Abstract
Recently, various bacterial components have been suggested as initiating and modulating immune activation, thereby substantially affecting the complex and dynamic host/pathogen interactions. Herein, we present a valuable and simple methodology for determining the capacity of bacteria as well as defined bacterial structures to stimulate cellular effectors of the innate and cognate immune system. This assay format is based on the exposure of freshly prepared single-cell cultures of splenic cells derived from naive mice with the immunogen of interest. Herein, the determination of exclusive panels of cytokines by the ELISA, ELISpot, and FACS technology will serve as an indicator for the activation of defined arms of the immune system. An increased knowledge about microbial components with immunomodulatory properties will substantially contribute to a more detailed understanding of the dynamic interplay between the host and potential pathogens and, based on this knowledge, to the development of novel substances for the prevention and therapy of microbial infections.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Janeway, C. A. Jr. (1989) PatThe role of CD4 in T-cell activation: accessory molecule or co-receptor? Immunol. Today 10, 234–238.
Janeway, C. A. Jr. (1992) The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol. Today 13, 11–16.
Medzhitov, R. and Janeway, C. A. Jr. (2000) Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173, 89–97.
Gronowski, A. M., Hilbert, D. M., Sheehan, K. C., Garotta, G., and Schreiber, R. D. (1999) Baculovirus stimulates antiviral effects in mammalian cells J. Virol. 73, 9944–9951.
Hayashi, F., Smith, K. D., Ozinsky, A., et al. (2001) The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103.
Smith, K. D. and Ozinsky, A. (2002) Toll-like receptor-5 and the innate immune response to bacterial flagellin. Curr. Top. Microbiol. Immunol. 270, 93–108.
Borges, M. M., Campos-Neto, A., Sleath, P., et al. (2001) Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein. Infect. Immun. 69, 5270–5277.
Rock, F. L, Hardiman, G., Timans, J. C., Kastelein, R. A, and Bazan, J. F. (1998) A family of human receptors structurally related to Drosophila Toll. Proc. Natl. Acad. Sci. USA 95, 588–593.
Aderem, A. and Ulevitch, R. J. (2000) Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787.
Hartmann, G., Weiner, G. J., and Krieg, A. M. (1999) CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc. Natl. Acad. Sci. USA 96, 9305–9310.
Dundon, W. G., de Bernard, M., and Montecucco, C. (2001) Virulence factors of Helicobacter pylori. Int. J. Med. Microbiol. 290, 647–658.
Atherton, J. C. (1998) H. pylori virulence factors. Br. Med. Bull. 54, 105–120.
Bodger, K. and Crabtree, J. E. (1998) Helicobacter pylori and gastric inflammation. Br. Med. Bull. 54, 139–150.
Shimoyama, T. and Crabtree, J. E. (1998) Bacterial factors and immune pathogenesis in Helicobacter pylori infection. Gut 43(suppl 1), S2–5.
Zevering, Y., Jacob, L., and Meyer, T. F. (1999) Naturally acquired human immune responses against Helicobacter pylori and implications for vaccine development. Gut 45, 465.
Suerbaum, S. and Michetti, P. (2002) Helicobacter pylori infection. N. Engl. J. Med. 347, 1175–1186.
Wunderlich, M. and Glockshuber, R. (1993) In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA). J. Biol. Chem. 268, 24547–24550.
Deml, L., Wolf, H., and Wagner, R. (1999) High level expression of hepatitis B virus surface antigen in stably transfected Drosophila Schneider-2 cells J. Virol. Methods 79, 191–203.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. New York, Cold Spring Harbor Laboratory.
Deml, L. and Wagner, R. (1998) Stable transfected Drosophila Schneider-2 cells as a novel tool to produce recombinant antigens for diagnostic, therapeutic and preventive purposes, in U Reischl (ed), Methods in Molecular Medicine. Molecular Diagnosis of Infectious Diseases. Humana, Totowa, NJ, pp. 185–200.
White, B. A. (1993) Methods in Molecular Biology: PCR Protocols, Current Methods and Applications. Humana, Totowa, NJ.
Inoue, H. and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Deml, L., Aigner, M., Eckhardt, A., Decker, J., Lehn, N., Schneider-Brachert, W. (2004). Application of Single-Cell Cultures of Mouse Splenocytes as an Assay System to Analyze the Immunomodulatory Properties of Bacterial Components. In: Decler, J., Reischl, U. (eds) Molecular Diagnosis of Infectious Diseases. Methods in Molecular Medicine™, vol 94. Humana Press. https://doi.org/10.1385/1-59259-679-7:159
Download citation
DOI: https://doi.org/10.1385/1-59259-679-7:159
Publisher Name: Humana Press
Print ISBN: 978-1-58829-221-6
Online ISBN: 978-1-59259-679-9
eBook Packages: Springer Protocols