Sepsis-damaged Organs-double-markers Identification of Organ Failure Using Fluorescent Nanoparticle Tracking Analysis
Condition: Sepsis With Multiple Organ Dysfunction (MOD)
MeSH terms: Sepsis
Study type: Observational

Summary: 
      Systematic establishment of exosome proteomics in co-culture medium and clinical sepsis
      specimens will be done. 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.
    

Detailed description: 
      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.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        All included patients will be classified according to the sepsis-3 criteria, also treat
        according to surviving sepsis campaign guidelines. Every enrolled patients will be traced
        the infection sources with positive culture results. Enrolled septic patients will have an
        "acute change in total SOFA score ≥ 2 points consequent to infection"
      
Biospecimen description: 
      blood, urine
    
Keywords: sepsis, nanoparticle tracking analysis