AIH sera recognize UNQ9419 and CHAD.

<p>(A), SDS-PAGE (left panel) and western blot against anti-His antibody analysis (right panel) of the purified UNQ9419 and CHAD recombinant proteins. (B), western blot analysis of the purified UNQ9419 and CHAD recombinant proteins against sera from AIH patients (left panel) and no AIH subjects (right panel), respectively.</p

Evaluation of feature stability.

<p>Robustness of the R-SVM and PLS-DA rankers across the different 50 datasets is plotted as heat maps. Columns and rows represent the independent 50 subsets and each square indicates the Tanimoto index between two subsets. The colour code of the heat map ranges from blue to red where a blue colour reflects a low similarity index suggesting few proteins in common between the subsets while a red colour denotes an high similarity index. For each considered selection method a similarity heat map is obtained. (A), The average similarity over all pair wise comparison is 69% for PLS-DA; (B), and 31% for R-SVM; thus PLS-DA outperforms the R-SVM.</p

Selection strategy of serum autoantigens.

<p>The Venn diagram shows the autoantigen selection obtained according to (i) VIP scores >1.0 and (ii) a delta difference recognition frequency of 25%. 70 autoantigens overlap between the two filter criteria. Then, final selection of autoantigens was based on relative frequency of autoantigens from all generated datasets. There were 31 variables that were present in all datasets.</p

Evaluation of AIH-associated autoantigens.

<p>(A) The heatmap summarizes the recognition frequencies among different sample groups (AIH, HD and the VH) for the new four autoantigens out of the 31 candidates selected with the proposed bioinformatic strategy after DELFIA screening. Colour intensity denotes the degree of recognition frequency within the sample group. (B) Recognition frequency for two of the four autoantigens, that were statistical significant. (C) Signals distribution detected for each of the new two proteins are displayed. Statistical differences in recognition frequency (ChiSquare test) and in signal intensity (t test) are denoted as single (p<0,05), double (p<0,001) or triple stars (p <0,0001). (D), ROC curve of the biomarker candidates exhibited AUCs of 0.899 (SE = 87.5% and SP = 77.7%), 0.782 (SE = 65.0% and SP = 81.5%) for UNQ9419 and CHAD, respectively. The arrows denote best cut-off points. Combo curve represents the combination, UNQ9419+CHAD, which exhibited AUC of 0.915 (SE = 95.0% and SP = 76.2%).</p

Overview of the PLS-DA analysis for the comparison between AIH and HD group.

<p>The normalized Mean Fluorescence Intensity (MFI) from microarray data was analyzed using PCA and PLS-DA models. (A) PCA shows that AIH (red circle) and HD (green triangle) have distinctive profiles with little overlap between the two groups of samples; the exception was the sample HD0088 (blue arrow) so this sample was omitted from the subsequently explorative analysis. (B) Plot of R²Y (explained variation) and Q²Y (predicted variation) shows how the considered parameters change as a function of increasing model complexity. Three components were calculated through cross-validation, R²Y and Q²Y were 74.28% and 62.18% and resulted significant in order to explain the relationship between the descriptor matrix and the class response. (C) PLS-DA 3D score plot reveals that each sample is found close to the samples belonging to the same subgroup. Samples are coloured according to the disease status (AIH- red circle, HD green triangle—the axes of the plot indicate PLS-DA component 1–3). (D) Density plot of the Q²Y values in the analysis of 1000 permutation tests, solid red line shows the real Q²Y value. Such reference distribution can be seen as sign of the degrees of overfit and overprediction of the model. The permutation test showed that the real PLS-DA model was not over-fitted and not over-predicted.</p

Overview of sensitivity and specificity in a validation step.

<p>^<b>a</b> Sensitivity is defined as the true positive rate in %.</p><p>^<b>b</b> Specificity is defined as the true negative rate in Healthy Donor (HD)subject in %.</p><p>^<b>c</b> Specificity is defined as the true negative rate in Viral Hepatitis (VH)patients in %.</p><p>Overview of sensitivity and specificity in a validation step.</p

Iron homeostasis is potentially deregulated by HGF driven suppression of hepcidin-25 in untreated AIH-1.

<p>(A) Spearman rank correlation (SR) analysis of serum ferritin in AIH-1 with alanine aminotransferase (ALT) at diagnosis (Dx) in patients with subsequent biochemical remission (BR, left panel, N = 24) and incomplete biochemical response (IR, right panel, N = 24) matched for ALT, gender and age as far as possible. (B) SR analysis of serum ferritin and hepcidin-25 in patients with IR (N = 8; left panel) and with BR (N = 21; right panel) upon standard therapy and (C) in patients with achieved BR after 6–12 months of therapy (M 6–12; right; N = 7). (D) The hepatocyte growth factor (HGF, red; autofluorescence in green and blue) is expressed in (top) the portal tracts, endothelium and (bottom) liver sinusoids in a representative liver biopsy of untreated AIH-1. White bars represent 100 μm. (E) SR analysis of HGF and hepcidin-25 in untreated AIH-1 (N = 12) with subsequent BR and high ferritin (>2,09x ULN). (F) HGF in patients with high (hSF, N = 30) and low serum ferritin (lSF, N = 37). (* p<0.05; ** p<0.01; *** p<0.001; not significant, p≥0.05; ULN = upper limit of normal)</p

Diagnostic performance of the treatment response score to predict incomplete treatment response in untreated AIH-1.

<p>Diagnostic performance of the treatment response score to predict incomplete treatment response in untreated AIH-1.</p

AUROC and univariate analysis for the prediction of incomplete biochemical remission upon standard therapy in untreated AIH-1 in the training cohort.

<p>AUROC and univariate analysis for the prediction of incomplete biochemical remission upon standard therapy in untreated AIH-1 in the training cohort.</p

Hyperferritinemia and hypergammaglobulinemia were associated with the subsequent treatment response upon standard therapy in untreated AIH-1.

<p>(A) Left panel: Serum ferritin (SF) in untreated AIH-1 patients with subsequent biochemical remission (BR; N = 83) and incomplete biochemical response (IR; N = 26). Right panel: Histological treatment response with complete remission (CR: N = 16), BR with incomplete histological response (BR/IHR: N = 7) and IR (N = 26). (B) The same analysis for immunoglobulin G (IgG). (C) The AUROC analysis for the prediction of IR before the treatment initiation for SF (dashed line), IgG (dotted line) and their combined treatment response score (black solid line) in the retrospective trainings cohort (N = 76). (D) Rates of BR according to the treatment duration for the treatment response score<1 (solid line) and score≥1 (dashed line) for the training and (E) validation cohort. (* p<0.05; not significant, p≥0.05)</p