Investigating mechanisms of epidermal cell death in Toxic Epidermal Necrolysis

项目摘要 Stevens约翰逊综合征/中毒性表皮坏死松解症（SJS/TEN）是一种重度药物超敏反应 表现为皮肤、眼睛、口腔、喉咙和生殖器的脱落，沿着全身炎症和内部炎症 器官参与。疾病可能发生在其他健康的人身上，并且在很大程度上是不可预测的。死亡率为20- 30%，没有已知的治疗方法。SJS/TEN是美国医疗保健系统的重大负担， 估计每年1.29亿美元。这是至关重要的阐明病理生物学的 SJS/TEN改善疾病预防、诊断和治疗。尽管有这种明确的需要，SJS/TEN是 研究严重不足。这一领域缺乏高质量的研究源于两个主要障碍： 缺乏可用于分析的患者样本，缺乏易于获得和可重现的模型 系统进行机械研究。我们对SJS/TEN发病机制的认识中的一个主要空白是 免疫细胞介导角质形成细胞死亡的假定机制。在很大程度上认为， 细胞毒性CD 8 + T细胞产生的颗粒诱导角质形成细胞凋亡。然而，矛盾的发现呼吁 这个假设受到质疑。这个提议的核心假设是炎症细胞死亡 途径坏死性凋亡和焦亡，而不是细胞凋亡，介导角质形成细胞死亡和表皮 在SJS/TEN中销毁。拟议的研究将直接测试这一假设，同时克服目前的障碍 在外地目的1采用两种新技术研究福尔马林固定石蜡包埋的大型银行 迄今为止研究用途有限的临床标本。临床标本是从一个精心制作的 开发并彻底审查了跨越三家主要三级保健医院的患者数据库。这些 技术是（i）OpalTM多光谱免疫荧光染色与MantraTM定量病理学 成像系统和inFORM®图像分析软件以及（ii）NanoString NCounter®基因分析平台 表达谱分析目的2通过与烧伤小组合作， 两个主要中心前瞻性地收集水疱液和受影响的表皮，这是一个独特的样本资源。分析 利用RNAseq、高参数流式细胞术和多重免疫测定，与传统实验室协同工作， 技术.目的3使用患者水疱液产生SJS/TEN的新型离体模型， 可用的3D全厚度组织工程人类皮肤，将用于研究提出的假设 同时探索潜在的新疗法。通过这项创新研究产生的数据 这对促进对基本疾病病理生物学和临床护理的理解具有重大潜力。

Project Summary Stevens Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is a severe drug hypersensitivity reaction manifesting as sloughing of skin, eyes, mouth, throat and genitals, along with systemic inflammation and internal organ involvement. Disease can occur in otherwise healthy people and is largely unpredictable. Mortality is 20- 30% and there is no known treatment. SJS/TEN is a substantial burden on the US healthcare system, costing an estimated 129 million dollars per year. It is of paramount importance to elucidate the pathobiology of SJS/TEN to improve disease prevention, diagnosis and treatment. Despite this clear need, SJS/TEN is significantly under-researched. The dearth of high-quality research in this field stems from two major barriers: a lack of patient samples available for analysis and the absence of an easily attainable and reproducible model system to perform mechanistic studies. One major gap in our knowledge of the pathogenesis of SJS/TEN is the putative mechanism(s) by which immune cells mediate keratinocyte death. It is largely assumed that cytolytic granules generated by cytotoxic CD8+ T cells induce keratinocyte apoptosis. However, contradictory findings call this assumption into question. The central hypothesis of this proposal is that the inflammatory cell death pathways necroptosis and pyroptosis, rather than apoptosis, mediate keratinocyte death and epidermal destruction in SJS/TEN. The proposed study will directly test this hypothesis while overcoming current barriers in the field. Aim 1 employs two novel technologies to study a large bank of formalin-fixed paraffin embedded clinical specimens that until now were of limited research utility. Clinical specimens are drawn from a meticulously developed and thoroughly vetted patient database that spans three major tertiary care hospitals. These technologies are (i) OpalTM multispectral immunofluorescence staining with MantraTM Quantitative Pathology Imaging System and inFORM® Image Analysis Software and (ii) NanoString NCounter® platform for gene expression profiling. Aim 2 interrogates mechanism(s) of keratinocyte death by partnering with burn teams at two major centers to prospectively collect blister fluid and affected epidermis, a unique sample resource. Analysis utilizes RNAseq, high parameter flow cytometry, and multiplex immunoassay in concert with traditional laboratory techniques. Aim 3 generates a novel ex vivo model of SJS/TEN using patient blister fluid with commercially available 3D full thickness tissue engineered human skin that will be used to investigate the proposed hypothesis while also exploring potential novel treatments. The data generated through this innovative research has significant potential to advance understanding of fundamental disease pathobiology and clinical care.