Number 41, July 2010 Dear Sens-it-iv Newsletter subscribers, Sens-it-iv is an integrated EU-funded research project involving 28 partners drawn from across Europe. They are joined together by the common goal of developing alternative strategies to animal testing for the assessment of skin and/or respiratory sensitizing potential of chemicals. This includes the development of predictive  in vitro methods. In previous Newsletters we addressed the work carried out by the different Workpackages (nrs 1-13), the scientific results generated in the various laboratories (nrs 14-21), and the work to develop assays based on dendritic cells (nrs 24 and 27), on a human keratinocyte cell line (nr 25), on T-cells (nr 26), on human Precision Cut Lung Slices (PCLS) (nr 28) and on an Epidermal Equivalent model (nr 29). We also reported on the induction of innate immune and stress responses by contact allergens (nr 30), the first Sens-it-iv Summer School (nr 31), the 'Sens-it-iv list of chemicals' (nr 32) and the Sens-it-iv database (nr 37), the tests chosen for further development in the 5th and final year of Sens-it-iv (nrs 33, 34 and 36) and our genomics, proteomics and metabonomics work (nr 35, 38 and 39). In Newsletter 40, we shared with you our visions of the world into which the Sens-it-iv results will ultimately have to be assimilated. In this Newsletter, nr 41, we present to you the decisions of the recently held last half-year General Assembly on the scientific and technical work to be done until the end of 2010. With kind regards - the 'Sens-it-iv Dissemination and Technology Transfer' Team Contact For contacts, please use the contact form on our website. Contact form Subscription To subscribe or unsubscribe from this newsletter, please use the form on our website. Subscription form

The outcome of the 4.5e General Assembly in Berlin, May 26-28, 2010 The last half-year General Assembly of the Sens-it-iv consortium was held in Berlin, Germany, from May 26-28, 2010 to discuss and decide on the procedure how to finalize and close up the project until the 5th and final General Assembly to be held on October 25-27 in Antwerp, Belgium. Even though it is planned to extend the 5-year period of Sens-it-iv by 6 months until March 31st 2011, the assembly decided to stop experimental work the latest by the end of the year 2010 and use the remaining time for closing reports, publications and management activities. One major management activity during this time will be to start the organization of an international congress planned for November 24-25, 2011 to present the outcome and perspectives of Sens-it-iv to an international industrial and academic scientific audience, as well as to regulators. Below we will review the decisions of the assembly on the scientific and technical work to be done until the end of 2010. Assays to identify skin sensitizers Major emphasis in the discussions concentrated on assays selected previously for further development, their practical application and attempts to transfer them to the state of pre-validation. Two-tiered test to assess skin sensitizers: Most advanced in this sense is the so called “two-tiered test” combining a yes/no identification of potential skin sensitizers by intracellular production of IL-18 in the NCTC2544 keratinocyte cell line, and the estimation of their sensitizing potency in an epidermal equivalent model (Figure 1). Additional molecular markers for specific keratinocyte responses to skin sensitizers may come from a large set of proteomic data presently under statistical evaluation (see Newsletter 38). Figure 1. Two-tiered integrated test system Current data generated within the Sens-it-iv project suggests that using human NCTC2544 keratinocyte cell line together with a reconstituted human epidermal skin model may generate an integrated test system to: 1) identify skin sensitizers from non-sensitizers and respiratory sensitizers; and 2) assess the potency of the identified skin-sensitizers. The first tier in the strategy determines the production of intracellular IL-18 by human-derived NCTC2544 keratinocytes and indicates the potential of a chemical to induce sensitization. The second tier in the strategy determines the viability (MTT) and production of IL-1α in a human reconstituted epidermal model as a measure for sensitizer potency. DC-based assays: Several such assays are under consideration. The DC-migration test, determining the differential migration of MUTZ-3 cells, differentiated into a Langerhans-like status, towards CXCL12 or CCL5 has also been described previously in Newsletter 34. This test, which represents the only truly functional correlation to the in vivo situation, is presently being tested for its technical transferability between different laboratories. Other assays determining the expression of DC-activation markers such as CD86 and IL-8 on MUTZ-3, U937 or THP-1 cells (Newsletter 27) have also been evaluated in inter- and intra-laboratory comparisons. Proteomic and genomic assays: Large data sets derived from proteomic and genomic experiments evaluating the reactivity of MUTZ-3 cells to sensitizers, irritants and controls are under way. Particularly promising are ongoing genomic experiments to define a robust signature of genes, the response of which in MUTZ-3 cells would allow the identification of skin sensitizers with high probability (>95%). As recently described in Newsletter 35, a set of about 100 genes has already been identified and a reduction of this set to 10-20 genes appears feasible without loss of predictive power. These experiments are presently being repeated with an enlarged and blinded set of chemicals and the results are expected to be available by October this year. Assays to identify respiratory sensitizers A human alveolar-endothelial cell based assay with cytokine expression as read-out This novel test model was developed outside the Sens-it-iv project, but within the Sens-it-iv sphere, by Dr. Iris Hermanns (Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Institut für Pathologie). The test is novel for sensitization testing since this is to our knowledge the only test system for the human alveolar compartment of the lung that builds upon human cell lines (H441 and ISO-HAS-1) and that reveals in vivo-like functionality (morphology, alveolar type 1 and type 2 cells, reconstituted epithelium with high trans-epithelial resistance (TEER), transport, in vivo-like responses to immunomodulators (e.g. LPS)) that is relevant for the purpose of Sens-it-iv. The need for endothelial cells to provide the alveolar cells with appropriate in vivo-functionality was demonstrated. As it was not developed for assessing sensitization, a first evaluation using the learning set of chemicals was performed. Dose-finding experiments were performed using reduction in TEER as read-out instead of increase in cytotoxicity. In general, test systems based upon lung tissue or lung cells are more responsive to respiratory sensitizers as compared to skin sensitizers and irritants. As for the skin, IL-8 seems to be able to discriminate both groups of sensitizers, but not between sensitizers and irritants. On the basis of these promising results, a larger evaluation was initiated including 3 skin sensitizers (cinnamaldehyde (CIN), 2,4-dinitrochlorobenzene (DNCB), eugenol (Eug), 3 chemical respiratory sensitizers (ammonium hexachloroplatinate (HCPt), diphenylmethane diisocyanate (MDI), trimellitic anhydride (TMA), 3 proteins (amylase 1, lipase 1, protease 1) and 2 controls (salicylic acid (SA), sodium lauryl sulphate (SLS)). The dose-finding experiments using TEER measurements were concluded. Exposure induced changes in the cytokine profiles were analyzed using a cytokine antibody array with focus on IL-8 and cytokines that were identified by other research groups in the project as potentially useful (e.g. TNF-α, IL-1α, RANTES, MIP-1β, M-CSF). Data analysis is currently in progress. An SOP draft was established. Supernatants and RNA samples were stored frozen for future –'omics' analysis and the assessment /confirmation of markers identified by other means. Chronic exposure of a human bronchial EC assay with cytokine expression as read-out An in house human bronchial EC model using primary bronchial EC was developed in collaboration with carcinoGENOMICS (www.carcinogenomics.eu) (Dr. Jan Boei, Leiden University Medical Center, Department of Toxicogenetics). Extensive genomic and functional (reconstituted epithelium with high TEER, CYP activity, responses to immunomodulators, mucus secretion, transport, beating cilia) characterization revealed that primary cells should be used within passage 4 after isolation from the human lung, and that the cells should be cultivated at an air-liquid interphase. In addition, donor-to-donor variability was described. In order to address the donor issue and to assure sustainability of the test system, a human bronchial cell line was developed using the telomerase (hTert) approach. The emerging cell line is currently being characterized, but will not be evaluated by the Sens-it-iv project. Sens-it-iv identified a commercial available human bronchial EC model (MucilAIR, Epithelix) with similar characteristics as the in house primary cell model described above. MucilAir has a shelf life of up to 1 year, assuring sustainability for the duration of the Sens-it-iv project. An additional advantage of this test model is that it allows for both acute and chronic exposure. As it was not developed for assessing sensitization, a first evaluation using the learning set of chemicals (MDI, TMA, HCPt, DNCB, CIN, tetramethyl thiuram disulfide (TMTD), SA, phenol (Phe)) and the prohapten cinnamic alcohol (CA) was performed. Dose-finding experiments were performed using reduction in TEER as read-out. The results of the quantification of the inflammatory mediators, before and after the exposure to the chemicals, revealed that each compound has very specific pharmacodynamics on the cytokine. Regarding respiratory sensitizers, even if the profile of IL-8 secreted after 24 hours exposure is not the same for MDI, TMA and HCPt, it is clear that the secretion increases 2-3 fold with increasing (subtoxic) concentration. With the as yet unexplained exception of the prohapten CA, dermal sensitizers did not show significant acute (< 72 hrs after exposure) effects on IL-8 secretion. Among the irritants tested, Phe stimulated IL-8 release. Interestingly, analysis of IL-8 responses after chronic (1-3 weeks) exposure to the chemicals revealed a further increase 5-10 fold in IL-8 levels upon stimulation with respiratory sensitizers. It was observed that at low concentrations chronic responses were more likely to occur than acute responses. The chemical haptens did not affect IL-8 secretion while CA (3-fold) and Phe (5-fold) still did. Within the period of Sens-it-iv these results will be confirmed and testing of the Sens-it-iv compounds will be initiated. Other assay-related R&D activities P38 as additional DC activation marker: Although IL-8 secretion by DC cell lines is in general a reliable marker for potentially sensitizing chemicals, several sensitizing pro- or pre-haptens fail to induce IL-8 secretion e.g. in THP-1 cells. Indications are that this might be due to compound-induced instability of IL-8 mRNA. Determination of enhanced P38 MAP kinase activity gave positive results in these cases and points to P38 as a valuable additional DC biomarker. The practical development of this method, however, will probably not be finalized within the lifespan of Sens-it-iv. T cell assays: significant progress was reported on the development of T cell assays. The decisive step in this procedure is the removal of regulatory as well as of memory T cells from the blood samples in the assay. Also the preparation of autologous, blood-derived DCs for antigen presentation was simplified. Loading of the DCs with sensitizers was achieved either by direct chemical modification in vitro or by addition of hapten-modified human serum albumin. The latter situation required an additional innate activation stimulus for the DCs (see Newsletter 30), e.g. by LPS. Readouts were either T cell proliferation or preferably cytokine production, e.g. IFN-γ. An expert meeting of numerous laboratories from within and outside Sens-it-iv developing T cell assays as alternatives to animal testing was held in Rome on November 7, 2009, and a review on the outcome of this meeting and developments in the field over the last two decades is in the process of being published. It was decided to repeat such meetings on a regular basis beyond the termination of Sens-it-iv. All participants declared their willingness to continue R&D efforts in the field beyond Sens-it-iv. Metabolism studies: studies on cellular metabolism of pro-haptens concentrated on co-cultures of THP-1 cells and neutrophils (to introduce metabolizing capacity into the DC test system, see Newsletter 39). Hoever, it was found that the co-culture system had no added value over studying both cell types separately. In fact, neutrophil preparations alone appeared as a very promising system to detect metabolism-dependent pro-haptens. These studies will be extended with more chemicals till the end of the project. The Sens-it-iv database: emphasis was put on the importance to complete and maintain the Sens-it-iv database concerning in vitro and in vivo information on sensitizing chemicals as well as on standard operation procedures (SOPs) for assays and chemicals. The discussion is ongoing as to how the database can be maintained and made publicly available after the termination of the Sens-it-iv project. Communication and technology transfer Sens-it-iv will continue communication and technology transfer by its monthly newsletter, publications, press releases and in particular by an attempt to set up an e-learning program to efficiently transfer Sens-it-iv’s accumulated knowledge on alternative test methods to interested industrial and academic laboratories. It is also planned to make the selected list of tutorial chemicals publicly available, including all SOPs for their solution and test application. A major task will then be the organization of the above mentioned Sens-it-iv congress on November 24-25, 2011. For more information: Please contact: Dr. Erwin Roggen, Sens-it-iv co-ordinator, at elro@novozymes.com Prof. Dr. Hans-Ulrich Weltzien, vice co-ordinator Sens-it-iv, at huweltzien@yahoo.de

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