Quantitative evaluation of interleukin-12 p40 gene expression in peripheral blood mononuclear cells.
The heterodimeric cytokine IL-12 (composed of a p35 and a p40 subunit) is produced primarily by monocytes, macrophages and B cells. In vitro and in vivo experiments have demonstrated the crucial role of IL-12 in initiating and establishing both innate immunity and T cell-mediated resistance to intracellular pathogens, including Leishmania donovani, Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis. Assessment of cytokine expression has thus become crucial to understand host responses to infections. In this study, by using the reverse transcriptase-real time PCR we developed a highly specific and sensitive assay to quantitatively evaluate IL-12p40 mRNA transcription levels in peripheral blood mononuclear cells (PBMCs) stimulated with PHA vs. unstimulated cells. We also used the ELISA to evaluate bioactive IL-12 release in culture supernatants. We provide evidence that IL-12 p40 mRNA levels were significantly up-regulated in PHA-activated PBMCs. These results were correlated with data of IL-12 levels obtained by ELISA.
Conte
Enrico
E
Department of Medical Specialties and Internal Medicine, Section of Infectious Diseases, University of Catania, Catania, Italy. econte@unict.it
Nigro
Luciano
L
Fagone
Evelina
E
Drago
Francesco
F
Cacopardo
Bruno
B
D000293:Adolescent; D000328:Adult; D000368:Aged; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D053762:Interleukin-12 Subunit p40 / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008875:Middle Aged; D010835:Phytohemagglutinins / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D053762:Interleukin-12 Subunit p40; D010835:Phytohemagglutinins; D012333:RNA, Messenger