The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation.

Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean +/- SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 +/- 0.02. A progressive and significant (P <.0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 +/- 0.1, +1 hour: 1.3 +/- 0.9, +2 hours: 4.1 +/- 0.9), peaking at +3 hours (10 +/- 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge. (Blood. 2000;96:554-559)

A novel quantitative multiplex NASBA method: application to measuring tissue factor and CD14 mRNA levels in human monocytes.

A new method to quantify two individual mRNAs in a single NASBA reaction is described. In this study, tissue factor and CD14 mRNAs were used as a model system. RNA ratios of -4 to +4 log units were determined with good precision (within 0.3 log) and accuracy (within 0.2 log). By measuring both mRNAs in human monocytes that were stimulated with LPS, the multiplex Q-NASBA proved to be a successful tool to monitor the expression levels of two individual mRNAs in a single-tube amplification system. The method has potential in all fields in which quantitative information is needed on two individual RNAs.

Serum HCV RNA levels assessed by quantitative NASBA: stability of viral load over time, and lack of correlation with liver disease. The Trent HCV Study Group.

BACKGROUND/AIMS: We used the hepatitis C virus quantitative NASBA technique to evaluate the stability of viral load within individuals with chronic hepatitis C, to determine the range of viraemic load between individuals, and to assess the usefulness of hepatitis C virus RNA quantitation in predicting the severity of underlying hepatitis C virus-induced liver disease. METHODS: Hepatitis C virus RNA was determined, using the quantitative NASBA assay, in multiple serum samples from 11 individuals with chronic hepatitis C over an average time period of 11 months (range = 3-23 months), and in single serum samples from a further 10 individuals. RESULTS: In 10/11 individuals the hepatitis C virus RNA titres were within one log10 copies/ml of each other during this time period. In the eleventh, there was a rise of 1.36 log10 copies/ml in two serum samples taken 8 months apart. The viraemic load varied by 2.79 log10 copies/ml serum between individuals. There were no correlations between mean RNA levels and total biopsy scores (either Knodell or Sheffield scores), or the individual components of the biopsy scoring systems, except the sinusoidal infiltration component of the Sheffield score. There was also no difference in viral RNA levels between those infected with type 1 as compared to type 3 virus, with a mean level in both groups of 7.2 log10 copies/ml. CONCLUSIONS: Hepatitis C virus serum RNA level is stable within individuals within the studied time period. Viral load varies between infected individuals but is not a useful prognostic indicator of the severity of virus-induced liver disease.