Immunology and Infectious Disease
Oral Presentations
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Muscarinic Receptors on Eosinophils Inhibit PAF-induced Eosinophil Activation
Norah Verbout, DB Jacoby, AD FryerKeywords: Parasympathetic, Muscarinic, Eosinophil, Asthma Abstract:In the lungs, eosinophils are clustered around parasympathetic nerves in asthmatic humans and animal models of asthma. Antigen challenge releases eosinophil major basic protein, an antagonist for M2 muscarinic receptors. Eosinophil mediated blockade of inhibitory M2 receptors on parasympathetic nerves increases acetylcholine release, which increases bronchoconstriction. Since eosinophils are associated with parasympathetic nerves, we tested whether they are reciprocally affected by acetylcholine. Eosinophils were isolated from guinea pig peritoneal lavage and human blood. RT-PCR and immunocytochemistry demonstrated that eosinophils express M3, M4 and M5, but not M1 or M2 muscarinic receptors. To test whether muscarinic receptors regulate eosinophil activation, pure eosinophils were treated with a nicotinic receptor antagonist hexamethonium (0.1uM), loaded with a calcium indicator (fluo-4; 5uM) and increases in intracellular calcium were measured via fluorescence microscopy. Eosinophil activation was measured as number of cells responding to an agonist within 1 minute. Platelet-activating factor (PAF) (0.01uM-10uM) increased the number of responding cells in a dose-dependent manner. Acetylcholine (1nM-10uM) alone did not increase intracellular calcium. However, the number of eosinophils activated by PAF (1uM) was dose-dependently decreased by acetylcholine (0.1-3uM; max response 49% of control at 3uM), an effect reversed by muscarinic blockade with atropine (1uM). Thus, eosinophils express 3 muscarinic receptors, one or more of which inhibit eosinophil activation in vitro. This inhibitory pathway may therefore be useful for treatment of asthma. Funding: AHA 0515486Z , NIH HL55543 , HL54659, ES014601 , HL071795
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The effect of murine norovirus on concomitant murine cytomegalovirus infection
Carmen Doom, H.Turula, A.B. HillKeywords: Reactivation, Interferon, Mnv, Mcmv Abstract:Norovirus is a human calicivirus that causes outbreaks of acute gastroenteritis, often in densely populated environments. A murine norovirus (MNV) was recently discovered and appears to be as highly infectious as the human strains. MNV is routinely present in specific pathogen-free mouse colonies and its impact on the immune system, particularly the response to other pathogens, is not known. We are interested in the effect of MNV infection on concurrent murine cytomegalovirus (MCMV) infection. MNV induces a vigorous interferon-α/β (IFN) response, and it grows in macrophages and dendritic cells, cells also infected by MCMV . We hypothesize that MNV infection will impact MCMV infection differently depending on the timing of MCMV exposure. Specifically, acute MNV infection will result in enhanced control of acute MCMV due to increased serum IFN -α/β levels, but acute MNV will cause reactivation of latent MCMV because it infects the cells in which MCMV latently resides. We demonstrate that MNV had no effect on MCMV titres in acute MCMV infection. Interestingly, however, the serum IFN -γ levels in BALB /c mice acutely infected with both MNV and MCMV were significantly lower than serum IFN -γ in mice infected with MCMV alone. Furthermore, acute MNV infection may subtly induce reactivation of latent MCMV in C57BL /6 mice, although the results are not yet conclusive. These data suggest that MNV infection dysregulates the systemic cytokine response to acute MCMV infection without impacting overall control of acute MCMV . MNV may also play a role in MCMV reactivation, and studies further addressing this are currently underway.
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Detection of Herpesviruses Aspirated from Endodontic Apical Abscesses and Cellulitis
Vicky Chen, Vicky P. Chen, Hong Li, J. Craig Baumgartner, and Curtis A. MachidaKeywords: Endodontic Apical Abscesses, Herpesviruses Abstract:Acute apical abscesses are severe endodontic diseases. These may be caused by co-infection with pathogenic bacteria and latent herpes viruses. This study screened for herpes viruses, including Human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), Varicella Zoster virus (VZV), and Herpes simplex virus (HSV-1), in aspirates obtained from patients presenting with acute apical abscesses of endodontic origin. 31 patients with acute apical abscess were clinically examined and clinical data were recorded. Samples of abscesses were aseptically aspirated from these patients and used in viral DNA extractions. Primary and nested polymerase chain reactions (PCR) were performed using virus-specific primers and DNA isolated from cell-free virus preparations. Abscess fluids were filtered to remove residual immune cells and treated with DNase I to degrade cellular DNA prior to extraction of nucleic acids from virus particles and use in PCR . PCR products were visualized using gel electrophoresis. From patients exhibiting concurrent spontaneous pain and periradicular rarefraction (n=29), 9 abscesses contained HCMV (31%) and 2 abscesses contained EBV (6.9%). One individual, who exhibited periradicular rarefraction but no spontaneous pain, contained HSV -1. No patients exhibited VZV . Primary PCR could only detect minimum viral DNA copy number of 10,000 copies. Due to the small copy numbers of virus particles present in the abscess samples, nested PCR was required for all viral detections. We conclude that herpes viruses, primarily HCMV and EBV , are replicated in patients exhibiting acute endodontic disease and are transmitted and released as virus particles in abscess fluid.
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Characterization of TtpC function in the TonB2-mediated iron transport in pathogenic Vibrios
Carole Kuehl, Claudia Lopez, Jorge CrosaKeywords: Vibrio, Iron Transport, Bacterial Genetics Abstract:A fourth required protein in the TonB2 energy transduction system in Vibrio anguillarum is necessary for iron transport mediated by the TonB2 system. The gene for this previously-unknown protein, TtpC, is in an operon encoding the genes for the TonB2 protein and its accessory proteins ExbB2 and ExbD2. TtpC is highly conserved in all pathogenic Vibrio species studied to date as well as several related marine organisms. We show here that the TtpC proteins from selected pathogenic Vibrio species can function with the TonB2 system of V. anguillarum to allow iron transport mediated by a chimeric TonB2 system where the native ExbB2, ExbD2 and TonB2 function with an episomally-expressed TtpC in trans from a different species. The discovery of a fourth component to the TonB2 system is of significant interest because it suggests a variation or possibly a new mechanism being used by marine organisms to transduce energy to the outer membrane receptor proteins. The discovery that inter-species complementation occurs can be used to identify the functional regions of the TtpC proteins and will lead to an investigation of the mechanism of interaction between the TtpC protein and the other members of the TonB2 system.
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Characterization of the ECF Sigma Factor MbaS from the Human Pathogen _Burkholderia pseudomallei_
Melissa Hauglund, Alehandro Alice, Carolyn Lowe, and Jorge CrosaKeywords: Sigma Factor, Human Pathogen, Bacteria Abstract:Burkholderia pseudomallei is a gram-negative human pathogen causing the disease known as melioidosis. We have identified the iron-responsive regulon of this bacteria and specifically focused on the iron-uptake system regulated by the extracytoplasmic sigma factor, MbaS. We have demonstrated that MbaS controls the expression of genes involved in the biosynthesis and uptake of the siderophore malleobactin. Current studies are underway to confirm the DNA sequence recognized by MbaS, elucidate the complete MbaS regulon, and generate a more detailed characterization of MbaS. We have used homologies to other sigma factor binding sites to identify a preliminary DNA binding site for MbaS and are currently constructing promoter fusions to further elucidate the interaction. We have completed a preliminary microarray using a strain of B. pseudomallei wherein MbaS has been deleted to elucidate additional genes regulated by MbaS and are presently in the process of confirming the expression changes observed. And finally, we are using both site-directed mutagenesis and error-prone PCR to generate mutants of MbaS to identify residues critical in this sigma factor’s function. So far we have shown that MbaS functions in a novel fashion when compared to its homolog, PvdS, in Pseudomonas aeruginosa and are planning on further characterizing its role in virulence.
Poster Presentations
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The Role of IL-5 in Sepsis
Stefanie Manzer, Ann Kelly, Jeff GoldKeywords: Macrophages, Neutrophils, Sepsis, Il-5 Abstract:Sepsis is controlled by a balance between numerous inflammatory cytokines. We have previously shown that transgenic mice overexpressing IL5 have improved survival in a model of polymicrobial sepsis induced by CLP . However, overexpression of IL5 in these mice is associated with profound eosinophilia. Consequently, we sought to specifically determine the role of IL5 in sepsis. Following CLP , we observed an upregulation of IL5R on PMNs and peritoneal macrophages compared to control mice by flow cytometry. These results were confirmed on thioglycollate-elicited PMNs, peritoneal macrophages and RAW264 .7 cells, with IL5R being upregulated by LPS stimulation. Functionality of the IL5R was confirmed by IL5 induced IL6 production and STAT1 nuclear translocation. In addition, treatment of WT mice with rIL5 (1ug) 4hrs pre-CLP, which resulted in PMN recruitment into the peritoneal cavity, improved survival as compared to controls (p=0.006). Mice administered rIL5 1hr post-CLP showed improved survival as compared to PBS injected mice (p=0.07). Moreover, humans with sepsis have increased levels of IL5 compared to healthy controls (p=0.0007). Interestingly, among septic individuals, those requiring mechanical ventilation have lower IL5 levels, compared to those who do not (5.4 vs. 2.1pg/ml;p=0.07) with a similar trend observed for those who died compared to survivors (1.97 vs. 4.6pg/ml;p=0.07). In conclusion, we document expression of the IL5R on granulocytes. The ability of IL5 to rescue mice from CLP combined with lower levels of IL5 in humans with more severe disease suggests a protective role for IL5 and its potential role as an immunomodulatory therapy for sepsis.
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Diversity of pol Sequence and Envelope Tropism of Plasma and Cervical Viruses Among HIV+ Pregnant Women in Zimbabwe
Jennifer Lint, Seble Kassaye, Sudeb Dalai, Elizabeth Johnston, Justen Manasa, Lynn Zijenah, and David KatzensteinKeywords: Env Tropism, Mother-To-Child Transmission, Hiv-1 Subtype C Abstract:Background: HIV -1 from genital secretions (GS) and plasma can infect an infant and the two compartments may differ with respect to drug resistance, immune escape mutation(s) and envelope tropism. We compared cervical and plasma consensus pol sequences among 43 women and diversity of env by clonal analysis among six women who vertically transmitted HIV -1. Methods: HIV RNA was measured with a modified Roche Amplicor v1.5 assay. A general time reversible model was used to determine genetic distances (PAUP) and Syn-SCAN (hivdb.stanford.edu) to determine evolutionary selection pressure. Twenty env sequences were obtained by TA cloning from plasma and GS virus from six transmitters. Env tropism was estimated by a subtype C specific X4 algorithm (Cootzee et al. J. Virol 2007). Results: In 43 women, plasma-GS evolution in pol was marginally greater in transmitters than non-transmitters (p =0.08). Clones from six transmitting women showed distinct X4 tropic clones; two had a mix of R5 and X4-tropic env clones in both compartments, three had only R5 viruses and one had X4-tropic env in GS and plasma. Compartmental clades existed among the three women with R5 viruses and mixtures of plasma and GS viruses among the X4 tropic viruses. Conclusions: Selection of viruses in GS relative to plasma in both pol and env genes provides evidence for independent compartmental replication in GS with a continuum of selection for CTL epitope evolution and viral tropism in each compartment. Identification of X4-tropism in transmitting women suggests that further studies are warranted as R5 inhibitors are developed.
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The TLR7 agonist R837 activates eosinophils in vitro, but inhibits bronchoconstriction in vivo.
Elad Kaufman, Elizabeth Bivins-Smith, David JacobyKeywords: Endosome, Ifnγ, Asthma, Tlr7, Eosinophils Abstract:Asthma is characterized by airway hyperreactivity and inflammation and is associated with accumulation of eosinophils around airway nerves. Activated eosinophils degranulate releasing major basic protein that antagonizes prejunctional inhibitory M2 muscarinic receptors. This increases acetylcholine (Ach) release in the junction and bronchoconstriction. Viruses are associated with asthma exacerbations. Toll-like receptor 7 (TLR7), which recognizes viral ssRNA, is expressed in eosinophils. We showed that eosinophils can be activated by ssRNA viruses and the synthetic TLR7 agonist R837 , and the response is potentiated by overnight treatment with Ifnγ, an anti-viral cytokine. This potentiation is due to translocation of TLR7 to the cell surface from the endosome, as observed by immunofluorescence (IF). Despite TLR7 -dependent eosinophil activation, R837 inhibited Ach- and vagally-mediated bronchoconstriction in an in vivo guinea pig model. In ovalbumin-sensitized guinea pigs, which have more airway eosinophils, the R837 inhibition of bronchoconstriction was less potent, possibly due to an opposing effect of eosinophil activation. We also showed that R837 inhibited in vitro airway smooth muscle contraction in response to electrical field stimulation, Ach, histamine, and KCl. We showed by immunostaining that TLR7 is expressed on cultured guinea pig airway parasympathetic neurons and smooth muscle. Stimulation of these receptors might decrease smooth muscle contraction. Thus TLR7 translocates to the cell surface of eosinophils in response to Ifnγ to increase activation of eosinophils in response to viruses. TLR7 -mediated activation of eosinophils increases vagally-mediated airway reactivity, but this increase in airway reactivity is countered by a direct smooth muscle relaxing effect of R837 .
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Genetic analysis of human cytomegalovirus glycoproteins that mediate entry
Paul Wille, David C. JohnsonKeywords: Virology Abstract:Herpesviruses enter cells by using several well-defined steps each involving different virus glycoproteins. The HCMV genome encodes herpesvirus entry glycoprotein homologues; however, little is known about how these glycoproteins mediate entry. HCMV expresses glycoproteins gH and gL that form a tight heterodimer. Other herpesvirus gH/gL complexes are essential for virus entry. We believe that gH and gL will be required for entry into all cell types, as is the case with other herpesviruses. My working hypothesis suggests that there are two HCMV gH/gL complexes: gH/gL/gO that mediates entry into fibroblasts and gH/gL/UL128/UL130/UL131 that mediates entry into epithelial and endothelial cells. We constructed HCMV mutants unable to express gL and gO in order to characterize their necessity for fibroblast entry. Propagating HCMV gL – or gO – mutants requires complementing cell lines that provide gL or gO in trans. Viruses produced on such cells should be able to enter non-complementing cells. Virus subsequently produced on non-complementing cells will lack gL or gO. gL – or gO – viruses will be used to define where mutants are blocked in virus replication. I am currently in the process of finding conditions in which gL or gO are produced at sufficient levels to complement gL – or gO – HCMV . I have screened greater than 1000 isolated clones for genome incorporation of either gL or gO, and am in the process of screening clones for their ability to produce gL or gO protein at sufficient levels for complementation.