Screening of BCR and TLR9 stimulated kinase activation pattern in BJAB Burkitt's lymphoma cells by MAPK protein profiler array.

<p>BJAB cells were treated with 5 µg/ml anti-Ig and/or 2 µg/ml CpG for 30 minutes. Relative phosphorylation values were calculated as the ratios of signal strengths for each kinases as compared to positive control spots. Only kinases that were activated are shown.</p

BCR and TLR9 induced signals synergistically activate TAK1 and p38 MAPK in human B cells that is independent of BAFF.

<p>(A) Purified human B cells were left untreated (0 h) or pretreated with 100 ng/ml BAFF for 2 h or 20 h, and then in the last 30 min of pretreatment were activated with anti-Ig (2.5 µg/ml) and/or CpG (1 µg/ml), (B) B cells were stimulated with anti-Ig (2.5 µg/ml), CpG-ODN (2 µg/ml) or the combination of both reagents as indicated for 30 min, in the absence (-) or presence of specific TAK1 inhibitor, (5Z)-7-Oxozeaenol, then samples were subjected to Western blot analysis using pTAK1 or pp38 MAPK specific antibodies. C) Control and TAK1-specific siRNA transfected BJAB cells were activated with 2.5 µg/ml anti-Ig and 1 µg/ml CpG for 30 minutes, and subjected to Western blot analysis to measure TAK1, pAKT and pp38 level. SHP1 was used as a loading control.</p

Plasma blast generation induced by single or combined stimuli of B cells via BCR, TLR9 and BAFF-R is partially inhibited by (5Z)-7-Oxozeaenol.

<p>Purified B cells (2×10⁵ cells/well) were cultured with anti-Ig (2.5 µg/ml), BAFF (100 ng/ml) and CpG (0.5 µg/ml) for 4 days in the presence of IL-2 (50 ng/ml) and IL-10 (50 ng/ml). Percentages of CD27⁺⁺ CD38⁺ plasma blast cells were evaluated by flow cytometry. Samples in the right column were treated with 5Z-7 Oxozeaenol (100 nmol).</p

Phospho-flow analysis of p38 MAPK activation in resting and activated B cells from healthy blood donors and from RA patients.

<p>PBMC (4×10⁶ cells) were left unstimulated (control) or stimulated with anti-Ig (7.5 µg/ml), CpG (4 µg/ml) or both agents for 30 min at 37°C. p38 phosphorylation was tested in the CD19⁺ CD27⁻ naive B cell subset. (A) The activation of p38 MAPK in healthy (square; <i>n</i> = 17) and RA (triangle; <i>n</i> = 13) B cells were analyzed by flow cytometry. (B) The relative activation of p38 MAPK was calculated based on the alteration of phosphorylated MAPKs expression in resting and stimulated naive B cells (stimulated sample MFI/resting sample MFI). Paired Student's t-test was used to assess the differences of phospho-p38 MAPK expression between control and stimulated samples. *<i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.0005.</p

TAK1 dependent activation of the MAPK and NFκB pathways in B cells stimulated with combinations of anti-IgG, CPG ODN and BAFF.

<p>Resting tonsill B cells were stimulated with combinations of anti-Ig (2.5 µg/ml), BAFF (100 ng/ml) and CpG (2 µg/ml) for 30 min with or without TAK1 inhibitor ((5Z)-7-Oxozeaenol), and the phosphorylation of various signaling molecules was tested by Western blot.</p

Synergistic stimulation of IgG producing plasma cell differentiation by anti-Ig and CpG ODN in the presence of TAK1 inhibitor.

<p>Purified human blood B cells (10⁵ cells/well) were cultured with anti-Ig (2.5 µg/ml), BAFF (100 ng/ml) and CpG (0.5 µg/ml) as indicated below the figure for 4 days in the presence of IL-2 (50 ng/ml) and IL-10 (50 ng/ml). Numbers of IgG producing plasma cells were determined by ELISPOT assay. Spot numbers in the presence of vehicle (white bars) and spots in the TAK1 inhibitor treated samples (black bars) are shown. Means ±SD of three different experiments.</p

BCR synergizes with TLR9 on a TAK1 dependent manner for cytokine production in primary human B cells.

<p>Secreted IL-6, IL-8, IL-10 and TNFα were measured after 48 h from culture supernatants of vehicle treated (white bars) and TAK1 inhibitor treated (black bars) purified human tonsill (A) and blood (B) B cells. Data represent the mean ±SD of three different experiments with resting tonsill B cells (A), while one experiment with blood B cells (B) is shown. *p<0.05.</p

Detection of the level and the complement activating properties of autoantibodies against citrullinated peptides.

<p>The arginine or citrulline-containing form of fibrinogen β chain peptide (SGSG⁶⁰RPAPPPISGGGYRAR⁷⁴ vs. SGSG⁶⁰XPAPPPISGGGYXAX⁷⁴) (A) and filaggrin peptides (⁴⁵⁴TRGRS⁴⁵⁸K vs. ⁴⁵⁴TXGRS⁴⁵⁸K) (B) were coupled on beads and incubated with serum samples to detect peptide-specific IgG, IgM, IgA autoantibody levels and their complement activating properties. Median fluorescent intensity (MFI) values for arginine or citrulline-containing forms of the peptides in RA patient group (red lines) or the control group (blue lines) are shown in the upper panel. Significance level of differences between controls and RA patients were calculated only for the citrullinated peptides by Mann-Whitney test and the p-values are indicated in the upper panel as follow: * p-value<0.05; ** p-value<0.01, ***p-value<0.001. Spearman's Rho correlation coefficients calculated between anti-C3, anti-IgG, anti-IgM and anti-IgA MFI values are shown for citrullined (middle panels) or native (lower panels) form of the peptides, where non-significant correlations are indicated by (n.s.).</p

Dependence of complement activation on serum dilution rate.

<p>For measuring the background, classical, lectin & alternative and only alternative pathway activation, a serially diluted serum sample (1∶1–1∶160) was applied to empty, human IgG, human IgA and properdin-coupled beads, respectively. The assay buffer for serum dilution contained either Ca²⁺-Mg²⁺, which promotes complement activation, or EDTA, which blocks complement activation. Complement activation-driven C3 fragment deposition on beads was detected by PE-conjugated anti-human C3 antibody. Plots display for each serum dilution the respective median fluorescence intensity (MFI) value against varying concentrations of human IgG, human IgA and properdin coupled on beads. AU - arbitrary units</p

Level of EBNA-1 specific antibodies and their complement activation.

<p>EBNA-1 coupled beads were incubated with sera of RA patients and controls. <b><i>A</i></b><i>)</i> Levels of bound human IgG, IgM and IgA antibodies along with the degree of complement activation are plotted for the two groups. Mann-Whitney non-parametric statistical test was used to calculate the statistical difference between the two groups. <b><i>B</i></b><i>)</i> Signal intensities for anti-IgM, anti-IgA and anti-C3 for the controls (upper panel) and RA patients (lower panel) are plotted in a 3D graph. The tables show the Spearman's Rho correlation coefficients between anti-C3, anti-IgG, anti-IgM and anti-IgA signal intensities calculated separately for the control and RA patient groups. Only statistically significant (p-value<0.05) correlation coefficients are shown and non-significant correlations are indicated by (n.s.).</p