Current Lung Biology T32 Trainees
MARAM ESSAWY, BS
Mentor: Dr. Colin Campbell
Program: Pharmacology Graduate Program
Research: It has been shown that lung tissue obtained from patients with diseases such as pulmonary fibrosis, asbestosis, and chronic airway disease possess elevated levels of mitochondrial DNA damage. It is likely that the elevated mitochondrial DNA damage results in mitochondrial dysfunction that contributes to the observed cellular pathology. Consequently, a detailed understanding of how mammalian cells maintain mitochondrial genome stability has the potential to identify molecular drivers of lung pathologies, and could result in the identification of biomarkers and therapeutic targets for lung pathologies.
As a first step towards these long term objectives, my project focuses on gaining insight into mechanisms of DNA damage detection and repair in mammalian mitochondria. I am studying the repair of synthetic DNA damage substrates electroporated into isolated mitochondria purified from cultured mammalian cells, by working to 1) Capture and characterize the biological intermediates formed in the repair of the electroporated DNA damage substrates, and 2) Use pharmacological inhibition to identify genes that are essential for the mitochondrial repair of the electroporated DNA damage substrates. This work will provide a fundamental understanding of how mitochondria maintain their genome integrity, which will thus provide insight into the molecular basis of lung pathologies associated with elevated mitochondrial DNA damage.
SHANLEY ROACH, BA
Mentor: Ryan Langlois, PhD
Program: Biochemistry, Molecular Biology and Biophysics, College of Biological Sciences
Research: The Role of Tuft Cells during Influenza Virus Infection
Tuft cells are a rare, chemosensory epithelial cell easily identifiable by their large size and thick microvilli. Their presence has been confirmed in the respiratory, gastrointestinal, and reproductive tracts of mice and humans. In the lung these have historically been termed brush cells. Several papers recently identified tuft cells of the intestine to be the primary epithelial source of IL-25 and key initiators of the type 2 immune response during parasite infections. When these cells were ablated in the gut there was reduced immunity and intestinal regeneration during parasite infection. Tracheal tuft cells have been shown to also express IL-25 but no links to the immune system have been explored. It is currently not understood if respiratory tuft cells have overlapping functions to those in the gut or if they have completely different roles in lung homeostasis and recovery from infection. The goal of my project is to study respiratory tuft cells and explore any potential immunological roles they may have during respiratory infection. In addition to pulmonary symptoms that occur during influenza infection, both mice and humans also exhibit gastroenteritis-like intestinal injury. Recent evidence suggests that gut dysbiosis and increased inflammatory Th17 cells in the gut may be responsible. A mechanism for how this gut dysbiosis occurs and is repaired has not been elucidated. My project also aims to better understand how intestinal tuft cells and gut injury may be impacted by pulmonary infection.
CALEB VOGT, BS
Mentor: Angela Panoskaltsis-Mortari
Program: MD/PhD program & Department of Biomedical Engineering
Research: 3D Bioprinting the Gastroesophageal Junction
Esophageal cancer is a leading cause of cancer death worldwide, with an incidence of almost 500,000 every year. The most common type of esophageal cancer in the United States is adenocarcinoma involving connection between the esophagus and the stomach, known as the gastroesophageal junction (GEJ). Our central hypothesis is that 3D bioprinting could be used to create an artificial, tissue replacement valve for the GEJ. Implantation of such a tissue construct would allow for smaller resection of the normal esophagus proximal to the cancer, improving morbidity outcomes. The scientific value of this work will lie in new knowledge around bioprinting of structured fibroelastic tissues, which we hope to apply in the future to our lab's ongoing work in pulmonary airway engineering.
Justine Delgado, PhD, BS
Mentor: Carston Wagner
Program: Medicinal Chemistry
Research: Assessing the Impact of Target Antigen Density and Spatial Expression on Prosthetic Antigen Receptor T cells in Three-Dimensional Models of Lung Cancer
Biography: I am completing my postdoctoral training at the University of Minnesota in Dr. Carston R. Wagner’s Lab. I am currently on the Lung Biology NHLBI T32 training grant. My current research is focused on the development of in vitro model systems to better recapitulate the in vivo tumor microenvironment. My focus is on developing three-dimensional cancer cell culture systems to better understand lymphocyte infiltration into lung tumors with non-genetically engineered T cells.BS Chemistry, University of the Incarnate Word.
NICHOLAS INGRAHAM, MD, BS
Mentor: Adams Dudley, MD
Program: Pulmonary, Allergy, Critical Care, and Sleep Medicine Fellowship
Interests: Critical Care Outcomes and Health Services Research
Research: Defining, Identifying, and Caring for the Chronically Critically Ill in the Modern Era
Biography: I am a 2nd-year pulmonary critical care fellow with an interest in critical care outcomes, specifically critical care survivorship. I am originally from Omaha Nebraska and moved to Minnesota to start residency after my wife and I both matched with University of Minnesota's Internal Medicine program. I enjoy collaborating on large research endeavors and continue to work towards pursuing an academic career after fellowship!
KATHLEEN MAHAN, MD
Mentor: Mikael Elias & Jordan Dunitz
Program: Pulmonary, Allergy, Critical Care & Sleep Medicine Fellowship
Research: Disrupting Bacterial Signaling: A New Era in the Treatment of Chronic Lung Infection
The recent increase in antibiotic-resistant bacterial strains represents an important challenge in health care. The frequent use of disinfectants and antibiotics selects for resistant strains that represent a threat to human health. This has resulted in increasing challenges in treating or preventing bacterial proliferation. There is a great need for new strategies to control bacterial infection. This is particularly important in cystic fibrosis (CF) and other chronic lung diseases. A promising strategy to control such infections relates to the interference in bacterial chemical communication, dubbed quorum sensing (QS). QS interference offers an alternative to limit the use of antibiotics and thereby the selection of antibiotic resistant pathogens. I have proposed to study and evaluate this new approach that controls bacterial behavior by interfering with their signaling. QS interference (QSI) can suppress both bacterial biofilm formation and virulence, and does not select for resistance because it does not kill bacteria.
We are investigating the ability of enzymatic quenchers to inhibit both virulence and biofilm formation of CF clinical isolates of P. aeruginosa. We are using two enzymes, which have been engineered to be highly stable and highly active against AHLs. Because P. aeruginosa is known to utilize two QS circuits, one using 3-oxo C12 AHL, the other C4 AHL, we are using two enzymes with different substrate specificity. For instance, one enzyme disrupts only the 3-oxo C12 AHL based circuit (Ssopox-W263I) and a second enzyme disrupts both circuits (GcL) used by P. aeruginosa for QS.
We are testing these enzymes on 39 clinical isolates of P. aeruginosa, and evaluation thus far has demonstrated that Ssopox treatment reduced biofilm formation in 23% of the strains by up to 88%, elastase in 54% of the clinical isolates by up to 63%, protease activity in 28% of the strains by up to 69% and pyocyanin production in 33% of strains. These results are consistent with recently published data using Ssopox-W263I on P. aeruginosa isolated from diabetic ulcers. Interestingly, as the enzymes target specific AHLs, we saw a separate pattern of quenching with treatment using an alternative lactonase, GcL.
This study demonstrates the ability of these engineered enzymes to disrupt the complex communication system utilized by P. aeruginosa so as to reduce virulence and biofilm formation. With future studies on mixed species communities and combination therapy with antibiotics, we expect to provide the key data to assess the potential of signal disruption to treat and prevent infections relevant to CF patients, transition to animal model studies, and delineate the importance of signaling in chronic lung infections.
DAVID MACDONALD, MD
Mentor: Ken Kunisaki, MD, MS
Program: Pulmonary, Allergy, Critical Care, and Sleep Medicine Fellowship
Research: I am studying how the heart rate response to stress affects the risk of acute exacerbations of COPD. We recently completed a secondary analysis of the Beta-Blockers for the Prevention of Acute Exacerbations of COPD trial where we showed that a higher increase in heart rate during exercise is associated with lower risk of COPD exacerbations. I am now working on validating those findings and have proposed a small clinical study evaluating associations between chronotropic reserve and other markers of cardiovascular function related to the autonomic nervous system. Additionally, I am working on two projects evaluating biomarkers in HIV related lung disease and am completing coursework towards a Masters of Science in Clinical Research.
MATTHEW MCELWEE, MD, PHD
Mentor: Marc Jenkins
Program: Rheumatology Fellowship/Microbiology and Immunology/PSTP
Research: Identification and Characterization of Pathogenic T-cells in Immune Mediated Disease
The adaptive immune system allows for extraordinary specificity in fighting infection, but this specificity has made it difficult to study the cells that drive immune-mediated diseases. With the advent of MHC tetramers, it is now possible to isolate and characterize the specific pathogenic T-cells. I intend to use this technique to characterize pathogenic T-cells in patients with antibody mediated lung transplant rejection and rheumatoid arthritis. Antibody-mediated rejection of transplanted lungs is chiefly caused by host antibodies to donor-specific HLA molecules and is the primary reason that 50% of lung transplants fail after 5 years. This antibody-mediated process requires help from T-cells. Previous work in our lab suggests that in organ-transplanted mice, T-cells are activated that recognize donor MHC binding CLIP. CLIP is a chaperone that prevents premature binding of peptide to the MHC molecule in the endoplasmic reticulum. However, 10-20% of MHC molecules that reach the cell surface still have CLIP in the peptide-binding groove. This represents a very attractive target to identify T-cells that recognized foreign HLA molecules, and thus mediate the rejection of transplanted lungs. The challenge in rheumatoid arthritis is different in that the autoimmune process is driven by peptides that are chemically modified (e.g. citrullinated). In my research, I will use MHC tetramers to identify and characterize pathogenic T-cells in patients with antibody mediated rejection of lung transplant as well as rheumatoid arthritis.
ANDREW SOERENS, PHD
Mentor: David Masopust, PhD
Program: Microbiology & Immunology
Research: CD8 T Cell Abundance and Early Antiviral Functions
We are studying the ability of CD8 T cells to eliminate flu-infected cells in the initial hours after an infection. Most research into possible universal flu vaccines has been focused on eliciting broadly effective antibodies. However, because antibodies mediate their effect through recognition of surface proteins, their effectiveness wanes as the surface proteins of the virus evolve. CD8 T cells can recognize internal, conserved proteins, which suggests that they have the potential to provide more durable protection, but the speed with which they can impact an incipient infection is not clear. By using novel tools that allow for the early detection and enumeration of flu-infected cells within the mouse lung, we can test whether an abundant population of memory CD8 T cells can eliminate infected cells in the initial hours after an infection.
Recent Past Trainees
RONY ARAUZ, MPH, PHD
Mentor: Drs. Jeffrey H. Mandel & Chris Wendt
Program: Environmental Health Sciences, School of Public Health
Research: Airborne Particle Exposure Assessments and Potential Impact on Inflammatory Markers and Lung Disease in Taconite Workers in Minnesota
It is well-established that dust components and particulate matter exposures increase the risk of developing lung disease. However, understanding of exposures to dust components and particulate matter (PM), and the process of inflammation and lung disease in certain occupational settings is limited. This dissertation addressed research gaps in this area by examining occupational dust components and PM exposures associated with inflammatory biomarkers. The primary objective of this dissertation was to assess exposure to respirable silica (RS), respirable dust (RD) and respirable dust without silica (RDw/oS), particulate matter with an aerodynamic diameter less than or equal to 1, 2.5 and 10 micrometers (PM1, PM2.5 and PM10, respectively) and a panel of inflammatory markers within a cohort of Minnesota taconite mining workers. Also, this dissertation investigated the relationship between levels of inflammatory markers and lung disease. Overall, alterations in inflammatory markers were associated with increased dust components and PM exposures. Additionally, changes in prevalence of lung disease were associated with circulating levels of several inflammatory markers.
ARIANNE BALDOMERO, MD
Mentors: Drs. Chris Wendt & Ken Kunisaki (Co-mentor)
Program: PACCS Fellowship
Research: The Relationship between Oral Health Status and COPD Exacerbations
INTRODUCTION: Poor oral health has been implicated as an independent risk factor for COPD, however, very few studies have evaluated the association between oral health and COPD exacerbations. We aimed to determine the association between poor oral health status and both COPD exacerbations and respiratory health status.
METHODS: Using a case-control design, we assessed the 1-item global oral health assessment, 5-item Oral Health Impact Profile (OHIP), dental symptoms, dental habits, and St. George's Respiratory Questionnaire (SGRQ). Cases were patients with frequent exacerbations defined as at least 1 exacerbation in the previous 12 months, while controls were patients with no exacerbations in the previous 24 months. Patients were contacted via phone interview or in-person visits at the Minneapolis VA Medical Center. In a subset, we additionally performed dental exams to measure bleeding on probing, probing depth, clinical attachment loss, periodontitis severity, plaque index, gingival index, and carries risk assessment. We evaluated associations between oral health and COPD exacerbation status using logistic regression, adjusted for smoking status, educational attainment, and inhaler use. Linear regression was used to assess relationships between oral health and SGRQ scores. RESULTS: Screened non-exacerbators (n=118) were significantly more likely to have <4 teeth, compared to screened exacerbators (n=100) (44% vs. 30% respectively; p=0.046). After excluding those with <4 teeth there were 70 cases (exacerbators) and 66 controls (non-exacerbators). Self-reported oral health status and objective dental exam measures did not vary significantly between cases vs. controls. However, the odds of severe COPD exacerbations requiring hospitalizations and/or emergency department visits trended higher in those with worse dental exam measures compared to those with better dental exam measures. Worse OHIP-5 scores were strongly associated with worse SGRQ scores.
CONCLUSIONS: Oral health status was not related to COPD exacerbation status, but was associated with patient-reported respiratory health status. Non-exacerbators were more likely to be edentate or have less than 4 teeth compared to exacerbators. Larger studies are needed to address oral health as a potential method to improve respiratory health status in patients with COPD.
My results were presented at the May 2018 ATS International conference and the June 2018 Aspen Lung Conference. I am attending the COPDGene working group meetings and I am writing both a first author primary research manuscript and a review article “Oral Health in COPD.” I am enrolled in the UMN Masters of Science in Clinical Research degree program and will complete this in May 2019. I will be writing a VA Career Development Award.
CHRISTOPHER BANEK, PHD
Mentor: John Osborn
Program: Center for Immunology & Dept of Pediatrics
Research: Targeted Sympathetic Nerve Ablation to Prevent and Treat Hypertension and Cardiovascular Disease
Recent clinical studies have shown that ablation of renal sympathetic nerves may be an effective, long-term treatment for resistant hypertension and related cardiovascular and renal diseases; however, the mechanisms behind this effect remain unclear. Dr. Banek’s current research with his mentor, Dr. John Osborn, is focused on delineating the role of efferent and afferent neural contributions to the effect of renal nerve ablation, and determining the role of brain-kidney crosstalk in the treatment of salt-sensitive hypertension. He published a first author manuscript in Hypertension titled “Resting afferent renal nerve discharge and renal inflammation: Elucidating the role of afferent and efferent renal nerves in DOCA-salt hypertension” (2016) and has since published two additional papers in this model with others in review. Dr. Banek presented his research multiple times during his training period including Conference on Neural, Hormonal, and Renal Interactions in Blood Pressure Control; Mussoorie, India; American Heart Association’s Council for Hypertension, Orlando, FL; and Experimental Biology 2016 and 2017. He won several awards for his research including the American Physiological Society’s Michael J. Brody New Investigator Award, American Heart Association Council on Hypertension New Investigator Travel Award, American Physiological Society’s Caroline tum Suden/Francis A. Hellebrandt Professional Opportunity Award, and American Physiological Society’s NCAR Postdoctoral and Early Career Research Recognition Award. He cycled off this T32 training grant in July of 2017 after receiving a post-doctoral fellowship from the American Heart Association. Dr. Banek subsequently was funded by his K99/R00 while continuing his research with mentorship from Dr. Osborn. He now is a Research Assistant Professor and is interviewing for tenure track faculty positions to launch his independent research program.
DANIEL BEISANG, MD, PHD
Mentor: Peter Bitterman, MD
Program: Pediatric Pulmonary Fellowship Program
Research: Mesenchymal Stem Cells in the Setting of Fibrotic Lung Disease
The Bitterman lab is focused on understanding the disease Idiopathic Pulmonary Fibrosis (IPF). IPF is a disease of unrelenting scarring of the lung, leading to two possible outcomes; death or lung transplant. Our lab focuses on the impact of mesenchymal cells with the extracellular matrix (ECM) and how this orchestrates the progression of IPF. Specifically, we are using single cell sequencing technologies to understand heterogeneity in the mesenchymal cell population, as well as molecular biology approaches to understand the impact of fibrotic extracellular matrix on gene regulatory mechanisms. Through these approaches we hope to understand the molecular steps dictating fibrotic progression, with the goal of identifying targets for the development of novel therapeutics for this devastating disease.
KELSEY BINDER, BA
Mentor: Ryan Hunter, PhD
Program: Predoc, MICaB
Research: Analyzing Newly Synthesized Proteins to understand Community Dynamics within Polymicrobial Respiratory Infections
Respiratory diseases such as Cystic Fibrosis, Asthma and Chronic Rhinosinusitus are characterized by chronic infections. Recent advances have pushed understanding of the intricate microbial communities that exist within these patients however, the role the microbes play in these diseases is still largely unestablished. Working in the lab of Dr. Ryan Hunter, they hypothesize that disease state is influenced not by which microbes are present but by what they are doing. Her work seeks to use Bio-orthogonal amino acid tagging (BONCAT) in order to identify actively growing bacteria and the proteins they are making. Her work focuses on using this technique to correlate bacterial activity with understanding of these chronic infections. She twice presented her research at the North American Cystic Fibrosis Conference and was a finalist in the Junior Investigator in Basic Sciences Best Abstract award in 2017. Kelsey was appointed to the training grant for three years before taking a medical leave of absence from her graduate studies. She is now working as a Scientist at MDBiosciences in Minnesota.
KATHARINE BLOCK, PHD
Mentor: Stephen Jameson, PhD
Program: Laboratory Medicine and Pathology
Research: Testing Susceptibility of Mice with Normal Microbial Experience to Induction of Allergic Airway Disease
Allergic asthma is a chronic condition that can result in acute, life-threatening episodes. Our understanding of what makes individuals susceptible to allergic responses is incomplete. Improvements in sanitation, vaccination programs, and other changes to individuals’ microbial exposure are hypothesized to contribute to the rise in allergy and asthma in recent decades. This ‘hygiene hypothesis’ has been difficult to test in animal models. Our laboratory has developed a murine model of normal microbial experience (NME) in which inbred laboratory mice are co-housed with pet store mice that harbor many pathogens and pathobionts that are absent in specific pathogen free (SPF) research animal colonies. I am using this NME model to test whether the level of microbial experience affects the immune response to airway allergens. I have developed or refined models of acute allergen exposure and chronic allergic airway inflammation to test whether early and late phases (or innate and adaptive arms of the immune system) are impacted differently by normal microbial exposure. Data suggest that early and late phases of the response to allergens overall are dampened by normal microbial experience and we are working to understand the mechanisms by which normal microbial experience decrease the immune system response towards allergens.
LISA CHESNER, MS
Mentor: Colin Campbell, PhD
Research: Understanding the cellular repair mechanisms of DNA-protein crosslinks
Dr. Lisa Chesner’s research goal was to gain a greater understanding of the mechanisms involved in DNA-protein crosslinks (DPCs) repair. From this work, a new assay termed strand-specific primer extension-quantitative polymerase chain reaction (SSPE-qPCR) was developed and used to quantify repair of DPC-containing plasmids (as well as other lesions) in mammalian cells. Under the mentorship of Dr. Colin Campbell, she exploited this assay to investigate the role of nucleotide excision repair (NER), homologous recombination (HR), and proteolysis in the repair of DPCs in the nucleus. Her research also examined DPC repair in the mitochondria. To do this, the SSPE-qPCR assay was used to quantify the repair of DPC-containing plasmids electroporated into purified mitochondria.. Results from these experiments provided a role for the homologous recombination pathway in mitochondrial DPC repair, providing the first evidence for DPC repair in this organelle. These experiments validate future use for this assay to gain a greater understanding of DPC and HR repair in mitochondria. During her two years on the training grant, Dr. Chesner authored three papers on this research, presented five posters and gave two talks at the University. She defended her thesis in June, 2018 and was offered several post-doctoral fellowship positions. In August, 2018, she began a fellowship in the lab of Felix Feng, MD, Vice Chair for Faculty Development & Translational Research, Dept of Radiation Oncology at the University of California, San Francisco.
Mentor: Dr. Peter Bitterman
Program: Biomedical Engineering
Research: Leveraging ECM-Based Droplet Microfluidics for the Development of a High-Throughput 3D Model of Idiopathic Pulmonary Fibrosis
Katherine's thesis involves leveraging the Wood lab's novel, hydrogel microtissue platform to produce a high-throughput idiopathic pulmonary fibrosis (IPF) model with applications in drug screening and molecular mechanism studies. She has established a variety of high-content, high-throughput metrics to quantify cell function as either fibrotic or nonpathogenic, including quantification of cell contractility, of increased extracellular matrix protein deposition, and of an imbalance in matrix stiffness regulation. In addition, she has demonstrated these upregulated fibrotic functions when comparing IPF derived primary cells to normal patient primary cells. Multiple pharmaceutical experiments have validated the utility of the platform for drug screening, including the dose dependent reduction in fibrotic behaviors using the 2 FDA approved therapeutics as well as a decrease in fibrosis when IPF patient cells are treated with epigenetic inhibitors in our system. Study of molecular mechanisms has begun through the encapsulation of single cells within microtissues, leading to single cell contractility analysis as well as studies of matrix protein affects on fibrotic behaviors. Future steps include sorting single cell collagen microtissues based on size in order to physically separate highly contractile (pathogenic) and lowly contractile (less fibrogenic) cells and perform transcriptome sequencing on recovered cells.
JESSICA FIEGE, PHD
Mentor: Ryan Langlois, PhD (co-mentored with Kris Hogquist)
Program: Department of Microbiology and Immunology
Research: Influenza a Virus Tropism in Hematopoietic and Non-hematopoietic Cells in the Lung
My research has focused on influenza A virus (IAV) tropism in non-hematopoietic cells in the lung and the impact of TCR signal strength on resident memory T cell (TRM) formation during influenza virus infection. We observed that lung epithelial cells can survive both IAV infection and the CD8+ T cell mediated-adaptive immune response to clear the virus. This was done by using an IAV-expressing Cre recombinase (Cre) and Cre-inducible fluorescent reporter mouse. We hypothesized that survivor cells either have a cell intrinsic mechanism of protecting against adaptive immune killing, or have cleared IAV and no longer express IAV antigen on major histocompatibility complex-I (MHC-I) surface molecules. When we performed RNA-seq of infected cells, we observed increased expression of programmed death-ligand 1 (PD-L1) and MHC-I RNAs. I confirmed surface PD-L1 and MHC-I expression on survivor cells by flow cytometry. We performed anti-PD-L1 blockade experiments to determine if survivor cells used PD-L1 to inhibit CD8+ T cell-mediated killing. We observed no difference in the number of survivor cells in the presence of PD-L1 blocking antibody, when compared to control treated mice. These results demonstrate that PD-L1 is not used by survivor cells to evade CD8+ T cell-mediated killing. We next assessed if survivor cells can be killed by antigen-specific CD8+ T cells. To perform these experiments, we used Cre-inducible GFP reporter mice and GFP-specific CD8+ T cells. If survivor cells can be killed, then reduced numbers of survivor cells will be observed in the presence of activated GFP-specific CD8+ T cells. We observed reduced numbers of survivor cells in the presence of activated GFP-specific CD8+ T cells, indicating survivor cells can be killed. These results demonstrate that survivor cells do not have a cell intrinsic mechanism to protect against CD8+ T cell-mediated killing. We also wanted to investigate the role of survivor cells after IAV clearance. We hypothesized that survivor cells are important for reduced pathology and promote efficient lung recovery after virus clearance. We first assessed the location of survivor cells in the lung after IAV clearance. We performed immunohistochemistry (IHC) and epifluorescent microscopy of lung sections. We observed that survivor cells were often present in clusters, and that the predominant location of survivor cells shifted from the large airways during infection, to small airways after clearance. To determine if survivor cells are present in clusters because survivor cells are proliferating in the lung, we performed experiments were the mice were treated with BrdU for 6 days prior to harvest. In all time after IAV clearance, we observed that the survivor cells were proliferating, and at later time points after clearance survivor cells were proliferating more than non-infected cells. These studies have greatly increased our understanding of how IAV infected lung epithelial cells can survive both IAV infection and the adaptive immune response, and the role these cells play after IAV clearance.
The second major project I worked on was assessing the role of T cell receptor (TCR) affinity and the generation of resident memory T cells (TRMs) in the lung after IAV infection. We have generated IAV-expressing altered peptide ligands (APLs) to the OT-I CD8+ T cell receptor, that are of reduced affinity for OT-I T cells. We transferred congenically distinct OT-I T cells into mice and infected with IAV_APLs. We observed that reduced affinity APL stimulated OT-I T cells underwent a smaller clonal burst after infection, than high affinity stimulated OT-I T cells. Of the reduced affinity expanded cells, a greater percentage of these are able to form TRMs in the lung. We next assessed the phenotype and functionality of high and low affinity stimulated cells by flow cytometry. Both high and low affinity cells upregulated CD69 and CD103 surface markers. Additionally, reduced affinity stimulated cells are functionally similar to high affinity stimulated cells based on interferon-g expression. As lung TRM cells are not stably maintained long term in the lung, we measured the number of high and low affinity stimulated cells in the lung at a late memory time point. We observed that both populations waned at a similar rate. These results show that low affinity T cells respond to an infection and are able to form a greater percentage of TRM cells, when compared to high affinity cells. These results are important when considering the generation of vaccines. New vaccines should include multiple CD8+ T cell epitopes to maximize the number and range of affinities of TCRs that respond.
JEREMY HERRERA, PHD
Mentor: Peter Bitterman, MD
Program: PACCS Division, Department of Medicine
Research: Loss of Dicer1 mediates suppression of lung fibroblast microRNA-29 in Idiopathic Pulmonary Fibrosis
Dr. Herrera’s postdoctoral work in Dr. Bitterman’s lab demonstrated that the lung extracellular matrix (ECM) in IPF mediates progression of fibrosis by decreasing fibroblast expression of microRNA-29 (miR-29), a master negative regulator of ECM production (PMID:24590289). Utilizing decellularized lung ECM as a scaffold for cell-matrix interactions, polyacrylamide hydrogels of defined stiffness, xenograft models, and IHC of IPF specimens we identify deregulation of Dicer1 (a core component of the microRNA processing machinery) as the molecular mechanism mediating miR-29 suppression in IPF (PMID:29579397).This pro-fibrotic ECM-mediated feedback loop can explain why IPF is a progressive disease. However, the properties of the fibrotic-ECM mediating this biology remain incompletely defined. Dr. Herrera published five manuscripts as a result of his post-doctorate training and in 2016, presented his work at the Gordon Research Conference – Signal Transduction by Engineered ECM and American Society for Matrix Biology. He left the University of Minnesota in late 2017 to do a second post-doctoral research fellowship as a Research Associate Fellow at Wellcome Trust Centre for Matrix-Cell Research at the University of Manchester, United Kingdom. There, he continues his work in IPF by (1) characterizing the ECM in IPF and (2) create a novel cell-ECM in vitro model that recapitulates disease. He has recently performed 3-D reconstructions of the fibroblastic focus in IPF using immunostains to register ECM proteins in and around the hallmark lesion in IPF, the fibroblastic focus (In Revision, likely to be published in JCI Insights); and is utilizing laser-capture microscopy coupled with mass spectrometry proteomics to characterize the fibroblastic focus, adjacent physiologically normal, and mature scar tissue in IPF. Guided by his findings in (1), he is developing cell/ECM constructs incorporating dimensionality, composition, and mechanics to determine how these ECM properties influence fibroblast biology.
KATHERINE JANSSEN, MD
Mentor: John Connett, PhD
Program: PACCS Fellowship
Research: The Impact of Palliative care on Quality of Life, Anxiety, and Depression in Patients with Idiopathic Pulmonary Fibrosis
My long term research goal is to identify therapies and interventions which will improve quality of life in patients with chronic lung disease. My residency and fellowship training has provided me with a strong background in pulmonary and critical care medicine. As a second year fellow, I began working with Dr. Hyun Kim, a successful investigator at the University of Minnesota Interstitial Lung Disease center. The Interstitial Lung Disease Center at the University of Minnesota serves a large population of patients with idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases (ILD). Patients are referred to us from Minnesota and throughout the upper Midwest and West regions. Together, Dr. Kim and I developed the concept for a pilot study to determine if visits with the University of Minnesota palliative care team improves patient related quality of life, anxiety, or depression in patients with idiopathic pulmonary fibrosis (IPF). With the assistance of palliative care and pulmonary experts, I designed this randomized controlled trial and protocol, as well as selected the instruments used to measure patient related quality of life. I am actively recruiting and enrolling patients in this study. To further develop my expertise in clinical research, I am a trainee on the Pulmonary NIH T32 Pulmonary Research Training Grant (PI D. Ingbar). As a third year fellow, I have begun coursework this Fall to pursue the certificate in Clinical Research at the University of Minnesota School of Public Health (consistently ranked in the top 10 public health programs in the nation), which includes coursework in biostatistics, research ethics, clinical trial design, and epidemiology.
EMILY JULIK, PHD
Mentor: Louis M. Mansky, PhD
Program: Institute for Molecular Virology
Research: HIV Mutation in Human Primary Cells
My research centers primarily on the application of next-generation sequencing technologies to understand natural and drug-induced mutation in HIV, with an emphasis on differences between HIV-1 and HIV-2. HIV cases are comprised of infections by two different viruses, designated HIV-1 and HIV-2. Of these, HIV-1 is more pathogenic and more widespread; among the 37 million cases of HIV infection globally, only ~2 million result from infection by HIV-2. Most drug development has focused on HIV-1, and a number of drugs approved for HIV-1 are not effective against HIV-2, such that there are fewer treatment options overall for HIV-2-infected individuals.
An important facet of fitness for all HIV is relatively high error rates of replication, attributable to large population sizes and the error-prone reverse transcriptase enzyme. This confers upon the viruses the ability to develop drug resistance mutations and evade adaptive immunity, but also renders them susceptible to loss of infectivity upon any further increase of the mutation rate, as demonstrated for example by the antiviral activity of cellular APOBEC enzymes that edit viral sequences to the point of loss of infectivity. Therapeutically, small molecule mutagens can impart a similar effect, introducing such a high mutation burden to the virus that it is rendered non-infectious.
Our group previously demonstrated that HIV-2 experiences significantly lower levels of mutation than HIV-1 in permissive cell lines, an intriguing finding given the importance of mutation rate to viral fitness. Our group also showed that HIV-2 exhibits greater susceptibility to a number of viral mutagens. My ongoing work is refining these findings in human primary CD4+ T cells. Using exquisitely sensitive sequencing techniques, I am defining the natural and drug-induced mutation frequencies and spectra of HIV-1 and HIV-2 in this most relevant primary cell type, which is the major target of HIV infection in vivo. These findings have important implications for understanding HIV evolution and for clinical translation of antiviral mutagens.
This work will increase understanding of the basic biology of HIV and support the development of a next generation of HIV therapies. HIV infection negatively impacts lung health and, of particular relevance here, T cells within the lung appear to represent a long-lived reservoir for HIV infection in the body. Understanding the biology and drug treatment of HIV in these cells thus has implications for clearance of HIV infection from the lungs. Future work can replicate these studies in alveolar macrophages, which are another potential long-lived reservoir for HIV infection.
JENNIFER MCCURTAIN, PHD
Mentors: Ryan Hunter and Garry Dunny
Program: Predoc, MICaB
Research: Survival Advantages of Pseudomonas Aeruginosa Cystic Fibrosis Clinical Isolates with AguA Deletion Mutations
Summary: In the Hunter Lab, Jennifer studied persistent lung infection by Pseudomonas aeruginosa in the Cystic Fibrosis (CF) lung. Previous studies have shown that agmatine increases biomass when P. aeruginosa UCBPP-PA14 (PA14) is grown as a biofilm; however, it remains unknown what this increase entails, such as more cells, a thicker biofilm, or an improved matrix. Jennifer’s project involves detecting PA14 agmatine metabolism in biofilms using a fluorescent reporting system and identifying the impact that agmatine has on the biofilm with regards to its structure and matrix components.
She has presented her research at the Pulmonary, Allergy, Critical Care and Sleep Medicine Citywide Conference and authored a manuscript published in Analytical and Bioanalytical Chemistry. She won the 2017 MICaB Spring Travel Award through which she attended ASM Microbe 2017 in New Orleans, LA. There, she presented a poster and was selected for and gave a Rapid Fire Talk. She defended her thesis in spring of 2018 and is now a Senior Scientist at Merck in New Jersey.
NATHAN SCHULDT, PHD
Mentor: Bryce Binstadt
Program: Postdoc, Center for Immunology & Dept of Pediatrics
Research: A Novel Dual TCR T Cell Reporter Mouse
Commonly when immunologists think of T cells they imagine them as having one T cell receptor (TCR) and therefore one specificity. While this simplification may have made it easier to comprehend thymic selection and T cell development, we now know that about one third of T cells actually have recombined a functional TCR from both alleles. These cells therefore have the potential to express two different TCR specificities on the cell surface. My research focuses on understanding how this phenomenon affects thymic selection and T cell development to ultimately impact several immune contexts, including: autoimmunity, allergy, alloreactivity, memory responses, and protective immunity. I developed a novel tagging method that allowed detection of surface-expressed TCR without interfering with TCR signaling in vitro. I have generated chimeric knock-in mice expressing the two tag constructs that each can be detected easily by flow cytometry without affecting TCR signaling. I am in the process of taking the two lines that have been bred to hozoygosity and breeding them together to obtain the dual TCR T cell reporter mice. These mice will be used for my studies in type 1 diabetes and asthma models.
STEVE SKOLASINSKI, MD
Mentor: Angela Panoskaltsis-Mortari
Program: PACCS Fellowship
Research: Pulmonary Bioengineering: Development of a novel cell sprayer device for lung bioengineering and directed cell therapy, and an automated bioreactor for more efficient decellularization of pulmonary tissue.
Steve works on a collaborative project incorporating device design, novel assessments of pulmonary ari flow using MRI, and bioengeering that will be applied to lung regeneration in Angela Panoskaltsis-Mortari’s lab. His T32 support ended after one year when he competed successfully for a NIH F32 grant. Steve has worked to refine and automate the process of cell removal from pulmonary tissue and designed a cell aerosol device that will be used to repopulate the decellularized airways with induced pluripotent stem cells. In addition to regenerating lungs, he is working on a novel method to assess air flow patterns in the pulmonary airway using MRI. This work has involved a multidisciplinary team composed of laboratory and medical scientists, biomedical and materials engineers, and physicists. He also is experiencing firsthand the interplay between academia and industry during device development with our industry partner. He has one first author publication thus far and given multiple oral presentations at national meetings. He received a travel award to participate in the National Jewish Respiratory Disease Young Investigators Forum in 2017 and was awarded first place at the Pediatrics Research, Education, and Scholarship Symposium in 2018. His NIH F32 grant was funded on initial submission and he is working towards submission of a K08 award in 2019.
THU TRUONG, PHD
Mentor: Carol Lange, PhD
Program: Postdoc, Masonic Cancer Center
Research: Steroid receptor co-activators PELP1 and SRC-3 cooperate to drive breast cancer stemness
Dr. Truong’s fellowship research focused on understanding how steroid receptors (SRs) and SR co-activators alter signaling pathways and transcriptional regulation to mediate cancer cell fate transitions. PELP1 (proline, glutamic acid, and leucine rich protein 1) is an essential nuclear protein that is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue where it serves as a potent SR co-activator. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. PELP1 is emerging as a viable therapeutic target and biomarker for women with breast cancer. She identified SRC-3 (steroid receptor co-activator 3) as a novel binding partner of cytoplasmic PELP1 in both normal and breast cancer cell models. Cytoplasmic PELP1 expression elevated SRC-3 activation levels, enhanced ALDH+ tumorsphere formation, and upregulated target genes involved in cell survival (e.g. AMIGO2, NELL2). Her work identified the cytoplasmic PELP1/SRC-3 complex as a novel protein-protein interaction that can be selectively targeted by potent inhibitors. These findings indicated that cytoplasmic PELP1 likely is involved in breast cancer stem cell expansion. She also showed that the cytoplasmic PELP1/SRC-3 interaction is required for tumorsphere growth in vitro and confirmed this with an in vivo mouse model. Her current studies are focused on further elucidating how the cytoplasmic PELP1/SRC-3 complex alters signal transduction pathways, target gene selection, and cell fate transitions relevant to therapy resistant metastasis and CSC outgrowth.
Collectively, this work resulted in a first-author research publication in Mol. Cancer Res. (2018), and was also a multi-PI investigator effort between three labs (Drs. Julie Ostrander, Carol Lange, and Kaylee Schwertfeger). She formed a number of collaborations that resulted in a second author research article (Journal of Hematology & Oncology), a second author review article (Journal of Steroid Biochemistry & Molecular Biology), and first author review article (Endocrinology). Dr. Truong ended her T32 support early when she received F32 funding (she was awarded, but declined 2 other career development awards) and submitted a K99/R00 proposal in October 2018 continuing her cytoplasmic PELP1/SRC-3 research. She has one additional first author research article currently under review based on her T32 research.
GERMÁN VÉLEZ REYES, BS
Mentor: David Largaespada, PhD
Program: MD/PhD program & MICaB
Research: Discovering novel Schwann cell tumor genes and the molecular pathways activated by RSPO2 in human cancers
As part of the Largaespada Lab I aim to study the molecular intricacies of the R-spondin and Wnt/β-catenin pathways in the development of human cancers, including lung adenocarcinoma. Here I have discovered that RSPO2 activates both the Hippo and Wnt pathways by using cellular and novel animal models, and that it is necessary for its oncogenic activity. Also, a problem that has fascinated me in the past couple of years is to understand how oncogene expression regulation occurs in cancer, especially how border elements keep oncogenic expression in control. I am also working in identifying novel Schwann cell tumor genes in order to find novel targeted therapies against MPNSTs.
JULIA WILLETT, PHD
Mentor: Gary Dunny
Program: Microbiology & Immunology
Research: A putative phosphatase regulates surface attachment and biofilm formation in Enterococcus faecalis
Enterococcus faecalis is an early colonizer of the infant gut and is a common nosocomial pathogen. E. faecalis readily infects surgically implanted devices, is the third leading cause of endocarditis, and is an emerging respiratory pathogen, especially in intubated patients. E. faecalis has high levels of intrinsic and acquired antibiotic resistance (even to "last resort" drugs such as vancomycin and linezolid) and can overtake the native microbiome upon antibiotic treatment. The ability of E. faecalis to thrive as a pathogen is due in part to its ability to form biofilms, which are surface-associated communities of bacteria surrounded by a complex extracellular matrix composed of DNA, proteins, and sugars. Biofilms render bacteria more resistant to antibiotics, thereby complicating treatment. Once established, biofilms are difficult to eliminate, leading to persistent and recurring infections. Therefore, understanding the mechanisms controlling the formation of E. faecalis biofilms is crucial for controlling the spread of and eradicating these infections.
We recently screened an E. faecalis transposon (Tn) mutant library and identified two Tn strains with highly attenuated biofilm production. Both insertions were in OG1RF_10435, a hypothetical gene that encodes a protein of unknown function. These mutants are specifically defective in surface attachment, the first step in biofilm formation. OG1RF_10435 is highly conserved throughout Gram-positive bacteria and is upregulated in the early stages of subdermal infections in a rabbit model. Based on preliminary in vitro and computational analysis, we hypothesize that OG1RF_10435 is a nucleoside phosphatase that is required for robust biofilm formation. The work proposed here will ascertain the molecular mechanism by which OG1RF_10435 regulates attachment and biofilm formation in E. faecalis. Because this protein is conserved throughout bacterial pathogens, including Staphylococcus and Streptococcus, this work will further our understanding of the basis of bacterial respiratory infections.