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1.
Arch Toxicol ; 94(7): 2435-2461, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32632539

RESUMO

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.


Assuntos
Documentação , Processamento Eletrônico de Dados/legislação & jurisprudência , Regulamentação Governamental , Testes de Toxicidade , Toxicologia/legislação & jurisprudência , Animais , Células Cultivadas , Europa (Continente) , Humanos , Formulação de Políticas , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco , Terminologia como Assunto , Peixe-Zebra/embriologia
2.
ALTEX ; 37(3): 409-428, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32150624

RESUMO

Human cell-based neural organoids are increasingly being used for investigations of neurotoxicity, and to study the pathophysiology of neurodegenerative diseases. Here, we present a fast and robust method to generate 3D cultured human dopaminergic neurons (LUHMES) for toxicity testing and long-term culture. Moreover, a plating step was introduced to allow generation of neurite networks with defined 2D orientation and several mm length, while all cell bodies (somata) remained in a 3D, dome-like structure. These cultures, named here 2.5D (for 2.5 dimensional), offer new approaches to quantify toxicant effects on organoids by standard technology and high throughput. For instance, the system reacted to the parkinsonian model toxicants MPP+, rotenone, MG-132 and the ferroptosis-inducer erastin. Moreover, stable incorporation of human stem cell-derived astrocytes or microglia was possible. Added astrocytes stabilized the post mitotic state of the LUHMES neurons and thereby allowed the formation of a stable micro-physiological system. We observed neuroprotection against the proteasome inhibitor MG-132 and the ferroptosis-inducer erastin by such glia. This exemplifies the crucial protective role of astrocytes in neurodegeneration. The modularity of the system was further employed to incorporate microglia together with astrocytes into the organoids. Such ratio-defined, three cell type-based organoids will allow new approaches to study human pathophysiology and toxicology of the nervous system.


Assuntos
Astrócitos/fisiologia , Neurônios/fisiologia , Organoides/fisiologia , Células-Tronco/fisiologia , Alternativas aos Testes com Animais , Técnicas de Cocultura , Humanos
3.
ALTEX ; 37(1): 164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31960940

RESUMO

In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Further, the reference to an article by Bal-Price et al. (2015) should have the following doi:10.1007/s00204-015-1464-2 .

4.
ALTEX ; 37(1): 155-163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31833558

RESUMO

Many toxicological test methods, including assays of cell viability and function, require an evaluation of concentration-response data. This often involves curve fitting, and the resulting mathematical functions are then used to determine the concentration at which a certain deviation from the control value occurs (e.g. a decrease of cell viability by 15%). Such a threshold is called the benchmark response (BMR). For a toxicological test, it is often of interest to determine the concentration of test compound at which a pre-defined BMR of e.g. 10, 25 or 50% is reached. The concentration at which the modelled curve crosses the BMR is called the benchmark concentration (BMC). We present a user-friendly, web-based tool (BMCeasy), designed for operators without programming skills and profound statistical background, to determine BMCs and their confidence intervals. BMCeasy allows simultaneous analysis of viability plus a functional test endpoint, and it yields absolute BMCs with confidence intervals for any BMR. Besides an explanation of the algorithm underlying BMCeasy, this article also gives multiple examples of data outputs. BMCeasy was used within the EU-ToxRisk project for preparing data packages that were submitted to regulatory authorities, demonstrating the real-life applicability of the tool.


Assuntos
Benchmarking , Sobrevivência Celular/efeitos dos fármacos , Interpretação Estatística de Dados , Testes de Toxicidade/normas , Incerteza , Alternativas ao Uso de Animais , Animais
5.
Arch Toxicol ; 94(2): 449-467, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31828357

RESUMO

While there are many methods to quantify the synthesis, localization, and pool sizes of proteins and DNA during physiological responses and toxicological stress, only few approaches allow following the fate of carbohydrates. One of them is metabolic glycoengineering (MGE), which makes use of chemically modified sugars (CMS) that enter the cellular biosynthesis pathways leading to glycoproteins and glycolipids. The CMS can subsequently be coupled (via bio-orthogonal chemical reactions) to tags that are quantifiable by microscopic imaging. We asked here, whether MGE can be used in a quantitative and time-resolved way to study neuronal glycoprotein synthesis and its impairment. We focused on the detection of sialic acid (Sia), by feeding human neurons the biosynthetic precursor N-acetyl-mannosamine, modified by an azide tag. Using this system, we identified non-toxic conditions that allowed live cell labeling with high spatial and temporal resolution, as well as the quantification of cell surface Sia. Using combinations of immunostaining, chromatography, and western blotting, we quantified the percentage of cellular label incorporation and effects on glycoproteins such as polysialylated neural cell adhesion molecule. A specific imaging algorithm was used to quantify Sia incorporation into neuronal projections, as potential measure of complex cell function in toxicological studies. When various toxicants were studied, we identified a subgroup (mitochondrial respiration inhibitors) that affected neurite glycan levels several hours before any other viability parameter was affected. The MGE-based neurotoxicity assay, thus allowed the identification of subtle impairments of neurochemical function with very high sensitivity.


Assuntos
Membrana Celular/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Biologia Molecular/métodos , Ácido N-Acetilneuramínico/metabolismo , Síndromes Neurotóxicas/patologia , Bortezomib/farmacologia , Linhagem Celular , Glicoconjugados/química , Glicoconjugados/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hexosaminas/química , Hexosaminas/metabolismo , Hexosaminas/farmacologia , Humanos , Neuritos/química , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Síndromes Neurotóxicas/metabolismo , Tunicamicina/farmacologia
6.
ALTEX ; 36(4): 682-699, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31658359

RESUMO

Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.


Assuntos
Testes de Toxicidade/métodos , Animais , Estudos de Avaliação como Assunto , Humanos , Organização para a Cooperação e Desenvolvimento Econômico , Reprodutibilidade dos Testes , Projetos de Pesquisa , Testes de Toxicidade/normas
7.
ALTEX ; 36(3): 505, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329254

RESUMO

In this manuscript, which appeared in ALTEX 35 , 268-271 ( doi: 10.14573/1803231 ), the Acknowledgements should read: This work was supported by BMBF and DFG (KoRS-CB) grants, and it has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681002 (EU-ToxRisk).

8.
ALTEX ; 35(2): 268-271, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29984806

RESUMO

Many types of assays in cell biology, pharmacology and toxicology generate data in which a parameter is measured in a reference system (negative control) and then also under conditions of increasing stress or drug exposure. To make such data easily comparable, they are normalized, i.e., the initial value of the system (e.g., viability or transport function) is set to 100%, and all data are indicated relative to this value. Then, curves are fitted through the data points and summary data of the system behavior are determined. For this, a benchmark response (BMR) is given (e.g., a curve drop by 15 or 50%), and the corresponding benchmark concentration (BMC15 or BMC50) is determined. Especially for low BMRs, this procedure is not very robust and often results in incorrect summary data. It is often neglected that a second normalization (re-normalization) is necessary to make the data suitable for curve fitting. It is also frequently overlooked that this requires knowledge of the system behavior at very low stress conditions. Here, good in vitro practice guidance for the re-normalization procedure is provided so that data of higher fidelity can be generated and presented.


Assuntos
Algoritmos , Técnicas In Vitro , Modelos Estatísticos , Distribuição Normal , Animais , Fenômenos Fisiológicos Celulares , Humanos , Software
9.
Arch Toxicol ; 91(11): 3613-3632, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28477266

RESUMO

Many in vitro tests have been developed to screen for potential neurotoxicity. However, only few cell function-based tests have been used for comparative screening, and thus experience is scarce on how to confirm and evaluate screening hits. We addressed these questions for the neural crest cell migration test (cMINC). After an initial screen, a hit follow-up strategy was devised. A library of 75 compounds plus internal controls (NTP80-list), assembled by the National Toxicology Program of the USA (NTP) was used. It contained some known classes of (developmental) neurotoxic compounds. The primary screen yielded 23 confirmed hits, which comprised ten flame retardants, seven pesticides and six drug-like compounds. Comparison of concentration-response curves for migration and viability showed that all hits were specific. The extent to which migration was inhibited was 25-90%, and two organochlorine pesticides (DDT, heptachlor) were most efficient. In the second part of this study, (1) the cMINC assay was repeated under conditions that prevent proliferation; (2) a transwell migration assay was used as a different type of migration assay; (3) cells were traced to assess cell speed. Some toxicants had largely varying effects between assays, but each hit was confirmed in at least one additional test. This comparative study allows an estimate on how confidently the primary hits from a cell function-based screen can be considered as toxicants disturbing a key neurodevelopmental process. Testing of the NTP80-list in more assays will be highly interesting to assemble a test battery and to build prediction models for developmental toxicity.


Assuntos
Movimento Celular/efeitos dos fármacos , Crista Neural/citologia , Testes de Toxicidade/métodos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , DDT/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Heptacloro/toxicidade , Humanos , Crista Neural/efeitos dos fármacos , Imagem com Lapso de Tempo
10.
Arch Toxicol ; 91(1): 407-425, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26928308

RESUMO

Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuro-selective cytotoxicity at concentrations ≥10 µM. 6-(2-((6-oxido-6H-dibenzo[c,e][1,2]oxaphosphinin-6-yl)amino)ethoxy)-6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (ETA-DOPO) and 6,6'-(ethane-1,2-diylbis(oxy))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 µM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6'-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents. The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 µM. Based on the superior flame retardation properties, biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants.


Assuntos
Retardadores de Chama/toxicidade , Queratinócitos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Compostos Organofosforados/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Pele/efeitos dos fármacos , Células A549 , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Óxidos P-Cíclicos/toxicidade , Células-Tronco Embrionárias Humanas/citologia , Humanos , Queratinócitos/citologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Neurônios/citologia , Neurônios/imunologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Pele/citologia , Pele/imunologia , Pele/metabolismo , Absorção Cutânea , Testes de Irritação da Pele , Sus scrofa , Alicerces Teciduais/química , Testes de Toxicidade
11.
Int J Cardiovasc Imaging ; 31(5): 947-56, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25744427

RESUMO

The purpose of this study was to analyze comprehensively the heart using modern and sensitive myocardial techniques in order to determine if structural or functional cardiac alterations are present in adult Pompe disease. Twelve patients with adult Pompe disease and a control group of 187 healthy subjects of similar age and gender were included. Structural and functional cardiac characteristics were analyzed by conventional and 2D speckle-tracking echocardiography. In addition, in a subgroup of adult Pompe patients, we analyzed the myocardial and musculoskeletal features by means of cardiac and whole-body muscle magnetic resonance imaging. Patients with Pompe disease had significant structural and functional musculoskeletal alterations such as atrophy with fatty replacement and weakness in trunk and extremities. In contrast, Pompe patients had similar structural and functional myocardial features to healthy subjects (LV strain -20.7 ± 1.9 vs. -21.3 ± 2.1%; RV strain -24.2 ± 5.3 vs. -24.8 ± 3.8%; LA strain 41.5 ± 10.3 vs. 44.8 ± 11.0%; P > 0.05; and no evidence of LV and RV hypertrophy or LA enlargement). In addition, there was no evidence of valvular cardiac alterations, electrocardiographic abnormalities, or myocardial fibrosis in Pompe patients. In the current study analyzing the heart with modern and sensitive myocardial techniques, we evidenced that functional and structural cardiac alterations are not present when Pompe disease begins in adulthood. Therefore, these findings suggest that adult Pompe disease should not be taken into consideration in the differential diagnostic of structural or functional cardiac disorders.


Assuntos
Diagnóstico por Imagem , Doença de Depósito de Glicogênio Tipo II/complicações , Cardiopatias/diagnóstico , Miocárdio/patologia , Função Ventricular Esquerda , Função Ventricular Direita , Adulto , Estudos de Casos e Controles , Diagnóstico por Imagem/métodos , Ecocardiografia Doppler , Feminino , Fibrose , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Cardiopatias/etiologia , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/diagnóstico , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Direita/diagnóstico , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Valor Preditivo dos Testes , Fatores de Risco , Disfunção Ventricular Esquerda/diagnóstico , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Direita/diagnóstico , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/fisiopatologia , Adulto Jovem
12.
Int J Cardiovasc Imaging ; 31(2): 301-13, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25315709

RESUMO

The aim of this study was to test the hypothesis that in patients with Fabry disease, 2D speckle-tracking echocardiography (2DSTE) could detect functional myocardial alterations such as left ventricular (LV), right ventricular (RV), and left atrial (LA) dysfunction, even when conventional cardiac measurements are normal. In addition, we hypothesized that these global cardiac alterations could be linked to worse symptomatic status in these patients. Fifty patients with Fabry disease and a control group of 118 healthy subjects of similar age and gender were included. The myocardial function and structural changes of the LV, RV, and LA were analyzed by 2DSTE and cardiac magnetic resonance imaging. Patients with Fabry disease had significantly lower functional myocardial values of the LV, RV, and LA than healthy subjects (LV, RV, and LA strain -18.1 ± 4.0, -21.4 ± 4.9, and 29.7 ± 9.9 % vs. -21.6 ± 2.2, -25.2 ± 4.0, and 44.8 ± 11.1 %, respectively, P < 0.001) and elevated rates of LV, RV, and LA myocardial dysfunction (24, 20, and 26 %, respectively), even when conventional cardiac measurements such as LVEF, TAPSE, and LAVI were normal. LV septal wall thickness ≥15 mm, RV free wall thickness ≥7 mm, and LV longitudinal dysfunction were the principal factors linked to reduced LV, RV, and LA strain, respectively. In addition, but to a lesser extent, LV and RV fibrosis were linked to reduced LV and RV strain. Patients with reduced LV, RV, and LA strain had worse functional class (dyspnea-NYHA classification) than those with normal cardiac function. In conclusion, in patients with Fabry disease, 2DSTE analyses detect LV, RV, and LA functional myocardial alterations, even when conventional cardiac measurements are normal. These functional myocardial alterations are common and significantly associated with worse symptomatic status in Fabry patients. Therefore, these findings provide important evidence to introduce global myocardial analyses using 2DSTE in the early detection of functional cardiac alterations in Fabry disease.


Assuntos
Função do Átrio Esquerdo , Ecocardiografia Doppler , Doença de Fabry/diagnóstico por imagem , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Direita/diagnóstico por imagem , Função Ventricular Esquerda , Função Ventricular Direita , Adulto , Estudos de Casos e Controles , Doença de Fabry/complicações , Doença de Fabry/fisiopatologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/fisiopatologia
13.
J Am Soc Nephrol ; 25(4): 837-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24556354

RESUMO

Because of the shortage of agalsidase-beta in 2009, many patients with Fabry disease were treated with lower doses or were switched to agalsidase-alfa. This observational study assessed end-organ damage and clinical symptoms during dose reduction or switch to agalsidase-alfa. A total of 105 adult patients with Fabry disease who had received agalsidase-beta (1.0 mg/kg body weight) for ≥1 year were nonrandomly assigned to continue this treatment regimen (regular-dose group, n=38), receive a reduced dose of 0.3-0.5 mg/kg (dose-reduction group, n=29), or switch to 0.2 mg/kg agalsidase-alfa (switch group) and were followed prospectively for 1 year. We assessed clinical events (death, myocardial infarction, severe arrhythmia, stroke, progression to ESRD); changes in cardiac, renal, and neurologic function; and Fabry-related symptoms (neuropathic pain, hypohidrosis, diarrhea, and disease severity scores). Organ function and Fabry-related symptoms remained stable in the regular-dose group. In contrast, estimated GFR decreased by about 3 ml/min per 1.73 m(2) (P=0.01) in the dose-reduction group, and the median albumin-to-creatinine ratio increased from 114 (0-606) mg/g to 216 (0-2062) mg/g (P=0.03) in the switch group. Furthermore, mean Mainz Severity Score Index scores and frequencies of pain attacks, chronic pain, gastrointestinal pain, and diarrhea increased significantly in the dose-reduction and switch groups. In conclusion, patients receiving regular agalsidase-beta dose had a stable disease course, but dose reduction led to worsening of renal function and symptoms. Switching to agalsidase-alfa is safe, but microalbuminuria may progress and Fabry-related symptoms may deteriorate.


Assuntos
Terapia de Reposição de Enzimas , Doença de Fabry/tratamento farmacológico , Isoenzimas/uso terapêutico , alfa-Galactosidase/uso terapêutico , Adulto , Idoso , Doença de Fabry/fisiopatologia , Feminino , Taxa de Filtração Glomerular , Humanos , Isoenzimas/administração & dosagem , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteínas Recombinantes , alfa-Galactosidase/administração & dosagem
14.
Exp Cell Res ; 323(1): 131-143, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24480576

RESUMO

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.


Assuntos
Antineoplásicos/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Esferoides Celulares/efeitos dos fármacos , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Transporte de Elétrons/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Glucose/metabolismo , Humanos , Estaurosporina/farmacologia , Células Tumorais Cultivadas , Microambiente Tumoral/fisiologia
15.
Europace ; 14(2): 224-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21946820

RESUMO

AIMS: Mortality in chronic heart failure (CHF) patients with left bundle branch block (LBBB) is high. Cardiac resynchronization therapy (CRT) reduces symptoms and mortality in CHF patients with LBBB. Whether CRT promotes or prevents ventricular tachycardia (VT)/ventricular fibrillation (VF) remains controversial, however. Therefore, we aimed to analyse arrhythmia-related CRT effects and characterized the VT/VF incidence in CRT-defibrillator patients and matched controls with conventional implantable cardioverter-defibrillators (ICDs) for primary prevention of sudden cardiac death. METHODS AND RESULTS: We enrolled 134 patients [110 men, left ventricular ejection fraction (LVEF) 24 ± 8%, 71 coronary artery disease, CRT-ICD 67, conventional ICD matched controls 67, follow-up 31 ± 17 months] and monitored overall survival and the time to a first VT/VF episode. Controls did not have LBBB. They were otherwise matched for age, LVEF, and follow-up duration. Gender and underlying disease did not differ between the groups. Kaplan-Meier analysis revealed more favourable arrhythmia-free survival in CRT-ICD vs. conventional ICD patients [hazard ratio (HR) 2.26, confidence interval (CI) 1.09-4.67, log rank P = 0.023]. The difference persisted in the multivariate Cox regression analysis (HR 3.25, CI 1.18-8.93, P= 0.022). Overall survival was similar in both groups (HR 1.45, CI 0.55-3.82, P = 0.45). CONCLUSIONS: Chronic heart failure patients with LBBB treated with CRT-ICD, experience less and delayed VT/VF episodes compared with matched controls without LBBB receiving conventional ICD. In the long-term, CRT appears to exert antiarrhythmic effects and to attenuate the particularly high arrhythmia-related risk of CHF patients with LBBB. The incremental benefit of adding the ICD option to CRT pacing in LBBB patients appears questionable.


Assuntos
Bloqueio de Ramo/prevenção & controle , Terapia de Ressincronização Cardíaca/estatística & dados numéricos , Desfibriladores Implantáveis/estatística & dados numéricos , Taquicardia Ventricular/prevenção & controle , Fibrilação Ventricular/prevenção & controle , Bloqueio de Ramo/mortalidade , Estudos de Casos e Controles , Terapia Combinada/estatística & dados numéricos , Comorbidade , Intervalo Livre de Doença , Feminino , Alemanha/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Prevalência , Medição de Risco , Fatores de Risco , Análise de Sobrevida , Taxa de Sobrevida , Taquicardia Ventricular/mortalidade , Fibrilação Ventricular/mortalidade
16.
Cardiovasc Ther ; 29(4): 243-50, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20337635

RESUMO

Evidence-based treatment for heart failure (HF) comprises beta-blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and aldosterone receptor antagonists (ARA). Diuretics (DR) are prescribed in acute and chronic HF, but their impact on survival and ventricular tachyarrhythmias (VT/VF) is unclear. The present observational study aims to examine the influence of DR and ARA on survival and appropriate cardioverter/defibrillator (ICD) treatment episodes in routine ICD patients. In 352 consecutive ICD patients (291 men, 60 ± 12 years, LVEF 34 ± 15%, follow-up 37 ± 19 months) overall survival and the time to a first appropriate VT/VF episode were assessed. Electrograms were validated. Potassium and creatinine serum levels and the medical treatment regimen for heart failure were documented at baseline. Multivariate Cox regression analyses revealed significantly worse survival for patients with DR compared to those without DR (OR 0.24, CI 0.08-0.76, P= 0.016), whereas the group with ARA had better survival compared to patients without (OR 2.05, CI 1.02-4.10, P= 0.04). Patient groups did not differ regarding survival without incident VT/VF (DR+ vs. DR- OR 1.10, CI 0.67-1.83, P= 0.70; OR 0.66, CI 0.40-1.09, P= 0.10). Long-term survival appears to be compromised in ICD patients receiving concomitant DR, but is favorably influenced by ARA, although VT/VF incidence does not differ. Randomized analyses are warranted to assess long-term prognostic effects of DR in HF.


Assuntos
Desfibriladores Implantáveis , Diuréticos/uso terapêutico , Insuficiência Cardíaca/mortalidade , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Taquicardia Ventricular/epidemiologia , Fibrilação Ventricular/epidemiologia , Adulto , Idoso , Doença Crônica , Creatinina/sangue , Feminino , Insuficiência Cardíaca/terapia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade
17.
Europace ; 11(7): 924-30, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19447808

RESUMO

AIMS: Data from previous defibrillator studies raised concern about right ventricular pacing (RVP) promoting heart failure progression and mortality in implantable cardioverter/defibrillator (ICD) patients. The present observational study re-examined the association of RVP, survival, and ventricular tachyarrhythmias/ventricular fibrillation (VT/VF) in routine ICD patients with restrictively programmed pacing. METHODS AND RESULTS: In 213 ICD patients [183 men, left ventricular ejection fraction (LVEF) 37 +/- 15%, follow-up 37 +/- 18 months, no advanced atrioventricular (AV) block], the RVP proportion, survival, and the time to a first appropriate VT/VF episode were assessed. Electrograms were validated and the overall survival was determined. The RVP prevalence was dichotomized at > or = 30% (high RVP) vs. <30% (low RVP). High RVP (RVP 94%, n = 24) and low RVP (RVP 0%, n = 189) patients had similar LVEF, underlying heart disease, ICD indication, and medication. Multivariate Cox regression showed no difference in survival without appropriate VT/VF treatment [odds ratio (OR): 0.92, 95% confidence interval (CI): 0.41-2.04, P = 0.83]. Overall survival was significantly more favourable in low RVP patients (OR: 0.34, CI: 0.13-0.91, P = 0.03). CONCLUSION: Frequent RVP is associated with impaired survival in ICD patients despite conservative pacing settings. Implantable cardioverter/defibrillator patients requiring concomitant bradycardia pacing should be cared for with particular attention to clinical worsening. Right ventricular pacing prevention and alternative modalities of ventricular pacing need prospective evaluation.


Assuntos
Desfibriladores Implantáveis/estatística & dados numéricos , Cardioversão Elétrica/mortalidade , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/prevenção & controle , Disfunção Ventricular Direita/mortalidade , Disfunção Ventricular Direita/prevenção & controle , Feminino , Alemanha/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Medição de Risco/métodos , Fatores de Risco , Análise de Sobrevida , Taxa de Sobrevida , Taquicardia Ventricular , Resultado do Tratamento
18.
Pacing Clin Electrophysiol ; 32(5): 604-13, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19422581

RESUMO

BACKGROUND: Most patients with symptomatic sinus node disease (SND) receive DDDR pacemakers (PM) in order to cover SND and atrioventricular (AV) block from the outset. But the concern about adverse effects of right ventricular pacing (RVP) is increasing. So far, data on the incidence of AV block in SND are based on clinical events. The study undertakes to assess and appraise AV block and atrial tachyarrhythmias (AT) from memory and electrograms of a dual-chamber PM set to an AAIR-DDDR switch mode (AAISafeR). METHODS: A dual-chamber PM incorporating the AAISafeR mode was implanted in 58 patients (70 +/- 10 years, 28 males) with SND, but without AV block >I. AV block and AT episodes were retrieved from the PM memory and validated from electrograms. AV block episodes were classified potentially relevant while comprising AV block III or AV block I/II during exercise. RESULTS: The patients experienced a median of 90 (interquartile range 7-1,084) commutations. Possibly relevant AV block occurred in 32 patients (55%). Validation revealed high-quality PM-based categorization. The RVP prevalence was 0% (0-16%). The median AT prevalence was 0.03 (0-26) min/day. RVP was the only multivariate predictor of AT (P = 0.001). CONCLUSIONS: Potentially relevant AV block occurs frequently in patients with SND. Nonetheless, the RVP prevalence is kept low through the AAISafeR mode. The protection of SND patients with demand-actuated ventricular pacing appears reasonable. The AT prevalence is low in SND patients treated by the AAISafeR mode. Even low RVP proportions appear to favor AT. Prospective evaluation is needed.


Assuntos
Bloqueio Atrioventricular/complicações , Bloqueio Atrioventricular/diagnóstico , Diagnóstico por Computador/métodos , Eletrocardiografia/métodos , Marca-Passo Artificial , Síndrome do Nó Sinusal/complicações , Síndrome do Nó Sinusal/diagnóstico , Idoso , Feminino , Alemanha , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
Pacing Clin Electrophysiol ; 32 Suppl 1: S16-20, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19250084

RESUMO

BACKGROUND: Implantable cardioverter-defibrillators (ICD) implanted after an episode of ventricular tachyarrhythmia (VTA) or in patients at high risk of VTA lower the long-term mortality. Comparisons of the clinical outcomes of the two indications are scarce. METHODS: The study enrolled 360 consecutive ICD recipients. The device was implanted for secondary prevention in 150 patients, whose mean age was 60 +/- 14 years, and mean left ventricular ejection fraction (LVEF) was 40 +/- 16%, and for primary prevention in 210 patients, whose mean age was 61 +/- 11 years, and mean LVEF was 31 +/- 13%. All-cause mortality and time to first appropriate ICD therapy were measured. RESULTS: The two study groups were similar with respect to age and prevalence of coronary artery disease. Mean LVEF was higher in the secondary prevention group (P = 0.001). Cox regression analysis revealed a significantly shorter time to first appropriate ICD therapy in the secondary prevention group (HR = 0.51, 95% CI = 0.30 - 0.87, P = 0.01). Over a mean follow-up of 37 +/- 19 months, the all-cause mortality in the overall population was 12.7%, and was similar in both subgroups (HR = 0.99, 95% CI = 0.55-1.77, P = 0.97). CONCLUSIONS: The long-term mortality in this unselected population of ICD recipients was low. Patients treated for secondary prevention received earlier appropriate ICD therapy than patients treated for primary prevention. Long-term mortality was similar in both groups. The higher VT incidence of VTA was effectively treated by the ICD and was not associated with a higher mortality.


Assuntos
Morte Súbita Cardíaca/epidemiologia , Morte Súbita Cardíaca/prevenção & controle , Desfibriladores Implantáveis/estatística & dados numéricos , Cardioversão Elétrica/mortalidade , Medição de Risco/métodos , Taquicardia Ventricular/mortalidade , Taquicardia Ventricular/prevenção & controle , Comorbidade , Cardioversão Elétrica/instrumentação , Feminino , Alemanha/epidemiologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Análise de Sobrevida , Taxa de Sobrevida , Resultado do Tratamento
20.
Pacing Clin Electrophysiol ; 31(1): 70-7, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18181912

RESUMO

BACKGROUND: Cardiac resynchronization therapy (CRT) reduces the left ventricular diameter (LVEDD) in heart failure (HF) patients with left bundle branch block (LBBB). The study compares structural and electrical remodeling in HF patients on CRT and matched HF controls without LBBB. METHODS: In 42 patients (64 +/- 9 years left ventricular ejection fraction [LVEF] 25 +/- 8%, 16 coronary artery disease, 26 nonischemic cardiomyopathy, 21 with LBBB and CRT indication vs 21 controls [matched for gender, age, LVEF, and underlying disease]) an unpaced electrocardiogram (ECG) and echocardiogram were recorded at baseline (bl) and after 20.6 +/- 13.8 months (fup). LVEDD, left atrial (LA) width, mitral regurgitation (MR), P-wave, PR interval, QRS width, QRS vector, and QT interval were analyzed. RESULTS: LVEDD diminished with CRT (bl 68.7 +/- 10.3 vs fup 62.0 +/- 7.7 mm, P = 0.002). Controls showed no change (bl 64.1 +/- 9.4 vs fup 64.8 +/- 8.4 mm, P = n.s.). MR improved with CRT (bl 1.2 +/- 0.6 vs fup 0.8 +/- 0.7, P = 0.02), but not among controls. LA width tended to decrease on CRT (CRT bl 48.9 +/- 4.4 vs fup 46.9 +/- 7.2 mm, P = 0.17, controls bl 48.5 +/- 5.1 vs fup 47.5 +/- 6.5 mm, P = 0.49). PR interval lengthened in both groups (CRT bl 175 +/- 29 vs fup 188 +/- 30 ms, P = 0.03, controls bl 177+/-25 vs fup 187 +/- 19 ms, P = 0.27). QRS increased in both groups (CRT bl 165 +/- 22 vs fup 171 +/- 20 ms, P = 0.07, controls bl 111 +/- 17 vs fup 118 +/- 19 ms, P = 0.01). Analyses revealed no significant association of echocardiographic and ECG parameters. CONCLUSIONS: Despite LVEDD reduction with CRT, electrical activation does not recover. Electrical remodeling does not differ between LBBB patients under CRT and matched controls without CRT indication.


Assuntos
Bloqueio de Ramo/terapia , Estimulação Cardíaca Artificial/métodos , Insuficiência Cardíaca/terapia , Remodelação Ventricular , Idoso , Bloqueio de Ramo/diagnóstico por imagem , Bloqueio de Ramo/fisiopatologia , Estudos de Casos e Controles , Ecocardiografia , Eletrocardiografia , Feminino , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Estatísticas não Paramétricas
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