PEPTIDE MEDIATED IMMUNOTHERAPY FOR AUTOIMMUNE DISEASE
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS028759-03 from National Institute Of Neurological Disorders And Stroke IRG: IMS
Abstract: The development of non-toxic, immunospecific therapy for autoimmune disease is a major goal of this work. The project is based on the demonstration in experimental autoimmune encephalomyelitis (EAE) that it is possible to prevent or reverse paralytic disease with monoclonal antibodies (mAb) specific for the T cell receptor (TcR) V region or for the class II major histocompatibility complex (MHC) molecule that presents myelin basic protein (MBP). Further, it is possible to design non-immunogenic peptides that block the ability of encephalitogenic myelin fragments to bind to MHC. In this project further development of blocking peptides for treatment of EAE will be undertaken. Peptides that not only block MHC but do not bind TcR will be tested. Finally, molecular vaccination with V-DJ and V-J peptides from the TcR sequence of encephalitogenic T cells will be used to treat EAE. These approaches using therapeutic peptides could lead to the rational design of drugs for autoimmune disease.
Keywords: DNA binding protein, autoimmune disorder, experimental allergic encephalomyelitis, immunotherapy, peptide, T cell receptor, T lymphocyte, antigen presentation, chemical binding, major histocompatibility complex, passive immunization, protein sequence, Freund s adjuvant, clone cell, laboratory mouse, myelin basic protein
Project start date: 1990-08-01
Project end date: 1993-11-30
5R01NS028759-03 (1993): $219022
Sponsored Links Excellgen http://Excellgen.com
PEPTIDE MEDIATED IMMUNOTHERAPY FOR AUTOIMMUNE DISEASE
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS028759-13 from National Institute Of Neurological Disorders And Stroke IRG: IMS
Abstract: This grant has funded work on altered peptide ligands (APL) for therapy of autoimmune disease, specifically multiple sclerosis (MS). Work undertaken during the previous five years has provided a framework for the first clinical trials on patients with relapsing remitting MS. In the next grant period we propose to carry out pre-clinical experiments that will optimize APL therapy in animal models of MS, and to develop the next generation of APL therapy utilizing techniques involving vaccination with naked DNA. We will also demonstrate how microbes have the capacity to modulate autoimmune disease via structural mimicry with self-molecules. The specific aims of this grant are to 1) subvert epitope spreading with APL. In this aim we will see whether after intermolecular epitope spreading has occurred, if an APL which targets a single epitope on a single myelin molecule, would be able to suppress ongoing EAE, and reverse paralysis. This is a stringent demand, but will be a useful paradigm to optimize therapeutic regimens in Phase II clinical trials with relapsing remitting MS patients. In Aim 2 we will analyse how APL pulsed dendritic cells will serve to reverse EAE. In Aim 3 we will attempt APL therapy via vaccination with DNA minigenes encoding myelin epitopes. We have succeeded in preventing EAE with DNA vaccination using a minigene encoding a myelin PLP epitope. We will optimize this approach utilizing myelin minigenes paired in tandem with cytokine and chemokine constructs. Finally in Aim 4 we will study APL as microbial mimics that modulate EAE. We have succeeded with preliminary studies, in demonstrating subversion of epitope spreading, with microbial sequences that mimic myelin epitopes. The optimization of such APL derived from microbes will be studied here. This work with APL may be applicable not only to MS, but to other autoimmune diseases including juvenile diabetes, rheumatoid arthritis, and inflammatory bowel disease.
Keywords: autoimmune disorder, experimental allergic encephalomyelitis, immunotherapy, myelin basic protein, peptide analog, vaccine development, T lymphocyte, antigen presentation, chemokine, circular DNA, cross immunity, cytokine, dendritic cell, epitope mapping, human papillomavirus, lymph node, neurotropic virus, vector vaccine, animal genetic material tag, clone cell, enzyme linked immunosorbent assay, histology, immunization, laboratory mouse, laboratory rat
Project start date: 1990-07-01
Project end date: 2006-06-30
5R01NS028759-13 (2003): $399728
5R01NS028759-12 (2002): $390270
5R01NS028759-11 (2001): $381086
5R01NS028759-10 (2000): $372172
5R01NS028759-02 (1992): $204301
PEPTIDE-MEDIATED IMMUNOTHERAPY FOR AUTOIMMUNE DISEASE
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS028759-07 from National Institute Of Neurological Disorders And Stroke IRG: EI
Abstract: Our goal is to design of T cell receptor and MHC blocking peptides with predictable properties that might be used therapeutically to treat multiple sclerosis. Originally we formulated a non-immunogenic peptide based on the based on the sequence of myelin basic protein (MBP) that binds to I-Au with much greater affinity relative to the encephalitogenic peptide Ac1-11 of MBP. This peptide reverses EAE, and even prevents subsequent relapses, if given at the time that signs of EAE first begin to appear. We shall extend these findings to develop inhibitors of MHC and of TCR specific for MBP peptide 87-99 in the Lewis rat. The reason for pursuing inhibitors of the TCR and of MHC-binding of peptide 87-99, follows from some serendipitous finding regarding a major set of TCR rearrangements in MS brain lesions. We analyzed T cell receptor (TCR) gene rearrangements directly from MS brain plaques. Rearrange Vbeta 5.2 genes were detected in the brains of all patients who were HLA DR2. A common Vbeta t.2-Dbeta-Jbeta sequence in these MS brain plaques was identical to that described for the VDJ region of a Vbeta 5.2 T cell clone. This clone from an MS patient, who was HLA DR2, was cytotoxic for targets with MBP peptide 89-106. The deduced amino acid sequence of this VDJ rearrangement, LRG, was also described previously in T Cells, cloned from EAE lesions, which were specific for MBP peptide 87-99. VDJ sequences with specificity for this MBP epitope constitute a large fraction (40%) of the TRC Vbeta 5.2N(D)N rearrangements in MS lesions. The capacity of T cells with these VDJ sequences to cause EAE, and the prevalence of such sequences in demyelinated lesions, indicated that T cells with this rearranged TCR, may be critical in MS. We have analyzed the MBP peptide 87-99 to determine the putative interaction sites with MHC and with TCR. Based on these studies we have designed peptide analogues of MBP 87-99 that interfere with either MHC or TCR binding, including the LRG motif found in the CDR3 of TCR commonly transcribed in MS lesions. Some of these TCR antagonists prevent EAE. We thus plan to develop MBP analogues that interfere with TCR and MHC recognition of MBP peptide 87-99. We will test these antagonists to determine if they can reverse EAE after the first signs of disease have appeared. We shall also develop CD4+ T cell clones specific for human MBP peptide 87-99 and restricted to HLA DRB1*1501(DR2), and test whether the blockers from the Lewis rat system also block their human counterparts. Ultimately we would hope to apply these results to clinical trials in MS.
Keywords: autoimmune disorder, experimental allergic encephalomyelitis, immunotherapy, multiple sclerosis, myelin basic protein, peptide, MHC class II antigen, T cell receptor, T lymphocyte, antigen presentation, cytokine, inhibitor /antagonist, peptide analog, animal genetic material tag, clone cell, human tissue, laboratory mouse, laboratory rat, peptide chemical synthesis, polymerase chain reaction
Project start date: 1990-08-01
Project end date: 1998-11-30
5R01NS028759-07 (1997): $218788
5R01NS028759-05 (1995): $209841
5R01NS028759-08 (1998): $227540
5R01NS028759-06 (1996): $216120
Grants awarded to Lawrence Steinman
TCR V GENE REPERTOIRE IN MS AND SELECTIVE IMMUNOTHERAPY
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS030201-06 from National Institute Of Neurological Disorders And Stroke IRG: IMS
Abstract: We have discovered VDJ beta and VJ alpha gene rearrangements in MS brain plaques that encode amino acid sequences identical to those seen in T cell clones from humans, mice and rats that have specificity for myelin basic protein (MBP) peptide 87-99. Using a high-efficiency expression system for TCR alpha and beta chains, we will rigorously prove that a major T cell response in the MS lesion is directed to MBPp87-99. We have developed potent T cell receptor antagonists for p87-99 that suppress ongoing paralysis in rodent models of EAE, and suppress proliferative and cytotoxic responses to p87-99 in humans. We will determine the putative contacts for amino acids in the TCR CDR3 regions with residues of p87-99. A core motif containing MBP87-91 (VHFFK) is found in this epitope. A number of microbes share sequence homology with this core motif-VHFFK, and stimulate MBPp87-99 specific T cells. We will determine whether microbial epitopes can trigger the transfected construct, as efficiently as the native MBP epitope. This would indicate how these T cells might become activated during infection, supporting the notion that self- reactivity arises from the molecular mimicry of self-antigens by microbes. Since T cells reactive to MBP p87-99 trigger EAE, we will test whether these microbial sequences either trigger EAE, or alternatively whether they serve as TCR or MHC antagonists and can block disease induction. Thus we will consider whether molecular mimics of MBP epitopes can induce disease or protect from pathology. We have demonstrated that a dominant antibody response found in the MS brain plaque and in MS cerebrospinal fluid is directed to an overlapping region of the MBP molecule from p86-99. This epitope is identical to the epitope restricted by HLA DR2 beta (HLA DRB1*1501), and overlaps with the epitope restricted by HLA DR2 alpha (HLA DRB5*0101). We will analyze immunoglobulin gene rearrangements in MS brain using RT-PCR, and in CSF using single cell PCR. We will see whether there are restricted Ig CDR3 motifs in MS. We will compare these CDR3 motifs to those found in murine monoclonal antibodies raised against the identical epitope p86-99. Our persistent goal is to develop selective immunotherapy for multiple sclerosis, a chronic disease of the central nervous system affecting approximately 250,000 Americans.
Keywords: T cell receptor, gene expression, gene rearrangement, immunoglobulin gene, multiple sclerosis, B lymphocyte, bacterial antigen, gene mutation, immunotherapy, myelin basic protein, receptor binding, human genetic material tag, human tissue, laboratory rat, polymerase chain reaction, postmortem, site directed mutagenesis, tissue /cell culture, transfection
Project start date: 1992-12-01
Project end date: 2000-04-30
5R01NS030201-06 (1998): $249835
5R01NS030201-05 (1997): $267182
DNA VACCINATION AS EAE IMMUNOTHERAPY
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5P01AI036535-080007 from National Institute Of Allergy And Infectious Diseases
Abstract: Vaccination was initiated empirically 200 years ago by Jenner and consolidated conceptually 100 years ago by Pasteur, and the procedure still provides surprises. Among the latest is vaccination with DNA. A particular variable region gene of the T cell receptor, Vbeta8.2, is rearranged and its product is expressed on pathogenic T cells that induce experimental autoimmune encephalomyelitis (EAE) and its product is expressed on pathogenic T cells that induce experimental autoimmune encephalomyelitis (EAE) in H-2/u mice following immunization with myelin basic protein (MBP). Vaccination of H-2/u mice with naked DNA pathogenic portion of the MBP molecule, indicated in the vaccinated mice there was a reversal of the autoimmune response from Th2 to Th2. This shift may make this approach attractive for treatment of recently of Th1 mediated diseases like multiple sclerosis, juvenile diabetes, and rheumatoid arthritis. We have recently extended this approach to the treatment of autoimmune diseases with altered peptide ligands. In this portion of the program project proposal, we aim to 1. Extend studies on the mechanism whereby DNA vaccination with TCR Vb constructs induces Th2 responses and suppresses EAE. 2. Test vaccination with DNA minigens encoding myelin epitopes for peptide- and APL-based treatment of EAE. 3. Investigate bacterial CpG sequences inducing gamma interferon and IL- 12, and their application for suppression of EAE. 4. Utilize DNA vaccination to chemokines for treatment of EAE. 5. Develop tandem cytokine and chemokine constructs for treatment of EAE.
Keywords: experimental allergic encephalomyelitis, gene therapy, immunotherapy, vaccine development, vector vaccine, T cell receptor, autoimmunity, chemokine, disease /disorder model, immune tolerance /unresponsiveness, interferon gamma, interleukin 10, interleukin 12, interleukin 4, multiple sclerosis, myelin basic protein, transfection /expression vector, laboratory mouse, laboratory rat, polymerase chain reaction
Project start date: 2001-09-01
Project end date: 2003-08-31
DELIVERY OF BIOPOLYMERS USING CATIONIC PEPTIDES
Lawrence Steinman, Professor
Neurology & Neurological Scisstanford University
stanford, Ca 94305
Grant 1R01AI040968-01 from National Institute Of Allergy And Infectious Diseases IRG: IMS
Abstract: The long term objective of this proposal is to use short, cationic peptides as a efficient method of delivering biopolymers, such as intact autoantigens, immunodominant peptides, and antisense oligonucleotides into the cytoplasm of antigen presenting cells and T lymphocytes to induce antigen specific anergy and/or affect the profile of secreted cytokines as a therapy for experimental allergic encephalomyelitis (EAE). We have demonstrated that conjugation of a peptide, corresponding to nine amino acids from the HIV tat protein, to protein antigens results in their rapid uptake by a variety of cells and permits the molecules to enter both the MHC class I and ll biosynthetic pathways, which results in a dramatic increase in their antigenicity and immunogenicity. In addition, to delivering antigens to the MHC class I and ll molecules, we propose to use the tat peptide to transport PNA antisense reagents into cells to modulate cytokine production characteristic of inflammation. The studies outlined in this project seek to explore the therapeutic potential of specifically suppressing proinflammatory cytokine expression by introducing antisense PNA for the transcriptional activator NF-kappaB into murine T cell clones. If successful, these methods could be used as therapeutic strategies in mice in vivo to reduce inflammation in the CNS of mice. This project has three specific aims, 1. demonstrate that conjugation of the tat peptide to intact myelin basic protein and proteolipid protein as well as their immunodominant determinants and altered peptide ligands dramatically increases their ability to induce antigen specific tolerance and/or modify the cytokine profile of the autoantigen specific clones, 2. demonstrate that tat conjugated proteins and peptides enter antigen presenting cells, such as dendritic cells and small resting B cells, and explore whether adoptive transfer of the antigen loaded cells more efficiently induces either immune stimulation or tolerance than immunization with the peptides or proteins, and 3. demonstrate that the expression of a variety of proinflammatory proteins can be dramatically reduced with HIV-1 tat conjugated antisense PNA against the 65 kilodalton subunit of NF-kappaB in vitro and that the antisense PNA constructs can reduce inflammation when directly injected into rodents with EAE
Keywords: experimental allergic encephalomyelitis, human immunodeficiency virus 1, immunoconjugate, immunotherapy, method development, transport protein, virus protein antigen presentation, antisense nucleic acid, cytokine, gene expression, immune tolerance /unresponsiveness, immunization, myelin basic protein laboratory mouse, laboratory rat, tissue /cell culture
Project start date: 1997-03-01
Project end date: 2000-02-29
1R01AI040968-01 (1997): $177813
5R01AI040968-03 (1999): $188329
5R01AI040968-02 (1998): $182843
Molecular And Cellular Immunobiology
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5T32AI007290-22 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: The Immunology Program at Stanford seeks to provide the best training possible for our pre- and postdoctoral students. The Program is interdepartmental in organization, with 46 faculty from 11 departments and 4 divisions (in the Department of Medicine), in the Schools of Medicine and Humanities and Sciences. The faculty s research is at the forefront of the key areas in immunology today - molecular, cellular, and clinical -- and they are leaders in the development of new technologies and in the application of current knowledge of basic immune mechanisms to the investigation of human immunological diseases and their treatment. The research and training activities in immunology benefit from this multidisciplinary approach and from our tradition of interactions and collaborations among the labs. Predoctoral trainees are required to develop strong background in basic biomedical sciences through coursework and they receive extensive and broad-based research training through laboratory rotations and thesis research. Both pre- and postdoctoral trainees develop professional skills and perspectives through participation in program activities including the weekly Immunology Seminar Series (bringing in over 30 scientists from around the world each year), 1 graduate student and 2 postdoctoral fellow journal clubs in immunology, the annual Stanford Immunology Scientific Conference at Asilomar, and the course in the Responsible Conduct of Research. Trainees are encouraged to present their research at local and national conferences. Trainees have access to modern laboratories, many in new or newly-renovated buildings, and to state-of-the-art specialized research facilities such as the cell-sorting and analysis (FACS) facility, the Protein and Nucleic Acid Facility, the Electron Microscope and Cell Imaging Facilities, the Molecular Modeling Facility, and the transgenic/gene knockout mouse facilities.
Project start date: 1985-09-01
Project end date: 2010-08-31
5T32AI007290-22 (2006): $1301635
Sponsored Links Excellgen http://Excellgen.com
2T32AI007290-21 (2005): $1591964
5T32AI007290-23 (2007): $1571338
2T32AI007290-26 (2010): $970818
TCR V GENE REPERTOIRE IN MS AND SELECTIVE IMMUNOTHERAPY
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS030201-03 from National Institute Of Neurological Disorders And Stroke IRG: NEUC
Abstract: Susceptibility to multiple sclerosis (MS) is linked to genes of the immunoglobulin supergene family, including the HLA class II genes, genes encoding the immunoglobulins, and genes encoding the T cell receptor (TCR). We and others have previously described germline polymorphisms in lg and TCR V genes associated with susceptibility to MS. Using PCR technology we have amplified rearranged TCR Valpha and Vbeta genes directly from MS brain plaque. There is limited diversity of TCR Valpha and Vbeta gene rearrangements in areas of demyelination. Sequencing further reveals limited junctional diversity. Finally, in a particular molecular phenotype HLA-DRB1 *1501, DQA1*0102, DQB1*0602, DPB1*0401, associated with the HLA-DR2Dw2 haplotype linked to MS, there is preferential rearrangement of Vbeta5.2 and Vbeta6. Based on animal models such observations may have clinical relevance. We have shown that antibodies to TCR V gene products, as well as to class II histocompatibility molecules, associated with susceptibility to experimental autoimmune encephalomyelitis have been used successfully to treat clinical paralysis in mice. Identification of HLA class II and TCR V genes involved in the pathogenesis of MS may allow us to design therapies in man, similar to that which we have used successfully in experimental animals.
Keywords: T cell receptor, gene expression, multiple sclerosis, genetic polymorphism, histocompatibility antigen, immunotherapy, meningitis, myelin basic protein, myelinopathy, nervous disorder, nucleic acid sequence, clone cell, human subject, human tissue, immunocytochemistry, polymerase chain reaction
Project start date: 1992-12-01
Project end date: 1995-11-30
5R01NS030201-03 (1995): $174521
5R01NS030201-02 (1994): $169618
ETHNIC VARIATION AND AUTOIMMUNITY
Lawrence Steinman
Institution:
Grant 5P01GM028428-150009 from National Institute Of General Medical Sciences
Abstract: Susceptibility to many diseases, suspected to be of autoimmune origin, is linked to genes of the immunoglobulin supergene family. These genes include the class II genes of the major histocompatibility complex, the immunoglobulin genes, and the genes encoding the antigen-specific receptor on T lymphocytes. We will study three diseases, including myasthenia gravis and pemphigus vulgaris, that are clear examples of autoimmune conditions mediated by antibody, and multiple sclerosis, a putative autoimmune neurologic disease. We will study associations between MHC class II genes, immunoglobulin genes, and TcR genes and these diseases using RFLP analysis and genomic sequencing, utilizing the polymerase chain reaction technology. We have seen that associations between class II HLA genes, immunoglobulin heavy chain allotype and T cell receptor genotype and susceptibility to disease, are often maintained in individuals with these diseases of different ethnic backgrounds. These data provide strong support for a direct participation for these genes in autoimmune disease, since it is unlikely that an allele linked to susceptibility would have remained in linkage with the same alleles across ethnic boundaries. We will study patients with MS, MG and PV in North America, Japan, Australia, France, Austria, and Israel (Arab and Jew). These associations between immunoglobulin supergenes and disease have been exploited in order to design new therapeutic approaches from human autoimmune disease that have shown results in treating autoimmune conditions in laboratory animals including rhesus monkeys.
Keywords: autoimmunity, biological polymorphism, ethnic group, genetic disorder, histocompatibility gene, molecular genetics, racial /ethnic difference, Australia, France, Israel, Japan, North America, antigen receptor, disease proneness /risk, epidemiology, gene expression, gene therapy, genetic manipulation, immunotherapy, myasthenia gravis, pemphigus, autoradiography, endonuclease, human genetic material tag, human subject, nucleic acid hybridization, nucleic acid sequence, polymerase chain reaction
Sponsored Links Excellgen http://Excellgen.com
IN VIVO TREATMENT OF EAE WITH ANTI I-A ANTIBODIES
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS018235-06 from National Institute Of Neurological Disorders And Stroke IRG: IMS
Abstract: Susceptibility to many autoimmune diseases is linked to the immune response genes (IR genes), found either in the I region of the murine major histocompatibility comples (H-2) or in the HLA region of the human major histocompatibility complex. This relationship between disease susceptibility and IR genes has been exploited to develop a novel immunosuppressive therapy for models of autoimmune disease including acute and chronic relapsing experimental allergic encephalomyelitis (EAE), experimental autoimmune myasthenia gravis (EAMG), NZB/W F1 lupus-nephritis, experimental autoimmune thyroiditis, streptozocin induced diabetes mellitus, and collagen induced arthritis. In EAE anti I-A antibodies can reverse ongoing paralytic disease. The mechanism whereby anti I-A suppresses EAE will be studied using T cell clones reactive to myelin basic protein that induce EAE. In addition, the effect of anti I-A on the general cellular and humoral status of the recipient will be examined. Finally, the role of IR gene products in the antigen specific suppression of EAE with myelin basic protein-spleen cell conjugates will be examined.
Keywords: GLOBULINS, GAMMA GLOBULINS, IMMUNOGLOBULIN A, IMMUNITY, IMMUNOTHERAPY, IMMUNIZATION, IMMUNOGENETICS, HISTOCOMPATIBILITY GENES, IMMUNE RESPONSE GENES, IMMUNOLOGICAL SCIENCES STUDY SECTION, IMMUNOLOGY, ANTI-ANTIBODIES (GENERAL), NERVOUS DISORDERS CENTRAL, ENCEPHALITIS, BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, B LYMPHOCYTES, BLOOD CELLS, T LYMPHOCYTES, BLOOD CELLS, T LYMPHOCYTES, SUPPRESSOR, GENETICS, GENETIC REGULATION, IMMUNITY, IMMUNOREGULATION, IMMUNOGENETICS (GENERAL), IMMUNOGENETICS, MAJOR HISTOCOMPATIBILITY COMPLEX (LOCUS), IMMUNOLOGICAL PREPARATIONS, MONOCLONAL ANTIBODIES, IMMUNOPATHOLOGY, AUTOIMMUNE DISORDERS, MAMMALS, RODENTS, MYOMORPHA, MICE (LABORATORY), TISSUE (CELL) CULTURE, TISSUE (CELL) CULTURE, CLONE CELLS
Project start date: 1982-04-01
Project end date: 1988-03-31
5R01NS018235-13 (1994): $289157
5R01NS018235-12 (1993): $276277
5R01NS018235-11 (1992): $238346
PEPTIDE-MEDIATED IMMUNOTHERAPY FOR AUTOIMMUNE DISEASE
Lawrence Steinman, Professor
Stanford University
stanford, Ca 94305
Grant 2R01NS028759-04 from National Institute Of Neurological Disorders And Stroke IRG: EI
Abstract: Our goal is to design of T cell receptor and MHC blocking peptides with predictable properties that might be used therapeutically to treat multiple sclerosis. Originally we formulated a non-immunogenic peptide based on the based on the sequence of myelin basic protein (MBP) that binds to I-Au with much greater affinity relative to the encephalitogenic peptide Ac1-11 of MBP. This peptide reverses EAE, and even prevents subsequent relapses, if given at the time that signs of EAE first begin to appear. We shall extend these findings to develop inhibitors of MHC and of TCR specific for MBP peptide 87-99 in the Lewis rat. The reason for pursuing inhibitors of the TCR and of MHC-binding of peptide 87-99, follows from some serendipitous finding regarding a major set of TCR rearrangements in MS brain lesions. We analyzed T cell receptor (TCR) gene rearrangements directly from MS brain plaques. Rearrange Vbeta 5.2 genes were detected in the brains of all patients who were HLA DR2. A common Vbeta t.2-Dbeta-Jbeta sequence in these MS brain plaques was identical to that described for the VDJ region of a Vbeta 5.2 T cell clone. This clone from an MS patient, who was HLA DR2, was cytotoxic for targets with MBP peptide 89-106. The deduced amino acid sequence of this VDJ rearrangement, LRG, was also described previously in T Cells, cloned from EAE lesions, which were specific for MBP peptide 87-99. VDJ sequences with specificity for this MBP epitope constitute a large fraction (40%) of the TRC Vbeta 5.2N(D)N rearrangements in MS lesions. The capacity of T cells with these VDJ sequences to cause EAE, and the prevalence of such sequences in demyelinated lesions, indicated that T cells with this rearranged TCR, may be critical in MS. We have analyzed the MBP peptide 87-99 to determine the putative interaction sites with MHC and with TCR. Based on these studies we have designed peptide analogues of MBP 87-99 that interfere with either MHC or TCR binding, including the LRG motif found in the CDR3 of TCR commonly transcribed in MS lesions. Some of these TCR antagonists prevent EAE. We thus plan to develop MBP analogues that interfere with TCR and MHC recognition of MBP peptide 87-99. We will test these antagonists to determine if they can reverse EAE after the first signs of disease have appeared. We shall also develop CD4+ T cell clones specific for human MBP peptide 87-99 and restricted to HLA DRB1*1501(DR2), and test whether the blockers from the Lewis rat system also block their human counterparts. Ultimately we would hope to apply these results to clinical trials in MS
Keywords: autoimmune disorder, experimental allergic encephalomyelitis, immunotherapy, multiple sclerosis, myelin basic protein, peptide MHC class II antigen, T cell receptor, T lymphocyte, antigen presentation, cytokine, inhibitor /antagonist, peptide analog animal genetic material tag, clone cell, human tissue, laboratory mouse, laboratory rat, peptide chemical synthesis, polymerase chain reaction
Project start date: 1990-08-01
Project end date: 1998-11-30
2R01NS028759-04 (1994): $181299
IN VIVO TREATMENT OF EAE WITH ANTI-IA ANTIBODIES
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS018235-03 from National Institute Of Neurological Disorders And Stroke IRG: IMS
Abstract: Experimental allergic enciphalitis (EAE) serves as a model of demyelinating disease of the central nervous system. The disease follows T cell sensitization to myelin basic protein, and is controlled in part by immune response genes in the major histocompatibility complex. We have recently prevented EAE in genetically susceptible mice by in vivo administration of antibodies reactive to products of immune response genes. A monoclonal anti I-A antibody was particularly effective. We propose to study the mechanisms whereby antibodies to producet of immune response genes prebent EAE. The mode of action of these antibodies on the homing of sensitized cells to brain, on the sensitization of T cells by myelin protein laden macrophages, and ont eh induction of suppressor cells to myelin protein will be evaluated. An antiserum directed against I-J determinants, found on suppressor T cells, will be used in vivo to try to induce passive relapsing EAE. Since the I region in mice is comparable to the HLA-D region in humans, the long range possibility exists for the use of this type of therapy in HLA-D linked diseases, like multiple sclerosis. This possibility is enhanced by the observation that in vivo treatment with an anti-I-A antibody in an F(1) hybrid produced haplotype specific suppression. Since individuals are usually heterozygous at the critical HLA locus associated with disease susceptibility, suppression of the haplotype controlling disease susceptibility might attenuate the illness, while leaving the immune system otherwise intact. Such therapy might be feasible with anti-HLA-D antibodies as they become available.
Keywords: IMMUNITY, IMMUNOTHERAPY, IMMUNIZATION, IMMUNOGENETICS, HISTOCOMPATIBILITY GENES, IMMUNE RESPONSE GENES, IMMUNOLOGICAL SCIENCES STUDY SECTION, IMMUNOLOGY, ANTIBODIES, ANTI-ANTIBODIES, NERVOUS DISORDERS CENTRAL, ENCEPHALITIS, BLOOD AND RE SYSTEM, MACROPHAGES, BLOOD CELLS, B LYMPHOCYTES, BLOOD CELLS, T LYMPHOCYTES, BLOOD CELLS, T LYMPHOCYTES, SUPPRESSOR, GENETICS, GENETIC REGULATION, IMMUNITY, IMMUNOREGULATION, IMMUNOLOGICAL PREPARATIONS, MONOCLONAL ANTIBODIES, MAMMALS, RODENTS, MYOMORPHA, MICE (LABORATORY), TISSUE (CELL) CULTURE
Project start date: 1982-04-01
Project end date: 1985-03-31
Sponsored Links Excellgen http://Excellgen.com
Large Scale Images Of Gene Transcription In MS And EAE
Lawrence Steinman, Professor
Neurology & Neurological Scisstanford University
stanford, Ca 94305
Grant 1R01NS041402-01A1 from National Institute Of Neurological Disorders And Stroke IRG: IMS
Abstract: EAE has served as a useful model for MS, yet many therapies which succeed in preventing or reversing paralysis in the animal model, do not succeed when applied to MS. However, approved drugs for MS like beta interferon and Copaxone have been successful in both EAE and MS. There are several convenient models of EAE in rodents with both acute and relapsing features along with demyelination. We shall examine transcription profiles in MS lesions using gene microarray technologies, comparing the transcriptional profiles of these lesions from six MS brains. Genes of interest found in microarray analysis will be corroborated with real time PCR RNAse protection, and Western blotting in selective cases. Lesions, where mRNA was isolated, will also be characterized histopathologically and immunohistochemically. We shall continue comparison of active and chronic lesions, where we have already identified key differences in transcription of immunoglobulin genes p38kinase and alpha-1-antichymotrypsin. Profiles from MS brain will be compared to those obtained from the CNS of rodents with relapse, remission, or acute attacks of EAE. We shall also study transcriptional profiles of pathogenic T cell clones that cause EAE, either stimulated with native myelin peptide or altered peptide ligands. APL´s have been successful in treating EAE, and have now been taken into phase II trials in MS. Finally we will study the role of osteopontin a gene identified in large scale transcriptional profiling of EAE and MS. We demonstrate that osteopontin is expressed in MS and EAE lesions, and that in animals with the osteopontin gene deleted there is a profound change in the regulation of disease relapses. The discovery of the role of osteopontin, first identified in large scale profiles, exemplifies how this approach may help us to understand MS and to develop new therapies
Keywords: experimental allergic encephalomyelitis, functional genomics, genetic transcription, molecular pathology, multiple sclerosis T lymphocyte, biological signal transduction, disease /disorder model, leukocyte activation /transformation, myelin basic protein, osteopontin, protein tyrosine kinase clinical research, high throughput technology, human tissue, laboratory mouse, microarray technology
Project start date: 2001-01-15
Project end date: 2004-12-31
1R01NS041402-01A1 (2002): $339305
5R01NS041402-03 (2004): $358124
5R01NS041402-02 (2003): $348579
DNA Vaccination To Prevent Autoimmune Disease
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5U19DK061934-020002 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZAI1
Abstract: DNA vaccination is an effective means of protecting experimental animals against infectious pathogens and cancer, and has more recently been used to prevent and reverse autoimmune disease. We have demonstrated that vaccination with DNA encoding TCR Vbeta elements, or with DNA sequences encoding myelin antigens can prevent or reverse experimental autoimmune encephalomyelitis. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by T cell-mediated destruction of the insulin-secreting Beta cells in the pancreas. The NOD mouse is an animal model of IDDM in which several autoantigens, including insulin, have been identified. In preliminary studies we have demonstrated that vaccination of NOD mice with DNA encoding an immunodominant peptide of insulin-residues 9-23 of the B chain-protects the animals from developing diabetes. Animals injected intramuscularly with a bacterial plasmid encoding the insulin B peptide show lower disease incidence and delayed onset of disease. Protection is mediated by insulin B (9-23)-specific downregulation of IFN-g. DNA immunization is explored here as a potential therapy in the treatment of autoimmune diabetes. We will rely on our growing experience with DNA vaccination in the prevention and treatment of EAE. These studies will complement the sections on therapy with genes introduced by retroviral vectors in the sections on in vitro gene transfer in Dr. Fathman s project, and studies of epitope spreading using a proteomic approach in Dr. Utz project. In particular, can we predict efficacious cDNAs using the autoantibody screen with peptide microarrays described in Project 3? These studies will attempt to correlate prevention with epitope selection in EAE as well as the studies described above on IDDM. In Aim 1, Reversal of Hyperglycemia, we will investigate whether diabetes in the NOD mouse can be reversed beginning immunization after the onset of glucosuria using DNA immunization to insulin residues 9-23 of the B chain. In Aims 2 and 3 we will utilize IL-4 or IL-12 p40, or NGF as adjuvants with cDNA vaccination. In Aim 4 we will test DNA vaccination with genes encoding multiple islet cell antigens, including insulin, GAD and hsp65. In Aim 5 we will use protein microarrays to identify potential cDNA vaccination candidates. These studies may lead to new therapies for prevention and treatment of autoimmune disease in man.
Keywords: active immunization, autoimmune disorder, disease /disorder prevention /control, experimental allergic encephalomyelitis, gene therapy, immunotherapy, vector vaccine, autoantibody, cooperative study, immunomodulator, insulin dependent diabetes mellitus, interleukin 12, interleukin 4, nerve growth factor, NOD mouse, biotechnology, microarray technology, polymerase chain reaction
DNA Vaccination For Autoimmunity Immunoinhibitory GpG Mo
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5P01AI036535-120007 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: Bacterial DNA and immunostimulatory CpG oligonucleotides (CpG-ODN) activate the innate immune system to produce proinflammatory cytokines. Shown to be potent Th1-like adjuvants, stimulatory CpG-motifs are currently utilized as effective therapeutic vaccines for various animal models of infectious diseases, tumors, allergies, and autoimmune diseases. We have shown that it is possible to induce antigen specific Th2 immunity to myelin, using a co-vaccination strategy with DNA encoding IL-4 and myelin proteins. This approach ameliorated and actually reversed ongoing EAE. We recently discovered that the application of an immunoinhibitory GpG-ODN, with a single base switch from CpG to GpG, can effectively inhibit the immunostimulatory response of its CpG-ODN counterpart. Moreover, this inhibitory GpG-ODN is not only capable of counteracting the stimulatory effect of CpG-ODN in vitro, it is also capable of suppressing the disease severity of experimental autoimmune encephalomyelitis (EAE) in mice, a Th1-mediated animal disease model for multiple sclerosis, and inducing a Th2 shift, much as DNA co-vaccination with genes encoding myelin and IL-4 [Garren et al, 2001]. We will explore the utility of the GpG motif for therapy of autoimmune disease, and examine the underlying mechanism whereby it exerts its effects. We will extend pre-clinical studies on the mechanism whereby GpG-ODN induces reduced expression of MHC class II, promotes a Th2 shift, and enhances antigen specific Th2 responses. We will test a co-vaccination strategy using GpG-ODN plus genes encoding myelin to prevent and reverse acute EAE, and to block further relapses, if given after the initial acute attack in chronic relapsing EAE. We will use our recently developed proteomic myelin array to monitor epitope spreading and the nature of the T cell response, when DNA vaccines encoding myelin proteins are used to treat relapsing remitting EAE after the acute attack.
Keywords: CpG island, autoimmunity, bacterial DNA, experimental allergic encephalomyelitis, vector vaccine, MHC class II antigen, T cell receptor, cell proliferation, gene expression, gene therapy, helper T lymphocyte, immune response, interleukin 4, multiple sclerosis, myelin, myelin basic protein, myelin proteolipid, phosphorylation, proteomics, SDS polyacrylamide gel electrophoresis, flow cytometry, laboratory mouse, polymerase chain reaction, western blotting
IN VIVO TREATMENT OF EAE WITH ANTI I-A ANTIBODIES
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS018235-20 from National Institute Of Neurological Disorders And Stroke IRG: ZRG5
Abstract: Adapted from s ) In this competing renewal application, the Principal Investigator wishes to expand his studies on immunotherapy for autoimmune disease by comparing selective anti-TCR therapy with less selective approaches targeting either class II MHC, adhesion molecules or cytokines. Three different models for the induction of relapsing EAE will be utilized; these include 1) spinal cord homogenate, 2) T-cell clones specific for MBP 1-11 and 3) superantigen. Five specific aims are proposed. Aim 1 is to determine whether highly selective anti-TCR therapy will succeed in clinical paradigms where "epitope spreading" might occur. Aim 2 is to determine whether non-selective anti-cytokine therapy or anti- adhesion molecule therapy will be beneficial in clinical paradigms where "epitope spreading" does occur. Aim 3 is to assess the role of the cytokines IL-4 and IL-10 in suppression of EAE. In Aim 4, the role of a variety of adhesion molecules, cytokines, class II and CD4 in selective and non- selective homing to the CNS will be assessed. Aim 5 is to assess the possibility of using DNA vaccination against TCR Vb for treatment of EAE. It is hoped that these studies will provide the basis for future clinical trials in MS patients.
Keywords: antireceptor antibody, experimental allergic encephalomyelitis, immunotherapy, nonhuman therapy evaluation, MHC class II antigen, T cell receptor, cell adhesion molecule, disease /disorder prevention /control, interleukin 10, interleukin 4, spinal cord, superantigen, laboratory mouse
Project start date: 1982-04-01
Project end date: 2002-02-28
5R01NS018235-20 (2001): $494981
5R01NS018235-19 (2000): $475945
Sponsored Links Excellgen http://Excellgen.com
5R01NS018235-18 (1999): $457640
5R01NS018235-17 (1998): $440040
5R01NS018235-16 (1997): $380272
3R01NS018235-19S1 (2000): $50000
AUTOANTIBODY ARRAYS GUIDE TOLERGENIC THERAPY FOR MULTIPLE SCLEROSIS
Lawrence Steinman, Professor
Stanford University, 340 Panama Street, Stanford, Ca 94305-6203
Grant 5R01NS055997-03 from National Institute Of Neurological Disorders And Stroke
Keywords: 2ar peptide; ATGN; Animal Model; Animal Models and Related Studies; Animals; Antibodies; Antigenic Determinants; Antigens; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Responses; Autoimmune Status; Autoimmunity; Autologous Antigens; B cell activating factor; B cell growth factor; B-Cell Differentiation Factor-1; B-Cell Growth Factor-1; B-Cell Growth Factor-I; B-Cell Proliferating Factor; B-Cell Stimulating Factor; B-Cell Stimulating Factor-1; B-Cell Stimulation Factor-1; B-Cell Stimulatory Factor-1; BAF; BCDF-1; BCGF; BCGF-1; BCSF 1; BSF-1; BSF1; BSF1 (B cell stimulating factor 1); Binding Determinants; Binetrakin; CNS Diseases; CNS disorder; Carbohydrates; Central Nervous System Diseases; Central Nervous System Disorders; Chemicals; Choline Chloride Dihydrogen Phosphate; Choline Phosphate; Choline Phosphate Chloride; Clinical; D-Mannose; Development; Disease; Disease Progression; Disorder; EAE; Encephalomyelitis; Encephalomyelitis, Allergic; Epitopes; Eta-1 protein; Eta-1-Op protein; Ethanaminium, N, N, N-trimethyl-2-(phosphonooxy)-, chloride; Experimental Allergic Encephalitis; Experimental Allergic Encephalomyelitis; Experimental Autoimmune Encephalitis; Experimental Autoimmune Encephalomyelitis; Goals; IL-4; IL4; IL4 Protein; Immune response; Immune system; Immunization; Immunologic Stimulation; Immunological Stimulation; Immunostimulation; Individual; Interleukin-4; Interleukin-4 Precursor; Laboratories; Lead; Lipids; Lymphocyte Stimulatory Factor 1; MCGF-2; MS (Multiple Sclerosis); Mannopyranose; Mannopyranoside; Mannose; Mast Cell Growth Factor-2; Measures; Microbe; Modeling; Multiple Sclerosis; Myelin; Myelin Sheath; Myeloencephalitis; Palsy; Paper; Paralysed; Pathway interactions; Pb element; Peptides; Phosphocholine; Phosphorylcholine; Phosphorylcholine Chloride; Plegia; Process; Proteins; Publishing; Relapse; Sclerosis, Disseminated; Self-Antigens; Sensitization, Immunologic; Sensitization, Immunological; Sphingomyelins; Structure; T-Cell Growth Factor 2; T-Cells; T-Lymphocyte; Thymus-Dependent Lymphocytes; Tolerogen; anergy; autoimmune antibody; autoimmune disorder; autoimmune encephalomyelitis; body system, allergic/immunologic; bone sialoprotein 1; bone sialoprotein I; cytokine; disease/disorder; early T-lympocyte activation-1 protein; gene product; heavy metal Pb; heavy metal lead; host response; immunogen; immunoresponse; improved; innovate; innovation; innovative; insular sclerosis; model organism; organ system, allergic/immunologic; osteopontin; paralysis; paralytic; pathway; phosphoprotein I, 2aR; pre-clinical; preclinical; response; secreted phosphoprotein 1; self reactive antibody; self recognition (immune); sialoprotein 1; thymus derived lymphocyte
Project start date: 2008-03-15
Project end date: 2013-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-07-070
5R01NS055997-03 (2010): $314553
MOLECULAR AND CELLULAR IMMUNOBIOLOGY
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5T32AI007290-20 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Project start date: 1985-09-01
Project end date: 2005-08-31
5T32AI007290-20 (2004): $1148856
5T32AI007290-19 (2003): $1127584
IN VIVO TREATMENT OF EAE WITH ANTI I-A ANTIBODIES
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 5R01NS018235-15 from National Institute Of Neurological Disorders And Stroke IRG: ZRG5
Project start date: 1982-04-01
Project end date: 2002-02-28
5R01NS018235-15 (1996): $365646
TCR V GENE REPERTOIRE IN MS AND SELECTIVE IMMUNOTHERAPY
Lawrence Steinman, Professor
Stanford University Stanford, Ca 94305
Grant 2R01NS030201-04 from National Institute Of Neurological Disorders And Stroke IRG: IMS
Project start date: 1992-12-01
Project end date: 1999-04-30
2R01NS030201-04 (1996): $230985
Sponsored Links Excellgen http://Excellgen.com