BD BIOSCIENCES RESEARCH GRANTS
2009 Research Grant Recipients Talk About Their Research
Peter Antinozzi, PhD
A Novel High-Content Assay for Renal Cell Biology
BD: Why did you become a scientist?
Peter Antinozzi: At a young age I was always fascinated by how things worked. The continuous supply of unanswered questions in the life sciences satisfied my curiosity and fostered my current career path into cell biology research. What excites me now is discovering previously unrecognized gene functions and pathways, and the innovative technologies used to get there.
BD: Tell us about your research.
Peter Antinozzi: Our lab concentrates on mechanisms underlying diabetes and complications associated with the disease. The project for which I was awarded the BD grant focuses on one of the most serious consequences of the disease, diabetic nephropathy, and attempts to overcome hurdles imposed by limited availability of primary kidney tissue. The key challenge is obtaining the most information possible from a very limited number of cells. We analyze these cells through direct imaging using an automated confocal fluorescence microscope.
BD: What are the advantages of this approach?
Peter Antinozzi: The kidney consists of several distinct cell types which may be implicated in diabetic nephropathy. Mechanical isolation of specific cell types results in losses that are unacceptable given the small tissue sample size normally obtained through biopsy. Imaging allows us to visualize individual cells directly, assess their function, and examine differences among all cell types in a kidney biopsy. What we’re doing is a type of “digital cell isolation,” where the imaging system and reagents identify cells by their protein expressions.
BD: How will the reagents awarded to you in the grant help you carry out your work?
Peter Antinozzi: The grant includes fluorescent probes, antibodies to TNF, Ki-67, and other proteins, Argutus test kits, and 384-well imaging plates. We have been using a BD imaging system for the last few years.
BD: What are the implications of your research, to science in general and in the treatment of kidney disease?
Peter Antinozzi: This project holds potential implications for laboratory operations, biology, and medicine. We hope to develop a novel discovery platform for analyzing heterogeneous cell populations within the context of kidney disease. The key here, given the diversity of kidney cells, is the ability to analyze all cell types from the same sample simultaneously.
Our second goal is to determine how genetic and transcriptional changes influence cell function within this disease context. Finally, we hope this work will lead to in vitro drug screening assays, and perhaps as a diagnostic or prognostic test based on biomarkers we identify.
Mary Cloud Ammons, PhD
Immunity to Bacterial Biofilm
BD: How did you become interested in molecular biology?
Mary Cloud Ammons: My parents always encouraged me to explore the world around me. Molecular biology fascinated me because I am intrigued with the scales of things. Biology studies smaller and smaller systems, but opens up worlds that are in some ways infinite. I am fortunate that the field has progressed so much since I began my higher education.
BD: Can you tell us about your research?
Mary Cloud Ammons: Most people think of bacteria as free-living cells that live and act independently, but 90% of the bacteria in the world exist in complex communities known as biofilms. In this form bacteria take on new characteristics, almost like bees in a hive. Biofilms can be beneficial, for example when they’re employed in bioremediation, but they are often harmful to host organisms, particularly when the host is immunocompromised.
Our bodies are covered with bacteria that are mostly harmless. Under the right conditions these establish themselves as biofilm infections, which give rise to serious problems like non-healing sores. Antibiotics are generally ineffective against biofilms.
My work examines the function of the innate immune system in the presence of biofilms. This branch of immunity, consisting of macrophages and neutrophils, is considered the body’s first line of defense against infection. We are working with in vitro and ex vivo models with biofilms derived from chronic wounds.
BD: What does your research mean to science and to our understanding of infectious diseases caused by biofilms?
Mary Cloud Ammons: Very little work has been carried out on the role of innate immune cells in combating biofilms. We know that macrophages exist in pro- and anti-inflammatory phenotypes, but their role in causing the collateral damage associated with biofilm infections is largely a mystery. If macrophages can be manipulated pharmacologically to enter one phenotype or another, it may possible to stop or slow down disease progression. This would be an entirely new approach to treating persistent biofilm infections.
We plan to use assays that detect proteins expressed in macrophages in response to biofilms. We will also be looking for antimicrobial peptides and at the bacteria themselves to determine changes at the transcriptional level in response to immune system activity.
BD: How will your reagent grant assist you in your work?
Mary Cloud Ammons: Throughout this project we will be employing quantitative PCR, quantitative cell sorting, and imaging as our primary methods. Imaging techniques can be very helpful in determining how macrophages penetrate into biofilms, and fluorescence cell sorting can detect phenotypic changes that occur when the host cell and the bacterial biofilm come into contact. We also expect to screen for a large number of peptides and proteins involved in this process using the BD™ Cytometric Bead Arrays.
Melanie Dart, PhD
Atherosclerosis and Collagen V
BD: How did you decide to become a scientist?
Melanie Dart: I originally wanted to become a physician. To aid my chances of getting into medical school, I began doing research for a Master’s degree in microbiology at the University of Wisconsin. I really enjoyed doing research and decided to go for a PhD in cellular and molecular pathology.
BD: Tell us about your research.
Melanie Dart: This project originated from the observations that lung transplant patients experiencing chronic rejection had autoimmunity to collagen V, and that the pathologies involved were similar to those in atherosclerosis. Plaque formation and destabilization are two critical components of atherosclerosis. We are examining if autoimmunity to collagen V is implicated in progression of atherosclerosis and in atherosclerotic plaque destabilization. Previous research has shown that collagen V exists in higher-than-normal levels in plaque, and that humans and test animals with severe coronary artery disease show T-cell immune responses to collagen V.
We expect that in patients with stable plaque and low inflammation, the response to collagen V will involve regulatory T cells, while the T-effector response will dominate in individuals with high inflammation and unstable plaque. Regulatory T cells suppress the inflammatory immune response, while T-effector cells promote it.
BD: How will you test your hypothesis?
Melanie Dart: We will analyze blood and plaque samples from patients undergoing carotid endarterectomy, or surgical removal of plaque from the carotid artery. We hope to uncover T-cell responses, if any, in patients with varying degrees and types of atherosclerosis, and to characterize those responses according to the type of plaque.
BD: What are some potential implications of your work?
Melanie Dart: If our hypothesis holds, we will learn a great deal more about atherosclerosis at the molecular level. Autoimmunity to collagen V could help diagnose or screen for unstable plaque, and thereby identify individuals likely to have a heart attack or stroke. Understanding the underlying autoimmune response could lead to therapies that specifically target T-effector cells, which could perhaps reverse or stabilize the inflammation believed to cause serious cardiac events. Based on experience with lung transplantation, the molecular signal involved in T-effector cell recruitment is interleukin-17, which could become a therapeutic target for a new class of drugs.
BD: What types of analyses will you run, and how will the BD reagents fit in?
Melanie Dart: Mostly the trans-vivo delayed type hypersensitivity assay, which involves injecting mice with collagen V and peripheral blood cells from patients undergoing carotid artery surgery, and measuring the resulting swelling response. We will also use enzyme-linked immunosorbent assays, ELISpot, and flow cytometry to characterize the T-cell response, and to correlate those results with plaque pathology. The BD reagents include antibodies to cytokines and cell surface markers, and bead arrays.
Nilufer Esen, MD
Infiltrating Myeloid Cells
BD: What is your background, and how did you become a scientist?
Nilufer Esen: I’d always planned to be a researcher. I earned my medical degree in Turkey and I did my residency and post-doc simultaneously. After completing my thesis in physiology I began a series of teaching and research positions in Turkey, and beginning in 1999, in the United States.
BD: Tell us about your research.
Nilufer Esen: My interest is in sindbis virus, an alphavirus that causes neuronal damage in mice and serves as a model for viral encephalitis in humans. While the virus infects neurons directly, the damage leading to paralysis and death is caused by an inflammatory response in the mouse brain and spinal cord. I hypothesize that nerve damage is caused by an immune response arising from the infiltration of mononuclear cells from the blood into the central nervous system (CNS).
BD: What types of assays are involved in this work?
Nilufer Esen: The pathology of sindbis infection begins in the spinal cord, so this is the first place to look. I plan to characterize white blood cells that invade the spinal cords and brains of infected mice at various time points post-infection. This will involve separating monocytes/macrophages from the other immune cells, and analyzing for various cell surface markers using multicolor flow cytometry and specific BD antibodies. We will also examine infiltrating T-helper and T-cytotoxic cells as well as natural killer cells. If T-helper cells are found to infiltrate preferentially over other T-cell types, we will home in on specific T-helper subtypes as well using antibodies appropriate to their intracellular cytokines.
Knowing which cells are infiltrating is the first step in understanding serious CNS disorders. Depletion experiments, in which specific populations of infiltrating cells are eliminated sequentially, will help identify which cell or cells are responsible for nerve damage and disease pathogenesis. We will use flow cytometry to verify depletion of relevant cells in both peripheral blood and in CNS samples, and correlate these to clinical symptoms and survival.
BD: What is the downstream potential of this research?
Nilufer Esen: Most serious neurologic diseases lack adequate treatment. If our hypothesis holds and our results apply to humans, it may be possible to deplete specific white blood cell populations pharmacologically, and thereby treat and/or modulate the disease outcome. Strategies may include administering antibodies against one or more macrophage cell surface markers, or clodronate-liposome therapy. When macrophages engulf liposome-coated clodronate, which is toxic and released in the cell following digestion of liposomes by intracellular lysosomal enzymes, a subsequent self-destruction occurs through a mechanism known as apoptotic killing.
Jeffrey Gold, MD
Associate Professor of Medicine
Novel Role for IL-5 in Sepsis
BD: Tell us about your educational background and why you became a scientist.
Jeffrey Gold: I did a double undergraduate major in biochemistry and philosophy, attended medical school, and did a residency in internal medicine. Trying to understand human disease has always intrigued me. When you treat patients, you realize there is so much you don’t know. During my med school days I was in a lab that researched tuberculosis using cells from the lungs of patients. Correlating what we saw in these cells with what was happening with the patients was exciting. That’s what got me started in research.
BD: What are you working on now?
Jeffrey Gold: We focus on sepsis, a systemic response to infection that is the number-one killer in intensive care units, and the tenth-leading cause of death in the United States. Sepsis is an infection that begins in one part of the body but for some reason becomes systemic. We’re trying to understand the underlying immunologic responses.
One would expect that as the infection spreads throughout the body, the immune-system cells that fight infection would be maximally activated. Paradoxically, during sepsis they can’t discriminate between helpful and unhelpful responses, and they lose their ability to kill bacteria. For example neutrophils, the predominant infection-fighting white blood cell, are weakened in septic patients, and eosinophils disappear completely. We have found that mice engineered to over-produce eosinophils survive sepsis, so perhaps eosinophil activity is the key to treating or at least understanding sepsis.
BD: What are the implications for human patients?
Jeffrey Gold: You can’t give eosinophils to human patients but it might be possible to increase native eosinophil populations by administering interleukin-5 (IL-5), a growth factor that stimulates eosinophil production. Unfortunately this does not work in mice. But IL-5 does increase the number of neutrophils and macrophages, which is sufficient to rescue mice that are very sick with sepsis. Interestingly, the IL-5 receptors on neutrophils and macrophages are only present when the mice are septic.
We found a similar up-regulation of IL-5 in human neutrophils and macrophages in humans with sepsis. This suggests that IL-5 therapy can be targeted to a narrow window when the patients are most likely to benefit. Our plan is to begin a phase I/II study in humans to test whether IL-5 therapy can treat sepsis.
BD: How will the reagent grant help you conduct your research?
Jeffrey Gold: We will be using BD antibodies that enable the identification and characterization of specific immune system cells and related proteins in septic patients. Part of this work will involve the use of flow methods to detect ingested apoptotic cells, and to quantify the expression of IL-5 receptors on monocyte subtypes during disease.
Celine S. Lages, PhD
Postdoctoral Research Fellow
BD: Tell us about your educational background.
Celine S. Lages: I have an undergraduate degree in genetics and a PhD in the biology of aging. I’m currently studying why immunity diminishes with aging, and perhaps how to improve the immune response in the elderly.
BD: What are some implications of an attenuated immune response in the elderly?
Celine S. Lages: We’ve observed, both in test animals and humans, the reactivation of persistent infections, inability to combat new infections, and less effective control of cancer. Another relevant effect is impaired response to vaccination.
BD: How does impaired immunity arise in the elderly?
Celine S. Lages: Impaired immunity during aging is undoubtedly multifactorial. We are studying the role of regulatory T cells, or Treg, which are a subset of CD4+ T cells. Treg cells control the intensity of the immune response by preventing activation and function of effector T cells and antigen-presenting cells. Treg expresses FoxP3, a gene transcription regulator that induces immune tolerance. FoxP3 normally prevents the immune system from being over-active, but when it is over-expressed, immunity decreases. Treg cells increase in numbers as people age. Why this occurs is the central question here. We know they arise in the thymus and peripheral blood from the conversion of non-Treg T cells. Determining the relative contribution from each source is impossible using conventional phenotyping since the cells appear to be identical.
BD: So how will you study this conversion?
Celine S. Lages: We hypothesize that some characteristic of non-Treg in the elderly makes these cells more readily convert to Treg compared with young individuals. To study this we will examine the in vitro conversion of aged and young non-Treg cells from mice using the transforming growth factor TGF-β1. For this work we will use BD antibodies attached to magnetic nanoparticles to sort non-Treg cells from mice lacking the FoxP3 gene. We expect that conversion will occur more easily in aged as opposed to young cells.
Since conversion to Treg also appears to depend on interactions between T-cells and specific dendritic cells, we will also examine conversion of young and old cells in the presence of different dendritic cell subsets. For this part of the study we will employ magnetic sorting and flow cytometry using BD antibodies.
BD: What are the implications of your work on human health?
Celine S. Lages: One could envision modulating the conversion to Treg, and thereby slowing down immune suppression, by inhibiting one or more of the biological factors responsible. The downstream benefits would include more efficient vaccination and better control of age-related disease associated with impaired immunity.
Scott Rapoport, PhD
BD: Your career path was somewhat unusual. Tell us what led you to your current position.
Scott Rapoport: I was always a scientist by disposition. As a child I liked to break things open to look inside them. Science demands a certain mindset, constant questioning, not being satisfied with answers. My undergraduate degree is in mechanical engineering. I then received a master’s in bioengineering and a PhD in marine biology. Everything I studied contributed to my success as a scientist.
BD: What is your primary research interest at Tengion?
Scott Rapoport: Our core competency, regenerative medicine, involves the regrowth of organs and tissues using a combination of the patient’s own cells and a biocompatible material, which together is called a “construct.” Regeneration occurs when the body converts the construct to a functional tissue or organ.
BD: What biological factors are involved in the success or failure of this technology?
Scott Rapoport: Although conceptually simple, the process of regenerating tissue is extremely complex. Low-abundance immune system cells known as macrophages have a profound impact on whether the graft is accepted or fails. Macrophages exhibit different activation states which either enhance the immune response, potentially leading to rejection, or subdue it. My work examines how constructs influence macrophages’ activation states, and hence the likelihood that the graft will be accepted.
BD: How does your research reagent grant fit into your experimental work?
Scott Rapoport: I will use BD’s microarrays to compare the activation states of “naïve” macrophages with those that have come into contact with the construct. We hope to learn at what point activation begins, and the contribution of the construct towards activation. For this part of the project we will use the BD OptEIA™ ELISA kit. I have previously used competitors’ kits so I am interested in seeing how the BD reagents compare. Each of the 96 wells in this kit contains an antibody pair that reacts with secreted proteins from macrophages.
BD: What could be some implications of your work?
Scott Rapoport: Regenerative medicine is a fairly new field, distinct from tissue engineering. We’re hoping that the tissue-plus-scaffold approach will stimulate the body’s regenerative process and help millions of people who suffer from organ failure, damage from degenerative diseases, and accidents. Understanding immune system responses to implants is a first step in learning which constructs work and which do not. At some point this knowledge may also lead to the discovery of rationally designed materials that modify the macrophage response, and thereby open up the field of regenerative medicine to a new generation of patients.