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

由于宿主反应失调，脓毒症可能导致危及生命的器官功能障碍。然而，目前还没有单一的生物标志物能够充分反映潜在败血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，我们希望将重点放在早期的物理变化和细胞效应上，这是复杂宿主反应的总和，而不是在脓毒症的早期阶段观察单一的标记物来表征细菌-宿主相互作用。具体来说，我们计划应用数字全息显微镜（DHM），这是一种基于干涉测量的无染色技术，为活细胞分析提供微创、高分辨率的定量相对比成像，以表征脓毒症背景下淋巴细胞和内皮细胞的物理变化。DHM可以在时间圈调查期间检测快速形态变化，并捕获物理细胞参数，如体积、折射率和干质量，例如指示细胞死亡过程。此外，我们希望使用原子力显微镜（AFM）分析内皮糖萼（eGC）的纳米力学变化，eGC是覆盖在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着关键作用，在全身性炎症和败血症患者中会降解。在目的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因素之间的定性和定量差异，这些反应预示着宿主反应失调的开始和全球净严重程度。在目标2中，我们将把我们的发现转化到人体系统中，研究暴露于微生物学证实的败血症患者保留的血液样本中的供体淋巴细胞和内皮细胞的细胞变化。样本将来自该联盟的EDGE研究（急诊室患者糖萼损伤的早期检测；Clinicaltrial.gov #: NCT03126032）。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，并在我们的急诊室就诊的疑似感染患者中进行分析。我们的目的是识别和验证特异性DHM模式和舌下eGC值，以早期预测即将发生的败血症器官衰竭（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，我们分别使用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调查。

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.