Ventilator–induced lung injury.

<p><b>A:</b> Pulmonary function is represented by the ratio of arterial oxygen partial pressure to fractional inspired oxygen concentration (PaO₂/FiO₂). <b>B:</b> Pulmonary edema is represented by wet to dry ratio of lung tissue (Wet/Dry). <b>C:</b> Alveolar–capillary permeability is represented by total protein levels in bronchoalveolar lavage fluid (BALF). <b>D:</b> On BALF cytospin preparations, differential cell counts were performed to determine neutrophil exudation into the alveolar space. <b>E:</b> In total lung homogenates, mRNA expression of the chemo–attractant keratinocyte–derived chemokine (KC) was determined by real–time RT–PCR. <b>F:</b> In total lung homogenates, protein expression of KC was determined by multiplex cytokine analysis. Expression levels were normalized for internal control concentrations (E), i.e. the average value of β–actin and glyceraldehyde 3–phosphate dehydrogenase (GAPDH), or total protein concentrations (F). Data are expressed as boxplot (min–max) of 18–22 (A) or 6–16 (B–F) mice per group (* p<0.05, ** p<0.01, *** p<0.001). E–F: Data are depicted relative to non–ventilated controls (NVC). REF = assumed PaO₂/FiO₂ ratio from mice with non–injured lungs; LV_T, HV_T = mechanically ventilated with low or high tidal volumes.</p

Vascular endothelial growth factor (VEGF).

<p>In total lung homogenates, protein expression of VEGF was determined by multiplex cytokine analysis. Levels were normalized for total protein concentrations. Data are expressed as boxplot (min–max) of 6–10 mice per group (* p<0.05, *** p<0.001). Data are depicted relative to non–ventilated controls (NVC). Veh, Dex = intravenously treated with either vehicle (sterile saline) or dexamethasone; HV_T = mechanically ventilated with high tidal volumes.</p

Hemodynamic and arterial blood gas variables.

<p>Veh, Dex = mechanically ventilated and treated with vehicle or dexamethasone; HR = heart rate in beats per minute; BP = systolic blood pressure in mmHg; PaO₂ = partial pressure of arterial oxygen in mmHg; PaCO₂ = partial pressure of arterial carbon dioxide in mmHg; BE = base excess in mmol/l. Data are presented as median [IQR] of 6–8 (A) or 10–16 (B) mice per group.</p

Alveolar–capillary barrier dysfunction.

<p><b>A:</b> Pulmonary function is represented by the ratio of arterial oxygen partial pressure to fractional inspired oxygen concentration (PaO₂/FiO₂). <b>B:</b> Pulmonary edema is represented by wet to dry ratio of lung tissue (Wet/Dry). <b>C:</b> Alveolar–capillary permeability is represented by total protein levels in bronchoalveolar lavage fluid (BALF). Data are expressed as boxplot (min–max) of 11–18 (A) or 9–12 (B–C) mice per group (*** p<0.001). REF = assumed PaO₂/FiO₂ ratio from mice with non–injured lungs; NVC = non–ventilated controls (NVC); Veh, Dex = intravenously treated with either vehicle (sterile saline) or dexamethasone; HV_T = mechanically ventilated with high tidal volumes.</p

Patient characteristics.

<p>Age, CPB, ACC and PICU durations represented as median ± SD. ACC: aortic crossclamping, ASD: atrial septum defect, AVSD: atrioventricular septum defect, CoA: Coarctation aorta, CPB: cardiopulmonary bypass, Extracardiac conduit change due to stenosis after Fontan procedure, PICU: pediatric intensive care unit, VSD: ventricular septum defect. No significant differences were found between both patient groups (Mann-Whitney test).</p

Post-perfusion plasma does not interfere with p38 MAPK or NF-κB activation.

<p>Representative examples (<b>A</b>) and densitometric analyses (<b>B–C</b>) of LPS-induced p38 MAPK and IκB-α phosphorylation in monocytes in the presence of 24 h (control) or 4 h post-surgery plasma. Tubulin: loading control. Mean ± SEM (n = 4). *<i>P</i><0.05 vs. 0 min (ANOVA).</p

Post-perfusion plasma suppresses LPS-induced TNF-α production by monocytes.

<p><b>A</b>. Percentage of TNF-α producing cells in the monocyte population after <i>ex vivo</i> LPS stimulation (100 ng/mL) of patient PBMC isolated at various time points (n = 4). <b>B</b>. Reduced TNF-α synthesis by monocytes after LPS (10 ng/mL) stimulation in whole blood assays with patient samples obtained at the indicated time points (n = 5). <b>C</b>. Experimental setup for experiments shown in D,E,G-I. In short, patient PBMC obtained before surgery (Pre-op) were mixed with control (pooled AB plasma from healthy donors) or autologous patient plasma samples obtained at indicated time points, followed by LPS (100 ng/mL) stimulation for 4 h. Monocyte populations (CD14/SSC gate) were then analyzed for intracellular TNF-α and IL-6 synthesis. <b>D</b>. Significantly reduced production of TNF-α by monocytes after LPS stimulation in the presence of plasma samples from different sources (n = 13). Shown are percentages of TNF-α producing monocytes relative to control (100%). *<i>P</i><0.05, **<i>P</i><0.001 vs. control (ANOVA). <b>E</b>. Percentages of IL-6 producing monocytes as in D. **<i>P</i><0.001 vs. control (ANOVA). <b>F</b>. Dexamethasone levels in patient plasma samples as measured by radio-immunoassay (n = 9). Median ± interquartile range. *<i>P</i><0.05 vs. pre-op (ANOVA). <b>G</b>. Production of TNF-α and IL-6 by monocytes after LPS stimulation in the presence of dexamethasone-free plasma samples (n = 4). *<i>P</i><0.05 vs. control (ANOVA). <b>H</b>. Mean fluorescence intensities (MFI) of TNF-α and IL-6 in monocytes after LPS stimulation in different plasma milieus (n = 7). *<i>P</i><0.05, **<i>P</i><0.001 vs. control (ANOVA). <b>I</b>. Representative flow cytometry results (contour plots) of the LPS-induced TNF-α production by monocytes in the presence of control or patient plasma (Pre-op, End-CPB, 4 h or 24 h post-perfusion plasma from a No-dexamethasone patient). Isotype control: mouse IgG1. Data represented as mean ± SEM, unless otherwise indicated.</p

STAT3 signaling is required for the suppressive effects of post-perfusion plasma on TNF-α production.

<p><b>A</b>. Pre-treatment of 4 h post-surgery plasma samples with anti-IL-10 partially restored TNF-α production by patient monocytes in response to LPS (n = 10). Control: plasma from healthy donors. <b>B</b>. Activation of STAT3 in monocytes by incubation with suppressive (4 h post-perfusion) but not control (24 h post-perfusion) plasma. Cells were incubated in the absence or presence of LPS to match the experimental setup as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035070#pone-0035070-g002" target="_blank">Fig. 2</a>. <b>C</b>. Pre-treatment of patient PBMC with active STAT3 inhibitor (pY-STAT3i) but not control peptide (STAT3i) before LPS stimulation in the presence of post-surgery plasma restored TNF-α synthesis (left panel), in contrast to IL-6 (right panel). Shown are percentages of TNF-α and IL-6 producing monocytes normalized to control (24 h post-surgery) plasma (n = 8). <b>D</b>. TNF-α and IL-6 levels measured in supernatants of LPS-stimulated mononuclear cells after pre-treatment with STAT3 inhibitor or control peptide, in the presence of 4 h post-surgery plasma (n = 8). Cytokine levels were normalized to LPS stimulation in control plasma from healthy donors due to interassay variability. All results are depicted as mean ± SEM. *<i>P</i><0.05 vs. control condition (ANOVA), ns: not significant.</p

Inflammatory events induced by CPB surgery.

<p>Increased mean neutrophil (<b>A</b>) and monocyte (<b>B</b>) counts after on-pump cardiac surgery (n = 21 and n = 24, respectively). <b>C</b>. Increased numbers of circulating CD14+CD16+ monocytes after CPB surgery (n = 14). <b>D</b>. Increased mean C-reactive protein (CRP) levels in patient blood samples post-surgery (n = 22). <b>E</b>. Lymphopenia was observed 4 h post-surgery (n = 27). Box-and-whiskers plots. *<i>P</i><0.01, **<i>P</i><0.001 vs. pre-op (ANOVA). <b>F</b>. Cyto- and chemokine color profiles of plasma samples (n = 12) obtained at indicated time points, represented as % change compared to baseline. MIF: Macrophage migration inhibitory factor.</p

Sequence variability in RSV proteins.

<p>The number of substitutions per site (black bars) and the sequence variability (%) in each RSV protein calculated per strain relative to the consensus.</p