The number of small RNAs in serum detected by Illumina sequencing.

<p>The number of small RNAs in serum detected by Illumina sequencing.</p

Validation of deep sequencing results for selected miRNAs.

<p>We have registered 10 samples in each group listed on Table S3. The threshold cycle for each miRNA primer/probe set were normalized with spiked in cel-miR-39 primer/probe pair and compared to CH-C-5. Result for normally distributed continuous variables are given as means and compared between groups by Student's t-test. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066086#s2" target="_blank">Results</a> for non-normally distributed continuous variables are summarized as medians and were compared by Mann-Whitney U test. Statistical significance indicates by one asterisk (p<0.05) and two (p<0.01).</p

Heat map and hierarchical clustering.

<p>Individual miRNA expression were calculated by R platform and heat map was computed and described using a function of heatmap.2 in gplots. It uses hierarchical clustering with Euclidean distance; Pearson Linear Correlation and Ward's method to generate the hierarchical tree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066086#pone.0066086-Ward1" target="_blank">[56]</a>. ANOVA was applied to extract differentially expressed miRNAs and adjustment of the p-value by multiple comparisons was performed by calculating FDR. Those miRNAs with FDR<0.1 were presented. The red indicates high level of miRNA expression and the blue shows low.</p

Differentially expressed miRNAs in serum from PBC patients compared with the second group (CH-C, CH-B, Healthy).

<p>Differentially expressed miRNAs in serum from PBC patients compared with the second group (CH-C, CH-B, Healthy).</p

Predicted target genes of 9 differentially expressed miRNA by Illumina sequencing in PBC.

*<p>miRIS :miRror Internal Score ranges from 0 top 1 by average 2 components (number of databases and input hits).</p

Clinical data for patients enrolled in Illumina sequencing analysis.

a<p>We used the Scheuer score in PBC and fibrosis score of histological activity index (HAI) in CH-B and CH-C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066086#pone.0066086-Knodell1" target="_blank">[53]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066086#pone.0066086-Ludwig1" target="_blank">[54]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066086#pone.0066086-Scheuer1" target="_blank">[55]</a>.</p>b<p>URSO is the abbreviation for ursodeoxycholic acid, IFN for interferon and NA for nucleos(t)ide analogue.</p

Clinical information of patients enrolled in the validation study.

a<p>The numbers of patients are indicated in the parentheses.</p>b<p>The range of laboratory data is indicated in the parentheses.</p

HCV Infection Enhances Th17 Commitment, Which Could Affect the Pathogenesis of Autoimmune Diseases

<div><p>Background</p><p>Various kinds of autoimmune diseases have been reported to have a significant relationship with persistent hepatitis c virus (HCV) infection and Th17 cells. Previously, our group reported that the existence of HCV in T lymphocytes could affect the development of CD4⁺ helper T cells and their proliferation, in addition to the induction of immunoglobulin hyper-mutation.</p><p>Methods</p><p>Therefore, we analyzed the relationship between persistent infection of HCV and the mechanism of Th17 cell induction <i>ex vivo</i> and <i>in vitro</i>.</p><p>Results</p><p>The prevalence of autoimmune-related diseases in chronic hepatitis c patients (CH-C) was significantly higher than in other types of chronic hepatitis (hepatitis B and NASH). A significantly higher frequency of IL6 and TGF-β double-high patients was detected in CH-C than in other liver diseases. Moreover, these double-high patients had significantly higher positivity of anti-nuclear antibody, cryoglobulinemia, and lymphotropic HCV and higher amounts of IL1-β, IL21, IL23. In addition to the previously reported lymphotropic SB-HCV strain, we found a novel, genotype 1b lymphotropic HCV (Ly-HCV), by deep sequencing analysis. Lymphotropic-HCV replication could be detected in the lymphoid cells with various kinds of cytokine-conditions including IL1β, IL23, IL6 and TGF-β in vitro. Infection by HCV could significantly enhance the development of Th17 cells. The HCV protein responsible for inducing the Th17 cells was HCV-Core protein, which could enhance the STAT-3 signaling and up-regulate the expression of RORγt as a Th17 master gene.</p><p>Conclusion</p><p>Infection by lymphotropic HCV might enhance the Th17 development and contribute to understanding the pathogenesis of autoimmune-related diseases.</p></div

The cytokine conditions affecting the positivity of ANA and Cryoglobulin, and Th17 development.

<p>A comparison of the amounts of IL6 and TGF-β among the CH-C, CH-B, NASH and healthy subjects is shown (A). The bar indicates the mean cytokine amounts. The frequency of TGF-β1 high, IL6 high, and TGF-β1 and IL6 double high patients among the 4 groups (CH-C, CH-B, NASH, and healthy subjects) is shown (B). The positive rate of ANA and Cryoglobulin in the double high CH-C patients (n = 9) and the other CH-C patients (n = 26) is shown(C). The IL6 and TGF-β1 mRNA expression of PBMCs in the double-high patients (n = 9) and other patients (n = 26) is shown in the bar graphs (D). The amounts of IL1β, IL17A, IL21 and IL23 in the serum were compared between double high CH-C patients (n = 9) and the other CH-C patients (n = 26) (E). The comparisons of serum cytokines before and after the Peg-interferon/Ribavirin treatment are shown (F). Serum samples were collected at just before the treatment and twelve month after the end of treatment. SVR indicates sustained virological treatment (n = 5).</p

The identification of proteins responsible for enhancing the Th17 development (A).

<p>The transfection of various kinds of plasmids expressing HCV-individual proteins (E1, E2, Core, NS3, NS4B, NS5A, NS5B and vector) was carried out by nucleofector. The cells were analyzed at 72 hours post-transfection. The bar graph indicates the IL17A-secreting cells among the sample's CD4⁺ cells/IL17A secreting cells and the vector's CD4⁺ cells×100. The obtained data were analyzed by Mann-Whitney U test. Three independent experiments were carried out. <b>The analysis of STAT-1 and STAT-3 signaling (B).</b> We used a pathscan to quantify sequentially the phosphor-STAT-1 and STAT-3. The dotted lines indicate data of the vector control. Three independent experiments were carried out. <b>Long-term culture affected the commitment of naïve T lymphocytes with HCV-core expressing Lenti-virus (C).</b> The gene expressions of T-bet, GATA-3 and ROR-γt were analyzed by real-time PCR. The relative amounts of mRNA were calculated by ΔΔCT methods. The target gene expressions were analyzed at pre-inoculation of Lent-virus and 10 days after the inoculation of lenti-virus. Three independent experiments were carried out.</p