PennScience Spring 2016
Volume 14 Issue 2
PennScience is a peer-reviewed journal of undergraduate research published by the Science and Technology Wing at the University of Pennsylvania and advised by a board of faculty members. PennScience presents relevant science features, interviews, and research articles from many disciplines, including the biological sciences, chemistry, physics, mathematics, geological sciences, and computer sciences. PennScience is funded by the Student Activities Council. For additional information about the journal including submission guidelines, visit www.pennscience.org or email firstname.lastname@example.org.
EDITORIAL STAFF EDITORS-IN-CHIEF DESIGN MANAGERS
Emily Chen Suzanne Knop
Richard Diurba Shelly Teng
Jane Chuprin Abhinav Suri
Tina Huang Alex Wong
WRITING Ritwik Bhatia Mia Fatuzzo Krisna Maddy Darsh Shah Evan Zou Paula Cauhy
DESIGN Alison Weiss Chigoziri Konkwo Sarah Cai
Samip Sheth Grace Ragi
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Dr. M. Krimo Bokreta Dr. Jorge Santiago-Aviles EDITING Angela Chang Joan Lim Jim Tse Andrew Wang Alex Wong Joyce Xu Zoe Daniels Rachel Levinson Abu-bakr Ahmed
Microbiota and the Development of the Adaptive Immune System
Antibiotics and Their Impact on the Microbiome
Vagal Pathways for Microbiome-BrainGut Axis Communication
The Metabolic Microbiome
Frederic D. Bushman, Ph.D. W. M. Measey Professor and Chair of the Department of Microbiology at the Perelman School of Medicine
The Role of Bafilomycin A1 in Autophagy and Apoptosis Pathways in Therapy-Resistant Neuroblastoma Sharon Y. Kim, Michael Hogarty, MD University of Pennsylvania
Exploring Quality of Life from the Perspective of Liver Transplant Patients Sophia Duque, David Goldberg, MD, MSCE University of Pennsylvania
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LETTER FROM THE EDITORS Dear Readers, We are pleased to present to you Volume 14, Issue 2, of the PennScience Journal of Undergraduate Research. In October 2015, the New York Times reported that leading scientists had “urged the creation of a major initiative to better understand the microbial communities critical to both human health and every ecosystem,” through two papers published concurrently in Science and Nature. The human body houses more than 100 trillion microorganisms, or microbes. Their genes, collectively termed the microbiome, outnumber our own genes one hundredfold and effectively serve as our second genome. This issue of PennScience addresses recent advances in our understanding of the human microbiome. In this issue, our writing committee considers the relationship between the microbiome and human health and disease. Ritwik Bhatia examines the role of the microbiome in Crohn’s disease, shedding light on the diagnosis and treatment of immune system dysregulation. Darsh Shah explores microbiome-brain-gut axis communication relying on the vagus nerve. Mia Fatuzzo explains the connection between overprescription of antibiotics and the rise of resistant bacteria. Paula Cauhy outlines the development of the adaptive immune system and its dependence on bacteria. Evan Zhou links the gut microbiome to human metabolic diseases, including obesity and Type I Diabetes. Finally, we are privileged to present an interview with Dr. Frederic Bushman, co-director of the Microbiome Program at the Children’s Hospital of Philadelphia, conducted by Krisna Maddy. This issue includes two research articles in the basic and clinical sciences, respectively. Sharon Kim investigates the role of Bafilomycin A1, an autophagy inhibitor, in therapy-resistant neuroblastoma, an extracranial solid cancer found in children. Her work may lead to greater understanding of specific cross-talk between the autophagy and apoptosis pathways in neuroblastoma. Sofia Duque studies liver transplant patients, evaluating their pre- and post-transplant quality of life and relationship to healthcare providers. Her work may impact the recovery outcomes for liver transplant patients. Our editing and design committees enhance our understanding of the microbiome and illuminate the work in this issue. We invite you to read through our journal and join a community of young investigators. PennScience continues to grow through coffee chats with professors, journal clubs with principal investigators, and collaboration with the Center for Undergraduate Research and Fellowships. Our business committee facilitates the scientific and professional enrichment of our membership, including a partnership with the Institute for Translational Medicine and Therapeutics Undergraduate Student Symposium. Please help us in thanking our faculty mentors, Krimo Bokreta and Jorge Santiago-Aviles, who guided the production of this issue; the Science and Technology Wing of the King’s Court College House, who launched PennScience; and the Student Activities Fund, who funded our organization. It is an honor to lead PennScience and promote scientific discourse at the University of Pennsylvania. We thank you for picking up our journal and hope you enjoy it cover-to-cover. Sincerely, Grace Ragi, C’18 and Samip Sheth, C’17 Co-Editors-in-Chief
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t h e d eve l o p m e n t o f t h e
ADAPTIVE IMMUNE SYSTEM by Paula Cauhy
he immune system present in most animals is absolutely essential in defending the organism against foreign substances. Despite its high efficiency, some microorganisms have found a way of bypassing the immune system and establishing themselves in hostsâ&#x20AC;&#x2122; bodies, creating a symbiotic relationship. These beneficial microorganisms constitute the microbiota. It is believed that they play an important role in the development and function of adaptive immunity in higher vertebrates, shaping certain subsets of T cells, namely Th17 and Treg.
soldiers assume different positions, for example, Th17 and Treg.
The cells of the adaptive immune system are called lymphocytes, which can be of two types: B and T. The T lymphocytes mature in the thymus and differentiate into various subsets. Helper T cells (CD4+) represent one of these types and act by influencing the behavior and activity of other cells. Furthermore, helper T cells also include different subsets, such as Th17 and Treg. Th17
It has been shown that many different beneficial bacteria in the gut induce Treg cells to express their specific transcription factor (Foxp3+) (1). Foxp3+ promotes regulatory phenotypes and functions by helper T cells. One of these bacteria, Bacteroides fragilis, is commonly used for microbiome studies. One of its bacterial polysaccharides (PSA) proved to be one of the microbial molecules that suppress inflammation pathologies in the host, and ultimately, to direct the development of the immune system (2). Studies in germ-free mice (born and raised in the absence of microbes, and consequently devoid of bacteria) demonstrated that Th17 development can be regulated by the microbiota (3). In these mice, less interleukin17, a small protein produced by Th17, was observed in the small intestine. This result is directly related to the fact that commensal bacteria in the intestine produce ATP, which drives the production of small proteins involved with cell signaling that induce Th17. Germ-free mice have this process impaired, affecting Th17 cell differentiation.
During infections, immature helper T cells rely on signals to promote their differentiation... this works as if the soldiers needed orders from the generals responsible for assigning positions and tasks. These generals would be the host and the microbiota. helps recruit other cells to sites of infection and is involved with extracellular bacteria and fungi. On the other hand, Treg cells (regulatory T cells) suppress and limit the immune response. Metaphorically speaking, the helper T cells would be the soldiers of a specific division of the army, and these 6
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The development of Treg and Th17 in the gut has been shown to be regulated by the microbiota. During infections, immature helper T cells rely on signals, which can come from the host and microbiota, to promote their differentiation into specific phenotypes. Recalling the previous metaphor, this works as if the soldiers needed orders from the generals responsible for assigning positions and tasks. These generals would be the host and the microbiota.
Evidence suggests that some of the gut microbiotaâ&#x20AC;&#x2122;s effects extend beyond the gastrointestinal tract. Experiments done with germ-
FEATURES free mice treated with PSA showed a reduced Th17 development and increased Treg cells in the central nervous system (4). Consequently, they showed decreased inflammation, since the Th17 cells promote inflammation and Treg prevents it. The capacity of some commensal bacteria to influence the two sides of a process like inflammation shows the magnitude of their effects. In fact, specific microbes that promote T cell differentiation are related to the development of autoimmune diseases in the host. Interestingly, some bacteria comprising the microbiota can predispose the host to self-reactivity. These bacteria intensify the production of Th17, which is usually associated with the promotion of autoimmunity. Thus, understanding the influence of the microbiome on the immune system may inspire alternative therapies for autoimmune diseases. Novel treatments could be developed from effectively using the ability of the microbiota to induce tolerance through Treg cells. The therapeutic advantages of using Treg cells are better understood, with a number of studies demonstrating their positive effects on the treatment of autoimmune rheumatic diseases, such as rheumatoid arthritis (5, 6).
Microbiota studies can shed a light on the coevolution between host and commensal microbes, as well as on new therapies for autoimmune diseases.
References: 1.Coombes, J. L., Siddiqui, K. R. R., Arancibia-Cรกrcamo, C. V., Hall, J., Sun, C.-M., Belkaid, Y. and Powrie, F. A functionally specialized population of mucosal CD103 DCs induces Foxp3 regulatory T cells via a T F- and retinoic acid dependent mec anism. T e Journal of xperimental Medicine. 2. Mazmanian, S. K., Liu, C. H., Tzianabos, A. O. and Kasper, D. L. An immunomodulatory molecule of symbiotic bacteria directs maturation of t e ost immune system. Cell. 3. Lee, Y. K. and Mazmanian, S. K. Has t e microbiota played a critical role in t e evolution of t e adaptive immune system? Science. 4. Lee, Y. K., Menezes, J. S., Umesaki, Y. and Mazmanian, S. K. (2011). Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encep alomyelitis. ational Center for Biotec nology nformation. . Miyara, M., to, Y. and Sakaguc i, S. (2014). Tregcell t erapies for autoimmune r eumatic diseases. ature.com. 6. Lan, Q., Fan, H., Quesniaux, V., Ryffel, B., Liu, Z. and Z eng, S. . (2011). nduced Foxp3 regulatory T cells: a potential new weapon to treat autoimmune and inflammatory diseases. Journal of Molecular Cell Biology.
Bacteria in the microbiota may have influenced features of the adaptive immune system, like the development of some effector T cells (such as Th17 and Treg cells), ameliorating the effectiveness nced microbial of immune responses. Balanced populations and Treg:Th17 proportions ng the are essential for avoiding colonization of bacteria thatt may eases. induce autoimmune diseases. Microbiota studies can shed light ween on the coevolution between bes, host and commensal microbes, as well as on new therapies for autoimmune diseases.
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Crohn’s Disease By Ritwik Bhatia
n America alone, over 600,000 individuals suffer from Crohn’s Disease, which, along with ulcerative colitis, comprises inflammatory bowel disease (IBD). Crohn’s disease is a chronic disease in which the immune system attacks the digestive tract, leading to severe abdominal pain, diarrhea, fatigue, and malnutrition. Currently, scientists and doctors are unsure what causes this disease, and attribute it to a combination of genetic, environmental, and bacterial factors. As such, while many treatment methods are available, there is no cure. However, recent breakthroughs in the study of microbiomes have offered insight regarding effective diagnosis and treatment for a variety of patients.
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FEATURES One tangible way of discerning if a patient is affected by IBD is to examine the microbiome, the entire ecological community of microorganisms influenced by environmental factors in the body, in his or her gut. The gastrointestinal tract bacteria in people with Crohn’s disease are significantly different than those in healthy individuals.
those of 26 healthy children. Upon analyzing fecal samples, the researchers found that all these therapies altered the abundance of gut microbes. Formula-based diet therapy subdued bacterial growth, but also led to the proliferation of fungi in the gut, which is usually associated with the aggravated state of Crohn’s. As a result, symptoms subsided and inflammation decreased, despite the fact that proper balance of the microbiome was For example, people diagnosed with Crohn’s disease are said not restored. Similarly, immunosuppressive therapy reduced to have “bad” bacteria (1). These “bad” bacterial organisms are inflammation, but without terminating fungal dysbiosis. The key part of the same family take-away remains that as Salmonella and E. coli, doctors do not need to which are known to cause The group, led by researchers Dr. James restore normal levels in severe reactions in human Lewis, Dr. Eric Chen, et al., concluded their patients in order bodily systems. Not for them to demonstrate only do these malicious that in order to lessen symptoms and therapeutic effects. bacteria flourish, but As novel therapies are beneficial bacteria are push the disease into remission, it developed, future studies found in much smaller is actually not necessary to restore will continue to monitor quantities. In addition, the condition of the gut patients with IBD have gut microbiome levels to normal. microbiome in response been noted to have to their administration. “lower levels of butyrate, and other short-chain fatty acids, decreased carbohydrate Ultimately, the goal of researchers and doctors is to find a cure for metabolism, and decreased amino acid biosynthesis” (2). this group of diseases. With increased funding being allocated These harmful populations of bacteria and associated loss to this cause, novel breakthroughs, and development of new of molecular function seem to be enhanced during times medicines, the future looks bright. Some of the most inspiring of flare-ups, when the condition and symptoms worsen. work has come from the Human Microbiome Project, an initiative by the National Institutes of Health, which aims to identify Currently, as there is no cure for the disease, doctors aim and analyze all microbiome populations and their genomes in to treat patients by keeping their symptoms in remission. individuals. These efforts will not only help our understanding Numerous methods are employed in the treatment of Crohn’s. of the human body, but will play an integral role in finding the For those with mild Crohn’s disease, medical professionals cure for IBD, and other diseases that affect human populations. often use mesalamine tablets and rectal suppositories. For those with worsening symptoms, various tumor necrosis factor (TNF) inhibitors, which suppress chemicals responsible for inflammation in the body, are available. A TNF binding protein is a monoclonal antibody that is controlled in healthy individuals such that adverse effects are not displayed. However, those with Crohn’s disease, as well as those suffering from forms of rheumatoid arthritis and other autoimmune diseases, have high levels of TNF in the blood. Since drugs using TNF inhibitors are immunosuppressants, patients are more susceptible to other infections. A microbial approach towards treatment involves drugs that specifically target gut bacteria. For example, antibiotics are used to eliminate harmful gut bacteria. However, this treatment is often used only as a first-line therapy, as “loss of protective microbes has the potential of triggering a proliferation of less beneficial taxa, exacerbating the inflammation” (2). References 1. Gevers, D, et. al (2014). The Treatment-Naive Microbiome This past year, a study conducted by researchers at the University in New-Onset Crohn’s Disease. Cell Host & Microbiome 15, of Pennsylvania made a remarkable discovery with great 382–392. implications for the treatment of Crohn’s. The group, led by 2. Wright EK, et. al (2015). Recent Advances in Characterizing researchers Dr. James Lewis, Dr. Eric Chen, et al., concluded that the Gastrointestinal Microbiome in Crohn’s Disease: A in order to lessen symptoms and push the disease into remission, Systematic Review. Inflammatory Bowel Diseases 21, 1219– it is actually not necessary to restore gut microbiome levels to 1228. normal (3). The researchers compared the gut microbiomes of 90 3. Lewis, J. D., et.al (2015). Inflammation, Antibiotics, and Diet children with Crohn’s disease, currently being treated with either as Environmental Stressors of the Gut Microbiome in Pediatric anti-TNF therapy or a formula-based diet of antibiotics, with Crohn’s Disease. Cell Host & Microbiome 18, 489-500.
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ANTIBIOTICS AND THEIR IMPACT ON THE MICROBIOME By Mi B Mia Fatuzzo F
oday, antibiotics render infections from strep throat to syphilis merely inconvenient. But one hundred years ago, most Americans died of infectious disease, such as pneumonia, influenza, and tuberculosis (1). Now, modern antibiotics offer cures for these previously devastating infections. The benefits of antibiotics cannot be understated—the drugs have lowered mortality rates and improved our quality of life. Recently, though, scientists have become concerned about the over-prescription of antibiotics. They link the overuse of these drugs to a worrisome rise in bacterial resistance to once impervious drugs
Could our use of antibiotics, the drugs we rely on to cure anything from strep throat to anthrax poisoning, be doing more harm than good? (2). In the United States alone, over 20,000 people die every year of now untreatable infections (2). In addition, new research has brought forth another concern regarding the over-prescription of antibiotics: they can alter our microbiota. Could our use of antibiotics, the drugs we rely on to cure anything from strep throat to anthrax poisoning, be doing more harm than good? The human microbiome includes all the microorganisms 10 PENNSCIENCE JOURNAL | SPRING 2016
that live symbiotically in and on our bodies. Although the specific functions of the microbiome are still being established, the overall structure of the system in a healthy human has been determined. We know that these bacteria help us to, for example, make vitamins, develop our immune system, and fight infection. The average human body hosts about one bacterial cell for every cell of its own, and the majority of these bacteria reside either in the colon or on the skin (1, 3). Several recent studies examined the impact of antibiotics on the human microbiome. In 2012, Carles Ubeda from the Center for Advanced Research in Public Health and Eric Pamer from the Sloan-Kettering Institute in New York demonstrated that antibiotics change the composition of the gut microbiota. The researchers administered a variety of antibiotics to healthy humans and compared the composition of their microbiota before and after dispensation. They found that, in general, the antibiotics decreased the “diversity, richness, and evenness of the fecal microbiota.” (4). The extent to which the effects remained after administration varied among antibiotics; for example, the microbiota proved more resistant to ciproflaxin (a strong antibiotic prescribed for anything from diarrhea to anthrax poisoning) than to clindamycin (a common antibiotic that targets anaerobic bacterial infections of the respiratory tract and soft
FEATURES tissue) (4). In 2015, a group of researchers from the Netherlands, Sweden, and the United Kingdom largely confirmed these results. The scientists explored the effects of four commonly prescribed antibiotics, clindamycin, ciprofloxacin, amoxicillin, and minocycline, on the oral and gut microbiomes. While the oral microbiome remained largely stable after exposure to antibiotics, the richness (number of species) of the gut microbiome was significantly decreased following their administration (5). These two groups of researchers also explored the connection between the altered gut microbiome and antibiotic resistance. Again, they came to similar conclusions. Ubeda and Pamer demonstrated that administration of an antibiotic often led to an increase in the frequency of bacterial resistance to that antibiotic. For example, when the researchers gave patients strong antibiotics such as metronidazole (Flagyl – typically used to treat C. difficile), neomycin (in Neosporin), and vancomycin (a strong antibiotic, typically a last resort), they saw an increase in vancomycin-resistant enterococcus (an infection which causes diarrhea, fever, and chills) in the colon (4). The Scandinavian researchers also observed an increase across all samples in genes associated with antibiotic resistance in the gut microbiome; the antibiotic resistance gene load increased from about 0.97 to 1.07 (relative abundance) (5). The researchers then looked at each antibiotic individually. They found that molecular mechanisms of resistance tended to increase in frequency after the administration of antibiotics. For example, the administration of amoxicillin was associated with a rise in beta-lactamases, a class of enzymes that break a critical 4-atom ring in amoxicillin (5). These results established that even one round of antibiotics significantly alters the symbiotic relationship between our microbiota and our digestive system. Furthermore, these antibiotic-induced shifts in the gut microbiome have been linked to increased susceptibility to infection. For example, Clostridium difficile, an inflammation of the large intestine, occurs when C. difficile bacteria supplant the normal gut bacteria. Individuals on antibiotics, whose gut bacteria are compromised, are more susceptible to infection. The defenseless gut microbiome is overrun by C. difficile (6). While mild antibiotic-associated cases are often resolved by stopping the offending antibiotic, more serious cases require another antibiotic such as Metronidazole or Vancomycin. Unfortunately, treating antibiotic-associated C. difficile with stronger antibiotics only further predisposes the patient to C. difficile infection. Gastroenterologists, doctors who specialize on the digestive system, are now hoping to use this newfound understanding of the gut microbiota to their advantage. In a 2009 paper, several doctors suggested capitalizing on the microbiome-altering effects of antibiotics to remove unwanted bacteria from the colon. This idea largely stems from research linking conditions such as Crohn’s disease and ulcerative colitis to aberrant gut microbiota (7). This personalized approach, combined with bacteria-introducing probiotics, would allow doctors to clean the colon of aberrant microbiota and, ultimately, return to the colon a normal and healthy composition of bacteria (8). More recently, doctors have used fecal transplantation to provide sick individuals with the gut bacteria of a healthy peer. This technique, in testing, has proven helpful for patients with intractable C. difficile and IBD (8).
Research has established that antibiotics both alter the composition of the gut microbiome and increase the frequency of antibiotic-resistant bacteria. The danger of entirely resistant bacteria, infections which neither our bodies nor our drugs can fight, is well-established. The solution to this problem, namely, reducing antibiotic prescription drastically, is theoretically simple but ethically and practically complicated. How would a doctor deem a patient “worthy” of antibiotics? The effects of altering the composition of our gut microbiome are somewhat less understood. Ideally, a healthy gut microbiota produces vitamins and regulates development. Aberrational gut microbiota have been linked to Crohn’s disease and ulcerative colitis, among other conditions (9). But the average patient simply does not experience any palpable short-term effects from a course of everyday antibiotics. Therefore, more research regarding the long-term, visceral effects of antibiotics on the gut microbiome is required. References: 1. Mortality and Cause of Death, 1900 v. 2010 (2014). Carolina Demography. 2. Antibiotic Resistance Threats in the United States, 2013 (2014). Centers for Disease Control and Prevention. 3. Sender, R., Fuchs, S. and Milo, R. (2016). Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host Cells in Humans. Cell. 4. Ubeda, C. and Pamer, E. (2012). Antibiotics, microbiota, and immune defense. Science Direct. 5. Zaura, E. et al. (2015). Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus LongTerm Microbial Shifts in Feces. mBio. 6. Chang, J. Y. and et al. (2008). Decreased Diversity of the Fecal Microbiome in Recurrent Clostridium difficile—Associated Diarrhea. The Journal of Infectious Disease. 7. Round, J. and Mazmanian, S. (2009). The gut microbiota shapes intestinal immuneresponses during health and disease. Nature Reviews Immunology. 8. Preidis, G. and Versalovic, J. (2009). Targeting the Human Microbiome WithAntibiotics, Probiotics, and Prebiotics: Gastroenterology Enters the Metagenomics Era . Science Direct.
9. Hofer, U. (2014). Bacterial imbalance in Crohn’s disease. Nature Reviews Microbiology.
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Vagal Pathways for Microbiome-Brain-Gut Axis Communication By Darsh Shah
id you know that your brain is as closely connected to your stomach as your arms or legs? It turns out that the stomach-brain link is more significant than we had ever thought, which has proven to be useful in diagnosing mental conditions and disorders, including depression and anxiety. Many scientists already understood the physiological mechanisms contributing to brain function, but only recently have they become acutely aware of another important factor: the gut microbiome. That is, there exists a matrix of bacteria supplied with nerves in the stomach, known as the enteric nervous system (ENS), which greatly influences the central nervous system (CNS), especially the brain. These vagal pathways that connect this microbiome directly to the CNS have been shown to modulate brain activity and impact behavior in surprising ways.
These vagal pathways that connect this microbiome directly to the CNS have been shown to modulate brain activity and impact behavior in surprising ways. The abundant exchange of sensory information between the stomach and the brain is due to the vagus nerve. Sensory neurons are the main messengers of the body, sending information along these vagal or spinal afferent routes from the ENS to the brain (1). In this way, the ENS is inseparably connected to the CNS. Furthermore, the innervation of the stomach indicates that an acidic environment brimming with numerous varied receptors will emerge. In particular, chemoreceptors and nociceptors figure prominently in this milieu and constantly send along sensory information to the brain (2). Because of this robust interconnectivity between the ENS and CNS, it becomes clear that a bacteria culture living in the gut would exert some force over the general functions of the brain and influence behavior in different ways.
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FEATURES If such a nexus exists between the gut microbiome and the brain via vagal pathways, then it follows that altering the microbiome would temper mental dysfunction. In fact, a recurring pattern is that any gut-related pathology may usher in a spate of mental health-related issues, including depression and anxiety (3). Recent studies have shown that patients with autism and depression also tend to have chronic gastrointestinal problems. Inflammatory bowel diseases, for example, occurred in such patients when the gut microbiome released host immune factors, including cytokines and inflammatory mediators, which targeted the CNS (1). Emotional stability is also associated with microbial balance in the gut microbiome. In other words, if the microbial structure of the gut microbiome were modulated, then mental disorders might be alleviated.
Emotional stability is also associated with microbial balance in the gut microbiome. In other words, if the microbial structure of the gut microbiome me were modulated, then mental disorders might be alleviated. lleviated. Specifically, electrostimulation of the vagus nerve has been shown to reduce depressive symptoms and behaviors, resulting in greater metabolism of the amino acid tryptophan into the neurotransmitter serotonin (2). This increase in serotonin can reduce anxiety and aggression by activating receptors on the presynaptic terminals of adjacent neurons. In addition to electrostimulation, ingestion of beneficial bacteria to smooth out any unevenness in the gut microbiome has been associated with the attenuation of chronic stress and anxiety syndromes (4). Vagal pathways connecting the gut microbiome in the ENS to the cortices of the brain in the CNS are taking on new importance with regard to mental and emotional states. Any imbalance in the gut microbiome would immediately affect the cerebral cortex and medulla oblongata, integral parts of the CNS. Certain techniques exploiting electrical connections may bring balance to a disturbed microbiome, ameliorating mental state and mental disorder. By modulating the gut microbiome, we can partially remedy illnesses such as major depressive disorder and anxiety.
References 1. Forsythe, P., Bienenstock, ienenstock, J. and Kunz Kunze, ze, W W.. A. (2014). Vagal Pathways for Microbiome-Brain-Gut Ax A Axis xis is Communication. perimental Medicine aand nd Biology Biology Microbial Advances in Experimental Endocrinology: The Microbiota-Gut Microbiota-Gut-Brain t-Bra rain A ra Axis x s in Health and xi Disease 115–133.. 2. Lyte, M. (2013).. Microbial Endocrinology Endo ocrin crinol cr inol in olo olog oggy in in the the MicrobiomeGut-Brain Axis: How Bacterial Prod Production oduc od duccti tion aand n Utilization nd of Neurochemicals PLoS cals Influence Behavior. Beh hav avior. v PLo L S Pathog Pathog PLoS Pathogens. 3. O’Mahony, S., Clarke, G., Borre, Y., Dinan, T. and Cryan, J. (2015). Serotonin, tryptophan metabolism and the brain-gutmicrobiome axis. Behavioural Brain Research 277, 32–48. 4. Flight, M. H. (2014). Neurodevelopmental disorders: The gut–microbiome–brain connection. Nature Reviews Drug Discovery Nat Rev Drug Discov 13, 104–104.
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The Metabolic G
astroenterology research has propelled the gut microbiome into the spotlight as a key indicator for metabolic disorders. A combination of metagenomics, the study of genes derived directly from the environment in which an organism functions, and correlative studies achieved groundbreaking success in identifying microbial bases for obesity. In this context, researchers are now exploring disorders associated with obesity, such as Type 1 Diabetes. Since an estimated 80% of all people suffering from Type 1 Diabetes are categorized as obese, researcher Ramnik Xavier from the University of Cambridge analyzed the microbiome’s role in Type 1 Diabetes (1). To identify bacteria in the microbiome warranting investigation, scientists began with cohort studies. By looking at differences among individuals, studies discovered correlations implicating specific bacteria in metabolic disorders. As trillions of bacteria reside in the microbiome, it is necessary to filter out those without metabolic roles. The studies have significantly contributed to more precise investigations. For instance, investigations into families have shown that a certain degree of the microbiome is conserved across generations, although varying lineages of
For instance, investigations families have shown that a certain degree of the microbiome is conserved across generations, although varying lineages of bacteria become introduced throughout one’s lifetime. bacteria become introduced throughout one’s lifetime. In these families, the observance of an obesity phenotype was associated with reduced bacterial diversity. This suggests that the sheer range of bacterial types can lead to metabolic disorders. In fact, when bacteria directly extracted from the human gut were analyzed taxonomically, researchers found a relationship between obesity and the ratio of Firmicutes to Bacteriodetes, two of the most abundant human gut bacteria. While normal weight individuals displayed an almost 1:1 ratio of the two bacterial phyla, obese individuals had guts containing a 7:1 Bacteroidetes to Firmicutes ratio(2). However, these taxonomic discoveries are not scientifically sound enough to explain the connection to obesity. Metagenomics has bridged this gap, clarifying taxonomic variability as an important indicator for bacterial genetic imbalances. Upon sequencing the genomes of Firmicutes and Bacteriodetes, scientists found significant differences between and even within the two phyla, especially metabolically (2). In fact, Katherine 14 PENNSCIENCE JOURNAL | SPRING 2016
Pollard at the Gladstone Institutes devised a method to deteration of different strains of bacteria, which can mine the variation % genetically, calling into question the validity of differ up to 30% mic variability (3). The results prompted Dr. Polsimple taxonomic lard and other scientists to scrutinize the genetic makeup of gut microbiomes. Using mouse models, Peter Turnbaugh induced th contrasting genomes, specifically targeted for microbiota with their enzymaticc and metabolic activity. Though 95% of the gut enomes were conserved, the small variations were microbiome genomes se a noticeable change in appearance. When they enough to cause inserted genes into the microbiome that encoded enzymes aking down otherwise indigestible polysaccapable of breaking charides, the Turnbaugh group found an obesity phee. The mouse intestine absorbed the bronotype in mice. rients rather than passing it through the ken-down nutrients digestive tract, showing that the microbiome’s activity altered mouse metabolism (4). After publication, additional studiess corroborated the idea that energy extraction by the gut microbiome can significantly abolism of the organism itself (5). alter the metabolism Extrapolating to other disorders, Ramnik Xavier ethodology from obesity investiapplied the methodology dy Type 1 Diabetes, an inherited gations to study n be diagnosed at around three disease that can years of age in humans. As microbial commued down, they potentially affect nities are passed the metabolic disorder’s heritability. Using huenetically predisposed to Type 1 man infants genetically Diabetes, the team from Cambridge sampled omes throughout the early years their microbiomes nt. As seen in the obesity studies, of development. ificant indicator of Type 1 diathe most significant op in bacterial diverbetes was a drop mpted genomic sity. This prompted sequencing, which did not indicate differences in the metabolic activity of nts diabetic infants during the stage of hdiagnosis. Rather, both the metabolite concentrationss in the gut and the metabolic uptake of thee microbiome remained constant further study revealed that the type (1). However, lites shifted significantly during this of metaboing a changing metabolic function stage, indicatof the bacterial community in general. Thus, Ramnik Xavi-er’s findings supported the genomic
Microbiome basis emphasized by Katherine Pollard and Peter Turnbaugh. While the aforementioned investigations have contributed much to our knowledge of the microbiome’s impact on human systems, additional steps must be taken to gauge the microbiome’s potential for medical applications. While research into related complications such as heart disease, high blood pressure, stroke, and cancer, have yet to warrant translational screenings, scientists have applied the methods from studying obesity to these disorders. Despite these limitations, research has already opened up pathways directly related to health. Metagenomics can contribute to a system of data analytics that identifies microbial factors that may predispose one to Type 1 Diabetes and associated disorders. Moreover, studies have already shown the effectiveness of treatments that modify the gut microbiome (6). Rather than invasively removing or replenishing gut bacteria, researchers have promoted fecal transplantations: extracting the microbiome from a healthy person and introducing it orally via pill to an affected individual. Such a potential solution allows for cheaper and simpler remedies to metabolic disorders that would otherwise require a lifetime of pills and tests to manage.
By Evan Zhou
References 1. Xavier, R.J., Kostic, A.D., Gevers, D., Siljander, H., Vatanen, T., Hyötyläinen, T., Hämäläinen, A., Peet, A., Tillmann, V., Pöhö, P., Mattila, I., Lähdesmäki, H., Franzosa, E.A., Vaarala, O., Goffau, M., Harmsen, H., Ilonen, J., Virtanen, S.M., Clish, C.B., Orešič, M., Huttenhower, C., Knip, M. (2015). The Dynamics of the Human Infant Gut Microbiome in Development and in Progression toward Type 1 Diabetes. Cell 17, 260-273. 2. Turnbaugh, P.J., Hamady, M., Yatsunenko T., Cantarel B.L., Duncan A., Ley R.E., Sogin ML., Jones W.J., Roe B.A., Affourtit J.P., Egholm M., Henrissat B., Heath A.C., Knight R., Gordon J.K. (2009). A core gut microbiome in obese and lean twins. Nature 457, 480-484. 3. S. Nayfach, K.S. Pollard. (2015). Population genetic analyses of metagenomes reveal extensive strain-level variation in prevalent human-associated bacteria. bioRxiv. 4.Turnbaugh,P.J.,LeyR.E.,Mahowald,M.A.,Magrini,V.,Mardis, E.R., Gordon, J.I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027-1031. 5. Holmes, E., Li J.V., Athanasiou, T., Ashrafian, H., Nicholson J.K. (2015). Understanding the role of gut microbiome–host metabolic signal disruption in health and disease. Trends in Microbiology 19, 349-357. 6. Vrieze, A., Groot P.F., Kootte, R.S., Knaapen, M., Nood, E., Nieuwdorp, M. (2013). Fecal transplant: A safe and sustainable clinical therapy for restoring intestinal microbial balance in human disease? Best Practice & Research Clinical Gastroenterology 27, 127-137.
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CONDUCTED BY Krisna S. Maddy In what ways have physician-scientists approached understanding host-microbiome interactions in the past decade?
Dr. Frederic D. Bushman serves as the W.M. Measey Professor and Chair of the Department of Microbiology at the Perelman School of Medicine at the University of Pennsylvania. Dr. Bushman completed his Ph.D. in Cell and Developmental Biology at Harvard University in 1988, and went on to complete his postdoctoral training at Harvard and the National Institutes of Health. He joined the faculty of Penn’s Perelman School of Medicine as a professor of Microbiology in 2003. His laboratory investigates hostmicrobe interactions in health and disease while focusing on the human microbiome, HIV pathogenesis, and DNA integration in human gene therapy. Dr. Bushman also serves as the co-director of the Penn CHOP Microbiome Program. He has published more than 250 scientific papers. His leading work in the field of microbiology has earned him accolades such as the Pioneer Award from the journal Human Gene Therapy.
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The “microbiome” is a relatively new nickname for research involving whole communities of organisms associated with the human body. Human poop, for example, has a hundred billion bacteria per gram. Enormous communities of microbes are associated with our bodies. One of the big things that has changed in recent years is the ability to measure this. Before, maybe 20 years ago, it was mostly not possible to study microbes associated with living humans in detail because we could not get them out of humans easily and grow them and identify them and do conventional bacteriology to study them. What’s changed is that we can do a lot of that now at the DNA level. If you want to know what kind of bugs are in someone’s lower gut, you can take a fecal sample, purify DNA, sequence the DNA, align those sequences to databases, and thereby read out the collection of bugs that are there. It’s this DNA-sequencing technology that has exploded in recent years that’s made possible the sort of efficient characterization of any kind of microbial community. We published a paper recently where the main data was half a trillion bases of DNA sequence, just enormous amounts of information, and that’s becoming increasingly routine. What are some cases in which studying the gut microbiome is particularly important? So the poster child for work in this area is a very nasty infection of the gut called Clostridium difficile. If you’re taking antibiotics, hospitalized, or elderly, you can lose your normal community, and instead have this bug come and lodge in your gut, which can make you very sick. It can be fatal. However, a stool transplant is curative. Approximately 95% of the time you can take the bacterial community from a healthy person’s poop, purify it, and give it to an infected person, and it allows you to become healthy again. So, stool transplantation is a dramatic example of how you can transform the community in your gut. One of your recent publications pertains to the microbiota of patients infected with Human Immunodeficiency Virus. How does the microbiome play a role in patients with immune deficiency disorders? The modern picture of you and your microbes is dynamic. Your immune system is sensing the bugs that are living associated with your body. There’s a dynamic equilibrium. For example, you make a type of immunoglobulin called Immunoglobulin A (IgA)1 and secrete a gram every day into your gut, where it binds
to the bacteria. So here’s this idea of immune tone. If you have too much immune activity, it can lead to disease or if you have too little, the bugs you are normally keeping stabilized and in control are now no longer stabilized, and so out of control. In Human Immunodeficiency Virus (HIV) infection, the immune system is suppressed, so you have numerous different kind of bugs growing out and harming patients due to opportunistic infections. Similarly, opportunistic infections are a big problem in organ transplantation. Previously, we looked at clinical assays and asked what grew, but sometimes, you can’t grow things efficiently or you don’t know what to look for. The DNA-based methods can tell you in a much more unbiased fashion what’s there. We’ve done a couple more experiments in that direction and we’re seeing a lot of new bugs in patients in the intensive care unit, for example. There’s a lot more to be done in that direction. How does microbiota affect cancer and related diseases? There’s been a lot of research and a bunch of suggestions. Inflammation is associated with cancer, and some bugs will promote inflammation in your body. Some bacteria encode specific toxins that are associated with cancers. The microbiota influences drug metabolism and it differs a lot between human individuals. The success of a drug is often modulated by the composition of the microbiome in your gut. You mention that the gut bacterium in an adult roughly stabilizes over time? Can we as humans do anything to engineer our own microbiome? One way that I think doesn’t work is drugstore probiotics2. They’re pretty much snake oil as far as I can tell. The amount of bacteria in those pills is extremely tiny compared to the amount of bugs that are already there. However, eating high fiber diets, fresh fruit, and vegetables can affect your gut microbiota and has numerous health effects. You are one of the directors of the CHOP Microbiome Program. Could you tell us more about the work of the program? Here at Penn and CHOP, we have a lot of activity in the microbiome area. We were a site for the Human Microbiome Project3. We’ve contributed a bunch of interesting work on Inflammatory Bowel Disease (IBD) and the lung microbiome in health and disease. For many of the diseases of the modern world, like cancer, autism, and autoimmune diseases, they’ve come on strongly in recent years, much more rapidly than changes in human genetics can explain. Attention turns to environmental influences, and one potential influence is the microbiome, the bugs that live associated with your body. Those clearly are different between people living traditional agrarian lifestyles and people living in modern environments. There are proposals for modulation by the microbiome for pretty much all of these diseases. There’s an association of disease and health with differences in the microbiome
structure, and that’s a lot of the reason that it’s such a popular research area right now. Where do you see the advancement of microbiome research going in the next decade? I think one area will be designing communities of microbes and installing them in your gut or elsewhere in order to improve our health. We had a paper that came out recently with my colleague Gary Wu in which we engineered the microbiota in mice to mitigate a metabolic disease. So if you’re having liver failure or a certain genetic disorder that makes too much ammonia, it turns out that some of the ammonia you make in your body comes from a compound called urea. Only bacteria encode the machinery for splitting urea to make ammonia and carbon dioxide. We did a study in which we took mice that were sick from hyperammonemia, removed the normal gut community, and put in a gut community that was low in urease activity. We were able to mitigate some of the disorder. This is an example of engineering our microbiome to benefit our health. Several companies are looking at ways to engineer microbiota. Could you tell us about your first research experience? I had a job as a technician between college and graduate school, and during that time recombinant DNA was just becoming available. As a technician, I worked with recombinant and I just really wanted to make things out of DNA. So I went to grad school, did some DNA-related work, and it was great—I really enjoyed it. I’ve always been interested in technology development, so more recently we wanted to take advantage of the new DNA sequencing methods to explore microbial communities. What advice do you have for undergraduates hoping to get more involved in research? Whatever excites you, do more of that—follow up on the things you think are really cool. Be open-minded and let your interests evolve. Expose yourself to novelty, try things, build skills, and follow up on what you think is the coolest thing. 1. Immunoglobulin is an antibody that plays a role in the immune function in mucous membranes. 2. Probiotics are live microorganisms that are believed to provide health benefits and prevent and treat some illnesses. 3. “The Human Microbiome Project (HMP) is developing research resources to enable the study of the microbial communities that live in and on our bodies and the roles they play in human health and disease.” —https://commonfund.nih.gov/hmp/index
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The Role of Bafilomycin A1 in Autophagy and Apoptosis Pathways in Therapy-Resistant Neuroblastoma Sharon Y. Kim, Michael Hogarty, MD Childrenâ&#x20AC;&#x2122;s Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine Neuroblastoma is the most prevalent extracranial solid cancer tumor found in children. Most high-risk cases involve the amplification of the MYCN oncogene, which results in poor outcomes due mainly to therapy resistance. Bafilomycin A1 is a successful inhibitor of autophagy, disrupting lysosomal degradation and the fusion between lysosomes and autophagosomes. We used the autophagic flux inhibitor Bafilomycin A1 to demonstrate that there may be higher levels of autophagic flux activity in therapy-resistant neuroblastoma cells than in therapy-sensitive neuroblastoma cells. Our results suggest that there may be higher levels of autophagosomes in therapy-resistant neuroblastoma than in therapy-sensitive neuroblastoma cells following Bafilomycin A1 treatment. Thus, autophagy may be used as a survival mechanism for therapy-resistant neuroblastoma cells to malignantly proliferate. We present a preliminary study in understanding the mechanistic crosstalk between autophagy and apoptosis in therapy-resistant neuroblastoma, alluding to potential therapeutic avenues.
Introduction Neuroblastoma is the most prevalent extracranial solid cancer tumor found in children. Originating from the neural crest of the sympathetic nervous system, it is a neuroendocrine tumor that not only forms in the adrenal glands but can also form in other nerve tissues, such as the pelvis, neck, chest, or abdomen. Neuroblastoma is known to have sporadic regression from an undifferentiated cell state to a benign cellular appearance and is a highly heterogeneous disease that is categorized into low, intermediate, and high risk disease. Nearly half of neuroblastoma cases can be categorized as high-risk disease with a poor survival rate outcome of only 40%-50%. Most high-risk cases involve the amplification of the MYCN oncogene and result in poor outcomes mainly due to therapy resistance. Consequently, it is critical to understand the biological mechanisms responsible for therapy-resistant high-risk neuroblastoma in order to offer new avenues for effective therapy. Apoptosis and autophagy have a complex relationship that has yet to be completely understood in neuroblastoma. While apoptosis is the programmed death of damaged or undesired cells, autophagy is a catabolic pathway that removes cellular debris through lysosomal degradation or recycles crucial proteins or organelles. Loss of apoptosis (Type I Cell Death Program) can contribute to therapy resistance of cancer tumors. Since autophagy is an alternative cell death program (Type II Cell Death Program), it is important to characterize the molecular cross-talk between autophagy and apoptosis and how this contributes to therapy-resistant tumor growth. The role of autophagy in cancer formation is complex due to its dual-functionality: it consists of both tumor-promoting and tumor-suppressing properties. On the one hand, autophagy is correlated with preventing cancer development with tumor suppressive gene regulators such as Beclin 1, p53, DAPk, p19ARF, 18 PENNSCIENCE JOURNAL | SPRING 2016
TSC, LKB, and PTEN. However, disruptions in autophagy may lead to tumor-progression due to the inability to eliminate damaged cells through type II cell death, in conjunction with the effects of dysfunctional type I apoptotic cell death program. Thus, the inhibition of autophagy may be effective in targeting therapy-resistant cancer cells. For instance, autophagy may protect tumor cells from cellular stress or continuously supplying nutrients. Ultimately, understanding how autophagy can induce cancer cell death or survival in therapy resistant neuroblastoma is important for the development of new cancer therapies. Bafilomycin A1 (BFA1), a macrolide antibiotic that originates from Streptomyces bacteria, inhibits vacuolar H+ ATPase (V-ATPase). It attaches to the V0 component c of the V-ATPase complex and blocks H+ translocation, thus causing an abnormal buildup of H+ in the cellâ&#x20AC;&#x2122;s cytoplasm. As such, Bafilomycin A1 can be used to block autophagic flux, thereby disrupting cell growth and inducing apoptosis and differentiation. The intracellular acidosis due to the bafilomycin A1 inhibiting V-ATPase is responsible for the anti-tumor effects of bafilomycin A1. In addition, Bafilomycin A1 is known to be a successful inhibitor of autophagy because of its role in disrupting lysosomal degradation and the fusion between lysosomes and autophagosomes. Bafilomycin A1 has been shown to affect both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Therefore, we propose that Bafilomycin A1 may be a candidate drug design for therapy-resistant neuroblastoma. Furthermore, Bafilomycin A1 has been shown to simultaneously induce apoptosis and inhibit autophagy in pediatric B-cell acute lymphoblastic leukemia through the formation of Beclin 1-Bcl-2 complex. We propose that targeting autophagy in apoptosis-resistant neuroblastoma could be an effective strategy to promote cell death in tumor cells and to obstruct cancer development in a therapy-resistant tumor environment.
RESEARCH In this paper, we attempt to characterize the function of autophagy in neuroblastoma after treatment with Bafilomycin A1, an autophagic flux inhibitor. Methods Chemicals Bafilomycin A1 from Sigma-Aldrich (St. Louis, MO, USA) was used at a concentration of 5 nM unless specified with alternate doses. Bafilomycin A1 was prepared as a 10 nM stock solution in DMSO (Sigma, D8418-1L) and stored at -20 degrees Celsius. Cells were plated onto 6 well-plates or T-25 flasks and subsequently treated with either DMSO vehicle (0 nM control) or Bafilomycin A1 (1 nM - 100nM) for 4h-48h or specified otherwise. Cell Culture Four human neuroblastoma cell lines of both pretherapy and post-therapy were used in our research. CHLA15 (pre-therapy tumor cells) and CHLA20 (post-therapy tumor cells) were acquired from Children’s Oncology Group of Texas Tech University School of Medicine (Texas, USA). Cells were maintained in complete IMDM (Iscove’s Modified Dulbecco’s Media): 20% FBS (Fetal Bovine Serum), 1% Penicillin Streptomycin, 1% L-glutamine, gentamycin, and 1x ITS-Growth Factor. Cells were cultured as monolayer
Figure 2. Autophagy in Cancer Promotes Therapeutic Resistance. Tumor cells revert apoptosis through the survival mechanism of autophagy in response to metabolic crisis. Cancer cells may be highly dependent on autophagy due to the strenuous metabolic demands in malignant proliferation. . Other studies have demonstrated promising therapies through the use of small-molecule autophagy inhibitors such as 3-methyladenine, Bafilomycin A, chloroquine, and hydroxychloroquine.
in a humidified atmosphere containing 5% CO2 at 37 degrees Celsius. Cells were used for experimentation once the confluency reached approximately 60-80%.
Figure 1. Overview of the molecular mechanisms underlying the autophagy–apoptosis crosstalk. Three mechanistic paradigms of apoptosis regulation by autophagy are shown. Blue lines indicate stimulatory interactions, the red lines indicate inhibitory interactions and dotted lines indicate interactions that are interrupted. PPI stands for protein-protein interaction; MOMP stands for mitochondrial outer-membrane permeabilization. Assaf D. Rubinstein, and Adi Kimchi J Cell Sci 2012;125:52595268.
Protein extraction and Immunoblot analysis After Bafilomycin A1 treatment for specified time, cells were collected, and plates were incubated in trypsin for 5 minutes at 37 degrees Celsius to detach cells. Whole cell lysates were extracted and protein concentrations were derived using BCA protein assay (Fisher Scientific, PI-23227). 30 ug of each protein sample were then loaded onto 12% NuPage Bis/Tris SDS-polyacrylamide precast gels (Fisher Scientific, PI-NW00120BOX) and transferred to polyvinylidene difluoride (PVDF) membranes through the Novex iBlot transfer system (Life Technologies, PI-IB24002). Blots were then washed with 1X tris-buffered saline with tween (TBST) and incubated at room temperature with 5% blocking milk (Bio-Rad, 170-6404). Membranes were then incubated in primary antibodies for immunodetection of the following proteins: LC3 (Microtubule-associated protein light chain 3)(rabbit anti-LC3, Abcam ab51520); Monoclonal Anti-b-Tubulin, Clone AA2 (Sigma Aldrich, T8328). Membranes were washed with 1X TBST containing 0.1% Tween 20 and then incubated with secondary IgG-HRP conjugated antibody for 1 hour at room temperature. After washing blots, enhanced chemilumescence (ECL; Thermo Scientific, PI32106) was used to detect LC3. Blots were then stripped with Restore Western Blot stripping buffer (Thermo Scien-
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RESEARCH tific, 21059) and probed for tubulin (Sigma Aldrich, T8328) to normalize for gel loading. Films that contained the desired bands were scanned and band intensities were quantified utilizing ImageJ gel digitizing software. LC3II/B-tubulin ratios were determined by densitometry (mean +- SD) P<0.05. The expression of anti-Beta-tubulin is used as a loading control. ImageJ LC3II/Beta-tubulin Ratio Calculation Autophagy can be measured by the conversion of LC3-1 to LC3-II, which is indicative of the amount of autophagosomes. Band intensities of LC3II were calculated through ImageJ software, and the averaged values were used to calculate the LC3II to Beta-tubulin band intensity ratios. A ratio was used to address the issue of unequal protein sample loading when carrying out the immunoblot experiment. Beta-tubulin is a protein loading control that was used to mark how much of the whole cell lysate sample was loaded. Thus, when comparing samples of unequal loading, the ratio would allow us to look definitively at LC3II levels. Results Bafilomycin A1 effectively inhibits autophagy, and higher levels of autophagic flux is observed in post-therapy neuroblastoma cells CHLA-15 pre-therapy cells with no treatment of Bafilomycin A1 did not exhibit anti-LC3I or anti-LC3II band signals in immunoblot analyses. Likewise, CHLA15 pre-therapy cells treated with 5 nM Bafilomycin A1 for 4h did not exhibit band signal for anti-LC3I or antiLC3II in immunoblot analyses (Figure 4). Interestingly, the opposite phenomenon occurred in CHLA-20 posttherapy tumor cells. There was band signal for antiLC3I in CHLA-20 post-therapy cells with no treatment of Bafilomycin A1. Additionally, both anti-LC3II and anti-LC3I bands were detected in CHLA-20 post-therapy cells treated with 5 nM of BFA1 for 4h (Figure 4).
Figure 4. BFA1 Treatment for 4 hours.
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A second experimental trial was performed with increased duration (24h) of Bafilomycin A1 treatment for both pre-therapy and post-therapy cell lines, and similar results to the first experimental trial were exhibited (Figure 5). There was little to no band detection of anti-LC3I and anti-LC3II in the CHLA-15 pre therapy cells and CHLA-15 pre-therapy cells treated with 5nM BFA1 for 24h. Furthermore, anti-LC3I band was detected in CHLA-20 post therapy cells, while both antiLC3I and anti-LC3II bands were detected in CHLA-20 post-therapy cells treated with 5nM of BFA1 for 24 h.
Figure 5. BFA1 Treatment for 24 hours.
Figure 6. Time course analysis of Bafilomycin A1 treatment across 4, 24, and 48 hours. LC3II bands, indicative of autophagosome accumulation, are darker in CHLA20 than in CHLA-15. This demonstrates that there may higher levels of autophagic activity in therapy-resistant neuroblastoma. Autophagy may contribute to the continued growth and survival of tumor.
RESEARCH ImageJ LC3II/Beta-tubulin Ratio Calculation Results suggest that BFA1 contributes to reduced autophagic activity due to an increase in autophagosome accumulation following an increased incubation time in both cell lines CHLA15 and CHLA20 (Figure 4). In the first experimental trial, pre-therapy neuroblastoma tumor CHLA-15 cells with no BFA1 treatment exhibited an LC3II/Beta-tubulin ratio of zero (Figure 5). Likewise, pre-therapy CHLA-15 cells with 5 nM BFA1 treatment for 4h exhibited an LC3II/Beta-tubulin ratio of zero. The average band intensity for CHLA-20 post-therapy cells exhibited an LC3II/ Beta-tubulin ratio of 0.287 (Figure 5), while CHLA-20 post-therapy cells treated with 5nM BFA1 for 4h exhibited an LC3II/Beta-tubulin ratio of 0.837 (Figure 5). Additionally, pre-therapy CHLA-15 cells with no BFA1 treatment exhibited an LC3II/Beta-tubulin ratio of zero (Figure 6). Likewise, pre-therapy CHLA-15 cells with 5 nM BFA1 treatment for 24h an LC3II/Beta-tubulin ratio of zero (Figure 6). The average band intensity for CHLA-20 post-therapy cells exhibited an LC3II/ Beta-tubulin ratio of 0.481 (Figure 6), while CHLA-20 post-therapy cells treated with 5nM BFA1 for 24h exhibited an LC3II/Beta-tubulin ratio of 0.940 (Figure 6). Discussion and Conclusions Bafilomycin A1 has been shown to inhibit the fusion of autophagosomes and lysosomes, thus inhibiting late-stage autophagic flux. This study suggests that Bafilomycin A1 may contribute to reduced autophagic activity in neuroblastoma cells. Our data shows that there are higher levels of autophagosomes, as indicated by the LC3 II bands, in CHLA-20 post therapy neuroblastoma cells, while there are no indications of autophagosomes in CHLA-15 pre-therapy neuroblastoma cells upon Bafilomycin A1 treatment. The presence of LC3II bands, or levels of autophagosomes, indicates that autophagic flux was properly inhibited following Bafilomycin A1 treatment. As such, the higher levels of autophagosomes in CHLA20 post-therapy neuroblastoma cells upon Bafilomycin A1 treatment suggest that there may be higher autophagic activity in therapy-resistant neuroblastoma cells. This would indicate that autophagy may be used as a survival mechanism in neuroblastoma. It is important to note that these conclusions are preliminary and there were a number of limitations. Firstly, LC3-II may not be an entirely reliable indicator of autophagic flux activity, since LC3-II itself is degraded by autophagy, thus complicating the interpretation of LC3 immunoblotting. In addition, LC3-II tends to be much more sensitive to immunoblot detection than LC3-I. The conversion of LC3-I to LC3-II is important in autophagic flux, so the fact that LC3-II can be more sensitive to detection may be problematic in data interpretation. Our experiment attempted to address this issue by comparing LC3-II to beta-tubulin ratios across various samples. A ratio was used to address the issue of unequal protein sample loading when carrying
out the immunoblot experiment. Secondly, testing the effect of Bafilomycin A1 across a larger sample size of pre-therapy and post-therapy tumor cell lines is crucial in confirming the role of autophagy in neuroblastoma. Future experiments should broaden the sample size to include the pre-therapy and post-therapy tumor cell pairs of CHLA-122 and CHLA-136, SMS-KCN and SMS-KCNR, SMS-KAN and KANR, and SK-N-BE(1) and SK-N-BE(2). Thirdly, our study only used Bafilomycin A1 to study autophagic flux in pre-therapy and post-therapy neuroblastoma cell lines, but it is important to take into consideration other methods to study autophagy through, for example, the autophagic inhibitor 3-methyladenine (3-MA) or the genetic disruption of autophagic ATG5 or ATG7 gene expressions. Future experiments should aim to study the specific cross-talk between autophagy and apoptosis in neuroblastoma. Inducing apoptosis activation in combination with autophagy inhibition may be an effective therapeutic strategy to neuroblastoma treatment. Previous studies have shown that Bafilomycin A1 activated apoptosis while inhibiting autophagy in BALL cells. This is done through the formation of Beclin 1- Bcl-2 complex, thereby averting Beclin 1 from the autophagic pathway and Bcl-2 from anti-apoptotic pathway. As such, it is important to study the role of Beclin 1, a bridge between autophagy and apoptosis, in therapy-resistant neuroblastoma. For example, a possible experiment might include the co-immunoprecipitation of Bcl-2 with Beclin 1 after Bafilomycin A1 treatment, assessing the Beclin1-Bcl2 complex formation. Apoptosis analysis in conjunction with autophagy analysis should also be done by probing for proteins responsible for cell cycle and apoptosis regulators (e.g. cyclin D1, cyclin D3, cyclin E1, cyclin E2, cytochrome C, etc.). Further understanding of Bafilomycin A1 may lead to future strategies to treat neuroblastoma. Elucidation of its direct binding target(s) in the complex autophagy and apoptosis cross-talk mechanisms in neuroblastoma cells will be important in future studies pertaining to Bafilomycin A1.
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RESEARCH References Djavaheri-Mergny, M., Maiuri, M. C., & Kroemer, G. (2010). Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1.Oncogene, 29(12), 1717-1719. Eisenberg-Lerner, A., & Kimchi, A. (2009). The paradox of autophagy and its implication in cancer etiology and therapy. Apoptosis, 14(4), 376-391. Goldsmith, K. C., Gross, M., Peirce, S., Luyindula, D., Liu, X., Vu, A., ... & Hogarty, M. D. (2012). Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma. Cancer research, 72(10), 2565-2577. Klionsky, D. J., Elazar, Z., Seglen, P. O., & Rubinsztein, D. C. (2008). Does BFA1 block the fusion of autophagosomes with lysosomes?.Autophagy, 4(7), 849-850. Letai, A., Bassik, M. C., Walensky, L. D., Sorcinelli, M. D., Weiler, S., & Korsmeyer, S. J. (2002). Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer cell,2(3), 183-192. Mangieri, L. R., Mader, B. J., Thomas, C. E., Taylor, C. A., Luker, A. M., Tse, T. E., ... & Shacka, J. J. (2014). ATP6V0C knockdown in neuroblastoma cells alters autophagy-lysosome pathway function and metabolism of proteins that accumulate in neurodegenerative disease. PloS one, 9(4), e93257. Mizushima, N., & Yoshimori, T. (2007). How to interpret LC3 immunoblotting.Autophagy, 3(6), 542-545. Oehme, I., Linke, J. P., Bรถck, B. C., Milde, T., Lodrini, M., Hartenstein, B., ... & Witt, O. (2013). Histone deacetylase 10 promotes autophagy-mediated cell survival. Proceedings of the National Academy of Sciences, 110(28), E2592-E2601. Ouyang, L., Shi, Z., Zhao, S., Wang, F. T., Zhou, T. T., Liu, B., & Bao, J. K. (2012). Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell proliferation, 45(6), 487-498. Park, J. R., Eggert, A., & Caron, H. (2010). Neuroblastoma: biology, prognosis, and treatment. Hematology/oncology clinics of North America, 24(1), 65-86. Wang, J., Gu, S., Huang, J., Chen, S., Zhang, Z., & Xu, M. (2014). Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells. BMC cancer, 14(1), 768. Yuan, N., Song, L., Zhang, S., Lin, W., Cao, Y., Xu, F., ... & Wang, J. (2015). BFA1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. haematologica, 100(3), 345-356. 22 PENNSCIENCE JOURNAL | SPRING 2016
Exploring Quality of Life from the Perspective of Liver Transplant Patients Sofia Duque, David Goldberg, MD, MSCE Hospital of the University of Pennsylvania Liver transplantation continues to be a highly successful procedure for patients with terminal liver illness. As patients who undergo the procedure live longer, more information is needed to understand their recovery and experience. Recovery rates for the procedure are well-documented, but the details underlying these numbers are hardly looked into. The transplant experience taken directly from the patient’s perspective can shed light on the difficulties patients endure during their recovery and the factors that interfere with their ability to execute a normal lifestyle. Three focus groups of eight to twelve participants were held in order to understand: 1) what defines a patient’s post-transplant experience and 2) which limitations directly affect patients’ quality of life (QOL). The patients’ post-transplant experiences were largely dominated by their pre-transplant state, relationship with healthcare provider, and how informed their expectations were prior to the procedure. The most significant limitations that interfered with QOL were medical side effects and strained relationships. The results of this study provide context for recovery rates and identify gaps in care provision that can be filled by healthcare professionals.
Introduction As the only curative treatment for terminal liver illness, liver transplantation surgery is a highly researched and effective procedure. About 17,000 individuals require a liver transplant each year, but only around 6,000 are able to receive one (unos.org). Since the need for liver transplant increases along with the longevity of patients who have undergone the procedure, more insight is needed regarding what they should expect following the transplant. The transplant experience from the patient’s perspective is important to understand, because the process is defined both by many limitations and strengths. As the core source of information regarding the transplant process, healthcare providers should obtain deeper insight into the multidimensionality of the patient’s experience. This could allow them to convey clearer expectations to patients and could enable patients to have have a more predictable and smooth recovery. Data gathered by the Organ Procurement and Transplantation Network between 2002 and 2004 shows that the recovery rate 1 year after transplant was 73.8% for patients with severe liver disease and 86.5% for patients with mild liver disease (optn.transplant.hrsa.gov). While a typical recovery can last from 6 months to about a year, the patient experience begins prior to the surgery. Despite the depth of research surrounding liver transplantation, a search of the patient experience finds limited content. Most published studies maintain a quantitative approach with a focus on survival rates and outcomes (Kilpe, Krakauer, and Wren, 1993; Adcock et al., 2010; Wallot et al., 2003), complications (Spicak and Bartakov, 2012; Ringe et al., 1991; ), and co-procedures (Mandell, Lockrem, and Kelley, 1997). This study addresses the lack of qualitative research focused on a patient’s transplant experience. Specifically, the two aims were to identify: 1) what defines a patient’s post-transplant experience and 2) which limitations directly affect quality of life (QOL), us-
ing qualitative research methods. As a secondary outcome, areas where healthcare providers could help improve the experience for their patients were identified. Results Dominant Themes One set of questions was asked during each focus group. The three sets were prepared with five to twelve questions each. The questions were verbalized in a contextually natural way and were not always asked in the set order. They covered a broad range of topics regarding the transplant experience with some eliciting long and emotional discussions and others remaining lighthearted and brief. Participants are referenced
The most signiwcant life-changing effect on the post-transplant experience, however, was job loss. according to the focus group session they participated in and their given participant identification number. For example, Participant 1.1 attended Focus Group I and was given participant identification number “1”. Analysis of the three focus group transcripts revealed several major themes. The theme touched upon most frequently was disability and limitations before transplant. Focus group participants described physical symptoms they experienced prior to their transplant such as bleeding, pain, inability to accomplish simple tasks on their own, and significant memory loss. They also described a desire to escape reality. Predictably, the attribution analysis demonstrated that almost all (seven of the eight) participants associated this theme with comments classified as strongly negative or negative. Simply put, the overall condition of transplant candidates can be
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RESEARCH poor and debilitated. When participants compared pretransplant limitations to life after the transplant, it appeared that the limitations lessened after they recovered from the procedure. The most significant life-changing effect on the post-transplant experience, however, was job loss. Five out of six participants who were working prior to the transplant were not able to preserve their job after surgery. Interestingly, most of them expected that they would be able to work again after recovering. The second most-discussed theme was reactions to healthcare providers. The majority of participants expressed strongly negative (two participants) or negative (three participants) sentiments, while only two participants conveyed positive statements. Most participants with negative comments did not feel prioritized by their
Most participants with negative comments did not feel prioritized by their healthcare providers. healthcare providers. Participant 1.6 described needing counsel and approval prior to a dental procedure and being unable to reach anyone easily, rendering him his “own advocate.” Other participants felt distant from their doctors, who seemed inconsiderate of their state or needs. Participant 1.2 spoke of being brushed off by a doctor despite daily diarrhea, Participant 1.7 spoke of the doctor neglecting to contemplate his/her medicine’s side effects, and Participant 1.8 spoke of not being forewarned by the doctor regarding reasonable expectations pre- and post-transplant, such as potential delusion and changing relationships, respectively. This same participant, 1.8, discussed feeling like a “lost soul” due to not knowing his/ her doctor and felt “disgusted” because doctors did not ask how patients were doing and left patients with the impression that they did not have time for them. Finally, Participant 1.6 expressed frustration because he/she was tended to by healthcare providers other than a doctor. Despite the overwhelmingly negative comments, a few participants gave positive statements. Participant 3.5 had a great recovery and support and was happy to keep coming back. Participant 1.1 was impressed that the doctor sat down and went through all of his/her patient history, and Participant 1.6 had an overall positive experience after the transplant. As trusted stewards and sources of support for transplant patients, it is not surprising that interactions with doctors (and other healthcare providers) arose as an important theme. It appeared that the majority of participants encountered behavioral interaction issues with their providers, rather than institutional ones. Most patients with unpleasant experiences seemed to wish that their providers were comprehensively considerate of them, that they were accessible, and that they conveyed all expectations and potential roadblocks clearly and early on. Physician-conveyed expectations vs. reality emerged as a third significant theme tying in neatly with the second one. The designation analysis showed six total 24 PENNSCIENCE JOURNAL | SPRING 2016
commenting participants. Four commenting participants were in the strongly negative and negative categories, one in neutral, and one in strongly positive. Of the strongly negative and negative commenters, the overall consensus was that physicians did not prepare patients for what they ultimately experienced following the transplant surgery. Participant 2.3 mentioned having “no orientation to what we should expect,” and Participant 2.1 specifically pointed out not being informed about the steroid side effects after surgery. Corticosteroids can be used in conjunction with immunosuppressants to minimize the effects of organ rejection. Unfortunately, they can have significant side effects such as hyperglycemia, diabetes, glaucoma, psychosis, and hallucinations, which many patients struggle with. Participant 2.4 also felt in the dark regarding expectations and mentioned having relied on the biweekly focus group to become informed about what lay ahead after transplant. One positive comment came from participant 2.5, who said that the amount of information was sufficiently detailed and explained that his/her transplant was very recent. Similar to the theme of reactions to healthcare providers, participant comments under physician-conveyed expectations vs. reality reflect a lack of connection and mutual understanding between providers and patients. Because many post-transplant obstacles are shocking and onerous, the information conveyed to patients on this matter can be relationship-determining. Post-transplant obstacles can cause confusion, panic, and a sense of defeat, and when patients know what to expect, they may feel more equipped to handle them. Without the information, however, the obstacles can be more overwhelming, and the patient may feel distanced and unaccounted for by his or her provider. Other themes that arose mainly encompassed lifestyle changes that emerged post-transplant. When
Post-transplant obstacles can cause confusion, panic, and a sense of defeat, and when patients know what to expect, they may feel more equipped to handle them. Without the information, however, the obstacles can be more overwhelming, and the patient may feel distanced and unaccounted for by his or her provider. questioned under the relationship with food and drink theme, participants claimed that they ate almost everything they wanted to, but that their cravings had changed. Three participants stated that they did not drink at all and three other ones explained that they did not drink immediately following the transplant but adjusted to small amounts of drinking as time passed. One participant drank wine while traveling, another for sleeping, and the last one for special occasions. Liver-transplant patients,
RESEARCH in particular, are told not to drink following their surgery to protect their new liver from the effects of alcohol. Prior to the transplant, most participants depicted a tumultuous relationship with their partner. These comments were categorized under relationship with partner. Even after the procedure, however, many participants’ relationships remained strained. One participant described her disease as also becoming her partner’s disease due to his role in her life and care. Another participant talked about his partner becoming consumed with his obligations such as doctor’s appointments, medicines, and lifestyle requirements and the toll it took on them. Regarding pre- and post-transplant sex life, many participants shared that sex was out of the question before the transplant. Participant 3.3 explained that it was the least of his worries because “you get depressed, sleep is all screwed up, you’re a mess.” After the transplant, sexual activity increased for participants, although not immediately. The transplant surgery process as a whole tends to bring a lot of confusion and disappointment, but it is clear that it has emotional and physical benefits as well. In terms of leisure, participants said that it was difficult to go on vacation. One participant discussed a past experience during which he/she bled rectally and became bloated. As a result, he/she had to run to the nearest emergency room and cut her vacation short. Another participant stated a fear of being too far from a hospital at all times and hesitation regarding transporting medication. The prescriptions for some medications are given for certain time periods, and being out of town during those time periods adds difficulty. For life-sustaining medicine, this means being unable to travel or resorting to trading pills with fellow transplantees. The medicines themselves, however, can also interfere with leisure time. Immunosuppressants are a strong type of medicine that is essential following any transplant to ensure the body’s acceptance of the new organ. While exploring this theme, namely the effects of immunosuppressants, it was found that all participants expressed negative side effects from the immunosuppressants. Participant 1.8 describes problems with his/her lungs linked to the immunosuppressants and having to remind him/herself that “someone died for me to get this [liver],” while Participant 1.6 claimed intense pain and difficulty standing up. Posttransplant maintenance requires significant attention and care that may interfere with a normal lifestyle and thus, pre-transplant patients should be made aware of it.
relationships with family members, this “pre- ” period weighed heavily on participants and must be considered when thinking of the overall experience. It sets a useful standard against which to measure post-transplant lifestyle and to form a definition of the transplant experience. Another equally dominating factor is their relationships with healthcare providers. It seemed that participants who had consistently positive interactions with providers thought of their overall experience in a better light than those who did not, reflecting the importance of this dynamic. In a simplified view, it makes sense that those patients who feel more supported due to a satisfying and reliable relationship with a provider would feel
Participants were asked what, if anything, they thought could be changed or adjusted to improve the patient experience. This information, along with building a holistic definition of the transplant experience and highlighting the limitations affecting patient QOL, is useful for identifying potential areas for improvement. The information gathered led to a few suggestions. The first is to build a stronger and more consistent focus on patient-provider relationships. This crucial dynamic can set the tone for the entire experience. Pa-
This qualitative study of what defines liver transplant patients’ experience and what significantly affects their QOL found various significant factors. The patient’s pre-transplant state seemed to partially dominate the experience.This period held a large stake in patients’ experiences due to the magnitude of the limitations that they endured. From hallucinations, extreme pain, paranoia, anxiety, and inability to work and complete simple tasks, to constrained
It seemed that participants who had consistently positive interactions with providers thought of their overall experience in a better light than those who did not, reyecting the importance of this dynamic. more in control and well-equipped to handle any mishap. It also leads to stronger trust, not only in the provider, but also in the process and the idea of consistent and sustainable self-improvement. The study did not explore whether those who were satisfied with their patient-provider relationship had better health outcomes, although this may be an important branch for further study. The third most significant factor seemed to be how well the patients’ post-transplant experiences matched their expectations. Participants who felt fully informed about the obstacles they would face during and after recovery seemed to have a more relaxed tone and attitude when speaking about their journey. Secondary contributors to the transplant experience were limitations due to medicine side effects and strained relationships. Based on the data collected from the three focus groups, the most significant factors that defined the transplant experience were pre-transplant status, patientprovider relationships, and well-informed expectations. The most significant limitations that affected patient QOL were the medical side effects and strained relationships. Areas of Potential Improvement
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RESEARCH tients rely on their providers for information, counsel, and relief. If the patient believes that he or she cannot trust his or her provider, the connection needed to accept the aforementioned benefits will be lacking. Further, for patients to maintain high morale, they must feel as if they have a reliable and accessible support system. A few specific issues arose with this concept. One such issue was that some patients would prefer to see a doctor rather than another healthcare provider, such as a nurse or assistant. The patient may need to be informed beforehand that other providers are sufficiently qualified and skilled to provide quality care. Another issue was continuity. Several participants were surprised to find that the doctor performing post-surgery check-ups was not the doctor who transplanted them, or even a doctor they had previously met. Perhaps it would strengthen the patient-provider relationship to preserve continuity. Lastly, the behavioral aspect of providers’ interactions with patients should be addressed on an individual basis. A few participants spoke of not feeling valued or considered by their doctor, so this may be something worth addressing as well. Another change that could lead to an improved experience for patients is emphasizing communication of post-transplant expectations. Having a clear view of the likely obstacles and low points be can help patients feel more prepared upon encountering them and minimize the shock and surprise factors that many participants experienced. Specifically, partici-
Lastly, participants themselves suggested that coming to the HUP Liver Transplant Support Group should be a requirement for all transplant candidates. pants described wishing they had known about the intense effects of steroids after surgery, potential hallucinations, and information on effects of co-morbidities and immunosuppressants. Participants seemed to have the hardest time with the surprises accompanying steroid use. Incorporating these subjects into the pre-transplant check-in could lead to participants having a smoother experience and a more positive retroactive outlook. Lastly, participants themselves suggested that coming to the HUP Liver Transplant Support Group should be a requirement for all transplant candidates. The Group is for both pre- and post-transplant patients. The post-transplant attendees explained that they will continue going back to the group even after they have recovered in order to continue giving back; understanding the pain and hassle involved, they want to streamline the process for others. One patient stated the importance of the group in his/ her experience by saying, “I’ve learned more from coming here than the hospital. For me, better to learn from someone who’s experienced it.” Another participant thought 26 PENNSCIENCE JOURNAL | SPRING 2016
that patients who attend the bi-weekly focus group “stand a better chance” because they come to know HUP employees personally, and in turn, feel less anonymous and more considered. It may be valuable to encourage or require liver transplant candidates to attend Group Support. Or, if the resources are available and people are willing, it could help to establish a mentorship program with posttransplant patients matched to pre-transplant patients. The findings presented here should be interpreted within the confines of the study. The study was conducted over three focus group sessions in one hospital, and enough data was acquired to identify gaps and patterns. While the experiences of the participating patients may be related or similar to the experiences of those across the nation, they are not necessarily wholly representative of or identical to others’. Further, because only one sample of a hospital’s patients was studied, it is important to keep in mind that other hospitals and their staff members may function differently and result in different patient experiences. While the quantitative dimension of this study measured the importance of certain themes, the qualitative branch offered a view of the themes associated with favorable and unfavorable attributions. Together, these approaches led to a big-picture view, which must also be kept in mind as another limitation. A third limitation that may be found in any observational study is the Hawthorne effect, in which participants may modify their responses to questions as a result of being observed. However, from participants’ apparent comfort and openness, this does concern does not seem to have played a significant role. In spite of these limitations, there was enough evidence to render the collected data reliable. In summary, the transplant experience is defined by multivariable themes, and many limitations affect QOL. Nevertheless, the process for liver transplantation at HUP should continue to be trusted as a high-quality system, due to its known success, national recognition, and the longevity of liver transplant patients. The gaps highlighted in this study neither determine life expectancy nor place patients at an unacceptable disadvantage. They are merely to be seen as windows of opportunity for improvement.
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