Tanya Tierney

Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface

Annexin A1 (ANXA1) has an important role in cell‐cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane‐bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo‐stellate) cells. LPS caused serine‐phosphorylation of ANXA1 (ANXA1‐S27‐PO4) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild‐type or mutated (S27/A) ANXA1 constructs tagged with enhanced green fluorescence protein. The trafficking of ANXA1‐S27‐PO4 to the cell surface was dependent on PI3‐kinase and MAP‐kinase. It also required HMG‐coenzyme A and myristoylation. The effects of HMG‐coenzyme A blockade were overcome by mevalonic acid (the product of HMG‐coen‐zyme A) and farnesyl‐pyrophosphate but not by geranyl‐geranylpyrophosphate or cholesterol. Together, these results suggest that serine‐27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters).—Solito, E., Christian, H. C., Festa, M., Mulla, A., Tierney, T., Flower, R. J., Buckingham, J. C. Post‐transla‐tional modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface. FASEB J. 20, E677–E687 (2006)

Annexin 1, Glucocorticoids, and the Neuroendocrine–Immune Interface

Abstract:  Annexin 1 (ANXA1) was originally identified as a mediator of the anti‐inflammatory actions of glucocorticoids (GCs) in the host defense system. Subsequent work confirmed and extended these findings and also showed that the protein fulfills a wider brief and serves as a signaling intermediate in a number of systems. ANXA1 thus contributes to the regulation of processes as diverse as cell migration, cell growth and differentiation, apoptosis, vesicle fusion, lipid metabolism, and cytokine expression. Here we consider the role of ANXA1 in the neuroendocrine system, particularly the hypothalamo‐pituitary‐adrenocortical (HPA) axis. Evidence is presented that ANXA1 plays a critical role in effecting the negative feedback effects of GCs on the release of corticotrophin (ACTH) and its hypothalamic‐releasing hormones and that it is particularly pertinent to the early‐onset actions of the steroids that are mediated via a nongenomic mechanism. The paracrine/juxtacrine mode of ANXA1 action is discussed in detail, with particular reference to the significance of the secondary processing of ANXA1, the processes that control the intracellular and transmembrane trafficking of the protein of the molecule and the mechanism of ANXA1 action on its target cells. In addition, the role of ANXA1 in the perinatal programming of the HPA axis is discussed.

Evidence from studies on co-cultures of TtT/GF and AtT20 cells that Annexin 1 acts as a paracrine or juxtacrine mediator of the early inhibitory effects of glucocorticoids on ACTH release.

Annexin 1 (ANXA1) is a key mediator of the inhibitory effects of glucocorticoids on adrenocorticotropic hormone (ACTH) release, which develop within 1–2 h of a steroid challenge. Our previous studies, which showed that (i) ANXA1 is expressed principally by the nonsecretory folliculo‐stellate cells in the pituitary gland; (ii) glucocorticoids cause the exportation of ANXA1 from these cells; and (iii) corticotrophs express specific ANXA1 binding sites, led us to propose that ANXA1 serves as a paracrine or juxtacrine mediator of glucocorticoids. To address this hypothesis, we examined ANXA1‐dependent glucocorticoid actions in co‐cultures of murine corticotroph (AtT20 clone D1) and folliculo‐stellate (TtT/GF) cell lines. ANXA1 mRNA and protein were found in abundance in TtT/GF cells but neither was detectable in the AtT20 cells. AtT20 cells (alone and in co‐culture with TtT/GF cells) responded to corticotropin‐releasing hormone (CRH) (0.1–1 µm) with increased ACTH release. The CRH‐stimulated release of ACTH from AtT20 cells cultured alone was unaffected by preincubation with dexamethasone (Dex, 100 nm); by contrast, in co‐cultures of AtT20 and TtT/GF cells, the steroid readily inhibited the secretory response to CRH. The effects of Dex on ACTH release were mimicked by N‐terminal ANXA1 fragments (ANXA1Ac2−26, 2 µg/ml and ANXA11‐188, 0.1 ng/ml) and reversed by mifepristone (1 µm) and by an antisense oligodeoxynucleotide (ODN) to ANXA1 (50 nm) but not by control ODNs. The antisense ODN also specifically blocked the Dex‐induced externalization of ANXA1 from TtT/GF cells. Immunofluorescence imaging of the co‐cultures localized the exported protein to the vicinity of the AtT20 cells and identified ANXA1 binding sites on these cells. These results provide functional and histological evidence to support our premise that the early inhibitory effects of glucocorticoids on ACTH release are dependent upon paracrine/juxtacrine actions of ANXA1 derived from folliculo‐stellate cells.

Real patient involvement in role development: evaluating patient focused resources for clinical procedural skills

Background: Simulated patients (SPs) are widely used in medical education. The literature offers little evidence for scenario or SP role development. Published materials describe guidelines for structuring roles but there is little information on process. Anecdotal evidence suggests that SP roles are usually created by health care professionals and teachers. Although this approach has advantages it places the role at risk of omitting or misrepresenting real patients’ experiences. Aim: We wanted to explore a systematic approach to role development that was based on individual patients experiences. Methods: Real patients were interviewed about their experiences of procedures they had undergone. This information formed the basis of our procedural skills SP roles. Results: Eight new roles were created. Evaluation by SPs (n = 22) showed more positive comments about realism on roles based on real patients’ experiences compared with those crafted by our multidisciplinary team although there were no statistically significant differences in numerical ratings. Conclusions: The approach to writing roles described here is not suited to all simulations. However, it offers guidance to those involved in writing scenarios and has led us to critically reflect on the ways in which we provide educational materials that are patient focused.

Learner-centred feedback using remote assessment of clinical procedures

This paper describes the Imperial College Feedback and Assessment System (ICFAS) and explores its feasibility in supporting the learning of practical clinical procedures. ICFAS is an innovative combination of existing technologies which brings together video recording, networked mobile computers (laptop and handheld) and content management software. The aim is to observe, record, assess and provide feedback on multiple simulated encounters. We have developed ICFAS within the framework of our Integrated Procedural Performance Instrument (IPPI) (Kneebone et al. ), providing a technology-based ‘feedback space’ for maximizing educational value.

Key challenges in simulated patient programs: An international comparative case study

BackgroundThe literature on simulated or standardized patient (SP) methodology is expanding. However, at the level of the program, there are several gaps in the literature. We seek to fill this gap through documenting experiences from four programs in Australia, Canada, Switzerland and the United Kingdom. We focused on challenges in SP methodology, faculty, organisational structure and quality assurance.MethodsWe used a multiple case study method with cross-case synthesis. Over eighteen months during a series of informal and formal interactions (focused meetings and conference presentations) we documented key characteristics of programs and drew on secondary document sources.ResultsAlthough programs shared challenges in SP methodology they also experienced differences. Key challenges common to programs included systematic quality assurance and the opportunity for research. There were differences in the terminology used to describe SPs, in their recruitment and training. Other differences reflected local conditions and demands in organisational structure, funding relationships with the host institution and national trends, especially in assessments.ConclusionThis international case study reveals similarities and differences in SP methodology. Programs were highly contextualised and have emerged in response to local, institutional, profession/discipline and national conditions. Broader trends in healthcare education have also influenced development. Each of the programs experienced challenges in the same themes but the nature of the challenges often varied widely.

Gaining Consent for Carotid Surgery: A Simulation-Based Study of Vascular Surgeons

AIM Despite no formal training in consenting patients, surgeons are assumed to be competent if they are able to perform an operation. We tested this assumption for carotid endarterectomy (CEA). METHODS Thirty-two surgeons [Group 1: junior surgical trainees--performed 0 CEAs (n=11); 2: senior vascular trainees--1-50 CEAs (n=11); 3: consultant vascular surgeons - > 50 CEAs (n=10)] consented two patients (trained actors) for a local anaesthetic CEA. The performance was assessed at post hoc video review by two independent assessors using a validated rating scale and checklist of risk factors. RESULTS There was no difference in performance between the junior and senior trainees (1: median 91 range 64-121; 2: median 100.5 range 66-125; p=0.118 1 vs. 2 Mann-Whitney). There was a significant improvement between senior trainees and consultant surgeons (3: median 120 range 89-1 142; p=0.001 2 vs. 3). Few junior (1/11) and senior (2/11) trainees, and most (8/11) consultants, were competent. Inter-rater reliability was high (alpha=0.832). Consultant surgeons were significantly more likely to discuss cranial nerve injuries (p<0.0001 Chi-square test) as well as personal or hospital specific stroke risk (p<0.0001) than their junior counterparts. They were less likely to discuss infection (p<0.0001). CONCLUSION Senior trainees, despite being able to perform a CEA, were not competent in consent. The majority of consultant surgeons had developed competence in consenting even though they had no formal training.

Design and validation of the surgical ward round assessment tool: a quantitative observational study.

BACKGROUND Ward round skills are essential for the best management of surgical inpatients, but assessment of their quality has received inadequate attention. This study aims to design and validate the surgical ward round assessment tool (SWAT). METHODS We used modified Healthcare Failure Mode and Effects Analysis to develop the SWAT by identifying ward round steps. We assessed the validity of the SWAT using simulated and real surgical ward rounds. RESULTS The Healthcare Failure Mode and Effects Analysis identified 30 ward round steps that were developed into the SWAT. Nineteen surgeons completed simulated surgical ward rounds. Eight fully trained surgeons scored significantly higher than 11 trainee surgeons when assessed with the SWAT (P = .001). On average, the participants thought the realism of the simulation was good. Forty-four surgeons completed real surgical ward rounds. Fifteen experts scored significantly higher than 29 trainee surgeons when assessed with SWAT (P = .001). Inter-rater reliability was .85 to .89, respectively. CONCLUSIONS The SWAT can be used to assess the quality of task-based and nontechnical surgical ward round skills.