Corneal HSV-1: Immunopathologic Mechanisms of HSK

我们的长期目标是了解关键的免疫病理学和分子机制 潜在的复发性HSV-1诱导的角膜瘢痕形成（复发性疱疹性角膜基质炎; R-HSK），是一种主要的 发达国家的视力下降原因。改良的HSV-1兔眼模型， 模拟人R-HSK，导致>70%的眼睛（而不是<2%）的角膜瘢痕形成， 首次对临床相关复发HSK进行研究。初步共聚焦显微镜（CM）研究期间 潜伏期显示了2种不同类型的丰富的亚临床角膜病变：1）EpiLs（上皮病变）：一过性 （~16-24小时），点状，小（直径100-200 μ m），与来自 三叉神经节; 2）SCF（亚临床病灶）：CD 45+（RA+/RO+）细胞的短暂（~3-7天）簇（即，免疫 在愈合的EpiL下，基底上皮和前基质中存在CD 45+细胞），这与CD 45+细胞被 被EpiL中的瞬时病毒Ag吸引到该位点，然后由于缺乏持续的病毒Ag而清除。在 无HSK的角膜，EpiLs和SCF没有重叠。偶尔，SCF似乎会演变成 炎性间质病变导致HSK，在这种情况下，总是看到覆盖的EpiL。我们 假设当活性SCF和活性EpiL重叠时，EpiL中的病毒Ag可以重新刺激SCF和EpiL。 SCF中的超免疫细胞导致炎症反应和HSK。我们的具体目标包括： 1.使用体内CM，证实了我们的假设，即上皮病变（EpiLs）导致 瞬时亚临床病灶（SCF），当第二个EpiL发生在现有的部位时，SCF导致HSK SCF，或者当原始EpiL持续到SCF完全形成之后。形成和发展 将每天检查EpiLs、SCF和HSK从急性感染到基质瘢痕形成的自然病程。我们将 并将病毒Ag微量注射到间质中以尝试诱导HSK。 2.证实了我们的假设，即SCF是由独特的免疫细胞亚群组成的 对病毒感染做出反应鉴定和表征CJLAT与CJLAT的SCF中的免疫细胞浸润和病毒Ag 在SCF前、SCF和HSK前（EpiL+SCF）时间的wt感染角膜（使用体内CM鉴定），通过： 体内CM; B）原位增殖分析（Ki 67 mAb染色/离体CM以识别和定量循环 c）激光显微解剖（LMD），然后进行RNA分析以检测兔的mRNA; 细胞因子和病毒蛋白。也将采用GFP、YFP、RFP标记的CJLAT和wt病毒。 3.证实了我们的假设，即形成EpiLs，SCF，因此，HSK需要复制在 从潜伏感染的三叉神经节（TG）返回的再活化HSV-1的角膜。HSK的变化和 因素目标1和2将在HSK的操作后重新检查：a）抗病毒治疗和切割 感觉神经至/自TG;和B）原发感染消退后的免疫抑制治疗。 所获得的见解应该有助于减轻感染性角膜失明的主要原因。项目负责人/主要研究者（最后，第一，中间）：Wechsler，Steven刘易斯 项目叙述 本项目旨在了解疱疹性眼病的免疫学和分子生物学 (HSK)这是发达国家失明的主要原因。

Our long term objective is to understand the critical immunopathologic and molecular mechanisms underlying recurrent HSV-1 induced corneal scarring (recurrent herpetic stromal keratitis; R-HSK), a major cause of vision loss in developed countries. Modification of an ocular HSV-1 rabbit model, which closely mimics human R-HSK, resulted in corneal scarring in >70% of eyes (instead of <2%), allowing systematic study of clinically relevant recurrent-HSK for the first time. Preliminary confocal microscopy (CM) studies during latency revealed 2 distinct types of abundant subclinical corneal lesions: 1)EpiLs (epithelial lesions): Transient (~16-24 h), punctate, small (100-200 um diameter), and consistent with replication of reactivated virus from the trigeminal ganglia; 2)SCF (sub-clinical foci): Transient (~3-7 days) clusters of CD45+(RA+/RO+) cells (i.e., immune cells) in the basal epithelium and anterior stroma under a healed EpiL, consistent with the CD45+ cells being attracted to the site by transient viral Ags in the EpiL and then clearing due to lack of continued viral Ag. In corneas without HSK, EpiLs and SCF did not overlap. Occasionally a SCF appeared to evolve into an inflammatory stromal lesion leading to HSK, in which case an overlying EpiL was always seen. We hypothesize that when an active SCF and an active EpiL overlap, viral Ags in the EpiL can restimulate the hyperimmune cells in the SCF resulting in an inflammatory response and HSK. Our Specific Aims include: 1. Using in vivo CM, confirm our hypothesis that epithelial lesions (EpiLs) lead to the formation of transient sub-clinical foci (SCF) and that SCF lead to HSK when a second EpiL occurs at the site of an existing SCF, or when the original EpiL persists until after the SCF is fully formed. The formation and progression (natural history) of EpiLs, SCF, and HSK from acute infection to stromal scarring will be examined daily. We will also microinject viral Ags into the stroma to try to induce HSK. 2. Confirm our hypothesis that SCF are composed of unique subpopulations of primed immune cells responding to viral infection. Identify and characterize immune cell infiltrates and viral Ag in SCF of CJLAT vs wt infected corneas at pre-SCF, SCF, and pre-HSK (EpiL+SCF) times (identified using in vivo CM), by: a) Ex vivo CM; b)In situ proliferation analyses (Ki67 mAb staining/ex vivo CM to identify and quantitate cycling [activated] immune cells); c)Laser MicroDissection (LMD) followed by RNA analysis to detect mRNAs for rabbit cytokines and viral proteins. GFP, YFP, RFP tagged CJLAT and wt viruses will also be employed. 3. Confirm our hypothesis that formation of EpiLs, SCF, and hence, HSK requires replication in the cornea of reactivated HSV-1 returning from latently infected trigeminal ganglia (TG). Changes to HSK and factors Aims 1 and 2 will be re-examined following manipulations of HSK by: a) Antiviral therapy and cutting sensory nerves to/from TG; and b) immuno-suppressive therapy after resolution of the primary infection. The insights obtained should help alleviate this leading cause of infectious corneal blindness. Program Director/Principal Investigator (Last, First, Middle): Wechsler, Steven Lewis Project Narrative This project is aimed at understanding the immunology and molecular biology of herpetic eye disease (HSK), a leading cause of blindness in developed countries.