Centre for Clinical Pharmacology and Therapeutics
Clinical Pharmacology and Therapeutics is part of the Division of Medicine
Published: Nov 20, 2014 9:40:05 AM
Published: Nov 17, 2014 11:14:07 AM
Published: Nov 17, 2014 8:18:03 AM
The Centre for Clinical Pharmacology and Experimental Therapeutics is a collection of scientist and clinician/scientists working on topics ranging from imaging to cardiovascular pharmacology; from development of the cardiovascular system to inflammation research, cell biology and genetics. The central theme is excellence in research and teaching with a track record in fostering independent scientific careers and mentoring of doctoral training students.
We are interested in understanding the endogenous pathways that bring about the resolution of acute inflammation. Resolution of acute inflammation is no longer considered a passive event where the response simply fizzles out, but an active process whose dysregulation may explain the complex aetiology of many chronic inflammatory diseases. Our overall objective, therefore, is to identify pro-resolution factors that help switch inflammation off and to develop drugs based on their mode of action in order to drive on-going, chronic inflammatory diseases down a pro-resolution pathway. We contend that this represents a novel approach to understanding and treating diseases driven by on-going inflammation. For detailed information on the Gilroy laboratory, its members, publications and research objectives go to derekgilroy.com
Technology in the Gilroy lab
Cantharidin skin blister
Following topical administration to the skin, cantharidin, a protein phosphatase inhibitor, induces atraumatic acantholysis and blister formation. This property is used to create defined skin windows which can be maintained for several days helping to profile the onset and resolving phases of inflammation.
Suction skin blister
In this model skin window is created by placing a suction cup on the fore-arm attached to negative pressure equipment. In addition to serving as a model of tissue injury, it can be modified to study in vivo cell function and effect of drugs on the onset and resolution phase.
Systemic inflammation in humans
In order to investigate critical illness-induced immune dysfunction (CIIID) the group has designed, and is currently characterizing a robust clinically relevant ‘two-hit’ human pre-clinical model of systemic inflammation (mirroring sepsis, trauma, burn injury or major surgery) coupled with a biologically valid secondary infective challenge (mimicking hospital-acquired infection, HAI). Intravenous endotoxaemia provides the primary insult, whilst the ‘second hit’ is induced by gram-positive bacterial challenge to suction-blister induced skin windows replicating central or peripheral venous access, surgical or traumatic wound infection, the second most common cause of HAI on intensive care (ICU). Application of specially developed blister wells uniquely allows in-vivo analysis of the local inflammatory and immune response (cellular component [via flow cytometry], humoral mediators [via multiplex cytokine ELISA, with lipidomics via mass spectroscopy]) and its functionality at the infective site (fluorochrome-tagged bacterial phagocytosis). Plasma and whole blood assays ex-vivo (stimulation and live bacterial killing) are complementary to the above, describing systemic disturbance in inflammo-immune homeostasis. Varying the time-point of the ‘second hit’ facilitates exploration of the time-course (and mediators) of immunosuppression, and sequential blister exudate aspiration the examination of different phases of inflammation. In sum, the model provide a unique, controlled, adaptable translational platform upon which to dissect the molecular, cellular and genetic drivers of CIIID in humans, and a means of evaluating the efficacy of immunomodulatory therapies.
Experimental models of inflammation
The Gilroy lab has extensive experience and expertise in a number of in vivo inflammatory models, which is shown in the group’s publication history. These included the air pouch, zymosan elicited peritonitis, immunisation, extravasation, monoarthritis, polyarthritis, inflammation, pharmacokinetics, airway inflammation, and liver inflammation and fibrosis. We use a range of stimuli to induce inflammation, including zymosan, live bacteria, carrageenan, and methylated BSA. We also have expertise in cardiac punctures, dissection of a variety of tissues for subsequent analysis by FACS, qRT-PCR, primary cell culture and proliferation studies. We maintain a good working relationship with the staff in our Biological Services Units to ensure our experimental systems are well cared for and monitored during the duration of our experiments, and always keep in mind the 3’R’s during experimental design.
In our pursuit of understanding B lymphocyte transdifferentiation, we have crossed transgenic B6.mb-1.iCre mouse harbouring a Cre recombinase gene integrated in the mb-1 locus (encodes the Ig-a signalling subunit of the B cell antigen receptor) with Rosa26R-eYFP (enhanced Yellow Fluorescent Protein) reporter mouse to obtain a B6.mb-1.iCre-Rosa26R-eYFP mouse. Hence, in Rosa26R-eYFP expression of florescent protein is dependent upon Cre recombinase, whose expression in B6.mb-1.iCre is restricted to B cell lineage enabling permanent labelling of these cells. Therefore B6.mb-1.iCre-Rosa26R-eYFP mice are a valuable tool for tracing of the B-1 cell-derived MΦs.
Our research focusses on small blood vessel remodelling with an emphasis on the role of transcription factors (calcineurin and PPARγ), growth factors and ATP-sensitive potassium channels in pulmonary hypertension and sepsis. A major focus of this work is to identify the receptors and the associated signalling pathways involved in the biological functions of prostacyclins and how pulmonary hypertension impacts on these pathways and the action of these agents. Another important aspect of our work is to elucidate the mechanisms underlying the cardiovascular collapse in septic shock and why patients are paradoxically hypersensitive towards the hormone vasopressin. We are pursuing these aims through in vitro and vivo models, small vessel myography and electrophysiology combining them with imaging and biochemical studies in tissues and cells derived from patients. Underpinning this research is the development of new therapeutic approaches to treat these life-threatening diseases, work which is funded in collaboration with various pharmaceutical companies.
Pulmonary hypertension; Prostacyclin signalling through membrane and nuclear receptors (PPARs); calcineurin, endothelin, vascular potassium channels, sepsis.
- Jabr RI, Wilson AJ, Riddervold MH, Jenkins AH, Perrino BA, Clapp LH. Nuclear translocation of calcineurin Aß but not A? by platelet-derived growth factor in rat aortic smooth muscle. Am J Physiol (Cell), 292:C2213-2225, 2007.
- Barrett LK, Orie NN, Taylor V, Stidwill RP, Clapp LH, Singer M. Differential effects of vasopressin and norepinephrine on vascular reactivity in a long-term rodent model of sepsis. Crit Care Med, 35:2337-2343, 2007.
- Orie NN, Thomas AM, Perrino BA, Tinker A, Clapp LH. Ca2+/calcineurin regulation of cloned vascular KATP channels: crosstalk with the protein kinase A pathway. Br J Pharmacol, 157:554-64, 2009.
- Falcetti,E, Hall SM, Phillips PG, Patel JA, Morrell NW, Haworth SG, Clapp LH. Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med, 182:1161-70, 2010.
- Chan Y, Orie NN, Dyson A, Taylor V, Stidwill RP, Clapp LH, Singer M. Inhibition of vascular adenosine triphosphate-sensitive potassium channels by sympathetic tone during sepsis, Crit Care Med 40:1261-8. 2012.
- Whittle BJ, Silverstein A, Mottola D, Clapp LH. Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: Treprostinil is a potent DP1 and EP2 agonist. Biochem Pharmacol. 84:68-75, 2012.
Technology in the Clapp laboratory
- Tissue culture & generation of primary cell lines (e.g. human lung smooth muscle, endothelial and fibroblasts).
- In vitro models of human pulmonary hypertension
- Cell proliferation, endothelin and cyclic nucleotide assays
- Confocal microscopy (smooth muscle)
- Human lung histology
- Small blood vessel myography
- Organ bath pharmacology
- Patch-clamp electrophysiology
- In vivo and ex-vivo rat models of sepsis
Professor Mark Lythgoe is the founder and Director of the UCL Centre for Advanced Biomedical Imaging, which is a new multidisciplinary research centre for experimental imaging. Mark leads a group of 35 researchers in a programme of study focusing on developing new imaging methods for identifying pathophysiology and its functional consequences in preclinical models of disease. Mark has established CABI as a world-class preclinical research centre, which offers researchers across the UK a range of platform imaging technologies to test their hypothesis in vivo. The Centre now hosts eight state-of-the-art imaging modalities: magnetic resonance imaging, photoacoustic imaging, ultrasound, bioluminescence and fluorescence imaging, CT/radiotherapy, SPECT/CT and confocal endoscopy. Mark has been awarded £24 million for his programme of research and has published in Nature, Nature Medicine and The Lancet. He is also the Director of the UCL Doctoral Training Programme in Biomedical Imaging. During Mark’s tenure as Director of the Cheltenham Science Festival, it has become one of the largest science festivals in the world. Mark has received the Davies Medal for a ‘significant contribution to imaging science’, as well as awards from the Biosciences Federation and British Associated for the Advancement of Science for his contributions to communicating science. . For detailed information on the Lythgoe laboratory, its members, publications and research objectives go to mlythgoe.com
Magnetic resonance imaging; nuclear imaging; bioluminescence; fluorescence imaging; photoacoustic imaging
Dr O’Brien is a Senior Clinical Lecturer in the UCL Division of Medicine and a Consultant Hepatologist at University College and The Royal Free Hospitals. He is investigating the role of prostaglandin E2 in the pathogenesis of immune suppression in liver cirrhosis. Liver disease is fast becoming one of the commonest causes of death in the UK and affects all socio-economic groups. In 50% of cirrhotic inpatients, infection is the precipitant for hospital admission and a further 15-35% develops nosocomial infections, compared to only 5-7% of general patients. Of those cirrhotic patients who develop sepsis and organ dysfunction, 80-90% will die. Dr O’Brien has have demonstrated that prostaglandin E2 is the key mediator of leukocyte in cirrhotic patientsHis findings have direct clinical implications and currently Dr O’Brien is expanding his work by trying to translate his work to the development of novel diagnostic and treatment paradigms for cirrhotic patients.
Dr O’Brien is collaborating with Professors Jules Wendon (King’s College, London), Dr Mark Thursz (Imperial College, London) and Dr Harry Antoniades (MRC clinician scientist, Imperial College).
Dr O’Brien is heavily involved in the teaching and training of undergraduate and postgraduate students in the UCL Department of Clinical Pharmacology and UCL Division of Medicine. Currently, he is co-supervising 2 PhD, 2BSc and 1 MSc students, who are investigating the role of lipid mediators in sepsis-induced immune suppression. In addition he runs the drug development module for the Neuroscience Physiology and Pharmacology BSc students, lectures on the MSc drug development course, as well as teaching the clinical medical students for the acute admissions firm, the junior doctors for the UCH PACES course and exams for MBBS finals.
Liver cirrhosis, Immune suppression, Eicosanoids, Albumin
I am interested in the adaptation of the mammalian fetal heart to hypoxia, adaptation of the newborn heart to the terrestrial environment and mouse models of heart failure.
The fetal heart is adapted to profoundly low levels of oxygen in utero. This adaptation is permanently lost immediately following birth leaving the adult cardiomyocyte vulnerable to hypoxia during cardiac ischaemia.
We have recently shown that the postnatal switch in cardiac energy metabolism form glycolysis to ß-oxidation of lipid is dependent on the increased ambient oxygen encountered by the neonate following birth, and mediated though a novel pathway centered on the transcription factor hand1. We have found that this increase in oxygenation also drives postnatal remodeling of the cardiac conduction system, and exposure of the neonate to low levels of oxygen can result in cardiac arrhythmia. We hypothesise that this is a novel mechanism underlying sudden infant death (SIDS).
Efforts are now underway to define the mechanisms underlying loss of cardiac hypoxic adaptation at birth, and possible biomarkers for SIDS.
Breckenridge R. A., et al., Hypoxic regulation of Hand1 controls the fetal-neonatal switch in cardiac metabolism (2013) PLOS Biology 11(9): e1001666. doi:10.1371/journal.pbio.1001666
Neary, M. T., Breckenridge R. A. Hypoxia at the Heart of Sudden Infant Death Syndrome? (2013)
Pediatric Research doi: 10.1038/pr.2013.122.
Neary MT, Mohun TJ, Breckenridge RA. A mouse model to study the link between hypoxia, long QT interval and sudden infant death syndrome. (2013) Dis Model Mech. Mar;6(2):503-7.
Breckenridge R.A. Heart Failure and Mouse models. (2010) Dis. Model Mech. 3:138-43
Technology in the Breckenridge Laboratory
- Conditional transgenic mouse line generation
- Hypoxic mouse rearing
- Hypoxic tissue culture
- Chromatin Immunoprecipitation/ChIP Seq
- Stable isotope label metabolomics
- Oxygen flux analysis
- Senior Lecturer/Honorary Consultant Physician, Division of Medicine, University College London Hospital
- Training Programme Director, Clinical Pharmacology London Rotation
- Programme Director, UCL Masters Programme in Clinical and Experimental Medicine
- Chair, UCLH Use of Medicine Committee
- Chair, UCL/UCLH Clinical Trials Safety Committee
- Member, UK Specialty Advisory Committee, Clinical Pharmacology
Dr Marina-Gonzalez is a cardiovascular neuroscientist based in the UCL Department of Clinical Pharmacology. He studied Medicine in Mexico where he did a Masters and a PhD in Neuroscience. He completed postdoctoral training in UCL and Cambridge before obtaining a British Heart Foundation Intermediate Basic Science Research Fellowship to study the interaction between circulating adipokines and peripheral and central chemoreceptors in the development of obesity-related hypertension.
He is organiser of the Heart and Circulation module for 3rd year undergraduate medical students.
In my lab we study the role of the autonomic nervous system in the pathogenesis of hypertension and heart failure. We employ a wide array of cutting-edge techniques, including optogenetics and pharmacogenetics for selective activation or inhibition of astrocytic and neuronal networks that control cardio-respiratory activity. This involves active collaboration with Prof Alexander Gourine (UCL), Profs Sergey Kasparov and Prof Julian Paton (University of Bristol), Prof Andyrew Tinker (William Harvey Heart Centre), Prof Michael Spyer (UCL), Dr Vidya Mohamed-Ali (ADL, Qatar) Dr Stefan Trapp (Imperial College London) and Prof Gregory Funk (University of Alberta).
In 2011, I was awarded a British Heart Foundation Intermediate Research Science Fellowship to investigate the role of peripheral and central adipokine sensors in the development of obesity-related hypertension.
In 2013 I became course organiser of the Heart & Circulation module
Lecturing in the following courses: Circulation & Breathing, Heart & Circulation, Respiration in health and disease.
Other academic activities: Workshops, practicals and tutorials for 1st and 3rd years MBBS students.
In 2010 I was awarded a Postgraduate certificate in learning and teaching in higher education by the Centre for Advancement of Learning and Teaching, UCL.
- Nephtali Marina, Natalie D. Bull & Keith R Martin. A semi-automated targeted sampling method to assess optic nerve axonal loss in a rat model of glaucoma. Nature Protocols 2010 Sep;5(10):1642-51. Citations: 12. Impact factor: 11.74
- Alexander V. Gourine, Vitaliy Kasymov, Nephtali Marina, Feige Tang, Melina F. Figueiredo, Samantha Lane, Anja G. Teschemacher, K. Michael Spyer, Karl Deisseroth, and Sergey Kasparov. Astrocytes Control Breathing Through pH-Dependent Release of ATP. Science. 2010; 329(5991): 571-575. Citations: 144. Impact factor: 31
- Nephtali Marina, Ana PL Abdala, Alla Korsak, Annabel E Simms, Andrew M Allen, Julian FR Paton and Alexander V Gourine. Control of sympathetic vasomotor tone by catecholaminergic C1 neurones of the rostral ventrolateral medulla oblongata. Cardiovascular Research 2011; 91(4):703-710. Citations: 12. Impact factor: 5.94
- Nephtali Marina, Melina Figueiredo, Vitaliy Kasymov, Feige Tang, Vidya Mohamed-Ali, Anja G Teschemacher, Sergey Kasparov and Alexander V Gourine. Purinergic signalling in the rostral ventro-lateral medulla controls sympathetic drive and contributes to the progression of heart failure following myocardial infarction in rats. Basic Research in Cardiology. 2013: 108(1) 317 Citations: 2. Impact factor: 5.9
- Nephtali Marina, Ana P Abdala, Stefan Trapp, Aihua Li, Eugene E. Nattie, Jeffrey C. Smith, Julian FR Paton, Alexander V Gourine. Essential Role of Phox2b-Expressing Ventrolateral Brainstem Neurons in the Chemosensory Control of Inspiration and Expiration. Journal of Neuroscience 2010 Sep 15;30(37):12466-73. Citations: 27. Impact factor: 7.12
Our group aims to better understand how genes are modulated by a subclass of lipid activated nuclear receptors (the LXRs) in the context of atherosclerosis to help develop improved LXR-based therapies for the treatment of this and other vascular and inflammatory diseases.
Summary of Research
Atherosclerosis is the hardening of major blood vessels resulting from the accumulation of cholesterol. This lesion eventually causes the majority of cardiovascular diseases, the leading cause of mortality worldwide. Abnormally high levels of cholesterol are a risk factor for atherosclerosis and in recent years inflammatory pathways have also been shown to extensively contribute to the development of atherosclerosis. Thus, molecules providing a tight control of cholesterol levels with additional anti-inflammatory properties represent ideal anti-atherosclerotic drugs. The Liver X receptors (LXRs) behave like cholesterol sensors: they are lipid-activated transcription factors that control the expression of key genes to tightly maintain cholesterol levels. Moreover, LXR activation inhibits inflammation in diverse settings. These actions contribute to the reduced atherosclerosis observed in several experimental models treated with LXR ligands which is why LXRs are considered promising targets to develop therapies against atherosclerosis.
Elucidating the signaling pathways that regulate its activity is critical to our understanding of LXR function. My group aims to better understand how genes are modulated by LXR in the context of atherosclerosis. We are particularly interested in pathways regulated by LXR in a non-canonical fashion, e.g. by modulating post-translational modifications of the receptor (such as phosphorylation) or through crosstalk with other transcriptional and/or signaling pathways. We are generating unique tools and experimental models to investigate whether changes observed in cultured cells are translated in vivo in a pathological context.
Main Research Projects
Impact of LXRalpha phosphorylation on vascular inflammation and atherosclerosis
Previous work identified a phosphorylation site in LXRalpha that is important to selectively inhibit the transcription of specific LXR target genes and expands the range of LXRalpha-responsive genes in macrophages (Pineda-Torra I, Mol Cell Biol 2008). We are now investigating whether changes in the phosphorylation status of LXR affect macrophage inflammation levels and the development of atherosclerosis. To this end, we have generated a novel experimental model that expresses a phosphorylation-deficient mutant of the receptor (LXRa-S196A) in a cell/tissue-selective manner in an atherosclerotic background. This innovative approach is at the forefront of nuclear receptor and atherosclerosis research and may lead to the development of novel and more efficient LXR activators to reduce atherosclerosis.
Funded by MRC New Investigator Award
Role of LXRalpha phosphorylation in lung inflammation
LXR agonists anti-inflammatory properties have also been shown in models of acute lung injury. LXRs are expressed in several alveolar cell types were they inhibit cytokine expression, diminish reactive oxygen species and reduce neutrophil recruitment. We now aim to
- Investigate LXR?lpha phosphorylation in lung as a means to modulate its activity alternatively to ligand activation
- Examine its impact on severe lung inflammation and
- Uncover how changes in LXR?lpha phosphorylation impact lung target gene expression during persistent severe inflammation.
We will employ our unique experimental model expressing a mutation that abolishes LXRa phosphorylation.
Funded by Grand Challenges PhD Studentship
Impact of interferon signaling on LXR anti-atherosclerotic actions
Our group at UCL (Pourcet B., Circulation Res 2011) demonstrated that LXR induces the expression of an important anti-inflammatory protein (arginase 1) in a novel way that involves two other transcription factors (IRF8 and PU.1). We now have evidence that in addition, other novel LXR target genes affecting pathways relevant to atherosclerosis are dependent on IRF8 for their response to LXR ligands. Thus we are exploring how IRF8 impacts the anti-atherosclerotic actions of LXR ligands by generating a novel experimental model deficient in IRF8 on an atherosclerotic background and determine how IRF8-dependent LXR regulated genes are regulated by the LXR/IRF8 crosstalk. We additionally aim to better understand how LXR ligands induce IRF8 interactions with other proteins to regulate LXR target gene expression.
Funded by a British Heart Foundation Project Grant
Technology in the Pineda-Torra Laboratory
- Isolation of bone marrow-preparation of murine (peritoneal and bone marrow derived) macrophages, isolation and culture of human macrophages
- murine and human macrophage-like cell lines (RAW 267.4, THP-1 cells)
- other cells lines commonly used to overexpress proteins and study nuclear receptor activity (Hek293, Cos)
- Real time quantitative RT-PCR
- RNA interference
- Microarray analysis of whole genome gene expression
Assays commonly used to study nuclear receptor/transcription factor activity:
- Luciferase reporter assays
- Cloning of promoters /enhancers into reporter vectors
- Promoter/enhancer mutagenesis
- Chromatin immunoprecipitation assays to study transcription factor binding to chromatin
Protein:protein interaction assays
- Immunoprecipitation assays
- GST pulldowns
- Generation of phosphospecific antibodies
- Detection of phosphorylation with phosphospecific antibodies (immunoblotting, immunocytochemistry and immunohistochemistry)
- Kinase assays
- Foam cell formation in vitro
- Dissection of aortic roots and arches, thoracic and abdominal aortas
- Oil Red O staining of lipids in foam cells (in vitro, atherosclerotic plaques)
- Immunohistochemistry to assess inflammatory markers and proliferation within atherosclerotic plaques
- Generation of experimental in vivo models
Present and Past Lab members
Ines Pineda-Torra (Group Leader)
Oscar Pello (Senior Research Associate)
Matthew Gage (Senior Research Associate)
Natalia Becares Salles (PhD student)
(currently at the Institut Pasteur de Lille, France email@example.com)
Main National and International Collaborations
Michael Garabedian, Prof in Urology and Microbiology, NYU Langone Medical Center, USA
Edward Fisher, Leon H Charney Prof of Cardiovascular Medicine, NYU Langone Medical Center, USA
Sidney Morris, Prof Emeritus in Microbiology and Molecular Genetics, University of Pittsburgh
Keyko Ozato, Prof in Universityof Maryland College Park
Xiaoyu Hu, Assistant Professor of Immunology, Department of Medicine, Weill Cornell
Antonio Castrillo, Group Leader in the Department of Metabolism and signaling, Instituto de Investigaciones Biomedics “Alberto Sols”
Ian Zachary, Prof in Cardiovascular Biology
Liz Jury, Principle Research Associate, Centre for Rheumatology, UCL
We are interested in the inflammatory response. Inflammation is an organism’s defensive response to foreign bodies and injury. While this is a beneficial, it can go into disarray resulting in tissue damage and the development of chronic inflammatory conditions - including arthritis, fibrosis, multiple sclerosis etc. We are interested in the cells of the mononuclear phagocyte system, which comprises blood monocytes, macrophages and dendritic cells. These cells are involved in tissue homeostasis and co-ordinate the immune response. Our research focuses on how tissue resident macrophages and blood monocytes interact during acute inflammation and following the resolution of inflammation. Understanding the differential roles of blood monocytes and resident macrophages under steady state and following inflammation may pave the way for future therapeutic purposes.
Monocyte, macrophage, inflammation.
Standard techniques: Flow cytometry, cell sorting, adoptive transfer.
Mice generated: Cx3cr1cre & Cx3cr1creER
Yona S, Kim K, Wolf Y, Mildner A, Breker M, Ayali D, Viukov S, Guilliams M, Misharin A, Hume D, Perlman A, Malissen B, Zelzer E & Jung S (2013) Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38 79-91