EPs 7630 stimulates human blood immune cells.

<p>(<b>A, B</b>) PBMCs, isolated from the blood of healthy donors, were treated with EPs 7630 as indicated, TLR3 and TLR4 ligands (T3L, T4L), a cytokine mixture (IL-1β, IL-2, IL-12), or anti-CD3 and anti-CD28 antibodies for 4 h and 24 h. TNF-α (4 h), IL-6 (24 h), and IL-10 (24 h) were quantified in respective culture supernatants by ELISA. Mean (± SEM) data from 6 donors are given as cytokine concentrations (A) or TNF-α/IL-10 ratio (B). (<b>C</b>) Healthy donor PBMCs were cultured in the presence of different concentrations of EPs 7630 as indicated for 48 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 12 donors are given. (<b>D</b>) Healthy donor PBMCs were stimulated with EPs 7630 and TLR4 ligand as indicated for 4 to 72 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 4 donors (except TNF-α 48h: n = 3) are given. (<b>A</b>, <b>B</b>) Significant differences compared to control group are indicated (* <i>p</i><0.05, ** <i>p</i><0.01, Wilcoxon matched-pairs signed-rank test).</p

TLR3- and TLR4-induced responses in immune cells are modulated by EPs 7630 pretreatment.

<p>Healthy donor PBMCs were treated with different concentrations of EPs 7630 as indicated for 24 h. Afterwards, TLR3 or TLR4 ligands (T3L, T4L) were added for additional 24 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 12 donors are given as percent of 0 μg/ml EPs 7630 group (control). Cytokine concentrations in the absence of EPs 7630 in TL3L and TL4L groups were: 1267±414 and 1534±324 pg/ml (TNF-α), 1278±369 and 2135±650 pg/ml (IL-6), 1011±127 and 1430±140 pg/ml (IL-10), respectively. Significant differences compared to 0 μg/ml EPs 7630 group are indicated (* <i>p</i><0.05, ** p<0.01, Wilcoxon matched-pairs signed-rank test).</p

CD4⁺ memory T cells are not the main responders to EPs 7630.

<p>(<b>A</b>) Memory T-helper cells (CD4⁺ CD45RO⁺) were isolated from healthy donor PBMCs by magnetic labeling-based cell sorting. Representative dot plots of the cells (live gate cells with CD4⁺ lymphocytes marked in blue), stained and analyzed by flow-cytometry before and after sorting, are given (<i>left panel</i>). Mean (± SEM) purity of isolated cells was 97.68 ± 0.4%. Isolated cells were then treated with different concentrations of EPs 7630 as indicated for 48 h, followed by quantification of TNF-α, IL-6, and IL-10 in respective culture supernatants by ELISA. Mean (± SEM) cytokine concentration data from 5 donors are given (<i>right panel</i>). (<b>B</b>) Memory T-helper cells, isolated as in (A), were treated with different concentrations of EPs 7630 as indicated for 24 h. Subsequently, cells were additionally stimulated with anti-CD3/anti-CD28 antibodies for another 24 h. Quantification of TNF-α, IL-6 and IL-10 in respective culture supernatants was carried out by ELISA. Mean (± SEM) cytokine concentration data from 5 donors are given as percent of 0 μg/ml EPs 7630 group (control). Cytokine concentrations in the absence of EPs 7630 were: 4753±608 pg/ml (TNF-α), 3.4±0.9 pg/ml (IL-6), 737±103 pg/ml (IL-10). (<b>C</b>) Healthy donor PBMCs were stimulated with different concentrations of EPs 7630 as indicated for 4 to 5 h. Brefeldin A was added for the last 3 to 4 h of culture, followed by antibody-based staining of intracellular TNF-α and surface markers and subsequent flow-cytometric analysis. Representative dot plots showing the proportions of TNF-α⁺ cells out of all CD4⁺ cells of one out of 3 independent experiments are given.</p

EPs 7630 mediates its effects via specific activation of MAPKs.

<p>(<b>A, B</b>) Healthy donor PBMCs were treated with different concentrations of EPs 7630 as indicated for 4 to 5 h. Brefeldin A was added for the last 3 to 4 h of culture, followed by antibody-based staining of surface markers and intracellular TNF-α and subsequent analysis by flow-cytometry. (A) Representative dot plots of CD14⁺ cells (monocytes) from one representative experiment are given, showing proportions of TNF-α⁺ cells out of all CD14⁺ cells. (B) Mean (± SEM) data of the proportions of TNF-α⁺ cells out of all CD14⁺ cells from 3 donors are given. Significant differences compared to control group (0 μg/ml EPs 7630) are indicated (* <i>p</i><0.05, Wilcoxon matched-pairs signed-rank test). (<b>C</b>) Healthy donor PBMCs, starved for 3.5 h, were treated or not (control) with different concentrations of EPs 7630, TLR3 and TLR4 ligands (T3L, T4L), cytokine mixture (IL-1β, IL-2 and IL-12), and anti-CD3/anti-CD28 coated Dynabeads for 10 and 30 min as indicated. Activation of the phosphorylated signaling molecules JNK1/2, p38, Erk1/2, Akt, STAT5, and p65 as well as of the standardization marker GAPDH was assessed by Western blot analysis. (<b>D</b>) Healthy donor PBMCs were pretreated with inhibitors targeting MAP kinase and NF-κB signaling pathways [SP600125 (JNK1/2/3), PD98059 (MEK), SB202190 (p38α/β), wedelolactone (IκB kinase)] or a respective control solvent for 45 min, followed by addition of 10 μg/ml EPs 7630 and culture for further 4 h. Brefeldin A was added for the last 3 h of culture, followed by antibody-based staining of surface markers and intracellular TNF-α, and analysis by flow-cytometry. Mean fluorescence intensity (MFI) data of TNF-α signal in CD14⁺ cells (monocytes) are given from 5 donors as mean ±SEM. Significant differences compared to control group are indicated (* <i>p</i><0.05, Wilcoxon matched-pairs signed-rank test).</p

Parameter levels and frequency of fulfilled criteria for metabolic syndrome in AI patients and control persons.

<p>(A) The average waist circumference, plasma HDL-cholesterol and TG levels, systolic and diastolic blood pressure, and fasting plasma glucose levels in AI patients and control participants are demonstrated as the mean ± SEM. Significance of differences was assessed by the Mann–Whitney U-test (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). (B) The frequency of central obesity, hypo-HDL-cholesterolemia, hypertriglyceridemia, hypertension, hyperglycemia in AI patients and controls are given. Significance of differences was assessed by the Chi-square test (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). (C) The percentages of AI patients and controls with metabolic syndrome are given. Significance of differences was assessed by the Chi-square test (**<i>P</i><0.01).</p

Correlation of disease severity and duration with parameters of the metabolic syndrome for AI patients.

<p>The correlation of Sartorius scores and duration of AI with each plasma TG levels and the number of positive metabolic syndrome criteria was investigated by Spearman's rank correlation analysis. No significant correlation was found.</p

Demographic characteristics of AI patients and controls.

<p>The <i>P</i>-values calculated by the Chi-square test (sex distribution) or Mann–Whitney U test (age) are indicated.</p

Percentages of AI patients and control participants that fulfilled the indicated number of metabolic syndrome criteria.

<p>The dashed vertical line marks the cutoff for the metabolic syndrome.</p

Correlation between age and metabolic syndrome for AI patients and control participants.

<p>(A) The correlation of age with the number of fulfilled metabolic syndrome criteria was investigated by Spearman's rank correlation analysis for AI patients and controls. Significant correlation was found only for controls (Spearman's rank correlation coefficient r_s = 0.363, ***<i>P</i><0.000). (B) The prevalence of the metabolic syndrome in AI patients and control participants in different age groups is given (≤34 years old AI patients: n = 22; ≤34 years old control participants: n = 36; 35 to 44 years old AI patients: n = 27, 35 to 44 years old control participants: n = 23; ≥45 years old AI patients: n = 31; ≥45 years old control participants: n = 41). Significance of differences was assessed by the Chi-square test (*<i>P</i><0.05, ***<i>P</i><0.001).</p

Correlation of severity and duration of AI with parameters of metabolic syndrome.

<p>The correlation was investigated by Spearman's rank correlation analysis. For each field, the Spearman's rank correlation coefficient and, in parenthesis, the <i>P</i>-values are indicated.</p