Anti tumor properties of lactobacilli are mediated by immuno modulation and direct cytotoxicity

ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG NATIONAL UNIVERSITY OF SINGAPORE 2010 ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements I would like to express my heartfelt gratitude to everyone who had helped me and made my pursuit for the PhD degree a pleasant and fulfilling experience. To my supervisor, Dr Ratha Mahendran, for her invaluable advice and guidance, without of which my PhD project would not have been so fruitful. To my co-supervisors, Prof Bay Boon Huat and A/Prof Lee Yuan Kun, for taking time off their busy schedules to sit through my updates and giving me advice and encouragement. To my fellow colleagues, Shih Wee, Mathu, Juwita and Rachel, thank you for all the support and patience you have shown me through the years. Your encouragement and help made my PhD journey a much sweeter one. Many thanks to Eng Shi, Kishore, Ms Chan Yee Gek and Mr Low Chin Seng, for all the technical help and advice given to me. To my CRCEC friends, Evelyn, Elaine, Delicia, Sally, Gaik Chin, Eric and Hafizah, thank you for your lovely company and for lending me a listening ear or a helping hand whenever I need it. I would like to thank my parents and brothers for their love, faith and support. Thank you for always believing in me and encouraging me to go a little further, dream a little bigger. Last but not least, my significant other, Terry, thank you for being my pillar of support and for being there with me always, through the high and lows of my PhD journey. i Table of Contents Page Acknowledgements……………………………………………………………………. i Table of Contents …………………………………………………………………… . ii Summary………………………………………………………………………………. x List of Tables.…………………………………………………………………………. xii List of Figures…………………………………………………………………………. xiv List of Abbreviations………………………………………………………………… xvi List of publications and conference papers…………………………………………… xix Chapter Introduction ……………………………………………………………… 1.1 Cancer…………………………………………………………………………… 1.1.1 Cancer and its prevalence……………………………………………… 1.1.2 Causes of cancer … ………………………………………………… 1.2 Cancer treatments.……………………………………………………………… 1.2.1 Surgery………………………………………………………………… 1.2.2 Chemotherapy…………………………………………………………. 1.2.3 Radiation………………………………………………………………. 1.2.4 Immunotherapy……………………………………………………… . 1.2.5 Future of cancer therapy………………………………………………. 1.3 Role of immune system in anti-tumor response……………………………… 1.4 Cell death pathways induced by chemotherapuetic drugs used in cancer therapy…………………………………………………………………… ii 1.5 Bacteria in cancer therapy………………………………………………………. 1.5.1 Bacteria as immunotherapeutic agents……………………………………… 1.5.2 Bacteria as a delivery vehicle……………………………………………… 1.5.3 Bacterial cytotoxic agents…………………………………………………… 1.5.4 Limitations………………………………………………………………… 10 1.6 Lactobacilli………………………………………………………………………. 10 1.6.1 Health benefits of Lactobacilli…………………………………………… . 11 1.6.2 Anti-tumor effects of lactobacilli………………………………………… . 14 1.6.2.1 Immunologically mediated anti-tumor effect……………………… 14 1.6.2.2 Non-immunologically mediated anti-tumor effect………………… 16 1.6.3 Lactobacilli immuno-modulatory potential.……………………………… 17 1.6.3.1 Lactobacilli modulate host immune response in vitro and in vivo…. 17 1.6.3.2 Receptor mediated interaction between lactobacilli and innate immune cells………………………………………………………. 20 1.6.3.2.1 Toll like receptors……………………………………… 20 1.6.3.2.2 Mannose receptors………………………………………. 21 1.6.3.2.3 NOD like receptors……………………………………… 21 1.6.3.3 Bridging innate and adaptive immunity………………………… . 22 1.6.3.3.1 Macrophages……………………………………………. 22 1.6.3.3.2 Dendritic cells………………………………………… . 22 1.6.3.3.3 Neutrophils…………………………………………… 23 1.6.3.3.4 Dendritic cell and neutrophil interaction……………… 23 1.7 Scope of study…………………………………………………………………. 25 iii Chaper Materials and Methods………………………………………………… 27 2.1 Bacteria Preparation………………………………………………………… 28 2.1.1 Live Lactobacilli………………………………………………………… 28 2.1.2 Lyophilized Lactobacilli………………………………………………… 29 2.1.3 Heat Killed LGG…………………………………………………………. 29 2.2 Ex vivo study of Lactobacilli interaction with immune cells……………… 30 2.2.1 Animals…………………………………………………………………… 30 2.2.2 Immune cells isolation……………………………………………………. 30 2.2.2.1 Isolation of bone marrow derived neutrophils and dendritic cells . 30 2.2.2.2 Isolation of T-cells……………………………………………… . 31 2.2.2.3 Splenocytes isolation…………………………………………… . 33 2.2.3 Co-culture of immune cells with Lactobacilli …………………………… 33 2.2.3.1 Co-culture of neutrophils or DCs with LGG …………………… 33 2.2.3.2 Study of neutrophil-neutrophil interaction……………………… 34 2.2.3.3 DC neutrophil co-culture…………………………………………. 35 2.2.3.4 DC or DC-neutrophil co-culture with T cells…………………… 35 2.2.3.5 Stimulation of splenocytes with live and lyophilized Lactobacilli 36 2.2.4 Interaction between immune cells and Lactobacilli……………………… 36 2.2.4.1 Uptake of Lactobacilli into immune cells………………………… 36 2.2.4.2 Blocking phagocytosis……………………………………………. 36 2.2.4.3 Cytokine and PGE2 ELISA………………………………………. 37 2.2.4.4 Blocking TLR2 and 9…………………………………………… 38 2.2.4.5 Flow cytometric analysis of surface markers and receptors on DCs and neutrophils……………………………………………… 39 iv 2.2.4.6 Blocking IL10 and COX2 in DC neutrophil co-culture…………. 40 2.2.4.7 Effect of LGG on neutrophil viability…………………………… 40 2.2.4.7.1 Annexin V-PI staining of neutrophils………………… 40 2.3 Cytotoxic effect of Lactobacilli on cancer cells……………………………… 41 2.3.1 Cancer and normal cell lines……………………………………………… 41 2.3.2 Direct co-culture of MGH with lactobacilli………………………………. 41 2.3.3 Production of cytotoxic molecule from LGG…………………………… 42 2.3.3.1 Cytotoxic molecule production in the culture supernatant……… 42 2.3.3.2 Extraction of LGG cytoplasmic fraction………………………… 42 2.3.3.3 Optimization of cytotoxic molecule production …………………. 42 2.3.3.4 Growth curve of LGG in media…………………………………… 43 2.3.3.5 Measurement of pH, lactate and glucose………………………… 43 2.3.4 Characterization of cytoxic molecule…………………………………… . 44 2.3.4.1 Stability of cytotoxic molecule……………………………………. 44 2.3.4.2 Molecular size of cytotoxic molecule…………………………… . 44 2.3.4.3 Nature of cytotoxic molecule……………………………………… 44 2.3.4.3.1 Proteinase K and trypsin digestion……………………… 44 2.3.4.3.2 Chloroform extraction…………………………………… 45 2.3.5 Effect of cytotoxic molecule on human cells……………………………… 45 2.3.5.1 Cell viability assays……………………………………………… 45 2.3.5.1.1 MTS and Multitox-fluor assay………………………… 45 2.3.5.1.2 Cell Count ………………………………………………. 46 2.3.5.2 Mechanism of cell death………………………………………… . 46 v 2.3.5.2.1 Caspase 3/7 activity……………………………………… 46 2.3.5.2.2 Lactate dehydrogenase (LDH) test………………………. 46 2.3.5.2.3 Cell cycle analysis……………………………………… 47 2.3.5.3 Effect of cytotoxic molecule on a panel of cancer and normal cells 47 2.3.5.4 Visualization of cell-lines…………………………………………. 48 2.3.5.4.1 Light and fluorescence microscopy…………………… . 48 2.3.5.4.2 Electron microscopy…………………………………… 48 2.3.5.5 Uptake of cytotoxic molecule…………………………………… . 49 2.3.5.6 Cellular Pathways activated by the cytotoxic molecule………… . 50 2.3.5.6.1 Total RNA extraction and cDNA conversion…………… 50 2.3.5.6.2 LDA…………………………………………………… . 51 2.3.5.6.3 Real-time PCR………………………………………… . 53 2.3.5.6.4 RT-PCR…………………………………………………. 53 2.3.5.6.5 Protein isolation from MGH cells………………………. 55 2.3.5.6.6 Western Blot of ACVR1C, pSMAD2 and SMAD.…… 55 2.3.6 Purification of cytotoxic molecule……………………………………… 56 2.3.6.1 High Performance Liquid Chromatography (HPLC)…………… 56 2.3.6.2 Gas chromatography TOF mass spectropmetry (GC-TOFMS)… 57 2.4 Statistical Analysis…………………………………………………………… 58 Chapter Results Part I: Immuno-stimulatory effect of lactobacilli………… 59 3.1 Interaction of Neutrophils and LGG………………………………………… 60 3.1.1 Internalization of LGG induces cytokine production in neutrophils…… . 60 vi 3.1.2 Role of toll-like receptor in LGG stimulation of neutrophils…………… 63 3.1.3 LGG induced cell death in neutrophils……………………………………. 64 3.1.4 Effect of LGG on surface marker expression in neutrophils……………… 66 3.1.5 Neutrophil-neutrophil interaction after exposure to LGG………………… 67 3.2 Effect of dose and exposure time of LGG on DC maturation and DC-neutrophils cross talk…………………………………………………… . 69 3.2.1 Maturation of dendritic cells is dependent on bacteria dose, exposure time and presentation by neutrophils…………………………… 70 3.2.2 Dose and duration of LGG exposure skews cytokine profile in DC and DC neutrophil co-culture…………………………………………………. 73 3.2.3 Effect of high LGG dose on IL12 production is dependent on IL10 levels but not Prostaglandin E2 (PGE2) levels…………………… 76 3.2.4 Downstream T-cell activation is dependent on the bacteria dose exposed to the DCs………………………………………………………. 78 3.3 Differential Immuno-stimulatory potential of live and lyophilized Lactobacillus species ……………………………………………………… 79 3.3.1 Different strains of lactobacilli induce different levels of TNF, IL10 and IL12p40 ………………………………………………………. 79 3.3.2 Lyophilized lactobacilli induced more TNF, IL10 and IL12p40 ……… 81 3.3.3 Contact is required for lactobacilli to stimulate spleen cells to produce cytokines……………………………………………………… . 83 3.3.4 Lactobacilli stimulate splenocytes through TLR2 but not TLR9……… . 84 3.3.5 Phagocytosis plays a role in cytokine induction by L.bulgaricus……… 85 Summary I……………………………………………………………………. 87 Chapter Results Part II: Direct cytotoxic effect of lactobacilli on cancer cells 89 3.4 Optimization of lactobacilli mediated direct cytotoxic effects on cancer cells…………………………………………………………………… . 90 vii 3.4.1 Effect of media pH on cytotoxic effect…………………………………… 90 3.4.2 Comparison of the cytotoxic effect of different lactobacillus strains ……. 91 3.4.3 LGG produced cytotoxic molecules are released into the culture supernatant 92 3.4.4 Other conditions that affect cytotoxicity of LGG………………………… 93 3.4.5 Glucose and amberlite enhances production of cytotoxic molecules by LGG 94 3.4.6 Culture media conditions at the end of 24 hours of incubation…………… 97 3.5 Characterizations of the cytotoxic molecule(s)………………………………… 98 3.5.1 Basic characterization of the cytotoxic molecule…………………………… 98 3.5.2 Purification of cytotoxic molecule from LGG supernatant…………………. 100 3.5.3 Possible identity of cytotoxic molecules, determined by GC-TOFMS ……. 101 3.6 Uptake of cytotoxic molecule into MGH cells…………………………………. 104 3.7 Cytotoxic and anti-proliferative effect of LGG supernatant and LCT……… 105 3.7.1 Cell cycle analysis with propidium iodide…………………………………. 106 3.7.2 LCT induced apoptosis in MGH but not LGG supernatant……………… . 106 3.7.3 Morphologies of MGH cells treated with LGG supernatant and LCT…… 109 3.8 LGG supernatant preferentially targets cancer cells and not normal cells… 112 3.9 Effect of LGG supernatant and LCT on gene expression in MGH cells…… 114 3.9.1 Confirmation of gene expression with real-time PCR ……………………. 114 3.9.2 Confirmation of gene expressions with RT-PCR…………………………. 116 3.10 Gene and protein expressions of ACVR1C…………………………………. 118 Summary II…………………………………………………………………… 120 Chapter Discussion………………………………………………………………. 122 4.1 Phagocytosis and TLR2 are important mediators of the interaction of lactobacilli with immune cells ………………………………………………. 123 viii 146. Braat, H., van den Brande, J., van Tol, E., Hommes, D., Peppelenbosch, M., van Deventer, S. (2004) Lactobacillus rhamnosus induces peripheral hyporesponsiveness in stimulated CD4+ T cells via modulation of dendritic cell function. Am J Clin Nutr 80, 1618-25. 147. Beauvillain, C., Delneste, Y., Scotet, M., Peres, A., Gascan, H., Guermonprez, P., Barnaba, V., Jeannin, P. (2007) Neutrophils efficiently cross-prime naive T cells in vivo. Blood 110, 2965-73. 148. Tvinnereim, A.R., Hamilton, S.E., Harty, J.T. (2004) Neutrophil involvement in cross-priming CD8+ T cell responses to bacterial antigens. J Immunol 173, 19942002. 149. Potter, N.S., Harding, C.V. (2001) Neutrophils process exogenous bacteria via an alternate class I MHC processing pathway for presentation of peptides to T lymphocytes. J Immunol 167, 2538-46. 150. Bennouna, S., Bliss, S.K., Curiel, T.J., Denkers, E.Y. (2003) Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection. J Immunol 171, 6052-8. 151. Megiovanni, A.M., Sanchez, F., Robledo-Sarmiento, M., Morel, C., Gluckman, J.C., Boudaly, S. (2006) Polymorphonuclear neutrophils deliver activation signals and antigenic molecules to dendritic cells: a new link between leukocytes upstream of T lymphocytes. J Leukoc Biol 79, 977-88. 152. Miyazaki, S., Ishikawa, F., Fujikawa, T., Nagata, S., Yamaguchi, K. (2004) Intraperitoneal injection of lipopolysaccharide induces dynamic migration of Gr1high polymorphonuclear neutrophils in the murine abdominal cavity. Clin Diagn Lab Immunol 11, 452-7. 153. Iking-Konert, C., Cseko, C., Wagner, C., Stegmaier, S., Andrassy, K., Hansch, G.M. (2001) Transdifferentiation of polymorphonuclear neutrophils: acquisition of CD83 and other functional characteristics of dendritic cells. J Mol Med 79, 464-74. 154. van Gisbergen, K.P., Sanchez-Hernandez, M., Geijtenbeek, T.B., van Kooyk, Y. (2005) Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN. J Exp Med 201, 1281-92. 155. Bennouna, S., Denkers, E.Y. (2005) Microbial antigen triggers rapid mobilization of TNF-alpha to the surface of mouse neutrophils transforming them into inducers of high-level dendritic cell TNF-alpha production. J Immunol 174, 4845-51. 164 156. Gorbach, S.L. (2000) Probiotics and gastrointestinal health. Am J Gastroenterol 95, S2-4. 157. Salminen, S., Bouley, C., Boutron-Ruault, M.C., Cummings, J.H., Franck, A., Gibson, G.R., Isolauri, E., Moreau, M.C., Roberfroid, M., Rowland, I. (1998) Functional food science and gastrointestinal physiology and function. Br J Nutr 80 Suppl 1, S147-71. 158. Benkerroum, N., Daoudi, A., Hamraoui, T., Ghalfi, H., Thiry, C., Duroy, M., Evrart, P., Roblain, D., Thonart, P. (2005) Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages. J Appl Microbiol 98, 56-63. 159. Mayordomo, J.I., Zorina, T., Storkus, W.J., Zitvogel, L., Celluzzi, C., Falo, L.D., Melief, C.J., Ildstad, S.T., Kast, W.M., Deleo, A.B., et al. (1995) Bone marrowderived dendritic cells pulsed with synthetic tumor peptides elicit protective and therapeutic antitumor immunity. Nat Med 1, 1297-302. 160. Sheehan, K.C., Pinckard, J.K., Arthur, C.D., Dehner, L.P., Goeddel, D.V., Schreiber, R.D. (1995) Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75. J Exp Med 181, 607-17. 161. Duramad, O., Fearon, K.L., Chang, B., Chan, J.H., Gregorio, J., Coffman, R.L., Barrat, F.J. (2005) Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation. J Immunol 174, 5193-200. 162. Maassen, C.B., van Holten-Neelen, C., Balk, F., den Bak-Glashouwer, M.J., Leer, R.J., Laman, J.D., Boersma, W.J., Claassen, E. (2000) Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains. Vaccine 18, 2613-23. 163. Carlos, T.M., Harlan, J.M. (1994) Leukocyte-endothelial adhesion molecules. Blood 84, 2068-101. 164. Kuijpers, T.W., Tool, A.T., van der Schoot, C.E., Ginsel, L.A., Onderwater, J.J., Roos, D., Verhoeven, A.J. (1991) Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation. Blood 78, 1105-11. 165. Banchereau, J., Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature 392, 245-52. 165 166. Kalinski, P., Vieira, P.L., Schuitemaker, J.H., de Jong, E.C., Kapsenberg, M.L. (2001) Prostaglandin E(2) is a selective inducer of interleukin-12 p40 (IL-12p40) production and an inhibitor of bioactive IL-12p70 heterodimer. Blood 97, 3466-9. 167. Kalinski, P., Hilkens, C.M., Snijders, A., Snijdewint, F.G., Kapsenberg, M.L. (1997) IL-12-deficient dendritic cells, generated in the presence of prostaglandin E2, promote type cytokine production in maturing human naive T helper cells. J Immunol 159, 28-35. 168. De Smedt, T., Van Mechelen, M., De Becker, G., Urbain, J., Leo, O., Moser, M. (1997) Effect of interleukin-10 on dendritic cell maturation and function. Eur J Immunol 27, 1229-35. 169. Stolina, M., Sharma, S., Lin, Y., Dohadwala, M., Gardner, B., Luo, J., Zhu, L., Kronenberg, M., Miller, P.W., Portanova, J., Lee, J.C., Dubinett, S.M. (2000) Specific inhibition of cyclooxygenase restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immunol 164, 361-70. 170. Bayer, A.S., Chow, A.W., Morrison, J.O., Guze, L.B. (1980) Bactericidal synergy between penicillin or ampicillin and aminoglycosides against antibiotic-tolerant lactobacilli. Antimicrob Agents Chemother 17, 359-63. 171. Kim, K.S., Morrison, J.O., Bayer, A.S. (1982) Deficient autolytic enzyme activity in antibiotic-tolerant lactobacilli. Infect Immun 36, 582-5. 172. Kapetanovic, R., Nahori, M.A., Balloy, V., Fitting, C., Philpott, D.J., Cavaillon, J.M., Adib-Conquy, M. (2007) Contribution of phagocytosis and intracellular sensing for cytokine production by Staphylococcus aureus-activated macrophages. Infect Immun 75, 830-7. 173. Hayashi, F., Means, T.K., Luster, A.D. (2003) Toll-like receptors stimulate human neutrophil function. Blood 102, 2660-9. 174. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., Kirschning, C.J. (1999) Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by tolllike receptor 2. J Biol Chem 274, 17406-9. 175. Harrington, N.P., Chambers, K.A., Ross, W.M., Filion, L.G. (1997) Radiation damage and immune suppression in splenic mononuclear cell populations. Clin Exp Immunol 107, 417-24. 176. O'Mahony, D.S., Pham, U., Iyer, R., Hawn, T.R., Liles, W.C. (2008) Differential constitutive and cytokine-modulated expression of human Toll-like receptors in primary neutrophils, monocytes, and macrophages. Int J Med Sci 5, 1-8. 166 177. Shida, K., Kiyoshima-Shibata, J., Nagaoka, M., Watanabe, K., Nanno, M. (2006) Induction of interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion. J Dairy Sci 89, 3306-17. 178. Daeron, M. (1997) Fc receptor biology. Annu Rev Immunol 15, 203-34. 179. Ross, G.D. (2000) Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/alphaMbeta2-integrin glycoprotein. Crit Rev Immunol 20, 197-222. 180. Ekman, A.K., Cardell, L.O. The expression and function of Nod-like receptors in neutrophils. Immunology 130, 55-63. 181. Smits, H.H., Engering, A., van der Kleij, D., de Jong, E.C., Schipper, K., van Capel, T.M., Zaat, B.A., Yazdanbakhsh, M., Wierenga, E.A., van Kooyk, Y., Kapsenberg, M.L. (2005) Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol 115, 1260-7. 182. Underhill, D.M., Ozinsky, A., Hajjar, A.M., Stevens, A., Wilson, C.B., Bassetti, M., Aderem, A. (1999) The Toll-like receptor is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401, 811-5. 183. Ozinsky, A., Underhill, D.M., Fontenot, J.D., Hajjar, A.M., Smith, K.D., Wilson, C.B., Schroeder, L., Aderem, A. (2000) The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between tolllike receptors. Proc Natl Acad Sci U S A 97, 13766-71. 184. Underhill, D.M., Ozinsky, A. (2002) Phagocytosis of microbes: complexity in action. Annu Rev Immunol 20, 825-52. 185. Kagan, J.C., Medzhitov, R. (2006) Phosphoinositide-mediated recruitment controls Toll-like receptor signaling. Cell 125, 943-55. 186. Gantner, B.N., Simmons, R.M., Canavera, S.J., Akira, S., Underhill, D.M. (2003) Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J Exp Med 197, 1107-17. 187. Hazeki, K., Masuda, N., Funami, K., Sukenobu, N., Matsumoto, M., Akira, S., Takeda, K., Seya, T., Hazeki, O. (2003) Toll-like receptor-mediated tyrosine phosphorylation of paxillin via MyD88-dependent and -independent pathways. Eur J Immunol 33, 740-7. 188. Peiser, L., Mukhopadhyay, S., Gordon, S. (2002) Scavenger receptors in innate immunity. Curr Opin Immunol 14, 123-8. adaptor 167 189. Hume, D.A., Ross, I.L., Himes, S.R., Sasmono, R.T., Wells, C.A., Ravasi, T. (2002) The mononuclear phagocyte system revisited. J Leukoc Biol 72, 621-7. 190. Baba, N., Samson, S., Bourdet-Sicard, R., Rubio, M., Sarfati, M. (2009) Selected commensal-related bacteria and Toll-like receptor agonist combinatorial codes synergistically induce interleukin-12 production by dendritic cells to trigger a T helper type polarizing programme. Immunology 128, e523-31. 191. Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin, R.L., Akira, S. (2002) Cutting edge: role of Toll-like receptor in mediating immune response to microbial lipoproteins. J Immunol 169, 10-4. 192. Morr, M., Takeuchi, O., Akira, S., Simon, M.M., Muhlradt, P.F. (2002) Differential recognition of structural details of bacterial lipopeptides by toll-like receptors. Eur J Immunol 32, 3337-47. 193. Buwitt-Beckmann, U., Heine, H., Wiesmuller, K.H., Jung, G., Brock, R., Akira, S., Ulmer, A.J. (2006) TLR1- and TLR6-independent recognition of bacterial lipopeptides. J Biol Chem 281, 9049-57. 194. Visintin, A., Mazzoni, A., Spitzer, J.H., Wyllie, D.H., Dower, S.K., Segal, D.M. (2001) Regulation of Toll-like receptors in human monocytes and dendritic cells. J Immunol 166, 249-55. 195. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S., Hartmann, G. (2002) Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168, 4531-7. 196. Miettinen, M., Veckman, V., Latvala, S., Sareneva, T., Matikainen, S., Julkunen, I. (2008) Live Lactobacillus rhamnosus and Streptococcus pyogenes differentially regulate Toll-like receptor (TLR) gene expression in human primary macrophages. J Leukoc Biol 84, 1092-100. 197. Vizoso Pinto, M.G., Rodriguez Gomez, M., Seifert, S., Watzl, B., Holzapfel, W.H., Franz, C.M. (2009) Lactobacilli stimulate the innate immune response and modulate the TLR expression of HT29 intestinal epithelial cells in vitro. Int J Food Microbiol 133, 86-93. 198. Faurschou, M., Borregaard, N. (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect 5, 1317-27. 199. Nauseef, W.M. (2007) How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 219, 88-102. 168 200. Quinn, M.T., Ammons, M.C., Deleo, F.R. (2006) The expanding role of NADPH oxidases in health and disease: no longer just agents of death and destruction. Clin Sci (Lond) 111, 1-20. 201. Kobayashi, S.D., Braughton, K.R., Whitney, A.R., Voyich, J.M., Schwan, T.G., Musser, J.M., DeLeo, F.R. (2003) Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils. Proc Natl Acad Sci U S A 100, 10948-53. 202. Yamamoto, A., Taniuchi, S., Tsuji, S., Hasui, M., Kobayashi, Y. (2002) Role of reactive oxygen species in neutrophil apoptosis following ingestion of heat-killed Staphylococcus aureus. Clin Exp Immunol 129, 479-84. 203. Watson, R.W., Redmond, H.P., Wang, J.H., Condron, C., Bouchier-Hayes, D. (1996) Neutrophils undergo apoptosis following ingestion of Escherichia coli. J Immunol 156, 3986-92. 204. Krysko, D.V., D'Herde, K., Vandenabeele, P. (2006) Clearance of apoptotic and necrotic cells and its immunological consequences. Apoptosis 11, 1709-26. 205. Lauber, K., Blumenthal, S.G., Waibel, M., Wesselborg, S. (2004) Clearance of apoptotic cells: getting rid of the corpses. Mol Cell 14, 277-87. 206. Silva, M.T. (2010) Bacteria-induced phagocyte secondary necrosis as a pathogenicity mechanism. J Leukoc Biol. Epub ahead of print June 2010. 207. Silva, M.T., Vale, A., dos Santos, N.M. (2008) Secondary necrosis in multicellular animals: an outcome of apoptosis with pathogenic implications. Apoptosis 13, 463-82. 208. Nathan, C. (2006) Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 6, 173-82. 209. Staali, L., Bauer, S., Morgelin, M., Bjorck, L., Tapper, H. (2006) Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes. Cell Microbiol 8, 690-703. 210. Staali, L., Morgelin, M., Bjorck, L., Tapper, H. (2003) Streptococcus pyogenes expressing M and M-like surface proteins are phagocytosed but survive inside human neutrophils. Cell Microbiol 5, 253-65. 211. Allen, L.A., Beecher, B.R., Lynch, J.T., Rohner, O.V., Wittine, L.M. (2005) Helicobacter pylori disrupts NADPH oxidase targeting in human neutrophils to induce extracellular superoxide release. J Immunol 174, 3658-67. 169 212. Soler-Rodriguez, A.M., Zhang, H., Lichenstein, H.S., Qureshi, N., Niesel, D.W., Crowe, S.E., Peterson, J.W., Klimpel, G.R. (2000) Neutrophil activation by bacterial lipoprotein versus lipopolysaccharide: differential requirements for serum and CD14. J Immunol 164, 2674-83. 213. Trevani, A.S., Chorny, A., Salamone, G., Vermeulen, M., Gamberale, R., Schettini, J., Raiden, S., Geffner, J. (2003) Bacterial DNA activates human neutrophils by a CpG-independent pathway. Eur J Immunol 33, 3164-74. 214. Mencacci, A., Montagnoli, C., Bacci, A., Cenci, E., Pitzurra, L., Spreca, A., Kopf, M., Sharpe, A.H., Romani, L. (2002) CD80+Gr-1+ myeloid cells inhibit development of antifungal Th1 immunity in mice with candidiasis. J Immunol 169, 3180-90. 215. Windhagen, A., Maniak, S., Gebert, A., Ferger, I., Wurster, U., Heidenreich, F. (1999) Human polymorphonuclear neutrophils express a B7-1-like molecule. J Leukoc Biol 66, 945-52. 216. Puellmann, K., Kaminski, W.E., Vogel, M., Nebe, C.T., Schroeder, J., Wolf, H., Beham, A.W. (2006) A variable immunoreceptor in a subpopulation of human neutrophils. Proc Natl Acad Sci U S A 103, 14441-6. 217. Venuprasad, K., Parab, P., Prasad, D.V., Sharma, S., Banerjee, P.R., Deshpande, M., Mitra, D.K., Pal, S., Bhadra, R., Mitra, D., Saha, B. (2001) Immunobiology of CD28 expression on human neutrophils. I. CD28 regulates neutrophil migration by modulating CXCR-1 expression. Eur J Immunol 31, 1536-43. 218. Fortin, C.F., Ear, T., McDonald, P.P. (2009) Autocrine role of endogenous interleukin-18 on inflammatory cytokine generation by human neutrophils. FASEB J 23, 194-203. 219. Borregaard, N., Sorensen, O.E., Theilgaard-Monch, K. (2007) Neutrophil granules: a library of innate immunity proteins. Trends Immunol 28, 340-5. 220. Yang, D., de la Rosa, G., Tewary, P., Oppenheim, J.J. (2009) Alarmins link neutrophils and dendritic cells. Trends Immunol 30, 531-7. 221. Oppenheim, J.J., Yang, D. (2005) Alarmins: chemotactic activators of immune responses. Curr Opin Immunol 17, 359-65. 222. Yang, D., Biragyn, A., Hoover, D.M., Lubkowski, J., Oppenheim, J.J. (2004) Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense. Annu Rev Immunol 22, 181-215. 170 223. Curran, C.S., Demick, K.P., Mansfield, J.M. (2006) Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways. Cell Immunol 242, 23-30. 224. Tian, J., Avalos, A.M., Mao, S.Y., Chen, B., Senthil, K., Wu, H., Parroche, P., Drabic, S., Golenbock, D., Sirois, C., Hua, J., An, L.L., Audoly, L., La Rosa, G., Bierhaus, A., Naworth, P., Marshak-Rothstein, A., Crow, M.K., Fitzgerald, K.A., Latz, E., Kiener, P.A., Coyle, A.J. (2007) Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 8, 487-96. 225. Tejada-Simon, M.V., Pestka, J.J. (1999) Proinflammatory cytokine and nitric oxide induction in murine macrophages by cell wall and cytoplasmic extracts of lactic acid bacteria. J Food Prot 62, 1435-44. 226. de Ambrosini, V.M., Gonzalez, S., Perdigon, G., de Ruiz Holgado, A.P., Oliver, G. (1996) Chemical composition of the cell wall of lactic acid bacteria and related species. Chem Pharm Bull (Tokyo) 44, 2263-7. 227. Heinzel, F.P., Sadick, M.D., Holaday, B.J., Coffman, R.L., Locksley, R.M. (1989) Reciprocal expression of interferon gamma or interleukin during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets. J Exp Med 169, 59-72. 228. Mitic, S. (1976) Transformation of amino acid composition in bacterial cells of Lactobacillus bulgaricus during freeze-drying. Cryobiology 13, 214-7. 229. Ludwig, I.S., Geijtenbeek, T.B., van Kooyk, Y. (2006) Two way communication between neutrophils and dendritic cells. Curr Opin Pharmacol 6, 408-13. 230. Veckman, V., Miettinen, M., Pirhonen, J., Siren, J., Matikainen, S., Julkunen, I. (2004) Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells. J Leukoc Biol 75, 764-71. 231. Konstantinov, S.R., Smidt, H., de Vos, W.M., Bruijns, S.C., Singh, S.K., Valence, F., Molle, D., Lortal, S., Altermann, E., Klaenhammer, T.R., van Kooyk, Y. (2008) S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions. Proc Natl Acad Sci U S A 105, 19474-9. 232. Fujimoto, Y., Tu, L., Miller, A.S., Bock, C., Fujimoto, M., Doyle, C., Steeber, D.A., Tedder, T.F. (2002) CD83 expression influences CD4+ T cell development in the thymus. Cell 108, 755-67. 171 233. Kuwano, Y., Prazma, C.M., Yazawa, N., Watanabe, R., Ishiura, N., Kumanogoh, A., Okochi, H., Tamaki, K., Fujimoto, M., Tedder, T.F. (2007) CD83 influences cell-surface MHC class II expression on B cells and other antigen-presenting cells. Int Immunol 19, 977-92. 234. Breloer, M., Kretschmer, B., Luthje, K., Ehrlich, S., Ritter, U., Bickert, T., Steeg, C., Fillatreau, S., Hoehlig, K., Lampropoulou, V., Fleischer, B. (2007) CD83 is a regulator of murine B cell function in vivo. Eur J Immunol 37, 634-48. 235. Cao, W., Lee, S.H., Lu, J. (2005) CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells. Biochem J 385, 85-93. 236. Hock, B.D., Kato, M., McKenzie, J.L., Hart, D.N. (2001) A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera. Int Immunol 13, 959-67. 237. Su, L.L., Iwai, H., Lin, J.T., Fathman, C.G. (2009) The transmembrane E3 ligase GRAIL ubiquitinates and degrades CD83 on CD4 T cells. J Immunol 183, 43844. 238. Kummer, M., Turza, N.M., Muhl-Zurbes, P., Lechmann, M., Boutell, C., Coffin, R.S., Everett, R.D., Steinkasserer, A., Prechtel, A.T. (2007) Herpes simplex virus type induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome. J Virol 81, 6326-38. 239. Lechmann, M., Krooshoop, D.J., Dudziak, D., Kremmer, E., Kuhnt, C., Figdor, C.G., Schuler, G., Steinkasserer, A. (2001) The extracellular domain of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand on dendritic cells. J Exp Med 194, 1813-21. 240. Senechal, B., Boruchov, A.M., Reagan, J.L., Hart, D.N., Young, J.W. (2004) Infection of mature monocyte-derived dendritic cells with human cytomegalovirus inhibits stimulation of T-cell proliferation via the release of soluble CD83. Blood 103, 4207-15. 241. Langenkamp, A., Messi, M., Lanzavecchia, A., Sallusto, F. (2000) Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat Immunol 1, 311-6. 242. Lanzavecchia, A., Sallusto, F. (2001) Regulation of T cell immunity by dendritic cells. Cell 106, 263-6. 172 243. Gilliet, M., Kleinhans, M., Lantelme, E., Schadendorf, D., Burg, G., Nestle, F.O. (2003) Intranodal injection of semimature monocyte-derived dendritic cells induces T helper type responses to protein neoantigen. Blood 102, 36-42. 244. Camporeale, A., Boni, A., Iezzi, G., Degl'Innocenti, E., Grioni, M., Mondino, A., Bellone, M. (2003) Critical impact of the kinetics of dendritic cells activation on the in vivo induction of tumor-specific T lymphocytes. Cancer Res 63, 3688-94. 245. Heufler, C., Koch, F., Stanzl, U., Topar, G., Wysocka, M., Trinchieri, G., Enk, A., Steinman, R.M., Romani, N., Schuler, G. (1996) Interleukin-12 is produced by dendritic cells and mediates T helper development as well as interferon-gamma production by T helper cells. Eur J Immunol 26, 659-68. 246. Nishimura, T., Nakui, M., Sato, M., Iwakabe, K., Kitamura, H., Sekimoto, M., Ohta, A., Koda, T., Nishimura, S. (2000) The critical role of Th1-dominant immunity in tumor immunology. Cancer Chemother Pharmacol 46 Suppl, S5261. 247. van der Pouw Kraan, T.C., Boeije, L.C., Smeenk, R.J., Wijdenes, J., Aarden, L.A. (1995) Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J Exp Med 181, 775-9. 248. Lee, K., Lee, H.G., Pi, K., Choi, Y.J. (2008) The effect of low pH on protein expression by the probiotic bacterium Lactobacillus reuteri. Proteomics 8, 162430. 249. Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H.D., Lang, H. (1974) Proteinase K from Tritirachium album Limber. Eur J Biochem 47, 91-7. 250. Lu, R., Fasano, S., Madayiputhiya, N., Morin, N.P., Nataro, J., Fasano, A. (2009) Isolation, identification, and characterization of small bioactive peptides from Lactobacillus GG conditional media that exert both anti-Gram-negative and Gram-positive bactericidal activity. J Pediatr Gastroenterol Nutr 49, 23-30. 251. Hall, E.A., Knox, K.W. (1965) Properties of the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei. Biochem J 96, 310-8. 252. Lebeer, S., Verhoeven, T.L., Francius, G., Schoofs, G., Lambrichts, I., Dufrene, Y., Vanderleyden, J., De Keersmaecker, S.C. (2009) Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase. Appl Environ Microbiol 75, 3554-63. 253. Sawada, H., Furushiro, M., Hirai, K., Motoike, M., Watanabe, T., Yokokura, T. (1990) Purification and characterization of an antihypertensive compound from Lactobacillus casei. Agric Biol Chem 54, 3211-9. 173 254. Majno, G., Joris, I. (1995) Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146, 3-15. 255. Krysko, D.V., Vanden Berghe, T., D'Herde, K., Vandenabeele, P. (2008) Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 44, 205-21. 256. Kerr, J.F., Wyllie, A.H., Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239-57. 257. Heilman, D.W., Green, M.R., Teodoro, J.G. (2005) The anaphase promoting complex: a critical target for viral proteins and anti-cancer drugs. Cell Cycle 4, 560-3. 258. Mork, C.N., Faller, D.V., Spanjaard, R.A. (2005) A mechanistic approach to anticancer therapy: targeting the cell cycle with histone deacetylase inhibitors. Curr Pharm Des 11, 1091-104. 259. Clarke, P.R., Allan, L.A. (2009) Cell-cycle control in the face of damage--a matter of life or death. Trends Cell Biol 19, 89-98. 260. McIlwrath, A.J., Vasey, P.A., Ross, G.M., Brown, R. (1994) Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity. Cancer Res 54, 3718-22. 261. Sangar, V.K., Cowan, R., Margison, G.P., Hendry, J.H., Clarke, N.W. (2004) An evaluation of gemcitabines differential radiosensitising effect in related bladder cancer cell lines. Br J Cancer 90, 542-8. 262. Moreno-Sanchez, R., Rodriguez-Enriquez, S., Marin-Hernandez, A., Saavedra, E. (2007) Energy metabolism in tumor cells. FEBS J 274, 1393-418. 263. Gatenby, R.A., Gillies, R.J. (2004) Why cancers have high aerobic glycolysis? Nat Rev Cancer 4, 891-9. 264. Warburg, O. (1956) On the origin of cancer cells. Science 123, 309-14. 265. Warburg, O. (1956) On respiratory impairment in cancer cells. Science 124, 26970. 266. Carew, J.S., Huang, P. (2002) Mitochondrial defects in cancer. Mol Cancer 1, 920. 267. Singh, K.K. (2004) Mitochondrial dysfunction is a common phenotype in aging and cancer. Ann N Y Acad Sci 1019, 260-4. 174 268. Brahimi-Horn, M.C., Pouyssegur, J. (2005) The hypoxia-inducible factor and tumor progression along the angiogenic pathway. Int Rev Cytol 242, 157-213. 269. Bustamante, E., Pedersen, P.L. (1977) High aerobic glycolysis of rat hepatoma cells in culture: role of mitochondrial hexokinase. Proc Natl Acad Sci U S A 74, 3735-9. 270. Munoz-Pinedo, C., Ruiz-Ruiz, C., Ruiz de Almodovar, C., Palacios, C., LopezRivas, A. (2003) Inhibition of glucose metabolism sensitizes tumor cells to death receptor-triggered apoptosis through enhancement of death-inducing signaling complex formation and apical procaspase-8 processing. J Biol Chem 278, 1275968. 271. Izyumov, D.S., Avetisyan, A.V., Pletjushkina, O.Y., Sakharov, D.V., Wirtz, K.W., Chernyak, B.V., Skulachev, V.P. (2004) "Wages of fear": transient threefold decrease in intracellular ATP level imposes apoptosis. Biochim Biophys Acta 1658, 141-7. 272. Xu, R.H., Pelicano, H., Zhou, Y., Carew, J.S., Feng, L., Bhalla, K.N., Keating, M.J., Huang, P. (2005) Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res 65, 613-21. 273. Maher, J.C., Krishan, A., Lampidis, T.J. (2004) Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs aerobic conditions. Cancer Chemother Pharmacol 53, 116-22. 274. Giancotti, F.G., Ruoslahti, E. (1999) Integrin signaling. Science 285, 1028-32. 275. Stupack, D.G., Cheresh, D.A. (2002) Get a ligand, get a life: integrins, signaling and cell survival. J Cell Sci 115, 3729-38. 276. Damiano, J.S., Cress, A.E., Hazlehurst, L.A., Shtil, A.A., Dalton, W.S. (1999) Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 93, 1658-67. 277. Schmidmaier, R., Baumann, P., Simsek, M., Dayyani, F., Emmerich, B., Meinhardt, G. (2004) The HMG-CoA reductase inhibitor simvastatin overcomes cell adhesion-mediated drug resistance in multiple myeloma by geranylgeranylation of Rho protein and activation of Rho kinase. Blood 104, 1825-32. 278. Kokubo, T., Uchida, H., Choi, E.T. (2007) Integrin alpha(v)beta(3) as a target in the prevention of neointimal hyperplasia. J Vasc Surg 45 Suppl A, A33-8. 175 279. Sakuma, T., Sari, I., Goodman, C.N., Lindner, J.R., Klibanov, A.L., Kaul, S. (2005) Simultaneous integrin alphavbeta3 and glycoprotein IIb/IIIa inhibition causes reduction in infarct size in a model of acute coronary thrombosis and primary angioplasty. Cardiovasc Res 66, 552-61. 280. Mitjans, F., Meyer, T., Fittschen, C., Goodman, S., Jonczyk, A., Marshall, J.F., Reyes, G., Piulats, J. (2000) In vivo therapy of malignant melanoma by means of antagonists of alphav integrins. Int J Cancer 87, 716-23. 281. Bewick, M.A., Lafrenie, R.M. (2006) Adhesion dependent signalling in the tumor microenvironment: the future of drug targetting. Curr Pharm Des 12, 2833-48. 282. Wilkinson-Berka, J.L., Jones, D., Taylor, G., Jaworski, K., Kelly, D.J., Ludbrook, S.B., Willette, R.N., Kumar, S., Gilbert, R.E. (2006) SB-267268, a nonpeptidic antagonist of alpha(v)beta3 and alpha(v)beta5 integrins, reduces angiogenesis and VEGF expression in a mouse model of retinopathy of prematurity. Invest Ophthalmol Vis Sci 47, 1600-5. 283. Maglott, A., Bartik, P., Cosgun, S., Klotz, P., Ronde, P., Fuhrmann, G., Takeda, K., Martin, S., Dontenwill, M. (2006) The small alpha5beta1 integrin antagonist, SJ749, reduces proliferation and clonogenicity of human astrocytoma cells. Cancer Res 66, 6002-7. 284. Kim, B.C., van Gelder, H., Kim, T.A., Lee, H.J., Baik, K.G., Chun, H.H., Lee, D.A., Choi, K.S., Kim, S.J. (2004) Activin receptor-like kinase-7 induces apoptosis through activation of MAPKs in a Smad3-dependent mechanism in hepatoma cells. J Biol Chem 279, 28458-65. 285. Xu, G., Zhong, Y., Munir, S., Yang, B.B., Tsang, B.K., Peng, C. (2004) Nodal induces apoptosis and inhibits proliferation in human epithelial ovarian cancer cells via activin receptor-like kinase 7. J Clin Endocrinol Metab 89, 5523-34. 286. Xu, G., Zhou, H., Wang, Q., Auersperg, N., Peng, C. (2006) Activin receptor-like kinase induces apoptosis through up-regulation of Bax and down-regulation of Xiap in normal and malignant ovarian epithelial cell lines. Mol Cancer Res 4, 235-46. 287. Avantaggiato, V., Dathan, N.A., Grieco, M., Fabien, N., Lazzaro, D., Fusco, A., Simeone, A., Santoro, M. (1994) Developmental expression of the RET protooncogene. Cell Growth Differ 5, 305-11. 288. Myers, S.M., Mulligan, L.M. (2004) The RET receptor is linked to stress response pathways. Cancer Res 64, 4453-63. Mosser, D.D., Caron, A.W., Bourget, L., Denis-Larose, C., Massie, B. (1997) Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Biol 17, 5317-27. 289. 176 290. Calderwood, S.K., Theriault, J.R., Gong, J. (2005) Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity. Eur J Immunol 35, 2518-27. 291. Pasquale, E.B. (2008) Eph-ephrin bidirectional signaling in physiology and disease. Cell 133, 38-52. 292. Pasquale, E.B. (2005) Eph receptor signalling casts a wide net on cell behaviour. Nat Rev Mol Cell Biol 6, 462-75. 293. Chiu, S.T., Chang, K.J., Ting, C.H., Shen, H.C., Li, H., Hsieh, F.J. (2009) Overexpression of EphB3 enhances cell-cell contacts and suppresses tumor growth in HT-29 human colon cancer cells. Carcinogenesis 30, 1475-86. 294. Noren, N.K., Foos, G., Hauser, C.A., Pasquale, E.B. (2006) The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway. Nat Cell Biol 8, 815-25. 295. Noblitt, L.W., Bangari, D.S., Shukla, S., Knapp, D.W., Mohammed, S., Kinch, M.S., Mittal, S.K. (2004) Decreased tumorigenic potential of EphA2overexpressing breast cancer cells following treatment with adenoviral vectors that express EphrinA1. Cancer Gene Ther 11, 757-66. 296. Kumar, S.R., Masood, R., Spannuth, W.A., Singh, J., Scehnet, J., Kleiber, G., Jennings, N., Deavers, M., Krasnoperov, V., Dubeau, L., Weaver, F.A., Sood, A.K., Gill, P.S. (2007) The receptor tyrosine kinase EphB4 is overexpressed in ovarian cancer, provides survival signals and predicts poor outcome. Br J Cancer 96, 1083-91. 297. Carles-Kinch, K., Kilpatrick, K.E., Stewart, J.C., Kinch, M.S. (2002) Antibody targeting of the EphA2 tyrosine kinase inhibits malignant cell behavior. Cancer Res 62, 2840-7. 298. Duxbury, M.S., Ito, H., Zinner, M.J., Ashley, S.W., Whang, E.E. (2004) EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma. Oncogene 23, 1448-56. 299. Yu, J., Bulk, E., Ji, P., Hascher, A., Tang, M., Metzger, R., Marra, A., Serve, H., Berdel, W.E., Wiewroth, R., Koschmieder, S., Muller-Tidow, C. (2010) The EPHB6 receptor tyrosine kinase is a metastasis suppressor that is frequently silenced by promoter DNA hypermethylation in non-small cell lung cancer. Clin Cancer Res 16, 2275-83. Truitt, L., Freywald, T., DeCoteau, J., Sharfe, N., Freywald, A. (2010) The EphB6 receptor cooperates with c-Cbl to regulate the behavior of breast cancer cells. Cancer Res 70, 1141-53. 300. 177 301. Cortina, C., Palomo-Ponce, S., Iglesias, M., Fernandez-Masip, J.L., Vivancos, A., Whissell, G., Huma, M., Peiro, N., Gallego, L., Jonkheer, S., Davy, A., Lloreta, J., Sancho, E., Batlle, E. (2007) EphB-ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells. Nat Genet 39, 1376-83. 302. Oki, M., Yamamoto, H., Taniguchi, H., Adachi, Y., Imai, K., Shinomura, Y. (2008) Overexpression of the receptor tyrosine kinase EphA4 in human gastric cancers. World J Gastroenterol 14, 5650-6. 303. Oshima, T., Akaike, M., Yoshihara, K., Shiozawa, M., Yamamoto, N., Sato, T., Akihito, N., Nagano, Y., Fujii, S., Kunisaki, C., Wada, N., Rino, Y., Tanaka, K., Masuda, M., Imada, T. (2008) Overexpression of EphA4 gene and reduced expression of EphB2 gene correlates with liver metastasis in colorectal cancer. Int J Oncol 33, 573-7. 304. Appelmann, I., Liersch, R., Kessler, T., Mesters, R.M., Berdel, W.E. (2010) Angiogenesis inhibition in cancer therapy: platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) and their receptors: biological functions and role in malignancy. Recent Results Cancer Res 180, 51-81. 305. D'Andrea, L.D., Del Gatto, A., De Rosa, L., Romanelli, A., Pedone, C. (2009) Peptides targeting angiogenesis related growth factor receptors. Curr Pharm Des 15, 2414-29. 306. Wang, B., Xiao, Y., Ding, B.B., Zhang, N., Yuan, X., Gui, L., Qian, K.X., Duan, S., Chen, Z., Rao, Y., Geng, J.G. (2003) Induction of tumor angiogenesis by SlitRobo signaling and inhibition of cancer growth by blocking Robo activity. Cancer Cell 4, 19-29. 307. Tole, S., Mukovozov, I.M., Huang, Y.W., Magalhaes, M.A., Yan, M., Crow, M.R., Liu, G.Y., Sun, C.X., Durocher, Y., Glogauer, M., Robinson, L.A. (2009) The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils. J Leukoc Biol 86, 1403-15. 308. Aruga, J., Mikoshiba, K. (2003) Identification and characterization of Slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth. Mol Cell Neurosci 24, 117-29. 309. Aruga, J. (2003) Slitrk6 expression profile in the mouse embryo and its relationship to that of Nlrr3. Gene Expr Patterns 3, 727-33. 310. Reissmann, E., Jornvall, H., Blokzijl, A., Andersson, O., Chang, C., Minchiotti, G., Persico, M.G., Ibanez, C.F., Brivanlou, A.H. (2001) The orphan receptor ACVR1C and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. Genes Dev 15, 2010-22. 178 311. Tsuchida, K., Nakatani, M., Yamakawa, N., Hashimoto, O., Hasegawa, Y., Sugino, H. (2004) Activin isoforms signal through type I receptor serine/threonine kinase ACVR1C. Mol Cell Endocrinol 220, 59-65. 312. Roberts, H.J., Hu, S., Qiu, Q., Leung, P.C., Caniggia, I., Gruslin, A., Tsang, B., Peng, C. (2003) Identification of novel isoforms of activin receptor-like kinase (ACVR1C) generated by alternative splicing and expression of ACVR1C and its ligand, Nodal, in human placenta. Biol Reprod 68, 1719-26. 313. Wang, H., Tsang, B.K. (2007) Nodal signalling and apoptosis. Reproduction 133, 847-53. 314. Kishimoto, H., Nakagawa, K., Watanabe, T., Kitagawa, D., Momose, H., Seo, J., Nishitai, G., Shimizu, N., Ohata, S., Tanemura, S., Asaka, S., Goto, T., Fukushi, H., Yoshida, H., Suzuki, A., Sasaki, T., Wada, T., Penninger, J.M., Nishina, H., Katada, T. (2003) Different properties of SEK1 and MKK7 in dual phosphorylation of stress-induced activated protein kinase SAPK/JNK in embryonic stem cells. J Biol Chem 278, 16595-601. 315. Zhang, N., Kumar, M., Xu, G., Ju, W., Yoon, T., Xu, E., Huang, X., Gaisano, H., Peng, C., Wang, Q. (2006) Activin receptor-like kinase induces apoptosis of pancreatic beta cells and beta cell lines. Diabetologia 49, 506-18. 316. Deveraux, Q.L., Takahashi, R., Salvesen, G.S., Reed, J.C. (1997) X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388, 300-4. 317. Deveraux, Q.L., Roy, N., Stennicke, H.R., Van Arsdale, T., Zhou, Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S., Reed, J.C. (1998) IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 17, 2215-23. 318. Wang, H., Jiang, J.Y., Zhu, C., Peng, C., Tsang, B.K. (2006) Role and regulation of nodal/activin receptor-like kinase signaling pathway in the control of ovarian follicular atresia. Mol Endocrinol 20, 2469-82. 319. Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P., Wang, X. (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-32. 179 [...]... Lactobacillus species are part of the commensal microflora in animals and humans and are commonly used as probiotics There are reports in the literature that lactobacilli have very promising anti- tumor effects both in animals as well as in clinical studies Administration of Lactobacillus reduced tumor growth, prevented recurrence of cancer and improved survival rates The anti- tumor effect of lactobacilli can... IIIB cervival cancer [75] Regular intake of lactobacilli can also reduce the risk of developing colorectal and bladder cancer [76, 77] 1.6.2.1 Immunologically mediated anti- tumor effect One explanation for the tumor suppressive effect of lactobacilli may be through immunomodulation of the host Intravesical instillations of lactobacilli recruited macrophages and neutrophils to the bladder mucosa [69,... reducing tumor growth in C3H mice [69] Aside from anti- tumor properties, lactobacilli also showed potential anti- metastatic properties as demonstrated by inhibition of lung and lymph node metastases by intrapleural and/ or intraveneous administration of L casei YIT9018 (LC 9018) [70, 71] in the tumor bearing mice Oral consumption of L casei strain Shirota (LcS) was found to suppress recurrence of bladder... cells like macrophages and neutrophils NK cells are able to provide surveillance against development of malignancies [13] and they can attack transformed tumor cells via NKG2D mediated cytotoxicity [14] CD8+ and NK cells are both cytotoxic lymphocytes and they induced apoptosis in cells similarly, by producing lytic molecules like perforin and granzymes and/ or trigger FasFas ligand (FasL) or TNF-related... LGG in media and the enhancement of cytotoxicity with increased glucose concentration and addition of amberlite……………… 96 3.15 Cytotoxicity of lactate on MGH cells………………………………………… 97 3.16 Purification of LGG supernatant using HPLC………………………………… 101 3.17 Comparison of GC-TOFMS chromatograms of LGG supernatant and control media…………………………………………………………………… 103 3.18 Effect of chemical inhibition of endocytotic... knowledge of tumor immunology and the immune system, other forms of cancer immunotherapy using monoclonal antibodies, cytokines (e.g interferon, interleukin-2), biological agents [e.g Mycobacterium bovis, Bacillus Calmette-Guérin (BCG)] and cancer vaccines have been developed Monoclonal antibodies used for cancer immunotherapy are raised against tumor antigens Once bound, the foreign cells are destroyed... with an anti- macrophage agent, carrageenan, and also in T-cell deficient athymic nude mice [66] These results suggest that the anti- tumor activity of lactobacilli may be macrophage and T cell dependent Recruitment and activation of immune cells is coupled with the production of cytokines that also contributes to the anti- tumor effect Matsuzaki et al showed that intrapleural injection of LcS into tumor. .. suppression in tumor bearing animals given BCG or Lactobacillus [29, 88] The former has potent anti- tumor and anti- metastatic effects against tumors by the stimulation of cytotoxic CD8+ T cells and natural killer cells while the latter activates macrophages to become cytotoxic to tumor cells [90] TNF is known to induce tumor cell apoptosis in vitro and enhance tumoricidal activity of macrophages [91,... host” They are part of the commensal microflora in animals and humans and they can be found in the oral, genital and gastrointestinal tracts They have also been conferred the GRAS (generally recognized as safe) status by the US FDA Many Lactobacillus species are associated with food production because of their nutritional benefits, ability to enhance flavor and preservation of food by production of lactic... acid Lactobacilli have also been associated with alleviating a host of diseases, ranging from a wide variety of gastrointestinal problems to allergies and the prevention of cancer More importantly, infection cases associated with use of lactobacilli are very rare and reported mostly in immuno- compromised individuals [43] As such, research on lactobacilli has much potential in both the food industry and . ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. ANTI-TUMOR PROPERTIES OF LACTOBACILLI ARE MEDIATED BY IMMUNO-MODULATION AND DIRECT CYTOTOXICITY CAI SHIRONG NATIONAL UNIVERSITY OF SINGAPORE. duration and dose of lactobacilli exposure to DCs and neutrophils affect Th1 polarization of T cells. The different strains of lactobacilli showed differential immunostimulatory potential and their