Microbiology / Infectious Disease

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21716

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    Regulation of Astrocyte Mitochondrial Function and Oxidative Stress by Trace Amine Associated Receptor 1 in the context of Methamphetamine Abuse and HIV-Associated Neurocognitive Disorders.
    (2016-03-23) Cisneros, Irma; Ghorpade, Anuja PhD; Borgmann, Kathleen
    As a psychostimulant, methamphetamine (METH) use leads to long-lasting, euphoric effects. Between 10- 15% of human immunodeficiency virus-1 (HIV-1) patients report METH abuse, which exacerbates HIV-1 infection, accelerating the onset of HIV-associated neurocognitive disorders (HAND) and immune dysfunction. Neuroinflammation, glial activation, oxidative stress and excitotoxicity contribute to METH and HIV neuropathogenesis. However, the mechanisms through which METH and HIV affect astrocyte function are unclear. Recently, we reported trace amine associated receptor 1 (TAAR1) as a novel astrocyte receptor for METH. Previous studies suggest TAAR1 activity may be regulated by G-protein promiscuity and desensitization by b-arrestin. We hypothesize that HIV-relevant stimuli upregulate astrocyte-TAAR1 expression and that METH exposure induces alterations in TAAR1 activation and intracellular localization, thus contributing to astrocyte dysfunction. To examine TAAR1 expression was assessed by real-time PCR and fluorescent microscopy in human astrocytes in the context of HIV and METH exposure. Changes in EAAT2, which could impair astrocyte ability to clear glutamate, were examined in parallel. To assess TAAR1 regulation by interacting partners, b-arrestin phosphorylation and co-immunoprecipitation studies were performed. Further, downstream outcomes of TAAR1-mediated cAMP and calcium signaling were evaluated, focusing on mitochondrial function and oxidative stress. TAAR1 expression is increased by HIV-relevant stimuli; while EAAT2 expression is concurrently decreased. TAAR1 localizes throughout the cell body in non-activated astrocytes and becomes perinuclear, increasing in the ER during gliosis. b-arrestin is activated by IL-1b and associates with astrocyte-TAAR1. Mitochondrial dysfunction increases with METH and HAND-relevant activation, leading to reactive oxygen species. These studies delineate how dysregulation of TAAR1 may contribute to astrocyte-mediated neurodegeneration during HAND and METH abuse, while also revealing a novel therapeutic target in astroglia.
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    HIV-1 TAT Induces MIR-132 Expression Leading to Neurotoxicity and Aberrant Dendritic Morphology
    (2016-03-23) He, Johnny; Rahimian, Pejman
    TITLE: HIV-1 Tat induces mir-132 expression leading to neurotoxicity and aberrant dendritic morphology Authors: Pejman Rahimian, Johnny He Presenter name: Pejman Rahimian Purpose: HIV-1 Tat is involved in the pruning of neurites and loss of synapses which are the most prominent pathological hallmarks of HIV-associated neurocognitive disorders (HAND). However the underlying molecular mechanisms of this synaptodendritic loss have not been elucidated. We report for the first time the induction of a brain-enriched microRNA by HIV-1 Tat protein leading to repression of significant regulating factors in dendritic arborization and synapse formation. Methods: Levels of miR-132 were quantitated in astrocyte cell lines (U373.MG), primary human and primary mouse astrocytes, and also in neurons (SH-SY5Y) following transfection with Tat plasmid and also in primary human astrocytes following infection with the VSVG-pseudotyped HIV-1 virus. Repression of miR-132 targets and involvement of CREB in miRNA induction were evaluated via Western blotting. Results: We observed significant miR-132 upregulation as the result of Tat expression in both neurons and astrocytes followed by the repression of miR-132 targets in both cell types. Activation of CREB as indicated by elevated p-CREB was observed along with Tat expression while using a Tat construct defective in CREB activation abrogated miR-132 induction by Tat. We also observed significant reduction in neuronal viability along with loss of dendritic arbor following Tat expression which correlate with the repression of miR-132 targets MecP2 and p250GAP and consequently BDNF loss. Conclusion: Our results indicate that HIV-1 Tat induces miR-132 in the brain through activation of CREB and stabilization of interaction between p-CREB and CREB-binding protein (CBP). Dysregulated miR-132 expression contributes to neurotoxicity and aberrant dendritic morphology witnessed in neurocognitive disorders associated with HIV-1 invasion of the central nervous system. Acknowledgments (ex. funding support, etc.) Neurobiology of Aging Training Grant-T32 AG020494 IACUC#: 2014/15-01-A04
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    The in vitro Virulence Phenotype of Clostridium difficile Ribotype 027 Impacts Disease Severity in the Mouse and Hamster CDAD Models
    (2016-03-23) Pulse, Mark; Simecka, Jerry; Vitucci, John
    C. difficile ribotype 027 (RT027) is the epidemic strain found primarily in North America. Studies have suggested an enhanced virulence phenotype for RT027 such as increased toxin production, but the impact on disease severity on in vivo models is not well understood. This study describes the in vitro characterization of important virulence characteristics for several RT027 and non-RT027 C. difficile clinical isolates, and how these factors may impact disease severity in the hamster and mouse C. difficile associated disease models. Six RT027 and six non-RT027 clinical isolates were evaluated in vitro for total spore counts and Toxin A/B titers in 72H broth cultures. Spore counts were generated from heat/ethanol shock culture samples and plated onto CCFA containing 0.1% taurocholate, and toxin A/B titers were determined from spent broth with the tgcBIOMICS ELISA assay. The mouse C. difficile model involved being administered for 5 days through oral gavage or drinking water. The mice were then placed on a non-antibiotic supplemented water for 48 hours IP administered 10 mg/kg of clindamycin, and orally inoculated with C. difficile spores 24H later. Survival was monitored for 10 days and fecal samples were taken each day to be processed for CFU/spore counts. The HCDAD studies involved inoculating male Golden Syrian hamsters with 72H broth cultures of two RT027 and two non-RT027 isolates, followed by subcutaneous administration of 10 mg/kg clindamycin 24H post-infection. One group of infected hamsters was orally treated with 20 mg/kg vancomycin once a day for 3 days following clindamycin administration, while the other group remained untreated. Survival was monitored for 11 days after infection and post-mortem cecal fluid samples were taken from 3 hamsters at set disease-associated time points to determine the CFU/spore counts and Toxin A/B titers. The RT027 and the non-RT027 strains generated similar mean CFU/mL in 72H broth cultures, while the mean spore counts were 548 spores/mL for the RT027 strains and 273 spores/mL for the non-RT027 strains. In addition, the 72H broth-associated mean toxin A/B titers were 2.8-fold higher for RT027 strains when compared to the 72H titers of non-RT027 strains. In the mouse model, 100% of the animals infected with the non-027 isolate survived regardless of how antibiotics were administered. In contrast, 13-26% morbidity was associated with mice infected with the RT027 isolate after being given antibiotics by oral administration or through supplemented water. In the HCDAD studies, 14% of the non-027 infected hamsters became moribund, while 71% of the hamsters infected with the RT027 isolates became moribund. The mean cecal fluid toxin A/B titers for RT027 infected hamsters were 2.3 to 9-fold higher than the titers for non-RT027 infected hamsters.
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    HIV-1 Tat adversely affects neurogenesis through Notch signaling
    (2016-03-23) Liu, Ying; He, Johnny PhD; Fan, Yan
    Alterations in adult neurogenesis appear to be a common hallmark in several neurodegenerative diseases including human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND). HIV-1 Tat is a major pathogenic factor in HIV-associated neuropathogenesis. In vitro study have revealed that Tat severely reduces the proliferation of NPCs and impacts neurogenesis. But adult neurogenesis is limited to specific brain regions in the mammalian brain, so further investigations on the in vivo effect of Tat on NPCs proliferation and differentiation, and the molecular mechanisms are still urgent and necessary. In this study, we took advantage of the Doxycycline (Dox)-inducible brain-specific HIV-1 Tat transgenic mouse model (iTat) to investigate how Tat affected neural progenitor cell (NPC) proliferation and differentiation in the dentate gyrus of hippocampus of the mouse brain. We found that Tat decreased NPC proliferation and impacted NPC differentiation by leading dynamic imbalance of neurogenesis and astrogliogenesis. When investigated the underlying mechanism, we demonstrated that HIV-1 Tat was sufficient for Hes1 activation, which is the critical molecular downstream pathway of Notch signaling. In this process, the cysteine-rich domain of Tat plays an essential role in Hes1 activation and modulating neurogenesis and astrogliogenesis. Lastly, we determined that Notch signaling was directly involved in HIV-1 Tat induced dynamic imbalance of neurogenesis and astrogliogenesis.
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    Development of a Polymerase Chain Reaction Assay to Assess Host Feeding Habits of Culex Mosquitoes in Fort Worth, Texas
    (2016-03-23) Lee, Joon; Lockwood, Laura
    Purpose: West Nile Virus (WNV) is endemic to the United States including Texas. The virus occurs in nature in a mosquito-bird-mosquito transmission cycle but mammals including humans can become incidental hosts. Mosquitoes of the genus Culex are considered the primary vector for WNV. The objective of this research is to develop an assay for detecting the presence of mammalian and/or avian DNA in the blood meal contents of blood-fed mosquitoes. Identifying the hosts and any patterns in feeding habits of the vector mosquitoes can provide more information on the natural transmission of West Nile Virus from avian reservoir hosts to Culex mosquito vectors to incidental mammalian hosts. Methods: Mosquitoes were collected weekly using gravid traps and CO2 light traps throughout Fort Worth. DNA was extracted from the abdomens of blood-fed mosquitoes, and the amount of blood meal was recorded. The DNA was amplified by PCR using universal vertebrate primers designed to generate 150-bp (mammals) or 120-bp (birds) 18S rDNA fragments. The PCR products were analyzed using gel electrophoresis. The PCR assay was confirmed using known mammalian and avian samples. Results: A total of 597 blood-fed mosquitoes were collected throughout Fort Worth in 2014. Results were recorded as avian host only, mammalian host only, or both avian and mammalian hosts. More mosquitoes with a blood meal ≤ 1/3 had no host identified compared to those with larger blood meals. Of those with the host identified, most were avian host only (76% of 292 tested with a host identified) followed by both avian and mammalian hosts (23% of 292) and only 2 of the 292 tested that had a host identified had a mammalian host only. Conclusions: The PCR assay using the universal vertebrate primers targeting 18S rDNA is a sensitive method that allows for the detection of both mammalian and avian host DNA in mosquito blood meals including the presence of both mammalian and avian DNA in a single vector blood meal. The assay will be used to analyze blood meal contents from mosquitoes collected in 2015 in addition to those collected in 2014 to identify any trends in feeding habits.
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    In Susceptible Mice Infected with M. pulmonis, Host Lung Damage is Associated with Recruitment of IL-17A+ Lymphocytes and Neutrophils into the Lung
    (2016-03-23) Simecka, Jerry Ph.D.; Mize, Maximillion
    Background: Possessing the smallest genomes, mycoplasma induce debilitating pneumonia in humans and animals resulting in chronic airway inflammation. Exacerbating previously acquired respiratory conditions (i.e. asthma), mycoplasma have evolved to resist antibiotics1-3. Current vaccines induce the same damage seen during actual infection. A proinflammatory cytokine contributing to chronic pathology and neutrophil-mediated host protection, IL-17A is secreted during infection with mycoplasma. Here, we investigate whether IL-17A can promote damage characteristic of mycoplasma disease. Our results will help development of vaccines that confer protection and lack side-effects. Methods: Murine pneumonia, induced by M. pulmonis, resembles the pulmonary pathogenesis seen in human mycoplasma diseases. Furthermore, BALB/c models have been well established for studying chronic respiratory mycoplasma infection4-6. Briefly, M. pulmonis was administered intra-nasally. At select time points post infection, mice were sacrificed and aspects of pulmonary pathogenesis analyzed. Results: Injecting neutralizing antibodies against IL-17A into BALB/c mice reduced inflammation during infection without influencing bacterial burden. Attenuating the effects of IL-17A reduced both airway cell numbers and total lung IL-17A+ lymphocytes by Day (14). The increase in IL-17A+ cells was associated with increased airway neutrophils, appearing as early as Day (1) post-infection with M. pulmonis. The presence of neutrophils appears alongside CD4+, CD8+, and γδ T-cells that secrete IL-17A early during infection. By Day (9) post-infection, the described T-cell populations are replaced by CD4+, SCA-1+, and NK cells that contribute to IL-17A levels. Although IL-17A production by CD4+ T-cells reaches its maximum response at Day (1) post-infection, this was the only T-cell population that persisted in their production of IL-17A by Day (14). Interesting, neutralizing the effect of IL-17A during early disease results in more severe inflammation when compared to both controls and animals starting treatment five days’ post-infection. The generation of IL-17A+ lymphocytes, and subsequent recruitment of neutrophils was associated with disease pathogenesis. Conclusions: During infection with M. pulmonis, neutrophil recruitment into the lungs is associated with the presence of IL-17A+ lymphocytes. Neutrophils and IL-17A+ cells drive host damage; neutralizing IL-17A reduces airway neutrophils, total IL-17A+ lung cells and host damage. Blocking IL-17A lowers lung lesion development, thus IL-17A and neutrophils promote respiratory damage. Surprisingly, blocking IL-17A during the innate response results in more severe inflammation when compared to neutralizing IL-17A once adaptive immunity takes over. Thus, IL-17A may have protective effects during the early phases of mycoplasma disease.
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    Safety and Efficacy of Ledipasvir plus Sofosbuvir with or without ribavirin in hepatitis C genotype 1 patients who failed previous treatment with Simeprevir plus Sofosbuvir
    (2016-03-23) Modi, Apurva; Gonzales, Gabriel
    Combination therapy with Simeprevir (SIM), NS3/4 protease inhibitor, with Sofosbuvir (SOF), NS5b polymerase inhibitor is an FDA approved treatment option for chronic hepatitis C genotype 1 patients with an over all SVR 12 rate of 85-95%. Single tablet fixed dose combination of Ledipasvir (LDV), NS5a inhibitor, with SOF is also FDA approved for treatment of hepatitis C genotype 1 with SVR 12 rates of ≥ 95%. However, there is no data on the efficacy of retreatment with LDV+SOF in patients who failed initial treatment with SIM+SOF. Methods: Data was collected from treatment cohorts at 2 large hepatology referral centers in Dallas-Fort Worth area. Patients included in the analysis were previously treated with SIM+SOF with or without RBV for 12 weeks but failed to achieve SVR 12 and then undergone re-treatment with LDV+SOF with or without RBV for 12-24 weeks. Patients with cirrhosis, including decompensated Child’s class B or C were included. Decompensation was defined by the presence of fluid overload, hepatic encephalopathy or variceal bleeding. Patients with HCC as the only event that defined decompensation were excluded.. Patients received singlet tablet fixed-dose combination of Ledipasvir 90 mg with Sofosbuvir 400 mg PO +/- wt based ribavirin (RBV) daily for 12-24 weeks at the discretion of the treating hepatologist. Baseline and end of treatment (EOT) laboratory tests & viral load were obtained on all patients. SVR 12 defined as undetectable viral load 12 weeks after EOT was collected on all patients who had reached that time point by Nov 10, 2015. Adverse effects during treatment were obtained on all patients. Data was analyzed using 2 sided t test for continuous variables and chi-quare test for categorical variables. Results: SVR 12 was achieved for 11/13 of all patients and 10/11 for patients who were cirrhotic. 100% (29/29) had achieved EOT response. 10/29 had no side effects on treatments. Of those who had side effects, none were considered severe enough to warrant discontinuation. Conclusions: Ledipasvir + Sofosbuvir is a viable treatment option with high SVR 12 rate in patients who have failed 12 weeks of treatment with SIM + SOF. High SVR 12 rates of 100% (4/4) & 86% (6/7) were achieved in patients with compensated and decompensated cirrhosis respectively •Treatment was generally well tolerated requiring no discontinuations including in those with cirrhosis