1998 — 2002 |
Klein, Robyn S |
K08Activity Code Description: To provide the opportunity for promising medical scientists with demonstrated aptitude to develop into independent investigators, or for faculty members to pursue research aspects of categorical areas applicable to the awarding unit, and aid in filling the academic faculty gap in these shortage areas within health profession's institutions of the country. |
Cytotoxic Lymphocyte Responses in Acute Hiv1 Infection @ Massachusetts General Hospital
The factors responsible for the initial fall in viremia during acute HIV-1 infection are incompletely understood. The appearance of HIV-1 specific cytotoxic lymphocytes (CTL) has been temporally correlated with the decline in viremia, before the appearance of detectable neutralizing antibodies, suggesting that the cellular immune response may initially contain viral replication. Alternatively, mathematical modeling suggests that the decline in viremia is the result of expected population dynamics of a lymphotropic cytopathic retrovirus, and that the sharp decline in viremia is a result of diminished numbers of infectable activated CD4 cells. Almost all studies of CTL responses during primary infection have been limited to analysis of recognition of laboratory strains of virus, such that the breadth and specificity of the initial response is not known. Although recent longitudinal studies in a single person with primary infection suggest that CTL can exert immune pressure, as evidenced by the emergence of sequence variation within a dominant CTL epitope, the ability of CTL or CD8 cells to actually control replication of the initial virus present has not been demonstrated. I propose to perform a detailed analysis of the antiviral cellular immune response in primary HIV-1 infection. We have developed in vitro assays which allow for the direct examination of antiviral activity of CD8 cells and CTL clones. We are also developing an adapted novel technique for the rapid expression of PCR amplified virus genes using a combination of vaccinia infection-plasmid transfection, such that responses to autologous virus can be assessed efficiently. Using these techniques, I will conduct a detailed study of the antiviral activity of CTL and CD8 cells during the critical period of primary infection when the viral load is declining, in order to determine the contribution of the immune system to this decline. Through a cohort of persons with primary infection in Boston and through an established and functional collaboration with UCSF, we have access to blood and tissue specimens from persons during primary infection, prior to seroconversion and prior to the establishment of the viral set point. An analysis of the antiviral immune response in these individuals will provide important information regarding the correlates of protective immunity and help guide the design of therapeutic strategies and vaccines. Specifically, I propose to: 1. Characterize the magnitude, breadth and specificity of the CTL response to autologous virus in blood and tissues at the time of primary infection, when peak viremia is declining. 2. Determine the ability of CD8 cells and CTL clones to inhibit replication of autologous virus at the time of declining viremia during acute infection.
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0.91 |
2003 — 2007 |
Klein, Robyn Sue |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Chemokines and Lymphocyte Trafficking Into the Cns
DESCRIPTION (provided by applicant): Lymphocytes are the predominant leukocyte found in central nervous system (CNS) inflammatory lesions in multiple sclerosis (MS) and other CNS inflammatory diseases. Lymphocytes may contribute both to the initiation and maintenance of CNS inflammation via antigen-specific interactions that occur during their baseline trafficking into normal CNS for the purpose of immune surveillance. Chemoattractant cytokines, or chemokines, are small secreted proteins that recruit lymphocytes into parenchymal tissues during both homeostatic and inflammatory states. Although the mechanisms that recruit lymphocytes specifically into the CNS during this baseline trafficking are unknown, our preliminary data that chemokines are constitutively expressed by the CNS and that activated lymphocytes traffic into normal CNS tissues suggests that chemokines may play an important role in this process. With the Independent Scientist Award (K02), the applicant will place an intensive research focus toward determining the chemokines and chemokine receptors that direct the trafficking of T lymphocytes into normal CNS tissues in vivo. Using an in vivo trafficking model, we will examine the role of chemokine receptors in the movement of T cells into normal CNS and assess the loss of chemokine receptor activity in this process using mice with targeted deletions of appropriate chemokine receptors. We will then go on to determine whether inhibition of the baseline trafficking of lymphocytes into the CNS affects the development of the murine CNS inflammatory disease, experimental autoimmune encephalomyelitis (EAE), which is a model for MS. The applicant is a principal investigator at the MGH Center for Immunology and Inflammatory Diseases, which provides a rich intellectual and experimental environment to establish the groundwork for these investigations. As evidenced by almost nine years of research in the neurosciences, the applicant remains firmly committed to a long-term career in basic science investigation.
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1 |
2005 — 2009 |
Klein, Robyn Sue |
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. |
Mechanisms of Cxcl10-Mediated Neuroprotection in Wnv Encephalitis
DESCRIPTION (provided by applicant): Encephalitis is a common and devastating consequence of infection with the fiavivirus West Nile virus (WNV), a rapidly emerging infectious disease. Although neurons are the primary target of central nervous system (CNS) infection, a hallmark of WNV encephalitis is the accumulation of inflammatory infiltrates extending from the meninges into the brain parenchyma that vary in severity between brain regions. This CNS inflammatory response is required for protection from lethal infection through the recruitment of leukocytes that clear virus through a variety of effector mechanisms. Some of these effects, however, may also be detrimental and contribute to the progression of neurologic disease. Thus, understanding the proinflammatory effects responsible for immune-mediated viral clearance during WNV infection of the CNS is an essential step for developing therapies that enhance clearance of virus in certain CNS compartments and limit inflammation in others. It is well established that inflammatory chemokines are upregulated in response to viral infections and that these molecules modulate the recruitment of leukocytes into infected tissues. In preliminary experiments, we have observed that the chemokine CXCL10 is highly upregulated in CNS tissues of mice with WNV encephalitis and localizes to subpopulations of neurons that are heavily infected with WNV. Moreover, neutralization of CXCL10 activity during WNV infection led to an increase in mortality, and treatment of neurons ex vivo with CXCL10 affects their survival. Based on these observations, the proposed research plans to directly test the role of CXCL10 in CD8+ T cell trafficking and neuronal injury in a well-defined mouse model of WNV encephalitis. In Specific Aim 1 we will determine the regional and cellular sources of CXCL10 and its receptor, CXCR3, in the CNS of mice infected with WNV. In Specific Aim 2, we will evaluate the effect of CXCL10 on different populations of neurons in vitro and on neuropathology in vivo after WNV infection. In Specific Aim 3, using CXCL10 sufficient and deficient mice, we will determine the role of CXCL10 in coordinating the CD8+ T cell response against WNV in the different regions of the CNS. Overall, the goal of this proposal is to determine the mechanisms of neuronal protection and, possibly, injury induced by CXCL10 in WNV encephalitis. An enhanced understanding of the molecular signals that govern neuroinflammation and neuronal injury after infection will be critical to the development of targeted anti-inflammatory agents that mitigate the morbidity and mortality associated with WNV encephalitis.
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1 |
2008 — 2012 |
Klein, Robyn Sue |
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. |
Animal Model Core
Acute; Adoptive Transfer; Animal Model; Animal Models and Related Studies; Apoplexy; Autoantigens; Autologous Antigens; Axon; Biscyclohexanone Oxaldihydrazone; Body Tissues; Bromide, Ethidium; CMV promoter; CNS processing; Cells; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Chronic; Clinical; Cuprizone; Data; Demyelinations; EAE; Electrons; Encephalomyelitis, Allergic; Engineering; Engineerings; Ethanedioic acid, bis(cyclohexylidenehydrazide); Experimental Allergic Encephalitis; Experimental Allergic Encephalomyelitis; Experimental Autoimmune Encephalitis; Experimental Autoimmune Encephalomyelitis; Folch-Lees Protein; Folch-PI Proteolipid Protein; Folch-Pi apoprotein; GFP; Gene Deletion; Gene Inactivation; Gene Silencing; Generations; Genetic; Genetics, in situ Hybridization; Goals; Green Fluorescent Proteins; Histology; Homidium Bromide; Image; Immune; Immune Cell Activation; In Situ; In Situ Hybridization; Inflammatory; Injury; Investigators; Lentivirinae; Lentivirus; MS (Multiple Sclerosis); Mammals, Mice; Measurement; Mice; Mice, Transgenic; Microscopic; Modeling; Molecular; Multiple Sclerosis; Murine; Mus; Myelin; Myelin PLP; Myelin Proteolipid Protein; Negative Beta Particle; Negatrons; Nerve Cells; Nerve Unit; Nervous; Neural Cell; Neurocyte; Neurons; Outcome; Outcome Assessment (Health Care); Outcomes Assessment; P01 Mechanism; P01 Program; PLP; Paper; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Process; Program Project Grant; Program Research Project Grants; Protocol; Protocols documentation; Publishing; Quelling; RNA Interference; RNA Silencing; RNA Silencings; RNAi; Relapse; Research Personnel; Research Program Projects; Researchers; Sclerosis, Disseminated; Score; Self-Antigens; Sequence-Specific Posttranscriptional Gene Silencing; Sorting - Cell Movement; Spinal Cord Contusions; Standards; Standards of Weights and Measures; Stroke; Subfamily lentivirinae; Suggestion; T-Cells; T-Lymphocyte; TXT; Technology; Text; Thymus-Dependent Lymphocytes; Tissues; Toxic effect; Toxicities; Transgenes; Transgenic Mice; Update; Vascular Accident, Brain; Virus-Lenti; Wallerian Degeneration; autoimmune encephalomyelitis; base; brain attack; central nervous system processing; cerebral vascular accident; experiment; experimental research; experimental study; feeding; gene deletion mutation; imaging; improved; in situ Hybridization Staining Method; in vivo; insular sclerosis; mRNA Expression; member; model organism; mouse model; myelin proteolipid; myelin proteolipid apoprotein; myelinopathy; neural; neuronal; proteolipid apoprotein; proteolipid protein of myelin; relating to nervous system; research study; retina ischemia; retinal ischemia; secondary degeneration; shRNA; short hairpin RNA; small hairpin RNA; sorting; stroke; thymus derived lymphocyte
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1 |
2008 — 2012 |
Klein, Robyn S. |
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. |
Neuroprotective Mechanisms of Cxcl12 in Cns Demyelinating Diseases
Project 2: "Neuroprotective mechanisms of CXCL12 during CNS demyelinating diseases" The molecular mechanisms responsible for leukocyte migration and activation within the CNS parenchyma are unknown, as are the mechanisms that orchestrate recovery and myelin repair during remitting phases of both MS and EAE. Blood-brain barrier (BBB) disruption and demyelination are consequences of white matter inflammatory infiltrates22"25. Utilizing confocal microscopy, Dr. Robyn Klein observed the loss of endothelial cell expression of the chemokine CXCL12, normally displays basolateral localization, during CNS autoimmune disease, suggesting CXCL12 plays a role in regulating the blood-brain barrier (BBB). Dr. Klein's team also observed that spinal cord motorneurons express CXCL12 and white matter oligodendrocyte precursors express its receptor CXCR4, suggesting a role for these molecules in remyelination. Thus, a novel, antiinflammatory role for CXCL12 whereby it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thus limiting their parenchyma! infiltration and activation is proposed in Project 2. Thus, Project 2 will (1) determine how CXCL12-mediated perivascular localization regulates mononuclear cell trafficking during CNS autoimmunity, (2) determine how perivascular localization regulates mononuclear cell activation during CNS autoimmunity, and (3) evaluate how CXCL12 regulates the remyelination process.
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1 |
2008 |
Klein, Robyn Sue |
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. |
Targeting Mononuclear Cell Trafficing For the Treatment of Viral Encephalitis
Cells; Encephalitis, Viral; Infectious Encephalomyelitis, Viral; Mononuclear; Viral Encephalitis
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1 |
2010 — 2014 |
Klein, Robyn S |
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. |
Chemokine-Mediated Neuroprotection and Repair During Wnv Encephalitis
DESCRIPTION (provided by applicant): The long term experimental objective of this competitive renewal is to evaluate the functional roles of chemokines and their receptors in contributing to viral clearance, immunopathology and repair in a murine model of WNV encephalitis. Murine WNV infection is well-accepted flavivirus encephalitis model whereby subcutaneous infection with low inoculum of virulent strains leads to neuroinvasive disease in individuals with depressed or dysregulated immune function. Thus, murine experimental studies have identified viral and host factors that determine the neuropathogenesis and outcome of WNV encephalitis in humans, including several chemokine receptors that determine the entry and fate of infiltrating immune cells. In published studies we have determined that the chemokines CXCL12 and CXCL10 control leukocyte access to the central nervous system (CNS) parenchyma at the blood-brain barrier (BBB) and within the CNS parenchyma in a region-specific manner, respectively. In preliminary studies we have also observed that WNV infection led to increased BrDU incorporation within neurogenic zones, suggesting that adult neurogenesis is affected by viral infection. In addition, we have determined that TLR3 signaling affects the expression of neural precursor cell transcription factors Pax6 and nestin in several brain regions, providing a link between viral infection and activation of innate immune molecules known to regulate both chemokine expression and neurogenesis. The present proposal seeks to understand the immunological basis of BBB trafficking for protective versus pathogenic leukocytes and for virus entry. Because chemokine up-regulation also recruits neural precursor cells for repair, we will additionally evaluate how chemokines influence neurogenesis within the WNV-damaged CNS. We will address these questions in three aims: 1) Identify the proinflammatory pathways responsible for the migration of protective versus pathogenic leukocytes across the BBB during WNV encephalitis. This aim will determine molecular cues for leukocyte entry at the BBB and trafficking within specific CNS regions. Additionally, we will determine the role of CXCR4 in T cell subset entry at the BBB; 2) Identify molecular mechanisms involved in WNV entry and effects at the BBB. This aim will use in vitro and in vivo approaches to determine WNV effects on CNS endothelium and viral invasion; and 3) Examine chemokine receptor-mediated strategies for neuroprotection and repair during WNV encephalitis. This aim will determine how WNV infection affects the generation of neural cells in the adult CNS and the chemokines that control this process. Together, these studies will extend our current understanding of how chemokines and their receptors control CNS inflammation and ultimately repair during WNV encephalitis.
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1 |
2014 — 2018 |
Klein, Robyn S |
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. |
Animal Models, Histology and Tissue Bank Core
The Animal Models, Histology and Tissue Bank Core will maintain availability and assistance in the induction of all murine models of experimental autoimmune encephalomyelitis (EAE) including monophasic and relapsing-remitting models induced by both active immunization and adoptive transfer of encephalitogenic T cells. Towards this latter goal, Core B will generate and maintain encephalitogenic and control T cell lines. Core B will also assist in all outcome measurements, histological analyses and immunohistochemical approaches. To support PPG investigators? exploration of alternative approaches and establish future goals for all Projects, Core B will also establish a murine tissue bank for screening purposes. Because the overall goal of all animals work is to translate findings to human tissues and, ultimately, patients, Core B will establish and maintain a human CNS tissue bank with histopathologic analyses from control and MS patients plus banked peripheral blood mononuclear cells, sera and cerebrospinal fluid. These samples will be utilized by all Projects to validate findings in murine models and to further investigate findings in murine systems using human tissues. We recently hired a dedicated technician to process, catalogue and coordinate the evaluation of CNS specimens for inflammation, demyelination, axonal injury and remyelination. In summary, the function of Core B is to provide technical support for a variety of in vivo experimentation using murine MS models and to facilitate studies for the translation of findings to MS patients via use of human specimens.
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1 |
2014 — 2018 |
Klein, Robyn S |
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. |
Targeting the Bbb to Treat Cns Inflammation
Project 2 will determine how targeting molecules that regulate permeability and polarity at central nervous system (CNS) endothelial barriers impacts neuroinflammation, as assessed via novel neuroimaging modalities with murine models of multiple sclerosis (MS). We have shown that endothelium in MS lesions exhibits altered polarity, displaying the abluminal chemokine CXCL12 aberrantly along luminal surfaces. Abluminal CXCL12 normally serves to limit leukocyte entry whereas luminal expression of CXCL12 is associated with increased activation of its signaling receptor CXCR4 on infiltrating leukocytes. We showed that the scavenging CXCL12 receptor, CXCR7 (also expressed by CNS endothelium), is a critical regulator of leukocyte entry via internalization of CXCL12 from abluminal surfaces. In preliminary studies, we have also found that sphingosine 1-phosphate (S1P) signaling via S1P2 disturbs abluminal expression of CXCL12, which then disrupts immune privilege. Thus, mice with targeted deletion of S1P2 or administered a specific antagonist, JTE-013, show reduced migration of leukocytes into the CNS parenchyma during EAE. We hypothesize that alterations in membrane polarity of CXCL12 at CNS endothelial barriers promote leukocyte capture and migration into the CNS, contributing to the establishment of disease cycles in relapsing-remitting forms of CNS autoimmunity. We have identified several molecules that regulate this process, leading to reversal of apicobasal expression of the localizing cue, CXCL12. In this proposal, we will examine the roles of CXCR7 and S1P2 in lymphocyte trafficking and inflammation in CNS autoimmune disease using novel imaging modalities including two-photon intravital microscopy and longitudinally using Diffusion Basis Spectrum Imaging (DBSI), which distinguishes and quantitates cellularity and edema in live mice (see Project 1) and examination of autopsied human MS patient and control CNS tissues. We expect DBSI to be more sensitive than Gd enhancement to inflammation, including in the setting of less profound blood-brain barrier alterations. To determine the relationships between endothelial and immune cell (Th1 versus Th17) interactions and loss of BBB integrity, we will directly compare results utilizing intravital imaging to DBSI and Gd enhancement in the same animals. To address mechanisms by which endothelial cell polarity and localizing cues are induced by interactions with inflammatory cells, we will utilize in vitro and in situ approaches with human tissues. Thus, Aim 1 will determine whether CXCR7-mediated internalization of CXCL12 requires infiltration with Th1 versus Th17 cells during EAE. Aim 2 will determine whether S1P2- mediated reversal of endothelial cell polarity leads to increased T cell capture and entry during EAE, and Aim 3 will examine the relationship between CXCR7 and S1PR2 activation and T cell migration at the BBB in MS. The experimental design of Project 2 will define how DBSI determined cellularity and edema changes responding to various degrees of BBB abnormality and cell infiltration in EAE mice.
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1 |
2016 — 2020 |
Diamond, Michael S (co-PI) [⬀] Klein, Robyn S |
UH2Activity Code Description: To support the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) UH3Activity Code Description: The UH3 award is to provide a second phase for the support for innovative exploratory and development research activities initiated under the UH2 mechanism. Although only UH2 awardees are generally eligible to apply for UH3 support, specific program initiatives may establish eligibility criteria under which applications could be accepted from applicants demonstrating progress equivalent to that expected under UH2. |
Effect of Aging On Neuroinvasion During West Nile Virus (Wnv) Infection.
Aging is associated with dysregulation of innate and adaptive immunity, which impairs an individual's ability to control infection by newly encountered pathogens. Aging also impacts neuroprotective mechanisms within the central nervous system (CNS), including the function of the blood-brain barrier (BBB), which limits the entry and replication of neutrotropic viruses. West Nile virus (WNV) is the leading cause of arthropod-transmitted viral infections in the United States. While most WNV infections are asymptomatic, individuals with symptomatic disease present either with a flu-like febrile illness that can progress to severe neuroinvasive diseases including meningitis, encephalitis, or flaccid paralysis. Severe WNV infection, especially fatal forms, predominate in the elderly. It is unclear whether this is due to effects of aging on viral invasion and/or virologic control within peripheral tissues or the CNS. To study many different aspects of immune regulation in adult mice, the Diamond and Klein laboratories have established a murine model of infection with a virulent WNV strain. However, interactions between antiviral immunity, neuroinflammation, and aging within the CNS have not been investigated. In this proposal, we will examine the impact of age on innate and adaptive immune mechanisms that control viral entry and clearance specifically within the CNS. The UH2 phase we will establish breeding cohorts and determine the feasibility of using 21 month-old aged animals in WNV infection studies. In the UH3 phase, we will explore how changes in skin immunity, BBB integrity, and CNS inflammation during aging influence neuroinvasion and disease progression associated with WNV infection.
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1 |
2016 — 2017 |
Klein, Robyn S |
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.) |
Mechanisms of Sex Differences in Blood-Brain Barrier Biology
? DESCRIPTION (provided by applicant): This proposal is a new direction for my laboratory, which is to define developmental origins of sex differences in susceptibility to multiple sclerosis (MS). MS is an autoimmune, demyelinating disease of the central nervous system (CNS) that has a strong sex bias, with the female to male ratio currently approaching 4:1. Relapsing- remitting MS RRMS, the most common form of the disease in women, is a condition in which recurrent episodes of new neurological dysfunction (relapses) are separated by periods of clinical stability (remission). The SJL strain of mice is a standard model for examining sex differences in MS, as it exhibits a similar sex bias in susceptibility and RREAE in female animals. In published studies we showed that cell intrinsic sex differences in the expression of the sphingosine 1-phosphate receptor 2 (S1PR2) on central nervous system (CNS) vasculature of SJL mice regulates endothelial cell polarity by destabilizing adherens junctions and contributes to the development of disease cycles in females compared with males. Not yet defined is how the process of normal sexual differentiation patterns activity in pathways that regulate blood-brain barrier (BBB) stability. In this proposal we will use sex differences in S1PR2 expression to develop approaches that will uncover molecular mechanisms that regulate sex differences in BBB biology. Because evidence suggests that acute effects of estrogens are not responsible for sex differences in RRMS and in S1PR2 expression, the focus of this proposal is the role of sex chromosome complement and the organizational effects of sex hormones on sex differences in S1PR2 expression at the BBB. We hypothesize that sex differences in endothelial cell expression of S1PR2 occur via organizational effects that occur during development. We further hypothesize that chromatin regulation establishes additional sex-specific effects through altered expression of genes involved in BBB function. In this proposal we will: 1) Determine whether sex-differences in S1PR2 expression occur via cell intrinsic endothelial cell alterations in gene expression; and 2) Determine whether sex chromosome complement and/or organizational effects of sex hormones underlie sex differences in BMEC S1PR2 expression. Our findings will define new interfaces between sexually dimorphic gene expression and BBB function, and may identify new strategies to treat disease in MS patients in a sex-specific fashion.
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1 |
2016 — 2020 |
Klein, Robyn S |
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. |
Neuroprotection and Repair During West Nile Virus Encephalitis
DESCRIPTION (provided by applicant): WNV is the leading cause of domestically acquired arboviral disease in the United States. In addition to the acute neuroinvasive syndromes and persistent motor deficits, patients that recover from WN- fever (WNF) and WN neuroinvasive disease (WNND) experience significant long-term cognitive sequelae, including high rates of memory impairment and abnormalities in executive function. While improved survival rates from WNND (~91%) have led to rising numbers of patients currently living with these impairments, few studies have addressed underlying mechanisms responsible for these deficits. To address this, we have developed a new murine model of recovery from intracranial infection with a mutant WNV (WNV-NS5-E218A), leading to uniform infection across CNS regions and measurable spatial learning deficits that persist after viral clearance. Using this model we have traced learning defects to hippocampal pathology including CNS tissue resident memory CD8 T cells (Trm), microglial activation and altered neurogenesis within this brain region. Microarray analyses of differentially expressed mRNAs within the hippocampi of mice with neurocognitive deficits revealed increased expression of a variety of immune molecules expressed by Trm (CD103, CD69, IFITM3), and those known to drive microglial effects on synaptic modeling and neurogenesis (complement proteins C1q and C3), and caspase-1, which drives expression of interleukin(IL)-1. In preliminary studies, Trm are CCR2+IFN-?+ and WNV- recovered IFN-?- and IFN-?R- deficient mice do not display learning defects. Activated microglia and defects in neurogenesis within the subgranular zone (SGZ) of the dentate gyrus, but not the subventricular zone (SVZ), were found to persist long after clearance of replicating virus. Moreover, microglia phagocytosis of synapses was observed to occur via a complement-mediated process. We hypothesize that innate immune responses of hippocampal microglia after WNV infection are driven by Trm and contribute to spatial learning defects via their effects on synaptic elimination and neurogenesis. To test these hypotheses we will 1) Define the role of Trm-derived IFN-? in microglia activation and neurocognitive dysfunction during recovery from WNND, 2) Define the innate immune role of microglia in neural correlates of spatial memory during recovery from WNND; and 2) Determine the role of IL-1R1 signaling in microglial modulation of synapse elimination and neurogenesis in the WNV-recovered CNS.
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1 |
2017 — 2021 |
Diamond, Michael S (co-PI) [⬀] Klein, Robyn S Lemke, Greg E [⬀] |
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. |
Tam Receptors and Virus Infection @ Salk Institute For Biological Studies
PROJECT SUMMARY/ABSTRACT The receptor tyrosine kinases of the TAM family ? Tyro3, Axl, and Mer ? are essential regulators of infection by enveloped viruses, and Axl and Mer are especially important to arbovirus infection of cells of the central nervous system (CNS). Infection of the CNS by encephalitic arboviruses, including West Nile virus (WNV), often has devastating consequences, both acutely and after recovery, and deciphering the molecular mechanisms through which TAM receptors control virus entry, propagation, and clearance is therefore a key objective. Genetic, molecular biologic, cell biologic, and behavioral assays will be used to elucidate these mechanisms. In Aim 1, a set of new conditional mouse mutants and cell-specific Cre drivers will be used to investigate the CNS cell types through which TAM receptors control infection by WNV and two other neurotropic enveloped arboviruses - La Crosse encephalitis virus and Venezuelan equine encephalitis virus. These studies will elucidate the specific roles played by Axl and Mer in brain microvascular endothelial cells (BMECs), microglia, astrocytes, and pericytes in neuroinvasion and CNS pathogenesis by these viruses. In Aim 2, a new mouse model of learning impairment after recovery from CNS infection by WNV will be used to probe the role that Axl and Mer in microglia and astrocytes play in spatial learning and memory after infection. These experiments also will assess the role that cell-specific TAM signaling plays in synapse elimination and neurogenesis subsequent to WNV infection of the brain. In Aim 3, molecular genetics and cell-based signaling assays will be used to elucidate the molecular architecture of TAM receptor-Interferon receptor (IFNAR) interaction, which is crucial to the phenomena of Aims 1 and 2, in BMECs, microglia, macrophages, and dendritic cells. These studies will identify the signaling pathways activated by cooperative TAM receptor/INFAR signaling in these cells, and assess the ability of these interacting receptor systems to reorganize actin cytoskeletons. Together, the experiments of this proposal will guide the formulation of novel strategies for inhibiting virus entry into the CNS, attenuating virus infection of neural cells, and promoting the repair and recovery of infected neural tissues.
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0.904 |
2018 — 2020 |
Klein, Robyn S |
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. |
Neuropathogenesis of Zika Virus Infections
SUMMARY Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus of global public health concern due to its ability to cause severe congenital abnormalities during infections of pregnant women2, and meningoencephalitis or post-infectious autoimmune demyelination in infected, non-pregnant adults. Host-viral interactions that control infection and dissemination of ZIKV remain poorly characterized, as are post-infectious effects on brain development and function in congenital and adult infections, respectively. We have developed both congenital and adult models of ZIKV infection that have been instrumental in defining many pathogenic features of ZIKV infection. In preliminary studies, mice with deficiencies in lymphocytes (Rag1-/-), B cells (?MT-/-) or T cell function (TCR-/-) exhibited differential abilities to clear virus depending on the manner of exposure. Thus, ?MT-/- mice exhibited increased pathogenesis in congenital models of ZIKV infection while Rag1-/- and TCR-/- adult mice exhibited decreased survival after intracranial infection with ZIKV. We hypothesize that antigen-specific T cells are required to eliminate ZIKV from the brain of adult mice, whereas humoral immunity plays the dominant role in clearance of ZIKV during congenital infection. Aim 1 we will define mechanisms of immune-mediated clearance during congenital and adult ZIKV infection of the CNS. While 30% of congenitally infected fetuses exhibit morphological abnormalities by ultrasound (e.g., microcephaly or brain calcifications)12, the majority of congenitally infected humans exhibit no morphological or structural abnormalities. Cognitive impairment and neurodevelopmental abnormalities are likely to occur after congenital ZIKV infection, but there may also be important effects on neurodevelopment after in utero infection. In addition, memory disturbances in adults that have recovered from acute ZIKV infection have recently been reported in the literature. One possible mechanism involves ZIKV infection of neural stem cells and early neural cell type progenitors. However, in preliminary studies of ZIKV-recovered adult mice, we observed persistent microglial activation, decreased SGZ neurogenesis and severe spatial learning defects, the latter of which is ameliorated by lack of signaling via the interferon-? receptor (IFN?R). These studies suggest adaptive immune mechanisms may also contribute to memory dysfunction in adults recovering from ZIKV encephalitis. We hypothesize that congenital infection with ZIKV leads to alterations in cognition due to direct effects on neuronal progenitors and indirect disruption of radial glial function, leading to abnormalities in functional connectivity. We further hypothesize that the persistence of CD8+IFN?+ T cells within the CNS of adult ZIKV- recovered animals promotes microglial activation, which alter neural correlates of learning and memory such as synaptic plasticity and adult neurogenesis. Aims 2 and 3 will define the mechanisms of spatial memory dysfunction during adult and congenital infection with ZIKV and determine the effects of congenital ZIKV infection on cerebral functional connectivity using optical neuroimaging.
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1 |
2021 |
Klein, Robyn S |
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. |
Astrocyte Innate Immune Mechanisms of Post-Viral Cognitive Dysfunction
ABSTRACT This proposal is focused on the pivotal role of astrocytes in molecular mechanisms underlying neurocognitive sequelae of flavivirus encephalitis. WNV is the leading cause of domestically acquired arboviral disease in the United States. In addition to the acute neuroinvasive syndromes and persistent motor deficits, patients that recover from WN neuroinvasive disease (WNND) experience significant long-term cognitive sequelae, including high rates of memory impairment and abnormalities in executive function. We recently established a novel murine model of recovery from intracranial infection with a mutant WNV (WNV-NS5-E218A), which leads to ongoing microglia activation with synapse elimination, decreased adult neurogenesis, and spatial learning defects that persist months after viral clearance These effects on synapses were also observed in patients who succumbed to WNND. While these data provide some molecular explanations for poor spatial learning in WNV-recovered subjects, the mechanisms underlying lack of recovery of the hippocampal circuit are unclear. We recently demonstrated that WNND is associated with T cell-derived interferon(IFN)?, and microglial-derived complement promote elimination of hippocampal CA3 presynaptic terminals, with lack of recovery due to the generation of interleukin(IL)-1?-expressing activated astrocytes. Indeed, inactivation of IL-1R1 signaling in WNV-recovered animals promotes adult neurogenesis, synaptic repair, and prevents defects in spatial learning. These reactive astrocytes also express recently identified markers of neurotoxicity (H2.D1, Gbp2, psmb8), suggesting viral-induced effects on cognition might be perpetuated by the generation of astrocytes with a redirected cell fate. Indeed, increased expression of some of these genes (GFAP, Gpb2, psmb8) is observed in human cortical astrocytes treated with IFN?, also highly expressed within the WNV-infected CNS. Prior research has suggested multiple cellular origins of reactive astrocytes with activated microglia being a potential key player in directing astrogliosis18,19. Similarly, the activation of the inflammasome complex within myeloid cells has been implicated in IL-1? production with IL-1 targeting multiple cell types including neural precursor cells20,21. We hypothesize that macrophage-derived IL-1 directs neuronal precursor cells toward an astrocyte fate during viral encephalitis. We further hypothesize that during recovery, activated microglia promote the differentiation of astrocytes towards neurotoxic phenotypes that inhibit hippocampal repair and recovery from neurocognitive deficits via effects of innate immune cytokines on neural precursor cells (NPC). Aim 1: Define the cellular targets of astrocyte-derived, anti-neurogenic cytokines in neurocognitive dysfunction during recovery from flavivirus encephalitis. Aim 2: Define innate immune mechanisms that direct and maintain astrogliosis during WNV infection and recovery. Aim 3: Define astrocyte-specific responses to IFN? that impact neural correlates of spatial memory during recovery from WNND.
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2021 |
Klein, Robyn S |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Research Program Award (R35 Clinical Trial Optional) - Dr. Robyn Klein Ninds
PROJECT SUMMARY/ABSTRACT Viral infections are now recognized as risk factors for diseases of progressive pathological forgetting, supporting a new paradigm in neuroimmunology whereby innate immune molecules that function as modulators of a variety of normal CNS functions induce neurodegenerative diseases during host-pathogen responses. Studying virus-mediated cognitive dysfunction through the multidisciplinary prism of immunology, neuroscience, and virology is critical for the identification of novel mechanisms of disease, discovery of neuroimaging tools and therapeutic treatments for a wide range of diseases of memory disorder. In my laboratory we made unanticipated, paradigm-shifting discoveries of the roles of CNS infiltrating mononuclear cells in microglial-mediated synapse elimination, disrupted adult neurogenesis, and generation of neurotoxic astrocytes using novel models of recovery from encephalitogenic flaviviruses, West Nile (WNV) and Zika (ZIKV) viruses. We identified classical complement proteins and cytokine receptor signaling as novel molecular mediators that regulate synapse elimination, neural stem cell (NSC) fates, neuron-microglia and microglia- astrocyte crosstalk within cortical structures that regulate memory formation and maintenance. We have also begun leveraging novel neuroimaging modalities to develop biomarkers that may be used to predict and monitor patients at risk for memory disorders. Our aim is to understand the mechanisms that induce alterations in synaptic connections and methods of repair that contribute to disruption of neuronal networks after recovery from viral infections. Our research program focuses on three broad areas related to the roles and regulation of innate immune molecules involved in spatial learning using novel murine models of post-infectious cognitive dysfunction. First, using genetic, pharmacologic and PET-MRI, we will identify and define molecular interactions between T cells and microglia or neurons that drive the generation and maintenance of resident memory T cells that promote cognitive dysfunction. We will use scRNAseq under BSL3 conditions to screen for genes and pathways to be targeted via cell-specific deletion of cytokine or chemokine receptors, or administration of agents that inhibit or enhance pathways. We will also develop diagnostic tools that employ ABSL3 PET-MRI. Second, we will define how microglia-astrocyte-NSC interactions in the context of recovery from CNS viral infections limit repair and recovery. We will use global and conditional gene targeting in mice to delineate the in vivo roles of cytokines in neural cell types that regulate astrocyte inflammasome activation and its relationship to neuronal and synapse recovery. We will also define innate immune mechanisms that direct and maintain astrogenosis during acute viral infection and recovery using PET-MRI detection of P2X7R, a marker of reactive astrocytes. Finally, will utilize reporter mice/fate mapping, bone marrow chimeras and scRNAseq to delineate the cytokine-mediated roles of myeloid cells in the generation of neurotoxic astrocytes during WNND recovery. Third, we will examine innate immune mechanisms triggered after viral infections that negatively impact cortical connectivity and determine whether neuroimaging can be used to predict and follow this process. Specifically, we will combine genetic approaches with functional optical intrinsic signal imaging of hemoglobin and calcium dynamics to define mechanisms that negatively impact cortical connectivity. Our research program will define new concepts in the molecular neuroimmunological regulation of synapses, T cell and glial interactions, inform studies of related processes throughout the nervous systems, and will likely enhance our understanding of neurodegenerative and other disorders of memory.
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