Allograft and patient survival after sequential HSCT and kidney transplantation from the same donor - A multicenter analysis

Tolerance induction through simultaneous hematopoietic stem cell and renal transplantation has shown promising results, but it is hampered by the toxicity of preconditioning therapies and graft-versus-host disease (GVHD). Moreover, renal function has never been compared to conventionally transplanted patients, thus, whether donor-specific tolerance results in improved outcomes remains unanswered. We collected follow-up data of published cases of renal transplantations after hematopoietic stem cell transplantation from the same donor and compared patient and transplant kidney survival as well as function with caliper-matched living-donor renal transplantations from the Austrian dialysis and transplant registry. Overall, 22 tolerant and 20 control patients were included (median observation period 10 years [range 11 months to 26 years]). In the tolerant group, no renal allograft loss was reported, whereas 3 were lost in the control group. Median creatinine levels were 85 μmol/l (interquartile range [IQR] 72-99) in the tolerant cohort and 118 μmol/l (IQR 99-143) in the control group. Mixed linear-model showed around 29% lower average creatinine levels throughout follow-up in the tolerant group (P < .01). Our data clearly show stable renal graft function without long-term immunosuppression for many years, suggesting permanent donor-specific tolerance. Thus sequential transplantation might be an alternative approach for future studies targeting tolerance induction in renal allograft recipients

Signaling properties of murine MPL and MPL mutants after stimulation with thrombopoietin and romiplostim

Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F_Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro

The Vaccines Consistency Approach Project: an EPAA Initiative

The consistency approach for release testing of established vaccines promotes the use of in vitro, analytical, non-animal based systems allowing the monitoring of quality parameters during the whole production process. By using highly sensitive non-animal methods, the consistency approach has the potential to improve the quality of testing and to foster the 3Rs (replacement, refinement and reduction of animal use) for quality control of established vaccines. This concept offers an alternative to the current quality control strategy which often requires large numbers of laboratory animals. In order to facilitate the introduction of the consistency approach for established human and veterinary vaccine quality control, the European Partnership for Alternatives to Animal Testing (EPAA) initiated a project, the “Vaccines Consistency Approach Project”, aiming at developing and validating the consistency approach with stakeholders from academia, regulators, OMCLs, EDQM, European Commission and industry. This report summarises progress since the project’s inception.JRC.I.5-Systems Toxicolog

Dobutamine-Stress Magnetic Resonance Microimaging in Mice

Abstract — The aim of this study was to assess the capability of MRI to characterize systolic and diastolic function in normal and chronically failing mouse hearts in vivo at rest and during inotropic stimulation. Applying an ECG-gated FLASH-cine sequence, MRI at 7 T was performed at rest and after administration of 1.5 μg/g IP dobutamine. There was a significant increase of heart rate, cardiac output, and ejection fraction and significant decrease of end-diastolic and end-systolic left ventricular (LV) volumes ( P &lt;0.01 each) in normal mice during inotropic stimulation. In mice with heart failure due to chronic myocardial infarction (MI), MRI at rest revealed gross LV dilatation. There was a significant decrease of LV ejection fraction in infarcted mice (29%) versus sham mice (58%). Mice with MI showed a significantly reduced maximum LV ejection rate ( P &lt;0.001) and LV filling rate ( P &lt;0.01) and no increase of LV dynamics during dobutamine action, indicating loss of contractile and relaxation reserve. In 4-month-old transgenic mice with cardiospecific overexpression of the β 1 -adrenergic receptor, which at this early stage do not show abnormalities of resting cardiac function, LV filling rate failed to increase after dobutamine stress (transgenic, 0.19±0.03 μL/ms; wild type, 0.36±0.01 μL/ms; P &lt;0.01). Thus, MRI unmasked diastolic dysfunction during dobutamine stress. Dobutamine-stress MRI allows noninvasive assessment of systolic and diastolic components of heart failure. This study shows that MRI can demonstrate loss of inotropic and lusitropic response in mice with MI and can unmask diastolic dysfunction as an early sign of cardiac dysfunction in a transgenic mouse model of heart failure. </jats:p