Protein expression of the immunoproteasome subunits PSMB8 and PSMB9 relative to beta actin in IM and NIM.

<p>The protein expression of the two subunits was analyzed by western blot in muscle biopsies from four non-inflammatory myopathies (NIM) and four inflammatory myopathies (IM). Intensity of chemiluminescent signals for PSMB8 and PSMB9 was normalized to corresponding signals of the housekeeping protein beta actin, which was detected in a second staining procedure on the same membrane. Only IM samples revealed PSMB8 and -9 protein expression.</p

Comparison of immunoproteosomal subunit expression between paired samples from isolated cells and muscle tissue:

<p>Gene expression analysis of immunoproteasomal subunits (PSMB8–10) in muscle biopsies vs. CD4+, CD8+, CD14+, CD19+, and DCs from patients with inflammatory myopathies (IM) and patients with non-inflammatory myopathies (NIM). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*) and p<0.01 (**).</p

Expression of immunoproteasomal subunits in immune cells:

<p>Gene expression of immunoproteasomal subunits (PSMB8–10) in CD4+, CD8+, CD19+, CD14+ and DCs of patients with myopathies (PM, DM, OM, NIM) and controls (HD). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*) and p<0.01 (**). Significantly higher expression of PSMB8 was observed in CD14+ cells and DC of PM patients compared to HD or DM, NIM and HDs, respectively. PSMB9 was increased in CD8+ and CD14+ of DM and in DCs of PM and DM patients compared to NIM. PSMB10 was found increased in PM patients compared to DM, NIM and HD in CD8+, compared to HD in CD19+ and CD14+ cells and compared to OM, NIM, and HD in DCs.</p

Quantification of cellular infiltration and confirmation of PSMB8/-9 gene activation in myositis muscle samples:

<p>Transcriptome data referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s007" target="_blank">table S2</a> were re-investigated. A) Cell type infiltration was quantified based on the expression of cell specific marker transcripts (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s005" target="_blank">figure S5</a>). Signal ratios between muscle biopsy and purified cell type were calculated for each cell type specific transcriptional markers. Taking the median of the ratios in each cell specific marker set and scaling their sum to 100% revealed an estimate of the cellular composition. Increase of monocyte, dendritic cell and T-cell transcription patterns corresponds to the molecular myositis score. (ND = normal donor; NM = necrotizing myopathy). B) The maximum of infiltration related signal intensity was calculated as described in materials and methods. Comparing these expected intensities (x-axis) with real intensities (y-axis) in each sample, PSMB8 and PSMB9 are higher expressed than expected in the majority of IBM and several of the PM muscle. In DM, PSMB8 expression also exceeds the expected signal intensity, although on a lower level. One out of the 8 DM samples (DM_13) showed consistently a pattern similar to the highly inflamed IBM samples. All other conditions including controls remained below the expected intensity. For PSMB10, the increased signal intensity in several samples of IBM seems to correspond to the level of infiltration.</p

Correlation of immunoproteasome with IFN and IFNR expression in myositis muscle tissue:

<p>Transcriptome data referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s007" target="_blank">table S2</a> were re-investigated. A) Correlation analysis was performed for 133P and 133A datasets independently. Only IFNγ revealed high correlation coefficients with PSMB8, PSMB9 and PSMB10. The corresponding constitutively expressed subunits PSMB5-7 were not or even negatively correlated. B) Comparing the association of IFNγ with PSMB8, -9 and -10 for each sample individually, the increase was much higher for PSMB8/-9 compared to PSMB10 as described before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone-0104048-g006" target="_blank">figure 6</a>).</p

Expression of catalytic proteasomal subunits in muscle:

<p>Gene expression analysis of constitutive and immunoproteasomal subunits (PSMB5–10) in muscle biopsies of patients with inflammatory myopathies (IM) and patients with non inflammatory myopathies (NIM). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*), p<0.01 (**) and p<0.001 (***). Comparing to NIM, mean relative expression levels in IM revealed 4-fold for PSMB8 [0.302±0.139 and 0.075±0.041] and about 5-fold increase for PSMB9 [0.049±0.029 and 0.009±0.002] but less than 2-fold for PSMB10 [0.065±0.064 and 0.036±0.022].</p

Correlation of proteasome subunit expression with myositis signatures and overlap with immune cell infiltration:

<p>Expression of all proteasome units were investigated in transcriptome data of muscle biopsies referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s007" target="_blank">table S2</a> and were correlated with genes increased in myositis muscle tissue. The heatmap of the probeset signal intensities (A) is opposed to the heatmap of the gene by gene correlation matrix (B) in 133P array experiments. Identical analysis in 133A arrays are shown in D and E. Expression of the 1209 probesets in reference transcriptomes of purified immune cells from the blood of healthy donors served as basis for clustering by probesets and cell types (C). Identification of cell type specific transcriptional patterns in the 1209 probesets demonstrates the extent of overlap of these probesets with immune cell related transcripts. Myositis samples include IBM (red), PM (orange), DM (yellow), NM (necrotizing myopathy)/IM (inflammatory myopathy) (light green) and HD (green). Immune cell types include neutrophils (dark blue), monocytes (blue), dendritic cells (light blue), CD4+ T-cells (dark violet), CD8+ T-cells (violet), NK-cells (light violet), B-cells (cyan), and healthy muscle (moss-green). A detailed graph with all probesets labelled is presented in supplemental material (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s004" target="_blank">figure S4</a>).</p

Additional file 2: Table S2. of Restrictive IgG antibody response against mutated citrullinated vimentin predicts response to rituximab in patients with rheumatoid arthritis

Sequence of selected mutated citrullinated vimentin (MCV) with epitopes from 1 to 88. The entire protein sequence of MCV was cut into 88 peptides consisting of 17 amino acids, with 12 overlapping amino acids to the neighbourhood peptide, with following amino acid code: X = citrulline; X = mutated glycine = citrulline; H = mutated serine = histidine. (DOCX 18 kb

Additional file 3: Figure S1. of Restrictive IgG antibody response against mutated citrullinated vimentin predicts response to rituximab in patients with rheumatoid arthritis

ROC curve analysis for AMCV IgA assay. Cut-off level at 20 U/ml resulted in a specificity of 100 % and sensitivity of 42 %. (DOCX 23 kb

Additional file 1 of Planning preclinical confirmatory multicenter trials to strengthen translation from basic to clinical research – a multi-stakeholder workshop report

Additional file 1: Figure S 1. Composition of workshop participants as per field of expertise. The organizing team mainly consists of meta-researchers with a clinical or preclinical background. Other refers to e.g., stakeholders from funding agencies that also attended the workshop. Methods S1. Multi-stakeholder workshop method-Workshop design. Glossary. Glossary of terms