title: A transcriptomic reporter assay employing neutrophils to measure immunogenic activity of septic patients' plasma.
abstract: BACKGROUND
  There are diverse molecules present in blood plasma that regulate immune functions and also present a potential source of disease biomarkers and therapeutic targets. Genome-wide profiling has become a powerful method for assessing immune responses on a systems scale, but technologies that can measure the plasma proteome still face considerable challenges. An alternative approach to direct proteome assessment is to measure transcriptome responses in reporter cells exposed in vitro to plasma. In this report we describe such a "transcriptomic reporter assay" to assess plasma from patients with sepsis, which is a common and severe systemic infectious process for which physicians lack efficient diagnostic or prognostic markers.
  
  
  METHODS
  Plasma samples collected from patients with culture-confirmed bacterial sepsis and uninfected healthy controls were used to stimulate three separate cell types - neutrophils, peripheral blood mononuclear cells, and monocyte-derived dendritic cells. Whole genome microarrays were generated from stimulated cells to assess transcriptional responses. Unsupervised analysis and enriched functional networks were evaluated for each cell type. Principal component analyses were used to assess variability in responses. A random K-nearest neighbor - feature selection algorithm was used to identify markers predictive of sepsis severity, which were then validated in an independent data set.
  
  
  RESULTS
  Neutrophils demonstrated the most distinct response to plasma from septic patients with 709 genes showing altered expression profiles, many of which are involved in established immunologic pathways. The amplitude of the neutrophil transcriptomic response was shown to be correlated with sepsis severity in two independent sets of patients comprised of 64 total septic patients. A subset of 30 transcripts selected using one set of patients was demonstrated to have a high degree of accuracy (82-90%) in predicting sepsis severity and outcomes in the other independent set. This subset included several genes previously established in sepsis pathogenesis as well as novel genes.
  
  
  CONCLUSIONS
  These results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for studying plasma, in this case from septic patients. The distinctive transcriptional signature we found could potentially help predict severity of disease and guide treatment. Our findings also shed new light on mechanisms of immune dysregulation in sepsis.
authors.forename: Prasong
authors.initials: P
authors.lastname: Khaenam
authors.forename: Darawan
authors.initials: D
authors.lastname: Rinchai
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Altman
authors.forename: Laurent
authors.initials: L
authors.lastname: Chiche
authors.forename: Surachat
authors.initials: S
authors.lastname: Buddhisa
authors.forename: Chidchamai
authors.initials: C
authors.lastname: Kewcharoenwong
authors.forename: Duangchan
authors.initials: D
authors.lastname: Suwannasaen
authors.forename: Michael
authors.initials: M
authors.lastname: Mason
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Whalen
authors.forename: Scott
authors.initials: S
authors.lastname: Presnell
authors.forename: Wattanachai
authors.initials: W
authors.lastname: Susaengrat
authors.forename: Kimberly
authors.initials: K
authors.lastname: O'Brien
authors.forename: Quynh-Ahn
authors.initials: QA
authors.lastname: Nguyen
authors.forename: Vivian
authors.initials: V
authors.lastname: Gersuk
authors.forename: Peter S
authors.initials: PS
authors.lastname: Linsley
authors.affiliation: Systems Immunology Division, Benaroya Research Institute, 1201 Ninth Avenue, Seattle, WA 98101, USA. ganja_le@kku.ac.th.
authors.forename: Ganjana
authors.initials: G
authors.lastname: Lertmemongkolchai
authors.forename: Damien
authors.initials: D
authors.lastname: Chaussabel
mesh_terms: D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D017930:Genes, Reporter; D056726:Genetic Association Studies; D006801:Humans; D058977:Molecular Sequence Annotation; D009504:Neutrophils / Q000378:metabolism*; D025341:Principal Component Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012372:ROC Curve; D018401:Sample Size; D018805:Sepsis / Q000097:blood* / Q000235:genetics / Q000276:immunology*; D049490:Systems Biology; D014158:Transcription, Genetic; D059467:Transcriptome / Q000235:genetics*
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger