Spring 2010 Research Grant Recipients Talk About Their Research

Angela Archambault, PhD
Senior Research Technician

Abstract Title:
The Role of Dendritic Cell Precursors in Experimental Autoimmune Encephalomyelitis

BD: What is your educational background?

Angela Archambault: I hold an undergraduate degree in biochemistry from the University of New Mexico, and a PhD in immunology from Washington University in St. Louis.

BD: How did you come to be a scientist, and become involved in this particular project?

Angela Archambault: I always knew I wanted to go into science. I was inspired to enter immunology by an undergraduate professor. In graduate school I did a rotation in a lab studying a mouse model of multiple sclerosis (MS), and I remained in that lab for my thesis. I believe that studying animal models of human disease provides opportunities to make a real, near-term impact.

BD: Tell us about your research.

Angela Archambault: Our lab studies experimental autoimmune encephalomyelitis (EAE), an inflammatory central nervous system disease of mice that serves as a model for multiple sclerosis. In EAE, CD4 T cells, which play a role in the autoimmune reactions characteristic of EAE and MS, become activated by dendritic cells, another immune system component. Dendritic cells accumulate in the brain and spinal cord during EAE, suggesting that they are actively recruited during CNS inflammation. We believe that the dendritic cells arise from precursor cells in the bone marrow.

The mechanism through which this occurs is complex, but our studies suggest that dendritic cells initiate the process by presenting antigens to the CD4 T cells. Animals that lack either CD4 T cells or the antigen-presenting components do not get sick.

BD: How is EAE induced?

Angela Archambault: By immunizing mice with a myelin protein, or by activating T cells ex vivo and introducing them into a mouse.

BD: How well does EAE mimic MS?

Angela Archambault: Although EAE is one of the best MS models we have, there is some debate about its relevance. While MS is often characterized by brain lesions, EAE affects mostly the lower spinal cord. Furthermore, various immune features in the mouse, such as cytokine involvement during disease, have not correlated well to human findings. EAE is actually a collection of several different animal models, some of which capture certain MS features quite well.

BD: How are you characterizing dendritic cells?

Angela Archambault: We stain the cells using BD antibodies, and characterize them with a BD flow cytometer. It’s a pretty complex stain, requiring seven colors. Some of the antibody stains, specific to pre-dendritic cell markers, are confirmatory for our target cells, while others are used to rule out cells we are not interested in.

BD: What are the near- and long-term goals of your work?

Angela Archambault: Short-term, we would like to understand the time course for the activation of dendritic cell proliferation and recruitment to the CNS. We are hoping to isolate these cells, transfer them to other animals, and see what roles they play in inducing EAE. Eventually, the goal is better treatments for multiple sclerosis.

Paul Ashwood, PhD
Assistant Professor

Abstract Title:
Innate Immunity and Autism

BD: What is your educational background?

Paul Ashwood: I received my undergraduate degree in pharmacology from University College, London, my master’s in pathology at Imperial College, London, and my PhD from King’s College, London in immunology.

BD: Why did you become a scientist?

Paul Ashwood: I was interested in science from an early age. Specifically, in middle school, I remember being fascinated by a book titled How Your Body Works.

BD: Tell us about your research project.

Paul Ashwood: Scientists have suspected for several years that immune system abnormalities may play a role in the development of autism or the exacerbation of associated behaviors. Among these abnormalities is activation of innate immune responses. Examination of post-mortem brain specimens has demonstrated active, ongoing inflammation in both young and adult individuals with autism. In particular, one notices microglia and astrocyte activation and increased levels of cytokines, that act as signaling molecules in both the nervous and immune systems.

Autism is difficult to study because no animal model for the disorder exists. We therefore decided to look for a system in the blood that might provide useful insight into what may be happening in the brain. It turns out that monocytes, which are thought to be precursors of brain macrophages and microglia, exist in abnormally high numbers in autistic patients. We are studying how various stimuli applied to monocytes affect the polarization of macrophages M1 and M2 subtypes. M1 macrophages are mediators of inflammation, while M2 cells attenuate the immune response and hence inflammation.

BD: What are the implications of your research?

Paul Ashwood: In the near term we hope to identify differences in innate immune function between autism and typically developing subjects. That will give us an idea of whether innate cells are implicated in the pathophysiology of the disorder and allow us to develop a cell-based model. Longer term, by identifying these cells and the stimuli that activate them, one could imagine a test for identifying children at risk, a tool for following the trajectory of the disorder, and/or monitoring the effectiveness of therapy.

BD: How will your reagent grant assist you in your work?

Paul Ashwood: The reagents will play into every aspect of this project, particularly with respect to characterizing monocyte populations by flow cytometry. Among the reagents we plan to use are BD GolgiPlug™ protein transport inhibitor, BD Cytofix/Cytoperm™ for fixing and permeabilizing cells, BD Pharmingen™ fluorescently conjugated antibodies, and BD OptEIA™ ELISA kits for confirming the presence of cytokines. We will also use flow cytometry and associated BD reagents to identify monocyte phenotypes.

Walter “Sunny” Dzik, MD
Co-Director of Blood Transfusion Service and Associate Professor

Abstract Title:
Flow Cytometric Measurement of P falciparum Erythrocyte Membrane Protein-1 (PfEMP-1)

BD: What is your educational background?

Sunny Dzik: I hold an AB degree from Princeton University and a medical degree from the University of Pennsylvania.

BD: What led you to enter medical research?

Sunny Dzik: My first academic interest was mathematics, but I ended up majoring in French and French literature. I went to medical school because I believed, and still do, that medicine was a mix of art and science. After my residency at Boston University I spent time at the National Institutes of Health, where I received more specialized training in blood diseases and transfusion. I became interested in malaria research through one of my trainees, who has since gone on to her own very promising career.

BD: Tell us about your research.

Sunny Dzik: When P. falciparum, the deadliest malaria parasite, invades red blood cells, it produces a protein, P. falciparum erythrocyte membrane protein-1 (PfEMP-1), which is expressed on the surface of the blood cell, appearing as bumps. These protein structures stick to proteins inside blood vessels, as well as to CD36, a platelet glycoprotein. Adhesion disrupts blood flow and, in serious cases, results in death. Our research involves detecting and quantifying the numbers of these bumps on red blood cells, and correlating their numbers to clinical severity.

We recently completed a study in which we used BD reagents to quantify CD36 in 2,000 malaria-infected Ugandan children. I was joined in this work by a group of dedicated researchers including Christine Cserti-Gazdewich of Canada and Isaac Ssewanyana, Aggrey Dhabangi, Charles Musoke, and Henry Ddungu of Uganda. Our data is currently undergoing statistical analysis. We believe that both PfEMP-1 and CD36 are necessary for adhesion of infected red cells to occur and to cause blood vessel blockage. Interestingly, about 10% of the African population carries no CD36 at all, suggesting a beneficial evolutionary adaptation.

BD: What would be the clinical significance of these findings?

Sunny Dzik: Knowing the correlation between PfEMP-1 and CD36 levels and illness should lead to tests for assessing risks when a child comes down with malaria. Our Uganda study measured CD36, ICAM-1, and other factors. Results should be available later this year.

If we can show that the interaction of PfEMP-1 and CD36 is an important aspect of malaria severity, then ultimately researchers could design drugs that will either inhibit formation of PfEMP-1 bumps, or prevent their binding to CD36. This will keep patients alive until the parasite infecting them is eradicated. This gives them a chance to develop immunity to further infection.

BD: What role do BD reagents and instrumentation play in your work?

Sunny Dzik: We used BD antibodies to CD36 in the Uganda project, plus fluorescently conjugated antibodies, a flow cytometer, lysing reagents, and consumables. We are very grateful to BD for the support the company has given to our work.

Julia Kirshner, PhD
Assistant Professor

Abstract Title:
Cancer Stem Cells in Myeloma

BD: What is your educational background?

Julia Kirshner: I hold a bachelor’s degree in genetics from the University of California, Davis, and a PhD from the City of Hope Irell and Manella Graduate School of Biological Sciences. I did post-docs at Lawrence Berkeley National Laboratory and the University of Alberta.

BD: How did you get into science?

Julia Kirshner: As a kid I thought about going into medicine, but after completing a summer job in a biology lab during high school I decided that research is really what I wanted to do. It was the first job I had where I did not mind coming in early or staying late.

BD: Tell us about your research.

Julia Kirshner: I’m working on multiple myeloma, an incurable white blood cell cancer. Chemotherapy works for a while by destroying rapidly reproducing cells, but the relapse rate of close to 100% suggests that the therapy misses cells that, after time, allow tumors to re-grow. Researchers believe that the culprits are cancer stem cells, which are drug resistant because they do not divide rapidly. Cancer stem cells are also difficult to isolate because they resemble other cells within the tumor.

Our lab identifies and isolates cancer stem cells through surface protein markers. We know cancer stem cells arise from a B-cell lineage. B cells, which secrete antibodies during infection, progress through several stages during their lifecycle. One of our aims is to determine from which stage cancer stem cells arise. The other is to isolate these cells, to see if they will produce tumors in test animals.

BD: What is the significance of your work?

Julia Kirshner: Immediate goals are a better understanding of which other cells cancer stem cells interact with, the pathways through which interactions occur, and the role of the microenvironment in cancer stem cell regulation. The ability to isolate and culture cancer stem cells will enable research into new treatments that prevent activation of cancer stem cells and subsequent regrowth of the malignancy. For example, a patient could take a combination of conventional chemotherapy to kill the main tumor, and a stem cell inhibitor to prevent recurrence. One could imagine down the line a diagnostic test based on cell surface markers, detecting cancer stem cells earlier, or monitoring the efficacy of drug treatments.

BD: How will BD reagents assist your research?

Julia Kirshner: We are planning to acquire a panel of antibodies to examine the different stages of B-cell differentiation through known surface markers. We also routinely use extracellular matrix proteins, including BD Matrigel™, a protein mixture that reproduces extracellular conditions. We don’t culture our cells directly on plastic because that environment does not represent the cancer stem cells’ biological niche very well. Instead, our cells grow within an extracellular matrix that resembles those found in the bone marrow of myeloma patients.

Georgia Perona-Wright, PhD
Postdoctoral Fellow

Abstract Title:
Cytokine Response Patterns in Influenza Infection

BD: Tell us about your educational background.

Perona-Wright: I earned my undergraduate degree in biology from the University of Cambridge and my PhD in immunology from the University of Edinburgh, both in the UK.

BD: How did you get into science?

Perona-Wright: I’ve wanted to be a scientist since age nine or ten. I was fascinated by medicine but decided that I wanted to make the remedies rather than administer them. I wrote to Sir Walter Bodmer, the famous Oxford geneticist, asking him about careers in science. Amazingly he wrote back, urging me to work hard and attend university.

BD: Tell us about your research.

Perona-Wright: Our ultimate goal is to improve the immune response generated by influenza vaccine. It’s known that virus-specific CD8+ memory T cells persist for months after the virus is cleared. These cells have a lower activation threshold than naïve T cells and respond differently to activation by cytokines. We hypothesize that this altered cytokine response defines the functions of these memory cells. We will test this by measuring how well memory cells respond to a range of different cytokines, examining which signaling cascades are initiated and how the cytokines affect the ability of the memory cells to proliferate, to kill virus-infected target cells, and to make cytokines of their own. We hope to understand enough about these signaling pathways to develop strategies useful in creating flu vaccines that work against every strain. The key will be to generate a cytokine response that induces not the strongest immune response, but the highest quality response.

BD: A universal flu vaccine would be a major breakthrough.

Perona-Wright: Yes. Currently, a new vaccine must be formulated every year based on estimated predictions of the predominant influenza strains. All seasonal vaccines today aim at generating antibody or T-cell responses to the virus surface proteins, which are constantly changing. At the Trudeau Institute we are investigating antibody responses toward core proteins which are common to all flu strains, and far less likely to change.

BD: Why do you stress quality of the immune response rather than magnitude?

Perona-Wright: It is possible to over-do immunity. You want T cells that kill enough infected cells to keep the infection at bay, but that leave healthy lung cells intact. It is thought that many people who died in the 1918 pandemic outbreak succumbed to an over-aggressive immune response.

BD: What BD reagents do you plan to acquire with your grant?

Perona-Wright: BD has recently introduced BD™ Phosflow reagents that enable the direct detection of intracellular signal transducers and activators of transcription, or STATs, in single cells. We will use these reagents, with flow cytometry, to identify and quantify cytokine responsiveness in naïve and memory CD8+ T cells from mice and humans previously infected with influenza virus. Previously analysis of this type was done via Western blot, and the ability of BD Phosflow to resolve individual cells is a significant advance.

Peter Walker, MD
Postdoctoral Fellow and General Surgery Resident

Abstract Title:
T Regulatory Cells as a Therapeutic Target for Progenitor Cells in Traumatic Brain Injury (TBI)

BD: What is your educational background?

Peter Walker: I received an undergraduate degree in mechanical engineering from the University of Missouri, Columbia, and my medical degree from the St. Louis University School of Medicine.

BD: Describe the transition from engineer to physician-researcher.

Peter Walker: Engineering is a valuable degree because it’s a hands-on field and opens a lot of doors. After doing two engineering internships during college, I decided to go into medicine. My general surgery residency program at the University of Texas is known for developing surgeon-scientists. The surgery part is quite demanding, therefore I am on a two year NIH sponsored postdoctoral research fellowship.

BD: Describe your research.

Peter Walker: Traumatic brain injury, TBI, is the leading cause of trauma death in children. Despite much effort there has been no real improvement in saving these patients. A few years ago stem cells came along as a potential treatment for TBI based on preliminary work with mesenchymal stem cells. Scientists originally thought that the stem cells were migrating to the brain, grafting, and differentiating into neurons. This mechanism has not been ruled out, however, since the degree of benefit observed is too great to be explained by the relatively small number of cells that actually undergo this process. We now believe that the benefit arises from a systemic or endocrine effect.

Research indicates that the immune system plays a role in neurological injury. After stroke the spleen, a large reservoir of T-regulatory cells (Tregs) increases in mass. These cells may provide protection by modulating anti-inflammatory responses. We hypothesize that adult stem cells alter levels of pro and/or anti-inflammatory cytokines through a T-regulatory cell mediated effect. Specifically, we believe that they increase the populations of neuroprotective brain microglia.

BD: What are the implications of your hypothesis?

Peter Walker: If our hypothesis is correct, and Tregs up-regulated in the spleen increase populations of microglia in the brain, this would explain the beneficial effects of stem cells in TBI. This result would be significant because after stem cell transplantation there are many, many more macrophages in the brain than there are stem cells.

BD: Is there a problem with immunogenicity of transplanted adult stem cells?

Peter Walker: No. Whether they are allografts or xenografts, these cells do not elicit immunogenicity.

BD: What are the implications of your work?

Peter Walker: If we can tease out a pathway, and show that the cells are safe, and that they do indeed alter the systemic inflammatory response to TBI, this could lead to treatments for many chronic or acute neurologic diseases. Macrophages are implicated in so many diseases that this could have widespread benefit.

BD: How will your BD reagent grant help?

Peter Walker: We plan on using BD antibodies to label and measure the populations of T regulatory cells based upon CD3/CD25/FOXP3 positivity with flow cytometry. Furthermore, we plan to use bead based BD™ Cytometric Bead Array Flex Sets to measure cytokine production from in vitro cultures of stem cells with naïve T cells.

Sheng Wu, PhD
Postdoctoral Researcher

Abstract Title:
Treg Cells in IL-2 Therapy

BD: Tell us about your educational background.

Sheng Wu: I received my undergraduate degree in biochemistry and molecular biology from Nanjing University in China. For my doctoral degree, also from Nanjing U, I studied cytokines against T-cell apoptosis and induction of T-cell mediated anti-tumor immunity. I’m currently a post-doc at M.D. Anderson.

BD: What factors influenced your decision to go into science?

Sheng Wu: When I was in high school my mother developed Parkinson’s syndrome. At the time the mechanism of that disease was unclear. We could alleviate her symptoms for a time, but there was and is no cure. Eventually my mother passed away from this disease. At that time I made up my mind to become a scientist.

BD: Please describe your research.

Sheng Wu: Interleukin-2 therapy has been used in renal cell carcinoma and melanoma for some time, but the response rate in late-stage melanoma is quite low. Only five percent of patients experience long-lasting remission, and ten percent more achieve a meaningful clinical response. We are looking for the difference between responders and non-responders.

BD: How do you plan to look for these differences?

Sheng Wu: We will systematically examine all the important immune system signaling pathways before and after patients receive IL-2 treatment. Classical methods like Western blot and ELISA are slow. More importantly, those techniques can only test the phosphorylation status of the bulk cell population, not individual cells. To test special cell subsets requires isolating them, but that changes the cells’ microenvironment in such a way that the results become unreliable. BD™ Phosflow flow cytometry reagents allow testing of individual cells without isolation, using monoclonal antibodies to more than a dozen signaling pathways. In these experiments the cells can be directly fixed in the microenvironments to replicate their natural niche, thus maintaining the original phosphorylation status. This method tests multiple cell subsets simultaneously, without the need to purify the cells. We are also using a BD FACSCanto™ II that reads eight colors, so we can test multiple signaling pathways simultaneously.

BD: What are the implications of your work?

Sheng Wu: The big question here is what are the differences between IL-2 responders and non-responders? Perhaps an event occurs early on in a critical pathway, where phosphorylation is turned on or off, and that affects all other events in that cascade. Once we learn the relevant events, it might be possible to develop a drug that will turn this critical step on or off. This could potentially turn more non-responders into responders, and perhaps bring about more long-lasting remissions.