Canadian National Medical Student Research Symposium June 12�14th, 2012 University of Manitoba
Table of Contents
WELCOME
5
JUDGING AND ORGANIZING COMMITTEE
6
SCIENTIFIC PROGRAM
7
SESSION 1: LIGHTNING ORAL PRESENTATION
18
Martin, Christopher
19
Eaton, Alison
20
Liu, Xiao-Yang
21
Kuzyk, Alexandra
22
Baxter, Laura
23
Benesch, Matthew
24
Bracey, Nathan
25
Skappak, Christopher
26
SESSION 2: POSTER DISCUSSION
27 28
Tan, Zachary
39
Verman, Sanam
30
Tulk, Sarah
31
Lother, Sylvain
32
Peden, Alexander
33
Wang, Michael
34
Miller, Corey
35
Morrissey, Laura
36
Stokes, William
37
Lachance, Audree 1
38
Reeson, Mark
39
Sivanathan, Lavarnan
40
Mullen, Sarah
41
Liu, Edward
42
Rhee, Chanseok
43
Martin, Christophe
44
Switzer, Noah
45
Benesch, Matthew
46
Bracey, Nathan
47
Bach, Paxton
48
Wadwani, Aman
49
Thornton, Christina
50
Skappak, Christopher
51
Elliott, Anna
52
Baxter, Laura
53
Basmaji, John
54
Cawthorn, Thomas SESSION 3: POSTER DISCUSSION
55
Richards, Ceri
56
Silaghi, Alex
57
Houston, Brett
58
Wieskopf, Jeffrey
59
Liu, Xiao-Yang
60
Eaton, Alison
61
Langdon, Christopher
62
Cooke-Hubley, Sandra
63 2
Bulir, David
64
LeMessurier, Jennifer
65
Chapleau, Julien
66
Harroud, Adil
67
Tremblay, Elsa
68
Savage, David
69
Armstrong, Susan
70
Costain, Gregory
71
Locke, Jennifer
72
Yeh, Calvin
73
Huynh, Melisa
74
Olah, Michelle
75
Chu, Jackson
76
Tsang, Erica
77
Min, Cynthia
78
Zhang, Charlie
79
Kaicker, Jaiten
80
Arsenault, Kyle
81
Yoo, Jeff
82
Liu, Yingwei
83
Yoon, Ju-Yoon
84
Lovat, Nicole
85
Kaye, Cameron
86
Westwell-Roper, Clara
87
SESSION 4: LIGHTNING ORAL PRESENTATION
88
Houston, Brett
89
Tsang, Erica
90
Tremblay, Elsa
91
Copley, Michael
92 3
Costain, Gregory
93
Armstrong, Susan
94
Silaghi, Alex
95
Westwell-Roper, Clara
96
PARTICIPATING SCHOOLS
97
PARTICIPANT CONTACT INFORMATION
98
SPONSORING AGENCIES
99
4
Welcome to the 4th Canadian National Medical Student Research Symposium. The opportunity to indulge your curiosity, make a discovery and thereby make a change in the world is an incredible privilege that few have. Researchers are one group that is very fortunate to do so. At the same time, there is no doubt that learning how to conduct and interpret research, how to develop it into a substantial portion of your career, and how to maintain a successful program is hard work that is fraught with uncertainly, insecurity and the occasional burst of excitement. Over the next several days you have the opportunity to share your experiences, your emerging research projects and your own experimental findings with others. This is an excellent way of helping you determine if research truly is a passion for you. For those individuals who can combine the superb breadth of biological understanding that a medical degree provides with the ability to ask well focused, targeted questions that develops in a graduate degree program, there are a great many professional opportunities. Future options range from leading large multicentre research groups, to running your own laboratory, to being a key collaborator with a sophisticated understanding of the benefits (and limitations) of biomedical research. The trainees this year represent medical schools, and research disciplines, from across Canada. About one third of our 65 attendees are in their first year of hands-on research, one third have intermediate experience and another third are engaged in joint MD PhD programs. As the abstracts demonstrate, your work ranges from studies of individual gene polymorphisms or molecules through to population and health systems research. Because so much groundbreaking research is highly multidisciplinary, we have designed the program to increase academic and social interactions between trainees such as yourselves with those enrolled in non-clinical graduate training programs. Make the most of the opportunity! I want to personally thank the individuals and organizations that have enabled us to offer you this special opportunity. Money is never is excess supply and they have gone to great effort to support this initiative. Contributors are listed at the back of the Symposium program. We welcome you and hope you enjoy the novel science, the professional development activities and the pleasures of meeting others with similar interests during your three days here.
Kent T. HayGlass PhD Canada Research Chair in Immune Regulation Director, Advanced Degrees in Medicine University of Manitoba
5
POSTER JUDGING TEAMS Internal
External
Team A
Cameron Kaye, University of Manitoba
Clara Westwell-Roper, University of British Columbia
Team B
Alexandra Kuzyk, University of Manitoba
Michael Copley, University of British Columbia
Team C
Nicole Lovat, University of Manitoba
Xiao-yang Liu, McGill University
Team D
Ju-Yoon Yoon, University of Manitoba
Susan Armstrong, University of Toronto
JUDGES: ORAL PRESENTATIONS Dr. Quim Madrenas, McGill University Dr. Meghan Azad, University of Alberta
Invited Chairs: Oral Presentations Julien Chapleau, University of Montreal Sarah Mullen, University of Toronto Christina Thornton, University of Calgary Charlie Zhang, University of British Columbia
ORGANIZING COMMITTEE Dr. Kent HayGlass – Director, Advanced Degrees in Medicine, CNMSRS Coordinator Kimberley Ponton – CNMSRS Coordinator, Program Coordinator, B.Sc.(Med) Ju-Yoon Yoon – Committee Member and MD/PhD student Alexandra Kuzyk – Committee Member and MD/PhD student Nicole Lovat - Committee Member and MD/PhD student Cameron Kaye – Committee Member and MD/PhD student
6
Canadian National Medical Student Research Symposium University of Manitoba Scientific Program Canadian National Medical Student Research Symposium DAY 1: Tuesday, June 12, 2012 1200 - 1800 Students check-in at the Delta Hotel throughout the day. Shuttle bus will loop between the Delta Hotel and the University of Manitoba, Brodie Centre (downtown campus). 1600 - 1800 Registration desk open – Inside front entrance Brodie Centre. Trainees to mount their posters in the Brodie Mezzanine area prior to 1800. 1750 Bus to St. Charles Country Club (Dress Code)Front of Brodie Centre 1820 Opening Reception – Mixer 1850 Welcome by Dr. Kent HayGlass CNMSRS Coordinator 1900 Dinner 2015 Keynote Speaker Dr. J. (Quim) Madrenas, MD, PhD, FCASH, Professor and Head, Department of Microbiology and Immunology, McGill University “A Career in Arts and Sciences: My Own Experience” 2200 Bus returns to Delta Hotel
DAY 2: Wednesday, June 13th, 2012 *Students to bring casual evening sweater/footwear to Conference as you will leave directly to the social event via bus from Brodie Centre. Locker storage for your goods will be provided to you for the day; please bring your own lock. 0730 (approx) Shuttle Bus starts at 0700, departing from the Delta Hotel to Brodie Centre. Bus runs in a continuous loop. 10-15 minutes between the two venues. 0745 – 0820 Continental Breakfast and Registration in Apotex Centre. Registration Desk open 0745 – 0945 for late arrivals. Breakfast on 2nd floor Apotex Atrium. 0825 Apotex Centre – Main Floor – Procurity Lecture Theatre Welcome by Dr. Kent HayGlass – Director Advanced Degrees in Medicine, Coordinator CNMSRS 0830 Apotex Centre – Main Floor – Procurity Lecture Theatre Keynote Speaker Dr. Julie Ho, Assistant Professor Sections of Nephrology and Biomedical Proteomics, University of Manitoba ‘Diagnostic – Hopefully Prognostic – Biomarkers of Acute Kidney Injury’ 0900 – 1030 Session I – Lightning Oral Presentations (6 minute presentation, 4 minutes for questions: 8 presentations in total. Procurity Lecture Theatre – Main Floor – Apotex Centre Chairs; Sarah Mullen, University of Toronto and Julien Chapleau, University of Montreal
7
Student
School
Abstract Title
University of Montreal
Evidence for a reference frame transformation of vestibular signal contributions to voluntary reaching movements
Eaton, Alison
Memorial University
Inflammation-induced changes in leukotriene receptor expression cause differential gene expression through nuclear calcium signaling in vascular smooth muscle cells
Liu, Xiao-Yang
McGill University
Driver mutations in histone H3.3 and chromatin remodeling genes in paediatric glioblastoma
Martin, Christophe
Selective telomere lengthening in mouse plasmacytoma supports a protective mechanism for functionally important chromosomes
University of Manitoba
Kuzyk, Alexandra
Baxter, Laura
Dalhousie University
An immunohistochemical investigation of C-bouton regulation of motoneuron excitability: Kv2.1 channels and local calcium regulation.
Benesch, Matthew
University of Alberta
Cholesterol oxidation leads to separation of lipid rafts: A biophysical model for protein plaque formation.
Bracey, Nathan
University of Calgary
The Nlrp3 Inflammasome promotes systolic dysfunction in structural cardiomyopathy through Il-1β.
Skappak, Christopher
University of Calgary
Diagnosing a severe neonatal intestinal disorder using nuclear magnetic resonance metabolomics
1030 – 1200 Session I Poster Discussion – Brodie Centre, Mezzanine (Coffee available 10:30 – 11:00) Poster presentation 4 minutes, discussion 4 minutes (8 minutes total)
Judging Team A
Asterisk indicates Trainee is in first 12 months of research training
Poster #
Student
School
Abstract Title
Time (estimate)
Tan, Zachary
University of Alberta
Characterization of PAX3 Activity in Melanoma
10:45
*2
Verman, Sanam
University of Alberta
Cardiac MRI volumetric assessment by shortaxis has better reproducibility than the axial orientation in single RV hearts.
10:55
*3
Tulk, Sarah
University of Calgary
Vitamin D3 induces release of IL-1β via the NLRP3 inflammasome: Implications for Crohn’s disease
11:05
*1
8
*4
Lother, Sylvain
Kaye, Cameron *5
6
Cawthorne, Thomas
University of Manitoba
-Estrogen Use and Survival of Women with Non-Small-Cell Lung Cancer in Manitoba
11:15
University of Manitoba
Feasibility Study of Microwave Tomography for In Vivo Diagnostic Characterization of Tissue in Human Disease
11:25
Queen’s University
Implementation of a novel curriculum for training medical students in the use and interpretation of hand-held echocardiography
11:35
Judging Team B
Asterisk indicates Trainee is in first 12 months of research training
Poster # *7
Student Wang, Michael
School
Abstract Title
Time (estimate)
McGill
Mast Cell Deficiency Significantly Delays Bone Tissue Regeneration
10:45
McGill
Meta-analysis of efficacy and safety of new oral anticoagulants (dabigatran, rivaroxaban, apixaban) versus warfarin in patients with atrial fibrillation
10:55
*8
Miller, Corey
*9
Morrissey, Laura
McMaster
Educating in child maltreatment: Mandatory reporting and medical student experiences.
11:05
*10
Stokes, William
Memorial University
Performance of a Commercial Urine Transport System for Stabilizing Bacterial Growth
11:15
11
Martin, Christophe
University of Montreal
Evidence for a reference fram transformation of vestibular signal contributions to N/A (ORAL) voluntary reaching movements
12
Switzer, Noah
University of Alberta
Does Electrical Remodeling Early After Myocardial Infarction Predict an Increased risk of Cardiac Arrest?
11:25
13
Benesch, Mathew
University of Alberta
Cholesterol oxidation leads to separation of lipid rafts: A biophysical model for protein plaque formation.
N/A (ORAL)
9
Judging Team C Asterisk indicates Trainee is in first 12 months of research training
Poster #
Student
School
Abstract Title
Time (estimate)
Sivanathan, Lavarnan
University of Toronto
Gene expression changes observed in cancer cells with acquired resistance to low-dose metronomic cyclophosphamide chemotherapy
10:45
*15
Mullen, Sarah
University of Toronto
Saccades as a Novel Tool for Patients with Traumatic Brain Injury?
10:55
*16
Liu, Edward
McMaster University
Microspheres releasing GDNF within fibrin gels improve peripheral nerve regeneration after delayed nerve repair
11:05
*17
Rhee, Chanseok
University of Western Ontario
Work Disability and Work Limitations in Rheumatoid Arthritis, Psoriatic Arthritis and Ankylosing Spondylitis
11:15
18
Thornton, Christina
University of Calgary
The Streptococcus Antibiotic Resistome of Adult Cystic Fibrosis Patients Results from Mutation and Horizontal Gene Transfer.
11:25
*14
19
Bracey, Nathan
University of Calgary
The Nlrp3 Inflammasome promotes systolic dysfunction in structural cardiomyopathy through Il-1β.
N/A (Oral)
20
Bach, Paxton
Queen’s University
Interferon-gamma release assays (IGRA) piloted as a latent TB screening tool in federal Canadian inmates
11:35
Judging Team D Asterisk indicates Trainee is in first 12 months of research training
Poster #
*21
Student
Peden, Alex
School
University of Manitoba
Abstract Title Canadians’ Satisfaction with Medicare: An Analysis of the Canadian Community Health Survey
Time (estimate)
10:45
10
*22
Lachance, Audree
University of Montreal
University of
Presence of inhibitory receptors at the surface of tumour-infiltrating T lumphocytes isolated from carcinomatous ascites.
10:55
Comparing health behaviours of internal medicine residents and medical students: an observational study.
11:05
11:15
*23
Reeson, Mark
*24
Basmaji, John
University of Western Ontario
Detection of circulating tumor cells in advanced head and neck cancer using the CellSearch® system
25
Wadhwani, Aman
University of Calgary
Mechanisms of the protease-induced increase in transepithelial resistance in intestinal epithelial Cells
11:25
26
Skappak, Christopher
University of Calgary
Diagnosing a severe neonatal intestinal disorder using nuclear magnetic resonance metabolomics
N/A (ORAL)
27
Elliott, Marie Anna
Dalhousie University
Hearing Preservation and Acoustic Neuromas: No Difference Found between Stereotactic Radiotherapy and Conservative Treatment
11:35
28
Baxter, Laura
Dalhousie University
An immunohistochemical investigation of Cbouton regulation of motoneuron excitability: Kv2.1 channels and local calcium regulation.
N/A (ORAL)
Saskatchewan
1200 – 1230 1245 - 1335
Luncheon – Joe Doupe Concourse Professional Development Workshop. Developing and Maintaining a Successful Research Career : What You Need to know (Theatre A/ Gaspard Lecture Theatre). Drs. P. Choy, J. Ho and Q. Madrenas. Moderator: K HayGlass.
1335 - 1345
Clinician Investigator Trainee Association of Canada (CITAC) information overview. (Theatre A/ Gaspard Lecture Theatre) Ju-Yoon Yoon, VP Internal Affairs ; Alexandra Kuzyk Secretary, Executive Committee.
1345 – 1515
Poster Discussion Session 2 – Brodie Centre Mezzanine Poster presentation 4 minutes, 4 minutes discussion (8 minutes in total)
Judging Team A Poster #
29
Student
Silaghi, Alex
School University of Manitoba
Abstract Title
Identification of Virulence Determinants in 1918 Spanish Flu Hemagglutinin
Time (estimate)
N/A (Oral)
11
30
Houston, Brett
University of Manitoba
Phenotypic and genotypic evaluation of two kindreds affected with hereditary xerocytosis
N/A (Oral)
13:45
31
Wieskopf, Jeffrey
McGill
The nicotinic Îą6 subunit gene affects chronic pain sensitivity via an interaction with P2X2/3 receptors
32
Liu, Xiao-Yang
McGill
Driver mutations in histone H3.3 and chromatin remodeling genes in paediatric glioblastoma
33
Eaton, Alison
N/A (Oral)
Memorial University
Inflammation-induced changes in leukotriene receptor expression cause differential gene expression through nuclear calcium signaling in vascular smooth muscle cells
13:55
14:05
34
Langdon, Kristopher
Memorial University
Prolonged, 24-h delayed peripheral inflammation increases short- and long-term functional impairment and histopathological damage after focal ischemia in the rat
35
Cooke-Hubley, Sandra
Memorial University
Medical Students’ Attitudes towards using Online Social Networking sites to Communicate with Patients
14:15
Bulir, David
McMaster University
Biochemical characterization of Chlamydia outer protein (Cop) B and D from Chlamydia pneumoniae.
14:25
37
Judging Team B Poster #
38
39
Student
School
Abstract Title
Time (estimate)
LeMessurier, Jennifer
Memorial University
The development of national indicators for the surveillance of osteoporosis in Canada
13:45
Chapleau, Julien
University of Montreal
Does malpositioning of the arm influence radiographic range of motion measurement?
13:55
12
Harroud, Adil
University of Montreal
Postoperative furosemide administration lowers transfusion rate in scaphocephaly surgery
Tremblay, Elsa
University of Montreal
Dorsal premotor cortex is involved in switching motor plans.
Savage, David
Northern Ontario School of Medicine
Developing seasonal physician shift schedules in the emergency department to meet patient demand
Armstrong, Susan
University of Toronto
Influenza infects lung microvascular endothelium leading to microvascular leak: Role of apoptosis and claudin-5
N/A (Oral)
Costain, Gregory
University of Toronto
Evaluating genetic counselling for multifactorial schizophrenia in the molecular age
N/A (Oral)
45
Locke, Jennifer
University of Toronto
NKX3.1 haploinsufficiency is prognostic for prostate cancer relapse following surgery or image-guided radiotherapy
14:25
46
Yeh, Calvin
McMaster University
"Activation complex assembly differentially protects coagulation factors Xa, IXa,
14:35
Huynh, Melissa
University of Western Ontario
40
41
42
43
44
47
Urinary Device Attachment of Staphylococcus saprophyticus in the Presence of Sub-Minimal Inhibitory Antimicrobial Concentrations
14:05
N/A (Oral)
14:15
14:45
Judging Team C Poster #
48
49
Student
School
Abstract Title
Time (estimate)
Olah, Michelle
University of Toronto
Discrimination in access to primary care on the basis of socioeconomic status
13:45
Chu, Jackson
University of British Columbia
Longitudinal assessment of bone mass and strength in perinatally HIV-infected youth
13:55
13
Tsang, Erica
University of British Columbia
Life-history Chronicle for a Patient with the Recently Described Chromosome 4q21 Microdeletion Syndrome
Min, Cynthia
University of British Columbia
Recognizing uncertainty in your knowledge: The impact of response options presentation on self-monitoring accuracy.
14:05
52
Zhang, Charlie
University of British Columbia
The Negative Predictive Value of US-Guided 14-Gauge Core Needle Biopsy of Breast Masses
14:15
53
Kaiker, Jatin
McMaster University
Assessing the Reliability and Quality of Online Uterine Fibroid Embolization Resources
14:25
Arsenault, Kyle
McMaster University
TCOM Study - Transcutaneous oximetry as a prediction tool for chronic wound and amputation healing outcomes
14:35
50
51
54
N/A (Oral)
Judging Team D Poster #
Student
School
Abstract Title
Time (estimate)
Yoo, Jeff
University of Ottawa
Development of a Symptom Specific Quality of Life Scale in Emergency Department Patients with Recent Onset Atrial Fibrillation/Flutter
13:45
Liu, Yingwei
University of Ottawa
Identification of Vulnerable Aortic Plaque Using Conventional FDG-PET Myocardial Viability Studies
13:55
Yoon, Ju-Yoon
University of Manitoba
Valproic Acid and Fludarabine induces a synergistic response against Chronic Lymphocytic Leukaemia
14:05
58
Lovat, Nicole
University of Manitoba
Characterization of Gestational Insulin Resistance in Normal Healthy Sprague Dawley Rats
14:15
60
Westwell-Roper, Clara
University of British Columbia
Islet amyloid polypeptide: a trigger for Toll-like receptor 2 in type 2 diabetes and islet transplantation?
N/A ORAL
55
56
57
14
61
62
Kuzyk, Alexandra
University of Manitoba
Selective telomere lengthening in mouse plasmacytoma supports a protective mechanism for functionally important chromosomes
Richards, Ceri
University of Manitoba
ROLE OF GLIA MATURATION FACTOR BETA (GMFβ) IN DIFFERENTIATION OF THE CHILDHOOD CANCER NEUROBLASTOMA
1530 – 1700
N/A (Oral)
14:25
Session 2 – Lightning Oral Presentations Procurity Lecture Theatre, Apotex Centre, Main Floor; 6 minute oral presentation, 4 minute questions, 8 in total Chairs; Christina Thornton, University of Calgary and Charlie Zhang, UBC
Student Armstrong, Susan
Silaghi, Alex
School University of Toronto University of Manitoba
Abstract Title Influenza infects lung microvascular endothelium leading to microvascular leak: Role of apoptosis and claudin-5 Identification of Virulence Determinants in 1918 Spanish Flu Hemagglutinin
University of British Columbia
Islet amyloid polypeptide: a trigger for Toll-like receptor 2 in type 2 diabetes and islet transplantation?
Houston, Brett
University of Manitoba
Phenotypic and genotypic evaluation of two kindreds affected with hereditary xerocytosis
Costain, Gregory
University of Toronto
Evaluating genetic counseling for multifactorial schizophrenia in the molecular age
Westwell-Roper, Clara
Tsang, Erica
Tremblay, Elsa
University of British Columbia University of Montreal
Life-history Chronicle for a Patient with the Recently Described Chromosome 4q21 Microdeletion Syndrome Dorsal premotor cortex is involved in switching motor plans.
1710 Preparation for departure from Brodie Centre to social event at Lower Fort Garry 1730 Depart from front of Brodie Centre via bus. CSHRF and CNMSRS Dinner and Mixer and Lower Fort Garry 2200 (estimate) Participants return via bus directly to the Delta Hotel
15
Symposium on:
Thursday, June 14, 2012 *Students check out of hotel; bring luggage to Joe Doupe Concourse. A luggage room will be provided. Frederic Gaspard Lecture Theatre A, Basic Medical Sciences Building 08:00
Registration & Cinnamon Bun Breakfast (Joe Doupe Concourse)
09:00
Welcome and Opening Remarks (Gaspard Theatre A) Dean, Faculty of Medicine, University of Manitoba Chair’s Overview
9:15
Dr. Joaquín (Quim) Madrenas McGill University, Montreal, QC “Pathobionts, Symbionts and Probiotics: Towards a new understanding of Infection and Immunity”
10:00
Coffee Break (Mezzanine)
10:30
Dr. Bruce McManus University of British Columbia, Vancouver, BC “Cardiovascular Disease and Inflammation”
11:15
Dr. Alexandre Prat Université de Montréal, Montreal, QC “Molecular Control of Immune Cell Trafficking to the CNS”
12:00
Lunch and Poster Session (Brodie Atrium)
16
12:30
• • • • • •
13:30
Dr. Gregory Gores
Presentations of Recognition (Brodie Atrium) Graduate Students Assoc. Award for Distinction in Mentorship Heart and Stroke Foundation Dr. Kenneth Hughes Young Investigator Award Dr. Aubie Angel Young Investigator Award Faculty Recognition for Outstanding Contribution to Research
Mayo Clinic, Rochester, MN “Nonalcoholic Steatohepatitis (NASH) as an Inflammatory Disease” 14:30
Coffee Break (Mezzanine)
15:00
Dr. Paul Kubes University of Calgary, Calgary, AB “Visualizing the Immune System in Blood Vessels”
16:00
Dr. Phillip Gardiner University of Manitoba – CIHR Strategic Overview
16:30
Awards Ceremony CNMSRS and CSHRF
17:15
Reception (Joe Doupe Concourse)
17
Lightning Oral SESSION 1
Procurity Lecture Theatre, Apotex Building Main Floor, Room 164 8:55 – 10:30 a.m. Wednesday, June 13th, 2012
18
Evidence for a reference frame transformation of vestibular signal contributions to voluntary reaching movements Christophe Martin, Ian Moreau-Debord, Marianne Landry, Andrea M. Green Dept Physiol, Univ. Montreal, Montreal, QC, Canada Rationale: Studies have shown that vestibular signals play a role in the execution of voluntary reaching movements by contributing to spatial, dynamic and postural compensations when reaching during body motion. However, because the vestibular sensors are fixed in the head whereas the arm is fixed to the trunk, to contribute appropriately vestibular signals must be transformed from a head- to a body-centered reference frame. To investigate the evidence for this transformation, we used galvanic vestibular stimulation (GVS) to stimulate vestibular afferents as human subjects made reaching movements in the horizontal plane. Methods: Bipolar GVS simulates net rotation about an axis inclined ~18o upward from the head naso-occipital axis (Fitzpatrick & Day, 2004). With the head upright it thus simulates mainly whole-body tilt about an earth-horizontal axis, whereas with the head pitched 72o forward the same stimulus simulates rotation about an earth-vertical axis (yaw). Thus, if vestibular signals contributing to reaching have been transformed from a head- to a bodycentered reference frame, the same stimulus should perturb horizontal-plane reach trajectories in a head- orientation-dependent way. With the head upright the stimulation should elicit mainly postural compensations and only small horizontal-plane trajectory deviations, whereas with the head pitched forward much larger trajectory deviations are predicted to compensate for a simulated yaw body rotation. To test this prediction, 35 subjects reached to a remembered target location in darkness with their right arm supported in the horizontal plane by a robotic exoskeleton (KINARM, B-Kin). GVS (3mA transmastoidal pulse) of unpredictable polarity was applied throughout or before the reaching movement in 16% of trials, chosen pseudo-randomly, during trial blocks in which the head was secured in either an upright or forward (50-60o) position. Results: GVS caused trajectories to deviate from non-stimulated controls about 300ms after movement onset. As predicted, with the head inclined forward average deviations were significantly larger than with the head upright (p<0.05) and were in the direction appropriate to provide spatial compensation for a simulated body yaw rotation. Conclusions: These results provide evidence that vestibular signals contributing on-line to reaching execution have been transformed from a head- to a body-centered reference frame. Funding: CIHR, NSERC Christophe_238@hotmail.com
19
Iflammation-induced changes in leukotriene receptor expression cause differential gene expression through nuclear calcium signaling in vascular smooth muscle cells a
a
a
Alison Eaton , Edit Nagy , Mathilde Pacault , Jérémy Fauconnier a
Department of Medicine
b, c
a,
, Magnus Bäck
*
b
and Department of Physiology & Pharmacology , Karolinska c
Institutet, Stockholm, Sweden and INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France. Aims: Leukotrienes are pro-inflammatory mediators that are locally produced in coronary atherosclerotic plaques. It is thought that the vasodilatory response induced by cysteinyl leukotrienes (CysLT) in healthy human coronary arteries may be altered under pathological conditions, such as atherosclerosis. This study aimed to elucidate CysLT signaling in vascular smooth muscle cells (SMCs), and the effects of inflammation on this process. Hypothesis: We tested the hypotheses that a) the CysLT1 receptor is upregulated in vascular SMCs in the context of atherosclerosis, and that b) CysLT1 receptor activation is coupled to nuclear calcium signaling, which c) leads to alterations in gene expression. Methods & Results: Immunohistochemical analyses revealed that CysLT1 receptor expression co-localized with a-smooth muscle actin in human carotid endarterectomy samples, and that the CysLT1 receptor exhibited a perinuclear expression in human coronary artery SMCs. Real-time quantitative PCR analysis demonstrated that lipopolysaccharide (LPS) significantly upregulated CysLT1 receptor mRNA levels in human coronary artery SMCs in a time-dependent fashion. Fluorescent-labeled calciumsignaling experiments showed that LPS significantly enhanced the changes in intracellular calcium induced by leukotriene C4 (LTC4) In these cells. LTC4 stimulation predominantly enhanced nuclear calcium increase, and this was inhibited by the CysLT1 receptor antagonist MK-571. Microarray analysis revealed that after 24h stimulation of human coronary artery SMCs with LTC4, there was a 5-fold increase in mRNA levels for Plasminogen Activator Inhibitor (PAI)-2, among a number of significantly upregulated genes. The LTC4-induced increase in PAI-2 expression was confirmed by real time quantitative PCR and ELISA, and was inhibited by the CysLT1 receptor antagonist MK571 and by calcium chelators. Conclusions: Pro-inflammatory stimulation of vascular SMCs upregulated CysLT 1 receptor expression, which was visualized in a peri-nuclear pattern. This receptor expression was coupled to nuclear calcium signaling and changes in gene expression, such as upregulation of PAI-2. Together, these findings suggest a role of nuclear CysLT 1 receptor signaling in vascular SMCs inducing gene expression patterns associated with atherosclerosis.
ajeaton@mun.ca
20
Driver mutations in histone H3.3 and chromatin remodeling genes in paediatric glioblastoma Xiao-Yang Liu1, Andrey Korshunov2, Jeremy Schwartzentruber3, David T.W.Jones4, Elke Pfaff4, Dominik Sturm4, Adam M. Fontebasso5, Dong-Anh Khuong Quang1, Steffen Albrecht6, Marcel Kool4, Zhifeng Dong7, Peter Siegel7, Andreas von Diemling2, Damien Faury1, Uri Tabori8, Peter Lichter4, Christoph Plass9, Jacek Majewski1, Stefan M. Pfister4, Nada Jabado1,10. 1 Department of Human Genetics, McGill University. 2Clinical Cooperation Unit Neuropathology, German Cancer Research Center, DKFZ, Heidelberg, Germany. 3McGill University and Genome Quebec Innovation Centre. 4Division Molecular Genetics, German Cancer Research Center, DKFZ, Heidelberg, Germany. 5Division of Experimental Medicine, McGill University. 6Department of Pathology, Montreal Children's Hospital, McGill University Health Center. 7 Rosalind and Morris Goodman Cancer Research Centre, McGill University. 8The Arthur and Sonia Labatt Brain Tumor Research Centre, Hospital for Sick Children Research Institute, University of Toronto. 9 Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center, DKFZ, Heidelberg. 10Department of Paediatrics, McGill University and McGill University Health Center Research Institute. Rationale: Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. Objectives and Methods: To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Results: Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (群-thalassaemia/ mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX- DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. Conclusions: This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.
Xiaoyang.liu@hotmail.ca
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Selective telomere lengthening in mouse plasmacytoma supports a protective mechanism for functionally important chromosomes Alexandra Kuzyka and Sabine Maia aThe Manitoba Institute of Cell Biology, Department of Biochemistry and Medical Genetics, University of Manitoba Rationale: Fast-onset mouse PCTs are induced in [T38HxBALB/c]N mice with pristane plus vabl/myc retrovirus and all tumors exhibit trisomy of cytoband 11E2. This cytoband contains all the genes necessary for the tumorâ&#x20AC;&#x2122;s accelerated tumorigenesis. This model is a unique opportunity to study 11E2 because it has a reciprocal translocation between chromosomes 11 and X (rcpT(X;11)) which generates a very small T(X;11) translocation chromosome containing only this cytoband and part of 11E1. Many studies have identified cancer cells to have overall shorter telomeres and a few have found that the length of individual telomeres can shorten, while others lengthen. This has led to the hypothesis that a chromosome-specific protective mechanism may lead to the lengthening of certain telomeres on chromosomes â&#x20AC;&#x2DC;keyâ&#x20AC;&#x2122; to the pathogenesis of the cancer. We hypothesize that fast-onset mouse PCTs will have an altered 3D telomere organization, compared to controls, and that chromosome T(X;11) will have lengthened telomeres compared to all other chromosomes. Methods: Primary B-cells are harvested from [T38HxBALB/c]N wild-type mice, with and without rcpT(X;11), and fast-onset PCT mice. Fluorescence in situ hybridizations are conducted using a telomere peptide nucleic acid probe. After high-resolution imaging of 2D metaphases and 3D interphase nuclei, quantitative telomere length measurements are made. Results: We find the chromosomes from the fast-onset PCTs to have significantly more low intensity or very short telomeres (p < 0.0001 for wild-type mice, with or without rcpT(X;11)). Contrastingly, the 2D telomere profiles of the wild-type mice are not significantly different. These results are consistent with the 3D nuclei studies in which the fast-onset PCT mice have a significantly different telomere profile than controls, with more very short telomeres than the wild-type nuclei (p < 0.0001). Although metaphases from the fast-onset PCTs illustrate many critically short telomeres, the T(X;11) translocation chromosomes have significantly longer telomeres (p = 4x10-16). This trend is not seen in wild-type mice with rcpT(X;11). Conclusions: The unique fast-onset PCT telomere profile supports the potential use of 3D telomere organization as a tumor biomarker. Significantly longer telomeres on chromosome T(X;11) in fast-onset PCTs provides support for a chromosome-specific protective mechanism on chromosomes that are functionally important for the tumorigenic process.
umkuzyka@cc.umanitoba.ca
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An immunohistochemical investigation of C-bouton regulation of motoneuron excitability: Kv2.1 channels and local calcium regulation. Laura J Baxtera and Robert M Brownstoneab a
Department of Anatomy and Neurobiology, bDepartment of Surgery, Dalhousie University,
Halifax, Canada. Rationale and Objectives: Spinal motoneurons (MNs) receive and integrate information from many sources. One input forms cholinergic C-bouton synapses, which facilitate MN output, and hence increase muscle contraction. Interestingly, in addition to the ligand receptor – type 2 muscarinic acetylcholine receptor (m2AChR) – the delayed rectifier potassium channel, Kv2.1, forms clusters at sites postsynaptic to C-boutons. The role of this voltage-gated channel in function of this synapse is not known. To address this question, we sought to determine whether m2AChR’s are associated with Kv2.1 channels at other locations in MNs. That is, because m2AChR’s are found on MN terminals at the neuromuscular junction (NMJ), we sought to determine whether Kv2.1 channels are likewise situated there. Furthermore, as Kv2.1 channels are modulated by phosphorylation, we sought to determine whether the calcium signaling molecules calcineurin (the B subunit, PP2Bβ) and/or inositol triphosphate receptor type 2 (IP3R2) are found in the nanodomain at this post-synaptic site. Methods: ChAT::eGFP mice (N=3) were used to identify MNs and their axons. Diaphragm, lumbar spinal cord, and gastrocnemius tissues were obtained following perfusion. Fluorescence immunohistochemistry was used to identify Kv2.1, green fluorescent protein, PP2Bβ, IP3R2, vesicular acetylcholine transferase, and m2AChRs, and rhodamine-conjugated bungarotoxin was used to visualize nicotinic receptors. Laser-scanning microscopy was used to obtain confocal images for analysis. Results: Although Kv2.1 clusters were found at C-bouton contacts on MNs, they were not present on NMJs in either the diaphragm or gastrocnemius muscles. Neither PP2Bβ nor IP3R2 were clustered at the post-synaptic site of C-boutons. Conclusions: (1) Kv2.1 channels are not necessary for m2AChR function in MNs, as these receptors are found at NMJs in the absence of Kv2.1 channels. A MN-specific Kv2.1 knockout would lead to understanding of the role of Kv2.1 in MN function. (2) Neither calcineurin nor IP3R2 were clustered at C-bouton sites. The molecules regulating local calcium metabolism remain to be identified.
Funding: CIHR, Dalhousie Medical Research Foundation
laurajbaxter@gmail.com
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Cholesterol oxidation leads to separation of lipid rafts: A biophysical model for protein plaque formation. Matthew G.K. Benescha, David A. Mannocka, and Ronald N. McElhaneya a
Department of Biochemistry, School of Translational Medicine, Faculty of Medicine & Dentistry, University of Alberta. Edmonton, Alberta, Canada. Rationale: Human and mammalian diseases have been associated with membrane sterol content and lipid rafts in particular. Lipid rafts are ordered microdomains within more-disordered cellular membranes containing structurally important lipids such as sphingomyelin, ceramide, and cholesterol. Such rafts may contain proteins that require spatial separation, and if the integrity of such rafts is compromised, these proteins may aggregate and form plaques similar to those found in Alzheimerâ&#x20AC;&#x2122;s disease. Oxidative damage has been implicated in such processes. Objectives: To investigate how the presence of cholesterone, an oxidized cholesterol metabolite, simulating the end metabolite of oxidative processes, leads to demixing of model mixtures containing physiological quantities of lipid raft components. Methods: Film casts of quaternary mixtures of C-16 sphingomyelin, C-16 ceramide, cholesterol and cholesterone in a 70:30 molar ratio of total sphingolipids to total sterol were dispersed in aqueous media and subjected to differential scanning calorimetry. Data collected was analyzed to determine changes in lipid phase transition behavior and identify demixing of mixture components. Results: Titration of cholesterone into mixtures of sphingomyelin:ceramide:cholesterol showed demixing with as little as 6-7 mol percent cholesterone. This demixing phenomenon occurs irrespective of the relative amounts of ceramide or cholesterol in sphingomyelin-rich mixtures. Even in binary mixtures of sphingomyelin:sterol (the simplest of lipid rafts) any cholesterone leads to a more rapid destabilization of membrane phase transitions than the corresponding quantities of cholesterol. Conclusions: Given that the presence of small amounts of oxidized cholesterol metabolites leads to measureable demixing of physiological-mimic lipid rafts, this proof-of-principle study highlights a novel approach for potential mechanisms behind protein aggregation and suggests a possible therapeutic role for inhibitors of sterol oxidase-type enzymes. Funding: Alberta Innovates â&#x20AC;&#x201C; Health Solutions, CIHR, NSERC, Vanier Canada Graduate Scholarships Contact Email: benesch@ualberta.ca
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The Nlrp3 Inflammasome promotes systolic dysfunction in structural cardiomyopathy through Il-1β. Nathan A. Bracey1, Paul L. Beck2, Daniel A. Muruve3, Henry J. Duff1 1 Libin Cardiovascular Institute, University of Calgary 2 Division of Gastroenterology, University of Calgary 3 Department of Medicine, University of Calgary Rationale: Heart failure is associated with persistent myocardial inflammation that impairs cardiac function. Il-1β, a pro-inflammatory cytokine is elevated in the serum from patients with heart failure and is independently and prospectively associated with poor outcomes. Regulation of Il-1β can occur downstream of the Nlrp3 pattern recognition receptor through formation of a caspase-1-activating danger sensing complex called the inflammasome. The specific regulation of Il-1b signaling in the heart, and the therapeutic utility of its blockade at the level of the inflammasome has not been fully addressed. We therefore sought to assess the role of Il-1β and Nlrp3 in a mouse model of structural heart disease. Methods: Cardiac-specific overexpression of the calcineurin transgene in mice (CNTg) results in progressive systolic dysfunction with hypertrophy and fibrosis. We assessed the impact of the Il-1 receptor antagonist (Il-1-ra) and Nlrp3 genetic deletion on cardiac function in CNTg mice using 2-D and M-mode echocardiography. Inflammasome activation and Il-1b signaling were assessed in heart tissue by immunoblotting and Luminex. Results: Inflammasome components Nlrp3, ASC and caspase-1 were all expressed in the heart, both in cardiac myocytes and fibroblasts to varying degrees. CNTg mice demonstrated elevated cardiac Nlrp3 with activation of the inflammasome, evidenced by increased Il-1β and maturation of caspase-1. Treatment of CNTg mice with Il-1-ra (10mg/kg/day) for 14 days by osmotic pump improved cardiac fractional shortening (p<0.05) and reduced myocardial infiltrate/apoptosis compared to vehicle (p<0.01). Il-1ra treatment additionally reduced Il-1β (p<0.05) and cardiac Nlrp3 protein, associated with reduced phosphorylation of IκBα. Lastly, Nlrp3 deletion in CNTg mice abrogated Il1β and caspase-1 maturation, resulting in improved cardiac structure and systolic function. Conclusions: Taken together, these results demonstrate that Il-1β signaling is activated in structural cardiomyopathy downstream of the Nlrp3 inflammasome. Blockade of Il-1β and/or Nlrp3 may offer novel therapeutic approaches for the treatment of chronic heart disease. Funding: Alberta HEART, Alberta Heart and Heart and Stroke Foundation of Alberta
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Diagnosing a severe neonatal intestinal disorder using nuclear magnetic resonance metabolomics Skappak C1, Saude E2, and Harabor A3. Department of Medicine1, Department of Emergency Medicine2, Department of Pediatrics and Division of Neonatology3, University of Calgary, Alberta.
Rationale: Rapid diagnosis of disease in pre-term and full-term neonates is a critical aspect of successful treatment. A major cause of morbidity and mortality in neonates is a gastrointestinal disease called necrotizing enterocolitis (NEC). The current diagnosis of NEC is based on a combination of clinical and radiological findings. Unfortunately, the clinical findings are sensitive but non-specific and can lead to over-diagnosis and iatrogenic effects of unnecessary treatment. Creation of a sensitive and specific diagnostic test for NEC and/or the other intestinal conditions of the neonate that can be mistaken for NEC could potentially decrease morbidity and mortality in this patient group. Nuclear magnetic resonance (NMR) metabolomics is a non-invasive method for identifying and measuring various bio-markers of cellular and disease processes. These measurements can be used to create a metabolomic signature of a disease state that differentiates it from the signature of a healthy individual. By applying metabolomics to biosamples from neonates with gastrointestinal conditions, we have been able to identify multiple metabolites related to the intestinal disease process and create a distinct metabolomic signature. With this unique signature we plan to create a computer model that is able to rule in / rule out NEC by analyzing metabolomic signatures from neonates with suspect symptoms.
Methods: Clinical information and fecal samples were collected at Calgary area Neonatal Intensive Care Units (NICU) from both neonates with NEC, and healthy premature infants. These samples were then stored at -80°C until analysis could be completed. The samples were thawed, weighed, homogenized and had the their pH standardized. NMR fecal spectra were collected on a 600MHz spectrometer and concentrations of metabolites were determined using Chenomx NMR Suite software version 7.1. Results: 35 Metabolites were identified and analyzed by partial least squares discriminate analysis (PLS-DA). A combination of metabolites excreted in the patientsâ&#x20AC;&#x2122; fecal matter allowed for the separation of neonates with NEC from healthy controls. Conclusions: Currently diagnosing NEC is based on subjective clinical signs, and radiological investigations. NMR metabolomics may allow for a faster more specific, and noninvasive diagnosis of neonates with NEC. Funding: AllerGen-NCE cdskappa@ucalgary.ca
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SESSION 2 Poster Discussion Brodie Centre Mezzanine 10:30 â&#x20AC;&#x201C; 12:00 p.m. Wednesday, June 13th, 2012
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CHARACTERIZATION OF PAX3 ACTIVITY IN MELANOMA a
ab
Zachary Tan and D. Alan Underhill b
a
Department of Experimental Oncology, Faculty of Medicine and Dentistry, University of Alberta. Edmonton, Canada. Rationale: The transcription factor PAX3 is critical for development of neural crest lineages including melanocytes. Prior to birth, PAX3 is required for the proliferation of melanocyte precursors and it is thought to maintain an `undifferentiated plastic state' in epidermal melanocytes after birth, as well in melanocyte stem cells. In addition, PAX3 is expressed throughout melanoma progression, from nevi to metastatic disease. Nevertheless, little is known about how PAX3 carries out these diverse roles. Objectives: To determine the role of PAX3 expression and phosphorylation in melanoma cell cycling. Methods: In the present study, we assessed the role of PAX3 in melanoma cell cycle progression and migration using an RNAi knockdown strategy. At the same time, we evaluated the potential role of Glycogen Synthase Kinase 3β (GSK3β) in modulating PAX3 activity using chemical inhibitors. To this end, fluorescence Activated Cell Sorting (FACS) was used to assess cell cycle distribution in B16F0 melanoma cells and the metastatic B16F10 derivative.
Results: In both melanoma cell lines, robust PAX3 knockdown was observed by immunoblotting following a 24hr treatment with PAX3 siRNA and resulted in significant G1 accumulation (p≤0.05). This result was validated by quantifying 5-bromo-2'-deoxyuridine (BrdU) incorporation and phosphoserine-10 histone H3 (H3S10p) by immunofluorescence to monitor cells in S or G2/M phase, respectively. In each case, there was a significant (p≤0.05) decline in staining upon knockdown, together indicating a defect in S phase entry. Treatment of cells with the GSK3β inhibitors lithium chloride (LiCl) or BIO caused decreased cell proliferation (p≤0.05) and G2/M accumulation (p≤0.05), and was associated with elevated PAX3 levels (p≤0.05). Last, inhibition of GSK3β as well as PAX3 knockdown was associated with markedly decreased cellular motility, and they may therefore modulate the metastatic phenotype. Together, these investigations establish an important role for PAX3 in regulating the G1 to S-phase transition in melanoma and identify GSK3β and as an important modulator of PAX3 levels and cell cycle progression in melanoma cells. Funding: Alberta Innovates Health Solutions, Alberta Cancer Foundation
zetan.du@ualberta.ca
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Cardiac MRI volumetric assessment by short-axis has better reproducibility than the axial orientation in single RV hearts. a
a
b
c
AUTHORS: Sanam Verma , Nee Khoo , Michelle Noga , Kimberley Myers and Edythe a Tham a
b
Division of Pediatric cCardiology, Department of Radiology and Diagnostic Imaging, University of Alberta, Department of Pediatric Cardiology, University of Calgary Rationale: Magnetic resonance imaging (MRI) is the standard for assessment of ventricular volumes and function in cardiac disease. However, few reports exist regarding methodology for measurements of single RV volumes in congenital heart disease. This study aims to determine which imaging plane, the short axis oblique (SAO) or the axial orientation (AX), provides greater reproducibility in evaluation of RV volumes in HLHS patients. Methods: A retrospective study of 23 patients (5moÂą3.4) with single RV (HLHS and variants) who underwent MRI prior to bidirectional cavopulmonary anastomosis. Cardiac MRI was performed on a Siemens 1.5T scanner under general anesthesia. Post-processing (CMR 42 - Circle Imaging, Calgary, Alberta) was performed by two independent observers to obtain enddiastolic (EDV) and end-systolic (ESV) volumes in both SAO (n=23) and AX (n=16) planes for inter-observer variability. Intra-observer variability was assessed by one observer reanalyzing 10 randomly selected datasets with a 14 days interval. Statistical analysis using paired t-tests, absolute differences (mean Âą SD), repeatability values, intraclass correlation coefficients (ICC) and Bland Altman plots to assess differences in reproducibility between methods. Results: No significant differences between the two planes for EDV and ESV (p>0.05) were noted. While inter-observer ICC showed higher correlation for ESV (SAO r=0.75, AX r=0.98) compared to EDV (SAO r=0.49, AX r=0.37). SAO inter-observer limits of agreement were narrower than those of the AX plane. AX EDV had poorer repeatability (11ml) than SAO EDV (6.4 ml), SAO ESV (3.2 ml), and AX ESV (3.5 ml). In addition AX EDV had the largest absolute difference between observers (p<0.01). Intra-observer testing showed a higher ICC for SAO assessment (EDV=0.88, ESV=0.87) than AX (EDV=0.43, ESV=0.55). SAO EDV, SAO ESV and AX ESV (2.7ml, 2.7ml, 3.5ml) had better repeatability than AX EDV (5.7ml). Conclusion: MRI assessment of single RV EDV had poorer inter-observer reproducibility than ESV. Better inter and intra-observer reproducibility suggests that the short axis orientation is the superior method in the assessment of single RV volumes. Funding: David and Beatrice Reidford Research Scholarship
sanam@ualberta.ca
29
Vitamin D3 induces release of IL-1β via the NLRP3 inflammasome: Implications for Crohn’s disease Sarah E Tulka,b, Simon A Hirotaa,b, Paul L Becka and Justin A MacDonaldb a
Departments of Medicine and b Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB Rationale: Recent studies have highlighted that the Crohn’s disease (CD) pathogenesis may involve hypoactive innate immune and inflammatory responses. Key to this is the fact that mutations in the CD susceptibility gene, NOD2, are hypofunctional. NOD2, a member of the NLR family, is an innate immune receptor tasked with activating inflammatory pathways following stimulation with the bacterial component LPS. Thus, an inability to initiate appropriate inflammatory responses may play a role in CD. Vitamin D has recently been shown to induce NOD2. By boosting NOD2 signalling, vitamin D may help compensate for the impaired innate immune responses in CD. NLRP3, another NLR, has also been shown to have hypofunctional mutations in CD. Vitamin D has been shown to induce production of the NLRP3 product precursor, pro-IL-1β. We therefore hypothesized that vitamin D could activate the NLRP3 inflammasome to induce release of mature IL-1β from macrophages. Methods: PMA-differentiated THP-1 cells were stimulated with 1,25(OH)2D3 or 25(OH)D3 for up to 24 hours and IL-1β release was assessed via ELISA. CYP27 gene expression was assessed via PCR.
Results: 1,25(OH)2D3 induced release of IL-1β from PMA-differentiated THP-1 cells after 24 hours of stimulation at concentrations above, but not below, 1 nM. This effect was blocked with the caspase-1 inhibitor yVAD and the NLRP3 inhibitor glyburide. Further, treatment with the 1,25(OH)2D3 precursor, 25(OH)D3, induced IL-1β release. CYP27 converts 25(OH)D3 to 1,25(OH)2D3 in the kidney, but monocytes have been shown to also express CYP27. PCR showed that PMA-differentiated THP-1 cells express CYP27. Treatment with the CYP27 inhibitor ICI 182 780 blocked 25(OH)D3-induced IL-1β release. Conclusions: We have shown that macrophages release IL-1β following stimulation with 1,25(OH)2D3 or 25(OH)D3. 25(OH)D3-induced IL-1β release is dependent on CYP27. 1,25(OH)2D3-induced IL-1β release is dependent on the NLRP3 inflammasome. We therefore propose a model whereby 25(OH)D3 is converted to 1,25(OH)2D3 by the macrophage, which in turn activates the NLRP3 inflammasome to induce release of IL-1β. We believe that this holds important implications for CD, as vitamin D could help ‘boost’ basal levels of the innate immune system to help compensate for the defective innate immunity seen in this disease. Funding: CIHR setulk@ucalgary.ca
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ANTI-ESTROGEN USE AND SURVIVAL OF WOMEN WITH NON-SMALL-CELL LUNG CANCER IN MANITOBA Sylvain Lother(1), Dr. Gary A Harding(1,3), Dr. Marshall W Pitz(1,3), Grace Musto(1,2), Dr. Sri Navaratnam(1,3) (1) Department of Medicine, University of Manitoba; (2) Department of Epidemiology, CancerCare Manitoba; (3) Department of Medical Oncology, CancerCare Manitoba BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. Gender differences in lung cancer outcomes are known. When compared to men, women have significantly better survival and women are more likely to develop lung cancer when nonsmokers. Research suggests estrogen plays a key role in the risk of development and outcomes of lung cancer. Accordingly, anti-estrogen use should also influence survival in female non-small cell lung cancer (NSCLC) patients. In this study we compared mortality among anti-estrogen users and non-users. METHODS: This population-based study had a retrospective study design. Using the Manitoba Cancer Registry (MCR) we identified all women diagnosed with NSCLC from 2000-2007. The Drug Program Information Network (DPIN) was accessed to establish patients that received anti-estrogens. Demographic data (e.g. smoking patterns, stage, histology) was gathered by chart review. Mortality rates for anti-estrogen users and non-users were compared using Kaplan-Meier survival functions and Cox regression models. RESULTS: 2320 women fit our patient criteria, of which 156 had received prior anti-estrogens. A positive smoking history was documented in 88%, 62% being former vs. 26% current smokers. A history of 30+ pack-years was seen in 55%. Exposure to anti-estrogen was associated with a significantly decreased mortality (HR 0.718, p = 0.0031). Overall survival with anti-estrogen vs. none resulted in median survival of 1.89 vs. 0.93 years, respectively (p < 0.0001). CONCLUSIONS: Our results demonstrate that anti-estrogens are associated with decreased mortality from NSCLC. These findings supplement and reinforce past evidence that estrogen plays a key factor in the biology and outcomes of NSCLC.
Funding: Manitoba Medical Service Foundation (MMSF), the Winnipeg Foundation (WF)
sa.lother@me.com
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Canadians’ Satisfaction with Medicare: An Analysis of the Canadian Community Health Survey Alexander Pedena a
Department of Economics, Department of Community Health Sciences, University of Manitoba. Winnipeg, Canada. Rationale: With the current Canadian Health Accord set to expire in 2014, policy makers must properly understand Canadians’ satisfaction with their health care system in order to find the specific areas where health care reform is most needed in the eyes of the public. Methods: The Canadian Community Health Survey, a biannual health survey contains several questions pertaining to respondents’ rating of both the accessibility and quality of the health care system. Microdata from the CCHS cycles 2.1, 3.1 and 4.1 was used in an ordered probit model to investigate the association between respondents’ individual characteristics (including age, gender, income, education, lifestyle and province of residence), their utilization of the health care system and their expressed satisfaction with various aspects of the health care system. Results: In terms of personal characteristics, males, students and minorities rate the system significantly better for both quality and access. Interestingly, age was associated with greater approval, but only in terms of quality, not access. Quebec residents were significantly more pessimistic towards their provincial health services than those from Ontario. In terms of selfrated factors, life satisfaction as well as general and mental health were positively associated with approval, while stress levels had a negative correlation. Both cancer and heart disease patients rated the system significantly better, as opposed to respondents suffering from back pain or migraines. Finally, health care utilization variables indicated that users of the system were generally satisfied with the system. Conclusions: The lower satisfaction of women, Quebec residents and patients with non-critical chronic conditions suggests the need for further research into potential unmet needs. The expressed dissatisfaction of high income Canadians is a particularly important issue for the future of Medicare, requiring further research to appropriately address the sources of their criticism.
umpedena@cc.umanitoba.ca
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Mast Cell Deficiency Significantly Delays Bone Tissue Regeneration Michael H. Wang1,3, Janet E. Henderson1,2,3, Paul A. Martineau1,2,3 1
McGill University Faculty of Medicine, 2McGill University Division of Orthopaedic Surgery, 3 McGill University Health Centre JTN Wong Labs for Bone Engineering Rationale: Skeletal reconstruction is currently limited by the availability of donor tissues used in autologous bone graft transplantation. Since mast cells have an innate ability to home in on injury sites, mast cells are a potential adjunct therapy and drug delivery mechanism to augment bone regeneration. However, previous research has neither established the role of mast cells in bone healing nor the timing of their involvement. Objectives: The current study has two objectives: 1) Demonstrate whether bone regeneration is significantly disturbed in mast cell-deficient (c-kit mutant) mice; and 2) Establish the timing of bone healing in wildtype and mutant. Methods: Wildtype and mutant C57Bl6/J mice underwent bilateral femoral cortical defect surgeries to investigate bone regeneration. Mice were euthanized at post-operative zero, two, four, and six weeks. Femurs were extracted for analysis using micro-computed tomography (Micro-CT) and histology to assess bone healing. Micro-CT examined the micro-architecture of the healing cortex, while histology identified cellular contributions, including osteoblast activity, mineralization, and the presence of mast cells. Quantification of bone healing was performed by analyzing bone volume fraction (BV/TV) on a region of interest within the cortical window defect where bone regeneration occurred. Results: At baseline post-operative zero weeks, wildtype and mutant have the same BV/TV. Compared to baseline, both genotypes showed significantly higher BV/TV at post-operative two and four weeks. Compared to mutants, wildtype femurs had significantly higher BV/TV at postoperative two weeks and both achieved similar BV/TV by post-operative four weeks. This indicates that wildtype regeneration was completed by week two and mutant regeneration by week four. Histology is preliminary but mineralization stains support Micro-CT data. Conclusions: In the absence of mast cells, bone regeneration still proceeds but is significantly delayed. This delay in bone regeneration warrants continued histological investigation to further strengthen Micro CT data demonstrating delayed bone healing in mast cell deficient mice, and possibly suggest a molecular explanation for this phenomenon, such as delayed osteoblast recruitment. Funding: CIHR, McGill University michael.wang@mail.mcgill.ca
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Meta-analysis of efficacy and safety of new oral anticoagulants (dabigatran, rivaroxaban, apixaban) versus warfarin in patients with atrial fibrillation Corey S. Millera,c, Sonia M. Grandia, Avi Shimonya,b,d, Kristian B. Filiona, Mark J. Eisenberga,b,c a
Division of Clinical Epidemiology, Lady Davis Institute for Medical Research, Jewish General Hospital, bDivision of Cardiology, Jewish General Hospital/McGill University, and cFaculty of Medicine, McGill University, Montreal, Canada; and dCardiology Department, Soroka University Medical Center, Ben-Gurion University, Beâ&#x20AC;&#x2122;er-Sheva, Israel. Rationale: New oral anticoagulants, including apixaban, dabigatran, and rivaroxaban, have been developed as alternatives to warfarin, the standard oral anticoagulation therapy for patients with atrial fibrillation (AF). A systematic review and meta-analysis of randomized controlled trials was performed to compare the efficacy and safety of new oral anticoagulants to those of warfarin in patients with AF. Methods: The published research was systematically searched for randomized controlled trials of >1 year in duration that compared new oral anticoagulants to warfarin in patients with AF. Random-effects models were used to pool efficacy and safety data across randomized controlled trials. Results: Three studies, including 44,563 patients, were identified. Patients randomized to new oral anticoagulants had a decreased risk for all-cause stroke and systemic embolism (relative risk [RR] 0.78, 95% confidence interval [CI] 0.67 to 0.92), ischemic and unidentified stroke (RR 0.87, 95% CI 0.77 to 0.99), hemorrhagic stroke (RR 0.45, 95% CI 0.31 to 0.68), all-cause mortality (RR 0.88, 95% CI 0.82 to 0.95), and vascular mortality (RR 0.87, 95% CI 0.77 to 0.98). Randomization to a new oral anticoagulant was associated with a lower risk for intracranial bleeding (RR 0.49, 95% CI 0.36 to 0.66). Data regarding the risks for major bleeding (RR 0.88, 95% CI 0.71 to 1.09) and gastrointestinal bleeding (RR 1.25, 95% CI 0.91 to 1.72) were inconclusive. Conclusion: The new oral anticoagulants are more efficacious than warfarin for the prevention of stroke and systemic embolism in patients with AF. With a decreased risk of intracranial bleeding, they appear to have a favorable safety profile, making them promising alternatives to warfarin. Funding: Corey Miller is supported by the Ivan Racheff and Clarke McLeod Scholarships funded through the McGill University Research Bursary Program. Avi Shimony is supported by the Azrieli Fellowship Fund for research at the Jewish General Hospital, Montreal, Quebec, Canada. Mark Eisenberg is a Chercheur-National of the Fonds de la Recherche en SantĂŠ du Quebec.
corey.miller@mail.mcgill.ca
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Educating in child maltreatment: Mandatory reporting and medical student experiences. Christine Wekerlea, Anne Nieca, Joyce Zazulaka, Laura Morrisseyc, Jessica Malachc, and Elise Wrightc. Child Advocacy and Assessment Program, Department of Pediatrics, McMaster Universitya. Family Medicine, McMaster Universityb. Michael G. Degroote School of Medicine, McMaster Universityc. Rationale: Child maltreatment increases the likelihood for impairment in physical, mental, and financial health domains across the lifespan16. Maltreatment victims are likely to present in an array of health clinics, particularly in higher-cost services (e.g., emergency, specialist), as well as have a greater likelihood of disability that prevents continued participation at work3,2,5,11. Despite this broad presentation profile, dedicated learning in maltreatment is limited in most health professional pre-service training. As compared to other health professionals, physicians are low-frequency reporters to child welfare authorities, with estimates in the range of 1015%14,9. Thus, there is a critical gap in medical education given existing mandatory reporting laws and profession policies. Methods: The objective was to assess medical undergraduates' experiences-to-date in mandatory reporting, as well as their comfort and knowledge of supportive resources. This study was a needs assessment for the development of an art- and case-based course in maltreatment, a partnership between McMaster Museum of Art, Family Medicine Department, and maltreatment specialty clinic, the Child Advocacy and Assessment Program. A 30-item questionnaire was developed to tap demographic characteristics, training on maltreatment content prior to and during medical school, the nature of reported cases and comfort with reporting. Results: N=150 students completed the questionnaire (68% female; Mean Age = 25.1 years; SD = 3.6 years). About half of the sample had been involved in a child maltreatment case in medical school, with significantly more in the latter two years. About a third of students had been involved in an adult case where there was a childhood maltreatment history. A minority of students (20%) had made a report to child welfare, with more in the third year of study. No students reported feeling comfortable with mandatory reporting; there was no difference in level of discomfort across the three study years of medical undergraduates. Conclusion: Most students have encountered cases where maltreatment was a feature and 20% initiated a report to child welfare. Despite experience, the majority report a lack of comfort with mandatory reporting. There is high interest in dedicated course learning to support the reporting role, identifying a potential learning need presently unavailable. Funding: Ontario Centre of Excellence for Child and Youth Mental Health Laura.morrissey@medportal.ca
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Performance of a Commercial Urine Transport System for Stabilizing Bacterial Growth William Stokes, Peter Daley, Elizabeth Parfitt and William Midodzi Memorial University. St. Johnâ&#x20AC;&#x2122;s, Newfoundland and Labrador. Rationale: Prolonged transportation of urine specimens from remote collection locations may lead to bacterial overgrowth and false culture results. Refrigeration is the gold standard for urine specimen preservation but it is difficult to maintain during transportation. Boric acid preparations such as the UriSwabTM (Copan, Italy) may preserve bacteria at room temperature. Our objective was to evaluate the effectiveness of this product in preserving bacteria at room temperature during prolonged storage times. Methods: We compared microbial population growth in consecutive clinical urine samples received in a hospital microbiology laboratory, after storage for 24, 48 and 72 hours in UriSwab at room temperature and in sterile container at 4oC. Growth was categorically quantified and interpreted according to laboratory protocol. Percent of specimens with significant growth was compared within and between groups using logistic regression. Results: 816 urine specimens were received during 7 days. 165 specimens (20.2%) demonstrated significant growth before storage, of which 108 (65.5%) were gram negative bacilli. There was no change in percent positives in urine stored in sterile container at 4oC at three time points (p = 0.346). OR for positivity in UriSwab at room temperature was 1.54, 2.11 and 2.49 at three time points (p = 0.014). Percent of samples with significant growth was significantly different between sterile container and UriSwab at all time points (p<0.001). Among 176 positives developing during storage in UriSwab, 128 (72.7%) were gram positive cocci. Conclusion: There was greater percent positivity in urine stored in UriSwab at room temperature compared to urine stored in sterile container at 4oC. Increased inhibition of gram positives might improve performance.
wstokes@mun.ca
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Presence of inhibitory receptors at the surface of tumour-infiltrating T lumphocytes isolated from carcinomatous ascites. Audrée Lachancea, Nathalie Demotteb and Pierre van der Bruggenb a
Biomedical sciences program, Université de Montréal, Québec, Canada Ludwig Institute for Cancer Research, de Duve Institute, Université Catholique de Louvain, Brussels, Belgium b
Rationale: The dysfunction of tumour-infiltrating T lymphocytes (TIL) that could explain the low efficiency of the immune system to eliminate cancer cells, is insufficiently documented in humans. Expression of inhibitory receptors like PD-1, Tim-3, BTLA and CTLA-4 has been suggested to explain T lymphocytes dysfunction. But the analysis of the expression of several inhibitory receptors on TIL is difficult because of the small number CD8+ T cells isolated from tumour samples. Objectives: The challenge is to analyse the expression of several inhibitory cell surface receptors expressed on a small number of CD8+ TILs. Methods: CD8+ T lymphocytes were isolated from various human carcinoma ascites and from blood. CD8+ T cells were labeled ex vivo with antibodies against PD-1, Tim-3, BTLA and CTLA4 and analysed by flow cytometry. A fraction of the cells were stimulated with beads coated with anti-CD3/CD28 antibodies and labeled 24h or 48h after stimulation. CD25 expression was used as a marker of activation. Results: A small number of cells (105) were enough to analyse the expression of four inhibitory receptors. When tested ex vivo, TILs expressed high level of PD-1 and low level of BTLA in comparison with blood CD8. After stimulation, expression of PD-1 and Time-3 increased slightly at the surface of TILs but increased strongly on blood CD8. Indeed, expression of PD-1 and Tim-3 seem to be characteristic of a particular TILs population into ascites because they were CD25 negative. Conclusions: The analysis of several inhibitory receptors on the same cell is possible on a reduced number of cells. We observed that the epression level of PD-1 is higher at the cell surface of TILs in comparison to blood CD8 and that part of the TILs expressed PD-1 and Tim=3 without being in an active state. PD=1 and Tim=3 expression on TILs could lead to the dysfunction of T cells, preventing tumour eradication. To really link the expression level of PD-1 and Tim-3 to the functional state of TILs from carcinomatous ascites, we will sort the different population and test them for their ability to release cytokines upon stimulation. Funding: WELBIO and Fondation contre le Cancer (Belgium)
Audree.lachance@umontreal.ca
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Comparing health behaviours of internal medicine residents and medical students: an observational study. Rye PL, Reeson ME, Pekrul CM, Asfour NA, Kundapur R, Wilson MP, Pausjenssen AM, Wilson TW. Department of Medicine and Cardiovascular Risk Factor Reduction Unit, University of Saskatchewan. PURPOSE: During residency, many physicians find it difficult to maintain a healthy lifestyle; however, there is little objective data available. In this study, residents' health behaviours and cardiovascular risk status were compared with those of medical students. METHODS: Medical residents (n=55, postgraduate years 1 to 4) were compared with medical students (n=62, years 1-4). The main dependent variable was the average number of steps per day (assessed using a pedometer) at work and leisure over three days, during which subjects were not on call or post-call. In addition, all subjects completed a three day food log. Frequency of vigorous exercise was assessed by a single question. Body mass index (BMI), waist circumference, blood pressure, total and high-density lipoprotein cholesterol, smoking habits and random blood glucose were measured, and Framingham Risk Score coronary artery disease 10 year probabilities (FRS) were calculated. RESULTS: Residents recorded 8344±3520 steps per day while students recorded 10703±3986 (p < 0.002). 35% of residents and 52% of students averaged more than 10,000 steps per day and senior residents took fewer steps than junior residents. Both groups frequently failed to achieve the recommended daily servings of fruits and vegetables; on average, 3.5±2.0 servings for residents and 5.4±2.2 for students (p < 0.0001). BMI and FRS were higher among the residents in comparison with the students. CONCLUSION: Medical residents at our institution appear less active and consume fewer servings of fruits and vegetables than undergraduate medical students. These differences are associated with higher BMI, waist circumference and cardiovascular risk. Funding: University of Saskatchewan College of Medicine Deans Summer Research Project
mer636@mail.usask.ca
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Gene expression changes observed in cancer cells with acquired resistance to low-dose metronomic cyclophosphamide chemotherapy a
b
b
Lavarnan Sivanathan , Annabelle Chow , Yutaka Amemiya and Urban Emmenegger a
ab
b
Faculty of Medicine, Sunnybrook Research Institute, University of Toronto and Sunnybrook Health Sciences Centre. Toronto, Canada Abstract Rationale: Advanced forms of prostate cancer, the most frequent cancer in Western males, are often treated with traditional maximum tolerated dose chemotherapy. Moreover, an alternative way of chemotherapy administration, i.e., low-dose metronomic chemotherapy (MC), is showing promising clinical results. However, resistance to MC usually develops within a few months, as is the case with maximum tolerated dose chemotherapy. Objective: To study molecular mechanisms of resistance to MC. Methods: We carried out whole transcriptome analysis on human PC3 prostate cancer cells made resistant to cyclophosphamide (CPA) MC (named LCR1.1), and on control cells (NS1.1). Next, 8 out of 589 â&#x2030;Ľ 1.5-fold differentially regulated genes were selected for validation using real-time PCR. Purposely, we focused on genes both involved in cellular functions postulated to contribute to resistance to MC, and coding for proteins detectable in plasma of cancer patients. Subsequently, we also performed real-time PCR studies in independently derived MC resistant human prostate and colorectal cancer cell lines. Results: We observed a close correlation between expression changes found in whole transcriptome analysis and real-time PCR. Insulin-like growth factor binding protein 3 (IGFBP3) was not only found to be downregulated in LCR1.1 but also in independently derived MC resistant prostate and colorectal cancer cell lines. Conclusion: Our analyses suggest a role of IGFBP3 in resistance to CPA MC. In fact, IGFBP3 modulates the insulin-like growth factor pathway, which is known to contribute to chemotherapy drug resistance. Furthermore, reduced IGFBP3 levels are associated with aggressive prostate cancer behavior. Thus, pharmacological targeting of IGFBP3 and/or the insulin-like growth factor pathway represents promising strategies to circumvent resistance to CPA MC. Since IGFBP3 can be measured in plasma of cancer patients, IGFBP3 plasma levels may serve as a predictive marker for CPA MC. Funding: Ontario Institute for Cancer Research (OICR), Prostate Cancer Canada (PCC), CREMS program at the University of Toronto Email: L.sivanathan@utoronto.ca
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Saccades as a Novel Tool for Patients with Traumatic Brain Injury? Sarah Mullena,b,c,e, Yeni Yucela,d,e, Tom Schweizerc,e, Michael Cusimanoc,e, Neeru Guptaa,b,e a
Department of Ophthalmology and Vision Sciences, bGlaucoma Unit, cDepartment of Surgery, Division of Neurosurgery, dDepartment of Laboratory Medicine and Pathobiology, University of Toronto. eLi Ka Shing Knowledge Institute of St. Michael’s Hospital. Toronto, Ontario, Canada. RATIONALE: Most traumatic brain injury leads to disability that is mild(mTBI), including concussion-type injuries. Diagnosis and monitoring is often subjective, not consistent with classic neuropsychological and neuroimaging criteria. Saccades are rapid intermittent eye movements allowing the eyes to fix on one point after another in the visual field, using multiple pathways from the eye to the brain. We hypothesize that this neural circuitry is affected in brain injury, and that measuring saccades may help to understand injury and recovery. METHODS: Patients with newly diagnosed mTBI(n=7), no history of eye disease, and agematched controls(n=4), were recruited. Participants underwent neurological exam, self-report measures, neuropsychological and eye movement testing within 1 and 3 weeks following injury. Saccades were measured using a head-mounted portable saccadometer performed to visual stimuli -10 or +10 degrees of a fixation point. Controls were similarly tested except for neuropsychological testing. Latency curve distributions were evaluated according to the LATER model(KS-2,p<0.05) in addition to measuring characteristic saccadic parameters, frequency of express saccades, and directional errors(t-test,p<0.05). Neuropsychological testing was scored against standardized norms and symptoms via self-report questionnaires(t-test,p<0.05). RESULTS: 5 of 7 patients with mTBI demonstrated significantly altered latency distributions between the two measurements (KS-2,p<0.05), with 4 showing increased median latency at week 1, resolving by week 3 (t-test,p<0.05). All 5 patients showed significantly increased directional errors at week 1, decreasing by week 3 (1.4±1.1 vs. 0.2±0.5,p<0.05). No changes were noted in controls (KS-2,p>0.05, t-test,p>0.05). However, 2 patients that reported increased symptoms, on questionnaire also showed increased median saccadic latencies (4.5±3.5 vs. 0.80±0.84,p=0.05). Compared to controls, lower mean neuropsychological scores of delayed recall and recognition were noted at week 1 (t-test,p<0.05), with no overall difference compared to standardized norms by week 3. CONCLUSIONS: Most patients with mTBI showed altered saccades with significantly increased median latency and directional errors, and decreased scores on neuropsychological testing. By 3 weeks, both measures normalized. Those patients with increased self-report symptoms by week 3 also showed increased latent saccades, indicating worsening disease. Saccades may be a useful tool to diagnose and monitor patients with mTBI. Acknowledgements Dorothy Pitts Research Fund Ontario Neurotrauma Foundation Research Internship sarah.mullen@mail.utoronto.ca
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Microspheres releasing GDNF within fibrin gels improve peripheral nerve regeneration after delayed nerve repair E. LIU1, M.D. WOOD1, T. GORDON2, S.W.P. KEMP2, H. KIM3, C. LAFONTAINE2, M.SHOICHET3, G.H. BORSCHEL2;1Div. of Plastic and Reconstructive Surgery, The Hosp. For Sick Children, Toronto, Canada; 2Div. of Plastic and Reconstructive Surgery, The Hosp. For Sick Children, Toronto, ON, Canada; 3Biomed. Engin., Univ. of Toronto, Toronto, ON, Canada
Rationale: Recovery following nerve cut and repair declines precipitously with prolonged denervation and axotomy. Glial cell-line derived neurotrophic factor (GDNF) has previously been shown to promote motor axon regeneration following delayed treatment. However, bioengineering strategies are needed to deliver GDNF to the site of nerve repair. Purpose: To determine the optimal duration of GDNF delivery to nerve following a 2-month period of delayed repair. Methods: We constructed polylactide-glycolic acid (PLGA) microspheres capable of extended GDNF release for 2 or 4 weeks. In vitro protein release assays (ELISA) were used to verify the time course of GDNF release. Rat common fibular nerve was chronically axotomized and denervated for 2 months. Experimental groups underwent delayed repair and treatment with GDNF microspheres in fibrin glue at the repair site or control treatments (empty microspheres, free GDNF without microspheres). An additional experimental group underwent nerve repair without a delay prior to nerve repair and without placement of fibrin glue or microspheres. Retrograde labeling of regenerating axons distal to the repair site was performed 8 weeks following treatment to quantify regenerating axons from motor and sensory neurons and contractile muscle force was measured 12 weeks following treatment. Results: Delivery of GDNF from microspheres to regenerating axons resulted in an increase in retrograde labeled motor and sensory neurons regenerating their axons compared to treatments without GDNF. Histomorphometric metrics of nerve cross-section distal to the repair site demonstrated differences in axon counts and fiber diameters due to GDNF microsphere delivery. Contractile muscle force production increased in both twitch and tetanic muscle force due to delivery of GDNF from microspheres compared to no GDNF or short-term release of GDNF to nerve following delayed nerve repair. Additionally, the GDNF microsphere groups were comparable in most outcome measures to immediate nerve repair. Conclusions: Delivery of free GDNF from fibrin glue had minimal effects on nerve regeneration suggesting a need for extended delivery of GDNF for at least 2 weeks to achieve beneficial nerve regeneration outcomes, which are comparable to the positive control group, following delayed nerve repair. Funding: The American College of Surgeons and the Plastic Surgery Foundation
edward.liu@medportal.ca
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Work Disability and Work Limitations in Rheumatoid Arthritis, Psoriatic Arthritis and Ankylosing Spondylitis Chanseok Rhee, BHSca, Janet Pope, MD, FRCPC, MPHb, Andrew E. Thompson, MD, FRCPC, MEd, Nicole Le Riche, MD, FRCPC, Gina Rohekar, MD, FRCPC, MSc, Sherry Rohekar, MD, FRCPC, MSc a
Schulich School of Medicine and Dentistry, bSt. Josephâ&#x20AC;&#x2122;s Health Care, Department of Rheumatology, London, Ontario
Objective: Few studies directly compare work disability (WD) and work productivity losses in different forms of inflammatory arthritis (IA). We measured the prevalence of WD and work productivity loss in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS) examining the relationship between WD and patient-reported measures of disease activity. Methods: WD was defined as the inability to work or early retirement due to arthritis. Work Limitations Questionnaire (WLQ) scores measure on-the-job work limitations as well as loss of productivity at work. Relationships between the WLQ scores, HAQ, Patient Global Assessment (PGA) and Functional Comorbidity Index (FCI) were analyzed in patients seen serially in a rheumatology clinic via standardized forms. Results: 846 responded (65% response rate with 332 RA, 88 PsA, 58 AS) and 289 had WLQ data as they were working (139 RA, 51 PsA, 35 AS). The mean age was 51.7 (SD 14.5), 74.3% female and 11.3 years (SD 11.3) of disease. WD due to arthritis was 22.8% (RA), 29.9% (PsA), and 27.6% (AS) (p<0.XX). WD was associated with HAQ, PGA, fatigue, pain score, and sleep score in all three IA conditions. The average loss of the productivity from WLQ was 4.45%, 4.71% and 5.63% for RA, PsA and AS, respectively (p<0.XX). The WLQ score was significantly correlated with HAQ, PGA, fatigue, pain score, sleep score, and with increasing number of comorbid conditions for all three IA conditions but not disease duration and gender. The association between the WLQ score and age was noted only in PsA (p=0.006). Conclusions: WD and productivity loss were not different in RA, PsA and AS. WD and WLQ scores were associated with HAQ, PGA, fatigue, pain, and sleep scores in all IAs. Comorbidities increased the likelihood of work productivity loss in RA, PsA and AS. Funding: SRTP rcs1535@gmail.com
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Evidence for a reference frame transformation of vestibular signal contributions to voluntary reaching movements Christophe Martin, Ian Moreau-Debord, Marianne Landry, Andrea M. Green Dept Physiol, Univ. Montreal, Montreal, QC, Canada Rationale: Studies have shown that vestibular signals play a role in the execution of voluntary reaching movements by contributing to spatial, dynamic and postural compensations when reaching during body motion. However, because the vestibular sensors are fixed in the head whereas the arm is fixed to the trunk, to contribute appropriately vestibular signals must be transformed from a head- to a body-centered reference frame. To investigate the evidence for this transformation, we used galvanic vestibular stimulation (GVS) to stimulate vestibular afferents as human subjects made reaching movements in the horizontal plane. Methods: Bipolar GVS simulates net rotation about an axis inclined ~18o upward from the head naso-occipital axis (Fitzpatrick & Day, 2004). With the head upright it thus simulates mainly whole-body tilt about an earth-horizontal axis, whereas with the head pitched 72o forward the same stimulus simulates rotation about an earth-vertical axis (yaw). Thus, if vestibular signals contributing to reaching have been transformed from a head- to a body-centered reference frame, the same stimulus should perturb horizontal-plane reach trajectories in a head- orientation-dependent way. With the head upright the stimulation should elicit mainly postural compensations and only small horizontal-plane trajectory deviations, whereas with the head pitched forward much larger trajectory deviations are predicted to compensate for a simulated yaw body rotation. To test this prediction, 35 subjects reached to a remembered target location in darkness with their right arm supported in the horizontal plane by a robotic exoskeleton (KINARM, B-Kin). GVS (3mA transmastoidal pulse) of unpredictable polarity was applied throughout or before the reaching movement in 16% of trials, chosen pseudo-randomly, during trial blocks in which the head was secured in either an upright or forward (50-60o) position. Results: GVS caused trajectories to deviate from non-stimulated controls about 300ms after movement onset. As predicted, with the head inclined forward average deviations were significantly larger than with the head upright (p<0.05) and were in the direction appropriate to provide spatial compensation for a simulated body yaw rotation. Conclusions: These results provide evidence that vestibular signals contributing on-line to reaching execution have been transformed from a head- to a body-centered reference frame. Funding: CIHR, NSERC Christophe_238@hotmail.com
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DOES ELECTRICAL REMODELING EARLY AFTER MYOCARDIAL INFARCTION PREDICT AN INCREASED RISK OF CARDIAC ARREST? Noah J. Switzer1 and Derek V. Exner1 1
Libin Cardiovascular Institute of Alberta, Calgary, AB, Canada
Rationale: Over 460,000 North Americans succumb to sudden death each year. Only one noninvasive technique has been shown to identify those at risk for sudden death after a myocardial infarction (MI); severe left ventricular dysfunction. The QT variability index (QTVI) is a new non-invasive tool that evaluates both ventricular repolarization and autonomic tone. Single measures of QTVI have been associated with a higher risk of adverse clinical outcomes. The early post-MI period is characterized by significant change (remodeling) in cardiac structure, autonomic tone, and electrical parameters. Thus, changes in QTVI may be useful in identifying patients at greatest risk of subsequent fatal or near fatal cardiac arrhythmias. Objectives: To explore the relationship of altered QTVI after MI with the later development of cardiac arrest. Methods: Ambulatory ECG (Holter) data from patients with a recent MI were evaluated earlier (2-4 weeks) and later (8-12 weeks) after the index MI. QTVI was measured using an algorithm developed by Berger et al. Holter data were assessed independent of adjudicated clinical outcomes, and vice versa. Results: Only a trend toward variation in QTVI in the earlier versus later post-MI periods was observed. However, significant variation of QTVI in the morning versus evening time periods was seen. QTVI, both earlier and later after MI, was associated with a higher risk of cardiac arrest. However, the change in QTVI between the earlier and later periods was not predictive of outcome. Impaired QTVI, using a pre-specified cut-point, predicted an increased risk of cardiac arrest, but the association was only modest and largely explained by other covariates. Abnormal QTVI had similar, modest, predictive value in the morning and evening time periods. Conclusions: Modest temporal variation and significant diurnal variation in QTVI was observed. QTVI, as a continuous variable, was associated with a higher risk of cardiac arrest both earlier and later after the index MI. However, change in QTVI between these times was not associated with an altered risk of cardiac arrest. Abnormal QTVI was associated with a higher risk of cardiac arrest earlier and later after MI. However, its predictive utility was largely explained by other factors. Funding: CIHR, AHFMR nswitzer@ualberta.ca
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Cholesterol oxidation leads to separation of lipid rafts: A biophysical model for protein plaque formation. Matthew G.K. Benescha, David A. Mannocka, and Ronald N. McElhaneya a
Department of Biochemistry, School of Translational Medicine, Faculty of Medicine & Dentistry, University of Alberta. Edmonton, Alberta, Canada.
Rationale: Human and mammalian diseases have been associated with membrane sterol content and lipid rafts in particular. Lipid rafts are ordered microdomains within more-disordered cellular membranes containing structurally important lipids such as sphingomyelin, ceramide, and cholesterol. Such rafts may contain proteins that require spatial separation, and if the integrity of such rafts is compromised, these proteins may aggregate and form plaques similar to those found in Alzheimerâ&#x20AC;&#x2122;s disease. Oxidative damage has been implicated in such processes. Objectives: To investigate how the presence of cholesterone, an oxidized cholesterol metabolite, simulating the end metabolite of oxidative processes, leads to demixing of model mixtures containing physiological quantities of lipid raft components. Methods: Film casts of quaternary mixtures of C-16 sphingomyelin, C-16 ceramide, cholesterol and cholesterone in a 70:30 molar ratio of total sphingolipids to total sterol were dispersed in aqueous media and subjected to differential scanning calorimetry. Data collected was analyzed to determine changes in lipid phase transition behavior and identify demixing of mixture components. Results: Titration of cholesterone into mixtures of sphingomyelin:ceramide:cholesterol showed demixing with as little as 6-7 mol percent cholesterone. This demixing phenomenon occurs irrespective of the relative amounts of ceramide or cholesterol in sphingomyelin-rich mixtures. Even in binary mixtures of sphingomyelin:sterol (the simplest of lipid rafts) any cholesterone leads to a more rapid destabilization of membrane phase transitions than the corresponding quantities of cholesterol. Conclusions: Given that the presence of small amounts of oxidized cholesterol metabolites leads to measureable demixing of physiological-mimic lipid rafts, this proof-of-principle study highlights a novel approach for potential mechanisms behind protein aggregation and suggests a possible therapeutic role for inhibitors of sterol oxidase-type enzymes.
Funding: Alberta Innovates â&#x20AC;&#x201C; Health Solutions, CIHR, NSERC, Vanier Canada Graduate Scholarships Contact Email: benesch@ualberta.ca
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The Nlrp3 Inflammasome promotes systolic dysfunction in structural cardiomyopathy through Il-1β. Nathan A. Bracey1, Paul L. Beck2, Daniel A. Muruve3, Henry J. Duff1 1 Libin Cardiovascular Institute, University of Calgary 2 Division of Gastroenterology, University of Calgary 3 Department of Medicine, University of Calgary Rationale: Heart failure is associated with persistent myocardial inflammation that impairs cardiac function. Il-1β, a pro-inflammatory cytokine is elevated in the serum from patients with heart failure and is independently and prospectively associated with poor outcomes. Regulation of Il-1β can occur downstream of the Nlrp3 pattern recognition receptor through formation of a caspase-1-activating danger sensing complex called the inflammasome. The specific regulation of Il-1b signaling in the heart, and the therapeutic utility of its blockade at the level of the inflammasome has not been fully addressed. We therefore sought to assess the role of Il-1β and Nlrp3 in a mouse model of structural heart disease. Methods: Cardiac-specific overexpression of the calcineurin transgene in mice (CNTg) results in progressive systolic dysfunction with hypertrophy and fibrosis. We assessed the impact of the Il-1 receptor antagonist (Il-1-ra) and Nlrp3 genetic deletion on cardiac function in CNTg mice using 2-D and M-mode echocardiography. Inflammasome activation and Il-1b signaling were assessed in heart tissue by immunoblotting and Luminex. Results: Inflammasome components Nlrp3, ASC and caspase-1 were all expressed in the heart, both in cardiac myocytes and fibroblasts to varying degrees. CNTg mice demonstrated elevated cardiac Nlrp3 with activation of the inflammasome, evidenced by increased Il-1β and maturation of caspase-1. Treatment of CNTg mice with Il-1-ra (10mg/kg/day) for 14 days by osmotic pump improved cardiac fractional shortening (p<0.05) and reduced myocardial infiltrate/apoptosis compared to vehicle (p<0.01). Il-1- ra treatment additionally reduced Il-1β (p<0.05) and cardiac Nlrp3 protein, associated with reduced phosphorylation of IκBα. Lastly, Nlrp3 deletion in CNTg mice abrogated Il1β and caspase-1 maturation, resulting in improved cardiac structure and systolic function. Conclusions: Taken together, these results demonstrate that Il-1β signaling is activated in structural cardiomyopathy downstream of the Nlrp3 inflammasome. Blockade of Il-1β and/or Nlrp3 may offer novel therapeutic approaches for the treatment of chronic heart disease. Funding: Alberta HEART, Alberta Heart and Heart and Stroke Foundation of Alberta nbracey@ucalgary.ca
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Interferon-gamma release assays (IGRA) piloted as a latent TB screening tool in federal Canadian inmates. Bach PJ1, Schwarz I1, Garrahan T2, Hume AM2, Smith J2, Barkley B2, Roscoe B2, Panaro L2,3, Majury A1,4, Jamieson F4,7, Ellis E5, Gemmill I1,6, Hopman WM1, Wobeser WL1. 1Queenʼs University, Kingston ON; 2Correctional Services Canada, Ottawa ON; 3University of Ottawa, Ottawa ON; 4Ontario Agency for Health Protection and Promotion, Ottawa ON; 5Public Health Agency of Canada, Ottawa ON; 6Kingston Frontenac Lennox & Addington Health Unit, Kingston ON; 7University of Toronto, Toronto ON. BACKGROUND: The prevalence of latent tuberculosis (TB) infection is disproportionately high in prison populations compared to the general public. In 1997, Correctional Services Canada established a Tuberculosis Tracking System (CSC TBTS) to offer comprehensive screening and monitoring services to control TB in the federal prison system. Screening has relied on the tuberculin skin test (TST), which is limited in part by false positive results, at least partly attributable to Bacille Calmette-Guérin (BCG) vaccination. IGRA tests should be negative in BCG vaccinated individuals who had a TST test with a low-risk for TB exposure and infection and may effectively increase the efficiency of comprehensive TB screening programs. INTERVENTION/RESPONSE: To ascertain whether this new tool could effectively be incorporated into the CSC TBTS screening process, 96 inmates with a positive (>=10mm induration) TST agreed to submit blood for testing with the Quantiferon®-TB Gold (QFT-G) IGRA assay. In addition, sputum cultures and chest x-rays were done to rule out active TB. RESULTS: Of the 96 inmates who had a history of being TST positive, 31 (32.3%) were QFT-G positive (concordant with their TST results). Outside of having a history of incarceration, birth in a country with moderate or high TB prevalence and age over 45 years were the main identified risk factors for TB acquisition. World region of birth did not appear to significantly affect the chance of having a positive IGRA. For Canadian-born inmates 9/29 (31.0%) were positive by IGRA compared to 18/67 (32.8%) foreign-born inmates (p = 0.862). Concordance was 12/50 (24.0%) for BCG positive individuals and 19/46 (41.3%) for BCG negative (p = 0.081). At the time of testing, no patients were known to be HIV positive. Treatment for LTBI was offered to the 31 inmates who were positive by IGRA. CONCLUSION: We found high rates of discordance between TST and QFT-G in our population. The incorporation of IGRA into the TB screening program for federal inmates may assist in identifying those who would benefit most from latent TB infection treatment, and help reduce the number of patients unnecessarily placed on isoniazid therapy. pbach@qmed.ca
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Mechanisms of the protease-induced increase in transepithelial resistance in intestinal epithelial cells A.Wadhwani, M. Dicay, W.K. MacNaughton University of Calgary, Calgary, AB Rationale: Apical addition of serine proteases like trypsin to intestinal epithelial cell (IEC) monolayers reduces epithelial permeability by increasing transepithelial resistance (TER) and reducing FITC-dextran flux. Given the central role of epidermal growth factor receptor (EGFR) in epithelial cell biology and homeostasis, we hypothesized that trypsin increases TER by activating members of the EGFR family. We also hypothesized that inflammation-induced increases in epithelial permeability were due to an imbalance between proteases and endogenous serine protease inhibitors (serpins). Methods: TER of confluent SCBN monolayers and mouse colonic tissue was measured in Ussing chambers. To determine a role for EGFR and associated signaling pathways, cells were pretreated with inhibitors of EGFR (PD153035), ErBb2 (AG879), ERK-1/2 (PD98059), PI3K (LY294002) and MMPs (marimastat, MMT), or a neutralizing anti-TGF antibody, prior to apical application of trypsin or EGF. Transepithelial flux of FITC-dextran was measured following treatment with PD153035 or AG879 and trypsin. Mucosal expression of serpin A1 was measured using western blots and immunohistochemistry in colons from DSS mice and untreated controls. Results: Apical trypsin and EGF increased TER in SCBN cells by 300% and 80% respectively within 60 min. PD153035 or AG879 caused a dose-dependent decrease in trypsin-induced ΔTER, with IC50s of 0.38 and 2.73 μM, respectively. PD98059, LY294002 and MMT also significantly reduced trypsin-induced ΔTER. Anti-TGF Ab re duce -induced TERd by tryps in 50%. Pretreatment of cells with PD153035 or AG879 prevented trypsin-induced reduction of dextran flux. As observed on western blots, mucosal expression of serpin A1 is increased in mouse colitis. Constitutive serpin A1 immunoreactivity was observed in the mouse colon and its mucosal expression was increased at 7 days of 2.5% DSS. Conclusions: Our data show that trypsin increases TER via processing membrane-bound TGF and activation of EGFR, ErBb2, ERK-1/2, PI3K and MMPs in vitro. In addition, the serine protease inhibitor serpin A1 is overexpressed in inflamed mouse colon. Serine proteases may strengthen epithelial barrier whereas overexpression of endogenous protease inhibitors may contribute to barrier dysfunction in inflammation. Funding: CIHR, NSERC awadhwan@ucalgary.ca
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The Streptococcus Antibiotic Resistome of Adult Cystic Fibrosis Patients Results from Mutation and Horizontal Gene Transfer. Christina S. Thorntona, Margot E. Grinwisa, Christopher D. Sibleya, Harvey R. Rabinab and Michael G. Suretteac. aDepartment of Microbiology and Infectious Diseases, bAdult Cystic Fibrosis Clinic, University of Calgary, Calgary, Alberta, Canada. cDepartments of Medicine and Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada. Rationale: Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. 90% of CF patients succumb to pulmonary failure due to chronic respiratory infections. Traditionally, research has focused on a narrow spectrum of microorganisms thought to be principal pathogens such as Pseudomonas aeruginosa. However, emerging species of bacteria, such as the Streptococcus genus, have been shown to be overlooked pathogens. Within the streptococci, there is the potential for an antibiotic resistome due to constant pressure from several prescribed antibiotics as well as the naturally competent nature of the bacteria. Earlier studies done on the microbiome within CF has shown the presence of multiple isolates from several novel species of streptococci, of which may play a role in CF exacerbation. Objectives: Determine rates of resistance among common antibiotics in CF and to elucidate molecular mechanisms by which this occurs. Methods: In this study, 459 streptococcal isolates from 68 adult CF patients comprising of 16 novel and typed species underwent susceptibility testing for nine antibiotics. Molecular mechanisms of resistance for the macrolides were determined by a PCR-based screen or DNA sequencing. Results: Resistance rates were the greatest for macrolide antibiotics at 51.6% for erythromycin and 56.4% for azithromycin, but with novel isolates closer to 80%. Due to these results, macrolide resistance was looked at in closer detail. The two most common mechanisms of macrolide resistance within the streptococci acquired by horizontal gene transfer, the mef (efflux pump) and erm (target site methylation) accounted for only 53% of resistant isolates. Interestingly, the rarely described mechanism of 23S ribosomal point mutations, the target site of macrolides, accounted for 47% of resistant isolates. Conclusions: The prevalence, species distribution and influence of therapy on resistance profiles suggest complex ecological interactions among the streptococci in the airways of CF patients with mutation, rather than solely horizontal gene transfer, being a significant mechanism of acquired antibiotic resistance in some species within this community. This is in contrast to similar studies done within CF demonstrating resistance solely by acquisition of virulence determinants. This study also illuminates the potential roles for the Streptococcus genus in CF disease progression. Funding: CIHR, AIHS, CFC. ceshaghu@ucalgary.ca
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Diagnosing a severe neonatal intestinal disorder using nuclear magnetic resonance metabolomics Skappak C1, Saude E2, and Harabor A3. Department of Medicine1, Department of Emergency Medicine2, Department of Pediatrics and Division of Neonatology3, University of Calgary, Alberta. Rationale: Rapid diagnosis of disease in pre-term and full-term neonates is a critical aspect of successful treatment. A major cause of morbidity and mortality in neonates is a gastrointestinal disease called necrotizing enterocolitis (NEC). The current diagnosis of NEC is based on a combination of clinical and radiological findings. Unfortunately, the clinical findings are sensitive but non-specific and can lead to over-diagnosis and iatrogenic effects of unnecessary treatment. Creation of a sensitive and specific diagnostic test for NEC and/or the other intestinal conditions of the neonate that can be mistaken for NEC could potentially decrease morbidity and mortality in this patient group. Nuclear magnetic resonance (NMR) metabolomics is a non-invasive method for identifying and measuring various bio-markers of cellular and disease processes. These measurements can be used to create a metabolomic signature of a disease state that differentiates it from the signature of a healthy individual. By applying metabolomics to biosamples from neonates with gastrointestinal conditions, we have been able to identify multiple metabolites related to the intestinal disease process and create a distinct metabolomic signature. With this unique signature we plan to create a computer model that is able to rule in / rule out NEC by analyzing metabolomic signatures from neonates with suspect symptoms. Methods: Clinical information and fecal samples were collected at Calgary area Neonatal Intensive Care Units (NICU) from both neonates with NEC, and healthy premature infants. These samples were then stored at -80°C until analysis could be completed. The samples were thawed, weighed, homogenized and had the their pH standardized. NMR fecal spectra were collected on a 600MHz spectrometer and concentrations of metabolites were determined using Chenomx NMR Suite software version 7.1. Results: 35 Metabolites were identified and analyzed by partial least squares discriminate analysis (PLS-DA). A combination of metabolites excreted in the patientsâ&#x20AC;&#x2122; fecal matter allowed for the separation of neonates with NEC from healthy controls. Conclusions: Currently diagnosing NEC is based on subjective clinical signs, and radiological investigations. NMR metabolomics may allow for a faster more specific, and noninvasive diagnosis of neonates with NEC. Funding: AllerGen-NCE
cdskappa@ucalgary.ca
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Hearing Preservation and Acoustic Neuromas: No Difference Found between Stereotactic Radiotherapy and Conservative Treatment Anna Elliotta, Dr. Manohar Bancea, Dr. David P Morrisa, Dr. Simon Wallingab, Linda Clarkeab a
Department of Otolaryngology, abDepartment of Neurosurgery, Dalhousie University. Halifax, Canada Rationale: Acoustic Neuromas (AN) are benign brain tumors on CN VIII that can cause symptoms such as hearing loss, tinnitius, vertigo, facial numbness and weakness, and life threatening conditions such as hydrocephalus and brainstem compression. Two treatment options include conservative management and stereotactic radiotherapy (SRT). It is unknown which method is superior with regards to hearing preservation. Our study aims to compare hearing outcomes associated with SRT vs. conservative treatment in the management of AN. Methods: We retrospectively compared 98 conservative and 25 SRT treated patients drawn from the Atlantic Tumor database. MRI imaging was performed to determine tumor location, size and growth. Audiometry was performed to determine Pure Tone Hearing Average (PTA) and Speech Discrimination Score (SDS). Only participants with serviceable hearing (AAOHNS1 class A/B) at the onset were included and loss of serviceable hearing was defined as a drop from class A/B to C/D. The four main hearing outcome measures were: 1) Hearing Preservation1 2) Hearing Survival in months 3) PTA Difference 4) SDS Difference Survival analysis and non-parametric tests were used to compare conservative and SRT treatment with regards to these hearing outcomes. Analyses took into account multiple demographic, tumor characteristics, and hearing predictor covariates. Results: Overall, serviceable hearing preservation among the 123 patients was 51%. The overall median hearing survival time was 46 months (mean 59 months). The overall PTA difference was 16 dB and SDS difference was 82% over a median follow-up time of 43 months. In either univariate or multivariate analysis, no significant difference (p>0.05) was found between the conservative and SRT groups as to either the maintenance of serviceable hearing, hearing survival time, or the PTA or SDS difference. However, hearing class at the outset was the greatest predictor of hearing class at the end of follow-up (p<0.001). Conclusions: In this case series of unilateral acoustic neuromas patients, no significant difference was demonstrated as to measures of hearing outcomes and survival time between patients receiving conservative versus SRT treatment. It appears that a superior hearing status at the outset is the greatest predictor of maintenance of serviceable hearing. 1American Academy of Otolaryngology-Head and Neck Surgery Funding: Dalhousie Medical Research Foundation and Music In Medicine anna.elliott@dal.ca
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An immunohistochemical investigation of C-bouton regulation of motoneuron excitability: Kv2.1 channels and local calcium regulation. Laura J Baxtera and Robert M Brownstoneab a
Department of Anatomy and Neurobiology, bDepartment of Surgery, Dalhousie University,
Halifax, Canada. Rationale and Objectives: Spinal motoneurons (MNs) receive and integrate information from many sources. One input forms cholinergic C-bouton synapses, which facilitate MN output, and hence increase muscle contraction. Interestingly, in addition to the ligand receptor – type 2 muscarinic acetylcholine receptor (m2AChR) – the delayed rectifier potassium channel, Kv2.1, forms clusters at sites postsynaptic to C-boutons. The role of this voltage-gated channel in function of this synapse is not known. To address this question, we sought to determine whether m2AChR’s are associated with Kv2.1 channels at other locations in MNs. That is, because m2AChR’s are found on MN terminals at the neuromuscular junction (NMJ), we sought to determine whether Kv2.1 channels are likewise situated there. Furthermore, as Kv2.1 channels are modulated by phosphorylation, we sought to determine whether the calcium signaling molecules calcineurin (the B subunit, PP2Bβ) and/or inositol triphosphate receptor type 2 (IP3R2) are found in the nanodomain at this post-synaptic site. Methods: ChAT::eGFP mice (N=3) were used to identify MNs and their axons. Diaphragm, lumbar spinal cord, and gastrocnemius tissues were obtained following perfusion. Fluorescence immunohistochemistry was used to identify Kv2.1, green fluorescent protein, PP2Bβ, IP3R2, vesicular acetylcholine transferase, and m2AChRs, and rhodamine-conjugated bungarotoxin was used to visualize nicotinic receptors. Laser-scanning microscopy was used to obtain confocal images for analysis. Results: Although Kv2.1 clusters were found at C-bouton contacts on MNs, they were not present on NMJs in either the diaphragm or gastrocnemius muscles. Neither PP2Bβ nor IP3R2 were clustered at the post-synaptic site of C-boutons. Conclusions: (1) Kv2.1 channels are not necessary for m2AChR function in MNs, as these receptors are found at NMJs in the absence of Kv2.1 channels. A MN-specific Kv2.1 knockout would lead to understanding of the role of Kv2.1 in MN function. (2) Neither calcineurin nor IP3R2 were clustered at C-bouton sites. The molecules regulating local calcium metabolism remain to be identified.
Funding: CIHR, Dalhousie Medical Research Foundation
laurajbaxter@gmail.com
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Detection of circulating tumor cells in advanced head and neck cancer using the CellSearch® system Student Name: John Basmaji Project Supervisor: Dr. Anthony Nichols ABSTRACT: Background: Head and neck cancer has seen many advances in surgery, chemotherapy, and radiation therapy over the past few decades, but with modest improvement in overall survival. Since one third of deaths are attributed to metastatic spread, early detection of circulating tumor cells (CTCs) offers the possibility of improved outcome for head and neck squamous cell cancer (HNSCC) patients by tailoring therapy based on each individual’s risk stratification for developing disseminated disease. Methods: Patients with advanced stage HNSCC (stage III/IV) were tested for CTCs using the CellSearch system, which has been approved by the US Food and Drug Administration (FDA) for monitoring CTCs in other cancers. Univariate logistical regression was used to assess associations between lung nodules and the presence of CTCs. Progression-free survival based on the presence of CTCs was assessed by a log-rank test. Results: CTCs were detected in six of 15 patients with advanced stage HNSCC (range 1-2 cells/7.5mL of blood). CTCs were significantly associated with patients with lung nodules > 1cm (p=0.04). There was also a suggestion of improved survival in the CTC negative versus the CTC positive patients (p=0.11). Conclusions: Circulating tumor cells can be successfully isolated in patients with advanced stage HNSCC using the CellSearch® system. CTC detection may be important for prognosis, evaluating treatment outcome and for determining efficacy of adjuvant treatments. jbasmaji2013@meds.uwo.ca
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Implementation of a novel curriculum for training medical students in the use and interpretation of hand-held echocardiography Thomas R. Cawthorn1, Curtis Nickel1, Michael O’Reilly1,2, Anthony J. Sanfilippo1,2, Amer M. Johri1,2. 1. School of Medicine, Queen’s University, Kingston, ON. 2. Division of Cardiology, Kingston General Hospital, Kingston, ON. Context/Setting The aim of this study was to examine three teaching modalities used in the introduction of a new hand-held echocardiography curriculum for junior medical students. We recruited 45 preclerkship medical students at Queen’s University and randomized them into one of three educational programs. Two novel electronic-learning programs were used in addition to a previously developed and validated lecture-based approach. Intervention A novel four-week hand-held echocardiography curriculum was developed through collaboration with expert echocardiographers and experienced medical educators. This curriculum was delivered via three educational programs: (1) a traditional lecture-based approach and hands-on training with a sonographer; (2) a novel electronic education module and hands-on training with a sonographer; (3) a novel electronic education module and hands-on training with an ultrasound simulation system. Observations Our results indicate that, following a defined educational program, junior medical students were able to competently and efficiently acquire standard echocardiographic images from volunteer patients. Fundamental echocardiography interpretation skills were assessed via pre-and postintervention tests and demonstrated that students were proficient in making basic interpretation of pathology from echocardiographic images. Discussion Echocardiography acquisition and interpretation are skills that are traditionally taught at the resident level. Therefore, the introduction of a new curriculum in focused hand-held echocardiography enables medical students to acquire knowledge and expertise previously not available until residency training. This work indicates that hand-held echocardiography may be introduced to students at earlier stages of their medical education through implementation of a novel curriculum. Key Words Echocardiography Curriculum Development Electronic Learning tcawthorn@qmed.ca
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SESSION 3 Poster Discussion Brodie Centre Mezzanine 13:45 â&#x20AC;&#x201C; 15:15 p.m. Wednesday, June 13th, 2012
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ROLE OF GLIA MATURATION FACTOR BETA (GMFβ) IN DIFFERENTIATION OF THE CHILDHOOD CANCER NEUROBLASTOMA Richards, Ceri1, Eisenstat, David D2,3 1
B.Sc. Medicine Program, 2Manitoba Institute of Cell Biology, 3Department of Biochemistry & Medical Genetics, Faculty of Medicine, University of Manitoba. Rationale: Prior studies from the lab demonstrated phosphorylation changes of GMFβ when comparing low grade tumor to higher grade tumors or when comparing adult brain to human fetal brain. This supported our hypothesis that phosphorylation status of GMFβ correlates with differentiation states in neural tissues. Objectives: The goal of the project was to characterize the role of glia maturation factor beta (GMFβ) as an actin depolymerizing factor (ADF) in neuroblastoma (NB), the second most common childhood solid tumour. Specific objectives were: (1) to determine the relationship between phosphorylation status of GMFβ and differentiation; (2) to assess morphological changes in NB cell lines after transfection with GMFβ; and (3) to determine the expression and phosphorylation of GMFβ in adult versus fetal mouse tissue. Methods: NB cultures were differentiated with 13-cis retinoic acid (RA). Western blotting, PCR and RT-PCR were used to quantify GMFβ expression. NB cells were transfected with GMFβ and GMFβ with a mutant candidate actin binding domain. Tissues were harvested from adult and fetal mice. Results: RA treatment was shown to result in GMFβ dephosphorylation. GMFβ was hypophosphorylated in adult mouse brain tissues compared to less differentiated tissue. GMFβ transfection showed modest increases in differentiation in NB cell lines as demonstrated in a neurite extension assay. A possible GMFβ dimer is formed with dephosphorylation of NB cells and in more differentiated mouse tissues. A MYCN-amplified NB cell line has increased GMFβ expression compared to non-amplified cells. Conclusions: Our results are consistent with the role of GMFβ as an ADF, since ADF’s function when dephosphorylated. These experiments support a relationship between GMFβ hypophosphorylation and differentiation in NB cell lines. Examination of the role of GMFβ as an ADF in NB differentiation may ultimately contribute towards novel biological therapies directed against this highly malignant childhood cancer.
richardsceri@gmail.com
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Identification of Virulence Determinants in 1918 Spanish Flu Hemagglutinin Alex Silaghi1,2,3,4, Tracy Taylor2, Darwyn Kobasa1,2,4 1
Department of Medical Microbiology, University of Manitoba
2
Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada
3
MD/PhD Program, University of Manitoba
4
International Infectious Diseases and Global Health Training Program
Spanish Flu caused the worst pandemic in history with 30-50 million deaths. Although this virus disappeared after the 1918 pandemic, viral RNA was isolated from histological samples and frozen cadavers, and used to generate live viruses containing all or some segments of Spanish influenza. The Hemagglutinin (HA) segment was identified as the major virulence determinant, yet the regions involved and mechanisms of virulence remain unknown. HA is a glycoprotein found on the surface of virions and catalyzes receptor binding and fusion of the viral and plasma membrane during viral entry. HA is cleaved by trypsin-like cellular proteases into HA1 and HA2, an essential process for activation of fusion. HA1 contains three structural/functional regions: Fusionâ&#x20AC;&#x2122; (Fâ&#x20AC;&#x2122;), Vestigial Esterase (VE) and Receptor-Binding Domain (RBD), while HA2 contains the main fusion peptide, transmembrane region and cytoplasmic tail. We generated chimera between Spanish influenza (Sp) HA and closely related H1N1 viruses: WSN/1933, WilsonSmith/1933, PR8/1934, USSR/1977, New Caledonia/1999 and a 2009 pandemic strain (Mexico/Indre4487/09; Mx10) by swapping known regions. The WSN/Sp chimera indicated that Spanish influenza HA virulence in mice is dependent on Serine (S) at residue 343, which is just upstream of the Arginine 344, the site of cleavage of HA into HA1 and HA2. A virus containing Sp HA with Tyrosine (Y) at 343 was attenuated in mice, but also grew poorly without exogenous trypsin in cell culture. Although most influenza viruses require exogenous trypsin for growth in tissue culture, Sp does not, but the relevance of this finding or pathogenesis was unknown. USSR/Sp HA chimera identified the N-glycosylation site at position 286 as another determinant of virulence in mice and trypsin-independent growth in cell culture, but there are likely more requirements of virulence spread across several structural/functional domains, which we are currently investigating. The other chimera systems are also currently being tested, with the hope of identifying more virulence determinants. In summary, trypsin-independent growth in cell culture correlates with virulence in mice, suggesting that the use of specific proteases in the lung by Sp may contribute to the severe disease not observed with other human influenza viruses. alex_silaghi@hotmail.com
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Phenotypic and genotypic evaluation of two kindreds affected with hereditary xerocytosis Brett L. Houstona, Teresa Zelinskib, Donald S. Houstonc, Sara J. Israelsd, Gail Coghlane, Bernie N. Chodirkerb, Patrick G. Gallagherf and Ryan Zarychanskic,g aUniversity of Manitoba, Winnipeg, MB, Canada; bDepartments of Biochemistry & Medical Genetics and Pediatrics & Child Health, University of Manitoba, Winnipeg, MB, Canada; cDepartment of Internal Medicine, Section of Hematology & Medical Oncology, CancerCare Manitoba, Winnipeg, MB, Canada; dDepartment of Pediatrics and Child Health and the Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, MB, Canada; eDepartment of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; fPediatrics and Genetics, Yale University School of Medicine, New Haven, CT; gDepartment of Community Health Sciences, University of Manitoba Rationale: The hereditary stomatocytoses are a rare, diverse group of clinical conditions associated with chronic red blood cell hemolysis and increased erythrocyte membrane permeability to monovalent cations. The most common form of hereditary stomatocytosis is hereditary xerocytosis (HX, OMIM: 194380). We studied two genetically unrelated kindreds with HX, one from Western Canada, and the other from upstate New York. Although the causative gene is unknown, previous studies have mapped the HX locus to 16q23 â&#x20AC;&#x201C; 16qter. Objectives: To systematically characterize the disease phenotype and identify the gene and causative mutation responsible for hereditary xerocytosis. Methods: A pedigree was constructed, a focused history was taken and the presence of splenomegaly was assessed by physical examination. Laboratory analysis included both biochemical and hematological parameters. DNA linkage studies and exome sequencing were used to identify novel gene mutations in two multigenerational HX kindreds. Results: The mode of inheritance was autosomal dominant. Affected family members were found to have well-compensated hemolysis, associated with an elevated MCHC, decreased osmotic fragility, decreased haptoglobin, and increased indirect hyperbilirubinemia. Cholelithiasis and progressive iron loading were common, despite normal hemoglobin levels. Using DNA linkage analysis, we confirmed the localization of the disease phenotype to chromosome 16q, and we refined the candidate region to 16q24.2 â&#x20AC;&#x201C; 16qter, a 2.4 million base pair interval containing 51 known or predicted genes. Exome sequencing identified mutations affecting PIEZO1 (encoded by FAM38A gene) in both HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and co-segregation with the disease phenotype in all affected individuals. Conclusions: In this family study, elevated percent reticulocyte counts were used to characterize the presence of a well compensated, autosomal dominant hemolytic process associated with an elevated MCHC and decreased osmotic fragility. Clinically this condition is associated with gallstones and progressive iron loading. Mutations in PIEZO1, encoded by the gene FAM38A, are responsible for hereditary xerocytosis. These findings, the first report of a mutation in a mammalian mechanosensory transduction channel associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte homeostasis. umhoustb@cc.umanitoba.ca
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The nicotinic α6 subunit gene affects chronic pain sensitivity via an interaction with P2X2/3 receptors Jeffrey Wieskopf1, Robert Sorge1, Susanna Sotocinal1, Jean-Sebastien Austin1, Peter Slepian1, Shad Smith2, Feng Dai2, Weike Lai2, Ryan Drenan3, Rahul Srinivasan4, Walrati Limapichat4, Chris Richards4, Jayanti Mathur4, Valerie Uzzell5, Jeff Janes5, Andrew Su5, Marshall Devor6, Michael McIntosh7, Uwe Maskos8, JP Changeux8, Henry Lester4, Luda Diatchenko2, Inna Belfer9, William Maixner2, Ardem Patapoutian5, Jeffrey Mogil1 1
The Integrated Program in Neuroscience & The Alan Edwards Centre for Research on Pain, McGill University, 2Carolina Center for Genome Sciences, University of North Carolina, 3Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 4Biology, California Institute of Technology, 5Genomics Institute for the Novartis Research Foundation, La Jolla, 6Department of Cell and Animal Biology, Hebrew University of Jerusalem 7 Department of Biology, University of Utah, 8 9 Institut Pasteur, France, Department of Anesthesiology, University of Pittsburgh Introduction: Chronic pain, an exceedingly costly health condition, is associated with increased mortality rates and a plethora of co-morbidities. Regarding an individual’s propensity to experience chronic pain following similar procedures or injuries, it is widely acknowledged that an immense amount of interindividual variability exists. Since much of this variability is likely genetic, identification of genes predisposing to severity of symptoms of chronic pain could be extremely valuable in terms of early intervention and drug development. Using null mutant (knockout) and gain-of-function (L9'S) mutant mice, and applying expression genomics, we identified the alpha6 subunit of nicotinic acetylcholine receptors and demonstrated its involvement in chronic pain conditions. Specifically, chronic pain behavioural testing revealed that Chrna6 knockout mice displayed higher overall levels of allodynia as compared to wild-type mice, while Chrna6 gain-of-function mice displayed significantly less allodynia, thereby demonstrating the protective role of Chrna6 in chronic pain. Here, we investigate the mechanism of Chrna6’s role in allodynia in various chronic pain models. Methods: Using Chrna6 knockout, gain-of-function, and wild-type mice, mechanical allodynia thresholds were recorded following spared nerve injury (SNI) and intraplantar complete Freund’s adjuvant (CFA) injection. Varying doses of nicotine (0.15 – 3.6 mg/kg) were subsequently administered systemically. To study the interaction between Chrna6 and purinergic receptors, α,β - MeATP, a selective P2X2/3 and P2X3 agonist, was administered, followed by nicotine injections 15 minutes later. Results: Both SNI and CFA caused reproducible allodynia in all groups. Systemic nicotine injections produced anti-allodynia following SNI or CFA in wild-type, CD-1, and gain-offunction mutant mice, but had no effect in Chrna6 knockout mice. α,β -MeATP injections also produced allodynia in all strains tested. When nicotine and α,β -MeATP were co-administered, nicotine produced robust anti-allodynia in the gain-of-function mice, with no effect in the knockout mice. Conclusions: These findings demonstrate the necessity of Chrna6 in nicotine induced antiallodynia. Furthermore, co-administration of nicotine and α,β -MeATP demonstrates a pharmacological interaction between Chrna6 and P2X2/3 receptors, suggesting a mechanism by which Chrna6 exerts a protective role in chronic pain. Ultimately, increased understanding of Chrna6’s mechanism of action could lead to novel drugs that target the symptoms of various pain conditions. jeffrey.wieskopf@mail.mcgill.ca
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Driver mutations in histone H3.3 and chromatin remodeling genes in paediatric glioblastoma Xiao-Yang Liu1, Andrey Korshunov2, Jeremy Schwartzentruber3, David T.W.Jones4, Elke Pfaff4, Dominik Sturm4, Adam M. Fontebasso5, Dong-Anh Khuong Quang1, Steffen Albrecht6, Marcel Kool4, Zhifeng Dong7, Peter Siegel7, Andreas von Diemling2, Damien Faury1, Uri Tabori8, Peter Lichter4, Christoph Plass9, Jacek Majewski1, Stefan M. Pfister4, Nada Jabado1,10. 1 Department of Human Genetics, McGill University. 2Clinical Cooperation Unit Neuropathology, German Cancer Research Center, DKFZ, Heidelberg, Germany. 3McGill University and Genome Quebec Innovation Centre. 4Division Molecular Genetics, German Cancer Research Center, DKFZ, Heidelberg, Germany. 5Division of Experimental Medicine, McGill University. 6 Department of Pathology, Montreal Children's Hospital, McGill University Health Center. 7 Rosalind and Morris Goodman Cancer Research Centre, McGill University. 8The Arthur and Sonia Labatt Brain Tumor Research Centre, Hospital for Sick Children Research Institute, University of Toronto. 9 Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center, DKFZ, Heidelberg. 10Department of Paediatrics, McGill University and McGill University Health Center Research Institute. Rationale: Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. Objectives and Methods: To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Results: Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (群-thalassaemia/ mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRXDAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. Conclusions: This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis. Xiaoyang.liu@hotmail.ca
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Inflammation-induced changes in leukotriene receptor expression cause differential gene expression through nuclear calcium signaling in vascular smooth muscle cells Alison Eatona, Edit Nagya, Mathilde Pacaulta, Jérémy Fauconnierb, c, Magnus Bäcka,* Department of Medicinea and Department of Physiology & Pharmacologyb, Karolinska c Institutet, Stockholm, Sweden and INSERM U1046, Université Montpellier 1, Université Montpellier 2, Montpellier, France. Aims: Leukotrienes are pro-inflammatory mediators that are locally produced in coronary atherosclerotic plaques. It is thought that the vasodilatory response induced by cysteinyl leukotrienes (CysLT) in healthy human coronary arteries may be altered under pathological conditions, such as atherosclerosis. This study aimed to elucidate CysLT signaling in vascular smooth muscle cells (SMCs), and the effects of inflammation on this process. Hypothesis: We tested the hypotheses that a) the CysLT1 receptor is upregulated in vascular SMCs in the context of atherosclerosis, and that b) CysLT1 receptor activation is coupled to nuclear calcium signaling, which c) leads to alterations in gene expression. Methods & Results: Immunohistochemical analyses revealed that CysLT1 receptor expression co-localized with a-smooth muscle actin in human carotid endarterectomy samples, and that the CysLT1 receptor exhibited a perinuclear expression in human coronary artery SMCs. Real-time quantitative PCR analysis demonstrated that lipopolysaccharide (LPS) significantly upregulated CysLT1 receptor mRNA levels in human coronary artery SMCs in a timedependent fashion. Fluorescent-labeled calcium- signaling experiments showed that LPS significantly enhanced the changes in intracellular calcium induced by leukotriene C4 (LTC4) In these cells. LTC4 stimulation predominantly enhanced nuclear calcium increase, and this was inhibited by the CysLT1 receptor antagonist MK-571. Microarray analysis revealed that after 24h stimulation of human coronary artery SMCs with LTC4, there was a 5-fold increase in mRNA levels for Plasminogen Activator Inhibitor (PAI)-2, among a number of significantly upregulated genes. The LTC4-induced increase in PAI-2 expression was confirmed by real time quantitative PCR and ELISA, and was inhibited by the CysLT1 receptor antagonist MK571 and by calcium chelators. Conclusions: Pro-inflammatory stimulation of vascular SMCs upregulated CysLT1 receptor expression, which was visualized in a peri-nuclear pattern. This receptor expression was coupled to nuclear calcium signaling and changes in gene expression, such as upregulation of PAI-2. Together, these findings suggest a role of nuclear CysLT1 receptor signaling in vascular SMCs inducing gene expression patterns associated with atherosclerosis.
ajeaton@mun.ca
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Prolonged, 24-h delayed peripheral inflammation increases short- and longterm functional impairment and histopathological damage after focal ischemia in the rat Kristopher D. Langdon1,2, Crystal L MacLellan3 and Dale Corbett1,2,4,5 1
Division of BioMedical Sciences, Faculty of Medicine, Memorial University, St. Johnâ&#x20AC;&#x2122;s NL Heart and Stroke Foundation Centre for Stroke Recovery, Ottawa ON 3 Dept of Physical Therapy, University of Alberta, Edmonton, AB 4 Dept Cellular and Molecular Medicine, University of Ottawa, Ottawa ON 5 Dept Medicine, University of Toronto, Toronto ON 2
Rationale: The incidence of infection among stroke patients is alarmingly high and both acute and delayed infections increase morbidity and mortality. Experimental studies support the acute clinical data, but little attention has focused on delayed systemic infections. Objectives: Here, we investigated the effects of prolonged systemic inflammation either before or 24-h after ischemia. Methods: Systemic inflammation was induced by injecting rats with three separate doses of lipopolysaccharide (LPS; 50 lg/kg, i.p.) with core temperature monitoring for 48h after middle cerebral artery occlusion (MCAo). Results: Lipopolysaccharide injected before MCAo increased injury by ~30%, whereas delayed injection increased injury by ~85% (30-day survival). Proinflammatory cytokines assessed repeatedly for 72h were significantly and persistently elevated with inflammation. This was accompanied by increases in microglia/macrophage and infiltrating leukocyte numbers in delayed LPS-treated animals. Behavioral assessments at 7 and 30 days revealed ~15% deficit in hindlimb function in animals treated with LPS 24h after ischemia. Conclusions: Clearly, delayed and prolonged postischemic systemic inflammation has devastating effects on stroke outcome, in the absence of a prolonged febrile response. These findings, together with corroborative clinical data, emphasize the importance of early intervention to counteract the deleterious consequences of stroke-associated inflammation and infection. klangdo@mun.ca
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Medical Students’ Attitudes towards using Online Social Networking sites to Communicate with Patients Sandra Cooke-Hubley, M.Sc (Med), MPHa, Michele Neary, PhD, R.Psychab and Paul Dancey, MD, FRCPCac a
Faculty of Medicine, Memorial University of Newfoundland, ab Counselling Centre, Memorial University of Newfoundland, ac Faculty of Medicine, Discipline of Paediatrics, Memorial University of Newfoundland Rationale: Use of Online Social Networking (OSN) sites such as Facebook and Twitter is arguably the fastest and most efficient way for people to communicate. Medical student and physician use of OSN sites has not yet been well researched, although there is a great interest in if and how future physicians will interact with patients online. Issues that may arise with online communication include protection of patient confidentiality and maintenance of professional boundaries. Objectives: The primary objective of this project aimed to investigate medical students’ attitudes and intentions towards using OSN sites to interact with patients. Secondly, this research aimed to examine whether there were differences in attitudes or intentions between pre-clerkship and clerkship students. Methods: Pre-clerkship (N=114) and clerkship (n=53) medical students from a Canadian Medical School completed a Social Media Questionnaire (all students who were distributed the questionnaire completed it). The questionnaire included 15 yes/no- type questions on the frequency of OSN site use and attitudes toward interacting with patients over OSN sites. The questionnaire included five clinical vignettes illustrating different physician behaviors related to interaction with patients using OSN sites. Students were asked to rank, using a Likert Scale, their agreement or disagreement with the physician behavior. Data was entered using Fluidsurveys, and was analyzed using SPSS. Preliminary Results: Preliminary results indicate that Facebook use by medical students is prevalent; 98.2% of respondents have an active account and 74.3% typically log on more than once per day, with pre-clerkship students significantly more likely than clerkship students to log on multiple times per day (p<0.00). Although the majority of students indicated they would not add a patient as a “Friend” on Facebook, (77.2%), pre- clerkship students were significantly more likely to consider adding a patient than clerkship students (22.8% vs. 9.4%, p=0.039). A large minority of students (46.1%) agreed that interacting with patients over a secure OSN sites could improve patient care, with significantly more preclerkship than clerkship students agreeing (52.6% vs. 32.1%%, p<0.013). Students identified a number of barriers to communication with patients online including breeching patient confidentiality and allowing patients to access their personal data online. z94smc@mun.ca
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Biochemical characterization of Chlamydia outer protein (Cop) B and D from Chlamydia pneumoniae. David C. Bulirab, Dr. Christopher B. Stonec, Tiffany Leightonb, Robert Claydenb and Dr. James Mahonyb a MD/PhD Program, McMaster University. bDepartment of Pathology and Molecular Medicine, McMaster University. cDepartment of Molecular Genetics, University of Toronto. Rationale: Chlamydia trachomatis is responsible for over 6 million cases of blindness in developing countries each year, and is the number one sexually transmitted bacterial infection in the United States. Chlamydia pneumoniae infections are limited to the respiratory tract, and have been noted to cause up to 10% of community-acquired pneumonia. It has also been implicated in other diseases, such as atherosclerosis, Alzheimer's disease, and the exacerbation of asthma and COPD. Chlamydia spp. use type III secretion (T3S) to facilitate invasion, and central to this process are proteins CopB and CopD which allow for translocaton of effector proteins from the bacterial cytoplasm to the host cell. However, the mechanism behind the function of these proteins remains unknown Objective: To elucidate the function of Chlamydia outer membrane proteins (Cop) B and D within the T3S apparatus of Chlamydia pneumoniae. Methods: To examine the function of CopB and CopD within the T3SS of Chlamydia we cloned fragments of each protein excluding transmembrane domains. We then performed in vitro interaction experiments using CopB/D fragments and other T3S components via glutathione-Stransferase (GST) pull-downs. Lastly, human RBC lysis experiments were used to explore the pore forming ability of CopB and CopD. Results: The putative needle filament protein, CdsF, interacts with both the N-terminal and middle fragments of CopB and CopD, under high salt conditions, whereas the chaperone, LcrH_1, interacted with only the N-terminal fragments. However, these fragments of the putative translocator proteins did not interact with known T3S-associated effector proteins, such as Cpn0803. Lastly, C. pneumoniae was able to lyse RBCs and preliminary results suggest that CopB and CopD may be responsible for this action. Conclusion: We found that the putative translocator proteins of the chlamydial T3SS, CopB and CopD, interact with similar orthologous proteins in other T3SS. Furthermore, preliminary RBC lysis experiments indicated that CopB and CopD may form pores in eukaryotic membranes. Together, this data suggests that CopB and CopD may function as a pair of translocator proteins in the T3SS. Although further data is required to understand their role during the replication cycle, these proteins may represent a novel antimicrobial target. David.Bulir@medportal.ca
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The development of national indicators for the surveillance of osteoporosis in Canada J. LeMessurier, MPHa; S. O’Donnell, MScb; P. Walsh, MScb; L. McRae, BScb; C. Bancej, PhDb; for the Osteoporosis Surveillance Expert Working Group a
Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada b Public Health Agency of Canada, Health Promotion and Chronic Disease Prevention Branch, Centre for Chronic Disease Prevention and Control, Chronic Disease Surveillance and Monitoring Division, Ottawa, Ontario, Canada Introduction: The Public Health Agency of Canada, in collaboration with bone health and osteoporosis experts from across Canada (n = 12), selected a core set of indicators for the public health surveillance of osteoporosis using a formal consensus process. Methods: A literature review identified candidate indicators that were subsequently categorized into an osteoporosis-specific indicator framework. A survey was then administered to obtain expert opinion on the indicators’ public health importance. Indicators that scored less than 3 on a Likert scale of 1 (low) to 5 (high) were excluded from further consideration. Subsequently, a majority vote on the remaining indicators’ level of public health importance was sought during a face-to-face meeting. Results: The literature yielded 111 indicators, and 88 were selected for further consideration via the survey. At the face-to-face meeting, more than half the experts considered 39 indicators to be important from the public health perspective. Conclusion: This core set of indicators will serve to inform the development of new data sources and the integration, analysis and interpretation of existing data into surveillance products for the purpose of public health action.
jlemessurier@mun.ca
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Does malpositioning of the arm influence radiographic range of motion measurement? Julien Chapleaua, Pierre-Yves Lagacéb,c, Fanny Canetd, Nicola Hagemeisterb,c, Dominique M Rouleaua,d a
Université de Montréal, b École de Technologie Supérieure (ETS), c Laboratoire d’Imagerie en Orthopédie (LIO- CRCHUM), d Hôpital du Sacré-Coeur de Montréal. Rationale: Radiographic range of motion measurement of the elbow has been shown to be both precise and reliable. For this method to be used routinely in research studies, it is important to describe its limits regarding: (1) rotation of the arm from the perfect lateral position and (2) the length of humerus and ulna visible on the radiograph. Methods: A 3D bone reconstruction was performed from an upper limb CT scan. Planar radiographs were simulated for rotations of the elbow within a range of ±30o from the perfect lateral position. The field of view was modified, ranging from five visible centimeters of diaphysis on the radiograph to full visibility of the upper limb. Results: The disparity was less than 2.5° (mean=0.68°, SD=0.43°) when the flexed arm was rotated between -30.0° (external rotation, ER) and + 18.0° (internal rotation, IR). When considering the extended arm, measured angles differed by less than 2.5° (mean=0.79°, SD=0.57°) within a range of -15.0° (ER) to +30.0° (IR). When a minimum of 12 cm of humerus and ulna, from the capitellum, were visible on the radiograph measured angles varied very slightly (mean disparity of 0.71°, SD= 0.71°). Finally a qualitative description of the appearance of the radiographs was included to help surgeons estimate acceptable degrees of rotation. Conclusion: Range of motion (ROM) measurement shows consistent results, despite 15to 30 degrees of internal or external rotation. At least 12 cm of the diaphysis of the humerus and ulna should be visible on the radiographs to ensure the validity of measurement. Radiographic ROM measurement is still recommended over the goniometer for research purposes because of its high reliability and precision. Moreover, malpositioning of the elbow should not jeopardize results since it will most likely be an angle measurement variation of less than 2.5°.
julien.chapleau @umontreal.ca
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Postoperative furosemide administration lowers transfusion rate in scaphocephaly surgery Adil Harroud1, Alexander G. Weil1, Jean Turgeon2, Claude Mercier1, Louis Crevier1 1
Section of Neurosurgery, Sainte-Justine Hospital, University of Montreal, Montreal, Canada 2 Department of Pediatrics, Sainte-Justine Hospital, University of Montreal, Montreal, Canada Rationale: One of the major challenges in scaphocephaly correction surgery is the high postoperative transfusion rate (15-100%) related to blood loss in these small pediatric patients. Several approaches have been proposed to prevent transfusion, including endoscopic surgery, erythropoietin or tranexamic acid administration, and preoperative hemodilution. We hypothesized that a significant proportion of postoperative anemia observed in these patients is actually dilutional. Consequently, since 2005, we have implemented a protocol of furosemide administration to treat 'dilutional anemia' and prevent transfuion. Objective: Evaluate the impact of the postoperative furosemide protocol on transfusion rate. Methods: This is a retrospective study of 96 consecutive scaphocephaly patients operated at our institution between January 2000 and April 2012. Mean age at surgery was 4.9 Âą 1.5 months (range: 2.8-8.7). Patients operated between 2005 and 2006 were considered part of an implementation phase during which furosemide administration was not systematic and were thus excluded from the statistical analysis. Transfusion rates before and after implementation of the protocol were compared. The impact of furosemide administration on transfusion requirement was also measured while accounting for other variables of interest in a multiple logistic regression model. Results: The postoperative transfusion rate was significantly reduced in the furosemide group compared to the control group (19% vs. 53.8% respectively; p=0.001). The lowest hemoglobine level tolerated without transfusing was higher in average in the furosemide group compared to the control group (74.5 vs. 65.9 respectively; p=0.011). The effect of furosemide administration on the probability of receiving a transfusion was evaluated in a multiple logistical regression model accounting fro age, sex, surgical procedure, peroperative blood loss, surgery duration, and preoperative hemoglobin. Only furosemide administration significantly predicted the need for postoperative transfusion (OR=0.214; p=0.008). Conclusions: Furosemide administration significantly reduces postoperative transfusion requirements in patients operated for correction of isolated scaphocephaly. Adil.harroud@gmail.com
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Dorsal premotor cortex is involved in switching motor plans. Elsa Tremblaya, Alexandre Pastor-Berniera and Paul Ciseka a
Department of Physiology and Groupe de recherche sur le système nerveux central, Université de Montréal. Montréal, Canada. Rationale: Previous studies have shown that neural activity in primate dorsal premotor cortex (PMd) can simultaneously represent multiple potential movement plans, and that activity related to these movement options is modulated by their relative subjective desirability. These findings support the hypothesis that decisions about actions are made through a competition within the same circuits that guide the actions themselves. This hypothesis further predicts that the very same cells that guide initial decisions will continue to update their activities if an animal changes its mind. Objectives: Determine whether cells in PMd involved in the selection of an initial movement choice update their activities to reflect a change in movement planning when a previously selected movement option suddenly becomes unavailable. Methods: We tested this prediction by recording neural activity in the PMd of a monkey performing an instructed-delay reach selection task. In the task, two targets were simultaneously presented and their border styles indicated whether each would be worth 1,2, or 3 juice drops. In a random subset of trials (FREE), the monkey was allowed a choice while in the remaining trials (FORCED) one of the targets disappeared at the time of the GO signal. In FORCED-LOW trials, the monkey was forced to move to the less valuable target and started moving either toward the new target (Direct) or toward the target that vanished and then curved to reach the remaining one (Curved). Results: Prior to the GO signal, PMd activity clearly reflected the monkey’s subjective preference, predicting his choices in FREE trials. In FORCED-LOW trials, PMd activity reflected the switch of the monkey’s plan, as early as 100 ms after the GO signal, will before movement onset (MO). These results were reproduced by a computational model. Conclusions: These findings confirm that the activity in PMd is not related to the feedback from the movement itself, and suggests that PMd continues to participate in action selection, even when the animal changes his mind online. These results suggest that switches between action plans can be explained by the same computational process responsible for initial decisions. Funding: CIHR, FRSQ, GRSNC. elsa.tremblay@umontreal.ca
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Developing seasonal physician shift schedules in the emergency department to meet patient demand Savage1, D.W., B. Weaver1, and D. Wood1,2 1. Northern Ontario School of Medicine, West Campus, Lakehead University, 955 Oliver Rd., Thunder Bay, ON, P7B 5E1 2. Thunder Bay Regional Health Sciences Centre, 980 Oliver Road, Thunder Bay, ON, P7B 6V4 Introduction: The Ontario government has developed performance guidelines for hospital emergency departments. One important factor in meeting the performance targets is proper scheduling of physicians to meet the demand by incoming patients. At the Thunder Bay Regional Health Sciences Centre (TBRHSC), the physician schedule in the emergency department (ED) is similar for all days of the year. However, the number of patients arriving hourly can vary greatly by day and season. This research will examine how physician scheduling in the ED (i.e., the time at which shifts start each day) can be improved to better meet patient demand. Methods: Using historical data, we intend to perform a cluster analysis on hourly patient arrival data in the ED. Individual days will be grouped by day of the week and season based on similar patterns of patient arrival. To develop optimal schedules which meet patient demand, a mathematical modeling technique known as mixed-integer programming will be used on the grouped patient arrival data. The new physician schedule will be compared to the old schedule to determine whether patient demand was better met. Results: Preliminary analysis using the model on a subset of the data show that physician start time at TBRHSC can be adjusted to better meet patient arrival time in the ED. As well, by scheduling more physicians on certain days of the week, the high demand by arriving patients on those days of the week can be better met. Conclusion: ED performance can be improved in many ways, and the results from this study show that having the proper resources available to meet incoming patient demand is one potential avenue for ED improvement. The techniques used in this study could be applied to other personnel in the ED to ensure that their services are not limiting system performance. dsavage@nosm.ca
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Influenza infects lung microvascular endothelium leading to microvascular leak: Role of apoptosis and claudin-5 Susan M Armstronga, Changsen Wangb, Jayesh Tigdib, Xiaoe Sic, Asela Gamagea, Theo J Moraesac, and Warren L Leeabd of Medical Science, University of Toronto; bKeenan Research Centre of the Li Ka Shing Knowledge Institute of St. Michael's Hospital (Toronto); cHospital for Sick Children, Toronto; the dDivision of Respirology and Interdepartmental Division of Critical Care Medicine, Faculty of Medicine, University of Toronto aInstitute
Rationale: Severe infections with human influenza are characterized by acute lung injury, a syndrome of pulmonary microvascular leak. Antiviral medications are only partially effective at reducing mortality, indicating that additional therapeutic strategies are needed. We hypothesized that influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. Methods: We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed spectrofluorimetrically and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. Results: We found that human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in endothelial permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced loss of the tight junction protein claudin-5; the adherens junction protein VE-cadherin was unaffected. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dosedependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Conclusions: We describe two mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza. Funding: WL Lee is supported by the Canadian Institutes of Health Research, the Ontario Thoracic Society, the Physician Services Incorporated Foundation and an Early Researcher Award from the Government of Ontario. SM Armstrong is supported by a MD/PhD Studentship award from the CIHR and an MD/PhD Studentship award from the McLaughlin Centre.
susanm.armstrong@mail.utoronto.ca
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Evaluating genetic counselling for multifactorial schizophrenia in the molecular age Gregory Costainac, Mary Jane Esplende, Brenda Tonerbcde, Kathleen Hodgkinsong, Anne S. Bassettacdf aClinical Genetics Research Program, bSocial Equity and Health Research, Centre for Addiction and Mental Health, Toronto, Ontario cInstitute of Medical Science, dDepartment of Psychiatry, University of Toronto, Toronto, Ontario eBehavioural Sciences and Health Research Division, fDepartment of Psychiatry, University Health Network, Toronto, Ontario gClinical Epidemiology and Genetics, Faculty of Medicine, Memorial University of Newfoundland and Labrador, St. John's, Newfoundland Rationale: Recent advances in schizophrenia genetics are shedding new light on etiopathogenesis, but issues germane to translation of findings into clinical practice are understudied. There is a longstanding proposal that genetic counselling for multifactorial schizophrenia may be an informative and therapeutic intervention. However, there are no data- driven reports of its desirability or worth. Objectives: Develop, assess interest in, and evaluate efficacy of a genetic counselling intervention for use in a community mental healthcare setting. Methods: We characterized rare copy number variation in a cohort of adults with schizophrenia using million-feature genotyping arrays, and subsequently approached 122 consecutive family members of patients without molecular diagnoses. A pre-post study design with longitudinal follow-up was used to assess the impact of the genetic counselling intervention on perceived recurrence risk and associated anxiety, relevant knowledge, and other pertinent variables. Results: Seventy-eight (63.9%) family members actively expressed an interest in the study, and 64 received genetic counselling during the overall study period. Participants greatly overestimated the risk of familial recurrence at baseline, with a significant reduction in mean qualitative and quantitative risk estimates post-intervention (p<0.0001). These changes were associated with a decrease in concern related to perceptions about recurrence risk for schizophrenia (p=0.0003). Significant and lasting improvements were also observed in knowledge (p<0.0001) and in measures of psychosocial functioning, including stigma (p=0.0047). Levels of blame were unexpectedly low at baseline but on average decreased further post-counselling. Satisfaction, including endorsement of the need for this intervention, was unanimous. Conclusions: This is the first study to investigate the impact of a standardized genetic counselling intervention that took into consideration the psychosocial impact of having a family history of schizophrenia. New m o l e c u l a r g e n e t i c i n f o r m a t i o n a b o u t s c h i z o p h r e n i a w a s successfully incorporated into the content of the intervention, while at the same time efficacy was displayed in the (majority) population of schizophrenia who have no known causal genetic variants. The findings provide preliminary evidence that supports the integration of genetic counselling for families into the general management of schizophrenia. The results may help in preparing for knowledge translation of emerging molecular genetic discoveries and personalized medicine in serious mental illness. Funding: MindCare New Brunswick, CIHR, Vanier CGS, CRC greg.costain@mail.utoronto.ca
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NKX3.1 haploinsufficiency is prognostic for prostate cancer relapse following surgery or image-guided radiotherapy Jennifer A. Locke1,2, Gaetano Zafarana1,2, Adrian S. Ishkanian1,2, Michael Milosevic1,2, John Thoms1,2, Cherry L. Have1, Chad A. Malloff4, Wan L. Lam4, Jeremy A. Squire3, Melania Pintilie1,2, Jenna Sykes2, Varune Rohan Ramnarine1,2, Alice Meng1,2, Omer Ahmed1,2, Igor Jurisica1,2, Theo van der Kwast1,2, and Robert G. Bristow1,2. 1 Departments of Radiation Oncology, Medical Biophysics, Laboratory Medicine and Pathology and Dalla Lana School of Public Health, University of Toronto; 2Ontario Cancer Institute/Princess Margaret Hospital-University Health Network, Toronto; 3Department of Pathology and Oncology, Queen's University, Kingston, Ontario; and 4Department of Cancer Genetics and Developmental Biology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada. Rationale: Despite the use of prostate specific antigen (PSA), Gleason-score, and T-category as prognostic factors, up to 40% of patients with intermediate-risk prostate cancer will fail radical prostatectomy or precision image-guided radiotherapy (IGRT). Additional genetic prognosticators are needed to triage these patients toward intensified combination therapy with novel targeted therapeutics. We tested the role of the NKX3.1 gene as a determinant of treatment outcome given its reported roles in tumor initiating cell (TIC) renewal, the DNA damage response, and cooperation with c-MYC during prostate cancer progression. Methods: Using high-resolution array comparative genomic hybridization (aCGH), we profiled the copy number alterations in TIC genes using tumor DNA from frozen needle biopsies derived from 126 intermediate-risk patients who underwent IGRT. These data were correlated to biochemical relapse-free rate (bRFR) by the Kaplan-Meier method and Cox proportional hazards models. Results: A screen of the aCGH-IGRT data for TIC genes showed frequent copy number alterations for NKX3.1, PSCA, and c-MYC. NKX3.1 haploinsufficiency was associated with increased genomic instability independent of PSA, T-category, and Gleason-score. After adjusting for clinical factors in a multivariate model, NKX3.1 haploinsufficiency was associated with bRFR when tested alone (HR = 3.05, 95% CI: 1.46-6.39, P = 0.0030) or when combined with c-MYC gain (HR = 3.88, 95% CI: 1.78-8.49, P = 0.00067). A similar association was observed for patients following radical prostatectomy with a public aCGH database. NKX3.1 status was associated with positive biopsies post-IGRT and increased clonogen radioresistance in vitro. Conclusions: Our results support the use of genomic predictors, such as NKX3.1 status, in needle biopsies for personalized approaches to prostate cancer management. Funding: This work was supported by the Ontario Institute for Cancer Research through funding provided by the Government of Ontario, the Canada Foundation for Innovation (grant numbers CFI #12301 and CFI #203383), the Ontario Research Fund (grant number GL2-01030), and IBM. Individuals were supported by the Canada Research Chair Program (I. Jurisica), the Comprehensive Research Experience for Medical Student program at the University of Toronto (J.A. Locke) and a Canadian Cancer Society Research Scientist Career Award (R.G. Bristow). jennifer.locke@mail.utoronto.ca
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Activation complex assembly differentially protects coagulation factors Xa, IXa, and VIIa from antithrombin inhibition
Calvin H. Yeh, Hashem Alshurafa, James C. Fredenburgh, Alan R. Stafford, Paul Y. Kim and Jeffrey I. Weitz MD/PhD Program, Michael G. DeGroote School of Medicine Department of Biochemistry and Biomedical Sciences The Thrombosis and Atherosclerosis Research Institute and McMaster University, Hamilton, Canada Rationale: Venous thromboembolic disease, such as deep vein thrombosis (DVT) causing pulmonary embolism, is the source of significant morbidity and mortality. Heparin is used for the treatment of DVT, and although effective, can fail to prevent clot accretion and extension. Previous studies suggest that clots harbour thrombogenicity that is resistant to heparin treatment because active coagulation factors are sequestered and protected from antithrombin (AT) when they are within the clot. Although these studies have shown with non-native proteins that assembly of the prothrombinase complex protects factor (F) Xa from inhibition by the heparin-antithrombin (AT) complex, it is unclear whether FIXa and FVIIa, the enzymes upstream to FXa, also are protected when incorporated into intrinsic or extrinsic tenase, respectively. Methods: To address this, we compared the effects of complex assembly on the heparincatalyzed rates of FXa, FIXa and FVIIa inhibition by AT using native proteins. When prothrombin or FX was present, we used hirudin or tick anticoagulant protein, respectively, to inhibit the activity of the generated enzymes. Results: The heparin-catalyzed rates of FXa and FIXa inhibition by AT were reduced 11.3- and 4.3-fold when the enzymes were incorporated into their respective activation complexes. Although addition of prothrombin produced a further 100-fold decrease in the rate of FXa inhibition, FX addition had no effect on the rate of FIXa inhibition. Tissue factor increased the heparin-catalyzed rate of FVIIa inhibition by AT 13.3-fold; an effect unchanged by FX addition. Conclusion: Our results indicate that like FXa in prothrombinase, FIXa in intrinsic tenase also is protected from inhibition by the heparin-AT complex, albeit to a lesser extent, whereas FVIIa in extrinsic tenase is not. Limiting protection to the common pathway ensures that sufficient FXa is generated to propagate clotting, while protection of FXa at the level of prothrombinase may contribute to the inability of heparin to fully suppress thrombus growth. Funding: CIHR
Contact: Calvin H. Yeh, yehch@mcmaster.ca
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Urinary Device Attachment of Staphylococcus saprophyticus in the Presence of SubMinimal Inhibitory Antimicrobial Concentrations Melissa Huynh, Varunkumar Bathini, Andrew Fuller, Lee W Goneau, Kyle W MacDonald, Hassan Razvi and Peter Cadieux Department of Surgery, Division of Urology, Western University, London, Ontario, Canada Background and Purpose: Staphylococcus saprophyticus is a frequent cause of both uncomplicated and complicated urinary tract infections (UTIs) and is the most prevalent Grampositive uropathogen. It is well established that sub-Minimal Inhibitory Concentrations of antimicrobials (MIC) are capable of modulating the expression of virulence determinants; however, little is known regarding how S. saprophyticus responds under such conditions. Recent work by our group has shown that this organism has an increased adherence to various abiotic surfaces and bladder cells in the presence of sub-MIC levels of several clinically-relevant antibiotics. This sub-MIC exposure also increases S. saprophyticus’ survival when challenged with bactericidal levels of the same agents. Collectively, these phenotypic changes may enhance the ability of this organism to persist within the urinary tract despite antimicrobial intervention. In this study, we investigated the effects of sub-MIC antimicrobial levels on S. saprophyticus attachment to urinary devices. Materials and Methods: S. saprophyticus 15305 was incubated with 1cm segments of latex Foley catheters (Bard Ltd.) or Sof-Flex ureteral stents (Cook Urological) in the presence or absence of ¼ MIC of either ciprofloxacin (CIP), ampicillin (AMP) or gentamicin (GEN) for 1 hour. Device segments were rinsed thoroughly and adherent organisms enumerated via sonication and dilution plating. Results: S. saprophyticus adherence to both devices was significantly increased following treatment with sub-MIC levels all 3 antimicrobial agents. Catheter adherence increased 5.9 fold (p<0.05), 6.1 fold (p<0.05) and 6.2 fold (p<0.05) and stent adherence increased 2.6 fold (p<0.01), 2.6 fold (p<0.05) and 2.7 fold (p<0.01) for CIP, AMP and GEN, respectively. Conclusions: Our results demonstrate that exposure to sub-MIC levels of several clinicallyrelevant antimicrobials increases S. saprophyticus’ surface adherence to urinary devices commonly used in clinical practice. This research highlights the need for clinicians to consider the impact of sub-inhibitory concentrations of antimicrobials on bacterial pathogens when designing treatment strategies for the prevention and management of UTI. mhuynh2013@meds.uwo.ca
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Discrimination in access to primary care on the basis of socioeconomic status Michelle E. Olah*(a), Gregory Gaisano*(a) and Stephen W. Hwang(ab) * These authors contributed equally to this work. (a)Centre for Research on Inner City Health, The Keenan Research Centre at the Li Ka Shing Knowledge Institute, St. Michael’s Hospital and (b)Division of General Internal Medicine, Department of Medicine, University of Toronto. Toronto, Canada.
Rationale: Health care providers may discriminate against people of low socioeconomic status, even in the absence of economic incentives to do so. Objective: To determine if an individual’s socioeconomic status affects the response he or she receives when seeking a primary care physician within a single-payer universal health insurance system. Methods: In this audit study, researchers made unannounced telephone calls to a random sample of family physician and general practitioner offices in Toronto, Ontario. Of the 568 offices selected, 375 offices were successfully contacted and eligible for the study. Using a standardized script, the caller played the role of an individual seeking a primary care physician. The prospective patient’s characteristics were randomized for each physician office to indicate high or low socioeconomic status and the presence or absence of chronic health conditions. The primary outcome measure was the proportion of calls resulting in the offer of an appointment. Results: The proportion of calls resulting in the offer of a primary care appointment was significantly higher for individuals of high vs. low socioeconomic status (22.6% vs.14.3%, p=0.04) and for individuals with vs. without chronic health conditions (23.5% vs. 12.8%, p=0.008). In an adjusted model, high socioeconomic status was associated with an odds ratio of 1.78 (95% CI, 1.02 – 3.08) and chronic health conditions were associated with an odds ratio of 2.10 (95% CI, 1.20 – 3.68) for the offer of an appointment. Socioeconomic status and chronic health conditions had independent effects on the likelihood of obtaining an appointment. Conclusion: Within a universal health insurance system in which physician reimbursement is the same for all patients, individuals of high socioeconomic status receive preferential access to primary care, regardless of whether or not they have chronic health conditions. This finding is consistent with discrimination by physicians’ offices on the basis of socioeconomic status.
michelle.olah@mail.utoronto.ca
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Longitudinal assessment of bone mass and strength in perinatally HIV-infected youth Jackson Chua, Ariane Alimentia, Lindsay Nettlefoldb, Evelyn Maana, Hélène Côtéc, Heather McKayd, Heather Macdonaldb and the CIHR Team Grant in HIV Therapy and Aging (CARMA) a
BC Women’s Hospital and Health Centre, Oak Tree Clinic, Vancouver, Canada, bUniversity of British Columbia, Orthopedics, Vancouver, Canada, cWomen’s Health Research Institute, Pathology and Laboratory Medicine, Vancouver, Canada, dCentre for Hip Health and Mobility, Vancouver, Canada Rationale: Highly active antiretroviral therapy (HAART) has dramatically improved the life expectancy of HIV-infected individuals such that perinatally HIV-infected (HIV+) children are now growing well into adolescence and adulthood. However, concerns arise that both HIV infection and prolonged HAART use could compromise healthy bone development. Objectives: We aimed to determine if bone mass, structure and strength are compromised HIV+ youth. Methods: At baseline we assessed bone mineral content (BMC) at the proximal femur (PF), lumbar spine (LS) and total body (TB), and TB lean mass using dual energy X-ray absorptiometry (DXA) in HIV+ youth (n=31; 12 girls; 8-18 yrs.) We used peripheral quantitative computed tomography (pQCT) to assess muscle cross-sectional area (MCSA) and bone parameters [cortical density (BMD), area and thickness, strength strain index (estimated bone strength)] at the mid-tibia shaft. We measured height and weight using standard methods. Thirty and 18 participants returned for the 12- and 24-month visits, respectively. We compared HIV+ measures to age- and sex-specific z-scores derived from the University of British Columbia Healthy Bones III study cohort (n=751; 386 girls) using multivariable regression adjusting for height and muscle mass (lean mass or MCSA) z-scores. Results: Four participants were ART-naïve while 27 took ART (median 75 months). Height, weight and MCSA z-scores were reduced in HIV+ youth (-0.71 to -0.30, p <0.05). Adjusted BMC z-scores at the PF< LS and TB were reduced HIV+ youth (-0.17 to 0.61, p<0.001) at baseline but did not decrease further over 24 months. Cortical BMD, thickness and area z-scores were higher (o.17 to 0.61, p<0.01) in HIV+ youth at baseline. Cortical BMD z-scores increased at 24 months (+0.77, p=0.003) in HIV+ youth who completed all 3 visits. Bone strength z-scores were not different from controls at baseline and did not change over time. Conclusions: We examined longitudinal changes in bone structure and strength in HIV+ youth. Deficits in BMC stabilized over 2 yrs whereas size-adjusted bone structure and strength were not compromised. Further study will determine possible causes of increased cortical BMD and its impact on bone strength of HIV+ youth. Funding: CIHR, CANFAR Chu88@interchange.ubc.ca
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Life-history Chronicle for a Patient with the Recently Described Chromosome 4q21 Microdeletion Syndrome Erica Tsang1, Rosemarie Rupps1, Barbara McGillivray1, Patrice Eydoux1, 3, Marco Marra1, 4, Laura Arbour1, Sylvie Langlois1, Jan M Friedman1, 2, Farah R Zahir1,2,4 1
Department of Medical Genetics, University of British Columbia, Vancouver, Canada Child and Family Research Institute, Vancouver, Canada 3 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada 4 BC Cancer Agency Genome Sciences Centre, Vancouver, Canada 2
Rationale: A novel 4q21 microdeletion syndrome with severe intellectual disability (ID), lack of speech, hypotonia, significant growth restriction, and distinctive facial features was reported recently by Bonnet et al. Overlapping the deleted regions of thirteen patients delineates a critical region, with RASGEF1B and PRKG2 emerging as candidate genes. Objectives: Previous case reports of this new syndrome have not detailed the prognosis or natural history of these patients. This is the first detailed clinical report and photographic life history of a patient with the emerging 4q21 microdeletion syndrome. Methods: We provide a chronological report of our patient’s clinical development. In addition, we compare our patient’s clinical features with that of other reported cases and investigate the involvement of both reported candidate genes in our patient using array genomic hybridization (AGH) and quantitative PCR (qPCR). Results: Our patient demonstrated severe ID, no speech, and growth failure. Our patient also had the characteristic facies of this emerging syndrome including frontal bossing, a broad forehead, hypertelorism, a short philtrum and downturned corners of the mouth. She exhibited sensorineural hearing loss and self-aggression, features seen in only two of the other cases. Array genomic hybridization (AGH) using the Affymetrix GeneChip® Mapping 100K Assay on the child and both parents revealed a de novo heterozygous deletion on chromosome 4q21.21q22.1 of ~11Mb between genomic co-ordinates 82,008,593 and 93,076,278bp (NCBI build 36.1). Subsequent AGH using the higher resolution 500K Assay narrowed down the affected region to be ~9Mb between genomic co-ordinates 82,429,949 and 91,434,337bp (NCBI build 36.1).The smaller ~9Mb deleted region identified by the higher resolution 500K AGH includes RASGEF1B but not PRKG2. However, qPCR testing demonstrated loss of PRKG2, thus confirming the deletion of both candidate genes in this syndrome. Conclusions: This natural history report is the first chronicled clinical and photographic description of a patient with the newly described 4q21 microdeletion syndrome. Using higher resolution AGH and qPCR, we define the deletion of two candidate genes for the phenotype characteristic of this syndrome and specific to our patient. This report is expected to provide guidance for anticipatory management of patients with this emerging 4q21 microdeletion syndrome. Funding: UBC Faculty of Medicine, Child and Family Research Institute ertsang@interchange.ubc.ca
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Recognizing uncertainty in your knowledge: The impact of response options presentation on self-monitoring accuracy. Cynthia Minab, Kevin W. Evabc a
MD/PhD Program, bCentre for Health Education Scholarship, cDepartment of Medicine, Faculty of Medicine, The University of British Columbia Rationale: To optimize patient care, physicians are expected to have a â&#x20AC;&#x153;moment-to-momentâ&#x20AC;? awareness of the likelihood of being correct. This type of self-assessment has been termed selfmonitoring and occurs when physicians recognize uncertainty in their clinical decision making and stop to seek additional information. It has been argued that self-monitoring is crucial in clinical practice because it carries the most immediate impact on patient safety. A series of studies using short answer questions have demonstrated a relationship between self-monitoring and response accuracy. The current study used multiple-choice questions to determine if eliminating the need to generate a response would impact the accuracy of self-monitoring. Methods: 40 participants were randomly allocated to answer 60 multiple-choice questions in which response options were presented before or after participants had to decide whether or not to answer. For each question, they were asked to answer if they were confident in their response and to pass otherwise. All deferred questions were then required to be answered when presented a second time. Measures of self-monitoring included accuracy of attempted versus deferred questions and the speed at which such decisions were made. Results: Respondents showed indications of accurate self-monitoring in both experimental conditions. When response options were provided prior to deciding to answer or pass respondents answered attempted questions with 52.4% accuracy and passed questions with 34.1% accuracy. This difference was equivalent when the decision to answer or pass was made prior to seeing the response questions (52.1% vs 27.7%, respectively). For both conditions, respondents had a faster response time (both answered and pass questions) when question were answered correctly compared to when questions were answered incorrectly. Conclusions: The findings indicate that the accuracy of self-monitoring was not influenced by the opportunity to review possible responses prior to judging the likelihood of being able to provide the correct answer. This suggests robust self-monitoring that is independent of the need to search oneâ&#x20AC;&#x2122;s knowledge base. Furthermore, the results suggest a relationship between response time the accuracy of responses. Further explorations should be undertaken to determine whether self-monitoring is a teachable skill that can aid in clinical decision making.
mincs2@interchange.ubc.ca
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The Negative Predictive Value of US-Guided 14-Gauge Core Needle Biopsy of Breast Masses Charlie Zhang, Darrell R. Lewis, BASc, Paola Nasute, MD, Malcolm Hayes, MD, FRCPC, Linda J. Warren, MD, FRCPC, Paula B. Gordon, MD, FRCPC Rationale: A number of biopsy techniques are available for the diagnosis and management of breast lesions, including core needle biopsy (CNB), fine needle aspiration biopsy, or open surgical biopsy. With the widespread implementation of screening mammography programs and subsequently the increased number of biopsies performed, continued assessment for safety and accuracy of all biopsy techniques is of great importance. Objective: To determine the negative predictive value of sonographically-guided 14-gauge core needle biopsy of breast masses, with detailed analysis of any false negative cases. Methods: All sonographically-guided 14-gauge core needle biopsies (669 cases) from March 2005 through April 2011 which had benign pathologic findings at our practice were reviewed. Lesions with malignant pathology were not evaluated as false positives are not considered to occur with core needle biopsies in clinical practice. We defined true negatives as lesions which had benign pathology on core biopsy and had either benign pathology upon surgical excision or at least 2 years of stable imaging and/or clinical follow-up. False negatives were defined as lesions which had benign pathology on core biopsy but malignant histology upon surgical excision. Results: Of the 339 ultrasonographically visible breast lesions for which follow-up was available, 117 were confirmed to be benign via surgical excision, and 220 were stable on â&#x2030;Ľ 2 years of imaging or clinical follow-up (mean follow-up time 33.1 months, range 24-64 months). The negative predictive value of sonographically -guided 14-gauge core needle biopsy was determined to be 99.4% (337 of 339 cases). There were 2 false negative cases, for a false negative rate of 0.1% (2 of 1388 cases of cancer). In both of these cases, there was no delay in diagnosis because the radiologist determined that there was discordance between imaging and core biopsy pathology, and recommended surgical excision despite the benign core biopsy pathology. Conclusions: Sonographically -guided 14-gauge core needle biopsy provides a high negative predictive value in assessing breast lesions. Radiologic/pathologic correlation should be performed to avoid delay in the diagnosis of carcinoma. For lesions with benign pathology on core needle biopsy and radiologic-pathologic correlation, routine clinical and imaging follow-up is sufficient. charliezhang825@hotmail.com
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Assessing the Reliability and Quality of Online Uterine Fibroid Embolization Resources Jatin Kaickera, Ke Wua, Sriharsha Athreyaa a Department of Medical Imaging, Michael G. Degroote School of Medicine, 1200 Main St West, MDCL 3111, Hamilton, Canada Rationale: Millions of patients undergo radiological examinations every year, using the internet to alleviate anxiety of medical procedures. The medium is effective at disseminating information about radiological tests, study preparation, benefits and risks of procedures and recovery time. Similarly, a recent survey has shown that 97% of radiologists use the internet for education. In 2009, Statistics Canada found that 80% of Canadians over the age of 16 use the Internet for personal reasons with searching for health information reported by 70% of home users. Despite widespread use, one concern with online health information is quality and reliability. Objective: To examine the best internet resources about Uterine Fibroid Embolization pertinent to medical trainees, radiologists, gynecologists, family physicians and patients. Materials and Methods: The terms ‘Uterine Fibroid Embolization’, ‘Uterine Fibroid Embolisation’ and ‘Uterine Artery Embolization’ were entered into Google, Yahoo, Bing search engines, with the top 20 hits assessed. The hits were categorized as organizational or nonorganizational. Additionally, 23 radiological and obstetrical organizations were assessed. The DISCERN instrument and JAMA benchmarks (authorship, attribution, currency, disclosure) were used to critically assess the information. The scope, strength, weaknesses and unique features were highlighted for the top 5 organizational and non-organizational websites. Results: A total of 203 websites were reviewed, 23 removed in accordance with the exclusion criteria and 146 duplicate websites, for a total of 34 unique sites. It was found that 35%(12/34 websites) were organizational (family medicine, radiology, obstetrics/gynecology) and 65%(22/34 websites) were non-organizational (teaching or patient resources). The overall mean DISCERN score was 49.60(10.70). Two tailed, unpaired T test demonstrated no statistically significant difference between organizational and non-organizational websites (p=0.101). JAMA benchmarks revealed 44%(15/34 websites) with authorship, 71%(24/34 websites) with attribution, 68%(23/34 websites) with disclosure and 47%(16/34 websites) with currency. Conclusion: The overall quality of websites for UFE is moderate, with important but not serious shortcomings. The best websites provided relevant information about the procedure, benefits/risks and were interactive. DISCERN scores were compromised by sites failing to provide resources for shared decision making, additional support and discussing consequence of no treatment. JAMA benchmarks revealed lack of authorship and currency. Funding: None jatin.kaicker@medportal.ca
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TCOM Study - Transcutaneous oximetry as a prediction tool for chronic wound and amputation healing outcomes Kyle A Arsenault,a Anthony Adili,b Abdullah Al-Otaibi,b Mohit Bhandari,b,c Hasib Hanif,b PJ Devereaux,c,d,e Jeremy R Harris,f Joey McDonald,b Sadeesh Srinathan,g Kristian Thorlund,c Jacques G Tittley,b Richard P Whitlockb,e a Michael G. DeGroote School of Medicine, bDepartment of Surgery, cDepartment of Clinical Epidemiology and Biostatistics, dDepartment of Medicine, McMaster University; Hamilton, Ontario, Canada e Population Health Research Institute, McMaster University/Hamilton Health Sciences; Hamilton, Ontario, Canada f Department of Surgery, University of Western Ontario; London, Ontario, Canada g Department of Surgery, University of Manitoba; Winnipeg, Manitoba, Canada Background: Researchers have proposed transcutaneous oximetry (TcPO2) as a method to predict non-healing of chronic wounds and the occurrence of healing complications for lower limb amputations. There is, however, uncertainty regarding the optimal threshold value for TcPO2 and its ability to independently predict these outcomes. Methods: We undertook two systematic reviews and meta-analyses, searching five major medical databases, relevant review articles and reference lists. One review addressed the ability of TcPO2 to predict non-healing of chronic wounds while the other focused on healing complications after lower limb amputation. We selected all studies that evaluated TcPO2 for its ability to predict these outcomes and conducted data abstraction independently and in duplicate. Results were pooled using a random-effects model. Results: In the first review, four studies, enrolling 901 patients with 910 lower extremity chronic wounds demonstrated that a peri-wound TcPO2 level below a cutoff of 20mmHg or 30mmHg was an independent predictor of chronic wound healing complications (OR 3.21, 95% CI 1.079.69). In the second review, fourteen prospective cohort studies, enrolling 626 patients with 658 amputations, reported data that allowed for the calculation of an unadjusted relative risk of amputation revision associated with a TcPO2 level below cutoffs of 10mmHg (1.80; 95% CI 1.19-2.72), 20mmHg (1.75; 95% CI 1.27-2.40), 30mmHg (1.41; 95% CI 1.22-1.62) and 40mmHg (1.24; 95% CI 1.13-1.35). There were an insufficient number of studies reporting risk-adjusted results to perform a pooled analysis. Conclusions: The first review suggests that TcPO2 measurements have independent prognostic value in the assessment of chronic wounds. The second review revealed significant associations of low TcPO2 levels with amputation healing complications, but highlighted the need for a large, sufficiently powered study to determine this toolâ&#x20AC;&#x2122;s independent predictive ability and an appropriate threshold value. We recently began a multicentre observational study that aims to evaluate TcPO2 and transcutaneous CO2 in this context. The results of this and future studies may significantly assist in the surgical and clinical care of this patient population by providing clinicians with a means to assess appropriate lower limb amputation levels. This will hopefully prevent inappropriate below-knee amputations and thus subsequent amputation revisions. Funding: Beamish Family Foundation Chair in Vascular Surgery, McMaster Surgical Associates, Canadian Institutes of Health Research Contact: kyle.arsenault@medportal.ca
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Development of a Symptom Specific Quality of Life Scale in Emergency Department Patients with Recent Onset Atrial Fibrillation/Flutter Jeff H. Yoo, Debra Eagles, Catherine M. Clement, Ian G. Stiell Department of Emergency Medicine, University of Ottawa, Ottawa, ON Canada Rationale: Recent onset atrial fibrillation/flutter (RAFF) causes significant morbidity and is the most common arrhythmia requiring management in the Emergency Department (ED). The incidence of RAFF is increasing due to an aging population and growing prevalence of chronic heart disease. Although the current treatment strategy for RAFF remains focused on improving or preserving quality of life (QoL), there are no validated QoL scales to quantify the severity of RAFF in the ED. The purpose of this study was to develop a symptom specific QoL measure as both a clinical and research tool for patients presenting to the ED with RAFF. Methods: We conducted a prospective cohort study in the EDs of 2 tertiary-care hospitals and enrolled adult patients presenting with palpitations. We adapted the ED RAFF Scale from the Canadian Cardiovascular Society Severity of AF Scale and created a 7 symptom questionnaire. Patients were asked to rate the severity of each symptom on a scale from 0 to 10, with 0 being none and 10 being worst possible. Patients were randomized to self or clinician administration of the ED RAFF Scale and the validated, generic SF-8 QoL Scale at time 0 before crossing over at time 15. We compared results with students t-test. Results: We enrolled 68 patients: male 58.8%, mean age 61.2 years, mean duration symptoms 11.7 hours. Individual items on the ED RAFF Scale had these means (SD): palpitations 2.8 (2.7), lightheadedness 2.0 (2.4), chest pain 1.8 (2.3), shortness of breath 2.2 (2.7), shortness of breath with exertion 6.3 (2.8), fatigue 3.9 (3.1), fatigue with exertions 6.7 (2.8). There was no difference between scores for self vs clinician completion (all p-values >0.40). High values (710) for all ED RAFF Scale questions showed good discrimination for both the physical and mental composite scores of the SF-8 (all p-values <0.05). Conclusions: The ED RAFF Scale is the first disease specific QoL measure for use in ED patients with RAFF. It showed good dispersion and stability, whether self- or clinicianadministered, and strong discrimination for the generic QoL SF-8. This scale will be used to judge the success of ED management strategies for RAFF patients. Funding: Mach-Gaensslen Research Scholarship, uOttawa Faculty of Medicine Research Scholarship, UROP Grant for Research Contact: jeffhyoo@gmail.com
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Identification of Vulnerable Aortic Plaque Using Conventional FDG-PET Myocardial Viability Studies Yingwei Liu BSc, Nina Ghosh MD, Girish Dwivedi MBBB PhD, Iftikar Ali MD, R. Glenn Wells PhD, Karen Vanderwerf MSc, Benjamin Chow MD, Robert DeKemp PhD, Jean DaSilva MD, Rob Beanlands MD, Terrence Ruddy MD University of Ottawa Heart Institute Rationale: Atherosclerotic plaque rupture carries a high burden of morbidity and mortality. Unfortunately, the plaques most vulnerable to rupture are highly metabolically active but only mildly stenotic and thus undetected using conventional imaging based on anatomy. However, 18-fluorodeoxyglucose (18-FDG) position emission tomography/computed tomography (PET/CT) relies on increased FDG uptake by inflammatory cells to visualize vulnerable plaque. Objective: We evaluated the feasibility of using clinically-indicated FDG-PET/CT myocardial viability scans to detect vulnerable plaque. Methods: Clinically indicated FDG-PET/CT myocardial viability scans from 103 patients were retrospectively analyzed for FDG uptake in the proximal ascending and descending thoracic aorta. The inferior vena cava blood pool FDG uptake was used as the background uptake. Hotspots were graded based on peak target-to-background ratio (TBR): Grade 0 – < 1, Grade 1 – 1.01-1.49, Grade 2 –1.5-1.99, and Grade 3 – > 2. Patient characteristics including age, gender, and medication use, as well as aortic and coronary calcification were also assessed. Results: The patients were on average 64.7 ± 9.4 years old, male (86/103), and on lipidlowering agents (87/103). Of the 103 studies, 66 (64.1%) demonstrated focal FDG uptake in the aortic wall with 20 (19.4%) having a Grade 1 plaque as the highest uptake, 35 (34.0%) Grade 2, and 11 (10.7%) Grade 3. Intraclass correlation coefficient (ICC) conveyed excellent interrater reproducibility (mean ICC = 0.89, 95% confidence interval = 0.84-0.93). There was no difference in age between patients with and without focal FDG uptake. Out of 103 studies, 59 (57.3%) and 92 (89.3%) demonstrated aortic and coronary calcification respectively. Patients with aortic (p < 0.0001) and coronary calcification (p < 0.05) were significantly older than those without calcification. Conclusion: This is the first study to demonstrate the feasibility of assessing vulnerable plaque using clinically-indicated myocardial viability scans. We found a high rate of focal FDG uptake in the aortic wall, likely reflecting a high prevalence of active atherosclerotic disease. However, the rate of very positive uptake in this population was low, possibly reflecting stabilization of atherosclerotic plaques by aggressive secondary risk factor modification including statin and ACE inhibitor use.
yingwei.liu@uottawa.ca
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Valproic Acid and Fludarabine induces a synergistic response against Chronic Lymphocytic Leukaemia Ju-Yoon Yoona,c, David Szwajcerb, Ganchimeg Ishdorjc, Pat Benjaminsonb, James Johnstonb,c, Spencer Gibsona,c. a. Department of Biochemistry and Medical Genetics, Faculty of Medicine, University Of Manitoba, Winnipeg, MB, Canada b. CancerCare Manitoba, Winnipeg, MB, Canada c. Manitoba Institute of Cell Biology, Winnipeg, MB, Canada Rationale: Chronic Lymphocytic Leukaemia (CLL) is the most common haematological malignancy in the western world. Patients with relapsed CLL are difficult to manage, and there is no consensus on the treatment regimen. We hypothesize that the addition of Valproic Acid (VPA), an inhibitor of histone deacetylases (HDACs), can improve fludarabine-based therapy. Methods: The pharmacodynamic relationship between VPA and fludarabine was examined in vitro in human leukaemic cell lines and primary CLL cells. The impact of the combination on the levels and post-translational modifications of various proteins were examined by immunoblotting. Results: The VPA-Fludarabine induced a synergistic response in human leukaemic cell lines and primary CLL cells in vitro. The combination induced a higher level of histone acetylation, and this was associated with increase in acetylation by VPA and upregulation of histones by fludarabine. The combination also downregulated class I HDACs, i.e. HDAC1 and HDAC3. VPA weakly downregulated the Ataxia Telangiectasia Mutated (ATM) kinase, a pivotal player in the DNA damage repair pathway, while fludarabine induced cleavage of ATM. Combined, VPA- Fludarabine induced stronger downregulation of ATM, and this was associated with higher levels of ÎłH2A.X and caspase-9 activation. The VPAFludarabine combination also suppressed a number of pro-survival signalling pathway targets, including the Akt kinase, resulting in strong downregulation of both phospho- and total Akt levels. The VPA-Fludarabine also resulted in enhanced downregulation of antiapoptotic proteins, including B-cell lymphoma-extra large (Bcl- xL), Myeloid Cell Leukemia1 (Mcl-1) and X-linked Inhibitor of Apoptosis Protein (XIAP). Different protein degradation pathways appear to be engaged in response to VPA-Fludarabine, and different proteins could be stabilized by blocking either the caspase, lysosome and/or the 26S proteasome pathway. Pre-treatment with inhibitors of the lysosomal pathway, namely chloroquine or NH4Cl, suppressed the level of apoptosis induced by VPAFludarabine, suggesting that the endo-lysosomal pathway is of particular importance. Conclusions: The VPA-Fludarabine targets multiple proteins for degradation, resulting in enhanced apoptotic pathways and suppressed survival pathways. Combined, the VPA- Fludarabine combination resulted in synergistic induction of apoptosis and represents a promising combination therapy for patients with CLL. Contact: umyoonj@cc.umanitoba.ca
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Characterization of Gestational Insulin Resistance in Normal Healthy Sprague Dawley Rats Nicole Lovat, Dallas Legare, and Wayne Lautt Department of Pharmacology & Therapeutics, University of Manitoba, Winnipeg, Canada. Rationale: Gestational diabetes is the most prevalent complication in pregnancy. It represents a high risk for birth complications, and multiple deleterious metabolic effects in mother and child. Healthy gestation is accompanied by insulin resistance. In order to develop an appropriate gestational diabetes model for further study, insulin resistance in healthy pregnancy must be characterized in the fed state in terms of direct (insulin-dependent) vs. indirect (Hepatic Insulin Sensitizing Substance (HISS)-dependent) insulin action. In the fed state, there is a dramatic Meal-induced Insulin Sensitization (MIS) mediated by HISS resulting in increased glucose uptake in skeletal muscle. Objectives: Determine what proportion of gestational insulin resistance in the fed animal is due to direct vs. indirect (HISS-dependent) insulin action. Methods: Animals were paired at 4 weeks of age with one bred 8 weeks later. After 13-18 days, the acute study was performed: blood samples were taken for plasma insulin and triglycerides, the Rapid Insulin Sensitivity Test (RIST) index measured, and fat pads weighed as a surrogate of adiposity. The RIST measures the dynamic response to insulin in the fed state. The RIST index is the administered glucose per kg body weight required to maintain a euglycemic state following an insulin bolus. Results: Total glucose uptake was (169.8 ± 17.2) mg/kg bw in virgins (N=7) vs. (111.8 ± 8.8) mg/kg bw in pregnancy (N=7). Insulin-dependent glucose uptake was (95.2 ± 9.8) mg/kg bw in virgins vs. (62.2 ± 7.8) mg/kg bw in pregnancy. HISS-dependent glucose uptake was (80.7 ± 8.8) mg/kg bw in virgins vs. (49.6 ± 8.6) mg/kg bw in pregnancy. Virginal insulin plasma concentration was (1.42 ± 0.10) ug/L vs. (2.37 ± 0.35) ug/L in pregnancy. Virginal triglyceride plasma concentration was (7.35 ± 0.64) mg/dL vs. (27.17 ± 7.63) mg/dL in pregnancy. Virgin fat pad mass was (16.8 ± 1.4) g vs. (23.3 ± 2.3) g in pregnancy. Conclusions: Insulin resistance marks healthy pregnancy attributable to both HISS-dependent and direct insulin dependent insulin resistance with resultant hyperinsulinemia. Hyperinsulinemia in this context alters metabolic balance to favor glucose storage as fat, contributing to hypertriglyceridemia and adiposity. Funding: SciMar, CIHR, Vanier CGS
nickielovat@gmail.com
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Feasibility Study of Microwave Tomography for In Vivo Diagnostic Characterization of Tissue in Human Disease Cameron Kayea and Joe LoVetria a Department of Electrical and Computer Engineering, University of Manitoba, Winnipeg, MB, Canada Rationale: Microwave tomography (MWT) is a promising low-cost, portable alternative or complementary biomedical diagnostic technique for conventional soft-tissue imaging modalities. MWT uses microwave scattering measurements in a safe, non-ionizing frequency range of a few hundred megahertz up to a few gigahertz to quantitatively reconstruct the bulk electrical properties, i.e., the permittivity and effective conductivity, of the tissue being imaged without the use of a contrast agent. These frequency-dependent properties are not only unique to different tissue types in the human body but also vary with the tissue's physiological state, offering a new quantifiable factor in the diagnosis and investigation of pathological conditions; human breast imaging applications of MWT are well established and a number of promising pilot studies have already been completed. Methods: A comprehensive feasibility study of the technology's potential use in relatively unexplored areas of diagnostic medicine will be carried out, reviewing publications focusing on many anatomical regions of the human body and detectable changes in their bulk electrical properties attributed to physiological distress or abnormality. Results: These studies will include those related to ischemic changes due to reduced tissue blood perfusion in extremities, simulating acute conditions such as compartment syndrome. Similarly, MWT may also present an effective supplement to current neuroimaging modalities for acute and chronic assessment of perfusion-related brain injuries such as ischemic or hemorrhagic stroke. As observable increases in tissue permittivity have shown to be strongly correlated with blood supply and ultimately water content, the metabolic changes in tissues exhibiting malignancy-associated angiogenesis, applicable to a wide range of cancers, have been measured in breast, lung, and liver tumours. Additional thoracic imaging applications, such as real-time cardiac monitoring, have been simulated and attempted on ex vivo excised samples. MWT may also have a role in rheumatological disorders, as preliminary research has shown that synovial fluid extracted from actively arthritic joints has a significantly higher permittivity than healthy samples. Conclusions: This ongoing study seeks to answer questions relating to the benefit of adding MWT, as a stand-alone modality or complementary imaging system, to supplement current gold standard diagnostic criteria for any of the investigated diseases or disorders. Funding: CIHR, NSERC. umkayecj@cc.umanitoba.ca
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Islet amyloid polypeptide: a trigger for Toll-like receptor 2 in type 2 diabetes and islet transplantation? Clara Westwell-Ropera, Jan A. Ehsesb and C. Bruce Verchereab a
Department of Pathology and Laboratory Medicine, bDepartment of Surgery, University of British Columbia. Vancouver, Canada.
Rationale: Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide co-secreted with insulin by pancreatic beta cells. Aggregation of IAPP to form amyloid fibrils contributes to beta cell dysfunction in patients with type 2 diabetes and in islet transplant recipients. IAPP aggregates share a common cross beta-sheet structure with other amyloids known to induce a potent proinflammatory response via activation of the membrane-bound pattern recognition receptor Tolllike receptor 2 (TLR2). Objective: To determine the role of TLR2 in IAPP-induced islet inflammation. Methods: HEK293 cells transfected with human TLR2, 3, 4, 7, or 9 were treated with human IAPP or non-amyloidogenic rodent IAPP and evaluated for NF-κB reporter gene activity. Bone marrow-derived macrophages (BMDMs) and isolated islets from wild-type and TLR2-deficient mice were treated with IAPP and assayed for pro-inflammatory cytokine expression by qRTPCR and ELISA. The contribution of resident macrophages to IAPP-induced islet gene expression was assessed by depleting C57BL/6 mouse islets of phagocytes using clodronatecontaining liposomes. Results: Human but not rodent IAPP induced expression of the pro-inflammatory cytokines IL1β (4.5±1.2 fold untreated; p<0.05), TNF-α (10.3±0.7; p<0.001), IL-6 (117±56; p<0.001), and CCL2 (10.2±0.6; p<0.001) in isolated islets after 4 h. Depletion of islet macrophages significantly reduced expression of both TLR2 and pro-inflammatory cytokines compared to treatment with empty liposomes, suggesting that macrophages are the major source of these mediators. Human IAPP induced NF-κB activation in TLR2-overexpressing HEK293 cells compared to the parental cell line (190±6 vs. 1.0±0.4 absorbance units; p<0.001). This effect was blocked in cells treated with anti-TLR2 or anti-TLR6 neutralizing antibody, suggesting that IAPP activates a TLR2/6 heterodimer. Upregulation of IL-1β, TNF-α, IL-6, and CCL2 in BMDMs and islets treated with IAPP was significantly attenuated in cells from TLR2-deficient mice, suggesting that IAPP-induced TLR2 signalling contributes to pro-inflammatory cytokine release by islet macrophages. Conclusions: These data suggest that islet macrophages are the major source of IAPPinduced pro-inflammatory cytokines, and that diverse amyloidogenic peptides – including IAPP – act as endogenous ligands for TLR2. Thus, manipulation of macrophage activation or blockade of TLR2 signalling may limit IAPP-induced islet inflammation and beta cell dysfunction in type 2 diabetes and islet transplantation. Funding: CIHR Vanier Canada Graduate Scholarship; Canadian Diabetes Association cwestwell@interchange.ubc.ca
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Lightning Oral SESSION 4
Procurity Lecture Theatre, Apotex Building Main Floor, Room 164 15:30 â&#x20AC;&#x201C; 17:00 Wednesday, June 13th, 2012
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Phenotypic and genotypic evaluation of two kindreds affected with hereditary xerocytosis Brett L. Houstona, Teresa Zelinskib, Donald S. Houstonc, Sara J. Israelsd, Gail Coghlane, Bernie N. Chodirkerb, Patrick G. Gallagherf and Ryan Zarychanskic,g aUniversity of Manitoba, Winnipeg, MB, Canada; bDepartments of Biochemistry & Medical Genetics and Pediatrics & Child Health, University of Manitoba, Winnipeg, MB, Canada; cDepartment of Internal Medicine, Section of Hematology & Medical Oncology, CancerCare Manitoba, Winnipeg, MB, Canada; dDepartment of Pediatrics and Child Health and the Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, MB, Canada; eDepartment of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; fPediatrics and Genetics, Yale University School of Medicine, New Haven, CT; gDepartment of Community Health Sciences, University of Manitoba Rationale: The hereditary stomatocytoses are a rare, diverse group of clinical conditions associated with chronic red blood cell hemolysis and increased erythrocyte membrane permeability to monovalent cations. The most common form of hereditary stomatocytosis is hereditary xerocytosis (HX, OMIM: 194380). We studied two genetically unrelated kindreds with HX, one from Western Canada, and the other from upstate New York. Although the causative gene is unknown, previous studies have mapped the HX locus to 16q23 â&#x20AC;&#x201C; 16qter. Objectives: To systematically characterize the disease phenotype and identify the gene and causative mutation responsible for hereditary xerocytosis. Methods: A pedigree was constructed, a focused history was taken and the presence of splenomegaly was assessed by physical examination. Laboratory analysis included both biochemical and hematological parameters. DNA linkage studies and exome sequencing were used to identify novel gene mutations in two multigenerational HX kindreds. Results: The mode of inheritance was autosomal dominant. Affected family members were found to have well-compensated hemolysis, associated with an elevated MCHC, decreased osmotic fragility, decreased haptoglobin, and increased indirect hyperbilirubinemia. Cholelithiasis and progressive iron loading were common, despite normal hemoglobin levels. Using DNA linkage analysis, we confirmed the localization of the disease phenotype to chromosome 16q, and we refined the candidate region to 16q24.2 â&#x20AC;&#x201C; 16qter, a 2.4 million base pair interval containing 51 known or predicted genes. Exome sequencing identified mutations affecting PIEZO1 (encoded by FAM38A gene) in both HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and co-segregation with the disease phenotype in all affected individuals. Conclusions: In this family study, elevated percent reticulocyte counts were used to characterize the presence of a well compensated, autosomal dominant hemolytic process associated with an elevated MCHC and decreased osmotic fragility. Clinically this condition is associated with gallstones and progressive iron loading. Mutations in PIEZO1, encoded by the gene FAM38A, are responsible for hereditary xerocytosis. These findings, the first report of a mutation in a mammalian mechanosensory transduction channel associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte homeostasis. umhoustb@cc.umanitoba.ca 89
Life-history Chronicle for a Patient with the Recently Described Chromosome 4q21 Microdeletion Syndrome Erica Tsang1, Rosemarie Rupps1, Barbara McGillivray1, Patrice Eydoux1, 3, Marco Marra1, 4, Laura Arbour1, Sylvie Langlois1, Jan M Friedman1, 2, Farah R Zahir1,2,4 1
Department of Medical Genetics, University of British Columbia, Vancouver, Canada Child and Family Research Institute, Vancouver, Canada 3 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada 4 BC Cancer Agency Genome Sciences Centre, Vancouver, Canada 2
Rationale: A novel 4q21 microdeletion syndrome with severe intellectual disability (ID), lack of speech, hypotonia, significant growth restriction, and distinctive facial features was reported recently by Bonnet et al. Overlapping the deleted regions of thirteen patients delineates a critical region, with RASGEF1B and PRKG2 emerging as candidate genes. Objectives: Previous case reports of this new syndrome have not detailed the prognosis or natural history of these patients. This is the first detailed clinical report and photographic life history of a patient with the emerging 4q21 microdeletion syndrome. Methods: We provide a chronological report of our patient’s clinical development. In addition, we compare our patient’s clinical features with that of other reported cases and investigate the involvement of both reported candidate genes in our patient using array genomic hybridization (AGH) and quantitative PCR (qPCR). Results: Our patient demonstrated severe ID, no speech, and growth failure. Our patient also had the characteristic facies of this emerging syndrome including frontal bossing, a broad forehead, hypertelorism, a short philtrum and downturned corners of the mouth. She exhibited sensorineural hearing loss and self-aggression, features seen in only two of the other cases. Array genomic hybridization (AGH) using the Affymetrix GeneChip® Mapping 100K Assay on the child and both parents revealed a de novo heterozygous deletion on chromosome 4q21.21q22.1 of ~11Mb between genomic co-ordinates 82,008,593 and 93,076,278bp (NCBI build 36.1). Subsequent AGH using the higher resolution 500K Assay narrowed down the affected region to be ~9Mb between genomic co-ordinates 82,429,949 and 91,434,337bp (NCBI build 36.1).The smaller ~9Mb deleted region identified by the higher resolution 500K AGH includes RASGEF1B but not PRKG2. However, qPCR testing demonstrated loss of PRKG2, thus confirming the deletion of both candidate genes in this syndrome. Conclusions: This natural history report is the first chronicled clinical and photographic description of a patient with the newly described 4q21 microdeletion syndrome. Using higher resolution AGH and qPCR, we define the deletion of two candidate genes for the phenotype characteristic of this syndrome and specific to our patient. This report is expected to provide guidance for anticipatory management of patients with this emerging 4q21 microdeletion syndrome. Funding: UBC Faculty of Medicine, Child and Family Research Institute ertsang@interchange.ubc.ca
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Dorsal premotor cortex is involved in switching motor plans. Elsa Tremblaya, Alexandre Pastor-Berniera and Paul Ciseka a
Department of Physiology and Groupe de recherche sur le système nerveux central, Université de Montréal. Montréal, Canada. Rationale: Previous studies have shown that neural activity in primate dorsal premotor cortex (PMd) can simultaneously represent multiple potential movement plans, and that activity related to these movement options is modulated by their relative subjective desirability. These findings support the hypothesis that decisions about actions are made through a competition within the same circuits that guide the actions themselves. This hypothesis further predicts that the very same cells that guide initial decisions will continue to update their activities if an animal changes its mind. Objectives: Determine whether cells in PMd involved in the selection of an initial movement choice update their activities to reflect a change in movement planning when a previously selected movement option suddenly becomes unavailable. Methods: We tested this prediction by recording neural activity in the PMd of a monkey performing an instructed-delay reach selection task. In the task, two targets were simultaneously presented and their border styles indicated whether each would be worth 1,2, or 3 juice drops. In a random subset of trials (FREE), the monkey was allowed a choice while in the remaining trials (FORCED) one of the targets disappeared at the time of the GO signal. In FORCED-LOW trials, the monkey was forced to move to the less valuable target and started moving either toward the new target (Direct) or toward the target that vanished and then curved to reach the remaining one (Curved). Results: Prior to the GO signal, PMd activity clearly reflected the monkey’s subjective preference, predicting his choices in FREE trials. In FORCED-LOW trials, PMd activity reflected the switch of the monkey’s plan, as early as 100 ms after the GO signal, will before movement onset (MO). These results were reproduced by a computational model. Conclusions: These findings confirm that the activity in PMd is not related to the feedback from the movement itself, and suggests that PMd continues to participate in action selection, even when the animal changes his mind online. These results suggest that switches between action plans can be explained by the same computational process responsible for initial decisions. Funding: CIHR, FRSQ, GRSNC. elsa.tremblay@umontreal.ca
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Hmga2 is cell-autonomously required for the high self-renewal activity of fetal hematopoietic stem cells Michael R. Copleya,b, Claudia Benza, David Knappa, David G. Kenta, Stefan W. Wohrera, Heidi Madera, Keegan Rowea, Chris Daya, David Treloara and Connie J. Eavesa,ca. Terry Fox Laboratory, BC Cancer Agency, b. MD/PhD Program, University of British Columbia, c. Department of Medical Genetics, University of British Columbia, Vancouver, Canada. Rationale: Hematopoietic stem cells (HSCs) are functionally defined by their ability to support life-long blood cell production in mice and humans. Although fetal and adult HSCs both fit these defining criteria, the manner in which they execute these functionalities (i.e. differentiation and self-renewal) are distinct. Specifically, fetal HSCs give rise to more myeloid-lineage cells and more daughter HSCs following transplantation. In a recent study we found that high-mobility group AT-hook 2 (Hmga2) is expressed 30-fold higher in fetal versus adult HSCs. Due to its differential expression and known roles as both a transcription regulator and chromatin architectural factor, we hypothesized that Hmga2 may be a key determinant of the high selfrenewal program of fetal HSCs. Methods: 1.7x105 nucleated cells (~4-5 HSCs) were isolated from E14.5 fetal livers (FL) of Hmga2+/+ (WT) or Hmga2-/- (KO) mouse embryos and transplanted into sublethally irradiated congenic W41/W41 mice by intravenous injection. 6-weeks after the primary transplantation the bone marrow was isolated and donor-derived stem/progenitor numbers were measured by determination of the donor-derved Lin-Sca1+c-Kit+ (LSK) population. Functional HSC-output was also measured using high-dose secondary transplantation and limiting-dilution HSC analysis. Results: Even though similar numbers of HSC were injected into primary mice, KO vs WT HSCs generated significantly fewer donor-derived LSK cells 6-weeks post-transplant. Highdose transplants from KO-transplanted mice produced fewer GM, B and T cells in secondary mice (p<0.01). Finally, Hmga2 KO cells were found to expand less (15-fold vs 165-fold; p<0.001) than WT as determined by limiting-dilution analysis. Conclusion: Fetal HSCs require Hmga2 to execute their high self-renewal program.
mcopley@bccrc.ca
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Evaluating genetic counselling for multifactorial schizophrenia in the molecular age Gregory Costainac, Mary Jane Esplende, Brenda Tonerbcde, Kathleen Hodgkinsong, Anne S. Bassettacdf aClinical Genetics Research Program, bSocial Equity and Health Research, Centre for Addiction and Mental Health, Toronto, Ontario cInstitute of Medical Science, dDepartment of Psychiatry, University of Toronto, Toronto, Ontario eBehavioural Sciences and Health Research Division, fDepartment of Psychiatry, University Health Network, Toronto, Ontario gClinical Epidemiology and Genetics, Faculty of Medicine, Memorial University of Newfoundland and Labrador, St. John's, Newfoundland Rationale: Recent advances in schizophrenia genetics are shedding new light on etiopathogenesis, but issues germane to translation of findings into clinical practice are understudied. There is a longstanding proposal that genetic counselling for multifactorial schizophrenia may be an informative and therapeutic intervention. However, there are no data- driven reports of its desirability or worth. Objectives: Develop, assess interest in, and evaluate efficacy of a genetic counselling intervention for use in a community mental healthcare setting. Methods: We characterized rare copy number variation in a cohort of adults with schizophrenia using million-feature genotyping arrays, and subsequently approached 122 consecutive family members of patients without molecular diagnoses. A pre-post study design with longitudinal follow-up was used to assess the impact of the genetic counselling intervention on perceived recurrence risk and associated anxiety, relevant knowledge, and other pertinent variables. Results: Seventy-eight (63.9%) family members actively expressed an interest in the study, and 64 received genetic counselling during the overall study period. Participants greatly overestimated the risk of familial recurrence at baseline, with a significant reduction in mean qualitative and quantitative risk estimates post-intervention (p<0.0001). These changes were associated with a decrease in concern related to perceptions about recurrence risk for schizophrenia (p=0.0003). Significant and lasting improvements were also observed in knowledge (p<0.0001) and in measures of psychosocial functioning, including stigma (p=0.0047). Levels of blame were unexpectedly low at baseline but on average decreased further post-counselling. Satisfaction, including endorsement of the need for this intervention, was unanimous. Conclusions: This is the first study to investigate the impact of a standardized genetic counselling intervention that took into consideration the psychosocial impact of having a family history of schizophrenia. New m o l e c u l a r g e n e t i c i n f o r m a t i o n a b o u t s c h i z o p h r e n i a w a s successfully incorporated into the content of the intervention, while at the same time efficacy was displayed in the (majority) population of schizophrenia who have no known causal genetic variants. The findings provide preliminary evidence that supports the integration of genetic counselling for families into the general management of schizophrenia. The results may help in preparing for knowledge translation of emerging molecular genetic discoveries and personalized medicine in serious mental illness. Funding: MindCare New Brunswick, CIHR, Vanier CGS, CRC greg.costain@mail.utoronto.ca
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Influenza infects lung microvascular endothelium leading to microvascular leak: Role of apoptosis and claudin-5 Susan M Armstronga, Changsen Wangb, Jayesh Tigdib, Xiaoe Sic, Asela Gamagea, Theo J Moraesac, and Warren L Leeabd of Medical Science, University of Toronto; bKeenan Research Centre of the Li Ka Shing Knowledge Institute of St. Michael's Hospital (Toronto); cHospital for Sick Children, Toronto; the dDivision of Respirology and Interdepartmental Division of Critical Care Medicine, Faculty of Medicine, University of Toronto aInstitute
Rationale: Severe infections with human influenza are characterized by acute lung injury, a syndrome of pulmonary microvascular leak. Antiviral medications are only partially effective at reducing mortality, indicating that additional therapeutic strategies are needed. We hypothesized that influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. Methods: We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed spectrofluorimetrically and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. Results: We found that human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in endothelial permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced loss of the tight junction protein claudin-5; the adherens junction protein VE-cadherin was unaffected. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dosedependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Conclusions: We describe two mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza. Funding: WL Lee is supported by the Canadian Institutes of Health Research, the Ontario Thoracic Society, the Physician Services Incorporated Foundation and an Early Researcher Award from the Government of Ontario. SM Armstrong is supported by a MD/PhD Studentship award from the CIHR and an MD/PhD Studentship award from the McLaughlin Centre.
susanm.armstrong@mail.utoronto.ca
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Identification of Virulence Determinants in 1918 Spanish Flu Hemagglutinin Alex Silaghi1,2,3,4, Tracy Taylor2, Darwyn Kobasa1,2,4 1
Department of Medical Microbiology, University of Manitoba
2
Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada
3
MD/PhD Program, University of Manitoba
4
International Infectious Diseases and Global Health Training Program
Spanish Flu caused the worst pandemic in history with 30-50 million deaths. Although this virus disappeared after the 1918 pandemic, viral RNA was isolated from histological samples and frozen cadavers, and used to generate live viruses containing all or some segments of Spanish influenza. The Hemagglutinin (HA) segment was identified as the major virulence determinant, yet the regions involved and mechanisms of virulence remain unknown. HA is a glycoprotein found on the surface of virions and catalyzes receptor binding and fusion of the viral and plasma membrane during viral entry. HA is cleaved by trypsin-like cellular proteases into HA1 and HA2, an essential process for activation of fusion. HA1 contains three structural/functional regions: Fusionâ&#x20AC;&#x2122; (Fâ&#x20AC;&#x2122;), Vestigial Esterase (VE) and Receptor-Binding Domain (RBD), while HA2 contains the main fusion peptide, transmembrane region and cytoplasmic tail. We generated chimera between Spanish influenza (Sp) HA and closely related H1N1 viruses: WSN/1933, WilsonSmith/1933, PR8/1934, USSR/1977, New Caledonia/1999 and a 2009 pandemic strain (Mexico/Indre4487/09; Mx10) by swapping known regions. The WSN/Sp chimera indicated that Spanish influenza HA virulence in mice is dependent on Serine (S) at residue 343, which is just upstream of the Arginine 344, the site of cleavage of HA into HA1 and HA2. A virus containing Sp HA with Tyrosine (Y) at 343 was attenuated in mice, but also grew poorly without exogenous trypsin in cell culture. Although most influenza viruses require exogenous trypsin for growth in tissue culture, Sp does not, but the relevance of this finding or pathogenesis was unknown. USSR/Sp HA chimera identified the N-glycosylation site at position 286 as another determinant of virulence in mice and trypsin-independent growth in cell culture, but there are likely more requirements of virulence spread across several structural/functional domains, which we are currently investigating. The other chimera systems are also currently being tested, with the hope of identifying more virulence determinants. In summary, trypsin-independent growth in cell culture correlates with virulence in mice, suggesting that the use of specific proteases in the lung by Sp may contribute to the severe disease not observed with other human influenza viruses. alex_silaghi@hotmail.com
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Islet amyloid polypeptide: a trigger for Toll-like receptor 2 in type 2 diabetes and islet transplantation? Clara Westwell-Ropera, Jan A. Ehsesb and C. Bruce Verchereab a
Department of Pathology and Laboratory Medicine, bDepartment of Surgery, University of British Columbia. Vancouver, Canada. Rationale: Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide co-secreted with insulin by pancreatic beta cells. Aggregation of IAPP to form amyloid fibrils contributes to beta cell dysfunction in patients with type 2 diabetes and in islet transplant recipients. IAPP aggregates share a common cross beta-sheet structure with other amyloids known to induce a potent proinflammatory response via activation of the membrane-bound pattern recognition receptor Tolllike receptor 2 (TLR2). Objective: To determine the role of TLR2 in IAPP-induced islet inflammation. Methods: HEK293 cells transfected with human TLR2, 3, 4, 7, or 9 were treated with human IAPP or non-amyloidogenic rodent IAPP and evaluated for NF-κB reporter gene activity. Bone marrow-derived macrophages (BMDMs) and isolated islets from wild-type and TLR2-deficient mice were treated with IAPP and assayed for pro-inflammatory cytokine expression by qRTPCR and ELISA. The contribution of resident macrophages to IAPP-induced islet gene expression was assessed by depleting C57BL/6 mouse islets of phagocytes using clodronatecontaining liposomes. Results: Human but not rodent IAPP induced expression of the pro-inflammatory cytokines IL1β (4.5±1.2 fold untreated; p<0.05), TNF-α (10.3±0.7; p<0.001), IL-6 (117±56; p<0.001), and CCL2 (10.2±0.6; p<0.001) in isolated islets after 4 h. Depletion of islet macrophages significantly reduced expression of both TLR2 and pro-inflammatory cytokines compared to treatment with empty liposomes, suggesting that macrophages are the major source of these mediators. Human IAPP induced NF-κB activation in TLR2-overexpressing HEK293 cells compared to the parental cell line (190±6 vs. 1.0±0.4 absorbance units; p<0.001). This effect was blocked in cells treated with anti-TLR2 or anti-TLR6 neutralizing antibody, suggesting that IAPP activates a TLR2/6 heterodimer. Upregulation of IL-1β, TNF-α, IL-6, and CCL2 in BMDMs and islets treated with IAPP was significantly attenuated in cells from TLR2-deficient mice, suggesting that IAPP-induced TLR2 signalling contributes to pro-inflammatory cytokine release by islet macrophages. Conclusions: These data suggest that islet macrophages are the major source of IAPPinduced pro-inflammatory cytokines, and that diverse amyloidogenic peptides – including IAPP – act as endogenous ligands for TLR2. Thus, manipulation of macrophage activation or blockade of TLR2 signalling may limit IAPP-induced islet inflammation and beta cell dysfunction in type 2 diabetes and islet transplantation. Funding: CIHR Vanier Canada Graduate Scholarship; Canadian Diabetes Association cwestwell@interchange.ubc.ca
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Participant Contact Information CNMSRS On-Site Contact Info Kimberley Ponton â&#x20AC;&#x201C; CNMSRS Coordinator 204-789-3558 204-333-7426 Office: A108 Chown Building 753 McDermot Avenue University of Manitoba Winnipeg, MB R3E 0T6 Email: kim.ponton@med.umanitoba.ca
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Dr. Paul H.T. Thorlakson Foundation 99