The GARD assay – assessment of skin sensitizing chemicals · The GARD assay – assessment of...
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The GARD assay – assessment of skin sensitizing chemicals Sandra Reynoso Paz 1 , Andy Forreryd 2 , Kathrin S. Zeller 2 , Tim Lindberg 2 , Olivia Larne 3 , Brian Rogers 1 , Ann-Sofie Albrekt 2 , Aakash Chawade 2 , Henrik Johansson 3 and Malin Lindstedt 2 1 SenzaGen Inc., Davis, CA, USA, 2 Department of Immunotechnology, Lund University, Lund, Sweden, 3 SenzaGen AB, Lund, Sweden The GARD assay is based of a human myeloid cell similar to DCs, which are immunologically active during sensitization. The GARD protocol include a cell line stimulation of the compound of interest and after incubation the transcripts are harvested. The gene expression is analysed through a bioinformatics multivariate technology (1). A brief overview of the GARD process During development of the GARD applications, total gene expression is analysed by microarrays, but after gene signature establishment, assays are transferred to the Nanostring platform (2). Depending on requested end-point data the GARD platform can be transformed to different applications. Today, GARDskin for skin sensitizers and GARDair for respiratory sensitizers are developed (3). THE GARD PLATFORM Depending on selected end-point gene expression panel measured the GARD platform is flexible. The GARDskin assay specifically assess the hazard of skin sensitizers estimating the expression of ~200 genes. The accuracy has been determined to ~90% analyzing 26 blinded chemicals (4). For comparison the accuracy of LLNA has been estimated to 72%. An example of tested chemicals is provided in table 1. A method evaluation on further chemicals provided by Cosmetics Europe Skin Task Force has been executed and the results are confirming that accuracies are constantly high (manuscript in preparation). Table 1. Examples of chemicals tested by GARDskin and LLNA. Blue = correct classification, pink = incorrect classification. GARDskin The cells are exposed to the substance of interest. 5 3 6 1 2 4 Their genomic products (transcripts) are isolated. The gene transcripts are quantified. Final report. The readout is processed to assess sensitizing ability. Human immunologically relevant cells are used. GARDskin in vitro model assess skin sensitizers genomic assay end-point measurement of ~200 genes Accuracy: ~90%. RESUME During skin sensitization dendritic cells (DCs) are key players linking the innate and acquired immune system. Located in dermis, they can get activated by foreign substances penetrating the skin or by molecules secreted by surrounding cells already stimulated by the foreign substance. THE BIOLOGY BEHIND Epidermis Dermis Blood vessel A dendritic cell activated by a foreign substance. Upon activation, their gene expression profile is changed, which is measurable by genomic techniques. The following alteration of the proteome facilitates a multitude of cell modifications. The DCs matures and their migratory capacity increase promoting their escape towards lymph nodes where they ultimately signaling to T cells to proliferate and defense the host against the chemical substance. References: 1) Johansson et al. BMC Genomics, 2011 2) Forreryd et al. BMC Genomics, 2014 3) Forreryd et al. PloSOne, 2015 4) Johansson et al. Toxicol Sci, 2014 Chemicals have significantly improved our welfare, however severe symptoms can arise in exposed persons. An adverse reaction is Allergic Contact Dermatitis (ACD), that can result after repeated contact. To decrease present chemicals inducing ACD (skin sensitizers), chemicals have to be tested. Previous tests included animals, but EU legislation, OECD test guidelines, TSCA and EPA today prioritize the use and development of alternative methods. Genomic Allergen Rapid Detection (GARD) is an in vitro assay for assessment of chemicals’ sensitizing capacity. GARD is based on a human cell line that is exposed to the chemical of interest and the as a gene expression panel is the end- point measurement. INTRODUCTION Chemical LLNA GARDskin Dinitrochlorobenzene (DNCB) extreme sensitizer sensitizer Cinnamal moderate sensitizer sensitizer Benzalkoniumchloride non-sensitizer sensitizer 7-Hydroxycitronella weak sensitizer sensitizer Phenyl Benzoate weak sensitizer non-sensitizer DMSO weak sensitizer non-sensitizer Xylene weak sensitizer non-sensitizer Menthol non-sensitizer non-sensitizer Salicylic acid non-sensitizer non-sensitizer Sodium lauryl sulphate (SLS) weak sensitizer non-sensitizer
Transcript of The GARD assay – assessment of skin sensitizing chemicals · The GARD assay – assessment of...
The GARD assay – assessment of skin sensitizing chemicalsSandra Reynoso Paz1, Andy Forreryd2, Kathrin S. Zeller2, Tim Lindberg2, Olivia Larne3, Brian Rogers1,
Ann-Sofie Albrekt2, Aakash Chawade2, Henrik Johansson3 and Malin Lindstedt2
1SenzaGen Inc., Davis, CA, USA, 2Department of Immunotechnology, Lund University, Lund, Sweden, 3SenzaGen AB, Lund, Sweden
The GARD assay is based of a human myeloid cell similar to DCs, which are immunologically active during sensitization.The GARD protocol include a cell line stimulation of the compound of interest and after incubation the transcripts areharvested. The gene expression is analysed through a bioinformatics multivariate technology (1).
A brief overview of the GARD process
During development of the GARD applications, total gene expression is analysed by microarrays, but after genesignature establishment, assays are transferred to the Nanostring platform (2).
Depending on requested end-point data the GARD platform can be transformed to different applications. Today,GARDskin for skin sensitizers and GARDair for respiratory sensitizers are developed (3).
THE GARD PLATFORM
Introduction
Depending on selected end-point gene expressionpanel measured the GARD platform is flexible.The GARDskin assay specifically assess thehazard of skin sensitizers estimating theexpression of ~200 genes.The accuracy has been determined to ~90%analyzing 26 blinded chemicals (4). Forcomparison the accuracy of LLNA has beenestimated to 72%. An example of tested chemicalsis provided in table 1.A method evaluation on further chemicalsprovided by Cosmetics Europe Skin Task Forcehas been executed and the results are confirmingthat accuracies are constantly high (manuscript inpreparation).Table 1. Examples of chemicalstested by GARDskin and LLNA. Blue = correct classification,pink = incorrect classification.
GARDskin
The cells are exposed to the substance of interest.
53 61 2 4
Their genomic products (transcripts) are isolated.
The gene transcripts are quantified.
Final report.The readout is processed to assess sensitizing ability.
Human immunologically relevant cells are used.
GARDskin in vitro model assess skin sensitizers genomic assay end-point measurement of ~200 genes Accuracy: ~90%.
RESUME
Introduction
During skin sensitization dendritic cells (DCs) are key playerslinking the innate and acquired immune system. Located indermis, they can get activated by foreign substancespenetrating the skin or by molecules secreted by surroundingcells already stimulated by the foreign substance.
THE BIOLOGY BEHIND
Epidermis
Dermis
Blood vessel
A dendritic cell activated by a foreign substance.
Upon activation, their geneexpression profile is changed, whichis measurable by genomictechniques. The following alterationof the proteome facilitates amultitude of cell modifications. TheDCs matures and their migratorycapacity increase promoting theirescape towards lymph nodes wherethey ultimately signaling to T cells toproliferate and defense the hostagainst the chemical substance.
References:1) Johansson et al. BMC Genomics, 20112) Forreryd et al. BMC Genomics, 20143) Forreryd et al. PloSOne, 20154) Johansson et al. Toxicol Sci, 2014
Introduction
Chemicals have significantly improved ourwelfare, however severe symptoms can arise inexposed persons. An adverse reaction is AllergicContact Dermatitis (ACD), that can result afterrepeated contact. To decrease present chemicalsinducing ACD (skin sensitizers), chemicals have tobe tested. Previous tests included animals, but EUlegislation, OECD test guidelines, TSCA and EPAtoday prioritize the use and development ofalternative methods.Genomic Allergen Rapid Detection (GARD) is an invitro assay for assessment of chemicals’sensitizing capacity. GARD is based on a humancell line that is exposed to the chemical of interestand the as a gene expression panel is the end-point measurement.
INTRODUCTION
Chemical LLNA GARDskin
Dinitrochlorobenzene (DNCB) extreme sensitizer sensitizer
Cinnamal moderate sensitizer sensitizer
Benzalkoniumchloride non-sensitizer sensitizer
7-Hydroxycitronella weak sensitizer sensitizer
Phenyl Benzoate weak sensitizer non-sensitizer
DMSO weak sensitizer non-sensitizer
Xylene weak sensitizer non-sensitizer
Menthol non-sensitizer non-sensitizer
Salicylic acid non-sensitizer non-sensitizer
Sodium lauryl sulphate (SLS) weak sensitizer non-sensitizer
