Characteristics of the Estimation, Second Gene and Validation groups.

<p>Data are expressed as Means ± SEM or N (%). AST: aspartate amino-tranferase; ALT: alanine amino-transferase; γGT: Gamma Glutamyl Transpeptidase.</p

Multivariate analysis of the Estimation group for the assessment of significant hepatic fibrosis.

<p>Patients were classified according to Fibrosis (F)<2 or ≥2. Multivariate analysis was realized using logistic regression.</p

The serum OPN level correlated with fibrosis and steatohepatitis in 109 alcoholic patients (estimation cohort).

<p>The circulating levels of OPN were measured in the serum of 109 alcoholic patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035612#pone-0035612-t001" target="_blank">Table 1</a>) and analyzed according to the grade of steatosis (S0, S1, S2, S3)(<b>A</b>), the presence of hepatic inflammation (I0, I1) (Hepatic inflammation was determined by the presence (I1) or absence (I0) of hepatocellular necrosis and ballooning degeneration, alcoholic Mallory's hyaline bodies, associated with an inflammatory reaction) (<b>B</b>) and the stage of fibrosis (F0, F1, F2, F3, F4)(<b>C</b>) (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035612#s2" target="_blank">Materials and Methods</a>). Results were expressed as the median (25^th, 75^th percentile). The Kruskal-Wallis test was used to compare the 4 groups S0, S1, S2 and S3, <i>P</i> = 0.900 and the 5 groups F0, F1, F2, F3 and F4, <i>P</i><0.001. The Mann Whitney test compared A0 A1: <i>P</i> = 0.047. The Mann Whitney test compared the two groups (C): &, <i>P</i><0.015, compared with F0; #, <i>P</i><0.028, compared with F1; $, <i>P</i> = 0.007, compared with F2. Correlations between the serum OPN level with hepatic steatosis, hepatitis or fibrosis were analyzed using the Spearman's rank correlation test.</p

Levels of serum OPN for diagnosis of significant fibrosis (F≥2) in cohorts of alcoholic patients.

<p>The area under the ROC curves are shown for the performance of serum OPN levels in evaluating significant fibrosis (F≥2) in estimation (109 patients) and validation (95 patients) cohorts.</p

Correlation between hepatic and adipose tissue OPN expression and hepatic steatosis, inflammation and fibrosis in alcoholic patients (the second gene group).

<p>Spearman's rank correlation test.</p

Univariate analysis of the Estimation group according to the severity of the liver disease.

<p>Patients were classified according to Fibrosis (F) <2 or ≥2.. Quantitative results are expressed as means ± standard deviations. AST: aspartate amino-tranferase; ALT: alanine amino-transferase; γGT: Gamma Glutamyl Transpeptidase; OPN: Osteopontin.</p

Levels of serum OPN in patients with chronic viral hepatitis C.

<p>(<b>A</b>) The circulating levels of OPN were measured in the serum of 86 patients with chronic hepatitis C (25 F1, 34 F2, 19 F3, 8 F4) and analyzed according to the stage of fibrosis. Results were expressed as the median (25^th, 75^th percentile). The Kruskal-Wallis test was used to compare the 4 groups F1, F2, F3 and F4: <i>P</i><0.001. The Mann-Whitney test compared the two groups: &, <i>P</i>≤0.048, compared with F1; #, <i>P</i>≤0.026, compared with F2. Correlation between the serum OPN level with hepatic fibrosis was analyzed using the Spearman's rank correlation test. (<b>B</b>) The areas under the ROC curves are shown for the performance of serum OPN levels in estimating significant fibrosis (F≥2) or advanced fibrosis (F≥3) in this cohort.</p

Hepatic OPN expression correlated with hepatic TGFβ expression, portal space neutrophils and the serum OPN level.

<p><b>A</b>) Hepatic OPN and TGFβ1 expression levels were analyzed by real-time quantitative PCR in 34 alcoholic patients from the estimation group without (<b>F0</b>) (N = 6) or with mild (<b>F1</b>) (N = 14), moderate (<b>F2</b>) (N = 6) or advanced (<b>F3/4</b>) (N = 8) fibrosis. The mRNA levels were normalized to the mRNA levels of RPLP0. Results are expressed relative to the expression levels in F0 patients and expressed as means ± SEM. The Kruskal-Wallis test compared the 4 groups F0, F1, F2 and F3/F4: OPN, <i>P</i> = 0.037; TGFβ1, <i>P</i> = 0.002. Mann Whitney test compared the two groups: &, <i>P</i>≤0.011, compared with F0; #, <i>P</i>≤0.029, compared with F1; $, <i>P</i> = 0.008, compared with F2. <b>B</b>) Correlation between hepatic OPN gene expression (ΔCt) and either the hepatic TGFβ1 expression level (ΔCt) or the serum OPN level were analyzed using the Spearman's rank correlation test. <b>C</b>) Correlation between the number of portal space neutophils (Cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035612#s2" target="_blank">Materials and methods</a>) and serum OPN level or hepatic OPN gene expression (ΔCt) in 11 alcoholic patients (2F0/3F1/1F2/5F4) were analyzed using the Spearman's rank correlation test.</p