Project description:Background: Community-acquired pneumonia (CAP) is defined as an acute lung infection involving the alveoli that occurs in a patient without recent health care exposure. A complication of CAP is severe sepsis, a syndrome of infection often accompanied by systemic inflammation and organ dysfunction. The aim of this study was to evaluate mRNA and miRNA in whole blood and to perform an integrative analysis to assess cellular signals that play a role in the pathogenesis of patients with CAP-associated sepsis. Methods: This was a prospective, observational, single-center study of patients transported to the Department of Traumatology and Acute Critical Medicine, Graduate School of Medicine, Osaka University. Patients with CAP-associated sepsis were analyzed. The diagnosis of pneumonia was made according to the clinical findings, including blood samples and chest computed tomography scan, and the diagnosis of sepsis followed the Sepsis-3 guidelines. Results: We included 14 critically ill patients with CAP-associated sepsis and 15 healthy control subjects (HCS). The median ages of the patient group and HCS were 78 and 55 years, and their body mass indexes were 22.8 and 21.7 kg/m2, respectively. All patients were treated at the critical care center, and 11 of the 14 patients received ventilatory management. All patients survived. These 14 patients met the diagnostic criteria of Sepsis-3 and were diagnosed as having CAP-associated sepsis. Of them, 6 patients met the diagnostic criteria for septic shock. RNA sequencing showed the number of genes with up:down (upregulated:downregulated) expression variation (false discovery rate [FDR] <0.05, |log2 fold change| >1.2) to be 1209:1461 for mRNA; 51:21 for microRNA; and 646:1274 for miRNA-targeted mRNA. Canonical pathway analysis using mRNA showed activation of the PD-1 and PD-L1 cancer immunotherapy signaling pathways and inhibition of the Th1 signaling pathway as well as that using miRNA-targeted mRNAs. Conclusions: Using integrated analysis of mRNA and miRNA, we elucidated for the first time, to our knowledge, that T-cell exhaustion occurred during the acute phase of CAP-associated sepsis and that miRNA regulated Th1 signaling and PD-1 and PD-L1 cancer immunotherapy signaling through the RNA interference action of mRNA.

Project description:Objective: It is unclear whether the host response of gram-positive sepsis differs from gram-negative sepsis at a transcriptome level. Using microarray technology, we compared the gene-expression profiles of gram-positive sepsis and gram-negative sepsis in critically ill patients. Design: A prospective cross-sectional study. Setting: A 20-bed general intensive care unit of a tertiary referral hospital. Patients: Seventy-two patients admitted to the intensive care unit. Interventions: Intravenous blood was collected for leukocyte separation and RNA extraction. Microarray experiements were then performed examing the expression level of 19,232 genes in each sample. Measurements and Main Results: There was no difference in the expression profile between gram-positive and gram-negative sepsis. The finding remained unchanged even when genes with lower expression level were included or after statistical stringency was lowered. There were, however, ninety-four genes differentially expressed between sepsis and control patients. These genes included those involved in immune regulation, inflammation and mitochondrial function. Hierarchical cluster analysis confirmed that the difference in gene expression profile existed between sepsis and control patients, but not between gram-positive and gram-negative patients. Conclusion: Gram-positive and gram-negative sepsis share a common host response at a transcriptome level. These findings support the hypothesis that the septic response is non-specific and is designed to provide a more general response that can be elicited by a wide range of different micro-organisms. The study included seventy-two critically ill patients admitted to the intensive care unit (ICU) of Nepean Hospital, Sydney, Australia. Of these, fifty-five patients were diagnosed to have sepsis, as confirmed by microbiological culture. The remaining seventeen patients did not have sepsis and were therefore used as controls. The study was approved by the hospital ethics committee and informed consent was obtained from all patients or their relatives. Patient Samples. Whole blood was taken from each patient on admission to ICU. Neutrophils were separated from whole blood using density-gradient separation with Ficoll-PaqueP P(Amersham). Subsequent neutrophil RNA extraction was performed using guanidinium thiocyanate (Ambion). Microarray Experiment. The neutrophil RNA was converted to cDNA, fluorescently labeled and hybridized to its complimentary sequences on the microarray (Invitrogen). The fluorescent signals on each micrroarray were captured using the GenePix 4000B laser scanner (Axon Instruments). Expression level of each gene was represented by the intensity of its fluorescent signal. Data Extraction. All signal intensity values were processed using background-subtraction method. Prior to analysis, all values were log-transformed and normalized by fitting a print-tip group Lowess curve. Normalization minimizes bias due to dye chemistry, signal intensity or location of a gene on the array. It ensures the detection of genes that are truly differentially expressed, instead of those caused by experimental artifacts or variation in the hybridization process. After normalization, genes that had more than 50% of data missing were removed. We then selected genes that had at least 80% of the data showing two-fold changes from the geneâs median values. After filtering, 1617 genes were available for further analysis.

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Quantitative N-glycoproteomic Analysis Reveals Plasma Glyco-Signatures in Sepsis

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