Fraunhofer Institute For Toxikologie Und Experimentelle Medizin

Hannover, Germany

Fraunhofer Institute For Toxikologie Und Experimentelle Medizin

Hannover, Germany
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Galuska S.P.,Justus Liebig University | Rollenhagen M.,Hannover Medical School | Kaup M.,Justus Liebig University | Eggers K.,Hannover Medical School | And 9 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths ofmore than 40 sialic acid residues was still observed in brain of newborn Ncam-/- mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam-/- brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam-/- background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam-/- brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings suggest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Wang J.,Leibniz Institute for Age Research | Wang J.,Friedrich - Schiller University of Jena | Lu X.,University of Regensburg | Sakk V.,Leibniz Institute for Age Research | And 4 more authors.
Blood | Year: 2014

Telomere shortening limits the proliferative capacity of human cells, and age-dependent shortening of telomeres occurs in somatic tissues including hematopoietic stem cells (HSCs). It is currently unknown whether genomic and molecular damage that occurs in HSCs induced by telomere shortening is transmitted to the progenitor cells. Here we show that telomere shortening results in DNA damage accumulation and gene expression changes in quiescent HSCs of aged mice. Upon activation, a subset of HSCs with elevated levels of DNA damage and p16 expression are blocked from cell cycle entry, and apoptosis is induced in HSCs entering the cell cycle. Activation of both checkpoints associates with normalization of DNA damage and gene expression profiles at early progenitor stages. These findings indicate that quiescent HSCs have an elevated tolerance to accumulate genomic alterations in response to telomere shortening, but the transmission of these aberrations to the progenitor cell level is prevented by senescence and apoptosis. © 2014 by The American Society of Hematology.

Koczulla A.R.,University of Marburg | Hattesohl A.,University of Marburg | Biller H.,Fraunhofer Institute For Toxikologie Und Experimentelle Medizin | Hofbauer J.,Ludwig Maximilians University of Munich | And 4 more authors.
Pneumologie | Year: 2011

Background: Volatile organic compounds (VOCs) can be used as biomarkers in exhaled air. VOC profiles can be detected by an array of nanosensors of an electronic nose. These profiles can be analysed using bioinformatics. It is, however, not known whether different devices of the same model measure identically and to which extent different set-ups and the humidity of the inhaled air influence the VOC profile. Methods: Three different measuring set-ups were designed and three healthy control subjects were measured with each of them, using four devices of the same model (Cyranose 320, Smiths Detection). The exhaled air was collected in a plastic bag. Either ambient air was used as reference (set-up Leipzig), or the reference air was humidified (100% relative humidity) (set-up Marburg and set-up Munich). In the set-up Marburg the subjects inhaled standardised medical air (Aer medicinalis Linde, AGA AB) out of a compressed air bottle through a demand valve; this air (after humidification) was also used as reference. In the set-up Leipzig the subjects inhaled VOC-filtered ambient air, in the set-up Munich unfiltered room air. The data were evaluated using either the real-time data or the changes in resistance as calculated by the device. Results: The results were clearly dependent on the set-up. Apparently, humidification of the reference air could reduce the variance between devices, but this result was also dependent on the evaluation method used. When comparing the three subjects, the set-ups Munich and Marburg mapped these in a similar way, whereas not only the signals but also the variance of the set-up Leipzig were larger. Conclusion: Measuring VOCs with an electronic nose has not yet been standardised and the set-up significantly affects the results. As other researchers use further methods, it is currently not possible to draw generally accepted conclusions. More systematic tests are required to find the most sensitive and reliable but still feasible set-up so that comparability is improved. © Georg Thieme Verlag KG Stuttgart New York.

Su Z.-H.,Peking Union Medical College | Zou G.-A.,Peking Union Medical College | Preiss A.,Fraunhofer Institute For Toxikologie Und Experimentelle Medizin | Zhang H.-W.,Peking Union Medical College | Zou Z.-M.,Peking Union Medical College
Journal of Pharmaceutical and Biomedical Analysis | Year: 2010

Chaihu-Shu-Gan-San (CSGS), a traditional Chinese medicine (TCM) formula containing seven herbal medicines, has been used in treatment of gastritis, peptic ulcer, irritable bowel syndrome and depression clinically. However, the chemical constituents in CSGS had not been studied so far. To quickly identify the chemical constituents of CSGS and to understand the chemical profiles related to antioxidant activity of CSGS, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC-LTQ-Orbitrap) mass spectrometry has been applied for online identification of chemical constituents in complex system, meanwhile, antioxidant profile of CSGS was investigated by the fraction collecting and microplate reading system. As a result, 33 chemical constituents in CSGS were identified. Among them, 13 components could be detected both in positive and in negative ion modes, 20 constituents were determined only in positive ion mode and 2 components were only detected in negative ion mode. Meanwhile, the potential antioxidant profile of CSGS was also characterized by combination of 96-well plate collection of elutes from HPLC analysis and microplate spectrophotometer, in which the scavenging activities of free radical produced by DPPH of each fraction could be directly investigated by the analysis of microplate reader. This study quickly screened the contribution of CSGS fractions to the antioxidant activity and online identified the corresponding active constituents. The results indicated that the combination of LC-MSn and 96-well plate assay system established in this paper would be a useful strategy for correlating the chemical profile of TCMs with their bioactivities without isolation and purification. © 2010.

Andersen G.,German Research Center for Food Chemistry | Burkon A.,German Research Center for Food Chemistry | Sulzmaier F.J.,German Research Center for Food Chemistry | Walker J.M.,University of Vienna | And 5 more authors.
Molecular Nutrition and Food Research | Year: 2011

Scope: trans-Resveratrol has been shown to improve insulin sensitivity and to enhance cellular glucose uptake. Evidence from recent studies indicates that these effects depend on SIRT1-pathways. Methods and results: Since ingestion of resveratrol leads to the presence of resveratrol and resveratrol metabolites in the body, we aimed at investigating (i) whether a daily dose of 300mg resveratrol/kg body weight in healthy male Wistar rats for a period of 8wk affects the selected parameters of glucose and lipid metabolism and (ii) whether the resulting plasma concentrations of resveratrol metabolites were effective in modulating SIRT1 expression. The dietary dose was based on the results from preceding toxicity studies. The results from the feeding experiment revealed plasma concentrations of resveratrol and its metabolites below 1μmol/L and showed that fasting glucose and insulin levels were decreased by 35 and 41%, respectively, in the resveratrol group compared with controls. Insulin sensitivity was enhanced by 70%, whereas liver SIRT1 protein expression was not affected. Treatment of HepG2 cells with 10μM resveratrol (1.49-fold) or its diglucuronides (1.21-fold) increased SIRT1 expression. Conclusion: These results suggest that the improved insulin sensitivity after dietary administration of 300mg resveratrol/kg body weight does not involve increased protein expression of SIRT1. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kornmann O.,University Hospital Mainz | Watz H.,Pulmonary Research Institute at LungenClinic Grosshansdorf | Fuhr R.,Parexel International | Krug N.,Fraunhofer Institute For Toxikologie Und Experimentelle Medizin | And 2 more authors.
Pulmonary Pharmacology and Therapeutics | Year: 2014

Background: When first approved in the European Union (EU), the omalizumab dosing table had upper bodyweight and IgE limits of 150kg and 700IU/mL, respectively. In this study, we assessed the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of omalizumab in patients with IgE/bodyweight combinations above those in the original dosing table. Methods: A multicentre, open-label, parallel-group study assessed the safety, PK and PD of omalizumab in 32 patients with mild-to-moderate allergic (IgE-mediated) asthma. Patients received two subcutaneous injections of omalizumab at one of three dosage levels (450, 525, or 600mg), chosen according to baseline IgE (300-2000IU/mL) and bodyweight (40-150kg), with a 14-day interval between injections. Results: Overall, 69 adverse events (AEs), none of them serious, were reported by 26 (81.3%) patients. Analysis of laboratory measurements, vital signs and ECG data revealed no adverse findings of clinical relevance. The PK profile was consistent with previous data for lower doses. Mean maximum decrease of free IgE from screening was ≥99% for all three doses, and mean free IgE concentrations remained <25ng/mL for at least 2 weeks after the second dose. The reductions in free IgE were consistent with levels previously associated with clinical improvements. Conclusions: The safety and PK/PD findings from this study are consistent with previous data, and supported the extension of the omalizumab dosing table to include those patients with higher IgE/bodyweight combinations.Clinical trial registry and registration number: (NCT00546143). © 2014 The Authors.

Ulmer A.,University of Tübingen | Dietz K.,University of Tübingen | Hodak I.,University of Regensburg | Polzer B.,University of Regensburg | And 15 more authors.
PLoS Medicine | Year: 2014

Background:Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.Methods and Findings:We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3-123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61-2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02-2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.Conclusions:Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically.Please see later in the article for the Editors' Summary. © 2014 Ulmer et al.

Batke M.,Fraunhofer Institute For Toxikologie Und Experimentelle Medizin | Gutlein M.,Johannes Gutenberg University Mainz | Partosch F.,Universitatsmedizin Gottingen | Gundert-Remy U.,Charité - Medical University of Berlin | And 5 more authors.
Frontiers in Pharmacology | Year: 2016

Interest is increasing in the development of non-animal methods for toxicological evaluations. These methods are however, particularly challenging for complex toxicological endpoints such as repeated dose toxicity. European Legislation, e.g., the European Union's Cosmetic Directive and REACH, demands the use of alternative methods. Frameworks, such as the Read-across Assessment Framework or the Adverse Outcome Pathway Knowledge Base, support the development of these methods. The aim of the project presented in this publication was to develop substance categories for a read-across with complex endpoints of toxicity based on existing databases. The basic conceptual approach was to combine structural similarity with shared mechanisms of action. Substances with similar chemical structure and toxicological profile form candidate categories suitable for read-across. We combined two databases on repeated dose toxicity, RepDose database, and ELINCS database to form a common database for the identification of categories. The resulting database contained physicochemical, structural, and toxicological data, which were refined and curated for cluster analyses. We applied the Predictive Clustering Tree (PCT) approach for clustering chemicals based on structural and on toxicological information to detect groups of chemicals with similar toxic profiles and pathways/mechanisms of toxicity. As many of the experimental toxicity values were not available, this data was imputed by predicting them with a multi-label classification method, prior to clustering. The clustering results were evaluated by assessing chemical and toxicological similarities with the aim of identifying clusters with a concordance between structural information and toxicity profiles/mechanisms. From these chosen clusters, seven were selected for a quantitative read-across, based on a small ratio of NOAEL of the members with the highest and the lowest NOAEL in the cluster ( < 5). We discuss the limitations of the approach. Based on this analysis we propose improvements for a follow-up approach, such as incorporation of metabolic information and more detailed mechanistic information. The software enables the user to allocate a substance in a cluster and to use this information for a possible read- across. The clustering tool is provided as a free web service, accessible at © 2016 Batke, Gütlein, Partosch, Gundert-Remy, Helma, Kramer, Maunz, Seeland and Bitsch.

Paranjpe M.,TU Braunschweig | Neuhaus V.,Fraunhofer Institute For Toxikologie Und Experimentelle Medizin | Braun A.,Fraunhofer Institute For Toxikologie Und Experimentelle Medizin | Mueller-Goymann C.C.,TU Braunschweig
European Journal of Lipid Science and Technology | Year: 2014

The aim of this study was to formulate sildenafil base-loaded liposomes using a solvent-free method, followed by the investigation of the potential cytotoxicity of the prepared liposomes in in vitro and ex vivo cell culture models. The liposomes were manufactured by dispersing 5%w/v phospholipid in aqueous phase at RT. The prepared dispersion was then homogenized using an Avestin extruder. The particle size of the prepared liposomes was between 100 and 110nm and was stable up to 6 months. For in vitro models, human alveolar epithelial cell line (A549) and mouse heart endothelium cell line (MHEC5-T) were used. For ex vivomodels, rat precision cut lung slices (PCLS) and rat heart slices (PCHS) were used. All the models were treated with liposomes loaded with or without sildenafil in a concentration range of 0- 2500μg/mL of phospholipid. The toxicity was assessed in vitro and ex vivo using MTT assay. Median lethal dose (LD50) values for A549 cells and PCLS were found between 800 and 1800μg/mL while for MHEC5-T cells and PCHS they were found between 1000 and 1200μg/mL. PCHS demonstrated higher LD50 values as compared to PCLS. Considering the high LD50 values, sildenafil-loaded liposomes may have the potential for the treatment of pulmonary hypertension via inhalation route. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Paranjpe M.,TU Braunschweig | Neuhaus V.,Fraunhofer Institute For Toxikologie Und Experimentelle Medizin | Finke J.H.,TU Braunschweig | Richter C.,TU Braunschweig | And 5 more authors.
Inhalation Toxicology | Year: 2013

The aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) loaded with sildenafil. The SLNs were tested as a new drug delivery system (DDS) for the inhalable treatment of pulmonary hypertension in human lungs. Solubility of sildenafil in SLN lipid matrix (30:70 phospholipid:triglyceride) was determined to 1% sildenafil base and 0.1% sildenafil citrate, respectively. Sildenafil-loaded SLN with particle size of approximately 180 nm and monomodal particle size distribution were successfully manufactured using a novel microchannel homogenization method and were stable up to three months. Sildenafil-loaded SLN were then used in in vitro and ex vivo models representing lung and heart tissue. For in vitro models, human alveolar epithelial cell line (A459) and mouse heart endothelium cell line (MHEC5-T) were used. For ex vivo models, rat precision cut lung slices (PCLS) and rat heart slices (PCHS) were used. All the models were treated with plain SLN and sildenafil-loaded SLN in a concentration range of 0-5000 μg/ml of lipid matrix. The toxicity was evaluated in vitro and ex vivo by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Median lethal dose 50% (LD50) values for A549 cells and PCLS were found to be in the range of 1200-1900 μg/ml while for MHEC5-T cells and precision cut heart slices values were found between 1500 and 2800 μg/ml. PCHS showed slightly higher LD50 values in comparison to PCLS. Considering the toxicological aspects, sildenafil-loaded SLN could have potential in the treatment of pulmonary hypertension via inhalation route. © 2013 Informa Healthcare USA, Inc.

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