Anthony L Defranco
University Of California San Francisco
Project start date: 2008-01-01
Project end date: 2012-12-31
Sponsored Links Excellgen http://Excellgen.com
CELL TYPE-SPECIFIC ROLES OF TLR SIGNALING IN IMMUNE RESPONSES
Anthony L Defranco, Associate Professor
University Of California San Francisco, 3333 California St., Ste 315, San Francisco, Ca 94143-0962
Grant 5R01AI072058-03 from National Institute Of Allergy And Infectious Diseases
Keywords: Address; Alleles; Allelomorphs; Animal Model; Animal Models and Related Studies; Animal Welfare; Bibliography; Breeding; Bypass; CD11b; CR3A; Comment; Comment (PT); Comment [Publication Type]; Commentary; Commentary (PT); Complication; Country; Data; Ecological impact; Editorial Comment; Editorial Comment (PT); Elements; Environment; Environmental Impact; Equipment; Ethics Committees, Research; Funding; Future; Genetic Alteration; Genetic Change; Genetic defect; IACUC; INFLM; IRBs; ITGAM; ITGAM gene; Immune response; Immunity, Innate; Immunity, Native; Immunity, Natural; Immunity, Non-Specific; Impact, Environmental; Infectious Agent; Inflammation; Institutional Animal Care and Use Committee; Institutional Review Boards; Intention; International; Knockout Mice; Ligands; MAC-1; MAC1A; MO1A; Mammals, Mice; Mice; Mice, Knock-out; Mice, Knockout; Mice, Mutant Strains; Modeling; Murine; Mus; Mutant Strains Mice; Mutation; Myeloid Cells; Natural Immunity; Null Mouse; Pathway interactions; Penetrance; Principal Investigator; Programs (PT); Programs [Publication Type]; Published Comment; Publishing; Receptor Signaling; Research; Research Ethics Committees; Research Resources; Resources; Role; Series; System; System, LOINC Axis 4; TLR protein; TXT; Text; Toll-like receptors; Update; Vertebrate Animals; Vertebrates; Viewpoint; Viewpoint (PT); Viral Diseases; Virus Diseases; ing; cell type; experiment; experimental research; experimental study; expiration; genome mutation; host response; human subject; immunoresponse; infectious organism; model organism; mouse mutant; pathway; programs; research study; social role; tool; vertebrata; viral infection; virus infection
Project start date: 2008-01-01
Project end date: 2012-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
5R01AI072058-03 (2010): $347625
Grants awarded to Anthony L Defranco
MOLECULAR AND CELLULAR IMMUNOLOGY
Anthony L Defranco, Associate Professor
University Of California San Francisco, 3333 California St., Ste 315, San Francisco, Ca 94143-0962
Grant 5T32AI007334-22 from National Institute Of Allergy And Infectious Diseases
Abstract: The UCSF Immunology Training Program encompasses 43 laboratories engaged in molecular and cellular immunology and actively training graduate and postdoctoral scientists. Areas of active research include lymphocyte cell surface receptor structure, signaling mechanism, and immune system function, histocompatability antigen expression and intracellular trafficking, roles of receptors and antigen specificity in lymphocyte development, lymphocyte and leukocyte cell adhesion molecule structure and function, immunoglobulin gene hypermutation and class-switch recombination, cytokine expression, mechanisms of autoimmunity, allergy, and defense against infectious agents, and various aspects of AIDS including pathogenesis, HIV interaction with lymphocytes, and mechanisms of HIV viral gene expression and genome packaging. Over the past 24 years, a vital graduate training program leading to the Ph.D. has been developed by the immunology program faculty and has been supported by this training grant for the past 20 years. This program is designed to provide a solid background in genetics, cell biology, molecular biology, and mammalian tissue and organ biology as well as thorough training in molecular and cellular immunology. The interdisciplinary nature of this training is enhanced by the affiliation of the Immunology Program with the UCSF Biomedical Sciences Program (BMS), an interdisciplinary program that also includes the study of infectious agents and inflammatory processes as well as other aspects of mammalian tissue/organ development, function, and disease. In addition to formal coursework and thesis research, the Immunology Program includes an active weekly seminar series of outside immunology speakers, both Immunology and BMS student-faculty journal clubs, an annual Immunology Program conference (held jointly with UC Berkeley immunologists), and seminar courses on advanced immunological topics. These activities provide an excellent training environment for postdoctoral fellows as well as for graduate students. Postdoctoral training is additionally enhanced by a postdoc research-in-progress seminar series and a new postdoctoral mentoring program
Keywords: Cellular Immunology; Molecular
Project start date: 1988-09-01
Project end date: 2014-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-08-226
5T32AI007334-22 (2010): $535634
2T32AI007334-21A1 (2009): $532018
5T32AI007334-20 (2007): $477412
5T32AI007334-19 (2006): $346275
5T32AI007334-18 (2005): $472992
5T32AI007334-17 (2004): $462648
2T32AI007334-16 (2003): $460227
Cytoskeleton And Signal Transduction In Host Defense
Anthony L Defranco, Professor
Medicineuniversity Of California San Francisco
Grant 5R01AI035811-15 from National Institute Of Allergy And Infectious Diseases IRG: CMI
Abstract: The leukocyte cytoskeleton is intimately involved in migration, adhesion, phagocytosis, and cell division; for these reasons, regulation of cytoskeletal assembly has an essential role in host defense. While cytoskeleton is broadly understood to be involved in the shape changes required for these functions, a less appreciated but equally fundamental role for the cytoskeleton is that it can act as a scaffold for the assembly of signaling cascades, particularly in response to cell adhesion. In recent years quite a lot has been learned about how signaling cascades affect actin polymerization. However, much less is known about how assembly of cytoskeleton affects signa ng cascades. Some years ago, we hypothesized that the leukocyte-specific actin crosslinking protein L-plastin (LPL) has an important role in crosstalk between cytoskeleton and signal transduction; this grant has supported all our studies to test this hypothesis. LPL is a prevalent and regulated component of the cortica cytoskeleton that can become phosphorylated on Ser5 near its aminoterminus in response to many leukocyte activators, including bacterial pathogen-associated molecular patters (PAMPs), immune complexes, chemotactic peptides, cytokines, and chemokines. In the last grant period, we have shown that cell-permeant peptides that recreate the LPL phosphorylation site can activate leukocyte integrins and that this effect requires phosphorylation of the peptide. For genetic approaches to understanding LPL in regulation of leukocyte function, we have created a mouse deficient in LPL by homologous recombination. These mice show that LPL is required for important aspects of integrin signaling in neutrophils and macrophages and that, as a consequence, absence of LPL causes a defect in host defense against Staphylococcus aureus. In the current application, we propose to continue our genetic and biochemical approaches to a molecular understanding of the role of LPL in integrin-dependent signaling in leukocytes. Specifically, we propose to determine 1) the molecular basis of the requirement for LPL in signaling following integrin ligation in PMN and macrophages ("outside-in signaling"); 2) the mechanisms and I biological roles of LPL phosphorylation relevant to signaling for integrin activation ("inside-out signaling"). From these studies we will obtain an increased understanding of molecular mechanisms involved in inflammation, immunity, and host defense, leading to increased opportunities for control of infectious and inflammatory diseases
Keywords: biological signal transduction, cytoskeleton, integrin, leukocyte activation /transformation, phagocyte actin, binding protein, cytoskeletal protein, gene mutation, phosphorylation, protein kinase, protein structure function clinical research, genetically modified animal, laboratory mouse, tissue /cell culture
Project start date: 1994-05-01
Project end date: 2010-01-31
5R01AI035811-13 (2007): $359123
5R01AI035811-12 (2006): $369849
Sponsored Links Excellgen http://Excellgen.com
TYROSINE KINASES AND B LYMPHOCYTE TOLERANCE
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5P50HL054476-080007 from National Heart, Lung, And Blood Institute
Abstract: The immune system must decide whether to mount an immune response to a particular entity or whether to ignore the latter outcome as referred to as immunological tolerance. A great deal has been learned about tolerance over the past decade, but our understanding is still fragmentary, despite its importance for understanding autoimmune disease, managing acceptance of organ transplants, promoting cancer immunotherapy, and decreasing the complication of allosensitization by transfusion of platelets and other blood products. In the previous project period, we unexpectantly found that mice deficient in the Lyn tyrosine kinase have B lymphocytes that exhibit elevated responsiveness to antigenic stimulation. In vivo, these mice make high levels of autoantibodies directed at nuclear components such as double-stranded DNA and some of them develop kidney disease. Double mutant mice defective in Lyn and another Src-family kinase, Fyn were found to develop a much more severe autoimmune lupus-like kidney disease, with 50% of the animals dying by 7 months of age. We hypothesize that the defect in lyn makes B cells hyperresponsiveness and also defective in tolerance induction, resulting in production of antibodies directed at nuclear components released by apoptotic cells. We further hypothesize that the defect in fyn contributes to more rapid disease, possibly by making the kidneys more susceptible to damage resulting from immune complex deposition. These hypotheses will be tested by four Specific Aims. 1) We shall define the cellular basis of defects leading to autoimmune diseases in lyn-/-,fyn-/- mice. This will be done by bone marrow transplantation and by adoptive transfer of mature lymphocytes. 2) We shall define the effects of the Fyn- deficiency on B cell phenotype, signaling ability, and activation, both in the context of otherwise normal mice and in the context of the Lyn- deficiency. We hypothesize that in contrast to Lyn, Fyn is a positive element in antigen receptor signaling, so its loss will decrease B cell responsiveness to antigen. 3) We shall determine the effects of Lyn and Fyn deficiencies on tolerance to double-stranded DNA using Ig transgenic mice developed by Martin Weigert and coworkers. 4) We shall determine the effects of Lyn and Fyn deficiencies on allosensitization by platelet transfusions to test the hypothesis that genetic affecting lymphocyte responsiveness may cause individuals to be more or less likely to make an alloantibody response following transfusions.
Keywords: B lymphocyte, immune tolerance /unresponsiveness, protein tyrosine kinase, antigen receptor, antinuclear autoantibody, autoimmune disorder, disease /disorder model, gene mutation, immune complex disease, immunogenetics, isoantibody, kidney disorder, platelet transfusion, protein structure function, laboratory mouse, tissue /cell culture, transgenic animal
Src Tyrosine Kinases And Autoimmunity.
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5P01AI035297-110008 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: The immune system must decide whether to mount an immune response to a particular entity or whether to ignore it. The latter outcome is referred to as immunological tolerance. A great deal has been learned about tolerance over the past decade, but our understanding is still fragmentary, despite its importance for understanding autoimmune disease, managing acceptance of organ transplants, and promoting cancer immunotherapy. We have unexpectedly found that mice deficient in the Lyn tyrosine kinase have B lymphocytes that exhibit elevated responsiveness to antigenic stimulation. In vivo, these mice make high levels of autoantibodies directed at nuclear components such as double-stranded DNA and some of them develop kidney disease. Double mutant mice defective in Lyn and another Src-family kinase, Fyn were found to develop a much more severe autoimmune lupus-like kidney disease, with 50% of the animals dying by 7 months of age. We hypothesize that the defect in Lyn makes B cells hyperresponsive and also defective in tolerance induction, resulting in production of antibodies directed at nuclear components released by apoptotic cells. We further hypothesize that the defect in fyn contributes to more rapid disease incidence, possibly by making the kidneys more susceptible to damage resulting from immune complex deposition. In addition, Fyn-deficiency may lead to defects in T cell tolerance. These hypotheses will be tested by three Specific Aims 1) We shall determine the effects of Lyn and Fyn deficiencies on tolerance to double-stranded (ds) DNA using Ig- transgenic mice developed by Martin Weigert and coworkers. 2) We shall determine the role of helper T cells in IgG anti-dsDNA production by Lyn-/- mice and examine the effects of lyn and fyn defects on T cell tolerance, and 3) We shall define the cellular basis of defects leading to autoimmune disease in lyn-/-fyn-/- mice. This will be done by bone marrow transplantation and by adoptive transfer of mature lymphocytes. The proposed studies may lead to significant insights into the nature of the severe autoimmune disease in lyn-/-fyn-/- mice, which in turn may aid in understanding the causes of human autoimmune diseases such as systemic lupus erythematosus.
Keywords: autoimmune disorder, autoimmunity, gene mutation, immune tolerance /unresponsiveness, immunoglobulin gene, protein tyrosine kinase, B lymphocyte, DNA, DNA binding protein, antibody formation, helper T lymphocyte, immunoglobulin G, nucleic acid structure, systemic lupus erythematosus, SDS polyacrylamide gel electrophoresis, bone marrow transplantation, enzyme linked immunosorbent assay, flow cytometry, laboratory mouse, polymerase chain reaction, tissue /cell culture, transgenic animal
Project start date: 2002-08-01
Project end date: 2003-05-31
SELECTIVE GENE ABLATION IN MATURE B LYMPHOCYTES
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 1F06TW002281-01 from Fogarty International Center IRG: ZRG2
Abstract: The goal of the proposed project is to develop a method of inactivating genes only in mature B cells. This will make it possible to study the function of genes in mature B cells in vivo, even if the gene in question is required for animal viability and/or development of mature B cells. Also, as other cell types will not be affected, interpretations of the phenotypes observed should be more straightforward. Therefore, the first Specific Aim is to express the Cre recombinase only in mature B cells. The primary strategy for doing this is to insert Cre and an internal ribosome entry site into the 3 untranslated region of the delta heavy chain locus. As this locus is only expressed following VDJ recombination of the IgH locus, expression should be tightly restricted to B cells. Moreover, the delta heavy chain is not expressed during B cell development, but is expressed at high levels in every mature resting B cell, prior to antigenic Stimulation. Thus, Cre will be inserted by homologous recombination into the delta locus of embryonic stem cells and mice will be made from these cells. These mice will be crossed to beta-galactosidase reporter mice to directly assess the tissue and cell-type expression pattern of Cre recombinase by a functional assay. In the second Aim, the gene for the ubiquitously expressed signaling component Shc will be altered by introducing loxP sites on either side of essential exons. This should not affect Shc expression except in cells expressing Cre, which will delete the DNA between the two loxP sites, inactivating the Shc gene. Thus, Shc should be lost in mature B cells, but should be normally expressed in all other cell types. The effects of this defect on mature B cell function will be assessed after the funding period.
Keywords: B lymphocyte, gene expression, gene targeting, protein tyrosine kinase, recombinase, erythroid stem cell, genetic library, genetic mapping, laboratory mouse, transgenic animal
1F06TW002281-01 (1998): $20000
Regulation Of B Lymphocyte Proliferation By Antigen
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5R01AI020038-23 from National Institute Of Allergy And Infectious Diseases IRG: CMI
Abstract: The fate of B cells is highly dependent on signals from the B cell antigen receptor (BCR) for development, tolerance, and activation decisions. The overall goal of this project is to understand the role of cholesterol-rich subdomains of the plasma membrane called lipid rafts in BCR signal transduction. Recent work in B cells has established that lipid rafts play a role in BCR signaling. Lipid rafts exist primarily as small, highly dynamic structures that coalesce into larger and more stable lipid rafts upon cell stimulation. BCR stimulation leads to its movement from the non-lipid raft plasma membrane into lipid rafts and induces coalescence of lipid rafts, eventually leading to a large lipid raft patch or cap on the cell surface. In Specific Aim 1, the mechanism of BCR-induced lipid raft coalescence will be studied. BCR stimulation causes ezrin to become dephosphorylated and to release its connection between some lipid raft proteins and the actin cytoskeleton. We shall test if this release promotes lipid raft coalescence and BCR signaling. We hypothesize that the later stages of lipid raft coalescence require active processes involving myosins and will attempt to identify the myosin isoform responsible. BCR stimulation also leads to outgrowth of long filopdia- like projections ("cytonemes") and in Specific Aim 2, we shall determine the mechanism of outgrowth of these projections. We shall explore the role of the B144 protein, the Rac1 and Cdc42 GTPases, and myosin 10 and its close relatives. In Specific Aim 3, we shall examine the connection of lipid rafts to the ability of the BCR to activate the key transcription factor, NF-kB. Carma1, a key scaffold molecule for this signaling pathway, is localized to lipid rafts and it recruits other components of this pathway to lipid rafts following BCR stimulation. The importance of Carma1 localization to lipid rafts and of lipid raft coalescence for activation of NF-kB will be determined. Finally, in Specific Aim 4, we shall examine lipid raft coalescence and NF-kB pathway activation in immature B cells and B cell lines of immature phenotype, which appear to differ from mature B cells in these processes. . Lay Language Statement The ability of the immune system to respond to infectious agents depends on the function of the antigen receptors of lymphocytes. This function appears to involve changes in a subdomain of the plasma membrane called lipid rafts and the mechanisms of these changes will be studied.
Keywords: B lymphocyte, antigen, lipid raft, lymphocyte proliferation, actin, antigen receptor, cAMP response element binding protein, cell, cell line, cell membrane, cholesterol, cytoskeleton, family, glycolipid, health /scientific organization, immune system, language, lead, lipid, lymphocyte, membrane, membrane lipid, muscle, myosin, phenotype, physical state, play, protein, receptor, role, sphingolipid
Project start date: 1984-01-01
Project end date: 2010-11-30
5R01AI020038-23 (2007): $367766
2R01AI020038-22A1 (2006): $374191
5R37AI020038-16 (1999): $314315
5R37AI020038-15 (1998): $305047
5R37AI020038-14 (1997): $290598
Sponsored Links Excellgen http://Excellgen.com
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5P01AI035297-119001 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: The Animal Facility Core (Core A) will meet the special animal needs of the Program Project members. In many cases, the Projects will require double or even triple transgenic/KO mice for the sophisticated experiments outlined in the proposal. The FIRST SPECIFIC AIM of this core is to breed unique and non-routine strains for use by all program project members. Core A will breed mice that are genetically altered in at least two ways (transgenes or knockouts) and genotype them to verify their genetic makeup. The Core will also maintain genetically altered strains used by at least two Projects. In addition, we will use a large number of immunocompomised and autoimmune mice that will require housing in a barrier facility providing a pathogen-free environment, an experienced animal husbandry staff capable of providing quality animal care, and a sophisticated quality assurance program. The availability of quality animals free from adventitious agents is absolutely essential for the success of the research effort proposed in this Program Project Grant. Thus, the SECOND SPECIFIC AIM of this core is to provide a specific pathogen-free environment to maintain mice for the studies proposed in the individual projects. In this regard, the Core will provide additional vigilance and monitoring to ensure that the SPF barrier maintained by the institution is functioning properly. The availability of the core ensures that sufficient mice shared by multiple investigators will be available and the appropriate genetic and health states will be maintained. Finally, the Core will assist investigators in creating bone marrow chimeras and adoptive transferred animals.
Keywords: animal breeding, biomedical facility, genetic strain, laboratory mouse, transgenic animal, veterinary science, flow cytometry, genotype, polymerase chain reaction
Project start date: 2002-08-01
Project end date: 2003-05-31
CELLULAR BASIS OF TLR SIGNALING FOR MUCOSAL IMMUNE RESPONSES
Anthony L Defranco, Associate Professor
University Of California San Francisco, 3333 California St., Ste 315, San Francisco, Ca 94143-0962
Abstract: In recent years, Toll-like receptors (TLRs) have emerged as critical recognition elements of innate immunity, both for induction of inflammation at the site of an infection and for induction of an adaptive immune response. These receptors are expressed on the three major types of immune cells in many tissues, immature dendritic cells, tissue macrophages, and mast cells, as well as on several other types of cells. In the proposed project, we shall define which type of cell is responsible for mediating several TLR-based immune responses, systemically and in the airways. In these studies, we shall take advantage of a conditional allele we have engineered into the mouse germ line for the key TLR signaling adaptor molecule MyD88. This allows us to delete the gene encoding MyD88 specifically in particular cell types including dendritic cells, macrophages and neutrophils, B cells, and T cells. In Aim 1, we shall determine the role of dendritic cells in a mouse model of allergic asthma in which antigen + TLR ligands are introduced via the airways. In Aim 2, we shall analyze the immune response of mice to systemic or airway exposure to zymosan, a yeast cell wall preparation that is composed of chitin, ligands for TLR2 and ligands for C-type lectin receptors (dectin-1, etc.). We shall dissect the roles that these different innate immune ligands play in directing the nature of the immune response between Th1, Th2 and Th17. The contributions of particular cell types will also be determined. Finally, in Aim 3, we´ll determine the cell type-specific roles of MyD88 in host defense to systemic infection by the fungal pathogen, Candida albicans. Lay Language Summary The proposed studies will determine which immune cells in tissues are responsible for initiating immune responses to inhaled antigens as a model of allergic asthma, to infections with yeasts and molds. This will be accomplished by the use of genetically modified mice, in which key immune cell types are unable to recognize the presence of fungal cell walls. These studies will be useful for improving vaccination strategies and for developing novel strategies to block inflammation for patients with inflammatory diseases
Keywords: ATGN; Ablation; Alleles; Allelomorphs; Allergic asthma; Antigens; Aspiration, Respiratory; B blood cells; B-Cells; B-Lymphocytes; Basophilic Histiocyte; Basophils, Tissue; Blood Neutrophil; Blood Polymorphonuclear Neutrophil; Blood Segmented Neutrophil; Body Tissues; Breathing; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; C Type Lectin Receptors; C. albicans; C.albicans; CD11c; CD154; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; CD40L; CD40LG; Candida albicans; Cell Communication and Signaling; Cell Maturation; Cell Signaling; Cell Wall; Cells; Cells, CD4; Chitin; DNA Recombination; DNA recombination (naturally occurring); Dendritic Cells; Development; Disease; Disorder; Elements; Engineering; Engineerings; Event; Exons; Exposure to; Extrinsic asthma; Fungi, Filamentous; Genes; Genetic Recombination; Germ Lines; Glucans; Glucose Polymer; Goals; Helper Cells; Helper T-Cells; Helper-Inducer T-Lymphocyte; Hematopoietic; Heterophil Granulocyte; Host Defense; Human; Human, General; INFLM; ITGAX; ITGAX gene; Immune; Immune response; Immunity; Immunity, Innate; Immunity, Native; Immunity, Natural; Immunity, Non-Specific; Individual; Inducer Cells; Infection; Inflammation; Inflammatory; Inhalation; Inhaling; Inspiration, Respiratory; Intracellular Communication and Signaling; Language; Ligands; Lung; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow Mast Cell; Marrow Neutrophil; Mediating; Mice; Modeling; Molds; Mucosal Immune Responses; Murine; Mus; Natural Immunity; Nature; Neutrophilic Granulocyte; Neutrophilic Leukocyte; Organism; Ovalbumin; Patients; Play; Polyglucoses; Polymorph; Polymorphonuclear Cell; Polymorphonuclear Leukocytes; Polymorphonuclear Neutrophils; Preparation; Property; Property, LOINC Axis 2; Receptor Protein; Receptor Signaling; Recombination; Recombination, Genetic; Relative; Relative (related person); Reporter; Respiratory System, Lung; Reticuloendothelial System, Spleen; Role; Signal Transduction; Signal Transduction Systems; Signaling; Site; Spleen; Surface; System; System, LOINC Axis 4; Systemic infection; T-Cells; T-Cells, Helper-Inducer; T-Lymphocyte; T-Lymphocytes, Helper; T-Lymphocytes, Inducer; T4 Cells; T4 Lymphocytes; TIL4; TLR protein; TLR2; TLR2 gene; TNFSF5; TNFSF5 gene; TRAP Gene; Testing; Thymus-Dependent Lymphocytes; Tissues; Toll-like receptors; Toll/Interleukin 1 Receptor-Like 4 Gene; Transgenes; Transgenic Organisms; Veiled Cells; Work; Yeasts; Zymosan; adaptive immunity; antigen challenge; atopic asthma; base; biological signal transduction; cell type; cytokine; dectin 1; disease/disorder; experiment; experimental research; experimental study; extrinsic allergic asthma; helper T cell; host response; immunogen; immunoresponse; improved; inspiration; interest; living system; macrophage; mast cell; mastocyte; mouse model; neutrophil; new approaches; novel approaches; novel strategies; novel strategy; pathogen; pulmonary; receptor; recombinase; research study; response; sensor; social role; thymus derived lymphocyte; transgenic; vaccination strategy
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5P01AI078869-03_0001 (2010): $351948
5P01AI078869-02_0001 (2009): $355161
INNATE IMMUNE REGULATION OF INFLAMMATION AND ADAPTIVE IMMUNITY
Anthony L Defranco, Associate Professor
University Of California San Francisco, 3333 California St., Ste 315, San Francisco, Ca 94143-0962
Grant 5P01AI078869-03 from National Institute Of Allergy And Infectious Diseases
Abstract: In recent years, Innate Immunity has gone from being the "immunologists´ dirty little secret" to being among the most active and exciting areas of immunology. Many recognition molecules of vertebrate innate immune cells have been defined and much is now known about their mechanisms of action. theless, much remains to be learned before we truly understand how to harness these mechanisms for vaccination and cancer immunotherapy or how to block them to treat autoimmune and inflammatory disease. This proposed Program Project combines 4 investigators with established expertise in the area of innate immunity to pursue related studies developing out of their independent research efforts, but containing numerous connections and great potential for combined effort. In Project #1, Dr. DeFranco will utilize his newly created conditional allele of myd88 to dissect the cellular basis of Toll-like receptor signaling for systemic and mucosal immune responses, with emphasis on airways and fungal infections. In Project #2, Dr. Ma will analyze the role of the ubiquitin-modifying regulator A20 in dendritic cells for restraining TLR responses and preventing inflammatory disease. In Project #3, Dr. Lowell will determine the mechanism by which myeloid cells contribute to lupus-like autoimmunity in the Lyn-deficient mouse model. Finally, in Project #4, Dr. Locksley will determine how chitin, a polysaccharide found in fungi and invertebrates, promotes type 2 immunity and how it interacts with TLR signaling pathways to regulate the type of immune response. Lay Language The immune system recognizes conserved elements of viruses, bacteria, fungi and multicellular invertebrates to allow it to detect infections and fight them. Immunologists are defining a number of the molecular mechanisms by which this is done, but much remains to be learned, particularly to understand how these reactions are controlled to avoid excessive inflammation and tissue injury, while directing the immune system toward the type of immune response most beneficial for fighting the infectious agent that is present. Better understanding of these issues will lead to improved vaccination procedures and better ability to control excessive inflammatory conditions. PROJECT 1 Cellular Basis of TLR signaling for Mucosal Immune Responses (DeFranco, A.) In recent years, Toll-like receptors (TLRs) have emerged as critical recognition elements of innate immunity, both for induction of inflammation at the site of an infection and for induction of an adaptive immune response. These receptors are expressed on the three major types of immune cells in many tissues, immature dendritic cells, tissue macrophages, and mast cells, as well as on several other types of cells. In the proposed project, we shall define which type of cell is responsible for mediating several TLR-based immune responses, systemically and in the airways. In these studies, we shall take advantage of a conditional allele we have engineered into the mouse germ line for the key TLR signaling adaptor molecule MyD88. This allows us to delete the gene encoding MyD88 specifically in particular cell types including dendritic cells, macrophages and neutrophils, B cells, and T cells. In Aim 1, we shall determine the role of dendritic cells in a mouse model of allergic asthma in which antigen + TLR ligands are introduced via the airways. In Aim 2, we shall analyze the immune response of mice to systemic or airway exposure to zymosan, a yeast cell wall preparation that is composed of chitin, ligands for TLR2 and ligands for C-type lectin receptors (dectin-1, etc.). We shall dissect the roles that these different innate immune ligands play in directing the nature of the immune response between Th1, Th2 and Th17. The contributions of particular cell types will also be determined. Finally, in Aim 3, we´ll determine the cell type-specific roles of MyD88 in host defense to systemic infection by the fungal pathogen, Candida albicans. Lay Language Summary The proposed studies will determine which immune cells in tissues are responsible for initiating immune responses to inhaled antigens as a model of allergic asthma, to infections with yeasts and molds. This will be accomplished by the use of genetically modified mice, in which key immune cell types are unable to recognize the presence of fungal cell walls. These studies will be useful for improving vaccination strategies and for developing novel strategies to block inflammation for patients with inflammatory diseases
Project start date: 2008-07-15
Project end date: 2013-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5P01AI078869-03 (2010): $1807133
5P01AI078869-02 (2009): $1817670
1P01AI078869-01 (2008): $1805168
LIPOPOLYSACCHARIDE-INDUCED SIGNALING IN MACROPHAGES
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5R01AI033442-03 from National Institute Of Allergy And Infectious Diseases IRG: BM
Abstract: Bacterial lipopolysaccharide (LPS) is a potent stimulator of the cells of the immune system, including B cells and macrophages. The responses of these cells are likely to represent an evolved mechanism for rapidly recognizing and responding to infection by Gram-negative bacteria. Although generally beneficial, the responses to UPS become life threatening when induced systemically during bacterial sepsis. The proposed experiments are concerned with understanding intracellular signal transduction reactions that are elicited in the macrophage upon contact with LPs. We have recently found that LPS rapidly induces tyrosine phosphorylation of several macrophage proteins. As tyrosine phosphorylation has recently emerged as a major receptor signaling mechanism, we propose to study these reactions further with the hope of gaining insight into the processes that mediate the macrophage responses to LPS. We have just identified two of the LPS-induced tyrosine phosphoproteins in the RAW264.7 macrophage cell line as two isoforms of mitogen-activated protein (MAP) kinase. These serine/threonine kinases are activated by tyrosine kinase growth factor receptors in many cell types, and may be involved in the regulation of a variety of cellular processes, including transcription and translation. The propose experiments will first examine the mechanism by which LPS activates MAP kinase. The possible roles of the GTP-binding protein Ras, and the protein kinases Raf-1 and MAP kinase in the activation of MAP kinase by LPS will be examined. If some or all of these signaling components play a role in mediating LPS action, that would bring us closer to understanding the initial signal transduction events triggered by the putative LPS receptor. We shall determine whether LPS activation of MAP kinases and upstream events is seen in macrophages and macrophage cell lines of varied differentiation or activation status, or whether it is restricted to certain macrophage subpopulations. The molecular identity of the two isoforms of MAP kinase expressed in macrophages will be determined, and then these molecules will be overexpressed by introducing the appropriate cDNAs encoding the MAP kinase isoforms into the RAW264.7 macrophage cell line. The effects of overexpression on MAP kinase activation and LPS- induced biological responses will be examined. These experiments may provide evidence for a role of MAP kinases in the responses of macrophages to LPS.
Keywords: bactericidal immunity, biological signal transduction, leukocyte activation /transformation, lipopolysaccharide, macrophage, antigen receptor, arachidonate, gene expression, guanine nucleotide binding protein, phosphoprotein, protein kinase, tumor necrosis factor alpha, immunoprecipitation, laboratory mouse, molecular cloning, tissue /cell culture, transfection
Project start date: 1993-08-01
Project end date: 1996-07-31
5R01AI033442-03 (1995): $112643
5R01AI033442-02 (1994): $111681
1R01AI033442-01A1 (1993): $99921
Sponsored Links Excellgen http://Excellgen.com
REGULATION OF B-LYMPHOCYTE PROLIFERATION BY ANTIGEN
Anthony L Defranco, Professor
Microbiology And Immunologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 5R01AI020038-11 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: B lymphocytes synthesize antibodies both as secreted proteins and as cell surface receptors for antigen. Crosslinkage of the membrane form of immunoglobulin (mIg) results in triggering of hydrolysis of phosphoinositides and the generation of second messengers including diacylglycerol and cytoplasmic free calcium ions. The goals of this project are 1.) to determine how mIg activates the phospholipase C enzyme that breaks down the phosphoinositides. This involves identifying and characterizing the phospholipase C, the G protein that mediates between mIg and phospholipase C, and putative accessory proteins that associate with crosslinked mIg and permit interaction with the G protein. A better understanding of the molecular mechanisms of mIg signal transduction should give us better insight into what molecular structures make particularly good or poor antigens, which would aid vaccine design. 2.) to determine whether phosphoinositide signaling reactions mediate the biological response of a B lymphoma cell line that is a model for an immature B cell undergoing tolerance induction. Phosphoinositide breakdown will be specifically interrupted by microinjection of antibodies that bind to these phospholipids or by genetic knockout of phospholipase C. In addition, other receptors that can trigger phosphoinositide breakdown will be introduced into the cell line by DNA-mediated gene transfer, with the expectation that they will also induce the growth inhibitory response normally induced by mIg, provided these signaling reactions actually mediate the growth inhibition. 3.) to examine the mechanisms by which phosphoinositide second messengers regulate the growth of B cells. These experiments will involve isolation of genes induced by mIg signal transduction, characterization of which second messengers are responsible for this induction, and use of these genes as molecular markers for characterizing the defects in mutants that have lost the growth inhibitory response to mIg signaling. 4.) to determine whether phosphoinositide breakdown is also triggered by mIg in authentic immature B cells undergoing tolerance induction, and whether these biochemical reactions are mediating the response here as well. These latter studies should give us additional insight into the process of "clonal deletion" whereby self-reactive lymphocytes are removed at an immature stage of their development. The failure of this process may play a role in the etiology of autoimmune diseases
Keywords: B cell receptor, B lymphocyte, biological signal transduction, immunoglobulin gene, immunoregulation, lymphocyte proliferation G protein, antibody, calcium metabolism, cell growth regulation, crosslink, gene expression, genetic manipulation, immune tolerance /unresponsiveness, membrane protein, phosphatidylinositol, phospholipase C, second messenger microinjection, molecular cloning, neoplastic cell culture for noncancer research, transfection
Project start date: 1984-01-01
Project end date: 1994-12-31
5R01AI020038-11 (1994): $204322
REGULATION OF B LYMPHOCYTE PROLIFERATION BY ANTIGEN
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5R01AI020038-10 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: B lymphocytes synthesize antibodies both as secreted proteins and as cell surface receptors for antigen. Crosslinkage of the membrane form of immunoglobulin (mIg) results in triggering of hydrolysis of phosphoinositides and the generation of second messengers including diacylglycerol and cytoplasmic free calcium ions. The goals of this project are 1.) to determine how mIg activates the phospholipase C enzyme that breaks down the phosphoinositides. This involves identifying and characterizing the phospholipase C, the G protein that mediates between mIg and phospholipase C, and putative accessory proteins that associate with crosslinked mIg and permit interaction with the G protein. A better understanding of the molecular mechanisms of mIg signal transduction should give us better insight into what molecular structures make particularly good or poor antigens, which would aid vaccine design. 2.) to determine whether phosphoinositide signaling reactions mediate the biological response of a B lymphoma cell line that is a model for an immature B cell undergoing tolerance induction. Phosphoinositide breakdown will be specifically interrupted by microinjection of antibodies that bind to these phospholipids or by genetic knockout of phospholipase C. In addition, other receptors that can trigger phosphoinositide breakdown will be introduced into the cell line by DNA-mediated gene transfer, with the expectation that they will also induce the growth inhibitory response normally induced by mIg, provided these signaling reactions actually mediate the growth inhibition. 3.) to examine the mechanisms by which phosphoinositide second messengers regulate the growth of B cells. These experiments will involve isolation of genes induced by mIg signal transduction, characterization of which second messengers are responsible for this induction, and use of these genes as molecular markers for characterizing the defects in mutants that have lost the growth inhibitory response to mIg signaling. 4.) to determine whether phosphoinositide breakdown is also triggered by mIg in authentic immature B cells undergoing tolerance induction, and whether these biochemical reactions are mediating the response here as well. These latter studies should give us additional insight into the process of "clonal deletion" whereby self-reactive lymphocytes are removed at an immature stage of their development. The failure of this process may play a role in the etiology of autoimmune diseases.
Keywords: B cell receptor, B lymphocyte, biological signal transduction, immunoglobulin gene, immunoregulation, lymphocyte proliferation, G protein, antibody, calcium metabolism, cell growth regulation, crosslink, gene expression, genetic manipulation, immune tolerance /unresponsiveness, membrane protein, phosphatidylinositol, phospholipase C, second messenger, microinjection, molecular cloning, neoplastic cell culture for noncancer research, transfection
Project start date: 1984-01-01
Project end date: 1994-12-31
5R01AI020038-10 (1993): $206346
IMMUNOGLOBULIN HEAVY CHAIN IN B LYMPHOCYTE DEVELOPMENT
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5P50HL054476-050007 from National Heart, Lung, And Blood Institute
Abstract: B lymphocytes develop from hematopoietic stem cells in a highly regulated series of steps, characterized by ordered rearrangements of immunoglobulin (Ig) genes. These rearrangements are required for subsequent progression to the next developmental stage. In particular, B cell precursors arrest at an S7+ c-kit+ CD2+ stage if they are unable to make a functional membrane mu heavy chain protein. Although these pre-B cells have not yet rearranged their Ig light chain genes, they do express two proteins, called gamma5 and Vpre=B1, which serve as surrogate light chains to combine with mu chain and forms an Ig-type structure. Mice deficient for gamma5 expression also exhibit a block in B cell development, although it is not as severe. Thus, it has been proposed that this pre-B cell form of membrane Ig sends a signal to induce developmental progression from the S7+ c-kit+ CD2+ stage to the subsequent S7+ c-Kit+ stage. The proposed experiments will test this hypothesis. We have recently developed a system for culturing pre-B cells in vitro and efficiently introducing proteins into them with retroviral vectors. In Aim 1, we shall determine whether introducing mu heavy chain into cultured Rag1-deficient pre=B cells will induce developmental maturation, as expected. Next, the structural requirements for mu chain to have developmental function will be determined. Mutant forms of muchain with characterized associations with the Ig-alpha/Ig-beta accessory proteins and with characterized signaling properties will be tested for developmental function. If these studies support eh hypothesis that the signaling function of mu chain is important for developmental progression, then in Aim 2 we shall tests the abilities of various chimeric proteins that have signaling regions of lg-alpha or Ig- beta grafted onto them. In particular, we hope to be able to introduce into the pre-B cells chimeric molecules with developmental function that is activated by exogenous crosslinking. Such molecules will allow us to study the signaling events activated in the pre-B cell. In Aims 3 and 4, alterations in signaling components will be analyzed for their effects on B cell development. Dr. Lowell Project 3 in this SCOR application will generate Lyn-deficient mice. Given the likely role of Lyn in membrane Ig signaling, the properties of pre-B cells from these mice will be quite interesting and will be analyzed in this project. These cells will be cultured and, if they exhibit may defect in development, we shall complement that by introducing wild type and altered forms of Lyn. In addition, we shall introduce a variety of dominant negative mutant forms of signaling components (including Lyn, Syk, and Ras) into cultured pre-B to examine their effect on mu heavey chain-induced developmental maturation.
Keywords: B lymphocyte, biological signal transduction, gene rearrangement, immunoglobulin, leukopoiesis, chimeric protein, crosslink, gene mutation, immunoglobulin gene, immunoglobulin structure, protein structure /function, protooncogene, laboratory mouse, tissue /cell culture, transfection /expression vector
Project start date: 2000-01-01
Project end date: 2000-12-31
LPS-INDUCED SIGNALING IN MACROPHAGES
Anthony L Defranco, Professor
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5R01AI033442-07 from National Institute Of Allergy And Infectious Diseases IRG: BM
Abstract: Adapted from applicant s ) The immune system is composed of both innate and adaptive components. The latter, involving antibodies and T cell-mediated cellular immunity is exquisitely sensitive, but requires time for expansion of the rare antigen-specific cells upon initial infection. In contrast, the innate immune system utilizes evolved recognition mechanisms and provides a first line of defense until the adaptive response can reach a significant magnitude. Among the innate immune responses are the responses of macrophages to bacterial cell wall components. The best studied of these is bacterial lipopolysaccharide (LPS), which is the major component of the outer leaflet of the outer membrane of gram-negative bacteria. Recent progress has identified CD14 as an important recognition component for responses to LPS and other bacterial cell wall components. While the responses of macrophages to LPS are ordinarily beneficial, in that they are pro-inflammatory and attract immune effector cells to the site of infection, when activated systemically, as in cases of bacterial sepsis, the result is life-threatening septic shock. This proposal is concerned with understanding the intracellular events that occur in macrophages upon contact with physiologically relevant concentration of LPS, with the view that such understanding may lead to improved methods of treating septic shock. Experiments of AIM 1 will determine the roles of the three MAP kinase-like pathways (classical MAP kinase, p38/HOG-1, and JNK) in mediating the responses to LPS. Experiments of AIM 2 will study the roles of the intracellular tyrosine kinases Syk, Hck, Lyn, and Fgr in initiating LPS signaling events by examining macrophages from mice made genetically defective in one or more of these tyrosine kinases. In Aim 3, the mechanism by which the signaling adapter protein Shc becomes tyrosine phosphorylated in response to LPS will be determined, as this may allow us to identify the putative signaling chain of the LPS receptor complex. Moreover, these experiments will assess the role of Shc in activation of Ras in response to LPS and in mediating other signaling events. Finally, in this aim, the role of the signaling component Vav in LPS responses will be assessed by studying LPS responses in macrophages from Vav-deficient mice.
Keywords: biological signal transduction, enzyme activity, lipopolysaccharide, macrophage, mitogen activated protein kinase, protein tyrosine kinase, bactericidal immunity, isozyme, leukocyte activation /transformation, phosphoprotein, protein structure /function, laboratory mouse, tissue /cell culture, transfection
Project start date: 1993-08-01
Project end date: 2001-07-31
5R01AI033442-07 (1999): $197969
5R01AI033442-06 (1998): $192203
5R01AI033442-05 (1997): $186605
Anthony L Defranco
University Of California San Francisco
Project start date: 1988-09-01
Project end date: 2014-05-31
Sponsored Links Excellgen http://Excellgen.com
Regulation Of B Lymphocyte Proliferation By Antigen
Anthony L Defranco, Professor
Microbiology And Immunologyuniversity Of California San Francisco
Grant 5R01AI020038-25 from National Institute Of Allergy And Infectious Diseases IRG: CMI
Project start date: 1984-01-01
Project end date: 2010-11-30
5R37AI020038-21 (2004): $426458
5R37AI020038-20 (2003): $414037
5R37AI020038-19 (2002): $401979
MOLECULAR AND CELLULAR IMMUNOLOGY
Anthony L Defranco, Professor
Microbiology And Immunologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 5T32AI007334-15 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Project start date: 1988-09-01
Project end date: 2003-05-31
5T32AI007334-15 (2002): $322044
REGULATION OF B LYMPHOCYTE PROLIFERATION BY ANTIGEN
Anthony L Defranco, Professor
George Williams Hooper Fdnuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 5R37AI020038-18 from National Institute Of Allergy And Infectious Diseases IRG: NSS
Keywords: B cell receptor, B lymphocyte, biological signal transduction, immunoregulation, lymphocyte proliferation apoptosis, enzyme activity, gene expression, phosphorylation, protein kinase, ribozyme autoradiography, laboratory mouse, laboratory rabbit, molecular cloning, nucleic acid sequence, tissue /cell culture, transfection
Project start date: 1984-01-01
Project end date: 2005-03-31
5R37AI020038-18 (2001): $390270
MOLECULAR AND CELLULAR IMMUNOLOGY
Anthony L Defranco, Professor
Microbiology And Immunologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 5T32AI007334-14 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Project start date: 1988-09-01
Project end date: 2003-05-31
5T32AI007334-14 (2001): $313315
REGULATION OF B LYMPHOCYTE PROLIFERATION BY ANTIGEN
Anthony L Defranco, Professor
George Williams Hooper Fdnuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 4R37AI020038-17 from National Institute Of Allergy And Infectious Diseases IRG: NSS
Project start date: 1984-01-01
Project end date: 2005-03-31
4R37AI020038-17 (2000): $378904
MOLECULAR AND CELLULAR IMMUNOLOGY
Anthony L Defranco, Professor
Microbiology And Immunologyuniversity Of California San Francisco
3333 California St., Ste 315
san Francisco, Ca 941430962
Grant 5T32AI007334-13 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Project start date: 1988-09-01
Project end date: 2003-05-31
5T32AI007334-13 (2000): $289249
5T32AI007334-12 (1999): $269118
Sponsored Links Excellgen http://Excellgen.com