title: A semi-quantitative reverse transcriptase polymerase chain reaction method for measurement of MRNA for TNF-alpha and IL-1 beta in whole blood cultures: its application in typhoid fever and exentric exercise.
abstract: Whole blood cultures are used to study cytokine stimulation and release ex vivo. In the present study this method was compared with a more direct approach and a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess mRNA expression for IL-1 beta and tumour necrosis factor alpha (TNF-alpha) and mRNA in whole blood. Stimulation of whole blood from normal donors with lipopolysaccharide (LPS) at various time intervals showed a parallel rise of immunogenic IL-1 beta and TNF-alpha as well as a rise of mRNA expression for IL-1 beta and TNF-alpha with peak levels for IL-1 beta after 4-6 h stimulation and for mRNA TNF-alpha expression after 2 h stimulation. These methods were used to explore cytokine production during the course of typhoid fever and after a 5 km run. In both conditions circulating cytokine concentrations were not influenced, but the TNF-alpha and IL-1 beta mRNA gene expression in circulating whole blood cells was increased in patients with typhoid fever. The LPS-stimulated production of TNF-alpha and IL-1 beta was decreased in both but there was no change for the mRNA content in whole blood for these cytokines. These findings demonstrate that RT-PCR is an attractive method to study the gene expression of cytokines in whole blood, an increased TNF-alpha and IL-1 beta gene expression is present in typhoid fever, and that the LPS stimulated downregulation of cytokines in exercise and typhoid fever may be mediated by post-transcriptional processes.
authors.affiliation: Department of Medicine, University Hospital St. Radboud, Nijmegen, The Netherlands.
authors.forename: M G
authors.initials: MG
authors.lastname: Netea
authors.forename: J P
authors.initials: JP
authors.lastname: Drenth
authors.forename: N
authors.initials: N
authors.lastname: De Bont
authors.forename: A
authors.initials: A
authors.lastname: Hijmans
authors.forename: M
authors.initials: M
authors.lastname: Keuter
authors.forename: E
authors.initials: E
authors.lastname: Dharmana
authors.forename: P N
authors.initials: PN
authors.lastname: Demacker
authors.forename: J W
authors.initials: JW
authors.lastname: van der Meer
mesh_terms: D002478:Cells, Cultured; D015444:Exercise; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000097:blood*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*; D014435:Typhoid Fever / Q000097:blood*
chemical_list: D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha