BAlloon versus Stenting in severe Ischaemia of the Leg-3 (BASIL-3): study protocol for a randomised controlled trial.

BACKGROUND: Severe limb ischaemia (SLI) is defined as the presence of rest pain and/or tissue loss secondary to lower extremity atherosclerotic peripheral arterial disease. The superficial femoral and popliteal arteries are the most commonly diseased vessels in such patients and are being increasingly treated using endovascular revascularisation techniques. However, it is currently unknown whether drug-eluting stents and drug-coated balloons confer additional clinical benefits over more established techniques using plain balloons and bare metal stents, or whether they represent a cost-effective use of NHS resources. METHODS: The BASIL-3 trial is a UK National Institute for Health Research, Health Technology Assessment Programme-funded, multicentre, randomised controlled trial (RCT) comparing the clinical and cost-effectiveness of plain balloon angioplasty with or without bail-out bare metal stenting, drug-coated balloon angioplasty with or without bail-out bare metal stenting, and primary stenting with drug-eluting stents for SLI secondary to femoro-popliteal disease. Patients with 'multilevel' disease may receive aorto-iliac and/or infrapopliteal treatments concurrently with their randomised femoro-popliteal intervention. The primary clinical outcome is amputation-free survival defined as the time to major (above the ankle) amputation of the index limb or death from any cause. The primary outcome for the economic analysis is cost per quality-adjusted life year. Secondary outcome measures include overall survival, major adverse limb events, major adverse cardiac events, relief of ischaemic pain, healing of tissue loss, and quality of life. The required sample size has been calculated at 861 participants (287 on each arm). These patients will be recruited over 3 years and followed-up for between 2 and 5 years. DISCUSSION: BASIL-3 is a pragmatic RCT designed to reflect current UK clinical practice. The results will inform decision-making regarding the appropriateness of funding the use of drug-coated balloons and drug-eluting stents, by the NHS, for the management of SLI due to femoro-popliteal disease. TRIAL REGISTRATION: ISRCTN Registry, identifier: ISRCTN14469736 . Registered on 22 October 2015.

Evaluation of the Healthy LifeCheck programme: a vascular risk assessment service for community pharmacies in Leicester city, UK.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death globally. Vascular risk assessment is recognized as playing a key role in reducing premature CVD-related morbidity and mortality. The current study evaluated the effectiveness of a pharmacy-led risk assessment service in Leicester City, UK. METHODS: The vascular risk assessment was offered opportunistically to individuals between 40 and 70 years without any prior diagnosis of CVD on attending their community pharmacist. Individuals were risk stratified using the Framingham score and those classified as high risk were referred to their general practitioner (GP). RESULTS: Overall, 2521 individuals were recruited from 39 pharmacies consisting of 1059 (42%) males, 1696 (67%) South Asians and 199 (7.9%) individuals not registered with a GP. A total of 462 (18%) individuals were referred to primary care and 52.6% of a representative subset were subsequently recorded as having attended an appointment with their GP; diagnoses and treatments commenced were recorded. CONCLUSIONS: Cardiovascular risk assessment led by community pharmacies can successfully assess people from large, multi-ethnic UK populations and identify those at high cardiovascular risk or with undiagnosed cardiovascular disease. The service may improve rates of assessments undertaken by individuals who do not access health care through traditional routes.

Ratiometric [Ca²âº]i measurements in adherent cell-lines using the NOVOstar microplate reader.

The control of free ionized intracellular calcium concentration ([Ca(2+)](i)) is an established mechanism of cellular activation, regulating a diverse range of cellular events. Consequentially, experimental measurement of [Ca(2+)](i) is a potent technique for the medical science laboratory. The NOVOstar microplate reader is a versatile system, which may be easily configured to measure [Ca(2+)](i). Moreover, the relatively low cost of this system makes it an attractive one for researchers adhering to a modest budget, whilst allowing medium throughput to be achieved.These methods serve as a starting point for researchers wishing to measure intracellular calcium concentration in adherent cell-lines using the NOVOstar plate reader. Briefly, adherent cells are seeded into well plates 1 day prior to calcium determinations being made. On the day of the experiment, autofluorescence values of individual wells of cells are determined prior to the cells being loaded with the fluorophore, fura-2. [Ca(2+)](i) determinations are acquired by activating a predefined program within the NOVOstar software; full parameters are provided within this chapter for this purpose. Fluorescence ratio values may be easily calibrated to give absolute intracellular calcium concentrations (nM). Calibration involves determining experimental fluorescence at calcium-saturating and calcium-free conditions; ionomycin and EGTA are used to produce these two conditions respectively. Finally, mathematical calculation of absolute intracellular calcium concentration is described by use of the Grynkiewicz equation.

In vitro siRNA-mediated knockdown of the UT receptor: implications of density on the efficacy of a range of UT ligands.

Urotensin-II (U-II) is the peptide agonist for the U-II receptor (UT). Putative UT antagonists, urantide and UFP-803, have been found to have variable efficacy in a range of assays. We have used siRNA-mediated RNA interference to probe the efficacy of these ligands compared to U-II. Knockdown of human UT occurs in the same cellular background with the same coupling machinery allowing relative efficacy to be probed. CHO cells stably expressing 1,110 fmol/mg protein of human UT (CHOhUT) were transfected with s194454, s194455 (UT-targeting), or a negative control siRNA using siPORT amine transfection reagent. After 48 h,silencing was assessed using quantitative PCR in a duplex assay format. Functional consequences of silencing were assessed by measuring [Ca2+]i in Fura-2 loaded cells using the NOVOstar plate reader. Silencing with s194455 was greater than that with s194454 (93.5±2.8% and 73.0±2.5%knockdown of UT mRNA respectively at 10−7 M, p00.006).Both s194455 and s194454 knocked down UT mRNA expression with equal potency (EC50 1.38 and 0.45 nM). The negative control did not affect UT mRNA expression. U-II(10−6M) increased [Ca2+]i 630±69, 402±49 and 190±14nM,urantide (10−6 M) increased [Ca2+]i 408±55, 191±40, and 131±10 nM and UFP-803 (10−6 M) increased [Ca2+]i 134±23, 83±11 and 53±3nM for negative control siRNA, s194454 and s194455, respectively.We have demonstrated silencing of UT mRNA and a reduction of absolute efficacy of three UT ligands. However, we were unable to resolve any changes in relative efficacy for urantide and UFP-803. This is likely to result from a high starting expression and retention of a receptor/coupling reserve.

A rat brain atlas of urotensin-II receptor expression and a review of central urotensin-II effects.

Urotensin-II (U-II) is an 11-amino acid cyclic peptide which exerts its actions through a G(q) protein-coupled receptor, UT. Much of the research focus of U-II is as a peptide of the periphery, particularly cardiovascular. Despite this, U-II was originally identified as a neuropeptide, and its expression is broad throughout the central nervous system. This brief review article catalogs the known sites of expression of UT within the CNS in the form of a diagrammatic rat brain atlas. Furthermore, the functional consequences of UT activation within specific brain regions are discussed along with the likely actions of synthetic UT ligands. Areas of high, medium, and low expression include the arcuate, paraventricular, and pedunculopontine tegmental nuclei, respectively. In the arcuate and paraventricular nuclei, where expression is high and moderate, U-II produces a pressor/tachycardic response in the former and a weaker response in the latter. Based on the known pharmacology of UT ligands (and assuming density is the primary determinant of efficacy in this case), we predict a weak response in the arcuate nucleus and possible antagonism of endogenous U-II response in the paraventricular nucleus by a low-efficacy partial agonist. These predicted responses lend themselves to relatively simple experimental verification.