Effect of linoleic acid on reactive oxygen species production in human monocytes and liver macrophages.

<p>(A) The stimulation index for reactive oxygen species production in monocytes was higher in patients with NAFLD (n = 12) than in control subjects (n = 10). The box and whiskers indicate the non-parametric statistics: the median, lower and upper quartiles and confidence interval around the median. A two-tailed Mann-Whitney U test was used; *p = 0.036. (B) DCF-MFI, 2', 7’-dichlorofluorescein median fluorescence intensity. Linoleic acid increased reactive oxygen species production in liver macrophages from patients with NAFLD (n = 12). Lines connect the “Basal” and “Linoleic acid” values for each patient. A Wilcoxon matched-pairs signed rank test was performed; *p = 0.001. (C) The stimulation index in monocytes and liver macrophages from patients with NAFLD were positively correlated. Spearman´s rank correlation coefficients test was used.</p

Diagram of the experimental design.

<p>Experimental design using human peripheral blood mononuclear cells (PBMCs), human or mouse liver cells. PMA: phorbol myristate acetate, H₂DCFDA: 2’7’-dichlorofluorescein diacetate.</p

Curcumin effects on linoleic acid- and leptin-induced the production of reactive oxygen species and cytokines as well as the infiltration of CD4⁺ cells in HFD-fed mice.

<p><b>(A)</b> After they were treated with linoleic acid ex vivo, liver macrophages from HFD-fed mice showed a higher stimulation index for reactive oxygen species production (*p<0.05 vs. normal chow-fed mice). In vivo curcumin administration of HFD-fed mice (HFD+curcumin) prevented the increase in the stimulation index (*p<0.05 vs. HFD-fed mice). <b>(B)</b> Ex vivo linoleic acid stimulation of hepatocytes from all the experimental groups resulted in similar stimulation indexes for reactive oxygen species production. <b>(C)</b> TNF-α production induced by ex vivo leptin treatment was higher in liver macrophages from HFD-fed mice (*p<0.05 vs. normal chow-fed mice). In vivo curcumin treatment of HFD-fed mice also prevented the increase in TNF- α production (*p<0.05, HFD+curcumin vs. HFD). <b>(D)</b> The percentage of CD4⁺ cells among the non-parenchymal cell populations was higher in HFD-fed mice (*p<0.01 vs. normal chow-fed mice). In vivo curcumin treatment also prevented the increase in CD4⁺ cell recruitment (*p<0.01, HFD+curcumin vs. HFD). The box and whiskers show the non-parametric statistics: the median, lower and upper quartiles and confidence interval around the median. The Kruskal-Wallis test with Dunn’s post-test was performed.</p

The reversal effects of curcumin on peripheral immunological cells.

<p>Ex vivo curcumin treatment of PBMCs from patients with NAFLD resulted in decreases in <b>(A)</b> linoleic acid-induced reactive oxygen species generation (n = 9, *p = 0.011) and <b>(B)</b> leptin-induced TNF-α production (n = 9, *p = 0.016) by monocytes. <b>(C)</b> Ex vivo curcumin treatment of PBMCs from patients with NAFLD resulted in decreased IFN-γ production in CD4⁺ cells (n = 9, *p = 0.048). Lines connect the “Linoleic acid” and “Linoleic acid+Curcumin” stimulation indexes or the “Leptin” and "Leptin+Curcumin" fold of increase indexes for each patient. A Wilcoxon matched-pairs signed rank test was performed.</p

Effect of leptin on TNF-α and reactive oxygen species production in human monocytes.

<p>(A) The fold of increase index for TNF-α production was higher in monocytes from patients with NAFLD (n = 10) than those from control subjects (n = 10); however, when monocytes were stimulated with leptin, the stimulation index for reactive oxygen species production (B) was similar in patients with NAFLD (n = 10) and control subjects (n = 10). The box and whiskers indicate the non-parametric statistics: median, lower and upper quartiles and confidence interval around the median. A two-tailed Mann-Whitney U test was used, *p = 0.004.</p

Demographic, anthropometric and biochemical baseline data of patients with NAFLD and control individuals.

<p>Demographic, anthropometric and biochemical baseline data of patients with NAFLD and control individuals.</p

The effects of leptin on IFN-γ production and T cell-associated alterations in liver samples from patients with NAFLD.

<p>(A) The fold of increase index for IFN-γ production in leptin-stimulated circulating CD4⁺ cells was higher in patients with NAFLD (n = 10) than in control subjects (n = 10; *p = 0.011). <b>(B)</b> The percentage of CD4⁺ cells among the total non-parenchymal cell population was higher in patients with NAFLD (n = 10) than in control subjects (n = 10), *p = 0.030. <b>(C)</b> Compared with control subjects (n = 9), patients with NAFLD (n = 9) showed increased hepatic mRNA expression levels of IFN-γ (*p = 0.012), T-bet (*p = 0.020) and CCL20 (*p = 0.007) as measured by quantitative PCR. The 2^-ΔΔCt method was used to calculate the mRNA fold change. The box and whiskers indicate the non-parametric statistics: the median, lower and upper quartiles and confidence interval around the median. A two-tailed Mann-Whitney U test was used for the statistical analysis.</p

Changes from baseline in HBV DNA by post-Week 24 treatment (efficacy population).

<p>Changes from baseline in HBV DNA by post-Week 24 treatment (efficacy population).</p

Most common (≥5%) all-cause adverse events through Week 52 (safety population).

*<p>Intensification patients may have had an event during both the telbivudine only and intensification periods. Overall numbers with an indicated event may therefore be less than the row total.</p><p>doi:10.1371/journal.pone.0054279.t003</p

Week 52 glomerular filtration rate (MDRD) changes, by baseline rate and treatment (efficacy population).

<p>Week 52 glomerular filtration rate (MDRD) changes, by baseline rate and treatment (efficacy population).</p