Cellular engineering of ventricular adult rat cardiomyocytes

Objective: Preparation of viable cultured adult cardiomyocytes (vARCs) is a prerequisite for cell-based transplantation and tissue engineering. Ectopic gene expression is important in this context. Here, we present an in vitro cell replating strategy using Accutase™ for cultured vARCs, allowing ectopic gene expression. Methods: Cultured vARCs from 6- to 8-week-old rats were used. Transfections with EGFP (enhanced green fluorescent protein) constructs, Mlc-3f-EGFP or α-actinin-EGFP were performed using adenovirus-enhanced transferrin-mediated infection (AVET). Accutase™ (PAA Laboratories, Linz, Austria) was used for the detachment of cultured cells. Immunohistochemical analysis, together with confocal laser microscopy was used for structural analysis of the cells. Results: Cultured vARCs could be detached with a high yield (40 to 60%) from primary cultures using Accutase™. The cultivation period plays an important role in the yield of viable cells. Resultant replated vARCs (rep-vARCs) rapidly (1-2 h) acquired a rounded up shape without degradation of their contractile apparatus, which is in contrast to the rod-shaped freshly isolated vARCs (fi-vARCs). The detached cells survived passage through a narrow syringe needle. After seeding, detached cells rapidly attached to various substrates, increased their content of the contractile apparatus, and formed cell-cell contacts within 3 days after reseeding. The detached cells survived passage through a narrow syringe needle. The high recovery of cells after replating enabled the use of the AVET system for gene delivery. AVET is free of infectious particles and does not lead to expression of viral proteins. Transfection of vARCs prior to detachment had a small effect on cell recovery and ectopically synthesized proteins were properly localized after replating. Conclusions: Detachment of cultured vARCs using Accutase™ is well compatible with ectopic gene expression and yields a viable transgenic population of vARCs that eventually may be suitable as transgenic cardiomyocyte graft

Cellular engineering of ventricular adult rat cardiomyocytes

ISSN:1755-3245ISSN:0008-636

Effects of Endotoxin and Catecholamines on Hepatic Mitochondrial Respiration

Catecholamines are frequently used in sepsis, but their interaction with mitochondrial function is controversial. We incubated isolated native and endotoxin-exposed swine liver mitochondria with either dopamine, dobutamine, noradrenaline or placebo for 1h. Mitochondrial State 3 and 4 respiration and their ratio (RCR) were determined for respiratory chain complexes I, II and IV. All catecholamines impaired glutamate-dependent RCR (p = 0.046), predominantly in native mitochondria. Endotoxin incubation alone induced a decrease in glutamate-dependent RCR compared to control samples (p = 0.002). We conclude that catecholamines and endotoxin impair the efficiency of mitochondrial complex I respiration in vitr

Effects of prolonged endotoxemia on liver, skeletal muscle and kidney mitochondrial function

INTRODUCTION: Sepsis may impair mitochondrial utilization of oxygen. Since hepatic dysfunction is a hallmark of sepsis, we hypothesized that the liver is more susceptible to mitochondrial dysfunction than the peripheral tissues, such as the skeletal muscle. We studied the effect of prolonged endotoxin infusion on liver, muscle and kidney mitochondrial respiration and on hepatosplanchnic oxygen transport and microcirculation in pigs. METHODS: Twenty anesthetized pigs were randomized to receive either endotoxin or saline infusion for 24 hours. Muscle, liver and kidney mitochondrial respiration was assessed. The cardiac output (thermodilution) and the carotid, superior mesenteric and kidney arterial, portal venous (ultrasound Doppler) and microcirculatory blood flow (laser Doppler) were measured, and systemic and regional oxygen transport and lactate exchange were calculated. RESULTS: Endotoxin infusion induced hyperdynamic shock and impaired the glutamate-dependent and succinate-dependent mitochondrial respiratory control ratio in the liver (glutamate, median (range) endotoxemia 2.8 (2.3–3.8) vs controls 5.3 (3.8–7.0); P < 0.001; succinate, endotoxemia 2.9 (1.9–4.3) vs controls 3.9 (2.6–6.3), P = 0.003). While the ADP added/oxygen consumed ratio was reduced with both substrates, the maximal ATP production was impaired only in the succinate-dependent respiration. Hepatic oxygen consumption and extraction, and the liver surface laser Doppler blood flow remained unchanged. Glutamate-dependent respiration in the muscle and kidney was unaffected. CONCLUSION: Endotoxemia reduces the efficiency of hepatic mitochondrial respiration but neither skeletal muscle nor kidney mitochondrial respiration, independent of regional and microcirculatory blood flow changes

Absence of Plekhg5 Results in Myelin Infoldings Corresponding to an Impaired Schwann Cell Autophagy, and a Reduced T-Cell Infiltration Into Peripheral Nerves

Lüningschrör P, Slotta C, Heimann P, et al. Absence of Plekhg5 Results in Myelin Infoldings Corresponding to an Impaired Schwann Cell Autophagy, and a Reduced T-Cell Infiltration Into Peripheral Nerves. Frontiers in Cellular Neuroscience. 2020;14: 185.Inflammation and dysregulation of the immune system are hallmarks of several neurodegenerative diseases. An activated immune response is considered to be the cause of myelin breakdown in demyelinating disorders. In the peripheral nervous system (PNS), myelin can be degraded in an autophagy-dependent manner directly by Schwann cells or by macrophages, which are modulated by T-lymphocytes. Here, we show that the NF-κB activator Pleckstrin homology containing family member 5 (Plekhg5) is involved in the regulation of both Schwann cell autophagy and recruitment of T-lymphocytes in peripheral nerves during motoneuron disease. Plekhg5-deficient mice show defective axon/Schwann cell units characterized by myelin infoldings in peripheral nerves. Even at late stages, Plekhg5-deficient mice do not show any signs of demyelination and inflammation. Using RNAseq, we identified a transcriptional signature for an impaired immune response in sciatic nerves, which manifested in a reduced number of CD4+ and CD8+ T-cells. These findings identify Plekhg5 as a promising target to impede myelin breakdown in demyelinating PNS disorders

final results of a noninterventional study

Background Data are limited regarding routine use of everolimus after initial vascular endothelial growth factor (VEGF)–targeted therapy. The aim of this prospective, noninterventional, observational study was to assess efficacy and safety of everolimus after initial VEGF-targeted treatment in patients with metastatic renal cell carcinoma (mRCC) in routine clinical settings. Methods Everolimus was administered per routine clinical practice. Patients with mRCC of any histology from 116 active sites in Germany were included. The main objective was to determine everolimus efficacy in time to progression (TTP). Progression-free survival (PFS), treatment duration, tumor response, adherence to everolimus regimen, treatment after everolimus, and safety were also assessed. Results In the total population (N = 334), median follow-up was 5.2 months (range, 0–32 months). Median treatment duration (safety population, n = 318) was 6.5 months (95% confidence interval [CI], 5–8 months). Median TTP and median PFS were similar in populations investigated. In patients who received everolimus as second-line treatment (n = 211), median (95% CI) TTP was 7.1 months (5–9 months) and median PFS was 6.9 months (5–9 months). Commonly reported adverse events (safety population, n = 318) were dyspnea (17%), anemia (15%), and fatigue (12%). Limitations of the noninterventional design should be considered. Conclusions This study reflects routine clinical use of everolimus in a large sample of patients with mRCC. Favorable efficacy and safety were seen for everolimus after previous therapy with one VEGF-targeted agent. Results of this study confirm everolimus as one of the standard options in second-line therapy for patients with mRCC. Novartis study code, CRAD001LD27: VFA registry for noninterventional studies (http://www.vfa.de/de/forschung/nisdb/ webcite)

Everolimus in metastatic renal cell carcinoma after failure of initial anti-VEGF therapy: final results of a noninterventional study

Background: Data are limited regarding routine use of everolimus after initial vascular endothelial growth factor (VEGF)-targeted therapy. The aim of this prospective, noninterventional, observational study was to assess efficacy and safety of everolimus after initial VEGF-targeted treatment in patients with metastatic renal cell carcinoma (mRCC) in routine clinical settings. Methods: Everolimus was administered per routine clinical practice. Patients with mRCC of any histology from 116 active sites in Germany were included. The main objective was to determine everolimus efficacy in time to progression (TTP). Progression-free survival (PFS), treatment duration, tumor response, adherence to everolimus regimen, treatment after everolimus, and safety were also assessed. Results: In the total population (N = 334),median follow-up was 5.2 months (range, 0-32 months). Median treatment duration (safety population, n = 318) was 6.5 months (95% confidence interval [CI], 5-8 months). Median TTP and median PFS were similar in populations investigated. In patients who received everolimus as second-line treatment (n = 211),median (95% CI) TTP was 7.1 months (5-9 months) and median PFS was 6.9 months (5-9 months). Commonly reported adverse events (safety population, n = 318) were dyspnea (17%),anemia (15%), and fatigue (12%). Limitations of the noninterventional design should be considered. Conclusions: This study reflects routine clinical use of everolimus in a large sample of patients with mRCC. Favorable efficacy and safety were seen for everolimus after previous therapy with one VEGF-targeted agent. Results of this study confirm everolimus as one of the standard options in second-line therapy for patients with mRCC

Machine learning in cardiovascular radiology:ESCR position statement on design requirements, quality assessment, current applications, opportunities, and challenges

Machine learning offers great opportunities to streamline and improve clinical care from the perspective of cardiac imagers, patients, and the industry and is a very active scientific research field. In light of these advances, the European Society of Cardiovascular Radiology (ESCR), a non-profit medical society dedicated to advancing cardiovascular radiology, has assembled a position statement regarding the use of machine learning (ML) in cardiovascular imaging. The purpose of this statement is to provide guidance on requirements for successful development and implementation of ML applications in cardiovascular imaging. In particular, recommendations on how to adequately design ML studies and how to report and interpret their results are provided. Finally, we identify opportunities and challenges ahead. While the focus of this position statement is ML development in cardiovascular imaging, most considerations are relevant to ML in radiology in general. KEY POINTS: • Development and clinical implementation of machine learning in cardiovascular imaging is a multidisciplinary pursuit. • Based on existing study quality standard frameworks such as SPIRIT and STARD, we propose a list of quality criteria for ML studies in radiology. • The cardiovascular imaging research community should strive for the compilation of multicenter datasets for the development, evaluation, and benchmarking of ML algorithms

Everolimus in Metastatic Renal Cell Carcinoma after Failure of Initial Vascular Endothelial Growth Factor Receptor-Tyrosine Kinase Inhibitor (VEGFr-TKI) Therapy: Results of an Interim Analysis of a Non-Interventional Study

Background: Everolimus is approved for treatment of anti-vascularendothelial growth factor (VEGF)-refractory patients with metastaticrenal cell carcinoma (mRCC). Clinical trials rarely mirror treatmentreality. Thus, a broader evaluation of everolimus is valuable forroutine use. Patients and Methods: A German multicenternon-interventional study documented mRCC patients starting everolimusafter failure of initial VEGF-targeted therapy. Primary endpoint waseffectiveness, defined as time to progression (TIP) according toinvestigator assessment (time from first dose to progression). Results:Of 382 documented patients, 196 were included in this interim analysis