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Departments of
1
Experimental and Clinical Pharmacology and Toxicology and
2
Biochemistry, Emil-Fischer-Center, University of Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany.
aAddress correspondence to this author at: Institut für Pharmakologie und Toxikologie, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany. Fax 49-9131-206119; e-mail pahl{at}pharmakologie.uni-erlangen.de.
Background: Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens.
Methods: We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-
from these monocytes were monitored by real-time reverse transcription-PCR.
Results: By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4 Asp299Gly and 6.0% for the TLR4 Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC50s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype.
Conclusions: Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.
The following articles in journals at HighWire Press have cited this article:
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  E. Shibuya, A. Meguro, M. Ota, K. Kashiwagi, F. Mabuchi, H. Iijima, K. Kawase, T. Yamamoto, M. Nakamura, A. Negi, et al. Association of Toll-like Receptor 4 Gene Polymorphisms with Normal Tension Glaucoma Invest. Ophthalmol. Vis. Sci., October 1, 2008; 49(10): 4453 - 4457. [Abstract] [Full Text] [PDF]
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 P. Rallabhandi, A. Awomoyi, K. E. Thomas, A. Phalipon, Y. Fujimoto, K. Fukase, S. Kusumoto, N. Qureshi, M. B. Sztein, and S. N. Vogel Differential Activation of Human TLR4 by Escherichia coli and Shigella flexneri 2a Lipopolysaccharide: Combined Effects of Lipid A Acylation State and TLR4 Polymorphisms on Signaling J. Immunol., January 15, 2008; 180(2): 1139 - 1147. [Abstract] [Full Text] [PDF]
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A. A. Awomoyi, P. Rallabhandi, T. I. Pollin, E. Lorenz, M. B. Sztein, M. S. Boukhvalova, V. G. Hemming, J. C. G. Blanco, and S. N. Vogel Association of TLR4 Polymorphisms with Symptomatic Respiratory Syncytial Virus Infection in High-Risk Infants and Young Children J. Immunol., September 1, 2007; 179(5): 3171 - 3177. [Abstract] [Full Text] [PDF]
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P. Rallabhandi, J. Bell, M. S. Boukhvalova, A. Medvedev, E. Lorenz, M. Arditi, V. G. Hemming, J. C. G. Blanco, D. M. Segal, and S. N. Vogel Analysis of TLR4 Polymorphic Variants: New Insights into TLR4/MD-2/CD14 Stoichiometry, Structure, and Signaling J. Immunol., July 1, 2006; 177(1): 322 - 332. [Abstract] [Full Text] [PDF]
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 C. Marsik, B. Jilma, C. Joukhadar, C. Mannhalter, O. Wagner, and G. Endler The Toll-Like Receptor 4 Asp299Gly and Thr399Ile Polymorphisms Influence the Late Inflammatory Response in Human Endotoxemia Clin. Chem., November 1, 2005; 51(11): 2178 - 2180. [Full Text] [PDF]
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S. Grannemann, O. Landt, S. Breuer, and B. Blomeke LightTyper Assay with Locked-Nucleic-Acid-Modified Oligomers for Genotyping of the Toll-Like Receptor 4 Polymorphisms A896G and C1196T Clin. Chem., August 1, 2005; 51(8): 1523 - 1525. [Full Text] [PDF]
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 R. S. Munford Invited review: Detoxifying endotoxin: time, place and person Innate Immunity, April 1, 2005; 11(2): 69 - 84. [Abstract] [PDF]
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 M Pierik, G De Hertogh, S Vermeire, G Van Assche, P Van Eyken, S Joossens, G Claessens, R Vlietinck, P Rutgeerts, and K Geboes Epithelioid granulomas, pattern recognition receptors, and phenotypes of Crohn's disease Gut, February 1, 2005; 54(2): 223 - 227. [Abstract] [Full Text] [PDF]
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 S. L. Zheng, K. Augustsson-Balter, B. Chang, M. Hedelin, L. Li, H.-O. Adami, J. Bensen, G. Li, J.-E. Johnasson, A. R. Turner, et al. Sequence Variants of Toll-Like Receptor 4 Are Associated with Prostate Cancer Risk: Results from the CAncer Prostate in Sweden Study Cancer Res., April 15, 2004; 64(8): 2918 - 2922. [Abstract] [Full Text] [PDF]
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 T. Illig, F. Bongardt, A. Schopfer, R. Holle, S. Muller, W. Rathmann, W. Koenig, C. Meisinger, H.-E. Wichmann, and H. Kolb The Endotoxin Receptor TLR4 Polymorphism Is Not Associated With Diabetes or Components of the Metabolic Syndrome Diabetes, November 1, 2003; 52(11): 2861 - 2864. [Full Text] [PDF]
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