Correlation of Protein and Gene Expression Profiles of Inflammatory Proteins After Endotoxin Challenge in Human Subjects

Abstract

Administration of endotoxin (LPS) in humans results in profound physiological responses, including activation of peripheral blood mononuclear cells and the release of inflammatory factors. The time course of the response of selected inflammatory proteins was examined in healthy subjects (n = 6) administered a single intravenous dose of the purified derivative of endotoxin (3.0 ng/kg). Microarray analysis demonstrated changes in the expression of a number of genes, which were confirmed in separate in vitro endotoxin stimulation experiments. Subsequent TaqMan analysis of genes of interest indicated time-dependent changes in the expression of many of these genes. This included pre-B cell enhancing factor, which was identified on microarray analysis as being markedly upregulated following endotoxin stimulation. Protein expression of the genes examined by TaqMan analysis was measured and demonstrated the appearance of tumor necrosis factor (TNF)-alpha and sTNF-R proteins in the plasma beginning within 1 h after dosing, followed by other cytokines/ inflammatory markers (e.g., IL-1ra, G-CSF, IL-6, IL-8, and IL-10) and suppressors of cytokine signaling (SOCS-1 and SOCS-3). In general, cytokine protein expression correlated well with gene expression; however, the temporal profile of expression of some genes did not correlate well with the protein data. For many of these proteins, the lack of correlation was attributable to alternate tissue sources, which were demonstrated on TaqMan analysis. Principal component analysis indicated that cytokines could be grouped according to their temporal pattern of response, with most transcript levels returning to baseline 24 h following endotoxin administration. The combination of cDNA microarray and TaqMan analysis to identify and quantify changes in gene expression, along with the analysis of protein expression, can be useful in investigating inflammatory and other diseases.

Establishment of a Low Dose Canine Endotoxemia Model to Test Anti-Inflammatory Drugs: Effects of Prednisolone

“…However, in contrast to observations in human endotoxemia (5,14), maximum IL-I0 concentrations are detected earlier (1 h vs 3 h) and show a second peak at 3-4 h. This indicates an initial release from preformed stores followed by rapid de novo synthesis in dogs. IL-l~, INF-y, IL-2 and IL-4 concentrations remained under the detection limit as described for human endotoxemia (15)(16)(17).…”

Lipid A Modification Systems in Gram-Negative Bacteria

“…even synthetic E. coli lipid A by itself, causes a similar pathology when injected into animals (26)(27)(28), supporting its proposed role in sepsis. The characteristic structural features of E. coli lipid A (Figure 2), especially its two phosphate and acyloxyacyl groups, are needed to trigger full TLR4/MD2 activation in human cells (26).…”

Clinical review: Sepsis and septic shock - the potential of gene arrays

“…More recently, the genome-level response during experimental human endotoxemia has been studied using microarray technology [16,22,23]. Talwar and colleagues [22] compared eight volunteers challenged with intravenous endotoxin to four controls challenged with saline.…”