Effects of cardiopulmonary bypass on NOP (A) and ppNOC (B) mRNA, N/OFQ peptide (C) concentrations.

<div><p>Panel D represents fold change relative to pre bypass sample. Samples from 40 patients taken immediately before, (t=0), 3h (t=3) and 24h (t=24) hours after the start of cardiopulmonary bypass. A fold change summary is presented in D where it can be seen that following bypass there was a reduction in ppNOC mRNA and an associated increase in N/OFQ. Data presented as median, interquartile and full range and for PCR are presented as change in PCR cycle threshold relative to the geometric mean of the two housekeepers used (ΔCt). Higher ΔCt values indicate more PCR cycles are required to detect the mRNA, and therefore less mRNA is being expressed. For NOP, in 2 samples there was insufficient material for analysis at t=24h. For ppNOC PCR 6 samples at t=0, 4 samples at t=3h and 6 samples at t=24 failed to amplify and there was insufficient material for analysis of a further 2 samples at t=24. Plasma N/OFQ measurements were made in samples from all patients at all time points (n=120), but for 5 samples at t=0, 4 samples at t=3h and 3 samples at t=24h, measurements were set at the lower limit of detection (1.25pg mL^-1). Data were analyzed using Kruskal-Wallis analysis of variance followed by Dunn’s post-hoc testing; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076682#pone-0076682-g004" target="_blank">Figure 4 A-C</a>: There was no change in mRNA expression for the N/OFQ receptor NOP up to 24 hours after cardiac surgery (panels A andD). However, there was a significant decrease (increased ΔCt) in mRNA for the N/OFQ precursor, ppNOC at 3 hours after the onset of cardiopulmonary bypass, which persisted at 24 hours (p<0.0001, panels B and D). This was associated with a significant (35%) increase in N/OFQ at 3h (p=0.0058) that returned to basal values at 24h (panels C and D).</p> <p>*significantly increased compared to t=0. Figure 4D: * significantly different from t=0.</p></div

Study recruitment diagram: cardiac surgical patients.

<p>Study recruitment diagram: cardiac surgical patients.</p

Study recruitment diagram: patients admitted to ICU with sepsis and matched volunteers.

<p>Study recruitment diagram: patients admitted to ICU with sepsis and matched volunteers.</p

Effects of sepsis on NOP mRNA, (panel A) and ppNOC (panel B) mRNA, and N/OFQ (panel C) concentrations.

<div><p>Panel D represents fold change relative to recovery samples. Data are median, interquartile and full range and for PCR are presented as change in PCR cycle threshold relative to the geometric man of the two housekeepers used (ΔCT). Higher ΔCT values indicate more PCR cycles are required to detect the mRNA, and therefore less mRNA is being expressed. For NOP PCR samples on Day 1, in 1 sample there was no polymorph prep and one failed to amplify; for Day 2 samples there was no polymorph prep in 4 samples and 2 failed to amplify; for recovery samples there was no polymorph prep in 2 and 4 failed to amplify. For ppNOC PCR on Day 1, in one sample there was no polymorph prep and in 12 there was no amplification; for Day 2 samples there was no polymorph prep in 4 and no amplification in 10; for recovery data there was no polymorph prep in 4 and no amplification in 3; for the volunteer group 16 failed to amplify. Plasma N/OFQ measurements were made in all samples but in the volunteer group 3 samples were set at the limit of detection (1.25pg mL^-1). Data were analyzed using Kruskal-Wallis analysis of variance followed by Dunn’s post-hoc testing; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076682#pone-0076682-g003" target="_blank">Figure 3 A-D</a>: NOP (p<0.001) and ppNOC (p=0.019) mRNA values were lower in patients with sepsis when compared to volunteers and this was more pronounced during the first day in ICU (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076682#pone-0076682-g003" target="_blank">Figure 3</a>, panels A and B). Plasma N/OFQ concentrations were higher on Days 1 & 2 of ICU admission compared to the recovery sample (p<0.0001, panels C and D). N/OFQ concentrations in recovery samples were reduced compared to volunteers (p<0.0001).</p> <p>*significantly different compared to volunteer; † significantly different compared to recovery samples.</p></div

The effect of UFP-101 on cardiovascular variables: mean arterial pressure (MAP, mmHg; upper panel) and heart rate (beats per minute, bpm; lower panel) were measured in anesthetised rats at baseline (T₀), this being 24 hours after i.v. injection with 1 mg kg^-1 + 0.5 mg kg^-1 (LPS, n=6; LPS + UFP-101, n=6) or saline (control, n=6; UFP-101, n=6).

<p>Measurements were repeated 40 minutes after baseline (T₄₀) in response to i.v. injection of 150 nM kg^-1 UFP-101 (LPS + UFP-101, UFP-101 groups) or saline (control; LPS, groups). Values are median, with open bars representing the 50^th-75^th percentile, grey bars the 25^th-50^th percentile and upper and lower error bars representing the maximum and minimum respectively.</p

Characterisation of FITC-N/OFQ (F-N/OFQ) at recombinant NOP and classical opioid receptors on CHO cells.

<p><b>A</b>: Binding affinity of F-N/OFQ and a range of opioid receptor subtype selective reference compounds. F-N/OFQ shows high selectivity for NOP over classical opioid receptors (mean (range) of up to 4 experiments.) *curves did not saturate K_i estimated between 1 and 10µM. <b>B</b>: Both N/OFQ and F-N/OFQ behave as full agonists on recombinant human NOP. These data are stimulation factor = agonist stimulated specific binding / basal specific binding (mean <u>+</u>SEM, <i>n</i>=8).</p

The effect of UFP-101 on macromolecular leak and leukocyte rolling <i>in vivo</i>: macromolecular leak (percentage change in interstitial FITC-BSA fluorescence from baseline, upper panel) and leukocyte rolling (per minute, baseline and after treatment, lower panel) in post capillary venules (<40 µm) within the anesthetised rat mesentery preparation.

<p>Measurements were taken in response to i.v. injection of 150 nM kg^-1 UFP-101 (LPS + UFP-101 and UFP-101 groups) or saline (control and LPS groups). Values are median, with open bars representing the 50^th-75^th percentile, grey bars the 25^th-50^th percentile and upper and lower error bars representing the maximum and minimum respectively. ^*increased compared to control.</p

Representative images of rat mesenteric arterioles and venules 5 seconds after injection of 200 nM kg^-1 FITC-N/OFQ into the mesenteric artery with (C,D) and without (A,B) N/OFQ (added 15 sec prior to FITC-N/OFQ and labelled T₁₅) competing for the same NOP receptor site.

<p>The areas highlighted by arrows indicate binding of FITC-N/OFQ to NOP (FITC-N/OFQ-NOP) and thus sparse distribution of NOP receptors on the endothelium in this animal.</p

GTPγ[³⁵S] Response Curves.

<p>Ligand stimulated GTPγ[³⁵S] binding by DeNo and reference ligands are shown at A) CHO_hMu, (B) CHO_hDelta, (C) CHO_hKappa and (D) CHO_NOP cell membranes. Graphs are represented as a stimulation factor, which is the fold change in activity when compared to basal. Data are mean (±SEM) for n = 5 experiments.</p

Calcium Mobilisation.

<p>Concentration-response curves to N/OFQ, dermorphin and DeNo in calcium mobilisation assay using CHO_hMu cells (A) and CHO_NOP cells (B). Data is represented as percentage increase over basal fluorescence intensity units (FIU). Data are the mean ± SEM of at least three separate experiments.</p