Linz 2003: Abstracts der Vorträge und Poster (alphabetisch nach Erstautor/inn/en geordnet, bei Vorträgen ist der Vortragende zuerst genannt)

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1 Linz 2003: Abstracts der Vorträge und (alphabetisch nach Erstautor/inn/en geordnet, bei Vorträgen ist der Vortragende zuerst genannt) Analysis of sensitizing chemicals in vitro by testing their potential to activate dendritic cells and to penetrate pig skin Pierre Aeby 1, Christoph Wyss 1, Heinz Beck 1 and Carsten Goebel 2 1 Cosmital SA (Research company of Wella AG), CH-Marly, 2 Department of product safety, Toxicology, Wella AG, D- Darmstadt In vitro sensitization tests are needed to identify the relevant aspects of the complex interactions of a chemical with the different compartments of the immune system. Furthermore, they receive public interest since animal testing should be avoided whenever possible. We propose a new approach, analyzing the following essential properties of sensitizing chemicals: skin penetration, protein binding, metabolism and dendritic cell (DC) activation. Activation of immature DCs derived from peripheral blood monocytes was evaluated by flow cytometric analysis of CD86 positive cells and quantitative measurement of the mrna expression of classical (interleukin-1ß) as well as newly identified activation markers (Aquaporin P3) using the Lightcycler real time PCR system. Bioavailability was evaluated by in vitro skin penetration measurement using the pig skin model, a protein binding assay and metabolic studies. Aromatic amine derivatives and the known sensitizer 2,4,6-trinitrobenzenesulfonic acid were tested according to that strategy and induced substantial modulation of DC activation markers reflecting their potential sensitizing properties. The irritant sodium lauryl sulfate did not trigger any relevant response. These in vitro data (DC activation, skin penetration, protein binding and metabolism study) were compared to the results of an in vivo murine local lymph node assay. We conclude that using adequate culture conditions and measurement time points, potential sensitizers can be identified due to their capacity to induce specific DC activation in vitro, when an estimation of the bioavailability is possible. Furthermore, we propose a concept to integrate in vitro protein binding and in vitro metabolism into a DC based in vitro test system. The effect of solvents on the embryonic stem cell differentiation Sarah Adler, Cristian Pellizzer, Martin Paparella, Thomas Hartung and Susanne Bremer ECVAM, Joint Research Centre, Institute for Health and Consumer Protection, I-Ispra DMSO is a common industrial solvent as it is an amphiphilic compound and therefore capable to solve a large range of substances. It is well known that DMSO has a differentiation inducing effect on different kind of cells including embryonic teratocarcinoma and embryonic stem cells. In this study we have analysed effects of DMSO and ethanol during in vitro differentiation of pluripotent cells. Cells of the teratocarcinoma cell line P19 have been transfected with the catalytical subunit of telomerase promoter linked to the green fluorescent protein gene (mtert_gfp/p19). Telomerase as well as mtert are well-known to be active in undifferentiated cells and both are down-regulated upon differentiation. In a previous study, it was found that DMSO induced cell differentiation of mtert_gfp/p19 cells in concentrations that are lower than the ones used in the validated embryonic stem cell test. In order to understand if similar effects are induced on embryonic stem cell differentiation we have tested different DMSO concentrations on the transgenic embryonic stem cell line D3 (mtert_gfp/d3). Differentiation inducing effects were detectable already in the lowest applied concentration of % DMSO in transgenic P19 cells as well as in transgenic D3 cells, while Ethanol did not show any differentiation inducing effects. In order to confirm these results we have investigated the expression of the marker gene OCT-4 in wildtype D3 cells after exposure to the solvents. OCT-4 is mainly expressed in undifferentiated cells and its expression is decreasing during the cell differentiation. Since embryonic stem cells will be an important tool for in vitro embryotoxicity testing effects of solvents on the cell system have to be excluded. This study shows that the use of most common solvent i.e. DMSO, needs to be carefully considered. Toxicogenomics: a novel perspective for 3R Hans J. Ahr

2 Bayer AG, D-Wuppertal During the recent years, powerful novel technologies became available in the areas of molecular biology and bioinformatics, which may also offer new opportunities for toxicology. When used in toxicological studies both in vitro and in vivo especially the DNA microarrays allow the in depth investigation of alterations of intracellular metabolic and regulatory pathways under the influence of chemical stressors. At the time being, this new discipline toxicogenomics - already contributes in an unprecedented manner to elucidate the mechanisms, which underly toxicological effects. By identifying specific pattern in the expression of characteristic genes the so-called fingerprints and correlating them to defined toxicological phenotypes, the toxicogenomic analysis will also allow predictions on a toxicological outcome, which may be faster, more sensitive, or more specific than those of conventional toxicological test systems. This may result in novel test systems utilizing toxicogenomic methods for the rapid and sensitive detection of toxicological effects. They bear the potential to refine the toxicological in vivo tests in making them shorter, use less animals, and provide more reliable data. The implementation of toxicogenomic techniques may also improve in vitro test systems. Mid term toxicogenomics may thus relevantly contribute to the realization of 3R. Bovine spermatozoa a suitable cell model for pharmacological studies: effects of some homeopathic drugs on mitochondrial activity and other important parameters of cells function Dhafer M. Aziz 1, Ulrich Janowitz 2, Ute Schnurrbusch 3 and Heinrich Enbergs 1 1 Institute of Physiology, Biochemistry and Hygiene of Animals, Bonn University, D- Bonn, 2 Rinder Union West, D-Borken, 3 Faculty of Veterinary Medicine, D-Leipzig We used bovine spermatozoa as a cell model for our pharmacological studies because these cells are adult differentiated mammalian cells with all important cell elements. The potential user can get them from artificial insemination stations, where they are stored and kept alive for many years. In this study we tested the effect of 3 commercial homeopathic drugs (Ubichinon Comp., Coenzyme Comp. and Selenium-Homaccord, Heel, Baden-Baden, Germany) on mitochondrial activity flowcytometrically using Rhodamine 123. To evaluate the possible side effects of the tested drugs on the integrity of the cell membrane, the acrosome and chromatin structure, SYBR-14, Lyso Tracker Green DNA-20 and Acridene orange were used, respectively. Frozen semen from 10 bulls was prepared using routine diluents + homeopathic drugs in two concentrations: 1:9 and 1:4. Sperm mitochondrial activity was increased significantly (P<0.05) in the following groups: 1:9 Coenzyme Comp., 1:4 Ubichinon Comp., 1:9 and 1:4 Selenium-Homaccord. There were no side effects of these drugs on the above mentioned cell parameters. In conclusion, the tested homeopathic drugs can be used to stimulate the mitochondrial activity and therefore the oxidative energy metabolism of sperms and also of other mammalian cells. So sperm cells offer an innovative, very sensitive and promising supplement or alternative to the use of cell cultures and laboratory animals for pharmacological studies. Comparison of drug uptake via Caco-2 cells and blood brain barriers cells Udo Bock and Thomas Flötotto Across Barriers, Science Park Saar, D-Saarbrücken Recently the German Federal Institute for Drugs and Medical Devices (BfArm) updated in an announcement the approval of drugs concerning 21 of the German AMG (Arzneimittelgesetz) for Bioavailability and Bioequivalence studies. The basis for the BfArm decisions was the EMEA Note for guidance, which is in coherence to the guidance of the U.S. Department of Health and Human Services, the Food and Drug Administration (FDA), which was published in 2000 as Waiver of In vitro Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System. The FDA and EMA guideline lists various in vitro methods acceptable to evaluate the permeability class of a drug substance. The list of methods includes monolayers of suitable epithelial cells. One epithelial cell line that has been widely used as a model system of intestinal permeability is the Caco-2 cell line. According to the recommendations of FDA and EMEA the Caco-2 cell culture model was validated for the classification of compounds in terms of permeability. By determining the permeability at the Caco-2 system a correlation was established between rank order of the permeability and the extent of fractional absorption in humans for a large number of model compounds. Absorption of model compounds is largely influenced by the activity of the ATP-binding cassette transporters (ABC-transporters)-efflux transporters. We therefore analysed the activity of ABC-transporters involved in Caco-2 cells, using substrates specific and unspecific for ABC-transporter subtypes. In addition, the active transport of model compounds was blocked by inhibitors specific for different ABC-transporter subtypes to analyse the role of different receptors in

3 transport of specific compounds. Similar experiments were performed using primary cells from porcine capillary endothelial cells, simulating the blood brain barrier. The transport mechanisms involved in both model systems were compared, to demonstrate the necessity of specific test systems for different epithelial drug barriers. EU-Weissbuch: Einsatz von in vitro Modellen und physikochemischen Methoden Udo Bock, Peter Lach and Eleonore Haltner Across Barriers GmbH, Science Park Saar, D-Saarbrücken Je nach Exposition einer Chemikalie bzw. des Fertigproduktes sind als Hauptaufnahmewege in den menschlichen Körper insbesondere der Gatrointestinaltrakt (Nahrung), der Respirationstrakt (Atmung) sowie die Haut zu nennen. Dabei müssen insbesondere Anwendungsprofil und Frequenz der Anwendung der Chemikalie berücksichtigt werden. Anhand dieser Randbedingungen sollte die Auswahl von in vitro Methoden zur Prüfung auf Absorption getroffen werden. Als wesentlichen Bestandteil dieser Prüfungen sind die Bestimmungen der physikochemischen Eigenschaften der Chemikalie im Vorfeld zur Planung und Absicherung der in vitro Versuche zu betrachten. In unseren Untersuchungen haben wir uns zunächst auf die Bestimmung der allgemeinen Exposition, d.h. nach sachgemäßer Anwendung beschränkt. Diese umfassen die Aufnahme über die Haut bzw. die Lunge. Der Applikationsweg über die orale Aufnahme bzw. als sekundäre Barriere, die Blut-Hirn- Schranke mittels in vitro Technologien sind als validierte Testmodelle bereits etabliert werden hier aber nicht näher beschrieben. Ein Schwerpunkt unserer Untersuchungen war die Einstufung der transdermalen Resorption der Referenzsubstanz Koffein entsprechend der Richtlinien der COLIPA und OECD an verschiedenen exzidierten Tierhäuten (z.b. Ratte, Katze, Hund, Schwein etc.) im Vergleich zu humaner Haut ex-vivo. Neben der in vitro Permeation an unterschiedlichen Spezies wurde insbesondere auf die Ermittlung der Konzentration in unterschiedlichen Hautschichten (Stratum Corneum, Epidermis und Dermis) der humanen Hautbiopsien Wert gelegt. Der Vergleich der Resorption der Referenzsubstanz an den Tierhäuten erlaubt eine Abschätzung und Hochrechung zur perkutanen Absorption am Menschen, sowie eine retrospektive Bewertung von Daten aus Tierversuchen an diesen Spezies. Ein wesentlicher Aspekt der in vitro Prüfung umfasst die physikochemische Charakterisierung der Testsubstanz, aber auch des Prüfmusters, d.h. die Testsubstanz eingebracht in das zu testende Produkt. Diese lassen insbesondere eine Bewertung der intrinsischen Eigenschaften der Testsubstanz selbst zu, aber auch der wesentlichen Inhalts- oder Begleitstoffe des Prüfmusters welche die Resorption beeinflussen können. Für die Testsubstanz und eine Auswahl von Wirkstoffen konnten die physikochemischen Eigenschaften wie Löslichkeit und Abhängigkeit vom ph Wert, pka, log P und Proteinbindung mittels validierter Methoden bestimmt werden. Zur Bestimmung der inhalativen Absorption von Testsubstanzen in vitro wurde das Zellkulturmodell Calu-3 etabliert und teilweise validiert. Die bisher durchgeführten Bestimmungen umfassten a) die Festlegung der Versuchsbedingungen (z.b. Kulturmedien, Puffer, Alter der Kultur), b) Auswahl geeigneter Referenzsubstanzen sowie c) Untersuchungen zur Robustheit und Reproduzierbarkeit der Methode. Ischemia-reperfusion injury in the isolated hemo-perfused bovine uterus a new in vitro model for the investigation of antiinflammatory substances? Michael Braun and Manfred Kietzmann Institut für Pharmakologie, Toxikologie und Pharmazie, Tierärztliche Hochschule, D-Hannover. In addition to previous studies on dermal inflammation in the isolated perfused bovine udder (Baeumer and Kietzmann, 2001), a new in vitro model of the isolated hemo-perfused bovine uterus was established for studies on acute inflammatory reactions (e.g. eicosanoid synthesis and regulation of inducible NO-synthase (inos), cyclooxygenase (COX) 1, and COX-2), caused by ischemia-reperfusion (I/R) injury. The organs and the blood used in this study were obtained at the slaughterhouse. Within two hours after slaughter, uterine perfusion was re-established using a mixture of homologous blood and tyrode solution (4:1). After equilibration, several deposits of arachidonic acid (5 mg and 0.1 mg, respectively) and arachidonoyl ethanolamide (0.1 mg) were injected into the myometrial tissue. 180 and 300 minutes after injection tissue biopsies from treated and untreated areas were taken for measurement of prostaglandin (PG) E 2 levels. Furthermore, the regulation of COX-1, COX-2, and inos mrna was determined by RT-PCR. The measurements of eicosanoid levels were performed with an enzyme immunoassay (ELISA). As both an increase in PGE 2 and an upregulation of COX-2 and inos mrna could be shown, inhibitory effects of dexamethasone, flunixin, and the selective COX-2 inhibitor DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)- phenyl-2-(5h)-furanone), added to the perfusion medium, were studied. All substances caused a significant decrease in tissue PGE 2 production, while none induced the downregulation of COX-2 mrna. Dexamethasone induced a slight decrease in the mrna level of inos after 300 minutes of perfusion.

4 In conclusion, the isolated hemo-perfused bovine uterus was introduced as an in vitro model of acute inflammation, induced by I/R-injury. Its suitability for the investigation of anti-inflammatory substances was demonstrated. By using the isolated hemo-perfused bovine uterus in pharmacological research and drug screening, it may contribute to the reduction of animal testing. Acknowledgements: These studies were kindly supported by the Zentralstelle zur Erfassung und Beurteilung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET), Berlin, Germany. 1 Baeumer, W. and Kietzmann, M. (2001). J. Pharm. Pharmacol., 53, Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern Susanne Bremer, Sarah Adler, Raffaella Corvi, Thomas Hartung, Cristian Pellizzer ECVAM, Institute for Health and Consumer Protection, Joint Research Centre, I-Ispra The development of in vitro methods for regulatory embryotoxicity testing is challenging since the understanding of chemical effects on the mammalian development is still poor. The aim of the project is to identify marker genes during in vitro cell differentiation of murine embryonic stem cells in order to predict chemical effects on cell differentiation of specific target tissues. The present study is focusing on the expression pattern by PCR of key genes involved in heart cell development; i.e. Oct-4, Brachyury, Nkx2.5 and alpha myosin heavy chain (α-mhc). Two reference chemicals with well-known in vivo data have been analysed by using this approach: retinoic acid and lithium chloride. Retinoic acid induces multiple malformations including heart defects whereas lithium chloride is interacting relatively selectively with heart development. We demonstrate that retinoic acid already affects in the early stage of germ layer formation, which was demonstrated by a change of Oct-4 and Brachyury gene expression. The expression of Oct-4 is maintained until day 8 versus day 4 in the control. In addition, Brachyury is expressed until day 10 versus until day 6 in the control. Both genes are not affected by lithium chloride exposure. In this case, we observed a modification in the normal gene expression pattern, for α-mhc, which is expressed from day 2 to day 10, versus day 4 to day 10 in the control. These data suggest that the inclusion of selective target organ genes in an established embryotoxicity test allows to deduce mechanistic actions of chemicals and drugs. Pharmacokinetics of acetyl-11-keto-ß-boswellic acid in two models of xenotransplantation, the chick chorioallantoic membrane and the NMRI mouse Berthold Büchele 1, Waltraud Zugmaier 1, Felicitas Genze 2, Karin Kunzi-Rapp 2, Thomas Simmet 1 1 Department of Pharmacology of Natural Products & Clinical Pharmacology and 2 Institute for Lasertechnologies, University of D-Ulm Extracts from the gum resin of Boswellia serrata have been used in traditional ayurvedic medicine for the treatment of various inflammatory disorders. It is believed that boswellic acids constitute the pharmacologically active ingredients. Only recently, we discovered that various pentacyclic triterpenes including several boswellic acids possess antitumor activity in vitro that might be related to the inhibition of DNA-processing enzymes. In order to evaluate the pharmacotherapeutic potential of compounds showing cytotoxic activity in vitro, their efficacy has to be proven in xenotransplantation models. We have used the chick chorioallantoic membrane (CAM) model that allows transplantation of human tumor cell lines, which remain accessible to local treatment. In this model various parameters, such as proliferation, invasiveness and angiogenesis can be monitored by immuno-histochemical techniques. Compounds tested in this model were further analyzed in nude NMRI mice. Whereas the in vitro testing revealed a large number of putative antitumor compounds, this number was clearly reduced in the CAM model. Compounds showing antitumor activity in the CAM model were active in the mouse model as well suggesting some correlating efficacy. Such a sequential testing approach has at least two advantages, i) it significantly reduces the number of mice required for screening, and ii) the amount of compound required for testing is dramatically reduced in the CAM model, which is particularly important with tediously isolated natural compounds. To strengthen the correlation between the CAM and the mouse model, we have developed analytical methods allowing the comparison of pharmacokinetic parameters between both models; we shall demonstrate the plasma pharmacokinetics of acetyl-11-keto-ßboswellic acid. The CAM model saves mice, is reproducible, quick, and cheap. However, it is imperative to use it strictly at the same time point after fertilization because the pharmacokinetic data change with the embryonic and CAM development. Further experiments with additional compounds are required to establish a clear correlation between both xenotransplantation models.

5 [Improving the embryonic stem cell test (EST) I: implementing molecular endpoints of ES cell differentiation into cardiomyocytes] Verbesserung des Embryonalen Stammzelltests (EST) I: Etablierung molekularer Endpunkte für die Entwicklung in Herzmuskelzellen Roland Buesen, Anke Visan, Birgitta Slawik, Elke Genschow, Horst Spielmann und Andrea Seiler Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET) im Bundesinstitut für Risikobewertung (BfR), D-Berlin Der Embryonale Stammzelltest (EST) ist ein in vitro Verfahren, das zur Abschätzung teratogener und embryotoxischer Eigenschaften chemischer Substanzen entwickelt worden ist. Das Prinzip des EST beruht auf dem Vermögen embryonaler Stammzellen (ES-Zellen) der Maus, unter definierten Kultivierungsbedingungen zu kontrahierenden Herzmuskelzellen zu differenzieren, welche lichtmikroskopisch detektiert werden können. Durch die Erstellung von Konzentrations-Wirkungs- Kurven wird die hemmende Wirkung einer Substanz auf diesen Differenzierungsweg untersucht und mit den zytotoxischen Eigenschaften auf ES-Zellen und 3T3-Fibroblasten verglichen (ID 50 - und IC 50 -Werte). Mit Hilfe eines statistischen Prädiktionsmodells kann dann eine Klassifizierung der Stoffe in nicht, schwach oder stark embryotoxisch vorgenommen werden. Der EST erwies sich als eine zuverlässige Methode zur Beurteilung embryotoxischer Potentiale von Testsubstanzen und wurde im Rahmen einer internationalen ECVAM-Studie wissenschaftlich validiert. Dennoch weist das Verfahren durch die relativ lange Testdauer von 10 Tagen und durch die kaum zu automatisierende mikroskopische Analyse der Herzmuskelzellifferenzierung Gründe für eine Weiterentwicklung auf. In Kooperation mit Vertretern der pharmazeutischen Industrie waren daher die Ziele unserer Arbeiten, die Testdauer zu verkürzen und die Analyse der ES-Zelldifferenzierung zu objektivieren. Die ZEBET befasste sich dabei mit der Etablierung und Standardisierung der FACS-Analyse zur Detektion differenzierter Herzmuskelzellen auf molekularer Ebene. Es werden die Ergebnisse der erfolgreichen Übertragung des EST auf die molekulare Ebene präsentiert. Der Einsatz der FACS-Analyse im EST stellt einen objektiven Endpunkt zur Bestimmung des embryotoxischen Potentials einer Substanz dar und ebnet den Weg zu einer möglichen Automatisierung im high-througput screening (HTPS). Alternatives to animal experimentation in undergraduate curricula at medical schools Miroslav Cervinka 1 and Zuzana Cervinkova 2 1 Dept. Medical Biology and Genetics and 2 Dept. Physiology, Charles Univ. Faculty of Medicine, CZ-Hradec Kralove Experiments with animals continue to be the standard part of curricula at many medical schools, particularly in physiology and pharmacology courses. Still, we believe that all undergraduate medical students should be both theoretically and practically informed about the existence of alternatives to the use of animals in research, and education. Therefore we prepared syllabi of a course based on the 3Rs concept. This course takes place in the first and second study year of Charles University Faculty of Medicine in Hradec Kralove. During twenty five practical sessions (three teaching hours once a week), the students learned and practically mastered, among others, the following topics: The 3Rs concept (scientific, ethical and legislative considerations) Experiments on mammalian cells cultivated in vitro (e.g. cytotoxicity testing of xenobiotics) as an alternative, Noninvasive (obligatory) self-experimentation (e.g. human buccal smear preparation, phenylthiocarbamide sensitivity testing, assessment of basic physiological parameters) Invasive (volunteered) self-experimentation with blood (e.g. DNA isolation from human blood, karyotyping from human blood) Screen-based alternatives (interactive computer programmes). No vertebrate animals were used during the course. In the academic year 150 students passed this course. We prepared a written anonymous questionnaire asking students to express their opinions regarding the quality of the course. 93 respondents volunteered the questionnaire. Results showed that our students were generally satisfied with our course, with mean grade being 1.4, modus grade 1 and median grade 2, respectively as evaluated by the grading scale 1(best) 5(poor). We suppose that the syllabi of our course suggest one suitable strategy how to acquaint medical students with the alternatives to animal experimentation.

6 A new bacterium suitable for egg yolk immunoglobulin (IgY) large-scale chromatographic purification Pablo Chacana 1, Rüdiger Schade 2 and Horacio Terzolo 1 1 INTA EEA Balcarce, Argentina, 2 Institute of Pharmacology and Toxicology, Humboldt University, D-Berlin Usage of egg yolk immunoglobulins (IgY) is increasingly been adopted in many laboratories all over the world, due to its advantages concerning animal welfare and economy. A joint project between Germany and Argentina was carried out to develop this technology in Latin America and to improve IgY purification methods. To achieve this aim, an Argentinean research group working on bacteriology joined to a German team with long-term experience in IgY-Technology. Standard purification of IgG is carried out using affinity chromatography techniques based on Gram positive cell wall proteins, such as protein A of Staphylococcus aureus Cowan and protein G of Streptococcus suis. As these proteins have no affinity for IgY, we screened by an ELISA-based test different Gram positive bacteria: Staphylococcus, Streptococcus, Listeria, Bacillus, Corynebacterium, etc. Besides we included 21 different unclassified bacteria randomly chosen from soil isolates. Among them we find one atypical strain, belonging to the Serratia liquefaciens group. This strain has strong nonimmunological IgY-binding. It was treated by sonication and protein extraction was performed to the soluble fraction. SDS Page and Western Blot were carried out and a single protein band was found to bind IgY. At present, further studies are being carried out searching for a new chromatographical IgY purification procedure based on this purified protein. Since this bacterium produces a luxuriant growth in standard culture media, it is a good candidate for the development of an inexpensive and specific affinity chromatography technique. This procedure could be applied at industrial scale, promoting a widespread use of IgY Technology. A simple and rapid procedure for the extraction of IgY from egg yolk using octanoic acid Pablo Chacana 1, Horacio Terzolo 1 and Rüdiger Schade 2 1 INTA EEA Balcarce, Argentina, 2 Institute of Pharmacology and Toxicology, Humboldt University, D-Berlin Egg yolk immunoglobulins (IgY) are a non-invasive alternative to usage of mammalian IgG, which implies the bleeding of mammals to obtain polyclonal antibodies. Additionally, IgY-Technology is feasible to be worldwide adopted due to development of the poultry industry. One of the main problems of IgY usage involves extraction from the egg yolk, procedures that usually require many laborious steps. Other ready-made available kits are easier to perform but they are costly and require laboratory equipment. Some of these procedures are cumbersome preventing the practical usage of IgY. Several methods have been developed for the isolation of IgY from the egg yolk. Basically, all of them include a first step, in which the yolk emulsion is disrupted and the lipids are separated from the aqueous fraction. Afterwards protein extraction methods are applied on this fraction. The aim of this work was the optimisation of a simple and rapid methodology for IgY purification based on the combination of different modified methods. This procedure consists of separation of lipids with 3.5% of polyethylenglycol 6000 followed by low speed centrifugation. The ph of the supernatant is lowered to 5.0 adding acetic acid. Immediately afterwards, 7% of octanoic acid is added to the acidified supernatant, incubated at room temperature for 1 hour and thereafter filtered through standard paper. The resulting filtrated liquid contains an enriched amount of IgY. Residual octanoic acid may be extracted by dialysis. This procedure was set up testing different percentages of octanoic acid (from 5% up to 10%) until the IgY yielding was optimised. The IgY amount was measured by an ELISA test and its purity was checked by means of SDS-PAGE. The use of octanoic acid is a rapid, inexpensive and specific methodology for IgY purification. IgY can be extracted in less than 3 hours. Furthermore, this procedure is very suitable to be performed by non-well equipped laboratories from developing countries. The network of the European Resource Centre for Alternatives in higher education (EURCA) Jasmijn De Boo 1 ; David Dewhurst 2 and Jan van der Valk 1 1 NCA, Dept. of Animals & Society, Faculty of Veterinary Medicine, Utrecht University, NL-Utrecht; 2 Learning Technology Section, Faculty group of Medicine and Veterinary Medicine, University of Edinburgh, UK-Edinburgh The idea of a European Resource Centre for Alternatives in higher education (EURCA) first arose in 1998, during a workshop on Alternatives to the use of animals in higher education sponsored by ECVAM. The idea has been progressed by David Dewhurst and Jan van der Valk, and EURCA has been in existence for 2.5 years. The aims of EURCA to effectively disseminate information about alternatives are achieved by several methods:

7 A Resource Centre with a collection of alternative models, which are taken to relevant scientific meetings in Europe where it would function as a drop-in advice centre for teachers An expansive Internet web site ( including an alternatives database with product descriptions and commissioned reviews, and background information Currently, EURCA holds nearly 60 alternative models in the Resource Centre, EURCA has demonstrated alternatives at 20 scientific meetings, and approximately 15,000 persons have visited the EURCA web site. To stimulate more input at the national level, lecturers are being recruited within Europe to act as EURCA s national contacts ( advocates ). Their role is seen as vital to the successful dissemination of information about alternatives in higher education. They will represent EURCA at local conferences in their country and inform EURCA about locally developed models and activities related to alternatives in education. In October 2003, a meeting with national contacts will be organised in Poland, to thoroughly brief them on the EURCA project, to share experiences and to discuss their activities for EURCA. This approach seems to contribute positively to extending the EURCA activities at the national level within the European community. [The isolated perfused human umbilical vein as in vitro model for toxicity testing] Die isoliert perfundierte menschliche Nabelschnurvene als in vitro Toxizitätsmodell Ulrike Eder 1, Tanja Schwarz 1, Martina Butter 1, Reinhold Wintersteiger 2 und Heinz Juan 1 1 Institut für Biomedizinische Forschung, Universität A-Graz, 2 Institut für Pharmazeutische Chemie und Technologie, Universität A-Graz In früheren Untersuchungen konnte gezeigt werden, dass eine Entzündung oder Gefäßschädigung neben einer erhöhten Prostaglandinbildung mit einer Steigerung der Phospholipid (PL)- und Isoprostaglandin-Freisetzung verbunden ist. Da Isoprostaglandine vorwiegend durch radikalische, nicht enzymatische Oxidation von Arachidonsäure gebildet werden, ist das Ausmaß ihrer Entstehung ein Indikator für oxidativen Streß. Weiters sind sie aufgrund ihrer ausgeprägten vasokonstriktorischen Wirkung auf eine Reihe von Gefäßen von pathophysiologischer Relevanz. Wir konnten auch zeigen, dass verschiedene PLA 2 -Aktivatoren, wie der Ca 2+ -Ionophor A23187, Histamin, Bradykinin und H zu einer vermehrten in vitro Isoprostanbildung führen (Sametz et al., 2000). Folglich sind Isoprostane Ausdruck einer entzündlichen Reaktion. Basierend auf diesen Ergebnissen sollte untersucht werden, ob die Toxizität von anorganischen Schwermetallsalzen (HgCl 2, CdCl 2, Pbac 2 ) auch auf einen radikalischen Reaktionsmechanismus zurückzuführen ist. Als in vitro Zellschädigungsmodell für die Bestimmung von PL und Isoprostanen wurde die isoliert perfundierte menschliche Nabelschnurvene verwendet. Die Schwermetallsalze wurden jeweils durch Perfusion verabreicht. Zur PL-Bestimmung wurde 14 C markierte Arachidonsäure mit Hilfe eines Recycling Verfahrens in die Gefäßzellmembranen eingebaut. Die nach Infusion mit dem jeweiligen Salz freigesetzten PL wurden mittels Szintillationszähler vermessen. Stellvertretend für die Schwermetallsalz-induzierte Isoprostaglandin-Bildung wurde 8-iso-PGF 2 mittels ELISA quantifiziert. Die Ergebnisse zeigen, dass die verwendeten Schwermetallsalze zu einer erhöhten PL- und Isoprostanfreisetzung führen. Die höchste Isoprostanfreisetzung erfolgte nach Infusion einer 1mM CdCl 2 -Lösung (10fache Erhöhung des Basalwertes). Die PL-Freisetzung hingegen wurde am stärksten durch eine 1mM HgCl 2 -Infusion angeregt (5fache Erhöhung des Basalwertes). Einige toxische Effekte der Schwermetalle könnten somit über eine erhöhte Radikalbildung erklärt werden. Zusammenfassend kann gesagt werden, dass sich die isoliert perfundierte Nabelschnurvene des Menschen in Kombination mit der Messung von PL und Isoprostanen als mögliches Modell zur Toxizitäts-Überprüfung erwiesen hat. Weitere Untersuchungen werden zeigen, ob auch toxische Effekte anderer Substanzen über diese Parameter zu bestimmen sind. Sametz et al. (2000). Brit. J. Pharmacol. 131, Replacement of sera for cell culture purposes: a survey Erwin Falkner 1, Harald Schöffl 2, Helmut Appl 2, Walter Pfaller 2,3, Karin Macfelda 1, Udo M. Losert 1 1 Institute for Biomedical Research, University of A-Vienna, 2 zet-center for Replacement and Refinement of Animal Testing, A-Linz, 3 Institute of Physiology, University of A-Innsbruck Aim of our work was an up to date study on aspects of animal serum usage for cell cultivation tasks and alternative approaches of nutrition medium supplementation. The final report should support researchers in making their best choices for specific in vitro studies. Data sheets, product descriptions, cell culture manuals and published papers on the subject were gathered/analysed by literature survey and contacting scientists working in the field at universities and industry research centres. A growing number of alternatives exists for cell lines and primary cultures derived from a broad range of tissues: e.g. chemically defined media, complementations of non-serum origin, non-animal derived proteins and also optimized

8 sampling/processing protocols and production systems/bioreactors for serum-free usage of all scales. A literature search structured for cell type/field of application was performed and paper abstract/citation-shortcuts were collected. The final document including the product guide can be downloaded from one of the following sites: or respectively. Regular updates are planned. In recent years various easily available alternatives to animal sera have been reported and are on sale. For optimal cell performance in vitro, to limit costs and, last but not least, following animal welfare considerations, the authors advise the gathering of exact information to make qualified choices possible. Translational extracts active biologically in vitro: a versatile method for the study of activity of the Hepatitis B Virus (HBV) polymerase in vitro Daniel Favre, Fabien Zoulim, and Christian Trépo Institut National de la Santé et de la Recherche Médicale INSERM U271, F-Lyon Recently, we have generated an in vitro translational extract obtained from eukaryotic cells that have grown as monolayers (Favre and Trépo, 2001). This translation (protein synthesis) system can be employed in place of the commercially available rabbit reticulocyte lysate. It allows both cap-dependent and cap-independent translation of the messenger ribonucleic acids (mrnas). Hepatitis B virus (HBV) does infect 350 million people worlwide, with about 1 million deaths due to the liver cancer (hepatocellular carcinoma) each year. Apart vaccination, new antiviral therapies are needed. To date, only the cognate duck HBV (DHBV) polymerase, but not the HBV polymerase itself, was biologically active in vitro in the rabbit reticulocyte lysate. This system served for the screening of potentially new antiviral molecules that are active against the DHBV polymerase, and by extension against the HBV polymerase. Other in vivo experiments do employ ducks, marmosets, transgenic mice and monkeys for the study of the fate of antiviral molecules against the (D)HBV polymerases. We have synthesized in vitro a biologically active HBV polymerase in extracts obtained from the human hepatocyte cell line HuH-7. The reverse transcriptase activity of the HBV polymerase was clearly established. This activity could be inhibited in vitro with the use of dideoxynucleotites and the nucleoside analogue lamivudine (3TC). These results will thus provide a major opportunity linked to this new tool for the selective screening of inhibitory molecules active against this medically important viral enzyme, the HBV polymerase, without the use of animals. This should lead to the discovery of new antiviral therapies, a features that might help the HBV-infected patients. The procedure might be extended to the polymerases of other relevant viruses. Favre, D. and Trépo, C. (2001). J. Virol. Methods 92, Infection of human hepatocyte cell lines with Hepatitits B and C viruses in vitro Daniel Favre, Pascale Berthillon, Marie-Anne Petit and Christian Trépo Institut National de la Santé et de la Recherche Médicale INSERM U271, F-Lyon Hepatitis B and C virus (HBV; HCV) infections are of the most prevalent viral diseases in the world, since around 350 and 170 million individuals are infected, respectively. Infection with HBV and HCV can lead to cirrhosis and hepatocellular carcinoma. Despite the use of the chimpanzee, there were no reliable models for the study of the many aspects of HBV and HCV infections. Although several alternative methods have been previously developed for the in vitro studies of these infections, there was still an urgent need for new in vitro infection models. Indeed, mostly chimpanzees, but also recombinant mice, marmosets, woodchucks and ducks have been used as animal models for the study of the HBV and HCV infections. It has been shown that cell-bound lipoproteins are playing a crucial role during the infection process in vitro. In order to obtain HBV and HCV infections in vitro, the cell-bound lipoproteins have first to be removed from their cellular receptor prior to the addition of viral inocula originating from human sera, the latter being made originally of a viruslipoprotein complex (Favre et al., 2001; Favre et al., 2003). The model of in vitro infection reported here may thus serve to look for new pharmacological targets to interfere with HCV replication and HBV DNA integration, and to screen in vitro for candidate inhibitors of therapeutic potential, without the use of animals. Favre, D., Berthillon, P. and Trépo, C. (2001). Removal of cell-bound lipoproteins: a crucial step for the efficient infection of liver cells with hepatitis C virus in vitro. C. R. Acad. Sci. III 324, Favre, D., Petit, M.-A. and Trépo, C. (2003). Hepatitis B virus (HBV) infection and HBV DNA integration associated with further transformation of hepatoma cells in vitro. ALTEX 20,3 in press.

9 Comparison and validation of novel pyrogen tests based on the human fever reaction summary of the successful EU-validation study Stefan Fennrich Human(e) pyrogen test group (QLRT ), University of D-Konstanz The absence of pyrogens (fever inducing contamination) in injectable drugs is an important safety control because pyrogens pose a life-threatening risk to the patient. The field of applications for pyrogen-testing is expanding and becoming more diverse due to innovative high-tech products and procedures such as medical devices (implants, medical plastics materials, dialysis machines), cellular therapies and species-specific agents (e.g. recombinant proteins). According to a ECVAM workshop (Hartung et al., 2001), the over all aim of the Human(e) pyrogen project, starting in February 2000, was to develop, evaluate and validate an in vitro method based on the human fever reaction to replace the rabbit pyrogen test (in vivo). All these methods are built upon responses of human leukocytes, particularly monocytes, which release inflammatory mediators (endogenous pyrogens in response to pyrogenic contaminants (exogenous pyrogens). However, the cell-based in vitro assay systems differ with regard to the cells employed (whole blood, isolated peripherial blood mononuclear cells or immortalised monocytic cell lines), the mediator determined (Interleukin-1β, Interleukin-6, Tumor Necrosis Factor α and Neopterin) and the precise set-up of the test. The network comprised ten partners from eight European countries and brought together test systems developed within the last years in Europe for trans-national comparison and subsequent validation of the models. For this purpose, a validation study was conducted. After intensive interaction and co-operation to evaluate and compare the methods which formed the basis for the study, a prevalidation and finally a validation study were carried out addressing several questions, but mainly relevance, transferability, precision and predictive capabilities. Overall, a successful study was conducted such that several methods are recommended for consideration to replace the rabbit pyrogen test. Based on this study, a suggestion for introduction into the European Pharmacopoeia will be developed. Additionally, this pre-competitive development initiates further applications and exploitations for new fields of pyrogen testing such as cellular therapies, medical devices and pyrogen pollution control in work places. Cultivation of PLA (adult pig kidney) cells in vitro on cellulose acetate wool as a substrate Bratko Filipič 1, Srećko Sladoljev 2, Sandor Toth 3, Srečko Koren 1 1 Institute of Microbiology and Immunology, Med. Fac., University of SLO-Ljubljana, 2 Institute of Immunology, CRO- Zagreb, 3 Institute of Biotechnology, University of Sciences H-Szeged Cultivation of different (primary and/or permanent) cells in vitro opens a broad spectrum of possible usage of these in different biomedical disciplines. The finding that practically any type of living eucariotic cell outside the organism as a "single unit" can under "proper conditions in vitro" introduce the propagation, development and differentiation of the named cell species, offers a quite universal tool to researcers. The research has become focused mainly on the conditions under which different cell types can survive in vitro, and produce varius biologicals. The PLA cell line shows characteristics of a stable cell line useful for production of porcine interferon - Beta. The question lies in the quantity of cells/cm3 grown in the shortest time provoking the analysis on how to cultivate the highest number of cells by the lowest price. The presented experiments were aimed to test the cellulose acetate woll of different length to cultivate the PLA cells for the induction of Po- IFN-Beta. Cells were cultivated in the bacteriological Petri dishes in Eagle's medium supplemented with 8% SR and antibiotics. In the Petri dishes sterile cellulose acetate woll of different length was added. Petri dishes were incubated in CO2 atmosphere at 37oC. After 4-5 days of incubation cells begin to grow in form of microtumors which are than adsorbed to the fibers and overgrown them. The form of owergrowth depends on the length of the fibre. When the fibers were overgrown the IFN was induced by Sendai virus. The output of monolayer culture of PLA cells and the culture grown on fiber was compared. The bigger number of cells on fibers gave the higher amount of IFN. Biological assays of interferons Bratko Filipič 1, Sandor Toth 3, Srećko Sladoljev 2, Srečko Koren 1 1 Institute of Microbiology and Immunology, Med. Fac., University of SLO-Ljubljana, 2 Institute of Immunology, CRO- Zagreb, 3 Institute of Biotechnology, University of Sciences H-Szeged Although more than a quarter of a century has passed since the discovery of the Interferons (IFN), a universally accepted method for its biological assay is still unavai- lable. One of the reason is definitely in the fact, that the IFNs are pleiotropic

10 molecules showing not only antiviral but also antiproliferative, cytocidal, antitumor, immunomodulatory and radioprotective activity. Presently the most widely used method for IFN quantitation is based on the measurment of various parameters of viral replication in IFN-treated cells. Among these parameters the one based on cytopathic effect (CPE)-inhibition offers the most useful assay to determine the biological activity of IFN. Such an activity is expressed in the I.U. (International units)/ ml. Although diploid fibroblast cells can be more sensitive, the use of different continous cell lines is more practical. The purpose of this study was to compare three established cell lines (WISH, HAC-3/T2 and MDBK) to ascertain which cellvirus system is more appropriate for qunatitation of both Human (Alpha, Beta and Gamma) and Porcine (Alpha, Beta and Gamma) Interferons. Subseqently, the antiviral activity was compared with the antiproliferative activity using the same cells and cytocidal activity using the PLA cells. The sensitivity and reproducibility of different test system was also determined, finnaly leading to the determining of the biological activity of IFNs. [Which cell lines can be adapted to chemically fully defined medium?] Welche Zelllinien können in definiertem synthetischen Medium kultiviert werden? René W. Fischer 1, Ferruccio Messi 2, Pablo Umena 1, Andreas H. Zisch 1, Susanne Scheiwiller 3 und Franz P. Gruber 3 1 ETH Zürich, 2 Messi Cell Culture Technologies, 3 FFVFF, CH-Zürich Vor zwei Jahren stellten wir an diesem Ort die Pläne für ein ambitioniertes Projekt mit dem Titel Welche Zellen können in definiertem synthetischen Medium kultiviert werden? vor. Die Arbeiten sind mittlerweile gestartet worden, so sind die Myelomzelllinien X63.Ag8 und Sp2/0, die als Fusionspartner bei der Herstellung monoklonaler Antikörper eingesetzt werden, erfolgreich an das Turbodoma Medium (1) gewöhnt worden. Serumfreie Fusionen konnten in der Folge, ohne Zusatz von tierischen Komponenten, mit grosser Ausbeute durchgeführt werden. Myelomas sowie die gebildeten Hybridoma zeigen ein identisches Wachstumverhalten im Vergleich zu ihren serumabhängigen parentalen Zelllinien. Die Umstellung der zweiten Gruppe der ausgewählten Zelllinien, die bei der Expression von rekombinanten Proteinen ihren Einsatz finden, gestaltete sich, wie erwartet, etwas schwieriger, handelt es sich dabei doch um adheränte Zelllinien, die bei der Adaption an serumfreies Medium oft ihre Morphologie ändern und teilweise ihre Adhärenz verlieren. Die Vero Zelllinie, eine Nieren-Fibroblasten Zelllinie des african green monkey konnte dennoch problemlos an das chemisch definierte Basalmedium Hektor (1) adapiert werden. Die Zellen behielten ihre Adhärenz, verlangsamten jedoch ihre Wachstumsrate signifikant. In einem zweiten Schritt versuchen wir nun, durch Zugabe von (rekombinant hergestellten) Wachstumsfaktoren das Wachstum auch bei anderen von uns umgestellten Zelllinien günstig zu beeinflussen, erste Resultate sollen vorgestellt werden. (1) Messi, Cell Culture Technologies, Buhnrain 14, CH-8052 Zürich Endocrine toxicology: in vitro methods contribute to 3R Alexius Freyberger and Gabriele Scholz Bayer AG, D-Wuppertal Concerns have been raised whether natural and man-made chemicals might have the potential to relevantly interfere with the endocrine system of humans and wildlife. This issue has been addressed by intensive research over the past years. Most studies focused on interactions with sex hormone receptors, as for a number of natural and anthropogenic compounds an estrogenic or anti-androgenic potency could be demonstrated. In vitro methods can substantially contribute to the detection of such effects. By performing hormone receptor binding studies affinities to various receptors over a broad concentration range can readily be determined, and recombinant, mostly human estrogen and androgen receptors that have become commercially available can now replace uterine and prostatic cytosol obtained from ovariectomized and castrated rats as receptor sources. More comprehensive result can be obtained from mammalian cellular systems such as MVLN- and PALM-cells. These cells express a functioning estrogen or androgen receptor and are stably transfected with the luciferase gene under the control of an estrogen- or androgen-responsive element as reporter gene. Increased expression of the luciferase gene correlates with hormone action. Modification of the assay system allows testing of receptor antagonists as well. In addition, cytotoxicity can be assessed in these cellular systems and can be related to hormonal activity. The metabolic competence of the described cellular systems is limited, accordingly their predictive power can be strengthened by testing known and anticipated metabolites as well. In general, in vitro methods are increasingly applied in endocrine toxicology. They are used for screening purposes, for mechanistic studies and to set research priorities and can thus contribute to the reduction of animal studies.

11 Evaluation of sensitising potential in chemical skin irritants using the Local Lymph Node Assay (LLNA) Armin O. Gamer, Edgar Leibold, Bennard van Ravenzwaay Product Safety, BASF AG, D-Ludwigshafen In the absence of promising in vitro methods for routine skin sensitisation testing, the LLNA was developed as refined method to replace the commonly used invasive guinea pig maximization test (Kimber et al., 2002). Recently the method was taken up into the OECD Guidelines for Testing of Chemicals (OECD 2002). It measures the cell proliferation response in the ear lymph nodes of mice after treatment of the ear skin with several concentrations of a test material in a suitable solvent. The published method uses ³H-thymidine incorporation into lymph node cell DNA as parameter and predicts a chemical to be a sensitizer if a 3-fold increase of radioactivity occurs in the lymph nodes of treated animal as compared to controls. However this cut-off value may also be exceeded after application of moderate to marked chemical irritants (Basketter et al., 1998). In order to avoid the use of radioactivity and to facilitate the differentiation of sensitisation and irritation, i.e. to refine the guideline method, a modified method was developed using lymph node cell count, lymph node weight and ear weight as parameters for evaluation (Vohr et al., 2000; Ulrich et al., 2001). So far, we used the latter method for testing of about 40 chemicals and products. About one third of the tests presented clear positive results i.e. statistically significant increased cell counts were detected in the absence of increased ear weights in at least one concentration tested. Another third of the tests were clearly negative as no increase in cell counts were found at concentrations exceeding the solubility of the test substance in the most suitable vehicle or those which induced some increase in ear weights. The last third showed a concomitant increase in lymph node cell counts and ear weight and thus did not allow for a final decision on the sensitising properties of the test substances. As indicated above, the evaluation of the results is based on statistics (Wilcoxon test). For reducing the fraction of equivocal outcomes we are developing cut off values for the measured parameters and the dose response of the relative increase of cell count in relation to the ear weights as additional evaluation tools. A further approach to reach final decisions is to perform challenge experiments, which compare the ear skin and lymph node response in naïve animals to a group pretreated at the clipped flank skin with a suitable concentration identified in the LLNA. [Prediction of embryotoxic Potential of Drugs and Chemicals applying the Embryonic Stem Cell Test (EST)] Vorhersage der embryotoxischen Eigenschaften von Arzneistoffen und Chemikalien im embryonalen Stammzelltest (EST) Elke Genschow, Andrea Seiler und Horst Spielmann Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET), Bundesinstitut für Risikobewertung (BfR), D-Berlin Der Embryonale Stammzelltest (EST), bei dem eine embryonale Stammzelllinie der Maus eingesetzt wird, wurde im Rahmen einer internationalen Ringstudie des Europäischen Zentrums für Alternativmethoden (ECVAM) unter blinden Bedingungen in vier Laboratorien erfolgreich validiert. Die Vorhersage der embryotoxischen Eigenschaften mit Hilfe eines Prädiktionsmodelles (PM) basiert auf der Klassifizierung der Prüfsubstanzen in drei Embryotoxizitätsklassen (in vivo stark, schwach und nicht embryotoxisch). Für die Vorhersage der embryotoxischen Eigenschaft werden Konzentrations- Wirkungskurven für drei Endpunkte ermittelt und in die Diskriminanzfunktionen eingesetzt. Für viele Anwendungen, wie z.b. die Entwicklung von Medikamenten, wäre lediglich die Vorhersage stark embryotoxischer Substanzen von Interesse, so dass eine Klassifizierung in zwei Klassen, in stark embryotoxische Chemikalien einerseits und schwach und nicht embryotoxische Substanzen andererseits, ausreichen würde. Dies macht die Entwicklung eines neuen Prädiktionsmodelles erforderlich. Unsere Ergebnisse zeigen, dass entweder der zytotoxische Effekt auf embryonale Zellen (IC 50 D3) oder die Hemmung der Differenzierung pluripotenter embryonaler Stammzellen (ID 50 ) als alleinige Endpunkte zur Vorhersage des stark embryotoxischen Potentials hervorragend geeignet sind. Der dritte Endpunkt, der zytotoxische Effekt auf adulte Zellen, erzielt eine deutlich geringere Sensitivität. Es wird gezeigt, dass die ES Zellen im Gegensatz zu den 3T3 Zellen ein sehr hohes Potential zur Identifizierung der stark embryotoxischen Substanzen haben. Embryotoxizitätstestungen in vitro könnten durch die Verwendung nur eines Endpunktes stark vereinfacht werden, wenn das Ziel in der Identifizierung stark embryotoxischer Substanzen liegt. [Collecting bovine fetal serum can suffering of the fetuses be avoided?]

12 Die Gewinnung von fötalem bovinen Serum kann ein Leiden der Föten ausgeschlossen werden? Franz P. Gruber 1, Vera Baumans 2,3, Ludo J. Hellebrekers 2, F. Herman Jonkers 2, David J. Mellor 3, Jan van der Valk 2, René Fischer 5 und Susanne Scheiwiller 1 1 FFVFF, CH-Zürich, 2 University NL-Utrecht, 3 Karolinska Institutet, S-Stockholm, 4 Massey University, NZ-Palmerston, 5 ETH Hönggerberg, CH-Zürich Wir müssen davon ausgehen, dass spätestens mit dem 200. Trächtigkeitstag Rinderföten voll schmerz- und leidensfähig sind. Vom 100. Trächtigkeitstag an bis kurz vor der natürlichen Geburt ( Tag, Median 283. Tag) werden Rinderföten zur Gewinnung von FBS überwiegend durch Herzpunktion ohne vorhergehende Narkose verwendet. Wir müssen weiterhin davon ausgehen, dass wir von dem weltweit geschätzten Verbrauch von 500'000 Litern pro Jahr nicht von heute auf morgen herunter kommen werden. Die Bemühungen, komplett auf die auch aus wissenschaftlichen Gründen dringend nötigen serumfreien Ersatzmedien umzusteigen, werden schätzungsweise 10 Jahre in Anspruch nehmen. Bis dahin müssen wir Mittel und Wege finden, Schmerzen und Leiden bei der Gewinnung von FBS zu vermeiden. Mit dem Blutentzug des Muttertieres bricht auch der Sauerstofftransport durch die Plazenta innerhalb weniger Sekunden zusammen. Fötales EEG und ECoG verflachen in Minutenfrist und zeigen damit tiefe Bewusstlosigkeit an. Es muss unter allen Umständen verhindert werden, dass der Fötus bei erneuter Sauerstoffzufuhr wieder erwacht. Dies kann durch verschiedene Maßnahmen erfolgen, die zu diskutieren sind (Belassen des fötalen Kopfes im Uterus, schneller Blutentzug durch Herzpunktion, Betäubung durch Bolzenschuss). Als Eckdaten sollen folgende Bestimmungen international eingeführt und strikt überwacht werden: Zwischen Blutentzug des Muttertieres und Herzpunktion des Föten muss ein Mindestzeitraum von fünf Minuten eingehalten werden. Der Fötus muss am Wiedererwachen gehindert werden (Vermeidung von Sauerstoffzufuhr oder Bolzenschuss). Dies sind Maßnahmen, die sich gut überprüfen lassen. Sie stellen einen Mindeststandard dar, der bei der Gewinnung von FBS eingehalten und von den entsprechenden Firmen garantiert werden muss. [AnimAlt-ZEBET internet database on alternatives to animal experiments] AnimAlt-ZEBET- Datenbank für Alternativmethoden zu Tierversuchen im Internet Barbara Grune, Antje Dörendahl, Dorothea Köhler-Hahn, Rainer Box, Heinz Wohlgemuth und Horst Spielmann Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET), Bundesinstitut für Risikobewertung (BfR), D-Berlin Die Europäische Richtlinie 86/609/EWG zum Schutz der für Versuche und andere wissenschaftliche Zwecke verwendeten Tiere bestimmt im Artikel 7, dass ein Tierversuch nicht durchgeführt werden darf, wenn zur Erreichung des angestrebten Ergebnisses eine Alternativmethode zur Verfügung steht. Das hat u.a. zur Folge, dass bei der Planung von Versuchsvorhaben geprüft werden muss, ob alle Informationsmöglichkeiten ausgeschöpft wurden und bereits Literatur über Alternativmethoden vorliegt. Das BfR bietet seit Februar 2000 die Datenbank AnimAlt-ZEBET über Alternativmethoden zu Tierversuchen in englischer Sprache über das Deutsche Institut für Medizinische Information und Dokumentation (DIMDI) online und lizenzfrei unter der Adresse an. AnimAlt-ZEBET enthält eine Dokumentation von sowohl experimentellen Methoden als auch computergestützten Verfahren, die gemäss dem 3R"-Konzept nach Russel und Burch die Durchführung von Tierversuchen vermeiden und/oder das Leiden der Versuchstiere verringern. Für jede Methode gibt es ein abrufbares Dokument mit einer kurzen Einführung in den thematischen Hintergrund, einer Beschreibung des zugrundeliegenden Prinzips, einer Darstellung des derzeitigen Standes der Entwicklung und einer wissenschaftlich fundierten Bewertung, d.h. einer durch Literatur belegte Einschätzung der wissenschaftlichen und behördlichen Akzeptanz. In AnimAlt-ZEBET sind insbesondere Methoden aus der Toxikologie, Pharmakologie, Pharmazie, Mikrobiologie, Immunologie, Krebsforschung und Gentechnik dokumentiert; weitere Bereiche sind Lebensmittelhygiene, Physiologie und Parasitologie. Gegenwärtig sind 112 Dokumente in englischer Sprache abrufbar; sie werden in regelmäßigen Abständen aktualisiert. Es werden laufend weitere Methodenberichte aufgenommen. Die systematische Suche in AnimAlt-ZEBET wird vorgestellt anhand von Beispielen zu aktuellen Themen wie RNA-Interferenz und Ersatz von Kälberserum in der Zellkultur. [Indexing of biomedical information on alternative methods]

13 Indexierung biowissenschaftlicher Informationen zu Alternativmethoden Barbara Grune, Antje Döhrendahl, Dorothea Köhler-Hahn, Ernst Höwer und Horst Spielmann Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET), Bundesinstitut für Risikobewertung (BfR), D-Berlin Entsprechend der geltenden Tierschutzgesetzgebung sind die Wissenschaftler aufgefordert, in die Recherche nach wissenschaftlicher Literatur zur Vorbereitung eines Forschungsprojektes die Recherche nach Alternativmethoden einzubeziehen. Eine Vielzahl an Literatur- und Faktendatenbanken und spezieller Webseiten wird im Internet angeboten. Die Wissenschaftler müssen sich den Zugang zu den Informationen durch geeignete Suchstrategien legen. Den Anbietern von Informationen kommt die Aufgabe zu, neben der Qualität der Informationen auch die Findbarkeit der Information für die Nutzer zu sichern. Für die Indexierung von Literatur bzw. Informationen zu Alternativmethoden in online Datenbanken gibt es gegenwärtig noch keine Anleitungen oder publizierte Erfahrungsberichte. Eine gute Indexierung von Literatur mit Deskriptoren (Schlagwörter) wird grundsätzlich als eine Voraussetzung für das Auffinden von Informationen betrachtet. Beim Indexieren wird der Inhalt eines Dokumentes mit Hilfe von Begriffen (Deskriptoren) gekennzeichnet, damit sie in zukünftigen Recherchen besser gefunden werden. In einem Sonderforschungsprojekt, gefördert durch das BfR, hat ZEBET bereits verfügbare und publizierte Indexierungssysteme in biomedizinischen Datenbanken hinsichtlich der Indexierung von Alternativmethoden untersucht. Es wurden in den Datenbanken MEDLINE, EMBASE, CAB Abstract, AGRIS, AGRICOLA und AnimAlt-ZEBET Recherchen nach Alternativmethoden mit ausgewählter Suchbegriffen durchgeführt. Die verwendeten Suchbegriffe wurden den Thesauri bzw. Schlagwortlisten der jeweiligen Datenbank entnommen. Dazu gehörten allgemeine Begriffe wie z.b. Animal Testing Alternatives, spezielle Begriffe für Alternativmethoden wie z.b. Limulus Amebocyte Lysate Test, spezielle Begriffe für Tierversuche wie z.b. Draize Test und auch allgemeine Begriffe wie z.b. in vitro. Die Auswertung der Ergebnisse zeigte, dass obwohl in den Thesauri der Datenbanken MEDLINE, EMBASE, CAB Abstract, AGRIS, AGRICOLA zutreffende Begriffe zur Indexierung von Alternativmethoden vorhanden sind, die Vergabe dieser Begriffe d.h. das eigentliche Indexieren problematisch ist. [Reduction of in vivo models in microsurgical training and education] Reduktion von Tierversuchen in der mikrochirurgischen Ausbildung: Ein Erfahrungsbericht über 10 Jahre Dietmar Hager 1,2, Harald Schöffl 1 und Oskar Kwasny 1 1 Allgemeines Krankenhaus der Stadt Linz und maz Mikrochirurgisches Ausbildungs- und Forschungszentrum, A-Linz, 2 zet Zentrum für Ersatz- und Ergänzungsmethoden zu Tierversuchen, A-Linz Während früher das narkotisierte Tier das Standardmodell zum Erlernen mikrochirurgischer Anastomosetechniken war, so wurden doch in den letzten Jahren zunehmend Ersatz- und Ergänzungsmethoden beschritten. Aus unserer eigenen Erfahrung der letzten zehn Jahren zeigt sich, dass etwa 90% aller Tierversuche durch andere Verfahren ersetzt werden können, wenn ein modular ausgebautes Ausbildungssystem verwendet wird. Wir verwenden ein modulares Ausbildungskonzept, das aus drei Kursstufen besteht, einerseits ein so genannter eintägiger Rookie-Kurs, bei dem einen Tag lang mit minimalistischer theoretischer Einführung vor allem erkundet wird, ob der an der Mikrochirurgie Interessierte diesem Fachgebiet wirklich näher treten will. In der nächsten Stufe ist ein dreitägiger Grundkurs zu absolvieren. Wir haben eine ausgewogene Verteilung zwischen Theorie und Praxis, wobei die praktischen operativen Teile zeitlich eher überwiegen. Es werden auch optimierte Verfahren verwendet, wie etwa die pulsatil perfundierten Schweineherzkoronarien, die uns als Hauptmodell für die mikrochirurgischen Anastomosetechniken dienen. Ferner verwenden wir miniendoskopische Verfahren, um transluminal Anastomosen kontrollieren zu können. Bis zu dieser Kursstufe kommt der Auszubildende nicht mit Tieren nicht in Kontakt. Unsere Hauptmodelle sind das Schweineherz und für die Nervenkoaptation der N. ischiadicus am Hühnerschenkel. Personen die nun schon erhebliche Grundkenntnisse in der Mikrochirurgie besitzen, absolvieren dann einen Aufbaukurs und erst im zweiten Teil des Aufbaukurses wird an der narkotisierten Ratte die Arterie carotis, die A. femoralis und die Aorta abdominalis präpariert und mit mikrochirurgischen Anastomosen versorgt. Dieses modulare Ausbildungssystem gewährt einerseits ein stressfreies Heranführen an die Mikrochirurgie, ferner sind die Auszubildenden erst in einer sehr späten Phase in Kontakt mit Tieren, somit reduzieren sich unnötige Tieropfer durch Präparation und operative Fehler. Rein rechnerisch konnten wir im Vergleich zu konventionellen Ausbildungssystemen in unseren Kursen die Tierversuchszahlen um ca. 90% reduzieren. ECVAM s response to the EU chemicals policy

14 Thomas Hartung ECVAM, Institute for Health & Consumer Protection, European Commission, Joint Research Centre, I-Ispra The field of alternatives is currently driven by the expectations from both cosmetics and chemicals policy: The 7 th amendment to the Cosmetics Directive (finally published in 3 03) foresees to phase out animal experiments within 10 years. A timetable for the individual tests to be published by the Commission in 18 months is currently developed by a taskforce of stakeholders chaired jointly by DG Enterprise and ECVAM. The legislation is reinforced by an immediate testing ban for finished products and any tests, where ECVAM has validated an alternative. Furthermore, a testing ban and a marketing ban, which cannot be postponed, applies in 6 years for topical and acute systemic toxicity, while all other tests shall be phased out in 10 years with a possible postponement by co-decision. The legislation for chemicals (REACH) is only emerging. It will foresee data requirements for more than substances produced at levels above 1 ton per year. Extensive in vivo data requirements are expected for a core of about 6000 substances with highest production and concern. Alternative methods shall first be considered throughout the testing and be predominantly used for the largest group (1-10 tons per year). The legislative process shall be completed by the end of the year. ECVAM has restructured its service directly targeting the animal tests to be replaced. Given the short time-lines to make available and implement validated methods, ECVAM is offering to steer the process by bundling the inputs of stakeholders and early involvement of regulators. In essence, steering groups from ECVAM s senior staff and complemented by external experts shall carry out the project management, which co-ordinates the various inputs. Use of medium supplemented with molecules of plant origin to culture epithelial cells Thomas Hartung, Patricia Pazos, Alessandra Gennari, Juan Casado and Pilar Prieto ECVAM, Institute for Health & Consumer Protection, European Commission, Joint Research Centre, I-Ispra To succeed in growing cells in media free of human or animal proteins is an interesting challenge in cell biology. The use of such media for in vitro cell culture work would contribute to the 3Rs concept, by avoiding the use of animals at any step. In addition, many problems related to virus infections coming from animal serum would be avoided. Prolifix is a new reagent of plant origin. It contains a molecule (GCR 1003) that has an activity similar to the one of mitogenic molecules in serum. It supports cell culture maintenance and proliferation, and it might therefore be used to replace fetal bovine serum (FBS). Two commonly used epithelial cell lines, LLC-PK1 and Caco-2 were progressively adapted to a special culture medium containing 10% Prolifix. After adaptation, cell cultures were characterized morphologically and functionally. Our results showed that both cell lines kept their typical epithelial morphology with formation of domes when grown in conventional cell plastic culture flasks. However, both cell lines grew slower in presence of Prolifix than in presence of FBS. When these cell lines were seeded in porous supports, the formation of an epithelial barrier was observed as shown by the trans-epithelial resistance (TER) measurement. LLC-PK1 cells reached similar stable TER values under both culture conditions. However, TER values of Caco-2 cells cultured in vegetal supplemented medium were 75% lowered than in FBS. In addition, the effects of CdCl 2 on barrier function integrity were evaluated. The results showed that CdCl 2 was more toxic when both cell lines were grown in medium supplemented with prolifix than medium with FBS. In summary, the preliminary work done with molecules of plant origin indicates that these reagents could be seen as a promising alternative to animal serum. However, more efforts should be put to improve cell adaptation, cell attachment, growing rates and freezing and thawing protocols. Advanced-Skin-Test 2000 (AST-2000). Rekonstituierte Haut als Werkzeug in der pharmako-toxikologischen und dermatologischen Forschung Jens J. Hoffmann, Petra Peters, Sonja Karpinski und Horst W. Fuchs CellSystems Biotechnologie Vertrieb GmbH, D-St. Katharinen Ein Hauptpunkt moderner Pharmakotoxikologie und Dermatologie besteht in der Entwicklung von in vitro Modellen mit hoher Reproduzierbarkeit und Vergleichbarkeit zur Situation in vivo. In den letzten Jahren haben sich eine Reihe von Epidermismodellen am Markt etabliert, die in den unterschiedlichsten Bereichen der Hautforschung eingesetzt werden und ihre Fähigkeit den Tierversucht zu ersetzen mehrfach unter Beweis gestellt haben. Hier präsentieren wir nun neueste Daten zu Advanced-Skin-Test 2000 (AST-2000), einem humanen Vollhautmodell bestehend aus einem dermalen Teil mit in eine Kollagenmatrix eingebetteten Fibroblasten und einer diesen Dermalteil bedeckenden Epidermis aus proliferierenden und differenzierten Keratinocyten. Immuncytochemische Untersuchungen zeigten die mit der in vivo Situation vergleichbare Expression verschiedener Marker (u.a. CK 1/10, CK 5/14, Involucrin, Transglutaminase Integrin ß1 sowie verschiedener Komponenten der Basalmembran (Laminin I, Kollagen IV). AST-2000 reagiert auf die Applikation verschiedener

15 Testsubstanzen wie z. B. SDS oder NiSO 4 mit einer zeit- und konzentrationsabhängigen Sekretion verschiedener Mediatoren. Nachgewiesen wurden hier u. a. IL-1 alpha, IL-8, PGE 2, GM-CSF. Die gefundenen Ergebnisse korrelieren mit Daten, die mit dem Human Patch Test oder Rabbit Skin Test erhalten wurden. Isolation and characterisation of a newly developed mouse pneumocyte type II cell line Paul Jennings, Cristina Bertocchi, Manfred Frick, Walter Pfaller and Paul Dietl Institute of Physiology, University of Innsbruck, A-Innsbruck Alveolar type II cells function primarily to secrete surfactant which maintains low alveolar surface tension and is therefore essential for normal lung function. Surfactant is a lipoprotein-like phospholipid rich material secreted via exocytosis of vesicles called lamellar bodies (LBs). The study of processes involved in surfactant secretion, on a cellular and molecular level, critically depends on the availability of type II pneumocytes isolated from the intact organ. Primary cultures of alveolar epithelial cells are subject to rapid dedifferentiation, and therefore can only be used within the first 48 h after isolation. Since the isolation of these cells is laborious and requires animal sacrifice, the availability of a well differentiated cell line would be advantageous. Here we describe the isolation and characterisation of a newly developed mouse pneumocyte type II cell line (MP2). Alveolar type II cells were isolated from the H-2K b tsa58 transgenic mouse lung using as previously described method (Dobbs et al. 1986). In these mice the tsa58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. After 3-4 days in culture, the temperature of the incubator was decreased to 33 C and interferon gamma was added to the culture medium. Thereby, we obtained cells stably maintaining their morphological features (vesicles located around the nucleus) which exhibit extended growth (have been grown for 14 passages, until now). The vesicles of MP2 were stained with lyso-tracker green, a dye exclusively accumulating in acidic compartments. LBs belong to a population of vesicles classified as secretory lysosomes, which have a highly acidic ph. Lamellar body secretion is dependent on an intracellular Ca 2+ increase. ATP induced a strong intracellular Ca 2+ release in these cells, as measured by fura-2 fluorescence. This event was followed by an increase in lamellar bodies secretion, detected by simultaneous measurement of FM 1-43, a dye which is highly specific for lipid material. These are important criteria for a type II cell phenotype. MP2 cells exhibit morphological and functional characteristics of alveolar type II cells and thus may represent a reliable mouse cell line to study alveolar type II cell function and hence would reduce the amount of animals used in such studies. [Tissue toxicity of antiseptics] Gewebetoxizität antiseptischer Spüllösungen Thomas Kalteis, Gerhard Guggler, Christian Lüring und Joachim Grifka Orthopädische Universitätsklinik Regensburg, D Regensburg Mit Hilfe eines Replacement-Verfahrens sollte die lokale Gewebeverträglichkeit gängiger antiseptischer Spüllösungen, die in der orthopädischen Chirurgie u.a. bei der Wundspülung und -reinigung angewendet werden, untersucht und verglichen werden. Die Verträglichkeitsuntersuchung der Spüllösungen erfolgte im Hühner-Ei-Test an der Chorionallantoismembran (HET-CAM). Der HET-CAM-Test ermöglicht neben der Beobachtung von vaskulären Veränderungen nach Aufbringen der Testsubstanzen die Berechnung der jeweiligen Irritationswerte (IS) sowie die Bestimmung der konzentrationsabhängigen Irritationsschwellen (IT). Getestet wurden Dibromol, Jodobac, Kodan, Octenisept, Lavasept 0,2%, Wasserstoffperoxid, Chlorhexidindigluconat 0,5% und 2-Propanol 60%. Typische vaskuläre Reaktionen an der Chorionallantoismembran waren u.a. ausgeprägte intra- und extravaskuläre Koagulationen (Chlorhexidindigluconat 0,5%, Kodan ), Kapillarabbrüche (Dibromol, 2-Propanol 60%) und Hämorrhagien (Octenisept, Jodobac ). Die höchsten Irritationswerte berechneten sich für Chlorhexidindigluconat 0,5% (IS 20 / IT 10%), Kodan (IS 18 / IT 5%) und Dibromol (IS 16 / IT 10%). Eine geringere Toxizität ergab sich für Octenisept (IS 12 / 20%), 2-Propanol 60% (IS 11) und Jodobac (IS 2 / 50%). Weder bei Lavasept 0,2% (IS 0) noch bei Wasserstoffperoxid (IS 0) konnten lokaltoxische Reaktionen erkannt werden. Angesichts der Ergebnisse des HET-CAM-Testes muss bei der unkritischen Anwendung von antiseptischen Spüllösungen von erheblichen gewebetoxischen Reaktionen ausgegangen werden. Hinsichtlich der lokalen Gewebeverträglichkeit scheinen Wasserstoffperoxid und Lavasept 0,2% die antiseptischen Lösungen der Wahl zu sein.

16 [Tissue-compatibility of methacrylate polymers] In vivo Untersuchung zur Gewebeverträglichkeit von PMMA- Knochenzementen Thomas Kalteis, Gerhard Guggler, Christian Lüring und Joachim Grifka Orthopädische Universitätsklinik Regensburg, D Regensburg Lokal-toxische Reaktionen werden als eine Ursache für die Bildung des bindegewebigen Interface zwischen Knochen und PMMA-Knochenzement angesehen. Im HET-CAM-Test als etabliertes Replacement-Verfahren für tierexperimentelle Untersuchungen an höher entwickelten Wirbeltieren sollte die in vivo Gewebeverträglichkeit handelsüblicher PMMA-Knochenzemente untersucht werden. Getestet wurden Polymere, Monomer-Extrakte und unterschiedliche Inhaltsstoffe (Polymerisationsinitiatoren, Stabilisatoren, u.a.). Kriterien für die lokale Toxizität waren innerhalb von 48h neu auftretende vaskuläre Veränderungen bzw. Gewebenekrosen. Für die jeweiligen PMMA-Polymere konnte eine gute Gewebeverträglichkeit nachgewiesen werden, solange die Knochenzemente in der Mitte bzw. am Ende der vom Hersteller angegebenen produktspezifischen Verarbeitungsphase auf das Gewebe aufgebracht wurde. Auch verursachten die von den PMMA-Polymeren über 72h freigesetzten Restmonomere keine toxischen Gewebereaktionen. Deutliche lokale Entzündungsreaktionen waren beim Auftragen einiger Knochenzemente zu Beginn der jeweiligen Verarbeitungsphasen zu erkennen. Ebenso werden konzentrationsabhängig durch unterschiedliche Inhaltsstoffe der PMMA-Knochenzemente z.t. erhebliche toxische Gewebereaktionen ausgelöst Gewebetoxische Reaktionen drohen bei PMMA-Knochenzementen v.a. durch noch ungebundene Inhaltsstoffe bzw. Monomere. Daher sollte eine zu frühe Verarbeitung der Knochenzemente vermieden werden. Auch sollte von Seiten der Hersteller der Versuch unternommen werden, potentiell toxische Inhaltsstoffe durch verträglichere Substanzen zu ersetzen. Use of quantitative gene expression data to detect embryotoxic effects on mouse embryonic stem cells Johanna Kaltenhäuser 1, Elke Genschow 2 and Klaus Becker 1 1 Schering AG, Research Laboratories, D-Berlin, 2 ZEBET, D-Berlin Adverse effects on development are among the most serious adverse effects of drugs. Up to now embryotoxic effects are detected in studies in vivo that require a large number of animals and a considerable amount of test substance. Accordingly, there is an increasing demand to use alternative tests in vitro to identify an embryotoxic potential early in drug development. One of the most promising in vitro approaches is the Embryonic Stem Cell Test (EST), which has been validated recently in a multicentre study supported by ECVAM. The EST takes advantage of the potential of the mouse embryonic stem cell line D3 to differentiate in vitro spontaneously into contracting cardiomyocytes. The morphological differentiation into cardiomyocytes is known to be correlated with the expression of tissuespecific genes. Further, it can be assumed that embryotoxic effects on the morphological differentiation are accompanied by alterations in gene expression of developmentally relevant genes. Thus, our investigations were aimed at defining expression profiles of marker genes as appropriate endpoints for the prediction of the embryotoxic potential of test compounds. The expression levels were quantified by means of Real-Time TaqMan-PCR. Here we present the expression profiles of several myocard specific genes during in vitro development into beating cardiomyocytes, e.g. α-mhc, β-mhc and MLC-1a/f. Moreover, expression of marker genes that are involved in early mesodermal cell diversification (e.g. MesP1 and MesP2) was also investigated. MesP1 which codes for a transcription factor of the basic HLH-type was found to be the earliest gene induced in cardiogenic development of ES cells. By use of such early marker genes it may be possible to shorten the duration of the test. To investigate the influence of reference substances on the expression of three molecular marker genes, differentiating ES cells were treated with compounds of known embryotoxic and non-embryotoxic potential. It could be shown that strong as well as weak embryotoxins exerted a dose-dependent effect on both the expression levels of α-mhc, MLC1 and MesP1 and the morphologic development of beating cardiomyocytes. [Investigations to establish a common skin model test protocol for the human skin models EPISKIN and EpiDerm for the upcoming ECVAM validation study on in vitro skin irritation tests] Untersuchungen zur Entwicklung eines gemeinsamen Testprotokolls für die Hautmodelle EPISKIN und EpiDerm im

17 Rahmen der geplanten ECVAM Validierung von in vitro Hautirritationstests Helena Kandárová, Manfred Liebsch und Horst Spielmann Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET), Bundesinstitut für Risikobewertung (BFR), D-Berlin Seit den nationalen Kommentierungen der OECD Testmethoden-Entwürfe der im Jahr 2002 von der OECD als Prüfrichtlinien anerkannten ersten in vitro Alternativmethoden ist klar, dass ein Test, der nur von einer kommerziellen Quelle angeboten wird, kaum Chancen hat als Test für die regulatorische Toxikologie international Anerkennung zu erlangen. So hatte der im Rahmen der ECVAM Validierungsstudie validierte EPISKIN Haut Korrosivitätstest (L ORÉAL, Frankreich) nur eine Chance in die später verabschiedete OECD Test Guideline 431 einzugehen, nachdem ZEBET in Zusammenarbeit mit BASF und Huntingdon Life Sciences zeigen konnte, daß ein Test mit gleichem Design mit dem Hautmodell EpiDerm (MatTek, USA) die gleichen Ergebnisse lieferte (sog. catch-up Validierung). Leider hatten die Vorhersagemodelle beider Tests historisch bedingte Unterschiede, so daß die OECD Test Guideline nur ein Vorhersagemodell exemplarisch benennt. ZEBET hat seit 1991 verschiedene Hautmodelle in Tests mit verschiedenen toxikologischen Fragestellungen geprüft und dabei immer festgestellt, daß die kommerziellen Hautmodelle sehr viel ähnlicher sind, als manchmal von den Herstellern behauptet. Im Bereich Phototoxizität war das für das Hautmodell Skin² entwickelte Protokoll direkt auf EpiDerm übertragbar (Liebsch et al., 1997). Für die bevorstehende ECVAM Validierungsstudie haben sich daher Wissenschaftler von L ORÉAL und ZEBET im Rahmen der ECVAM Task Force Skin Irritation darauf geeinigt, im Rahmen der nach einer Prävalidierung notwendig gewordenen Testverbesserungen für die Hautmodelle EPISKIN und EpiDerm ein gemeinsames, identisches Testprotokoll zu entwickeln (Zuang et al., 2002). Die im Rahmen dieser Entwicklung durchgeführten Untersuchungen und Testoptimierungen werden vorgestellt. Liebsch et al. (1997). Entwicklung eines neuen in vitro Tests auf dermale Phototoxizität mit einem Modell menschlicher Epidermis (EpiDerm TM ). ALTEX 14, Zuang et al. (2002). Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ECVAM skin irritation task force report 2. ATLA 30, Rahmenbedingungen zur Verwendung humaner embryonaler Stammzellen (hes) in der Industrieforschung Identifikation humaner Neurotoxizitätsmarker mit einem in vitro hes- Zellscreeningsystem Martina Klemm und André Schrattenholz ProteoSys AG, D-Mainz Mit der humanen Stammzellforschung werden enorme Hoffnungen verknüpft, die zum besseren Verständnis von Entwicklungsprozessen, der Heilung von Gewebedefekten und der beschleunigten, sichereren Entwicklung von Medikamenten beitragen sollen. Nach gegenwärtigem Stand der Wissenschaft haben sowohl die humanen embryonalen, als auch die humanen adulten Stammzellen ihre Vorzüge und Einschränkungen in den verschiedenen Einsatzbereichen der Pharmaforschung, welche eingangs kurz dargestellt werden. Im Rahmen der Arzneimittelsicherheit müssen pharmakologisch aktive Substanzen hinsichtlich ihres potenziellen embryotoxischen und teratogenen Gefahrenpotenzials überprüft werden. Die aussergewöhnlichen Vorzüge des Einsatzes der humanen embryonalen (hes)-zellen für diesen Forschungsbereich werden anhand eines proprietären, murinen in vitro Embryo/Neurotox-ES-Zellscreeningsystem der Firma ProteoSys aufgezeigt. Das von ProteoSys entwickelte in vitro Testverfahren basiert auf neuronal differenzierten murinen embryonalen Stammzellen und wird zur Identifikation von Neurotoxizitätsmarkern mit Hilfe einer quantitativen differentiellen Proteinmusteranalyse eingesetzt. Unter Einsatz fortschrittlichster Proteomics-Techniken wird der Aktivitätsstatus von Proteinen sowohl qualitativ, als auch quantitativ direkt erfasst. Somit ist dieses Verfahren den rein nukleinsäurebasierten Testsystemen an Aussagekraft weit überlegen. Durch die Fortschritte in der humanen Stammzellforschung und die erfolgte gesetzliche Regelung (StZG) zu Import und Umgang mit humanen embryonalen Stammzellen (hes Zellen) ergibt sich jetzt auch in Deutschland erstmals die Möglichkeit, das embryo- und neurotoxische Gefährdungspotenzial einer Substanz humanspezifisch ohne Extrapolationsunsicherheiten vom Tier auf den Menschen erkennen und humane Toxizitätsmarker direkt identifizieren zu können. A novel method for the in vitro screening of UV-B protective substances Hans-Peter Klöcking 1, Kerstin Hübner 1, 3, Simone Kühn 1, Renate Klöcking 2 and Alfred Wegener 3

18 1 Institute of Pharmacology and Toxicology/Working Group Erfurt, University of D-Jena, 2 Institute for Virology and Antiviral Therapy, University of D-Jena, 3 Department of Experimental Ophthalmology, University of D-Bonn The aim of this study was to set up an in vitro screening method for UV-B protective substances. For this purpose, we used human promonocytic U937 cells (ECACC No ), which were UV-B irradiated for 20 seconds (140 J/cm 2 ) in the presence and in the absence of several UV-B protective agents. After a postexposure period of 24 h and 48 h, cell viability was measured using the XTT reduction assay EZ4U. The assay is based on the ability of living cells to reduce the colourless tetrazolium salt to an orange, water-soluble formazan product. Taking in consideration a possible interference between the cytotoxicity of test substances and their UV-B protective effect, cell damage was measured in irradiated as well as in nonirradiated cells. The UV-B protective effect was calculated for each substance concentration from the following equation, PC UV-B (%) = [(x' i x' o /(x o x' o )] 100, where x o is the mean absorbance of non-irradiated and non-treated control cells, x' o is the mean absorbance of irradiated, non-treated cells and x' i is the mean absorbance of substance-treated irradiated cells. To compare the effectiveness of test substances, the half-maximum protective concentration (PC 50 ) and the halfmaximum cytotoxic concentration (CC 50 ) were calculated from the percentage UV-B protective activity in irradiated cells and the cytotoxicity in non-irradiated cells, respectively. According to the therapeutic index (TI = CC 50 /PC 50 ), a ranking order of the test substances will be received. For maximal UV-B protection and safety, the TI should be as high as possible. The application of the test procedure to several UV-B protective agents is demonstrated. An approach to study the mode of UV-B protective action is presented in the poster of R. Klöcking et al. (see next abstract). In vitro evaluation of photoprotective agents using Cytomorph -b microtest plates Renate Klöcking 1, Simone Kühn 2 and Hans-Peter Klöcking 2 1 Institute for Virology and Antiviral Therapy, 2 Institute of Pharmacology and Toxicology/Working Group Erfurt, Friedrich Schiller University of D-Jena The aim of this study was to develop an in vitro assay for evaluating the mode of action of photoprotective agents. To discriminate between physical, chemical, pharmacological and toxic effects of the test substances, we used Cytomorph microtest plates as cultivation, reaction and measuring vessels. Cytomorph -b is a microplate with 3x3 flat-bottomed cylindrical wells, the arrangement of which corresponds to 3x3 congruent wells of the lid. Usually employed for phase contrast microscopy and multipoint reading, Cytomorph -b is applied for the first time as test device for evaluating the UV filter effect of photoprotective agents. Promonocytic U937 cells (ECACC No ) were used as indicator cells of irradiation damages and the XTT reduction assay EZ4U as the measure of cell viability. The following test designs were applied: (1) Graduated concentrations of the test substance were added to cells immediately before irradiation (140 J/cm²). (2) The cells were kept separate in the wells of the plate and the test substance solution was poured in the corresponding wells of the lid immediately before irradiation. (3) The test substance was added to cells 1 hr after irradiation. (4) The test substance was added to cells 1 hr before irradiation. (5) Non-irradiated cells were exposed to graduated substance concentrations (cytotoxicity assay). Half-maximum protective concentrations (PC 50 ) and half-maximum cytotoxic concentrations (CC 50 ) of the test substances were calculated from the percentage UV-B protective activity in irradiated cells and from the cytotoxicity in nonirradiated cells, respectively. The examination of a synthetic melanin (DOPA oxidation product, DOPAOP), a synthetic phenolic polymer (hydrocaffeic acid oxidation product, HYKOP) and a well-known photoprotective agent (p-aminobenzoic acid, PABA) demonstrate that the UV-B protective effect of these substances is caused by UV-B absorption and scattering (positive in experiment 1, 2 and 4) rather than by anti-oxidative and antiphlogistic effects (negative in experiment 3). Our experimental model is recommended for in vitro evaluation of photoprotective agents including their primary mode of action. Ready-to-use 96-well plates containing pre-seeded and cryopreserved cells for standardised cytotoxicity assays Oliver Klotzsche, Claudia Gelli and Alexander Loa CCS Cell Culture Service GmbH, D-Hamburg Standardisation of the cell culture still is a problem in the validation of in vitro toxicity assays. Even within a given laboratory, system-inherent use of different serum charges and variability of cells of different passages make it difficult to define a valid basis for a cell-based assay. Up to now this problem only insufficiently could be encountered by costly tests of different serum charges and a standardised propagation of cells from a working cell bank. We have developed in our laboratory a method enabling direct cryopreservation of cells in 96-well plates. Large batches of identical test plates can be manufactured and stored at -80 C for several months without loss of activity. So, even

19 in case of long-term assays it is always possible to access identical test material. The bottleneck of cell cultivation in the preparative phase is eliminated thereby enabling an increase in sample throughput. Various cell lines, even those that are adapted to grow in a serum-free medium (e.g. L929, Vero and several human tumour lines) are frozen in 96-well plates without toxic cryoprotectants and can be used directly for toxicity testing and other cell-based assays without a single washing step. This DMSO-free freezing procedure additionally provides for a rapid revitalisation of the cells. Already a few hours after thawing the activity of the frozen cells is restored nearly completely, exhibiting very low variation of vitality from well to well within a given plate and also between plates of a given batch. The frozen assay plates therefore are distinguished by their high conformity and reproducibility. Using established protocols IC 50 values for various reference substances were determined in comparison with non-frozen control plates. The results demonstrated the cryoconserved assay plates to be as reliable and expressive as assay plates with freshly seeded cells. The use of the assay plates requires no cell culture facility. So even less equipped laboratories are able to perform toxicity assays with these ready-to-use plates. [IgY-technology: benefit and chances for diagnostics and (immun-) therapies by humans and animals actually applicability and projects] Die IgY-Technologie: Nutzbarkeit und Chancen für Diagnostik und (Immun-) Therapien bei Mensch und Tier aktuelle Anwendungen und Projekte Hartmut Kobilke 1, Josef Krejci 2, Wolfgang Bergter 3 1 Egg-Immun Damsdorf, D-Damsdorf, 2 Veterinary Research Institute (VUVel), CZ-Brno, 3 Altmann-Therapie GmbH und Co KG D-Salzgitter Das von Klemperer 1883 entdeckte Prinzip, nach dem Hennen zum Schutz ihrer Nachkommen via Eidotter IgY-Antikörper gegen Antigene mitgeben, mit denen sie sich auseinandergesetzt haben, fand in dem Begriff IgY-Technologie (Schade et al., 1996) zunehmend Beachtung in Medizin, Veterinärmedizin, Landwirtschaft und Umweltschutz sowohl auf dem Gebiet der Diagnostik als auch der passiven Immuntherapie. Die IgY-Antikörper der Eidotter = IgG im Serum, stellen eine erfreuliche Alternative zu den bisher jährlich mehreren Millionen getöteten Labor- und Kleinsäugern dar, die zur Gewinnung polyklonaler und monoklonaler Antikörper verwendet werden. Im Sinne der 3R (Russel und Burch, 1959) leistet diese innovative Methode der jüngsten Branche der Biotechnologie / Biomedizin auch einen gewichtigen Beitrag im Sinne des praktizierten Tierschutzes. Die IgY-Technologie stellt ein Gesamtsystem mit mehreren Teilgebieten dar: 1) Auswahl und Vorbereitung der Junghennen einschließlich ihrer tierartgerechten Haltung und Fütterung, 2) Präparation und Applikation der Immunisierungsantigene beim Huhn, Antigenauswahl, 3) Auswahl und Prüfung verschiedener geeigneter (alternativer) Adjuvantien und Applikationsarten, 4) Antigenaufbereitung, Dosis und geeigneter Immunisierungsablauf (Immunisierungsprotokoll), 5) Aufbereitung, Reinigung, Mengenbestimmung der spezifischen IgY- Antikörper, 6) Anwendungs- und Applikationsstrategien für diese IgY-Antikörper, 7) Aufbau eines Qualitätssicherungssystems (Öko- Audit, EN-ISO 2000, GLP, GMP u.a.) 8) Erforderliche Aus- und Fortbildung des Personals. Die Anwendungsmöglichkeiten für IgY-Antikörper umfassen die veterinär-, humanmedizinische und toxikologische Diagnostik, die orale, parenterale und lokale passive Immuntherapie sowie die Therapie von Autoimmunerkrankungen. Aus bisher realisierten über 50 Forschungs- und Entwicklungsprojekten und praktischen Anwendungen, fundiert durch bisher 3 Patente, ergeben sich weitreichende Chancen für Nutzanwendungen der IgY-Antikörper auf den Gebieten der Diagnostik oraler, lokaler, parenteraler und extrakorporaler passiver Immuntherapien bei Mensch und Tier. Amendments to Directive 86/609 on animal experimentation an animal welfare perspective Roman Kolar and Alexandra Achenbach Deutscher Tierschutzbund Akademie für Tierschutz (German Animal Welfare Federation Animal Welfare Academy), D- Neubiberg The scientific and political basis for Council Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes was set approximately 20 years ago. New knowledge on ethological and other aspects of housing laboratory animals, new scientific achievements and initiatives in the field of the 3Rs (replacement, reduction, and refinement of animal experiments), but also new applications of laboratory animal use and generation/production (in particular in the area of genetic engineering) of animals result in an urgent need for a revision of the Directive in order to

20 ensure that it truly meets its goal to protect animals. Additionally, many critical aspects that were not considered when the Directive was drafted should to be taken into account in the discussions to come. The European Commission has acknowledged the need for an amendment of Directive 86/609 and therefore in 2000 initiated the procedure to do so. The animal welfare community has precise ideas on how the Directive should be modified, including a broadening of the scope of the Directive, more precise demands on an ethical review scheme, amended housing guidelines that should receive a compulsory status, special protection of non-human primates, and other topics. The outlines the major questions on these issues and gives an overview of the political process related to the amendment of the Directive. Assessment of telomere shortening as a novel end-point for chronic in vitro nephrotoxicity testing Christian Koppelstaetter 1, Paul Jennings 2, Walter Pfaller 2 und Gert Mayer 1 1 Internal Medicine, University of A-Innsbruck, 2 Institute of Physiology, University of A - Innsbruck Telomeres, the non-coding region at the end of chromosomes, are required for the stability of chromosome ends and non genome erosive cell-replication. Telomeres are shortened by each cell division and environmental stress factors and hence have been implicated in processes such as aging and cancer development. We have investigated the effect of the widely used immunosuppressive reagent Cyclosporine A (CsA) on telomere length in a renal proximal tubular epithelial cell line (HK-2). HK-2 cells were cultured in growth medium containing 0, 1 and 10 µg/ml CsA. Since these concentrations have been shown in our laboratory to be non-toxic to confluent HK-2 monolayers, experiments were done on proliferating cells. HK-2 cells were passaged every other day for 6 days (total of three passages), using a splitting ratio of 1 to 3 each time. Genomic DNA was isolated on the sixth day (DNeasy Tissue Kit, Quiagen) and analysed by quantitative PCR for relative telomere length. CsA treatment caused a dose dependent decrease in total DNA content over the three concentrations and 10 µg/ml CsA resulted in a significant telomere shortening as compared to control cells (p value = , students T-test, n=3). We conclude that telomere length shortening is a potential endpoint for assessing the chronic toxic potential of compounds in vitro. Hydrogen peroxide alters the migration of polymorph nuclear leukocytes through monolayers of the human proximal tubular epithelial cell line HK-2 Marius Koppler 1, Paul Jennings 1, Klaudija Bijuklic 2, Michael Ioannidis 2 and Walter Pfaller 1 1 Institute of Physiology, University of A-Innsbruck, 2 Department of Internal Medicine, University of A-Innsbruck Hydrogen peroxide (H 2 O 2 ) is a member of the reactive oxygen species (ROS) and is generated as a by product of certain metabolic pathways and has a potential role in selected signalling pathways. Oxygen radicals cause lipid peroxidation, protein denaturation and direct DNA damage due to strand breaks. The first part of this study was to establish a toxicity model of hydrogen peroxide in the human renal proximal tubular cell line HK-2. To find out the harming potential of H 2 O 2 to epithelium, dose-time and dose-effect relations were investigated. To this end transepithelial electrical resistance (TEER) and lactate dehydrogenase (LDH) release were utilised. The second part of this study was to asses whether H 2 O 2 affects the transmigration ratio of human polymorph nuclear leucocytes (PMN s) through HK-2 monolayers. HK-2 cells were seeded onto microporous membrane inserts and cultured to confluence. After pre-treating the monolayers with H 2 O 2 the PMN s were added and the transmigration measured 5 hours later using a myelo-peroxidase detection assay. There is a H 2 O 2 dose dependent decrease in TEER. Recovery experiments demonstrated 300µM H 2 O 2 treated cells recovered full barrier function after 2 h, and 600µM H 2 O 2 treated cells recovered during the following 12 h. LDH release also increased in a dose dependent manner. The results of the transmigration experiments suggest that within a sublethal concentration range of H 2 O 2 the PMN activity is stimulated and the migration ratio increased, what could not be shown in higher H 2 O 2 concentrations. Thus H 2 O 2 may contribute to the expression of signalling molecules in HK-2 cells that affect binding and migration of neutrophils. Another point to consider is that this in vitro model allowed us to examine a single subset of leucocytes under specific conditions, what would not have been possible in a standard animal experiment. PMN migration as an endpoint in toxicity testing may further provide the possibility to gain information on the on the potency of a toxic chemical to trigger inflammatory processes. [Toxicoproteomics: first experience in a case study]

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