Corneal HSV-1: Immunopathologic Mechanisms of HSK

我们的长期目标是了解关键的免疫病理和分子机制。 潜在的复发HSV-1诱导的角膜瘢痕形成(复发性疱疹间质角膜炎；R-HSK)是一种主要的 发达国家视力丧失的原因。兔单纯疱疹病毒1型眼部模型的改进 模拟人类R-HSK，导致70%的眼睛(而不是2%)的眼睛形成角膜疤痕，允许系统性 首次对临床相关的复发性单纯疱疹病毒性角膜炎进行研究。共聚焦显微镜(CM)的初步研究 潜伏期显示了两种不同类型的丰富的亚临床角膜病变：1)EpiL(上皮病变)：一过性 (~16-24小时)，点状，小(100-200微米直径)，与从 三叉神经节；2)SCF(亚临床病灶)：一过性(~3-7天)CD45+(RA+/RO+)细胞簇(即免疫 细胞)在愈合的EPIL下，与CD45+细胞 被EPIL中的瞬时病毒AGS吸引到该部位，然后由于缺乏持续的病毒抗原而清除。在……里面 无HSK、EpiL和SCF的角膜不重叠。偶尔，SCF似乎会演变为 炎性间质病变导致HSK，在这种情况下，总是可以看到覆盖的EPIL。我们 假设当活跃的SCF和活跃的EPIL重叠时，EPIL中的病毒AGS可以重新刺激 SCF中的高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，即上皮损伤(EpiL)导致了 暂时性亚临床病灶(SCF)，当第二个EPIL发生在现有的 SCF，或者当原始EPIL持续到SCF完全形成之后。它的形成和发展 (自然病史)从急性感染到间质瘢痕形成的EpiL、SCF和HSK将每天进行检查。我们会 同时，向间质内微量注射病毒AGS，试图诱发HSK。 2.确认我们的假设，即SCF由独特的免疫细胞亚群组成 对病毒感染做出反应。CJLAT VS小鼠SCF中免疫细胞和病毒抗原的鉴定与鉴定 WT感染的角膜在SCF前、SCF前和HSK前(Epil+SCF)时间(使用活体CM识别)，由：A)Ex 活体CM；b)原位增殖分析(Ki67单抗染色/体外CM以识别和定量周期 [激活的]免疫细胞)；c)激光显微切割(LMD)，然后进行RNA分析以检测兔的mRNAs 细胞因子和病毒蛋白。GFP、YFP、RFP标记的CJLAT和WT病毒也将被使用。 3.确认我们的假设，即EpiL、SCF的形成以及HSK的形成需要在 从潜伏感染的三叉神经节(TG)返回的重新激活的HSV-1角膜。对HSK和 目标1和2因子将在HSK操作后重新检查：a)抗病毒治疗和切割 感觉神经往返于TG；以及b)原发炎消退后的免疫抑制治疗。 所获得的见解应该有助于减轻这一感染性角膜失明的主要原因。项目主任/首席调查员(最后、第一、中间)：韦克斯勒，史蒂文·刘易斯 项目叙事 本项目旨在了解疱疹病毒性眼病的免疫学和分子生物学。 (HSK)，这是发达国家导致失明的主要原因。

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.