Polymerase Chain Reaction–Based Method for Quantifying Recruitment of Monocytes to Mouse Atherosclerotic Lesions In Vivo

The critical role of monocyte recruitment in atherogenesis has been appreciated for some time. However, until recently, there have been no sufficiently sensitive methods for measuring rates of monocyte recruitment to the arterial wall in vivo. We have developed a novel highly sensitive method, based on the polymerase chain reaction, for quantitatively tracking DNA-marked monocytes and have adapted it for use in mice. We use the uniquely male gene, SRY:, on the Y chromosome as a gene marker. We transfuse monocytes from a male donor into a congenic female mouse, euthanize the mouse after 24 to 48 hours, and then quantify the arterial uptake of monocytes by quantitative polymerase chain reaction. This study describes the techniques used and their sensitivity and reproducibility and demonstrates the approach by assessing the effects of cytokines. In control low density lipoprotein receptor-negative mice, monocyte recruitment decreased slightly but significantly as lesions progressed. Intraperitoneal injection of a combination of tumor necrosis factor-alpha and interleukin-1 beta more than doubled the rate of monocyte recruitment into developing lesions. However, the response to the cytokines was much greater in younger mice with less advanced lesions than in older animals with more advanced lesions

Polymerase chain reaction-based method for quantifying recruitment of monocytes to mouse atherosclerotic lesions in vivo: enhancement by tumor necrosis factor-alpha and interleukin-1 beta.

The critical role of monocyte recruitment in atherogenesis has been appreciated for some time. However, until recently, there have been no sufficiently sensitive methods for measuring rates of monocyte recruitment to the arterial wall in vivo. We have developed a novel highly sensitive method, based on the polymerase chain reaction, for quantitatively tracking DNA-marked monocytes and have adapted it for use in mice. We use the uniquely male gene, SRY:, on the Y chromosome as a gene marker. We transfuse monocytes from a male donor into a congenic female mouse, euthanize the mouse after 24 to 48 hours, and then quantify the arterial uptake of monocytes by quantitative polymerase chain reaction. This study describes the techniques used and their sensitivity and reproducibility and demonstrates the approach by assessing the effects of cytokines. In control low density lipoprotein receptor-negative mice, monocyte recruitment decreased slightly but significantly as lesions progressed. Intraperitoneal injection of a combination of tumor necrosis factor-alpha and interleukin-1 beta more than doubled the rate of monocyte recruitment into developing lesions. However, the response to the cytokines was much greater in younger mice with less advanced lesions than in older animals with more advanced lesions

Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications