Outcome in patients perceived as receiving excessive care across different ethical climates: a prospective study in 68 intensive care units in Europe and the USA

Purpose: Whether the quality of the ethical climate in the intensive care unit (ICU) improves the identification of patients receiving excessive care and affects patient outcomes is unknown. Methods: In this prospective observational study, perceptions of excessive care (PECs) by clinicians working in 68 ICUs in Europe and the USA were collected daily during a 28-day period. The quality of the ethical climate in the ICUs was assessed via a validated questionnaire. We compared the combined endpoint (death, not at home or poor quality of life at 1 year) of patients with PECs and the time from PECs until written treatment-limitation decisions (TLDs) and death across the four climates defined via cluster analysis. Results: Of the 4747 eligible clinicians, 2992 (63%) evaluated the ethical climate in their ICU. Of the 321 and 623 patients not admitted for monitoring only in ICUs with a good (n = 12, 18%) and poor (n = 24, 35%) climate, 36 (11%) and 74 (12%), respectively were identified with PECs by at least two clinicians. Of the 35 and 71 identified patients with an available combined endpoint, 100% (95% CI 90.0–1.00) and 85.9% (75.4–92.0) (P = 0.02) attained that endpoint. The risk of death (HR 1.88, 95% CI 1.20–2.92) or receiving a written TLD (HR 2.32, CI 1.11–4.85) in patients with PECs by at least two clinicians was higher in ICUs with a good climate than in those with a poor one. The differences between ICUs with an average climate, with (n = 12, 18%) or without (n = 20, 29%) nursing involvement at the end of life, and ICUs with a poor climate were less obvious but still in favour of the former. Conclusion: Enhancing the quality of the ethical climate in the ICU may improve both the identification of patients receiving excessive care and the decision-making process at the end of life

Liquid chromatography-tandem mass spectrometric assay for the cyclin-dependent kinase inhibitor AT7519 in mouse plasma

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the cyclin-dependent kinase inhibitor AT7519 in mouse plasma was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing rucaparib as internal standard. After dilution with water, the extract was directly injected into the reversed-phase LC system. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 5-10,000ng/ml calibration range using double logarithmic calibration, 5ng/ml was the lower limit of quantification. Within day precisions (n=6) were 2.9-5.6%, between day (3 days; n=18) precisions 3.2-7.2%. Accuracies were between 95.9 and 99.0% for the whole calibration range. The drug was stable under all relevant analytical conditions. Finally, the assay was successfully used to determine plasma pharmacokinetics after intraperitoneal administration of AT7519 in mice with neuroblastoma xenograft

Targeted BCL2 inhibition effectively inhibits neuroblastoma tumour growth

Genomic aberrations of key regulators of the apoptotic pathway have hardly been identified in neuroblastoma. We detected high BCL2 mRNA and protein levels in the majority of neuroblastoma tumours by Affymetrix expression profiling and Tissue Micro Array analysis. This BCL2 mRNA expression is strongly elevated compared to normal tissues and other malignancies. Most neuroblastoma cell lines lack this high BCL2 expression. Only two neuroblastoma cell lines (KCNR and SJNB12) show BCL2 expression levels representative for neuroblastoma tumours. To validate BCL2 as a therapeutic target in neuroblastoma we employed lentivirally mediated shRNA. Silencing of BCL2 in KCNR and SJNB12 resulted in massive apoptosis, while cell lines with low BCL2 expression were insensitive. Identical results were obtained by treatment of the neuroblastoma cell lines with the small molecule BCL2 inhibitor ABT263, which is currently being clinically evaluated. Combination assays of ABT263 with most classical cytostatics showed strong synergistic responses. Subcutaneous xenografts of a neuroblastoma cell line with high BCL2 expression in NMRI nu/nu mice showed a strong response to ABT263. These findings establish BCL2 as a promising drug target in neuroblastoma and warrant further evaluation of ABT263 and other BCL2 inhibiting drugs. (C) 2012 Elsevier Ltd. All rights reserved

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour model

GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain.

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2(+/-) mice with an approximately 50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre-Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Na(v)1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2(+/-) mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2(+/-) mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor alpha (TNF-alpha) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-alpha activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2(+/-) mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia

Cyclin-Dependent Kinase Inhibitor AT7519 as a Potential Drug for MYCN-Dependent Neuroblastoma

MYCN-dependent neuroblastomas have low cure rates with current multimodal treatment regimens and novel therapeutic drugs are therefore urgently needed. In previous preclinical studies, we have shown that targeted inhibition of cyclin-dependent kinase 2 (CDK2) resulted in specific killing of MYCN-amplified neuroblastoma cells. This study describes the in vivo preclinical evaluation of the CDK inhibitor AT7519. Preclinical drug testing was performed using a panel of MYCN-amplified and MYCN single copy neuroblastoma cell lines and different MYCN-dependent mouse models of neuroblastoma. AT7519 killed MYCN-amplified neuroblastoma cell lines more potently than MYCN single copy cell lines with a median LC50 value of 1.7 compared to 8.1 μmol/L (P = 0.0053) and a significantly stronger induction of apoptosis. Preclinical studies in female NMRI homozygous (nu/nu) mice with neuroblastoma patient-derived MYCN-amplified AMC711T xenografts revealed dose-dependent growth inhibition, which correlated with intratumoral AT7519 levels. CDK2 target inhibition by AT7519 was confirmed by significant reductions in levels of phosphorylated retinoblastoma (p-Rb) and nucleophosmin (p-NPM). AT7519 treatment of Th-MYCN transgenic mice resulted in improved survival and clinically significant tumor regression (average tumor size reduction of 86% at day 7 after treatment initiation). The improved efficacy of AT7519 observed in Th-MYCN mice correlated with higher tumor exposure to the drug. This study strongly suggests that AT7519 is a promising drug for the treatment of high-risk neuroblastoma patients with MYCN amplificatio