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Research Interests: Ubiquitous nucleic-acid binding proteins in cell nuclei are major targets of the autoimmune response that is mounted in systemic autoimmune diseases such as lupus. However, little is known regarding the mechanisms that govern tolerance to these types of antigens, particularly with respect to the T-cell compartment. The primary focus of my laboratory is to use mouse models to delineate mechanisms of immune tolerance against autoantigens targeted in systemic autoimmune diseases and to understand how these pathways are breached in individuals who are susceptible to disease. Using mice transgenic (Tg) for the human La/SS-B gene, which encodes an RNA-binding nuclear protein that is commonly targeted in lupus and Sjögren’s syndrome, we showed that T helper cells are tolerant to endogenous levels of La. To understand the cellular and molecular mechanisms of this tolerance, we have begun experiments using a transgenic mouse line that we created that has a T-cell receptor (3B5.8) specific for an I-Ek-restricted immunodominant T-cell epitope of human La. The epitope targeted by the 3B5.8 receptor (human La 68-76) differs from the endogenous mouse La sequence by a single amino acid residue that is required for reactivity of the parent hybridoma and is predicted to contact the TCR. The 3B5.8 TCR Tg mice were produced on the C57BL/6 background in the OMRF Transgenic Core Facility and are being maintained by crossing to C57BL/6 mice that are congenic for H-2k. The figure depicts essentially complete allelic exclusion at the beta locus that is characteristic of 3B5.8 TCR transgenic mice and skewed development of T cells into the CD4+ compartment. Preliminary studies reveal T-cell tolerance in double transgenic mice expressing both the 3B5.8 TCR and the hLa transgene. These mice will be a valuable resource for delineating how lupus susceptibility genes mediate loss of T-cell tolerance to ribonucleoproteins. Constructing a comprehensive model of immune tolerance to the La/SS-B antigen will expedite identification of molecular pathways that perturb immune tolerance in SLE. |
Michael Centola Ph.D. E-mail: Mike-Centola@omrf.ouhsc.edu
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Research Interests Using genomics- and proteomics-based biomedical discovery technology and bioinformatics, our lab helps to define the molecular mechanisms mediating human inflammatory diseases including rheumatoid arthritis, juvenile rheumatoid arthritis, spondyloarthropathies, and inflammatory bowel disease. Patients’ samples are collected in ongoing clinical research programs in collaboration with local physicians and clinical researchers. Gene and protein-based “biomarkers” of disease activity are then identified in broad-based screening assays and the massive data sets integrated with clinical data. Our studies have led to a refined understanding of the etiopathology of these complex disorders by defining novel disease mediators and the mechanisms in which they participate. A principal focus of our laboratory is to utilize this discovery pipeline to create the next generation of diagnostic and therapeutic response tests that will enhance treatment through broad-based monitoring of disease regulators. These tests will provide information on a patient-specific basis helping to usher in the age of personalized medicine, wherein the diagnosis and treatment of a given patient is performed with agents and procedures tailored to that patient’s genetics, physiology, and pathology. A section of the lab develops novel bioinformatics methodologies necessary to present these complex data in a manner that can be used practically by clinicians. Another section of the lab develops the robotic and database technologies necessary to perform these tests. |
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J. Donald Capra, M.D.
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Research Interests: Our laboratory employs an arsenal of innovative molecular and cellular techniques to study fundamental B cell biology, differentiation and mechanisms of tolerance. Our interests primarily focus on factors that influence antibody repertoire establishment and B cell differentiation, using all known maturational subpopulations that exist in the peripheral and tonsillar compartments. While our major efforts are directed toward human B cell biology, we routinely employ murine models to broaden our understanding of proper B cell development, exemplified in our ability to reproduce human B cell development in NOD/SCID/b2m-/- mice. Of major interest, we are currently investigating the association between autoimmunity and lymphomagenesis as they pertain to tolerance and the apparent breakdown thereof. We are addressing regulatory and developmental issues such as the induction of apoptosis in autoreactive B cells and the potential mechanisms responsible for their avoidance and/or escape from an anergic-like state. Most of the experiments in our laboratory begin with sophisticated cell sorting techniques. We use both magnetic and fluorescent activated cell sorting on the MOFLO to separate human B cells into an increasingly complex number of “stages” for further in vitro analysis. Other techniques in use include confocal imagery, immunohistochemistry, numerous PCR applications, DNA sequencing (especially Ig transcripts) and protein expression in multiple systems. The results of our studies are bridging the gap between fundamental academic research and the development of therapeutic agents that will ultimately control, or eradicate, a variety of immunological disorders and aid in the development and delivery of mucosal vaccines. Joined OMRF Scientific Staff in 1997. |
Mark Coggeshall, M.D. |
Research Interests We also study how B lymphocytes are activated by their antigen receptors. B lymphocytes respond to pathogenic proteins through surface immunoglobulin (sIg). Our work in signal transduction by sIg has shown recruitment of several proteins to the sIg receptor when it is stimulated by antigens. The enzyme phosphatidylinositol 3-kinase (PI3K) is central to protein recruitment since inhibiting PI3K blocks all aspects of B-cell activation. We use mice deficient in the enzymes that regulate PI3K to study the role of PI3K in B cells. Our current work focuses on how sIg-recruited proteins contribute to antigen internalization and B lymphocyte activation, with emphasis on the key role of PI3K. The third project involves signal transduction by a receptor tyrosine kinase (c-fms) that triggers macrophage migration. We are studying how PI3K regulates small GTPases Rho, Rac and CDC42. Primary macrophages from the bone marrow of mice that have altered metabolism of PI3K products show elevated chemotactic responses. We have traced the elevation to constitutively-active Rac and Vav in these same macrophages. By retroviral transduction and fluorescence microscopy, we determined how Vav is regulated by PI3K products. Our future studies will examine how these proteins involved in the signal transduction process are able to regulate assembly of the actin cytoskeleton and movement of the cell. |
John B. Harley, P.h. D Link to Dr. Harley's OMRF site |
Research Interests: Genetics and immunology of systemic rheumatic diseases are the areas of our research emphasis. We have been collecting pedigrees multiplex for systemic lupus erythematosus for a decade and now have over 1,500 participants from 350 nuclear families and 250 pedigrees. We have built a database of over 500,000 genotypes. Analysis of these data strongly supports or confirms ten different genetic linkages. Now, we are entering the very exciting time of trying to indentify the genes responsible for the linkages. |
Mary Beth Humphrey, M.D., Ph.D.
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Research Interests: Bone is a dynamic tissue undergoing a constant remodeling process through out life. During remodeling, osteoclasts resorb bone and osteoblasts form new bone. Abnormal bone remodeling, secondary to increased osteoclast maturation or activation, contributes to bone destruction in inflammatory arthritis and osteoporosis. RANK Ligand, a TNF family member produced by both T cells and osteoblasts, is a differentiation and activation signal for osteoclasts. Discerning other immunomodulatory signals that govern osteoclast development, function, or survival is essential to understanding both normal as well as pathological bone processes. Towards this end our research focuses on the molecular mechanisms that lead to osteoclast differentiation and activation. In particular, we are studying the roles of ITAM (immunoreceptor tyrosine-based activation motif) signaling adapter proteins DAP12 and FcRgamma and their associated receptors in osteoclast development and function. DAP12 and FcRgamma are transmembrane proteins found in myeloid cells that transmit activation signals upon ligation of associated receptors. Osteoclasts derive from monocyte/macrophage myeloid cells and were recently shown to express DAP12 and several DARs. Our studies to date reveal that DAP12 and FcRgamma signaling is required for normal in vitro osteoclast development and that mice genetically deficient in DAP12 have increased bone mass secondary to decreased activity of osteoclasts. Additionally, mice deficient in DAP12 and FcRgamma are severely osteopetrotic secondary to abnormal osteoclast development and function. We subsequently showed that Syk tyrosine kinase mediates the ITAM activation signals in osteoclasts. Ongoing studies continue to focus on the roles of DAP12 and several DAP12-associated receptors during in vitro and in vivo osteoclast activation in several mouse models of osteoporosis and inflammatory bone loss. Additionally, we are studying the negative regulation of ITAM and RANK signals and in osteoclasts. Future studies will aim at determining the role of ITAM signals in cancer metastasis to bone and in mouse models of Pagets disease. Techniques that students might gain experience with in our lab includes Western Blotting, Immunoprecipitation, Cell culture, Elisa, Molecular Biology, Protein Expression, Fusion Protein production, Flow cytometry, Micro array, Immunofluorescent microscopy, Animal experiments, Osteoclast migration and resorption assays, Micro Computed tomography (microCT) of mouse bones, bone histology, and bone histomorphometry.
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Linda F. Thompson |
Research Interests: Research in my laboratory concerns lymphocyte development and the function of two purine metabolizing enzymes, ecto-5’-nucleotidase (CD73) and adenosine deaminase (ADA). Ecto-5’-nucleotidase is a glycosyl phosphatidylinositol (GPI)-anchored enzyme that catalyzes the dephosphorylation of extracellular AMP to adenosine and, therefore, can regulate adenosine receptor signaling. We are using CD73-deficient mice, created in our lab, to learn more about the function of this enzyme. CD73-/- mice appear healthy and reproduce normally but show exaggerated responses to a variety of inflammatory stimuli. For example, they exhibit a vascular leak syndrome characterized by neutrophil accumulation in tissues, especially the lung, when exposed to hypoxia. They also show elevated cytokine responses in experimental models of colitis sepsis, and pulmonary inflammation and fibrosis. CD73-deficient mice exhibit increased lymphocyte migration to the draining lymph nodes after exposure to an inflammatory stimulus. These findings suggest that CD73-generated adenosine plays an important role in regulating leukocyte migration across endothelial barriers in a variety of physiological or pathological circumstances. Future studies will investigate the importance of the GPI anchor of CD73 in its ability to provide adenosine for adenosine receptor signaling using newly-created transgenic mice expressing CD73 with a conventional transmembrane anchor. ADA mutations cause severe combined immunodeficiency in humans. We used murine fetal thymic organ cultures to show that ADA-deficient fetal thymuses exhibit a block in thymocyte differentiation due to the accumulation of dATP. Our results are consistent with the hypothesis that dATP, derived from the ADA substrate deoxyadenosine, induces cytochrome c release from mitochondria followed by the initiation of the apoptotic cascade. Future experiments will focus on the consequences of ADA deficiency on the development of human thymocytes. The mechanism by which T cells commit to either the ab or gd lineage is a fundamental question in T-cell biology. We are using a combination of molecular and cell culture approaches to define the role of the T-cell receptor (TCR) in this process. Our results using human thymocytes suggest that gd development is the default pathway and that ab lineage cells are derived from those thymocytes that are unable to rearrange their g and/or d loci productively. Compared to murine T cell development, human thymocytes lose gd potential and commit to the ab lineage gradually throughout several phenotypic stages. Knowledge of the pathways of thymocyte differentiation is important for understanding the pathogenesis of immunodeficiency diseases, autoimmunity and T lymphoid malignancies. Finally, we are involved in a multi-investigator project to understand why some lupus patients fail to make adequate responses to immunization with the influenza vaccine. |
Michell C. Callegan, Ph.D. |
Research Interest: Bacillus cereusis an insidious ocular pathogen, causing a rapid and fulminant endophthalmitis that invariably leads to blindness within one to two days. Despite aggressive antibiotic and surgical intervention, B. cereus endophthalmitis has a relatively poor prognosis. Clinicians and researchers have attributed the virulence of B. cereus endophthalmitis to its toxin production; however, surprisingly little specific data relating to ocular virulence and B. cereus toxin production exists. Relevant studies identifying factors necessary for the ocular virulence of other ocular pathogens compared infections produced by strains isogenically lacking one or more potential virulence determinants. The study of the relationship of B. cereus toxins to fulminant endophthalmitis will involve such an approach. The results gained from these studies will lead to the identification of specific factors contributing to fulminant B. cereus endophthalmitis. These studies will facilitate our understanding of this blinding disease and could lead to the development of target-based therapies to be used in combination with antibiotics and surgery. Other Research Interests: The primary objective of treating any type of infection is to kill the offending organism and stop tissue damage. Staphylococcus aureusliberates in excess of 30 exoproteins that could contribute to its virulence during staphylococcal keratitis. Identifying these important virulence factors and their mechanisms of action during infection is essential in pinpointing possible targets for the development of new chemotherapeutic agents. Experiments designed to compare the corneal virulence of S. aureus isogenic mutants defective in specific proteins6 led to the discovery that alpha-toxin and sphingomyelinase (i.e., beta-toxin) contributed to ocular tissue damage during infection, but protein A and coagulase did not. The results of these studies suggested potential tissue-specificities for alpha-toxin and sphingomyelinase, and coagulase as a possible mediator of fibrin accumulation during infection. Understanding the exact mechanisms of action of these toxins could provide the foundation for development of therapies designed to inhibit toxin activity during infection and could provide insight into the activities of other functionally related toxins on ocular cell types during other bacterial ocular infections. |
Xiao-Hong Sun, P.h.D. Email:xh-sun@omrf.ouhsc.edu |
| Research Interests: The basic helix-loop-helix family of proteins represented by the E2A transcription factor is crucial for the development of both B and T lymphocytes, as well as for the suppression of leukemogenesis. Therefore, mechanisms controlling the function of E2A proteins are important subjects of study. Previously, we have found two classes of proteins that function as inhibitors of E2A proteins, namely Id and Tal proteins. We have developed transgenic mouse models, in which these inhibitors are expressed, to study the role of E2A in T cell development. Data accumulated in the last decade allowed us to formulate a hypothesis that E2A controls the threshold of pre-T or T cell receptor stimulation and thereby ensures proper T cell development and prevents leukemogenesis. We continue to use these mouse models to address the molecular mechanisms by which E2A controls the threshold of stimulation. We have also identified new E2A target genes by combining inducible gene expression and DNA microarray. Recently, we have also made a novel connection between the Notch signaling pathway and the ubiquitin-mediated degradation of several important regulatory proteins including E2A transcription factors and JAK kinases. Since the Notch signaling pathway is crucial for several lineage choices during lymphocyte development, our finding of Notch-induced E2A degradation could explain how these decisions are implemented. This finding also shed light on the mechanism by which abnormal activation of Notch signaling pathways causes leukemia. Along this line of investigator, we have also demonstrated that one of the Notch target genes, HES1, synergizes with loss of E2A function in leukemogenesis. Furthermore, we have created a mouse model in which green fluorescent protein (GFP) is expressed in place of Id1. Using this mouse model, we have shown that Id1 is important for the maintenance of the size of hematopoietic stem cell pool and for the differentiation of myeloid lineage cells. Using GFP as a marker, we were able to visualize various stem or progenitor cells, which will greatly facilitate our investigators in the areas of hematopoiesis, angiogenesis and hair regeneration.. |
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Research Interests: T cells represent one of the important defenses of the host against foreign pathogens. However, faulty regulation of T cells can lead to autoimmune diseases, where T cells mount a response against self. Accordingly, understanding the mechanisms by which T cells are stimulated and regulated represents an important topic of research towards developing strategies to protect against autoimmune diseases. My laboratory studies the role of glycolipid rafts in these events. Rafts are discrete regions within membranes that have specialized functions in T-cell signaling, such as concentrating signaling proteins on the cell surface in order to enhance and sustain T-cell activation. To measure rafts in T-cell signaling, my group employs a combination of cell imaging, molecular biology and genetic approaches. The cell imaging experiments consist of visualizing and measuring rafts within cells by expressing fluorescent proteins that are selectively targeted to these structures. In our molecular biology experiments, we employ protein detection to assay for activation of proteins that reside in rafts, as well as identify the signals that allow proteins to associate with them. We also seek to identify protein and lipid interactions that are important in forming and stabilizing the rafts. Finally, in our genetic approach, we are constructing and expressing signaling proteins that have mutations in their association with rafts. Our most recent experiments with this approach include generating transgenic mice so that we can determine whole animal responses to mutated raft proteins. The future goals of my laboratory are to apply what we learn in our current studies of rafts and T-cell signaling towards designing rationale strategies to better protect hosts from autoimmune diseases. Specifically, we hypothesize that by identifying the factors that lead to the assembly and the maintaining of rafts in cell membranes, we can disrupt these events in order to attenuate T-cell activation and thereby enhance tolerance of the host to self. |
Carol Webb, Ph. D. E-mail: Carol-Webb@omrf.ouhsc.edu |
Research Interests: My laboratory has long performed basic research on antibody production and B cell differentiation. Most of our recent studies have focused upon a B lymphocyte-restricted transcription factor called Bright (B cell regulator of immunoglobulin heavy chain transcription) and how it regulates antibody heavy chain production. Several years ago, we discovered that Bright associates with Bruton’s tyrosine kinase, or Btk. Btk was identified as the defective kinase in X-linked immunodeficiency diseases in both mice and humans. X-linked agammaglobulinemia primarily affects boys and is characterized by blocks in B-cell development that result in abnormally low production of B lymphocytes and serum immunoglobulin. Immunodeficient patients are treated with intravenous immunoglobulin to ward off infections. Although the specific genetic defect of this disease was identified in 1993, the underlying mechanisms that lead to early blocks in B-cell development remain unclear. Our experiments indicate that the ability of Bright to activate transcription of an immunoglobulin promoter in vitro critically requires both Btk and another protein that is a substrate for Btk. These data provide a direct link between Btk function and immunoglobulin expression. Furthermore, studies using transgenic mice that express a dominant negative form of Bright in B cells suggest that the transgenic mice share characteristics with Btk deficient mice. These data suggest that Bright functions in the same molecular pathway as Btk. Because Btk deficiency results in a more severe phenotype in man than in mice, we are using retroviral transduction to determine the effects of dominant negative Bright on human B-cell progenitors. In addition, transgenic mice that over-express wild type Bright were generated and resulted in mice with an autoimmune phenotype that resembles lupus. Data from these mice has helped to define specific stages in B-cell development important for maintenance of B-cell tolerance. Studies to explore the effects of Bright over-expression in human systems on antibody production are also in progress. The long-term goal of these studies is to develop a better understanding of the mechanisms controlling immunoglobulin production so that new drugs can be developed to treat both autoimmune and immunodeficiency diseases. |
Paul W. Kincade, Ph.D. E-mail: Paul-Kincade@omrf.ouhsc.edu |
Research Interests: An understanding of leukemias, lymphomas, immunodeficiency and autoimmune diseases requires fundamental information about immune system development. In addition, hematopoietic stem cells (HSC) represent an excellent model for studying stem cell properties, plasticity and utility for tissue regeneration. Our laboratory explores developmental relationships between HSC and progenitors of B, T, NK and plasmacytoid dendritic cells. High-speed cell sorting and RAG-1/GFP knock-in mice are being exploited to identify the most primitive of lymphoid progenitors in fetal and adult tissues. We can identify even earlier lymphopoietic cells in E8.5 embryos and direct them to B or T lineage fates by differential ligation of Notch family receptors. Additionally, we have charted and extensively characterized the first waves of B and T lymphocyte formation. Fetal and adult lymphoid progenitors are quite different in many respects, and we are trying to determine if adult progenitors can reacquire fetal characteristics. Adult bone marrow contains plasmacytoid dendritic cells (pDC), a population that has attracted considerable interest because of their exceptional ability to produce type I interferon. We have recently studied their turnover, determined their origins and found that there are actually two, functionally distinct subsets of pDC. While both diverge from HSC at a very early stage and are developmentally unrelated to T lymphocytes, one shares a number of properties with B cells. Since pDC are thought to participate in regulation of immune responses and autoimmune disease, it will now be important to attribute these functions to one of the subsets. Blood cell formation has traditionally been described in terms of a series of binary fate decisions. Recent findings are not compatible with that view, and a new model was constructed to describe lympho-hematopoiesis. It appears that HSC progressively and gradually lose differentiation options. Their progeny have more plasticity than previously thought and can be reprogrammed to different fates. In fact, we now believe this may happen during life-threatening infections. |
Susan Kovats , Ph. D. E-mail: susan-kovats@omrf.org |
Research Interests: Sex biases in autoimmunity and infection suggest that steroid sex hormones directly modulate the function of immune cells expressing estrogen receptors (ER). Antigen specific T-cell responses depend on the function of dendritic cells (DC) to initiate innate and adaptive immunity. Dendritic cells regulate protective immunity to pathogen proteins or tumor antigens but also may elicit detrimental autoimmune responses. A primary focus of my lab has been the study of the role of estrogen in DC differentiation and function. Other projects focus on the role of antigen-presenting cells during the autoimmune diseases, rheumatoid arthritis and lupus. We have found that estrogen promotes the differentiation of functional DC from undifferentiated myeloid progenitors in murine bone marrow. Estrogen acting via ER is required for the development of a subset of DC exhibiting features of epidermal Langerhans cells. We also studied the effects of selective ER modulators (SERM), such as tamoxifen, used for breast cancer prevention, which may act as ER agonists or antagonists, depending on the target cell type. We found that inclusion of tamoxifen reduces DC numbers during in vitro differentiation, and those DC that do develop are hyporesponsive to activation signals, suggesting that systemic SERM treatment might alter DC function in response to inflammatory stimuli. Current studies involve definition of estrogen-responsive DC progenitors in bone marrow, which will facilitate future studies to distinguish among the potential effects of estrogen on survival, proliferation or induction of a specific differentiation program in DC precursors. Secondly, ongoing studies are testing the hypothesis that variable systemic estrogen levels or exposure to SERM result in differences in DC number or function in vivo. These studies are being done in a model system in which DC-mediated antigen presentation and T-cell activation in vivo may be monitored after systemic estrogen response manipulation. The long-term goals of these studies are to define the molecular mechanisms by which estrogen promotes DC differentiation from bone marrow progenitors and to understand how variable ER-mediated responses to estrogen or SERM modulate antigen-specific immune responses in vivo. Ultimately, we will apply the information gained from such studies to murine models of autoimmunity. |
Last updated: 07/16/08
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