Experimental design.

<p>(A) During the acquisition phase in conditioning <i>experiment A</i>, subjects of the experimental group received four times cyclosporin A (CsA) as an US together with a green-colored, novel tasting drink, the CS. During evocation, subjects were re-exposed to the drink four times but received identically looking placebo capsules instead of CsA. The control group was treated in an identical way but received placebo capsules throughout the study. Blood was drawn on the first day (baseline), on day 3 to determine the CsA-effect, on day 8 to analyze possible residual drug effects and on day 10 in order to determine the conditioned effect on IL-2 production <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049477#pone.0049477-Wirth1" target="_blank">[19]</a>. (B) During the acquisition phase in conditioning <i>experiment B</i> subjects were identically treated as in <i>experiment A</i>. However, during evocation, subjects were re-exposed to the drink and the placebo capsules only once. Blood was drawn on the first day (baseline), on day 3 to determine the CsA-effect, on day 8 to analyze possible residual drug effects and on day 10 in order to determine the conditioned effect on IL-2 production. (C) In <i>experiment C</i>, subjects were told to have a probability of either 25%, 50%, 75% or 100% of receiving CsA to manipulate subjects’ expectation of receiving an active drug. Capsules were given at four time points on 3 consecutive days. Blood was drawn on the first day for baseline measurement and on day 3 to determine the potential effect of expectation on IL-2 production of anti-CD3 stimulated PBMC.</p

Placebo Effects on the Immune Response in Humans: The Role of Learning and Expectation

<div><p>Placebo responses are primarily mediated via two neuropsychological mechanisms: patients’ expectation towards the benefit of a treatment and associative learning processes. Immune functions, like other physiological responses, can be modulated through behavioral conditioning. However, it is unknown whether learned immune responses are affected by the number of re-expositions to the conditioned stimulus (CS) during evocation. Moreover, it is unclear whether immune functions can also be modulated through mere verbally induced expectation. In the experiments reported here, we investigated in healthy male volunteers with an established model of learned immunosuppression whether a single re-exposition to the CS is able to induce a behaviorally conditioned immunosuppression. This conditioned immunosuppression is reflected through a significantly decreased interleukin (IL)-2 production by anti-CD3 stimulated peripheral blood mononuclear cells. Our data revealed that in contrast to four CS re-expositions (control group n = 15; experimental group n = 17), a single CS re-exposition was not sufficient to significantly suppress IL-2 production (control group n = 9, experimental group n = 10). Furthermore, we could demonstrate that mere expectation of taking an immunosuppressant did not cause an immunosuppressive response (n = 8–9 per expectation condition). Together, these findings extend our knowledge about the kinetics and mechanisms of placebo-induced immunosuppression and provide therewith information for designing conditioning protocols, which might be employed as a supportive therapy in clinical settings.</p> </div

Experimental design.

<p>During acquisition, patients in the conditioned group received desloratadine (US) in combination with the CS. During evocation, the drug was replaced by placebo pills. Sham-conditioned patients received the CS together with placebo pills throughout the study. During 3 subsequent days during acquisition (2–4) and evocation (17–19) patients were instructed to intake the pills together with the drink (CS) at home.</p

Expectation induced cytokine response.

<p>(A) Expectation did not induce a significant reduction in IL-2 production in any of the four expectation groups differing in the probability of receiving CsA. IL-2 (pg/ml) concentration in supernatants of anti-CD3 stimulated PBMC was analyzed before and after placebo pill intake (25% n = 9, 50% n = 8, 75% n = 8, 100% n = 8). Data are shown as mean ± SEM. (B) Expectation did not reduce the percentage of IL-2 producing CD4⁺T cells. IL-2 producing CD4⁺T cells were analyzed as percent of total CD4⁺T cells before and after placebo pill intake. Data are shown as mean ± SEM.</p

Behavioral conditioning induced cytokine response.

<p>Behaviorally conditioned suppression of IL-2 release was observed after four CS re-expositions (control group n = 15, experimental group n = 17) (Fig. 2A). In contrast, a single CS re-exposition did not induce a significant inhibition in IL-2 production (control group n = 9, experimental group n = 10) (Fig. 2B). Data are expressed as percental changes from baseline. Bars represent mean ± SEM; ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Sociodemographic and psychological characteristics.

<p>BDI = Becks Depression Inventory; STAI X2 = Trait anxiety form of the State-trait anxiety inventory.</p>*<p>Results of Chi² Test or univariate ANOVA.</p

Conditioned and sham-conditioned patients show reduced allergic reactions during evocation.

<p><b>A</b> After skin prick test wheal size (mm) was analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in the natural history group. <b>B</b> Symptom scores after nasal provocation were analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in natural history group. Data are presented as mean ±SEM. *p<0.05 **p<0.01 ***p<0.001, comparison against natural history group.</p

The distribution of Noradrenaline response to the Trier Social Stress Test among individuals.

<p>The distribution of Noradrenaline response to the Trier Social Stress Test among individuals.</p

(A) Experimental design. (B) Noradrenaline and (C) salivary alpha-amylase response to the Trier Social Stress Test.

<p>Results are presented as mean ± SEM.</p

Noradrenaline (A), Salivary alpha-amylase (B), Plasma cortisol (C), Salivary cortisol (D), Nervousness (E), State anxiety (F) and Heart rate (G) in response to the Trier Social Stress Test for 2 groups with high-NA and low-NA response.

<p>Results are presented as mean ± SEM.</p