1. Human immunodeficiency virus facilitates infection/replication of hepatitis C virus in native human macrophages
by Tomasz Laskus, Marek Radkowski, Joanna Jablonska, Karen Kibler, Jeffrey Wilkinson, Debra Adair, and Jorge Rakela
Blood
Volume 103(10):
May 15, 2004
©2004 by American Society of Hematology

2. Real-time strand-specific quantitative assay for the detection of HCV RNA–negative and –positive strands.
Real-time strand-specific quantitative assay for the detection of HCV RNA–negative and –positive strands. Reverse transcription was done at 70° C using thermostable enzyme Tth and the first round of PCR amplification was limited to 20 cycles. The nested round employed LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics). (A) Detection of negative-strand HCV RNA synthetic template. (B) Detection of positive-strand HCV RNA synthetic template. (C) Specificity of the assay for the detection of negative-strand HCV RNA: reverse transcription was done with positive-sense primer using serial dilution of positive-strand HCV RNA as template. (D) Nonnested real-time PCR detection of HCV RNA–positive strand using MMLV for the reverse transcription step.
Tomasz Laskus et al. Blood 2004;103:
©2004 by American Society of Hematology

3. Real-time strand-specific quantitative assay for the detection of HCV RNA–negative and –positive strands in liver tissue.
Real-time strand-specific quantitative assay for the detection of HCV RNA–negative and –positive strands in liver tissue. One microgram of total RNA was extracted from an infected liver and serially diluted in water; one μg of RNA extracted from uninfected liver was added to each dilution to keep the quantity of RNA constant. (A) Detection of HCV RNA–negative strand: sense primer present in the reverse transcription step. (B) Detection of HCV RNA–positive strand: negative-antisense primer present in the reverse transcription step.
Tomasz Laskus et al. Blood 2004;103:
©2004 by American Society of Hematology

4. Quantitative detection of HCV RNA by real-time RT-PCR in cell culture supernatants from HIV/HCV-coinfected macrophage cultures.
Quantitative detection of HCV RNA by real-time RT-PCR in cell culture supernatants from HIV/HCV-coinfected macrophage cultures. Supernatants were collected at day (d) 3, 6, 10, and 14 after exposure to 8 different HCV-positive sera.
Tomasz Laskus et al. Blood 2004;103:
©2004 by American Society of Hematology

5. Correlation between quantitative parameters of HIV and HCV infection in macrophage cultures.
Correlation between quantitative parameters of HIV and HCV infection in macrophage cultures. (A) Amplification of synthetic HIV RNA template with real-time quantitative RT-PCR: the assay detected 100 template copies and remained linear up to 109 template copies. (B-D) Relationship between HCV RNA cellular load at 2 weeks and HIV RNA quantity in culture supernatant at time 0 (B; right before exposure to HCV), 7 days (C), and 14 days (D) after HCV exposure. There was significant correlation (r = 0.96; P < .01) for HCV RNA and HIV RNA quantity at time 0. (E) Relationship between quantity of HCV RNA and proviral HIV DNA in cells at 2 weeks. (F) Relationship between the quantity of HCV RNA in cells and the percent of cells expressing p24 at 2 weeks. All correlations were calculated by nonparametric Spearman rank test.
Tomasz Laskus et al. Blood 2004;103:
©2004 by American Society of Hematology

6. Staining of HIV/HCV-coinfected macrophages for HCV and HIV antigens.
Staining of HIV/HCV-coinfected macrophages for HCV and HIV antigens. (A) Infected macrophages stained with monoclonal antibodies against nonstructural protein 3 (NS3) and visualized with DAB. Two of 5 macrophages shown demonstrate brown granular cytoplasmic staining (original magnification, × 400). Cell shown in the bottom insert is positive for both p24 (left) and NS3 (right). (B) Relationship between HCV RNA cellular load at 2 weeks and the proportion of cells staining for NS3 (r = 0.94; P < .01 by Spearman rank test).
Tomasz Laskus et al. Blood 2004;103:
©2004 by American Society of Hematology