Characterization of the interaction of bacterial determinants and host cells during systemic inflammation for sepsis prediction

全身炎症过程中细菌决定簇与宿主细胞相互作用的表征，用于脓毒症预测

• 批准号: 427775125
• 负责人: Professorin Dr. Stefanie Kampmeier
• 金额: --
• 依托单位: Institut für Hygiene
• 依托单位国家: 德国
• 项目类别: Clinical Research Units
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/427775125?language=en
• 关键词: Characterization; interaction; bacterial; determinants; host

Sepsis may result in life-threatening organ dysfunction due to a dysregulated host response. However, no single biomarker has turned out to adequately reflect the rapidly evolving nature of a potentially septic patient’s status and to predict subsequent organ dysfunction. Hence, in this project, we want to focus on early physical changes and cellular effects, which are the sum of the complex host reactions rather than to look at singular markers to characterize bacteria-host interactions in the early stages of sepsis. Specifically, we plan to apply digital holographic microscopy (DHM), which is an interferometry-based stain-free technique that provides minimally-invasive, high resolution quantitative phase contrast imaging for live cell analysis, to characterize physical changes of lymphocytes and endothelial cells in the context of sepsis. DHM can detect fast morphological changes during time-laps investigations and captures physical cell parameters like volume, the refractive index and dry mass, indicating for example cell death processes. Furthermore, we want to use atomic force microscopy (AFM) to analyze nanomechanical changes of the endothelial glycocalyx (eGC), an up to 3 μm thick gel-like layer coating the luminal surface of the entire vascular endothelium. The eGC plays a pivotal role in the maintenance of microcirculatory homeostasis and is degraded in patients with systemic inflammation and sepsis. In aim 1 we want to use DHM andAFM to characterize host cell reaction in vitro with focus on morphological/physical changes of lymphocytes and damage of the eGC. We hypothesize that host cells react depending on qualitative and quantitative differences among the bacterial determinants and that these reactions herald the onset and global net severity of a dysregulated host response. In aim 2, we will translate our findings to the human system and investigate cellular changes of donor lymphocytes and endothelial cells exposed to retain blood samples from patients with microbiologically confirmed sepsis. Samples will be derived from the EDGE study (Early Detection of Glycocalyx Damage in Emergency Room Patients; Clinicaltrial.gov #: NCT03126032) within this consortium. Furthermore, we will prospectively measure sublingual eGC dimensions using intravital microscopy (GlycoCheckTM) and isolate lymphocytes for DHM analysis and in patients presenting to our ER with suspected infection. Our aim is to identify and validate specific DHM patterns and sublingual eGC values for early prediction of impending septic organ-failure (especially kidney and lung) and clinically relevant endpoints. Finally, we will validate our findings in a second, prospective study, in which patients with a suspected infection requiring hospitalization are investigated for eGC-changes and morphological alterations in lymphocytes using GlycoCheckTM and DHM, respectively.

由于宿主反应失调，败血症可能会导致危及生命的器官功能障碍。然而，没有一个单一的生物标志物已被证明能够充分反映潜在脓毒症患者状态的快速演变性质，并预测随后的器官功能障碍。因此，在这个项目中，我们希望专注于早期的物理变化和细胞效应，这是复杂的宿主反应的总和，而不是着眼于单一的标志物来表征脓毒症早期阶段的细菌-宿主相互作用。具体而言，我们计划应用数字全息显微镜（DHM），这是一种基于干涉的无染色技术，可为活细胞分析提供微创，高分辨率的定量相衬成像，以表征脓毒症背景下淋巴细胞和内皮细胞的物理变化。DHM可以在时间重叠调查期间检测快速形态学变化，并捕获物理细胞参数，如体积，折射率和干质量，指示例如细胞死亡过程。此外，我们希望使用原子力显微镜（AFM）来分析内皮糖萼（eGC）的纳米力学变化，eGC是覆盖整个血管内皮管腔表面的厚达3 μm的凝胶状层。eGC在维持微循环稳态中起关键作用，并且在全身性炎症和脓毒症患者中降解。在目标1中，我们希望使用DHM和AFM来表征体外宿主细胞反应，重点关注淋巴细胞的形态/物理变化和eGC的损伤。我们假设宿主细胞的反应取决于细菌决定簇之间的定性和定量差异，并且这些反应预示着失调的宿主反应的发作和全球净严重程度。在目标2中，我们将把我们的发现转化到人体系统中，并研究暴露于微生物学证实的脓毒症患者保留血液样本的供体淋巴细胞和内皮细胞的细胞变化。样本将来自该联盟内的EDGE研究（急诊室患者糖萼损伤的早期检测; Clinicaltrial.gov #：NCT 03126032）。此外，我们将使用活体显微镜（GlycoCheckTM）前瞻性地测量舌下eGC尺寸，并分离淋巴细胞用于DHM分析，以及在疑似感染的患者中进行我们的ER。我们的目的是识别和验证特定的DHM模式和舌下eGC值，用于早期预测即将发生的脓毒性器官衰竭（特别是肾脏和肺）和临床相关终点。最后，我们将在第二项前瞻性研究中验证我们的发现，在该研究中，分别使用GlycoCheckTM和DHM对疑似感染需要住院治疗的患者进行eGC变化和淋巴细胞形态学改变的研究。