IntroductionAtherosclerosis is a chronic inflammatory disease characterized by endothelial cell damage, infiltration, proliferation and accumulation of macrophages, lymphocytes and transformed vascular smooth muscle cells within the vascular wall and procoagulation processes involving activation of plasmatic coagulation events and platelets. Numerous studies suggested a close interaction between thrombin action and atherogenesis, but possibly underlying mechanisms are multiple and specific treatment options were missing until now.Material and methodsAtherosclerosis prone 12 weeks old ApoE–/– mice were fed a cholesterol rich diet for 4 weeks and were concomitantly treated orally with placebo or the thrombin inhibitor dabigatran (1.2 g/kg/day).ResultsThe thrombin time (HEMOCLOT®) was significant extended in dabigatran treated animals. Vascular oxidative stress was significantly reduced during thrombin inhibition, as assessed by L012 chemiluminescence in aortic segments (212 ±84 vs. 69 ±21 RLU/s/mg dry weight, p = 0.048). Organ chamber experiments of isolated aortic rings showed that dabigatran treatment significantly improved endothelium-derived vasorelaxation (p < 0.001). Dabigatran treated mice developed less atherosclerotic lesions (6.2 ±0.2% vs. 9 ±1.1%, p = 0.037) and showed less infiltration of atherosclerotic lesions with macrophages (2.59 ±0.3% vs. 5.14 ±0.7%, p = 0.0046), as determined by systematic histological and immunohistological analyses of the aortic root. Blood pressure, body weight and food intake were not altered by the treatment.ConclusionsThe thrombin inhibitor dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function and decreases atherosclerosis in mice.

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Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.

Up-regulation of tissue factor (TF) expression in circulating blood cells, especially monocytes, plays a key role in the pathogenesis of various thromboembolic diseases (1)(2)(3)(4). Measurement of TF expression in monocytes therefore might be helpful in the diagnosis of a hypercoagulable state (5-7 ). Monocytic TF expression can be measured on both the protein and RNA levels. Testing on the RNA level seems to be more appropriate than antigen testing to detect a procoagulant state because monocytes that already express functionally active TF on their membrane surfaces are rapidly cleared from the circulation (8 ).The aim of the present study was to develop a protocol for quantitative determination of TF mRNA transcripts in monocytes and other circulating blood cells. The method should be rapid, robust, and applicable in whole blood without the need for cell isolation. We developed a one-step quantitative reverse transcription (qRT)-PCR assay based on the real-time TaqMan ® technology and evaluated the preanalytical conditions required for the use of TF mRNA measurements on a routine clinical basis as well as several normalization strategies, including normalization based on CD14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene expression. Finally, we measured baseline TF expression in healthy individuals and in thrombophilic patients.RNA calibrators for quantification were prepared by in vitro transcription of DNA hybrids that combined the T7 promoter sequence with the following sequences of TF, CD14, or GAPDH cDNA: for TF, bp 265-848 (GenBank accession no. M16553); for CD14, bp 18 -564 (GenBank accession no. M86511); and for GAPDH, bp 8 -525 (GenBank accession no. M33197). Generated RNAs were treated with DNase I (Roche) to remove the added DNA templates and purified by use of the RNeasy Mini Kit (Qiagen). RNA was quantified by photometric measurement (A 260 reading of 1 ϭ 44 mg/L). Oligonucleotide primers and probes for TF and CD14 qRT-PCR were designed by use of Primer Express software, Ver. 1.5 (Perkin-Elmer), and were purchased from Eurogentec. All sequences shown in Table 1 were chosen to prevent amplification of genomic DNA. Primer and probe sequences for amplification of GAPDH mRNA were taken from the "TaqMan Gold RT-PCR Kit" protocol (PerkinElmer).PCRs were performed in a final volume of 20 L using the QuantiTect Probe RT-PCR-Mix (Qiagen). Optimum reaction conditions were as follows: 1ϫ Mastermix (including PCR buffer, deoxynucleotide triphosphates, 4 mM MgCl 2 , and Rox reference dye); TF or CD14 forward and reverse primers (200 and 150 nM, respectively); GAPDH forward and reverse primers (100 nM); TF or CD14 probe (200 nM); GAPDH probe (100 nM); 1 U of QT Probe RT Mix; and 5 L (TF/GAPDH PCR) or 1 L (CD14/GAPDH PCR) of calibrator or sample preparation. Calibrators and samples were run in duplicate.Thermal cycling was performed in a 96-well spectrofluorometric thermal cycler (Prism SDS 7700; Applied Biosystems) with the following profile: 50°C for 20 min for the reverse transcription ...