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.

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.

HLA-DRB1*12 is associated with protection against complicated typhoid fever, independent of tumour necrosis factor alpha.

We investigated whether HLA-DR2 or -DR12 alleles in 63 Javanese patients with complicated or non-complicated typhoid fever were associated with severity of disease. No association was observed between HLA type and susceptibility to disease. However, in patients we did find a negative association of DR12 (DRB1*12021) with complicated typhoid fever (P = 0.05; OR = 0.3; 95% CI: 0.1-1.0). No effect of DR2 (DRB1*1502) on outcome (P = 0.46; OR = 1.5; 95% CI: 0.5-4.5) was demonstrated. The odds ratio for DR12 remained unchanged after adjusting for DR2. Tumour necrosis factor alpha (TNF-alpha) production capacity in lipopolysaccharide (LPS)-stimulated whole blood culture, as measured by non-equilibrium radioimmunoassay, was significantly lower in complicated than in non-complicated cases (P = 0.02), confirming previous data. No significant correlation of either DR12 (P = 0.47) or DR2 (P = 0.89) was found with TNF-alpha production capacity. Apparently, protection against complications by DR12 is attributable to other mechanisms.

Patterns of proinflammatory cytokines and inhibitors during typhoid fever.

Cytokines and inhibitors in plasma were measured in 44 patients with typhoid fever. Ex vivo production of the cytokines was analyzed in a whole blood culture system with and without lipopolysaccharide (LPS). Acute phase circulating concentrations of cytokines (+/- SD) were as follows: interleukin (IL)-1 beta, < 140 pg/mL; tumor necrosis factor-alpha (TNF alpha), 130 +/- 50 pg/mL; IL-6, 96 +/- 131 pg/mL; and IL-8, 278 +/- 293 pg/mL. Circulating inhibitors were elevated in the acute phase: IL-1 receptor antagonist (IL-1RA) was 2304 +/- 1427 pg/mL and soluble TNF receptors 55 and 75 were 4973 +/- 2644 pg/mL and 22,865 +/- 15,143 pg/mL, respectively. LPS-stimulated production of cytokines was lower during the acute phase than during convalescence (mean values: IL-1 beta, 2547 vs. 6576 pg/mL; TNF alpha, 2609 vs. 6338 pg/mL; IL-6, 2416 vs. 7713 pg/mL). LPS-stimulated production of IL-1RA was higher in the acute than during the convalescent phase (5608 vs. 3977 pg/mL). Inhibited production of cytokines during the acute phase may be due to a switch from a proinflammatory to an antiinflammatory mode.

Phospholipase A2 is a circulating mediator in typhoid fever.

Circulating proinflammatory mediators have not been found in studies on typhoid fever, although the patients suffer from a systemic disease with characteristic protracted fever. The 14-kDa group II extracellular phospholipase A2 (PLA2) is induced by interleukin-1 and tumor necrosis factor and may mediate some of the effects of these cytokines. Circulating PLA2 concentrations were measured in 12 typhoid fever patients on various days after admission and after recovery. On admission, mean concentrations of PLA2 were elevated (1444 +/- 1560 ng/mL) and decreased gradually and significantly to day 14 (55 +/- 48 ng/mL). patients with complicated disease had significantly higher PLA2 levels on admission. PLA2 was not produced in a lipopolysaccharide-stimulated whole blood culture, indicating that PLA2 originates from other types of cells. These data indicate that PLA2 may be a mediator of disease in protracted inflammatory diseases such as thyroid fever.