Differentiate Gram Positive from Gram Negative Sepsis:Gene Expression Analysis in Peripheral Blood Mononuclear Cells from Patients with Early Sepsis
Date Issued
2004
Date
2004
Author(s)
DOI
zh-TW
Abstract
Sepsis is one of the leading causes of death in critically ill patients. Early identification of pathogens and treatment with appropriate antibiotics are crucial for patients’outcome. However, difficulties remain for clinicians to timely identify the causative pathogens of sepsis, either by presenting symptoms or by laboratory studies. Plenty of evidences disclosed that Gram-positive sepsis differed substantially from Gram-negative sepsis in clinical manifestations, immunopathogenesis, host response, and response to the treatment with anti-inflammatory agents. Hence, we proposed that gene expression profiles involving the response of host immune system were different between Gram-positive and Gram-negative sepsis. We used cDNA microarry technique to analyze the gene expression patterns of peripheral blood mononuclear cells (PBMC) from patients in the early phase of Gram-positive or Gram-negative sepsis. Once identifying pathogen-specific genes, we anticipated to develop a diagnostic tool for early identification of the causative pathogen, and to explore the complex pathogenesis of sepsis.
Blood samples were prospectively collected from three groups of subjects: the Gram-positive group, the Gram-negative group, and the non-sepsis control group. Patients who fit the definition of sepsis had their blood sampled in the early phase (within 24 hours) of disease, and were later classified into Gram-positive or -negative group based on the result of blood culture. Only those with positive blood culture results were enrolled into the study. Non-sepsis control group included people who were older than 40 years of age and were undertaking health examinations in the Health Management Center of NTUH. The peripheral blood mononuclear cells were immediately isolated after blood sampling, followed by extraction of total RNA and amplification. cDNA microarrays with 9600 non-redundant EST clones and with 346 angiogenesis-related genes were used. The signals developed during the hybridization process were scanned with a high-resolution (3000dPi) platform scanner, and digitalized with image processing software (GenePix, Axon instruments). To standardize and normalize the gene expression on each membrane for comparison, the intensities of genes were rescaled with the sum of total signal intensities on the individual membrane, followed by log transformation and standardization. The difference of gene expression was analyzed with student t test.
The results demonstrated a substantial difference of gene expression profile among the Gram-positive (n=6), the Gram-negative (n=6) and the control groups (n=10). More than 2000 genes were differentially expressed between sepsis and non-sepsis patients. Among these, sixty genes were statistically different between the Gram-positive and the Gram-negative sepsis, including those involved in signal transduction pathway (eg. NF-kB p65 subunit, zinc finger proteins and phosphatidylinositol 3-kinase catalytic subunit), cell-cell interaction (eg. Thrombomodulin, Platelet glycoprotein IX, MAdCAM-1), ubiquitin-proteasome proteolysis (eg. 26S proteasome subunit, ubiquitin specific protease 8 and von Hippel-Lindau syndrome), chemokines (Homo sapiens CC chemokine gene cluster), and genes participating in transcription or translation processes. We later quantified the expression of von Hippel-Lindau syndrome gene by RT-QPCR and revealed a coherent trend found by cDNA microarray. The mean expression amount of VHL gene was 2.10 folds of control group in Gram-negative group, and it was 1.52 folds of controls in Gram-positive group.
Our preliminary results had shown that most genes were regulated similarly in either Gram-positive or Gram-negative sepsis, which was in agree with the assumption of final common pathway in the pathogenesis of sepsis. However, several genes did have a pathogen-specific expression, which was also in agree with our hypothesis that Gram-positive and Gram-negative sepsis differed in the pathogenesis. Further studies in these pathogen-specific genes may help to elucidate the complex pathogenic mechanism of sepsis, to develop a rapid diagnostic tool for identifying causative pathogens and, perhaps, to assist establishing strategies for treating sepsis.
Subjects
cDNA微陣列
敗血症
基因表現
sepsis
gene expression
cDNA microarray
Type
text
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