Salivette sampling device

Saliva cortisol samples were collected directly before the first treatment session (T1), immediately after the first one (T2) and immediately after the second treatment session (T3), using Salivette® sampling devices (Sarstedt, Germany). On doing so the patients chewed on a cotton roll for the duration of about two minutes. The samples were immediately chilled on ice and centrifuged at 2400 x g for 2 minutes at 4°C. Then the samples were chilled on ice again and after the procedure of the presentation of the two treatment methods they were frozen in aliquots at -80°C until assayed. Cortisol levels (nmol/l) were determined using the commercially available immunoassay ELISA-Kit Parameter™ Cortisol Assay (R&D Systems, Inc., USA). According to the manufacturer the assay sensitivity for the kit was 0.111 nmol/l. Intra-assay and inter-assay coefficients of variation were 5.4% and 9.3%, respectively.

Salivary free cortisol is a reliable and valid measure of the biologically active fraction of cortisol (Vining et al., 1983 (link); Kirschbaum and Hellhammer, 1994 (link)). Salivary free cortisol gradually increases within about 10 min, and peaks around 10–30 min after stressor cessation (Foley and Kirschbaum, 2010 (link)). Saliva was collected using Salivette sampling devices (Sarstedt, Rommelsdorf, Germany). Participants were asked to keep the samples collected on the three morning prior to the test day in their freezers and return them to the laboratory on their test day. On that day, before and after the TSST, eight saliva samples were collected from each participant. The baseline was calculated by the mean of the first three samplings before the onset of the TSST (-20, -10, -2 min), the endocrine response to the psychosocial stressor was computed from the samples obtained after the onset of the TSST (+15, +30, +45, +60, +75 min), resulting in six repeated cortisol measures. Saliva samples were stored at -20°C and sent to the University of Trier (Germany) for biochemical analysis of free cortisol concentration. Cortisol was analyzed by a time-resolved immunoassay with fluorescence detection (Dressendörfer et al., 1992 (link)). Intra- and interassay coefficients of variation were below 9.0%.

Saliva was collected to assess free cortisol concentrations [36 (link)] as a marker of HPA axis activity. The samples were collected using Salivette sampling devices (Sarstedt, Nümbrecht, Germany). Participants collected saliva in the late evening (23:00h) and the next morning of the day following the university appointment. Saliva was collected upon awakening and thirty minutes later in order to assess the cortisol awakening response [37 (link)]. Free cortisol concentrations were analyzed without prior extraction using a commercial Chemoluminescence Immunoassay (CLIA; IBL International, Hamburg, Germany). All inter- and intra-assay variations were below 10%. A complete set of cortisol data for all 5 session could be obtained from 29 participants.
Immunological phenotyping was performed using blood samples as recently described [38 (link)]. Using the methods described in this publication we determined the absolute number of T cells, B cells, NK cells and monocytes in whole blood. Using flow cytometry we performed an analysis of NK and T lymphocyte subpopulations. We stimulated whole blood samples with Lipopolysaccharide (LPS) and measured the production of IL-6 and TNF-α. Finally, we determined the concentrations of cytokines in serum samples. The biological functions of the different immune parameters investigated in this study are detailed in a recent publication [38 (link)].