Escherichia coli Nissle 1917
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1 Immunoregulation in Inflammatory Bowel Diseases New therapeutic approaches Probiotics Escherichia coli Nissle 1917 Andreas Sturm Medizinische Klinik m.s. Hepatologie, Gastroenterologie, Endokrinologie und Stoffwechsel Universitätsmedizin Berlin
2 Escherichia coli Nissle 1917 (DSM 6601)
3 Escherichia coli Nissle 1917 (DSM 6601) Alfred Nissle ( )
4 Comprehensive genomic map of E. coli Nissle 1917 Grozdanov L. J Bacteriol 2004
5 Characteristics of E. coli Nissle 1917 Microcins Iron acquisition systems Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Fe 3+ Small cryptic plasmids 3 kb 5 kb chu aer cbt ent ybt cit mch chromosome STOP rfb mcm kps fla fim foc csg Fimbriae Flagellae of H1 type Special LPS of O6 type K5 capsule O6 : K5 :: : H1 serotype
6 Schematic structure of E. coli Nissle LPS E. coli 536 UPEC Lipid A Core About 40 repeating units wzy functioning Smooth colonies Serumresistant E. coli Nissle 1917 wzy with genetic deficiency Semi-rough colonies Serumsensitive Acc. to Grozdanov L. J Bacteriol 2002
7 Protein coding sequences specific in E. coli Nissle 1917 Sun, J Biotechnol. 2005
8 Evidence linking enteric Why should probiotics work in IBD? or enteric bacterial flora with pathogenesis of IBD The bacterial flora affect development, structure, and function of the mucosal immune system (Berg, Trends Micobiol 1995; Gordon, Am J Physiol 1997) Greatest incidence of inflammation in the area with the highest concentration of luminal bacteria, high number of pathological adherent bacteria in IBD (Darfeuille-Michaud, Gastroenterology 2004; Swidsinski, Gastroenterology 2002) Crohn s-like disease can be transferred with T cells reactive against enteric bacteria in animals with the disease (Cong, J Exp Med 1998) Diversion of faecal stream is associated with distal improvement in patients with Crohn's disease and the disease consistently relapses upon restoration of faecal stream (D'Haens, Gastroenterology 1998)
9 Evidence linking enteric Why should probiotics work in IBD? or enteric bacterial flora with pathogenesis of IBD Lesions can be induced experimentally in susceptible individuals by direct instillation of faecal material into non-inflamed loops of bowel (Harper, Gut 1985; D'Haens, Gastroenterology 1998) Immune reactivity against enteric bacteria can be seen in patients with Crohn's disease (Duchmann, Clin Exp Immunol 1995) Colonisation with normal enteric bacterial flora is required for expression of disease in animals with Crohn's disease (Blumberg, Curr Opin Immunol 1999; Hoffmann, Pathobiology 2002) Alteration of the flora with probiotics and antibiotic strategies have beneficial effects in human beings with IBD (Shanahan, Gastroenterology 2001)
10 E. Coli Nissle inhibits the adhesion of pathogenic bacteria enteroinvasive bacteria (black) + E. coli Nissle (light-grey) + E. coli DH5α (dark-grey) adherent invasive E. coli (AIEC) LF82 (black) + E. coli Nissle (grey) Altenhöfer, FEMS Immunol Med Microbiol 2004 Boudeau, Aliment Pharmacol Ther 2003
11 E. coli Nissle 1917 induce Defensin-2 mrna expression Wehkamp J. Infect Immun 2004
12 EcN-CM inhibits T-cell activation by down-regulation of co-stimulatory molecules -EcN-CM + EcN-CM % positive cells M1 M2 35 % 7 % CD69 -EcN-CM + EcN-CM -EcN-CM + EcN-CM % positive cells 13.2% 3.1% FITC FITC CD % 2.5% FITC FITC CD86 Sturm, Infect Immun 2005
13 EcN-CM inhibits T cell cycling 0vol% EcN-CM unstim 0vol% EcN-CM stim 10 vol% EcN-CM stim 25 vol% EcN-CM stim 50 vol% EcN-CM stim counts G0/G1: 99% S: 0% G2/M: 1% counts G0/G1: 71% S: 13% G2/M: 16% counts G0/G1: 79% S: 9% G2/M: 12% counts G0/G1: 86% S: 5% G2/M: 9% counts G0/G1: 91% S: 3% G2/M: 6% DNA DNA DNA DNA DNA Propidium iodide vol% EcN-CM Cyclin D2 prb Rb E2F-1 Cyclin A GAPDH Sturm, Infect Immun 2005
14 EcN-CM inhibits pro-inflammatory cytokine release 0vol%EcN-CM CD3 + 0 vol% EcN-CM CD vol% EcN-CM CD vol% EcN-CM ng/ml * * IL-2 * TNF-α * * IFN-γ * * IL-10 Sturm, Infect Immun 2005
15 EcN up-regulates TLR-2 2 and TLR-4 4 surface expression counts w/o EcN w/ EcN counts w/o EcN w/ EcN TLR-2 TLR-4 Grabig, Infect Immun (in press)
16 EcN reduces disease activity indices in wt, but not TLR-2 2 or TLR-4 4 deficient mice 10 8 p< % NaCl p.o. 0.9% NaCl i.r. EcN p.o. EcN i.r. DAI 6 * * wildtype TLR-2 -/- TLR-4 -/- Grabig, Infect Immun (in press)
17 EcN ameliorates DSS induced colitis in wt, but not TLR-2 2 or TLR-4 4 deficient mice Wildtyp TLR-2 ko TLR-4 ko control EcN Grabig, Infect Immun (in press)
18 EcN reduces disease activity indices in wt, but not TLR-2 2 or TLR-4 4 deficient mice Clinical score IFN-γ (pg/ml) PBS untreated EcN 0 DSS DSS + EcN DSS + V/I CD4 + CD62L SCID mice Schultz M. Clin Diag Lab Imm 2004
19 EcN distinctively modulates cytokine secretion dependent on the TLR status ng/ml * * * * * * * * ng/ml * * 0.9% NaCl p.o. 0.9% NaCl i.r. EcN p.o. EcN i.r. ng/ml pg/ml TNF-α IFN-γ MCP-1 IL-10 IL-6 0 TNF-α IFN-γ MCP-1 IL-10 IL-6 0 TNF-α IFN-γ MCP-1 IL-10 IL-6 0 TNF-α IFN-γ MCP-1 IL-10 IL-6 wildtype TLR-2 -/- TLR-4 -/- Grabig, Infect Immun (in press)
20 EcN secreting an HIV fusion inhibitor peptide Colonization of the mouse GI Tract with a genetically modified EcN that secrete anti-hiv peptides secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells Cmr HlyB,HlyD black: green: orange: without ampicillin 1 day ampicillin cont. ampicillin Ampicillin stop Mouse colonization after 3 days Rao J, PNAS 2005
21 Passage and Concentration of EcN in the GI Tract Intestinal passage of EcN-GFP following single oral administration Concentration of fluorescent E. coli colonies in faecal samples Schultz M, J Microbiol Methods. 2005
22 RCT s s in IBD with E. coli Nissle 1917 Kruis et al., Aliment Pharmacol Therapy 1997 Rembacken et al., Lancet 1999 Kruis et al., Gut 2004 Double uble-blind, blind, placebo-controlled controlled trials to test the efficiency of Mutaflor in the maintenance therapy of ulcerative colitis
23 EcN (Mutaflor( ) and ulcerative colitis - clinical studies patients with inactive ulcerative colitis Randomized to 3 x 500 mg mesalazine/d or EcN (2 x 2,5-25x10 9 viable bacteria) for 12 weeks Kruis W, Aliment Pharmacol Ther. 1997
24 EcN (Mutaflor( ) and ulcerative colitis - clinical studies patients with active ulcerative colitis Standard medical therapy together with a 1-week course of oral gentamicin After remission, patients were maintained on either 3 x 800 mg mesalazine or EcN (2 x 2,5x10 10 viable bacteria) for 1 year Rembacken BJ, Lancet 1999
25 EcN (Mutaflor( ) and ulcerative colitis - clinical studies patients with inactive ulcerative colitis Randomized to 3 x 500 mg mesalazine/d or EcN (2 x 2,5-25x10 9 viable bacteria) for 1 year Kruis W, Gut 2004
26 Summary E. coli Nissle have a genomic peculiarity encoding a distinct semi-rough LPS E. coli Nissle inhibits activation, cell cycle progression and expansion of T cells by a TLR-2 dependent mechanism E. coli Nissle inhibits adhesion and invasion of various adherent-invasive bacteria to intestinal epithelial cells E. coli Nissle down-regulates pro-inflammatory cytokine secretion and induces human Defensin-2 expression E. coli Nissle ameliorates acute and chronic intestinal inflammation in different murine models of colitis E. coli Nissle can serve as carrier for therapeutic peptides into the GI tract E. coli Nissle is effective in the maintenance therapy of ulcerative colitis
27 Thank you very much for your attention!
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