Corneal HSV-1: Immunopathologic Mechanisms of HSK

角膜 HSV-1：HSK 的免疫病理学机制

• 批准号: 7755352
• 负责人: STEVEN L WECHSLER
• 金额: $ 37.87万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7755352
• 关键词: Acute; Address; Anterior; Antiviral Therapy; Apoptosis; Biological Assay; Blindness; CD8B1 gene; Caliber; Cells; Cicatrix; Clinical; Confocal Microscopy; Cornea; Data; Developed Countries; Epithelial; Epithelium; Eye; Eye diseases; Fluorescein; Fluoresceins; Fluorescence; Grant; Healed; Herpesvirus 1; Human; Image; Immune; Immune response; Immunity; Immunology; In Situ; Individual; Infection; Inflammatory; Inflammatory Response; Keratitis; Label; Lasers; Lead; Lesion; Measures; Microdissection; Modeling; Modification; Molecular; Molecular Biology; Natural History; Neurons; Oryctolagus cuniculus; PTPRC gene; Principal Investigator; Proteins; Published Comment; RNA analysis; Recombinants; Recurrence; Resolution; Simplexvirus; Site; Staining method; Stains; Structure; Structure of trigeminal ganglion; Suggestion; T-Lymphocyte; Time; Viral; Viral Proteins; Virus; Virus Diseases; afferent nerve; cell type; clinically relevant; corneal scar; cytokine; healing; in vivo; insight; programs; protein function; reactivated HSV-1; recombinant virus; research study; response; Acute; Address; Anterior; Antiviral Therapy; Apoptosis; Biological Assay; Blindness; CD8B1 gene; Caliber; Cells; Cicatrix; Clinical; Confocal Microscopy; Cornea; Data; Developed Countries; Epithelial; Epithelium; Eye; Eye diseases; Fluorescein; Fluoresceins; Fluorescence; Grant; Healed; Herpesvirus 1; Human; Image; Immune; Immune response; Immunity; Immunology; In Situ; Individual; Infection; Inflammatory; Inflammatory Response; Keratitis; Label; Lasers; Lead; Lesion; Measures; Microdissection; Modeling; Modification; Molecular; Molecular Biology; Natural History; Neurons; Oryctolagus cuniculus; PTPRC gene; Principal Investigator; Proteins; Published Comment; RNA analysis; Recombinants; Recurrence; Resolution; Simplexvirus; Site; Staining method; Stains; Structure; Structure of trigeminal ganglion; Suggestion; T-Lymphocyte; Time; Viral; Viral Proteins; Virus; Virus Diseases; afferent nerve; cell type; clinically relevant; corneal scar; cytokine; healing; in vivo; insight; programs; protein function; reactivated HSV-1; recombinant virus; research study; response

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.

我们的长期目标是了解关键的免疫病理和分子机制 潜在的复发性HSV-1引起的角膜疤痕（复发性疱疹性基质角膜炎； R-HSK），这是一个主要的 发达国家的视力丧失原因。 Ocular HSV-1兔模型的修改，该模型紧密 模拟人类R-HSK，导致角膜疤痕> 70％的眼睛（而不是<2％），允许系统 首次研究临床相关的复发性-HSK。初步共聚焦显微镜（CM）研究 潜伏期揭示了2种不同类型的丰富亚临床角膜病变：1）Epils（上皮病变）：瞬态 （〜16-24 h），点状，小（直径100-200 um），并且与重新激活病毒的复制一致 三叉神经节； 2）SCF（亚链型焦点）：CD45+（RA+/RO+）细胞的瞬态（〜3-7天）簇（即免疫 细胞）基底上皮和前基质中的细胞，与CD45+细胞一致 由于缺乏持续的病毒Ag而被静态病毒AG吸引到该部位，然后清除。在 没有HSK，EPIL和SCF的角膜没有重叠。有时SCF似乎演变成一个 炎症性基质病变导致HSK，在这种情况下，总是会看到上覆的Epil。我们 假设当活动的SCF和主动EPIL重叠时，Epil中的病毒AG可以重新刺激 SCF中的高免疫细胞导致炎症反应和HSK。我们的具体目的包括： 1。使用体内CM，证实我们的假设，即上皮病变（EPILS）导致形成 当第二个EPIL发生在现有的位置时 SCF，或者原始EPIL持续存在直到SCF充分形成后。形成和进展 （自然史）将每天检查从急性感染到基质疤痕的Epils，SCF和HSK的（每天检查）。我们将 还会对基质中的病毒AG进行微型病毒AG，以试图诱导HSK。 2。证实我们的假设，即SCF由引发免疫细胞的独特亚群组成 反应病毒感染。识别和表征CJLAT与SCF中的免疫细胞浸润和病毒Ag WT在pre-SCF，SCF和Pre-HSK（Epil+SCF）时感染了WT的角膜时间（使用体内CM鉴定），通过：a）EX Vivo CM; b）原位增殖分析（Ki67 mAb染色/离体CM以识别和定量循环 [激活]免疫细胞）； C）激光显微解剖（LMD），然后进行RNA分析以检测mRNA的兔子 细胞因子和病毒蛋白。还将使用GFP，YFP，RFP标记的CJLAT和WT病毒。 3。确认我们的假设是，epils，scf和因此，HSK需要在 重新激活的HSV-1的角膜从潜在感染的三叉神经节（TG）返回。更改HSK和 在操纵HSK之后，将重新检查目标1和2的因素：a）抗病毒疗法和切割 感官神经往返TG； b）分辨原发性感染后的免疫抑制治疗。 获得的见解应有助于减轻传染性角膜失明的主要原因。计划主任/首席研究员（最后，第一，中间）：韦克斯勒，史蒂文·刘易斯 项目叙述 该项目旨在了解疱疹眼病的免疫学和分子生物学 （HSK），发达国家失明的主要原因。