Additional file 1: Table S1. of A novel DNA/histone H4 peptide complex detects autoantibodies in systemic lupus erythematosus sera

Number of positive sera in the different control diseases. (DOC 31 kb

Expression of Ig receptors on the U937-NF-κB reporter cell line.

<p>U937 cells express FcγRI, FcγRII, FcαRI of Fc receptors (A). Transfection with NF-κB-GFP did not alter the Fc receptor expression patterns in the U937 cell line (B). U937 and U937-NF-κB cells were labeled with PE-conjugated anti-FcγRI, FcγRII, FcγRIII and FcαRI, antibodies. The histograms show the unlabeled cells (filled histogram), the corresponding isotype control labeled cells (dashed line) and the specific antibody stained cells (solid line).</p

Activation of U937-NF-κB cells occurs mainly through FcγRI.

<p>The U937-NF-κB cells were activated on anti-FcγR-coated surfaces (A). FcγRI cross-linking induces strong, while FcγRII results in weaker activation. The cell activation was measured on IgG4-coated surfaces (B, C, D) in the presence of soluble IgG1(B), IgG3 (C) and IgG4 (D) in three doses (10, 20, 30 μg/ml). The untreated cells on IgG4 coat served as control (black bars).Results are mean % ± SEM % (n = 9) *p<0.05 versus control (untreated cells).</p

Detection of NF-κB activation by U937-NF-κB cells.

<p>Cells were treated with 10 μg/ml LPS (A), and the activation was detected for 24 h. Cells subjected to the medium only served as a negative control. The activation reached the maximum at 12 h. Cells were also activated on 10 μg/ml human immunoglobulin G (hIgG) coated surface (B). For negative controls, cells subjected to the medium only or to 10 μg/ml soluble human immunoglobulin (shIgG) on uncoated surface were used. The activation was tested on human immunoglobulin coat in the presence of 10 μg/ml goat-anti human immunoglobulin (F(ab’)₂).</p

Time- and dose-dependent activation of U937-NF-κB cells.

<p>The activation of U937-NF-κB cells was screened on plates coated with BSA, human IgG1, IgG2, IgG3, IgG4, IgA, or IgM in two-fold serial dilution from 20 μg/ml to 1.25 μg/ml concentration. Following a one hour adhesion period, the cells were imaged for 12 hours by HCS (A-F). The results were normalized to the fluorescence intensities of cells measured on BSA coat. Soluble form of LPS served as positive control (G).</p

Design, synthesis, conformational analysis, and biological activity of Cα¹-to-Cα⁶ 1,4- and 4,1-disubstituted 1<i>H</i>-[1,2,3]triazol-1-yl-bridged oxytocin analogues

Oxytocin (OT) is a neurohypophyseal peptide hormone containing a disulphide-bridged pseudocyclic conformation. The biomedical use of OT peptides is limited amongst others by disadvantageous pharmacokinetic parameters. To increase the stability of OT by replacing the disulphide bridge with the stable and more rigid [1,2,3]triazol-1-yl moiety, we employed the Cu2+-catalysed side chain-to-side chain azide-alkyne 1,3-cycloaddition. Here we report the design, synthesis, conformational analysis, and in vitro pharmacological activity of a homologous series of Cα1-to-Cα6 side chain-to-side chain [1,2,3]triazol-1-yl-containing OT analogues differing in the length of the bridge, location, and orientation of the linking moiety. Exploiting this macrocyclisation approach, it was possible to generate a systematic series of compounds providing interesting insight into the structure-conformation-function relationship of OT. Most analogues were able to adopt similar conformation to endogenous OT in water, namely, a type I β-turn. This approach may in the future generate stabilised pharmacological peptide tools to advance understanding of OT physiology.</p

Designed Glucopeptides Mimetics of Myelin Protein Epitopes As Synthetic Probes for the Detection of Autoantibodies, Biomarkers of Multiple Sclerosis

We previously reported that CSF114­(Glc) detects diagnostic autoantibodies in multiple sclerosis sera. We report herein a bioinformatic analysis of myelin proteins and CSF114­(Glc), which led to the identification of five sequences. These glucopeptides were synthesized and tested in enzymatic assays, showing a common minimal epitope. Starting from that, we designed an optimized sequence, SP077, showing a higher homology with both CSF114­(Glc) and the five sequences selected using the bioinformatic approach. SP077 was synthesized and tested on 50 multiple sclerosis patients’ sera, and was able to detect higher antibody titers as compared to CSF114­(Glc). Finally, the conformational properties of SP077 were studied by NMR spectroscopy and structure calculations. Thus, the immunological activity of SP077 in the recognition of specific autoantibodies in multiple sclerosis patients’ sera may be ascribed to both the optimized design of its epitopic region and the superior surface interacting properties of its C-terminal region

Heatmap and principal component analysis of the functional antibody profiling data.

<p>Samples are ordered as study groups. Variables are grouped by the molecular composition and the nature of the detected serum immunoglobulin or complement protein, the order of the particular antigens is shown and applies for each detected protein. PCA plot of samples shows the distribution of samples in the space of the first two components. The variable plot shows the contribution of the variables to the generation of this space, where distance and orientation from the zero origin define a particular interaction’s load in the first two principal components. Percentages in parenthesis indicate contribution of the indicated principal component to overall variability in the dataset. xdsDNA, ultrasound-fragmented dsDNA; CENPB, centromere protein B; Sm-p, peptide of Smith antigen D polypeptide</p

Association of organ involvement with ITGAM genotype and immune complex composition.

<p><b>a</b>. Percentage of the SLE patients with the involvement of the indicated three organs is shown for rs1143679 genotypes. The presence of single, double or triple organ involvement is illustrated by Euler ellipses with areas proportional to the percentages shown. <b>b</b>. Association with organ involvement of the level and composition (IgG, IgM, C4 and IgG/IgM) of immune complexes formed on dsDNA and nucleosome is indicated. Odds ratios for patients falling into the indicated percentile groups (upper 10^th, 25^th, 50^th and lower 10^th, 25^th, 50^th percentiles) for the given measurements are shown. Positive odds (>1) are in red, negative odds (<1) are in blue.</p

Clinical and serological characteristics of the SLE patient cohorts.

<p>Clinical and serological characteristics of the SLE patient cohorts.</p