Issue 33 by The Meducator

APRIL 2018 | ISSUE 33

EXPLORING PIECE CAUGHT THEANOTHER FLU? EOF X P LOTHE R I N G T PUZZLE H E N OV E L R O L E O F H I G H M O B I L I TY G R O U P B OX 1 P R OT E I N I N S E V E R E

ICNOFML O U ER NB ZI DA M I NEFNETA CTLI OHNE A LT H C H A L L E N G E S I N C H I L D R E N W I T H A U T I S M S P E CT R U M D I S O R D E R

THE POWER OF POOP PHYSICIAN-ASSISTED DYING F E CA L M I C R O B I O TA T RA N S P LA N TAT I O N A N E W O P T I O N I N CA N A D I A N E N D O F L I F E CA R E

BENCH TO BABY BELLY SHINING LIGHT ON BIOMEDICINE: I N T E RV I E W W I T H D R. D E B O RA H S LO B O D A, IEW R UDCEEPA WA AI NS TS EORV C I AT E PWRI OT FHEDS R.B S O R, RTI N MM E NA TN,ODFI R E CT O R O F O NAPNRDOBGIRA A NLATO BT IHOECEHDEUMCAT I S T IRY O MME DI NI CA S C IM E NYC E S

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table of

April 2018 | Issue 33

CONTENTS

1 INTRODUCTION 3 MEDPULSE 5 MEDBULLETIN 7 PATHOPROFILE 9 ABSTRACTS 10 SPOTLIGHT 11 OPINION 13 FORUMSPACE

table of contents

RESEARCH INSIGHTS 15 Caught the flu? Exploring the novel role of high mobility group box 1 protein in severe influenza infection

19 Identifying genes associated with biofilm production in Pseudomonas aeruginosa

CRITICAL REVIEW 23 Fecal microbiota

transplantation: the power of poop

M E D U CATO R

A P R I L 2018

27 INTERVIEW SPOTLIGHT 30 CONTRIBUTORS

COVER ARTIST BOB YANG TOC ARTIST SUFFIA MALIK

dear reader,

INTRODUCTION ISSUE 33

Welcome to Issue 33 of The Meducator! Over the years, the profound evolution of medical technologies has allowed for the continual transcendence of medical knowledge, transforming chronic conditions from incurable to manageable. This idea is exemplified in our cover, designed by Bob Yang, which alludes to the history of surgery in medical practice. Within its pages, our issue continues to follow this central theme with articles featuring the emerging topics of stem cell donations, fecal transplantation, and the practice of biohacking. Issue 33 begins with our MedPulse piece, in which staff editors Devin Roshan and Eva Liu collaborate with graphic designer Candy Niu to explore recent scientific breakthroughs. In Pathoprofile, Deeksha Kundapur, Sheila Yu, and Sylvia Mohanraj elucidate the physiology behind congenital insensitivity to pain. In the latest rendition of Spotlight, Angela Dong provides insight into “biohacking,” a movement that has raised significant biosecurity and ethical concerns among the general and medical populace. In the Opinion piece, members of the McMaster Stem Cell Club present an evidence-informed commentary on misconceptions associated with stem cell donations.

introduction

Continuing their biannual collaboration with The Meducator, the McMaster Health Forum examines the multifaceted implications of adverse childhood experiences in public health. We are also excited to feature two Research Insight pieces within this issue. In the first, Danny Ma explores the protein high mobility group box 1 and its role as a potent proinflammatory cytokine in influenza infections. In the second, Jessica Chee identifies a novel gene associated with Pseudomonas aeroginosa biofilm production. With their Critical Review, Eva Liu and Maggie Hou assess the impact of fecal microbiota transplantation on the field of medicine. Finally, this issue features an interview with Dr. Deborah Sloboda, who shares insights from her research on maternal and child health. Over the past year, we were able to collaborate with an incredible team who worked tirelessly to produce this publication before you. In particular, we would like to thank Adrian Marcuzzi, Cathy Lu, Owen Luo, and Valerie Kim for their outstanding work as team managers. The Meducator has been an inspiration for us over the past three years. As we step down from our roles, we would like extend a huge welcome to Owen Luo and Eva Liu, The Meducator’s incoming Editors-in-Chief. We have the utmost confidence that these two will not only uphold, but elevate, The Meducator’s continuing legacy within the McMaster community.

M E D U CATO R

All the best, Sabrina Lin & Sarah Ge

SABRINA LIN

A P R I L 2018

SARAH GE

Bachelor of Health Sciences (Honours) Class of 2019

MEDBULLETIN TOXINS

medbulletin

OUABAIN: FROM POISON TO CONTRACEPTIVE

A NEW PROMISE IN CANCER IMMUNOTHERAPY

KEVIN C HE N

J AMES YU

Despite the myriad of contraceptive methods designed for women, the options for men have generally relied on vasectomy or use of condoms. 1 Recently, however, researchers from the American Chemical Society have investigated the potential of introducing a third option based on analogues of ouabin, a plant-derived cardiovascular toxin traditionally used as a poison. 2,3

The immune system is naturally programmed to target and attack tumours using T lymphocytes. As tumours grow, however, they can inhibit immune cells and escape immune detection, creating an internal reservoir of inhibited T cells. 1 Immunotherapy, or the introduction of biological agents to boost immune function, has long been heralded as the holy grail of cancer treatment. 2 Unfortunately, many current approaches incur high costs, involve long wait-times, and present the possibility of overdriving the immune system. 3

Ouabain inhi bits Na + /K + -ATPase, a protein expre sse d in almo st al l vertebrate cel ls in various isoforms to maintain re sting membrane p otential. 4,5 The purp o se of this protein is to maintain re sting membrane p otential through so d ium/p otassium home o stasis. Of particular intere st is the α4 isoform lo cate d only in mature sp erm cel ls, where its activity maintains home o static cond itions ne ce ssary for sp erm survival. 5 Despite these attributes, ouabain remains a poor cand idate for cont raception due to serious cl inical conse quence s of Na+/K+-ATPase inhi bition, which may include hyperkalemia, cardiac arrhythmias, or death. 5 In a study publ ishe d in The Journal of Me dicinal Chemistry , 32 hetero gene ous ouabain analo gue s were create d. 2 In vitro exp eriments with rat sp erm reveale d that the analo gue with the highe st affinity for the α4 isoform cause d a re duction in the moti l ity of sp erm, hindering its abi l ity to reach the e gg. 2 The drug also de crease d hyp eractivation, compromising the sp erm’s abi l ity to p enet rate the zona p el lucida, a thick membrane surround ing the e gg. 2 Furthermore, in vivo exp erimentation reveale d that oral administ ration to rats had no toxic effe cts and re duce d b oth sp erm count and moti l ity. 2 Despite these promising results, additional experiments are required, especially considering the potentially dangerous clinical consequences of oubain and its inhibition of Na + /K + -ATPase. Further research on the oubain analogue’s safety, as well as its exact mechanism of action and its efficacy in human subjects will be needed to confirm or reject the possibility of a male contraceptive derived from this plant extract.

Using mice models, researchers at Stanford Universit y developed a combination therapy of two immune-stimulating agents to be injected into solid tumours.1 One agent, CpG, upregulates the expression of a receptor called OX40 on the sur face of specific T cells. The other agent, an anti- OX40 antibody, stimulates the OX40 receptor and activates T cells within the tumour, allowing them to destroy cancerous cells. 4 These T cells can then leave the original tumour and at tack other identical, cancerous cells in distant, untreated metastases. Remarkably, the therapy resulted in complete elimination of cancer in 87 of the 90 mice subjects and regression af ter a second injection in the three remaining mice.1 This success can be balanced with safet y, as the local administration of the treatment enables researchers to avoid overdriving the immune system and eliciting systemic side ef fects. T h e s e re s u l t s , d e s p i te t h e i r p ro m is e, m u s t co n si d e r t h e si g n i f i c a nt d i f fe re n ce s b et we e n s p o nt a n e o u s t u m o u r s a n d t ra n s p l a nte d t u m o u r s , w h i c h t h e i n i tia l ex p e r i m e nt s u s e d . 3 A l t h o u g h t h e re s e a rc h e r s eve nt u a l l y m i m i c ke d s p o nt a n e o u s t u m o u r i g e n e sis w i t h m i ce l a c k i n g a n o n co g e n e, ef fects in these animal models may not necessarily translate to humans. Nonetheless, the impressive preliminar y success achieved with this combination therapy and the well-characterized nature of its two agents have encouraged its clearance for a clinical trial of 15 patients with low-grade lymphoma.1

M E D U CATO R

A P R I L 2018

CANCER

1. 2. 3. 4. 5.

National Health Service. What is the male pill?. 11 Jul 2017. Available from: https://www.nhs.uk/conditions/contraception/male-pill/ [Accessed: 10th February 2018]. Syeda SS, Sánchez G, Hong KH, Hawkinson JE, Georg GI, Blanco G. Design, synthesis, in vitro and in vivo evaluation of ouabain analogs as potent and selective Na, K-ATPase α4 isoform inhibitors for male contraception. Journal of Medicinal Chemistry. 2018; 61(5):1800-1820. Cassels BK. Analysis of a Maasai arrow poison. Journal of Ethnopharmacology. 1985;14(2-3):273-81. Available from: doi: 10.1016/0378-8741(85)90094-7. Fürstenwerth H. Ouabain–the insulin of the heart. International Journal of Clinical Practice. 2010;64(12):1591-4. Liu J, Shapiro JI. Regulation of sodium pump endocytosis by cardiotonic steroids: molecular mechanisms and physiological implications. Pathophysiology. 2007;14(3):171-81.

1. 2. 3. 4.

Staveley-O’Carroll K, Sotomayor E, Montgomery J, Borrello I, Hwang L, Fein S et al. Induction of antigenspecific T cell anergy: An early event in the course of tumor progression. Proc Natl Acad Sci U S A. 1998;95(3):1178–83. Sagiv-Barfi I, Czerwinski DK, Levy S, Alam IS, Mayer AT, Gambhir SS et al. Eradication of spontaneous malignancy by local immunotherapy. Sci Transl Med. 2018;10(426):eaan4488. Dempke WCM, Fenchel K, Uciechowski P, Dale SP. Second- and third-generation drugs for immuno-oncology treatment—The more the better? Eur J Cancer. 2017;74:55–72. Voo KS, Bover L, Harline ML, Vien LT, Facchinetti V, Arima K et al. Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function. J Immunol. 2013;191(7):3641– 50.

ANTIBIOTICS UNEARTHING NEW ANTIBIOTICS

DIABETES

DIABETES – BLOOD, SWEAT, AND TEARS K EVIN CHEN

International ly, more than 700,000 p e ople d ie from antimicrobial-re sistant bacterial d isease s each year. 1 W ith su p e r b u gs d eve l o pin g resis t a n ce to eve n th e a ntibiotic of las t res o r t , va n co mycin , s cie ntis t s a re racin g to ex p a n d o ptio ns . Cu r re ntl y, res ea rch is inves tigatin g th e p ote ntia l of a ntibiotic s d e r ive d fro m natu ra l p ro d u c t s of b ac te r ia residin g within p etr i dish es . H oweve r, th e dive rsit y of bacteria being examined within cultures is limited, and rediscovery rates for antibiotics are high. 2

Diabetes mellitus (DM), a metabolic disorder characterized by irregular insulin production or insulin resistance, affects over 350 million people worldwide. 1 Diabetic individuals must monitor their blood glucose levels several times a day, using a process that commonly involves pricking the skin and obtaining a small blood sample to test. Although effective, the frequency of self-monitoring has been reported to decrease over time, largely due to inconvenience. 2 Hence, there is a need for the development of a need to develop more pragmatic and continuous monitoring methods.

Currently, researchers from Rockefeller University are extracting antibiotics from soil microorganisms. 3 Since only 1% of bacterial species in soil can be cultured in labs, the team has developed a method to extract and sequence DNA from soil without growing bacteria. 4 Specifically, the researchers inserted clusters corresponding to the production of antibiotics into bacteria; with this method, they discovered malacidin - a novel molecule similar to daptomycin. Experiments with Staphylococcus aureus (MRSA), a superbug resistant to many antibiotics, detected no resistance to malacidin. In fact, this antibiotic completely eliminated MSRA in rat subjects. 3 Although malacid in is a promising solution for cont rol l ing gram-p o sitive bacteria, it is ineffe ctive against gram-ne gative bacteria, which are re sp onsi ble for pneumonia and Lyme d isease, for instance. New anti biotics for gram-ne gative b a c te r ia n e e d e d , e s p e cia l l y b e c a u s e t h e i r re l ati ve l y i m p e r m e a b l e ce l l wa l l g ra nt s a l l ow s t h e m to b e m o re a nti b i oti c re sis t a nt . 3 A l t h o u g h i t is i m p o s si b l e to co n c l u d e w h et h e r m a l a ci d i n w i l l b e c l i n i c a l l y u s ef u l , t h e m et h o d o l o g y of this study may prove valuable to other scientists searching for antibiotics in unexplored environments, from deserts to oceans.

1. 2. 3. 4. 5.

Lotfy M, Adeghate J, Kalasz H, Singh J, Adeghate E. Chronic complications of diabetes mellitus: A mini review. Current Diabetes Reviews. 2017;13(1):3-10. Peel E, Douglas M, Lawton J. Self monitoring of blood glucose in type 2 diabetes: longitudinal qualitative study of patients’ perspectives. BMJ. 2007;335(7618):493. Liu Q, Liu Y, Wu F, Cao X, Li Z, Alharbi M et al. Highly sensitive and wearable In2O3 nanoribbon transistor biosensors with integrated on-chip gate for glucose monitoring in body fluids. ACS nano. 2018;12(2):1170-1178. Shockley W. A unipolar” field-effect” transistor. Proceedings of the IRE. 1952;40(11):1365-76. Rouse, M. Field-Effect Transistor (FET). 2005. Available from: http://whatis.techtarget.com/definition/field-effect-transistor-FET [Assessed 2nd February 2018].

| A P R I L 2018

2. 3.

O ’neill J. Tackling drug-resistant infections globally: final report and recommendations the review on antimicrobial resistance. 2016; Available from: https://amr-review.org/sites/default/files/160518_ Final paper_with cover.pdf [Assessed 2nd February 2018]. Reddy BVB, Kallifidas D, Kim JH, Charlop-Powers Z, Feng Z, Brady SF. Natural Product Biosynthetic Gene Diversity in Geographically Distinct Soil Microbiomes. Appl Environ Microbiol. 2012;78(10):3744–52. Hover BM, Kim S-H, Katz M, Charlop-Powers Z, Owen JG, Ternei MA et al. Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Grampositive pathogens. Nat Microbiol. 2018;1. Tringe SG, von Mering C, Kobayashi A, Salamov AA, Chen K, Chang HW et al. Comparative Metagenomics of Microbial Communities. Science. 2005;308(5721):554–7.

In a potentially revolutionary advancement, however, researchers have developed a new indium oxide nanoribbon field-effect transistor (FET) biosensor. 3 Used to amplify weak signals, a FET is a special type of transistor in which current flow is modulated by a transverse electric field and used to amplify weak signals. 4,5 Incorporating an on-chip gold side gate electrode, a natural chitosan film, and carbon nanotubes, this new device relies on the reactions between glucose in the fluid sample and glucose oxidase in the biosensor; the ensuing process produces an electrical signal that can be analyzed by the sensor for the detection of glucose concentrations from 10 nanomolar to 1 millimolar in the sweat, saliva, and tears of both diabetic and non-diabetic individuals. As researchers modify the design of the biosensor to fit the surface of an artificial eye and an artificial arm, this technology may be potentially worn as contact lenses or skin patches for the continuous monitoring of glucose levels. 3

M E D U CATO R

In recent years, the advent of wearable biosensor technology has allowed users to monitor their heart rates and physical activity levels more easily, even though current commercially available wearables are still unable to monitor the body ’s status on a molecular level. 3

medbulletin

JA ME S YU

CONGENITAL INSENSITIVITY TO PAIN Pathoprofile AUTHORS: Deeksha Kundapur & Sheila Yu ARTIST: Sylvia Mohanraj

INTRODUCTION

GENERAL NOCICEPTION

Pain is an essential sensation that has developed in complex organisms as an evolutionary mechanism to signal impending danger. Without pain, day-to-day functions become incredibly compromised. Pain is responsible for triggering the adoption of protective behaviours, such as physical withdrawal from painful stimuli to for tissue protection.1 Hereditary sensory and autonomic neuropathy type V (HSAN V), generally known as congenital insensitivity to pain (CIP), is a rare autosomal recessive sensory neuropathy. It is caused by defective nociceptive mechanisms, which result in an inability to experience pain.2,3 HSANs range from type I to type V, encompassing varying degrees of sensory impairment at each level. Only a handful of individuals experience the clinical symptoms associated with HSAN V, as it is the rarest of the HSAN disorders.2

Pain is perceived through nociception: central and peripheral neurological events that encode information regarding noxious stimuli. When the intensity of a stimulus reaches the noxious threshold, primary nociceptive peripheral nerve endings located in the skin, muscles, visceral tissue, corneas, and mucosa depolarize to send signals to their cell bodies in the dorsal root ganglia. There, primary nociceptive neurons synapse with interneurons, which transmit the neural impulse to the thalamus and cortex for pain perception. A spinal nerve contains different types of pain fibres, which are categorized based on anatomical and functional criteria. Of particular interest are the thinly myelinated C fibres, which conduct rapid, sharp and acute pain, and the thicker, unmyelinated Aẟ fibres, which facilitate delayed, diffused, and dull pain.1,4

To thalamus and cortex

Acute pain Secondary sensory neuron

Aẟ ﬁbre

C ﬁbre Dorsal root ganglion Dull pain

Spinal cord cross-section

MOLECULAR MECHANISMS Neuron growth, including that of Aẟ and C fibres, is stimulated by NGFβ, a protein encoded by the NGF gene (1p13.2).2,5 NGFβ regulates the transduction of anti-apoptotic and differentiative signals by binding to the high affinity TrkA receptor and the low-affinity p75NTR receptor; these form a co-receptor system that positively modulates neuronal growth.6 More specifically, the binding of NGFβ to TrkA triggers the phosphorylation of PLC-γ1 and RAS/ERK, initiating signalling pathways necessary for cell growth, survival, proliferation, and differentiation. Additionally, the binding of NGFβ to p75NTR activates JNK/C-jun, resulting in a cascade that transmits noxious stimuli.6-8 In contrast to the normal nociceptive mechanism described above, HSAN V is characterized by mutations in the NGF gene, producing mutant NGFβ that does not undergo normal post-translational processing events.2,9 As a result, it remains as immature proNGFβ that binds to a related, but different co-receptor system: p75NTR and sortilin.9 The activation of this system triggers a signal cascade responsible for apoptosis, which, in the absence of cell survival and growth factors, results in neurodegeneration. Nerve biopsies demonstrate that the resulting loss of the aforementioned Aẟ and C fibres leads to a significant loss in pain perception.10

CLINICAL IMPLICATIONS With their inability to experience pain, patients with HSAN V are more vulnerable to wounds, injuries, and self-mutilating behaviour, which may result in autoamputation in extreme cases.6 Additionally, the absence of deep pain perception—the feeling of pain from injuries to bones, ligaments, or muscles—can lead to fractures, bone necrosis, osteochondritis, and osteomyelitis, ultimately necessitating neuropathic arthroplasty.10 Less common symptoms include anhidrosis (the inability to sweat), fevers, and mental retardation. This latter group of symptoms occurs primarily in cases that symptomatically overlap with HSAN IV, otherwise known as CIP with anhidrosis.11

Current treatments for CIP are largely supportive and oriented to prevent self-mutilation or to manage orthopedic problems. Examples may include continual monitoring for injuries, special footwear for orthopedic complications, and surgery. However, many difficulties arise when treating CIP patients. For instance, due to their absence of pain awareness, they lack incentive to wear braces or other orthoses.12 Despite the severity of the disease, there is no effective standard for CIP treatment, in part due to the limited amount of research that has been conducted regarding the condition. Additionally, the poor delineation between HSAN IV and HSAN V has further complicated the diagnosis and treatment of each condition.3

L MA Unaffected NGF gene

Mutant NGF gene

NGFβ protein

proNGFβ protein

NGFβ/TrkA/p75NTR co-receptor system

proNGFβ/sortilin/p75NTR co-receptor system

Neuronal cell growth and survival

Neuronal cell death including C and Aẟ ﬁbres

FUTURE RESEARCH In order to achieve a more thorough understanding of NGF/proNGF receptor distribution and function, a more accurate model for HSAN V is needed, as existing mouse models lack validity. Compared to humans, current NGF knockout mice exhibit more severe phenotypes, as demonstrated by the massive loss of sensory fibres and the higher risk of death within a few weeks of birth.13,14 A more accurate model for testing NGF function is crucial to a better understanding of CIP and its consequences.6 Despite our general aversion to pain, the sensation of pain is a basic physiological necessity. Those with CIP are deprived of the sensory input necessary to navigating the dangers of their environment, leaving them unable to identify—let alone avoid—these threats. With an improved in vivo model, research may further inform the development of pharmacological treatments or genetic therapies as early interventions for CIP, ultimately helping to prevent or better manage its harmful and long-lasting effects.

REVIEWED BY DR. ERIC SEIDLITZ Dr. Seidlitz is both a researcher and educator, with specific interest in the fields of pain, novel cancer therapeutics, and drug development. He completed his Doctorate in Physiology and Pharmacology at McMaster University, where he is now a faculty member.

REFERENCES 1. 2.

Basbaum AI, Bautista DM, Scherrer G, Julius D. Cellular and molecular mechanisms of pain. Cell. 2009;139(2):267–84. Einarsdottir E, Carlsson A, Minde J, Toolanen G, Svensson O, Solders G. A mutation in the nerve growth factor beta gene (NGFB) causes loss of pain perception. Human Molecular Genetics. 2004;13(8):799–805. Carvalho OP, Thornton GK, Hertecant J, Houlden H, Nicholas AK, Cox JJ. A novel NGF mutation clarifies the molecular mechanism and extends the phenotypic spectrum of the HSAN5 neuropathy. Journal of Medical Genetics. 2011;48(2):131–5. Milner R, Doherty C. Pathophysiology of Pain in the Peripheral Nervous System. In: Tubbs RS, Rizk E, Shoja MM, Loukas M, Barbaro N, Spinner RJ, editors. Nerves and Nerve Injuries. San Diego: Academic Press; 2015. p. 3–22. Available from: https://www.sciencedirect.com/science/article/pii/ B9780128026533000506. OMIM Entry 162030 - Nerve Growth Factor NGF [Internet]. Omim.org. Available from: https://www.omim.org/en-

6. 7. 8.

10.

try/162030 [Accessed 9th February 2018]. Capsoni S. From genes to pain: nerve growth factor and hereditary sensory and autonomic neuropathy type V. European Journal of Neuroscience. 2014;39(3):392-400. Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends in Biochemical Sciences. 2011;36(6):320-8. Tang J, Zhu C, Li ZH, Liu XY, Sun SK, Zhang T et al. Inhibition of the spinal astrocytic JNK/MCP-1 pathway activation correlates with the analgesic effects of tanshinone IIA sulfonate in neuropathic pain. Journal of Neuroinflammation. 2015;12(1):57. Capsoni S, Covaceuszach S, Marinelli S, Ceci M, Bernardo A, Minghetti L et al. Taking pain out of NGF: a “painless” NGF mutant, linked to hereditary sensory autonomic neuropathy type V, with full neurotrophic activity. Public Library of Science One. 2011;6(2):e17321. Minde J, Toolanen G, Andersson T, Nennesmo I, Remahl IN, Svensson O et al. Familial insensitivity to pain (HSAN V) and a

11.

12. 13.

14.

mutation in the NGFB gene. A neurophysiological and pathological study. Muscle & Nerve. 2004;30(6):752-60. Eichler F. Hereditary sensory and autonomic neuropathies [Internet]. UpToDate. Available from: https://www.uptodate. com/contents/hereditary-sensory-and-autonomic-neuropathies [Accessed 9th February 2018]. Kalaskar R, Kalaskar A. Hereditary sensory and autonomic neuropathy type V: Report of a rare case. Contemporary Clinical Dentistry. 2015;6(1):103–6. Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armaninl MP. Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell. 1994;76(6):1001– 11. Larsson E, Kuma R, Norberg A, Minde J, Holmberg M. Nerve growth factor R221W responsible for insensitivity to pain is defectively processed and accumulates as proNGF. Neurobiology of Disease. 2009;33(2):221–8.

ABSTRACTS

2017 International Women’s and Children’s Health Conference AMPK REGULATES EXTRAVILLOUS TROPHOBLAST INVASION GURRATTAN K. CHANDHOKE1, PATRICK J.A. RODRIGUEZ1,2, ANSON CHEUNG1, SANDEEP RAHA1,2 Department of Pediatrics, McMaster University 2 The Graduate Program in Medical Sciences, McMaster University Correspondence: chandgk@mcmaster.ca

abstracts

Background

The placenta is involved in the transport and exchange of gases, nutrients, and waste products at the mother-fetus interface. During the early stages of pregnancy, trophoblasts, the primary stem cell lineage in the placenta, play a crucial role in embryo implantation and in spiral artery remodeling. Improper progression of these initial stages can lead to various adverse effects for both mother and fetus. At a cellular level, AMP-activated protein kinase (AMPK) contributes not only to the maintenance of cellular homeostasis, but also possibly to the regulation of cell invasion, an important step in spiral artery remodeling. Hypothesis

It is proposed that AMPK regulates extravillous trophoblast cell invasion.

Using a transwell invasion assay, we determined that Metformin and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) – an AMPK activator – increase trophoblast invasion. Conversely, Compound C, an AMPK inhibitor, decreases the cells’ ability to invade over a 32 hour period. Manipulation of AMPK expression via siRNA technology reduced trophoblast invasion across all of the pharmacological treatments. Altered expression of genes linked to trophoblast cell invasion, such as TIMP-1 .TIMP-2, MMP2 and MMP-9 were also observed. Conclusion

AMPK was shown to play a role in altered trophoblast invasion through various invasion markers. Future steps include evaluating integrin expression, involved in cellular movement, to further elucidae this relationship.

Method/Results

MATERNAL HIGH-FAT DIET ALTERS MATERNAL AND FETAL GLUCONEOGENESIS AT E14.5 IN THE LIVER. YU FEI XIA,1 JESSICA G. WALLACE,1 DEBORAH M. SLOBODA1-3 Department of Biochemistry and Biomedical Sciences, McMaster University 2 Department of Obstetrics and Gynecology, McMaster University 3 Department of Pediatrics, McMaster University Correspondence: xiayf@mcmaster.ca

M E D U CATO R

A P R I L 2018

Background insulin and leptin were elevated in high-fat dams. mRNA of maternal Maternal obesity is associated with increased risk of offspring metabolic cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), a key enzyme dysfunction in adulthood. Lipotoxicity, a metabolic syndrome, has been in the gluconeogenic pathway, was significantly reduced (p<0.0001) in linked with gluconeogenic changes in clinical and animal studies. The livers of high-fat dams. Maternal hepatic nuclear factor 4 alpha (HNF4α) impact of maternal obesity on fetal gluconeogenesis at embryonic day mRNA, a known transcription activator of PEPCK, was decreased (E)14.5, is unknown. We hypothesize that a maternal high fat diet (HFD) (p=0.0004), along with mRNA of the upstream insulin receptor substrate will result in increased fetal liver gluconeogenesis. 2 (IRS2) (p=0.0018) in high-fat livers. Fetal PEPCK-C (p=0.0068) and pyruvate carboxylase (p=0.0003) mRNA levels were also decreased in Methodology the high-fat livers. Four-week-old C57BL/6 genetically-standardized laboratory mice were fed a control standard purified diet (CON; 17% kcal fat) (n=7) or HFD Conclusion (60% kcal fat) (n=9) for 6 weeks prior to mating with control fed C57BL/6 Maternal HFD is associated with downregulated mRNA transcript levels of males. They were maintained on this diet throughout gestation. Observation major gluconeogenic enzymes in the maternal and fetal liver at E14.5. This of a vaginal plug confirmed pregnancy and designated E0.5. Dams were does not support the hypothesis, and is likely due to an overabundance sacrificed at E14.5, and maternal and fetal livers were collected. Transcript of triglycerides and altered maternal-fetal glucose gradient. The altered levels of key liver gluconeogenic enzymes were investigated by RT-qPCR. gluconeogenic profile in maternal and fetal liver at E14.5 may underlie the increased risk for offspring metabolic dysfunction later in life. Future Results research should investigate lipid metabolism and the expression of High-fat females weighed more than controls at the time of mating and gluconeogenic factors. throughout gestation. At E14.5, maternal blood glucose and circulating

Newsgeek. Baby in the Womb. 2017. Available from: https://www.thenewsgeeks.com/wp-content/uploads/2017/06/baby-in-the-womb-1024x500-e1502005407940.jpg [Accessed: 12th February 2018]

Biohacking: The next scientific revolution? ANGELA HONG TIAN DONG

ARTIST MATILDA KIM

Bachelor of Health Sciences (Honours) Class of 2020, McMaster University Correspondence: dongh13@mcmaster.ca

The biohacking movement was catapulted into mainstream attention when AIDS patient Tristan Roberts livestreamed a self-injection with an untested gene therapy vector.1 Biohacking, also known as Do-ItYourself Biology, is the technological manipulation of biological systems outside of traditional academic and industrial settings for the purposes of self-improvement, innovation, art, and political expression. Encompassing everything from dieting to the genetic engineering of bacteria, biohacking may seem to lack focus.3 However, the movement is unified by its ethos: to bring science into the tinkering hands of the general populace.2 Nascent and replete with biosecurity controversy, biohacking poses a challenge for policymakers.

ORIGINS

Policy approaches to biohacking have been polarizing, with extremes exemplified by Germany and the United States. Germany punishes scientific experimentation outside of registered laboratory spaces with a fine of up to $72 000 USD and 3 years in prison.10-11 This approach may be effective at popularizing community laboratories, yet critics of the German system maintain that such punitive measures deter innovation and drive amateur science underground. Meanwhile, in the U.S., the Federal Bureau of Investigation (FBI) holds a cooperative alliance with the biohacking community via a neighbourhood watch program relying on community members to report suspicious activity.10 Critics of the American system question the effectiveness of this self-monitoring. Although the Code of Conduct established by the International Association of Synthetic Biology states that safety, transparency, and peaceful purposes are amongst its core values, there is little tangible community enforcement to punish violations.11 Although the FBI also spreads awareness on legality and safety, there remains inadequate incentive to shift from garage labs to community laboratories.

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THE FUTURE OF BIOHACKING Biohacking—with its diverse, collaborative populace, renegade philosophy, and growing community—remains a conundrum for policymakers. Realizing biohacking’s potential in furthering the public good requires its proactive integration with current institutions. Ultimately, efforts to properly address pressing security and safety issues should focus on establishing more community laboratories and mandating biosafety training rather than criminalizing layperson scientific curiosity.

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In 2016, the U.S. Director of National Intelligence, James Clapper, named genetic engineering as a Weapon of Mass Destruction (WMD).7 This decision was based on concerns that allowing non-scientists to tinker with DNA without regulation and formal training could lead to contamination, accidents, or even deliberate bioterrorism. However, contrary to the notion of a rogue bioterrorist operating out of a garage lab, 92% of biohackers carry out experiments under the watchful supervision of community laboratories. 8 Biotechnology companies have also increasingly adopted protocols for screening safe custom-

POLICY APPROACHES

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SECURITY AND SAFETY

Potential plans to address safety education may include biosafety workshops, formal lab safety certification programs accessible to non-scientists, and community enforcement of safe behaviour.

Lussenhop J. Why I injected myself with an untested gene therapy. BBC. November 21 2017. Available from: http://www.bbc.com/news/worldus-canada-41990981 [Accessed 15th January 2018]. Keulartz J, van den Belt H. DIY-Bio – economic, epistemological and ethical implications and ambivalences. Life Sciences, Society and Policy. 2016;12(1). DOI:10.1186/s40504016-0039-1. Woo G. The Evolution of the Biohacking Ecosystem. TechCrunch. 2015. Available from: https://techcrunch. com/2015/11/19/the-evolutionof-the-biohacking-ecosystem/ [Accessed 15th January 2018]. Frazzetto G. The changing identity of the scientist. EMBO Reports. 2004;5(1):18-20. Available from: doi: 10.1038/sj.embor.7400061. Landrain T, Meyer M, Perez A, Sussan R. Do-it-yourself biology: challenges and promises for an open science and technology movement. Systems and Synthetic Biology. 2013;7(3):115126. DOI:10.1007/s11693-0139116-4 Mair L, Harrison P, Jönsson M, Löbel S, Nordén J, Siitonen J et al. Evaluating citizen science data for forecasting species responses to national forest management. Ecology and Evolution. 2016;7(1):368-378. DOI:10.1002/ ece3.2601 Senate Armed Services Committee. Statement for the Record Worldwide Threat Assessment of the US Intelligence Community. Government of the United States. 2016. Available from: https://www.dni.gov/files/documents/SASC_Unclassified_2016_ ATA_SFR_FINAL.pdf [Accessed 15th January 2018]. Nature. The DIY dilemma. 2013;503(7477):437-438. Available from: https://www.nature.com/ news/the-diy-dilemma-1.14240 [Accessed 15th January 2018]. Hayden EC. Keeping genes out Nature. of terrorists’ hands. 2009;461(7260):22-22. Available from: doi: 10.1038/461022a. Wolinsky H. The FBI and biohackers: an unusual relationship. EMBO Reports. 2016;17(6):793-796. Available from: doi: 10.15252/embr.201642483. DIYbio. Draft DIYbio Code of Ethics from North American Congress [Internet]. 2011. Available from: https:// diybio.org/codes/code-of-ethicsnorth-america-congress-2011/[Accessed 15th January 2018].

spotlight

The modern academic and industrial institutions’ monopoly on science has created barriers to layperson participation. Biohacking’s inclusive philosophy, however, may harken the dawn of a new era in which hands-on science is open to everyone. Since its origins in the open science movement—a societal shift towards increased layperson involvement in research—accessible resources have emerged including community laboratories, online education, and affordable equipment.4-5 This inclusiveness may enhance current science advocacy and dispel the ivory tower stereotype through its creative and interdisciplinary approach. More lay participants may also drive innovation and competition. The flip side of decentralization is that it may reduce the reliability of results obtained by untrained researchers and devalue formal scientific education. 6

ers and gene synthesis orders. 9 Despite this progress, biosafety education remains under-addressed; almost 30% of biohackers lack a post-secondary education in biology, and they often use improvised equipment. This puts them at higher risk for accidents and contamination.10-11

EDITED BY PARNIKA GODKHINDI & KEVIN ZHAO

Dispelling Potential Fears Associated with Stem Cell Donation AN EVIDENCE-BASED COMMENTARY ALEXANDER E. ANAGNOSTOPOULOS 1 , OWEN BARIBEAU 2 , ALI ESHAGHPOUR 3 , YUJIA (JULIA) GUO 2 , ANNA LEE 3 , OWEN DAN LUO 2

ABSTRACT

Bachelor of Health Sciences (Honours) Class of 2018, McMaster University Bachelor of Health Sciences (Honours) Class of 2019, McMaster University Bachelor of Health Sciences (Honours) Class of 2020, McMaster University Correspondence: luoo@mcmaster.ca

1 2 3

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opinion

An increasing number of patients require life-saving stem cell transplants, often from unrelated donors. In order to facilitate this process, bone marrow and stem cell registries have been established to genetically catalog potential donors and can be used to find matches for patients in need. Given the wide genetic variability in populations and significant ethnic disparities in donor registries worldwide, there are substantial gaps in the availability of compatible unrelated stem cell donors. Limited understanding of the procedures involved in stem cell donation, along with potential misconceptions of associated risks, may discourage prospective donors. Many people are unaware that there are two established methods for stem cell donation from adult donors, either through bone marrow harvest or—more commonly—through peripheral blood stem cell harvest. This evidence-based commentary explores these two procedures, deconstructs misconstrued fears associated with stem cell donation, and subsequently encourages readers to consider registering as stem cell donors.

Passweg JR, Halter J, Bucher C, Gerull S, Heim D, Rovó A et al. Hematopoietic stem cell transplantation: a review and recommendations for follow-up care for the general practitioner. Swiss Med Wkly. 2012;142:w13696. 2. Grulke N, Albani C, Bailer H. Quality of life in patients before and after haematopoietic stem cell transplantation measured with the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Core Questionnaire QLQ-C30. Bone Marrow Transplant. 2012;47(4):473. 3. Kopolovic I, Turner R. Donation and transplantation of allogeneic hematopoietic stem cells. CMAJ. 2011;183(17):2014. 4. Adler J. Donation & Transplantation at Canadian Blood Services. [Presentation] Nova Scotia Provincial Blood Coordinating Program. 6th November 2015. Available from: https://novascotia.ca/dhw/ nspbcp/docs/Donation-and-Transplantation-at-Canadian-Blood-Services.pdf [Accessed 3rd January 2018]. 5. Smith S. OneMatch Network Overview and Stem Cell/Bone Marrow Transplantation [Presentation]. Canadian Blood Services; Oct 2010. Available from: https:// novascotia.ca/dhw/nspbcp/docs/OneMatch-Network-Overview-and-Transplantation.pdf [Accessed 3rd January 2018]. 6. Fingrut W, Parmar S, Cuperfain A, Rikhraj K, Charman E, Ptak E, Kahlon M, Graham A, Luong S, Wang YG, Yu J. The Stem Cell Club: a model for unrelated stem cell donor recruitment. Transfusion. 2017 Dec 1;57(12):2928-36. Available from: https://www.ncbi.nlm.nih.gov/ pubmed/28944484 [Accessed 3rd January 2018]. 7. Institute for Justice. Bone Marrow Statistics. Available from: http://ij.org/ bonemarrowstatistics/ [Accessed 3rd January 2018]. 8. Onitilo AA, Lin YH, Okonofua EC, Afrin LB, Ariail J, Tilley BC. Race, education, and knowledge of bone marrow registry: indicators of willingness to donate bone marrow among African Americans and Caucasians. Transplant Proc. 2004; 36(10):3212-9. 9. Kollman C, Weis T, Switzer GE, Halet M, Kitajima D, Hegland J et al. Non-HLA barriers to unrelated donor stem cell transplantation. Bone Marrow Transplant. 2001;27(6):581. 10. Garcia MC, Chapman JR, Shaw PJ, Gottlieb DJ, Ralph A, Craig JC et al. Motivations, experiences, and perspectives of bone marrow and peripheral blood stem cell donors: thematic synthesis of qualitative studies. Biol Blood Marrow Transplant. 2013;19(7):1046-58. 11. Mclaren PJ, Hyde MK, White KM. Exploring the role of gender and risk perceptions in people’s decisions to register as a bone marrow donor. Health Educ Res. 2011;27(3):513-22.

INTRODUCTION

may be explained by 2017 data revealing that only 1% of Canadians are currently registered as stem cell donors.6 Additionally, although patients are more likely to find matches within their own ethnic group, there is a lack of ethnic diversity in donor registries, with non-caucasians comprising only 31% of the OneMatch Registry, as of January 2017. 7 As a result, the probability of finding a suitable donor varies greatly depending on patient ethnicity, from 75% for Caucasian patients to 27% for AfricanAmerican patients. 7 The resulting discrepancy highlights the need for more ethnically diverse donors.7

COMMON FEARS ASSOCIATED WITH STEM CELL DONATION

Literature reports that fear and lack of underFor patients with a range of conditions - in- standing of stem cell donation represent barriers cluding blood cancers, autoimmune disorders, for donor registration.8,9 Individuals informed and metabolic diseases - hematopoietic stem about the donation process are more likely to cell transplants may be an integral component participate as stem cell donors; thematic synof their treatment.1 Medical literature has theses of qualitative investigations on donor extensively detailed the benefits of these proce- motivations reveal that fear of invasive procedures, namely their potential to significantly in- dures is a primary concern in their decision.10 crease patient survival and long-term quality of According to psychosocial assessments of prolife2 Unfortunately, the requirement of genetic spective bone marrow donors, the possibility compatibility between donor and recipient and of postoperative paralysis or death during the the reality of related donors found by fewer operation induces substantial anxiety.11 This is than 30% of patients leave the remaining 70% evident from statements such as, “I thought of of patients to search for a compatible unrelated an operation on the spine, that I could not walk donor in a national registry.3 These donors will anymore,” or “I thought that you might have altruistically provide stem cells through one of died through it. I didn’t know that you could two methods: bone marrow transplantation make more bone marrow.” These fears are per(BMT) or peripheral blood stem cell transplan- petuated by the popular misconception that the tation (PBSC-T). In Canada, the OneMatch bone marrow is the sole source of stem cells for Stem Cell and Bone Marrow transplantation; in reality, stem cells registry collaborates with 75 can be collected from the periphery registries in 53 other counwith the more-common and less intries to facilitate hundreds of vasive method of PBSC-T.12 annual matches.4 Nonetheless, the idea of Despite these efPBSC-T elicits its own fears. forts, nearly half of The drug granulocyte colonythe Canadian pastimulating factor (G-CSF) is tients in need of stem instrumental in PBSC donation, cell transplants are for which blood is filtered through still unable to find a matchan apheresis machine to extract ing donor.5 This disconnect stem cells. Before the two to six between supply and demand hour donation procedure, G-CSF is

administered to greatly increase the count of hematopoietic stem cells in the bloodstream.13 Prospective PBSC donors are reportedly wary of side effects associated with G-CSF drug administration.14,15 Furthermore, an online survey exploring public awareness of stem cell donation found that approximately 31% of the 1,471 participants against joining a stem cell registry worried that the PBSC donation process would be painful.16

“Evidence indicates a low overall risk for bone marrow donors, suggesting that many reported fears may be exaggerated.” ADDRESSING FEARS ASSOCIATED WITH STEM CELL DONATION

The long-term health benefits of stem cell transplants, compared to those of the non-transplant alternative, are substantial, but they are unfortunately unattainable for many patients. Given that the number of Canadian patients in need of unrelated donors is only increasing, we hope that our review, with its examination of the low overall risks associated with stem cell donation and the promising outcomes for recipients, dispels potential fears associated with the procedure and inspires individuals to register as stem cell donors. In fact, the number of patients in need of unrelated donors is only increasing in Canada.27 We hope that our review outlining the low overall risks associated with stem cell donation and the promising outcomes for recipients of stem cell therapies dispels potential fears associated with donation procedures and inspires individuals to register as stem cell donors.

ARTIST MATILDA KIM EDITED BY TAKHLIQ AMIR & SHIKHA PATEL

Dr. Warren Fingrut is an internal medicine resident physician at the University of Toronto. He is the founder and director of Stem Cell Club, a non-profit organization that aims to improve membership on Canada’s stem cell donor database. He has published on topics pertaining to unrelated donor recruitment in the journals, Transfusion and Vox Sanguinis.

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REVIEWED BY DR. WARREN FINGRUT

Regardless of the method for stem cell donation, recipient prognosis significantly improves, whereas life expectancies for individuals unable to receive stem cell therapies deteriorates.25 Among a sample of adult patients with previous blood cancer who became cancer-free two years after transplantation, after transplantation, the probability of five-year survival was 89%. Additionally, for the patients who remained disease-free, rate of survival 20 years after transplantation was estimated at 80.4%.26

M E D U CATO R

Furthermore, PBSC-T employs an apheresis procedure that is well-documented to be safe with a very low risk of serious adverse effects.20 Most recipients experience short-term side effects from G-CSF injections, such as bone pain, nausea, and fatigue.18,21,22 However, the median recovery time for PBSC donors is one week, with 94% of donors fully recovering after 30 days.18 Although the long-term (>10 years) risk profile of G-CSF treatment and PBSC donation remains to be elucidated, prospective studies suggest that G-CSF does not increase the risk for leukemia or other hematopoietic malignancies

STEM CELL DONATION MATTERS

12. D’Souza A, Fretham C. Current Uses and Outcomes of Hematopoietic Cell Transplantation (HCT): CIBMTR Summary Slides. Center for International Blood & Marrow Transplant Research. 2017. Available from: http://www.cibmtr.org [Accessed 15th February 2018]. 13. Billen A, Madrigal J, Scior K, Shaw B, and Strydom A. Donation of peripheral blood stem cells to unrelated strangers: A thematic analysis. PLoS One. 2017;12(10):e0186438. 14. van Walraven SM., Ball L.M, Koopman, HM et al. Managing a dual role—experiences and coping strategies of parents donating haploidentical G-CSF mobilized peripheral blood stem cells to their children. Psycho-Oncol. 2012; 21: 168–175. 15. Pillay B, Lee, SJ, Katona, L. et al. The psychosocial impact of haematopoietic SCT on sibling donors. Bone Marrow Transplant. 2012; 47: 1361–1365. 16. Anthony Nolan Stem Cell Donation Poll. ComRes; 2011 p. 15-16. Available from: http://www.comresglobal.com/polls/anthonynolan-stem-cell-donation-poll/ [Accessed 11th February 2018]. 17. Nishimori M, Yamada Y, Hoshi K, Akiyama Y, Hoshi Y, Morishima Y et al. Health-related quality of life of unrelated bone marrow donors in Japan. Blood. 2002;99(6):1995-2001. 18. Miller JP, Perry EH, Price TH, Bolan CD, Karanes C, Boyd TM et al. Recovery and safety profiles of marrow and PBSC donors: experience of the National Marrow Donor Program. Biol Blood Marrow Transplant. 2008;14(9 Suppl):29-36. 19. Halter J, Kodera Y, Ispizua AU, Greinix HT, Schmitz N, Favre G et al. Severe events in donors after allogeneic hematopoietic stem cell donation. Haematologica. 2009;94(1):94-101. 20. Philip J, Sarkar RS, Pathak A. Adverse events associated with apheresis procedures: Incidence and relative frequency. Asian J Transfus Sci. 2013(1):37. 21. Anderlini P, Przepiorka D, Seong D, Miller P, Sundberg J, Lichtiger B et al. Clinical toxicity and laboratory effects of granulocyte-colony- stimulating factor (filgrastim) mobilization and blood stem cell apheresis from normal donors, and analysis of charges for the procedures. Transfusion. 1996;36(7):590-595. 22. McCullough J, Kahn J, Adamson J, Anderlini P, Benjamin R, Confer D et al. Hematopoietic growth factors-use in normal blood and stem cell donors: clinical and ethical issues. Transfusion. 2008;48(9):2008-2025. 23. Confer D, Miller J. Long-term safety of filgrastim (rhG-CSF) administration. Br J Haematol. 2007;137(1):77-78. 24. Anderlini P, Chan F, Champlin R, Körbling M, Strom S. Long-term follow-up of normal peripheral blood progenitor cell donors treated with filgrastim: no evidence of increased risk of leukemia development. Bone Marrow Transplant. 2002;30(10):661-663. 25. Socié G, Stone JV, Wingard JR, Weisdorf D, Henslee-Downey PJ, Bredeson C et al. Longterm survival and late deaths after allogeneic bone marrow transplantation. Late Effects Working Committee of the International Bone Marrow Transplant Registry. N Engl J Med. 1999;341(1):14–21. 26. Martin PJ, Counts Jr GW, Appelbaum FR, Lee SJ, Sanders JE, Deeg HJ et al. Life expectancy in patients surviving more than 5 years after hematopoietic cell transplantation. J Clin Oncol. 2010;28(6):1011-6. 27. Allan DS, Takach S, Smith S, Goldman M. Impact of declining fertility rates in Canada on donor options in blood and marrow transplantation. Bio Blood and Marrow Transplant. 2009;15(12):1634-7.

opinion

Evidence indicates a low overall risk for bone marrow donors, suggesting that many reported fears may be exaggerated. Bone marrow donation involves the extraction of stem cells from the hip bone, rather than the spine.17 Moreover, bone marrow donation is a day surgery performed under general anaesthetic, with most donors discharged from the hospital within 24 hours.17,18 An investigation into the major adverse events associated with bone marrow donation revealed only one death and zero cases of paralysis in a large sample of 27,770 donors.19 Although pain at the anatomical site of donation is self-reported by 82% of donors, this symptom is amenable to anti-inflammatory drug therapy, with a median recovery time of three weeks.17,18 This evidence suggests that the risks associated with bone marrow donation are overall low.

relative to unexposed controls.23,24

FORUMSPACE 1.

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Kuh D and Ben-Shlomo Y. A life course approach to chronic disease epidemiology. New York: Oxford University Press; 1997. Nurius PS, Green S, Logan-Greene P, and Borja S. Life course pathways of adverse childhood experiences toward adult psychological wellbeing: A stress process analysis. Child Abuse & Neglect. 2015;45:143–53. Denese A and McEwan B. Adverse childhood experiences, allostasis, allostatic load, and age-related disease. Physiology and Behaviour. 2012;106(1):29–39. Dube SR, Anda RF, Felitti VJ, Chapman DP, Williamson DF, and Giles WH. Childhood abuse, household dysfunction, and the risk of attempted suicide throughout the life span: Findings from the adverse childhood experiences study. JAMA. 2001;286(24):3089. McDonald S and Tough S. The 2013 Alberta Adverse Childhood Experiences (ACE) study - key findings [Internet]. 2013. Available from: http://www.foothillsnetwork.ca/ files/file/2015-06jun-10-sheilamcdonald-and-suzanne-tough-abace-presentat.pdf [Accessed 13th February 2018]. Centers for Disease Control. Adverse Childhood Experiences - looking at how ACEs affect our lives & society [Internet]. 2014. Available from: https://vetoviolence.cdc.gov/apps/ phl/resource_center_infographic. html [Accessed 13th February 2018]. Bellis M, Hughes K, Hardcastle K, Ashton K, Ford K, Quigg Z et al. The impact of adverse childhood experiences on health service use across the life course using a retrospective cohort study. Journal of Health Services Research & Policy. 2017;22(3):168–77. Merrick M, Leeb R, and Lee R. Examining the role of safe, stable, and nurturing relationships in the intergenerational continuity of maltreatment. Journal of Adolescent Health. 2013;53(5):1-3. Watamura S, Phillips D, Morrissey T, McCartney K, and Bub K. Double jeopardy: poorer social-emotional outcomes for children in the NICHD SECCYD experiencing home and child-care environments that confer risk. Child Development. 2011;82(1):48-65. Prinz R, Sanders M, Shapiro C, Whitaker D, and Lutzker J. Population-based prevention of child maltreatment: The U.S. Triple P system population trial. Prevention Science. 2009;10(1):1-12. Available from: doi: 10.1007/s11121-009-0123-3. Metzler M, Merrick M, Klevens J, Ports K, and Ford D. Adverse childhood experiences and life opportunities: Shifting the narrative. Children and Youth Services Review. 2017; 72:141–149. Haglund M, Nestadt P, Cooper N, Southwick S, and Charney D. Psychobiological mechanisms of resilience: Relevance to prevention and treatment of stress-related psychopathology. Development and Psychopathology. 2007;19(3):889–920. Soleimanpour S, Geierstanger S, and Brindis C. Adverse childhood experiences and resilience: Addressing the unique needs of adolescents. Academic Pediatrics. 2017;17(7):S108–S114. Evans G and Schamberg M. Childhood poverty, chronic stress, and adult working memory. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(16):6545-6549.

Adverse Childhood Experiences: A Public Health Challenge FANNY CHENG1 , ANGEL HUANG 2 , PURU PANCHAL 1 , SANDY TAT 3

Bachelor of Health Sciences (Honours) Class of 2019, McMaster University 2 Bachelor of Arts and Science Class of 2020, McMaster University 3 Honours Bachelor of Health Sciences in Biomedical Discovery and Commercialization Class of 2018, McMaster University Correspondence: panchalp@mcmaster.ca 1

INTRODUCTION The life-course approach to health and the critical period model of disease causation posit that adult health is shaped by experiences in early life, specifically during critical developmental periods.¹ Adverse Childhood Experiences (ACEs) are stressful contextual exposures faced in childhood and adolescence that can have lifelong health effects.² Examples of ACEs include physical abuse, emotional neglect, parental substance abuse, and economic hardship.² ACEs elevate levels of stress hormones and impair homeostatic feedback mechanisms, negatively affecting neurodevelopment, as well as emotional and social well-being.³ As a result, ACEs are primordial risk factors for poor mental health outcomes in adulthood, such as depression.³ Individuals with seven or more ACEs are over 50 times more likely to attempt suicide than those without ACEs.4 ACEs also increase the risk of developing many physical chronic diseases later in life, including diabetes, cancer, and cardiovascular disease.4

The McMaster Health Forum strives to be a leading hub for improving health outcomes at the regional and provincial levels in Canada. Through problem-solving and discussion, they harness information, convene stakeholders, and prepare action-oriented leaders to meet pressing health issues creatively.

profound public health implications of ACEs, systems-level strategies are required to reduce future disease burden and healthcare demands.

SOCIAL SYSTEM RESPONSES Social interventions play an important role in preventing and minimizing the impact of ACEs. Early childhood adversity is cyclical in nature; parents with ACEs often have children who experience ACEs as well.8 High-quality childcare, characterized by well-educated, emotionally-sensitive caregivers in wellresourced environments with higher staff-child ratios, can mitigate the intergenerational nature of ACEs.9 For instance, the Triple P-Positive Parenting Program, a parenting support system that prevents and treats behavioural problems in children, has been shown to reduce child abuse and foster care placements.10

Integrating trauma-informed and resiliencebuilding practices into social services and school settings may address the detrimental behavioural outcomes of ACEs, which are often trauma-related coping mechanisms.11 By Over half of all Canadians and nearly two- utilizing trauma-sensitive language to maintain thirds of all Americans have at least one ACE, an empowering environment and promote and in the United States, it is estimated that cognitive reframing (e.g. using the term “survivor” ACEs place a $25 billion burden on annual instead of “victim”), these programs build public health spending.5,6 Individuals with more resilience and improve emotional regulation than one ACE use emergency care services up skills to help those with ACEs successfully to 29% more than the general population.7 manage stressors.12,13 Given the complex environmental origins and Long-term results will require interventions

CONCLUSION

Systems-level interdisciplinary interventions may also be effective in addressing ACEs. These include reducing prenatal and early childhood adversities through effective public health campaigns and school curricula that target sources of family stress.21 Additionally, preventative measures, such as increasing LEGAL SECTOR RESPONSES screening through trauma-informed primary care and improving timely access to mental Individuals within the criminal justice system health specialists, may decrease the costs tend to have experienced more ACEs. A study of of ACE-related illnesses in adulthood by over 64,000 Florida juvenile offenders revealed addressing unmet developmental needs.22 The that they typically experienced three times Washington State Family Policy Council’s more ACEs than youth not in contact with the community development strategy has been justice system.19 Another study indicated that particularly effective at integrating these approximately 90% of juvenile detainees have practices. In this initiative, families’ medical, experienced at least one ACE.20 Also, a strong social service, and educational needs are provided correlation exists between certain ACEs and by 42 community public health networks.23 criminal offenses. For example, youth who have Leaders within local government, business, experienced physical abuse are more likely to be faith-based, and neighborhood organizations violent and to damage property.19 Additionally, have improved juvenile adjudication by formally involvement in the justice system can itself serve as recognizing ACE status during legal proceedings, an ACE, contributing to cycles of unemployment, advocating for changes in school disciplinary poverty, homelessness, and repeated convictions.20 and suspension procedures, and arranging crisis Despite the strong association between ACEs nursing facilities to stop the intergenerational and criminal offense, programs recognising and propagation of ACEs.23,24 With these networks, addressing ACEs remain absent in juvenile and Washington State has saved over $50 million in criminal justice systems. the span of two years.23,24

ACEs represent a pressing health and social systems challenge that is further complicated by the stigma surrounding childhood adversities. Moving forward, decision-makers must coordinate strategies across social, legal, and health sectors to lessen the extensive individual and public harms associated with ACEs.

More discussions on current healthcare topics are available at http://www.mcmasterhealthforum.org/

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Acknowledging the role of ACEs within the context of the justice system may mitigate their social consequences and their role in future criminal behaviour. Evidence suggests that it is important for justice system employees to understand that survivors of ACEs often use maladaptive or antisocial behaviours to cope with stress.19 These tendencies are unlikely to dissipate without targeted intervention.19 Screening for ACEs may therefore help identify

MULTI-SECTOR RESPONSES INVOLVING THE HEALTH SYSTEM

15. Evans G and Kim P. Multiple risk exposure as a potential explanatory mechanism for the socioeconomic status-health gradient. Annals of the New York Academy of Sciences. 2010;1186:174–189. 16. Harper C, Marcus R, and Moore K. Enduring poverty and the conditions of childhood: Lifecourse and intergenerational poverty transmissions. World Development. 2003;31(3):535-554. 17. Chartier M, Walker J, and Naimark B. Separate and cumulative effects of adverse childhood experiences in predicting adult health and health care utilization. Child Abuse & Neglect. 2010;34(6):454-464. 18. Saxena S, Jane-Llopis E, and Hosman C. Prevention of mental and behavioural disorders: implications for policy and practice. World Psychiatry. 2006;5(1):5–14. Available from: https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC1472261/ [Accessed 13th February 2018]. 19. Epps N, Baglivio MT, Swartz K, and Hardt NS. The prevalence of adverse childhood experiences (ACE) in the lives of juvenile offenders. Journal of Juvenile Justice. 2014;3(2):12-34. Available from: https://www.ncjrs. gov/pdffiles/246951.pdf [Accessed 13th February 2018]. 20. The Illinois ACEs Response Collaborative. Justice brief: juvenile and criminal justice systems. [Internet]. 2012. Available from: http://www.hmprg. org/assets/root/ACEs/Justice%20 Policy%20Brief.pdf [Accessed 10th February 2018]. 21. National Scientific Council on the Developing Child. The foundations of lifelong health are built in early childhood [Internet]. 2010. Available from: http://developingchild.harvard.edu/ wp-content/uploads/2010/05/ Foundations-of-Lifelong-Health.pdf [Accessed 13th February 2018]. 22. World Health Organization. Addressing adverse childhood experiences to improve public health: expert consultation [Internet]. 2009. Available from: http://www.who.int/ violence_injury_prevention/violence/ activities/adverse_childhood_experiences/global_research_network_may_2009.pdf [Accessed 13th February 2018]. 23. Hall J, Porter L, Longhi D, BeckerGreen J, and Dreyfus S. Reducing adverse childhood experiences (ACE) by building community capacity: A summary of Washington family policy council research findings. Journal of Prevention & Intervention in the Community. 2012;40(4):325–34.

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those who need trauma-informed treatments and case-appropriate considerations.20

forumspace

that address the social determinants of health. Social and economic disadvantage is heavily correlated with early life adversity.14 Compared to their more affluent peers, children below or near the poverty line are over twice as likely to experience three or more ACEs.14 By addressing poverty, structural social policies can promote safe and healthy home environments for children, and reduce ACEs related to homeless and community violence.11,15-16 This serves to reduce the intergenerational propagation of ACEs.17,18

ARTIST PERI REN

RESEARCH INSIGHT

CAUGHT THE FLU?

Exploring the novel role of high mobility group box 1 protein in severe influenza infection

DANNY MA

Bachelor of Health Sciences (Honours) Class of 2019, McMaster University Correspondence: mad1@mcmaster.ca

ABSTRACT

Severe influenza infections are a common cause of acute respiratory distress syndrome (ARDS). ARDS is characterized by acute lung injury as well as pulmonary edema or “leakage,” which is caused by endothelial dysfunction and subsequent alveolar flooding from circulating blood vessels. High mobility group box 1 (HMGB1) is a protein released from lung endothelial cells that acts as a proinflammatory cytokine during bacterial and viral infections. However, the effect of HMGB1 on the endothelium during influenza viral infection is unknown. This study demonstrates that HMGB1 released from primary human microvascular lung endothelial cells (HLMECs) during influenza infection increases endothelial activation and apoptosis, while also disrupting adherens junctions between cells. Sequestering extracellular HMGB1 using the small molecule glycyrrhizin (GCZ) attenuates these effects, suggesting that HMGB1 signaling occurs in a paracrine manner. Inhibition of the Toll-like receptor (TLR) 4 pathway, a downstream effector of HMGB1, with peptide P5779 did not substantially decrease endothelial apoptosis. Therefore, HMGB1 likely elicits its effects on the endothelium in a TLR4-independent manner. These results will elucidate a partial mechanism that potentially explains ARDS symptoms seen in patients with severe influenza infections.

INTRODUCTION

research insight M E D U CATO R | A P R I L 2018

Cell culture and influenza infection Severe respiratory infections cause increased morbidity and Primary human lung microvascular endothelial cells mortality in vulnerable populations including infants, elders, 1 (HLMECs) were grown to confluency to establish an and patients with chronic diseases. Influenza viruses are comintact monolayer in cultures of endothelial growth memon causes of respiratory infections, producing an estimated dium-2 (EGM-2) and fetal bovine serum at 37°C. 200,000 hospitalizations and 36,000 deaths within a typical “flu 2 season” in the U.S. Previous studies have shown that patients with novel swine-origin influenza A (H1N1) virus infection had A mouse-adapted human influenza strain A (H5N1) was pulmonary inflammation, acute lung injury, and acute respira- used for influenza infection. Prior to infection, cells were tory distress syndrome (ARDS) – a syndrome characterized by washed with phosphate buffered saline, followed by the adincreased microvascular permeability that can cause respiratory dition of serum-free media. Influenza particles were then failure.3 Due to the mutability of influenza viruses and their de- added to the cells in serum-free media, facilitating viral entry. velopment of resistance to antiviral medication, there is a need to Following 1-hour incubation at 37°C, EGM-2 with serum monitor influenza response to existing drugs and develop novel was added to establish normal culture conditions. Cells were therapeutic approaches.4 Better understanding of the molecular incubated with influenza virus for 24 hours along with any players and mechanisms underlying ARDS may decrease mor- drug co-administration before further experimentation. Spebidity and mortality and improve outcomes in affected patients. cifically, glycyrrhizin (GCZ), a direct inhibitor of extracellular HMGB1, and peptide P5779, an inhibitor of lymphocyte Influenza A virus can infect multiple cell types in the respira- antigen 96 which is required for TLR4 signalling, were used. tory tract. It primarily binds the respiratory epithelium, which Immunoblotting lines the respiratory tract and functions as a physical barrier Levels of intercellular adhesion molecule 1 (ICAM-1) and to potential pathogens. However, in ARDS patients, the virus cleaved caspase-3 expression, which are markers of endothelial is hypothesized to infect the lung endothelium – a polarized activation (a proinflammatory state) and apoptosis, respectively, monolayer that lines blood vessels. ARDS patients have pulmowere measured using standard immunoblotting techniques. nary edema, hypothesized to be caused by a ‘leaky’ endothelium that allows liquid from the capillaries to move into the alveolar Immunofluorescence space.5 In this case, the virus is suggested to contribute to en6,7 To visualize the distribution of vascular endothelial cadherin dothelial dysfunction, microvascular leakage, and lung injury. (VE-cadherin), which are cell-cell junction proteins, standard Since resistance to antiviral therapies continues to increase, immunofluorescent techniques were used. Images were acquired novel treatments targeting host responses may be better at with the Quorum Diskovery Multi-Modal Imaging System. preserving lung function. A commonly damaged host pathRESULTS way involves high mobility group box 1 (HMGB1), a nuclear 8 HMGB1 inhibition decreases endothelial activation of protein present in eukaryotic cells that stabilizes chromatin. ICAM-1 expression during influenza infection Recent studies have revealed its role as a key regulator of patho8 The endothelium can be activated by influenza infection to inlogical immune responses, such as unrestrained inflammation. crease expression of cell adhesion proteins such as ICAM-1.6 HMGB1 is secreted as a delayed mediator of inflammation Circulating leukocytes adhere to the activated endothelium and transduces signals via receptor for advanced glycation end8,9 and are recruited to proximal tissues. This enables immune products (RAGE), Toll-like receptor (TLR) 2, and TLR4. cells to mediate antiviral or antibacterial responses and reBecause systemic inflammatory responses are mediated through 9 solve infection, but excessive leukocyte infiltration in the these receptors, HMGB1 is now recognized as a cytokine. lung can lead to injury.8 Via western blotting, we observe that HLMECs infected with influenza have increased ICAM-1 RESEARCH DESIGN expression, suggesting endothelial activation. In infected cells Hypothesis HMGB1 is released from lung endothelial cells fol- treated with GCZ, a direct inhibitor of extracellular HMGB1, lowing influenza infection, compromising the endo- ICAM-1 expression is decreased closer to basal levels, demthelial barrier and promoting subsequent cell injury. onstrating significant attenuation of activation (Figure 1).

Inhibition of HMGB1, but not TLR4 signalling, decreases influenza-induced endothelial cell apoptosis Influenza infection of endothelial cells induces apoptosis.7 Cleavage of caspase-3 is among the final steps of the apoptotic pathway, enabling cleaved caspase-3 to be used as a marker of apoptosis.7 As seen using western blotting, infected endothelial cells treated with GCZ had significantly reduced cleaved caspase-3 compared to non-GCZ-treated infected endothelial cells (~50%). As GCZ is an inhibitor of extracellular HMGB1, this suggests that paracrine HMGB1 signalling may promote apoptosis in influenza-infected endothelial cells. Treatment of infected endothelial cells with peptide P5779 did not substantially reduce cleaved caspase-3 levels. This suggests HMGB1 may elicit its effects independent of TLR4.

partment, development of ARDS, and death in some cases.14 Specifically, influenza infection can affect the endothelial barrier – a key component of the alveolar-capillary membrane that normally prevents vascular leakage into the alveolar compartment.14 During disease states, endothelial leakage is characterized by pulmonary edema, where fluid reduces gas exchange and can result in respiratory failure.15 Influenza infection can also upregulate the expression of endothelial adhesion molecules, enabling recruitment of leukocytes to the alveolus for an immune response.16 However, if the response is too extensive, lung injury can result.16

This study examines the role of extracellular HMGB1 during influenza infection of the pulmonary endothelium. Inhibiting extracellular HMGB1 with GCZ was shown to decrease the resulting endothelial activation by reducing ICAM-1 expression on influenza-infected HMGB1 inhibition partially restores VEendothelial cells. Interestingly, multiple groups cadherin integrity during influenza infection Infection of the endothelium by influenza vi- have shown that recombinant HMGB1 induces ruses enhances endothelial permeability, reflecting a proinflammatory phenotype by increasing exendothelial apoptosis or remodeling of endo- pression of leukocyte adhesion molecules, such 17,18 thelial cell-cell junctions such as tight junctions as ICAM-1, in a dose-dependent manner. and adherens junctions.11 To visualize changes in These results corroborate our finding that junction integrity, immunofluorescence was used HMGB1 inhibition decreases ICAM-1 exto assess the distribution of VE-cadherin. This pression in influenza-infected endothelial cells. endothelial-specific adherens junction protein plays a key role in barrier function and paracel- During influenza infection, endothelial cell apop8 lular permeability at the plasma membrane.7,12 tosis increases permeability of the endothelium. Fluorescent staining showed that VE-cadherin in We show that influenza-infected cells treated with control endothelial cells were continuous and had GCZ had attenuated cleaved caspase-3. These retight cell-cell contacts without intercellular gaps. sults show that inhibiting extracellular HMGB1 However, endothelial cells infected with influ- attenuates influenza-induced endothelial cell enza had discontinuous VE-cadherin, which was apoptosis, suggesting that, when not inhibited, partially rescued through extracellular HMGB1 HMGB1 acts in a paracrine manner to induce inhibition with GCZ treatment (Figure 2). endothelial cell apoptosis. In unpublished work, our group has observed that HMGB1 knockdown (i.e. blocking intracellular HMGB1) also DISCUSSION Severe influenza infections cause lung damage and yields similar effects, suggesting that HMGB1 affected patients often require admission to inten- is released by influenza-infected cells to elicit its sive care for respiratory failure. There, they ex- effects on neighbouring cells. Immunoblotting hibit a mortality rate of ~20% despite mechanical revealed that peptide P5779 does not attenuate ventilation and antiviral therapies.13 The respira- cleaved caspase-3 levels, suggesting HMGB1 acts tory deterioration is often caused by virus-induced through a TLR4-independent pathway. Studies acute lung injury, which leads to flooding of the have demonstrated that recombinant HMGB1 can cause endothelial apoptosis by eliciting stress alveolar comsignaling pathways other than TLR4, which

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Figure 2: Inhibition of HMGB1 partially restores VE-cadherin integrity in influenza infected HLMECs. HLMECs were grown to confluency on coverslips, infected with influenza, and then treated with either GCZ (100 μM) or nothing at all. Through immunofluorescence, VE-cadherin was stained to visualize endothelial cell barrier integrity. In uninfected cells, there is continuous VEcadherin (green) surrounding the cells. In cells infected with influenza, there is induced thinning of VE-cadherin borders and dissociation of junctions, leading to increased intercellular gap formation. This would suggest increased endothelial permeability and an impaired endothelial barrier. With influenza infection and GCZ treatment, there is partial restoration of VE-cadherin surrounding cells and less intercellular gap formation. “GCZ alone” is a control that shows that treatment alone does not affect the cells and the integrity of VE-cadherin (n = 2).

Figure 3: Possible mechanism by which HMGB1 acts to modulate endothelial activation, apoptosis, and junction disruption during influenza infection. Influenza virus infects lung endothelial cells and causes release of HMGB1 into the extracellular space. HMGB1 then acts on a receptor, possibly RAGE, to cause downstream signalling and activate stress signaling pathways, thereby causing the observed effects of increased endothelial activation, apoptosis, and junction disruption. The consequences of these effects in vivo would be vascular leakage, which would lead to pulmonary edema, and greater immune infiltration. These combined effects would then contribute to the observed lung injury during severe influenza infections in ARDS patients.

Victoria Mintsopoulos is a M.Sc. candidate in the Institute of Medical Science at the University of Toronto, where her research focuses on mechanisms of endothelial leakage in acute lung injury. Dr. Warren Lee is an Associate Professor in the Department of Medicine at the University of Toronto and is a critical care physician at St. Michael’s Hospital. As a Canada Research Chair in Mechanisms of Endothelial Permeability, his research looks at contributions from leaking between endothelial cells (paracellular leak) and through individual endothelial cells (transcytosis). EDITED BY KELVIN NG AND ADAM WADE-VALLANCE

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by RAGE and increases the production of TNF-α in human umbilical vein endothelial cells. Immunobiology. 2010;215(12):956–62. Available from: doi: 10.1016/j.imbio.2009.11.001. 21. Hiyoshi M, Indalao IL, Yano M, Yamane K, Takahashi E, Kido H. Influenza A virus infection of vascular endothelial cells induces GSK-3β-mediated β-catenin degradation in adherens junctions, with a resultant increase in membrane permeability. Archives of Virology. 2015;160:225–34. Available from: doi: 10.1007/s00705-014-2270-5. 22. London NR, Zhu W, Bozza FA, Smith MCP, Greif DM, Sorensen LK, et al. Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza. Science Translational Medicine. 2010;2(23):23ra19. Available from: doi: 10.1126/ scitranslmed.3000678. 23. Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O, Erlandsson-Harris H, et al. High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. Journal of Experimental Medicine. 2000;192(4):565–70. Available from: doi: 10.1084/jem.192.4.565.

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REVIEWED BY VICTORIA MINTSOPOULOS

Taubenberger JK, Morens DM. The pathology of influenza virus infections. Annual Review of Pathology. 2008;3:499–522. Available from: doi: 10.1146/annurev.pathmechdis.3.121806.154316. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. Journal of American Medicine. 2003;289(2):179–86. Available from: doi: 10.1001/jama.289.2.179. Yang ML, Wang CT, Yang SJ, Leu CH, Chen SH, Wu CL, et al. IL-6 ameliorates acute lung injury in influenza virus infection. Scientific Reports. 2017;7:43829. Available from: doi: 10.1038/srep43829. Govorkova EA, Baranovich T, Seiler P, Armstrong J, Burnham A, Guan Y, et al. Antiviral resistance among highly pathogenic influenza A (H5N1) viruses isolated worldwide in 2002–2012 shows need for continued monitoring. Antiviral Research 2013;98(2):297– 304. Available from: doi: 10.1016/j.antiviral.2013.02.013. Maniatis NA, Orfanos SE. The endothelium in acute lung injury/acute respiratory distress syndrome. Current Opinion in Critical Care. 2008;14(1):22–30. Available from: doi: 10.1097/ MCC.0b013e3282f269b9. Armstrong SM, Darwish I, Lee WL. Endothelial activation and dysfunction in the pathogenesis of influenza A virus infection. Virulence. 2013;4(6):537–42. Available from: doi: 10.4161/viru.25779. Armstrong SM, Wang C, Tigdi J, Si X, Dumpit C, Charles S, et al. Influenza infects lung microvascular endothelium leading to microvascular leak: role of apoptosis and claudin-5. PLoS ONE. 2012;7(10):e47323. Available from: doi: 10.1371/journal.pone.0047323. Yanai H, Taniguchi T. Nucleic acid sensing and beyond: virtues and vices of high-mobility group box 1. Journal of Internal Medicine. 2014;276(5):444–53. Available from: doi: 10.1111/joim.12285. Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nature Reviews Immunology. 2005;5(4):331. Available from: doi: 10.1038/nri1594. Yang H, Wang H, Ju Z, Ragab AA, Lundbäck P, Long W, et al. MD-2 is required for disulfide HMGB1–dependent TLR4 signaling. The Journal of Experimental Medicine. 2015;212(1):5–14. Available from: doi: 10.1084/jem.20141318. Wang S, Le TQ, Kurihara N, Chida J, Cisse Y, Yano M, et al. Influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza. The Journal of Infectious Diseases 2010;202(7):991–1001. Available from: doi: 10.1086/656044. Zou M, Dong H, Meng X, Cai C, Li C, Cai S, et al. Storeoperated Ca2+ entry plays a role in HMGB1-induced vascular endothelial cell hyperpermeability. PLoS ONE. 2015;10(4):e0123432. Available from: doi: 10.1371/journal.pone.0123432. Domínguez-Cherit G, Lapinsky SE, Macias AE, Pinto R, Espinosa-Perez L, de la Torre A, et al. Critically Ill patients with 2009 influenza A(H1N1) in Mexico. The Journal of the American Medical Association. 2009;302(17):1880–7. Available from: doi: 10.1001/jama.2009.1536. Herold S, Becker C, Ridge KM, Budinger GRS. Influenza virus-induced lung injury: pathogenesis and implications for treatment. European Respiratory Journal. 2015;45(5):1463–78. Available from: doi: 10.1183/09031936.00186214. Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiological Reviews. 2006;86(1):279–367. Available from: doi: 10.1152/physrev.00012.2005. Short KR, Kroeze EJBV, Fouchier RAM, Kuiken T. Pathogenesis of influenza-induced acute respiratory distress syndrome. The Lancet Infectious Diseases. 2014;14(1):57–69. Available from: doi: 10.1016/ S1473-3099(13)70286-X. Fiuza C, Bustin M, Talwar S, Tropea M, Gerstenberger E, Shelhamer JH, et al. Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood. 2003;101(7):2652–60. Available from: doi: 10.1182/blood-2002-05-1300. Luo Y, Li S-J, Yang J, Qiu Y-Z, Chen F-P. HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway. Biochemical and Biophysical Research Communications. 2013;438(4):732–8. Available from: doi: 10.1016/j.bbrc.2013.07.098. Huang Q, Yang Z, Zhou J-P, Luo Y. HMGB1 induces endothelial progenitor cells apoptosis via RAGEdependent PERK/eIF2 pathway. Molecular and Cellular Biochemistry. 2017;431(1–2):67–74. Available from: doi: 10.1007/s11010-017-2976-2. Luan Z-G, Zhang H, Yang P-T, Ma X-C, Zhang C, Guo R-X. HMGB1 activates nuclear factor-κB signaling

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Figure 1: Inhibition of HMGB1 causes lower expression of endothelial activation marker ICAM-1 in influenzainfected HLMECs:HLMECs were grown to confluency, infected with influenza, and then treated with either GCZ (100 μM) or nothing at all. Through western blotting, ICAM-1 was probed to detect endothelial activation. A: Visual representation of one of the western blots. B: Analysis of blots shows that treatment with GCZ to influenza-infected HLMECs decreases influenza-induced endothelial activation of ICAM-1 closer to basal levels. n = 3; *, P < 0.05 versus control and Flu + GCZ groups.

research insight

apoptosis, activation, and barrier function. Our study suggests that HMGB1 induces these effects independently of TLR4. Using inhibitors or siRNA treatment, we can explore whether HMGB1 acts through other receptors, such as We also investigated how influenza infection RAGE or TLR2. Furthermore, although the of endothelial cells affects adherens junctions data suggests that HMGB1 affects adherens such as VE-cadherin, which regulates paracel- junctions during infection, future experiments lular permeability to circulating leukocytes.21 should address whether it affects claudins, which Immunofluorescence staining revealed that are tight junction proteins that contribute to influenza infection of endothelial cells disrupts the paracellular barrier. Future work should also VE-cadherin, and that GCZ treatment partially progress to a mouse model of influenza infection prevents this disruption. Interestingly, a study to determine the contributing role of HMGB1 shows that a recombinant Slit protein fragment, to lung injury and pulmonary edema. A better Slit2N, is able to improve microvascular bar- understanding of HMGB1’s mechanism of rier integrity and decrease vascular leakage in a action in ARDS patients may establish it as a murine model of influenza infection. Slit2N acts therapeutic target in severe influenza infection. to promote association between p120-catenin and VE-cadherin, thereby stabilizing adherens CONCLUSION junctions and improving barrier integrity during During severe influenza infections, HMGB1 inflammatory states.22 These results highlight can compromise the endothelium, leading to inthe importance of improving destabilized junc- creased leakage of circulating fluid into the alveotions in infected states as the influenza-infected lar space, as well as lung injury from the immune mice that received Slit2N had reduced mortality. response to surrounding tissues. Although our This is consistent with the loss of VE-cadherin findings require confirmation in vivo, they imply integrity during influenza infection as well as that endothelial dysfunction is critical to the vasvascular leakage seen in our results and ARDS cular leakage that contributes to mortality among patients, suggesting that therapies rescuing ARDS patients. Understanding the mechanisms endothelial barrier integrity may be effective. by which influenza impedes endothelial barrier function would allow for the further development In summary, the presented data provides evi- of therapeutics targeting harmful host pathways. dence that HMGB1 plays a role in endothelial Our study highlights how HMGB1 signaldysfunction during influenza infection. When ling may serve as a novel target for influenza HMGB1 is released from an infected cell, it therapeutics to reduce endothelial activation and likely binds to its receptors on neighbouring apoptosis, and enhance cell junction continuity. cells in a paracrine fashion to induce endothelial activation, increase apoptosis, and redistribute ACKNOWLEDGMENTS adherens junction proteins. These effects present I would like to sincerely thank Victoria Minta partial mechanism that provides insight into sopoulos for her incredible support and menthe causes of ARDS symptoms (Figure 3).23 toring, Dr. Warren Lee for his guidance, and my NSERC Undergraduate Research Student Future investigations should elucidate the mech- Award that allowed me to pursue this opportunity. anism by which HMGB1 modulates endothelial further supports the lack of effect of P5779.19,20 Thus, inhibiting the ability of HMGB1 to elicit these pathways may attenuate the apoptosis of lung endothelial cells during influenza infection.

ARTIST CATHY YAQIONG REN

RESEARCH INSIGHT

Identifying genes associated with biofilm production in Pseudomonas

aeruginosa

JESSICA CHEE Bachelor of Health Sciences (Honours) Class of 2020, McMaster University Correspondence: cheej1@mcmaster.ca

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen associated with a variety of life-threatening diseases. Infections caused by P. aeruginosa can be nearly untreatable because of its multidrug-resistance. One of the characteristics of P. aeruginosa that helps it survive in high drug concentrations is its ability to form biofilmsâ&#x20AC;&#x201C;large communities of cells encompassed by extracellular polymeric substances that defend against many antibiotics. In fact, sub-minimum inhibitory concentrations of antibiotics stimulate biofilm production. This project aims to identify genes associated with biofilm induction in P. aeruginosa by screening a transposon mutant library for mutants that fail to show increased biofilm production when exposed to sub-minimum inhibitory concentrations of cefixime, tobramycin, and thiostrepton. So far, we have identified one gene, PA2714, that encodes a predicted molybdopterin oxidoreductase required for biofilm production. Because of the strong association between biofilm production and antibiotic tolerance in P. aeruginosa, the gene identified in this screen may be a useful therapeutic target for novel antimicrobials that can disrupt biofilm formation.

INTRODUCTION Pseudomonas aeruginosa, a gram-negative, rod-

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Sub-minimum inhibitory concentrations of antibiotics (sub-MIC), or concentrations below the lowest concentration that tprevents growth,

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The severity of P. aeruginosa infections is driven by its ability to form biofilms.5 A biofilm is a complex community of microorganisms embedded in an extracellular polymeric substance (EPS) that adheres to a surface. The EPS consists of a variety of macromolecules, including polysaccharides that act as a protective barrier against antimicrobials, proteins that provide a nutrient source via digestion of exogenous macromolecules, and extracellular DNA that facilitates horizontal gene transfer and provides structural integrity to the biofilm matrix.6 The benefits conferred by biofilms offer a more suitable environment, compared to planktonic growth, for bacteria.5

It would be useful to investigate the genes associated with biofilm production in antibiotic conditions to gain insight into molecular mechanisms behind the antibiotic tolerance conferred by biofilms and to determine novel therapeutic targets for antimicrobials. We aim to identify genes that are essential to biofilm production by screening a library of transposon mutants for their ability to form biofilms in sub-MIC levels of the antibiotics cefixime, tobramycin, and thiostrepton. Genes associated with biofilm production can be elucidated by comparing biofilm production in mutants relative to wild-type P. aeruginosa. Furthermore, we will investigate the link between biofilms and antimicrobial activity by demonstrating that sub-MIC levels of antibiotics can stimulate biofilm production in wild type P. aeruginosa.

research insight

shaped bacteria, is a leading cause of hospital acquired infections; it remains difficult to treat because of its multidrug-resistance, especially in susceptible cystic fibrosis (CF) patients.1 CF is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.2 One characteristic of CF, increased viscosity of mucoid secretions, leads to environments conducive to the growth of P. aeruginosa biofilms in the lungs, resulting in inflammation and life-threatening chronic infections.3 The prevalence of P. aeruginosa infection in CF patients is 60%, with acquired infections almost always becoming chronic.4 In CF patients, de novo emergence of antibiotic resistance has been observed after continual exposure to antimicrobials.2 In combination with patient-to-patient transmission of resistant strains in healthcare settings, this leads to the emergence of multidrugresistant strains.2 It is important to continue studying the development of antibiotic resistance in P. aeruginosa to inform potential interventions.

stimulate biofilm production in P. aeruginosa.7 Although the protective effect of the EPS is primarily structurally-related, the efficacy of antibiotics that can diffuse through this barrier is reduced against biofilms. One possible explanation for this phenomenon is that antimicrobial activity in biofilms can be quenched through diffusion-reaction inhibition via chelation of metal antimicrobials or enzymatic degradation of antimicrobials by proteins in the EPS.8 By lowering the concentration of antimicrobials to sub-lethal levels, cell survival within the biofilm community is promoted. In addition, there is a high degree of physiological heterogeneity within these cellular communities; in response to their local microenvironment some cells are more resistant than others due to their growth state or patterns of gene expression. The diverse set of genetic traits found in biofilm communities can be disseminated through horizontal gene transfer in order to increase overall antibiotic tolerance within the community. The acquisition of resistant genes can occur through uptake of extracellular DNA.5

RESEARCH DESIGN A transposon mutant library of P. aeruginosa PAO1 KP containing the Himar1 Mariner transposon element was generated. A transposon is a mobile genetic element that can create mutations within the genome. The mutants in this library were created by mating PAO1 KP with Escherichia coli containing a plasmid carrying the Himar1 Mariner transposon, which inserts itself between TA dinucleotides, and a transposase that excises the transposon element from the plasmid.9 Containing a gene that encodes for gentamicin resistance, the transposon is inserted into a suicide vector unable to replicate in P. aeruginosa.9 Mutants that contained the transposon element in their genomic DNA were then selected by gentamicin screening, yielding ~13,500 mutants.

acetic acid for 5 minutes. Absorbance of the crystal violet in acetic acid was measured at 600 nm to quantify the relative amounts of biofilm.10

research insight

False-positives were eliminated by retesting mutants identified from the initial screen through a dose-response experiment, with a concentration range spanning the MIC of cefixime, tobramycin, and thiostrepton to confirm that the mutants showed inhibited biofilm formation when compared with PAO1 KP in varying concentrations of three different antibiotics. Reduced biofilm formation would suggest that the gene disrupted by the Himar1 Mariner transposable element is essential for biofilm stimulation in the presence of sub-MIC antibiotic. The transposon insertion sites were then identified using two rounds of polymerase chain reaction (PCR). The first round was a touchdown PCR performed with a The library is currently being screened for mu- Himar1 Mariner-specific primer and an arbitrary tants demonstrating a lack of biofilm stimulation primer. The second round of PCR was performed in the presence of sub-MIC (5 μM) cefixime. The using the Himar1 Mariner-specific primer and cut-off for increased biofilm production in wild a primer specific to the arbitrary primer used in type PAO1 KP was established to be a >200% the first round. PCR products were gel-purified increase in biofilm in the presence of sub-MIC and sequenced. cefixime, compared to the vehicle control (dimethyl sulfoxide). Thus, hits from the screen were RESULTS defined as all mutants that did not meet this cutoff (i.e. produced less than 200% of biofilm when Of the 4,200 mutants screened, one displayed exposed to the antibiotic, compared to DMSO). reduced biofilm production in the presence of The bacteria were grown overnight in 150 μL of cefixime, tobramycin, and thiostrepton compared 10% lysogeny broth (LB) in a 96-well plate and to the PAO1 KP wild type control, hereafter resubcultured for 2 hours in a 1:25 dilution. They ferred to as BBTn41_G1. We also demonstrated were then grown overnight in a 96-well plate that the highest levels of biofilm were observed at with a 96-peg lid with 5 μM cefixime or DMSO the highest concentration below the MIC of ceadded. Biofilm was allowed to form on the pegs. fixime (5 μM) for both wild type and the mutant. After rinsing with 1X PBS to remove non-adher- The results from the second round of screening ent bacteria, the biofilms on the pegs were stained (false positive elimination) for BBTn41_G1 is with 150 μL of 0.1% (w/v) crystal violet for 15 shown in Figure 1. minutes and de-stained into 200 μL of 33% (v/v)

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FIGURE 1: Effects of cefixime on PAO1 KP and BBTn41_G1 growth and biofilm production. A. Effects of cefixime on PAO1 KP planktonic growth and biofilm formation as a percentage of PAO1 KP in DMSO (n=1, 3 technical replicates). B. Effects of cefixime on BBTn41_G1 planktonic growth and biofilm formation as a percentage of BBTn41_G1 in DMSO (n=1, 3 technical replicates). Biofilm production increased for BBTn41_G1 and PAO1 KP with increasing concentration of drug until the lethal dose (10 μM). Both BBTn41_G1 and PAO1 KP showed maximum biofilm production at 5 μM of cefixime. However, at this concentration, BBTn41_G1 produced less than 600% more biofilm than the mutant in no drug, while PAO1 KP produced around 800% more biofilm than with no drug. Similar reduction in biofilm production was seen in tobramycin and thiostrepton.

Touchdown PCR determines that the transposable element in BBTn41_G1 is located at the start of the gene PA2714. Although this gene has yet to be characterized, the nucleotide sequence suggests that it encodes a molybdopterin oxidoreductase. Activated by binding to the cofactor molybdopterin, this family of enzymes catalyzes electron transfer reactions.11

of antimicrobial activity may be a more useful screening method, as we have demonstrated that relative amounts of biofilm production can be observed and measured at concentrations above and below the MIC of antibiotics.

CONCLUSION

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Dr. Lori Burrows, PhD, is a professor in the Department of Biochemistry and Biomedical Sciences with a joint appointment in the Department of Pathology and Molecular Medicine. As the Principal Investigator of Burrows Lab, she researches antibiotic resistance, Type IV pili, and biofilms, with a particular focus on the opportunistic pathogen Pseudomonas aeruginosa.

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REVIEWED BY DR. LORI BURROWS

EHA Consulting Group, Inc. [Internet]. What is Pseudomonas Aeruginosa? Available from: http://www.ehagroup.com/resources/ pathogens/pseudomonas-aeruginosa/ [Accessed 12th January 2018]. Aloush V, Navon-Venezia S, Seigman-Igra Y, Cabili S, Carmeli Y. Multidrug-resistant Pseudomonas aeruginosa: risk factors and clinical impact. Antimicrobial agents and chemotherapy. 2006;50(1):43-8. Bjarnsholt T, Jensen PØ, Fiandaca MJ, Pedersen J, Hansen CR, Andersen CB et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatric pulmonology. 2009;44(6):547-58. Cystic Fibrosis Foundation. Patient Entry Annual Data Report 2006. [Accessed 12th January 2018]. Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nature Reviews Microbiology. 2016;14(9):563-75. Flemming HC, Wingender J. The biofilm matrix. Nature Reviews Microbiology. 2010;8(9):623-33. Andersson DI, Hughes D. Microbiological effects of sublethal levels of antibiotics. Nature Reviews Microbiology. 2014;12(7):465-78. Oubekka SD, Briandet R, Fontaine-Aupart MP, Steenkeste K. Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms. Antimicrobial agents and chemotherapy. 2012;56(6):3349-58. Lampe DJ, Akerley BJ, Rubin EJ, Mekalanos JJ, Robertson HM. Hyperactive transposase mutants of the Himar1 mariner transposon. Proceedings of the National Academy of Sciences. 1999;96(20):11428-33. O’Toole GA. Microtiter dish biofilm formation assay. Journal of visualized experiments: JoVE. 2011;(47);2437. Pseudomonas Genome DB. [Internet]. Pseudomonas aeruginosa PAO1, PA2714. Available from: http://pseudomonas.com/ feature/show?id=108214 [Accessed 12th January 2018]. Kritikos G, Banzhaf M, Herrera-Dominguez L, Koumoutsi A, Wartel M et al. Typas A. A tool named Iris for versatile high-throughput phenotyping in microorganisms. Nature microbiology. 2017;2:17014. Moy TI, Conery AL, Larkins-Ford J, Wu G, Mazitschek R, Casadei G et al. High-throughput screen for novel antimicrobials using a whole animal infection model. ACS chemical biology. 2009;4(7):527-33. World Health Organization. [Internet]. Antibiotic resistance. [last modified 2017]. Available from: http://www.who.int/mediacentre/factsheets/antibiotic-resistance/en/ [Accessed 12th January 2018]. World Health Organization. [Internet]. WHO publishes list of bacteria for which antibiotics are urgently needed. [last modified 2017 Feb 27]. Available from: http://www.who. int/mediacentre/news/releases/2017/ bacteria-antibiotics-needed/en/ [Accessed 12th January 2018].

research insight

Antibiotic resistance represents a major global health threat. The deteriorating effectiveness of antibiotics renders both common and lifeSo far, we have identified one possible gene that threatening diseases more difficult to treat.14 may contribute to biofilm formation. Based on Accordingly, it is of paramount importance its nucleotide sequence, PA2714 is likely to be for the scientific community to better una gene encoding a molybdopterin oxidoreduc- derstand the molecular mechanisms behind tase.11 This improves our understanding of the antibiotic tolerance, so that novel and reliable mechanism of molybdopterin-induced biofilm antimicrobial treatments can be developed. production. Loss-of-function mutations in molybdopterin synthases–an enzyme family that The World Health Organization has classified synthesizes the molybdopterin cofactor–increase P. aeruginosa in the critical category of “priority biofilm formation.12 A possible explanation for pathogens” to target because of its multidrugthis is that molybdopterin synthases consume resistance and its threat in healthcare settings.15 guanine triphosphate (GTP), thereby reducing In this project, we have demonstrated that P. the amount of intracellular GTP available to aeruginosa has the ability to form biofilms in form c-di-GMP, an important component of bio- sub-MIC levels of antibiotics. In addition to films.12 Thus, decreased molybdopterin synthesis identifying genes associated with biofilm produccaused by mutations in molybdopterin synthases tion, we hope this project can serve as a proofcould increase intracellular GTP and c-di-GMP of-concept that biofilm production is a reliable production, ultimately increasing biofilm produc- indicator of antimicrobial activity in P. aeruginosa, tion. In our experiment, we observed a decrease a possibility that may be useful for drug screening. in biofilm formation with a molybdopterin With the dearth of effective antibiotics, scientists oxidoreductase mutated by a transposon element. may consider novel antimicrobials that target biofilm production as their mechanism of action. Moving forward, we will continue screening the rest of the library for genes that affect bioACKNOWLEDGEMENTS film formation at sub-MIC antibiotic levels. Our findings may be useful in designing novel I would like to thank all the members of the Burantimicrobials. With a better understanding of rows Lab, especially Michael Ranieri, who created genetic factors affecting biofilm production, we the transposon mutant library and aided me in can design drugs that inhibit biofilm formation labwork and manuscript preparation. I would also and subsequently reduce antibiotic resistance. like to thank my supervisor, Dr. Lori Burrows, for her guidance. In addition, many of the current high-throughput screens for new antimicrobials involve testing a huge library of drugs at arbitrary concentrations for indications of cell death.13 These screens could yield false negatives because MICs of new molecules are unknown; therefore, many potential antimicrobials are missed simply because they were screened at a concentration that was too low. Using biofilm production as an indicator

DISCUSSION

EDITED BY DANIEL DIATLOV & ANGELA DONG

ARTIST LAURA NGUYEN

CRITICAL REVIEW

Fecal microbiota transplantation: the power of poop

MAGGIE HOU1 & EVA LIU1 Bachelor of Health Sciences (Honours) Class of 2020, McMaster University Correspondence: houy14@mcmaster.ca

ABSTRACT

Fecal microbiota transplantation (FMT), a procedure involving the transfer of stool from healthy donors to patients, has demonstrated success in re-establishing gut microbiota homeostasis and facilitating the recovery of metabolic and immune function. Based on positive results from numerous clinical trials, current North American and European clinical guidelines recommend FMT as a treatment option for recurrent Clostridium difficile infections. Ongoing investigations into FMTâ&#x20AC;&#x2122;s efficacy in the treatment of other conditions, such as multiple sclerosis and inflammatory bowel disease, may expand the application of FMT to a myriad of diseases. Nonetheless, challenges regarding biosafety concerns and public perception need to be addressed before FMTs can be considered for broader applications.

INTRODUCTION

The first step of FMT is to collect a stool sample from a healthy donor. The donor may be a relative, a spouse, or an unrelated stranger.1 After a donor has been recruited, their stool and serum will be screened for the presence of infectious agents such as human immunodeficiency virus to reduce the risk of disease transmission. Donor history will also be assessed; donors with active infections, recent exposure to antibiotics, or chronic gastrointestinal conditions will be excluded. If the donor meets the eligibility criteria, the collected stool sample will then be emulsified in a sterile saline solution and administered to patients after removing particulates.1,2

The human gut microbiota is comprised of over 35,000 bacterial species and microbes, including archaea, viruses, and protozoa.5,6 These microbes play important roles in nutrient metabolism, immune development, and immune protection.7 Under normal conditions, the gut microbiota maintains a homeostatic equilibrium that rests on a symbiotic relationship between the microbial community and the animal host.6 Disruption of the commensal flora is associated with inflammatory gastrointestinal disease, colon cancer, and diabetes.6 However, evidence has yet to identify a causal relationship between dysbiosis and the aforementioned conditions. Nevertheless, there has been a building interest from the medical community to treat these diseases by restoring the normal gut microbiota through FMT.8 This interest is promoted by the potential for FMT to act as an alternative to antibiotic treatments, which has been shown to collaterally disrupt and decrease the biodiversity of the gut microbiome, ultimately placing patients at higher risk for future infections.9,10

FECAL TRANSPLANTS & CLOSTRIDIUM DIFFICILE INFECTION

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C. difficile infection (CDI) manifests as abdominal pain, fever, and diarrhea secondary to colon inflammation and damage from toxins excreted by the bacteria.11 C. difficile is transmitted primarily through spores in feces, a process that can be facilitated by cross-contamination in healthcare institutions and between healthcare professionals.11 In fact, CDI is the most common hospital-acquired infection in Canada.12 Patients can become susceptible to CDI as a result of indiscriminate use of protonpump-inhibitors and antibiotics harmful to the gut microbiota. For example, reductions in bifidobacteria populations have been shown to decrease host immune protection.5 Additionally, older patients are more likely to contract CDIs and require hospital admissions in order to manage the increasing complexity of their conditions. In Canada, an estimated 37,900

M E D U CATO R

Currently, the main application of FMT focuses on the treatment of recurrent Clostridium difficile infections (rCDI), a condition characterized by chronic gastrointestinal inflammation unresponsive to antibiotic treatments.1 Numerous clinical guidelines from North America and Europe have recommended FMT as a treatment option against rCDI.3,4 Additionally, the applicability of FMT for the management of many other gastrointestinal and non-gastrointestinal disorders, including irritable bowel disease (IBD), slow-transit con-

THE HUMAN GUT MICROBIOTA

critical review

Fecal microbiota transplantation (FMT) is a procedure that transfers stool from a healthy donor to the gastrointestinal tract of a patient with altered gut microbiota. This process aims to restore the normal gut microbiome and to suppress pathogenic bacteria without the use of antibiotics.1

stipation, and multiple sclerosis, is currently under investigation.1 Despite its potential, FMT also faces challenges such as biosafety precautions and stigma within the general population.

cases of CDI were reported in 2012, with $281 million in direct and opportunity costs.12 Case mortality rates range from 6-30% with a general upward trend since as early as 1995.13

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critical review

For mild cases of CDI, individuals can reasonably recover without medical intervention.10 In severe cases with risks of organ failure or toxic megacolon, surgery is needed to remove diseased and necrotized portions of the colon.13 For the majority of cases gauged as moderate in severity, antibiotics are considered first-line therapies.13 However, in recent years, more CDI patients have presented as antibioticresistant, with higher rates of severe, recurrent, and chronic cases. Accordingly, FMTs were introduced as a safe, inexpensive, and more effective alternative to the standard of care.11,13 Randomized control trials with a combined pool of over 600 participants determined that FMTs, compared to placebo or first-line treatment with the antibiotic vancomycin, cured diarrhea in 59% more CDI patients.14 Under normal conditions, the gut microbiota exhibits “colonization resistance,” a phenomenon by which normal bacteria outcompete and directly attack invading microorganisms to taper their growth.1 In CDI treatments, antibiotics such as vancomycin kill both C. difficile and healthy strains of bacteria.11 The resulting loss of healthy, commensal bacteria can contribute to rCDI, as pockets of resistant C. difficile may remain even after symptoms recede. Consequently, the infection is likely to return with increased severity as well as with an increased likelihood for antibiotic resistance.11,13 FMT reintroduces commensal microbiota, such as bifidobacteria populations, via healthy feces, thereby re-establishing colonization resistance as a part of the normal immune function in warding off recurrent infections.1,11 Since numerous North American and European clinical guidelines already call for the use of FMTs in antibiotic-resistant CDI, there is mounting support for the adoption of FMT as the first-line treatment for CDI.11 This establishes a need for future clinical trials to focus on optimizing FMT’s preparation and administration for effective large-scale deployment.11

FECAL TRANSPLANT IN OTHER DISORDERS FMT’s efficacy in treating CDI has drawn attention to its potential to treat similar disorders,

including inflammatory bowel disease (IBD), diabetes, and slow-transit constipation.1 IBD is a chronic inflammatory disorder of the gastrointestinal system.8 The pathophysiology of IBD is complex, and has been associated with a decrease in the diversity of the gut microbiota and depletion of commensal bacteria.8,15 Several clinical studies have been conducted evaluating the effectiveness of FMT in treating IBD, but the results are not conclusive. One parallel RCT conducted by Moayyedi and colleagues found a statistically significant difference in remission between patients treated with FMT versus patients treated with a placebo enema.16 However, another RCT comparing FMT with placebo found no statistically significant differences in remission rates between the groups.17 Although more research is needed to determine FMT’s effectiveness in treating IBD, current research suggests that dysbiosis, or microbial imbalance, is not the sole pathophysiology under IBD. Thus, FMT will likely be limited as an adjunct therapy.11 Patients with diabetes have also been found to have lower diversity in their gut microbiota, but a causal path for this association has yet to be elucidated.18 In humans, FMT appeared to improve insulin sensitivity in insulinresistant patients when assessed 6 weeks after treatment, which sheds light to its therapeutic potential in treating diabetes.19 However, further developments in the FMT process are needed before it can be reliably used in clinical settings for conditions other than rCDI.18

PUBLIC PERCEPTION While researchers view FMT as an innovative and successful treatment that could be more effective at eradicating infections than firstline antibiotics, there is still significant stigma ftom the general population on the treatment. The media plays a crucial role in educating the public and improving their understanding of FMT. Currently, the media presents FMT as a disgusting but necessary treatment.20 The “ick factor” is often the first topic discussed when addressing FMT instead of the positive impact it can have on patients. This bias poses significant hurdles to the recruitment of healthy donors.20 As FMT is being considered as a first-line therapy over antibiotics, there is no longer a need to portray FMT as an outlandish idea. Instead, more rational public discussions regarding the merits and challenges of FMT are required to increase public acceptance and overcome patient non-compliance. Additionally, physician education is also needed as stigma remains among healthcare professionals.

There have been reports in which patients have been turned down by physicians who protested the “ick factor” of the treatment despite emerging evidence that the treatment is superior to antibiotics against rCDI.20 Education on and destigmatization of FMT are necessary to entertain future discussions on biosafety regulations and policy implementations.

tration, preparation, and storage. This new data will hopefully allow for more nuanced and targeted approaches for both specific diseases and patient types. Internationally, many are following in the footsteps of Western guidelines that have adopted FMTs as a treatment for CDI.12

CONCLUSION Despite its relative anonymity and stigma among the general public, FMT is quickly becoming a mainstay treatment for rCDI. There are currently several ongoing clinical trials gauging FMT’s effectiveness against various other diseases with consideration of routes of adminis-

REVIEWED BY AADIL BHARWANI Aadil Bharwani is an MD/PhD Candidate at the Michael G. Degroote School of Medicine with a research focus on the functional relationship between commensal microbes and the brain in chronic stress, in particular through perturbations of neuro-immune signaling along the gut-brain axis underlying psychiatric conditions. EDITED BY JESSICA CHEE & JIM XIE

Clostridium difficile infections. American Journal of Gastroenterology. 2013;108(4):478-98. Available from: doi: 10.1038/ajg.2013.4. Yang H, Duan Z. The local defender and functional mediator: Gut microbiome. Digestion. 2018;97(2):137-145. Available from: doi: 10.1159/000484687. Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Nageshwar Reddy D. Role of the normal gut microbiota. World Journal of Gastroenterology. 2015;21(29):8787-8803. Available from: doi: 10.3748%2Fwjg.v21. i29.8787. O’Hara AM, Shanahan F. The gut flora as a forgotten organ. EMBO Reports. 2006;7(7):688693. Available from: doi: 10.1038%2Fsj. embor.7400731. Nishida A, Inoue R, Inatomi O, Bamba S, Naito Y,

Andoh A. Gut microbiota in the pathogenesis of inflammatory bowel disease. Clinical Journal of Gastroenterology. 2017;11(1):1-10. Available from: doi: 10.1007/s12328-017-0813-5. 9. Panda S, El khader I, Casellas F, Lopez Vivancos J, Garcia Cors M, Santiago A, et al. Short-term effect of antibiotics on human gut microbiota. PLoS One. 2014;9(4):e95476. Available from: doi: 10.1371/journal.pone.0095476. 10. Sekirov I, Tam NM, Jogova M, Robertson ML, Li Y, Lupp C, et al. Antibiotic-induced perturbations of the intestinal microbiota alter host susceptibility to enteric infection. Infection and Immunity. 2008;76(10):4726-4736. Available from: doi: 10.1128/IAI.00319-08. 11. Bakken JS, Borody T, Brandt LJ, Brill JV, Demarco DC, Franzos MA, et al. Treating Clostridium difficile Infection With Fecal Microbiota Transplantation. Clinical Gastroenterology and Hepatol-

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Vindigni SM, Surawicz CM. Fecal Microbiota Transplantation. Gastroenterology Clinics of North America. 2017;46(1):171-185. Available from: doi: 10.1016/j.gtc.2016.09.012. 2. Tauxe WM, Dhere T, Ward A, Racsa LD, Varkey JB, Kraft CS. Fecal Microbiota Transplant Protocol for Clostridium Difficile Infection. Laboratory Medicine. 2015;46(1):e19-23. Available from: doi: 10.1309/LMCI95M0TWPDZKOD. 3. Debast SB, Bauer MP, Kuijper EJ. European Society of Clinical Microbiology and Infectious Diseases: Update of the Treatment Guidance Document for Clostridium Difficile Infection. Clinical Microbiology and Infection. 2014;20 Suppl 2:1-26. Available from: doi: 10.1111/14690691.12418. 4. Surawicz CM, Brandt LJ, Binion DG, Ananthakrishnan AN, Curry SR, Gilligan PH, et al. Guidelines for diagnosis, treatment, and prevention of

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ogy. 2011;9(12):1044-1049. Available from: doi: 10.1016/j.cgh.2011.08.014. 12. Verbeke F, Janssens Y, Wynendaele E, Spiegeleer BD. Faecal microbiota transplantation: a regulatory hurdle? BMC Gastroenterology. 2017;17(1):128. Available from: doi: 10.1186/s12876017-0687-5. 13. Kelly CP, Pothoulakis C, LaMont JT. Clostridium difficile Colitis. The New England Journal of Medicine. 1994; 2018;330(4):257-262. Available from: doi: 10.1056/ NEJM199401273300406 14. Moayyedi P, Yuan Y, Baharith H, Ford AC. Faecal microbiota transplantation for Clostridium difficile-associated diarrhoea: a systematic review of randomised controlled trials. The Medical Journal of Australia. 2017;207(4):166-172. Available from: doi: 10.5694/mja17.00295. 15. Frank DN, St Amand AL, Feldman RA, Boedeker EC, Harpaz N, Pace NR. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(34):1378013785. Available from: doi: 10.1073/ pnas.0706625104. 16. Moayyedi P, Surette MG, Kim PT, Libertucci J, Wolfe M, Onischi C, et al. Fecal Microbiota Transplantation Induces Remission in Patients With Active Ulcerative Colitis in a Randomized Controlled Trial. Gastroenterology. 2015;149(1):102109.e6. Available from: doi: 10.1053/j. gastro.2015.04.001. 17. Rossen NG, Fuentes S, van der Spek MJ, Tijssen JG, Hartman JH, Duflou A, et al. Findings From a Randomized Controlled Trial of Fecal Transplantation for Patients With Ulcerative Colitis. Gastroenterology 2015;149(1):110-118.e4. Available from: 10.1053/j.gastro.2015.03.045. 18. Meijnikman AS, Gerdes VE, Nieuwdorp M, Herrema H. “Evaluating Causality of Gut Microbiota in Obesity and Diabetes in Humans”. Endocrine Reviews. 2017. Available from: doi: /10.1210/er.201700192/4772276. 19. Vrieze A, Van Nood E, Holleman F, Salojärvi J, Kootte RS, Bartelsman JF, Dallinga–Thie GM, Ackermans MT, Serlie MJ, Oozeer R, Derrien M. Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. Gastroenterology. 2012;143(4):913-6. Available from: 10.1053/j.gastro.2012.06.031 20. H Chuong ,Kim, O’Doherty K, Secko D. Media Discourse on the Social Acceptability of Fecal Transplants. Qualitative Health Research. 2015; 25(10):1359-71. Available from: doi: 10.1177/1049732314568199. 21. Guidance Document: Fecal Microbiota Therapy Used in the Treatment of Clostridium difficile Infection Not Responsive to Standard Therapies Ministry of Health Government of Canada Available from: https://www.canada.ca/en/health-canada/services/drugs-health-products/ public-involvement-consultations/ b i o l o g i c s - ra d i o p h a rm a c e u t i c a l s genetic-therapies/guidance-documentregulation-fecal-microbiota-therapy.html [Accessed 29th January 2018].

critical review

As FMTs move forward into larger and broader applications, there needs to be more policy considerations regarding its use and distribution. BIOSAFETY With potential for biohazardous contamination As FMT is a transfer of biomatter between and implications in human genetic material, dedonor and recipient, it carries the risks analo- cisions need to be made regarding how FMTs gous to that of a blood transfusion. Inadequate are to be handled in various legal and societal screening of donors may contribute to the contexts.12 The scientific community is only transmission of parasites, pathogens, or dis- beginning to grasp the full identity and power eases that have fecal links such as colorectal of the human microbiota. Moving forward, it is cancer.20 As the impact of fecal matter in the increasingly important to uphold the dissemipathology of some long-term illnesses is still nation of accurate information and its use in not fully understood, there exists the pos- supporting decisions. While clinical guidelines sibility that FMT may collaterally introduce are bound to evidence-based support, the public new conditions into immunocompromised must be mindful of media representation that patients. Currently, Health Canada includes a falsely interprets scientific findings for sensanon-exhaustive list of diseases to be screened tionalism or fraudulent conflicts of interest.12 for as a part of their donation protocol.21 More research into the link between fecal matter and various conditions needs to be conducted to prevent FMT-based disease transmissions.

INTERVIEW SPOTLIGHT

DR.DEBORAH SLOBODA BENCH TO BABY BELLY MILENA CIOANA1 & EDWARD CUI2

Bachelor of Health Sciences (Honours) Class of 2020, McMaster University Bachelor of Health Sciences (Honours) Class of 2021, McMaster University

 DR. DEBORAH

SLOBODA IS AN ASSOCIATE PROFESSOR IN THE DEPARTMENT OF BIOCHEMISTRY AND BIOMEDICAL SCIENCES AT MCMASTER UNIVERSITY. DEDICATED TO IMPROVING MATERNAL AND CHILD HEALTH, SHE EXPLORES THE RELATIONSHIP BETWEEN PRENATAL RISK FACTORS, SUCH AS POOR MATERNAL NUTRITION, AND LONG -TERM DISEASE RISK IN HOPES OF TRANSLATING HER BASIC SCIENCE RESEARCH INTO TANGIBLE IMPROVEMENTS FOR WOMEN’S HEALTH SERVICES IN HAMILTON.

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CAN YOU TELL US ABOUT HOW YOU BEGAN CONDUCTING RESEARCH ON THE DEVELOPMENTAL ORIGINS OF HEALTH AND DISEASE?

In my undergrad, I was always really interested in developmental embryology. I did my Masters of Kinesiology,

looking at how exercise changes maternal metabolism. During that time, I took a course that I found extremely interesting. I was going in to dispute a mark on the course’s exam, and I started asking my professor a lot of questions. His answer was, “Well, we don’t know the answer to any of that, but you could do a PhD and find out!” Afterwards, he offered ... me to do a PhD to find out the answers to those questions. I was fascinated by the topic as I had so many questions, and I decided to pursue the opportunity — I really liked it.



WHY IS IT IMPORTANT FOR US TO UNDERSTAND DEVELOPMENTAL BIOLOGY? WHAT DRIVES YOU TO DO THIS RESEARCH?

The biggest thing is the modifiable developmental window. Most of our research on non-communicable diseases targets areas that are not necessarily correctable. We try to find treatments. Prevention, however, can actually save lots of money for governments and also save individuals [from] stress, improve quality of life, and decrease inequity. I am really quite focused on how a preventative approach can improve the lives of kids growing up. We also know that there are transgenerational effects; improving the life of one fetus can have massive effects for many generations down the road. There is

a cycle of inequity, and if you can break it, you’ll allow those individuals to have a more positive outcome. It is really the long term ideology that fascinates me the most. I think that prevention can do a lot for how we live as a community.

 BEYOND BASIC BIOMEDICAL RESEARCH, YOU ARE HEAVILY ENGAGED IN COM MUNITY TRANSLATIONAL STUDIES LIKE YOUR MOTHERS2BABIES INITIATIVE. COULD YOU SPEAK MORE ABOUT THE LINK BETWEEN BIOMEDICAL AND TRANSLATIONAL RESEARCH?

Part of the Mothers2Babies study is based on the fact that Hamilton has a lot of disparity in a very small area. There are very “desperate” neighbourhoods in downtown Hamilton located very close to neighbourhoods with a much higher quality of life. Life expectancy is 85 in some areas and 70 in other areas just a few kilometres away. Mothers2Babies is a community-based knowledge translation study that aims to better understand the impact of these disparities on the barriers and enablers of pregnant women in Hamilton. We have an accessible online survey that is concerned with [barriers] preventing women from being healthy. The answers [are very] different based on where they live, their age, and other factors. We’re also engaged in focus group interviews with pregnant women, their partners, as well as community healthcare workers. What we want to do is gather all this information and work together with Hamilton Public Health to try to modify or extend the current pregnant programs in the city, so they are accessible to everybody.

I think it’s an important issue. I think the disparity in the scientific community is in part due to the fact that women’s health and preventative measures in women’s health are low on the priority list. That’s all wrapped up in promoting women in science. Now, more than ever, there are just as many, if not more, women engaged in scientific activities at the elementary or undergraduate level as compared to men. However, something is stopping them from going on. There are few women at higher level positions. This, I think, is largely a historical issue. Historically, white men were employed at the higher levels in many institutions and we, in general, are more comfortable hiring people like ourselves. Sometimes it’s conscious [and] sometimes it’s unconscious. We’re all human and implicit bias is a part of us. I think it’s important that we call it out wherever we see it. “Hang on, if all qualifications are comparable, perhaps we should give equal consideration to this lady from southeast Asia instead of the white male who grew up in Toronto.” Whether it be business or science, we know that having a diverse range of voices at the table leads to improved outcomes. I feel obligated to make sure that the [women’s health] equity I advocate through the scientific research I do is translated into real life.



DO YOU THINK ACHIEVING GENDER EQUITY IN THE SCIENTIFIC COMMUNITY DEPENDS MORE ON MACROSCOPIC CHANGE OR INDIVIDUAL ACTION?

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I think it’s a combination of both. [Gender equity in science] is like littering. The government can say that it’s illegal to litter, but it doesn’t necessarily cause everyone to stop littering. In the same way, universities can mandate that there shouldn’t be racism or sexism, but each one of us still has to acknowledge our own participation. Me chucking something on the ground may have little impact, but I have to acknowledge that my piece, plus your piece, plus your piece, really changes what the playground looks like. So I think it’s both. The policy-related changes must be in place, but we can’t just sit back and assume we have no role to play. Nowadays, we would be appalled to see somebody chuck something on the street, right? [Littering] is no longer acceptable in this society because we’ve called each other out by saying, “Excuse me, I think you dropped this piece of garbage.” All of us need to continually work on [the gender bias] by saying, “Actually, I’m not okay with this panel being entirely male. You’re going to have to change this or I’m not going to participate.” The policy changes don’t negate our personal responsibility of pointing it out to people who haven’t noticed.

M E D U CATO R

Ultimately, the qualitative data in Hamilton will tell us about the needs of those women on the community level. The basic science [component] lets me translate what the women know into science. For example, we found that a lot of women know that they must eat healthy during pregnancy. But, what they may not know, which we found in the lab, is that [their diet] during pregnancy will even affect their grandchildren. If [the women] understand that the program in which they are participating is fueled by hardcore science, the ideas conveyed through the program will be better accepted.

A STRONG ADVOCATE FOR GENDER EQUITY IN SCIENCE, WHAT DO YOU THINK IS THE MAIN REASON WHY WOMEN AREN’T SEEN AT HIGHER LEVELS IN SCIENCE? WHY ARE YOU SO PASSIONATE ABOUT THIS ISSUE?

interview spotlight

As much as I love investigating signalling pathways and understanding the minutia [within] developmental biology, I am not interested in studying pregnancy in mice or sheep — I want a bigger role to play. The majority of what I’m invested in doing is centred on the belief that we have significant disparities in the community. I think that if we can shed some light on these disparities through the use of science, we can improve community relationships. Many people, for example, blame obese individuals for their eating habits, but they fail to understand that obesity is really a disease embedded in early life. I am hoping that through biomedical research, we can try and change people’s views on how to treat this postnatally. We’re also looking at non-communicable diseases from the standpoint that it is all of our jobs to make sure that families, mothers, and babies grow up healthy, since they are our future.

 AS



GIVEN YOUR ACHIEVEMENT IN THE SCIENTIFIC COMMUNITY, WHAT DOES IT TAKE TO BE A GREAT SCIENTIST? IS IT ABOUT PUBLISHING THE MOST PAPERS OR IS THERE SOMETHING ELSE THAT ALLOWS YOU TO PRODUCE AMAZING WORK?

M E D U CATO R

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interview spotlight table of contents

As much as we like to write down numbers — how many papers have you published? What’s the impact factor, hindex and citation index? — I think what makes a great scientist is the passion you have for what you’re doing. If you lose the passion, then you’re done. How am I going to drag myself into the office and dedicate my life to reading papers about something I hate? I can’t [even] imagine it.

The Mothers2Babies study is so important to me because I am dedicated to those [underprivileged] women and to making their lives as good as mine, since I’ve been incredibly privileged. That’s what drives me. I’m sure every scientist would say that [about passion]. My ideal holiday would be sitting on the beach with a research paper. Who wants to do that? Often, my brother asks, “Are you working? Why are you working?” And I say, “Oh, I’m not working. It’s not work to me!” I’m as interested in reading a research paper as I am a fiction novel because I think it’s cool and I want to learn more about it. I think that’s the number one thing that makes a great scientist. Once you lose the passion, I can’t see how you could possibly dedicate the time or the effort to do what we do. [You must also be willing] to take risks, calculated risks. I think art and science are very similar in the fact that you have to be creative, and sometimes “just go with it” — see where it takes you. You don’t always have to be committed to exactly what you think should be happening. That becomes dangerous because you start making your data fit something predetermined. You [have to] be flexible, open, and willing to listen to the things happening around you. I just had this conversation with my PhD student. He wants to do something that I disagree with, but I said, “If you can convince me with some data, we’ll do it.” When it comes to taking risks with new things, you have to be open and listening.

 HOW HAVE YOU OVERCOME

THE CHALLENGES YOU’VE ENCOUNTERED ON THE ROAD TO BECOMING A SCIENTIST?

I had a strong support network and amazing mentors. That was the most important thing — having people around me that

supported what [I was] doing. Both my husband and my PhD supervisor have supported me every step of the way. There’s a lot to battle for women scientists, but it’s a hard road regardless of who you are. I tell my graduate students that graduate work is half scientific growth, half emotional battle. There’s [often] a lot of disappointment and a lot of happiness all at the same time. Sometimes, there’s a lot of disappointment and no happiness. Experiments [might not] work or your hypotheses [are] totally wrong. How are you going to overcome that? I often thought to myself, “Why does it always take me twice as long to get to where I think others are already at? Twice as many experiments, twice as many post-docs…” So, having people around you that you can talk to [is so critical]. Overcoming failure is a big thing that you have to master.

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Jessica Chee Jim Xie Kelvin Ng Kevin Chen Kevin Zhao Milena Cioana Parnika Godkhindi Sheila Yu Shikha Patel Takhliq Amir

Bachelor of Health Sciences (Honours) Program

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