Unknown,Transcriptomics,Genomics,Proteomics

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A Transcriptomic Reporter Assay Employing Neutrophils to Measure Immunogenic Activity of Septic Patients' Plasma (Expt. 3)

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Damien Chaussabel

PROVIDER: E-GEOD-49757 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Melioidosis, caused by Gram negative bacteria Burkholderia pseudomallei, is a major type of community-acquired septicemia in Southeast Asia and Northern Australia with high mortality and morbidity rate. More accurate and rapid diagnosis is needed for improving the management of septicemic melioidosis. We previously identified 37-gene candidate signature to distinguish septicemic melioidosis from sepsis due to other pathogens. The aims of this current study were to independently validate our previous biomarker and consolidate gene selection from each of our microarray data set for establishing a targeted assay for the differential diagnosis of melioidosis. Blood samples were collected from patients who presented with severe inflammatory response syndromes from 3 provincial hospitals in Northeast of Thailand during September 2009 and November 2011. Only culture-confirmed sepsis were included in the study (n=166). We generated a new microarray dataset comprising of 29 patients with septicemic melioidosis and 54 patients with sepsis due to other pathogens. Validation of the 37-gene signature using this new dataset demonstrated the prediction accuracy of approximately 80% for detecting type of sepsis. In order to develop a nanoliter-scale high throughput PCR technology, we further identified additional gene signature from this new microarray dataset and by revisiting our published data. Altogether 85 genes including 6 housekeeping genes were selected. Using multi-steps iteration approach we could reduce the number of biomarkers to 12 genes while the performance is comparable to that of the full panel. The high performance (accuracy >70%) of this 12-gene signature could be validated in a second independent set of samples. The 12-gene panel identified by our study provides high performance for the differential diagnosis of septicemic melioidosis. This finding will be useful for improving the management of septicemic melioidosis in term of diagnosis, treatment and follow up. Total RNA from whole blood obtained from patients with sepsis caused by B.pseudomallei (n=29) or other pathogens (n=54) and uninfected controls (28 healthy and 27 subjects with type 2 diabetes mellitus) were collected. In order to validate the published signature, microarray data were generated from these samples. This dataset was also used for an independent selection of signature for septicemic melioidosis. The same RNA samples were used for validation by a high throughput real-time PCR technique, Fluidigm.

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A Transcriptomic Reporter Assay Employing Neutrophils to Measure Immunogenic Activity of Septic Patients' Plasma (Expt. 3)

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