Sepsis-damaged Organs-double-markers Identification of Organ Failure Using Fluorescent Nanoparticle Tracking Analysis

Background: Sepsis, defined as life-threatening organ dysfunction due to a dysregulated host response to infection, continues to be a source of considerable morbidity and mortality. 19 million cases are seen worldwide each year. Many animal sepsis models had found that sepsis induced multiple organ failure. Ubiquitination, autophagy, apoptosis may involve the process of sepsis related multiple organ failure. Mass spectrometry-based proteomics studies in clinical populations and in rodent and mammalian animal models had started with discovered many novel biomarkers of sepsis. Esoxomes had been found in blood or urine presented the signal of autophagy and apoptosis. Recently, nanoparticle tracking analysis (NTA) was used as a new method for direct and real-time analysis of exosomes. These make it possible to study the exosome biomarkers to analyze septic patients with multiple organ failure.

Aims of the study: This research will be the first study not only to set up macrophage co-cultured with human organ cell models for exosomes secretions but also collect purified exosomes in blood and urine from septic patients. Proteomics studies in exosomes from cell culture and clinical specimens. Analyze ubiquitination, autophage, and apoptosis related biomarkers of exosomes by bioinformatics. Use NTA to set up newly diagnostic methods, to judge the specific organ damage of septic patient by exosomes autophagy-apoptosis biomarkers.

Materials and Methods: In 1st year, LPS (lipopolysaccharide) stimulated macrophage co-cultured with human organ cells will be set up. Exosomes will be isolated and purified from different groups. ScFv (single chain fragments of antibody) will be selected for blocking infection related exosome's transmission. The 2nd year, a total of 60 patients with infection and positive culture results will be included, of whom 30 septic patients had at least one organ failure, others will have only infection. All patients included and classified according to the sepsis-3 criteria. Clinical specimens will be collected from August 2017 to July 2019. Exosome will be isolated and purified. Magnetic beads purification, 2D gel electrophoresis, MALDI-TOF, and bioinformatics will be used to analyze proteomics of exosomes and association of organ-specific markers, autophagy, and apoptosis markers. Western blotting will be done to prove the proteins found by proteomics. Cytokines array in blood also confirm and correlate to autophagy. Finally, we will use nanoparticle tracking analysis with double markers identification to understand the exosome subpopulations of specific organ and autophagy-apoptosis biomarkers.

Possible effect: Systematic establishment of exosome proteomics in co-culture medium and clinical sepsis specimens will be done. In vitro study, ScFv exploring will help to block exsomes uptake as a possible therapeutic method. Ubiquitination-autophagy-apoptosis biomarkers in exosomes will be detected and correlated to specific organ failure in sepsis. The inflammatory process also will be validated by cytokines analysis. NTA double markers identification will be a smart method to understand the exosome subpopulations.

Inclusion criteria

Septic patients will have an "acute change in total SOFA score ≥ 2 points consequent to infection" Sepsis with organ failure Sepsis without organ failure

Exclusion criteria

patients who had chronic respiratory failure with ventilator dependence chronic renal failure with regular hemodialysis