Isolation of CD56⁺ T and CD56^- T cells from PBMCs.

<p>The purity of enriched cell population was determined by flow cytometry using antibodies to CD56 (PE) and CD3 (FITC) markers.</p

Effect of heroin use or heroin use plus HCV infection on the key element expression in IL-12 pathway.

<p>CD56⁺ T cells (unstimulated) were isolated from healthy subjects (n = 17) and heroin users with (n = 17) or without (n = 20) HCV infection. Total cellular RNA extracted from freshly isolated CD56⁺ T cells was subjected to the real-time RT PCR for key element (IL-12Rβ1, IL-12Rβ2, STAT-1, 3, 4, 5, JAK-2, TYK-2) in IL-12 pathway and GAPDH mRNA quantification. The data are expressed as the relative ratio of the key element mRNA level to GAPDH mRNA level. Median values are indicated by horizontal bars and statistical significance was calculated by the Mann-Whitney <i>U</i>-test.</p

Heroin use or heroin use plus HCV infection down-regulate IFN-γ expression in IL-12-stimulated CD56⁺ T cells.

<p>CD56⁺ T cells isolated from the healthy subjects (n = 17) and the heroin users with (n = 17) or without (n = 20) HCV infection were stimulated by IL-12 <i>in vitro</i> for 48 h. (A) Total cellular RNA extracted from IL-12-stimulated CD56⁺ T cells was subjected to the real-time RT PCR for the mRNA levels of IFN-γ and GAPDH. The data are expressed as the relative ratio of the IFN-γ to GAPDH mRNA levels. Mean values are indicated by horizontal bars and statistical significance was calculated by the Mann-Whitney <i>U</i>-test. (B) IFN-γ protein levels in IL-12-stimulated CD56⁺ T cell cultures were determined by ELISA. Median values are indicated by horizontal bars and statistical significance was calculated by the Mann-Whitney <i>U</i>-test.</p

Primers for Real-Time RT-PCR.

<p>Primers for Real-Time RT-PCR.</p

Schematic diagram of possible molecular mechanisms underlying heroin use/HCV infection-mediated suppression of IL-12 pathway.

<p>Although heroin use or heroin use plus HCV infection has little impact on the gene expression of key elements (IL-12Rβ1, IL-12Rβ2, STAT-1, 3, 4, 5, JAK-2, TYK-2) in IL-12 pathway in CD56⁺ T cells. Heroin use or heroin use plus HCV infection induces the expression of SOCS-3 or PIAS-3, the key negative regulators of IL-12 pathway, resulting in the downregulation of IL-12-mediated IFN-γ production.</p

Demographics of Study Subjects.

<p>Demographics of Study Subjects.</p

Heroin use or heroin use plus HCV infection induces the expression of the suppressors of IL-12 pathway.

<p>CD56⁺ T cells (unstimulated) were isolated from the healthy subjects (n = 17), heroin users with (n = 17) or without (n = 20) HCV infection. Total cellular RNA extracted from freshly isolated CD56⁺ T was subjected to the real-time RT PCR for the suppressor and GAPDH mRNA quantification. The data are expressed as the relative ratio of mRNA levels of suppressor (SOCS-1, 2, and 3, or PIAS-1, 3, x, and y) to those of GAPDH. Median values are indicated by horizontal bars and statistical significance was calculated by the Mann-Whitney <i>U</i>-test.</p

Comparison of anti-HCV activity of CD56⁺ T cells among three study groups.

<p>HCV JFH-1-infected Huh 7 cells (day 3 post infection) were cultured for 48h in the presence or absence of pooled SN from IL-12-stimulated CD56⁺ T cells from the healthy subjects and the heroin users with or without HCV infection as indicated. (A) Real-time RT-PCR analysis of HCV RNA. Total cellular RNA extracted from Huh7 cells treated with or without pooled SN of CD56⁺ T cells from three study groups as indicated was analyzed by the real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative (%) to control (without SN treatment, which is defined as 100). The results are mean ±SD of triplicate cultures, representative of three repeated experiments using pooled CD56⁺ T SN from three study groups. Statistical significance was performed using student <i>t</i>-test. (B) Immunofluoresence analysis of HCV NS3 protein expression in Huh 7 cells. HCV NS3 protein expression in Huh7 cells treated with or without pooled CD56⁺ T SN from three study groups as indicated was determined by immunofluoresence staining with antibody against HCV NS3 (green). The nuclei were stained with Hoechst 33258 (blue). One representative result of 3 experiments is shown (200×).</p

Effect of heroin use or heroin use plus HCV infection on CD56⁺ T cells.

<p>(A) Heroin use or heroin use plus HCV infection has little impact on CD56⁺ T cell frequency (%) in PBMCs. The CD56⁺ T cell frequency (%) in PBMCs from healthy subjects (n = 17), heroin users with (n = 17) or without (n = 20) HCV infection were shown by scatter plots. Median values are indicated by horizontal bars. Statistical significance was calculated by the Mann-Whitney <i>U</i>-test. There is no significant difference in CD56⁺ T cell numbers in PBMCs between normal subjects, heroin users with or without HCV infection (P>0.05). (B) Heroin use down-regulates IFN-γ gene expression in unstimulated CD56⁺ T cells. CD56⁺ T cells were isolated from healthy subjects (n = 17) and heroin users with (n = 17) or without (n = 20) HCV infection. Total cellular RNA extracted from freshly isolated CD56⁺ T cells was subjected to the real-time RT PCR for IFN-γ and GAPDH mRNA quantification. The data are expressed as the relative ratio of the IFN-γ mRNA levels to GAPDH mRNA levels. Median values are indicated by horizontal bars and statistical significance was calculated by the Mann-Whitney <i>U</i>-test.</p

Plasma HCV viral loads is negatively associated with IFN-γ produced by IL-12-stimulated CD56⁺ T cells.

<p>Spearman correlation (r and p-value) was calculated to determine the relationship between the levels of IL-12-induced IFN-γ protein by CD56⁺ T cells and the levels of HCV RNA in plasma from HCV-infected heroin users (n = 17). The levels of IFN-γ protein produced by IL-12-stimulated CD56⁺ T cells were negatively correlated with those of plasma HCV RNA (P<0.01).</p