title: Transcriptional profiling of TLR-4/7/8-stimulated guinea pig splenocytes and whole blood by bDNA assay.
abstract: Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development. Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens.
authors.affiliation: Infectious Disease Research Institute, 1124 Columbia Street, Suite 400, Seattle, WA 98104, USA.
authors.forename: Lance K
authors.initials: LK
authors.lastname: Ching
authors.forename: Farah
authors.initials: F
authors.lastname: Mompoint
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Guderian
authors.forename: Alex
authors.initials: A
authors.lastname: Picone
authors.forename: Ian M
authors.initials: IM
authors.lastname: Orme
authors.forename: Rhea N
authors.initials: RN
authors.lastname: Coler
authors.forename: Steven G
authors.initials: SG
authors.lastname: Reed
authors.forename: Susan L
authors.initials: SL
authors.lastname: Baldwin
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D018932:Chemokine CCL2 / Q000235:genetics; D054360:Chemokine CXCL1 / Q000235:genetics; D018925:Chemokines / Q000235:genetics; D016207:Cytokines / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006168:Guinea Pigs; D007093:Imidazoles / Q000494:pharmacology; D000077271:Imiquimod; D018664:Interleukin-12 / Q000235:genetics; D053583:Interleukin-1beta / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000097:blood / Q000502:physiology; D051199:Toll-Like Receptor 7 / Q000819:agonists / Q000097:blood / Q000502:physiology; D051200:Toll-Like Receptor 8 / Q000819:agonists / Q000097:blood / Q000502:physiology; D051193:Toll-Like Receptors / Q000819:agonists / Q000097:blood / Q000502:physiology*; D059467:Transcriptome; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D000276:Adjuvants, Immunologic; D000634:Aminoquinolines; D018932:Chemokine CCL2; D054360:Chemokine CXCL1; D018925:Chemokines; D016207:Cytokines; D007093:Imidazoles; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D051197:Toll-Like Receptor 4; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; C546771:gardiquimod; D018664:Interleukin-12; D000077271:Imiquimod; C402365:resiquimod