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Cases and Mortality Rates for Infectious Diseases

Cases and Mortality Rates for Infectious Diseases. Disease. Worldwide Annual Deaths. U.S. Annual Cases. Annual Deaths. All Infectious Diseases. 17,000,000 (1995). n/a. 165,750 (1992) 2. HIV/AIDS. 2,300,000 (1997). 71,293 (1995). 32,655 (1996). Acute respiratory diseases.

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Cases and Mortality Rates for Infectious Diseases

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  1. Cases and Mortality Rates for Infectious Diseases Disease WorldwideAnnualDeaths U.S Annual Cases Annual Deaths All Infectious Diseases 17,000,000(1995) n/a 165,750(1992)2 HIV/AIDS 2,300,000(1997) 71,293(1995) 32,655(1996) Acute respiratory diseases 4,400,000(1995) 90,400,000(1994) 607(1996) Foodbourne illnesses 3,100,000(1995) 6,000,000(1983) 9,000(1983) Malaria 2,000,000(1995) n/a n/a Tuberculosis 3,100,000(1995) 22,860(1995) 1,194(1996) Hepatitis B 1,100,000(1995) 10,805(1995) 3,811(1996) Chlamydia n/a 4,000,000(1996)3 n/a 1 Sources include the World Health Organization and the Centers for Disease Control and Prevention (CDC).

  2. Ghost E. coli

  3. Bacterial Cell Envelopes, LPS, Lipid A partial- structures Immune stimulation- adjuvans- activity Carrier-Targeting Inflammation, Schock Major immunological questions: Endotoxic activities, therapeutic window, routes of administration Routes of activation comparing LPS, uptake by APCs Stimulation of immune responses Possibility for packaging, targeting to specific cells

  4. Detoxified Lipid A monophosphoryl derivative endotoxically inactive acting as an adjuvant inducing tolerance to Salmonella enteritidis LPS and tumor necrosis factor alpha (TNF-alpha) Avanti'sNew Potent Vaccine Adjuvant Natural (Salmonella Minnesota, R595

  5. Structure LPS

  6. Limulus Test, Endotoxicity EU/mg S-layer ( B.sphaericus) S-layer ( B. stear.) Ghosts (E.coli O26:B6) Ghosts (S.typhim.) LPS (E.coli O26:B6) LPS ( S. ab. equi ) Control

  7. Stimulation of TNFa by bacterial ghostsin Mo

  8. Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines. Bacterial ghosts prepared from Escherichia coli O26:B6 and Salmonella typhimurium C5 induce dose-dependent antibody responses against bacterial cells or their corresponding lipopolysaccharides (LPS) in doses 25 ng kg-1 when administered intravenously to rabbits. No significant fever responses in rabbits have been recorded in doses of < 250 ng kg-1 E. coli O26:B6 ghosts and up to doses of 250 ng kg-1 S. typhimurium C5 ghosts when applying test methods recommended by the US pharmacopoeia. Vaccine 1997 Feb;15(2):195-202

  9. LPS, CD14 Hypothesis: transmembrane integrin glycoproteins e.g. Mac-1 serve as a signaling partner for glycolipid-linked glycoproteins that lack trans-membrane and cytoplasmic domains.

  10. Inhibition of TNFa by anti CD14 TNF[ng/ml] LPS FCS aCD14 p< (ng/ml) (ug/ml) 10 + - 10 - - 10 + 0,5 0,05 10 - 0,5 10 + 0,1 0,02 10 - 0,1 100 + - 100 - - 100 + 0,5 0,02 100 - 0,5 0,02 100 + 0,1 0,05 100 - 0,1 0,05 - +/- - - +/- +/-

  11. Pathways for LPS stimulation LPS- LBP HSP60? ? IL-6, IFNy,... CD14 GPI Toll- like R-4 CR3 MAC1 ? R Janus Kinase PLC ? Stat 3,1,4 MAPK/ p38 JNK ERK MAPK P CREB, ATF1 NFkB ...,myk,Elk, Mnk eIF4E AH,10/2000

  12. Uptake of ghosts in RAW-Mo

  13. GFP labelled Ghost, RAW cells

  14. FISH, GFP mRNA in RAW Mo

  15. Inhibition of LPS stimulated TNFa Inhibition of Ghost stimulated TNFa by kinase inhibitors in supernatant by kinase inhibitors packed inside ghosts SB/G5 LPS LPS: Co, 0,001; 0,01; 0,1 ug/ml , Co:50-200 pg/ml TNFa, 100%: 5-20 ng/ml TNFa Ghosts: E. Coli, 5, 0,5 ul; 100 %: 5-20 ng/ml TNFa SB203580: 5, 10, 20 uM; G5: Protein kinase CK2 inhibitor, 5,20, 50 uM

  16. APCs and T-cells

  17. Quantitative Real Time PCR( Light Cycler System ) Reverse transcripton reaction RT- PCR: LightCyclerTM System (Roche), FastStart DNA Master SYBR Green I (Roche) Quantification: external cDNA standards with known amounts of initial copy number over a 105 - fold range were coamplified calculation with LightCycler Quantification Software v3.39 during the log- linear phase of the amplification ß- actin was used to normalize for inefficiencies in cDNA synthesis mRNA Isolation: Quick PrepMicro mRNA Purification Kit mRNA semiquantification: Nuleic dotMetricTM Basic Kit (Geno Technology)

  18. Amplification in RT- PCR A B 24 32 30 20 28 26 16 24 22 -dF/dT 20 Fluorescence 12 18 16 106 105 104 103 102 8 14 12 no template 10 4 8 no template 6 0 4 2 0 -4 -2 71 73 75 77 79 81 83 85 87 89 91 93 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Temperature (°C) Cycle number

  19. IL-12, TNFa mRNA in Ghost- stimulated THP-1 MO (RT- PCR) IL-12 TNFa Eif2-by M Co 3 6 16 16 hrs

  20. Stimulation IL-12 and TNFa in THP1 cellsELISA TNFa LPS  IL-12 Ghosts o

  21. Stimulation of IL-12 and TNFa in PBM TNFa IL-12

  22. Immune mediators in DC derived from PBM ( IL4, GMCSF, 5-7 d ) secrete large amounts of inflammatory cytokines as precursors high Ag capture capacity at their immature stage stimulation of quiescient, B and T lymphocytes low levels of antigen to induce strong T cell response

  23. Adherence of ALEXA labeled ghosts

  24. Stimulation of TNFa in PBM and DC

  25. Stimulation of IL-12 ( p40, p70) protein in PBM and DC

  26. Stimulation of IL-12 p40 mRNA in DC

  27. Stimulation of IL-18 relative mRNA in DC

  28. Ghosts and DC What are the mechanisms for the good activation of IL-12 in APC, especially DC ? Does this have a clinical value ?

  29. Microbial stimuli that promote DC maturation Bacteria ( E. coli, M. tuberculosis, Staph. aureus, Strept. ,... Protozoa LPS Bacterial DNA, Viral and doubled-stranded RNA Bacterial heat shock proteins

  30. Activation of DC functions by microbial stimuli. Migration Exit of activated DCs from peripheral sites Entry into the T cell areas of secondary lymphoid tissues Antigen presentation Upregulation of antigen presenting molecules (MHC class I and class II, CD1) Delivery of antigen to the MHC class I pathway Upregulation of molecules involved in interaction with T lymphocytes Costimulatory molecules (B7-1, B7-2) Adhesion molecules (ICAM-1, VLA-4) Signalling molecules (CD40) Production of cytokines Induction of IL-12, TNF, IL-10, IL-6, IFN-/ Recruitment of DC precursors to peripheral sites Increased transient survival of DCs in the absence of growth signals Irreversible maturation followed by apoptotic death

  31. Mature DCs and ghosts induce strong T-cell responses, MLR Pavol KUDELA maturation mix (MM)-TNF-; IL-1; PGE2; IL-6; GM-CSF; IL-4 MM + bacterial ghosts+ GM-CSF + IL-4

  32. Induce ghosts differentiation and activation of DC functions ? MHC class I and II, CD11a,b,c, CD50, CD54, CD58 Activation of T- cells IL-12, T cell attractant chemokine +++ + + +++ + +++

  33. Gut 2000 Jul;47(1):79-87 Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures.Haller D, Bode C, Hammes WP, Pfeifer AM, Schiffrin EJ, Blum S.Institute of Biological Chemistry and Nutrition Science, University Hohenheim, Germany.BACKGROUND AND AIM: Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1beta, and TNF-alpha mRNA in the presence of underlying leucocytes. Leucocyte sensitised CaCO-2 cells produced TNF-alpha and IL-1beta whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells. CaCO-2 cells alone remained hyporesponsive to the bacterial challenge. CONCLUSION: The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.

  34. Microbiol Immunol 1999;43(10):925-35 Cytokine secretion by stimulated monocytes depends on the growth phase and heat treatment of bacteria: a comparative study between lactic acid bacteria and invasive pathogens.Haller D, Bode C, Hammes WP.Hohenheim University, Stuttgart, Germany. dirk.haller@rdls.nestle.comThe challenge of monocytes with three LAB strains, Listeria monocytogenes or enterohaemorrhagic Escherichia coli (EHEC) elicited a strain specific, dose-dependent biphasic TNF-alpha secretion. LPS exhibited a higher capacity to stimulate monocytes than purified gram positive cell walls or muramyldipeptide. In comparison to pathogenic bacteria, the maximal secretory TNF-alpha response (TNFmax) was up to 2 fold higher with LAB strains. In general, the amount of bacteria (EDmax) necessary to induce maximal TNF-alpha secretion (TNFmax) was approximately 1 to 3 log higher for heat killed bacteria when compared to live bacterial cells illustrating the significant lower potential of heat killed bacteria to activate monocytes.

  35. Bacterial cell envelopes for vaccine development • No endotoxic limitations • lipid A activation pathways, CD14, p38 MAP kinase • provide adjuvans activity comparable to lipid A structures • effective uptake by phagocytes ( others by different mechanisms ?) • potent inducers of IL-12, ( IL-18 ) • may propagate maturation of dendritic cells • may enable packaging of systemic toxic compounds and targeting • to specific cells or organs AH, 10/2000

  36. Ghosts and Immunology Ghosts, preparation and ... M. Szostak Horst Mader M. Schmalnauer Gudrun Kohl Fürst Ladani Beate Mayr Margit Weghofer

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