Cox proportional hazard model for prognosis of HD patients at a four-year follow-up.

a<p>Values are expressed as mean ± SD or percent.</p>b<p>ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker.</p>c<p>CI, confidence interval.</p>d<p>HR, hazard ratio.</p>*<p><i>p</i><0.10 in univariate Cox regression analysis.</p

Box plots of plasma vitamin D binding protein and clusterin levels in HD patients.

<p>ELISA validation of vitamin D binding protein and clusterin concentration for patients’ plasma originally analyzed by 2-DE. Long-term HD survivors have (a) higher vitamin D binding protein (204.5±63.9 mg/L vs. 149.0±71.6 mg/L, p = 0.036) and (b) lower clusterin (282.3±105.8 mg/L vs. 438.5±174.2 mg/L, p = 0.011) than the short-term HD patients, which are concordant with 2-DE expression pattern.</p

Kaplan-Meier analysis of plasma DBP level in HD patients.

<p>HD patients with the lowest plasma vitamin D binding protein level were at the highest risk for mortality than those with the top tertile plasma levels (p = 0.03; log-rank test).</p

Representative gel sections of protein alterations related to HD duration.

<p>Each bar represents the intensity of the squared spot quantitatively analyzed by PDQuest software. Circles were used to indicate the α1-antitrypsin and fibrinogen γ chain respectively in (h) and (i) according to SWISS-2DPAGE (<a href="http://tw.expasy.org/ch2d/" target="_blank">http://tw.expasy.org/ch2d/</a>).</p

Demographics and biochemistry of the studied HD population.

<p>Demographics and biochemistry of the studied HD population.</p

Plasma Protein Characteristics of Long-Term Hemodialysis Survivors

<div><p>Hemodialysis (HD) patients are under recurrent circulatory stress, and hemodialysis has a high mortality rate. The characteristics of plasma proteomes in patients surviving long-term HD remain obscure, as well as the potential biomarkers in predicting prognoses. This study reports the proteome analyses of patient plasma from non-diabetic long-term HD (LHD, dialysis vintage 14.9±4.1 years, n = 6) and the age/sex/uremic etiology-comparable short-term HD (SHD, dialysis vintage 5.3±2.9 years, n = 6) using 2-DE and mass spectrometry. In addition, a 4-year longitudinal follow-up of 60 non-diabetic HD patients was subsequently conducted to analyze the baseline plasma proteins by ELISA in predicting prognosis. Compared to the SHD, the LHD survivors had increased plasma vitamin D binding proteins (DBP) and decreased clusterin, apolipoprotein A-IV, haptoglobin, hemopexin, complement factors B and H, and altered isoforms of α1-antitrypsin and fibrinogen gamma. During the 45.7±15 months for follow-up of the 60 HD patient cases, 16 patients died. Kaplan-Meier analysis demonstrated that HD patients with the lowest tertile of the baseline plasma DBP level have a significantly higher mortality rate. Multivariate Cox regression analysis further indicated that DBP is an independent predictor of mortality. In summary, the altered plasma proteins in LHD implicated accelerated atherosclerosis, defective antioxidative activity, increased inflammation/infection, and organ dysfunction. Furthermore, lower baseline plasma DBP in HD patients is related to mortality. The results suggest that the proteomic approach could help discover the potential biomarker in HD prognoses.</p> </div

Lists of differentially expressed proteins in the plasma of long-term HD compared to the short-term HD patients.

a<p>Protein identification was done by the peptide mass fingerprinting (pmf) using MALDI-TOF MS, or by sequencing amino acids using Q-TOF tandem MS, as shown in the supplementary materials including <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040232#pone.0040232.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040232#pone.0040232.s002" target="_blank">Data S1</a>.</p

Comparison between the surviving and deceased HD patients in a four-year follow-up.

*<p>Values are expressed as mean ± SD or percent unless otherwise listed.</p

A representative 2D gel of plasma proteins from a long term HD patient.

<p>Plasma proteins were pre-treated with an albumin depletion kit and then separated according to their <i>p</i>I (IEF) and mass (SDS-PAGE). An average of 598 spots were detected per gel and further analyzed by MS or 2D database comparisons. The identified protein spots with differential expression related to HD duration were numbered and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040232#pone-0040232-t002" target="_blank">Table 2</a>.</p

Fisetin Inhibits Lipopolysaccharide-Induced Macrophage Activation and Dendritic Cell Maturation

Macrophages and dendritic cells are required for initiating innate immunity and adaptive immunity. Aberrant activation of macrophages and dendritic cells can cause detrimental immune responses; thus, agents effectively modulating their functions are of great clinical value. We herein investigated whether fisetin, a flavonoid prevalently present in fruits and vegetables, could inhibit macrophage activation and dendritic cell maturation. Fisetin suppressed LPS-induced NF-κB activation, expression of pro-inflammatory proteins (TNF-α and iNOS), MMP-9 activity, and phagocytic activity in macrophages. Furthermore, upon LPS-induced dendritic cell maturation, fisetin at nontoxic concentrations suppressed the expression of costimulatory molecules (CD80 and CD86), the production of cytokines (IL-12, IL-6, and TNF-α), and the endocytic activity of dendritic cells. Fisetin treatment significantly attenuated migration of dendritic cells into spleens and dendritic cell-mediated T cell activation in LPS-treated mice. Collectively, our data reveal that fisetin inhibits macrophage activation and impairs functional maturation of dendritic cells