In vitro and in vivo evidence that the switch from calcineurin to mTOR inhibitors may be a strategy for immunosuppression in Epstein-Barr virus-associated post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder is a life-threatening complication of immunosuppression following transplantation mediated by failure of T cells to control Epstein-Barr virus (EBV)-infected and transformed B cells. Typically, a modification or reduction of immunosuppression is recommended, but insufficiently defined thus far. In order to help delineate this, we characterized EBV-antigen-specific T cells and lymphoblastoid cell lines from healthy donors and in patients with a kidney transplant in the absence or presence of the standard immunosuppressants tacrolimus, cyclosporin A, prednisolone, rapamycin, and mycophenolic acid. Phenotypes of lymphoblastoid cell-lines and T cells, T cell-receptor-repertoire diversity, and T-cell reactivity upon co-culture with autologous lymphoblastoid cell lines were analyzed. Rapamycin and mycophenolic acid inhibited lymphoblastoid cell-line proliferation. T cells treated with prednisolone and rapamycin showed nearly normal cytokine production. Proliferation and the viability of T cells were decreased by mycophenolic acid, while tacrolimus and cyclosporin A were strong suppressors of T-cell function including their killing activity. Overall, our study provides a basis for the clinical decision for the modification and reduction of immunosuppression and adds information to the complex balance of maintaining anti-viral immunity while preventing acute rejection. Thus, an immunosuppressive regime based on mTOR inhibition and reduced or withdrawn calcineurin inhibitors could be a promising strategy for patients with increased risk of or manifested EBV-associated post-transplant lymphoproliferative disorder.

Risk factors for Epstein-Barr virus reactivation after renal transplantation: Results of a large, multi-centre study.

Epstein-Barr virus (EBV) reactivation is a very common and potentially lethal complication of renal transplantation. However, its risk factors and effects on transplant outcome are not well known. Here, we have analysed a large, multi-centre cohort (N = 512) in which 18.4% of the patients experienced EBV reactivation during the first post-transplant year. The patients were characterized pre-transplant and two weeks post-transplant by a multi-level biomarker panel. EBV reactivation was episodic for most patients, only 12 patients showed prolonged viraemia for over four months. Pre-transplant EBV shedding and male sex were associated with significantly increased incidence of post-transplant EBV reactivation. Importantly, we also identified a significant association of post-transplant EBV with acute rejection and with decreased haemoglobin levels. No further severe complications associated with EBV, either episodic or chronic, could be detected. Our data suggest that despite relatively frequent EBV reactivation, it had no association with serious complications during the first post-transplantation year. EBV shedding prior to transplantation could be employed as biomarkers for personalized immunosuppressive therapy. In summary, our results support the employed immunosuppressive regimes as relatively safe with regard to EBV. However, long-term studies are paramount to support these conclusions.

Correlational analysis of the regulatory interplay between molecules and cellular components mediating angiogenesis in wound healing under normal and hyperglycemic conditions.

AIM: The aim of this study was to correlate the content of cells with regulatory molecules associated with angiogenesis in wound healing in a rat model of hyperglycemia. We hypothesize that blood neutrophils are the main VEGF source and can stimulate FLT-1 receptor expression, which is the perquisite for efficient neoangiogenesis. MATERIALS AND METHODS: Kinetic studies of the healing dynamics (3, 7, 14, 21 days) of burn wounds on the skin were conducted in white adult male rats. The content of nuclear factor kappa B (NF-κB), vascular endothelial growth factor (VEGF), its receptor (Flt-1) in the regenerated tissue was analyzed by western blot. Numbers of cells associated with the regenerative process and from peripheral blood (PB) were determined. Additionally a bone marrow (BM) myelogram was conducted. RESULTS: The relative number of peripheral blood (PB) neutrophils was found to be associated with the level of VEGF (R = 0.708) and Flt-1 (R = 0.472). The relative number of fibroblasts was also associated with VEGF (R = 0.562), but not with Flt-1. A negative association was found between the number of neutrophils in the regenerated tissue with VEGF (R = -0.454) and FLT-1 (R = -0.665). This confirms our hypothesis, that blood neutrophils are the main VEGF producer that stimulate the expression of the FLT-1 receptor subsequently inducing neoangiogenesis.Furthermore, that under hyperglycemic conditions fibroblasts were highly associated with VEGF (R = 0.800), while negatively associated with FLT-1 (R = -0.506). There was a high association between PB neutrophils and newly generated tissue cells: neutrophils (R = 0.717) and macrophages (R = 0.622), as well as the association between neutrophils and macrophages (R = 0.798). This is an indication of chronic inflammation and increased transmigration of blood cells to the burned tissue. CONCLUSION: Blood neutrophils are the main producer of VEGF and stimulate the expression of the FLT-1 receptor. In the context of hyperglycemia the imbalance of receptor and ligand associated with angiogenesis indicates for chronic inflammation: VEGF and FLT-1, which facilitates hypoxia, prevents the physiological course of burn wound healing and may be an important factor in impaired tissue regeneration in diabetes.

The role of soluble mediators in the clinical course of EBV infection and B cell homeostasis after kidney transplantation.

Epstein-Barr virus (EBV) reactivation can lead to serious complications in kidney transplant patients, including post-transplant lymphoproliferative disorder (PTLD). Here, we have assessed the impact of EBV on B cell homeostasis at cellular and humoral level. In a multicenter study monitoring 540 kidney transplant patients during the first post-transplant year, EBV reactivation was detected in 109 patients. Thirteen soluble factors and B cell counts were analyzed in an EBV+ sub-cohort (N = 54) before, at peak and after EBV clearance and compared to a control group (N = 50). The B cell activating factor (BAFF) was significantly elevated among EBV+ patients. No additional soluble factors were associated with EBV. Importantly, in vitro experiments confirmed the proliferative effect of BAFF on EBV-infected B cells, simultaneously promoting EBV production. In contrast, elevated levels of BAFF in EBV+ patients did not lead to B cell expansion in vivo. Moreover, diminished positive inter-correlations of soluble factors and alterations of the bi-directional interplay between B cell and soluble factors were observed in EBV+ patients at peak and after clearance. Our data suggest that such alterations may counteract the proliferative effect of BAFF, preventing B cell expansion. The role of these alterations in lymphoma development should be analyzed in future studies.

BKV, CMV, and EBV Interactions and their Effect on Graft Function One Year Post-Renal Transplantation: Results from a Large Multi-Centre Study.

BACKGROUND: BK virus (BKV), Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivations are common after kidney transplantation and associated with increased morbidity and mortality. Although CMV might be a risk factor for BKV and EBV, the effects of combined reactivations remain unknown. The purpose of this study is to ascertain the interaction and effects on graft function of these reactivations. METHODS: 3715 serum samples from 540 kidney transplant recipients were analysed for viral load by qPCR. Measurements were performed throughout eight visits during the first post-transplantation year. Clinical characteristics, including graft function (GFR), were collected in parallel. FINDINGS: BKV had the highest prevalence and viral loads. BKV or CMV viral loads over 10,000 copies·mL-1 led to significant GFR impairment. 57 patients had BKV-CMV combined reactivation, both reactivations were significantly associated (p = 0.005). Combined reactivation was associated with a significant GFR reduction one year post-transplantation of 11.7 mL·min-1·1.73 m-2 (p = 0.02) at relatively low thresholds (BKV > 1000 and CMV > 4000 copies·mL-1). For EBV, a significant association was found with CMV reactivation (p = 0.02), but no GFR reduction was found. Long cold ischaemia times were a further risk factor for high CMV load. INTERPRETATION: BKV-CMV combined reactivation has a deep impact on renal function one year post-transplantation and therefore most likely on long-term allograft function, even at low viral loads. Frequent viral monitoring and subsequent interventions for low BKV and/or CMV viraemia levels and/or long cold ischaemia time are recommended. FUND: Investigator Initiated Trial; financial support by German Federal Ministry of Education and Research (BMBF).

Differential T cell response against BK virus regulatory and structural antigens: A viral dynamics modelling approach.

BK virus (BKV) associated nephropathy affects 1-10% of kidney transplant recipients, leading to graft failure in about 50% of cases. Immune responses against different BKV antigens have been shown to have a prognostic value for disease development. Data currently suggest that the structural antigens and regulatory antigens of BKV might each trigger a different mode of action of the immune response. To study the influence of different modes of action of the cellular immune response on BKV clearance dynamics, we have analysed the kinetics of BKV plasma load and anti-BKV T cell response (Elispot) in six patients with BKV associated nephropathy using ODE modelling. The results show that only a small number of hypotheses on the mode of action are compatible with the empirical data. The hypothesis with the highest empirical support is that structural antigens trigger blocking of virus production from infected cells, whereas regulatory antigens trigger an acceleration of death of infected cells. These differential modes of action could be important for our understanding of BKV resolution, as according to the hypothesis, only regulatory antigens would trigger a fast and continuous clearance of the viral load. Other hypotheses showed a lower degree of empirical support, but could potentially explain the clearing mechanisms of individual patients. Our results highlight the heterogeneity of the dynamics, including the delay between immune response against structural versus regulatory antigens, and its relevance for BKV clearance. Our modelling approach is the first that studies the process of BKV clearance by bringing together viral and immune kinetics and can provide a framework for personalised hypotheses generation on the interrelations between cellular immunity and viral dynamics.