Expression of surface receptors by secretions-differentiated macrophages.

<p>Results, expressed as the mean fluorescence intensity (MFI), are means±SEM of 6–11 experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for control cells. ND: not detectable.</p

Light microscopy analysis of the effect secretions on the differentiation of monocytes to macrophages.

<p>Monocytes were differentiated to MØ-1 in the presence of GM-CSF (A) or in the presence of GM-CSF and 35 µg of secretions/ml (B) and their morphology evaluated. Similarly, monocytes were differentiated to MØ-2 in the presence of M-CSF (C) and in the presence of M-CSF and secretions (D). Results indicated in days are from a representative experiment.</p

LPS-induced chemokine production by secretions differentiated macrophages.

<p>The results for control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the chemokine production by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS. ND: not detectable.</p

LPS-induced growth factor production by secretions differentiated macrophages.

<p>The results for control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the production of growth factors by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS. ND: not detectable.</p

LPS-induced cytokine production by secretions differentiated macrophages.

<p>The results for the control macrophages are expressed as the median value and range, and are set at 100%. The effect of secretions is expressed as a percentage relative to the cytokine production by control cells. Results are means±SEM of at least six experiments. Values are significantly (*p<0.05 and **p<0.005) different from those for macrophages stimulated with LPS.</p

Cytokine production in response to LTA.

<p>We measured the production of IL-12p40 (A,B), TNF-α (C,D), IL-6 (E,F) and IL-10 (G,H) by control and secretions-differentiated MØ-1 and MØ-2 induced by a range of LTA. The results, expressed in ng/ml, are means±SEM of 12 experiments. Open bars: control macrophages; filled bars: secretions differentiated macrophages. *p<0.05 for differences from control macrophages.</p

Cytokine production in response to LPS.

<p>We measured the production of IL-12p40 (A,B) and IL-10 (C,D) by control and secretions-differentiated MØ-1 and MØ-2 in response to a range of LPS. The results, expressed in ng/ml, are means±SEM of 9–10 experiments. Open bars: control macrophages; filled bars: secretions differentiated macrophages. *p<0.05 for differences from control macrophages.</p

Effects of KYE28 against LPS <i>in vivo.</i>

<p>(<b>A-E</b>) Septic shock in C57BL/6 mice was induced by intraperitoneal (i.p.) injection of <i>E. coli</i> LPS (18 mg/kg) followed by i.p. injection of 0.5 mg of KYE28 or buffer 30 min later. (<b>A</b>) Survival of the animals challenged with LPS and buffer (Control n = 6) or KYE28 (n = 8) was monitored for 7 days. (<b>B</b>) Diagram presenting the weight development during the experiment in (A) for KYE28 treated mice. (<b>C</b>) Measurement of cytokines 8 and 20 h after LPS injection in mouse plasma (8 h: LPS n = 12, LPS+KYE28 n = 8; 20 h LPS n = 14, LPS+KYE28 n = 10). (<b>D</b>) Number of platelets were determined 8 and 20 h after LPS, as well as in survivors at day 7 (8 h: LPS n = 11, LPS+KYE28 n = 8; 20 h LPS n = 14, LPS+KYE28 n = 7, day 7 LPS+KYE28 n = 7, Control n = 8). (<b>E</b>) Lung sections of healthy (Control), LPS treated and LPS+KYE28 treated mice were analysed 20 h after LPS injection. Left panel illustrates representative light microscopy images stained with haematoxylin-eosin (original magnification 20x, scale bar: 100 µM) and the right panel shows representative scanning electron micrographs (scale bar: 20 µM).</p

MIC values of KYE28 and LL-37 for the indicated bacteria.

<p>MIC values of KYE28 and LL-37 for the indicated bacteria.</p

Modulation of NF-κB/AP-1 activation by KYE28.

<p>(<b>A</b>) Determination of NF-kB/AP-1 activation in supernatants of THP1-X-Blue CD14 cells after stimulation with 100 ng/mL <i>E. coli</i> LPS and increasing concentrations of KYE28 (n = 6). The HCII-derived peptide LGK23 (5 µM) was used as control (n = 3). (<b>B</b>) NF-kB/AP-1 activity was measured in supernatants of RAW-Blue cells stimulated with 10 ng/mL <i>E. coli</i> LPS and increasing concentrations of KYE28 (n = 4). (<b>C</b>) RAW-Blue cells stimulated with 10 ng/mL <i>E. coli</i> LPS and KYE28 (10 µM). (Together: LPS and peptide added at the same time; no removal: Addition of KYE28 1 h before addition of LPS; removal: Addition of KYE28 for 1 h, removal of the peptide followed by addition of LPS; Control  =  buffer only) (n = 4). (<b>D</b>) RAW-Blue cells were stimulated with 10 ng/mL <i>E. coli</i> LPS and KYE28 (10 µM) was added after indicated times (n = 5).</p