Chronic obstructive pulmonary disease (COPD) includes emphysema and chronic bronchitis, which are characterised by a progressive airflow limitation and chronic inflammation. The pathogenesis of COPD involves different cells, mainly epithelial cells, macrophages, neutrophils, and CD8 lymphocytes. Bronchial epithelium lines the mucosal surface of the airways, forming a mechanical barrier that separates the external environment from the internal milieu. Recently, substantial evidence has emerged indicating that airway epithelial cells are able to liberate a number of chemokines fundamental to both inflammatory and immune responses. Therefore, we established an in vitro model by showing that cigarette smoke is able to induce the release of chemokines by lung epithelial cells. Furthermore, we show that cigarette smoke induced chemokine expression is resistant to dexamethasone, mimicking the clinical situation. In contrast, pyrrolidinedithiocarbamic acid, an experimental antioxidant compound, inhibited smoke induced chemokine expression. These results suggest that this epithelial cell culture model may allow the evaluation of novel anti-inflammatory compounds for the treatment of COPD directly on the relevant target cells in vitro. This approach may result in the replacement of animal experimentation in screening of new therapeutics for COPD.
On 29 October 2003, the European Commission forwarded the "Proposal for a Regulation of the European Parliament and of the Council concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency and amending Directive 1999/45/EC and Regulation (EC) on Persistent Organic Pollutants" to the European Parliament and the Council for adoption under the so-called co-decision procedure. From the point of view of animal welfare it is to be welcomed that it is envisaged better to protect humans and the environment from unknown risks through chemical substances. However, in order to ensure that this goal can be met, extensive amendments to the proposed draft are requested: Instead of continuing to perform animal tests, relevant and reliable non-animal testing strategies should be implemented. Before considering testing, all existing data must be made available, shared and evaluated. This request should be enforced in the new Regulation through an adequate mandatory system. Data requirements must be tailored to the specific substance, to ensure that only such information is collected that is necessary for its safe handling by workers and consumers. Missing data should be determined with non-animal test methods. Finally, the European Union and its Member States are called to provide adequate funding for alternative method research without delay and to call for tenders on those information-gaps that currently prevent the implementation of an entirely non-animal testing strategy, so that such new methods will be available in time for application under the REACH system.
The embryonic stem cell test (EST) is an in vitro assay that has been developed to assess the teratogenic and embryotoxic potential of drugs and chemicals. It is based on the capacity of murine ES cells (cell line D3) to differentiate into contracting myocardial cells under specific cell culture conditions. To assess the embryotoxic potential of a test substance the appearance of beating cardiomyocytes in embryoid body (EB) outgrowths is used as an toxicological endpoint. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study the EST predicted the embryotoxic potential of chemicals and drugs in the same manner as two other in vitro embryotoxicity tests employing embryonic cells and tissues from pregnant animals. In a joint research project with German drug companies we have successfully improved the EST by establishing molecular endpoints of differentiation in cultured ES cells. The quantification of cardiac-specific protein expression by intracellular flow cytometry has been studied in the presence of chemicals with different embryotoxic potentials. The results obtained using molecular endpoints specific for differentiated cardiomyocytes employing FACS (fluorescence activated cell sorting) analysis will be presented in comparison to the validated endpoint - the microscopic analysis of beating areas. FACS analysis provides a more objective endpoint for predicting the embryotoxic potential of chemicals than the validated method. Furthermore, flow cytometry holds promise to be suitable for high-throughput screening systems (HTS). In addition, our partners from the joint project have improved the EST by developing protocols that stimulate differentiation of ES cells into neural and endothelial cells, chondrocytes and osteoblasts because some substances might have embryotoxic effects on specific cell-types other than cardiomyocytes. These protocols have been successfully established at ZEBET and in the participating laboratories. Additionally, molecular endpoints have been established for the detection of specific differentiation pathways. Furthermore, new prediction models (PMs) have been developed using single endpoints of the EST.
Botulinum toxin, a nervous poison produced by bacteria, is increasingly being used - besides its medical application - as a beauty product for smoothing facial wrinkles. It is unknown in public that each batch of the toxin has to undergo a quality control before marketing. The test used is a LD50 test using mice that is very animal consuming and causes extreme suffering. Although several alternative methods exist, none of these are have yet been adopted by the European pharmacopoeia. Consumers in the EU do not accept animal experiments for cosmetic purposes. However, Botulinum toxin does not fall under the definition of a "cosmetic product" and therefore the bans on animal experiments laid down in the EU Cosmetic Directive do not apply. Therefore on a short term scale, only the voluntarily renouncement of the use of this toxin as an anti-wrinkling agent can prevent the suffering and death of animals for a beauty product.