Study flowchart in accordance to the STARD criteria.

<p>In phase 0 diagnostic criteria were evaluated in 164 patients undergoing DE and RHC. In phase 1 the calculated cut-off value for mPAP was validated in a cohort of patients with the suspicion of PH.</p

Characteristics of 164 patients and indications for RHC.

<p>BMI indicates body mass index (kg/m²), RHC indicates right heart catheterization.</p

Analysis of relative differences and ROC analysis of mPAP for diagnosis of PH.

<p>A, the relative positive and negative deviation between DE and RHC were larger for sPAP than mPAP. B, ROC analysis reveal an excellent diagnostic accuracy of mPAP for the diagnosis of PH with an area under the curve (AUC) of 0.95.</p

DE versus RHC correlations.

<p>DE mPAP (B) was better correlated with RHC than sPAP (A). Dotted lines mark virtual best correlation of 1 and solid lines mark the real correlation. r indicates the correlation coefficient, sPAP indicates systolic pulmonary artery pressure.</p

Ex vivo HLA-DR1-HCV-1806-1818 tetramer staining in stable resolved and chronic HCV infection.

<p>Tetramer staining was performed in five HLA-DR1-positive patients with chronic hepatitis C in comparison to three HLA-DR1-positive resolved patients. In all PBMC from control groups (five individuals with acute HCV who did not possess HLA-DR1, and four healthy and four HIV1+ individuals with HLA-DR1, respectively), tetramer staining was <0.001%. In all patients and healthy controls, proliferation assays following stimulation with recombinant NS4-antigen were performed. The SI for healthy controls was 1.2±0.48 (mean±SD, range 0.71 to 1.66), for chronic hepatitis C patients 1.7±0.9 (range 0.76 to 3.7), and for recovered patients 22.1±22.1 (range 1.74 to 52.9).</p

Sequence of epitope 1806-1818 in chronic hepatitis C in relation to the presence of the HLA-DR1 allele.

<p>The consensus sequence for the epitope aa 1806-1818 as used in the tetramer and functional assays is shown in full, and identity at a residue indicated by a dash. Z and X represent ambiguous residues within the bulk sequence.</p><p>Any mutation in epitope associated with DR1, p = NS</p

Clinical data of patients with acute hepatitis C

<p>IFN = interferon-alpha; RBV = ribavirin; NR = non-responder to antiviral therapy; SR = sustained responder to antiviral therapy.</p

Examples of ex vivo HLA-DR1-HCV-1806-1818 tetramer staining from the acute hepatitis C cohort.

<p>PBMC from one acute resolving patient (AR1) and one patient with acute hepatitis C and chronic evolution (AC1) were stained with the HLA-DR1-HCV-1806-1818 tetramer as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000649#s2" target="_blank">methods</a>. For each patient the upper panels represent pre-enrichment stainings and the lower-panels the post-enrichment stainings. The numbers in the upper right corner of the post-enrichment panels are calculated according to the input CD4+ T cell number. The insert on top of the figure shows in detail the calculation method. These figures are plotted over time in both cases in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000649#pone-0000649-g002" target="_blank">Figure 2</a> (lower panels).</p

Clinical course of acute hepatitis C cohort in correlation to HCV specific CD4+ T cell response.

<p>The clinical course of each individual is shown in the upper panel in each case. Note the time scale (days) is different in each case. The upper panel shows the ALT (IU/L; open boxes; left hand y axis) and the serum HCV RNA level (closed triangles; right hand y axis). For AR1, AR2 and AC3, at some time points only qualitative assays for HCV-RNA were available which have been marked with a +. Interferon alpha therapy is marked with a bar. In patients AC1 and AC2 time points where the sequence of epitope 1806-1818 was determined are indicated by arrows. The middle panel in each case corresponds to functional CD4+ T cell assays: proliferation in response to NS3/4-antigen (open boxes) and peptide-1806-1818 (open triangles) is shown as stimulation index (SI) on the left hand y axis and HCV-antigen induced interferon-gamma secretion as determined by Elispot (solid diamonds) is shown as spot forming units (SFU) on the right y-axis. The lower panel of each case shows the frequency of HLA-DR1-HCV-1806-1818 tetramer positive CD4+ T cells (grey circles). The cut-off for detection was 0.001%. The upper limit of normal for ALT is indicated in panel AR2 as dotted line.</p

CD38 expression of HLA-DR1-HCV-1806-1818-tetramer positive CD4+ T cells.

<p>Longitudinal phenotypic analysis of the DR1 tetramer positive cells was performed for the activation marker CD38. The dot plots shown are gated on the DR1 tetramer positive cell population. Representative examples of the staining for AR1, AC1, and AC3 at early and late time points are shown and the percentage of CD38 positive cells is given for each plot. The inserted histograms show CD38 staining on all CD4+ T cells pre-enrichment and have been used to set the quadrants for the DR1 tetramer staining.</p