STROKE-CARD care to prevent cardiovascular events and improve quality of life after acute ischaemic stroke or TIA: A randomised clinical trial.

Background Patients with ischaemic stroke or transient ischaemic attack (TIA) are at high risk of recurrent stroke and other cardiovascular diseases and commonly suffer from reduced quality of life. We aimed to determine whether the disease management programme STROKE-CARD can prevent cardiovascular diseases and improve quality of life in these patients. Methods In this pragmatic open-label two-centre randomised controlled trial with blinded outcome assessment, we randomly assigned patients with acute ischaemic stroke or TIA (ABCD2 score ≥3) in a 2:1 ratio to receive STROKE-CARD care or standard care. STROKE-CARD care is a disease management programme by a multidisciplinary stroke team that comprises a standardised 3-month visit and access to a web-based patient portal targeting risk factor management, post-stroke complications, comorbidities and cardiovascular warning signs, rehabilitation demands, and patient education, counselling, and self-empowerment. Co-primary outcomes were analysed on an intention-to-treat basis and were: (i) major cardiovascular disease events defined as nonfatal ischaemic or haemorrhagic stroke, nonfatal myocardial infarction, or vascular death occurring between hospital discharge and 12 months; and (ii) health-related quality of life at 12 months quantified with the EuroQol-5-Dimensions-3-Levels (EQ-5D-3L) overall utility score. This trial is registered with ClinicalTrials.gov, number NCT02156778. Findings Of 2149 patients enrolled between January 2014 and December 2017 (mean age 69 years, 41% female, 83% with ischaemic stroke, 17% with TIA), 1438 were assigned to STROKE-CARD care and 711 to standard care. Major cardiovascular disease events occurred in 78 patients in the STROKE-CARD care group (5.4%) and in 59 patients in the standard care group (8.3%) (hazard ratio, 0.63; 95% confidence interval: 0.45-0.88; P=0.007). STROKE-CARD care also led to a better EQ-5D-3L overall utility score at 12 months (P<0.001). Among pre-specified secondary outcomes, STROKE-CARD care improved all individual EQ-5D-3L dimensions and functional outcome on the modified Rankin Scale at 12 months. Post hoc explanatory analyses identified considerable demands for additional rehabilitation and refinement of preventive therapy regimes at the 3-month visit and high proportions of post-stroke complications and warning signs of imminent cardiovascular diseases within the first three months. Interpretation The pragmatic and easily implementable STROKE-CARD care programme reduced cardiovascular risk and improved health-related quality of life and functional outcome in patients with acute ischaemic stroke or TIA. Funding Tirol Kliniken, Tyrolean Health Insurance Company, Tyrol Health Care Funds, Boehringer Ingelheim, Nstim Services, Sanofi, Bayer Healthcare

Point mutations of the human parathyroid calcium receptor gene are not responsible for non-suppressible renal hyperparathyroidism

Point mutations of the human parathyroid calcium receptor gene are not responsible for non-suppressible renal hyperparathyroidism. The calcium-dependent secretion of parathyroid hormone (PTH) is mediated through an extracellular G protein-coupled calcium receptor (CaR). Inactivating point mutations of this receptor have been found in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. These diseases feature a decreased calcium sensitivity of the parathyroid glands, resulting in a rightward shift of the Ca2+-PTH relationship. Severe, non-suppressible renal hyperparathyroidism (rHPT) is often characterized by similar setpoint shifts to the right. Thus, point mutations of the CaR gene could contribute to non-suppressible rHPT. We examined genomic DNA of hyperplastic or mainly nodular tissues of 39 parathyroids from 25 rHPT-patients with resistance to calcitriol therapy. Amplification of the six exons of the CaR gene was followed by single-strand conformation polymorphism (SSCP) analysis. DNA sequencing was performed where band shifts were observed. No point mutations in the coding sequence of the CaR gene were detected using the PCR-SSCP strategy. Point mutations in the coding regions of the CaR gene probably play no role in the evolution of renal HPT and are not responsible for the calcitriol resistance of PTH secretion

Abstract 1388A: Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system

Abstract Angiogenesis is a hallmark of most cancers, and is thus an attractive target for the treatment of cancer. One of the easiest screening and target validation strategies for anti-angiogenic target identification involves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation in Corning's Matrigel product. The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector™ Technology. In the current study we used the Lonza 4D-Nucleofector™ X-Unit to transfect single-donor Clonetics™ HUVEC cultured in EGM™-2 Endothelial Cell Growth Media. Cells were transfected with siRNA directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Transfection conditions were fine-tuned using pmaxGFP™ vector, and it was shown that the Nucleofection™ process had no deleterious effect on the tube formation potential of HUVEC on Corning's Matrigel product. Efficient knock-down of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency was observed 24 hours after transfection. VEGFR2-siRNA transfected cells demonstrated significant inhibition of tube formation on Corning's Growth Factor Reduced Matrigel product in comparison to control samples. Since the efficient knock-down of the VEGFR2 protein in Clonetics™ HUVEC using the Nucleofector™ Technology can be demonstrated at time points as early as 24 hours after transfection, this is an efficient approach for target identification, validation and screening of siRNA based anti-angiogenesis therapeutics for cancer treatment. Citation Format: Srinivasan Kokatam, Kanchan Tiwari, Jenny Schroeder, Andrea Toell, Lubna Hussain, Preeti Kapoor. Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1388A. doi:10.1158/1538-7445.AM2015-1388A</jats:p

Abstract 3485: Efficient transfection of cancer cell lines using the 4D-NucleofectorTM System

Abstract Cell lines isolated from tumors are an important tool to study cancer in vitro. They can be used for drug development as well as for understanding the basic mechanisms underlying cancer. Transfection of cancer cell lines with different molecules like plasmid DNA, siRNA or mRNA is often an integral part for this kind of research. Lonza's 4D-Nucleofector™ System is a modular system for the efficient transfection of primary cells and cell lines with a variety of substrates including plasmid DNA and siRNA. The 4D-Nucleofector™ X Unit supports the Nucleofection™ in two different formats. The aluminum-free 20µl Nucleocuvette™ Strip allows the transfection of low cell numbers down to 2x104 cells per reaction. As 16 reactions can be performed in parallel it is well suited for optimizing Nucleofection™ Conditions for cells lacking a ready-to-use Optimized Protocol. For higher cell numbers of up to 2x107 cells per reaction the same Nucleofection™ Conditions can be applied in the 100μl single Nucleocuvette™ Vessel. For higher throughput needs the 96-well Shuttle™ Add-on can be connected to the 4D-Nucleofector™ System. With this add-on six 20µl Nucleocuvette™ Strips can be processed in parallel allowing for screening applications or accelerating the optimization of transfection parameters for many cell types. In this study we used the 4D-Nucleofector™ System in combination with the 96-well Shuttle™ Add-on for optimizing transfection conditions for multiple cancer cell lines. An exemplary optimization process is depicted for the human prostate carcinoma cell line DU 145 and for the human colorectal adenocarcinoma cell line COLO 205. Optimization steps included the selection of the appropriate Nucleofector™ Solution, Nucleofector™ Program and plasmid DNA concentration. Transfection parameters were thereby optimized for a variety of adherent and non-adherent human cancer cell lines resulting in transfection efficiencies of up to 99%; while maintaining high cell viability. Citation Format: Jenny Schroeder, Ludger Altrogge, Elke Lorbach, Srinivasan Kokatam, Sabine Schaepermeier, Meike Weigel, Gina Andretta-Beu, Stefanie Buesch, Tamara Grabeck, Alexandra Krumnow, Sonja Spicker, Sampada Kallol, Preeti Kapoor, Andrea Toell. Efficient transfection of cancer cell lines using the 4D-NucleofectorTM System. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2014-3485</jats:p

The dimension of preventable stroke in a large representative patient cohort.

OBJECTIVE To analyze the frequency of inadequately treated risk factors in a large representative cohort of patients with acute ischemic stroke or TIA and to estimate the proportion of events potentially avertable by guideline-compliant preventive therapy compared to the status quo. METHODS A total of 1,730 patients from the Poststroke Disease Management STROKE-CARD trial (NCT02156778) were recruited between 2014 and 2017. We focused on 8 risk conditions amenable to drug therapy and 3 lifestyle risk behaviors and assessed pre-event risk factor control in retrospect. RESULTS The proportion of patients with at least 1 inadequately treated risk condition was 79.5% (95% confidence interval [CI] 77.6%-81.4%) and increased to 95.1% (95% CI 94.1%-96.1%) upon consideration of the lifestyle risk behaviors. Risk factor control was worse in patients with recurrent vs first-ever events (p 75 years of age (p < 0.001). The estimated degree of stroke preventability ranged from 0.4% (95% CI 0.2%-0.6%) to 13.7% (95% CI 12.2%-15.2%) for the individual risk factors. Adequate control of the 5 most relevant risk factors combined (hypertension, hypercholesterolemia, atrial fibrillation, smoking, and overweight) would have averted ≈1 of 2 events or 1 in 4 with a highly conservative computation approach. CONCLUSIONS Our study confirms the existence of a considerable gap between risk factor control recommended by guidelines and real-world stroke prevention. Our study intends to increase awareness among physicians about stroke preventability and provides a quantitative basis for the emerging discussion on how to best tackle this challenge