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.

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.

Factors influencing short- and long-term mortality after lower limb amputation.

Mortality after lower limb amputation is high, with UK 30-day mortality rates of 9-17%. We performed a retrospective analysis of factors affecting early and late outcome after lower limb amputation for peripheral vascular disease or diabetic complications at a UK tertiary referral vascular centre between 2003 and 2010. Three hundred and thirty-nine patients (233 male), of median (IQR [range]) age 73 (62-79 [26-92]) years underwent amputation. Thirty-day mortality was 12.4%. On regression modelling, the risk of 30-day mortality was increased in patients of ASA grade ≥ 4 (OR 4.23, 95% CI 2.07-8.63), p < 0.001 and age between 74 and 79 years (OR 3.8, 95% CI 1.10-13.13), p = 0.04 and older than 79 years (OR 4.08, 95% CI 1.25-13.25), p = 0.02. Peri-operative (30-day) mortality for these groups was 23.2%, 13.7% and 18.8%, respectively. Survival and Cox regression analysis demonstrated that long-term mortality was associated with: age 74-79 years (HR 2.15, 95% CI 1.38-3.35), p = 0.001; age > 79 years (HR 2.78, 95% CI 1.82-4.25), p < 0.001; ASA grade ≥ 4 (HR 2.04, 95% CI 1.51-2.75), p < 0.001; out-of-hours operating (HR 1.51, 95% CI 1.08-2.10), p = 0.02; and chronic kidney disease stage 4-5 (1.57, 95% CI 1.07-2.30), p = 0.02. Anaesthetic technique was associated with long-term mortality on survival analysis (p = 0.04), but not when analysed using regression modelling. Mortality after lower limb amputation relates to patient age, ASA, out-of-hours surgery and renal dysfunction. These data support lower limb amputations' being performed during daytime hours and after modification replace with 'of ' correctable risk factors.

Opioids and immune modulation: more questions than answers.

Opioid addicts are more likely to present with infections suggesting opioids are immune modulators. The potential sites/mechanism(s) for this modulation are controversial and on close inspection not well supported by the current literature. It has long been assumed that opioid-induced immune modulation occurs via a combination of direct actions on the immune cell itself, via the hypothalamic-pituitary-adrenal (HPA) axis, or both. Opioid receptors are classified as MOP (µ, mu), DOP (δ, delta), and KOP (κ, kappa)--classical naloxone sensitive receptors--or NOP (the receptor for nociceptin/orphanin FQ), which is naloxone insensitive. Opioids currently used in clinical practice predominantly target the MOP receptor. There do not appear to be classical opioid receptors present on immune cells. The evidence for HPA activation is also poor and shows some species dependence. Most opioids used clinically or as drugs of abuse do not target the NOP receptor. Other possible target sites for immune modulation include the sympathetic nervous system and central sites. We are currently unable to accurately define the cellular target for immune modulation and suggest further investigation is required. Based on the differences observed when comparing studies in laboratory animals and those performed in humans we suggest that further studies in the clinical setting are needed.