1990 — 1994 |
Flavell, Richard A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Autoimmunity in Aids Related Immunodeficiency
The mechanism of pathogenesis leading to immunosuppression and death in AIDS is unclear. At any given time only a small fraction of T cells appear to be productively infected with HIV, yet ultimately the CD4+ T cell population is essentially eliminated. While most attention has focused on the role of virus induced cytopathic effects, some in vitro data have suggested possible autoimmune mechanisms leading to T cell destruction. Whether these play a role in vivo is, however, unclear. In this proposal a murine model to test the hypothesis that HIV induced immunosuppression results from gp120 mediated autoimmunity will be tested. Direct effects of gp120 on T cell function will be measured. Transgenic mice have been generated expressing the human CD4 gene. These mice will also be immunized with gp120, and it will be determined whether class II specific cytotoxic T cell responses or ADCC responses that are gp120 specific can be demonstrated in vitro using cells and antibodies from these immunized mice. If these mechanisms can be demonstrated in vitro, transgenic mice will be chronically immunized with gp120 to determine whether immunosuppression caused by destruction of lymphocytes occurs. Since these mice express human CD4 on both B and T cells, ADCC mechanisms will lead to destruction of both cell populations, whereas class II specific cytotoxic T cells (CTL) responses will cause destruction of B cells only. If the CTL mechanism can be demonstrated to occur, we will generate a second series of transgenic mice which express class II MHC on T cells using constructions that we have previously validated. These mice will be crossed with CD4 transgenic mice and again immunized with gp120. Under these conditions it is to be predicted that class II CTL responses should destroy both T cells and B cells. If the results obtained lead to immunosuppression and loss of lymphocytes in these mice,, then they suggest that a similar mechanism may well occur in vivo in man and suggests further that intervention and treatment of the disease may well be effected by inhibition of these processes. These transgenic mice should then provide a system which can be bred and analyzed in large numbers unlike the SCID-human T cell model.
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0.958 |
1994 — 1997 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Dis/Regulation of the Immune System in Autoimmunity
The goal of this program is to understand the regulation and disregulation of the immune system in autoimmunity. The program involves collaborative interactions between members of three Departments, and is organized into four projects supported by three Core facilities. Expertise in the field of immunology, molecular biology, and biochemistry will focus on the events that initiate and sustain autoimmune responses, and the regulatory processes which contain autoimmunity. We will address the following questions? What are the requirements to initiate autoimmune responses? Are autoimmune responses regulated, and if so, by what mechanisms? Does immune regulation contain autoimmune responses under normal circumstances? Finally, do sustained autoimmune responses remain chronic because they diversify from a single initiating response to responses to other autoantigens from the same tissue? These questions will be addressed by collaborative interactions between the Principal Investigators of these projects, which are as follows: (1) R.A. Flavell, PI - Using transgenic mice expressing SV40-T antigen in the acinar pancreas, and T cell receptor transgenic mice which recognize antigen, mechanisms that lead to autoimmunity in this system will be determined, as well as mechanisms which regulate that autoimmune response. The role of diversification of the immune response from SV40-T antigen to other acinar antigens will be investigated. (2) C. A. Janeway, Jr. PI - This project will test the hypothesis that regulation of autoimmune T cells can prevent a monoclonal T cell attack on tissues, but cannot contain a polyclonal attack. It will also test the hypothesis that the autoimmune T cells response diversifies through the action of autoreactive B cells that recruit T cells of novel specificities to the response. Finally, it will characterize the interplay between regulation and diversification in generating autoimmunity. (3) M. J. Shlomchik, PI - Transgenic mouse models will be used to study the regulation of B cells expressing a disease-related autoantibody, rheumatoid factor (RF), in normal and autoimmune mice. In contrast to some other autoantibody models, RF B cells from the first series of transgenics appear competent to initiate an immune response. Thus, studies will focus on why RF B cell escape tolerance and what prevents chronic autoimmunity in RF transgenic mice. (4) M. J. Mamula, PI - This project will examine the role of self peptides in the initiation and perpetuation of both B and T cell autoimmunity in models of systemic lupus erythematosus (SLE). Autoreactive T cells will be investigated for their fine specificity to SLE autoantigens in spontaneous and antigen-induced autoimmunity. Finally, we will study how autoimmunity may diversify from a single initiating determinant to other sites on autoantigens; a mechanism that may exacerbate lupus autoimmunity. These four projects will be supported by a genetically modified mouse core to provide gene targeted and transgenic rodents essential to most of these studies, and a FACS core, to allow us to separate cells for analysis and to analyze cells in all of these projects. The program is coordinated by frequent meetings of the program facility bringing together these diverse approaches to address a common goal.
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0.958 |
1996 — 2011 |
Flavell, Richard A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pathogenesis and Prevention of Autoimmune Diabetes
DESCRIPTION (Adapted from Investigator's abstract): The NOD mouse provides one of the best animal models for autoimmune diabetes. TNF, when administered to adult animals in pharmacologic doses, mediates protection from autoimmune diabetes in NOD mice, and exacerbates disease when administered neonatally. Islet-specific transgene encoded TNF mediates inflammatory infiltrates in normal mice, but surprisingly the investigators have shown that it is also protective against the development of autoimmune diabetes. In this project they will elucidate the mechanism whereby islet-specific transgene encoded TNF on an NOD background prevents diabetes. They will determine whether the mechanism whereby TNF prevents the anti-islet autoimmune response is mediated by the elimination or inactivation of autoaggressive T cells. They will also test for possible immune deviation of the anti-islet response, for example, from a TH1-like to a TH2-like response. They will test whether protection is mediated by dominant regulatory mechanisms by performing adoptive transfer studies. To facilitate these analyses they will utilize T-cell clones in adoptive transfer experiments, and T cells from TCR transgenic mice carrying either CD4 or CD8 islet-specific T cells. Finally, they will determine whether TNF expression initiating early in the life of the NOD mouse, protects or exacerbates from disease, and, if exacerbation is observed, they will determine the mechanism by which that occurs.
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0.958 |
1996 — 1997 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Containment of Spontaneous Autoimmune Pancreatitis in Transgenic Mice
The goal of these studies is to elucidate the mechanisms of induction and prevention of autoimmunity to a peripheral antigen. The elastase gene shows onset of gene expression, which is delayed until after T cells are first exported from the thymus to the periphery. We use the elastase promoter to direct SV 40-T antigen transgene synthesis; the expression of T antigen mimics that of the endogenous elastase gene and therefore permits the study of tolerance and autoimmunity to a protein exhibiting delayed expression. Transgenic mice (ACI-T) expressing SV40-T antigen do not exhibit autoimmunity, despite this delayed expression, although they do show perivascular infiltration in the acinar pancreas. However, simplification of the T cell repertoire by crossing ACI-T mice with mice transgenic for rearranged T cell receptors specific for SV40-T antigen, leads to the early development of autoimmune pancreatitis. As the animals age, tolerance develops, anergic T cells are detectable in the thymus, and depletion of peripheral SV40-Tag reactive T cells occurs in the periphery, coupled with changes in the cytokine profile synthesized by these T cells; cytotoxic T cell activity can, however, be elicited at approximately the same level in double transgenic mice, or in TCR single transgenic mice. We will determine mechanisms that lead to tolerance or autoimmunity in TCR/ACI-T double transgenic mice, and how this process is prevented in single transgenic ACI-T transgenic mice, which express the same antigen but a different T cell repertoire. We will address the following questions: 1. When double transgenic TCR/ACI-T transgenic mice become tolerant, where is this tolerance mediated, in the thymus, in the periphery, or both? 2. Tolerant TCR/ACI-T double transgenic mice exhibit drastic alterations in the nature of CD8 SV40-Tag reactive T cells, with the disappearance of IL-2 producing cells, the reduction of IFN-gamma producing cells, and appearance of IL-4 producing CD8 positive cells; CTL are apparently unaltered. We will determine the relationships between the cells of these different phenotypes to determine if they are the same cells or different cells, with overlapping properties, and we will use ablation techniques, using transgenic mice to determine the lineage relationships between these different CD8 T cell subsets. 3. Why do ACI-T transgenic mice show only mild indications of autoimmunity, while TCR/ACI-T transgenic mice show a dramatic autoimmune phenotype. We hypothesize that this is mediated by immune regulation. We will test this hypothesis and, if true, will characterize the regulatory T cells. 4. A key hypothesis to be tested in this Program Project is that diversification of the autoimmune response is responsible for the maintenance of this chronic disorder. We will test whether diversification of the autoimmune response initiated to SV40-Tag occurs to other acinar antigens. 5. Subsets of T cells producing IL-4 (like TH2 cells), or IFN-gamma (like TH1 cells), inhibit each other. We will test the hypothesis that IL-4 producing CD8 T cells can play a causal role in the prevention and containment of autoimmunity in TCR/ACI-T double transgenic mice. We will elicit large numbers of IL-4 producing cells in vivo, followed by analysis of the capability of these cells in vivo and in vitro.
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0.958 |
1996 — 1997 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Animal Resource
The overall objective of this Program Project is to determine the nature and diversity of mechanisms active in the regulation and dysregulation of the immune system in autoimmunities. Such study presupposes that the tools to identify, characterize and manipulate cells, genes and molecules of this system are available to Program Project. Key tools in this analysis are highly specialized transgenic, mutant, homozygous deficient ('gene knock-out') congenic, recombinant and other mutant mouse strains. These strains provide the key reagents for each of the projects in this Program Project, and the availability of this type of genetically altered animal has in general revolutionized the study of immunobiology. These strains will permit clear study of tolerance in vivo and in vitro. Key strains will include TCR+ and Ig-transgenic mice to permit direct analysis of autoreactive T and B cells, 'antigen' transgenic mice in which the tissue-specific expression of a given peripheral antigen provides the experimental system, and 'gene knock-out' mice to provide genetic backgrounds deficient in specific significant aspects of immunity. These strains are crucial to our studies of autoimmunity. This core mouse breeding facility is thus an essential resource for the research proposed in projects 1 to 4.
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0.958 |
1996 — 1997 |
Flavell, Richard A |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core--Molecular Biology Facility
molecular biology; diabetes mellitus; biomedical facility; animal breeding; tissue mosaicism; antisense nucleic acid; genetically modified animals; laboratory mouse; tissue /cell culture; in situ hybridization; genetic library; nuclear runoff assay; site directed mutagenesis;
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0.958 |
1996 — 1999 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Mechanisms of Immune Evasion by B Burgdorferi
Infection of mice by Borrelia burgdorferi can be prevented by the prior immunization of the animal with outer surface protein (Osp) A and OspB. Protection cannot readily be afforded, however, by immunization with these proteins post infection, and animals and humans frequently remain chronically infected by Borrelia for years unless treated. This, and several lines of evidence suggest that changes in borrelial gene expression occur at different stages of its life cycle in the tick and the mammalian host. These changes are likely to be of importance for mechanisms of pathogenesis and immune evasion. We will utilize a variety of approaches to identify proteins that are differentially expressed in vector and host, to clone and express the genes that encode these proteins, and to assess the role of these proteins in protective immunity. These proteins will be provided to Barthold and Malawista for pathogenesis studies in their proposals, and to Fikrig for studies of immune selection. We will address the following specific aims. First we will demonstrate the antigenic differences between Borrelia in mammalian host, culture, and ticks by generating antibodies against proteins expressed specifically in ticks and infected mice. Second we will isolate borrelial genes differentially expressed in ticks, in BSK culture, or in the mammalian host in vivo by using immunologic screening and differential hybridization techniques. Such genes obtained will be characterized, their DNA sequences determined, and expression patterns validated. The proteins, and the immune response for those proteins, that are expressed differentially in B. burgdorferi derived from ticks, or culture in vitro, or the mammalian host, will then be characterized. The DNA sequence of these genes will be determined in Borrelia of diverse origin to assess the level of sequence conservation. Finally, the role of proteins differentially expressed in tick and host will be assessed in protection from infection by active and passive immunization of C3H mice with these proteins, followed by infection of these mice with either tick or syringe challenge, initially with B. burgdorferi sensu stricto, and if protection is afforded, subsequently with B. afzelii and B. garinii strains.
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0.958 |
1997 — 2002 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Role of Mhc Class 2 Molecules On the Development of T Cell Tolerance
The hallmark of insulin dependent diabetes mellitus is the activation of autoreactive T ells and, as a consequence, the destruction of the islets of Langerhans. Most individuals do to develop this disease from which we conclude that normal mechanisms exist which prevent autoimmunity and that these are circumvented in autoimmune predisposed individuals. We hypothesize that in normal mice and humans peripheral tolerance mechanisms prevent the destruction of islets, although the level at which this prevention occurs is not yet established. We further hypothesize that in autoimmune predisposed NOD mice, that these mechanisms fail and, as a consequence, autoimmunity results. Our previous studies have established that sch peripheral tolerance mechanisms exist in mice of the B6 genetic background, though the mechanisms have not been elucidated. In this grant we will use covalently linked MHC-peptide complexes in conjunction with T cells from TCR transgenic mice to elucidate the mechanisms of peripheral tolerance of CD4 T cells in congenic mice of the B6B10 background. We will also determine the nature of the defects in peripheral tolerance in autoimmune predisposed NOD mice. We will elucidate which cells mediate peripheral tolerance mechanisms and specifically we will test the hypothesis that tissue cells and/or tolerogenic antigen presenting cells mediate peripheral tolerance.
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0.958 |
1997 — 2002 |
Flavell, Richard A |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Core--Transgenic
NOD mouse; laboratory mouse
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0.958 |
1998 — 2001 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Selectin in Experimental Allergic Encephalomyelitis
Experimental allergic encephalomyelitis (EAE) is a model for the human autoimmune disease multiple sclerosis, and results from the activation of myelin-reactive CD4 T cells. The mechanisms underlying this disease model are poorly understood. Selectins play a key role in the entry of lymphocytes into lymphoid organs where priming of autoreactive T- lymphocytes takes place, and leukocytes to sites of inflammation where tissue destruction occurs in autoimmunity. We will employ selectin- deficient mice to determine the role of these molecules in EAE. Our preliminary data show that L-selectin is required for EAE to develop. We will determine the nature of the L-selectin deficiently in EAE and upon which cell populations L-selectin must be expressed for disease. We will further specificity determine whether L-selectin is required for the entry of inflammatory cells into the CNS or for the effector mechanisms of tissue destruction within the CNS. Finally, we will determine whether endothelial selectins (E/P selectins) are required for EAE, and if so, why.
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0.958 |
1998 — 2001 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Dysregulation of the Immune System in Autoimmunity
The goal of this program is to understand the regulation and dysregulation of the immune system in autoimmunity. The program involves collaborative interaction between members of three Departments, and is organized into four projects supported by three Core facilities. Expertise in the field of immunology, molecular biology, and biochemistry will focus on the vents that initiate and sustain autoimmune responses, and the regulatory processes. which contain autoimmunity. We will address the following questions. What are the requirements to initiate autoimmune responses? Are autoimmune responses regulated, and if so, by what mechanisms? Does immune regulation contain autoimmune responses under normal circumstances? Finally, do sustained autoimmune responses remain chronic because they diversity from a single initiating response to responses to other autoantigens from the same tissue? These questions will be addressed by collaborative interactions between the Principal Investigators of these projects, which are as follows: (1)R.A. Flavell- Using transgenic mice expressing a T cell receptor specific for myelin basic protein (MPB) and gene targeted mice lacing L- selectin or E- and P-selectin, the role of selectins in the development of EAE will be determined. The requirement of selectins for the development of disease, as well as the mechanisms which underlie this requirement will be determined, focusing on the cell types which must express L-selectin, the role of selectins in the entry of leukocytes into the CNS and the potential role of selectins within the CNS. (2) C.A. Janeway Jr.- This project will investigate four aspects of the regulation of experimental allergic encephalomyelitis (EAE): Why are mice lacking B cells unable to fully resolve their disease; why does the inability to form cells with other receptors lead to spontaneous disease in mice transgenic for a TCR that recognizes myelin; why do mice cells with other receptors lead to spontaneous disease in mice transgenic for a TCR that recognizes myelin; why do mice with the same receptor who are heterozygous for gld get spontaneous disease; and what is the role of L- selectin in EAE, in collaboration with project 1. (3) M.J. Shlomchik- Transgenic mouse models will be used to study the regulation of B cells expressing a disease-related autoantibody, rheumatoid factor (RF), in normal and autoimmune mice. In contrast to some other autoantibody models, RF B cells from these transgenics are competent to initiate an immune response. Thus, studies will focus on how RF B cells are regulated after Ag stimulation in normal mice and propagated in autoimmune mice, and what prevents chronic autoimmunity in RF transgenic mice. (4) M.J. Mamula, PI- This project will examine the role of self-peptides in the initiation and perpetuation of both Band T cell autoimmunity in models of systemic lupus erythematosus (SLE) and multiple sclerosis (EAE). The role of B cells as autoantigen in models of systemic lupus erythematosus (SLE) and multiple sclerosis (EAE). The role of B cells as autoantigen presenting cells will be examined with relevance to mechanisms that lead to epitope spreading in autoimmunity. Finally, this work will study a novel post-translational peptide modification that arises naturally in cells and confers immunity to self peptides. These four projects will be supported by an administrative core to coordinate the project as a whole, a genetically modified mouse core to provide gene targeted and transgenic rodents essential to most of these studies, and a FACS core, to allow us to separate cells for analysis and to analyze cells in all of these projects. The program is coordinated by frequent meetings of the program faculty bringing together these diverse approaches to address a common goal.
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0.958 |
1998 — 2001 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Transgenic Animal Resource
The overall objective of this Program Project is to determine the nature and diversity of mechanism active in the regulation and dysregulation of the immune system in autoimmunities. Such study presupposes that the tools to identify, characterize and manipulate cells, genes and molecules of this system are available to the Program Project. Key tools in this analysis are highly specialized transgenic, mutant, homozygous deficient ("gene knock-out") congenic, recombinant and other mutant mouse strains. These strains provide the key reagents for each of the projects in this Program Project, and the availability of this type of genetically altered reagents for each of the projects in this Program Project, and the availability of this type of genetically altered animal has in general revolutionized the study of immunobiology. These strains will permit clear study of autoimmunity in vivo and in vitro. Key strains will include TCR+ and Ig- transgenic mice to permit direct analysis of autoreactive T and B cells, and "gene knock-out" mice to provide genetic backgrounds deficient in specific significant aspects of immunity. These strains are crucial to our studies of autoimmunity. This core mouse breeding facility is thus an essential resource for the research projects.
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0.958 |
1999 |
Flavell, Richard A |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core C--Transgenic |
0.958 |
2001 — 2005 |
Flavell, Richard A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Immunoregulatory Mechanisms in Autoimmune Diabetes
DESCRIPTION (provided by applicant): Insulin Dependent Diabetes Mellitus affects a substantial part of the human population. It is an autoimmune disease mediated by autoaggressive T cells. In our previous studies we have shown that TNFa expressed ectopically in the islets of Langerhans, accelerates and exacerbates the development of diabetes in NOD mice. The same transgene accelerates the development of diabetes in B6 mice with a CD80 transgene on their islets. In the previous funding cycle we established that the mechanism that underlies this exacerbation is the potentiation of presentation of islet antigen to autoreactive T cells. Regulatory mechanisms serve naturally to impede the progress of IDDM, and probably in many individuals prevent the development of disease. In the previous grant period we have found that regulatory CD4 T cells also develop in our models, and substantially slow the time course of diabetes. Prolonged expression of TNFa, however, overcomes the ability of these cells to prevent disease. In the present application we will elucidate which cells mediate the protective affects against TNFa exacerbated diabetes, the potency of these cells and the mechanism whereby they prevent CD8 T cells from promoting IDDM. We have also shown that these regulatory T cells depend on signals from CD40/CD154 and RANK/TRANCE. We will determine how these signals lead to the accumulation of Tregs in transgenic mice. It is likely that several overlapping regulatory mechanisms exist which together collaborate to inhibit autoimmunity. We have recently found that ICOS -/- mice are hyper-susceptible to the development of the autoimmune model EAE. We will determine whether this is also true for IDDM, and if so we will determine the mechanism whereby this occurs. The candidate mechanism immune deviation, for which we have evidence in the EAE system, will be explored in the current application. TGF beta has also been shown to inhibit IDDM. We will use two approaches to examine how TGF beta contributes to regulation of IDDM in our models. We will use regulated expression of TGF beta in the islets and a new T cell transgene which renders T cells resistant to inhibition to TGF beta to ask how TGF beta blocks IDDM and what happens in conditions when it cannot act.
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0.958 |
2002 — 2006 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Dys/Regulation of the Immune System in Autoimmunity
DESCRIPTION (provided by applicant): The goal of this program is to understand the regulation and disregulation of the immune system in autoimmunity. The program involves collaborative interactions between members of three departments, and is organized into four projects supported by three core facilities. Expertise in the field of immunology, molecular biology, and biochemistry will focus on the events that initiate and sustain autoimmune responses, and the regulatory processes, which contain autoimmunity. We will address the following questions. What are the requirements to initiate autoimmune responses? Are autoimmune responses regulated, and if so, by what mechanisms? Does immune regulation contain autoimmune responses under normal circumstances? Finally, do sustained autoimmune responses remain chronic because they diversify from a single initiating response to responses to other autoantigens from the same tissue? These questions will be addressed by collaborative interactions between the Principal Investigators of these projects, which are as follows: (1) R.A. Flavell -Dr. Flavell will address the role of IL-10 in autoimmune disease. First, he will develop a novel model system to visualize IL-10 production in vitro and in vivo. Using a GFP knock-in strategy, IL-10 producing cells will be detectable through GFP expression. This will be used to visualize IL-10 production during EAE and IBD and can be used to monitor the effects on IL-10 of a number of interventional strategies, including Dr. Janeway's "therapeutic vaccination" using skin patch. He will also address whether IL-10 acts on T cells or antigen presenting cells in mediating its effects on EAE and IBD using mice transgenic for receptors that block IL-10 action. He has previously used this approach successfully for the other regulatory cytokine TGF-beta. This approach therefore appears promising. (2) C.A. Janeway Jr. -This project will examine the ability to generate suppressor or regulatory T cells in mice that are transgenic for the TCR of the MBP specific clone on a B10.PL background. They have recently observed that they can generate such cells in both induced and spontaneous disease. They will determine if they can observe the same phenotype in normal B10.PI mice, and mice reactive to other autoantigens. (3) M.J. Shlomchik -Transgenic mouse models will be used to study the regulation of B cells expressing a disease- related autoantibody, rheumatoid factor (RF), in normal and autoimmune mice. These B cells are activated spontaneously in autoimmune-prone mice, yet remain quiescent in normal mice. This system will be used to: 1 ) determine the factors -environmental and genetic -that are required for the onset and early propagation of disease; 2) define the cascade of events that leads from an initial nidus of proliferating autoreactive B cells to chronic, ongoing disease; and 3) determine the identities, at the phenotypic and molecular level, of the unique B lineage cells that have escaped peripheral tolerance. (4) M.J. Mamula -Overall, this proposal will examine B cell- T cell interactions in systemic autoimmune disease. The work will make use of autoantibody transgenic mice to determine their role in the maintenance of immune tolerance in normal strains of animals versus the induction of autoimmunity and pathology in autoimmune prone mice. Additional studies will examine how the levels and exposure to self antigens in vivo control the induction and perpetuation of B and T cell autoimmunity. These four projects will be supported by an Administrative Core (Core A) to coordinate the project as a whole, a genetically modified mouse core (Core B) to provide gene targeted and transgenic rodents essential to most of these studies, and a Cell Separation Core (Core C) to allow us to isolate cells and to analyze cells in all of these projects. The program is coordinated by frequent meetings of the program faculty and research workers bringing together these diverse approaches to address a common goal.
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0.958 |
2002 — 2005 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Icos in Allergic Airway Responses
DESCRIPTION (provided by applicant): Asthma appears to be a disease that is driven by a predominantly Th2 immune response to environmental antigens. CD4 T cell effector responses are, however, extensively regulated under normal circumstances with Th2 responses being modulated by Th1 CD4 T cells, as well as by other mechanisms. Costimulatory molecules play a critical role in the activation and differentiation of CD4 T cells. Recently, the costimulator ICOS has been discovered to be expressed on activated T cells, where it seems to play a critical role in the differentiation of CD4 T cells to effector cells producing a number of critical cytokines such as IL4 and IL13. In this proposal, we will utilize ICOS deficient mice that were recently generated in the laboratory of the PI to elucidate the cellular and molecular mechanisms whereby ICOS regulates Th2 cytokine gene expression, and the role that ICOS plays in regulating the immune response and pathology, in ovalbumin and HDI mouse models of asthma.
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0.958 |
2002 — 2005 |
Flavell, Richard A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Transgenic and Gene Targeting
DESCRIPTION (provided by applicant): An overall aim of the Program Project is to elucidate mechanisms of asthma using, in part, animal models. A critical feature of the SCOR is to provide transgenic and knockout and knock-in mice as appropriate models. The Transgenic Core will offer a comprehensive genetic modified animal facility including transgenic and knockout animals to the Program Project. It will also provide a "speed backcrossing" service whereby DNA polymorphisms will be used to facilitate rapid backcrossing of any given transgene or knockout onto an appropriate inbred background. The Core will also provide cryopreservation (and reconstitution of mice from frozen embryos) capabilities for the cost-effective storage of genetically modified mice. Finally, the Core will develop regulated transgenes that permit the expression of given gene products in the airways to be controlled by exogenous manipulation by the investigator.
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0.958 |
2003 |
Flavell, Richard A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Il-2 in Autoimmune Diabetes
DESCRIPTION (provided by applicant): Type 1 insulin-dependent diabetes mellitus is caused by an autoimmune attack on the islets of Langerhans. The susceptibility to this disease is determined both genetically and by environmental events. The elucidation of the underlying mechanisms of this disease has been greatly facilitated by the mapping of genes conferring susceptibility or resistance to diabetes in humans. Further, the availability of animal models, including the non-obese diabetic (NOD) mouse has made it possible to perform fine mapping of the corresponding mouse genes. Idd3 is a critical diabetes-susceptibility gene, which has profound effects on development of disease. NOD mice carrying an Idd3 gene from the diabetes-resistant B6 mouse show a substantially reduced incidence of insulitis and diabetes as well as greatly retarded kinetics of disease development. The Idd3 locus has been mapped to a 780 kb region of chromosome 3. The most likely candidate gene in this region is the IL-2 gene. This gene shows several structural differences between alleles that are derived from resistant or susceptible strains of mice. However, definitive evidence for or against a role of IL-2 has not yet been obtained, since it has not been possible until recently to target genes in the NOD genetic background. In this study we will establish gene knock-in technology on the NOD genetic background by using NOD/129 F1 ES (embryonic stem) cells, which we have recently found to be very effective in all the steps of gene targeting. Specifically, to establish or refute a role of IL-2 in T1 DM, we will use gene targeting to replace the IL-2 gene of the NOD mouse with the IL-2 gene of the diabetes-resistant B6 mouse. We hypothesize that the NOD allele of IL-2 confers susceptibility to diabetes in one of two ways. First, IL-2 is required for antigen induced cell death (AICD) and we propose that AICD in NOD mice is deficient as a result of a defective IL-2 gene. Our second hypothesis proposes that suppressor (also called regulatory) T-cells, which are IL-2 dependent, are defective in the NOD mouse as a result of an IL-2 deficiency in this strain. We will utilize our IL-2 knock-in mice to test these two mutually nonexclusive hypotheses.
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0.958 |
2003 — 2007 |
Flavell, Richard A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Vaccine Responsiveness to Borrelia Burgdorferi Ospa
DESCRIPTION (provided by applicant): The Lyme disease vaccine is based on OspA, a Borrelia burgdorferi lipoprotein, and immunity is contingent upon high levels of OspA antibodies. Toll-like receptors (TLRs) are important for the initiation of immune responses to pathogens and TLR2 recognizes bacterial lipoproteins, including OspA. Our preliminary data now demonstrate that TLR1 is also involved in recognizing OspA. Therefore defects in TLR-mediated signaling could result in ineffective lipoprotein-recognition and influence responsiveness to OspA-vaccination. In this proposal we will explore the association between TLR1, TLR2, and responsiveness to vaccination with OspA in mice and humans. We have now identified 7 persons with very low OspA antibody titers after vaccination. Macrophages from these individuals produced less TNF-a after stimulation with OspA - but not peptidoglycan - than controls; suggesting a defect in signaling that is partially associated with TLR2 (because peptidoglycan is also recognized by TLR2). In vitro transfection studies then demonstrated that dominant-negative TLR1 could inhibit TLR2-mediated OspA responsiveness, implying that TLR1 and TLR2 cooperate for lipoprotein recognition. This was further examined in Tlr2-/- mice, and Tlr1-/-mice that we have created. Tlr1-/- or Tlr2-/- mice made lower levels of OspA antibodies, after immunization with OspA, than controls. Macrophages from Tlr2-/- mice responded poorly to both OspA and peptidoglycan whereas cells from Tlr1-/- mice responded to peptidoglycan - but not OspA. These preliminary data suggest that TLR1 and TLR2 are required for OspA recognition, that the absence of TLR1 or TLR2 results in impair antibody responses to OspA immunization in mice, and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination. We will now fully characterize the response to OspA in Tlr1-/- or Tlr2-/- mice, and in humans that do not develop significant OspA antibodies following vaccination. These studies may provide an understanding of the molecular mechanisms for variability in antibody responses to the OspA vaccine, a first glimpse into TLR deficiencies in humans, and lead to new approaches to augment immune responses to OspA and other vaccines.
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0.958 |
2004 — 2007 |
Flavell, Richard A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Il-2 in Autoimmune Diabetes
DESCRIPTION (provided by applicant): Type 1 insulin-dependent diabetes mellitus is caused by an autoimmune attack on the islets of Langerhans. The susceptibility to this disease is determined both genetically and by environmental events. The elucidation of the underlying mechanisms of this disease has been greatly facilitated by the mapping of genes conferring susceptibility or resistance to diabetes in humans. Further, the availability of animal models, including the non-obese diabetic (NOD) mouse has made it possible to perform fine mapping of the corresponding mouse genes. Idd3 is a critical diabetes-susceptibility gene, which has profound effects on development of disease. NOD mice carrying an Idd3 gene from the diabetes-resistant B6 mouse show a substantially reduced incidence of insulitis and diabetes as well as greatly retarded kinetics of disease development. The Idd3 locus has been mapped to a 780 kb region of chromosome 3. The most likely candidate gene in this region is the IL-2 gene. This gene shows several structural differences between alleles that are derived from resistant or susceptible strains of mice. However, definitive evidence for or against a role of IL-2 has not yet been obtained, since it has not been possible until recently to target genes in the NOD genetic background. In this study we will establish gene knock-in technology on the NOD genetic background by using NOD/129 F1 ES (embryonic stem) cells, which we have recently found to be very effective in all the steps of gene targeting. Specifically, to establish or refute a role of IL-2 in T1 DM, we will use gene targeting to replace the IL-2 gene of the NOD mouse with the IL-2 gene of the diabetes-resistant B6 mouse. We hypothesize that the NOD allele of IL-2 confers susceptibility to diabetes in one of two ways. First, IL-2 is required for antigen induced cell death (AICD) and we propose that AICD in NOD mice is deficient as a result of a defective IL-2 gene. Our second hypothesis proposes that suppressor (also called regulatory) T-cells, which are IL-2 dependent, are defective in the NOD mouse as a result of an IL-2 deficiency in this strain. We will utilize our IL-2 knock-in mice to test these two mutually nonexclusive hypotheses.
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0.958 |
2004 — 2008 |
Flavell, Richard A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Roles of Nod2 in the Pathogenesis of Crohn's Disease
[unreadable] DESCRIPTION (provided by applicant): Crohn's disease is a common illness of the gastrointestinal tract with a prevalence of approximately 0.1-0.2% in many developed countries; it affects approximately half a million Americans. It had been postulated that genetic mechanisms contribute to the pathogenesis of Crohn's disease. The recent finding that mutations of the Nod2 gene are observed in Crohn's disease patients at a high frequency has been one of the great successes of molecular medicine in the inflammatory bowel disease (IBD) field. However this finding by itself cannot make possible prevention or treatment of Crohn's disease since we do not know the mechanism whereby mutation of Nod2 gene leads to susceptibility to the disease. Recently, we found that Nod2 signaling requires the kinase, Rip2/Rick using Rip2 deficient mice. Our goal in this proposal is to elucidate IBD disease mechanisms by using genetically manipulated mice. There are two conceivable scenarios through which mutation of Nod2 could play a role in disease pathogenesis, namely loss or gain of function of Nod2. At this stage both mechanisms are possible. Therefore, by using two different strains of mice, Nod2 deficient mice and Nod2 transgenic mice, we will address this question and try to reveal the underlying mechanisms whereby Nod2 mutation contributes to Crohn's disease. Furthermore, by using mice in which Rip2, the kinase downstream of Nod2, is deficient, we will try to reveal if the IBD pathology caused by Nod2 mutation is dependent on Rip2. [unreadable] [unreadable]
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0.958 |
2004 |
Flavell, Richard A |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Roles of Bacterial Flora in Endotoxin Shock Mediated Vi*
DESCRIPTION (provided by applicant) Approximately 20000 people die from septic shock every year alone in this country as an outcome of gram negative bacterial infection. Lipopolysaccharide (LPS) or endotoxin, a structural component of outer cell wall, is a major effector and can activate the innate immune system via Toll-like receptors (TLRs) and lead to multiple organ damage, termed endotoxin shock. Human bodies are cohabitating with commensal bacterial flora. The largest observed is the one in the intestinal tract which is approximately 1 kg of bacteria/adult of weight. These bacterial flora continuously yield large amounts of bacterial products such as LPS. It has been shown that previous exposure to bacterial products change the sensitivity to LPS challenge in vitro and in vivo. Since LPS produced by commensal bacterial flora in the intestine is actually observed in blood circulation in the portal vein in laboratory animals, it is conceivable that commensal bacterial flora simulate the immune system, leading to the change of sensitivity to endotoxin shock via TLRs. We will test if commensal bacterial flora alter TLR signaling and sensitivity to endotoxin shock in vivo, using mutant strains of mice with increased TLR signaling and decreased TLR signaling. This research is performed primarily in the Institute of Microbiology AS CR in the Czech Republic in collaboration with Helena Tlaskalova as an extension of NIH grant # P01 AI36529.
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0.958 |
2006 — 2010 |
Flavell, Richard A. |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Enhancement of Anti-Tumor Immunity by Inhibition of Tgf-B Signaling in Patiens Wi
Treatment for patients with metastatic melanoma is inadequate. A subset of metastatic melanoma patients can undergo meaningful tumor regression in response to agents that modify lymphocyte activation and/or expansion, for example, IL-2, anti-CTLA4, or IL-2 in combination with transfer of ex vivo expanded tumor-infiltrating lymphocytes (TIL). However, most patients receiving these therapies fail to respond or to achieve lasting benefit. Preclinical studies conducted in our laboratories and confirmed by other investigators provide strong evidence that inhibition of TGF-beta signaling can markedly enhance the anti-tumor activity of CD8+ cytotoxic T-lymphocytes (CTL) in animal models. We propose to extend these studies to determine optimal approaches for clinical development of agents that inhibit TGF-beta signaling in combination with IL-2, IL-2 + TIL, anti-CTLA4, or other related immunotherapeutic manipulations. The goal of the clinical trials proposed in this application is to improve the rate and quality of tumor responses in patients with metastatic melanoma and to reduce the morbidity and mortality from this disease. In Aim 1 we propose to confirm the improved anti-tumor effects of CD8+ TGFRII-DNR cells (CD8+ lymphocytes with the transgene for the dominant negative transforming growth factor beta receptor II) in mouse models and to determine if the anti-tumor activity of CD8+ TGFRII-DNR cells can be improved by addition of IL-2, addition of anti-CTLA4, and/or addition of anti-CD137. Furthermore, we propose to further characterize the mechanisms by which TGF-betaattenuates anti-tumor lymphocyte responses, in particular, to determine if the effects of TGF-beta are directly on CD8+ CTL or indirectly through induction of Treg (T regulatory cells which inhibit CD8+ and CD4+ anti-tumor lymphocyte responses), to determine the role of tumor driven TGF- beta in induction of Treg, and to determine whether and how Treg attenuate CD8+ CTL mediated tumor rejection. In Aim 2, we propose to create a retroviral vector carrying the TGFRII-DNR gene suitable for use in human clinical trials, to develop methods for transduction and expansion of human melanoma-derived TIL, and to characterize the cell product. We will also use mouse melanoma models to determine the optimal conditions necessary to maximize the anti-tumor effects of TGFRII-DNR CTL, and will use the results of the experiments to guide the design of a clinical trial. In Aim 3, we propose to evaluate non-genetic (pharmacologic) approaches to inhibit TGF-beta combined with an immunotherapy. Finally, in Aim 4, we propose to conduct proof of concept clinical trials in which TGF-beta inhibition is combined with an immune therapy. We propose that one of the clinical trials will involve adoptive transfer of melanoma TIL carrying the gene for TGFRII-DNR.
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0.958 |
2009 — 2010 |
Flavell, Richard A. |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Understanding the Role of Amcase in Asthma
DESCRIPTION (provided by applicant): Asthma is characterized by chronic airway inflammation. It is well established that Th2 inflammation and the downstream responses that occur provide much of the underlying basis of asthma pathogenesis. The biologic function of Th2 inflammation is believed to be the recognition and elimination of parasites and fungi. As such, the asthmatic immune response is thought to be a misdirected anti-parasite response. Chitin is the second-most abundant carbohydrate of our planet. It is found in the walls of parasites, crustaceans and fungi, and its role seems to be to protect them from noxious events in their environment, such as predators. The enzymes that degrade chitin, chitinases, are encoded by organisms that express chitin as well as the hosts of parasites. Chitinase is therefore believed to play an essential role in the innate immune response to parasites, fungi and other chitin expressing infectious organisms. Mammals, including humans, do not synthesize chitin but all mammals studied encode chitinases. Recent studies have shown that a large number of chitinase and chitinase-related genes are encoded in the mammalian genome. While some of these genes encode true chitinases that have enzymatic activity, others have the ability to bind but not to hydrolyze chitin. Chitinases and chitin have been shown to both induce and inhibit features of allergic asthma, depending on the model studied. In this project we will define the function of AMCase, a chitinase with true enzymatic activity by generating and characterizing conditional knockout AMCase mice with both chitin and non-chitin airway exposure models. Further we will elucidate the role of AMCase enzymatic activity as well as chitin binding activity in a murine model of asthma using a novel AMCase knockin mouse with a loss of enzymatic activity while preserving the chitin-binding function. Control of diseases such as Asthma is a critical challenge for the health care system in the U.S. Work proposed in this application should provide a better understanding of the events leading to the unintended activation of the immune response leading to immunopathology. In addition, this funding will allow continued support for a very talented technician, who would otherwise have to leave and retention of one and hiring of a second postdoctoral associate. PUBLIC HEALTH RELEVANCE: Our laboratory has in hand mice conditionally targeted for AMCase as well as mice with a knockin of an enzymatically defective AMCase. For that reason we are in a unique position to test the role of AMCase in lung disease and, moreover to determine if and how the chitinase activity of AMCase affects this response.
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0.958 |
2009 — 2013 |
Flavell, Richard A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Bcl-Rambo in Thymic Involution
DESCRIPTION (provided by applicant): Thymic involution is one the major changes that occur in the immune system with age (reviewed in (1)). The reduction of the size and function of the thymus lead to a decreased number of naive T cells in the periphery, compensated by homeostatic proliferation of peripheral memory T cells. As a consequence, the diversity of T cell antigen receptors and the immune responsiveness are reduced in aged individuals. Although the impact of aging on thymocytes, thymic progenitors and thymic microenvironment have been well characterized during the past few years, the genetic determinants and the molecular mechanisms responsible for thymic involution are still poorly understood. We recently discovered that Bcl-Rambo, a member of the Bcl-2 family of apoptotic regulators, is involved in the regulation of thymic involution. Notably, mice lacking this protein display a slower reduction of thymic size with age. The proposed project intends to characterize the role of Bcl-Rambo in the regulation of thymic involution. This study could lead to the identification of potential targets for pharmacological intervention to mitigate thymic involution and its physiological effects. Bcl-Rambo is one of the first proteins described to date, that positively regulates the process of age-related involution of the thymus. With this application we have the opportunity to assess the relative contributions of age-related deficiencies in lymphohematopoietic progenitor function;peripheral or intrathymic signals that regulate involution;and changes in the thymic microenvironment that contribute to the decline in na[unreadable]ve immune cell production, differentiation, and function.
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0.958 |
2010 — 2014 |
Dhodapkar, Madhav V Flavell, Richard A. Manz, Markus Gabriel |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Understanding Hemotopoietic Neoplasias Using Humanized Mice
DESCRIPTION (provided by applicant): The ultimate interest of biomedical research is the study of humans and the goal is the improvement of human health. However, research on humans is severely compromised by the inability to perform invasive experimental work in order to establish the cause and effect relationships between processes. Mouse models play an important role in characterizing key aspects of the driving forces of malignant transformation and disease in humans. However, they rarely represent the genetic complexity and clinicopathologic characteristics of human disease. Xenotransplantation of human cells into mice is regularly used to study the biology of human cancer and test the efficacy of novel anti-cancer therapies in vivo. But its value is limited because scientists are currently mainly limited to aggressively growing primary tumors or cell lines that are less susceptible to xenorejection and less dependent on critical signals provided by the microenvironment. Therefore there is an unmet need to develop methods to reliably grow primary human tumor cells in mice. In the here proposed project, humanization of the microenvironment will be achieved by replacement and controlled expression of essential, but not cross-reactive, growth and survival factors in the mouse genome with their human counterparts. Furthermore, novel approaches to reduce xenorejection by myeloid cells and to generate space in the bone marrow niche complete the proposed genetic manipulations. These novel in vivo models will then be used to test and compare as a benchmark acute myeloid leukemia (AML) engraftment, and to establish models for primary chronic myeloid leukemia and myeloproliferative neoplasias, as well as multiple myeloma - diseases in which the lack of in vivo models faithfully reflecting these diseases curtails research. Successful establishment of these models would allow detailed studies of biology as well as validation of approved and experimental therapies. The development of a versatile platform allowing the study of human primary malignancies in vivo in genetically modified mice would represent a major advance for the field. In summary, this model will serve as platform for preclinical therapy testing with increased predictive value and allow studies on biology of the dynamic evolution of neoplasias with an immediate impact on clinical practice.
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1 |
2013 — 2017 |
Flavell, Richard A. |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Molecular Genetic Mouse Core
The main purpose of this combined core is to provide a comprehensive service using state-of-the-art molecular and genetic methods to generate a variety of sophisticated knock-in, knock-out and transgenic mice for DRC investigators in their type 1 and type 2 diabetes research. We have successfully generated many lines of knock-in, knock-out and transgenic mice related to diabetes studies. It is noteworthy three ofthe highlights among all the animals generated during the previous funding cycle. Firstly, humanized mice, which carry 5 human genes by sequential re-targeting the same ES cells. The engraftment of human fetal liver-derived CD34+ hematopoietic stem cells can reach nearly 100% and last up to 9 months in this humanized mouse. Secondly, lines of innate immunity deficient NOD mice for the studies of type 1 diabetes, especially in association with exogenous and endogenous environmental factors. Using MyD88 deficient NOD mice generated by the core, DRC investigators demonstrated the importance of commensal flora in type 1 diabetes development and gut flora is an environmental modifier that influence diabetes onset Thirdly, lines of inflammsome deficient B6 mice for the studies of metabolic syndrome and type 2 diabetes. DRC investigators found that inflammasomes NLRP3 and NLRP6 negatively regulate NAFLD progression and obesity via modulation ofthe gut flora. This combined core consists of three subcores - Molecular, Gene targeting and Mouse breeding (MGM) components. Each subcore has its distinct function while all is interconnected. We will continue to provide our outstanding service for Yale DRC community. Moleculai: subcore will increase the use of improved high efficiency BAC mutagenesis to facilitate the generation of customized constructs for both type 1 and type 2 diabetes studies. Together with Molecular subcore. Gene targeting subcore will extend the humanized mouse model systems to include diabetes-related genes in order to evaluate preclinical testing of new therapies and/or vaccines, by our DRC investigators, for treatment and prevention type 1 diabetes. Mouse breeding subcore will backcross the improved humanized mouse lines to NOD background and several lines of infammasome deficient mice to pure NOD background forthe studies of type 1 diabetes.
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0.958 |
2014 — 2015 |
Flavell, Richard A. Williams, Adam (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Identifying Lincrnas Critical in Asthma Pathogenesis
DESCRIPTION (provided by applicant): Asthma is an allergic disease of the airways that has reached epidemic proportions and represents a significant burden on health services globally. Asthma is characterized by a dysregulated adaptive immune response initiated by Th2 cells~ however, our understanding of why the disease develops is limited. Delineating the mechanisms which regulate the gene expression programs that define Th2 differentiation and effector function will be essential if we are to fully understand the etiology of asthma or develop new therapies. The recently identified family of large intergenic non coding RNAs (lincRNAs) has been shown to regulate core gene expression programs. Hundreds of lincRNAs are differentially expressed within cells of the immune system highlighting the exciting potential for an important role for lincRNAs in defining immune cell identity and function, as well as contributing to immunopathologies such as asthma. However, very little is known about the function of lincRNAs in vivo and their role in asthma is completely unknown. We hypothesize that Th2 cells express specific lincRNAs that are instrumental in regulating the gene expression programs that determine Th2 differentiation, Th2 effector function and susceptibility to asthma. Using state-of-the-art methodologies this proposal seeks to identify and characterize lincRNAs specifically expressed in Th2 cells and evaluate their biological function in vivo by using a murine model of allergic airway disease. Aim 1 will identify specific lincRNAs expressed in Th2 cells generated in vitro and in vivo and determine at which stage in the Th2 differentiation program they are expressed~ this will reveal lincRNAs most likely to be involved in directing Th2 differentiation. Aim 2 will use knockdown and overexpression studies to define lincRNAs with the ability to modulate Th2 cytokine production in vitro. In parallel, high-throughput RNA-sequencing combined with robust network and guilt-by-association computational analyses will be utilized to generate testable predictions for the function of each lincRNA. Aim 3 will use the recently developed Cas9 gene targeting technology to generate mice deficient in Th2 specific lincRNAs. By modeling allergic airway disease in these animals the potential role for lincRNAs in the etiology of asthma will be dissected in vivo for the first time. In summary this proposal will test the previously unexplored function of lincRNAs in asthma and represents a novel avenue in allergic airway disease research. These studies will not only identify previously unstudied lincRNAs but will aso provide critical information on their function in Th2 cells. Furthermore, as the majoriy of murine lincRNAs exist as syntenic orthologues in humans the work established in this proposal will ultimately serve as a platform for future analysis of human lincRNAs in asthma.
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1 |
2017 — 2019 |
Fields, Ryan C Flavell, Richard A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Towards True Precision Oncology: Validation of a Comprehensively Humanized, Autologous Mouse Model
PROJECT SUMMARY/ABSTRACT Progress in the early detection and treatment of cancer requires accurate model systems to further evaluate new, promising discoveries. Small animal, and in particular mouse, model systems are attractive to researchers for numerous reasons, including their ease of use and well-described platforms. Immunotherapy has revolutionized clinical oncology, but lacks pre-clinical models of the human immune system and human cancer to investigate new modalities and limitations/toxicities of treatment regimens. The ability to grow human tumors in immunodeficient mice (so-called patient-derived xenografts, or PDXs) allows researchers to work directly with human cancer tissue in a controlled setting. However, PDX models are limited by their lack of an intact immune system. The broad objective of this proposal is to validate an in vivo model to evaluate human tumors in the context of a complete and intact human immune system in a completely personalized and autologous fashion. Herein, we propose to: (1) validate the ability to establish humanized mice from patients with melanoma; (2) evaluate tumor growth and lymphocyte development in autologous human melanoma tumors established in humanized mice; and (3) validate the ability of this humanized system to serve as a model for cancer immunotherapy treatment response and toxicity. In each of these areas, we will leverage our multi- institutional team's individual expertise along with our institutional infrastructure to maximize the success of the experimental aims. Furthermore, we will participate in the Oncology Models Forum and utilize the NCIP Hub resources to ensure that the methodology, data, and biologic model systems that result from this project are made widely available to the general research community for future, hypothesis-driven research. Taken together, the studies described in this research proposal will meet multiple goals and address several unmet needs identified in this grant opportunity, thus significantly enhancing the applicability of a fully autologous and immunocompetent precision model system for use in translational oncology research.
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0.97 |
2017 — 2019 |
Craft, Joseph Edgar [⬀] Flavell, Richard A. |
R33Activity Code Description: The R33 award is to provide a second phase for the support for innovative exploratory and development research activities initiated under the R21 mechanism. Although only R21 awardees are generally eligible to apply for R33 support, specific program initiatives may establish eligibility criteria under which applications could be accepted from applicants demonstrating progress equivalent to that expected under R33. R61Activity Code Description: As part of a bi-phasic approach to funding exploratory and/or developmental research, the R61 provides support for the first phase of the award. This activity code is used in lieu of the R21 activity code when larger budgets and/or project periods are required to establish feasibility for the project. |
An in Vivo Crispr-Cas9 Genetic Screen in Murine Primary T Cells to Discover Metabolic Regulators of Follicular B Helper T (Tfh) Cell Differentiation
Follicular B helper T (Tfh) cells are required for normal immune responses, promoting development of memory B cells and long-lived plasma cells. When aberrantly regulated, such as in systemic lupus erythematosus (SLE, lupus), they drive maturation of autoreactive memory B cell and pathogenic plasma cell formation. Modulation of T-B cell interactions in murine models of lupus ameliorates disease, with promise that such intervention will be therapeutically beneficial in human lupus. Thus, it is reasonable to develop a more comprehensive understanding of the mechanisms that govern Tfh cell differentiation, survival, and collaboration with B cells in normal and disease settings. Upon activation, CD4 T cells exhibit dynamic changes in metabolism to meet their proliferative and effector needs. The understanding of how metabolism is regulated in Tfh cells is currently insufficient, although recent work has shown that interference with T cell metabolic programming is as beneficial in lupus models as it is in cancer immunotherapy, albeit without identification to-date of the specific T cell target(s). As Tfh cells operate in the unique GC niche in comparison to their T helper effector counterparts, we hypothesize that these cells utilize different programs of metabolism to fuel their function, with targeting such nodes proposed as a strategy for reprogramming cytotoxicity in tumor-infiltrating T lymphocytes. In the first, R61 phase of this project, we will establish in vivo, high-throughput sgRNA library screening system that supports Tfh cell generation from adoptively transferred T cells, transduced with a metabolome sgRNA library which allows for sufficient library coverage; provides enough sensitivity such that Cas9-mediated genetic lesions lead to observable Tfh cell phenotypes; and permits tracking of individual viral integration events. To achieve these goals, we will take advantage of an acute Armstrong LCMV (lymphocytic choriomeningitis virus) infection model we have used to interrogate Tfh cell differentiation, in which we can track development and differentiation of adoptively transferred T cells genetically manipulated with shRNA expressing retroviruses. This model will be adapted to our retroviral sgRNA system. We will use sgRNA against genes known to critically regulate Tfh cell development to establish benchmarks against which to evaluate the pooled screen and to define the dynamic range of the experimental setup. We also will generate a new barcoded vector to track individually transduced T cells, which will both enhance resolution of the screen and provide robust statistical power for the analysis. Once these goals have been achieved, we will proceed to generating the barcoded sgRNA library and conducting the in vivo screen. In the second, R33 phase, we will generate novel knockout animal models using using Cas9 technology to validate relevance of screen hits, with verified targets bred to lupus-prone mice in order to directly test the role of candidate metabolic genes in autoimmune disease.
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0.958 |
2018 — 2021 |
Flavell, Richard A. |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Molecular Genetics Mouse Core
The overall goal of this Core is to enable diabetes-related research through the generation of unique, genetically-modified mutant mouse models. Our current organizational structure is composed of three distinct, but tightly interrelated Subcores: 1) the Molecular Subcore generates custom designed DNA and RNA constructs for genetic modification in mice and provides PCR genotyping as well as quantitation and localization of gene expression; 2) the Gene Targeting Subcore generates genetically modified mouse strains on a number of genetic backgrounds; and 3) the Diabetic Mouse Breeding Subcore maintains and interbreeds key strains of mutant (such as NOD) and/or genetically modified mice and distributes them to diabetes researchers within the DRC, including novel humanized models and genetically modified NOD strains generated by the Molecular and Gene Targeting Subcores. While the current structure of the Molecular Genetic Mouse Core remains ideally suited to the generation and characterization of animal models, our methodology has undergone a transformational shift in the last funding period with the development of CRISPR/Cas9 gene editing technology, which has, to a great extent, replaced our prior methods for generating animal models. The MGM Core has successfully generated a great many lines of genetically-modified mice related to diabetes studies, most recently using CRISPR-based technology, and its continuing focus on technological development will ensure that services to the diabetes community remain cutting-edge. In addition, a unique feature of this core is that the interests of the subcore directors have fueled a critical mass of resources for investigation of the rapidly developing research areas of the mechanistic relationships between gut microbiota, inflammasome biology and obesity, NAFLD, and type 1 and type 2 diabetes. The MGM Core has developed mouse models with altered innate immunity genes, including inflammasome-deficient NOD mice for the studies of type 1 diabetes, especially in association with the role of gut microbiota. Using these models, DRC investigators demonstrated the importance of commensal bacteria in type 1 diabetes development and implicated gut microbiota as an environmental modifier that influences diabetes onset. Also, mouse models of inflammasome-deficient B6 mice have been developed for studies of metabolic syndrome and type 2 diabetes. DRC investigators found that NLRP3 and NLRP6 inflammasomes negatively regulate NAFLD progression and obesity via modulation of the gut microbiota. To further enable such studies, the core has established gnotobiotic mouse strains in both B6 and NOD backgrounds, including some inflammasome-deficient strains. The gnotobiotic mouse is perhaps the ultimate tool for testing the effects of commensal bacteria on diabetes (type 1 and type 2) and metabolic syndrome development and intervention. Finally, the core has a continuing investment in extending and improving the development and application of technologies for humanization of the mouse genome for engraftment of CD34+ human HSC, and the reconstitution of a human hematopoietic and immune system. These ?humanized? mouse models will be invaluable tools for the dissection of the genetic components regulating the immunologic and metabolic responses in both type 1 and type 2 diabetes, especially with respect to human disease.
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1 |
2018 — 2021 |
Flavell, Richard A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Inflammasome as a Novel Mediator and Therapeutic Target of Gi Syndrome
Project Summary/Abstract High doses of ionizing radiation causes massive cell death and severe damage to the gastrointestinal (GI) tract which is referred to as GI syndrome. The GI syndrome is a common and severe complication in cancer patients undergoing radiotherapy or chemotherapy, but the cellular and molecular mechanisms are unclear and there is no effective therapy. Absent in melanoma 2 (AIM2) forms an inflammasome with DNA and the adapter protein ASC to initiate caspase-1 activation, secretion of inflammatory cytokines and a form of cell death known as pyroptosis. However, the functional role of AIM2 in the GI syndrome was not appreciated. Our recent studies using mouse models of subtotal body irradiation (SBI) induced GI syndrome showed (i) Aim2-/-, Asc-/- Casp-1-/- and mice had reduced GI syndrome and mortality. (ii) Remarkably, nuclear AIM2 bound DNA at sites of DNA double-strand breaks (DSBs) in a complex with ?-Histone 2AX foci to initiate inflammasome activation in response to radiation, suggesting AIM2 may monitor genomic instability through sensing DSBs. The rationale to our approach is based on the scientific premise supporting AIM2 as a DNA sensor in detecting DSB initiated signals to facilitate cell death in GI syndrome. Thus, we hypothesize that AIM2 sensing DSBs signal is a newly identified core feature of the sensing of genomic integrity and inflammasome coordinated pyroptotic IEC death. We will therefore test following three specific aims: Aim1: Determine the mechanism of cell death mediated by the AIM2 inflammasome in response to DNA damage; 1). Determine the contributions of different forms of cell death to GI syndrome. 2). Identify the dynamic subcellular localization of AIM2 inflammasome components during both steady state and activation by radiation. 3). Test the efficacy of a clinically relevant Caspase-1 inhibitor VX-765 to mitigate GI syndrome in APCmin mouse model. Aim2: Determine the intersection of the AIM2 inflammasome with canonical DSB response; 1) Investigate the DSB response in Aim2-, Asc- and Gsdmd-deficient mice. 2). Identify the role of the AIM2 inflammasome in the canonical DNA damage response. 3). Characterize the dynamic interaction of AIM2 with the DSB repair proteins of the ATM/DNA-PK/p53 pathway. Aim3: Identify novel AIM2 partner(s) as a potential radio-/chemo-sensitizing target in GI syndrome. 1). Define the molecular composition of the AIM2 interactome that responds to radiation. 2). Determine the novel genetic AIM2 interactome in the control of radiation-induced DSB response and cell death in vitro through genome-wide CRISPR/cas9 library screening. 3). Validate and determine the functions of the novel AIM2 partner(s) in GI syndrome both in vitro and in vivo. Collectively, our proposed research will elucidate the novel regulatory mechanisms underlying the GI syndrome, and lay the foundation for developing novel therapeutics for GI syndrome by manipulation of DSBs sensing AIM2 activation.
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1 |
2018 — 2021 |
Fikrig, Erol [⬀] Flavell, Richard A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Nlrp6 and Dhx15 in Control of Infection by Rna Viruses
ABSTRACT Pattern recognition receptors sense pathogen-associated molecular patterns and mediate the earliest host innate immune response to infection. The cytosolic nucleotide oligomerization domain (NOD) like receptors (NLRs) are a highly conserved group of cytosolic proteins that play a central role in the immune response to diverse microorganisms, environmental insults and cellular danger signals. We have recently demonstrated a role for Nlrp6 in the control of enteric virus infection. Nlrp6 controls enteric virus infection in the intestine by interacting with a RNA sensor, Dhx15. Here we propose to define the mechanisms of NLRP6-mediated anti-viral pathways, to identify whether other helicases may also interact with NLRP6 (Aim 1), whether other relevant RNA viruses are recognized through this pathway, and whether mutations may underlie certain human cases of vaccine failures (Aim 2). A fuller understanding of the triggers and physiologic function and signaling pathways for NLRs will provide key insights to immune mechanisms involved in host defense and immune-mediated diseases.
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1 |
2020 |
Flavell, Richard A. |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Animal Modeling Core
PROJECT SUMMARY/ The mission of the Animal Modeling Core (AMC) is to provide research groups within the YCCEH and the larger scientific community access to the latest technologies for hematologic studies in animal models. In vivo studies allow researchers to ask questions about the function of molecules, cell populations, and tissues within the relevant spatial and temporal contexts. The YCCEH will continue to provide scientists training and facilitate research in mouse models of hematologic diseases. Importantly, the AMC will continue to develop and propagate the most innovative humanized mouse models for the study of human hematopoiesis and diseases thereof with the goal to overcome major shortcomings of current models. The AMC consists of two cores, the Humanized Mouse Core (HMC) and the Murine Stem Cell Transplantation Core (mSCTC). The Animal Modeling Core will be directed by Dr Richard Flavell, Sterling Professor of Immunobiology and co-directed by Dr. Stephanie Halene, Associate Professor and Acting Section Chief of Hematology. The HMC will offer expertise, technical assistance, and mice for human-into-mouse xenotransplantation studies. The mSCTC will offer training and technical assistance in the study of hematopoiesis and benign hematologic questions in mice. The Humanized Mouse Core will facilitate human-into-mouse xenotransplantation studies into immunodeficient mice. As a unique feature the Yale HMC will offer hematology researchers access to novel humanized immunodeficient mice optimized to support human hematopoietic stem cells, trilineage hematopoiesis, mature myeloid cells, red cells and platelets in circulation, and a functional human immune system. The Murine Stem Cell Transplantation Core will provide technical expertise in the study of hematologic questions in mice. The mSCTC will offer training and expertise for the study of hematopoiesis and hematologic diseases in mice using hematopoietic cell transplantation and functional assays. The Animal Modeling Core will be an integral part of the YCCEH. It will provide Hematology researchers with access to state-of-the-art technologies for in vivo studies of hematopoiesis and hematologic diseases. It will provide teaching, expertise, and resources in an economical way. Its cores will support basic science as well as translational studies, key to understanding and cure of hematologic diseases.
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1 |
2020 — 2021 |
Fields, Ryan C Flavell, Richard A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Advancing Precision Oncology in a Humanized, Fully Autologous Mouse Model
Progress in the early detection and treatment of cancer requires accurate model systems to further evaluate new, promising discoveries. Small animal, and in particular mouse, model systems are attractive to researchers for numerous reasons, including their ease of use and well-described platforms. Immunotherapy has revolutionized clinical oncology, but lacks pre-clinical models of the human immune system and human cancer to investigate new modalities and limitations/toxicities of treatment regimens. The ability to grow human tumors in immune-deficient mice (so-called patient-derived xenografts, or PDXs) allows researchers to work directly with human cancer tissue in a controlled setting. However, PDX models are limited by their lack of an intact immune system. The broad objective of this proposal is to validate an in vivo model to evaluate human tumors in the context of a complete and intact human immune system in a completely personalized and autologous fashion to study cancer immunotherapy. Herein, we propose to: (1) validate the ability of our humanized system to serve as a model for cancer immunotherapy treatment response and toxicity in patients with melanoma, including immunotherapy checkpoint blockade and vaccine strategies, and to (2) extend our current work in melanoma by validating the ability to establish humanized mice and evaluate tumor growth and leukocyte development in autologous human pancreatic and colorectal tumors established in humanized mice. In each of these areas, we will leverage our multi-institutional team's individual expertise along with our institutional infrastructure to maximize the success of the experimental aims. The results from this project will made widely available to the general research community for future, hypothesis-driven research. Taken together, the studies described in this research proposal will meet multiple goals and address several unmet needs identified in this grant opportunity, thus significantly enhancing the applicability of a fully autologous and immunocompetent precision model system for use in translational oncology research.
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0.97 |
2021 |
Flavell, Richard A. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Generation and Characterization of a Humanized Mouse Model of Alcoholic Liver Disease
Generation and characterization of a humanized mouse model of Alcoholic liver disease Project Summary/Abstract Alcoholic Liver Disease (ALD) is the first cause of liver related deaths in USA. However, effective treatment options for ALD are very limited due to the lack of suitable in vivo models that recapitulate the full spectrum of ALD. In human ALD, there is marked inflammation, liver damage and steatosis. On the other hand, mouse models of ALD display very mild phenotype. Thus, there is an urgent but still unmet need to develop a clinical relevant ALD model that can capture the key features of human disease. We have already developed a humanized murine system in which mice have been humanized at key loci by knock-in of human genes (MISTRG-6-Fah-KO mouse) and have been further humanized at a cellular level by engraftment of human hepatocytes and CD34+ stem cells. These mice support human liver hepatocytes, immune, endothelial and stellate cell populations. Crucially, we have shown that the treatment of humanized MISTRG-6-Fah-KO mice with Lieber-DeCarli ethanol liquid plus a single binge ethanol induced higher liver inflammation and liver damage than in non-humanized. Therefore, we hypothesize that the exposure of human cells to alcohol in vivo can better mimic the ALD pathology of humans. To test this hypothesis, we are proposing to treat the humanized MISTRG- 6-Fah-KO mice with alcohol diet and to examine disease pathology in comparison to humans. Next, we will examine the transcriptional state and the composition of human liver cells of MISTRG-6-Fah-KO mice upon alcohol treatment and how much is the extent of their overlap with the human ALD. With the proposed experiments, we aim to develop and characterize the first human-clinical relevant alcoholic liver damage model in the humanized MISTRG-6-Fah-KO mice. In that model, human immune cells and human effector cytokines will drive the development of inflammation and liver damage and human hepatocytes will drive the development of steatosis.
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1 |