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Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQATTO594) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24–48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1μM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1μM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis.

BackgroundVascular adhesion protein 1 (VAP-1) has been implicated in a wide range of clinical conditions. Moreover, serum levels are associated with disease prediction and progression in several clinical studies. There is a paucity of data on VAP-1 and pregnancy. Given the emerging role of VAP-1 in pregnancy, the aim of this study was to examine sVAP-1 as an early biomarker of pregnancy complications, especially hypertension during pregnancy. The objectives of the study are to associate sVAP-1 levels with other pregnancy complications, patient demographics and blood tests performed throughout pregnancy.MethodsWe conducted a pilot study in a cohort of pregnant women (gestational week lower than 20 at the time of recruitment) attending their first antenatal ultrasound scan at the Leicester Royal Infirmary (LRI, UK). Data were both prospectively generated (from blood sample analysis) and retrospectively collected (from hospital records).ResultsFrom July and October 2021, a total of 91 participants were enrolled. Using ELISA (enzyme-linked immunosorbent assay), we found reduced serum levels of sVAP-1 in pregnant women with either pregnancy induced hypertension (PIH) (310 ng/mL) or GDM (366.73 ng/mL) as compared to controls (427.44 ng/mL and 428.34 ng/mL, respectively). No significant difference was found between women with FGR compared to controls (424.32 ng/mL vs 424.52 ng/mL), and patients with any pregnancy complications compared to healthy pregnancies (421.28 ng/mL vs 428.34 ng/mL).ConclusionFurther studies are needed to establish whether or not sVAP-1 might be considered as an early, non-invasive, and affordable biomarker to screen women who will develop PIH or GDM. Our data will aid sample size calculations for such larger studies.

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.

INTRODUCTION:Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ. METHODS:We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats. RESULTS:DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats. CONCLUSION:Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.

Microvascular inflammation occurs during sepsis and the endogenous opioid-like peptide nociceptin/orphanin FQ (N/OFQ) is known to regulate inflammation. This study aimed to determine the inflammatory role of N/OFQ and its receptor NOP (ORL1) within the microcirculation, along with anti-inflammatory effects of the NOP antagonist UFP-101 (University of Ferrara Peptide-101) in an animal model of sepsis (endotoxemia). Male Wistar rats (220 to 300 g) were administered lipopolysaccharide (LPS) for 24 h (-24 h, 1 mg kg(-1); -2 h, 1 mg kg(-1) i.v., tail vein). They were then either anesthetised for observation of the mesenteric microcirculation using fluorescent in vivo microscopy, or isolated arterioles (~200 µm) were studied in vitro with pressure myography. 200 nM kg(-1) fluorescently labelled N/OFQ (FITC-N/OFQ, i.a., mesenteric artery) bound to specific sites on the microvascular endothelium in vivo, indicating sparse distribution of NOP receptors. In vitro, arterioles (~200 µm) dilated to intraluminal N/OFQ (10(-5)M) (32.6 + 8.4%) and this response was exaggerated with LPS (62.0 +7.9%, p=0.031). In vivo, LPS induced macromolecular leak of FITC-BSA (0.02 g kg(-1) i.v.) (LPS: 95.3 (86.7 to 97.9)%, p=0.043) from post-capillary venules (

BACKGROUND AND OBJECTIVES:Nociceptin/Orphanin FQ (N/OFQ) is a non-classical endogenous opioid peptide that modulates immune function in vitro. Its importance in inflammation and human sepsis is unknown. The objectives of this study were to determine the relationship between N/OFQ, transcripts for its precursor (pre-pro-N/OFQ [ppNOC]) and receptor (NOP), inflammatory markers and clinical outcomes in patients undergoing cardiopulmonary bypass and with sepsis. METHODS:A prospective observational cohort study of 82 patients admitted to Intensive Care (ICU) with sepsis and 40 patients undergoing cardiac surgery under cardiopulmonary bypass (as a model of systemic inflammation). Sixty three healthy volunteers, matched by age and sex to the patients with sepsis were also studied. Clinical and laboratory details were recorded. Polymorph ppNOC and NOP receptor mRNA were determined using quantitative PCR. Plasma N/OFQ was determined using ELISA and cytokines (TNF- α, IL-8, IL-10) measured using radioimmunoassay. Data from patients undergoing cardiac surgery were recorded before, 3 and 24 hours after cardiopulmonary bypass. ICU patients with sepsis were assessed on Days 1 and 2 of ICU admission, and after clinical recovery. MAIN RESULTS:Plasma N/OFQ concentrations increased (p