Free serum or saliva cortisol was measured at the Institute of Clinical Chemistry, Hospital of the
University of Munich, Germany by an electrochemiluminescence immunoassay (Elecsys 2010; Roche, Mannheim, Germany). Using the same test set-up as described above, whole blood (n = 7) was incubated in the presence of antigens together with increasing concentrations of hydrocortisone (HC). For each of the antigen PLX4032 cost compositions, three different hydrocortisone concentrations (20, 40 and 60 μg/dl) were added and the assay was incubated at 37°C. After 48 h of incubation the supernatants were harvested as described. After basal blood-drawing, 100 mg hydrocortisone was injected intravenously (n = 7). Blood was collected 1 h and 24 h thereafter. At each time-point whole blood was drawn for serum cortisol measurement and incubation with the new in-vitro test. Supernatant was collected after 48 h of incubation. In the acute stress model of free fall during parabolic flight, pre- and post-flight
saliva was collected in Salivettes® (Sarstedt, Nümbrecht, Germany) for cortisol measurement, and blood was drawn for the in-vitro test at the same time-points. Parabolic flights were performed with an Airbus A300 ZeroG (Novespace, Paris, France). One parabolic flight manoeuvre results in approximately 22 s of free fall. In total, 31 parabolas were flown in 1 flight day. Normal distribution check details of sample data was tested using the Kolmogorov–Smirnov test. All normal distributed data were tested with a paired t-test. Concerning multiple comparisons, Bonferroni’s correction was applied. For repeated measurements within groups, repeated-measures analysis of variance (one-way RM-anova) was calculated followed by Fisher’s post-hoc least significant difference (LSD) test. Results were statistically significant if P < 0·05. Results are expressed as mean ± standard error of the
mean (s.e.m.) (spss version 15·0; SPSS, Inc., Chicago, IL, USA). The proinflammatory cytokine IL-2 showed a significant increase over time when challenged Pregnenolone with bacterial, viral and fungal antigens as well as with the positive control PWM and peaked after 24–48 h of incubation. IFN-γ concentrations doubled after 24 h with bacterial and viral antigen stimulation, but remained low following stimulation with fungal antigens. In contrast, TNF-α peaked earlier and significantly at 12 h after viral and 24 h after bacterial antigen challenges (see Table 1). The antigen-free negative control showed only minor cytokine release at all time-points, whereas the positive selective control with ConA and the overall control with PWM resulted in an appropriate cytokine release, with peak concentrations for IL-2 and IFN-γ at 48 h and for TNF-α at 12 h.