This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Deering, R. P. Articles by Orange, J. S. Search for Related Content PubMed PubMed Citation Articles by Deering, R. P. Articles by Orange, J. S.

 Previous Article  |  Next Article 

Clinical and Vaccine Immunology, January 2006, p. 68-76, Vol. 13, No. 1
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.1.68-76.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Development of a Clinical Assay To Evaluate Toll-Like Receptor Function

Division of Immunology, Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received 6 July 2005/ Returned for modification 25 August 2005/ Accepted 21 October 2005

Toll-like receptors (TLRS) recognize pathogen-associated molecular patterns to enable innate immune responses. A number of genetic defects influencing the function of these receptors have been identified and are associated with recurrent and/or severe infection. Our goal was to develop a reproducible assay of TLR function in order to evaluate patients with recurrent infection who would be suspected of having a genetic defect affecting TLR signaling. We chose to study peripheral blood mononuclear cells (PBMCS) to avoid potential influences of soluble factors contained in whole blood, and we utilized ligands for TLRS 1/2, 2/6, 3, 4, 5, 6, 7, and 9. Tumor necrosis factor (TNF) production in PBMC supernatants was measured by an enzyme-linked immunosorbent assay after TLR ligand stimulation and was dependent on gene transcription and NF-B activation. Some variables affecting the assay were assessed, including the effects of: blood anticoagulant, serum-containing media, incubation time, ligand storage, blood storage time, and cell cryopreservation. By using optimized assay conditions, effective concentrations of individual ligands and mean responses to those ligands were established for healthy control donors. Finally, three patients with a mutation in the IKBKG gene, encoding the NF-B essential modulator (NEMO) protein, were evaluated as disease controls and were almost uniformly below the standard deviation of healthy donors for all ligands tested. Although a number of variables influence TLR ligand-induced TNF responses, this assay can be optimized for potential clinical use to screen patients with primary immunodeficiencies affecting TLR function.

This article has been cited by other articles:

• von Bernuth, H., Ku, C.-L., Rodriguez-Gallego, C., Zhang, S., Garty, B.-Z., Marodi, L., Chapel, H., Chrabieh, M., Miller, R. L., Picard, C., Puel, A., Casanova, J.-L. (2006). A Fast Procedure for the Detection of Defects in Toll-like Receptor Signaling. Pediatrics 118: 2498-2503 [Abstract] [Full Text]