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Nadine Rodriguez February 02, 2009 Biol 520. Automatic regulation system Branch of the Autonomic nervous system 2 sets of nerve bodies Preganglionic in.

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Presentation on theme: "Nadine Rodriguez February 02, 2009 Biol 520. Automatic regulation system Branch of the Autonomic nervous system 2 sets of nerve bodies Preganglionic in."— Presentation transcript:

1 Nadine Rodriguez February 02, 2009 Biol 520

2 Automatic regulation system Branch of the Autonomic nervous system 2 sets of nerve bodies Preganglionic in the CNS Postganglionic, which leads to effector organ Active at basal level – More active in times of stress – Part of the fight-or-flight response Up regulates and down regulates homeostatic mechanisms

3 Action Potential Ca 2+    ACH Na + Preganglionic neuron Postganglionic neuron Nicotinic Receptor ach ing/lectures/ans%20lecture1.ppt Contribute to release of Ach

4 Action Potential Ca 2+ NE G Effector Organ Postganglionic neuron Adrenergic Receptor ach ing/lectures/ans%20lecture1.ppt

5  G protein-coupled  Binds to catecholamines  Epinephrine  Norepinephrine  Binds to endogenous ligands  Found on many cells including immune cells and bacterial cells  Subtypes  alpha receptor  Beta receptor Adrenal binds to the adrenergic receptor and associates with the G.protein. The G protein associates with adenylate cyclase to convert ATP to cAMP.

6  Released by the postganglionic neuron  May be excitatory or inhibitory depending on what gland or muscle it is acting on.  Stimulates the heart  Raises blood pressure  Dilates pupils, trachea and bronchi  Stimulates liver glycogen to glucose  Decreases blood from skin and viscera to skeletal muscles, brain and heart  Inhibits movement in the GI tract  Inhibits contraction of bladder and rectum

7 Oberbeck et al 2006

8 Immune CellFunction B-cellMake Antibodies against antigens, role in APC CD4+ T cellpre-defined role as a helper T cell CD8+ T cellMature cytotoxic T cell. Destroy infected host cells Natural Killer (NK) cellRole in rejection of tumors and virus infected cell MacrophagePhagocytose cellular debris and pathogens, stimulate other immune cells

9  Effector T cells or Th cells  Sub group of lymphocytes  Role in the immune system  No cytotoxic or phagocytic activity  Cannot kill infected cells or pathogens  Activate and direct other immune cells  Mature Th cells express CD4.

10 Th1Th2 Cell typeMacrophageB-cell Cytokines producedIFN- γ, TNF- α IL-4, IL-5, IL-6, IL-10, IL- 13 Immune stimulation promoted Cellular immune system Increased killing efficacy of macrophages and proliferation of cytotoxic CD8+ T cells Humoral immune system Stimulates B-cells to proliferate, Induce B-cell Ab class switching, increase Ab production Other functionsIFN- γ increases production of IL-12 by DC and Mac, IL-12 stimulates IFN- γ in helper T cells and inhibits production of IL-4 Il-4 acts on helper t cells to promote th2 cytokine. IL-10 inhibits IL-2 and IFN- γ in helper T cells and IL-12 in DC.

11  Also known as gastroenteritis  Causes inflammation of the gastrointestinal tract  Resulting in acute diarrhea  Inflammation caused by viruses, bacteria, toxins from bacteria or parasites  Kills millions world wide

12  Gram-negative rod shape enterobacteria  Typhoid fever, Paratyphoid fever, Salmonellosis  Via contaminated food and water sources  Symptoms range from gastroenteritis to death  Children, immunocompromised patients, and elderly are at much higher risk for mortality. Salmonella typhimurium invading human cells

13  Gram negative rod  Found in the lower intestine of mammals  Usually harmless  Normal flora  Serotype O157:H7  Gastroenteritis, UTI and neonatal meningitits  Rare: haemolytic-uremic syndrome (HUS), peritonitis, mastitis and septicemia  Involved in product recalls  Micrograph of E. coli 10,000X  E. coli cartoon depicting flagella

14  The sympathetic nervous system plays an important role in the innate response to bacterial infections. Release of the neurotransmitter norepinephrine, recruit immune cells to induce the host inflammatory response.

15 In Support of the Hypothesis

16  Thermal injured mice are susceptible to infections  CCL2 (monocyte chemoattractant) is produced with in one day of trauma  Leads to an up-regulation of type 2 T cell response  Study investigates NE on CCL2 production in mice after burn trauma.

17  6-OHDA was given to mice at 100mg/kg for 5 days, one time a day. Mice were then subjected to thermal injury. 3 rd degree burns were applied to 15% of the mice body surface. NE was measured from the sera of the mice using HPLC.  PBMC and PM was prepared form normal mice and TI mice and treated with saline or 6-OHDA. The cells were culture without stimulation and the culture fluid was collected after 48 hours and assayed for CCL2 using ELISA. Takahashi et al (2004)

18  PBMC’s taken from normal mice and stimulated with different concentrations of NE for 48 hr. Culture fluid assayed for CCL2 using an ELISA Takahashi et al (2004)

19  Torres et al’s group studied the effects of norepinephrine, dopamine and dexamethasone on cellular activation and expression of cytokines and chemokines on human PBMC  Torres’ group found that NE and DA increase lymphocyte activation and augments Th1 and Th2 type cytokine production.

20  Blood was taken from healthy men and women, n=9. PBMC’s were collected and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. PBMC’s were cultured with NE (1x10-7 M). Cultures harvested after 18 hr of stimulation. Cells then stained with anti-surface markers and anti-cytokine mAbs and analyzed by flow cytometry. Increase frequency of CD69 expressing lymphocytes in total lymp and CD4+ T cells Decrease expression of CD62L in CD8+ T cells Pro- inflammatory cytokine IFN- γ increased by NE. Pro- inflammatory cytokine TNF- α increased by NE. IL-6 pro and anti- inflammat ory cytokine is increased by NE. NE increased immunomodula troy cytokine, IL-10 Torres et al 2005

21  Goyart’s group found that NE modulates dendritic cells in animal models  Not fully understood in humans  Studied the effects of NE on cytokine response on an mixed human dendritic cell population

22  Cells taken from donor n=1, and seeded at 4x10 5 per well. Birbeck granule-containing dendritic cells (BG-DC) stimulated with LPS (11, 250U/750ul) with NE (10-6M), Yoh, ICI, Pro or Forsk. Three independent experiments. Assayed in triplicate using ELISA.  Alpha receptor antagoinst, yohimbine (Yoh)  Beta receptor antagonist  ICI-118 (ICI)  Propranolol (Pro) Goyarts et al 2007

23 Showing Direct Effects of Norepinephrine on Bacteria

24  Authors found that NE caused upregulation of flagellar and chemotaxis genes.  Used motility assays to test motility of Salmonella enterica serovar Typhimurium (ST)  Blocking of the alpha – adrenergic receptor inhibits motility of ST.  Mutation if QseC sensor kinase leads to decrease in motility of ST.

25  ST grown o/n in Dulbecco’s modified Eagle medium (DMEM )at 37 ° C. Strains diluted 1:50 in DMEM @ 37 ° C. At 7 hr, ST pelleted and re- suspended in equal vol. of DMEM. 1 μ l of ST spotted on DMEM @ 37 ° C for 16-18hr. NE added at 50 μ M. Phentolamine added at 200 μ M. Cultures grown and assayed at 37 ° C. Bearson and Bearson (2007)

26  1 ul of Wildtype ST and qseC mutated ST spotted on DMEM agar with 50 uM of NE. Zone diameter measuered after 16 h of growth at 37C. (Bearson and Bearson 2007)

27  Quorum sensing – Bacteria use this cell-to-cell signaling mechanism to respond to hormone- like molecules  Involved with eukaryotic hormones, epinephrine and norepinephrine  Activates virulence genes of E. coli O157:H7  Not shown in vivo  Bacterial receptors not identified  Clarke et at found that bacterial QseC sensor kinase is a receptor for NE

28  5 or 10 μ M of NE alone or with PE and added to QseC liposomes. Twenty ml of liposomes adjusted to 10mM. [ γ 32P] dATP added to each reaction. SDS loading buffer was added at 10 min. Samples were run on a SDS/PAGE and visualized with a phosphorImager. Bands quantified using Imagequant version 5.0 software. Clarke et al (2006) 2.254 Fold Increase 10 uM NE +50 uM PE? 6 fold decrease SDS/PAGE

29  NE was found to increase adherence of E. coli to the mice intestinal mucosa.  Chen’s group found that NE promoted adherence of non-O157H:7 and E. coli O157:H7

30 Chen et al (2006) Dot Plot. Tissue taken from Yorkshire-Landrace pigs, 6-10 weeks old. Pigs anesthetized with Telazol. Midline laparotomy to expose intestine. Explants obtained from pigs and placed in ice-cold oxygenated physiological tissue preservation solution. Bacteria added for 90 min to tissue. Tissue was then weighed and washed with PBS to remove free bacteria. Tissue homogenized and plated on Fluorocult E. coli O157:H7 agar. Plates incubated at 37 ° C for 24h, colonies were then counted. Unfilled circles represent untreated. Filled circles represent tissue exposed to 10 μ M NE. NE – norepinephrine, CM – porcine cecum, PC – proximal colon, SC – spiral colon, DC – distal colon.

31  It is known from previous findings that NE augments E. coli O157:H7.  In Vlisidou et al paper, NE promotes adherence of E. coli on intestinal mucosa.

32 Vlisidou et al 2004  Confocal laser scanning micrograph. Bovine mid-ileal mucosa inoculated with E. coli O157:H7. Four separate experiments, bull calf subjected to fasting for 12h, anesthetized with Thiovet and intubated during the experiment. Laparotomy preformed, mid ileum flushed with saline, 6 cm loops ligated with surgical silk. E. coli grown to stationary phase in LB broth at 37 ° C for 16 h. Bacterial cultures supplemented with NE at a conc. of 50 μ M or 5 μ M and injected into loops. Tissue was fixed ante mortem and stained for E. coli (red) and F-actin (green). 5 mM NEControl E. coli

33  Bansal’s group exposed E. coli to many signaling molecules  NE, EPI and Indole (a bacterial signal molecule)  Bansal’s group compared NE and EPI to indole  Had effects on E. coli chemotaxis and motility.

34 A. Construct a microscope slide bridge using two strips of plastic B. Agarose plug is placed in the middle of strips C. Glass slip covers plug within 1 second D. Cell suspension is pipetted between slide and cover slip until space is completely filled Yu and Alam (1997)

35  Agarose plug chemotaxis assay. O/n culture of E. coli O157:H7 use to inoculate 10 ml of growth medium. Cheomtaxis compound added to medium during growth. Kanamycin-killed E. coli TG1 added to cell suspension where live to dead cells was 2:1. 10ul of agarose added to slide and 200 μ l of cell suspension added. Migration imaged on flurescence microscope. Images taken every 5 min for 30 min. Bansal et al (2007) 500 uM EPI500 uM NE 2% Casamino Acids 5,000 uM indoleGlycerolM9 Salt solution Chemoattractant ring Clearance zone Chemorepellant band


37 AuthorKey FindingsCritic Takahashi et al (2004)NE has a role in CCL2 production in burn trauma Type 2 T cell response No n values Torres et al (2005)NE increase lymph act. Increase Th1 and Th2 cytokine production Negative CRTL missing in flow cytomety analysis Fold increase 1.6-2.7 Goyarts et al (2008)NE downreg. Proinflamm. Cytokine by LPS stimulation Decrase Th1 cell response Used only 2 donors Bearson and Bearson (2007)NE enhances motility of ST QseC mutant, decrease in motility of ST Only two figures, Fold increase between 1.17 and 15.4. Clarke et al (2006)QseC is a bacterial receptor to EPI and NE Act. Virulence genes Manipulate figures? Missing y-axis legend Chen et al (2006)QseC is a bacterial receptor to EPI and NE and act. Virulence genes Vlisidou et al (2004)NE enhances early attachment of E. coli Used only one cow. No materials and method section. Bansal et al (2007)NE and EPI is sensed by E. coli, initiation of infectionUsed HeLa cells in some of their experiments.

38 SNS Lymph node activation NE E. Coli Vlisidou et al (2004) Chen (2006) Bansal (2007) Increase adhesion Salmonella Bearson and Bearson (2008) Enhanced motility NE E. Coli Clarke et al (2006) QseC bacterial receptor, increase virulence Increase monocytes CCL2 Increase Th2 Takahashi et al (2003) NE Increase Dendrtic cells Increase in Th2 not Th1 Goyarts et al (2008) Increase in lymphocyte activation and Th2 and Th1 Torres et al (2005) NE stimulus Brain.

39  Use a intestinal epithelial human cell line  Test with E. coli O157:H7 and Salmonella  Use various concentrations of Norepinephrine  10 μ M, 50 μ M and 100 μ M  Measure the amount of E. coli and ST that adhere  Use qRT-PCR to measure genes up-regulated by cells when infected with E. coli and ST.

40  Norepinephrine increases lymphocyte activation  Increase in pro-inflammatory and anti-inflammatory cytokines.  In the presence of LPS, NE decreases IL-23 and IL- 12 p40 in dendritic cells.  Therefore does not promote stimulation of memory T cells and naïve T cells.  NE enhances the motility of E. coli an S. Typhimurim through Qsec sensor kinase  E. coli and ST take advantage NE to increase its survival

41  According to the data I found, these results refute my hypothesis.  NE promotes the adherence of E. coli and the motility of Salmonella.  Not enough evidence supporting that NE promotes the recruitment of my immune cells.

42 1. Bansal, T., Englert, D., Lee, J., Hegde, M., Wood, T.K., Jayaraman, A. (2007) Differential effects of epinephrine, norepinephrine, and indole on Escherichia coli O157:H7 chemotaxis, colonization, and gene expression. Infect Immun. 75,4597-607. 2. Bearson, B.L., Bearson, S.M.(2008) The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine- enhanced motility of Salmonella enterica serovar Typhimurium. Microb Pathog. 44,271-8 3. Chen, C., Lyte, M., Stevens, M.P., Vulchanova, L., Brown, D.R. (2006) Mucosally-directed adrenergic nerves and sympathomimetic drugs enhance non-intimate adherence of Escherichia coli O157:H7 to porcine cecum and colon. Eur J Pharmacol. 539, 116-24 4. Clarke, M.B., Hughes, D.T., Zhu, C., Boedeker, E.C., Sperandio, V. (2006) The QseC sensor kinase: a bacterial adrenergic receptor. Proc Natl Acad Sci U S A. 103, 10420-5 5. Goyarts, E., Matsui, M., Mammone, T., Bender, A.M., Wagner, J.A., Maes, D., Granstein, R.D. (2008) Norepinephrine modulates human dendritic cell activation by altering cytokine release. Exp Dermatol. 17, 188-96 6. Green, B.T., Lyte, M., Kulkarni-Narla, A., Brown, D.R. (2003) Neuromodulation of enteropathogen internalization in Peyer's patches from porcine jejunum. J Neuroimmunol.141, 74-82. 7. Straub, R.H., Wiest, R., Strauch, U.G., Härle, P., Schölmerich, J. (2006) The role of the sympathetic nervous system in intestinal inflammation. Gut. 55, 1640-9. 8. Takahashi, H., Tsuda, Y., Kobayashi, M., Herndon, D.N., Suzuki, F.(2004) Increased norepinephrine production associated with burn injuries results in CCL2 production and type 2 T cell generation. Burns. 30, 317-21 9. Torres, K.C., Antonelli, L.R., Souza, A.L., Teixeira, M.M., Dutra, W.O., Gollob, K.J. (2005) Norepinephrine, dopamine and dexamethasone modulate discrete leukocyte subpopulations and cytokine profiles from human PBMC. J Neuroimmunol. 166, 144-57 10. Vlisidou, I., Lyte, M., van Diemen, P.M., Hawes, P., Monaghan, P., Wallis, T.S., Stevens, M.P. (2004) The neuroendocrine stress hormone norepinephrine augments Escherichia coli O157:H7-induced enteritis and adherence in a bovine ligated ileal loop model of infection. Infect Immun. 72, 5446-51 11. Yu, H.S., Alam,M. (1997) An agarose-in-plug bridge method to study chemotaxis in the Archaeon Halobacterium salinarum. FEMS Microbiol Lett. 156, 265-9. 12. 13. 14. 15. 16.

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