2006 Progress towards novel testing strategies for in vitro assessment of allergens Erwin L Roggen, HansUlrich Weltzien, Helma Hermans for The Sensitiv Consortium
2006 Overview on the project activities and WP110 10 interactions WP1: Compound Selection Technology module (Months 1860) WP7: Data Management WP 10: Management Science module (Months 042) WP3: DC Tcell interactions and biology WP4: Genomics WP2: EC DC interactions and biology WP6: Metabonomics WP5: Proteomics WP9: Technology transfer & Dissemination (Months 060) WP8: In vitro assay development isub Brugge 2225.04.2008 2
2006 Addressed topics Precision cut lung slices Standardised immunologically and metabolically competent human cell lines for hazard assessment Assays addressing bioactivation and hapten formation Culture systems suitable for assessing the sensitising potential of compounds Relevant markers for the sensitising potency of different classes of chemicals isub Brugge 2225.04.2008 3
2006 Ex Vivo: Precisioncut cut lung slices (PCLS) PCLS react to immunomodulary compounds, including sensitisers. Relevant changes in membrane marker expression are observed. Calcein staining of untreated PCLS ICAM1 staining of untreated PCLS Untreated mouse PCLS, stained with anticd86 antibodies mouse PCLS incubated with LPS, staining with anticd86 antibodies Calcein staining of LPS treated PCLS ICAM1 staining of LPS treated PCLS isub Brugge 2225.04.2008 4
2006 Ex Vivo: Precisioncut cut lung slices (PCLS) PCLS react to immunomodulary compounds, including sensitisers. Relevant changes in membrane marker expression are observed. Calcein staining of untreated PCLS ICAM1 staining of untreated PCLS Calcein staining of LPS treated PCLS ICAM1 staining of LPS treated PCLS Untreated mouse PCLS, stained with anticd86 antibodies mouse PCLS incubated with ovalbuminspecific Tcells, staining with CD86 after exposure to ovalbumin and LPS isub Brugge 2225.04.2008 5
2006 Ex Vivo: Precisioncut cut lung slices (PCLS) PCLS react to sensitising compounds by upregulating the expression of cytokines (intra and extracellular). PCLS seem to discriminate between respiratory and skin sensitisers. Eotaxin GCSF IFNγ IL1α IL5 IL8 IL10 IL12 (p40) MCP1 MIP1ß RANTES TNF α ()control: LPS Lung: TMA HCpt Skin: DNCB CA ()control: SA Ph isub Brugge 2225.04.2008 6
2006 Addressed topics Precision cut lung slices Standardised immunologically and metabolically competent human cell lines for hazard assessment Assays addressing bioactivation and hapten formation Culture systems suitable for assessing the sensitising potential of compounds Relevant markers for the sensitising potency of different classes of chemicals isub Brugge 2225.04.2008 7
2006 Identification of in vivolike epithelial cell (EC) lines Criteria adherance to the surface; differentiation; viability/sustainability; polarisation; tightjunction formation (TJ): metabolic activity (constitutive, inducible); responsiveness to immunomodulating stimuli.. microscopy, histology MTT, Alamar Blue retention of macromolecules, TEER, presence of TJ proteins substrate cocktail MS ELISA isub Brugge 2225.04.2008 8
2006 Identification of in vivolike epithelial cell (EC) lines Cell culture engineering soluble factors; growth factors interaction with matrix constituents; collagen IV, hyaluronic acid enhanced cellcell interaction; position, shape and polarity; biomaterials isub Brugge 2225.04.2008 9
2006 Identification of in vivolike epithelial cell (EC) lines Skin Alveolar Bronchial Primary cells keratinocytes alveolar EC (type 1 and type 2) 9HTE16o, HBE14o, 1HAEo, BEAS2B, CF/T43, CFBE41o, CFBE45otracheobronchial and bronchial EC Cell lines NCTC 2544 A427, A549, H292 Calu1, Calu3, Calu6, H441, HBE1, IB31 Steimer, Haltner and Lehr, Journal of Aerosol Medicine 18; 137182 (2005) isub Brugge 2225.04.2008 10
2006 Identification of in vivolike dendritic cell (DC) lines Hierarchical clustering and principle component analysis (PCA) revealed populationspecific profiles. PCA of 80 DC marker genes revealed subtype similarities. A. primary DC from tonsils (N=3) and blood (N=4) B. C. A. C. primary DC from skin (N=2) B. DC derived from precursor cells (N=8) MUTZ3 Cell line Expressing most characteristic markers Most in vivolike functionality: differentiation maturation antigenpresentation isub Brugge 2225.04.2008 11
2006 Addressed topics Precision cut lung slices Standardised immunologically and metabolically competent human cell lines for hazard assessment Assays addressing bioactivation and hapten formation Culture systems suitable for assessing the sensitising potential of compounds Relevant markers for the sensitising potency of different classes of chemicals isub Brugge 2225.04.2008 12
2006 In Vitro: Air/liquid phase A549 models for sensitisation Proteinspecific cytokine profiles (33 tested): Protease Lipase BSA GCSF polycarbonate insert GMCSF MCSF 750 6well tissue culture plate I309 IFNγ IL1β pg/ml MCSF 500 250 IL6 IL8 MCP1 0 0.01 0.1 1 10 µg/ml enzyme RANTES Alcalase Savinase BPN' Coronase Inactive Savinase isub Brugge 2225.04.2008 13
2006 In Vitro: Executive summary of sensitiser induced cytokine responses in A549 EC PCLS Eotaxin GCSF GM CSF IFNγ IL1 IL5 IL6 IL8 IL10 IL12 (p40) MCP1 MCSF MIP1ß RANTES TNF α () control) n.d. n.d. n.d. Lung Skin () control) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. A549 EC line Eotaxin GCSF GM CSF IFNγ IL1 IL5 IL6 IL8 IL10 IL12 (p40) MCP1 MCSF MIP1ß RANTES TNF α () control) Lung Skin () control) n.d. isub Brugge 2225.04.2008 14
2006 An in vitro coculture culture model of the human alveolocapillary barrier using the H441 cell line stimulation apical. cytokine [pg/ml] 200µl 1000µl stimulation basolateral apical basolateral Iris Hermanns, Johannes Gutenberg University, Mainz, Germany isub Brugge 2225.04.2008 15
2006 An in vitro coculture culture model of the human alveolocapillary barrier using the H441 cell line apical basolateral Iris Hermanns, Johannes Gutenberg University, Mainz, Germany isub Brugge 2225.04.2008 16
2006 In Vitro: Air/liquid phase model using primary human lung EC (Epithelix( Epithelix) After 12 months: 1. Appearing of ciliated, mucus and basal cells 2. Establishment of absorption / secretion properties 3. Stabilisation of electrophysiological properties Exposure regimes: Basal: the cells can be incubated during 24, 48 or 72 h Apical: only a short time exposure immersion (> 1 hr) induced inflammatory response chronic exposure (10x 1hr/day) Readouts: viability cytokine profiles (IL1, IL6 and IL8) isub Brugge 2225.04.2008 17
2006 In Vitro: The Sensit itiv iv epidermal equivalent penetration/cytokine release high barrier competency impaired barrier sumerged MDI no/no no/no n.a./yes TMA no/no no/no n.a./yes HCPt no/no no/no n.a./yes DNCB yes/yes yes/yes n.a./yes CIN yes/yes yes/yes n.a./yes TMDT no/no no/no n.a./yes SLS poor/poor yes/yes n.a./yes SA Phe isub Brugge 2225.04.2008 18
2006 In Vitro: DC models for sensitisation MUTZ3, THP1, U937, MoDC SOPs IL1β and IL8 (ELISA and QPCR) CD54 and CD86 (flow cytometry) Evaluation in progress isub Brugge 2225.04.2008 19
2006 In Vitro: EC DC cultures supporting in vivolike cellcell interactions Lung Development of ECDC airlifted assays is experiencing problems: Nonpolarising cell lines (A549, BEAS2B) do not form the tightjunctions (TJ) required for assay development (ECDC) Calu3 form TJ but shows weak responsiveness to stimuli. ttert immortalised human bronchial cells available isub Brugge 2225.04.2008 20
2006 In Vitro: EC DC cultures supporting in vivo like cellcell interactions Skin keratinocytemutz3 interactions do not affect viability nor phenotype (preliminary) Progenitor MUTZ3 cells differentiate into MUTZLC in epidermis and MUTZDC in the dermis keratinocytes required for stimulation (IL8 release) of DC by prohaptens progenitor DC respond stronger than differentiated MUTZ3 are responsive. isub Brugge 2225.04.2008 21
2006 In Vitro: EC DC cultures supporting in vivolike cellcell interactions Any relevance for ECDC based assays? Learnings from the skin sensitisation area (TeSens): loosefit coculture of activated keratinocytes and DCrelated cells sensitivity allows for testing without general cytotoxicity discrimination of sensitisation and inflammation Cocultures of A549 and MUTZ3 demonstrated an intensive communication between these cells. no sensitiserinduced changes isub Brugge 2225.04.2008 22
2006 Addressed topics Precision cut lung slices Standardised immunologically and metabolically competent human cell lines for hazard assessment Assays addressing bioactivation and hapten formation Culture systems suitable for assessing the sensitising potential of compounds Relevant markers for the sensitising potency of different classes of compounds isub Brugge 2225.04.2008 23
2006 Identification of specific endpoints and in vivo relevant markers (EC) Genomic analysis: immune reponses IL6, IL6R, IL16, IL16R, IL27R, IL28R cell cycle genes energy (phosphate) metabolism Proteomic analysis: GCSF, GMCSF, IL1, IL6, IL8, MCSF, RANTES isub Brugge 2225.04.2008 24
2006 Identification of specific endpoints and in vivo relevant markers (EC) Signaling pathway analysis: JakStat signaling SAPK/JNK signaling PI3K/AKT signaling NotchJagged signaling integrinmediated signaling leukocyte extravasation signaling axonal guidance signaling neuregulin signaling IGF1 signaling Tolllike receptor campmediated signaling isub Brugge 2225.04.2008 25
2006 Identification of specific endpoints and in vivo relevant markers (DC) Proteomics IL1β, IL8 (CXCL8) CD11c, CD40, CD54, CD86 HLADR IL8 (CXCL8) and CD86 induced by most sensitisers progenitor MUTZ3 cells > differentiated MUTZ3 and DC isub Brugge 2225.04.2008 26
2006 Identification of specific endpoints and in vivo relevant markers (DC) PepChip Kinase array p38 signaling pathway CD analysis Proteins: CD30, CD49d, CD71, CD81, CD86, CD95, CD123 Chemicals:CD40, CD43, CD54, CD80, CD83, CD86 isub Brugge 2225.04.2008 27
Status LSHBCT 2006 An in vivolike lung EC was not yet identified: EC lines discriminate lung from skin sensitisers A catalogue of biomarkers An in vivolike DC line was identified (MUTZ3): A catalogue of biomarkers Development of ECDC airlifted assays experiences problems: Lung: nonpolarising cell lines do not form the tightjunctions (TJ) required for ECDC assay development. Skin: model established. Submerged cocultures under investigation isub Brugge 2225.04.2008 28
2006 to be continued iv.eu IVTIP, ECOPA, ECVAM, COLIPA Monthly newsletters Meetings, Congresses Workshops/Courses (Hogeschool Utrecht, Poster 68) Publications 2nd year: 11 manuscriptes published, accepted or in press isub Brugge 2225.04.2008 29