BD BIOSCIENCES RESEARCH GRANTS

Fall 2011 Research Grant Recipients Talk About Their Research


Stephan Borte
Post-Graduate Student

Abstract Title:
Functional Immunophenotyping for Primary Immunodeficiencies


BD: Tell us about your educational background and training.

Stephan Borte: I attended Humboldt Gymnasium in Leipzig, Germany, followed by the University of Leipzig, Germany, where I completed my medical studies with a thesis. I was a German National Academic Foundation Scholar from 2003 to 2009, and then undertook further medical training in clinical immunology and transfusion medicine at Leipzig. I am currently in the MD/PhD program at the Karolinska Institute.

BD: What are your broad research interests, and what drew you to them?

Stephan Borte: My research interests arose from the clinical orientation of the Department of Clinical Immunology at Leipzig, where I undertook four years of research during my medical studies. An MD degree requires a written thesis in Germany. The close interaction between diagnostics, research, and clinical practice prompted me to study primary immunodeficiency further, and to try to improve the scope of diagnostic and therapeutic approaches.

My broad research interests include three areas: the immune system in health and disease, particularly in pediatric patients; neuroimmunologically-derived characteristics in multiple sclerosis and the immunomodulatory potential of IFN-beta therapies; and early recognition of primary immunodeficiencies, including severe forms characterized by the absence of T or B lymphocytes (SCID and XLA), by means of neonatal screening tests.

BD: Describe the research project for which you won the BD grant.

Stephan Borte: Defects of the innate and specific immune response are commonly referred to as primary immunodeficiency diseases, or PIDs. Since a broad variation of genotypes and clinical phenotypes resembles PIDs, there is high demand for diagnostic tools to identify and classify patients. Since most PIDs affect the lymphoid lineage, flow cytometric analysis of peripheral blood samples conveniently provides diagnostic information. My project involves both the strategy of a polychromatic flow cytometric pattern for phenotyping cell populations, and tests of lymphocyte function to interpret their clinical relevance. The project will coordinate with neonatal screening for lymphoid PIDs in Sweden.

BD: What are the project's scientific goals?

Stephan Borte: To improve validated protocols for broad-based early identification and characterization of PIDs, to unravel novel defects and disease mechanisms that account for the clinical phenotype of PIDs, and to develop and evaluate novel technical platforms for earlier diagnosis, for example neonatal screening, and treatment.

BD: What are the implications for human health?

Stephan Borte: This project seeks to broaden the view on immunophenotyping in PID patients in the specific setting of the tracking process of neonatal screening, to allow earlier and personalized treatment to reduce the morbidity and mortality related to these diseases.

BD: Which BD reagents do you expect to use, and how will you use them?

Stephan Borte: Immunophenotyping by flow cytometry relies on the use of well characterized fluorochrome-labeled antibodies from BD. These will be combined in 8-color panels to be run on the BD FACSCanto™ II and BD™ LSR II instruments. Moreover, these antibodies will also be used together with optimized buffer systems from BD for intracellular protein and phosphorylation status analysis. In addition, calcium mobility, cytokine release in vitro, and phosphorylation of cellular intermediates will be analyzed using BD kits or reagents for functional studies in PID samples.


Brad Hoffman, PhD
Assistant Professor

Abstract Title:
Unraveling the Tissue Specific Antigen Presentation That Results in the Systemic Induction of Tolerance during Hepatic Gene Therapy


BD: Tell us about your educational background and training.

Brad Hoffman: I received my BS degree in chemistry and biology from the University of Central Florida, and my PhD in immunology at Temple University School of Medicine, where I studied the immune response in a mouse model of multiple sclerosis. I then moved to the University of Florida as a postdoctoral fellow, where I studied organ-specific immunology during viral gene therapy.

BD: What are your broad research interests, and what drew you to them?

Brad Hoffman: I chose immunology because of its direct connection to and impact on clinical treatments. My research interests involve exploiting the unique molecular and cellular mechanisms of liver-directed viral gene therapy to modulate immunological tolerance to specific antigens as a preventative or therapeutic intervention in autoimmune and neurological diseases.

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

Brad Hoffman: The naturally tolerogenic nature of the liver makes it an ideal location for targeted gene transfer. Under the right conditions, antigens expressed by hepatocytes are more likely to be tolerogenic than those expressed in the periphery. However, the specific details that determine the balance between intrahepatic immunity and tolerance are poorly understood.

Within the liver there are several types of cells, such as Kupffer cells, dendritic cells, and liver sinusoidal epithelial cells, which are capable of presenting antigen and initiating an immune response. We hypothesize that there is a competition between these intrahepatic antigen presenting cells that determines or directs the induction of tolerance.

This project seeks to characterize the specific contribution that these cells have in antigen presentation and the subsequent immune response following liver directed viral gene therapy. We will first assess the hepatic microenvironment and phenotypically characterize the various cells involved, identify tissues where antigen presentation and early proliferation of T cells occur, and track the cells' migration following intrahepatic gene transfer.

BD: What are the project's scientific goals?

Brad Hoffman: The objective is to improve the understanding of how to induce or prevent unresponsiveness to a transgene product, which is a key requirement for successful gene therapy. Long term, I will continue to investigate the complex immune regulatory mechanisms that lead to the induction of tolerance, and apply that knowledge to developing treatment strategies for autoimmune diseases.

BD: What are the implications for human health?

Brad Hoffman: Immune responses to therapeutic gene products are a major concern in gene therapy, so maintaining immunological unresponsiveness to the intrahepatically expressed protein is a key requirement. Better understanding of the underlying immune interactions will help us develop more efficient therapeutic protocols.

BD: Which BD reagents do you expect to use, and how will you use them?

Brad Hoffman: We will rely heavily on BD's fluorescently conjugated monoclonal antibodies. Using BD IMag™ cell enrichment kits and BD™ Cytometric Bead Arrays, we will perform phenotypic and functional analyses of isolated cells using specific panels of BD antibodies and cytometry reagents by multiparametric flow cytometry using a BD LSR II flow cytometer with BD FACSDiva™ software. BD ELISA reagents will be used to determine transgene expression levels and profile the microenvironment of the liver in mice following gene transfer.


Makio Iwashima, PhD
Associate Professor

Abstract Title:
Neonatal Immunity and CD36hi Monocytes


BD: Tell us about your educational background and training.

Makio Iwashima: I attended the University of Tokyo, where I received bachelor's and master's degrees in immunology and maternal and child health. I came to the United States as a Fulbright Scholar and earned my PhD in immunology at Stanford University. I completed my training through a postdoctoral fellowship at the University of California, San Francisco, where I studied signal transduction.

BD: What are your broad research interests, and what drew you to them?

Makio Iwashima: Chemistry was my original academic interest—my father is a chemist. But while in college I took a biology course in which one of the topics was the H-Y antigen, a surface antigen found only on cells from males. H-Y was originally thought to determine differentiation between male and female, but now we know it plays a different role. This is what interested me in developmental biology and immunology.

Broadly speaking, I'm interested in how the immune system discriminates foreign objects from "self," what is known as immunological self-tolerance. Everyone knows about transplant rejection, but this process does not occur in all situations. During pregnancy the mother tolerates the fetus and vice versa. Similarly, in some species such as cows, pseudo-twins, which have different fathers, do not reject each other, although one would expect them to. We are trying to figure out why this occurs.

BD: Describe the research project for which you won the BD grant.

Makio Iwashima: Most researchers are studying fetal tolerance from the perspective of the mother's immune system. Our approach looks at the fetus side, using cells harvested from umbilical cord blood. We found that monocytes in fetal blood that carry the CD14 antigen are immunoregulatory or immunosuppressant, meaning they help attenuate immune responses rather than provoking them. We hope to identify these cells in fetal blood and understand their function.

A striking difference between cord blood and adult blood is the ratio of monocytes to lymphocytes. Where most mononuclear cells in adult peripheral blood are CD3+ lymphocytes, most mononuclear cells in cord blood are CD14+ monocytes, which in culture induce powerful immunosuppressive responses in naïve T cells. We hypothesize that a subset of CD14+ monocytes are signaling the differentiation of naïve T cells into immunosuppressive regulatory T cells. Our project will attempt to identify and characterize the mechanism by which this occurs.

BD: What are the project's scientific goals?

Makio Iwashima: Specifically we aim to unravel the molecular mechanism of membrane localization of a key regulatory complex in CD14hi monocytes, determine if CD14 helps to induce the development of immunosuppressive T cells, and understand the TGF-beta-induced signaling process following TCR stimulation.

BD: What are the implications for human health?

Makio Iwashima: The translational medical aspects of this project are intriguing. We hope our work will lead to the development of treatments, particularly small-molecule drugs that will duplicate the immunosuppressive molecular mechanisms we are studying. Such drugs could be used to control autoimmune diseases and perhaps to combat organ transplant rejection.

BD: Which BD reagents do you expect to use, and how will you use them?

Makio Iwashima: We will use antibodies against human surface antigens such as GARP, CD14, and LAP. We plan to use BD's Cytometric Bead Array to quantify cytokines, and the BD FACSAria™ system for flow cytometry. We will also require tissue culture reagents and disposables.


Kang Liu, PhD
Assistant Professor

Abstract Title:
In vivo Detection of Duration of Antigen Presentation by Human Dendritic Cells Using Flow Cytometry in Humanized Mice


BD: Tell us about your educational background and training.

Kang Liu: I received my undergraduate degree in biochemistry from Wuhan University, in China, followed by a master's degree in biology from Fordham University and a PhD in immunology from Rockefeller University. I stayed at Rockefeller for my postdoctoral fellowship in immunology.

BD: What are your broad research interests, and what drew you to them?

Kang Liu: Dendritic cells interest me because they are the most potent stimulators of the immune system. They are the best cells to exploit in vaccine design, or for controlling immune reactions that "tolerize" self-antigens in the case of autoimmune diseases. In contrast to B and T cells, which are very well characterized, much is still unknown about dendritic cells and their stages of development. Acquiring knowledge of every step in the development of dendritic cells could enable us to manipulate them early, before they become final dendritic cells, and thereby direct immune responses more effectively.

BD: Describe the research project for which you won the BD grant.

Kang Liu: We are using a mouse model that recapitulates human dendritic cell development in vivo to detect the proliferation and lifetime of human dendritic cells in vivo. This type of experiment cannot be conducted in humans, nor is it possible in vitro because dendritic precursor cells do not behave the same way in vitro as in vivo.

Our experiment involves several steps in sequence. First we inject a donor mouse with bromo-deoxyuridine (BrdU), a nucleotide analog that acts as a chemical label and is taken up only by dividing cells. This particular mouse has a human-like immune system and accepts human dendritic cell precursors. These cells reside in the bone marrow and spleen, and give rise to human dendritic cells in the peripheral blood. We then harvest dendritic cells from the blood, bone marrow, and spleen and measure their uptake of BrdU. To determine the decay, or lifetime, of dendritic cells we join the circulatory systems of the human-like donor mouse with an immunodeficient recipient mouse that does not have a functioning immune system, and therefore no dendritic cells of its own. After the populations of dendritic cells in the two animals balance, we disconnect them and measure the disappearance of dendritic cells in the recipient. This protocol is known as a "pulse-chase" experiment.

BD: What are the project's scientific goals?

Kang Liu: We hope this work will result in techniques that allow measurement of the proliferation and lifetimes of human dendritic cells, in vivo, in our mouse model. One could then test various adjuvants used in vaccine formulations to see which ones affect the proliferation and turnover of dendritic cells–the two parameters that directly determine the outcome of immunity. Ultimately, one might arrive at a set of conditions that optimize dendritic cell-based immune stimulation and apply that knowledge to developing human vaccines.

BD: What are the implications for human health?

Kang Liu: Improving the efficacy of vaccines, and possibly designing new vaccines based on manipulation of dendritic cell proliferation and turnover.

BD: Which BD reagents do you expect to use, and how will you use them?

Kang Liu: We will draw on BD's antibodies conjugated with BD fluorochromes, plus disposables and reagents for analysis with the BD™ LSR II flow cytometer.


Matthias Marti, PhD
Assistant Professor

Abstract Title:
Role of RBC Derived Microparticles in Human Malaria


BD: Tell us about your educational background and training.

Matthias Marti: I received my diploma, equivalent to a masters of science degree, at the Swiss Tropical Institute in Basel, where I studied in vitro drug assays for the intestinal parasite Giardia lamblia. I trained at the University of Texas, El Paso for one and a half years under a Roche Research Fellowship, then completed my PhD at the University of Zurich, where I worked on mechanisms of protein secretion in Giardia. For my postdoctoral studies I studied protein secretion in the human malaria parasite Plasmodium falciparum at the Walter and Eliza Hall Institute in Melbourne, Australia.

BD: What are your broad research interests, and what drew you to them?

Matthias Marti: I am generally interested in infectious diseases, and malaria is one that has a major impact on human health worldwide. Working on fundamental and translational aspects of human malaria may contribute to reducing malaria's health burden. The biology of malaria parasites in mosquitoes and human hosts is poorly understood and may serve as a model for other infectious organisms.

Our laboratory is interested in malaria virulence and transmission. Specifically, we are interested in the function of parasite antigens at the host-parasite interface, and their contribution to virulence during infection. We also investigate the biology of malaria transmission stages, both in vitro and during human infection. Another interest is identifying how to block transmission as part of a global strategy to reduce and eventually eradicate malaria. In that context we are particularly interested in the identification of transmission-blocking drugs.

BD: Describe the research project for which you won the BD grant.

Matthias Marti: Malaria parasites develop in red blood cells, which both normally and during malaria infection release small vesicles known as microparticles measuring 200–800 nanometers across. We aim to characterize red blood cell-derived microparticles and study their roles in malaria infection. We know that microparticles (MPs) derived from infected red blood cells contain parasite antigens and probably other parasite-derived materials.

Microparticles therefore represent a significant pool of parasite material in the blood. We hypothesize that they modulate the host's immune response, and our preliminary data support this hypothesis. We will further test this hypothesis by a series of in vitro and in vivo assays.

BD: What are the project's scientific goals?

Matthias Marti: Our immediate goal is to characterize infected red blood cell-derived MPs. In the longer term I hope that this project will help us further understand the interaction of the parasite with the host immune system.

BD: What are the implications for human health?

Matthias Marti: Identification of parasite molecules in red blood cell-derived particles is of great relevance. First, the particles could potentially activate the innate immune system during infection, making them promising candidates for vaccine adjuvants. Second, the presence of a mixture of parasite antigens, some of them known vaccine candidates, suggests MPs may represent a valid alternative vaccination strategy. Third, their presence in the plasma of malaria patients may be exploited for diagnostic purposes in prediction of disease outcome and transmission and to monitor treatment.

BD: Which BD reagents do you expect to use, and how will you use them?

Matthias Marti: We will mainly use antibodies to define the composition of microparticles in vitro and in vivo. Specific BD reagents include multiple fluorochrome-conjugated antibodies to characterize maturation and activation of MF, DC, T cells, and endothelial cells including CD14, CD45, CD3, CD25, CD19, cell culture reagents including TNF, IL-4, TGF-β, and GM-CSF, viability dyes, cell enrichments kits, and cell fixation and permeabilization kits.


Sallie Permar, PhD
Assistant Professor

Abstract Title:
Isolation of HIV Envelope-Specific Mucosal B Cells from Colostrum of HIV-Infected, Lactating Women


BD: Tell us about your educational background and training.

Sallie Permar: I received my undergraduate degree in biology from Davidson College, followed by a year under a Fulbright fellowship studying viral immunology in Zambia. I received my PhD in microbiology and immunology from the Johns Hopkins School of Public Health. I then entered Harvard Medical School and completed my MD and PhD concurrently. I completed training in pediatrics and pediatric infectious diseases at Children's Hospital Boston and performed initial studies of HIV and SIV immune responses in human breast milk at Beth Israel Deaconess Hospital. I recently accepted a junior faculty position in the Department of Pediatrics and the Human Vaccine Institute at Duke University Medical Center.

BD: What are your broad research interests, and what drew you to them?

Sallie Permar: My interest in immunology and prevention of childhood infections goes back to my experiences in sub-Saharan Africa. One cannot help but notice the huge disparity in healthcare infrastructures between wealthy countries and developing nations where HIV has reached epidemic proportions. I was drawn to working on ways to prevent infections, especially in neonates.

My general research interest is determining what maternal or infant immune responses are needed to prevent mother-to-child transmission of perinatal viral pathogens. Knowing the immune correlates of protection against mother-to-child transmission of viral pathogens illuminates the design of vaccines to protect infants against pathogenic viral infections.

BD: Describe the research project for which you won the BD grant.

Sallie Permar: We will study HIV-specific antibody responses produced by B cells found in colostrum of lactating, HIV-infected women. This is important because breast feeding is the most common mode of mother-to-neonate HIV transmission. Most infants of these women breast feed for up to two years and are exposed to the virus multiple times daily, yet only ten percent of these infants become infected. This suggests that some HIV-specific immune response in breast milk is protective against HIV transmission. If this is true, then enhancing that immune response might protect the ten percent of infants who eventually become infected.

The HIV vaccine trial in Thailand in 2010 suggested that virus-specific antibody responses could protect against mucosal HIV transmission. While mucosal B cells are normally difficult to isolate, breast milk is a rich source of these cells and related antibodies. We know that B cells and antibodies in milk contribute to passive protection of infants from neonatal pathogens, including respiratory and diarrheal infections and, perhaps, HIV. So isolation and characterization of HIV Envelope (Env)-specific monoclonal antibodies in milk of HIV-infected women should elucidate the infection-blocking capabilities of Env-specific antibodies at the mucosal surface.

We propose to isolate HIV Env-specific B cells through fluorescently tagged antigen labeling, colostrum B-cell staining, and flow cytometric cell sorting. The antibodies produced by the isolated HIV Env-specific B cells will be recombinantly produced and used to study the specificity and function of HIV Env-specific antibodies in breast milk.

BD: What are the project's scientific goals?

Sallie Permar: In the short term we will isolate and characterize HIV envelope-specific memory B cells and the monoclonal antibodies they produce from colostrum. We will then amplify antigen-specific regions on antibodies produced by each sorted B cell, and eventually clone the reactive variable regions onto an IgG backbone to produce enough antibodies for further study. We hope that this work may eventually illuminate immunologic strategies for preventing HIV transmission from breast milk, or for blocking the virus from infecting the infant.

BD: What are the implications for human health?

Sallie Permar: An effective HIV vaccine could prevent a large number of HIV infections in developing countries where a third to one half of pediatric infections arise from breast feeding. A maternal vaccine may be more effective than a neonatal vaccine for several reasons: vaccinating the mother might only need to be done once, it avoids problems with infants' immature immune systems, and eliminates some vaccine safety issues in young infants. The model for effective maternal vaccination has been validated with tetanus and influenza vaccination during pregnancy, both of which provide robust protection for the infant.

BD: Which BD reagents do you expect to use, and how will you use them?

Sallie Permar: We will use multiple fluorescently labeled monoclonal antibodies and streptavidin tags, B-cell enrichment kits, cell culture media, and consumables. BD fluorescently labeled monoclonal antibodies have been paramount to my work evaluating both human and nonhuman primate HIV/SIV-specific maternal immune responses.


Yogesh Singh, PhD
Post-Doctoral Fellow

Abstract Title:
Role of microRNAs (miRNA) in Development and Functions of Differentiation of T Helper Cells and Induced Regulatory T Cells


BD: Tell us about your educational background and training.

Yogesh Singh: I received my undergraduate degree in zoology, botany, and chemistry at Dr. B. R. Ambedkar University, India, before moving to Banaras Hindu University, where I received a postgraduate degree in biotechnology. I received my PhD in immunology from Imperial College London and Royal Veterinary College London. During my first postdoctoral fellowship, at the International Center for Genetic Engineering and Biotechnology in New Delhi, I investigated microRNA in Mycobacterium tuberculosis infections. I am currently a postdoctoral researcher at the Royal Veterinary College, London, where I study the effects of microRNA on regulatory T cells and T helper cells.

BD: What are your broad research interests, and what drew you to them?

Yogesh Singh: My understanding of how microRNAs (miRNAs) control survival of Mycobacterium tuberculosis-infected dendritic cells piqued my interest in miRNA, dendritic cells, and T-cell biology cross-talk. MicroRNA affects T-cell development and differentiation, especially regulatory T cells, and plays a role in cancer, autoimmunity, and infections. Previous work found that DICER, an RNase polymerase that processes miRNA, is essential for regulatory T-cell development in the thymus and peripheral organs. This led me to explore how individual miRNAs affect the T helper subsets and induce development of regulatory T cells (Tregs).

BD: Describe the research project for which you won the BD grant.

Yogesh Singh: MicroRNAs are an abundant class of small, highly conserved non-coding RNAs. They suppress gene expression by binding to the 3′ untranslational region (UTR) of target messenger RNAs, and are known to regulate adaptive and innate immunity. We have hypothesized that controlled miRNA expression in CD4+ T cells differentiates these cells into effector T helper cells and suppressive induced regulatory T cells by changing transcriptional regulation. To test this idea, we have produced ten miRNA vectors to explore their effects on differentiation and function of T helper cells such as Th1, Th2, Th17, and iTregs. We expect that some of these miRNAs, when over-expressed or suppressed, should convert CD4+ T cells into T helper effector cells or iTregs.

To explore the effects of miRNAs on over-expression or inhibition, we will characterize various cytokines, chemokines, and transcription factors by flow cytometry. We will also investigate whether miRNAs target the gene relevant to cytokines (IFN-γ, IL-4, IL-17, and TGF-β) and transcriptional activation and repression genes of T helper subsets or Tregs.

BD: What are the project's scientific goals?

Yogesh Singh: Our goals are to identify novel miRNAs involved in the development of various T helper cell subsets and iTregs. Once we identify them, they will be over-expressed or inhibited in CD4 T helper cells for identification of different surface markers, transcription factors, and cytokine gene expressions that are essential for T helper cell subset commitment. Finally, individual miRNAs will be over-expressed or inhibited in bone-marrow cells and used to characterize specific T-cell subtypes. These goals will enable us to decipher the role of miRNAs in the development of T helper and regulatory T cells.

BD: What are the implications for human health?

Yogesh Singh: We are just beginning to understand the role of microRNAs in human immune system. Individual miRNAs recognize 6-8-nucleotide sequences found in hundreds of messenger RNAs. Therefore, a miRNA has the potential to alter the global protein profile of any cell type. Furthermore, since miRNAs interact directly with messenger RNAs, not with DNA, their action in raising or lowering levels of disease-mediating proteins is rapid. This makes small miRNAs, molecules that behave similarly, or miRNA inhibitors prime drug candidates for treating cancer, autoimmune diseases, and serious infections.

BD: Which BD reagents do you expect to use, and how will you use them?

Yogesh Singh: We will use cytokines (BD™ Cytometric Bead Array kit), chemokines (BD antibodies) and transcription factors (BD antibodies) to identify the role of different miRNA expression/decoy vectors in T helper cell development. We will characterize various T-cell surface markers, intracellular transcription factors, chemokines and cytokines with multicolor flow cytometry (BD FACSCanto™ II) techniques using BD Pharmingen™ antibodies and BD Cytometric Bead Array kits.