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See detailIn Silico Modeling for the Prediction of Dose and Pathway-Related Adverse Effects in Humans From In Vitro Repeated-Dose Studies.
Klein, Sebastian; Maggioni, Silvia; Bucher, Joachim et al

in Toxicological sciences : an official journal of the Society of Toxicology (2016), 149(1), 55-66

Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Nowadays advanced human in vitro systems, eg, metabolically competent HepaRG ... [more ▼]

Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Nowadays advanced human in vitro systems, eg, metabolically competent HepaRG cells, are used for toxicity screening. Extrapolation of in vitro toxicity to in vivo effects is possible by reverse dosimetry using pharmacokinetic modeling. We assessed long-term repeated-dose toxicity of bosentan and valproic acid (VPA) in HepaRG cells under serum-free conditions. Upon 28-day exposure, the EC50 values for bosentan and VPA decreased by 21- and 33-fold, respectively. Using EC(10) as lowest threshold of toxicity in vitro, we estimated the oral equivalent doses for both test compounds using a simplified pharmacokinetic model for the extrapolation of in vitro toxicity to in vivo effect. The model predicts that bosentan is safe at the considered dose under the assumed conditions upon 4 weeks exposure. For VPA, hepatotoxicity is predicted for 4% and 47% of the virtual population at the maximum recommended daily dose after 3 and 4 weeks of exposure, respectively. We also investigated the changes in the central carbon metabolism of HepaRG cells exposed to orally bioavailable concentrations of both drugs. These concentrations are below the 28-day EC(10) and induce significant changes especially in glucose metabolism and urea production. These metabolic changes may have a pronounced impact in susceptible patients such as those with compromised liver function and urea cycle deficiency leading to idiosyncratic toxicity. We show that the combination of modeling based on in vitro repeated-dose data and metabolic changes allows the prediction of human relevant in vivo toxicity with mechanistic insights. [less ▲]

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See detailFluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.
Priesnitz, C.; Sperber, S.; Garg, R. et al

in Cytotechnology (2016), 68(4), 1647-53

Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the ... [more ▼]

Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10 % standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably normalized to the exact viable cell number at a given time to allow meaningful interpretation and understanding of the biological processes. Manual counting of cell number is very laborious and not possible in certain experimental setups. We therefore, developed a simple and reliable fluorescence based method with an accuracy of 95-98 % for the determination of the viable cell number in situ. We optimized the seeding cell densities for primary rat hepatocytes for optimal cell adhesion. This will help in efficient use of primary cells which are usually limited in availability. The method will be very useful in the application of "-omics" techniques, especially metabolome analysis where the specific rates of uptake/production of metabolites can be reliably calculated. [less ▲]

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See detailA shift in paradigm towards human biology-based systems for cholestatic-liver diseases.
Noor, Fozia UL

in The Journal of physiology (2015), 593(23), 5043-55

Cholestatic-liver diseases (CLDs) arise from diverse causes ranging from genetic factors to drug-induced cholestasis. The so-called diseases of civilization (obesity, diabetes, metabolic disorders, non ... [more ▼]

Cholestatic-liver diseases (CLDs) arise from diverse causes ranging from genetic factors to drug-induced cholestasis. The so-called diseases of civilization (obesity, diabetes, metabolic disorders, non-alcoholic liver disease, cardiovascular diseases, etc.) are intricately implicated in liver and gall bladder diseases. Although CLDs have been extensively studied, there seem to be important gaps in the understanding of human disease. Despite the fact that many animal models exist and substantial clinical data are available, translation of this knowledge towards therapy has been disappointingly limited. Recent advances in liver cell culture such as in vivo-like 3D cultivation of human primary hepatic cells, human induced pluripotent stem cell-derived hepatocytes; and cutting-edge analytical techniques such as 'omics' technologies and high-content screenings could play a decisive role in deeper mechanistic understanding of CLDs. This Topical Review proposes a roadmap to human biology-based research using omics technologies providing quantitative information on mechanisms in an adverse outcome/disease pathway framework. With modern sensitive tools, a shift in paradigm in human disease research seems timely and even inevitable to overcome species barriers in translation. [less ▲]

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See detailFinal report of the effectiveness study in Virtual Coach Reaches Out to me project
Aleksic, Gabrijela UL; Roelofsma, Peter

Report (2014)

One out of three persons in our society suffers from social or emotional loneliness. This percentage is even higher in the population of people who suffer from chronic diseases. Loneliness has pervasive ... [more ▼]

One out of three persons in our society suffers from social or emotional loneliness. This percentage is even higher in the population of people who suffer from chronic diseases. Loneliness has pervasive effects on mental health but it also has negative effects on physical well-being. The aim of this paper is to present a series of pilot studies evaluating the development of an ambient virtual coaching system. This system, called V2me (Virtual Coach reaches Out to Me) offers a friendship enrichment course for people in need for improving their social network. Several pilot studies were performed with about 50 participants in total who evaluated the system that was developed using a living lab approach. In this approach new health media was developed from a user-centered process that allows frequent iterations of user evaluation and involvement. The paper presents the results of the first five iterations. The reactions on receiving the system and experiences during instruction were increasingly positive over these iterations of the system development phase. Over time the system has passed the user’s choice selection criteria. Participants have been expressing a clear interest for choosing and wanting to use the system in their daily lives. However, the system did not pass the persistent use selection criteria when the system was brought and left for independent use. It appeared that participants did not use it very much during the day as expected. Moreover, participants did not perform all the tasks (i.e., messaging, Skyping) that they were asked to achieve on a daily basis. The final goal of the V2me system, i.e. establishing social relations between elderly persons, was not achieved as well, although this might be due to the limited time space of using the system and its facilities. Given these observation, it is concluded that more iterations in the system development are needed for the system in order to pass the habitual use criteria which is needed for its effectiveness. [less ▲]

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See detailState-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.
Alepee, Natalie; Bahinski, Anthony; Daneshian, Mardas et al

in ALTEX (2014), 31(4), 441-77

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk ... [more ▼]

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing. [less ▲]

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See detailLong-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study.
Klein, Sebastian; Mueller, Daniel; Schevchenko, Valery et al

in Journal of applied toxicology : JAT (2014), 34(10), 1078-86

Chronic repeated-dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species-specific differences in biotransformation. The ... [more ▼]

Chronic repeated-dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species-specific differences in biotransformation. The human hepatoma cell line HepaRG has been used for human relevant toxicity assessment. However, HepaRG cells are commonly maintained in serum containing medium which limits their use in 'omics'-based toxicology. In this study, we compared the maintenance of HepaRG cells in standard serum-supplemented and serum-free conditions. Viability and Cytochrome P450 (CYP) activity during long-term cultivation were assessed. Liver-specific albumin and urea production was measured. The extracellular metabolome (amino acids, glucose, lactate and pyruvate) was measured to compare different cultivation conditions using metabolic flux analysis. Although metabolic flux analysis reveals differences in certain parts of the metabolism, e.g. production of urea, the overall metabolism of serum-free and serum-supplemented cultured HepaRG cells is similar. We conclude that HepaRG cells can be maintained in optimized serum-free conditions for 30 days without viability change and with high CYP activity. We also tested the acute (24 h) and long-term repeated-dose (7 doses, every second day) toxicity of valproic acid. We calculated an EC50 value of 1.4 mM after repeated exposure which is close to the cmax value for valproic acid. Maintenance of HepaRG cells in serum-free conditions opens up the opportunity for the use of these cells in human long-term repeated-dose hepatotoxicity studies and for application in systems toxicology. [less ▲]

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See detail3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies.
Mueller, Daniel; Kramer, Lisa; Hoffmann, Esther et al

in Toxicology in vitro : an international journal published in association with BIBRA (2014), 28(1), 104-12

Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between ... [more ▼]

Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between experimental in vitro and in vivo data. Using 3D HepaRG organotypic cultures we tested four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) for toxic effects and compared the results with 2D HepaRG and HepG2 cultures. We show that 3D HepaRG cultures are more sensitive than the other tested cultures to aflatoxin B1 which is only toxic upon metabolic activation in the liver. We observed that CYP3A4 activity is higher in the 3D HepaRG cultures compared to the 2D HepaRG cultures. Furthermore, we investigated repeated dose toxicity of chlorpromazine and assessed its effects on glucose and lactate metabolism. Sub-toxic concentrations of chlorpromazine induced significant metabolic changes in both 2D and 3D HepaRG cultures upon acute and repeated dose (3 doses) exposure. In summary, our data support the hypothesis that 3D cell culture models better mimic the in vivo tissue and improve cellular functionality. The 3D HepaRG organotypic cultures represent a high throughput system for drug toxicity screening. This system is therefore a promising tool in preclinical testing of human relevance which can allow reducing and/or replacing animal testing for drug adverse effects. [less ▲]

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See detailEvaluation of V2me: A Virtual Coaching System for Loneliness Prevention and Intervention
Roelofsma, Peter; Ferring, Dieter UL; Aleksic, Gabrijela UL

in Medicine 2.0: Social Media, Mobile Apps and Internet/Web 2.0 (2013, September)

One out of three persons in our society suffers from social or emotional loneliness. This percentage is even higher in the population of people who suffer from chronic diseases. Loneliness has pervasive ... [more ▼]

One out of three persons in our society suffers from social or emotional loneliness. This percentage is even higher in the population of people who suffer from chronic diseases. Loneliness has pervasive effects on mental health but it also has negative effects on physical well-being. The aim of this paper is to present a series of pilot studies evaluating the development of an ambient virtual coaching system. This system, called V2me (Virtual Coach reaches Out to Me) offers a friendship enrichment course for people in need for improving their social network. Several pilot studies were performed with about 50 participants in total who evaluated the system that was developed using a living lab approach. In this approach new health media was developed from a user-centered process that allows frequent iterations of user evaluation and involvement. The paper presents the results of the first five iterations. The reactions on receiving the system and experiences during instruction were increasingly positive over these iterations of the system development phase. Over time the system has passed the user’s choice selection criteria. Participants have been expressing a clear interest for choosing and wanting to use the system in their daily lives. However, the system did not pass the persistent use selection criteria when the system was brought and left for independent use. It appeared that participants did not use it very much during the day as expected. Moreover, participants did not perform all the tasks (i.e., messaging, Skyping) that they were asked to achieve on a daily basis. The final goal of the V2me system, i.e. establishing social relations between elderly persons, was not achieved as well, although this might be due to the limited time space of using the system and its facilities. Given these observation, it is concluded that more iterations in the system development are needed for the system in order to pass the habitual use criteria which is needed for its effectiveness. [less ▲]

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See detailImportance de la langue maternelle pour le development des enfants au Luxembourg
Aleksic, Gabrijela UL

Article for general public (2013)

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See detailA l'école les programmes bilingues sont très efficaces
Aleksic, Gabrijela UL

Article for general public (2013)

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See detailVirtual Coach Reaches Out To Me
Aleksic, Gabrijela UL

Conference given outside the academic context (2013)

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See detailVirtual Coach Reaches Out To Me
Aleksic, Gabrijela UL

Conference given outside the academic context (2013)

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See detailMetabolomics in toxicology and preclinical research.
Ramirez, Tzutzuy; Daneshian, Mardas; Kamp, Hennicke et al

in ALTEX (2013), 30(2), 209-25

Metabolomics, the comprehensive analysis of metabolites in a biological system, provides detailed information about the biochemical/physiological status of a biological system, and about the changes ... [more ▼]

Metabolomics, the comprehensive analysis of metabolites in a biological system, provides detailed information about the biochemical/physiological status of a biological system, and about the changes caused by chemicals. Metabolomics analysis is used in many fields, ranging from the analysis of the physiological status of genetically modified organisms in safety science to the evaluation of human health conditions. In toxicology, metabolomics is the -omics discipline that is most closely related to classical knowledge of disturbed biochemical pathways. It allows rapid identification of the potential targets of a hazardous compound. It can give information on target organs and often can help to improve our understanding regarding the mode-of-action of a given compound. Such insights aid the discovery of biomarkers that either indicate pathophysiological conditions or help the monitoring of the efficacy of drug therapies. The first toxicological applications of metabolomics were for mechanistic research, but different ways to use the technology in a regulatory context are being explored. Ideally, further progress in that direction will position the metabolomics approach to address the challenges of toxicology of the 21st century. To address these issues, scientists from academia, industry, and regulatory bodies came together in a workshop to discuss the current status of applied metabolomics and its potential in the safety assessment of compounds. We report here on the conclusions of three working groups addressing questions regarding 1) metabolomics for in vitro studies 2) the appropriate use of metabolomics in systems toxicology, and 3) use of metabolomics in a regulatory context. [less ▲]

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See detail3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies.
Gunness, Patrina; Mueller, Daniel; Shevchenko, Valery et al

in Toxicological sciences : an official journal of the Society of Toxicology (2013), 133(1), 67-78

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a ... [more ▼]

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity. [less ▲]

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See detailReal-time in situ viability assessment in a 3D bioreactor with liver cells using resazurin assay.
Mueller, Daniel; Tascher, Georg; Damm, Georg et al

in Cytotechnology (2013), 65(2), 297-305

Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is ... [more ▼]

Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is very difficult in such systems. Commonly applied indirect methods such as glucose consumption, albumin or urea production are greatly affected by culture conditions, stress and time of cultivation and do not reflect the real time viability of the cells. In this study we established a real-time in situ viability assay namely; resazurin assay, in a 3D hollow-fiber bioreactor using human liver cells. Resazurin assay is based on the conversion of resazurin to a fluorescent dye by cytoplasmatic and mitochondrial enzymes. We show that the resazurin reagent in concentrations used in this study is non-toxic and could be rapidly removed out of the system. Moreover, we observed that dead cells do not affect the results of the assay. We optimized the assay on HepG2 cells and tested it with primary human hepatocytes. Moreover, we maintained primary human hepatocytes in the 3D bioreactor system in serum-free conditions and also assessed viability before and after the exposure to amiodarone using the resazurin assay. We show that this approach is applicable during long-term cultivation of cells in bioreactors under different conditions and can moreover be applied to pharmacological studies, e.g. investigation of chronic drug effects in such 3D bioreactors. [less ▲]

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See detailDoppelt halt besser
Aleksic, Gabrijela UL

Article for general public (2013)

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See detailPortugiesen im Nachteil
Aleksic, Gabrijela UL

Article for general public (2013)

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Peer Reviewed
See detailBiotransformation of diclofenac and effects on the metabolome of primary human hepatocytes upon repeated dose exposure.
Mueller, Daniel; Muller-Vieira, Ursula; Biemel, Klaus M. et al

in European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences (2012), 45(5), 716-24

In vitro repeated dose testing for the assessment of chronic drug-induced effects is a huge challenge in preclinical pharmaceutical drug development. Chronic toxicity results in discontinuation of therapy ... [more ▼]

In vitro repeated dose testing for the assessment of chronic drug-induced effects is a huge challenge in preclinical pharmaceutical drug development. Chronic toxicity results in discontinuation of therapy or post-marketing withdrawal of drugs despite in vivo preclinical screening. In case of hepatotoxicity, due to limited long term viability and functionality of primary hepatocytes, chronic hepatic effects are difficult to detect. In this study, we maintained primary human hepatocytes in a serum-free cultivation medium for more than 3 weeks and analyzed physiology, viability and drug metabolizing capacities of the hepatocytes. Moreover, we assessed acute (24 h) diclofenac toxicity in a range of (10-1000 muM) concentrations. The chronic (9 repeated doses) toxicity at one clinically relevant and another higher concentration (6.4 and 100 muM) was also tested. We investigated phase I and II metabolism of diclofenac upon repeated dose exposure and analyzed effects on the cellular exometabolome. Acute 24 h assessment revealed toxicity only for the highest tested concentration (1 mM). Upon repeated dose exposure, toxic effects were observed even at a low, clinically relevant concentration (6.4 muM). Biotransformation pathways were active for 3 weeks and diclofenac-acylglucuronide was detected as the predominant metabolite. Dose dependent diclofenac-induced effects on exometabolome, such as on the production of lactate and 3-hydroxybutyric acid as well as glucose and galactose metabolism, were observed upon nine repeated doses. Summarizing, we show that repeated dose testing on long-term functional cultures of primary human hepatocytes may be included for the assessment of long term toxic effects in preclinical screening and can potentially help replace/reduce in vivo animal testing. [less ▲]

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