 |
|
The Final General Assembly in
Edegem, Belgium, October 25-27, 2010 |
|
|
|
|
| The Sens-it-iv project
aims at the replacement of the local lymph node assay (LLNA) by non-animal in vitro tests to identify potentially allergenic chemicals. Originally scheduled to terminate by September 30th, 2010, the project has been extended
by the European Commission until March 31st, 2011. Nevertheless, the final General Assembly was held after the end of year five from October 25-27 in Edegem, Belgium. |
|
| Unlike previous General Assemblies,
the meeting was not subdivided into reports of the individual workpackages, but rather into the topics “Scientific Side Effects or Spin-offs”, “Biomarkers”, “Immature tests”, “Prevalidation Efforts”, “Data Storage and Dissemination”, and what is considered the “Sens-it-iv Toolbox” after five years of input. |
|
|
Scientific spin-offs
As spin-offs or side effects we consider findings resulting in new insights into the basic molecular mechanisms of skin or lung sensitization without necessarily being directly transferable into practical assays (see also Newsletter 42). |
|
The Consortium as well as the Scientific Advisory Board acknowledged the great importance of such efforts within the Sens-it-iv project. |
|
|
Pivotal role innate immune mechanisms
The group of partner 28b (University of Freiburg/Germany) used in vitro and in vivo gene knockout systems to uncover a pivotal role of innate immune mechanisms in the initiation of contact sensitivity (Newsletter 30,
Martin and Esser, 2010). As summarized in Figure 1, these involve Toll-like (TLR) or NOD-like receptors, reactive oxygen species (ROS) and the NLRP3 inflammasome, reflecting closely the signalling cascades induced by microbial infections. |
|
Chemical sensitizers like TNCB activate TLR2 and TLR4 indirectly via TLR ligands produced e.g. by enzymatic degradation of high-molecular weight hyaluronic acid (Martin et al., 2008).
In contrast, nickel ions can activate human (but not murine) TLR4 by direct binding to accessible histidine residues (Schmidt et al., 2010). Moreover, evidence was provided for activation of the NLRP3 inflammasome by chemical
sensitizer-induced release of ATP from skin cells and triggering of the ATP receptor P2X7 on dendritic cells (Weber et al., 2010). |
|
|
|
|
Figure 1. Innate immune and stress response pathways
The initiation of contact sensitization involves Toll-like (TLR) or NOD-like receptors, reactive oxygen species (ROS) and the NLRP3 inflammasome, reflecting closely the signalling cascades induced by microbial infections. |
|
|
Pre- and
pro-haptens fail to induce IL-8 secretion in DC cell line
Another spin-off example originates from the finding of partner 13 (University of Milano/Italy) that unlike typical haptens most pre- and pro-haptens (i.e. chemicals requiring oxidative or enzymatic metabolism to become
chemically reactive haptens) fail to induce secretion of IL-8 in the human DC line THP-1 within 24 hours. This failure to produce IL-8 protein appears to result from chemical-induced destabilization of IL-8 mRNA. |
|
Consequently, when measured at earlier time points (i.e. 3 hours), elevated levels of mRNA for IL-8 as well as for p38 MAP kinase can be detected after treatment with pre- or pro-haptens (Newsletter 42, Mitjans et al., 2010).
Verification of these findings with more chemicals would be of great value because most other assays fail to detect pre-and pro-haptens. |
|
|
Neutrophils
respond to pro-haptens
The question of metabolic competence for the detection of pro-haptens was intensively addressed by partners 10 (University of Liverpool, UK) and 24 (University Hospital Aachen, Germany). The Aachen group concentrated on the
role of P450 enzymes and membrane transporter systems for metabolizing pro-haptens (Newsletters 16 and 39). Recombinant individual enzymes were combined in vitro to mimic e.g. the situation of human skin. |
|
Analysis of various cell lines employed within Sens-it-iv by the group at the University of Liverpool showed negligible P450 levels in all of them. However, expression of myeloperoxidase (MPO) was found high in
neutrophils from peripheral human blood. Neutrophils respond to contact sensitizers by IL-8 secretion and, consequently, responded positively to several pro-haptens as well. Studies are under way to broaden the basis of a
neutrophil assay by testing more chemicals. |
|
|
Interaction
dinitrobenzene-derivatives with THP-1 cells
Interesting data have also emerged from studying the interaction of allergenic haptens with cellular proteins (P2-University of Mannheim/Heidelberg and P10-University of Liverpool) revealing partially very specific types of
interaction. Particularly detailed experiments were conducted comparing the interaction of dinitrobenzene-derivatives of different intrinsic chemical reactivity with THP-1 cells (P10, Megherbi et al., 2009). |
|
The studies included the detection of specific protein modifications, effects on GSH depletion and identification of the signalling pathways involved. These findings are to be analysed in the context of the newly
defined genomic and proteomic markers and pathways (see below). |
|
|
Identification of signalling pathways
Proteomic and genomic studies designed to identify new biomarkers, which differentiate sensitizers from irritants and non-sensitizers have become highly successful during the last year of the project. Proteome analyses
by partners 2 (University of Mannheim/Heidelberg, Germany) and 32 (Proteome Sciences R&D, Frankfurt, Germany) of primary human keratinocytes as well as of MUTZ-3 dendritic cells (Newsletter 38), and gene-chip based
analyses by partner 7 (University of Lund, Sweden, Newsletter 39) of MUTZ-3 cells revealed large numbers of clearly sensitizer-specific markers. |
|
So far these studies predominantly employed skin sensitizers, but the methodology is open also for detection of respiratory sensitizers. |
|
|
Identification
of signalling pathways
Identified markers were analysed in both systems according to their assignment to defined cellular signalling pathways (Newsletter 42). The most prominent of these pathways for keratinocytes and MUTZ-3 cells are listed in
Figure 2, revealing several interesting overlaps. Particularly markers relating to the NRF2-mediated oxidative response and oxidative stress in general were identified repeatedly, very much in line with the prominent
position that oxidative stress holds in the hapten sensitization scheme in Figure 1. |
|
It is planned to use the remaining time to investigate which of these markers could be verified by other methods (FACS, ELISA, Western blot etc.) and how they might be integrated into the assays under development. |
|
|
|
|
Figure 2.
Signalling pathways involved in the reaction to skin sensitizers identified by proteomic and genomic analyses in keratinocytes and MUTZ-3 cells. |
|
|
Genomic
markers for respiratory sensitizers
Genomic markers indicating respiratory sensitizers are being studied by partner 14 (VITO, Belgium) using the human lung cell line BEAS-2B aiming at defining a genomic marker signature for lung sensitizers. |
|
Presently, a collaborative study with partners 1 (Novozymes, Copenhagen, Denmark) and 12 (Fraunhofer Institute, Hannover, Germany) evaluates the robustness of discriminating gene expression by RT-qPCR in
the 3 different laboratories. |
|
|
Immature tests
The term immature tests is used for promising assay developments, which cannot be expected to become functional within the time scale of Sens-it-iv. The borders between this group and “spin-offs”
or “Toolbox” are floating.
A typical example was reported by partner 9 (VU Medical Center, Amsterdam) who developed a full thickness immunocompetent skin equivalent model incorporating MUTZ-3 derived Langerhans cells (LC). |
|
The model (Figure 3) allows to determine sensitizer-induced LC migration as well as secretion of pro-inflammatory mediators (e.g. IL-1ß) or the maturation-dependent expression of cell surface markers such
as chemokine receptors (e.g. CCR7). Although surely not a high-throughput test this may expand the number of truly functional in vitro assays. |
|
|
|
|
Figure 3. MUTZ-3 derived LC
integrated into a full thickness skin equivalent
Staining by CD1a, Langerin or HLA-DR identifies Langerhans cells (in red) in a full thickness immunocompetent skin equivalent (lower panel) as compared to control human skin (top panel). |
|
|
Prevalidation efforts
Partner 3 (ECVAM) gave an outline of the pre-validation and validation processes by ECVAM (see also Newsletter 40). R. Pieters from ZonMW (Utrecht, Netherlands) reported on the status of a prevalidation study
(Newsletter 36) of Sens-it-iv’s 2-tiered assay, consisting of the NCTC2544 keratinocyte test and the epidermal equivalent potency assay (see under „toolbox“ for details). |
|
The project is joined by several groups from outside Sens-it-iv, who in addition to the Sens-it-iv training set of chemicals contribute further compounds to the system. The tests are being
established in 3 independent laboratories and actual testing is supposed to start by 2011. |
|
|
Data
Storage and Dissemination
Planned transfer of database
Partner 28a (FDM, University Freiburg) referred on the status of the Sens-it-iv data bank and how to access and use the various parts of it (Newsletter 37). The problem of keeping the data bank open after
the termination of Sens-it-iv was addressed. The consortium agreed to transfer the Sens-it-iv data bank after March 2011 to the more recent European data banks ToxBank and OpenTox. |
|
It will have
to be decided at a later time point, whether this would just concern an independent storage of the Sens-it-iv data base or rather it’s firm integration and merging with the ToxBank data and analysis system. |
|
|
Dissemination and technology transfer
Recent efforts concerning Dissemination and Technology transfer were reported by Partner 23 (SW&C, Pedreguer, Spain). It was acknowledged that Sens-it-iv has the exceptional record of publishing now 44
Newsletters within the past 4 years, covering up-to-date research reports from individual partners as well as general aspects and reports on General Assemblies.
In the remaining months, efforts will concentrate on a trial to set up an e-learning program to support public access to the experimental knowhow on assays available within the final Sens-it-iv toolbox
(see below). |
|
Furthermore, a final international congress to summarize the outcome of the Sens-it-iv project is scheduled for November 23-25, 2011 at the Crowne Plaza Airport Hotel in Brussels. A preliminary
program fort his meeting will be made available on the Sens-it-iv website in December 2010; the website can also be used to be kept informed about the congress and for registration (see www.sens-it-iv.eu). |
|
|
The Sens-it-iv Toolbox
The Sens-it-iv Toolbox (Figure 4) is the major deliverable of the project. It contains the most advanced and promising assays developed within or with contribution of Sens-it-iv, which are worth to
find financial support beyond the end of the project. |
|
Because, in contrast to the LLNA, lung and skin sensitizers very often are not detected by the same in vitro test system, different assay systems had to be developed for the two types of allergens. |
|
|
|
|
Figure 4. The Sens-it-iv Toolbox
Cell-based assays developed within the Sens-it-iv project for the assessment of lung and/or skin sensitizers |
|
|
Respiratory allergens
Precision Cut Lung Slices (PCLS)
As reported by Partner 12 (Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover), human PCLS represent the material of choice to correlate ex vivo chemical toxicity with in vitro data
(Switalla et al., 2010). The method is prone to verify in vitro detected induction of mediators and biomarkers under ex vivo conditions in human lung tissue. |
|
Moreover, the system allows to validate the significance of markers detected in proteomic or genomic analyses of cultured lung cells such as BEAS-2B (partner 14, Vito, Belgium). Initial studies on inter-laboratory transferability are under way. |
|
|
Cell culture methods
At present 4 different cell culture methods to test for respiratory allergens are under developmet
• An A549-based test to assess allergenicity of proteases (partner 1, Novozymes), which however is not applicable to other enzymes (Baccam, M. et al., 2010)
• An Alveolar-endothelial cell line model (Newsletter 43), developed at the University of Mainz, Germany in cooperation with partner 1 (Novozymes) (Figure 5) |
|
• An in house model employing primary human bronchial epidermal cells (Newsletter 43) developed at the University Medical Center, Leiden, Netherlands in cooperation with partner 1. Recently, the
primary cells have been replaced in this model by the bronchial cell line MucilAir. The characteristics of MucilAir are similar to the primary cells, but they exhibit a shelf life of up to 1 year, allowing to study both acute and chronic exposure.
• The BEAS-2B cell culture model (partner 14, Vito, Belgium) has been mentioned above under Biomarkers. |
|
|
|
|
Figure 5. Testing for
respiratory allergens:
Testing for respiratory allergens using an alveolar-endothelial cell line model consisting of alveolar (NCI-H441 cells, top compartment) and endothelial (ISO-HAS-1) cells (lower compartment). The allergenic potential of a
compound is determined using measurement of trans-epithelial resistance (TEE). |
|
|
Contact allergens
• The human epidermal equivalent (EE) assay (Dos Santos et al., 2010) determines the strength of irritancy of a compound by determining the concentration reducing the viability of a human epidermal equivalent
culture by 50% as well as by IL-1α secretion. This test does not distinguish between irritants and sensitizers. However, when applied on compounds identified as sensitizers in any of the tests below it allows to group
them according to their sensitizing potency. It can be performed with different commercial EE cultures. |
|
• The NCTC2544 IL-18 assay (Corsini et al., 2009) determines intracellular production of IL-18. The assay is selective for skin sensitizers including several pre- and pro-haptens and does not detect respiratory
sensitizers or irritants. A two-tiered assay (Newsletter 36) combining the NCTC and the EE tests allows for qualitative and quantitative characterization of skin allergens and is presently undergoing a multi-laboratory
pre-validation study (see above). |
|
|
| • The DC migration assay uses the Langerhans cell (LC) like maturation form of MUTZ-3 cells in a 2-chamber culture system (Ouwehand et al. 2010). The LC are fluorescently labelled and deposited in the upper chamber. Upon treatment with skin sensitizers or
irritants the cells migrate towards the recombinant chemokines CXCL12 or CCL5, respectively, and are quantified by fluorescence in the lower chamber. Technology transfer between 3 laboratories (Partners 9-VU University Medical Center,
Amsterdam, P11-University of the West of England, Bristol and P13-University of Milan) has been successfully established. |
|
| • The genomic prediction
assay. The number of about 200 genes found to be affected by skin sensitizers in MUTZ-3 cells (Newsletter 35) can be reduced to 10-20 without significant loss of selectivity. Such a final gene signature is presently
being tested for its robustness with the aim to use it on custom-made gene chips for the detection of potential allergens. |
|
|
| • The T cell priming
assay (Dietz, L. et al., 2010; Martin et al., 2010) addresses the final and specificity-determining steps of allergic sensitization, i.e. the hapten-specific activation of T cells. Developments of standard operation
procedures (SOP) for the preparation of effector, memory, and regulatory depleted human T cells, of short cuts for the generation of autologous dendritic cells and the application of haptens either free or coupled to human
serum albumin are quite advanced. For readout the determination of IFN-γ and TNF-α appears superior to T cell proliferation. |
|
| • The neutrophil IL-8
assay mentioned above may prove an important tool for the identification of chemically non-reactive pro-haptens. |
|
|
Conclusions
When the Sens-it-iv project will be terminated in March 2011 its contribution to reduction and replacement of animal experimentation for allergen testing will be twofold: First, the funding of basic research
in the field of skin and respiratory sensitization has led to significant contributions for a better understanding of the underlying cellular and molecular mechanisms. Secondly, on this basis Sens-it-iv can
deliver a number of promising assays for non-animal testing of allergenic sensitizers at various stages of development, the most advanced and promising of which are part of what is termed the Sens-it-iv Toolbox.
All of these methods will be made accessible to academic and industrial researchers. Although probably none of these tests on its own will be able to replace the present regulatory need for animal testing, combinations, also
with other in vitro methods, may eventually even surpass the informative value of the established animal tests.
However, none of all possible in vitro
replacements for the LLNA within or outside Sens-it-iv have yet gone through the rigid procedures for scientific validation and (hopefully) regulatory acceptance by national and
international authorities. This will require further financial input and time-consuming experimental efforts. |
|
Nevertheless, a number of these tests could be ready for non-regulatory use earlier, specifically when used for research purposes or in-house in the compound discovery and development process, resulting in a significant
reduction of experimental animals. The Sens-it-iv toolbox was already applied in a weight-of-evidence approach together with other data sources. This exercise demonstrated that Sens-it-iv tools
can be particular useful for the purpose of registration under REACH and classification under the CLP Regulation. The REACH Regulation allows for such use of weight-of-evidence for filling certain endpoints, in
accordance with Annex XI of the legislation and the corresponding ECHA guidance. Similarly, the CLP Regulation allows for weight-of-evidence with expert opinion as part of the classification of substances and mixtures. |
|
|
References
Baccam, M. et al., 2010. Initial interactions of protease allergens with airway epithelial cells: secretion of M-CSF. Toxicol In Vitro, submitted.
Corsini, E. et al., 2009. Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. Toxicol in Vitro 23: 789-96.
Dietz, L. et al., 2010. Tracking Human Contact allergens_From Mass Spectrometric Identification of Peptide-Bound Reactive Small Chemicals to Chemical-Specific Naive Human T-Cell Priming. Toxicol. Sciences 2010, 117:336-47.
Dos Santos, G.G. et al., 2010. A potential in vitro epidermal equivalent assay to determine sensitizer potency. Toxicol. In Vitro. Oct 19 [Epub ahead of print] PMID: 20940038 [PubMed - as supplied by publisher].
Martin, S.F. et al., 2008. Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity. J. Exp. Med. 205:2151-2162.
Martin, S.F. and Esser, P.R., 2010: Innate immune mechanisms in contact dermatitis and the resulting T cell responses. ALTEX 27: 293-295.
Martin, S.F., et al. 2010. T cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays. Cell. Mol. Life Sci. 67:4171-4184.
Megherbi, R. et al., 2009. Role of protein haptenation in triggering maturation events in the dendritic cell surrogate cell line THP1. Toxicol. Appl. Pharmacol. 2009, 238: 120-32. |
|
Mitjans, M. et al., 2010. Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro. Toxicol. In Vitro 24(6):1803-9 doi:10.1016/j.tiv.2010.06.001
Ouwehand, K. et al., 2010. Epidermis-to-dermis migration of immature Langerhans cells upon topical irritant exposure is dependent on CCL2 and CCL5. Eur. J. Immunol. 40(7):2026-34.
Schmidt, M. et al., 2010. A crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel. Nat. Immunol. 11:814-819. doi:10.1038/ni.1919.
Switalla, S. et al., 2010. Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices. Toxicol Appl Pharmacol. 246(3): 107-115.
Weber F.C., et al. 2010. Lack of the purinergic receptor P2X7 results in resistance to contact hypersensitivity. J. Exp. Med. 207:2609-2619.
MucilAir™: A Premium 3D Human Airway Epithelia reconstituted in vitro with Long Shelf-Life
http://www.epithelix.com/
For a full list of publications generated within the Sens-it-iv project, please see the website: www.sens-it-iv.eu |
| |
|
|
For more information
For more information on the scientific results and tests developed, please contact the persons mentioned in the accompanying Newsletters (see text for reference). The Newsletters can be downloaded from the Newsletter archive on the Sens-it-iv website (www.sens-it-iv.eu) |
|
For more information
For general questions on the end results of
the Sens-it-iv project, please contact the
co-ordinator Dr. Erwin Roggen
elro@novozymes.com
or the vice-co-ordinator,
Prof Hans-Ulrich Weltzien
(huweltzien@yahoo.de). |
|
