2001 — 2003 |
Kielian, Tammy L |
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 Pathogenesis of Brain Abscess
DESCRIPTION: (adapted from applicant's abstract) The focus of this proposal is to understand the immune response to pyogenic bacteria, such as S. aureus, in the pathogenesis of experimental brain abscess. This study may lead to identification of host and bacterial factors that play a key role in brain abscess development and pathology. First, they will evaluate, using S. aureus (one of the main etiological agents of brain abscess in humans,) the host immune responses in the central nervous system (CNS) parenchyma. They will use a rodent experimental model, and examine cytokines and chemokines elicited in response to bacterial challenge, the cellular origin(s) of these mediators within the CNS, and the response of primary astrocytes and microglia. Next, they will evaluate the relation of bacterial virulence factors in abscess pathogenesis. They will evaluate the role of virulence factors expressed in the CNS and what effect they have on the host immune response to infection.
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0.94 |
2002 — 2010 |
Kielian, Tammy L |
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. |
Receptors Involved in Microglial Responses to S. Aureus @ University of Arkansas Med Scis Ltl Rock
DESCRIPTION (provided by applicant): Microglia are one of the resident mononuclear phagocyte populations within the central nervous system (CNS). These cells share many phenotypical and functional characteristics with macrophages, indicating that microglia participate in innate immune responses in the brain. We have recently demonstrated that microglia are capable of recognizing S. aureus and respond by elaborating numerous inflammatory mediators and exhibit bactericidal activity. As such, microglia are uniquely poised to provide an initial line of defense against invading microorganisms into the CNS prior to leukocyte infiltration. However, the receptor(s) responsible for mediating microglial activation in response to S. aureus have not been identified. The pattern recognition receptors (PRRs) Toll-like receptor 2 (TLR2) and CD 14 play a pivotal role in macrophage activation in response to the gram-positive cell wall products peptidoglycan (PGN) and lipoteichoic acid (LTA). We have recently revealed that microglia express both of these PRRs which may be responsible for mediating cell activation in response to S. aureus. With the goal of defining the role(s) of TLR2 and CD14 in microglial responses to pyrogenic bacteria in the CNS, the following specific aims are proposed: I) To characterize the response of microglia to intact S. aureus organisms, PGN, LTA, and secreted virulence factors in terms of inflammatory mediator production; II) To examine the expression and regulation of TLR2 and CD14 on microglia; III) To delineate the functional significance of TLR2 and CD14 expression on microglial activation using microglia from receptor knockout mice, receptor blocking antibodies, and transient transduction of dominant negative receptor constructs; and IV) To examine the importance of microglial TLR2 and CD14 expression in the pathogenesis of S. aureus-induced brain abscesses using receptor knockout mice and radiation bone marrow chimeras. Although we are examining microglial activation in response to S. aureus, it is likely that our findings will extend to other gram-positive organisms by virtue of their conserved structural components. Understanding the mechanisms by which microglia recognize and respond to microbial products could have a significant impact on a broad range of bacterial infectious diseases in the CNS.
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1 |
2004 |
Kielian, Tammy L |
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 of Brain Abscess @ University of Arkansas Med Scis Ltl Rock
DESCRIPTION (provided by applicant): Brain abscesses represent an important medical problem despite recent advances made in detection and therapy. Because of the emergence of multi-drug resistant strains and the ubiquitous nature of bacteria, these CNS infections are likely to persist. The size of a developing abscess normally extends well beyond the original site of infection leading to damage of surrounding normal brain parenchyma. This finding suggests that the CNS antibacterial response is not down regulated in an efficient manner, resulting in chronic inflammation and large abscess lesions. They propose that a balance exists between sufficient and over-compensatory responses to S. aureus in the CNS, which dictates the outcome of brain abscess development; therefore, therapies aimed at attenuating chronic CNS inflammation subsequent to effective bacterial neutralization may result in smaller abscesses and subsequent improvements in cognitive and neurological functions. The objective of the proposed work is to examine the influences of minocycline and PPAR-gamma agonists on the pathogenesis of brain abscess development. Recently, these compounds have been found to exhibit neuroprotective effects in several models of CNS disease; however, their roles in regulating CNS infectious disease has not yet been examined. To address this objective, the following Specific Aims will be addressed: (I) to evaluate the dose- and time-dependent effects of PPAR-gamma agonists and minocycline on S. aureus-induced brain abscess development; (II) to investigate the effects of PPAR-gamma agonists and minocycline on cell migration and neuronal cell death induced by S. aureus-stimulated microglia; and (III) to examine the mechanism(s) responsible for impaired neutrophil infiltration into brain abscesses of CXCR2 KO mice and the potential effects of PPAR-gamma agonists and minocycline in the CNS compartment. In addition to its anti-inflammatory properties, the bacteriostatic activity of minocycline may augment its effects on brain abscess development. The potential multifactorial effects of these compounds suggest that they may be more efficacious compared to traditional therapies developed to counteract a single pathway in CNS diseases. These experiments should provide meaningful insights into how minocycline and PPAR-gamma agonists influence brain abscess development and will reveal whether their ability to modulate non-infectious CNS conditions extends to infectious diseases.
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0.94 |
2005 — 2013 |
Kielian, Tammy L |
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 Pathogenesis of Brain Abscess @ University of Nebraska Medical Center
DESCRIPTION (provided by applicant): The host anti-bacterial immune response that ensues during brain abscess development not only neutralizes pathogens but also contributes to the destruction of surrounding normal brain parenchyma. Limiting this pathological damage could result in long-term improvements for brain abscess patients. During the previous funding period, we found that the PPAR-3 agonist ciglitazone accelerated abscess encapsulation/fibrosis, which coincided with a significant reduction in proinflammatory mediator expression as well as bacterial burdens. Since fibrosis typically ensues subsequent to the dampening of immune responses, it is possible that ciglitazone facilitates brain abscess wall formation by its ability to limit ongoing inflammation. Indeed, recent studies have demonstrated that PPAR-3 agonists can transition classically activated (M1) macrophages into an alternatively activated (M2) phenotype as well as enhance the production of Th2 cytokines such as IL-4, IL-5, and IL-13, both of which are pro-fibrotic. However, the signal(s) responsible for regulating fibrosis during brain abscess development and the pathways influenced by ciglitazone to accelerate this process remain unknown. The overall hypothesis of this proposal is that the PPAR-3 agonist ciglitazone accelerates brain abscess encapsulation through its ability to transition activated microglia/macrophages into a M2 alternative phenotype that, in turn, triggers a pro- fibrotic (Th2) cytokine profile to accelerate fibrosis. We will also utilize magnetic resonance imaging (MRI) modalities to monitor the effects of ciglitazone on brain abscess size, perfusion, and edema formation as well as density and organization of the abscess wall throughout disease development. These measures will provide a serial non-invasive assessment of brain abscess formation and how ciglitazone modulates these processes. MRI-based methods will include diffusion tensor imaging (DTI) for wall density and edema formation as well as high-resolution T1 and T2 MRI maps for clearly delineating the abscess wall from surrounding tissue. Perfusion maps will be used to monitor regional changes in blood flow along the brain abscess wall using arterial spin labeled (ASL) perfusion MRI. The power of these approaches is that MRI enables a longitudinal assessment of wall formation and perfusion throughout the course of brain abscess development in an individual animal. To address these objectives, the following specific aims will be investigated: 1) to investigate the mechanism(s) responsible for the PPAR-3 agonist ciglitazone to accelerate brain abscess wall formation and fibrosis;and 2) to define the events influencing regulated fibrosis along the developing brain abscess wall. By understanding the mechanisms responsible for its ability to accelerate abscess encapsulation, ciglitazone may prove to be an effective adjunct therapy, in concert with conventional antibiotics, for the rapid containment of brain abscesses. Conceivably, this approach could lead to significant improvements in clinical outcomes and quality of life for patients recovering from brain abscesses. PUBLIC HEALTH RELEVANCE: Brain abscesses represent a serious infection, especially due the recent emergence of antibiotic-resistant strains of bacteria, and can cause long-term deficits including seizures and cognitive loss. In this proposal, we will study a synthetic compound that causes brain abscesses to become walled off more rapidly, which may help to protect surrounding brain tissue from damage. This compound may prove beneficial, in combination with conventional antibiotics, for the treatment of brain abscess patients by facilitating rapid abscess containment, effectively limiting bacterial spread throughout the infected hemisphere.
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1 |
2006 — 2010 |
Kielian, Tammy L |
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. |
Effects of Neuroinflammation On Gap Junction Communication in Glia @ University of Nebraska Medical Center
DESCRIPTION (provided by applicant): Gap junctions represent direct intercellular conduits between contacting cells that permit the passage of small molecules (>1 kDa) including ions, metabolic precursors, and second messengers. The observation of extensive intercellular coupling and large numbers of gap junctions in the central nervous system (CNS) suggests a syncytium-like organization of glial compartments. One CNS infectious disease in which nothing is known regarding its impact on glial gap junction communication (GJC) is parenchymal infection with pyogenic bacteria leading to the establishment of brain abscess. Recent studies have revealed that several proinflammatory mediators detected in developing brain abscesses and produced by S. aureus activated glia, including interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-a), and nitric oxide (NO) are capable of modulating GJC in astrocytes and microglia. Specifically, these molecules attenuate GJC in astrocytes whereas activated microglia become functionally coupled. We have coined this phenomenon a "syncytial switch" and propose that the inflammatory milieu that develops during the course of brain abscess may be important for remodeling the types of interactions between resident glia and that deviation from physiological coupling may impact the integrity of brain regions distant from the primary focus of infection. These changes may be dictated by regional variations in Cx expression within the abscess. The objective of the proposed work is to investigate the functional importance of IL-1, TNF-a, and NO in regulating the glial syncytial switch and the role of Cx43 in brain abscess pathogenesis. To address this objective the following Specific Aims will be addressed: (1), to evaluate the consequences of S. aureus and its cell wall product PGN on homocellular GJC in primary astrocytes and microglia and the signaling pathways responsible for the syncytial switch;(2), to establish the functional importance of the proinflammatory mediators IL-1, TNF-a, and NO on modulating glial GJC in response to S. aureus stimulation using primary glia from knockout (KO) mice;and (3), to investigate the role of proinflammatory mediators on connexin expression and the functional importance of Cx43 in disease pathogenesis in a mouse model of S. aureus-induced experimental brain abscess using genetic KO models. Due to the extensive gap junctional coupling of glial cell populations in the normal CNS, neuroinflammatory disruption of normal glial syncytial networks could contribute, in part, to some of the long-term effects observed in patients following brain abscess resolution including seizures and cognitive deficits. These experiments will provide meaningful insights into how proinflammatory mediators influence the extent of glial GJC in brain abscess.
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1 |
2009 — 2013 |
Kielian, Tammy L |
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. |
Innate Immunity to S. Aureus Biofilm @ University of Nebraska Medical Center
PROJECT SUMMARY (See instructions): Biofilm infections on body surfaces or medical devices represent a serious therapeutic challenge since organisms are recalcitrant to conventional antibiotics. Therefore, an urgent need exists to approach biofilm biology from unique perspectives and employ collaborative approaches to facilitate the identification of novel treatment paradigms. The current consensus is that biofilms evade the host immune response; however, the available reports have focused on classical anti-bacterial mechanisms operative during planktonic growth and have neglected to examine the possibility that biofilms induce an immune deviation towards alternative anti-inflammatory pathways. To date, only one report has examined the immune response to S. aureus biofilm and further in-depth studies are warranted given the propensity of these infections to disseminate and colonize other sites in the body. The hypothesis of this proposal is that S. aureus biofilm skews the host innate immune response from a classical pro-inflammatory bactericidal phenotype towards an anti-inflammatory, pro-fibrotic response to favor bacterial persistence. We will focus on the functional importance of key bacterial recognition molecules (Toll-like receptor 2 (TLR2) and TLR9) as well as anti-microbial mediators (INOS) by studying the pathogenesis of S. aureus biofilm using a foreign body infection model in mice deficient for these various molecules. We will also investigate the effects of purified neutrophils, macrophages, and dendritic cells on biofilm survival and whether these immune cells induce changes in biofilm gene expression by transcriptional profiling. In addition, we will employ in vivo bioluminescence imaging (IVIS) to monitor the immune response throughout biofilm development utilizing reporter mouse strains engineered to express luciferase under the control of promoters pivotal in host immunity to gram-positive bacteria (i.e. INOS, TLR2, and NF-KB). Finally, we will examine the host immune response to S. aureus murein hydrolase {cidA and IrgAB). nuclease {nuc), and nitric oxide reductase {nor) mutants from Projects 1 and 3 of this PPG in the mouse foreign body infection model. To address these objectives, the following specific aims will be investigated: 1) compare host innate immune responses to S. aureus biofilm versus planktonic infection; 2) define the role of extracellular bacterial DNA (eDNA) and peptidoglycan (PGN) in modulating host innate immunity to S. aureus biofilm; and 3) examine the immune mechanisms leading to fibrotic encapsulation of S. aureus biofilms. The studies described in this proposal will employ a comprehensive approach to investigate the innate immune response to biofilm both in vitro and in vivo taking advantage of our experience in studying innate immunity. Understanding the cross-talk between S. aureus biofilm and innate immune cells may identify novel candidates to target for drug discovery to disrupt biofilm growth.
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1 |
2009 — 2010 |
Kielian, Tammy L |
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. |
The Role of Nuclease in Biofilm Development and Disease @ University of Nebraska Medical Center
Staphylococcus aureus is emerging as the most problemafic bacterial pathogen facing our community and healthcare settings. An effective strategy for S. aureus to survive in the host is to attach to a surface and develop into an encased community of cells called a biofilm. We recenfiy discovered that quorum-sensing can control the balance between a planktonic or biofilm lifestyle, suggesfing that modulafion of this dispersal mechanism could be an effective therapeutic strategy. In collaboration with Dr. Kenneth Bayles (the PI of this PPG), we demonstrated that a deletion of the S. aureus secreted nuclease (Nuc) caused an overall thickening of the biofilm and inhibited secondary structure formation, and we have confirmed a recent report that S. aureus possesses a second extracellular nuclease activity (Nuc2). Based on these findings, our central hypothesis is that control over extracellular nuclease activity is a critical determinant of biofilm maturafion and dispersal. To address this quesfion, in Specific Aim 1 we will (i) define the role of Nuc and Nuc2 in biofilm maturation;(ii) determine whether nuclease activity is important for biofilm dispersal;and (iii) modulate biofilm integrity with controlled exposure to nuclease. We further propose that S. aureus nuclease is an important virulence factor. To investigate the nuclease funcfion in disease, we will work with Dr. Tammy Kielian (Project 4 leader) and (i) examine the role of nuclease acfivity in evasion of neutrophil extracellular traps (NETs);(ii) define the significance of nuclease in mouse models of planktonic versus biofilm infecfion;and (iii) compare the host inflammatory response to nuclease in planktonic versus biofilm infection. Finally, we speculate that small-molecule inducers of nuclease activity could serve as anfi-biofilm therapeufics. Towards this end, in Specific Aim 3, we will employ new technology to generate cyclic peptide libraries in S. aureus that are amenable to high-throughput screening methods. More specifically, we will (i) screen for cyclic pepfides that induce nuclease expression through FACS;(ii) perform molecular and biochemical studies to identify pepfide targets;(iii) characterize the best candidates as dispersal agents in a biofilm infection model;and (iv) compare results to transposon mutants with increased nuclease activity. Overall, the goal of this Project is to understand how these S. aureus biofilm structures form and disassemble, the contribufion of extracellular DNA to this process, and the relevance in disease. RELEVANCE (See instructions):
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1 |
2014 — 2021 |
Kielian, Tammy L |
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. |
Innate Immune Response to S. Aureus Biofilm @ University of Nebraska Medical Center
Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infections (PJIs) typified by an anti-inflammatory cellular milieu. The inflammatory phenotype of leukocytes is intimately tied to their metabolic status, where anti-inflammatory M?s primarily rely on oxidative phosphorylation (OxPhos) and pro-inflammatory M?s utilize glycolysis. Monocytes are also polarized toward an anti-inflammatory state during S. aureus PJI, which our preliminary data shows coincides with an OxPhos bias. We have devised an innovative approach to metabolically re-reprogram biofilm-associated monocytes to promote glycolysis using cell-targeted nanoparticles containing the OxPhos inhibitor oligomycin. Treatment of established biofilms with oligomycin nanoparticles promoted monocyte pro-inflammatory activity concomitant with increased neutrophil recruitment, leading to biofilm clearance. During the recent PPG cycle, our laboratory was the first to identify that myeloid- derived suppressor cells (MDSCs) skew biofilm-associated monocytes toward an anti-inflammatory state, in part, through IL-10 production. Therefore, we screened the Nebraska Transposon Mutant Library to identify mutants impaired in their ability to trigger IL-10 production. Significant hits involved in lactate biosynthesis were identified, and our preliminary data support a role for S. aureus-derived lactate in organizing the anti-inflammatory biofilm milieu, progressing from MDSCs/monocytes as a target to defining the molecular mechanism of action. First, during PJI, D- and L-lactate levels are reduced in S. aureus ddh1 and ldh1/ldh2 mutants, respectively, concomitant with significant reductions in MDSC infiltrates and IL-10 production, which translates into enhanced leukocyte recruitment, and biofilm clearance. Second, the IL-10 promoter is activated by acetylation and our ChIP-Seq data demonstrate that histone promoter acetylation is dramatically increased genome-wide in leukocytes recovered from WT vs. ldh1/ldh2 infected mice, providing molecular evidence that S. aureus biofilm- derived lactate functions as a histone deacetylase inhibitor (HDACi). Our central P01 hypothesis is that S. aureus biofilm development creates unique metabolic niches that promote an immune suppressive environment. In Project 4, we will explore the existence of a metabolic triad between S. aureus biofilm, MDSCs, and monocytes, whereby biofilm-derived lactate promotes leukocyte anti-inflammatory properties, in part, via IL- 10 production, contributing to biofilm persistence. This metabolic crosstalk and the molecular mechanisms responsible for this interplay will be explored in the following Specific Aims. 1) Establish that leukocyte metabolism can be targeted in vivo to promote pro-inflammatory activity and biofilm clearance; 2) Investigate the role of S. aureus biofilm-derived lactate in promoting MDSC and monocyte anti-inflammatory activity by stimulating IL-10 production; and 3) Determine whether S. aureus biofilm-derived lactate regulates IL-10 production by inhibiting histone deacetylase (HDAC) activity. These studies will inform our long-term goal of targeting metabolic pathways that disarm anti-bacterial innate immune defenses to facilitate biofilm eradication.
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1 |
2014 — 2015 |
Kielian, Tammy L |
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.) |
Therapeutic Targeting of Aberrant Glial Function During Juvenile Batten Disease @ University of Nebraska Medical Center
DESCRIPTION (provided by applicant): Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in the CLN3 gene. JNCL presents between 5-10 years of age, with progressive vision loss, seizures, cognitive and motor decline, and death by late teens-early 20s. There is no treatment for JNCL, which underscores the significance of identifying novel therapeutics to improve lifespan and quality-of-life for children suffering from this deadly disease. Recent work from our laboratory suggests that aberrant glial activation during early JNCL may contribute to neuronal loss. In particular, CLN3?ex7/8 microglia are primed to produce numerous proinflammatory mediators with known neurotoxic effects, whereas wild type (WT) cells are non-responsive. Astrocyte hemichannel (HC) opening is also enhanced in numerous brain regions of CLN3?ex7/8 mice, which allows the non-discriminant passage of molecules from the intra- to extracellular milieus and disruption of physiologic gradients. The combination of early HC opening and aberrant microglial activation in JNCL likely disrupts the brain metabolome, contributing to the pathological chain of events that culminates in neuronal loss. Our hypothesis is that targeting aberrant glial activation with two classes of compounds that affect multiple pathways will significantly delay JNCL progression. The first, INI-0602, is a novel HC inhibitor that reduces glutamate accumulation in CLN3?ex7/8 mice to levels typical of WT animals. The other group includes the second generation phosphodiesterase-4 (PDE4) inhibitors Roflumilast and PDE4 subtype specific inhibitors provided by Pfizer that attenuate proinflammatory mediator production by CLN3?ex7/8 microglia and also increase astrocyte glutamate transporter expression. Importantly, both INI-0602 and PDE4 inhibitors reduce inflammation and neuronal loss in numerous disorders, including AD and HD. This R21 proposal will identify the optimal neuroprotective regimens for INI-0602 and PDE4 inhibitors, by evaluating effects on the brain metabolome, behavior, and neuronal survival in CLN3?ex7/8 mice. We will establish optimal dose-response profiles for each drug, the ideal therapeutic window for intervention, and whether INI-0602 and PDE4 inhibitors display additive effects in a combinational therapy approach. The preclinical assessment of INI-0602 and PDE4 inhibitors as novel therapeutics to delay JNCL progression will be examined in the following specific aims: 1) The hemichannel inhibitor INI-0602 attenuates glutamate accumulation during early JNCL, leading to significant neuronal sparing in thalamocortical structures; 2) PDE4 inhibitors reduce neuronal loss in JNCL by attenuating proinflammatory mediator release and glutamate accumulation; and 3) A combinational therapy with INI-0602 and PDE4 inhibitor provides superior efficacy to impede JNCL progression due to distinct mechanisms of drug action. Together, our novel rationale for compound selection, supporting preliminary data for INI-0602 and PDE4 inhibitors as JNCL therapeutics, and existing intellectual property for these compounds in JNCL, form a solid foundation for the preclinical testing outlined in this R21 application.
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1 |
2016 |
Kielian, Tammy L |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Faseb Src On Translational Neuroimmunology: From Mechanisms to Therapeutics @ Federation of Amer Soc For Exper Biology
? DESCRIPTION (provided by applicant): The purpose of this R13 submission is to request funds to partially support the 2016 FASEB Translational Neuroimmunology conference and its associated pre-meeting course. This will be the 13th FASEB Neuroimmunology conference, with the first being held in 1988. Importantly, it will be the 2nd pre-meeting course on neuroimmunology, the specific purpose of which is to introduce graduate students, postdoctoral fellows, and young investigators to basic neuroimmunology concepts and the active participation of these investigators and their integration into the broader research community. These meetings will be held at the Big Sky Resort in Big Sky, MT, with the pre-meeting course on neuroimmunology being held the day of July 24, with the main FASEB meeting beginning the evening of July 24 with two internationally renowned keynote speakers, and continuing until July 29, 2016. The last FASEB meeting was also held in Big Sky, MT in 2014, where Dr. Monica Carson was elected Co-Chair, to work with Dr. Tammy Kielian, Chair of the 2016 meeting. The overall goal of the FASEB Translational Neuroimmunology meeting and its associated pre-meeting course is to bring the world's leading scientists and the most promising young investigators and trainees together in a collegial and supportive environment. The specific goals are to: 1) provide a forum for the presentation of unpublished, cutting-edge research and both formal and informal discussions of the manner in which these results advance the field; 2) include investigators focusing on diverse topics in the neuroimmunology field and highlight translational aspects of this work; 3) include investigators focusing on all aspects of neuroimmunology and provide these investigators with a forum to expand the boundaries of their own work; and 4) ensure development and integration of young investigators into the broader research community in a meaningful and interactive way. The collegial atmosphere will include organized discussion sessions as well as opportunities for informal gatherings in the afternoons, thus providing an ideal setting for scientists from different disciplines to exchange ideas and foster cross-disciplinary collaborations both among themselves and the next generation of neuroimmunologists. Thus, the FASEB Translational Neuroimmunology conference will provide a unique and fundamentally important opportunity to bring together the world's leading investigators in a format designed for the free exchange of research results and ideas covering all aspects of neuroimmunology and its relationship to disease and, together with the pre-meeting course on neuroimmunology, it will do so in a manner specifically designed to promote the development of promising young investigators in the field and their integration into the larger research community. In the short term, this will significantly enhance the ability of investigators around the world to address critical problems in neuroimmunology, while in the long term it will ensure the continued success of their efforts and those of the next generation of investigators.
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0.909 |
2016 |
Kielian, Tammy L |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Mechanistic Insights Into Juvenile Neuronal Ceroid Lipofuscinosis Using Targeted Gene Delivery @ University of Nebraska Medical Center
Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a neurodegenerative lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Symptom onset occurs between 5-10 years of age with blindness and intractable seizures, followed by progressive cognitive and motor deterioration, and premature death (late teens-early 20s). JNCL is typified by neuronal apoptosis; however, neuronal death in the CLN3?ex7/8 mouse model occurs later in the disease process (12 months) compared to astrocyte activation, which is an early event (1-3 months) and predicts regions of eventual neuronal loss. Our preliminary data reveal reduced Ca2+ signaling, glutamate transporter, and glutamine synthetase expression in CLN3?ex7/8 astrocytes, which manifests as impaired glutamate clearance. This is expected to influence neuronal apoptosis, since excessive extracellular glutamate has been implicated in neuron excitotoxicity during JNCL. This suggests that aberrant astrocyte activity may impact neuronal dysfunction and late- stage apoptosis, which mandates a systematic assessment to alleviate this confound and separate the cell autonomous contributions of CLN3 mutation in neurons vs. astrocytes. To this end, our group has developed novel self-complementary (sc) adeno-associated virus 9 (scAAV9) constructs to probe cell type-specific effects of CLN3 action, where human CLN3 (hCLN3) is preferentially targeted to neurons (synapsin-hCLN3), astrocytes (GFAP-hCLN3), or both populations using the methyl-CpG-binding protein 2 (MeCP2) promoter (MeCP2-hCLN3) to examine effects on disease mechanisms and pathology. We will utilize these viruses as tools to test the hypothesis that neuron loss in JNCL is influenced by distinct cell autonomous actions of CLN3 in neurons and astrocytes. The effects of cell type selective CLN3 expression on disease attributes will be examined in the following Specific Aims: 1) Examine the effects of restoring CLN3 expression in neurons vs. astrocytes on behavioral, pathological, and physiological deficits in JNCL; 2) Examine the effects of CLN3 mutation in neurons vs. astrocytes on neurotransmitter perturbations in JNCL; and 3) Examine cell autonomous effects of CLN3 on proteostasis. This work will be the first to directly demonstrate cell autonomous effects of CLN3 mutation and how this contributes to neuronal loss in JNCL. This would significantly expand our understanding of the major cell types driving neuropathology and delineate how/where therapies should be targeted to exert the greatest impact on JNCL pathogenesis.
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1 |
2018 — 2020 |
Kielian, Tammy L |
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. |
Immune Mechanisms That Promote S. Aureus Persistence During Craniotomy-Associated Biofilm Infection @ University of Nebraska Medical Center
Neurosurgery to relieve life-threatening edema (decompressive craniectomy) or gain temporary access to the brain for tumor resection (craniotomy) requires removal of a portion of the skull (i.e. bone flap). The infection incidence after craniotomy/craniectomy ranges from 0.8-12%, with a significant number caused by methicillin- resistant S. aureus (MRSA), which forms a biofilm on both surfaces of the bone flap. Biofilms are bacterial communities encased in a self-produced matrix that are recalcitrant to antibiotics due to their metabolic dormancy. Our laboratory has developed a mouse model of S. aureus craniotomy-associated biofilm infection that shares important ultrastructural and MRI attributes with human disease, which can be exploited to identify mechanisms for infection persistence. We have identified a unique immune compartmentalization in the S. aureus craniotomy model, namely preferential neutrophil (PMN) recruitment and chemokine expression in the subcutaneous galea, whereas monocytes are more prominent in the brain. Myeloid-derived suppressor cells (MDSCs), an immature granulocyte population with anti-inflammatory properties, are present in both compartments, but most abundant in the galea. Our preliminary studies have identified a role for IL-10 in promoting S. aureus survival in both the galea and brain, suggesting that IL-10 may be critical for programming glia and infiltrating leukocytes towards an anti-inflammatory state to promote biofilm persistence. Although IL- 10 is expressed in both the galea and brain, the cytokine is poised to inhibit the antibacterial activity of cell types that are enriched at either site, namely PMNs and MDSCs (galea) vs. microglia, astrocytes, and monocytes (brain). In terms of molecular mechanisms, our preliminary data demonstrate that S. aureus- derived lactate induces IL-10 production in MDSCs. Since lactate is a histone deacetylase inhibitor (HDACi) and the IL-10 promoter is regulated by histone acetylation, this supports the hypothesis that S. aureus biofilm- derived lactate is an exogenous HDACi that promotes IL-10 expression and inhibits antimicrobial responses in the galea and brain by targeting MDSCs/PMNs vs. microglia/astrocytes/monocytes, respectively, to promote infection persistence. The crosstalk between IL-10 and galeal vs. brain populations and the molecular mechanisms responsible for this compartmentalized specificity will be tested in the following Specific Aims. 1) Identify the role of MDSC-derived IL-10 on PMN antimicrobial activity in the galea during S. aureus craniotomy- associated biofilm infection; 2) Determine whether IL-10 promotes S. aureus persistence in the brain during craniotomy-associated biofilm infections by polarizing glia and monocytes towards an anti-inflammatory state; and 3) Establish that S. aureus biofilm-derived lactate enhances IL-10 production by inhibiting HDAC activity. An improved understanding of the immune compartmentalization during craniotomy biofilm infection may be leveraged to enhance antimicrobial activity and biofilm clearance in both the galea and brain. Our findings suggest that IL-10 represents an attractive candidate to explore for this purpose.
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1 |
2019 — 2021 |
Kielian, Tammy L |
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. |
Contribution of Extracellular Enzymes to Staphylococcus Aureus Biofilm Development @ University of Nebraska Medical Center
PROJECT SUMMARY Staphylococcus aureus is one of the most problematic bacterial pathogens in our healthcare settings. S. aureus can survive and persist in the host by developing into an encased community of cells called a biofilm, and numerous studies have demonstrated that biofilms are resistant to host immune defenses and chemotherapies. Our central PPG hypothesis is that S. aureus biofilm development creates unique metabolic niches that promote an immune suppressive environment. In this proposal (Project 3), we are focusing on the contribution of S. aureus extracellular enzymes to biofilm growth and maturation, persistence in the host, and ultimately dissemination to a new site. Of the many enzymes that S. aureus secretes, we will prioritize hyaluronidase (HysA) and nuclease (Nuc1), as they have commonalities in biofilm-host interaction phenotypes and regulatory schemes. We recently demonstrated that hyaluronan accumulates in an S. aureus biofilm infection and that HysA can degrade this host glycosaminoglycan to disaccharides (HA-DS). Our preliminary studies indicate that HA-DS can serve as a carbon source through an unknown catabolic pathway, and this disaccharide has additional anti-inflammatory properties that could be contributing to the persistent nature of S. aureus biofilm infections. In Specific Aim 1, we will determine the role and regulation of hyaluronan metabolism in S. aureus biofilm maturation. We will characterize the HA-DS catabolic pathway using genetic analysis and labeling studies in collaboration with the Metabolomics Core. We will also test catabolic pathway knockouts in biofilm maturation and foreign-body infections, and investigate the contribution of CodY and CcpA to regulation of hyaluronan catabolism. Additionally, S. aureus will be grown on HA-DS and RNAseq performed to identify global transcriptomic changes. In Specific Aim 2, we will investigate how S. aureus enzymatic degradation of host polymers impacts the biofilm anti-inflammatory state. In collaboration with Dr. Tammy Kielian (PPG Project 4), we will assess the effect of HA-DS, as well as Nuc1 and HysA enzymes and their regulators, on immune cell function. We will also determine the impact of HA-DS and HysA inhibitors on biofilm infection, and test whether HA-DS is a biomarker for S. aureus in human synovial fluid from patients with prosthetic joint infection (PJI). In Specific Aim 3, we will examine S. aureus exo-enzyme regulation and function in biofilm dispersal. We hypothesize that CodY controls dissemination from S. aureus biofilms in an enzyme and nutrient dependent manner. We will investigate the contribution of Nuc1 and HysA, along with CodY and SaeRS regulators, to biofilm dispersal in vitro and during foreign body infection. We will also examine the impact of nutritional status on CodY activity during biofilm formation and dispersal in collaboration with Dr. Ken Bayles (PPG Project 1) and the Bioimaging Core. Finally, we will determine the role of aureusimine molecules in S. aureus biofilm development. Collectively these studies will define the contribution of S. aureus exo-enzymes to biofilm metabolism, development and persistence, potentially leading to innovative therapies for biofilm infections.
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