Winner Interviews

Spring 2015 Research Grant Recipients Talk About Their Research


James Ankrum, PhD
Assistant Professor
University of Iowa

Abstract Title:
Elucidating Disease-Specific Mechanisms of Mesenchymal Stem Cell-Mediated Polarization of Macrophages from Diabetic and Healthy Donors to an Anti-Inflammatory M2 Phenotype


BD: What is your educational background?

James Ankrum: I received my undergraduate degree in biomedical engineering from the University of Iowa, followed by an MPhil degree in engineering design from Cambridge University in the UK. Under the direction of Prof. Jeff Karp at MIT, I received my PhD in medical engineering and medical physics. My thesis was titled A Microparticle Engineering Approach to Enhance the Potency of Mesenchymal Stem Cells. For my postdoctoral work I was Senior Innovation Fellow at the Medical Devices Center at the University of Minnesota.

BD: How and when did you become interested in science?

James Ankrum: Growing up in rural Iowa, I was always fascinated with how things worked. I used to take everything apart to figure out its operation. My interest in science definitely started with mechanics and machines, and has evolved over the years as I’ve become interested in understanding biological systems.

BD: How did you become interested in your broad field of study?

James Ankrum: I became interested in cell-based therapies during my first semester of graduate school at MIT. I had intentionally sought out the program at MIT because it combined rigorous training in biological sciences with in-depth training in classical engineering, which allowed me to deepen my understanding of human health and disease. I took a course from Prof. Jeff Karp, who ended up being my PhD mentor, and was fascinated by the prospects of cell-based therapies. Compared to traditional therapies that utilize single molecules to elicit a therapeutic effect, a cell’s ability to sense and respond to its environment provides a feedback loop that other therapies simply lack. Since seeing the potential of cells as therapeutics, I have devoted my research to understanding how mesenchymal stem cells (MSCs) exert their therapeutic effect, and trying to augment their potency.

BD: Describe the project for which you received the BD grant.

James Ankrum: In our proposed project we will attempt to elucidate disease-specific mechanisms of interaction between MSCs and immune cells within the context of diabetes. While many groups have analyzed the ability of MSCs to suppress various immune cell populations, nearly all of the studies are performed with immune cells collected from healthy donors. However, cells sense and respond to their environments, and differences in immune cell function in the context of specific diseases could alter the way MSCs are triggered and respond to resident immune cells. In this proposal, we are specifically looking at MSC interactions with macrophages from healthy and diabetic patients. MSCs have been shown to polarize macrophages away from an M1 phenotype into less inflammatory or anti-inflammatory phenotypes. We will perform a series of experiments first to characterize differences in macrophage cytokine secretion profile between healthy and diabetic donors, and then to measure the ability of MSCs from healthy donors to polarize the macrophages to anti-inflammatory subtypes. Since MSC-mediated immune suppression is mediated through the expression of multiple factors, we will perform a series of blocking experiments to determine the role of individual immunomodulatory factors.

BD: What are the long- and short-term scientific goals of this project?

James Ankrum: The short-term goals of this project are to test the assumptions that the immunosuppressive mechanisms of MSCs can be determined in assays utilizing immune cells from healthy individuals, and if immune cells harvested from diseased patients alter MSC-immune cell interactions. In this initial study, we are using immune cells from patients with diabetes, a major comorbidity in the US, as a case study. Long-term we hope to develop disease-specific potency assays and techniques for enhancing the survival and potency of MSC therapies.

BD: What are the implications of your project for human health?

James Ankrum: MSC-based therapies have enormous potential to restore function to diseased and damaged tissues. The ability of MSCs to interact with host immune cells and remodel inflammatory environments to allow regeneration makes them applicable to dozens of diseases including diabetes, and diabetic complications such as diabetic wounds and myocardial infarction. However, to date, potency assays that predict MSC function and efficacy in disease models have been elusive. A possible contributor to our lack of predictive potency assays could lie in the way MSCs interact with local immune cells to become activated and exert an effect. By understanding MSC-immune cell interactions within the context of a specific disease, we might be able to generate disease specific potency assays that better predict whether a batch of MSCs will be therapeutic or if a specific patient will respond to a given MSC therapy.

BD: Which BD reagents do you plan to use, and for what purposes?

James Ankrum: Using BD Pharmingen™ antibodies and BD OptEIA™ kits, we will monitor the conversion of macrophages into M2 phenotypes in co-cultures with MSCs. Macrophages exist in a spectrum of states ranging from highly inflammatory to moderate to anti-inflammatory in their repertoire of secreted factors. Using BD OptEIA kits to measure secreted factors in addition to measuring cell surface marker expression will enable detailed characterization of macrophages harvested from each donor. The BD™ CBA bead-based human inflammatory cytokine kit will allow for unprecedented characterization of the signaling molecules derived from macrophages of healthy or diabetic origin and for the understanding of the repertoire of secreted cytokines’ changes upon exposure to MSCs. Finally, for the blocking studies, BD blocking antibodies will be used to inactivate secreted factors in the media.


Manuela Colucci, PhD
Postdoctoral Fellow
Bambino Gesù Children’s Hospital, Rome

Abstract Title:
Evaluating the Reconstitution of the B Cell Repertoire and the T Cell Homeostasis Following Rituximab Treatment to Predict Disease Relapse in Pediatric Idiopathic Nephrotic Syndrome


BD: What is your educational background?

Manuela Colucci: I earned my bachelor’s degree at “La Sapienza” University and my PhD in immunology at Tor Vergata University in Rome. As a student at the Istituto Superiore di Sanità, I first studied the upregulation of regulatory T cells by inducing tolerogenic dendritic cells using bacterial products, which was the basis of my PhD thesis. Today, as a postdoctoral fellow at the Division of Nephrology and Dialysis of the Bambino Gesù Children’s Hospital, I focus on the role of B and T cells in pediatric renal autoimmune diseases and on specific immunomodulatory treatment. This work is performed under the supervision of Dr. Marina Vivarelli, a pediatrician with expertise in immunology, and Dr. Francesco Emma, who is Chief of the Division of Nephrology. Together they have opened a new front of translational research in pediatric immune-mediated renal diseases at the Bambino Gesù Children’s Hospital. My expertise in immunomodulation has contributed to understanding some aspects of glomerular diseases such as idiopathic nephrotic syndrome (INS) and systemic lupus erythematosus.

BD: How and when did you become interested in science?

Manuela Colucci: I decided to become a biologist when I was only twelve years old, as a result of being inspired by my biology teacher. In addition, my grandfather was a prominent cardiologist who transmitted to me an interest in medicine and caring for sick persons.

BD: How did you become interested in your broad field of study?

Manuela Colucci: During my undergraduate studies in a microbiology course, I was intrigued by the role of the immune system in fighting infections. On that basis I decided to increase my knowledge of this extraordinary and relatively young field of study by working in an immunology laboratory as a student researcher and, later, by completing a PhD in immunology.

BD: Describe the project for which you received the BD grant.

Manuela Colucci: INS is the most frequent glomerular disease in childhood. It is characterized by heavy proteinuria, associated with edema, hyperlipidemia, and hypoalbuminemia. Non-genetic forms have an immune pathogenesis mediated by a not yet defined circulating factor that causes modifications of the selective glomerular permeability barrier. T-cell alterations have been described in INS for a long time, and the role of B cells is still widely discussed. Recently it was demonstrated that rituximab, a monoclonal antibody that depletes B cells, is effective in inducing prolonged remission in nephrotic syndrome patients. However, the maintenance of remission after rituximab infusion is often transient, and the mechanisms responsible for rituximab’s effectiveness remain elusive.

We hope to characterize by fluorescence activated cell sorting analysis the presence and reappearance of different B-cell subpopulations, and the subsequent effect on T-cell homeostasis after rituximab infusion in INS patients. We believe that in addition to inducing depletion of autoreactive B-cell clones, rituximab treatment could modify antigen presentation and cytokine production, with a subsequent alteration of T-cell homeostasis and homing.

BD: What are the long- and short-term scientific goals of this project?

Manuela Colucci: The characterization of the effect exerted by rituximab on B-cell subsets and on T-cell homeostasis could allow us to elucidate the role of B cells in the pathogenesis of nephrotic syndrome. More importantly, the reconstitution of different B-cell subsets could be predictive of the response to rituximab treatment and could permit identification of patients with major risks of relapse. Indeed, to date no biologically reliable readout predicts relapse following rituximab treatment in INS.

BD: What are the implications of your project for human health?

Manuela Colucci: Current therapy for INS is based on steroid treatment for 8–12 weeks, which brings about complete remission in up to 80% of patients. However, 40–50% of patients experience frequent relapsing-remitting episodes and acquire steroid dependency or, rarely, develop resistance to this treatment. Concurrent treatment with immunosuppressive agents such as calcineurin inhibitors and mycophenolate mophetil, or replacement of steroids with these agents, is also effective, but 30–40% of the patients experience treatment-related toxicity. The recently introduced rituximab therapy is effective not only in inducing prolonged remission but also in allowing tapering of concomitant steroid and calcineurin inhibitor treatment, despite the transient nature of the remission. By monitoring the reappearance of specific B-cell subsets following rituximab infusion, we hope to characterize new pathogenic mechanisms of INS, and finally have a means of predicting the risk of relapse. We also hope, through this approach, to optimize the concomitant immunosuppressive treatment regimen in pediatric patients who experience significant side effects from immunosuppressive drugs.

BD: Which BD reagents do you plan to use, and for what purposes?

Manuela Colucci: To discriminate the different B-cell subpopulations and effector and regulatory T cells, we will stain peripheral blood mononuclear cells with several BD Pharmingen™ fluorochrome-conjugated anti-human antibodies such as anti-CD19, CD24, CD27, CD38, IgM, and IgD for B-cell subsets, and anti-CD3, CD4, CD8, CD25, CD45RO, CD127, and FoxP3 for T cells. Stained cells will be then analyzed using a BD FACSCanto™ II or BD LSRFortessa™ flow cytometer.


Raquel Margarida da Silva Ferreira, PhD
Postdoctoral Fellow
University of Beira Interior, Portugal

Abstract Title:
Anti-Inflammatory Therapy for Functional Repair in Stroke


BD: What is your educational background?

Raquel Ferreira: I completed my undergraduate degree in applied biology at the University of Minho in Braga, Portugal. I was awarded my PhD in the Department of Cellular Biology at the University of Coimbra, also in Portugal. My thesis work involved neuroinflammation and microglial activity. Since receiving my degree I have worked in two postdoctoral positions. The first was at the University of Southern California, where I worked on human cerebral arteriovenous malformations and aberrant brain angiogenesis. That was followed by my return to Portugal and the University of Beira Interior, which is in Covilhã. Here I research stroke, biomaterials, and cell-based therapies under the direction of Prof. Liliana Bernardino.

BD: How and when did you become interested in science?

Raquel Ferreira: I was an inquisitive child. My older sister taught me the various subjects she was learning, topics someone of my age would not normally study, including science. My father also has a very creative mind. When I was young he told me extraordinary stories that instilled in me the need to think outside the box and challenge everything. Finally, I had the most amazing teacher in primary school, a woman who taught all subjects. She motivated me to be always eager to learn.

BD: How did you become interested in your broad field of study?

Raquel Ferreira: My interest in immunology began during my college years. It was then that I solidified my awareness of the immune system’s intricacy and efficiency. I became particularly interested in studying the central immune system and, for my doctoral studies, dedicated myself to studying microglial responses. What appeals to me is the growing body of evidence for an inflammatory component in almost every disease, which further highlights the importance of this research and my proposed project.

BD: Describe the project for which you received the BD grant.

Raquel Ferreira: My project focuses on restoring the integrity of brain vasculature through the promotion of balanced inflammatory and pro-angiogenic environments, to ensure an efficient recovery in stroke. We know that an immune response carried out mainly by microglial cells is a key process set in motion by stroke. Microglial cells have the potential both to aggravate cell damage in the ischemic area and to promote its recovery. We aim to control endothelial cell activation to prevent microglial-mediated inflammation, to shift the immune response towards the protection of the blood-brain barrier.

Retinoic acid is a known antioxidant and has been described as an inducer of stem cell differentiation and a regulator of angiogenesis. To exploit this therapeutic potential, we developed retinoic acid nanoparticles, which protect retinoic acid from degradation and allow a gradual and efficient release of the molecule. We have preliminary data indicating that these nanoparticles stimulate endothelial cell growth in an ischemic environment. Since cerebral ischemia strongly triggers a massive inflammatory response at the blood-brain barrier, we want to test the effect of retinoic acid nanoparticles on the inflammatory component of stroke via activated endothelium.

BD: What are the long- and short-term scientific goals of this project?

Raquel Ferreira: In the short term we expect to fully characterize the direct effect of retinoic acid nanoparticles on endothelial cell activity and focus on blood-brain barrier integrity from a vascular perspective. Then, we will focus on the inflammatory processes that occur at the blood- brain barrier that affect its integrity and neuronal survival. In the long term we will test the multirestorative potential of retinoic acid nanopoarticles more comprehensively in an experimental model of stroke. Those experiments will allow us to assess the effects of nanoparticles on angiogenic and immune responses after middle cerebral artery occlusion, a procedure that transiently interrupts blood flow to the brain. The ultimate aim is to encourage a suitable inflammatory environment that allows rebuilding the blood-brain barrier while minimizing neuronal death.

BD: What are the implications of your project for human health?

Raquel Ferreira: Cardio- and cerebrovascular diseases are the leading causes of death worldwide. Stroke alone kills nearly 62 people per 100,000 inhabitants in Portugal. This number skyrockets in the elderly population, reaching 720 people of 100,000 individuals over the age of 70. Increasing life expectancy will likely expand this number. Stroke-associated economic costs total $69 billion in Europe and $48 billion in the US, and current therapies benefit only a small number of patients. The standard of treatment is based mainly on the administration of thrombolytic drugs, with no more than a four-hour therapeutic window and life-threatening side effects. Our project fits the global aim of developing a safer and more inclusive therapeutic platform that enhances motor and cognitive recovery and decreases time of admission and post-hospital care. This project could potentially help patients with other vascular disorders as well, thus benefiting human health and the healthcare economy.

BD: Which BD reagents do you plan to use, and for what purposes?

Raquel Ferreira: Our experiments will use significant quantities of Matrigel matrix to evaluate endothelial cell migration and network formation. Our migration studies will require Boyden chambers. For the quantification of soluble factors secreted by endothelial cells, we plan to purchase human and/or mouse BD™ Cytometric Bead Arrays. Primary antibodies will be necessary to evaluate proliferation, neuronal differentiation, endothelial cell permeability, and the development of inflammatory processes associated with nitric oxide formation. Finally, we will employ ELISA kits to quantify the release of inflammatory mediators.


Rebecca Ingram, PhD
Lecturer
Queen’s University Belfast

Abstract Title:
Innate Memory to Bacterial Infection


BD: What is your educational background?

Rebecca Ingram: I received my BSc in zoology from the University of Aberdeen, MSc in parasitology from the Universities of Manchester, Salford and Keele, and a PhD in parasitology and pharmacy from Kings College London. My first postdoctoral position was at Imperial College London, but I spent extended periods working at the Uganda Virus Research Institute. There, under the direction of Prof. Frances Gotch, I studied immune correlates of protection from progression in HIV. Returning full time to England, I took another position at Imperial College, where I researched T-cell epitope mapping for potential organisms of bioterror. I’m now at the Centre of Infection and Immunity, Queens University Belfast, establishing my own research group with a focus on the role of lymphocytes in responses to respiratory bacterial infections.

BD: How and when did you become interested in science?

Rebecca Ingram: I have been interested in science for as long as I can remember. One of my favorite activities as a child was moving stones in the garden and watching the insects that emerged from under them. Later I had a chemistry set and a microscope, and even kept a lab book of all my experiments. Other than a brief and hugely delusional interest in becoming a ballerina, I don’t remember ever wanting to be anything other than a scientist.

BD: How did you become interested in your broad field of study?

Rebecca Ingram: My research focuses on the immune responses to infections, an interest that began while I was an undergraduate, a result of attending my first parasitology lecture. Ever since, I have been awestruck at evolutionary processes that hide, divert, and generally outsmart our immune system. I initially believed I wanted to study the organisms themselves. However, the further I got into my research the more intrigued I became at the battlefront where the infectious organism and our immune systems meet. I have worked on parasites, viral, and bacterial infections, and this diversity has shaped my view of the immune system. Immunology is a fast-paced subject, and there is always more to learn. With almost every new discovery, another layer of immune regulation is revealed—the exquisite control with which the immune system identifies and eliminates its enemies amongst a constant barrage of infectious organisms.

BD: Describe the project for which you received the BD grant.

Rebecca Ingram: Vaccines are arguably the greatest medical achievement, exemplified by the world-wide eradication of smallpox, a 99% decrease in polio cases, and a 74% reduction in global measles deaths. Immunologic protection is achieved eight to ten days following vaccination. However, with the threat of global pandemics or bioterrorism, a need exists for new vaccination strategies that induce rapid immunity. Increasing evidence suggests that the early, innate, lymphocyte responses to pathogens develop a “memory” response, but this memory and its potential to be exploited in vaccines remain uncharted.

I have shown that exposure to bacteria induces an enhanced memory response in a recently identified immune cell population known as innate lymphoid cells (ILCs). These ILC memory cells demonstrate a potent ability to clear bacterial infections upon subsequent reinfection with the same bacteria. Through this project I will examine if this enhanced response is specific or if it offers protection against other types of bacteria, too. We hope to identify the effector molecules that mediate this effect, and determine if these memory cells expand to overcome subsequent bacterial infections.

BD: What are the long- and short-term scientific goals of this project?

Rebecca Ingram: The field of innate memory is currently exploding. It becomes increasingly apparent that a number of innate immune responses are modulated by previous exposure to antigens. The short-term goals for this project are to understand the completely novel innate memory responses and how they mediate their potent ability to clear infection. Longer term, I intend for innate memory to become a major focus of my laboratory. I will explore the underlying mechanisms by which innate memory occurs. For example, does it up-regulate specific receptors, epigenetic changes, or modulations in RNA regulatory machinery? I also plan to study how we can induce this innate immunity via vaccination, and the implications on classic adaptive memory responses.

BD: What are the implications of your project for human health?

Rebecca Ingram: This research has the potential to influence how we prevent and/or treat bacterial infections. Innate memory is likely to be induced earlier than traditional T- and B-cell adaptive immunity. Therefore, inclusion of antigens inducing ILC memory in vaccines should provide more rapid protection. In addition to providing essential early immunity during a pandemic, it is possible that this strategy will allow vaccinating patients before surgery or as part of critical care to prevent hospital-acquired infections. Furthermore, the lack of functional adaptive immune responses makes vaccines less effective in infants, the elderly, and in immune-compromised individuals. This research has the potential to open new avenues for inducing specific innate immune responses in these populations.

BD: Which BD reagents do you plan to use, and for what purposes?

Rebecca Ingram: The effector molecules released by the ILC memory cells during infection will be established through experiments employing BD™ CBA Flex Sets (IL-17A, IL-17F, IL-23 p19/p40, IL-6, IL-21, IL-1b TNF, G-CSF, and KC). We will also use BD Horizon™ Violet Proliferation Dye to determine if memory ILC cells transferred into a naive host proliferate during infection.


Tania Konry, PhD
Assistant Professor
Northeastern University

Abstract Title:
Development of Novel Lab on a Chip Based Technology for Immune Regulation Studies in Blood Related Cancers


BD: What is your educational background?

Tania Konry: I have a BMedSc in medical science and an MSc in biotechnology engineering, both from Ben Gurion University in Israel. After completing a PhD in biotechnology engineering, my interest in the development of high throughput measurement techniques, such as sensors and microarrays, led to work as a postdoctoral fellow at Tufts University under the supervision of Prof. David Walt, a world-renowned specialist in microarray technology. In the Walt lab I developed a method to detect nucleic acid sequences and proteins in a single test using an optical fiber microsphere-array platform combined with immuno-rolling circle amplification. This work resulted in two well received papers in top journals. Next I was invited to join the laboratory of Dr. Martin Yarmush, Professor of Surgery and Bioengineering at Harvard Medical School and Director of the Center for Engineering in Medicine (CEM). I served as Instructor at Harvard Medical School, an Assistant in bioengineering, and co-leader of the nano- and microsystems bioengineering group.

BD: How and when did you become interested in science?

Tania Konry: In my undergrad classes, where I was introduced to biomedical courses and became fascinated by my professors’ research. I was very fortunate to meet and work closely with incredible scientists like Profs. Smadar Cohen, David Walt, Martin Yarmush, and David Avigan, who influenced my own research interests and opened my mind to new perspectives.

BD: How did you become interested in your broad field of study?

Tania Konry: During grad school my PhD advisor, Prof. Robert Marks, introduced me to biosensor and immunoassay technology. He has a very futuristic approach, which allowed me to develop novel immunoassays and bioassays that combined very well established methods with nano- and micro-engineering tools. Then, during my graduate studies in biotechnology engineering, I developed a novel type of highly sensitive analytical assays and devices, useful in biomarker detections, clinical profiling of patients in disease screening, and diagnosis or disease monitoring. This work, which was completed within my PhD dissertation, has been summarized in eight peer-reviewed papers and received the Prof. Shemona Geresh award for “outstanding research thesis” in the Department of Biotechnology at Ben Gurion University. I also received a Women in Science Award from Israel’s Ministry of Science.

I am currently Assistant Professor at Northeastern University in the Department of Pharmaceutical Science. My laboratory is primarily oriented towards single-cell studies in cancer immunology and in the development of diagnostic tools, assays, and bioassays for biomarkers and cell detection. We are also involved in engineering new materials for future biological and chemical application in artificial organs, drug delivery, and diagnostic tools.

BD: Describe the project for which you received the BD grant.

Tania Konry: Evaluating the subtypes, numbers, and functionality of immune cells as well as characterizing the activity of immune regulators is a complex scientific and technological challenge. In this context, developing and validating sample-sparing assays is essential. The rationale of those assays involves simultaneous multiparameter assessments of immune function. These are in turn critical for establishing a correlation between the immunological mechanism of the activation or inhibition of immune effectors, and the targeted clinical response in diseases such as allergy, asthma, autoimmunity, acquired and primary immunodeficiency, transplantation, and infection.

Recent evidence from studies of single cells reveals that the assumption that all cells of a particular type used in immunoassays on groups of cells is incorrect. These assays are often used to study cell-cell interactions and secretions. We propose to develop new sample-sparing approaches to single-cell analyses and live cell-cell interaction at the single-cell level to uncover fundamental biological principles and ultimately improve the detection and treatment of disease. The micro-technology proposed in this application will allow us to conduct sample-sparing simultaneous multiparameter analysis of both live- and fixed-cell surfaces and secretions. This will be achieved through the controlled delivery of picoliter volumes of immunoregulatory agents and therapeutics to cells of interest to study their effect on the functional phenotype of the cell. We expect as well to be able to monitor live cell-cell interactions at the level of single cells, and make secretion measurements during this interaction. Finally, we will use fluorescence-activated droplet sorting for specific cell phenotype isolation.

BD: What are the long- and short-term scientific goals of this project?

Tania Konry: The short-term goal is development of a robust single-cell phenotyping method or platform. In the long term, we would like to integrate our platform as a novel research and diagnostic tool compatible with currently available technologies for cell phenotyping.

BD: What are the implications of your project for human health?

Tania Konry: The proposed approach should have a broad impact on diverse biological systems for the study of cell surface and secretion proteins as potential biomarkers and targets for diagnostics and therapeutics, as well as the imaging of cell-cell interactions for immunotherapy and biomarker discovery.

BD: Which BD reagents do you plan to use, and for what purposes?

Tania Konry: We plan to use BD Lyoplate™ Human Cell Surface Marker Screening Panels and BD fluorescently labeled antibodies, BD™ CBA bead-based immunoassays, and a fluorescence-activated droplet sorting system for the isolation of specific cell phenotypes.


Alexander Misharin, MD, PhD
Research Assistant Professor
Northwestern University, Feinberg School of Medicine

Abstract Title:
Multiparameter Flow Cytometric Analysis of Human Alveolar Macrophages in Patients with Idiopathic Pulmonary Fibrosis: a Guide to Molecular Taxonomy of the Rare Disease and Personalized Treatment


BD: What is your educational background?

Alexander Misharin: I received my MD degree, with a concentration in pediatrics, from the Russian State Medical University, and my PhD from the Institute for General Pathology, both in Moscow, Russia. I did my first postdoctoral fellowship in thyroid autoimmunity from 2006 to 2008 in the laboratory of Drs. Sandra McLachlan and Basil Rapoport, Cedars-Sinai Medical Center, Los Angeles. In 2009 I joined the laboratory of Dr. Harris Perlman in the Division of Rheumatology, Northwestern University, where I concentrated on macrophage biology and its role in various diseases. I am currently switching departments, from Rheumatology to Pulmonary and Critical Care Medicine, where I will study the role of pulmonary macrophages in the development of age-related lung pathology, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis. My mentors are Dr. Harris Perlman and Dr. GR. Scott Budinger.

BD: How and when did you become interested in science?

Alexander Misharin: I became interested in science during high school, where I studied molecular biology, human anatomy, and evolution. The Double Helix, by James D. Watson, was one of the most influential and inspiring books for me.

BD: How did you become interested in your broad field of study?

Alexander Misharin: From my first studies of the immune system, I was intrigued at how deeply it penetrates every tissue of the body. It controls tissue and organ development and homeostasis, and initiates (or sometimes fails to initiate) the response to tissue damage, infection, and cancers. The immune system connects local tissue context with more global responses at the organism level. Most human diseases involve the immune system in some way. Understanding it will allow us to answer global questions with profound impact on human health. It is normal for researchers to be ambitious and want to solve big problems.

BD: Describe the project for which you received the BD grant.

Alexander Misharin: Idiopathic pulmonary fibrosis is a relentlessly progressive and usually fatal disorder for which no treatments exist except lung transplantation. Current classification of the disease is based on clinical features and does not reflect disease pathogenesis. Moreover, molecular markers to predict prognosis and guide therapy are limited. We and others have demonstrated that lung macrophages play key roles in the development of lung fibrosis in murine models. Data from patients with idiopathic pulmonary fibrosis and systemic sclerosis-associated lung fibrosis support this hypothesis. Our preliminary results suggest that, in contrast to a healthy lung, the population of alveolar macrophages during pulmonary fibrosis is heterogeneous. We suggest that proteins expressed by subpopulations of alveolar macrophages might serve as biomarkers to predict clinical outcomes or guide therapy for patients with lung fibrosis. In addition, some of these proteins or their downstream targets might serve as novel therapeutic targets.

To uncover the role of the pulmonary macrophages in the development and progression of pulmonary fibrosis, we will obtain lung tissue from healthy donor lung and from patients with idiopathic pulmonary fibrosis. We plan to do this through our ongoing collaboration with the transplant team at our hospital. We will then prepare a single-cell suspension of the lung tissue using our established protocol and then will process samples for multiparameter flow cytometric analysis and cell sorting. We will sort different populations of alveolar macrophages and isolate RNA from them to perform gene expression profiling. We will then compare gene expression profiles in macrophages from healthy subjects and from patients with idiopathic pulmonary fibrosis.

BD: What are the long- and short-term scientific goals of this project?

Alexander Misharin: This study has three major goals. First, to uncover alveolar macrophage heterogeneity in patients with idiopathic pulmonary fibrosis or in healthy individuals using high-throughput multiparameter flow cytometry. We expect that alveolar macrophages from patients with idiopathic pulmonary fibrosis will exhibit more heterogeneity than alveolar macrophages from controls and that some subpopulations of monocyte-derived macrophages will exhibit a profibrotic phenotype.

Next we will identify novel biomarkers expressed by profibrotic alveolar macrophages and begin to create molecular classification of idiopathic pulmonary fibrosis. To accomplish this, we will sort those individual subpopulations of alveolar macrophages and assess their gene expression profiles. Proteins, encoded by the genes that are differentially expressed between subpopulations of alveolar macrophages, will be incorporated into the diagnostic flow cytometric panel.

Finally we hope to develop a diagnostic flow cytometry panel based on molecular taxonomy of alveolar macrophages to predict clinical outcomes and guide therapy in patients with idiopathic pulmonary fibrosis.

BD: What are the implications of your project for human health?

Alexander Misharin: Successful realization of our goals will lead to revising our understanding of the mechanisms leading to the development of idiopathic pulmonary fibrosis, and allow better prediction of outcomes and development of a personalized approach to therapy. Moreover, these findings can be applied to the other forms of lung fibrosis.

BD: Which BD reagents do you plan to use, and for what purposes?

Alexander Misharin: We will utilize fluorochrome-conjugated antibodies to identify macrophages using multiparameter flow cytometry using a BD LSRFortessa™ cell analyzer, and will use the BD FACSAria™ III instrument to sort these cells.


Ellen Wehrens, PhD
Postdoctoral Fellow
University of California, San Diego

Abstract Title:
T-Helper Cells Take Center Stage Defining the Role of a Recently Discovered Cytotoxic CD4 T-Cell Subset in Chronic Infection


BD: What is your educational background?

Ellen Wehrens: After receiving my bachelor’s degree in biology from Utrecht University, I remained there for my graduate studies. My master’s and PhD degrees are both in immunology. For my doctoral work I studied factors interfering with immune regulation in juvenile arthritis patients. Having studied autoimmune diseases for five years, I decided to change both research subject and working environment. So I moved to the US to study T-cell regulation during chronic infection under the supervision of Dr. Elina Zuniga at the University of California, San Diego. My financial support comes from the Dutch Society for Scientific Research.

BD: How and when did you become interested in science?

Ellen Wehrens: As a child I was always curious about the natural world and about how things worked, but honestly I was not interested in science—at least not in conducting research myself. Even in college, where I studied biology, I was certain that I would never perform research or work in a laboratory. To convince myself of this and to be able to cross scientific research off my list, I felt that I had to try it at least once. That’s how I ended up doing an internship in an immunology lab studying mechanisms of food allergy. I loved it and decided to pursue science as a career.

BD: How did you become interested in your broad field of study?

Ellen Wehrens: I studied many fields of biology in college, including plant biology and animal behavior, but quickly realized that subjects directly relating to human health interested me most. Immunology has many implications for human diseases, including infectious disease, autoimmune disease, and cancer. What I love about immunology is the complexity and ingenuity of the immune system. All its workings are regulated at multiple levels; something beneficial in one setting, say activation of T cells to protect against invading pathogens, can be completely detrimental under different circumstances. An example of this is when these same activated T cells start to attack the body’s own tissues, resulting in autoimmune disease.

BD: Describe the project for which you received the BD grant.

Ellen Wehrens: My project deals with the fine line between protective immune responses and effects that are detrimental to the host. We are studying the role and function of a recently discovered cytotoxic CD4 T-cell subset in chronic infection. More specifically, we investigated whether these cells contribute to viral control by killing virus-infected cells. To study this phenomenon, we generated a genetic knockout model in which we can specifically delete the transcription factor responsible for cytotoxic function in CD4 T cells. As a model for chronic infection, we will use the natural mouse pathogen LCMV, which resembles human persistent viruses such as human immunodeficiency virus (HIV), hepatitis B virus, and hepatitis C virus (HCV). Although we are investigating the contribution of these cells to viral control and their therapeutic potential in enhancing antiviral immunity, we will also consider possible detrimental effects, like tissue damage, that these cells might have on the host.

BD: What are the long- and short-term scientific goals of this project?

Ellen Wehrens: Short term we hope to understand more completely the function of recently discovered cytotoxic CD4 T cells in chronic infection and whether they contribute to viral control. Persistent viruses such as HIV and HCV in humans induce a severely immune suppressive environment, which allows the viruses to persist in the infected host. Our long-term goals are to uncover how this inhibitory environment during infection influences cytotoxic CD4 T cells, and whether we can manipulate it to enhance their function and subsequent viral control.

BD: What are the implications of your project for human health?

Ellen Wehrens: By studying the function of cytotoxic CD4 T cells in chronic infection, we hope to identify methods and therapeutic targets for enhancing viral control. This approach could be beneficial for patients suffering from chronic viral infections such as HIV and HCV. Our results might extrapolate to cancer as well, since cytotoxic CD4 T cells have been shown to improve the survival of tumor-bearing mice by killing malignant cells. The same inhibitory environment during infection is also observed in the tumor microenvironment.

BD: Which BD reagents do you plan to use, and for what purposes?

Ellen Wehrens: Among others, we will use a multicolor panel of BD flow cytometry antibodies to identify cytotoxic CD4 T cells at different time points during infection. The same antibodies, together with BD magnetic cell separation kits, will be used to sort cytotoxic CD4 T cells. This will allow us to study their cytotoxic function in vitro, as well as their therapeutic potential in vivo, after performing adoptive transfers. We will also use BD recombinant cytokines or blocking antibodies to study the role of cytokines in regulating cytotoxic CD4 T cells during chronic infection.