Maja-Theresa Dieterlen

Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR-inhibitors in peripheral human blood.

Therapeutic drug monitoring (TDM) of immunosuppressive drugs after organ transplantation is based on measuring blood levels alone, which often results in under‐ or over‐immunosuppression. Previous studies have shown the potential of measuring pharmacodynamic drug effects for TDM, but assessment of biomarkers for individual drugs is still not clinical routine. Therefore, we validated a specific assay to measure the pharmacodynamic effects of mammalian target of rapamycin (mTOR)‐inhibitors on phosphorylated S6 ribosomal protein (p‐S6RP), a downstream target of mTOR.

Flow cytometry-based pharmacodynamic monitoring after organ transplantation.

Conventional therapeutic drug monitoring based on measuring immunosupressive drug concentrations in blood is important in the clinical management of immunosuppressive therapy in transplantation medicine. Since rejection or infection occurs at irregular drug concentrations immunosuppressive drug therapy is often empiric and prophylactic in nature. In addition, blood immunosuppressant levels are only indirect predictors of the pharmacologic effects on immune cells, because the genetic heterogeneity the immune systems of transplant recipients are not equally sensitive to drug effects. Therefore, therapeutic drug monitoring requires the application of reliable and effective methods to study the pharmacodynamic variability by direct measurements of drug effects on immune cell functions. Flow cytometry offers a multiplicity of quantitative analysis possibilities, from detection of phosphorylated molecules up to complex multicolor analysis of whole blood samples. A large spectrum of flow cytometry-based applications for pharmacodynamic monitoring is available and allows detection and analysis of diverse function of T cells and dendritic cell subsets. By combining several assays, it is possible to generate a broad picture of the immune status of every single transplanted recipient. Furthermore, it is even possible to differentiate between synergistic and antagonistic pharmacodynamic effects of immunosuppressive drug combination therapy in vitro and to predict the pharmacodynamic drug effects in transplanted recipients. Such a pharmacodynamic drug monitoring may offer the opportunity to complete conventional therapeutic drug monitoring and, therefore, to tailor immunosuppressive therapy more individually.

Dendritic Cells and Their Role in Cardiovascular Diseases: A View on Human Studies.

The antigen-presenting dendritic cells (DCs) are key to the immunological response, with different functions ascribed ranging from cellular immune activation to induction of tolerance. Such immunological responses are involved in the pathophysiological mechanisms of cardiovascular diseases, with DCs shown to play a role in atherosclerosis, hypertension, and heart failure and most notably following heart transplantation. A better understanding of the interplay between the immune system and cardiovascular diseases will therefore be critical for developing novel therapeutic treatments as well as innovative monitoring tools for disease progression. As such, the present review will provide an overview of DCs involvement in the pathophysiology of cardiovascular diseases and how targeting these cells may have beneficial effects for the prognosis of patients.

Extracorporeal photopheresis after heart transplantation

The addition of extracorporeal photopheresis (ECP) to a standard immunosuppressive drug therapy after heart transplantation in clinical studies has shown to be beneficial, for example, by reducing acute rejection, allograft vasculopathy or CMV infection. However, the protocols varied considerably, have a predetermined finite number of ECP treatments and adjuvant immunosuppressive regimens used in combination with ECP have differed significantly. Furthermore, there are scarce data to guide which patients should be treated with ECP and when or who would benefit further if ECP were to be continued long term to increase the safety by reducing immunosuppressive drug toxicities without losing efficacy. The knowledge of the tolerance-inducing effects of ECP-like upregulation of regulatory T cells and of dendritic cells may allow to develop a strategy to monitor immunomodulation effects of ECP to further identify ECP responders, the optimal individual ECP schedule and whether ECP therapy can replace or reduce immunosuppressive drug therapy.

Role of dendritic cells in the context of acute cellular rejection: Comparison between Tacrolimus- or Cyclosporine A-treated heart transplanted recipients.

Background: In the last years many studies have been designed to predict risk of acute rejection and to adapt the immunosuppressive therapy. The importance of dendritic cells (DCs) in the immune response, especially their role in tolerance is known. Thus, we investigated the influence of tacrolimus (TAC)‐based and of cyclosporine A (CsA)‐based immunosuppressive therapies on dendritic cells and the incidence of rejection in heart transplant recipients.

Effect of confounding factors on a phospho-flow assay of ribosomal S6 protein for therapeutic drug monitoring of the mTOR-inhibitor everolimus in heart transplanted patients

Abstract Context: Several assays of monitoring immune cell function have been developed to enhance therapeutic drug monitoring. Objective: An in vitro-validated whole-blood assay of phosphorylated ribosomal protein S6 (pS6RP) was evaluated for confounders to monitor the mTOR-inhibitor everolimus (ERL). Materials and methods: Whole blood samples from 87 heart transplant recipients were analyzed for pS6RP-expression in CD3-positive T-cells by phospho-flow analysis. Results: ERL blood concentration, laboratory parameters, co-medications, demographic and clinical data were reviewed. Conclusion: Evaluating the pS6RP-assay revealed that pS6RP is influenced by cyclosporine A (CsA) blood concentration, duration of ERL treatment, co-medication with thiazide diuretics and different metabolic parameters.

Clinical value of flow cytometric assessment of dendritic cell subsets in peripheral blood after solid organ transplantation

OVER the last years, the clinical value of flow cytometric monitoring of circulating dendritic cells (DCs) following solid organ transplantation attains more and more importance in transplantation medicine. The central role of DCs in transplantation immunology can be attributed to their function as professional antigenpresenting cells: firstly, DCs capture and present antigens and present these antigens to antigen-specific T cells. This process is enriched by bidirectional communication between DCs and T cells leading to T cell polarization into various effector and regulatory types (1). Secondly, within the scope of tolerance induction, DCs present self or foreign antigens to immature CD4CD8 thymocytes or induce anergy in peripheral T cells (2). This central role in the immunological processes makes DCs valuable to monitor immune function status after organ transplantation. Our study group recently published results in Cytometry B about the prognostic value of flow cytometric monitoring of the plasmacytoid DCs (pDCs) and myeloid DC (mDCs) subsets in peripheral whole blood of long-term heart transplanted patients (3). Patients were classified in two study groups regarding their calcineurin-inhibitor immunosuppressive drug, which is the cornerstone of an immunosuppressive drug therapy after transplantation. One group was treated with tacrolimus (TAC), and the other group with cyclosporine A (CsA)-based drug regimen, respectively. The TAC group had a higher expression of pDCs in peripheral blood compared to the CsA group. In general, TAC-treated patients had less biopsy proven acute rejection (BPAR) in their medical history compared to CsA-treated patients. Moreover, patients with higher values of pDCs had less events of BPAR as patients with low pDC values. Our results indicate that DC subsets could serve as useful biomarkers for detecting patients at risk of acute cellular rejection after heart transplantation. Interestingly, a small study in 20 heart transplanted (HTx) patients showed that expression of mDCs in peripheral blood was lower in patients with BPAR in contrast to patients without rejection. These results suggest that DC subsets may have the potential to serve as surrogate markers of rejection in the early period after HTx (4). In summary, this study results in organ transplanted patients revealed that analysis of circulating DCs enables (i) monitoring of an optimized immunosuppressive therapy following HTx (3,5), (ii) analysing tolerance-inducing effects following transplantation (6), and (iii) drawing conclusions on the incidence of rejection (3). However, little is known about the development of immunological cell populations and especially about DCs within the first year post organ transplantation. Thus, we designed a study, which was approved by the local Ethic Committee of Leipzig, to monitor expression of pDCs and mDCs

Indication-specific immunomodulatory effects of extracorporeal photopheresis: A pilot study in heart transplanted patients

The clinical use of extracorporeal photopheresis (ECP) is based on its ability to induce cell‐mediated immune tolerance towards foreign and self‐antigens. Up‐to‐date, no clear consensus consists on how to use ECP after heart transplantation (HTx). In this pilot study, we evaluated the stimulatory effects of ECP on immune cells in HTx patients.

Evaluation of conventional and frozen elephant trunk techniques on spinal cord blood flow in an animal model

OBJECTIVES The treatment of patients with extensive thoracic aortic disease involving the arch and descending aorta is often performed using the frozen elephant trunk technique (FET). Spinal cord blood flow (SCBF) in cervical, thoracic and lumbar sections prior, during and after aortic arch surgery were compared in conventional elephant trunk (cET) and FET technique in a pig model. METHODS German Landrace pigs (75‐85 kg) underwent aortic arch surgery using the FET (n = 8) or cET (n = 8) techniques. The E‐vita Open hybrid stent graft was applied in all FET animals. Regional SCBF was measured 4 times: (i) before cardiopulmonary bypass, (ii) after 1 h, (iii) after 3 h, and (iv) after 6 h of reperfusion using fluorescence microspheres. Spinal cord segments were examined histopathologically and by immunohistochemistry. RESULTS SCBF in FET decreased significantly from 0.13 ± 0.03 to 0.05 ± 0.02 ml/min/g after 1 h (P = 0.047). While at 3 h of reperfusion, SCBF increased and was comparable to baseline (0.09 ± 0.01 ml/min/g), beyond this time SCBF decreased again (0.05 ± 0.02 ml/min/g). A similar trend was found for SCBF in the cET group (baseline: 0.16 ± 0.04 ml/min/g, 1 h reperfusion: 0.02 ± 0.01 ml/min/g, 3 h reperfusion: 0.03 ± 0.01 ml/min/g and 6 h reperfusion: 0.02 ± 0.01 ml/min/g, P = 0.019). Cervical, thoracic and lumbar SCBF were also comparable in both groups. Histological analyses of spinal cord showed no differences in necrosis between cET and FET, while no differences were found for hypoxia‐inducible factor‐1&agr; and apoptosis‐inducing factor. In contrast, oxidative stress and caspase‐induced apoptosis were higher in cET versus FET. CONCLUSIONS The SCBF changed significantly during extensive aortic arch surgery with circulatory arrest and moderate hypothermia, but such changes were comparable between the FET and cET groups. The implantation of hybrid stent graft did not influence SCBF in thoracic and lumbar segments of the spinal cord. The immunohistological examination showed no differences between cET and FET regarding ischaemic damage and hypoxia‐induced effects in spinal cord segments.

Assessment of Immunological Biomarkers in the First Year after Heart Transplantation.

Background. Pharmacodynamic biomarkers that detect changes of immunological functions have been recognized as a helpful tool to increase the efficacy of immunosuppressive drug therapies. However, physiological changes of immunological biomarkers following transplantation are not investigated. Therefore, we assessed frequently used immunological biomarkers of the circulating blood in the first year following heart transplantation (HTx). Methods. Activation markers CD25 and CD95, intracellular cytokines IL-2 and IFNγ, chemokines IP10 and MIG, and subsets of dendritic cells as well as antibodies against human leukocyte antigens (HLA) and major histocompatibility complex class I-related chain A (MICA) antigens were analyzed at different time points using flow cytometry and Luminex xMAP technology. Results. Expression of IL-2, IFNγ, and plasmacytoid dendritic cells (pDCs) significantly increased (p < 0.01) during the first year. Anti-HLA antibodies decreased continuously, while anti-MICA antibodies showed minor increase within the first year. An association between percentage of pDCs and anti-MICA antibody positivity was proven. pDCs, IFNγ-producing T cells, and IP10 concentration were associated in a stronger way with age and gender of HTx recipients than with antibodies against HLA or MICA. Conclusions. We conclude that certain immunological biomarkers of the circulating blood change during the first year after HTx. These changes should be considered for interpretation of biomarkers after transplantation.