Alexandra Brinkhoff

Learned immunosuppressive placebo responses in renal transplant patients

Significance Akin to other physiological responses, immune functions can be modified in humans through associative conditioning procedures as part of a learned placebo response. However, it is unclear whether learned immune responses can be produced in patient populations already receiving an immunosuppressive regimen. In the present study, we demonstrate in renal transplant patients who were already receiving immunosuppressive treatment that learned immunosuppressive placebo responses increased efficacy of immunosuppressive medication. These data demonstrate that behavioral conditioning of drug responses may be a promising tool that could be used as a placebo-based dose-reduction strategy in an ongoing immunopharmacological regimen, the aim being to limit unwanted drug adverse effects and to improve treatment efficacy. Patients after organ transplantation or with chronic, inflammatory autoimmune diseases require lifelong treatment with immunosuppressive drugs, which have toxic adverse effects. Recent insight into the neurobiology of placebo responses shows that associative conditioning procedures can be employed as placebo-induced dose reduction strategies in an immunopharmacological regimen. However, it is unclear whether learned immune responses can be produced in patient populations already receiving an immunosuppressive regimen. Thus, 30 renal transplant patients underwent a taste-immune conditioning paradigm, in which immunosuppressive drugs (unconditioned stimulus) were paired with a gustatory stimulus [conditioned stimulus (CS)] during the learning phase. During evocation phase, after patients were reexposed to the CS, T cell proliferative capacity was significantly reduced in comparison with the baseline kinetics of T cell functions under routine drug intake (ƞp2 = 0.34). These data demonstrate, proof-of-concept, that learned immunosuppressive placebo responses can be used as a supportive, placebo-based, dose-reduction strategy to improve treatment efficacy in an ongoing immunopharmacological regimen.

Granzyme B producing B-cells in renal transplant patients.

OBJECTIVES A separate subset of Granzyme B (GrB) producing B-cells regulating T-cell mediated immunity has been identified. In the present study, we investigated the role of GrB+ B-cells in renal transplant patients (RTX). METHODS 12 healthy controls (HC) and 26 RTX patients were enrolled. In addition, 19 healthy volunteers treated with cyclosporine A (CsA) were enrolled. GrB+ B-cells were determined via flow cytometry. RESULTS RTX Patients showed a diminished fraction of GrB+ B-cells as compared to HC. CsA treatment of healthy volunteers had no impact on the development of GrB+ B-cells. RTX patients with a history of allograft rejection showed an increased frequency of GrB+ B-cells. RTX patients with at least one episode of CMV viremia tended to have lower GrB+ B-cells as compared to patients without viremic episodes. CONCLUSION We demonstrate that treatment with CsA does not impair the development of GrB+ B-cells. GrB+ B-cells may have a dual role in renal transplantation as regulatory cells to maintain allospecific tolerance and as effector cells enhancing viral control.

Pro-inflammatory Th1 and Th17 cells are suppressed during human experimental endotoxemia whereas anti-inflammatory IL-10 producing T-cells are unaffected

Objective Sepsis is one of the leading causes of the deaths in hospitals. During sepsis, patients are exposed to endotoxemia, which may contribute to the dysregulation of the immune system frequently observed in sepsis. This dysregulation leads to impaired pro-inflammatory responses and may increase the risk for secondary infections in sepsis. The experimental human endotoxemia model is widely used as a model system to study the acute effects of endotoxemia. Under physiological circumstances, the immune system is tightly regulated. Effector T-cells exert pro-inflammatory function and are restrained by regulatory T-cells (Tregs), which modulate pro-inflammatory effector responses. Endotoxemia may induce inadequate Treg activity or render effector T-cells dysfunctional. It was the aim of the study to investigate effector T-cell and Treg responses in an experimental human endotoxemia model. Methods In a cross-over designed placebo-controlled study, 20 healthy male volunteers received an intravenous injection of either lipopolysaccharide (LPS) (0.8 ng/kg body weight) or a placebo (saline 0.9%). CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and intracellular cytokine profiles were measured with flow cytometry at baseline and at repeated points after LPS/placebo injection. Complete blood cell counts were obtained with an automated hematology analyzer and cytokines were quantified by ELISA. Results Circulating neutrophils were significantly increased 2 h after LPS injection (p < 0.001) while absolute number of CD3+ T-cells, CD4+ T-cells, and CD8+ T-cells decreased (p < 0.001). Effector T-helper-cells (THs) showed a significant—but transient—decrease of pro-inflammatory IFNγ, interleukin (IL)-2, TNFα, and IL-17A production after LPS injection (p < 0.001). In contrast, the frequency of Treg and the capacity to produce IL-10 were unchanged (p = 0.21). Conclusion Effector THs fail to produce pro-inflammatory Th1-/Th17-associated cytokines after LPS challenge. In contrast, IL-10 production by Treg is not affected. Thus, endotoxemia-induced suppression of pro-inflammatory THs might be considered as a contributing factor to immunoparalysis in sepsis.

Does Human Experimental Endotoxemia Impact Negative Cognitions Related to the Self

A role of inflammatory processes in the pathophysiology of depression is increasingly recognized. Experimental endotoxemia offers an established model to induce transient systemic inflammation in healthy humans, and has been proposed as an experimental paradigm of depression. Indeed, different symptoms of depression can be observed during experimental endotoxemia, including negative mood or dysthymia as key symptoms of depression. Hopelessness and low self-esteem constitute common cognitive symptoms in depression, but have not been specifically assessed during endotoxemia. Thus, we pooled data from healthy volunteers who received low-dose endotoxin (i.e., 0.4 or 0.8 ng/kg lipopolysaccharide, LPS) or placebo in three randomized, controlled studies to investigate the effects of LPS on cognitive schemata related to depression. Validated questionnaires were used to assess self-esteem, hopelessness and the vulnerability factor intolerance of uncertainty after intravenous injection of LPS or placebo. Plasma tumor necrosis factor (TNF)-α and interleukin (IL)-6 were repeatedly assessed, along with self-reported mood. Because not all questionnaires were available from primary studies, data were analyzed in two separate data sets: In data set 1, self-esteem and intolerance of uncertainty were assessed in N = 87 healthy volunteers, who randomly received either 0.4 or 0.8 ng/kg LPS or placebo. In data set 2, hopelessness was measured in N = 59 volunteers who randomly received either LPS (0.8 ng/kg) or placebo. In both data sets, LPS-application led to significant increases in TNF-α and IL-6, reflecting systemic inflammation. Positive mood was significantly decreased in response to LPS, in line with inflammation-induced mood impairment. General self-esteem, intolerance of uncertainty and hopelessness did not differ between LPS- and placebo groups, suggesting that these negative cognitive schemata are not responsive to acute LPS-induced systemic inflammation. Interestingly, LPS-treated volunteers reported significantly lower body-related self-esteem, which was associated with increased TNF-α concentration. Thus, certain aspects of self-esteem related to physical attractiveness and sportiness were reduced. It is conceivable that this effect is primarily related to physical sickness symptoms and reduced physical ability during experimental endotoxemia. With respect to cognitive symptoms of depression, it is conceivable that LPS affects cognitive processes, but not negative cognitive schemata, which are rather based on learning and repeated experiences.

Impact of immune suppressive agents on the BK-Polyomavirus non coding control region

Background: Reactivation of the BK‐Polyomavirus (BKPyV) can cause a polyomavirus associated nephropathy in approx. 10% of kidney transplant recipients. In these cases, current therapy is based on the reduction of immunosuppression. Since BKPyV‐transcription is driven by the Non‐Coding‐Control‐Region (NCCR) we were interested whether NCCR‐activity is affected by immunosuppressive agents. Methods: Plasma samples from 45 BKPyV‐positive patients after renal transplantation were subjected to PCR‐analysis. NCCR‐amplicons were cloned into a plasmid that allows the quantification of early and late NCCR‐activity by tdTomato and eGFP expression, respectively. HEK293T‐cells were transfected with the reporter‐plasmids, treated with immunosuppressive agents, and subjected to FACS‐analysis. In addition, H727‐cells were infected with patient derived BKPyV, treated with mTOR‐inhibitors, and NCCR activity was analysed using qRT‐PCR. Results: While tacrolimus and cyclosporine‐A did not affect NCCR‐promoter‐activity, treatment with mTOR1‐inhibitor rapamycin resulted in the reduction of early, but not late‐NCCR‐promoter‐activity. Treatment with dual mTOR1/2 inhibitors (INK128 or pp242) led to significant inhibition of early, however, concomitantly enhanced late‐promoter‐activity. In BKPyV infected cells both rapamycin and INK128 reduced early expression, however, INK128 resulted in higher late‐mRNA levels when compared to rapamycin treatment. Conclusions: Our results demonstrate that mTOR1‐inhibitors are able to reduce early‐expression of wildtype and rearranged NCCRs, which might contribute to previously described inhibition of BKPyV‐replication. Dual mTOR1/2‐inhibitors, however, additionally might shift viral early into late‐expression promoting synthesis of viral structural proteins and particle production. HighlightsA dual fluorescence reporter was constructed to quantify the non‐coding‐control‐region (NCCR) driven BKPyV gene expression.NCCRs from patient material obtained after kidney transplantation were amplified and cloned into the reporter plasmid.The impact of immunosuppressive drugs on the bidirectional BKPyV‐promoter activity was analysed.mTOR1‐inhibitors were able to reduce early‐expression of archetypical and rearranged NCCRs.Dual mTOR1/2‐inhibitors, however, shifted viral early‐into late‐expression.