Project description: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.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.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.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.

Project description:The wide array of molecules carried by plasma regulates critical immune functions and constitutes valuable biomarkers and therapeutic targets. In recent years the introduction of “systems approaches” has provided investigators with powerful means for assessing immune responses in patient samples on a global scale. However, while the use of genome-wide profiling technologies has become widespread, measuring the plasma proteome still presents considerable challenges. An alternative approach that consists in measuring transcriptome responses in reporter cells exposed in vitro to patient plasma has been successfully employed in a limited number of studies. Here we devised such a “Transcriptomic Reporter Assay” system to assess the immunogenicity of plasma from septic patients and evaluate its potential for biomarker discovery. Sepsis is a common, severe systemic infectious process for which physicians still lack efficient diagnostic or prognostic tools. Of the three different cell reporter systems tested, neutrophils were identified as the most capable “plasma sensor”. Compared to peripheral blood mononuclear cells and dendritic cell preparations neutrophils were best able to discriminate between plasma from septic and control subjects and responded by upregulating a robust immune transcriptional program. Additionally, the amplitude of the neutrophil transcriptomic response was shown to be associated with disease severity in two additional sets of patients. Overall, our results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for assessing immunopathogenic processes in a complex and severe condition such as sepsis. Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy donors. Plasma samples were obtained from patients with culture-confirmed sepsis (n=12) and from uninfected controls (n=12). PBMCs were cultured for 6 h in medium alone, plasma from patients with sepsis, plasma from uninfected controls, and LPS using a final concentration of 20%. Transcriptional profiles were acquired using Illumina HumanHT12 V4 BeadChips.

Project description:The wide array of molecules carried by plasma regulates critical immune functions and constitutes valuable biomarkers and therapeutic targets. In recent years the introduction of “systems approaches” has provided investigators with powerful means for assessing immune responses in patient samples on a global scale. However, while the use of genome-wide profiling technologies has become widespread, measuring the plasma proteome still presents considerable challenges. An alternative approach that consists in measuring transcriptome responses in reporter cells exposed in vitro to patient plasma has been successfully employed in a limited number of studies. Here we devised such a “Transcriptomic Reporter Assay” system to assess the immunogenicity of plasma from septic patients and evaluate its potential for biomarker discovery. Sepsis is a common, severe systemic infectious process for which physicians still lack efficient diagnostic or prognostic tools. Of the three different cell reporter systems tested, neutrophils were identified as the most capable “plasma sensor”. Compared to peripheral blood mononuclear cells and dendritic cell preparations neutrophils were best able to discriminate between plasma from septic and control subjects and responded by upregulating a robust immune transcriptional program. Additionally, the amplitude of the neutrophil transcriptomic response was shown to be associated with disease severity in two additional sets of patients. Overall, our results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for assessing immunopathogenic processes in a complex and severe condition such as sepsis. Monocytes were isolated from two healthy donors. Monocytes were cultured with interleukin 4 (IL4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate monocyte derived dendritic cells (MoDCs). Plasma samples were obtained from patients with culture-confirmed sepsis (n=12) and from uninfected controls (n=12). MoDCs were cultured for 6 h in medium alone, plasma from patients with sepsis, plasma from uninfected controls, and LPS using a final concentration of 20%. Transcriptional profiles were acquired using Illumina HumanHT12 V4 BeadChips.

Project description:The wide array of molecules carried by plasma regulates critical immune functions and constitutes valuable biomarkers and therapeutic targets. In recent years the introduction of “systems approaches” has provided investigators with powerful means for assessing immune responses in patient samples on a global scale. However, while the use of genome-wide profiling technologies has become widespread, measuring the plasma proteome still presents considerable challenges. An alternative approach that consists in measuring transcriptome responses in reporter cells exposed in vitro to patient plasma has been successfully employed in a limited number of studies. Here we devised such a “Transcriptomic Reporter Assay” system to assess the immunogenicity of plasma from septic patients and evaluate its potential for biomarker discovery. Sepsis is a common, severe systemic infectious process for which physicians still lack efficient diagnostic or prognostic tools. Of the three different cell reporter systems tested, neutrophils were identified as the most capable “plasma sensor”. Compared to peripheral blood mononuclear cells and dendritic cell preparations neutrophils were best able to discriminate between plasma from septic and control subjects and responded by upregulating a robust immune transcriptional program. Additionally, the amplitude of the neutrophil transcriptomic response was shown to be associated with disease severity in two additional sets of patients. Overall, our results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for assessing immunopathogenic processes in a complex and severe condition such as sepsis. Polymorphonuclear neutrophils (PMNs) were isolated from two healthy donors. Plasma samples were obtained from patients with culture-confirmed sepsis (n=12) and from uninfected controls (n=12). PMNs were cultured for 6 hours in medium alone, plasma from patients with sepsis, plasma from uninfected controls, and LPS using a final concentration of 20%. Transcriptional profiles were acquired using Illumina HumanHT12 V4 BeadChips.

Project description:The wide array of molecules carried by plasma regulates critical immune functions and constitutes valuable biomarkers and therapeutic targets. In recent years the introduction of “systems approaches” has provided investigators with powerful means for assessing immune responses in patient samples on a global scale. However, while the use of genome-wide profiling technologies has become widespread, measuring the plasma proteome still presents considerable challenges. An alternative approach that consists in measuring transcriptome responses in reporter cells exposed in vitro to patient plasma has been successfully employed in a limited number of studies. Here we devised such a “Transcriptomic Reporter Assay” system to assess the immunogenicity of plasma from septic patients and evaluate its potential for biomarker discovery. Sepsis is a common, severe systemic infectious process for which physicians still lack efficient diagnostic or prognostic tools. Of the three different cell reporter systems tested, neutrophils were identified as the most capable “plasma sensor”. Compared to peripheral blood mononuclear cells and dendritic cell preparations neutrophils were best able to discriminate between plasma from septic and control subjects and responded by upregulating a robust immune transcriptional program. Additionally, the amplitude of the neutrophil transcriptomic response was shown to be associated with disease severity in two additional sets of patients. Overall, our results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for assessing immunopathogenic processes in a complex and severe condition such as sepsis. Polymorphonuclear neutrophils (PMNs) were isolated a healthy donor. Plasma samples were obtained from a first set of patients with culture-confirmed sepsis (n=29) and from uninfected controls (n=17). PMNs were cultured for 6 h in medium alone, plasma from patients with sepsis, plasma from uninfected controls, and LPS using a final concentration of 20%. Transcriptional profiles were acquired using Illumina HumanHT12 V4 BeadChips.

Project description:The wide array of molecules carried by plasma regulates critical immune functions and constitutes valuable biomarkers and therapeutic targets. In recent years the introduction of âsystems approachesâ has provided investigators with powerful means for assessing immune responses in patient samples on a global scale. However, while the use of genome-wide profiling technologies has become widespread, measuring the plasma proteome still presents considerable challenges. An alternative approach that consists in measuring transcriptome responses in reporter cells exposed in vitro to patient plasma has been successfully employed in a limited number of studies. Here we devised such a âTranscriptomic Reporter Assayâ system to assess the immunogenicity of plasma from septic patients and evaluate its potential for biomarker discovery. Sepsis is a common, severe systemic infectious process for which physicians still lack efficient diagnostic or prognostic tools. Of the three different cell reporter systems tested, neutrophils were identified as the most capable âplasma sensorâ. Compared to peripheral blood mononuclear cells and dendritic cell preparations neutrophils were best able to discriminate between plasma from septic and control subjects and responded by upregulating a robust immune transcriptional program. Additionally, the amplitude of the neutrophil transcriptomic response was shown to be associated with disease severity in two additional sets of patients. Overall, our results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for assessing immunopathogenic processes in a complex and severe condition such as sepsis. Polymorphonuclear neutrophils (PMNs) were isolated a healthy donor. Plasma samples were obtained from a first set of patients with culture-confirmed sepsis (n=35) and from uninfected controls (n=19). PMNs were cultured for 6 h in medium alone, plasma from patients with sepsis, plasma from uninfected controls, and LPS using a final concentration of 20%. Transcriptional profiles were acquired using Illumina HumanHT12 V4 BeadChips.

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A Transcriptomic Reporter Assay Employing Neutrophils to Measure Immunogenic Activity of Septic Patients' Plasma

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