Now showing items 1-20 of 1960

    • Implementation Guide to Support Use of Elastomeric Half Mask Respirators in Healthcare

      Hines, Stella; THURMAN, PAUL; McDiarmid, Melissa A. (2021-02-03)
      Elastomeric half mask respirators (EHMRs) provide similar or higher level of protection as do N95 filtering facepiece respirators (N95 FFRs) and are designed to be reused. Supply chain shortages of respirators at the onset of COVID-19 surge prompted a large, urban, US academic medical center to rapidly expand use of EHMRs. A scaled fit-testing operation completed over 7000 fit-tests over six weeks. A centralized EHMR decontamination program was established in the Central Sterile Processing department. EHMR users obtained and returned their respirators each day from a central distribution center using a shared-supply model. This EHMR-based model of respiratory protection was cost effective. All healthcare personnel that needed respiratory protection during COVID-19 surge had access to NIOSH-approved, employer-provided respirators.
    • What's the Buzz? 2021

      University of Maryland, Baltimore. School of Medicine, 2021
    • Probiotic Use for the Primary Prevention of Clostridioides difficile infection

      Heil, Emily; Leekha, Surbhi; 0000-0002-6644-6684 (2020)
      Primary prevention of C. difficile infection (CDI) is a priority for hospitals and probiotics have the potential to interfere with colonization and/or infection with C. difficile offering an opportunity to enhance ongoing primary prevention strategies. The overall objective of this study was to evaluate the impact of a hospital-wide computerized clinical decision support system tool to prescribe probiotics to eligible adult patients receiving antibiotics for the primary prevention of CDI. After implementation of the tool, the odds of CDI was 1.41 in eligible patients compared to the pre-intervention time period (aOR 1.41, 95% CI 1.11, 1.79). A propensity score matched analysis showed that patients who received probiotics did not have lower rates of CDI compared to those who did not (OR 1.46, 95% CI 0.87, 2.45). Based on these findings, the use of probiotics for the primary prevention of CDI is not supported.
    • Discovery of a thalamic integrator for action reinforcement

      Cover, Kara; Mathur, Brian N. (Brian Neil); 0000-0003-3938-9669 (2020)
      The pursuit and acquisition of rewards is essential for survival. The input nucleus of the basal ganglia, the striatum, facilitates the selection, execution, and reinforcement of reward obtaining actions. The contributions of excitatory input from cortical regions and the influence of dopaminergic signaling on corticostriatal synapses to these vital functions has been studied extensively. A relative underappreciation for thalamic contributions to striatal signaling has consequently excluded this major excitatory input from basal ganglia models of action selection and reinforcement. I investigated the contributions of the rostral intralaminar thalamic nuclei to striatal-mediated action sequence performance using a combination of ex vivo physiological and in vivo behavioral assays. In the present dissertation, I characterize mechanisms by which this thalamic projection interacts with striatal neuronal populations and modulates dopaminergic signaling to sculpt output activity. I further identify that the rostral intralaminar nuclei causally contribute to action execution in the pursuit of rewards. This work serves to further understanding of how thalamic inputs participate in action selection and reinforcement.
    • Melanocyte stem cell subpopulations show distinct pigmentation and regenerative potential

      Tandukar, Bishal; Hornyak, Thomas; 0000-0002-9650-1664 (2020)
      Melanocyte stem cells (McSCs) are key components of the hair follicle (HF) stem cell system. They are derived from neural crest during embryogenesis and are responsible for regeneration of differentiated melanocytes during successive HF cycles. We have described McSC subsets that can be distinguished by CD34 expression. CD34+ McSCs are located within the bulge/lower permanent portion (LPP) while CD34- McSCs are in the secondary hair germ (SHG) during the resting stage (telogen). Whether these two cell subpopulations are maintained separately or exist in a developmental hierarchy is not yet known. The goal of my thesis is to explore whether (a) the two McSC subpopulations are functionally distinct, (b) if they are maintained independently throughout the HF cycle and (c) their role in generation of mature melanocytes. To study McSCs, we engineered the Dct-H2BGFP bitransgenic mouse. We confirm that this animal model accurately identifies melanoblasts, McSCs and mature melanocytes by constitutive GFP expression that can be regulated by doxycycline. Using our Dct-H2BGFP mouse, we compared the transcriptomes of bulge and SHG McSC subsets by genome-wide expression profiling (RNA-seq). This study, along with functional in vitro and in vivo assays, confirms that CD34+/bulge share characteristics of neural crest stem cells with multilineage potential while CD34-/SHG McSCs represent a stem cell population that is more committed to melanocyte differentiation. To further understand the relationship between two McSC subpopulations, we traced their proliferation throughout the HF cycle and found that proliferation of SHG McSCs gives rise to mature melanocytes. The analysis also surprisingly revealed quiescent CD34- melanocytes maintained outside of the HF bulge region throughout anagen retaining the stem cell phenotype, identifying a SHG McSC-like population outside telogen suggesting independent or quasi-independent maintenance of the two McSC subpopulations. Taken together, our study identifies heterogeneous McSC subpopulations with distinct pigmentation and regenerative potential for the first time. Strikingly, CD34+/bulge McSCs exhibited the ability to myelinate neurons in vivo, revealing a novel therapeutic possibility for demyelinating disorders and traumatic nerve injury.
    • Characterization of Ketamine’s (2,6)-Hydroxynorketamine Metabolites: Pharmacokinetic and Behavioral Considerations for Antidepressant Applications

      Highland, Jaclyn; Gould, Todd D.; 0000-0002-9600-2830 (2020)
      Despite numerous available treatments for depression, most are not only slow to take effect but also ineffective in many patients, highlighting the need for novel and effective antidepressant treatments. While (R,S)-ketamine (ketamine) has gained attention for its rapid-acting antidepressant effects in previously treatment-resistant patients, its widespread antidepressant use is limited by adverse effects and abuse potential. The ketamine metabolite (2R,6R;2S,6S)-hydroxynorketamine (HNK), and most potently the (2R,6R)-HNK stereoisomer, shares the antidepressant-like behavioral effectiveness, but lacks the adverse effect burden and abuse potential, of ketamine in rodents, suggesting that (2R,6R)-HNK, and possibly other HNKs (12 have been identified, but only (2R,6R)- and (2S,6S)-HNK were previously studied), may be favorable antidepressant drug candidates. However, several important considerations for the development of HNKs as antidepressants were not previously evaluated. Such considerations were addressed in the present study. First, the pharmacokinetic profiles of the 12 HNKs were characterized, revealing robust differences in plasma and brain concentrations among the various HNKs, despite similar brain-to-plasma ratios and rapid elimination profiles. Second, it was demonstrated that (2R,6R)-HNK has favorable oral bioavailability (45-52%) and orally administered (2R,6R)-HNK exerts antidepressant-relevant behavioral effects, but not overt adverse effects, in mice. Third, the sex-dependent differences in the metabolism of ketamine and (2R,6R)-HNK in mice were characterized. It was demonstrated that, following ketamine administration, female mice have lower levels of ketamine and higher levels of HNK than male mice. In addition, following direct dosing, female mice have higher levels of (2R,6R)-HNK than males. Male gonadal hormones at least partly mediate these differences. Further, the relative effectiveness of the four (2,6)-HNKs in the mouse forced swim test were compared, revealing that both (2R,6S)- and (2S,6R)-HNK exert effects at lower doses compared to either (2R,6R)- or (2S,6S)-HNK, and establishing a relative rank-order of effectiveness of (2R,6R)- > (2S,6R)- > (2R,6R)- > (2S,6S)-HNK. Finally, it was identified that the novel compound, (5R)-methyl-(2R,6R)-HNK recapitulates the antidepressant dose-response relationship of (2R,6S)-HNK, with which it shares a similar three-dimensional structure, suggesting a critical role of three-dimensional structure in mediating antidepressant-relevant effectiveness. Altogether, these studies represent several important insights which may support the development of (2,6)-HNKs as antidepressant treatments.
    • Impact of Colonic Inflammation During Chronic Experimental Traumatic Brain Injury in Mice on Long-Term Outcomes

      Hanscom, Marie; Shea-Donohue, Terez; Faden, A. I.; 0000-0001-8308-135X (2020)
      Disruptions in the bidirectional communications of the brain-gut axis are increasingly implicated in the onset and progression of a variety of gastrointestinal and neurological disorders, diseases, and injuries including traumatic brain injury (TBI). In addition to the effects in the brain, TBI can result in gastrointestinal dysfunction, potentially affecting TBI pathogenesis and outcomes. This preclinical study examined the effects of colonic inflammation induced during chronic (long-term) experimental TBI on TBI-associated neurobehavioral and neuropathological outcomes. Additionally, the potential involvement of the neural and immunological pathways of the brain-gut axis in colonic inflammation induced changes in neurobehavior and neuropathology was examined. Dextran sodium sulfate (DSS) was administered to adult male C576Bl/6 mice 28 days following craniotomy (Sham) or TBI for 7 days to induce colonic inflammation (DSS injury phase), followed by a return to normal drinking water for an additional 7 to 28 days for recovery (DSS recovery phase). Uninjured animals (Naïve) served as an additional control group. Colonic inflammation during chronic experimental TBI in mice persistently exacerbated deficits in fine motor coordination and anxiety-like behavior and induced deficits in social behavior in TBI-injured mice. These behavioral changes were associated with an induction, or exacerbation, of ipsilateral hippocampal neuronal cell loss and microglial activation in Sham and TBI mice subjected to acute DSS administration, respectively. Colonic inflammation resulted in a sustained systemic immune response with increases in myeloid cells in blood and spleen, as well as myeloid cells and lymphocytes in mesenteric lymph nodes (mLN) in all DSS experimental groups. A sustained increase in spleen and mLN weights in all DSS groups, and thymus weight in Sham and TBI mice administered DSS, was observed up to 28 days following DSS administration. Dysautonomia was also induced in Sham and TBI mice subjected to acute colonic inflammation, with increased sympathetic tone beginning during DSS injury phase and persisting through the end of the first DSS recovery week. These data show that acute colonic inflammation during chronic experimental TBI results in enhanced neurocognitive deficits, neurodegeneration, microglial-related neuroinflammation, and a sustained systemic immune response with altered autonomic balance.
    • Investigating the pathophysiological significance of obscurin immunoglobulin domains Ig58/59 in the heart

      Grogan, Alyssa; Kontrogianni-Konstantopoulos, Aikaterini; 0000-0002-1505-3311 (2020)
      Obscurin (720-870 kDa) is a giant cytoskeletal protein that surrounds the periphery of myofibrils at M-bands and Z-disks where it plays key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin mediate binding to several important modulators of muscle structure and function, including canonical titin, a smaller splice isoform of titin termed novex-3, and phospholamban (PLN). Importantly, missense mutations identified within obscurin Ig58/59 that affect binding to titins and/or PLN are linked to cardiac and skeletal myopathies in humans. To assess the pathophysiological role of Ig58/59 in vivo, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59. Our studies revealed that young Obscn-ΔIg58/59 animals, which do not exhibit cardiac pathologies under sedentary conditions, experience tachycardia and severe arrhythmia following acute β-adrenergic stimulation and develop enlarged atria following prolonged exercise stress. Moreover, sedentary Obscn-ΔIg58/59 mice develop compensatory left ventricular hypertrophy that progresses to dilation, atrial enlargement, contractile dysfunction, and arrhythmia through aging in which males are more affected than females. Together, these findings demonstrate that deletion of obscurin Ig58/59 leads to both ventricular and atrial pathologies as a function of aging and in response to stress that manifest to different extents between sexes. We therefore aimed to decipher the cellular and molecular impact of the Ig58/59 deletion in Obscn-ΔIg58/59 ventricular and atrial myocardia, specifically focusing on males due to their enhanced disease severity. Our findings revealed that ventricular and atrial cardiomyocytes isolated from sedentary aging Obscn-ΔIg58/59 mice are enlarged and display tissue-specific alterations in Ca2+ cycling kinetics compared to controls, associated with distinct changes in the expression and phosphorylation status of major Ca2+ cycling proteins including PLN, the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and the ryanodine receptor 2 (RyR2). Lastly, proteomics and phospho-proteomics studies conducted on aged Obscn-ΔIg58/59 atria revealed significant changes in the levels of cytoskeletal proteins, Ca2+ regulators, and Z-disk associated complexes. Taken together, our studies demonstrate that obscurin Ig58/59 is a key regulatory module in the heart, and its deletion leads to age- and sex-dependent cardiac remodeling and arrhythmia associated with tissue-specific molecular alterations in the ventricles and atria.
    • The PAX-SIX-EYA-DACH Network regulates GATA factors and drives human erythropoiesis

      Creed, Timothy M.; Kingsbury, Tami (2020)
      Erythropoiesis is orchestrated by the coordinated action of multiple transcription factors. The master erythropoietic-regulator GATA1 is itself modulated via interactions with multiple co-regulatory factors. Though the PAX-SIX-EYA-DACH network (PSEDN) of conserved transcription factors has been well characterized in human organogenesis, a role for PSEDN members in hematopoietic systems has only recently been recognized (Liu et al., Nature 2019). Here we studied the PSEDN member SIX and discovered its ability to drive erythropoiesis of human hematopoietic cells. Overexpression of SIX1 or SIX2 in human TF1 erythroleukemia or primary CD34+ hematopoietic stem-progenitor cells (HSPCs) stimulated the generation of erythroid cells, as measured by flow cytometry, qPCR, and Western blot. Conversely, SIX1 knockout in TF1 cells or primary HSPCs reduced erythroid cell generation in response to erythropoietin. By gene set enrichment analysis of RNA-seq data, SIX1/SIX2 overexpression stimulated heme metabolism genes as well as genes known to be regulated by GATA1. SIX1/SIX2 overexpression reduced GATA2 and increased GATA1 expression, resembling GATA switching downstream of EPO signaling. To determine whether GATA1 was necessary for SIX1 to stimulate erythropoiesis, we generated GATA1-knockout cells using CRISPR/Cas9. In contrast to control cells, SIX1 OE in GATA1-knockout cells failed to stimulate erythropoiesis, indicating that SIX1 stimulation of erythropoiesis requires GATA1. To gain further insight into the mechanism by which SIX1/SIX2 stimulates erythropoiesis we generated a BirA*-SIX fusion protein to determine its interactome. Streptavidin-enrichment of biotinylated proteins in BirA*-SIX overexpression lysates revealed GATA1 and FOG1 as proximal interactors of BirA*-SIX. When co-expressed in HEK293T cells GATA1 and SIX1 were found to coimmunoprecipitate, suggesting the two proteins can physically interact in a complex. We demonstrated the functional consequence of the SIX1/SIX2 and GATA1 using a GATA1-dependent luciferase reporter construct. Cells in which SIX1/SIX2 and GATA1 were co-expressed exhibited significantly higher levels of luciferase expression compared to cells expressing only GATA1, suggesting SIX1 could stimulate GATA1-dependent transcription. Together our results suggest that SIX1 can stimulate erythropoiesis via multiple mechanisms. This thesis provides the first demonstration of a role for the PSEDN in erythropoiesis and reveal physical and functional interactions between two central developmental transcriptional networks (GATA/FOG and PSEDN).
    • In vitro and in vivo Models of Biofilm-Mediated Infections

      Brao, Kristen; Ernst, Robert K.; 0000-0003-0804-7105 (2020)
      Biofilms are microbial communities encased in a matrix of polysaccharides, extracellular DNA, and proteins. When growing in biofilms, bacteria display increased resistance to immune clearance and antibiotic tolerance. For these reasons, biofilms are responsible for many chronic infections. Treatment of biofilm-mediated infections is challenging, and often requires surgical debridement and/or lengthy courses of antibiotics. In order to develop more effective treatment strategies and therapeutics, it is important to develop and utilize model systems that incorporate the biofilm phenotype. In this dissertation, we describe the use of a rabbit model of internal fixation to evaluate the use of prophylactic antibiotic powders in surgery, characterize Scnn1b-Tg mice as models of Pseudomonas aeruginosa and Staphylococcus aureus infections in cystic fibrosis lungs, and use a continuous flow biofilm reactor to study the interactions of S. aureus and P. aeruginosa in chronic biofilms. We found that tobramycin powder is able to prevent surgical site infection with tobramycin-resistant Enterobacter cloacae, demonstrating the potential utility of antibiotic powders in preventing infections in orthopedic surgery. We also found that Scnn1b-Tg mice clear infections with P. aeruginosa and S. aureus more slowly than wildtype littermates. When Scnn1b-Tg mice were infected with a mucoid CF isolate, bacterial aggregates like those observed in cystic fibrosis patients’ sputum were found in the lungs, illustrating the potential of Scnn1b-Tg mice as models of cystic fibrosis lung infection. Finally, we found that P. aeruginosa modifies the structure of its lipid A by adding 4-amino-4-deoxy-L-arabinose (Ara4N) to the terminal phosphates when introduced to established S. aureus biofilms. The addition of Ara4N is associated with resistance to polymyxin antibiotics, such as colistin. This finding represents a potential mechanism by which interactions in polymicrobial biofilms can lead to changes in antibiotic resistance. These studies illustrate the importance of incorporating biofilm models when developing treatments for biofilm-mediated infections.
    • LRP1 as a Regulator of the Vasculature and Extracellular Matrix

      Arai, Allison Lindsay; Strickland, Dudley K.; Muratoglu, Selen C.; 0000-0002-0431-1856 (2020)
      The arterial wall and extracellular matrix (ECM) constantly remodel in response to physiological and pathophysiological signals including aging, atherosclerosis, and aneurysm formation. Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic and signaling receptor capable of binding and internalizing over 100 structurally unrelated ligands. LRP1 has an important role in vascular maintenance and homeostasis. Global knock-out of LRP1 in mice results in embryonic lethality due to failure to recruit mural cells to developing vessels. Smooth muscle cell (SMC)-specific knock-out of LRP1 (smLRP1-/-) causes fully penetrant aortic aneurysm formation in mice. Collaborators at University of Texas Health Science Center and Johns Hopkins University have identified multiple rare human LRP1 variants associated with aortic disease. We hypothesize that genetic variants of LRP1 associated with aneurysm formation will impact ligand binding, trafficking, or signaling of LRP1. We used biochemical, cell-based, and mouse studies to determine the functional impact of rare LRP1 variants. We used surface plasmon resonance to investigate the binding relationship between LRP1 and matrix metalloprotease-1 (MMP-1), an ECM protease associated with aneurysm formation. We found that MMP-1 was a novel LRP1 ligand and the endogenous inhibitory MMP-1 complex was the preferred MMP-1 form for LRP1 binding. Complex formation of MMP-1 with tissue inhibitor of metalloproteases 1 (TIMP-1) resulted in a 30- to 40-fold increase in the binding affinity for LRP1 compared to other forms of MMP-1. Next, we introduced rare LRP1 variants by site-directed mutagenesis into truncated forms of LRP1 called mini-receptors to examine LRP1 trafficking. Specific LRP1 mutations impacted LRP1 turnover and/or maturation. We also found that LRP1 mini-receptors have a trafficking impairment that results in targeted, proteasomal degradation and limited LRP1 cell surface delivery. We suggest future caution when using mini-receptors in assays dependent on LRP1 trafficking. Finally, our mouse-based studies examined the impact of loss of LRP1 in SMCs (smLRP1-/-) on the cerebrovasculature and found that the vascular branching pattern and geometry was unaffected. In summary, our results demonstrate that LRP1 rare variants associated with aneurysm formation have functional deficits in LRP1 and that LRP1 is an important regulator of the vasculature and ECM.
    • Quest for New Drugs Against HIV-1 Multi-Drug Resistant Proteases

      Vernon, Kasey; Zhao, Richard Y.; 0000-0002-6294-503X (2020)
      HIV-1 protease inhibitors (PIs) are the most potent class of drugs in combinational antiretroviral therapies (cART). However, the current therapies are still not adequate due to the emergence of drug resistance. There is still a need for development of more potent PIs. The objective of this study was to search for multi-drug resistant inhibitors to combat HIV-1 and viral resistant proteases. Our lab developed a fission yeast (Schizosaccharomyces pombe) cell-based system that allows large-scale or high-throughput drug screening. We first used a cell growth-based assay to test a drug candidate. We then used a HIV-1 enzymatic assay to confirm it. We explored four batches of potential anti-HIV compounds as well as nine FDA approved PIs. All FDA-approved PIs potentially inhibited HIV-1 PR with 3 small molecule compounds showed minimal inhibitory effects. Results of this study demonstrated the use of fission yeast cell-based system as a means to discover new PIs.
    • Estradiol Action at the Median Preoptic Nucleus Modulates Adenosinergic Homeostatic Sleep Pressure

      Smith, Philip C.; Mong, Jessica Aurora; 0000-0001-6726-2781 (2020)
      To further our understanding of how gonadal steroids impact sleep biology, we sought to address the mechanism by which proestrus levels of cycling ovarian steroids, particularly estradiol (E2), suppress sleep in female rats. We showed that steroid replacement of proestrus levels of E2 to ovariectomized female rats, suppressed sleep to similar levels as those reported by endogenous ovarian hormones. We further showed that this suppression is due to the high levels of E2 alone, and that progesterone did not have a significant impact on sleep behavior. We found that E2 action within the Median Preoptic Nucleus (MnPN), which contains estrogen receptors (ERs), is necessary for this effect; antagonism of ERs in the MnPN attenuated the E2-mediated suppression of both non-Rapid Eye Movement (NREM) and Rapid Eye Movement (REM) sleep. Finally, we found E2 action at the MnPN is also sufficient for sleep suppression, as direct infusion of E2 into the MnPN suppressed sleep. Based on our findings, we predict proestrus levels of E2 alone, acting at the MnPN, mediate sex-hormone driven suppression of sleep in female rats. Furthermore, our findings have demonstrated that E2 has different effects on sleep time and slow wave activity (NREM-SWA), a measure of the homeostatic efficiency of sleep, under conditions of normal sleep and sleep deprivation. E2 serves to decrease NREM-SWA, but not sleep time, in the light phase under normal sleep, while in recovery, E2 decreases sleep time but not NREM-SWA following deprivation. We further found that E2 increased levels of extracellular adenosine, a measure of homeostatic sleep need, under both normal and deprivation sleep conditions. To resolve the discrepancy between the decrease in behavioral markers associated with homeostatic sleep need and increase in levels of adenosine, we employed an agonist of the A2A receptor to stimulate the adenosinergic sleep pressure system. These studies show that E2 blunts the ability of the A2A agonist to drive sleep behavior, showing an interposition of E2 into the adenosinergic sleep pressure system. Overall, these experiments show an interaction between E2 and adenosinergic homeostatic sleep pressure at the level of the MnPN.
    • FOXO1 Nucleo-Cytoplasmic Distribution and Regulation

      Russell, Sarah; Schneider, Martin F. (2020)
      Skeletal muscle atrophy is regulated at the cellular level by several signaling pathways that modulate the balance between protein degradation and protein synthesis. A primary pathway regulating skeletal muscle atrophy is the IGF1 or Insulin/PI3K/Akt/Foxo1 pathway. IGF1/insulin lead to activation of a cascade of kinases, ultimately activating Akt which phosphorylates specific sites on Foxo1 and causes Foxo1 to translocate out of myofiber nuclei. When present in myofiber nuclei, Foxo1 functions as a transcription factor that activates transcription of several pro-atrophic genes, such as MurF1 and MAFbx/atrogin1. Here we use fluorescence time lapse imaging of the nuclear cytoplasmic distribution of Foxo1-GFP in adult isolated skeletal muscle fibers a to investigate (a) variability of FOXO1 nucleo-cytoplasmic distribution in mature muscle fibers as well as FOXO1 nuclear influx and efflux, and (b) the effects of IGF1 and insulin on Foxo1-GFP translocation in the presence of pharmacological inhibitors that target specific sections of the canonical IGF1 or insulin/PI3K/Akt/Foxo1 pathway. For our first aim, our results demonstrate that FOXO1 N/C and apparent rate coefficient (kI’) of FOXO1 nuclear influx are nearly identical for nuclei within the same fiber, they are highly varied for nuclei belonging to different fibers. These results indicate that variability in cellular factors, but not extracellular factors, determine FOXO1 distribution from fiber to fiber. Additionally, we show that application of IGF1 is able to reduce variation in kI’ and N/C in all fibers while AKT-inhibitor application does not, indicating other pathways are involved. For our second aim, our results demonstrate Akt is absolutely necessary for the dramatic IGF1 or insulin-induced rapid and near complete nuclear efflux of Foxo1, PI3K is not. Insulin induced movement of the PIP3 biosensor PH ARNO-GFP to transverse tubules is prevented by PI3K inhibitor BKM120, demonstrating that PI3K inhibition was effective. We have also found that two additional protein kinases, Ack1 and ATM, contribute to IGF1-induced Akt activation and Foxo1-GFP nuclear efflux. These results indicate the presence of PI3K-dependent -independent parallel pathways from IGF1 to Akt activation in adult muscle that are not well understood in Foxo1 regulation.
    • The impact of the non-immune chemiome on T cell activation

      Rosenberg, Kenneth; Singh, Nevil; 0000-0002-1231-7836 (2020)
      T cells are critical organizers of the immune response and rigid control over their activation is necessary for balancing host defense and immunopathology. It takes 3 signals provided by dendritic cells (DC) to fully activate a T cell response – T cell receptor (TCR) engagement of antigen on MHC (Signal 1), co-stimulatory signals (Signal 2) and cytokines (Signal 3). Yet, even before activation T cells are typically exposed to a universe of chemicals (a “chemiome”) including drugs, metabolites, hormones etc. which are not typically ascribed an immunological role. In this thesis, we hypothesized that members of this non-immune chemiome acting on T cells, prior to antigen encounter, flavor specific signaling pathways to differentially influence subsequent T cell activation and fate. Unraveling these signals, which we termed “Signal 0”, could help us understand and manipulate tissue and time specific flavoring of immunity. In this thesis we first developed a pharmacological model for signal 0, by treating T cells with drugs that activate only subsets of the TCR-signaling network prior to full antigen exposure. We found that pharmacological pre-activation of the PKCƟ/ERK pathways modulates long time survival of T cells without changing proliferation or cytokine production. Next, we examined receptors for the non-immune chemiome that resting T cells express and identified neurotransmitter receptors (NR) as a major family. All T cells expressed a core NR signature, but very few NR were also modulated in a T cell lineage-specific fashion. Of these, we focused on VPAC1, the receptor for vasoactive intestinal peptide (VIP). We found that VIP signaling attenuates ERK phosphorylation, but paradoxically drives increased differentiation towards IL-17 and IL-22 secretion. In addition ERK signaling induced by drugs (phorbol esters) versus the TCR followed differential kinetics and recruited non-overlapping negative feedback mechanisms, suggesting that even the same branch of TCR signaling is subject to different localization and temporal controls. Taken together, our data suggest that the branches of the TCR-signaling network integrate pre-existing signals (Signal 0) into the activation program of T cells, allowing localized cues, including neurotransmitter levels, to modify the long-term trajectory of the immune response.
    • Regulation of intrinsic activation thresholds of T cells

      Matson, Courtney; Singh, Nevil; 0000-0002-9817-4986 (2020)
      T cells are activated when their T cell receptor (TCR) senses peptide-MHC (pMHC) molecules from pathogens and tumors. A network of signaling molecules downstream of the TCR drives the extent and nature of subsequent cellular responses. The activation threshold (AT) of these signaling pathways is a critical checkpoint for T cell responses. Here, we examined mechanisms by which the AT of a T cell is first determined and how it changes during the course of responding to antigen. The initial AT of a T cell is set during development by calibrating to how strongly it senses pMHC in the thymus. This calibration affects the surface levels of a receptor CD5, whose subsequent role is poorly defined. We found that CD5, independent of the TCR, sets basal levels of IκBα in T cells. Since IκBα critically modulates the transcription factor NFκB, which regulates multiple T cell functions including cell-survival, we hypothesized that variations in basal AT of T cells stem from varying NFκB depots maintained by CD5. Indeed, blocking NFκB abolished differences in cell-survival of thymocytes with different CD5 levels. The initial heterogeneities are further modified when peripheral T cells encounter antigen. Resulting memory T cells acquired higher CD5 levels and continuously required CD5 expression to maintain higher IκBα expression. If the stimulating antigen was not cleared efficiently, peripheral T cells further ‘tuned’ their AT in the opposite direction and resulted in loss of sensitivity to antigen (as seen in exhausted T cells). Importantly, this AT tuning involved additional regulation of TCR-proximal kinases such as Zap70 and was reversible in vivo, but not in vitro. We also found that rather than just the duration of antigen exposure, AT-tuning was potentially influenced by the rate at which antigen changes in vivo. This can help us understand how different persistent pathogens or tumors affect T cell responses, potentially based on the rates at which they replicate in the host. Finally, we characterized compounds that can target a T cell’s AT, identified in a high-throughput pharmacological screen, to potentially isolate drugs for altering T cell function during these physiological contexts.
    • “Interest-STING”: Inhibition of Innate Immune Signaling by Prostaglandin E2

      Mathena, Reilley; Vogel, Stefanie N.; 0000-0002-2049-8139 (2020)
      The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic double-stranded DNA from microbial or host cells. cGAS-STING activation elicits an inflammatory response, including production of type I interferons, through activation of TBK1 kinase and the transcription factor IRF3. Therefore, negative regulation of cGAS-STING activity would be predicted to prevent microbial-induced or autoimmune inflammatory damage. Based on prior work, we tested the hypothesis that the inflammatory product prostaglandin E2 (PGE2) acts on immune cells to control inflammation induced by cGAS-STING. STING pathway activating agents, 5,6 dimethylxanthenone-4-acetic acid (DMXAA) and cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), were used to initiate STING signaling. Exogenous PGE2 suppressed DMXAA- and cGAMP-induced STING signaling in murine embryonic fibroblasts and primary murine macrophages. Cells treated with PGE2, followed by DMXAA or cGAMP, exhibited decreased activation of TBK1 and IRF3, and decreased inflammatory gene expression, arguing that PGE2 signaling may be a mechanism restricting cGAS-STING activation.
    • Psilocybin as a Rapid Acting Antidepressant: Are Hallucinations Necessary?

      Hesselgrave, Natalie; Thompson, Scott M., Ph.D.; 0000-0001-8076-1413 (2020)
      Major depressive disorder (MDD) is a leading cause of mental illness world-wide. Economists estimate MDD to be one of the leading causes of disability worldwide. The cost is not just due to treatment, but comorbidities, lost work and suicide, too. However, treatments are available and range from psychotherapy, pharmacotherapy or more invasive treatments like electroconvulsive therapy or deep brain stimulation. For decades, the standard of pharmacotherapy care has been selective serotonin reuptake inhibitors (SSRIs). Yet, SSRIs are only effective in 60% of MDD patients and require weeks of treatment before reaching therapeutic efficacy. Thus, a large proportion of individuals experiencing MDD endure symptoms for weeks without guaranteed relief. In 2000, low doses of ketamine, a dissociative anesthetic, was found to have rapid acting antidepressant effects. Prior to this, electroconvulsive therapy was the only treatment known to induce rapid antidepressant effects. The use of ketamine as an antidepressant has been met with resistance since it is a known drug of abuse, has an addictive potential and psychotomimetic side effects. Findings from recent human studies suggest a single oral administration of psilocybin has antidepressant effects within 7 days. However, the hallucinatory effects of psilocybin, like ketamine, severely limit its clinical use. Currently, psilocybin is administered in a controlled environment with psychological support, which greatly increases the cost of care and patient burden. Administration of ketanserin, a serotonin 2 receptor (5HT2R) antagonist, prior to psilocybin attenuates the psychedelic side effects. But whether ketanserin would also block the antidepressant effect is unknown. I sought to test the hypothesis that psilocybin has an antidepressant-like effect in rodents independent of the 5HT2R. To this end, I have demonstrated that psilocybin has a rapid antidepressant effect in anhedonic mice and increases synaptic strength in hippocampus following chronic stress. Prior administration of ketanserin blocks neither psilocybin’s antidepressant-like effect nor the increase in synaptic strength. In vitro electrophysiology experiments failed to show acute effects of psilocin, possibly due to psilocin’s rapid oxidation. While the antidepressant mechanism of action of psilocybin remains unknown, my data suggest the 5HT2R may not be necessary for the antidepressant effects.