Publications by authors named "Mark J McVey"

31 Publications

Achieving sustained extrauterine life: Challenges of an artificial placenta in fetal pigs as a model of the preterm human fetus.

Physiol Rep 2021 Mar;9(5):e14742

Division of Cardiovascular Surgery, The Labatt Family Heart Centre, The Hospital for Sick Children, University of Toronto, Toronto, Canada.

Artificial placenta (AP) technology aims to maintain fetal circulation, while promoting the physiologic development of organs. Recent reports of experiments performed in sheep indicate the intrauterine environment can be recreated through the cannulation of umbilical vessels, replacement of the placenta with a low-resistance membrane oxygenator, and incubation of the fetus in fluid. However, it remains to be seen whether animal fetuses similar in size to the extremely preterm human infant that have been proposed as a potential target for this technology can be supported in this way. Preterm Yucatan miniature piglets are similar in size to extremely preterm human infants and share similar umbilical cord anatomy, raising the possibility to serve as a good model to investigate the AP. To characterize fetal cardiovascular physiology, the carotid artery (n = 24) was cannulated in utero and umbilical vein (UV) and umbilical artery were sampled. Fetal UV flow was measured by MRI (n = 16). Piglets were delivered at 98 ± 4 days gestation (term = 115 days), cannulated, and supported on the AP (n = 12) for 684 ± 228 min (range 195-3077 min). UV flow was subphysiologic (p = .002), while heart rate was elevated on the AP compared with in utero controls (p = .0007). We observed an inverse relationship between heart rate and UV flow (r = .4527; p < .001) with progressive right ventricular enlargement that was associated with reduced contractility and ultimately hydrops and circulatory collapse. We attribute this to excessive afterload imposed by supraphysiologic circuit resistance and augmented sympathetic activity. We conclude that short-term support of the preterm piglet on the AP is feasible, although we have not been able to attain normal fetal physiology. In the future, we propose to investigate the feasibility of an AP circuit that incorporates a centrifugal pump in our miniature pig model.
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http://dx.doi.org/10.14814/phy2.14742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923578PMC
March 2021

Frequency and timing of all-cause deaths in visits involving suspected transfusion reactions, and the significance of cardiopulmonary disturbances.

Vox Sang 2021 Feb 26. Epub 2021 Feb 26.

Utilization, Efficacy, & Safety of Transfusion (QUEST) Research Program, University of Toronto Quality, Toronto, ON, Canada.

Background/objectives: Transfusion reactions (TRs) may cause or contribute to death. Cardiopulmonary TRs are distressing, and collectively account for most transfusion fatalities, though the degree to which they alter survival more broadly is unclear. Deaths (and their timing) after TRs may provide further insights.

Materials/methods: Adult (tri-hospital network) haemovigilance data (2013-2016) recorded referrals with conclusions ranging from unrelated to transfusion (UTR) to entities such as: septic TRs, serologic/haemolytic reactions, transfusion-associated circulatory overload (TACO), transfusion-associated dyspnoea (TAD), transfusion-related acute lung injury (TRALI), allergic transfusion reaction (ATR), and others. For (in- or out-patient) visits involving suspected TRs (VISTRs), all-cause mortalities (% [95% confidence interval]) and associated time-to-death (TTD) (median days, [interquartile range]) were compared. Diagnoses were defined inclusively (possible-to-definite) or strictly (probable-to-definite).

Results: Of 1144 events, rank order VISTR mortality following (possible-to-definite) TRs, and associated TTDs, were led by: DHTR 33% [6-19], 1 death at 123d; TRALI 32% [15-54], 6 deaths: 3d [2-20]; BaCon 21% [14-31], 17 deaths: 10d [3-28]; TACO 18% [12-26], 23 deaths: 16d [6-28]; TAD 17% [11-26]: 18 deaths, 6d [3-12]. Higher-certainty TRs ranked similarly (DHTR 50% [9-91]; BaCon 29% [12-55], 4 deaths: 12d [3-22]; and TACO 25% [16-38], 15 deaths: 21d [6-28]). VISTR mortality after TACO or TRALI significantly exceeded ATR (3·3% [2·4-5·8], P < 0·00001) but was not different from UTR events (P = 0·3).

Conclusions: Only half of cardiopulmonary TRs constituted high certainty diagnoses. Nevertheless, cardiopulmonary TRs and suspected BaCon marked higher VISTR mortality with shorter TTDs. Short (<1 week) TTDs in TAD, BaCon or TRALI imply either contributing roles in death, treatment refractoriness and/or applicable TR susceptibilities in the dying.
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http://dx.doi.org/10.1111/vox.13086DOI Listing
February 2021

General medical publications during COVID-19 show increased dissemination despite lower validation.

PLoS One 2021 2;16(2):e0246427. Epub 2021 Feb 2.

Department of Anesthesia and Pain Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada.

Background: The COVID-19 pandemic has yielded an unprecedented quantity of new publications, contributing to an overwhelming quantity of information and leading to the rapid dissemination of less stringently validated information. Yet, a formal analysis of how the medical literature has changed during the pandemic is lacking. In this analysis, we aimed to quantify how scientific publications changed at the outset of the COVID-19 pandemic.

Methods: We performed a cross-sectional bibliometric study of published studies in four high-impact medical journals to identify differences in the characteristics of COVID-19 related publications compared to non-pandemic studies. Original investigations related to SARS-CoV-2 and COVID-19 published in March and April 2020 were identified and compared to non-COVID-19 research publications over the same two-month period in 2019 and 2020. Extracted data included publication characteristics, study characteristics, author characteristics, and impact metrics. Our primary measure was principal component analysis (PCA) of publication characteristics and impact metrics across groups.

Results: We identified 402 publications that met inclusion criteria: 76 were related to COVID-19; 154 and 172 were non-COVID publications over the same period in 2020 and 2019, respectively. PCA utilizing the collected bibliometric data revealed segregation of the COVID-19 literature subset from both groups of non-COVID literature (2019 and 2020). COVID-19 publications were more likely to describe prospective observational (31.6%) or case series (41.8%) studies without industry funding as compared with non-COVID articles, which were represented primarily by randomized controlled trials (32.5% and 36.6% in the non-COVID literature from 2020 and 2019, respectively).

Conclusions: In this cross-sectional study of publications in four general medical journals, COVID-related articles were significantly different from non-COVID articles based on article characteristics and impact metrics. COVID-related studies were generally shorter articles reporting observational studies with less literature cited and fewer study sites, suggestive of more limited scientific support. They nevertheless had much higher dissemination.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246427PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853485PMC
February 2021

Inflammasome activation in acute lung injury.

Am J Physiol Lung Cell Mol Physiol 2021 02 9;320(2):L165-L178. Epub 2020 Dec 9.

Department of Anesthesiology and Pain Medicine, University of Toronto, Toronto, Ontario, Canada.

Inflammasomes are multiprotein complexes tasked with sensing endogenous or exogenous inflammatory signals and integrating this signal into a downstream response. Inflammasome activation has been implicated in a variety of pulmonary diseases, including pulmonary hypertension, bacterial pneumonia, COPD, and asthma. Of increasing interest is the contribution of inflammasome activation in the context of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Inflammasome activation in both the lung parenchyma and resident immune cells generates intereukin-1β (IL-1β) and IL-18, both of which drive the cascade of lung inflammation forward. Blockade of these responses has been shown to be beneficial in animal models and is a focus of translational research in the field. In this review, we will discuss the assembly and regulation of inflammasomes during lung inflammation, highlighting therapeutically viable effector steps. We will examine the importance of IL-1β and IL-18, two key products of inflammasome activation, in ALI, as well as the contribution of the pulmonary endothelial cell to this process. Finally, we will explore translational research moving toward anti-inflammasome therapies for ALI/ARDS and speculate toward future directions for the field.
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http://dx.doi.org/10.1152/ajplung.00303.2020DOI Listing
February 2021

Platelet extracellular vesicles mediate transfusion-related acute lung injury by imbalancing the sphingolipid rheostat.

Blood 2021 Feb;137(5):690-701

Keenan Research Centre for Biomedical Science, St Michael's Hospital, Toronto, ON, Canada.

Transfusion-related acute lung injury (TRALI) is a hazardous transfusion complication with an associated mortality of 5% to 15%. We previously showed that stored (5 days) but not fresh platelets (1 day) cause TRALI via ceramide-mediated endothelial barrier dysfunction. As biological ceramides are hydrophobic, extracellular vesicles (EVs) may be required to shuttle these sphingolipids from platelets to endothelial cells. Adding to complexity, EV formation in turn requires ceramide. We hypothesized that ceramide-dependent EV formation from stored platelets and EV-dependent sphingolipid shuttling induces TRALI. EVs formed during storage of murine platelets were enumerated, characterized for sphingolipids, and applied in a murine TRALI model in vivo and for endothelial barrier assessment in vitro. Five-day EVs were more abundant, had higher long-chain ceramide (C16:0, C18:0, C20:0), and lower sphingosine-1-phosphate (S1P) content than 1-day EVs. Transfusion of 5-day, but not 1-day, EVs induced characteristic signs of lung injury in vivo and endothelial barrier disruption in vitro. Inhibition or supplementation of ceramide-forming sphingomyelinase reduced or enhanced the formation of EVs, respectively, but did not alter the injuriousness per individual EV. Barrier failure was attenuated when EVs were abundant in or supplemented with S1P. Stored human platelet 4-day EVs were more numerous compared with 2-day EVs, contained more long-chain ceramide and less S1P, and caused more endothelial cell barrier leak. Hence, platelet-derived EVs become more numerous and more injurious (more long-chain ceramide, less S1P) during storage. Blockade of sphingomyelinase, EV elimination, or supplementation of S1P during platelet storage may present promising strategies for TRALI prevention.
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http://dx.doi.org/10.1182/blood.2020005985DOI Listing
February 2021

Association between preoperative hemoglobin levels after iron supplementation and perioperative blood transfusion requirements in children undergoing scoliosis surgery.

Paediatr Anaesth 2020 10 29;30(10):1077-1082. Epub 2020 Aug 29.

Department of Anesthesiology and Pain Medicine, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada.

Background And Aims: In this study, we assessed the association between preoperative hemoglobin and red blood cell transfusion in children undergoing spine surgery after the implementation of our preoperative iron supplementation protocol.

Method: We performed a retrospective analysis of patients who underwent posterior spinal fusion surgery between January 2013 and December 2017 and received preoperative iron supplementation. We used uni- and multivariable logistic regression to determine the association between preoperative hemoglobin level and red blood cell transfusion in patients receiving iron supplementation.

Results: A total of 382 patients treated with preoperative oral iron were included. Of these, 175 (45.5%) patients were transfused intraoperatively. Multivariable logistic regression analysis revealed nonidiopathic etiology of the scoliosis (OR 4.178 [95% CI: 2.277-7.668], P < .001), the Cobb angle (OR 1.025 [95% CI: 1.010-1.040], P = .001), and number of vertebrae fused (OR 1.169 [95% CI: 1.042-1.312], P = .008) were associated with red blood cell transfusion. In addition, patients with a preoperative hemoglobin ≥ 140 g/L (OR 0.157 [95% CI: 0.046-0.540], P = .003), and hemoglobin between 130 and 140 g/L (OR 0.195 [95% CI: 0.057-0.669], P = .009) were less likely to be transfused compared with patients with preoperative hemoglobin between 120 and 130 g/L (OR 0.294 [95% CI: 0.780-1.082], P = .066) or <120 g/L (reference).

Conclusion: Our study suggests that higher preoperative hemoglobin levels (>130 g/L) are associated with a reduced need for red blood cell transfusion in pediatric patients who have received iron supplementation before undergoing posterior spinal fusion in our institution. The effect of iron supplementation, the optimal dosing, and duration of supplemental iron therapy remains unclear at this time.
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http://dx.doi.org/10.1111/pan.13987DOI Listing
October 2020

A regional massive hemorrhage protocol developed through a modified Delphi technique.

CMAJ Open 2019 Jul-Sep;7(3):E546-E561. Epub 2019 Sep 3.

Departments of Laboratory Medicine and Molecular Diagnostics (Callum, Chin, Viveiros), Surgery (Nathens, Nascimento), Emergency Services (McDonald), Critical Care Medicine (Adhikari) and Anesthesia (Margarido), Sunnybrook Health Sciences Centre; Departments of Laboratory Medicine and Pathobiology (Callum, Pendergrast, Skeate, Pavenski), Anesthesia (McVey, Karkouti, Alam, Margarido), Surgery (Nathens, Nascimento, Rizoli) and Paediatrics (Beno), University of Toronto; Division of Emergency Medicine (Yeh, Petrosoniak, McDonald, MacDonald), Department of Medicine, University of Toronto; Departments of Emergency Medicine (Petrosoniak), Surgery (Rizoli) and Laboratory Medicine (Sholzberg, Pavenski), St. Michael's Hospital; Department of Anesthesia and Pain Medicine (McVey, Skelton), The Hospital for Sick Children; Ontario Regional Blood Coordinating Network (Cope, Thompson, Collins, Owens); Department of Anesthesia and Pain Management (Karkouti), Sinai Health System, University Health Network, and Women's College Hospital, Toronto, Ont.; Department of Anesthesiology and Pain Medicine (Murto), Children's Hospital of Eastern Ontario; Department of Anesthesiology and Pain Medicine (Murto), University of Ottawa, Ottawa, Ont.; Paediatric Emergency Medicine (Beno), The Hospital for Sick Children; Department of Clinical Pathology (Pendergrast), University Health Network, Toronto, Ont.; Ornge Transport Medicine (McDonald, MacDonald), Mississauga, Ont.; Interdepartmental Division of Critical Care Medicine (Adhikari), University of Toronto; Department of Anesthesia (Alam, Arnold), North York General Hospital, Toronto, Ont.; McMaster Centre for Transfusion Research (Arnold, Pai, Zeller); Departments of Medicine (Pai, Zeller) and Pathology and Molecular Medicine (Pai), McMaster University, Hamilton, Ont.; Canadian Blood Services (Arnold, Skeate, White); St. Michael's Hospital (Barratt, Chaudhry, Harvey), Toronto, Ont.; Department of Surgery (Beckett), McGill University, Montréal, Que.; Canadian Forces Health Services (Beckett), Ottawa, Ont.; Sunnybrook Health Sciences Centre (Brenneman), Toronto, Ont.; General Surgery, Acute Care and Trauma (Lampron), The Ottawa Hospital; Departments of Surgery (Lampron), Medicine (Tinmouth) and Laboratory Medicine and Pathology (Tinmouth), Faculty of Medicine, University of Ottawa, Ottawa, Ont.; Trauma Program and Quality Assurance (McFarlan), St. Michael's Hospital, Toronto, Ont.; Departments of Pathology (Ruijs) and Surgery (Van Heest), William Osler Health Centre, Brampton, Ont.; Lakeridge Health Corporation (Syer), Oshawa, Ont.; Department of Critical Care (Theriault), Health Sciences North, Sudbury, Ont.; Division of Hematology (Tinmouth), The Ottawa Hospital; University of Ottawa Centre for Transfusion Research (Tinmouth), Ottawa Hospital Research Institute, Ottawa, Ont.; Canadian Blood Services (Zeller), Ancaster, Ont.

Background: A massive hemorrhage protocol (MHP) enables rapid delivery of blood components in a patient who is exsanguinating pending definitive hemorrhage control, but there is variability in MHP implementation rates, content and compliance owing to challenges presented by infrequent activation, variable team performance and patient acuity. The goal of this project was to identify the key evidence-based principles and quality indicators required to develop a standardized regional MHP.

Methods: A modified Delphi consensus technique was performed in the spring and summer of 2018. Panellists used survey links to independently review and rate (on a 7-point Likert scale) 43 statements and 8 quality indicators drafted by a steering committee composed of transfusion medicine specialists and technologists, and trauma physicians. External stakeholder input from all hospitals in Ontario was sought.

Results: Three rounds were held with 36 experts from diverse clinical backgrounds. Consensus was reached for 42 statements and 8 quality indicators. Additional modifications from external stakeholders were incorporated to form the foundation for the proposed MHP.

Interpretation: This MHP template will provide the basis for the design of an MHP toolkit, including specific recommendations for pediatric and obstetrical patients, and for hospitals with limited availability of blood components or means to achieve definitive hemorrhage control. We believe that harmonization of MHPs in our region will simplify training, increase uptake of evidence-based interventions, enhance communication, improve patient comfort and safety, and, ultimately, improve patient outcomes.
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http://dx.doi.org/10.9778/cmajo.20190042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726467PMC
September 2019

Transfusion-related Acute Lung Injury in the Perioperative Patient.

Anesthesiology 2019 09;131(3):693-715

From the Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Canada (M.J.M., J.W.S., W.M.K) Departments of Physiology and Anesthesia (M.J.M.) Pharmacology (J.W.S.) Laboratory Medicine-Pathobiology (C.C.-G.) Medicine-Division of Hematology (C.C.-G.) Anesthesia and Pain Management, University Health Network (K.K.) Physiology and Surgery (W.M.K.) University of Toronto, Toronto, Canada (C.C.-G.) Sickkids Department of Anesthesia and Pain Medicine (M.J.M.) Division of Hematology and Transfusion Medicine (R.K., J.W.S.), Lund University, Lund, Sweden Department of Experimental Immunohematology, Sanquin Research, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands (R.K.) Departments of Laboratory Hematology (Blood Transfusion Laboratory), Laboratory Medicine Program (C.C.-G.) Medical Oncology and Hematology (C.C.-G.) University Health Network, Toronto, Canada (K.K.) Department of Clinical Pathology, Blood and Tissue Bank, Sunnybrook Health Sciences Centre, Toronto, Canada (C.C.-G.) Institute of Physiology, Charité University Berlin, Berlin, Germany (W.M.K.).

Transfusion-related acute lung injury is a leading cause of death associated with the use of blood products. Transfusion-related acute lung injury is a diagnosis of exclusion which can be difficult to identify during surgery amid the various physiologic and pathophysiologic changes associated with the perioperative period. As anesthesiologists supervise delivery of a large portion of inpatient prescribed blood products, and since the incidence of transfusion-related acute lung injury in the perioperative patient is higher than in nonsurgical patients, anesthesiologists need to consider transfusion-related acute lung injury in the perioperative setting, identify at-risk patients, recognize early signs of transfusion-related acute lung injury, and have established strategies for its prevention and treatment.
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http://dx.doi.org/10.1097/ALN.0000000000002687DOI Listing
September 2019

Extracellular vesicles in lung health, disease, and therapy.

Am J Physiol Lung Cell Mol Physiol 2019 06 20;316(6):L977-L989. Epub 2019 Mar 20.

Keenan Research Centre for Biomedical Science, St. Michael's Hospital , Toronto, Ontario , Canada.

Both physiological homeostasis and pathological disease processes in the lung typically result from complex, yet coordinated multicellular responses that are synchronized via paracrine and endocrine intercellular communication pathways. Of late, extracellular vesicles have emerged as important information shuttles that can coordinate and disseminate homeostatic and disease signals. In parallel, extracellular vesicles in biological fluids such as sputum, mucus, epithelial lining fluid, edema fluid, the pulmonary circulation, pleural fluid, and lymphatics have emerged as promising candidate biomarkers for diagnosis and prognosis in lung disease. Extracellular vesicles are small, subcellular, membrane-bound vesicles containing cargos from parent cells such as lipids, proteins, genetic information, or entire organelles. These cargos endow extracellular vesicles with biologically active information or functions by which they can reprogram their respective target cells. Recent studies show that extracellular vesicles found in lung-associated biological fluids play key roles as biomarkers and effectors of disease. Conversely, administration of naïve or engineered extracellular vesicles with homeostatic or reparative effects may provide a promising novel protective and regenerative strategy to treat lung disease. To highlight this rapidly developing field, the is now launching a special Call for Papers on extracellular vesicles in lung health, disease, and therapy. This review aims to set the stage for this call by introducing extracellular vesicles and their emerging roles in lung physiology and pathobiology.
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http://dx.doi.org/10.1152/ajplung.00546.2018DOI Listing
June 2019

The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques.

Sci Rep 2019 02 12;9(1):1837. Epub 2019 Feb 12.

Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada.

Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity.
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http://dx.doi.org/10.1038/s41598-018-38320-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372651PMC
February 2019

A suspected case of anesthesia-induced rhabdomyolysis in a child undergoing strabismus surgery.

J AAPOS 2019 06 5;23(3):167-169. Epub 2019 Feb 5.

Department of Ophthalmology and Vision Sciences, University of Toronto, Toronto, Ontario, Canada; Department of Ophthalmology and Vision Sciences, The Hospital for Sick Children, Toronto, Ontario, Canada. Electronic address:

We report a case of acute rhabdomyolysis following general anesthesia for strabismus surgery in a previously healthy 11-year-old girl. The patient received a depolarizing muscle relaxant (succinylcholine) and halogenated volatile anesthetic agent (sevoflurane) during surgery. In rare cases, these classes of drugs can trigger malignant hyperthermia (MH) or anesthesia-induced rhabdomyolysis (AIR), which can cause significant morbidity and mortality if not recognized and treated promptly. Pathophysiology, early recognition, and special considerations in strabismus patients are discussed.
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http://dx.doi.org/10.1016/j.jaapos.2019.01.006DOI Listing
June 2019

Extracellular vesicles: biomarkers and regulators of vascular function during extracorporeal circulation.

Oncotarget 2018 Dec 14;9(98):37229-37251. Epub 2018 Dec 14.

Keenan Research Centre for Biomedical Science, St Michael's Hospital, Toronto, ON, Canada.

Extracellular vesicles (EVs) are generated at increased rates from parenchymal and circulating blood cells during exposure of the circulation to abnormal flow conditions and foreign materials associated with extracorporeal circuits (ExCors). This review describes types of EVs produced in different ExCors and extracorporeal life support (ECLS) systems including cardiopulmonary bypass circuits, extracorporeal membrane oxygenation (ECMO), extracorporeal carbon dioxide removal (ECCOR), apheresis, dialysis and ventricular assist devices. Roles of EVs not only as biomarkers of adverse events during ExCor/ECLS use, but also as mediators of vascular dysfunction are explored. Manipulation of the number or subtypes of circulating EVs may prove a means of improving vascular function for individuals requiring ExCor/ECLS support. Strategies for therapeutic manipulation of EVs during ExCor/ECLS use are discussed such as accelerating their clearance, preventing their genesis or pharmacologic options to reduce or select which and how many EVs circulate. Strategies to reduce or select for specific types of EVs may prove beneficial in preventing or treating other EV-related diseases such as cancer.
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http://dx.doi.org/10.18632/oncotarget.26433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324688PMC
December 2018

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

Authors:
Clotilde Théry Kenneth W Witwer Elena Aikawa Maria Jose Alcaraz Johnathon D Anderson Ramaroson Andriantsitohaina Anna Antoniou Tanina Arab Fabienne Archer Georgia K Atkin-Smith D Craig Ayre Jean-Marie Bach Daniel Bachurski Hossein Baharvand Leonora Balaj Shawn Baldacchino Natalie N Bauer Amy A Baxter Mary Bebawy Carla Beckham Apolonija Bedina Zavec Abderrahim Benmoussa Anna C Berardi Paolo Bergese Ewa Bielska Cherie Blenkiron Sylwia Bobis-Wozowicz Eric Boilard Wilfrid Boireau Antonella Bongiovanni Francesc E Borràs Steffi Bosch Chantal M Boulanger Xandra Breakefield Andrew M Breglio Meadhbh Á Brennan David R Brigstock Alain Brisson Marike Ld Broekman Jacqueline F Bromberg Paulina Bryl-Górecka Shilpa Buch Amy H Buck Dylan Burger Sara Busatto Dominik Buschmann Benedetta Bussolati Edit I Buzás James Bryan Byrd Giovanni Camussi David Rf Carter Sarah Caruso Lawrence W Chamley Yu-Ting Chang Chihchen Chen Shuai Chen Lesley Cheng Andrew R Chin Aled Clayton Stefano P Clerici Alex Cocks Emanuele Cocucci Robert J Coffey Anabela Cordeiro-da-Silva Yvonne Couch Frank Aw Coumans Beth Coyle Rossella Crescitelli Miria Ferreira Criado Crislyn D'Souza-Schorey Saumya Das Amrita Datta Chaudhuri Paola de Candia Eliezer F De Santana Olivier De Wever Hernando A Del Portillo Tanguy Demaret Sarah Deville Andrew Devitt Bert Dhondt Dolores Di Vizio Lothar C Dieterich Vincenza Dolo Ana Paula Dominguez Rubio Massimo Dominici Mauricio R Dourado Tom Ap Driedonks Filipe V Duarte Heather M Duncan Ramon M Eichenberger Karin Ekström Samir El Andaloussi Celine Elie-Caille Uta Erdbrügger Juan M Falcón-Pérez Farah Fatima Jason E Fish Miguel Flores-Bellver András Försönits Annie Frelet-Barrand Fabia Fricke Gregor Fuhrmann Susanne Gabrielsson Ana Gámez-Valero Chris Gardiner Kathrin Gärtner Raphael Gaudin Yong Song Gho Bernd Giebel Caroline Gilbert Mario Gimona Ilaria Giusti Deborah Ci Goberdhan André Görgens Sharon M Gorski David W Greening Julia Christina Gross Alice Gualerzi Gopal N Gupta Dakota Gustafson Aase Handberg Reka A Haraszti Paul Harrison Hargita Hegyesi An Hendrix Andrew F Hill Fred H Hochberg Karl F Hoffmann Beth Holder Harry Holthofer Baharak Hosseinkhani Guoku Hu Yiyao Huang Veronica Huber Stuart Hunt Ahmed Gamal-Eldin Ibrahim Tsuneya Ikezu Jameel M Inal Mustafa Isin Alena Ivanova Hannah K Jackson Soren Jacobsen Steven M Jay Muthuvel Jayachandran Guido Jenster Lanzhou Jiang Suzanne M Johnson Jennifer C Jones Ambrose Jong Tijana Jovanovic-Talisman Stephanie Jung Raghu Kalluri Shin-Ichi Kano Sukhbir Kaur Yumi Kawamura Evan T Keller Delaram Khamari Elena Khomyakova Anastasia Khvorova Peter Kierulf Kwang Pyo Kim Thomas Kislinger Mikael Klingeborn David J Klinke Miroslaw Kornek Maja M Kosanović Árpád Ferenc Kovács Eva-Maria Krämer-Albers Susanne Krasemann Mirja Krause Igor V Kurochkin Gina D Kusuma Sören Kuypers Saara Laitinen Scott M Langevin Lucia R Languino Joanne Lannigan Cecilia Lässer Louise C Laurent Gregory Lavieu Elisa Lázaro-Ibáñez Soazig Le Lay Myung-Shin Lee Yi Xin Fiona Lee Debora S Lemos Metka Lenassi Aleksandra Leszczynska Isaac Ts Li Ke Liao Sten F Libregts Erzsebet Ligeti Rebecca Lim Sai Kiang Lim Aija Linē Karen Linnemannstöns Alicia Llorente Catherine A Lombard Magdalena J Lorenowicz Ákos M Lörincz Jan Lötvall Jason Lovett Michelle C Lowry Xavier Loyer Quan Lu Barbara Lukomska Taral R Lunavat Sybren Ln Maas Harmeet Malhi Antonio Marcilla Jacopo Mariani Javier Mariscal Elena S Martens-Uzunova Lorena Martin-Jaular M Carmen Martinez Vilma Regina Martins Mathilde Mathieu Suresh Mathivanan Marco Maugeri Lynda K McGinnis Mark J McVey David G Meckes Katie L Meehan Inge Mertens Valentina R Minciacchi Andreas Möller Malene Møller Jørgensen Aizea Morales-Kastresana Jess Morhayim François Mullier Maurizio Muraca Luca Musante Veronika Mussack Dillon C Muth Kathryn H Myburgh Tanbir Najrana Muhammad Nawaz Irina Nazarenko Peter Nejsum Christian Neri Tommaso Neri Rienk Nieuwland Leonardo Nimrichter John P Nolan Esther Nm Nolte-'t Hoen Nicole Noren Hooten Lorraine O'Driscoll Tina O'Grady Ana O'Loghlen Takahiro Ochiya Martin Olivier Alberto Ortiz Luis A Ortiz Xabier Osteikoetxea Ole Østergaard Matias Ostrowski Jaesung Park D Michiel Pegtel Hector Peinado Francesca Perut Michael W Pfaffl Donald G Phinney Bartijn Ch Pieters Ryan C Pink David S Pisetsky Elke Pogge von Strandmann Iva Polakovicova Ivan Kh Poon Bonita H Powell Ilaria Prada Lynn Pulliam Peter Quesenberry Annalisa Radeghieri Robert L Raffai Stefania Raimondo Janusz Rak Marcel I Ramirez Graça Raposo Morsi S Rayyan Neta Regev-Rudzki Franz L Ricklefs Paul D Robbins David D Roberts Silvia C Rodrigues Eva Rohde Sophie Rome Kasper Ma Rouschop Aurelia Rughetti Ashley E Russell Paula Saá Susmita Sahoo Edison Salas-Huenuleo Catherine Sánchez Julie A Saugstad Meike J Saul Raymond M Schiffelers Raphael Schneider Tine Hiorth Schøyen Aaron Scott Eriomina Shahaj Shivani Sharma Olga Shatnyeva Faezeh Shekari Ganesh Vilas Shelke Ashok K Shetty Kiyotaka Shiba Pia R-M Siljander Andreia M Silva Agata Skowronek Orman L Snyder Rodrigo Pedro Soares Barbara W Sódar Carolina Soekmadji Javier Sotillo Philip D Stahl Willem Stoorvogel Shannon L Stott Erwin F Strasser Simon Swift Hidetoshi Tahara Muneesh Tewari Kate Timms Swasti Tiwari Rochelle Tixeira Mercedes Tkach Wei Seong Toh Richard Tomasini Ana Claudia Torrecilhas Juan Pablo Tosar Vasilis Toxavidis Lorena Urbanelli Pieter Vader Bas Wm van Balkom Susanne G van der Grein Jan Van Deun Martijn Jc van Herwijnen Kendall Van Keuren-Jensen Guillaume van Niel Martin E van Royen Andre J van Wijnen M Helena Vasconcelos Ivan J Vechetti Tiago D Veit Laura J Vella Émilie Velot Frederik J Verweij Beate Vestad Jose L Viñas Tamás Visnovitz Krisztina V Vukman Jessica Wahlgren Dionysios C Watson Marca Hm Wauben Alissa Weaver Jason P Webber Viktoria Weber Ann M Wehman Daniel J Weiss Joshua A Welsh Sebastian Wendt Asa M Wheelock Zoltán Wiener Leonie Witte Joy Wolfram Angeliki Xagorari Patricia Xander Jing Xu Xiaomei Yan María Yáñez-Mó Hang Yin Yuana Yuana Valentina Zappulli Jana Zarubova Vytautas Žėkas Jian-Ye Zhang Zezhou Zhao Lei Zheng Alexander R Zheutlin Antje M Zickler Pascale Zimmermann Angela M Zivkovic Davide Zocco Ewa K Zuba-Surma

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Kraków, Poland.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

Gastrointestinal microbiota contributes to the development of murine transfusion-related acute lung injury.

Blood Adv 2018 07;2(13):1651-1663

Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden.

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress upon blood transfusion and is the leading cause of transfusion-related fatalities. Whether the gut microbiota plays any role in the development of TRALI is currently unknown. We observed that untreated barrier-free (BF) mice suffered from severe antibody-mediated acute lung injury, whereas the more sterile housed specific pathogen-free (SPF) mice and gut flora-depleted BF mice were both protected from lung injury. The prevention of TRALI in the SPF mice and gut flora-depleted BF mice was associated with decreased plasma macrophage inflammatory protein-2 levels as well as decreased pulmonary neutrophil accumulation. DNA sequencing of amplicons of the 16S ribosomal RNA gene revealed a varying gastrointestinal bacterial composition between BF and SPF mice. BF fecal matter transferred into SPF mice significantly restored TRALI susceptibility in SPF mice. These data reveal a link between the gut flora composition and the development of antibody-mediated TRALI in mice. Assessment of gut microbial composition may help in TRALI risk assessment before transfusion.
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http://dx.doi.org/10.1182/bloodadvances.2018018903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039664PMC
July 2018

The Pathogenesis of Choanal Atresia.

JAMA Otolaryngol Head Neck Surg 2018 08;144(8):758-759

Department of Otolaryngology-Head and Neck Surgery, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.

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http://dx.doi.org/10.1001/jamaoto.2018.1246DOI Listing
August 2018

Improved resolution in extracellular vesicle populations using 405 instead of 488 nm side scatter.

J Extracell Vesicles 2018 22;7(1):1454776. Epub 2018 Mar 22.

Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Canada.

Improvements in identification and assessment of extracellular vesicles (EVs) have fuelled a recent surge in EV publications investigating their roles as biomarkers and mediators of disease. Meaningful scientific comparisons are, however, hampered by difficulties in accurate, reproducible enumeration and characterization of EVs in biological fluids. High-sensitivity flow cytometry (FCM) is presently the most commonly applied strategy to assess EVs, yet its utility is limited by variant ability to resolve smaller EVs. Here, we propose the use of 405 nm (violet) wavelength lasers in place of 488 nm (blue) for side scatter (SSC) detection to obtain greater resolution of EVs using high-sensitivity FCM. To test this hypothesis, we modelled EV resolution by violet versus blue SSC and compared resolution of reference beads and biological EVs from plasma and bronchoalveolar lavage (BAL) fluid using either violet or blue wavelength SSC EV detection. Mie scatter modelling predicted that violet as compared to blue SSC increases resolution of small (100-500 nm) spherical particles with refractive indices (1.34-1.46) similar to EVs by approximately twofold in terms of light intensity and by nearly 20% in SSC signal quantum efficiency. Resolution of reference beads was improved by violet instead of blue SSC with two- and fivefold decreases in coefficients of variation for particles of 300-500 nm and 180-240 nm size, respectively. Resolution was similarly improved for detection of EVs from plasma or BAL fluid. Violet SSC detection for high-sensitivity FCM allows for significantly greater resolution of EVs in plasma and BAL compared to conventional blue SSC and particularly improves resolution of smaller EVs. Notably, the proposed strategy is readily implementable and inexpensive for machines already equipped with 405 nm SSC or the ability to accommodate 405/10 nm bandpass filters in their violet detector arrays.
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http://dx.doi.org/10.1080/20013078.2018.1454776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912191PMC
March 2018

Targeting Transfusion-Related Acute Lung Injury: The Journey From Basic Science to Novel Therapies.

Crit Care Med 2018 05;46(5):e452-e458

Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden.

Objectives: Transfusion-related acute lung injury is characterized by the onset of respiratory distress and acute lung injury following blood transfusion, but its pathogenesis remains poorly understood. Generally, a two-hit model is presumed to underlie transfusion-related acute lung injury with the first hit being risk factors present in the transfused patient (such as inflammation), whereas the second hit is conveyed by factors in the transfused donor blood (such as antileukocyte antibodies). At least 80% of transfusion-related acute lung injury cases are related to the presence of donor antibodies such as antihuman leukocyte or antihuman neutrophil antibodies. The remaining cases may be related to nonantibody-mediated factors such as biolipids or components related to storage and ageing of the transfused blood cells. At present, transfusion-related acute lung injury is the leading cause of transfusion-related fatalities and no specific therapy is clinically available. In this article, we critically appraise and discuss recent preclinical (bench) insights related to transfusion-related acute lung injury pathogenesis and their therapeutic potential for future use at the patients' bedside in order to combat this devastating and possibly fatal complication of transfusion.

Data Sources: We searched the PubMed database (until August 22, 2017).

Study Selection: Using terms: "Transfusion-related acute lung injury," "TRALI," "TRALI and therapy," "TRALI pathogenesis."

Data Extraction: English-written articles focusing on transfusion-related acute lung injury pathogenesis, with potential therapeutic implications, were extracted.

Data Synthesis: We have identified potential therapeutic approaches based on the literature.

Conclusions: We propose that the most promising therapeutic strategies to explore are interleukin-10 therapy, down-modulating C-reactive protein levels, targeting reactive oxygen species, or blocking the interleukin-8 receptors; all focused on the transfused recipient. In the long-run, it may perhaps also be advantageous to explore other strategies aimed at the transfused recipient or aimed toward the blood product, but these will require more validation and confirmation first.
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http://dx.doi.org/10.1097/CCM.0000000000002989DOI Listing
May 2018

Mature murine megakaryocytes present antigen-MHC class I molecules to T cells and transfer them to platelets.

Blood Adv 2017 Sep 8;1(20):1773-1785. Epub 2017 Sep 8.

Toronto Platelet Immunobiology Group, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, ON, Canada.

Megakaryocytes (MKs) are bone marrow-derived cells that are primarily responsible for generating platelets for the maintenance of hemostasis. Although MK can variably express major histocompatibility complex (MHC) class I and II molecules during their differentiation, little is known whether they can elicit nonhemostatic immune functions such as T-cell activation. Here, we demonstrate that mature CD34 MHC class II CD41 MKs can endocytose exogenous ovalbumin (OVA) and proteolytically generate its immunogenic peptide ligand, which is crosspresented on their surface in association with MHC class I molecules. This crosspresentation triggered in vitro and in vivo OVA-specific CD8 T-cell activation and proliferation. In addition, the OVA-MHC class I complexes were transferred from MK to pro-platelets upon thrombopoiesis in vitro. MK could also present endogenous MK-associated (CD61) peptides to activate CD61-specific CD8 T cells and mediate immune thrombocytopenia in vivo. These results suggest that, in addition to their hemostatic role, mature MKs can significantly affect antigen-specific CD8 T-cell responses via antigen presentation and are able to spread this immunogenic information through platelets.
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http://dx.doi.org/10.1182/bloodadvances.2017007021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728336PMC
September 2017

Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets.

Am J Physiol Lung Cell Mol Physiol 2017 05 10;312(5):L625-L637. Epub 2017 Mar 10.

Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada;

Pulmonary complications from stored blood products are the leading cause of mortality related to transfusion. Transfusion-related acute lung injury is mediated by antibodies or bioactive mediators, yet underlying mechanisms are incompletely understood. Sphingolipids such as ceramide regulate lung injury, and their composition changes as a function of time in stored blood. Here, we tested the hypothesis that aged platelets may induce lung injury via a sphingolipid-mediated mechanism. To assess this hypothesis, a two-hit mouse model was devised. Recipient mice were treated with 2 mg/kg intraperitoneal lipopolysaccharide (priming) 2 h before transfusion of 10 ml/kg stored (1-5 days) platelets treated with or without addition of acid sphingomyelinase inhibitor ARC39 or platelets from acid sphingomyelinase-deficient mice, which both reduce ceramide formation. Transfused mice were examined for signs of pulmonary neutrophil accumulation, endothelial barrier dysfunction, and histological evidence of lung injury. Sphingolipid profiles in stored platelets were analyzed by mass spectrophotometry. Transfusion of aged platelets into primed mice induced characteristic features of lung injury, which increased in severity as a function of storage time. Ceramide accumulated in platelets during storage, but this was attenuated by ARC39 or in acid sphingomyelinase-deficient platelets. Compared with wild-type platelets, transfusion of ARC39-treated or acid sphingomyelinase-deficient aged platelets alleviated lung injury. Aged platelets elicit lung injury in primed recipient mice, which can be alleviated by pharmacological inhibition or genetic deletion of acid sphingomyelinase. Interventions targeting sphingolipid formation represent a promising strategy to increase the safety and longevity of stored blood products.
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http://dx.doi.org/10.1152/ajplung.00317.2016DOI Listing
May 2017

T regulatory cells and dendritic cells protect against transfusion-related acute lung injury via IL-10.

Blood 2017 05 15;129(18):2557-2569. Epub 2017 Feb 15.

Keenan Research Centre for Biomedical Science and.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following blood transfusion. Donor antibodies are frequently involved; however, the pathogenesis and protective mechanisms in the recipient are poorly understood, and specific therapies are lacking. Using newly developed murine TRALI models based on injection of anti-major histocompatibility complex class I antibodies, we found CD4CD25FoxP3 T regulatory cells (Tregs) and CD11c dendritic cells (DCs) to be critical effectors that protect against TRALI. Treg or DC depletion in vivo resulted in aggravated antibody-mediated acute lung injury within 90 minutes with 60% mortality upon DC depletion. In addition, resistance to antibody-mediated TRALI was associated with increased interleukin-10 (IL-10) levels, and IL-10 levels were found to be decreased in mice suffering from TRALI. Importantly, IL-10 injection completely prevented and rescued the development of TRALI in mice and may prove to be a promising new therapeutic approach for alleviating lung injury in this serious complication of transfusion.
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http://dx.doi.org/10.1182/blood-2016-12-758185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418638PMC
May 2017

Anesthesia for Complex Cardiovascular Surgery in a Patient With PHACES Syndrome and Review of the Literature.

J Cardiothorac Vasc Anesth 2017 06 11;31(3):1042-1047. Epub 2016 Jul 11.

Department of Anesthesia, University of Toronto, Toronto, ON, Canada; Department of Diagnostic Imaging, The Hospital for Sick Children, Toronto, ON, Canada.

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http://dx.doi.org/10.1053/j.jvca.2016.07.010DOI Listing
June 2017

Microparticles as biomarkers of lung disease: enumeration in biological fluids using lipid bilayer microspheres.

Am J Physiol Lung Cell Mol Physiol 2016 05 4;310(9):L802-14. Epub 2016 Mar 4.

Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada; Departments of Physiology, Surgery, University of Toronto, Toronto, Ontario, Canada; Institute of Physiology, Charité-Universitätsmedizin Berlin, Germany; and German Heart Institute, Berlin, Germany.

Extracellular vesicles, specifically microparticles (MPs), are rapidly gaining attention for their capacity to act as biomarkers for diagnosis, prognosis, or responsiveness to therapy in lung disease, in keeping with the concept of precision medicine. However, MP analysis by high-sensitivity flow cytometry (FCM) is complicated by a lack of accurate means for MP enumeration. To address this gap, we report here an enhanced FCM MP gating and enumeration technique based on the use of novel engineered lipid bilayer microspheres (LBMs). By comparison of LBM-based MP enumeration with conventional bead- or fluorescent-based FCM enumeration techniques and a gravimetric consumption gold standard, we found LBMs to be superior to commercial bead preparations, showing the smallest fixed bias and limits of agreement in Bland Altman analyses. LBMs had simultaneous capacity to aid FCM enumeration of MPs in plasma, BAL, and cell culture supernatants. LBM enumeration detected differences in MP counts in mice exposed to intraperitoneal lipopolysaccharide or saline. LBMs provided for 1) higher sensitivity for gating MPs populations, 2) reduced background within MP gates, 3) more appropriate size, and 4) an inexpensive alternative amenable to different fluorescent tags. LBM-based MP enumeration was useful for a series of different FCM systems assessed, whereas LBM gating benefited high- but not low-sensitivity FCM systems compared with fluorescence gating. By offering exclusive advantages over current means of gating and enumerating MPs, LBMs are uniquely suited to realizing the potential of MPs as biomarkers in biological lung fluids and facilitating precision medicine in lung disease.
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http://dx.doi.org/10.1152/ajplung.00369.2015DOI Listing
May 2016

Microparticles as biomarkers of lung disease: enumeration in biological fluids using lipid bilayer microspheres.

Am J Physiol Lung Cell Mol Physiol 2016 05 4;310(9):L802-14. Epub 2016 Mar 4.

Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada; Departments of Physiology, Surgery, University of Toronto, Toronto, Ontario, Canada; Institute of Physiology, Charité-Universitätsmedizin Berlin, Germany; and German Heart Institute, Berlin, Germany.

Extracellular vesicles, specifically microparticles (MPs), are rapidly gaining attention for their capacity to act as biomarkers for diagnosis, prognosis, or responsiveness to therapy in lung disease, in keeping with the concept of precision medicine. However, MP analysis by high-sensitivity flow cytometry (FCM) is complicated by a lack of accurate means for MP enumeration. To address this gap, we report here an enhanced FCM MP gating and enumeration technique based on the use of novel engineered lipid bilayer microspheres (LBMs). By comparison of LBM-based MP enumeration with conventional bead- or fluorescent-based FCM enumeration techniques and a gravimetric consumption gold standard, we found LBMs to be superior to commercial bead preparations, showing the smallest fixed bias and limits of agreement in Bland Altman analyses. LBMs had simultaneous capacity to aid FCM enumeration of MPs in plasma, BAL, and cell culture supernatants. LBM enumeration detected differences in MP counts in mice exposed to intraperitoneal lipopolysaccharide or saline. LBMs provided for 1) higher sensitivity for gating MPs populations, 2) reduced background within MP gates, 3) more appropriate size, and 4) an inexpensive alternative amenable to different fluorescent tags. LBM-based MP enumeration was useful for a series of different FCM systems assessed, whereas LBM gating benefited high- but not low-sensitivity FCM systems compared with fluorescence gating. By offering exclusive advantages over current means of gating and enumerating MPs, LBMs are uniquely suited to realizing the potential of MPs as biomarkers in biological lung fluids and facilitating precision medicine in lung disease.
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http://dx.doi.org/10.1152/ajplung.00369.2015DOI Listing
May 2016

IL-7 receptor recovery on CD8 T-cells isolated from HIV+ patients is inhibited by the HIV Tat protein.

PLoS One 2014 17;9(7):e102677. Epub 2014 Jul 17.

Ottawa Hospital Research Institute, Chronic Disease, Ottawa, Ontario, Canada; Division of Infectious Diseases, Ottawa Hospital General Campus, Ottawa, Ontario, Canada; Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.

Expression of the IL-7 receptor α-chain (CD127) is decreased on CD8 T-cells in HIV infected patients and partially recovers in those receiving antiretroviral therapy with sustained viral suppression. We have shown that soluble HIV Tat protein down regulates CD127 expression on CD8 T-cells isolated from healthy HIV-negative individuals. Tat is taken up by CD8 T-cells via endocytosis, exits the endosome and then translocates to the inner leaflet of the cell membrane where it binds to the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. This down regulation of CD127 by Tat results in impaired CD8 T-cell function. Interestingly, suppression of CD127 by Tat is reversible and requires the continual presence of Tat in the culture media. We thus questioned whether the low IL-7 receptor expression evident on CD8 T-cells in HIV+ patients was similarly reversible and if suppression of the receptor could be maintained ex vivo by Tat protein alone. We show here that when CD8 T-cells isolated from HIV+ patients are incubated alone in fresh medium, low CD127 expression on the cell surface recovers to normal levels. This recovery of CD127, however, is completely inhibited by the addition of HIV Tat protein to the culture media. This study then provides evidence that soluble factor(s) are responsible for low CD127 expression on circulating CD8 T-cells in HIV+ individuals and further implicates Tat in suppressing this receptor essential to CD8 T-cell proliferation and function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102677PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102547PMC
December 2015

Soluble HIV Tat protein removes the IL-7 receptor alpha-chain from the surface of resting CD8 T cells and targets it for degradation.

J Immunol 2010 Sep 26;185(5):2854-66. Epub 2010 Jul 26.

Ottawa Hospital Research Institute.

IL-7 signaling is essential to CD8 T cell development, activation, and homeostasis. We have previously shown decreased expression of the IL-7R alpha-chain (CD127) on CD8 T cells in HIV(+) patients and that this downregulation is mediated at least in part by the HIV Tat protein. We show in this study that CD127 has a prolonged t(1/2) in resting CD8 T cells and continuously recycles on and off the cell membrane. We also demonstrate soluble Tat protein significantly decreases the t(1/2) of CD127. Soluble Tat is taken up from the medium and accumulates in CD8 T cells with a peak of 6 h. Once inside the cell, Tat exits the endosomes during their normal acidification and enters the cytosol. Tat then translocates to the inner leaflet of the cell membrane, where it binds directly to the cytoplasmic tail of CD127, inducing receptor aggregation and internalization through a process dependent on microtubules. Tat appears to then target CD127 for degradation via the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is thus able to reduce IL-7 signaling and impair CD8 T cell proliferation and function.
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http://dx.doi.org/10.4049/jimmunol.0902207DOI Listing
September 2010

Effects of dietary fats and proteins on rat testicular steroidogenic enzymes and serum testosterone levels.

Food Chem Toxicol 2008 Jan 11;46(1):259-69. Epub 2007 Sep 11.

Toxicology Research Division, Health Products and Foods Branch, Food Directorate, Health Canada, Sir Fredrick G. Banting Research Centre, 2202D1 Tunney's Pasture, Ottawa, ON, Canada K1A 0L2.

It is known that certain dietary fats can modulate rat testosterone metabolism. In the current study we have investigated testicular steroidogenic enzyme activities and serum testosterone levels in rats fed diets containing either different protein sources (casein, fishmeal, whey) or different lipid sources (soybean oil, docosahexaenoic acid (DHA), seal oil, fish oil, lard). The diets examined reflect different marine oils and proteins which are significant components of Northern Canadian diets. Male rats (42-45 days old, 6 per group), were assigned to specific diets for 42 days. On the 43rd day of the study, rats were sacrificed and blood plasma and testes frozen (-80 degrees C) until analysis. Microsomal steroidogenic enzyme activities (3beta-HSD, 17-OHase, C-17,20-lyase, 17beta-HSD) were measured radiometrically. There were no differences in enzyme activities between the three dietary protein sources. In contrast, compared with the standard casein diet, all lipid sources caused reductions in C-17,20-lyase activity (>50%); seal oil and fish oil reduced 17-OHase activity (approximately 30%) and soybean oil, DHA fish oil and lard reduced 17beta-HSD activity (approximately 30%). No effect on 3beta-HSD activity was evident. Serum testosterone levels were determined using ELISA kits and were not affected by any diet with the exception of the soybean oil diet which was significantly elevated compared with the casein protein diet. Body and testis weights were not affected by diet. In conclusion, these data demonstrate that some dietary lipid sources caused reductions in testicular 17-OHase and C-17,20-lyase activities but not to the extent that serum T levels were affected, while soybean oil caused elevated serum testosterone in the absence of elevated steroidogenic enzyme activities.
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http://dx.doi.org/10.1016/j.fct.2007.08.045DOI Listing
January 2008

An investigation of the effects of methylmercury in rats fed different dietary fats and proteins: testicular steroidogenic enzymes and serum testosterone levels.

Food Chem Toxicol 2008 Jan 14;46(1):270-9. Epub 2007 Aug 14.

Toxicology Research Division, Health Products and Foods Branch, Food Directorate, Health Canada, Sir Fredrick G. Banting Research Centre, 2202D1 Tunney's Pasture, Ottawa, ON, Canada K1A 0L2.

Methylmercury (MeHg) is a testicular toxicant causing reduced steroidogenic enzyme activity, reduced serum testosterone (T) and abnormal spermatogenesis in mammals and fowl. It is also known that certain diets can alter androgen metabolism in rats. Previously we have shown that diets used in the current study impact circulating androgen levels and testicular steroidogenic enzyme activities in Sprague Dawley rats in the absence of MeHg. In the present study, we have investigated the impact of imposing an environmental contaminant (MeHg) commonly found in marine mammals and fish onto the rats' dietary intake of different proteins and lipids in order to determine if the different diets could modify MeHg toxicity in rats. Therefore, we examined the effects of MeHg on testicular steroidogenic enzymes and serum testosterone in rats fed diets containing either different protein sources (casein, fishmeal, whey) or different lipid sources (soybean oil, docosahexaenoic acid (DHA), seal oil, fish oil, lard). Male rats 42-45 days of age (18 per group) were assigned to different experimental diets for 28 days after which 6 rats in each group were gavaged daily with 0, 1 or 3 mg/kg body weight (BW)/day MeHg chloride in 5 mM Na(2)CO(3) solution for 14 days while being maintained on their diets. On the 43rd day of dosing, rats were sacrificed and blood plasma and testes frozen (-80 degrees C) until analysis. Microsomal steroidogenic enzyme activities (3beta-HSD, 17-OHase, C-17, 20-lyase, 17beta-HSD) were measured radiometrically. Serum testosterone was determined using ELISA kits. Testis weights were not affected by MeHg. MeHg at 3 mg/kg BW/day caused a reduction (>50%) in the activity of C-17, 20-lyase in all three protein diets and similar reductions in 17-OHase activity were seen in the casein and whey protein fed rats. At 3 mg/kg BW/day, MeHg reduced 17-OHase activity in the DHA diet but had no effect on 3beta-HSD activity and no inhibitory effects on 17beta-HSD activity. MeHg (3 mg/kg BW/day) caused significant reductions in serum T in the whey, soybean oil and fish oil groups. Interestingly, fishmeal protein but not fish oil offered some protection with respect to maintaining steroidogenic enzyme activities and serum T levels in rats dosed with MeHg. In conclusion, these studies show that different lipid diets can alter the toxic effects of MeHg on male rat steroidogenesis in terms of serum testosterone and steroidogenic enzyme activities.
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http://dx.doi.org/10.1016/j.fct.2007.08.004DOI Listing
January 2008

Interleukin-7 receptor expression on CD8 T-cells is downregulated by the HIV Tat protein.

J Acquir Immune Defic Syndr 2006 Nov;43(3):257-69

Ottawa Health Research Institute, Ottawa, Ontario, Canada.

We have previously shown decreased expression of the interleukin (IL)-7 receptor alpha-chain (CD127) on CD8 T-cells in HIV-infected patients and an apparent recovery of this receptor in those receiving antiretroviral therapy with sustained viral suppression. Here, we demonstrate that the HIV Tat protein specifically downregulates cell surface expression of CD127 on human CD8 T-cells in a dose- and time-dependent manner. The effects of Tat on CD127 expression could be blocked with anti-Tat monoclonal antibodies or by preincubating Tat with heparin. Tat had no effect on the expression of other cell surface proteins examined, including CD132, or on cell viability over 72 hours. Further, CD127 expression was not altered by other HIV proteins, including gp160 or Nef. Preincubation of purified CD8 T-cells with Tat protein inhibited CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Because IL-7 signaling is essential for optimal CD8 T-cell proliferation and function, the downregulation of CD127 and apparent inhibition of cytotoxic activity by Tat may play an important role in HIV-induced immune dysregulation and impaired cell-mediated immunity.
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http://dx.doi.org/10.1097/01.qai.0000230319.78288.f4DOI Listing
November 2006

Altered testicular microsomal steroidogenic enzyme activities in rats with lifetime exposure to soy isoflavones.

J Steroid Biochem Mol Biol 2004 Dec 21;92(5):435-46. Epub 2004 Dec 21.

Department of Cellular and Molecular Medicine and Obstetrics and Gynecology, Reproductive Biology Unit, University of Ottawa, Ont., Canada.

Androgen production in the testis is carried out by the Leydig cells, which convert cholesterol into androgens. Previously, isoflavones have been shown to affect serum androgen levels and steroidogenic enzyme activities. In this study, the effects of lifelong exposure to dietary soy isoflavones on testicular microsomal steroidogenic enzyme activities were examined in the rat. F1 male rats were obtained from a multi-generational study where the parental generation was fed diets containing alcohol-washed soy protein supplemented with increasing amounts of Novasoy, a commercially available isoflavone supplement. A control group was maintained on a soy-free casein protein-based diet (AIN93G). The diets were designed to approximate human consumption levels and ranged from 0 to 1046.6 mg isoflavones/kg pelleted feed, encompassing exposures representative of North American and Asian diets as well as infant fed soy-based formula. Activities of testicular 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17 (CYP17), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) were assayed on post natal day (PND) 28, 70, 120, 240 and 360 while 5alpha-reducatase was assayed on PND 28. At PND 28, 3beta-HSD activity was elevated by approximately 50% in rats receiving 1046.6 mg total isoflavones/kg feed compared to those on the casein only diet. A similar increase in activity was observed for CYP17 in rats receiving 235.6 mg total isoflavones/kg feed, a level representative of infant exposure through formula, compared to those receiving 0mg isoflavones from the casein diet. These results demonstrate that rats fed a mixture of dietary soy isoflavones showed significantly altered enzyme activity profiles during development at PND 28 as a result of early exposure to isoflavones at levels obtainable by humans.
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http://dx.doi.org/10.1016/j.jsbmb.2004.08.002DOI Listing
December 2004

Increased serum and testicular androgen levels in F1 rats with lifetime exposure to soy isoflavones.

Reprod Toxicol 2004 Jul;18(5):677-85

Reproductive Biology Unit, Departments of Cellular and Molecular Medicine and Obstetrics and Gynecology, University of Ottawa, Ottawa, ON, Canada.

The consequences of dietary soy isoflavones on serum and testicular androgen levels were examined in F1 male rats from a multigeneration study investigating the effects of diets varying in isoflavone content. Rats were fed either a soy-free casein based diet (AIN93G) or a diet in which alcohol-washed soy protein replaced casein as the protein source and to which increasing amounts of Novasoy, a commercially available isoflavone supplement were added. Analysis of these diets showed that the isoflavone content in each diet was 0 (diet 1; casein based control), 31.7 (diet 2; alcohol-washed soy-based diet control), 36.1 (diet 3), 74.5 (diet 4), 235.6 (diet 5) and 1046.6 (diet 6) mg total isoflavones/kg pelleted diet. The levels of isoflavones in diet 1 would represent a daily intake level of 0 mg isoflavones, diets 2 and 3 estimate a low soy-containing human diet (e.g. North American), diet 4 would correspond to Asian diets (e.g. Japanese) or adult humans taking isoflavone supplements, diet 5 approximates the isoflavone intake by babies fed soy based infant formula and diet 6 approximates fivefold the intake levels by babies or 10-fold the intake levels of adults consuming high isoflavone containing diets. Serum testosterone (T) from F1 male rats sacrificed on postnatal days (PND) 28, 70, 120, 240 and 360 were low at PND 28 (0.4 ng/ml), increased approximately five to sixfold at PND 70 (2.5-3.0 ng/ml) and thereafter declined to a steady state level of approximately 1 ng/ml by PND 120. However, rats on diets 5 and 6 demonstrated altered serum testosterone profiles such that at days 120, testosterone levels remained significantly elevated at approximately 3 ng/ml (P < 0.05). Serum dihydrotestosterone levels exhibited similar profiles and the levels in PND 120 rats on diet 5 or 6 were also significantly elevated (two to threefold, P < 0.05). The intra-testicular testosterone concentration in rats on diet 5 was also elevated at PND 120 compared with diet 1 (P < 0.05). These findings show that F1 male rats continuously exposed to a mixture of dietary soy isoflavones from conception onwards exhibit altered serum and testicular androgen profiles.
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http://dx.doi.org/10.1016/j.reprotox.2004.04.005DOI Listing
July 2004