Using nanopocket membranes to capture bacterial outer membrane vesicles from biofluids

项目摘要 败血症不仅是美国医院治疗费用最高的疾病， 死亡之当宿主的促炎免疫反应变得异常时， 由于宿主对感染的反应失调或异常而升高。诊断方法 脓毒症在不同的医院可能有所不同，但通常涉及评分系统（例如APACHE II和SOFA） 对病人的病情进行分级。许多改变的生理参数 通过这些评分系统测量的不一定是脓毒症特有的，这使得很难 早期诊断败血症患者脓毒症诊断的时间通常可以预测 他们的临床结果，强调需要一个更明确的分子诊断测试。 然而，最近的一项研究发现，在大多数（70.1%）脓毒症病例中， 无法确定微生物，可能是由于侵袭性抗生素或局部感染。 细菌外膜囊泡（OMV）是有吸引力的诊断生物标志物，因为： 它们的丰富性和在整个身体中循环的能力以及更容易通过组织屏障 B）它们的鲁棒性-与它们的细菌细胞“亲本”不同，OMV可以 能够抵抗广谱抗生素的泛滥;以及C）它们的独特功能， 允许细菌物种之间的区分。 这项提议的目的是双重的：确定细菌OMV是否可能是一种分子， 因此，本发明提供了用于脓毒症的诊断生物标志物，并开发了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的高纯度和快速分离技术， 从包括血浆在内的生物流体中分离和纯化OMV。我们将实施一项修改后的 切向流过滤方法，类似于生物制药公司用于高容量和高 产量纯化，采用新的纳米口袋膜，有效地捕获和释放 细菌OMV，损失最小。这些目标的成功将为未来铺平道路 使用细菌OMV作为败血症的一步式即时诊断试验的开发 分子生物标志物，以及更复杂的诊断测试，更准确地量化OMV 患者的生物液体中的水平，以帮助指导早期治疗和降低死亡率。该项目将 由一个跨学科的专家小组领导，在E.大肠杆菌; 膜，材料和生物分离;和重症监护医生科学家。

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.