A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells.

Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

Effect of the UK Psychoactive Substances Act 2016 on episodes of toxicity related to new psychoactive substances as reported to the National Poisons Information Service. A time series analysis.

BACKGROUND: There have been recent increases in use of new psychoactive substances (NPS) associated with acute health harms including hospital presentations due to toxicity and increasing numbers of deaths. In response, the UK Government enacted generic legislation on 26th May 2016 (the Psychoactive Substances Act) making it an offence to produce, possess with intent to supply, supply, import or export, or possess within a custodial setting a psychoactive substance. We studied the impact of this Act on monthly frequency of enquiries made by health professionals to the UK National Poisons Information Service (NPIS) about NPS. We also studied five commonly used 'conventional' drugs of misuse that had been controlled prior to January 2009. METHOD: Anonymised clinical enquiries to the NPIS and accesses to the poisons information database TOXBASE were reviewed retrospectively from January 2009 to December 2018 to ascertain the trends in reported toxicity for NPS, cocaine, heroin, cannabis, amphetamines and MDMA. Data were analysed using interrupted time series analysis with the date of the PSA used as an independent predictor. RESULTS: Over the period of study there were 3,866 NPIS telephone enquiries and 79,271 TOXBASE user accesses made by UK health professionals concerning NPS. There were increases in monthly TOXBASE accesses (t = 7.408, P < 0.0001) and telephone enquiries (t = 4.74, P < 0.001) over the pre-specified period January 2009 to May 2016. Comparing the period after the PSA with that before, there were significant reductions in TOXBASE accesses (t = -3.327, P < 0.001) and telephone enquiries (t = -6.97, P < 0.001), although reductions started before May 2016. There were no significant changes for the five conventional drugs. There were significant reductions in telephone enquiries (t = -3.418, P < 0.001) and non-significant reductions in TOXBASE accesses (t = -1.713, P = 0.089) for NPS between June 2016 and December 2018. Increases in telephone enquiries for cocaine and reductions TOXBASE accesses for MDMA were also observed over that period. CONCLUSIONS: There have been significant recent reductions in NPIS enquiry activity relating to NPS; although these began before enactment of the PSA in May 2016.

Features reported after exposure to automatic dishwashing rinse aids.

INTRODUCTION: Automatic dishwashing rinse aids are drying aids which contain non-ionic surfactants, usually ethoxylated alcohols, typically at concentrations of ≤30%. OBJECTIVE: To assess the reported toxicity of rinse aids. METHODS: Telephone enquiries to the UK National Poisons Information Service were analysed from January 2008 to June 2019. RESULTS: Ingestion: Ingestion was involved in 976 cases and produced gastrointestinal features, coughing and central nervous system depression, particularly in young children. In those in whom the amount ingested was known, the majority (56%) of children <18 years and of adults (57%) ingested <50 mL of rinse aid. Although moderate or severe exposures (Poisoning Severity Score (PSS) ≥ 2) were uncommon, they occurred significantly (p < 0.0008) more often in adults (9.0%) than in children (1.8%); however, three of the four adults with PSS ≥ 2 co-ingested other substances. Eye exposure: Ocular exposure was reported in 35 cases, of whom 29 developed features. Eye irritation (n = 10, 28.6%) and eye pain (n = 10, 28.6%) were reported most commonly, and three patients (8.6%) developed corneal abrasions (PSS 2). Dermal exposure: Thirty-four patients were exposed dermally, and six (17.6%) reported features, including rash, numbness, pruritus and burns (PSS 1). CONCLUSIONS: Overall, clinical features developed in 47% of patients exposed to rinse aids, but more severe features (PSS ≥ 2) were rare (<3%) following exposure by any route.

Toxicity of traditional and soluble film automatic dishwashing tablets.

INTRODUCTION: Detergents used in automatic dishwashing machines are of two main types: traditional tablets that require removal from an external wrapper and newer soluble film tablets. OBJECTIVE: To determine the toxicity of automatic dishwashing tablets. METHODS: Telephone enquiries to the UK National Poisons Information Service were analysed for the period January 2008 to June 2019. RESULTS: Ingestion: Ingestion was involved in 798 traditional tablet exposures and 725 soluble film exposures. Clinical features (Poisoning Severity Score ≥ 1) developed in 22.2% of patients ingesting traditional tablets and in 28.8% ingesting soluble film tablets; moderate or severe toxicity was rare (<0.5% for both traditional and soluble film tablets). Children (≤5 years) significantly (p < 0.0001) more often developed features following ingestion of soluble film (n = 193, 28.2%) than traditional tablets (n = 134, 19.1%). In contrast, adults more often developed features following ingestion of traditional than soluble film tablets, although this difference was not statistically significant. Eye exposure: The eye was involved in only 26 of 1539 exposures; 17 of 26 exposures resulted in ocular features. The most commonly reported features were conjunctivitis, eye pain and blurred vision, although one patient sustained a corneal abrasion and developed loss of vision. Skin exposure: Thirty-four of 1539 exposures involved the skin but only 3 developed dermal features which were minor. CONCLUSIONS: Children (≤5 years) significantly more often developed features following ingestion of soluble film than traditional tablets, although the likelihood of a child developing features was relatively low (<30%) and features that did develop were almost always mild. In contrast, adults more often developed features following the ingestion of traditional than soluble film tablets. Overall, the eye was involved in only 1.7% of exposures and only one patient sustained a corneal abrasion.

Resistance of Wheat Genotypes to Root-Lesion Nematode (Pratylenchus thornei) Can be Used to Predict Final Nematode Population Densities, Crop Greenness, and Grain Yield in the Field.

The root-lesion nematode Pratylenchus thornei is a major pathogen of wheat (Triticum aestivum) in many regions globally. Resistance of wheat genotypes to P. thornei can be determined from final nematode population densities in glasshouse experiments but combining results across multiple experiments presents challenges. Here, we use a factor analytic method for multiexperiment analysis of final population densities of P. thornei for 1,096 unique wheat genotypes in 22 glasshouse experiments. The resistance to P. thornei of the genotypes was effectively represented by a two-factor model with rotation of the axes to a principal components solution. Principal axes 1 and 2 (PA1 and PA2) accounted for 79 and 11% of the genetic variance, respectively, over all experiments. Final population densities of P. thornei as empirical best linear unbiased predictors (PA[1+2]-eBLUPs) from the combined glasshouse experiments were highly predictive (P < 0.001) of final nematode population densities in the soil profile, crop canopy greenness (normalized difference vegetation index), and grain yield of wheat genotypes in P. thornei-infested fields in the Australian subtropical grain region. Nine categories of resistance ratings for wheat genotypes from resistant to very susceptible were based on subdivision of the range of PA(1+2)-eBLUPs for use in growers' sowing guides. Nine genotypes were nominated as references for future resistance experiments. Most (62%) Australian wheat genotypes were in the most susceptible three categories (susceptible, susceptible to very susceptible, and very susceptible). However, resistant germplasm characterized in this study could be used in plant breeding to considerably improve the overall resistance of Australian wheat crops.

Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.

BACKGROUND AND PURPOSE: The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594 ) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. EXPERIMENTAL APPROACH: We assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). KEY RESULTS: N/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594 , movement of ligand and receptor from the cell surface to the cytosol can be measured. CONCLUSIONS AND IMPLICATIONS: In the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ-driven cellular responses.

Hospital usage of TOXBASE in Great Britain: Temporal trends in accesses 2008 to 2015.

AIM: To examine temporal trends in accesses to the UK's National Poison Information Service's TOXBASE database in Britain. METHODS: Generalized additive models were used to examine trends in daily numbers of accesses to TOXBASE from British emergency departments between January 2008 and December 2015. Day-of-the-week, seasonality and long-term trends were analysed at national and regional levels (Wales, Scotland and the nine English Government Office Regions). RESULTS: The long-term trend in daily accesses increases from 2.8 (95% confidence interval (CI): 2.6-3.0) per user on 1 January 2008 to 4.6 (95% CI: 4.3-4.9) on 31 December 2015, with small but significant differences in population-corrected accesses by region ( p < 0.001). There are statistically significant seasonal and day of the week patterns ( p < 0.001) across all regions. Accesses are 18% (95% CI: 14-22%) higher in summer than in January and at the weekend compared to weekdays in all regions; there is a 7.5% (95% CI: 6.1-8.9%) increase between Friday and Sunday. CONCLUSIONS: There are consistent in-year patterns in access to TOXBASE indicating potential seasonal patterns in poisonings in Britain, with location-dependent rates of usage. This novel descriptive work lays the basis for future work on the interaction of TOXBASE use with emergency admission of patients into hospital.

Post-Carotid Endarterectomy Hypertension. Part 1: Association with Pre-operative Clinical, Imaging, and Physiological Parameters.

OBJECTIVE/BACKGROUND: Post-endarterectomy hypertension (PEH) is a well recognised, but poorly understood, phenomenon after carotid endarterectomy (CEA) that is associated with post-operative intracranial haemorrhage, hyperperfusion syndrome, and cardiac complications. The aim of the current study was to identify pre-operative clinical, imaging, and physiological parameters associated with PEH. METHODS: In total, 106 CEA patients undergoing CEA under general anaesthesia underwent pre-operative evaluation of 24 hour ambulatory arterial blood pressure (BP), baroreceptor sensitivity, cerebral autoregulation, and transcranial Doppler measurement of cerebral blood flow velocity (CBFv) and pulsatility index. Patients who met pre-existing criteria for treating PEH after CEA (systolic BP [SBP] > 170 mmHg without symptoms or SBP > 160 mmHg with headache/seizure/neurological deficit) were treated according to a previously established protocol. RESULTS: In total, 40/106 patients (38%) required treatment for PEH at some stage following CEA (26 in theatre recovery [25%], 27 while on the vascular surgical ward [25%]), while seven (7%) had SBP surges > 200 mmHg back on the ward. Patients requiring treatment for PEH had a significantly higher pre-operative SBP (144 ± 11 mmHg vs. 135 ± 13 mmHg; p < .001) and evidence of pre-existing impairment of baroreceptor sensitivity (3.4 ± 1.7 ms/mmHg vs. 5.3 ± 2.8 ms/mmHg; p = .02). However, PEH was not associated with any other pre-operative clinical features, CBFv, or impaired cerebral haemodynamics. Paradoxically, autoregulation was better preserved in patients with PEH. All four cases of hyperperfusion associated symptoms were preceded by PEH. Length of hospital stay was significantly increased in patients with PEH (p < .001). CONCLUSION: In this study, where all patients underwent CEA under general anaesthesia, PEH was associated with poorly controlled pre-operative BP and impaired baroreceptor sensitivity, but not with other peripheral or central haemodynamic parameters, including impaired cerebral autoregulation.

Late Survival in Nonoperated Patients with Infrarenal Abdominal Aortic Aneurysm.

OBJECTIVE/BACKGROUND: Historical studies report high rupture rates in patients with nonoperated abdominal aortic aneurysms (AAAs) of > 5.5 cm diameter, although a recent audit has questioned this. METHODS: This was a retrospective review of 138/764 (18%) patients with AAAs evaluated in a preassessment anaesthetic clinic (PAC) between 2006 and 2012, who either did not undergo elective AAA repair or who underwent deferred repair. The remaining 626 underwent repair. Patients with severe comorbidities (dementia, advanced malignancy, life-expectancy < 1 year) and not referred to PAC were excluded. RESULTS: At a median of 27 months, 71 (52%) died, 36 (51%) following rupture. Cumulative survival, free from rupture or surgery for acute symptoms, was 96% at 1 year, 84% at 3 years, and 64% at 5 years, where baseline AAA diameters were 5.5-6.9 cm. For diameters ≥ 7 cm, survival, free from rupture, was 65% at 1 year, 29% at 3 years, and 0% at 5 years. Median interval to rupture was 47 months (AAA diameter 5.5-6.9 cm) and 21 months where baseline diameters were ≥ 7 cm. Rupture accounted for 32% of late deaths in patients with AAAs of 5.5-5.9 cm diameter, 46% in those with AAAs measuring 6.0-6.9 cm in diameter, and 71% in patients with AAA measuring ≥ 7 cm in diameter. CONCLUSION: Approximately half of all late deaths in this nonoperated cohort were not AAA related, suggesting that even had repair been undertaken, it would not have prolonged patient survival. The incidence of rupture in "high-risk" patients with an AAA < 7 cm diameter was < 5% at 1 year, thereby giving ample time to optimise risk factors and improve pre-existing medical conditions prior to undertaking a deferred intervention. Even if these patients did not undergo surgical repair, the risk of late rupture was relatively low. By contrast, nonoperated patients with AAAs ≥ 7 cm in diameter face a very high risk of rupture and will probably benefit from elective surgery, with the caveat that a higher procedural risk might have to be incurred.

Evidence for nociceptin/orphanin FQ (NOP) but not µ (MOP), δ (DOP) or κ (KOP) opioid receptor mRNA in whole human blood.

BACKGROUND: While it is well known that opioids depress the immune system, the site(s) of action for this depression is highly controversial. Immune modulation could occur directly at the immune cell or centrally via the hypothalamic-pituitary-adrenal axis. In a number of studies using individual enriched immune cell populations we have failed to detect classical µ (MOP), δ (DOP) and κ (KOP) receptors. The non-classical nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is expressed on all cells examined thus far. Our hypothesis was that immune cells do not express classical opioid receptors and that using whole blood would definitively answer this question. METHODS: Whole blood (containing all immune cell types) was incubated with opioids (morphine and fentanyl) commonly encountered in anaesthesia and with agents mimicking sepsis [lipopolysaccharide (LPS) and peptidoglycan G (PepG)]. Opioid receptor mRNA expression was assessed by endpoint polymerase chain reaction (PCR) with gel visualisation and quantitative PCR. RESULTS: Classical MOP, DOP, and KOP receptors were not detected in any of the samples tested either at rest or when challenged with opioids, LPS or PepG. Commercial primers for DOP did not perform well in quantitative PCR, so the absence of expression was confirmed using a traditional gel-based approach. NOP receptors were detected in all samples; expression was unaffected by opioids and reduced by LPS/PepG combinations. CONCLUSIONS: Classical opioid receptors are not expressed on circulating immune cells.

Taking stock: UK national antidote availability increasing, but further improvements are required.

BACKGROUND: A 2010/2011 audit of the Royal College of Emergency Medicine (RCEM) National Poisons Information Service (NPIS) UK guidelines on antidote availability demonstrated variable stocking of antidotes for the management of poisoned patients; the guidelines were updated and republished in 2013. AIM: To assess if antidote stocking has improved since the 2010/2011 audit and introduction of the 2013 guidelines. METHODS: Questionnaires were sent to Chief Pharmacists at all 215 acute hospitals in England, Wales and Northern Ireland in October 2014. Data were collected on the timing of availability (category A antidotes should be available immediately, category B within 1 h and category C can be held supraregionally) and stock levels. RESULTS: 169 (78.6%) responses were received. Atropine, calcium gluconate and flumazenil (category A) were the only antidotes available in all hospitals within the recommended time and stock levels. Forty-one (24.3%) hospitals held every category A antidote; this increased to 81 (47.9%) for those holding at least one cyanide antidote and all other category A antidotes. The proportion of hospitals stocking category A/B antidotes within the recommended time increased for 20 (90.9%) category A/B antidotes. Fomepizole (category B) availability increased to 62.1% of hospitals from 11.4% in 2010/2011. Other than penicillamine (63.3% hospitals), there was poor availability (2.4%-36.1%) of category C antidotes. CONCLUSIONS: Availability of category A and B antidotes has improved since the 2010/2011 audit and 2013 guidelines. However, there remains significant variability particularly for category C antidotes. More work is required to ensure that those treating poisoned patients have timely access to antidotes focusing particularly on category C antidotes.