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

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

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

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

Cell-Penetrating Bisubstrate-Based Protein Kinase C Inhibitors

Although protein kinase inhibitors present excellent pharmaceutical opportunities, lack of selectivity and associated therapeutic side effects are common. Bisubstrate-based inhibitors targeting both the high-selectivity peptide substrate binding groove and the high-affinity ATP pocket address this. However, they are typically large and polar, hampering cellular uptake. This paper describes a modular development approach for bisubstrate-based kinase inhibitors furnished with cell-penetrating moieties and demonstrates their cellular uptake and intracellular activity against protein kinase C (PKC). This enzyme family is a longstanding pharmaceutical target involved in cancer, immunological disorders, and neurodegenerative diseases. However, selectivity is particularly difficult to achieve because of homology among family members and with several related kinases, making PKC an excellent proving ground for bisubstrate-based inhibitors. Besides the pharmacological potential of the novel cell-penetrating constructs, the modular strategy described here may be used for discovering selective, cell-penetrating kinase inhibitors against any kinase and may increase adoption and therapeutic application of this promising inhibitor class

Chemical inhibition of HSF1 synergizes with VX809 to improve F508del-CFTR function in patient-derived primary epithelium.

<p>(A) Short-circuit current analysis of human primary hBE cells (F508del/F508del, patient code CF006) treated with DMSO, 3 µM VX809, and 25 nM triptolide or a combination of VX809 and triptolide, for 96 h (daily dosing). The data is presented as fold change relative to the basal current seen with DMSO treatment, and shown as mean ± SD, <i>n</i>≥3 (replicated multiple times); * and # indicate <i>p</i><0.05 relative to DMSO or VX809, respectively. (B) Representative short-circuit current (I_sc) traces for DMSO, VX809, triptolide, or triptolide + VX809 treatment of primary hBE cells from (A). (C) Quantitative analysis of organoid swelling (shown in D) that is indicative of CFTR function over the period of 60 min. Organoids were obtained from two distinct F508del/F508del CF patients (CF4, CF22), and treated with DMSO, 3 µM VX809, 25 nM triptolide, or a combination of VX809 and triptolide. Experiments were repeated once and results are shown as a mean ± SD, <i>n</i>≥2; * and # indicate <i>p</i><0.05 relative to DMSO or VX809, respectively. (D) Representative images of organoids derived from patients (CF4 and CF22) at T = 0 or after stimulus with Forskolin/Genistein at T = 60 min treated with the indicated compounds. Scale bar represents 110 µm. The underlying data used to make (A–C) in this figure can be found in the supplementary file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998.s008" target="_blank">Data S1</a>.</p

Expression of F508del induces chronic proteotoxic stress that affects cellular protein folding.

<p>(A) Immunoblots of WT-CFTR, F508del-CFTR, and HSF1-P during HS time course (42°C for a total of 60 min). (B) Quantification of total CFTR during HS, relative to pre-HS (T = 0) (<i>n</i> = 4). (C) Immunoblots of indicated proteins in CFBE41o- cellular lysates following the co-expression of F508del-CFTR with the constitutively active ΔHSF1^186–201 or control empty plasmid. (D) Immunoblots of HSF1-P and I-Hsp70 in WT or F508del expressing cells (<i>n</i> = 4). (E) Quantification of the expression of HSR markers in WT-CFTR, F508del-CFTR at 37°C or 30°C, and CFTR null (CFTR−/−) expressing cells. (F) Quantification of HFS-1 trimer levels in WT-, F508del- and CFTR null–CFBE cells. (E,F) Results are shown as percentage of that seen in WT-expressing cells, as a mean ± standard error of the mean (SEM), n≥3. qRT-PCR of I-Hsp70 (HspA1A, or HspA6), I-Hsp90 (Hsp90α), and I-Hsp40 (DNAJB1) in (G,J) or of cancer-related HSF1 responsive genes (CKS2, LY6K, EIF4A2) shown in (J) from mRNA isolated from WT-, F508del- and CFTR null–CFBE cells (G) or from mRNA obtained from hBE primary cells obtained from homozygous patients for WT- or F508del-CFTR (J). qRT-PCR data was normalized to the housekeeping gene beta-glucuronidase (GUS). Results are shown as percentage of WT-expressing cells set to a 100 (mean ± standard deviation [SD] or SEM, n≥3, and * indicates <i>p</i><0.05 relative to WT). Immunoblot (H) and quantification (I) of CFTR, HSF1-P, and I-Hsp70 from hBE primary cell lysates of WT or F508del patients. Data is shown as the relative protein expression normalized to actin (mean ± SD, n≥2). (K) Quantification of I-Hsp70 and I-Hsp40 protein level in F508del-expressing cells at 37°C (MSR) or following acute HS (shown as a percentage of the level seen in WT-expressing cells; mean ± SD, n≥2). (L) Firefly luciferase (FLuc) activity in WT- and F508del-CFTR expressing cells following siCFTR silencing. Results represent normalized specific activity of FLuc (luminescence/relative FLuc expression) for each condition. Data is shown as percentage of WT-CFTR expressing cells, mean ± SEM, n≥3, and *, # indicate <i>p</i><0.05 relative to WT and F508del (0 nM siCFTR), respectively. (M) (1) Diagram showing the proteostatic environment of WT folding, where substrates are properly managed by the physiological Q-state (yellow cloud). (2) Representation of the transient stress level observed during acute stress responses (dotted red line). (3) The MSR state induced in misfolding diseases (abnormal Q-state, gray cloud) that results in a continuous elevated (subacute) stress affecting global folding and cellular function. All experimental data was repeated at least once. The underlying data used to make (B), (E–G), and (I–L) in this figure can be found in the supplementary file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998.s008" target="_blank">Data S1</a>.</p

Q-state management of MSR to correct human disease.

<p>Illustrated is the activation state of the HSR in response to acute stress (red) or to the MSR (blue) seen in disease. Acute HSR activation, seen during acute stress insults, protects from and/or corrects misfolding and rapidly returns to basal levels, allowing normal biology to resume. In misfolding disease, chronic activation of the HSR alters the normal, physiologic Q-state (Qⁿ) because of the continued expression of misfolded protein. Once chronically elevated (Q*), the folding environment becomes maladaptive as it fails to return to the Qⁿ (light yellow area). Down-regulation of the MSR by siHSF1, sip23, or triptolide promotes a reduction of the Q*, which now falls within the proteostasis buffering capacity (green line), promoting a more normal cellular folding environment. This effect can be further improved (purple line) when combined with protein fold correctors (pharmacologic chaperones; PCs) which impart improved thermodynamic stability to the fold, or proteostasis regulators (PRs) that improve protein Q-state biology, improving function of disease-related misfolded protein and its proteome's associated environment, promoting abrogation of the chronic stress and improving health.</p

Schematic representation of the study procedure.

<p>Schematic representation of the study procedure.</p