Study population characteristics and antiretroviral therapeutic situation
Four groups of patients were recruited into this study: HIV/HCV co-infected, HIV mono-infected, HCV mono-infected, and healthy controls. The demographics and laboratory parameters of the four groups are summarized in Table . The mean age of HIV/HCV co-infections was older than HIV mono-infections (p > 0.05), as well as significantly younger than HCV mono-infections (p < 0.05). The main transmission route of HIV/HCV co-infected patients was IDU (80%). The HIV mono-infections has the lowest nadir CD4 T cell count (p < 0.05) and highest HIV-1 RNA levels in plasma (p < 0.05) comparing with the other three study groups. The HCV RNA levels in plasma of HIV/HCV co-infection group were higher than HCV mono-infection group without statistical difference (p > 0.05).
Baseline demographic and immunological parameters of study participants
All patients infected with HIV were treated with combined ART according to currently accepted guidelines (2NRTI + PI or 2NRTI + NNRTI). Patients infected with HCV were treated for 48 week with pegylated IFN alfa-2a (180 μg/week) and ribavirin (900 to 1,200 mg/day, depending on body weight). HIV and HCV RNA levels were measured at six time points: before the start of therapy (baseline) and at 4, 12, 24, 36 and 48 weeks of antiretroviral therapy (Figure ). HIV/HCV co-infected patients were more sensitive to HAART comparing with HIV mono-infected patients. However, after 48 weeks of HAART all patients had reached lower HIV-1 RNA level which could be considered as a great viral response to antiretroviral therapy (Figure ). HCV antiretroviral therapeutic situation indicated that HIV/HCV co-infected patients were slower to reach sustained virologic response (SVR) than HCV mono-infected patients (Figure ).
Figure 1 Changes in plasma viral RNA during antiretroviral therapy between the HIV/HCV co-infection and the HIV or HCV mono-infection group. Each symbol represents the log10 HIV-1 or HCV RNA copies per milliliter of plasma. HIV-1 and HCV RNA levels were measured (more ...)
Th1 and Th2 cytokine concentration between four groups at baseline
Six cytokines were detected using CBA Th1/Th2 cytokine kit, Th1/Th2 cytokine standard curves were shown in Figure . Cytokine concentration of plasma in patients was calculated according to the standard curves with 20 pg/ml ~ 5000 pg/ml quantifiable ranges. Results of six cytokines' concentration of plasma in four study groups were shown in Figure . IFN-γ concentration was significantly lower in HIV/HCV co-infection group comparing with HIV mono-infection group (p < 0.001), while higher comparing with both HCV mono-infection (p < 0.001) and healthy control group (p < 0.001). IL-10 concentration was highest in HIV/HCV co-infection group when compared with the other three groups, but it was only shown a statistical raise when compared with healthy control group (p < 0.01). In HIV/HCV co-infection group, IL-2 concentration was the highest without statistical difference (p > 0.05). Co-infection group and HIV mono-infection group revealed a slightly elevation of IL-5 concentration when compared with HCV mono-infection and healthy control group (p > 0.05), while the same level between HIV mono-infection. TNF-α concentration was detectable in the people who were infected with HIV. Co-infection group has lower IL-4 concentration compared with both HIV mono- and HCV mono-infection group, while higher than healthy control group.
Figure 2 Th1/Th2 Cytokine Standard curves generated by BD CBA Software. Human Th1/Th2 Cytokine Standards were reconstituted in 2.0 ml of Assay Diluent; dilute the Standards primary liquid in a serial dilution rate as described: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, (more ...)
Figure 3 Comparison of Th1 and Th2 cytokine levels between different groups at baseline. Horizontal axis represents different kind of cytokines; vertical axis represents cytokine concentration (pg/ml) in plasma. Four coloured bar represents different study groups: (more ...)
Furthermore, we estimated the ratio of Th1 and Th2 by calculating Th1/2 cytokines' concentration, thus to evaluate the change of Th1/Th2 expression balance. The ratio of Th1 and Th2 cytokine concentration was presented in Figure , illustrating the HIV mono-infection group had the highest ratio than the others (p < 0.001); the ratio of HIV/HCV co-infection group was significantly lower than HIV mono-infection group (p < 0.001) and higher than HCV mono-infection (p < 0.05). Comparing with healthy controls, infected with HIV could induce higher Th1 cytokine levels, however, infected with HCV could induce higher Th2 cytokine level. Considering these two opposite effects, we observed a coordinative result in the HIV/HCV co-infection group, which was higher than HCV mono-infection and lower than HIV mono-infection group.
Figure 4 Ratio of Th1 and Th2 cumulative concentration at baseline in different groups. Horizontal axis represents four different study groups: HIV/HCV co-infection group (pink rectangle), HIV mono-infection group (orange rectangle), HCV mono-infection group (olive (more ...)
Dynamic changes of Th1 and Th2 cytokines during HAART
After initiating HAART, we estimated Th1 and Th2 cytokine levels of the HIV mono-infection and the HIV/HCV co-infection group at series time-points (baseline, 4 weeks, 12 weeks, 24 weeks and 48 weeks). Concentration of IFN-γ at 48 weeks after HAART in HIV/HCV co-infected patients indicated a significant decrease comparing with the other time points (p < 0.01) (Figure ), while the other cytokine levels presented no statistical differences. However, concentration of IFN-γ in HIV mono-infection group was more variable during HAART (Figure ). IFN-γ was secreted at the highest level at the baseline (p < 0.001), fell sharply at 4 weeks after HAART (p < 0.05), rebounded at 24 weeks (p < 0.001), and fell down again at 48 weeks (p < 0.001). Concentrations of IL-2 shown peak level at baseline, descended quickly at 4 weeks (p < 0.01), and ascended slightly as time-varying (p > 0.05). IL-4 was secreted at first (p < 0.05), descended progressively after HAART in 12 weeks (p < 0.05), rebounded abruptly at 24 weeks (p > 0.05), and fell off at 48 weeks (p < 0.05). TNF-α, IL-10 and IL-5 levels remained the same level of baseline as HAART was proceeding.
Figure 5 Comparison of Th1 and Th2 cytokine concentration during HAART between HIV/HCV co-infection and HIV mono-infection group. Horizontal axis represents six time points with different colour bars: before the start of therapy (0 W, Paris blue bars) and at 4 (more ...)
The change of Th1/Th2 ratio of mono- and co-infection group was demonstrated in Figure , which indicated that co-infection group has a complete different pattern during HAART; Th1/Th2 ratio in co-infection group was stabilized at first then elevated at 12 weeks, and descended after 12 weeks; while in HIV mono-infection group Th1/Th2 ratio fell sharply in 4 weeks then kept stable after with a slightly ascending trend. After 48 weeks treatment of HAART, Th1/Th2 ratio of HIV/HCV co-infected patients stayed in the same level of healthy control group. However, HIV mono-infection group was still higher than the control group.
Figure 6 Comparison of the dynamic change of Th1/Th2 cytokine ratio during HAART between HIV/HCV co-infection group and HIV mono-infection group. Vertical axis represents the ratio of Th1 and Th2 cytokine cumulative concentration at baseline; two coloured lines (more ...)
Furthermore, we evaluated the changes of Th1 and Th2 cytokine levels of the entire HIV infected patients who had received HAART. There was an obviously time-varying descending pathway (Figure ). Th1 cytokine levels seemed more sensitive to HAART than Th2.
Figure 7 Changes of Th1 and Th2 cytokine expression levels during HAART in both HIV mono-infection and HIV/HCV co-infection group. Vertical axis represents cytokine concentration (pg/ml) in plasma. The orange line represents Th1 cytokine cumulative concentration (more ...)
Relationships between Th1/Th2 cytokines, CD4+ T lymphocyte count and virus load
We analyzed the correlations between the main factors concerned, included CD4+ T lymphocyte count, HIV viral load, concentration of Th1 and Th2 cytokines (IL-2, IL-4, IL-5, IL-10, TNF-α and IFN-γ) and Th1/Th2 ratio, the results were exhibited on Table . Statistic results indicated that HIV-1 viral load had strong positive correlation with most Th1 and Th2 cytokine levels, especially in IFN-γ (r = 0.537; p < 0.01), IL-2 (r = 0.592; p < 0.01), IL-10 (r = 0.381; p < 0.01) and IL-5 (r = 0.360; p < 0.01), while negative correlation with TNF-α (r = -0.405; p < 0.05). CD4+ T lymphocyte count had significant negative correlation with HIV viral load (r = -0.519; p < 0.01), IL-2 (r = -0.335; p < 0.01) and IL-5 (r = -0.317; p < 0.01) concentration, and Th1/Th2 ratio (r = -0.236; p < 0.05). IFN-γ, IL-2, IL-10, Th1 cytokines levels and Th2 cytokines levels all had strong positive correlations with the cytokine concentration except TNF-α. Moreover, TNF-α was the most insensitive immune cytokine which did not correlated with the others except a negative correlation with HIV viral load (r = -0.405; p < 0.05). As the proceeding of HAART, HIV viral load was inhibited, CD4+ T lymphocyte count aroused, TNF-α expression raised, the concentration of IFN-γ, IL-2, IL-5 and IL-10 progressively decreased. However, Th1 cytokine behaved more sensitive than Th2 cytokine under the effect of HAART. IFN-γ secretion had a strong and intense relationship with Th1 cytokine levels (r = 0.969; p < 0.01), which indicated that IFN-γ might play an important role in Th1/Th2 balance.
Correlation between the concentration of Th1/Th2 cytokines, CD4+ T lymphocyte count, virus load and Th1/Th2 cytokine ratio