Michael W Pfaffl
Division of Animal Physiology & Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Germany
Extracellular vesicles (EVs) circulate in body liquids and are involved in intercellular communication. They have important regulative functions in almost any physiological or pathological process. In recent time especially exosomes have gained huge scientific interest because of their molecular diagnostic potential, mainly based on the contained microRNA pattern. The past decade has brought about the development and commercialization of a multitude of extraction methods to isolate EVs and exosomes, primarily from blood compartments. Exosome purity and which subpopulations of EVs are captured strongly depend on the applied isolation method, which in turn determines how suitable resulting samples are for potential downstream applications and biomarker discovery. Herein we compared the overall performance of various optimized isolation principles for serum EVs/exosomes in healthy individuals and critically ill patients suffering mainly from pneumonia and sepsis. The isolation methods were benchmarked regarding their suitability for biomarker discovery as well as biological characteristics of captured vesicles, according to the latest MISEV 2018 guidelines. To analyze the small-RNA deep sequencing results, a self-established bioinformatics pipeline for microRNA (based on R) and a deeper analysis of their isoforms (via isomiRROR) was applied. Final goal was the development of a microRNA/isomiR biomarker signature for an early diagnosis and a valid classification of critical ill patients. Various patient cohorts were investigated: healthy volunteers, sepsis (referred to mild or severe pneumonia), acute pulmonary failure (ARDS) and septic shock. Distinct miRNA signatures were identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS or sepsis shock. The study results indicate that EV miRNA biomarkers have future potential for early diagnosis of pneumonia and to indicate disease progression towards severe inflammation events. Further the methodological findings provide guidance for navigating the multitude of EV/exosome isolation methods available, and help researchers and clinicians in the field of molecular diagnostics to make the right choice about the EV/exosome isolation strategy.
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