Using nanopocket membranes to capture bacterial outer membrane vesicles from biofluids

项目总结 败血症不仅是美国医院治疗费用最高的疾病，也是主要原因 对死亡的恐惧。当宿主的促炎免疫反应异常时，就会发生脓毒症 由于宿主对感染的反应不正常或异常而升高的。用于诊断的方法 败血症因医院而异，但通常涉及评分系统(如APACHE II和SOFA) 对病人病情的严重程度进行分级。许多改变后的生理参数 通过这些评分系统测量的结果不一定是脓毒症的特异性，这使得它很难 对脓毒症进行早期诊断。患者脓毒症诊断的时机通常预示着 他们的临床结果，强调了更明确的分子诊断测试的必要性。 然而，最近的一项研究发现，在大多数(70.1%)脓毒症病例中，特定的原因 细菌无法确定，可能是由于侵略性的抗生素或局部感染。 细菌外膜囊泡(OMV)是很有吸引力的诊断生物标志物，因为：a) 它们的数量和能力在全身循环，更容易通过组织屏障 细菌本身；B)它们的健壮性--不像它们的细菌细胞“母体”，OMV可以 抵御广谱抗生素的淹没；以及c)它们的独特功能，可以 允许不同细菌物种之间的差异。 这项提议有两个目的：确定细菌OMV是否可能是一个分子 脓毒症的诊断生物标志物，并开发一种从患者血浆中分离它们的快速方法。 我们寻求开发一种简单、高纯度和快速有效的分离技术 从生物体液中分离和提纯OMV，包括血浆。我们将实现修改后的 切向流过滤方法，类似于生物制药用于大容量和高密度过滤的方法。 产量净化，使用一种新的纳米囊膜，有效地捕获和释放 损失最小的细菌型OMV。这些目标的成功将为未来铺平道路 以细菌OMV为基础的一步法、一站式败血症诊断试验的研制 分子生物标记物，以及更复杂的诊断测试，更准确地量化OMV 患者体液中的水平，以帮助指导早期治疗和降低死亡率。这个项目将 由一个跨学科的专家小组领导，以确定大肠杆菌中潜在的生物标志物； 膜、材料和生物分离；以及重症监护内科科学家。

PROJECT SUMMARY Sepsis is not only the most expensive condition treated in US hospitals, but also a leading cause of death. Sepsis occurs when host pro-inflammatory immune responses become abnormally elevated due to a dysregulated or aberrant host-response to infection. Diagnostic methods for sepsis can vary between hospitals, but often involve scoring systems (e.g. APACHE II and SOFA) that grade the severity of illness in patients. Many of the altered physiological parameters measured by these scoring systems are not necessarily specific to sepsis, which makes it difficult to diagnose sepsis in early stages. Timing of a patient’s sepsis diagnosis is often predictive of their clinical outcome, underlining the need for a more definitive molecular diagnostic test. However, a recent study found that in the majority (70.1%) of sepsis cases, a specific causal organism could not be determined, likely due to aggressive antibiotics or localized infections. Bacterial outer membrane vesicles (OMVs) are attractive diagnostic biomarkers because of: A) their abundance and ability to circulate throughout the body and pass tissue barriers more easily than bacteria themselves; B) their robustness - unlike their bacterial cell “parent,” OMVs can withstand the inundation of broad-spectrum antibiotics; and C) their unique features that could allow for differentiation between bacterial species. The objective of this proposal is two-fold: Identify whether bacterial OMVs could be a molecular diagnostic biomarker for sepsis and develop a rapid approach to isolate them from patient plasma. We seek to develop a straightforward high-purity and rapid separation technology that effectively isolates and purifies OMVs from biofluids, including plasma. We will implement a modified tangential flow filtration approach, similar to those used by biopharma for high-capacity and high- yield purification, with a new nanopocket membrane that effectively captures and releases bacterial OMVs with minimal loss. Success of these aims will pave the way for the future development of a one-step, point-of-care diagnostic test for sepsis using bacterial OMVs as the molecular biomarker, as well a more complex diagnostic test that more accurately quantifies OMV levels in the patient’s biofluids to help direct early treatment and reduce mortality. This project will be led by an interdisciplinary team of experts in identifying potential biomarkers in E. coli; membranes, materials, and bioseparations; and a critical care physician scientist.