Proteomic Analysis of Implant Surfaces in Athroplasty Failure

项目总结 我们的团队已经开发出一种策略，通过使用以下方法对移除的骨科植入物表面的材料进行采样 超声化，并表明衍生的生物膜采样“超声液”具有最高的产量的任何样本。 可用于微生物检测类型，并可进一步用于评估植入物表面的宿主反应。而当 对宿主反应的评估已经在临床上被用来区分感染和未感染的骨科 到目前为止，植入、基于宿主的方法都是简单的，检测α-防御素或剖析 例如，中性粒细胞数量，没有定义感染的关节成形术的微生物学或潜在的 非感染性人工关节置换失败的原因分析。我们假设，除了区分传染性和 非感染性关节置换术失败，使用蛋白质组学捕捉声化液中的宿主反应 方法将识别假体周围关节感染的微生物病因和非关节感染的个体原因 感染性关节置换术失败。鉴于假体周围关节感染中的致病微生物的检测和 定义非感染性关节置换术失败的个别原因，需要直接治疗，而不是 用现有的诊断方法普遍实现，这将在临床上有用。 我们首次提出了对声化液进行全局性的、无偏倚的蛋白质组学分析，以评估体内的人类免疫。 对感染性与非感染性关节置换术失败的反应及其子集。我们进一步建议 假体周围关节感染声化液中微生物蛋白的无偏倚全球分析。声化液 已在初步工作中被证明适用于拟议的蛋白质组分析。超声化液来自 失败的全膝关节置换术将使用新的高效液相色谱串联技术进行研究 用质谱法确定宿主蛋白质组分析是否能区分金黄色葡萄球菌， 表皮葡萄球菌、鲁顿葡萄球菌、粪肠球菌和无乳链球菌 假体周围关节感染和非感染性关节置换术失败的无菌性松动，假体周围 骨折、不稳定和骨溶解/不良组织反应。除了质谱学之外，还有一种互补的 下一代基于测序的邻近延伸分析方法，测量3,072个水平 细胞因子和其他宿主蛋白将被用来测量低丰度的人类蛋白遗漏使用 仅用质谱仪，就可以更详细地检查宿主的反应。最后，微生物 将对假体周围关节感染患者的超声液进行蛋白质组学分析，以确定 微生物蛋白质组学分析能否区分金黄色葡萄球菌、表皮葡萄球菌、卢顿葡萄球菌、粪肠球菌和 无乳链球菌感染。

PROJECT SUMMARY Our team has developed a strategy that samples materials on surfaces of removed orthopedic implants using sonication and shown that the derived biofilm-sampling “sonicate fluids” have the highest yield of any specimen type for microbial detection and can furthermore be used to assess host response at implant surfaces. While assessment of host response has been clinically leveraged to differentiate infected from non-infected orthopedic implants, host-based approaches deployed to date have been simplistic, detecting alpha-defensin or profiling neutrophil numbers, for example, with none defining the microbiology of infected arthroplasties or underlying etiologies of non-infectious arthroplasty failure. We hypothesize that, beyond differentiating infectious from non-infectious arthroplasty failure, capturing host response in sonicate fluid using a proteomic approach will discern microbial etiologies of periprosthetic joint infection and individual causes of non- infectious arthroplasty failure. Given that detection of causative organisms in periprosthetic joint infection and definition individual causes of non-infectious arthroplasty failure, needed to direct management, are not universally realized with extant diagnostic approaches, this will be clinically useful. We propose the first-ever global, unbiased proteomic analysis of sonicate fluid, assessing in vivo human immune response to infectious compared to non-infectious arthroplasty failure and subsets thereof. We further propose an unbiased global analysis of microbial proteins in sonicate fluid in periprosthetic joint infection. Sonicate fluid has been shown in preliminary work to be suitable for the proposed proteomic analyses. Sonicate fluid from failed total knee arthroplasties will be studied using the new technology of liquid chromatography tandem mass spectrometry to determine whether host proteomic analysis can differentiate Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Enterococcus faecalis and Streptococcus agalactiae periprosthetic joint infection, and non-infectious arthroplasty failure subsets of aseptic loosening, periprosthetic fracture, instability, and osteolysis/adverse tissue reaction. In addition to mass spectrometry, a complementary next generation sequencing-based proximity extension assay approach that measures levels of 3,072 cytokines and other host proteins will be used to measure low abundance human proteins missed using mass spectrometry alone, allowing examination of host response in even greater detail. Finally, microbial proteomic analysis of sonicate fluid from patients with periprosthetic joint infection will be performed, to determine whether microbial proteomic analysis can differentiate S. aureus, S. epidermidis, S. lugdunensis, E. faecalis and S. agalactiae infections.