Arne Trummer

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Cytoreductive treatment with clofarabine/ara‐C combined with reduced‐intensity conditioning and allogeneic stem cell transplantation in patients with high‐risk, relapsed, or refractory acute myeloid leukemia and advanced myelodysplastic syndrome

The combination of cytoreductive chemotherapy with reduced‐intensity conditioning (RIC) is a highly effective antileukemic therapy. Purpose of this retrospective analysis was to evaluate the antileukemic efficacy and toxicity of clofarabine‐based chemotherapy followed by RIC and allogeneic stem cell transplantation (SCT) for high‐risk, relapsed, or refractory acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). From May 2007 until October 2009, a total of 27 patients underwent allogeneic SCT after treatment with clofarabine and ara‐C for 5 d and RIC (4 Gy TBI/cyclophosphamide/ATG). Prophylaxis of graft‐versus‐host disease (GvHD) consisted of cyclosporine and mycophenolate mofetil. Unmanipulated G‐CSF mobilized PBSC (n = 26) or bone marrow cells (n = 1) were transplanted from unrelated (n = 21) or matched related (n = 6) donors. Non‐hematological toxicities of this regimen mainly affected liver and skin and were all reversible. Seven patients relapsed within a median time of 5.7 months. The overall survival (OS) and relapse‐free survival rates were 56% and 52% at 2 yr, respectively. In this cohort of patients, cytoreduction with clofarabine/ara‐C (ClAraC) followed by RIC allogeneic SCT was well tolerated and showed good antileukemic efficacy even in patients with high‐risk AML or MDS, with engraftment and GvHD‐incidence comparable to other RIC regimens.

Recovery and composition of microparticles after snap-freezing depends on thawing temperature

Consent regarding the correct processing and storage of blood microparticles is lacking and different protocols for the freeze–thaw cycle exist. Therefore, three different thawing procedures were evaluated regarding their influence on recovery and composition of microparticles. Microparticles were prepared by TRAP-6 or A23187 stimulation of platelet-rich plasma from smokers and nonsmokers (n = 8), from an endothelial cell line or directly obtained from platelet-free plasma of septic patients (n = 5). After snap-freezing in liquid nitrogen platelet-free samples were thawed at 37°, at room temperature or on ice and staining of microparticles was carried out with Annexin V-Cy5 as well as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Microparticle concentrations were determined by means of Trucount tubes. Recovery of platelet microparticles was significantly reduced when samples were thawed on ice (P = 0.001 for all antigens) compared with the two other techniques (P = 0.6 for 37° and P = 0.7 for room temperature, respectively) whereas microparticles of endothelial origin appeared to be less influenced. There was a strong trend towards altered microparticle composition as microparticle counts detected by CD41 staining showed a stronger decrease on ice than Annexin V enumeration (P = 0.07). For microparticle detection thawing of snap-fozen, platelet-free plasma samples should be carried out at room temperature or at 37°C in a water bath but not on ice.

Thrombelastographic monitoring of recombinant factor VIIa in acquired haemophilia

Summary.  Monitoring of the global haemostatic capacity is desired to optimize the treatment with bypassing agents in inhibitor patients. Thrombelastographic methods have been used in ex vivo studies and were suggested useful to evaluate the individual response to bypassing agents. This study aimed at assessing changes in thrombelastographic profiles and their association to clinical outcome in patients treated with recombinant factor VIIa (rFVIIa). Ten patients with acquired haemophilia were treated with rFVIIa for acute bleeding. Thrombelastography was performed after activation with a small amount of tissue factor in samples obtained before and after in vivo administration of rFVIIa. In patients studied before and after a first dose, correction of the thrombelastographic profile was observed but did not predict cessation of bleeding. During steady‐state dosing, the median Alpha angle tended to be higher in patients with a good clinical treatment response as compared with patients with a partial or poor response. Similar trends were observed for clotting time and clot formation time. A good clinical treatment response was more frequent in patients with a fully corrected trough‐level thrombelastographic profile as compared with patients with an abnormal profile. However, a poor treatment response was observed also in a surgical patient with a normal thrombelastographic profile during steady‐state dosing. In conclusion, thrombelastographic monitoring was sensitive to haemostatic changes in response to treatment with rFVIIa. In the limited number of patients studied here, a better clotting profile during steady‐state dosing was associated with a better clinical treatment response.

Competitive Cytokeratin 19 RT-PCR for Quantification of Breast Cancer Cells in Blood Cell Suspensions

Detection of residual tumor cells in BM and PBPC products has been correlated with worse outcome of breast cancer patients. Still, there is a considerable demand for studies investigating the influence of the actual tumor cell number on prognosis, as quantification routinely has been cumbersome and time consuming and, thus, was evaded. We developed and evaluated a competitive RT-PCR-ELISA assay for cytokeratin 19 (CK19) with standard curve quantification that allows quantification of multiple samples within a working day; mRNA isolation, RT-PCR reaction, and automated ELISA detection were carried out using commercial kits. Results were expressed as OD420nm ratios of CK19 and an internal competitor. Values were then converted into tumor cell numbers using a standard curve of MCF-7 tumor cells. The assay had high specificity because of primers and capture probes with great heterogeneity to both published pseudogenes, which was confirmed by BLAST sequence alignment. We achieved a sensitivity of detecting 1 tumor cell per 10(6) mononuclear cells (MNC). Between-batch precision (n = 8) for quantification was consistent and reasonable, with a coefficient of variation around 25%. Therefore, this assay should be suitable and sufficient for routine quantification of tumor cell numbers in BM or PBPC samples.

Epithelial and Erythrocyte Microvesicles From Bronchoalveolar Lavage Fluid Are Elevated and Associated With Outcome in Chronic Lung Allograft Dysfunction.

Background Chronic lung allograft dysfunction (CLAD) is the major outcome limitation for lung transplant recipients (LTR) after the first year, and therapies targeting immunological pathways show only limited success. Because microvesicles (MV) are biomarkers of endothelial dysfunction and coagulation but are also involved in immunological responses, we hypothesized that MV, found in bronchoalveolar lavage (BAL) fluids (BALF) of LTR at CLAD diagnosis, are elevated and potential prognostic biomarkers. Methods The BALF was collected from 37 LTR at time point of CLAD diagnosis and 37 LTR without any complication at routinely performed BAL. The MV concentration and origin were determined by flow cytometry by detection of different antigens. Patient- and transplant-related risk factors were included in a retrospective statistical analysis. Results The BALF-MV levels of epithelial and red blood cell (RBC) origin were significantly higher in CLAD patients (mean: 1533/&mgr;L and 158/&mgr;L) compared to controls (436/&mgr;L, 57/&mgr;L). The LTR with high levels of epithelial MV >580/&mgr;L showed a significantly shorter overall survival at 4 years after BAL (39.5%) compared to patients with low MV (66.4%) and this proofed to be an independent prognostic factor in multivariate Cox analysis (hazards ratio = 3.05). Furthermore, LTR with high levels of RBC MV ≥225/&mgr;L were also associated with worse disease-specific survival, with probabilities at 4 years after BAL of 85.8% vs. 36.0%. Conclusions Epithelial and RBC BALF-MV are elevated at CLAD diagnosis, have a potential as biomarkers, and support the hypothesis of a pathway, including activation of coagulation and complement, endothelial barrier dysfunction, and microangiopathy.

Increased amounts of von Willebrand factor are bound to microparticles after infusion of desmopressin.

Effects of desmopressin (DDAVP) in platelet disorders and primary haemostasis cannot be attributed solely to the increase in FVIII/VWF (von Willebrand factor), as VWF/FVIII concentrates have no effect in these circumstances. Microparticles (MP) can support haemostasis by expression of phospholipids, tissue factor and VWF on their surface. We hypothesized that significant amounts of VWF are bound to MP after DDAVP administration and that consequently depletion of MP should influence VWF:Ag and VWF:RCo plasma levels. Platelet‐poor plasma was either obtained well from healthy controls or before and after DDAVP administration from patients with von Willebrands disease (type 1 or possible type 1) or patients with other bleeding disorders as controls. Concentrations of MP and VWF parameters were determined before and after MP depletion by different methods (magnetic bead selection, plasma microfiltration, ultracentrifugation). Platelet MP and VWF‐bearing MP were significantly increased after DDAVP. MP depletion by magnetic bead selection led to a significant reduction in VWF:Ag (−18.0%) and VWF:RCo (−27.7%) plasma levels without changes in VWF multimer composition. As results were similar for DDAVP control subjects, the amount of VWF bound to circulating microparticles was significantly higher after DDAVP administration compared with healthy controls (reduction −11.7%). DDAVP leads to a release of microparticles and increases the amount of VWF bound to microparticles which might explain the clinical efficacy of DDAVP in platelet disorders.

Evaluation of tissue factor bearing microparticles as biomarkers in allogeneic stem-cell transplantation.

Background. There is compelling evidence that blood-borne tissue factor that is predominantly found on circulating microparticles (MPs) plays an important role in both, cancer biology and organ and stem-cell transplantation (SCT). Therefore, we hypothesized that numbers of tissue factor bearing MPs might be associated with complications and outcome in allogeneic SCT (allo-SCT). Materials and Methods. In a prospective study, we enumerated total, platelet, endothelial, and tissue factor bearing MPs in plasma samples obtained from up to 60 patients with hematologic diseases at different time-points during the course of allo-SCT by flow cytometry. Patient- and transplant-related risk factors were included in statistical analysis. Results. Mean follow-up time was 968 days (0–1981 days). Thirty-four (56.7%) patients died, 17 due to transplant-related mortality (28.3%). High numbers of tissue factor positive MPs more than 500/&mgr;L before conditioning were predictive for shorter overall survival (P=0.017, hazard ratio=4.5) in multivariate analysis. This was mainly caused by an increase in transplant-related mortality (P=0.010, hazard ratio=11.0) with cumulative incidences at 1 year of 68.8% compared with patients with lower values (20.1%; P=0.002). Conclusions. Tissue factor bearing MPs might be useful biomarkers for risk stratification in allo-SCT patients and further studies should investigate their origin, functional properties, and optimal cut-off values.

Deletion or inhibition of Fc gamma receptor 2B (CD32) prevents FVIII-specific activation of memory B cells in vitro

Development of inhibitory antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in haemophilia A. Patients with inhibitors are treated with high FVIII doses in the context of immune tolerance therapy (ITT). Data from haemophilia A mouse model suggest that high FVIII concentrations prevent the formation of antibody secreting cells (ASCs) from memory B cells (MBCs) by inducing apoptosis. Fc gamma receptor 2B (CD32) is an important regulator of B cell function, mediating inhibitory signals after cross-linking with the B cell receptor. Here, the role of CD32 in the regulation of FVIII-specific MBCs was investigated using F8-/- and F8-/-CD32-/- knockout mice and monoclonal antibodies (mAbs). The initial immune response was similar between F8-/- and F8-/-CD32-/- mice, including concentration of anti-FVIII antibodies and number of FVIII-specific ASCs in spleen and bone marrow. In contrast, formation of ASCs from MBCs upon rhFVIII re-stimulation in vitro was abolished in F8-/-CD32-/- mice, whereas FVIII/anti-FVIII immune complexes significantly enhanced ASC formation in F8-/- mice. Inhibition of CD32 by mAbs or F(ab)2 fragments prevented ASC formation in a dose-dependent manner. Transfer of B cell-depleted splenocytes using CD45R (B220) depletion from CD32-competent mice did not restore ASC formation in F8-/-CD32-/- cells confirming that CD32 is required on B cells. We conclude that CD32 is a crucial regulator of FVIII-specific B cells and is required for the differentiation of MBCs into ASCs. Inhibition of CD32 could potentially improve the efficacy of FVIII in the context of ITT.

Recombinant human antithrombin prevents xenogenic activation of hemostasis in a model of pig‐to‐human kidney transplantation

Xenogenic activation of hemostasis (XAH) represents a major hurdle for the transplantation of discordant animal organs into humans as it results in thrombotic microangiopathy (TMA). We have previously shown that recombinant human‐activated protein C (rhAPC) mitigates XAH and TMA in an ex vivo model of pig‐to‐human kidney transplantation. However, the use of rhAPC may not be feasible in a perioperative setting due to possible bleeding complications.