The Use of Real Time rtPCR to Quantify Inflammatory Mediator Expression in Leukocytes from Patients with Severe Sepsis

Abstract

Real-time reverse transcriptase polymerase chain reaction (RT rtPCR) was used to quantify the pattern of inflammatory mediator mRNA expression in circulating leukocytes from adult patients diagnosed with severe sepsis. We analysed 29 blood samples from 26 severely septic patients with different septic sources and eight samples from eight healthy adult volunteers. RT rtPCR was used to quantify mRNA expression of 21 different inflammatory mediators in peripheral leukocytes. The median variability in gene expression in the sepsis patients was 10.5 times greater than the variability of the healthy comparison group. We found a significant change in the regulation for the following genes: C5aR (20-fold, P<0.001), IL-8 (29-fold, P<0.001), MMP9 (72-fold, P<0.001), HSP70 (2.4-fold, P=0.02), and RIP2 (1.8-fold, P<0.04) were up-regulated. Conversely the median expression of IFNγ, and IL-6 were zero (P<0.001), and mtHSP (0.4-fold, P=0.02) was significantly down-regulated. Using linear discriminant analysis, IFNγ, IL-12, and TLR4 were correlated to a negative outcome. Different septic sources (peritonitis, burn, pneumonia and musculo-skeletal infections) resulted in significantly different mRNA patterns. The RT rtPCR is a useful tool to monitor the immune response in septic patients. We found a very high variability in inflammatory mediator expression among septic patients compared to healthy volunteers. This suggests that any future immune-modulatory therapy may need to be individualized to the patient's requirements as monitored by RT rtPCR. Different sources of sepsis may result in markedly different activation patterns.