病毒）在眼附属器肿瘤中的作用。 多种微生物可能通过类似的肿瘤发生机制（包括慢性抗原刺激和感染性癌基因的作用）在某些眼附属器肿瘤的病因学中发挥作用。 然而，与其他人类恶性肿瘤相似，感染因子在眼附属器肿瘤中的最终作用最有可能是作为其他遗传和环境风险因素的辅助因素。 2.视网膜相关黄斑变性（AMD）的分子病理学： 虽然AMD的临床和组织病理学特征已得到很好的表征，但其病因和发病机制仍不清楚。 最近的发现表明免疫过程在AMD发病机制中的作用，包括布鲁赫膜内和视网膜色素上皮（RPE）下的细胞外沉积物（玻璃疣）、用于清除这些沉积物的巨噬细胞（M2）的募集、补体活化、组织破坏性巨噬细胞（M1）的募集、小胶质细胞活化和积累，以及微生物如衣原体引起的慢性炎症的促炎作用。 需要进一步研究以阐明AMD发展的确切机制和时间进程，进而开发用于预防和治疗AMD的新疗法。 3.眼科疾病的新病理和发病机制： 我们报道了与von Hippel-Lindau病（VHL）相关的眼部血管母细胞瘤中簇表达的显著减少。 在VHL蛋白的抑瘤作用中，白蝶呤具有潜在的重要作用，有望成为眼VHL肿瘤的一种新的生物标志物。 我们还发现高水平的血管内皮生长因子在视网膜新生血管作为眼部VHL病变的表现。 我们检测了急性环形外视网膜病变患者血清中抗视网膜自身抗体。 我们首次提供了组织病理学证据，证明氟达拉滨（一种嘌呤类似物）直接诱导视网膜神经节和双极细胞毒性，导致迅速进行性失明。 4.各种眼部疾病的实验模型： 我们证明了Ccl 2-/-/Cx 3cr 1-/-（DKO）小鼠表现出广泛的AMD病理，具有早期发作和高复发率，并且可能暗示某些炎症分子、A2 E和内质网蛋白在AMD发病机制中的作用。 DKO小鼠表现出相似的免疫病理学特征。 先天性和适应性免疫在DKO视网膜变性中起重要作用。 与NEI的Caspi，Gery，Hooks和Nussenblatt博士以及NCI的Restifo博士合作，发现并发表了几种新的眼部炎症和黑色素瘤免疫治疗模式和机制。这些活动在其年度报告中作了进一步说明。

The identity and topographic location of ocular inflammatory, degenerated, and tumor cells and their products in patient specimens and animal samples are analyzed by routine pathology, pathophysiology, immunohistochemistry, and molecular pathology. The application of cutting-edge technology such as microdissection combined with molecular techniques such as PCR, RT-PCR, and RFLP, allows us to provide more accurate pathological diagnosis (assessment) of the disease, and guide us in selecting an appropriate therapy for the patient. We generate and/or apply what we learn from various animal models with ocular disorders, so we can know the mechanisms of different ocular diseases. Using these animal models we can also access the efficacies of different therapeutic agents for various ocular diseases. This helps us to better understand disease pathogenesis and to better select therapies targeting specific diseases. In FY2008, we continued and accomplished the following in our research: 1. Ocular Lymphoma and Adnexal Tumors: We continue to confirm elevation of interleukin-10 in ocular fluids in patients with primary intraocular B-cell lymphoma and IgH gene rearrangements of ocular B-cell lymphoma cells. Chemotherapy regimens with high dose methotrexate are recommended for primary CNS lymphoma. Intraocular chemotherapy has the advantage of limited side effects and has been shown to be useful in primary intraocular lymphoma patients. We found that alterations in the transport of methotrexate across the cellular membrane might contribute to resistance following repeated intravitreal injection. The vast majority of primary intraocular lymphomas are malignant B-cells, while intraocular T-cell lymphomas are rare. We reported a case of metastatic T-cell lymphoma to the eye and demonstrated the utility of immunophenotyping and molecular analysis using microdissection and PCR, as critical adjunctive studies in patients presenting with masquerade syndrome. Vitreoretinal involvement, with no uveal involvement, was seen in this case, similar to many ocular metastatic T-cell lymphomas in the literature. This is intriguing since the uvea, rather than the retina, is the typical ocular tissue invaded in the majority of metastatic tumors, including systemic B-cell lymphoma. We also explore the role of infectious microorganisms (human papilloma virus, HIV, human herpes simplex virus-8, Helicobacter pylori, Chlamydia and hepatitis C virus) in ocular adnexal neoplasms. Multiple organisms may play a role in the etiology of certain ocular adnexal neoplasms by acting through similar mechanisms of oncogenesis, including chronic antigenic stimulation and the action of infectious oncogenes. However, similar to other human malignancies, the ultimate role of infectious agents in ocular adnexal neoplasms is most likely as a cofactor to the other genetic and environmental risk factors. 2. Molecular Pathology of Age-Related Macular Degeneration (AMD): While the clinical and histopathological features of AMD are well characterized, its etiology and pathogenesis remain unclear. Recent discoveries suggest a role for immunologic processes in AMD pathogenesis, including extracellular deposits (drusen) inside the Bruch membrane and beneath the retinal pigment epithelium (RPE), recruitment of macrophages (M2) for clearance of these deposits, complement activation, recruitment of tissue-destructive macrophages (M1), microglial activation and accumulation, and proinflammatory effects of chronic inflammation by microorganisms such as Chlamydia. Further studies are necessary to clarify the precise mechanisms and time course underlying AMD development and, in turn, develop novel therapies for the prevention and treatment of AMD. 3. New Pathology and Pathogenesis of Ocular Diseases: We reported a sizeable decrease of cluster expression in ocular hemangioblastomas associated with von Hippel-Lindau disease (VHL). Clusterin has potential important functions in tumor suppression by VHL protein, and thus may become a novel biomarker in ocular VHL tumors. We also found high vascular endothelial growth factor levels in retinal neovascularization as a manifestation of ocular VHL lesion. We detected serum anti-retinal autoantibodies in acute annular outer retinopathy. We provided the histopathologic evidence, for the first time, that fludarabine (a purine analog) directly induce toxicity to retinal ganglion and bipolar cells causing a rapidly progressive blindness. 4. Experimental Models for Various Ocular Diseases: We proved that Ccl2-/-/Cx3cr1-/- (DKO) mice showed a broad spectrum of AMD pathologies with early onset and high penetrance and possibly imply a role for certain inflammatory molecules, A2E, and endoplasmic reticulum proteins in AMD pathogenesis. The DKO mice present similar immunopathological profiles. Both innate and adaptive immunities play an important role in DKO retinal degeneration. In collaboration with Drs. Caspi, Gery, Hooks, and Nussenblatt of the NEI, and Dr. Restifo of the NCI, several new modes and mechanisms of ocular inflammation and immunotherapy in melanoma have been discovered and published. These are further described in their annual reports.